MSC-EVs-CHIP have been shown to promote Smad2/3 degradation, anti-fibrotic-like change of renal tubular cell, all of which play a critical role in the development and procession of RIF. We further evaluated the therapeutic efficacy of MSC-EVs-CHIP in RIF rat model established by unilateral UUO rat model. First, we assessed the organizational distribution of MSC-EVs-CHIP. CM-DIR labeled MSC-EVs and MSC-EVs-CHIP were injected into rat intravenously for 48 h, then the organ fluorescence was evaluated by PerkinElmer IVIS Lumina II ex vivo imaging. As shown in Figure ##SUPPL##0##S4##, MSC-EVs and MSC-EVs-CHIP was enriched in liver, hardly enriched in the kidney of control groups, while accumulated in kidney of UUO treated rat (Supplementary Fig. ##SUPPL##0##4##). In addition, the fluorescence imaging of the kidney tissue of RIF-rat receiving MSC-EVs-CHIP exhibited significant CM-DIR signals relative to the control (Supplementary Fig. ##SUPPL##0##5a##). Western blotting analysis further showed that MSC-EVs treatment resulted in a high CHIP level in kidney tissue (Supplementary Fig. ##SUPPL##0##5b##). These data suggested that under RIF pathological conditions, MSC-EVs-CHIP have an ability to target the injury kidney.</p>","<p id=\"Par10\">We next investigated whether MSC-EVs-CHIP promote smad2/3 degradation and inhibits inflammation in the kidney of RIF model. Rats were raised for 7 days after unilateral ureteral obstruction, and were then intravenously treated with MSC-EVs or MSC-EVs-CHIP (2 mg per rat/every three days). After one week, we performed immunofluorescence analysis of the renal tissue. In UUO rats, Smad2/3 is highly expressed in the nucleus of renal tubular epithelial cells, and fibrosis-related proteins (Collagen I, α-SMA) are significantly increased (Supplementary Fig. ##SUPPL##0##6##). While the renal of rat receiving MSC-EVs-CHIP, we observed a significantly reduced expression of Smad2/3 in renal tubular nucleus compared to MSC-EVs treatment, suggesting the downregulation of Smad2/3 in the renal tissue of RIF rat (Fig. ##FIG##2##3a##). In addition, the fibrosis related indicators α-SMA and Fibronectin in the kidneys of RIF rats receiving MSC-EVs-CHIP were significantly reduced (Fig. ##FIG##2##3b##). We further observed the reduced Smad2/3 accumulation and tubular cells fibrotic like change in the renal of MSC-EVs-CHIP-treated rat by immunofluorescence analysis, suggesting that MSC-EVs-CHIP can significantly rescue RIF pathology by promoting Smad2/3 degradation and recovering normal homeostatic processes (Fig. ##FIG##2##3c, d##).</p>","<p id=\"Par11\">Given the critical role of inflammation in the progression of RIF, its effect on inflammation in renal was also evaluated after MSC-EVs-CHIP intervene. Persistent mechanical damage can stimulate the inflammatory response in tubular cells, especially the CD66 positive neutrophil accumulation, as shown in the HE staining and immunofluorescence (Fig. ##FIG##2##3e–g##). Encouragingly, the induced inflammation and cytokine (IL-6, IL-1β, TNF-α) release was remarkably inhibited by MSC-EVs-CHIP treatment (Fig. ##FIG##2##3h##). These data suggested that CHIP overexpression mediated by MSC-EVs inhibited Smad2/3 and decreased fibrosis index, further remitted the inflammation in RIF-rat.</p>","<title>MSC-EVs-CHIP reduced interstitial collagen deposition in UUO model</title>","<p id=\"Par12\">Inject the prepared MSC-EVs-CHIP and MSC-EVs into the tail vein of the UUO model, and collect large mouse notebooks from each group on the 15th day. The biochemical analyzer detected renal function indicators such as creatinine and urea nitrogen, and found that the MSC-EVs-CHIP group significantly downregulated creatinine and urea nitrogen compared to the MSC-EVs group, significantly improving renal function (Fig. ##FIG##3##4a, b##). The appearance of kidneys in the UUO group significantly larger and lighter in color, while the kidney tissue significantly returned to control after MSC-EVs-CHIP treatment (Fig. ##FIG##3##4c##). Further through the HE staining results of renal tissue pathology showed that the UUO injury group had an increase in tubular vacuolar degeneration, a significant thickening of the glomerular basement membrane, and a tissue damage score of 70%. Compared with the MSC-EVs group, the MSC-EVs-CHIP group restored normal renal tissue structure after intervention, and reduced tubular vacuolar degeneration. The damage score was reduced to 18% (Fig. ##FIG##3##4d, e##). The results of Sirius red staining and Masson staining showed significant fibrosis in UUO renal tissue. The change area reached 64%, while the MSC-EVs-CHIP group showed a significant reduction in renal interstitial collagen fibers and a significant decrease in fibrosis degree after treatment, which was better than the MSC-EVs treated group alone (Fig. ##FIG##3##4f-h##). Extract kidney tissue proteins from each group for Western blot detection, displaying fibrosis related proteins Fibronectin, Collagen I, and α-SMA were significantly reduced (Fig. ##FIG##3##4i##). The above research results indicate that high expression of CHIP enhances MSC-EVs in improving renal function and reducing renal interstitial collagen.</p>","<title>In vivo renal-targeting and antifibrotic therapeutic effect of SPION-EVs</title>","<p id=\"Par13\">In order to further enhance the anti-fibrotic effect of MSC-EVs, we used the SPION with surface modified transferrin (Tf) to combine with the transferrin receptor on the surface of MSC EVs CHIP for engineering modification to enhance its ability to target the injured site. Scanning electron microscope observation shows that the size of the nanospheres is about 5 nm, and dynamic light scattering shows that the size of the nanoparticle is about 10 nm (Fig. ##FIG##4##5a##). The superparamagnetic modification of transferrin binds to the transferrin receptor on the surface of the MSC-EVs-CHIP membrane after co incubation at 4 °C for 4 h, and SPION-EVs are collected and dissolved in PBS by external magnetic field adsorption (Fig. ##FIG##4##5b##). The elliptical vesicle structure combined with multiple SPION was observed by transmission electron microscopy (Fig. ##FIG##4##5c##). Analyze the stability and magnetic characteristics of SPION-EVs in aqueous solutions using hysteresis loops and PDI polymer dispersion index (Fig. ##FIG##4##5d, e##). The average particle size of SPION-EVs measured by dynamic light scattering (DLS) is 115 ± 11.3 nm, compared to MSC-EVs-CHIP, the average particle diameter increases by about 7 nm, indicating that SPION not obvious change the size of extracellular vesicles (Fig. ##FIG##4##5f##). The zeta potential of SPION-EVs was ≈−16 mV (Supplementary Fig. ##SUPPL##0##7##). Western blot was used to detect the expression of surface positive marker proteins CD9, CD81, Alix and negative marker Calnexin in SPION-EVs, and strong positive expression of CHIP and transferrin receptor TfR (Fig. ##FIG##4##5g##). This indicates that we have successfully prepared engineered SPION-EVs.</p>","<p id=\"Par14\">Construct the renal fibrosis treatment model using prepared engineered SPION-EVs under external magnetic field intervention (Fig. ##FIG##4##5h##). By using a small animal live imaging system, it was shown that under the action of an external magnetic field, the signal intensity of DIR labeled SPION-EVs was enriched by targeting the liver to the kidneys (Fig. ##FIG##4##5i##). The pathological tissue showed a decrease in tubular vacuolar degeneration and a return to normal structure in the SPION-EVs intervention group. Sirius red and Masson staining confirmed that compared to the MSC-EVs -CHIP group, the SPION-EVs group showed a decrease in interstitial collagen fibers and a significant decrease in fibrotic areas (Fig. ##FIG##4##5j##). Immunofluorescence showed that SPION-EVs significantly inhibited the nuclear uptake of Smad2/3 in renal tubular cells and decreased α-SMA was superior to the MSC-EVs-CHIP group (Fig. ##FIG##4##5k##). The above results indicate that the targeted enrichment of engineered SPION-EVs under magnetic field significantly enhances the anti-fibrotic effect.</p>","<title>SPION-EVs attenuated renal interstitial fibrosis in the DKD model</title>","<p id=\"Par15\">To further prove that SPION-EVs is a promising nanoplatform for the delivery of CHIP and treatment of renal fibrosis, we constructed another renal interstitial fibrosis model in diabetic kidney disease (DKD) rat. The DKD injury model was established by feeding high-fat combined with streptozotocin (STZ) for 18 weeks, intervened by tail vein injection of SPION-EVs. Firstly, we assessed the alleviation of SPION-EVs against interstitial fibrosis in DKD rat. As expected, histological analysis of kidney sections at 24 weeks after STZ injection revealed significant tubulointerstitial damage, including tubular atrophy, cast formation, infiltrates of leukocytes, and fibrosis, all of which were markedly ameliorated by SPION-EVs treatment (Fig. ##FIG##5##6a, b##). To fully assess the efficacy of SPION-EVs, kidney sections were subjected to Masson and Sirius red staining to determine the fibrosis index and stained with anti-α-SMA/Fibronectin antibody for the detection of fibrosis-like tubules. The results demonstrated that the renal fibrosis was reduced in DKD rat treated with SPION-EVs (Fig. ##FIG##5##6c–f##), and reduction of α-SMA, Collagen I, and Fibronectin deposition in renal tissues of SPION-EVs treated DKD rat (Fig. ##FIG##5##6g–j##). Moreover, immunofluorescence shows that SPION-EVs can significantly inhibit the nuclear expression of Smad2/3 in renal tubules stimulated by high glucose environment and reduce α-SMA (Fig. ##FIG##5##6k##). These data suggested that SPION-EVs could attenuate renal fibrosis and retarding the progress of DKD (Fig. ##FIG##6##7##).</p>","<title>Assessment of toxicity and inflammation in SPION-EVs–treated rat model</title>","<p id=\"Par16\">We next examined the concentrations of hepatic enzyme such as alanine aminotransferase and aspartate aminotransferase in the serum of SPION-EVs treated rats showed no obvious differences from those of phosphate-buffered saline (PBS) treated rats (Supplementary Fig. ##SUPPL##0##8a##). Similarly, SPION-EVs and MSC-EVs-CHIP group toxicity on the major organs, including the heart, liver, spleen and lung. The histological analysis showed no evidence of in vivo toxicity of SPION-EVs (Supplementary Fig. ##SUPPL##0##8b##). Consistent with previous studies that engineered cell-derived EVs are biocompatible and not significantly toxic.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Characteristics of MSC derived extracellular vesicles with CHIP high expression</title>\",\n \"<p id=\\\"Par6\\\">Umbilical derived MSCs with high purity were isolated from rat according to previous protocols (Supplementary Fig. ##SUPPL##0##1##). We next infected MSCs with lentivirus encoding the CHIP gene and selected with puromycin to obtain CHIP high-expressing MSCs, and confocal graph shows that CHIP is mainly expressed in the cytoplasm (Fig. ##FIG##0##1a, b##). The morphology of MSCs was not affected by high CHIP expression levels (Fig. ##FIG##0##1c##), lipogenesis and osteogenesis experiment slightly induced stem-cell differentiation compared to non-transfected MSCs (Supplementary Fig. ##SUPPL##0##2##). Then MSCs derived EVs were then isolated and purified according to previously described protocols. Transmission electron microscopy (TEM) imaging shows then cup shaped vesicle morphology of MSC-EVs-CHIP (Fig. ##FIG##0##1d##). And the average particle diameter of MSC-EVs-CHIP measured by dynamic light scattering (DLS) is 108 ± 10.6 nm (Fig. ##FIG##0##1e##). The zeta potential of MSC-EVs-CHIP was ≈−15 mV (Fig. ##FIG##0##1f##). Finally, we confirmed the expression of CHIP in MSCs-EVs by western blotting assay. The results showed that CHIP was also highly expressed in engineered EVs containing EVs-associated proteins, such as CD63, CD9, Alix and Calnexin was expressed negatively (Fig. ##FIG##0##1g##), and GFP signals was also found in EVs-CHIP. These results suggested that the genetically engineered MSC-EVs with high CHIP expression were successfully prepared.</p>\",\n \"<title>MSC-EVs-CHIP reduced fibrotic change by inducing Smad2/3 degradation in renal tubular cells</title>\",\n \"<p id=\\\"Par7\\\">Evidence suggests that activation of the Smad2/3 pathway and inflammatory infiltration exacerbate fibrosis in CKD. To test whether MSC-EVs-CHIP induces Smad2/3 degradation and reduce renal interstitial fibrosis, MSC-EVs with GFP-tagged CHIP high expression were incubated with renal tubular cells NRK-52E. As shown by confocal imaging, a high GFP fluorescence signal was observed in NRK-52E (Fig. ##FIG##1##2a##). Imaging flow cytometry showed that GFP MSC-EVs-CHIP can be internalized by NE-52E cells (Fig. ##FIG##1##2b##). Western blot experiment confirmed high expression of CHIP in NRK-52E (Fig. ##FIG##1##2c##). Furthermore, the GFP signal was colocalized with Smad2/3 (Red) in the cells, suggesting that CHIP shifted into the cytoplasm with MSC-EVs (Fig. ##FIG##1##2d##).</p>\",\n \"<p id=\\\"Par8\\\">Next, we used fibrosis stimulating factor TGF-β<sub>1</sub> (10 nM) to induce fibrosis-like change in NRK-52E cells and establish in vitro RIF model. In our experiment, it was found that TGF-β<sub>1</sub> stimulates α-SMA overexpress and transform into myofibroblasts cells (Supplementary Fig. ##SUPPL##0##3a##), MSC-EVs-CHIP significantly inhibited Smad2/3 and fibrotic of TGF-β<sub>1</sub> incubated cells, as shown by the confocal microphage and western blot (Fig. ##FIG##1##2e, f##). Additionally, Co-immunoprecipitation observed that MSC-EVs-CHIP promoted ubiquitin molecules expression and decreased the expression of Smad2/3 in TGF-β<sub>1</sub>-incubated cells (Fig. ##FIG##1##2g##). In order to simulate the mechanical pressure environment in <italic>vivo</italic> by using stiff gel (30Kpa) to stimulate NRK-52E (Fig. ##FIG##1##2h##, Supplementary Fig. ##SUPPL##0##3b##), confocal microscopy also showed that the number of Smad2/3 in nucleus was decreased obviously in NK-52E cells treated with MSCEVs-CHIP relative to that in controls (Fig. ##FIG##1##2i##), and statistical analysis confirms the results (Fig. ##FIG##1##2j##). These results demonstrated that MSC-EVs-CHIP could alleviate TGF-β<sub>1</sub> and stiff gel induced renal tubular cell fibrotic like change by promoting Smad2/3 ubiquitination degradation.</p>\",\n \"<title>MSC-EVs-CHIP migrated to the kidney and ameliorated inflammatory infiltration by reduced Smad2/3 accumulation</title>\",\n \"<p id=\\\"Par9\\\">MSC-EVs-CHIP have been shown to promote Smad2/3 degradation, anti-fibrotic-like change of renal tubular cell, all of which play a critical role in the development and procession of RIF. We further evaluated the therapeutic efficacy of MSC-EVs-CHIP in RIF rat model established by unilateral UUO rat model. First, we assessed the organizational distribution of MSC-EVs-CHIP. CM-DIR labeled MSC-EVs and MSC-EVs-CHIP were injected into rat intravenously for 48 h, then the organ fluorescence was evaluated by PerkinElmer IVIS Lumina II ex vivo imaging. As shown in Figure ##SUPPL##0##S4##, MSC-EVs and MSC-EVs-CHIP was enriched in liver, hardly enriched in the kidney of control groups, while accumulated in kidney of UUO treated rat (Supplementary Fig. ##SUPPL##0##4##). In addition, the fluorescence imaging of the kidney tissue of RIF-rat receiving MSC-EVs-CHIP exhibited significant CM-DIR signals relative to the control (Supplementary Fig. ##SUPPL##0##5a##). Western blotting analysis further showed that MSC-EVs treatment resulted in a high CHIP level in kidney tissue (Supplementary Fig. ##SUPPL##0##5b##). These data suggested that under RIF pathological conditions, MSC-EVs-CHIP have an ability to target the injury kidney.</p>\",\n \"<p id=\\\"Par10\\\">We next investigated whether MSC-EVs-CHIP promote smad2/3 degradation and inhibits inflammation in the kidney of RIF model. Rats were raised for 7 days after unilateral ureteral obstruction, and were then intravenously treated with MSC-EVs or MSC-EVs-CHIP (2 mg per rat/every three days). After one week, we performed immunofluorescence analysis of the renal tissue. In UUO rats, Smad2/3 is highly expressed in the nucleus of renal tubular epithelial cells, and fibrosis-related proteins (Collagen I, α-SMA) are significantly increased (Supplementary Fig. ##SUPPL##0##6##). While the renal of rat receiving MSC-EVs-CHIP, we observed a significantly reduced expression of Smad2/3 in renal tubular nucleus compared to MSC-EVs treatment, suggesting the downregulation of Smad2/3 in the renal tissue of RIF rat (Fig. ##FIG##2##3a##). In addition, the fibrosis related indicators α-SMA and Fibronectin in the kidneys of RIF rats receiving MSC-EVs-CHIP were significantly reduced (Fig. ##FIG##2##3b##). We further observed the reduced Smad2/3 accumulation and tubular cells fibrotic like change in the renal of MSC-EVs-CHIP-treated rat by immunofluorescence analysis, suggesting that MSC-EVs-CHIP can significantly rescue RIF pathology by promoting Smad2/3 degradation and recovering normal homeostatic processes (Fig. ##FIG##2##3c, d##).</p>\",\n \"<p id=\\\"Par11\\\">Given the critical role of inflammation in the progression of RIF, its effect on inflammation in renal was also evaluated after MSC-EVs-CHIP intervene. Persistent mechanical damage can stimulate the inflammatory response in tubular cells, especially the CD66 positive neutrophil accumulation, as shown in the HE staining and immunofluorescence (Fig. ##FIG##2##3e–g##). Encouragingly, the induced inflammation and cytokine (IL-6, IL-1β, TNF-α) release was remarkably inhibited by MSC-EVs-CHIP treatment (Fig. ##FIG##2##3h##). These data suggested that CHIP overexpression mediated by MSC-EVs inhibited Smad2/3 and decreased fibrosis index, further remitted the inflammation in RIF-rat.</p>\",\n \"<title>MSC-EVs-CHIP reduced interstitial collagen deposition in UUO model</title>\",\n \"<p id=\\\"Par12\\\">Inject the prepared MSC-EVs-CHIP and MSC-EVs into the tail vein of the UUO model, and collect large mouse notebooks from each group on the 15th day. The biochemical analyzer detected renal function indicators such as creatinine and urea nitrogen, and found that the MSC-EVs-CHIP group significantly downregulated creatinine and urea nitrogen compared to the MSC-EVs group, significantly improving renal function (Fig. ##FIG##3##4a, b##). The appearance of kidneys in the UUO group significantly larger and lighter in color, while the kidney tissue significantly returned to control after MSC-EVs-CHIP treatment (Fig. ##FIG##3##4c##). Further through the HE staining results of renal tissue pathology showed that the UUO injury group had an increase in tubular vacuolar degeneration, a significant thickening of the glomerular basement membrane, and a tissue damage score of 70%. Compared with the MSC-EVs group, the MSC-EVs-CHIP group restored normal renal tissue structure after intervention, and reduced tubular vacuolar degeneration. The damage score was reduced to 18% (Fig. ##FIG##3##4d, e##). The results of Sirius red staining and Masson staining showed significant fibrosis in UUO renal tissue. The change area reached 64%, while the MSC-EVs-CHIP group showed a significant reduction in renal interstitial collagen fibers and a significant decrease in fibrosis degree after treatment, which was better than the MSC-EVs treated group alone (Fig. ##FIG##3##4f-h##). Extract kidney tissue proteins from each group for Western blot detection, displaying fibrosis related proteins Fibronectin, Collagen I, and α-SMA were significantly reduced (Fig. ##FIG##3##4i##). The above research results indicate that high expression of CHIP enhances MSC-EVs in improving renal function and reducing renal interstitial collagen.</p>\",\n \"<title>In vivo renal-targeting and antifibrotic therapeutic effect of SPION-EVs</title>\",\n \"<p id=\\\"Par13\\\">In order to further enhance the anti-fibrotic effect of MSC-EVs, we used the SPION with surface modified transferrin (Tf) to combine with the transferrin receptor on the surface of MSC EVs CHIP for engineering modification to enhance its ability to target the injured site. Scanning electron microscope observation shows that the size of the nanospheres is about 5 nm, and dynamic light scattering shows that the size of the nanoparticle is about 10 nm (Fig. ##FIG##4##5a##). The superparamagnetic modification of transferrin binds to the transferrin receptor on the surface of the MSC-EVs-CHIP membrane after co incubation at 4 °C for 4 h, and SPION-EVs are collected and dissolved in PBS by external magnetic field adsorption (Fig. ##FIG##4##5b##). The elliptical vesicle structure combined with multiple SPION was observed by transmission electron microscopy (Fig. ##FIG##4##5c##). Analyze the stability and magnetic characteristics of SPION-EVs in aqueous solutions using hysteresis loops and PDI polymer dispersion index (Fig. ##FIG##4##5d, e##). The average particle size of SPION-EVs measured by dynamic light scattering (DLS) is 115 ± 11.3 nm, compared to MSC-EVs-CHIP, the average particle diameter increases by about 7 nm, indicating that SPION not obvious change the size of extracellular vesicles (Fig. ##FIG##4##5f##). The zeta potential of SPION-EVs was ≈−16 mV (Supplementary Fig. ##SUPPL##0##7##). Western blot was used to detect the expression of surface positive marker proteins CD9, CD81, Alix and negative marker Calnexin in SPION-EVs, and strong positive expression of CHIP and transferrin receptor TfR (Fig. ##FIG##4##5g##). This indicates that we have successfully prepared engineered SPION-EVs.</p>\",\n \"<p id=\\\"Par14\\\">Construct the renal fibrosis treatment model using prepared engineered SPION-EVs under external magnetic field intervention (Fig. ##FIG##4##5h##). By using a small animal live imaging system, it was shown that under the action of an external magnetic field, the signal intensity of DIR labeled SPION-EVs was enriched by targeting the liver to the kidneys (Fig. ##FIG##4##5i##). The pathological tissue showed a decrease in tubular vacuolar degeneration and a return to normal structure in the SPION-EVs intervention group. Sirius red and Masson staining confirmed that compared to the MSC-EVs -CHIP group, the SPION-EVs group showed a decrease in interstitial collagen fibers and a significant decrease in fibrotic areas (Fig. ##FIG##4##5j##). Immunofluorescence showed that SPION-EVs significantly inhibited the nuclear uptake of Smad2/3 in renal tubular cells and decreased α-SMA was superior to the MSC-EVs-CHIP group (Fig. ##FIG##4##5k##). The above results indicate that the targeted enrichment of engineered SPION-EVs under magnetic field significantly enhances the anti-fibrotic effect.</p>\",\n \"<title>SPION-EVs attenuated renal interstitial fibrosis in the DKD model</title>\",\n \"<p id=\\\"Par15\\\">To further prove that SPION-EVs is a promising nanoplatform for the delivery of CHIP and treatment of renal fibrosis, we constructed another renal interstitial fibrosis model in diabetic kidney disease (DKD) rat. The DKD injury model was established by feeding high-fat combined with streptozotocin (STZ) for 18 weeks, intervened by tail vein injection of SPION-EVs. Firstly, we assessed the alleviation of SPION-EVs against interstitial fibrosis in DKD rat. As expected, histological analysis of kidney sections at 24 weeks after STZ injection revealed significant tubulointerstitial damage, including tubular atrophy, cast formation, infiltrates of leukocytes, and fibrosis, all of which were markedly ameliorated by SPION-EVs treatment (Fig. ##FIG##5##6a, b##). To fully assess the efficacy of SPION-EVs, kidney sections were subjected to Masson and Sirius red staining to determine the fibrosis index and stained with anti-α-SMA/Fibronectin antibody for the detection of fibrosis-like tubules. The results demonstrated that the renal fibrosis was reduced in DKD rat treated with SPION-EVs (Fig. ##FIG##5##6c–f##), and reduction of α-SMA, Collagen I, and Fibronectin deposition in renal tissues of SPION-EVs treated DKD rat (Fig. ##FIG##5##6g–j##). Moreover, immunofluorescence shows that SPION-EVs can significantly inhibit the nuclear expression of Smad2/3 in renal tubules stimulated by high glucose environment and reduce α-SMA (Fig. ##FIG##5##6k##). These data suggested that SPION-EVs could attenuate renal fibrosis and retarding the progress of DKD (Fig. ##FIG##6##7##).</p>\",\n \"<title>Assessment of toxicity and inflammation in SPION-EVs–treated rat model</title>\",\n \"<p id=\\\"Par16\\\">We next examined the concentrations of hepatic enzyme such as alanine aminotransferase and aspartate aminotransferase in the serum of SPION-EVs treated rats showed no obvious differences from those of phosphate-buffered saline (PBS) treated rats (Supplementary Fig. ##SUPPL##0##8a##). Similarly, SPION-EVs and MSC-EVs-CHIP group toxicity on the major organs, including the heart, liver, spleen and lung. The histological analysis showed no evidence of in vivo toxicity of SPION-EVs (Supplementary Fig. ##SUPPL##0##8b##). Consistent with previous studies that engineered cell-derived EVs are biocompatible and not significantly toxic.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par17\">In this study, we reported a method for manufacturing engineered SPION-EVs and evaluated the feasibility, safety, and effectiveness of MSC-EVs with CHIP overexpressed in treating renal interstitial fibrosis in UUO and DKD models. Impressively, provide packed CHIP protein into MSC-derived EVs by genetic engineering with lentivirus transfection (Fig. ##FIG##0##1##), then through nanomaterials SPION decorated MSC-EVs with CHIP overexpressing target and enrich the damaged kidneys, resulting in notable renal protective effects, specifically by promoting Smad2/3 ubiquitination and degradation to reverse the transformation of renal tubular cells into myofibroblast. Our findings not only demonstrate the role of engineered MSC extracellular vesicles in the anti-fibrotic activity, but also as a new renal fibrosis therapeutic agent and target delivery nanoplatform. In a word, engineered SPION-EVs constitute an effective nanotherapeutic for the renal fibrosis treatment.</p>","<p id=\"Par18\">Over the past several years, the clinical studies of RIF therapies have concentrated on antifibrotic, including anti-fibrosis antibodies and YAP inhibitors<sup>##REF##32382019##24##,##REF##37328872##25##</sup>. Unfortunately, these treatment strategies have not achieved good results and were not significantly reduce fibrosis in clinical tests. Furthermore, the pathogenesis of RIF is complex and includes inflammation, tubular injury, oxidative stress and metabolic dysfunction<sup>##REF##28388958##26##</sup>. There is increasing evidence that Smad2/3 ubiquitination and degradation in renal interstitial is a promising treatment method for CKD therapy<sup>##UREF##3##27##</sup>. we developed an SPION decorated engineered MSC-EVs platform by CHIP expression to reduced renal fibrosis and inflammatory infiltration in RIF rats. In vitro, we observed that MSC-EVs-CHIP inhibit Smad2/3 expression in rat renal tubular epithelial cells and relieved cells myofibroblast change induced by TGF-β<sub>1</sub>, and inhibited inflammasome activation. In UUO and DKD rat model, MSC-EVs-CHIP promoted Smad2/3 ubiquitination and degradation, further alleviated tubular cells fibrosis and inflammation.</p>","<p id=\"Par19\">CHIP as an E3 Ubiquitin ligase, that induces protein folding and degradation balance by upregulating ligase expression or activation<sup>##REF##35020437##28##</sup>. And which is an important component of the protein quality control system, preventing and treating diseases related to protein metabolism disorders<sup>##REF##32494669##29##</sup>. CHIP protein could be used as a biological drug for the treatment of RIF. However, due to the distribution of metabolism in <italic>vivo</italic>, and poor renal target efficacy, the poor stability, lead to the CHIP protein can hardly be used in clinical practice. Moreover, we verified the low enrichment of CHIP protein in kidney tissue by intravenous injection in the RIF rat model. According to our previous works on genetically engineering decorated MSC-EVs for delivery of the key protein molecules, which have packed CHIP protein into MSC-EVs by genetic engineering with lentivirus transfection, then use SPION decorated MSC-EVs-CHIP that resolved the challenges for enrichment and renal-target.</p>","<p id=\"Par20\">Mesenchymal stem cell derived extracellular vesicles as cell-free long-distance delivery systems have displayed apparent advantages, for instance high injury tissue target ability, tissue repairment capacity and low immunogenicity, which have been applicated in clinical therapies<sup>##REF##35787632##30##</sup>. It has reported that MSC-EVs have strong tissues target capacity to realize drug delivery<sup>##UREF##4##31##</sup>. Our previous research confirmed that MSC-EVs attenuate renal interstitial fibrosis through the kinase ubiquitin system CK1δ/β-TRCP mediated YAP ubiquitination and degradation<sup>##REF##32382019##24##</sup>. MSC-EVs could to some extent reach the site of damaged kidney tissue and exert the effective on tissues fibrosis, however, the fluorescence statistical distribution of MSC-EVs labeled with DIR membrane dyes, we found that MSC-EVs were mainly concentrated in the liver and lungs. This has prompted us to think deeply about the need to enhance the targeted enrichment of extracellular vesicles through engineering transformation.</p>","<p id=\"Par21\">Superparamagnetic iron oxide nanoparticles (SPION) have been widely used in disease therapy for excellent superparamagnetism property. The conjugation of SPIONs with extracellular vesicles has many advantages, including magnetic targeted functionalization, magnetic thermotherapy, and delivery of anti-fibrosis agents<sup>##REF##36858264##32##</sup>. Furthermore, in order to reduce the uptake of MSC-EVs in the liver and lungs after tail vein injection, nanomaterials SPION modified MSC-EVs were used to enhances the penetration and enrichment of MSC-EVs to renal under external magnetic field.</p>","<p id=\"Par22\">In conclusion, we have exploited a therapeutic strategy for renal fibrosis using nanomaterials SPION decorated MSC-EVs-CHIP to increase target the concentration of CHIP in renal tissue. We have constructed a platform for targeted delivery of CHIP by using MSC-EVs and highlighted the potential of SPION-EVs as a promising nanotherapeutic for renal interstitial fibrosis treatment. Our platform may provide potential for clinical application in the future owing to the advantages of engineered MSC-EVs and the novel inducing Smad2/3 protein degradation strategy in RIF treatment.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par17\\\">In this study, we reported a method for manufacturing engineered SPION-EVs and evaluated the feasibility, safety, and effectiveness of MSC-EVs with CHIP overexpressed in treating renal interstitial fibrosis in UUO and DKD models. Impressively, provide packed CHIP protein into MSC-derived EVs by genetic engineering with lentivirus transfection (Fig. ##FIG##0##1##), then through nanomaterials SPION decorated MSC-EVs with CHIP overexpressing target and enrich the damaged kidneys, resulting in notable renal protective effects, specifically by promoting Smad2/3 ubiquitination and degradation to reverse the transformation of renal tubular cells into myofibroblast. Our findings not only demonstrate the role of engineered MSC extracellular vesicles in the anti-fibrotic activity, but also as a new renal fibrosis therapeutic agent and target delivery nanoplatform. In a word, engineered SPION-EVs constitute an effective nanotherapeutic for the renal fibrosis treatment.</p>\",\n \"<p id=\\\"Par18\\\">Over the past several years, the clinical studies of RIF therapies have concentrated on antifibrotic, including anti-fibrosis antibodies and YAP inhibitors<sup>##REF##32382019##24##,##REF##37328872##25##</sup>. Unfortunately, these treatment strategies have not achieved good results and were not significantly reduce fibrosis in clinical tests. Furthermore, the pathogenesis of RIF is complex and includes inflammation, tubular injury, oxidative stress and metabolic dysfunction<sup>##REF##28388958##26##</sup>. There is increasing evidence that Smad2/3 ubiquitination and degradation in renal interstitial is a promising treatment method for CKD therapy<sup>##UREF##3##27##</sup>. we developed an SPION decorated engineered MSC-EVs platform by CHIP expression to reduced renal fibrosis and inflammatory infiltration in RIF rats. In vitro, we observed that MSC-EVs-CHIP inhibit Smad2/3 expression in rat renal tubular epithelial cells and relieved cells myofibroblast change induced by TGF-β<sub>1</sub>, and inhibited inflammasome activation. In UUO and DKD rat model, MSC-EVs-CHIP promoted Smad2/3 ubiquitination and degradation, further alleviated tubular cells fibrosis and inflammation.</p>\",\n \"<p id=\\\"Par19\\\">CHIP as an E3 Ubiquitin ligase, that induces protein folding and degradation balance by upregulating ligase expression or activation<sup>##REF##35020437##28##</sup>. And which is an important component of the protein quality control system, preventing and treating diseases related to protein metabolism disorders<sup>##REF##32494669##29##</sup>. CHIP protein could be used as a biological drug for the treatment of RIF. However, due to the distribution of metabolism in <italic>vivo</italic>, and poor renal target efficacy, the poor stability, lead to the CHIP protein can hardly be used in clinical practice. Moreover, we verified the low enrichment of CHIP protein in kidney tissue by intravenous injection in the RIF rat model. According to our previous works on genetically engineering decorated MSC-EVs for delivery of the key protein molecules, which have packed CHIP protein into MSC-EVs by genetic engineering with lentivirus transfection, then use SPION decorated MSC-EVs-CHIP that resolved the challenges for enrichment and renal-target.</p>\",\n \"<p id=\\\"Par20\\\">Mesenchymal stem cell derived extracellular vesicles as cell-free long-distance delivery systems have displayed apparent advantages, for instance high injury tissue target ability, tissue repairment capacity and low immunogenicity, which have been applicated in clinical therapies<sup>##REF##35787632##30##</sup>. It has reported that MSC-EVs have strong tissues target capacity to realize drug delivery<sup>##UREF##4##31##</sup>. Our previous research confirmed that MSC-EVs attenuate renal interstitial fibrosis through the kinase ubiquitin system CK1δ/β-TRCP mediated YAP ubiquitination and degradation<sup>##REF##32382019##24##</sup>. MSC-EVs could to some extent reach the site of damaged kidney tissue and exert the effective on tissues fibrosis, however, the fluorescence statistical distribution of MSC-EVs labeled with DIR membrane dyes, we found that MSC-EVs were mainly concentrated in the liver and lungs. This has prompted us to think deeply about the need to enhance the targeted enrichment of extracellular vesicles through engineering transformation.</p>\",\n \"<p id=\\\"Par21\\\">Superparamagnetic iron oxide nanoparticles (SPION) have been widely used in disease therapy for excellent superparamagnetism property. The conjugation of SPIONs with extracellular vesicles has many advantages, including magnetic targeted functionalization, magnetic thermotherapy, and delivery of anti-fibrosis agents<sup>##REF##36858264##32##</sup>. Furthermore, in order to reduce the uptake of MSC-EVs in the liver and lungs after tail vein injection, nanomaterials SPION modified MSC-EVs were used to enhances the penetration and enrichment of MSC-EVs to renal under external magnetic field.</p>\",\n \"<p id=\\\"Par22\\\">In conclusion, we have exploited a therapeutic strategy for renal fibrosis using nanomaterials SPION decorated MSC-EVs-CHIP to increase target the concentration of CHIP in renal tissue. We have constructed a platform for targeted delivery of CHIP by using MSC-EVs and highlighted the potential of SPION-EVs as a promising nanotherapeutic for renal interstitial fibrosis treatment. Our platform may provide potential for clinical application in the future owing to the advantages of engineered MSC-EVs and the novel inducing Smad2/3 protein degradation strategy in RIF treatment.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<p id=\"Par1\">Renal interstitial fibrosis (RIF) is a fundamental pathological feature of chronic kidney disease (CKD). However, toxicity and poor renal enrichment of fibrosis inhibitors limit their further applications. In this study, a platform for CKD therapy is developed using superparamagnetic iron oxide nanoparticles (SPION) decorated mesenchymal stem cells derived extracellular vesicles with carboxyl terminus of Hsc70-interacting protein (CHIP) high expression (SPION-EVs) to achieve higher renal-targeting antifibrotic therapeutic effect. SPION-EVs selectively accumulate at the injury renal sites under an external magnetic field. Moreover, SPION-EVs deliver CHIP to induce Smad2/3 degradation in renal tubular cells which alleviates Smad2/3 activation-mediated fibrosis-like changes and collagen deposition. The extracellular vesicle engineering technology provides a potential nanoplatform for RIF therapy through CHIP-mediated Smad2/3 degradation.</p>","<title>Subject terms</title>"],"string":"[\n \"<p id=\\\"Par1\\\">Renal interstitial fibrosis (RIF) is a fundamental pathological feature of chronic kidney disease (CKD). However, toxicity and poor renal enrichment of fibrosis inhibitors limit their further applications. In this study, a platform for CKD therapy is developed using superparamagnetic iron oxide nanoparticles (SPION) decorated mesenchymal stem cells derived extracellular vesicles with carboxyl terminus of Hsc70-interacting protein (CHIP) high expression (SPION-EVs) to achieve higher renal-targeting antifibrotic therapeutic effect. SPION-EVs selectively accumulate at the injury renal sites under an external magnetic field. Moreover, SPION-EVs deliver CHIP to induce Smad2/3 degradation in renal tubular cells which alleviates Smad2/3 activation-mediated fibrosis-like changes and collagen deposition. The extracellular vesicle engineering technology provides a potential nanoplatform for RIF therapy through CHIP-mediated Smad2/3 degradation.</p>\",\n \"<title>Subject terms</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary information</title>","<p>\n\n\n</p>"],"string":"[\n \"<title>Supplementary information</title>\",\n \"<p>\\n\\n\\n</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Supplementary information</title>","<p>The online version contains supplementary material available at 10.1038/s41536-024-00348-0.</p>","<title>Acknowledgements</title>","<p>C.J. and J.Z. contributed equally to this work. The authors thank the members of Qian lab for helpful discussion and paper preparation. This work was supported by the National Natural Science Foundation of China (82172102, 81871496), the Jiangsu Province’s Major Project in Research and Development (BE2021689), the Natural Science Foundation of Jiangsu Province (BK20220527), China Postdoctoral Science Foundation (2023M731376), Zhenjiang Key Laboratory of High Technology Research on Exosomes Foundation and Transformation Application (Grant ss2018003).</p>","<title>Author contributions</title>","<p>C.J. performed experimental design, tissue procurement, data generation pathological assessments, data analysis and interpretation, and manuscript preparation; J.Z. performed experimental design, data generation, and data analysis; H.S. and L.S. performed tissue procurement, data generation, interpretation, and intellectual contribution. W.X., J.J. provided intellectual contribution and critically appraised the manuscript; H.Q. conceived the study, designed experiments, interpreted data, and prepared the manuscript.</p>","<title>Data availability</title>","<p>All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.</p>","<title>Competing interests</title>","<p id=\"Par36\">The authors declare no competing interests.</p>"],"string":"[\n \"<title>Supplementary information</title>\",\n \"<p>The online version contains supplementary material available at 10.1038/s41536-024-00348-0.</p>\",\n \"<title>Acknowledgements</title>\",\n \"<p>C.J. and J.Z. contributed equally to this work. The authors thank the members of Qian lab for helpful discussion and paper preparation. This work was supported by the National Natural Science Foundation of China (82172102, 81871496), the Jiangsu Province’s Major Project in Research and Development (BE2021689), the Natural Science Foundation of Jiangsu Province (BK20220527), China Postdoctoral Science Foundation (2023M731376), Zhenjiang Key Laboratory of High Technology Research on Exosomes Foundation and Transformation Application (Grant ss2018003).</p>\",\n \"<title>Author contributions</title>\",\n \"<p>C.J. performed experimental design, tissue procurement, data generation pathological assessments, data analysis and interpretation, and manuscript preparation; J.Z. performed experimental design, data generation, and data analysis; H.S. and L.S. performed tissue procurement, data generation, interpretation, and intellectual contribution. W.X., J.J. provided intellectual contribution and critically appraised the manuscript; H.Q. conceived the study, designed experiments, interpreted data, and prepared the manuscript.</p>\",\n \"<title>Data availability</title>\",\n \"<p>All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.</p>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par36\\\">The authors declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Characteristics of MSC-derived extracellular vesicles with CHIP high-expression.</title><p><bold>a</bold> MSCs were transfected with lentivirus encoding the CHIP gene. The expression of CHIP (green) with GFP-tagged in MSCs was determined by immunofluorescence (left). Nuclei were stained with DAPI (Blue). Scale bar: 50 µm. Representative quantification of the mean fluorescence intensity (MFI) of CHIP (<italic>n</italic> = 3) (right). <bold>b</bold> Flow cytometry analysis of CHIP expression in MSCs (left) and representative quantification of the mean fluorescence intensity (MFI) of CHIP (<italic>n</italic> = 3) (right). <bold>c</bold> Morphology of MSCs with lentivirus transfection or non-transfection. Scale bar: 50 µm. <bold>d</bold> Representative transmission electron microscope (TEM) images of MSC-EVs and MSC-EVs- CHIP. Scale bar: 100 nm. <bold>e</bold> The size distribution of MSC-EVs and MSC-EVs-CHIP was measured by dynamic light scattering (DLS). <bold>f</bold> Surface zeta potential of MSC-EVs and MSC-EVs-CHIP (<italic>n</italic> = 3). <bold>g</bold> The expression of CHIP, CD73, CD63, and CD9 in MSCs and MSC-EVs with lentivirus was determined by western blotting (left). Relative values of CHIP/GAPDH and CHIP/CD9 stipe gray (<italic>n</italic> = 3) (right). Data are represented as the mean ± standard error of the mean (SEM). Statistical significance was calculated by a student’s t test. ns non-significance, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001.</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>MSC-EVs-CHIP induced Smad2/3 degradation in renal tubular cells.</title><p><bold>a</bold> MSC-EVs and MSC-EVs-CHIP were incubated with NRK-52E cells for 12 h. Confocal observed the expression of GFP-CHIP in cells. Nuclei were stained with DAPI (Blue). Scale bar: 25 µm. <bold>b</bold> Flow cytometry shows that NRK-52E cells internalize GFP-CHIP high expression of MSC-EVs. <bold>c</bold> The expression of CHIP in NRK-52E cells was determined by western blotting (left). Representative quantification of CHIP (right) (n = 3). <bold>d</bold> Confocal observed the co-localization of CHIP (Green) with Smad2/3 (Red) of NRK-52E cells. Nuclei were stained with DAPI (Blue). Scale bar: 25 µm. <bold>e</bold> The expression of Smad2/3 in NRK-52E was determined by western blotting. <bold>f</bold> The renal tubular cells (NRK-52E cell line) were pretreated with 10 nM TGF-β<sub>1</sub> protein in the absence or presence of MSC-EVs and MSC-EVs-CHIP for 24 h. The fibrosis index α-SMA (Red) was observed. Nuclei were stained with DAPI (Blue). Scale bar: 25 µm. <bold>g</bold> Determination of ubiquitin binding on Smad2/3 protein by CO-IP after MSC-EVs and MSC-EVs-CHIP treatment. <bold>h</bold> Diagrammatic of NRK-52E cells were stimulated with 30Kpa stiff gel in the presence of MSC-EVs and MSC-EVs-CHIP for 24 h. <bold>i</bold> The co-localized of CHIP (Green, stained by GFP) with Smad2/3 (Red, stained by Cy3) was observed. Nuclei were stained with DAPI (Blue). Scale bar: 25 µm. <bold>j</bold> Representative quantification of nuclear cytoplasmic ratio of Smad2/3. Data are represented as the mean ± SEM. Statistical significance was calculated by a student’s t-test. ns non-significance, *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01.</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>MSC-EVs-CHIP inhibited Smad2/3 and reduced inflammatory infiltration.</title><p><bold>a</bold> RIF was established by ureteral ligation of SD rat for 14 days, and intravenously injected with MSC-EVs and MSC-EVs-CHIP for three times in 7 days. Representative images of Smad2/3 in renal interstitial were demonstrated by immunohistochemistry (<italic>n</italic> = 3). Scale bar: 100 µm. <bold>b</bold> The localized of fibrosis marker α-SMA (Red) with renal tubular cell marker Slc5a1 (Green) in renal was showed by immunofluorescence (left). Nuclei were stained with DAPI (Blue). And representative images of Fibronectin were demonstrated by immunohistochemistry. Scale bar: 100 µm. <bold>c</bold> The co-localized of Smad2/3 (Green) with α-SMA (Red) in renal was showed by immunofluorescence (left). Nuclei were stained with DAPI (Blue). Scale bar: 100 µm. <bold>d</bold> The expression of Smad2/3, α-SMA and β-actin in kidney was determined by western blotting (left). Relative values of Smad2/3 and α-SMA stipe gray (<italic>n</italic> = 3) (right). <bold>e</bold> Representative images of HE staining of renal inflammatory infiltration. Scale bars: 100 µm. <bold>f</bold> The representative quantitation of Inflammatory infiltration was demonstrated by CD66 (Red) immunofluorescence staining. Nuclei were stained with DAPI (Blue). Scale bar: 100 µm. <bold>g</bold> Representative number of CD66 (Red) positive cell (<italic>n</italic> = 6). Nuclei were stained with DAPI (Blue). Scale bar: 100 µm. <bold>h</bold> The production of cytokines (IL-6, IL-1β, TNF-α) in the rat serum was measured using enzyme-linked immunosorbent assay (ELISA) kits (<italic>n</italic> = 3). Data are represented as the mean ± SEM. Statistical significance was calculated by a student’s t test. ns non-significance, *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001.</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>MSC-EVs-CHIP reduced collagen deposition in UUO model.</title><p><bold>a</bold> Effects of MSC-EVs-CHIP on serum urea nitrogen and <bold>b)</bold> creatinine. <bold>c</bold> Representative images of renal tissues established by UUO rats (<italic>n</italic> = 6 per group) that received different treatments including sham, PBS, MSC-EVs (10 mg/kg of body weight), MSC-EVs-CHIP (10 mg/kg of body weight). <bold>d</bold> Representative images of HE staining in MSC-EVs and MSC-EVs-CHIP group (<italic>n</italic> = 3). Scale bars: 100 µm. <bold>e</bold> The quantification of tubular injury based on HE staining (<italic>n</italic> = 5). <bold>f</bold> Representative images of Sirius red staining (<italic>n</italic> = 3). The bottom was a higher magnification of the boxed region. Scale bars: 1 mm (top) and 100 µm (bottom). <bold>g</bold> The quantification of fibrotic area based on Sirius red staining (<italic>n</italic> = 5). <bold>h</bold> Representative images of Masson trichrome staining on renal tissues sections (<italic>n</italic> = 3). Scale bars: 100 µm. <bold>i</bold> Western blotting analysis of Fibronectin, α-SMA, and Collagen I in kidney tissues.</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>In <italic>vivo</italic> renal-targeting and antifibrotic therapeutic effect of SPION-EVs.</title><p><bold>a</bold> TEM and dynamic light scattering (DLS) detect the graphic and nanoparticle size of SPION. Scale bar: 500 nm. <bold>b</bold> Separation and preparation of SPION-EVs flowchart. <bold>c</bold> Representative TEM images of MSC-EVs-CHIP and SPION-EVs. Scale bar: 100 nm. <bold>D</bold> Stability analysis of SPION-EVs using polymer dispersibility index (PDI). <bold>e</bold> Hysteresis loop analysis of SPION-EVs. <bold>f</bold> DLS detect the nanoparticle size of SPION-EVs. <bold>g</bold> The expression of CHIP, TfR, Alix, CD63, and CD9 in MSC-EVs-CHIP and SPION-EVs was determined by western blotting. <bold>h</bold> Schematic diagram of the experimental design. Briefly, rats were concurrently treated with MSC-EVs-CHIP and SPION-EVs (2 mg) every 3 days after renal UUO injury 7 days and were euthanized at 7 days after disease induction. <bold>i</bold> Intravenously injected with CM-DIR-labeled MSC-EVs-CHIP and SPION-EVs for 24, 48,72 h in UUO model. Ex vivo fluorescence images of CM-DIR-labeled EVs in major organs (left). Fluorescence intensity per gram of tissue in heart, liver, spleen, lung and kidney (right) (n = 3). <bold>j</bold> The role of SPION EVs in anti-fibrotic were demonstrated by HE and Masson staining, Sirius red staining (n = 3). Scale bar: 100 µm. <bold>k</bold> The expression of Smad2/3 (Green) with α-SMA (Red) after MSC-EVs-CHIP and SPION-EVs treatment in renal was showed by immunofluorescence. Nuclei were stained with DAPI (Blue). Scale bar: 100 µm. Data are represented as the mean ± S.E.M. Statistical significance was calculated by a student’s t test. ns, non-significance, *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001.</p></caption></fig>","<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>The anti-fibrotic effect of SPION-EVs in DKD model.</title><p><bold>a</bold> Representative image of HE staining with MSC-EVs-CHIP and SPION-EVs treatment in DKD model (<italic>n</italic> = 3). The bottom was a higher magnification of the boxed region. Scale bars: 1 mm (top) and 100 µm (bottom). <bold>b</bold> The quantification of tubular injury score based on HE staining (<italic>n</italic> = 5). <bold>c</bold> Representative images of Sirius red staining (<italic>n</italic> = 3). Scale bars: 100 µm. <bold>d</bold> The quantification of Masson positive area based on Masson staining (n = 5). <bold>e</bold> Representative images of Sirius red staining (<italic>n</italic> = 3). Scale bars: 100 µm. <bold>f</bold> The quantification of fibrotic area based on Sirius red staining (<italic>n</italic> = 5). <bold>g</bold> The expression of Smad2/3, α-SMA, Fibronectin and Collagen I in DKD model was determined by western blotting (left). <bold>h</bold> Relative values of Smad2/3, α-SMA, Fibronectin and Collagen I stipe gray (<italic>n</italic> = 3) (right). <bold>i</bold> Representative images of α-SMA were demonstrated by immunohistochemistry. Scale bar: 100 µm. <bold>j</bold> Representative images of Fibronectin in DKD model were demonstrated by immunohistochemistry. Scale bar: 100 µm. <bold>k</bold> The expression of Smad2/3 (Green) with α-SMA (Red) after MSC-EVs-CHIP and SPION-EVs treatment in DKD model was showed by immunofluorescence. Nuclei were stained with DAPI (Blue). Scale bar: 100 µm. Data are represented as the mean ± SEM. Statistical significance was calculated by a student’s t test. ns, non-significance, *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001.</p></caption></fig>","<fig id=\"Fig7\"><label>Fig. 7</label><caption><title>Schematic illustrations of engineered extracellular vesicle as novel nanotherapeutics against renal fibrosis.</title><p>A platform for RIF therapy using SPION decorate nanosized mesenchymal stem cells-derived extracellular vesicles with carboxyl terminus of Hsc70-interacting protein (CHIP) high-expression (SPION-EVs) is developed. SPION-EVs significantly enrichment of renal injury sites and induces Smad2/3 degradation of renal tubular cells, which alleviates fibrosis and inflammation, accompanying with rescued collagen deposition in RIF.</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><title>Characteristics of MSC-derived extracellular vesicles with CHIP high-expression.</title><p><bold>a</bold> MSCs were transfected with lentivirus encoding the CHIP gene. The expression of CHIP (green) with GFP-tagged in MSCs was determined by immunofluorescence (left). Nuclei were stained with DAPI (Blue). Scale bar: 50 µm. Representative quantification of the mean fluorescence intensity (MFI) of CHIP (<italic>n</italic> = 3) (right). <bold>b</bold> Flow cytometry analysis of CHIP expression in MSCs (left) and representative quantification of the mean fluorescence intensity (MFI) of CHIP (<italic>n</italic> = 3) (right). <bold>c</bold> Morphology of MSCs with lentivirus transfection or non-transfection. Scale bar: 50 µm. <bold>d</bold> Representative transmission electron microscope (TEM) images of MSC-EVs and MSC-EVs- CHIP. Scale bar: 100 nm. <bold>e</bold> The size distribution of MSC-EVs and MSC-EVs-CHIP was measured by dynamic light scattering (DLS). <bold>f</bold> Surface zeta potential of MSC-EVs and MSC-EVs-CHIP (<italic>n</italic> = 3). <bold>g</bold> The expression of CHIP, CD73, CD63, and CD9 in MSCs and MSC-EVs with lentivirus was determined by western blotting (left). Relative values of CHIP/GAPDH and CHIP/CD9 stipe gray (<italic>n</italic> = 3) (right). Data are represented as the mean ± standard error of the mean (SEM). Statistical significance was calculated by a student’s t test. ns non-significance, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001.</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><title>MSC-EVs-CHIP induced Smad2/3 degradation in renal tubular cells.</title><p><bold>a</bold> MSC-EVs and MSC-EVs-CHIP were incubated with NRK-52E cells for 12 h. Confocal observed the expression of GFP-CHIP in cells. Nuclei were stained with DAPI (Blue). Scale bar: 25 µm. <bold>b</bold> Flow cytometry shows that NRK-52E cells internalize GFP-CHIP high expression of MSC-EVs. <bold>c</bold> The expression of CHIP in NRK-52E cells was determined by western blotting (left). Representative quantification of CHIP (right) (n = 3). <bold>d</bold> Confocal observed the co-localization of CHIP (Green) with Smad2/3 (Red) of NRK-52E cells. Nuclei were stained with DAPI (Blue). Scale bar: 25 µm. <bold>e</bold> The expression of Smad2/3 in NRK-52E was determined by western blotting. <bold>f</bold> The renal tubular cells (NRK-52E cell line) were pretreated with 10 nM TGF-β<sub>1</sub> protein in the absence or presence of MSC-EVs and MSC-EVs-CHIP for 24 h. The fibrosis index α-SMA (Red) was observed. Nuclei were stained with DAPI (Blue). Scale bar: 25 µm. <bold>g</bold> Determination of ubiquitin binding on Smad2/3 protein by CO-IP after MSC-EVs and MSC-EVs-CHIP treatment. <bold>h</bold> Diagrammatic of NRK-52E cells were stimulated with 30Kpa stiff gel in the presence of MSC-EVs and MSC-EVs-CHIP for 24 h. <bold>i</bold> The co-localized of CHIP (Green, stained by GFP) with Smad2/3 (Red, stained by Cy3) was observed. Nuclei were stained with DAPI (Blue). Scale bar: 25 µm. <bold>j</bold> Representative quantification of nuclear cytoplasmic ratio of Smad2/3. Data are represented as the mean ± SEM. Statistical significance was calculated by a student’s t-test. ns non-significance, *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01.</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><title>MSC-EVs-CHIP inhibited Smad2/3 and reduced inflammatory infiltration.</title><p><bold>a</bold> RIF was established by ureteral ligation of SD rat for 14 days, and intravenously injected with MSC-EVs and MSC-EVs-CHIP for three times in 7 days. Representative images of Smad2/3 in renal interstitial were demonstrated by immunohistochemistry (<italic>n</italic> = 3). Scale bar: 100 µm. <bold>b</bold> The localized of fibrosis marker α-SMA (Red) with renal tubular cell marker Slc5a1 (Green) in renal was showed by immunofluorescence (left). Nuclei were stained with DAPI (Blue). And representative images of Fibronectin were demonstrated by immunohistochemistry. Scale bar: 100 µm. <bold>c</bold> The co-localized of Smad2/3 (Green) with α-SMA (Red) in renal was showed by immunofluorescence (left). Nuclei were stained with DAPI (Blue). Scale bar: 100 µm. <bold>d</bold> The expression of Smad2/3, α-SMA and β-actin in kidney was determined by western blotting (left). Relative values of Smad2/3 and α-SMA stipe gray (<italic>n</italic> = 3) (right). <bold>e</bold> Representative images of HE staining of renal inflammatory infiltration. Scale bars: 100 µm. <bold>f</bold> The representative quantitation of Inflammatory infiltration was demonstrated by CD66 (Red) immunofluorescence staining. Nuclei were stained with DAPI (Blue). Scale bar: 100 µm. <bold>g</bold> Representative number of CD66 (Red) positive cell (<italic>n</italic> = 6). Nuclei were stained with DAPI (Blue). Scale bar: 100 µm. <bold>h</bold> The production of cytokines (IL-6, IL-1β, TNF-α) in the rat serum was measured using enzyme-linked immunosorbent assay (ELISA) kits (<italic>n</italic> = 3). Data are represented as the mean ± SEM. Statistical significance was calculated by a student’s t test. ns non-significance, *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001.</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><title>MSC-EVs-CHIP reduced collagen deposition in UUO model.</title><p><bold>a</bold> Effects of MSC-EVs-CHIP on serum urea nitrogen and <bold>b)</bold> creatinine. <bold>c</bold> Representative images of renal tissues established by UUO rats (<italic>n</italic> = 6 per group) that received different treatments including sham, PBS, MSC-EVs (10 mg/kg of body weight), MSC-EVs-CHIP (10 mg/kg of body weight). <bold>d</bold> Representative images of HE staining in MSC-EVs and MSC-EVs-CHIP group (<italic>n</italic> = 3). Scale bars: 100 µm. <bold>e</bold> The quantification of tubular injury based on HE staining (<italic>n</italic> = 5). <bold>f</bold> Representative images of Sirius red staining (<italic>n</italic> = 3). The bottom was a higher magnification of the boxed region. Scale bars: 1 mm (top) and 100 µm (bottom). <bold>g</bold> The quantification of fibrotic area based on Sirius red staining (<italic>n</italic> = 5). <bold>h</bold> Representative images of Masson trichrome staining on renal tissues sections (<italic>n</italic> = 3). Scale bars: 100 µm. <bold>i</bold> Western blotting analysis of Fibronectin, α-SMA, and Collagen I in kidney tissues.</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><title>In <italic>vivo</italic> renal-targeting and antifibrotic therapeutic effect of SPION-EVs.</title><p><bold>a</bold> TEM and dynamic light scattering (DLS) detect the graphic and nanoparticle size of SPION. Scale bar: 500 nm. <bold>b</bold> Separation and preparation of SPION-EVs flowchart. <bold>c</bold> Representative TEM images of MSC-EVs-CHIP and SPION-EVs. Scale bar: 100 nm. <bold>D</bold> Stability analysis of SPION-EVs using polymer dispersibility index (PDI). <bold>e</bold> Hysteresis loop analysis of SPION-EVs. <bold>f</bold> DLS detect the nanoparticle size of SPION-EVs. <bold>g</bold> The expression of CHIP, TfR, Alix, CD63, and CD9 in MSC-EVs-CHIP and SPION-EVs was determined by western blotting. <bold>h</bold> Schematic diagram of the experimental design. Briefly, rats were concurrently treated with MSC-EVs-CHIP and SPION-EVs (2 mg) every 3 days after renal UUO injury 7 days and were euthanized at 7 days after disease induction. <bold>i</bold> Intravenously injected with CM-DIR-labeled MSC-EVs-CHIP and SPION-EVs for 24, 48,72 h in UUO model. Ex vivo fluorescence images of CM-DIR-labeled EVs in major organs (left). Fluorescence intensity per gram of tissue in heart, liver, spleen, lung and kidney (right) (n = 3). <bold>j</bold> The role of SPION EVs in anti-fibrotic were demonstrated by HE and Masson staining, Sirius red staining (n = 3). Scale bar: 100 µm. <bold>k</bold> The expression of Smad2/3 (Green) with α-SMA (Red) after MSC-EVs-CHIP and SPION-EVs treatment in renal was showed by immunofluorescence. Nuclei were stained with DAPI (Blue). Scale bar: 100 µm. Data are represented as the mean ± S.E.M. Statistical significance was calculated by a student’s t test. ns, non-significance, *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001.</p></caption></fig>\",\n \"<fig id=\\\"Fig6\\\"><label>Fig. 6</label><caption><title>The anti-fibrotic effect of SPION-EVs in DKD model.</title><p><bold>a</bold> Representative image of HE staining with MSC-EVs-CHIP and SPION-EVs treatment in DKD model (<italic>n</italic> = 3). The bottom was a higher magnification of the boxed region. Scale bars: 1 mm (top) and 100 µm (bottom). <bold>b</bold> The quantification of tubular injury score based on HE staining (<italic>n</italic> = 5). <bold>c</bold> Representative images of Sirius red staining (<italic>n</italic> = 3). Scale bars: 100 µm. <bold>d</bold> The quantification of Masson positive area based on Masson staining (n = 5). <bold>e</bold> Representative images of Sirius red staining (<italic>n</italic> = 3). Scale bars: 100 µm. <bold>f</bold> The quantification of fibrotic area based on Sirius red staining (<italic>n</italic> = 5). <bold>g</bold> The expression of Smad2/3, α-SMA, Fibronectin and Collagen I in DKD model was determined by western blotting (left). <bold>h</bold> Relative values of Smad2/3, α-SMA, Fibronectin and Collagen I stipe gray (<italic>n</italic> = 3) (right). <bold>i</bold> Representative images of α-SMA were demonstrated by immunohistochemistry. Scale bar: 100 µm. <bold>j</bold> Representative images of Fibronectin in DKD model were demonstrated by immunohistochemistry. Scale bar: 100 µm. <bold>k</bold> The expression of Smad2/3 (Green) with α-SMA (Red) after MSC-EVs-CHIP and SPION-EVs treatment in DKD model was showed by immunofluorescence. Nuclei were stained with DAPI (Blue). Scale bar: 100 µm. Data are represented as the mean ± SEM. Statistical significance was calculated by a student’s t test. ns, non-significance, *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001.</p></caption></fig>\",\n \"<fig id=\\\"Fig7\\\"><label>Fig. 7</label><caption><title>Schematic illustrations of engineered extracellular vesicle as novel nanotherapeutics against renal fibrosis.</title><p>A platform for RIF therapy using SPION decorate nanosized mesenchymal stem cells-derived extracellular vesicles with carboxyl terminus of Hsc70-interacting protein (CHIP) high-expression (SPION-EVs) is developed. SPION-EVs significantly enrichment of renal injury sites and induces Smad2/3 degradation of renal tubular cells, which alleviates fibrosis and inflammation, accompanying with rescued collagen deposition in RIF.</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":[],"string":"[]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>","<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\",\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM2\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Cheng Ji, Jiahui Zhang, Linru Shi.</p></fn></fn-group>"],"string":"[\n \"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Cheng Ji, Jiahui Zhang, Linru Shi.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"41536_2024_348_Fig1_HTML\" id=\"d32e388\"/>","<graphic xlink:href=\"41536_2024_348_Fig2_HTML\" id=\"d32e452\"/>","<graphic xlink:href=\"41536_2024_348_Fig3_HTML\" id=\"d32e575\"/>","<graphic xlink:href=\"41536_2024_348_Fig4_HTML\" id=\"d32e658\"/>","<graphic xlink:href=\"41536_2024_348_Fig5_HTML\" id=\"d32e740\"/>","<graphic xlink:href=\"41536_2024_348_Fig6_HTML\" id=\"d32e845\"/>","<graphic xlink:href=\"41536_2024_348_Fig7_HTML\" id=\"d32e853\"/>"],"string":"[\n \"<graphic xlink:href=\\\"41536_2024_348_Fig1_HTML\\\" id=\\\"d32e388\\\"/>\",\n \"<graphic xlink:href=\\\"41536_2024_348_Fig2_HTML\\\" id=\\\"d32e452\\\"/>\",\n \"<graphic xlink:href=\\\"41536_2024_348_Fig3_HTML\\\" id=\\\"d32e575\\\"/>\",\n \"<graphic xlink:href=\\\"41536_2024_348_Fig4_HTML\\\" id=\\\"d32e658\\\"/>\",\n \"<graphic xlink:href=\\\"41536_2024_348_Fig5_HTML\\\" id=\\\"d32e740\\\"/>\",\n \"<graphic xlink:href=\\\"41536_2024_348_Fig6_HTML\\\" id=\\\"d32e845\\\"/>\",\n \"<graphic xlink:href=\\\"41536_2024_348_Fig7_HTML\\\" id=\\\"d32e853\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"41536_2024_348_MOESM1_ESM.pdf\"><caption><p>Supplementary material</p></caption></media>","<media xlink:href=\"41536_2024_348_MOESM2_ESM.pdf\"><caption><p>Reporting-summary</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"41536_2024_348_MOESM1_ESM.pdf\\\"><caption><p>Supplementary material</p></caption></media>\",\n \"<media xlink:href=\\\"41536_2024_348_MOESM2_ESM.pdf\\\"><caption><p>Reporting-summary</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"9.\"], \"surname\": [\"Jin\"], \"given-names\": [\"Q\"], \"article-title\": [\"Therapeutic potential of artemisinin and its derivatives in managing kidney diseases\"], \"source\": [\"Front Pharm.\"], \"year\": [\"2023\"], \"volume\": [\"14\"], \"fpage\": [\"1097206\"], \"pub-id\": [\"10.3389/fphar.2023.1097206\"]}, {\"label\": [\"13.\"], \"surname\": [\"Zipkin\"], \"given-names\": [\"M\"], \"article-title\": [\"Big pharma buys into exosomes for drug delivery\"], \"source\": [\"Nat. Biotechnol.\"], \"year\": [\"2020\"], \"volume\": [\"8\"], \"fpage\": [\"1226\"], \"lpage\": [\"1228\"], \"pub-id\": [\"10.1038/s41587-020-0725-7\"]}, {\"label\": [\"17.\"], \"surname\": [\"Zhang\"], \"given-names\": [\"X\"], \"article-title\": [\"Engineered extracellular vesicles for cancer therapy\"], \"source\": [\"Adv. Mater.\"], \"year\": [\"2021\"], \"volume\": [\"28\"], \"fpage\": [\"e2005709\"], \"pub-id\": [\"10.1002/adma.202005709\"]}, {\"label\": [\"27.\"], \"surname\": [\"Ni\"], \"given-names\": [\"JY\"], \"article-title\": [\"Deubiquitinating enzyme USP11 promotes renal tubular cell senescence and fibrosis via inhibiting the ubiquitin degradation of TGF-\\u03b2 receptor II\"], \"source\": [\"Acta Pharm. Sin.\"], \"year\": [\"2023\"], \"volume\": [\"44\"], \"fpage\": [\"584\"], \"lpage\": [\"595\"], \"pub-id\": [\"10.1038/s41401-022-00977-5\"]}, {\"label\": [\"31.\"], \"surname\": [\"Kutchy\"], \"given-names\": [\"NA\"], \"article-title\": [\"Extracellular vesicle-mediated delivery of ultrasmall superparamagnetic iron oxide nanoparticles to mice brain\"], \"source\": [\"Front Pharm.\"], \"year\": [\"2022\"], \"volume\": [\"13\"], \"fpage\": [\"819516\"], \"pub-id\": [\"10.3389/fphar.2022.819516\"]}, {\"label\": [\"33.\"], \"mixed-citation\": [\"Li Q., et al. Requirements for human mesenchymal stem cell-derived small extracellular vesicles. Interdisciplinary Medicine, 10.1002/INMD.20220015.\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":33,"string":"33"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"NPJ Regen Med. 2024 Jan 13; 9:3"},"package_file":{"kind":"string","value":"oa_package/0b/12/PMC10787844.tar.gz"}}},{"rowIdx":152,"cells":{"accession_id":{"kind":"string","value":"PMC10787845"},"pmid":{"kind":"string","value":"38222223"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Diarrhea is one of the top three infectious diseases responsible for ~8%-10% of total deaths among children below the age of five years; 90% of these deaths are estimated to occur in South Asia and sub-Saharan Africa [##UREF##0##1##, ##UREF##1##2##]. Rotavirus and diarrheagenic <italic>Escherichia coli</italic> (<italic>E. coli</italic>) are the two most common pathogens responsible for acute diarrhea in children below the age of five years. <italic>Shigella</italic>, <italic>Cryptosporidium</italic>, <italic>Campylobacter spp.</italic>, and <italic>Vibrio cholerae</italic> are a few other pathogens frequently associated with diarrhea in children under five [##REF##15825143##3##], with variable region-specific incidence.</p>","<p>The Global Enteric Multicentric Study (GEMS), a large case-control study, determined the etiology of childhood diarrhea in South Asia and sub-Saharan Africa. Rotavirus, <italic>Cryptosporidium</italic>, <italic>E. coli</italic> producing heat-stable toxin (ST-ETEC), and <italic>Shigella </italic>were identified as major attributable organisms for the cause of diarrhea [##REF##23680352##4##]. Later, stool samples from GEMS were re-analyzed using quantitative polymerase chain reaction (PCR) and revised pathogen-specific burdens were calculated. Pathogen-attributable burden increased significantly, and <italic>Shigella </italic>was reported as the most common causative agent [##REF##27673470##5##]. Customarily, <italic>Shigella </italic>infection is associated with bloody diarrhea, but this re-analysis using PCR techniques documented that most such children have only loose stools without blood. Despite <italic>Shigella </italic>being reported as the most common cause of acute diarrhea in GEMS re-analysis, there is a paucity of studies evaluating <italic>Shigella </italic>by molecular methods and comparing the isolation rate with conventional culture techniques. Also, the clinical characteristics and outcomes of children with non-bloody <italic>Shigella </italic>diarrhea managed as per current WHO diarrhea treatment guidelines are not known.</p>","<p>In this study, we determined the frequency of <italic>Shigella </italic>detected by molecular and conventional methods in under-five children with diarrhea and the clinical profile and outcome of children with <italic>Shigella </italic>diarrhea over the next three months.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Diarrhea is one of the top three infectious diseases responsible for ~8%-10% of total deaths among children below the age of five years; 90% of these deaths are estimated to occur in South Asia and sub-Saharan Africa [##UREF##0##1##, ##UREF##1##2##]. Rotavirus and diarrheagenic <italic>Escherichia coli</italic> (<italic>E. coli</italic>) are the two most common pathogens responsible for acute diarrhea in children below the age of five years. <italic>Shigella</italic>, <italic>Cryptosporidium</italic>, <italic>Campylobacter spp.</italic>, and <italic>Vibrio cholerae</italic> are a few other pathogens frequently associated with diarrhea in children under five [##REF##15825143##3##], with variable region-specific incidence.</p>\",\n \"<p>The Global Enteric Multicentric Study (GEMS), a large case-control study, determined the etiology of childhood diarrhea in South Asia and sub-Saharan Africa. Rotavirus, <italic>Cryptosporidium</italic>, <italic>E. coli</italic> producing heat-stable toxin (ST-ETEC), and <italic>Shigella </italic>were identified as major attributable organisms for the cause of diarrhea [##REF##23680352##4##]. Later, stool samples from GEMS were re-analyzed using quantitative polymerase chain reaction (PCR) and revised pathogen-specific burdens were calculated. Pathogen-attributable burden increased significantly, and <italic>Shigella </italic>was reported as the most common causative agent [##REF##27673470##5##]. Customarily, <italic>Shigella </italic>infection is associated with bloody diarrhea, but this re-analysis using PCR techniques documented that most such children have only loose stools without blood. Despite <italic>Shigella </italic>being reported as the most common cause of acute diarrhea in GEMS re-analysis, there is a paucity of studies evaluating <italic>Shigella </italic>by molecular methods and comparing the isolation rate with conventional culture techniques. Also, the clinical characteristics and outcomes of children with non-bloody <italic>Shigella </italic>diarrhea managed as per current WHO diarrhea treatment guidelines are not known.</p>\",\n \"<p>In this study, we determined the frequency of <italic>Shigella </italic>detected by molecular and conventional methods in under-five children with diarrhea and the clinical profile and outcome of children with <italic>Shigella </italic>diarrhea over the next three months.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<p>This hospital-based prospective observational study was conducted between November 2017 and April 2019 at the University College of Medical Sciences, Delhi, an urban hospital catering mainly to the low-income population of eastern Delhi and adjoining areas of the state of Uttar Pradesh in India. Approval from the Institutional Ethics Committee for Human Research (IEC-HR) of the institute was obtained before enrolling the study participants (approval number: IEC-HR/2017/32/88). Informed consent was obtained from the parents or caregivers of all the participants enrolled in the study.</p>","<p>Participants</p>","<p>All consecutive children aged between one month and five years, presenting with acute diarrhea to the outpatient department or pediatric emergency services of the hospital and requiring hospitalization due to any reason (such as dehydration, severe malnutrition, septicemia, etc.), were eligible for inclusion. Acute diarrhea was defined as diarrhea of < seven days duration with or without blood in stools [##UREF##2##6##]. Children who received antibiotic therapy seven days prior and those with any other known cause of blood in stools (rectal polyp, bleeding diathesis, inflammatory bowel disease, etc.) were excluded.</p>","<p>At the time of enrollment, detailed clinical history, including the treatment prescribed (oral rehydration solution (ORS), zinc, paracetamol, etc.) during the current diarrheal episode, was elicited from the parents or guardian. A detailed clinical examination, including an assessment of hydration status, was performed. Anthropometry was performed on all children after the correction of dehydration. All findings were recorded in a case record form.</p>","<p>Collection and processing of stool samples</p>","<p>A freshly passed stool sample was collected in a wide-mouthed, clean container. A part of the stool sample was concentrated by the formal ether sedimentation method for wet-mount microscopy for ova, parasites, and cysts. The second part of the sample was enriched in selenite F broth (SFB) and alkaline peptone water (APW) for four to six hours at 37º C and cultured on MacConkey’s medium, xylose lysine deoxycholate (XLD) medium, and bile salt agar (BSA) medium for the isolation of pathogenic enteric bacteria by standard laboratory methods. Antibiotic sensitivity testing of isolates was performed as per Clinical and Laboratory Standards Institute (CLSI) guidelines [##UREF##3##7##]. The third part of the sample was stored at -80 °C for PCR. The DNA was extracted from a stool sample using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) and stored at -20 °C. The extracted DNA was subjected to PCR amplification using specific primers for the invasion plasmid antigen H (ipaH) gene at 424 bp for <italic>Shigella </italic>[##REF##11478669##8##]. Forward 5’- GCTGGAAAAACTCAGTGCCT-3’ and reverse 5’- CCAGTCCTGTAAATTCATTCT-3’ amplification was performed using a reaction mixture of Taq polymerase, deoxynucleotide triphosphate (dNTP), and buffer (QIAGEN). Thirty-five amplification cycles were performed with the following steps: Initial denaturation at 95 °C for five minutes, cycling denaturation at 95 °C for 30 seconds, annealing at 56 °C for 30 seconds, extension at 72 °C for 40 seconds, and final extension at 72 °C for 10 minutes. The PCR amplicons were loaded onto a 2% agarose gel in a specific order for electrophoresis and run at 150 volts for 20-25 minutes. After electrophoresis, the 0.5 mM ethidium bromide-stained gel was visualized and interpreted for specific amplification patterns on the UV transilluminator.</p>","<p>Patient management</p>","<p>Hydration status was assessed, classified, and managed with ORS and intravenous fluids as per WHO guidelines [##UREF##4##9##]. Oral zinc was administered (10 mg/day for two- to six-month-olds, 20 mg/day for six- to 59-month-olds) for a total duration of 14 days. All children with visible blood in their stools received antimicrobial therapy as per WHO and Indian Academy of Pediatrics (IAP) guidelines [##UREF##4##9##, ##UREF##5##10##]. Children with bloody diarrhea, aged more than one year, with no risk factors (no malnutrition, no dehydration, preserved appetite) received oral antibiotics (cefixime), while those with malnutrition, severe dehydration, or poor oral acceptance received injectable antibiotics. Enrolled children were evaluated for comorbidities like hypoglycemia, hypothermia, and electrolyte imbalance and managed accordingly. Children with severe acute malnutrition (SAM) were stabilized and managed as per WHO guidelines [##UREF##6##11##].</p>","<p>Outcome variables</p>","<p>Outcomes were assessed as a percentage of children with a diagnosis of <italic>Shigella </italic>infection by molecular (PCR) and conventional methods (stool microscopy, culture). Children were discharged once the dehydration was corrected, stool frequency was reduced, and oral acceptance improved. After discharge, follow-up for persistence or recurrence of diarrhea was done on days three and seven, and then fortnightly for three months with simultaneous evaluation for growth faltering. Any recurrence of diarrhea or dysentery over three months after discharge was noted. The need for follow-up was emphasized by personal communication at each visit or by telephonic communication to minimize the attrition rate during follow-up.</p>","<p>Sample size</p>","<p>In an earlier study [##REF##27061990##12##], <italic>Shigella </italic>infection was diagnosed by molecular methods in 11 out of 60 children with acute watery diarrhea (18.33%) and 34 out of 60 children with dysentery (56.66%). Assuming 10% of our enrolled cases to have dysentery (visible blood in stools), the estimated proportion of <italic>Shigella </italic>positivity by molecular methods was 22.2%. With an estimated proportion of 22.2%, the calculated sample size was 150 children with a relative precision of 30% (absolute precision of 6.66%) at a 95% confidence level. Thus, we planned to enroll 150 children with acute diarrhea.</p>","<p>Statistical analysis</p>","<p>Data collected were entered in a Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA) and analyzed using IBM Statistical Package for the Social Sciences (SPSS) software version 25 (IBM Corp., Armonk, NY). Outcome parameters such as frequency of isolation, duration of diarrhea, and recurrence of diarrhea were measured using descriptive statistics. Categorical variables were compared between <italic>Shigella</italic>-positive and <italic>Shigella</italic>-negative cases by the chi-square test to identify clinical characteristics and the outcome of <italic>Shigella </italic>infection. For normally distributed data, continuous variables between the two groups were compared using the Student's t-test, and for data that were not normally distributed, the Mann-Whitney U test was used. Odds ratios and 95% CI were calculated to determine risk factors (e.g., age < one month, socioeconomic status, malnutrition status) and clinical predictors (e.g., blood in stools, vomiting, dehydration) by univariate analysis.</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<p>This hospital-based prospective observational study was conducted between November 2017 and April 2019 at the University College of Medical Sciences, Delhi, an urban hospital catering mainly to the low-income population of eastern Delhi and adjoining areas of the state of Uttar Pradesh in India. Approval from the Institutional Ethics Committee for Human Research (IEC-HR) of the institute was obtained before enrolling the study participants (approval number: IEC-HR/2017/32/88). Informed consent was obtained from the parents or caregivers of all the participants enrolled in the study.</p>\",\n \"<p>Participants</p>\",\n \"<p>All consecutive children aged between one month and five years, presenting with acute diarrhea to the outpatient department or pediatric emergency services of the hospital and requiring hospitalization due to any reason (such as dehydration, severe malnutrition, septicemia, etc.), were eligible for inclusion. Acute diarrhea was defined as diarrhea of < seven days duration with or without blood in stools [##UREF##2##6##]. Children who received antibiotic therapy seven days prior and those with any other known cause of blood in stools (rectal polyp, bleeding diathesis, inflammatory bowel disease, etc.) were excluded.</p>\",\n \"<p>At the time of enrollment, detailed clinical history, including the treatment prescribed (oral rehydration solution (ORS), zinc, paracetamol, etc.) during the current diarrheal episode, was elicited from the parents or guardian. A detailed clinical examination, including an assessment of hydration status, was performed. Anthropometry was performed on all children after the correction of dehydration. All findings were recorded in a case record form.</p>\",\n \"<p>Collection and processing of stool samples</p>\",\n \"<p>A freshly passed stool sample was collected in a wide-mouthed, clean container. A part of the stool sample was concentrated by the formal ether sedimentation method for wet-mount microscopy for ova, parasites, and cysts. The second part of the sample was enriched in selenite F broth (SFB) and alkaline peptone water (APW) for four to six hours at 37º C and cultured on MacConkey’s medium, xylose lysine deoxycholate (XLD) medium, and bile salt agar (BSA) medium for the isolation of pathogenic enteric bacteria by standard laboratory methods. Antibiotic sensitivity testing of isolates was performed as per Clinical and Laboratory Standards Institute (CLSI) guidelines [##UREF##3##7##]. The third part of the sample was stored at -80 °C for PCR. The DNA was extracted from a stool sample using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) and stored at -20 °C. The extracted DNA was subjected to PCR amplification using specific primers for the invasion plasmid antigen H (ipaH) gene at 424 bp for <italic>Shigella </italic>[##REF##11478669##8##]. Forward 5’- GCTGGAAAAACTCAGTGCCT-3’ and reverse 5’- CCAGTCCTGTAAATTCATTCT-3’ amplification was performed using a reaction mixture of Taq polymerase, deoxynucleotide triphosphate (dNTP), and buffer (QIAGEN). Thirty-five amplification cycles were performed with the following steps: Initial denaturation at 95 °C for five minutes, cycling denaturation at 95 °C for 30 seconds, annealing at 56 °C for 30 seconds, extension at 72 °C for 40 seconds, and final extension at 72 °C for 10 minutes. The PCR amplicons were loaded onto a 2% agarose gel in a specific order for electrophoresis and run at 150 volts for 20-25 minutes. After electrophoresis, the 0.5 mM ethidium bromide-stained gel was visualized and interpreted for specific amplification patterns on the UV transilluminator.</p>\",\n \"<p>Patient management</p>\",\n \"<p>Hydration status was assessed, classified, and managed with ORS and intravenous fluids as per WHO guidelines [##UREF##4##9##]. Oral zinc was administered (10 mg/day for two- to six-month-olds, 20 mg/day for six- to 59-month-olds) for a total duration of 14 days. All children with visible blood in their stools received antimicrobial therapy as per WHO and Indian Academy of Pediatrics (IAP) guidelines [##UREF##4##9##, ##UREF##5##10##]. Children with bloody diarrhea, aged more than one year, with no risk factors (no malnutrition, no dehydration, preserved appetite) received oral antibiotics (cefixime), while those with malnutrition, severe dehydration, or poor oral acceptance received injectable antibiotics. Enrolled children were evaluated for comorbidities like hypoglycemia, hypothermia, and electrolyte imbalance and managed accordingly. Children with severe acute malnutrition (SAM) were stabilized and managed as per WHO guidelines [##UREF##6##11##].</p>\",\n \"<p>Outcome variables</p>\",\n \"<p>Outcomes were assessed as a percentage of children with a diagnosis of <italic>Shigella </italic>infection by molecular (PCR) and conventional methods (stool microscopy, culture). Children were discharged once the dehydration was corrected, stool frequency was reduced, and oral acceptance improved. After discharge, follow-up for persistence or recurrence of diarrhea was done on days three and seven, and then fortnightly for three months with simultaneous evaluation for growth faltering. Any recurrence of diarrhea or dysentery over three months after discharge was noted. The need for follow-up was emphasized by personal communication at each visit or by telephonic communication to minimize the attrition rate during follow-up.</p>\",\n \"<p>Sample size</p>\",\n \"<p>In an earlier study [##REF##27061990##12##], <italic>Shigella </italic>infection was diagnosed by molecular methods in 11 out of 60 children with acute watery diarrhea (18.33%) and 34 out of 60 children with dysentery (56.66%). Assuming 10% of our enrolled cases to have dysentery (visible blood in stools), the estimated proportion of <italic>Shigella </italic>positivity by molecular methods was 22.2%. With an estimated proportion of 22.2%, the calculated sample size was 150 children with a relative precision of 30% (absolute precision of 6.66%) at a 95% confidence level. Thus, we planned to enroll 150 children with acute diarrhea.</p>\",\n \"<p>Statistical analysis</p>\",\n \"<p>Data collected were entered in a Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA) and analyzed using IBM Statistical Package for the Social Sciences (SPSS) software version 25 (IBM Corp., Armonk, NY). Outcome parameters such as frequency of isolation, duration of diarrhea, and recurrence of diarrhea were measured using descriptive statistics. Categorical variables were compared between <italic>Shigella</italic>-positive and <italic>Shigella</italic>-negative cases by the chi-square test to identify clinical characteristics and the outcome of <italic>Shigella </italic>infection. For normally distributed data, continuous variables between the two groups were compared using the Student's t-test, and for data that were not normally distributed, the Mann-Whitney U test was used. Odds ratios and 95% CI were calculated to determine risk factors (e.g., age < one month, socioeconomic status, malnutrition status) and clinical predictors (e.g., blood in stools, vomiting, dehydration) by univariate analysis.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p>Enrollment and baseline characteristics</p>","<p>During the study period, 209 children aged between one month and five years with acute diarrhea were hospitalized, out of which 150 were included in the present study. Figure ##FIG##0##1## depicts the flow of participants in the study, along with the reasons for the exclusion of cases not enrolled in the study.</p>","<p>The median (interquartile range (IQR)) age of enrolled children (72 (48%) males and 78 (52%) females) was 10 (5-23) months; 87 (58%) were infants (age <1 year). Only 94 (63%) were completely immunized for age. The majority (142, 94.7%) of the children’s families belonged to the lower middle, upper lower, or lower socioeconomic class on the Kuppuswamy scale [##UREF##7##13##]. The median (IQR) duration of diarrhea at hospitalization was two (one to three) days, and the number of stools passed in the previous 24 hours was 15 (10-20). Out of 150 children, 141 (94%) had watery, mucoid, or loose stools, and nine (6%) had blood in their stools. The most common associated symptom with diarrhea was vomiting (85%), followed by decreased oral acceptance (80%), and fever (40%).</p>","<p>Clinical characteristics</p>","<p>Undernutrition was prevalent in the enrolled children; 58 (38.7%) were stunted (height-for-age Z-score (HAZ) <−2SD) and 64 (42.7%) were wasted (weight-for-height Z-score (WHZ) <−2SD). Among these, 28 (19%) were severely stunted (HAZ <−3SD), while 40 (26.7%) children had SAM (weight for height Z-score <-3SD or mid-upper arm circumference <11.5 cm for children aged between six months and five years). At admission, dehydration was present in 138 (92%) children; 89 (59%) were severely dehydrated, and 15 (10%) had features suggestive of circulatory shock. All enrolled children received ORS during the hospitalization with a mean (SD) duration of 89.0 (31.3) hours, while IV fluids were administered to 95% of children for a mean (SD) duration of 44.1 (31.6) hours. Antibiotics were used in 101 (67.3%) cases for a mean (SD) of 5.3 (2.8) days. The presence of SAM (37%) and suspected or confirmed sepsis (33%) were the most common indications for the use of antibiotics in children admitted with acute diarrhea, followed by clinically suspected cholera (22%), dysentery (9%), pneumonia (5%), and meningitis (2%).</p>","<p>Stool microscopy</p>","<p><italic>Shigella sonnei</italic> was diagnosed by stool culture in only one child. <italic>Escherichia coli</italic> was cultured from 67 stool samples, whereas <italic>Klebsiella pneumonia</italic>, trophozoites of <italic>Giardia lamblia</italic> and ova of <italic>Ascaris lumbricoides</italic>, and motile <italic>Vibrio cholera</italic> in hanging drop were detected in one stool sample each. <italic>Shigella </italic>was detected in 13 (8.7%) out of 150 stool samples by PCR amplification using the ipaH gene. The only culture-positive case was also detected by PCR. The sensitivity of stool culture was 7.7% against PCR for the diagnosis of <italic>Shigella </italic>infection. Out of 13 children in whom the <italic>Shigella </italic>PCR was positive, 11 (84.6%) had non-bloody diarrhea, and only two (15.4%) had dysentery. In 11 children with <italic>Shigella </italic>PCR positivity and non-bloody diarrhea, the characteristics of the stool did not change to bloody diarrhea until the resolution of the diarrhea episode, but eight of these children had received antibiotics due to associated comorbidities. Among three children with PCR positivity and non-bloody diarrhea managed without antibiotics, two had vomiting and poor oral acceptance at presentation, whereas none of them had fever or abdominal pain. All three children presented with some or severe dehydration, and none of these children developed bloody diarrhea or persistent diarrhea, despite no antibiotic usage.</p>","<p>Comparison of the characteristics and outcomes of children who were PCR-positive vs. PCR-negative for <italic>Shigella</italic>\n</p>","<p>Table ##TAB##0##1## compares the clinical and anthropometric characteristics of children with stool PCR positivity for <italic>Shigella </italic>with those who had a negative test result on PCR.</p>","<p>There was no statistically significant difference between the two groups. The mean (SD) duration of diarrhea, as monitored after enrolment, was not significantly different (P=0.94) between PCR-positive participants (2.2 (1.54) days) and PCR-negative participants (2.3 (1.45) days). On univariate analysis, none of the evaluated risk factors or clinical predictors had a significant association with the <italic>Shigella </italic>infection (Table ##TAB##1##2##).</p>","<p>Follow-up</p>","<p>Out of 150 enrolled children, 23 were lost to follow-up, whereas 43 had a recurrence of diarrhea over the next three months. Among these, 14 patients required hospitalization, and antibiotics were used in 12 patients. During follow-up, only two patients had dysentery, and one patient developed persistent diarrhea. The recurrence of diarrhea episodes over three months was seen slightly more often among the children who had initial PCR (5, 45.4%) positivity than in PCR-negative participants (38, 37.8%), but this was not statistically significant (P=0.507). Also, there were no statistically significant differences in outcome in terms of growth faltering, recurrence of diarrhea, or hospitalization (Table ##TAB##2##3##).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p>Enrollment and baseline characteristics</p>\",\n \"<p>During the study period, 209 children aged between one month and five years with acute diarrhea were hospitalized, out of which 150 were included in the present study. Figure ##FIG##0##1## depicts the flow of participants in the study, along with the reasons for the exclusion of cases not enrolled in the study.</p>\",\n \"<p>The median (interquartile range (IQR)) age of enrolled children (72 (48%) males and 78 (52%) females) was 10 (5-23) months; 87 (58%) were infants (age <1 year). Only 94 (63%) were completely immunized for age. The majority (142, 94.7%) of the children’s families belonged to the lower middle, upper lower, or lower socioeconomic class on the Kuppuswamy scale [##UREF##7##13##]. The median (IQR) duration of diarrhea at hospitalization was two (one to three) days, and the number of stools passed in the previous 24 hours was 15 (10-20). Out of 150 children, 141 (94%) had watery, mucoid, or loose stools, and nine (6%) had blood in their stools. The most common associated symptom with diarrhea was vomiting (85%), followed by decreased oral acceptance (80%), and fever (40%).</p>\",\n \"<p>Clinical characteristics</p>\",\n \"<p>Undernutrition was prevalent in the enrolled children; 58 (38.7%) were stunted (height-for-age Z-score (HAZ) <−2SD) and 64 (42.7%) were wasted (weight-for-height Z-score (WHZ) <−2SD). Among these, 28 (19%) were severely stunted (HAZ <−3SD), while 40 (26.7%) children had SAM (weight for height Z-score <-3SD or mid-upper arm circumference <11.5 cm for children aged between six months and five years). At admission, dehydration was present in 138 (92%) children; 89 (59%) were severely dehydrated, and 15 (10%) had features suggestive of circulatory shock. All enrolled children received ORS during the hospitalization with a mean (SD) duration of 89.0 (31.3) hours, while IV fluids were administered to 95% of children for a mean (SD) duration of 44.1 (31.6) hours. Antibiotics were used in 101 (67.3%) cases for a mean (SD) of 5.3 (2.8) days. The presence of SAM (37%) and suspected or confirmed sepsis (33%) were the most common indications for the use of antibiotics in children admitted with acute diarrhea, followed by clinically suspected cholera (22%), dysentery (9%), pneumonia (5%), and meningitis (2%).</p>\",\n \"<p>Stool microscopy</p>\",\n \"<p><italic>Shigella sonnei</italic> was diagnosed by stool culture in only one child. <italic>Escherichia coli</italic> was cultured from 67 stool samples, whereas <italic>Klebsiella pneumonia</italic>, trophozoites of <italic>Giardia lamblia</italic> and ova of <italic>Ascaris lumbricoides</italic>, and motile <italic>Vibrio cholera</italic> in hanging drop were detected in one stool sample each. <italic>Shigella </italic>was detected in 13 (8.7%) out of 150 stool samples by PCR amplification using the ipaH gene. The only culture-positive case was also detected by PCR. The sensitivity of stool culture was 7.7% against PCR for the diagnosis of <italic>Shigella </italic>infection. Out of 13 children in whom the <italic>Shigella </italic>PCR was positive, 11 (84.6%) had non-bloody diarrhea, and only two (15.4%) had dysentery. In 11 children with <italic>Shigella </italic>PCR positivity and non-bloody diarrhea, the characteristics of the stool did not change to bloody diarrhea until the resolution of the diarrhea episode, but eight of these children had received antibiotics due to associated comorbidities. Among three children with PCR positivity and non-bloody diarrhea managed without antibiotics, two had vomiting and poor oral acceptance at presentation, whereas none of them had fever or abdominal pain. All three children presented with some or severe dehydration, and none of these children developed bloody diarrhea or persistent diarrhea, despite no antibiotic usage.</p>\",\n \"<p>Comparison of the characteristics and outcomes of children who were PCR-positive vs. PCR-negative for <italic>Shigella</italic>\\n</p>\",\n \"<p>Table ##TAB##0##1## compares the clinical and anthropometric characteristics of children with stool PCR positivity for <italic>Shigella </italic>with those who had a negative test result on PCR.</p>\",\n \"<p>There was no statistically significant difference between the two groups. The mean (SD) duration of diarrhea, as monitored after enrolment, was not significantly different (P=0.94) between PCR-positive participants (2.2 (1.54) days) and PCR-negative participants (2.3 (1.45) days). On univariate analysis, none of the evaluated risk factors or clinical predictors had a significant association with the <italic>Shigella </italic>infection (Table ##TAB##1##2##).</p>\",\n \"<p>Follow-up</p>\",\n \"<p>Out of 150 enrolled children, 23 were lost to follow-up, whereas 43 had a recurrence of diarrhea over the next three months. Among these, 14 patients required hospitalization, and antibiotics were used in 12 patients. During follow-up, only two patients had dysentery, and one patient developed persistent diarrhea. The recurrence of diarrhea episodes over three months was seen slightly more often among the children who had initial PCR (5, 45.4%) positivity than in PCR-negative participants (38, 37.8%), but this was not statistically significant (P=0.507). Also, there were no statistically significant differences in outcome in terms of growth faltering, recurrence of diarrhea, or hospitalization (Table ##TAB##2##3##).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>In this hospital-based study on under-five children with acute diarrhea, we documented that stool culture's diagnostic yield was very low compared to stool PCR amplification using a specific primer for the ipaH gene for the diagnosis of <italic>Shigella </italic>infection. The majority of <italic>Shigella </italic>infections presented with watery diarrhea rather than bloody diarrhea, and a history of blood in stools was a poor marker for the diagnosis of shigellosis. We documented that the molecular diagnostic approach performed better than conventional culture methods for the diagnosis of shigellosis. We could not find any significant clinical predictors associated with <italic>Shigella </italic>infection. There was no statistically significant difference in the overall outcome between PCR-positive and PCR-negative children at three months of follow-up.</p>","<p>The conventional methods of detection of <italic>Shigella </italic>infection rely on the stool culture, which turns out positive in a small fraction of actual cases of shigellosis due to multiple reasons like low bacterial load, loss of bacterial viability due to changes in ambient temperature and pH during specimen transport and storage, and use of antibiotics before specimen collection, transport, and storage [##REF##1184731##14##]. Molecular diagnostic methods seem to overcome some of the shortcomings of conventional diagnostic methods, thus improving the diagnosis of <italic>Shigella </italic>infection. The use of PCR assays based on the amplification of the ipaH gene sequence is one such molecular method for diagnosis in cases of dysentery, as it is carried by all four species of <italic>Shigella </italic>[##REF##8421181##15##].</p>","<p>Recent data from other settings also suggest that conventional culture yield is poor in episodes of <italic>Shigella</italic> diarrhea in young children [##UREF##8##16##]. The PCR-derived incidence for <italic>Shigella </italic>surpassed the original estimates by two-fold in the GEMS re-analysis, thus making it the most attributable pathogen of diarrhea among children under five [##REF##27673470##5##]. In a re-analysis by PCR of stool samples from the multi-country Malnutrition and Enteric Disease (MAL-ED) Study, <italic>Shigella</italic>-attributable incidence increased from 4% to 41.3% in children aged between 12-24 months [##UREF##9##17##]. Earlier, Dutta et al. from Kolkata, India, reported a <italic>Shigella </italic>detection rate of 15.3% (46 of 300) by stool PCR as against 7.7% (23 of 300) by stool culture [##REF##11478669##8##]. The positivity rate in the study by Dutta et al. was higher than that seen in our study, which is likely to be due to the inclusion of a very high proportion (42%; 126 out of 300) of children with dysentery. Similarly, Aggarwal et al. reported that the prevalence of <italic>Shigella </italic>increased from 8% (17 of 207) to 18.3% (11 of 60) in diarrhea and from 33% (39 of 118) to 56.7% (34 of 60) in dysentery when stool samples were analyzed by culture and PCR, respectively [##REF##27061990##12##].</p>","<p>In two large population-based surveillance studies from Vietnam and China, an ipaH gene amplification-based PCR assay was used to detect <italic>Shigella spp</italic>. from the rectal swab specimens of cases presenting with dysentery. Results showed the presence of the ipaH gene in approximately 93%-97% of randomly selected <italic>Shigella </italic>culture-positive specimens and 46%-58% of randomly selected <italic>Shigella </italic>culture-negative specimens [##REF##15131166##18##, ##REF##20951728##19##]. In a study by Von Seidlein et al. on the analysis of pooled data from studies from six Southeast Asian countries, i.e., Bangladesh, China, Pakistan, Indonesia, Vietnam, and Thailand, <italic>Shigella </italic>was isolated from 2,927 (5%) of 56,958 diarrhea episodes, and more than half of these were in children under the age of five, and PCR detected the ipaH gene in 33% of samples of culture-negative stool specimens [##UREF##10##20##]. In a study from Bangladesh, Islam et al. documented that a PCR assay based on an ipaH probe improved the rate of detection of <italic>Shigella </italic>in stool samples to 61% from 44% by the conventional culture method [##REF##10453122##21##]. They also tested 123 strains of <italic>E. coli</italic> by PCR to identify enteroinvasive <italic>E. coli</italic>, but none yielded any positive results. Though these studies have included variable age groups, the high detection rates of the ipaH gene in culture-negative stool specimens suggest that earlier estimates of shigellosis burden measured by conventional culture may have underestimated the true disease burden.</p>","<p>Blood in stools has been used as a surrogate marker for <italic>Shigella </italic>diarrhea. The WHO's diarrhea management guidelines recommend antibiotics effective against <italic>Shigella </italic>only when visible blood is present in stools. In our study, 84.6% (11 of 13) of the children with stool PCR positivity for <italic>Shigella </italic>presented with non-bloody diarrhea. In the GEMS re-analysis, <italic>Shigella spp</italic>. was the second highest cause of watery diarrhea, and overall, 40.3% (~527 of 1,310) of cases due to S<italic>higella spp.</italic> were non-dysenteric [##REF##27673470##5##]. Likewise, 86.2% of the attributable incidence for <italic>Shigella </italic>was non-dysenteric in the re-analysis of the MAL-ED cohort study [##UREF##9##17##]. The results of a recent meta-analysis showed that the proportion of <italic>Shigella </italic>infections presenting as dysentery has been decreasing [##UREF##11##22##]. This may be related to the diagnosis of such cases by molecular methods or early treatment with antibiotics. However, it is unclear whether <italic>Shigella </italic>infection without blood in stools needs therapy on similar lines. In our series of cases, antibiotics were administered for other reasons in cases where <italic>Shigella </italic>was later detected by molecular methods, except in three children in whom the infection resolved without any use of antibiotics.</p>","<p>The strength of our study was the prospective nature of enrollment, with a focus on the clinical presentation and outcome of <italic>Shigella </italic>infection detected with molecular methods. Our study also attempted to evaluate the clinical profile (resolution of diarrhea, change to bloody diarrhea in those with watery diarrhea, and recurrence) during a three-month follow-up period for children with <italic>Shigella </italic>infection managed as per current diarrhea treatment guidelines, which has not been evaluated in any of the studies comparing detection rates of <italic>Shigella </italic>between conventional and molecular methods. However, because of antibiotic use for other indications, we were unable to validly comment on the outcome of PCR-positive cases when they are managed without antibiotics. A relatively low <italic>Shigella </italic>prevalence in our series precluded the identification of any significant clinical marker of <italic>Shigella </italic>infection. Another limitation of our study was the use of the ipaH gene as a marker for <italic>Shigella </italic>detection, which may also be found in enteroinvasive <italic>E. coli</italic>. We enrolled hospitalized patients who primarily had moderate-to-severe diarrhea and concurrent illnesses; therefore, results from our study may not be generalizable to community settings with less severe diarrheal illnesses.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>In this hospital-based study on under-five children with acute diarrhea, we documented that stool culture's diagnostic yield was very low compared to stool PCR amplification using a specific primer for the ipaH gene for the diagnosis of <italic>Shigella </italic>infection. The majority of <italic>Shigella </italic>infections presented with watery diarrhea rather than bloody diarrhea, and a history of blood in stools was a poor marker for the diagnosis of shigellosis. We documented that the molecular diagnostic approach performed better than conventional culture methods for the diagnosis of shigellosis. We could not find any significant clinical predictors associated with <italic>Shigella </italic>infection. There was no statistically significant difference in the overall outcome between PCR-positive and PCR-negative children at three months of follow-up.</p>\",\n \"<p>The conventional methods of detection of <italic>Shigella </italic>infection rely on the stool culture, which turns out positive in a small fraction of actual cases of shigellosis due to multiple reasons like low bacterial load, loss of bacterial viability due to changes in ambient temperature and pH during specimen transport and storage, and use of antibiotics before specimen collection, transport, and storage [##REF##1184731##14##]. Molecular diagnostic methods seem to overcome some of the shortcomings of conventional diagnostic methods, thus improving the diagnosis of <italic>Shigella </italic>infection. The use of PCR assays based on the amplification of the ipaH gene sequence is one such molecular method for diagnosis in cases of dysentery, as it is carried by all four species of <italic>Shigella </italic>[##REF##8421181##15##].</p>\",\n \"<p>Recent data from other settings also suggest that conventional culture yield is poor in episodes of <italic>Shigella</italic> diarrhea in young children [##UREF##8##16##]. The PCR-derived incidence for <italic>Shigella </italic>surpassed the original estimates by two-fold in the GEMS re-analysis, thus making it the most attributable pathogen of diarrhea among children under five [##REF##27673470##5##]. In a re-analysis by PCR of stool samples from the multi-country Malnutrition and Enteric Disease (MAL-ED) Study, <italic>Shigella</italic>-attributable incidence increased from 4% to 41.3% in children aged between 12-24 months [##UREF##9##17##]. Earlier, Dutta et al. from Kolkata, India, reported a <italic>Shigella </italic>detection rate of 15.3% (46 of 300) by stool PCR as against 7.7% (23 of 300) by stool culture [##REF##11478669##8##]. The positivity rate in the study by Dutta et al. was higher than that seen in our study, which is likely to be due to the inclusion of a very high proportion (42%; 126 out of 300) of children with dysentery. Similarly, Aggarwal et al. reported that the prevalence of <italic>Shigella </italic>increased from 8% (17 of 207) to 18.3% (11 of 60) in diarrhea and from 33% (39 of 118) to 56.7% (34 of 60) in dysentery when stool samples were analyzed by culture and PCR, respectively [##REF##27061990##12##].</p>\",\n \"<p>In two large population-based surveillance studies from Vietnam and China, an ipaH gene amplification-based PCR assay was used to detect <italic>Shigella spp</italic>. from the rectal swab specimens of cases presenting with dysentery. Results showed the presence of the ipaH gene in approximately 93%-97% of randomly selected <italic>Shigella </italic>culture-positive specimens and 46%-58% of randomly selected <italic>Shigella </italic>culture-negative specimens [##REF##15131166##18##, ##REF##20951728##19##]. In a study by Von Seidlein et al. on the analysis of pooled data from studies from six Southeast Asian countries, i.e., Bangladesh, China, Pakistan, Indonesia, Vietnam, and Thailand, <italic>Shigella </italic>was isolated from 2,927 (5%) of 56,958 diarrhea episodes, and more than half of these were in children under the age of five, and PCR detected the ipaH gene in 33% of samples of culture-negative stool specimens [##UREF##10##20##]. In a study from Bangladesh, Islam et al. documented that a PCR assay based on an ipaH probe improved the rate of detection of <italic>Shigella </italic>in stool samples to 61% from 44% by the conventional culture method [##REF##10453122##21##]. They also tested 123 strains of <italic>E. coli</italic> by PCR to identify enteroinvasive <italic>E. coli</italic>, but none yielded any positive results. Though these studies have included variable age groups, the high detection rates of the ipaH gene in culture-negative stool specimens suggest that earlier estimates of shigellosis burden measured by conventional culture may have underestimated the true disease burden.</p>\",\n \"<p>Blood in stools has been used as a surrogate marker for <italic>Shigella </italic>diarrhea. The WHO's diarrhea management guidelines recommend antibiotics effective against <italic>Shigella </italic>only when visible blood is present in stools. In our study, 84.6% (11 of 13) of the children with stool PCR positivity for <italic>Shigella </italic>presented with non-bloody diarrhea. In the GEMS re-analysis, <italic>Shigella spp</italic>. was the second highest cause of watery diarrhea, and overall, 40.3% (~527 of 1,310) of cases due to S<italic>higella spp.</italic> were non-dysenteric [##REF##27673470##5##]. Likewise, 86.2% of the attributable incidence for <italic>Shigella </italic>was non-dysenteric in the re-analysis of the MAL-ED cohort study [##UREF##9##17##]. The results of a recent meta-analysis showed that the proportion of <italic>Shigella </italic>infections presenting as dysentery has been decreasing [##UREF##11##22##]. This may be related to the diagnosis of such cases by molecular methods or early treatment with antibiotics. However, it is unclear whether <italic>Shigella </italic>infection without blood in stools needs therapy on similar lines. In our series of cases, antibiotics were administered for other reasons in cases where <italic>Shigella </italic>was later detected by molecular methods, except in three children in whom the infection resolved without any use of antibiotics.</p>\",\n \"<p>The strength of our study was the prospective nature of enrollment, with a focus on the clinical presentation and outcome of <italic>Shigella </italic>infection detected with molecular methods. Our study also attempted to evaluate the clinical profile (resolution of diarrhea, change to bloody diarrhea in those with watery diarrhea, and recurrence) during a three-month follow-up period for children with <italic>Shigella </italic>infection managed as per current diarrhea treatment guidelines, which has not been evaluated in any of the studies comparing detection rates of <italic>Shigella </italic>between conventional and molecular methods. However, because of antibiotic use for other indications, we were unable to validly comment on the outcome of PCR-positive cases when they are managed without antibiotics. A relatively low <italic>Shigella </italic>prevalence in our series precluded the identification of any significant clinical marker of <italic>Shigella </italic>infection. Another limitation of our study was the use of the ipaH gene as a marker for <italic>Shigella </italic>detection, which may also be found in enteroinvasive <italic>E. coli</italic>. We enrolled hospitalized patients who primarily had moderate-to-severe diarrhea and concurrent illnesses; therefore, results from our study may not be generalizable to community settings with less severe diarrheal illnesses.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>The present study concludes that <italic>Shigella </italic>is an important cause of diarrhea in children under five, often missed by conventional laboratory methods. Blood in stools as a syndromic indicator for <italic>Shigella </italic>infection needs to be reconsidered as the majority of <italic>Shigella </italic>diarrhea cases have non-bloody stools. As the current management guidelines for childhood diarrhea recommend antibiotics for only bloody diarrhea, studies evaluating other clinical predictors of <italic>Shigella </italic>infection and faster and more cost-effective techniques for its molecular diagnosis are required. Large, community-based longitudinal studies are needed to evaluate the outcome of non-dysenteric <italic>Shigella </italic>infections diagnosed by molecular methods so that management guidelines for such infections can be formulated.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>The present study concludes that <italic>Shigella </italic>is an important cause of diarrhea in children under five, often missed by conventional laboratory methods. Blood in stools as a syndromic indicator for <italic>Shigella </italic>infection needs to be reconsidered as the majority of <italic>Shigella </italic>diarrhea cases have non-bloody stools. As the current management guidelines for childhood diarrhea recommend antibiotics for only bloody diarrhea, studies evaluating other clinical predictors of <italic>Shigella </italic>infection and faster and more cost-effective techniques for its molecular diagnosis are required. Large, community-based longitudinal studies are needed to evaluate the outcome of non-dysenteric <italic>Shigella </italic>infections diagnosed by molecular methods so that management guidelines for such infections can be formulated.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Background and objectives: <italic>Shigella </italic>is an important cause of diarrhea in children under five, often missed by conventional laboratory methods. Blood in stools has always been a syndromic indicator for <italic>Shigella </italic>diarrhea, but most cases present with watery diarrhea without blood. This study aimed to determine the frequency of <italic>Shigella </italic>detected by molecular and conventional methods in children under five. Additionally, we aimed to study the clinical profile and outcome of children with <italic>Shigella </italic>diarrhea managed as per current diarrhea treatment guidelines.</p>","<p>Methods: In this hospital-based prospective observational study, stool samples from 150 children (age range: one month to five years) with acute diarrhea (duration < seven days) were subjected to routine microscopic examination, stool culture, and DNA extraction. The extracted DNA from stored stool samples was subjected to polymerase chain reaction (PCR) amplification using a specific primer for the invasion plasmid antigen H gene sequence (ipaH) gene at 424 bp. Results were interpreted in the context of the percentage of isolation of <italic>Shigella </italic>by molecular (PCR) and conventional methods (stool microscopy and culture) and the follow-up outcome in terms of recurrence of diarrhea or dysentery and growth faltering over three months after discharge.</p>","<p>Results: <italic>Shigella </italic>infection was diagnosed in stool samples by PCR from 13 (8.7%) children, whereas it was isolated by conventional stool culture in only one (0.7%) child. The sensitivity of culture was only 7.7% against PCR for the diagnosis of <italic>Shigella </italic>infection, whereas blood in stools had a sensitivity of 15.4%. The majority of <italic>Shigella </italic>PCR-positive cases (11 out of 13) presented with non-bloody diarrhea. None of the evaluated clinical predictors had a significant association with the <italic>Shigella </italic>infection. No statistically significant difference was found between PCR-positive and PCR-negative children at the end of follow-up (P>0.05).</p>","<p>Conclusion: The majority of children with <italic>Shigella </italic>infection present with watery diarrhea rather than bloody diarrhea, and a history of blood in stools is a poor marker for the diagnosis of shigellosis. The diagnostic performance of stool culture is also very low compared to stool PCR for the diagnosis of <italic>Shigella </italic>diarrhea.</p>"],"string":"[\n \"<p>Background and objectives: <italic>Shigella </italic>is an important cause of diarrhea in children under five, often missed by conventional laboratory methods. Blood in stools has always been a syndromic indicator for <italic>Shigella </italic>diarrhea, but most cases present with watery diarrhea without blood. This study aimed to determine the frequency of <italic>Shigella </italic>detected by molecular and conventional methods in children under five. Additionally, we aimed to study the clinical profile and outcome of children with <italic>Shigella </italic>diarrhea managed as per current diarrhea treatment guidelines.</p>\",\n \"<p>Methods: In this hospital-based prospective observational study, stool samples from 150 children (age range: one month to five years) with acute diarrhea (duration < seven days) were subjected to routine microscopic examination, stool culture, and DNA extraction. The extracted DNA from stored stool samples was subjected to polymerase chain reaction (PCR) amplification using a specific primer for the invasion plasmid antigen H gene sequence (ipaH) gene at 424 bp. Results were interpreted in the context of the percentage of isolation of <italic>Shigella </italic>by molecular (PCR) and conventional methods (stool microscopy and culture) and the follow-up outcome in terms of recurrence of diarrhea or dysentery and growth faltering over three months after discharge.</p>\",\n \"<p>Results: <italic>Shigella </italic>infection was diagnosed in stool samples by PCR from 13 (8.7%) children, whereas it was isolated by conventional stool culture in only one (0.7%) child. The sensitivity of culture was only 7.7% against PCR for the diagnosis of <italic>Shigella </italic>infection, whereas blood in stools had a sensitivity of 15.4%. The majority of <italic>Shigella </italic>PCR-positive cases (11 out of 13) presented with non-bloody diarrhea. None of the evaluated clinical predictors had a significant association with the <italic>Shigella </italic>infection. No statistically significant difference was found between PCR-positive and PCR-negative children at the end of follow-up (P>0.05).</p>\",\n \"<p>Conclusion: The majority of children with <italic>Shigella </italic>infection present with watery diarrhea rather than bloody diarrhea, and a history of blood in stools is a poor marker for the diagnosis of shigellosis. The diagnostic performance of stool culture is also very low compared to stool PCR for the diagnosis of <italic>Shigella </italic>diarrhea.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Flowchart depicting the selection of participants for the study</title><p>mo: month; y: year; d: days; TB: tuberculosis</p></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>Flowchart depicting the selection of participants for the study</title><p>mo: month; y: year; d: days; TB: tuberculosis</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Comparison of clinical and anthropometric parameters at presentation between children with or without stool PCR Shigella positivity</title><p><sup>a</sup>Data in number (%) unless stated otherwise; <sup>b</sup>n=7 missing cases as the measurement taken for children >6 months; <sup>c</sup>n=136 as the value could not be calculated for children with a length less than 45 cm; <sup>d</sup>n=96 missing cases as the measurement taken for children >6 months</p><p>n: number of patients; IQR: interquartile range; SD: standard deviation; PCR: polymerase chain reaction; SAM: severe acute malnutrition</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Parameter</td><td rowspan=\"1\" colspan=\"1\">Stool PCR-positive (n=13, 8.7%)<sup>a</sup>\n</td><td rowspan=\"1\" colspan=\"1\">Stool PCR-negative (n=137, 91.3%)<sup>a</sup>\n</td><td rowspan=\"1\" colspan=\"1\">P-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Diarrhea duration (days); median (IQR)</td><td rowspan=\"1\" colspan=\"1\">2.00 (1.0, 3.0)</td><td rowspan=\"1\" colspan=\"1\">2.00 (1.0,3.0)</td><td rowspan=\"1\" colspan=\"1\">0.908</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">No. of stools in last 24 hours; median (IQR)</td><td rowspan=\"1\" colspan=\"1\">15.00 (8.0, 19.0)</td><td rowspan=\"1\" colspan=\"1\">15.00 (11.0, 20.0)</td><td rowspan=\"1\" colspan=\"1\">0.390</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Blood in stools</td><td rowspan=\"1\" colspan=\"1\">2 (15.4%)</td><td rowspan=\"1\" colspan=\"1\">7 (5.1%)</td><td rowspan=\"1\" colspan=\"1\">0.176</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Vomiting</td><td rowspan=\"1\" colspan=\"1\">11 (84.6%)</td><td rowspan=\"1\" colspan=\"1\">116 (84.7%)</td><td rowspan=\"1\" colspan=\"1\">0.996</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Fever</td><td rowspan=\"1\" colspan=\"1\">6 (46.2%)</td><td rowspan=\"1\" colspan=\"1\">54 (39.4%)</td><td rowspan=\"1\" colspan=\"1\">0.636</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Abdominal pain</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">8 (5.8%)</td><td rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Poor oral acceptance</td><td rowspan=\"1\" colspan=\"1\">10 (76.9%)</td><td rowspan=\"1\" colspan=\"1\">89 (65%)</td><td rowspan=\"1\" colspan=\"1\">0.544</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Convulsions</td><td rowspan=\"1\" colspan=\"1\">1 (7.7%)</td><td rowspan=\"1\" colspan=\"1\">8 (5.8%)</td><td rowspan=\"1\" colspan=\"1\">0.568</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Rash</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">5 (3.6%)</td><td rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Cough</td><td rowspan=\"1\" colspan=\"1\">1 (7.7%)</td><td rowspan=\"1\" colspan=\"1\">18 (13.1%)</td><td rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Immunized for age</td><td rowspan=\"1\" colspan=\"1\">7 (53.8%)</td><td rowspan=\"1\" colspan=\"1\">87 (63.5%)</td><td rowspan=\"1\" colspan=\"1\">0.679</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Dehydration</td><td rowspan=\"1\" colspan=\"1\">11 (84.6%)</td><td rowspan=\"1\" colspan=\"1\">127 (92.7%)</td><td rowspan=\"1\" colspan=\"1\">0.465</td></tr><tr><td colspan=\"4\" rowspan=\"1\">Anthropometry at enrolment</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Weight for age (WFA) (kg); mean ± SD</td><td rowspan=\"1\" colspan=\"1\">6.62 (2.30)</td><td rowspan=\"1\" colspan=\"1\">6.76 (2.68)</td><td rowspan=\"1\" colspan=\"1\">0.847</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Height for age (HFA) (cm); mean ± SD</td><td rowspan=\"1\" colspan=\"1\">69.10 (12.91)</td><td rowspan=\"1\" colspan=\"1\">71.05 (13.63)</td><td rowspan=\"1\" colspan=\"1\">0.620</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Weight for age Z-score (WAZ); mean ± SD</td><td rowspan=\"1\" colspan=\"1\">-2.62 (1.60)</td><td rowspan=\"1\" colspan=\"1\">-2.98 (1.35)</td><td rowspan=\"1\" colspan=\"1\">0.359</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Height for age Z-score (HAZ); mean ± SD</td><td rowspan=\"1\" colspan=\"1\">-1.62 (1.63)</td><td rowspan=\"1\" colspan=\"1\">-1.79 (1.69)</td><td rowspan=\"1\" colspan=\"1\">0.724</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Weight for height Z-score (WHZ); mean ± SD</td><td rowspan=\"1\" colspan=\"1\">-2.10 (1.97)</td><td rowspan=\"1\" colspan=\"1\">-2.68 (1.58)<sup> c</sup>\n</td><td rowspan=\"1\" colspan=\"1\">0.219</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Mid upper arm circumference (MUAC) (cm); mean ± SD</td><td rowspan=\"1\" colspan=\"1\">12.36 (1.53)<sup> b</sup>\n</td><td rowspan=\"1\" colspan=\"1\">12.48 (1.21)<sup> d</sup>\n</td><td rowspan=\"1\" colspan=\"1\">0.803</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Wasting</td><td rowspan=\"1\" colspan=\"1\">2 (15.4%)</td><td rowspan=\"1\" colspan=\"1\">31 (22.8%)</td><td rowspan=\"1\" colspan=\"1\">0.607</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Severe wasting</td><td rowspan=\"1\" colspan=\"1\">4 (30.8%)</td><td rowspan=\"1\" colspan=\"1\">27 (19.9%) <sup>c</sup>\n</td><td rowspan=\"1\" colspan=\"1\">-</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Stunting</td><td rowspan=\"1\" colspan=\"1\">2 (15.4%)</td><td rowspan=\"1\" colspan=\"1\">28 (19%)</td><td rowspan=\"1\" colspan=\"1\">0.890</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Severe stunting</td><td rowspan=\"1\" colspan=\"1\">3 (23.1%)</td><td rowspan=\"1\" colspan=\"1\">25 (18.3%)</td><td rowspan=\"1\" colspan=\"1\">-</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">SAM</td><td rowspan=\"1\" colspan=\"1\">5 (38.5%)</td><td rowspan=\"1\" colspan=\"1\">35 (25.7%)</td><td rowspan=\"1\" colspan=\"1\">0.336</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Analysis of risk factors and clinical predictors of Shigella infection</title><p>n: number of patients; PCR: polymerase chain reaction; SAM: severe acute malnutrition.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Parameter</td><td rowspan=\"1\" colspan=\"1\">Stool PCR-positive cases (n=13, 8.7%)</td><td rowspan=\"1\" colspan=\"1\">Stool PCR-negative cases (n=137, 91.3%)</td><td rowspan=\"1\" colspan=\"1\">Odds ratio (95% CI)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Age <1 year</td><td rowspan=\"1\" colspan=\"1\">9 (69.2%)</td><td rowspan=\"1\" colspan=\"1\">78 (56.9%)</td><td rowspan=\"1\" colspan=\"1\">1.70 (0.50-5.80)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Male gender</td><td rowspan=\"1\" colspan=\"1\">7 (53.8%)</td><td rowspan=\"1\" colspan=\"1\">65 (47.4%)</td><td rowspan=\"1\" colspan=\"1\">1.29 (0.41-4.04)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Lower socioeconomic status</td><td rowspan=\"1\" colspan=\"1\">12 (92.3%)</td><td rowspan=\"1\" colspan=\"1\">130 (94.9%)</td><td rowspan=\"1\" colspan=\"1\">0.65 (0.07-5.70)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Blood in stools</td><td rowspan=\"1\" colspan=\"1\">2 (15.4%)</td><td rowspan=\"1\" colspan=\"1\">7 (5.1%)</td><td rowspan=\"1\" colspan=\"1\">3.38 (0.62-18.26)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Vomiting</td><td rowspan=\"1\" colspan=\"1\">11 (84.6%)</td><td rowspan=\"1\" colspan=\"1\">116 (84.7%)</td><td rowspan=\"1\" colspan=\"1\">1.00 (0.21-4.82)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Poor oral acceptance</td><td rowspan=\"1\" colspan=\"1\">10 (76.9%)</td><td rowspan=\"1\" colspan=\"1\">89 (65%)</td><td rowspan=\"1\" colspan=\"1\">1.8 (0.47-6.85)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Convulsion</td><td rowspan=\"1\" colspan=\"1\">1 (7.7%)</td><td rowspan=\"1\" colspan=\"1\">8 (5.8%)</td><td rowspan=\"1\" colspan=\"1\">1.34 (0.15-11.67)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Cough</td><td rowspan=\"1\" colspan=\"1\">1 (7.7%)</td><td rowspan=\"1\" colspan=\"1\">18 (13.1%)</td><td rowspan=\"1\" colspan=\"1\">0.55 (0.07-4.50)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dehydration</td><td rowspan=\"1\" colspan=\"1\">11 (84.6%)</td><td rowspan=\"1\" colspan=\"1\">127 (92.7%)</td><td rowspan=\"1\" colspan=\"1\">0.43 (0.08-2.23)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Wasting</td><td rowspan=\"1\" colspan=\"1\">6 (46.2%)</td><td rowspan=\"1\" colspan=\"1\">58 (42.3%)</td><td rowspan=\"1\" colspan=\"1\">1.17 (0.37-3.66)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Stunting</td><td rowspan=\"1\" colspan=\"1\">5 (38.5%)</td><td rowspan=\"1\" colspan=\"1\">51 (37.2%)</td><td rowspan=\"1\" colspan=\"1\">1.05 (0.33-3.40)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">SAM</td><td rowspan=\"1\" colspan=\"1\">5 (38.5%)</td><td rowspan=\"1\" colspan=\"1\">35 (25.7%)</td><td rowspan=\"1\" colspan=\"1\">1.82 (0.56-5.94)</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB3\"><label>Table 3</label><caption><title>Outcome of enrolled children with acute diarrhea at the three-month follow-up</title><p><sup>a</sup>n=127 (11 stool PCR-positive cases, 116 stool PCR-negative cases); <sup>b</sup>n=43 (5 stool PCR-positive cases, 38 stool PCR-negative cases)</p><p>PCR: polymerase chain reaction</p><p>Binomial analysis was not done for persistent diarrhea, bloody diarrhea, and mortality as none of the PCR cases who completed the three months of follow-up had any of these outcomes.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Parameter</td><td rowspan=\"1\" colspan=\"1\">Stool PCR-positive cases (n=13, 8.7%)</td><td rowspan=\"1\" colspan=\"1\">Stool PCR-negative cases (n=137, 91.3%)</td><td rowspan=\"1\" colspan=\"1\">Odds ratio (95% CI)</td><td rowspan=\"1\" colspan=\"1\">P-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Duration of diarrhea ≥7 days</td><td rowspan=\"1\" colspan=\"1\">4/13 (30.8)</td><td rowspan=\"1\" colspan=\"1\">50 (36.1)</td><td rowspan=\"1\" colspan=\"1\">0.77 (0.23-2.64)</td><td rowspan=\"1\" colspan=\"1\">0.68</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Recurrence in the next 3 months<sup> a</sup>\n</td><td rowspan=\"1\" colspan=\"1\">5 (45.5)</td><td rowspan=\"1\" colspan=\"1\">38 (32.8)</td><td rowspan=\"1\" colspan=\"1\">1.71 (0.49-5.96)</td><td rowspan=\"1\" colspan=\"1\">0.51</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hospitalization in the next 3 months <sup>b</sup>\n</td><td rowspan=\"1\" colspan=\"1\">1 (20)</td><td rowspan=\"1\" colspan=\"1\">13 (34.2)</td><td rowspan=\"1\" colspan=\"1\">0.48 (0.05-4.75)</td><td rowspan=\"1\" colspan=\"1\">1.00</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB1\\\"><label>Table 1</label><caption><title>Comparison of clinical and anthropometric parameters at presentation between children with or without stool PCR Shigella positivity</title><p><sup>a</sup>Data in number (%) unless stated otherwise; <sup>b</sup>n=7 missing cases as the measurement taken for children >6 months; <sup>c</sup>n=136 as the value could not be calculated for children with a length less than 45 cm; <sup>d</sup>n=96 missing cases as the measurement taken for children >6 months</p><p>n: number of patients; IQR: interquartile range; SD: standard deviation; PCR: polymerase chain reaction; SAM: severe acute malnutrition</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Parameter</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stool PCR-positive (n=13, 8.7%)<sup>a</sup>\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stool PCR-negative (n=137, 91.3%)<sup>a</sup>\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">P-value</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Diarrhea duration (days); median (IQR)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.00 (1.0, 3.0)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.00 (1.0,3.0)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.908</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">No. of stools in last 24 hours; median (IQR)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">15.00 (8.0, 19.0)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">15.00 (11.0, 20.0)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.390</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Blood in stools</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2 (15.4%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7 (5.1%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.176</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Vomiting</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">11 (84.6%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">116 (84.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.996</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Fever</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6 (46.2%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">54 (39.4%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.636</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Abdominal pain</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8 (5.8%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.000</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Poor oral acceptance</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 (76.9%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">89 (65%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.544</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Convulsions</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1 (7.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8 (5.8%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.568</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Rash</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5 (3.6%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.000</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cough</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1 (7.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">18 (13.1%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.000</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Immunized for age</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7 (53.8%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">87 (63.5%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.679</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Dehydration</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">11 (84.6%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">127 (92.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.465</td></tr><tr><td colspan=\\\"4\\\" rowspan=\\\"1\\\">Anthropometry at enrolment</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Weight for age (WFA) (kg); mean ± SD</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.62 (2.30)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.76 (2.68)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.847</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Height for age (HFA) (cm); mean ± SD</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">69.10 (12.91)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">71.05 (13.63)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.620</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Weight for age Z-score (WAZ); mean ± SD</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-2.62 (1.60)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-2.98 (1.35)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.359</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Height for age Z-score (HAZ); mean ± SD</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-1.62 (1.63)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-1.79 (1.69)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.724</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Weight for height Z-score (WHZ); mean ± SD</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-2.10 (1.97)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-2.68 (1.58)<sup> c</sup>\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.219</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Mid upper arm circumference (MUAC) (cm); mean ± SD</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12.36 (1.53)<sup> b</sup>\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12.48 (1.21)<sup> d</sup>\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.803</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Wasting</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2 (15.4%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">31 (22.8%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.607</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Severe wasting</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4 (30.8%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">27 (19.9%) <sup>c</sup>\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stunting</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2 (15.4%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">28 (19%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.890</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Severe stunting</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3 (23.1%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">25 (18.3%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">SAM</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5 (38.5%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">35 (25.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.336</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB2\\\"><label>Table 2</label><caption><title>Analysis of risk factors and clinical predictors of Shigella infection</title><p>n: number of patients; PCR: polymerase chain reaction; SAM: severe acute malnutrition.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Parameter</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stool PCR-positive cases (n=13, 8.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stool PCR-negative cases (n=137, 91.3%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Odds ratio (95% CI)</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Age <1 year</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">9 (69.2%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">78 (56.9%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.70 (0.50-5.80)</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Male gender</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7 (53.8%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">65 (47.4%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.29 (0.41-4.04)</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Lower socioeconomic status</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12 (92.3%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">130 (94.9%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.65 (0.07-5.70)</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Blood in stools</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2 (15.4%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7 (5.1%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.38 (0.62-18.26)</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Vomiting</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">11 (84.6%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">116 (84.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.00 (0.21-4.82)</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Poor oral acceptance</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 (76.9%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">89 (65%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.8 (0.47-6.85)</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Convulsion</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1 (7.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8 (5.8%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.34 (0.15-11.67)</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cough</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1 (7.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">18 (13.1%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.55 (0.07-4.50)</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Dehydration</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">11 (84.6%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">127 (92.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.43 (0.08-2.23)</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Wasting</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6 (46.2%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">58 (42.3%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.17 (0.37-3.66)</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stunting</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5 (38.5%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">51 (37.2%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.05 (0.33-3.40)</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">SAM</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5 (38.5%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">35 (25.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.82 (0.56-5.94)</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB3\\\"><label>Table 3</label><caption><title>Outcome of enrolled children with acute diarrhea at the three-month follow-up</title><p><sup>a</sup>n=127 (11 stool PCR-positive cases, 116 stool PCR-negative cases); <sup>b</sup>n=43 (5 stool PCR-positive cases, 38 stool PCR-negative cases)</p><p>PCR: polymerase chain reaction</p><p>Binomial analysis was not done for persistent diarrhea, bloody diarrhea, and mortality as none of the PCR cases who completed the three months of follow-up had any of these outcomes.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Parameter</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stool PCR-positive cases (n=13, 8.7%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stool PCR-negative cases (n=137, 91.3%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Odds ratio (95% CI)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">P-value</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Duration of diarrhea ≥7 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4/13 (30.8)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">50 (36.1)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.77 (0.23-2.64)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.68</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Recurrence in the next 3 months<sup> a</sup>\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5 (45.5)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">38 (32.8)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.71 (0.49-5.96)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.51</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hospitalization in the next 3 months <sup>b</sup>\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1 (20)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">13 (34.2)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.48 (0.05-4.75)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.00</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study. Institutional Ethics Committee for Human Research, University College of Medical Sciences, Delhi issued approval IEC-HR/2017/32/88</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study. Institutional Ethics Committee for Human Research, University College of Medical Sciences, Delhi issued approval IEC-HR/2017/32/88</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Animal Ethics</title><fn fn-type=\\\"other\\\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0015-00000050546-i01\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0015-00000050546-i01\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"1\"], \"article-title\": [\"Diarrhoeal disease\"], \"date-in-citation\": [\"\\n\"], \"month\": [\"11\"], \"year\": [\"2022\", \"2017\"], \"uri\": [\"https://www.who.int/news-room/fact-sheets/detail/diarrhoeal-disease\"]}, {\"label\": [\"2\"], \"article-title\": [\"Levels and trends in child mortality 2019\"], \"date-in-citation\": [\"\\n\"], \"month\": [\"11\"], \"year\": [\"2022\", \"2019\"], \"uri\": [\"https://www.unicef.org/reports/levels-and-trends-child-mortality-report-2019\"]}, {\"label\": [\"6\"], \"article-title\": [\"Diarrhoea\"], \"date-in-citation\": [\"\\n\"], \"month\": [\"12\"], \"year\": [\"2023\", \"2018\"], \"uri\": [\"https://www.who.int/health-topics/diarrhoea#tab=tab_1\"]}, {\"label\": [\"7\"], \"article-title\": [\"Performance Standards for Antimicrobial Susceptibility Testing, 33rd Edition\"], \"year\": [\"2011\"], \"uri\": [\"https://clsi.org/m100?gad=1&gclid=Cj0KCQjwnrmlBhDHARIsADJ5b_l8l6hcAhpuFmBtCxzNQQo2kCFfcnYxy9KKCXd-huus2GJMsqf10dYaAnieEALw_wcB\"]}, {\"label\": [\"9\"], \"article-title\": [\"World Health Organization. \\\"The treatment of diarrhoea: a manual for physicians and other senior health workers.\\\" (2005). Accessed: November 10, 2022: http://apps.who.int/iris/bitstream/10665/43209/1/9241593180.pdf\"], \"source\": [\"The Treatment of Diarrhoea: A Manual for Physicians and Other Senior Health Workers, 4th Rev\"], \"person-group\": [\"\\n\"], \"surname\": [\"World Health\"], \"given-names\": [\"Organization\"], \"publisher-loc\": [\"Geneva\"], \"publisher-name\": [\"World Health Organization\"], \"year\": [\"2005\"], \"uri\": [\"https://apps.who.int/iris/handle/10665/43209\"]}, {\"label\": [\"10\"], \"article-title\": [\"World Health Organisation. Guidelines for the Control of Shigellosis, Including Epidemics due to Shigella Dysenteriae 1.2005. Accessed: November 10\"], \"source\": [\"Guidelines for the Control of Shigellosis, Including Epidemics Due to Shigella dysenteriae Type 1\"], \"person-group\": [\"\\n\"], \"surname\": [\"World Health\"], \"given-names\": [\"Organisation\"], \"publisher-loc\": [\"Geneva, Switzerland\"], \"publisher-name\": [\"World Health Organization\"], \"year\": [\"2005\"], \"uri\": [\"http://whqlibdoc.who.int/int/publications/2005/9241592330.pdf\"]}, {\"label\": [\"11\"], \"article-title\": [\"Guidelines for the inpatient treatment of severely malnourished children\"], \"source\": [\"Guidelines for the Inpatient Treatment of Severely Malnourished Children\"], \"date-in-citation\": [\"\\n\"], \"month\": [\"11\"], \"year\": [\"2022\", \"2003\"], \"person-group\": [\"\\n\"], \"surname\": [\"Ashworth\", \"Khanum\", \"Jackson\", \"Schofield\"], \"given-names\": [\"A\", \"S\", \"A\", \"C\"], \"publisher-loc\": [\"Geneva, Switzerland\"], \"publisher-name\": [\"World Health Organization\"], \"uri\": [\"https://iris.who.int/bitstream/handle/10665/42724/9241546093.pdf?sequence=1\"]}, {\"label\": [\"13\"], \"article-title\": [\"Socio-economic status scales updated for 2017\"], \"source\": [\"Int J Res Med Sci\"], \"person-group\": [\"\\n\"], \"surname\": [\"Singh\", \"Sharma\", \"Nagesh\"], \"given-names\": [\"T\", \"S\", \"S\"], \"fpage\": [\"3264\"], \"lpage\": [\"3267\"], \"volume\": [\"7\"], \"year\": [\"2017\"]}, {\"label\": [\"16\"], \"article-title\": [\"Epidemiology of Shigella infections and diarrhea in the first two years of life using culture-independent diagnostics in 8 low-resource settings\"], \"source\": [\"PLoS Negl Trop Dis\"], \"person-group\": [\"\\n\"], \"surname\": [\"Rogawski McQuade\", \"Shaheen\", \"Kabir\"], \"given-names\": [\"ET\", \"F\", \"F\"], \"fpage\": [\"0\"], \"volume\": [\"14\"], \"year\": [\"2020\"]}, {\"label\": [\"17\"], \"article-title\": [\"Use of quantitative molecular diagnostic methods to assess the aetiology, burden, and clinical characteristics of diarrhoea in children in low-resource settings: a reanalysis of the MAL-ED cohort study\"], \"source\": [\"Lancet Glob Health\"], \"person-group\": [\"\\n\"], \"surname\": [\"Platts-Mills\", \"Liu\", \"Rogawski\"], \"given-names\": [\"JA\", \"J\", \"ET\"], \"fpage\": [\"0\"], \"lpage\": [\"18\"], \"volume\": [\"6\"], \"year\": [\"2018\"]}, {\"label\": [\"20\"], \"article-title\": [\"A multicentre study of Shigella diarrhoea in six Asian countries: disease burden, clinical manifestations, and microbiology\"], \"source\": [\"PLoS Med\"], \"person-group\": [\"\\n\"], \"surname\": [\"von Seidlein\", \"Kim\", \"Ali\"], \"given-names\": [\"L\", \"DR\", \"M\"], \"fpage\": [\"0\"], \"volume\": [\"3\"], \"year\": [\"2006\"]}, {\"label\": [\"22\"], \"article-title\": [\"Identification and management of Shigella infection in children with diarrhoea: a systematic review and meta-analysis\"], \"source\": [\"Lancet Glob Health\"], \"person-group\": [\"\\n\"], \"surname\": [\"Tickell\", \"Brander\", \"Atlas\", \"Pernica\", \"Walson\", \"Pavlinac\"], \"given-names\": [\"KD\", \"RL\", \"HE\", \"JM\", \"JL\", \"PB\"], \"fpage\": [\"0\"], \"lpage\": [\"48\"], \"volume\": [\"5\"], \"year\": [\"2017\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":22,"string":"22"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50546"},"package_file":{"kind":"string","value":"oa_package/45/9d/PMC10787845.tar.gz"}}},{"rowIdx":153,"cells":{"accession_id":{"kind":"string","value":"PMC10787846"},"pmid":{"kind":"string","value":"38222208"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Mucoid degeneration (MD) of the anterior cruciate ligament (ACL) is a rare condition, with a prevalence rate ranging from 0.2% to 1.2% [##REF##11372618##1##]. The cause of this condition is not well understood and has been the subject of vigorous debate, with only a limited number of reports in the literature detailing its origin [##REF##20811733##2##,##REF##22265045##3##]. It is characterized by the infiltration of a mucoid-like substance within the ACL, which can cause knee pain and restricted motion [##REF##19089409##4##]. Although it was once considered rare, recent reports suggest that it may be more common than previously thought, implying that it is underdiagnosed or misdiagnosed [##REF##18210315##5##]. We describe a successful treatment approach for MD of ACL, which involves the application of arthroscopic debridement.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Mucoid degeneration (MD) of the anterior cruciate ligament (ACL) is a rare condition, with a prevalence rate ranging from 0.2% to 1.2% [##REF##11372618##1##]. The cause of this condition is not well understood and has been the subject of vigorous debate, with only a limited number of reports in the literature detailing its origin [##REF##20811733##2##,##REF##22265045##3##]. It is characterized by the infiltration of a mucoid-like substance within the ACL, which can cause knee pain and restricted motion [##REF##19089409##4##]. Although it was once considered rare, recent reports suggest that it may be more common than previously thought, implying that it is underdiagnosed or misdiagnosed [##REF##18210315##5##]. We describe a successful treatment approach for MD of ACL, which involves the application of arthroscopic debridement.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>MD of ACL is an uncommon condition that leads to knee pain and discomfort, primarily impacting individuals in their fourth decade of life [##REF##18997652##6##]. Kumar et al. [##REF##10204939##7##] initially coined the term mucoid cystic degeneration to describe this condition affecting the ACL. It is characterized by significant disruption of the ligament's normal appearance and can contribute to knee pain. The predominant symptom associated with MD of ACL is knee pain, primarily located in the posterior aspect [##UREF##0##8##,##REF##19288177##9##]. This pain is caused by mechanical impingement on the posterior cruciate ligament and the posterior capsule or bone erosions [##REF##19288177##9##]. Additional clinical manifestations include a mechanical hindrance to extension, varying degrees of swelling, and audible clicking sounds [##REF##14997351##10##].</p>","<p>MRI serves as a valuable tool for evaluating MD of ACL during preoperative assessment. It shows an ACL that is indistinctly defined, exhibiting an enlarged dimension while retaining a typical orientation, and presenting heightened signal intensity interspersed among visible intact ACL fibers, creating a distinctive \"celery stalk\" appearance [##REF##11465770##11##]. On T1-weighted images, MD of ACL typically appears with intermediate signal intensity, while on T2-weighted images, it shows high signal intensity [##REF##11465770##11##,##REF##14760345##12##]. Hodler et al. correlated MRI appearances with histological findings and found that 29 out of 38 ligaments had focal areas of signal increase, suggesting a correlation between the focal MRI signal changes and the presence of degenerative changes in the ligaments [##REF##1632355##13##].</p>","<p>Arthroscopic observations suggest that MD of ACL involves a hypertrophied, fibrillated ligament with the presence of yellowish mucinous material interspersed among the fibers. This is often accompanied by the absence of the ligamentum mucosum [##REF##14760345##12##]. Additionally, a lack of smooth synovial lining is typically noted [##REF##21331652##14##]. Arthroscopic surgery is a discretionary treatment option for MD of ACL. This procedure involves the partial removal of lesions within the ACL, leading to rapid pain relief and improved range of motion, without any persistent symptoms of instability. The reduction in volume and tension within the ACL is often attributed to the notable pain relief [##REF##27299081##15##]. Debridement of mucinous substance, along with partial resection of ACL, is a recommended and effective therapy that does not cause instability, according to many authors [##REF##18629461##16##].</p>","<p>While certain authors have underscored the significance of notchplasty as a supplementary step in the procedure, Motmans and Verheyden have challenged this idea by arguing against the necessity of notchplasty. They contend that a comprehensive debridement alone is sufficient to resolve impingement and effectively address the underlying pathology [##REF##19089409##4##].</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>MD of ACL is an uncommon condition that leads to knee pain and discomfort, primarily impacting individuals in their fourth decade of life [##REF##18997652##6##]. Kumar et al. [##REF##10204939##7##] initially coined the term mucoid cystic degeneration to describe this condition affecting the ACL. It is characterized by significant disruption of the ligament's normal appearance and can contribute to knee pain. The predominant symptom associated with MD of ACL is knee pain, primarily located in the posterior aspect [##UREF##0##8##,##REF##19288177##9##]. This pain is caused by mechanical impingement on the posterior cruciate ligament and the posterior capsule or bone erosions [##REF##19288177##9##]. Additional clinical manifestations include a mechanical hindrance to extension, varying degrees of swelling, and audible clicking sounds [##REF##14997351##10##].</p>\",\n \"<p>MRI serves as a valuable tool for evaluating MD of ACL during preoperative assessment. It shows an ACL that is indistinctly defined, exhibiting an enlarged dimension while retaining a typical orientation, and presenting heightened signal intensity interspersed among visible intact ACL fibers, creating a distinctive \\\"celery stalk\\\" appearance [##REF##11465770##11##]. On T1-weighted images, MD of ACL typically appears with intermediate signal intensity, while on T2-weighted images, it shows high signal intensity [##REF##11465770##11##,##REF##14760345##12##]. Hodler et al. correlated MRI appearances with histological findings and found that 29 out of 38 ligaments had focal areas of signal increase, suggesting a correlation between the focal MRI signal changes and the presence of degenerative changes in the ligaments [##REF##1632355##13##].</p>\",\n \"<p>Arthroscopic observations suggest that MD of ACL involves a hypertrophied, fibrillated ligament with the presence of yellowish mucinous material interspersed among the fibers. This is often accompanied by the absence of the ligamentum mucosum [##REF##14760345##12##]. Additionally, a lack of smooth synovial lining is typically noted [##REF##21331652##14##]. Arthroscopic surgery is a discretionary treatment option for MD of ACL. This procedure involves the partial removal of lesions within the ACL, leading to rapid pain relief and improved range of motion, without any persistent symptoms of instability. The reduction in volume and tension within the ACL is often attributed to the notable pain relief [##REF##27299081##15##]. Debridement of mucinous substance, along with partial resection of ACL, is a recommended and effective therapy that does not cause instability, according to many authors [##REF##18629461##16##].</p>\",\n \"<p>While certain authors have underscored the significance of notchplasty as a supplementary step in the procedure, Motmans and Verheyden have challenged this idea by arguing against the necessity of notchplasty. They contend that a comprehensive debridement alone is sufficient to resolve impingement and effectively address the underlying pathology [##REF##19089409##4##].</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>The clinical and radiological characteristics of MD of ACL can be often inconclusive, potentially leading to a misdiagnosis of a torn ACL. Arthroscopists must be cognizant of this uncommon condition, as they may encounter a mucoid ACL during surgical procedures. This awareness is crucial for an accurate diagnosis and appropriate management of the condition.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>The clinical and radiological characteristics of MD of ACL can be often inconclusive, potentially leading to a misdiagnosis of a torn ACL. Arthroscopists must be cognizant of this uncommon condition, as they may encounter a mucoid ACL during surgical procedures. This awareness is crucial for an accurate diagnosis and appropriate management of the condition.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Mucoid degeneration (MD) is an uncommon pathological phenomenon that specifically affects the anterior cruciate ligament (ACL). This condition arises from the infiltration of yellowish material within the fibers of the ACL, contributing to the clinical presentation characterized by discomfort and limited mobility. MRI has proven to be the foremost diagnostic modality in effectively distinguishing MD of ACL from other potential pathologies. Preoperative recognition of this condition facilitates straightforward diagnosis, particularly via characteristic findings observed during knee arthroscopy. We present a case of MD of ACL, review prior studies about the condition, and outline its clinical features and symptoms, including those observed in our specific case.</p>"],"string":"[\n \"<p>Mucoid degeneration (MD) is an uncommon pathological phenomenon that specifically affects the anterior cruciate ligament (ACL). This condition arises from the infiltration of yellowish material within the fibers of the ACL, contributing to the clinical presentation characterized by discomfort and limited mobility. MRI has proven to be the foremost diagnostic modality in effectively distinguishing MD of ACL from other potential pathologies. Preoperative recognition of this condition facilitates straightforward diagnosis, particularly via characteristic findings observed during knee arthroscopy. We present a case of MD of ACL, review prior studies about the condition, and outline its clinical features and symptoms, including those observed in our specific case.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Case presentation</title>","<p>The patient was a 57-year-old female who presented to our hospital with a two-year history of right knee pain and limited flexion following a minor injury. A thorough examination was conducted. Notably, there was no apparent swelling or instability observed. The knee exhibited a restricted range of motion, spanning from 0° to 90°, with pain occurring during terminal flexion. Tenderness was localized to the lateral joint line of the knee, and the McMurray test yielded a positive result. Significantly, no clinical signs or symptoms suggestive of instability, ligamentous laxity, or patellofemoral pathology were evident (Figure ##FIG##0##1##).</p>","<p>A plain X-ray examination provided valuable insights, revealing subtle degenerative changes, particularly in the medial compartment (Figure ##FIG##1##2##). The patient underwent conservative treatment for several months; however, there was no improvement in the condition.</p>","<p>MRI of the knee was performed, which revealed specific findings in the right knee. In the coronal view, a clear thickening of the ACL was evident, characterized by preserved fibers, and a consistent increase in signal intensity. Noteworthy findings on the sagittal view revealed an indistinct ACL presenting a distinctive \"celery stalk\" appearance (Figure ##FIG##2##3##).</p>","<p>Arthroscopy was performed, uncovering femoropatellar arthritis marked by advanced osteocartilaginous lesions and degenerative changes in the lateral and medial meniscus. The continuous fibers, notably the anterolateral bundle, exhibited enlargement and displayed a yellowish degenerative appearance, confirming MD. Additionally, degenerative vertical lesions were evident in the external meniscus. A decision was made to perform debridement of the yellowish lesion of the ACL, and subsequent stability testing through the anterior drawer maneuver yielded satisfactory results (Figure ##FIG##3##4##). At the 10-month follow-up, the patient demonstrated a complete range of knee motion without experiencing pain or instability.</p>"],"string":"[\n \"<title>Case presentation</title>\",\n \"<p>The patient was a 57-year-old female who presented to our hospital with a two-year history of right knee pain and limited flexion following a minor injury. A thorough examination was conducted. Notably, there was no apparent swelling or instability observed. The knee exhibited a restricted range of motion, spanning from 0° to 90°, with pain occurring during terminal flexion. Tenderness was localized to the lateral joint line of the knee, and the McMurray test yielded a positive result. Significantly, no clinical signs or symptoms suggestive of instability, ligamentous laxity, or patellofemoral pathology were evident (Figure ##FIG##0##1##).</p>\",\n \"<p>A plain X-ray examination provided valuable insights, revealing subtle degenerative changes, particularly in the medial compartment (Figure ##FIG##1##2##). The patient underwent conservative treatment for several months; however, there was no improvement in the condition.</p>\",\n \"<p>MRI of the knee was performed, which revealed specific findings in the right knee. In the coronal view, a clear thickening of the ACL was evident, characterized by preserved fibers, and a consistent increase in signal intensity. Noteworthy findings on the sagittal view revealed an indistinct ACL presenting a distinctive \\\"celery stalk\\\" appearance (Figure ##FIG##2##3##).</p>\",\n \"<p>Arthroscopy was performed, uncovering femoropatellar arthritis marked by advanced osteocartilaginous lesions and degenerative changes in the lateral and medial meniscus. The continuous fibers, notably the anterolateral bundle, exhibited enlargement and displayed a yellowish degenerative appearance, confirming MD. Additionally, degenerative vertical lesions were evident in the external meniscus. A decision was made to perform debridement of the yellowish lesion of the ACL, and subsequent stability testing through the anterior drawer maneuver yielded satisfactory results (Figure ##FIG##3##4##). At the 10-month follow-up, the patient demonstrated a complete range of knee motion without experiencing pain or instability.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Examination of the patient at presentation</title><p>A: Range of flexion of the right knee 90°. B: Range of extension of the right knee 0°</p></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Plain radiographs show degenerative changes on the medial aspect of the knee</title><p>A: Anteroposterior radiographs of the right knee. B: Lateral radiographs of the right knee</p></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG3\"><label>Figure 3</label><caption><title>MRI of the knee at presentation</title><p>A: MRI Sagittal T1-weighted image showing the high signal intensity of the anterior cruciate ligament with a \"celery stalk\" appearance. B: MRI Coronal T2-weighted image showing a thickening of the anterior cruciate ligament with meniscus lesions</p><p>MRI: magnetic resonance imaging</p></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG4\"><label>Figure 4</label><caption><title>Arthroscopic images of the anterior cruciate ligament</title><p>A: Enlarged anterior cruciate ligament occupying the intercondylar notch. B: Yellowish material and sclerotic lesions within the anterior cruciate ligament were subsequently removed through debridement</p></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>Examination of the patient at presentation</title><p>A: Range of flexion of the right knee 90°. B: Range of extension of the right knee 0°</p></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG2\\\"><label>Figure 2</label><caption><title>Plain radiographs show degenerative changes on the medial aspect of the knee</title><p>A: Anteroposterior radiographs of the right knee. B: Lateral radiographs of the right knee</p></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG3\\\"><label>Figure 3</label><caption><title>MRI of the knee at presentation</title><p>A: MRI Sagittal T1-weighted image showing the high signal intensity of the anterior cruciate ligament with a \\\"celery stalk\\\" appearance. B: MRI Coronal T2-weighted image showing a thickening of the anterior cruciate ligament with meniscus lesions</p><p>MRI: magnetic resonance imaging</p></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG4\\\"><label>Figure 4</label><caption><title>Arthroscopic images of the anterior cruciate ligament</title><p>A: Enlarged anterior cruciate ligament occupying the intercondylar notch. B: Yellowish material and sclerotic lesions within the anterior cruciate ligament were subsequently removed through debridement</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":[],"string":"[]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Oussama El Alaoui, Adnane Lachkar, Najib Abdeljaouad, Hicham Yacoubi</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Oussama El Alaoui, Ousama Jelti, Adnane Lachkar</p><p><bold>Drafting of the manuscript:</bold> Oussama El Alaoui, Ousama Jelti</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Oussama El Alaoui, Ousama Jelti, Adnane Lachkar, Najib Abdeljaouad, Hicham Yacoubi</p><p><bold>Supervision:</bold> Adnane Lachkar, Najib Abdeljaouad, Hicham Yacoubi</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Concept and design:</bold> Oussama El Alaoui, Adnane Lachkar, Najib Abdeljaouad, Hicham Yacoubi</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Oussama El Alaoui, Ousama Jelti, Adnane Lachkar</p><p><bold>Drafting of the manuscript:</bold> Oussama El Alaoui, Ousama Jelti</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Oussama El Alaoui, Ousama Jelti, Adnane Lachkar, Najib Abdeljaouad, Hicham Yacoubi</p><p><bold>Supervision:</bold> Adnane Lachkar, Najib Abdeljaouad, Hicham Yacoubi</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0015-00000050545-i01\" position=\"float\"/>","<graphic xlink:href=\"cureus-0015-00000050545-i02\" position=\"float\"/>","<graphic xlink:href=\"cureus-0015-00000050545-i03\" position=\"float\"/>","<graphic xlink:href=\"cureus-0015-00000050545-i04\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0015-00000050545-i01\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0015-00000050545-i02\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0015-00000050545-i03\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0015-00000050545-i04\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"8\"], \"article-title\": [\"Mucoid degeneration of the anterior cruciate ligament\"], \"source\": [\"Arthroscopy\"], \"person-group\": [\"\\n\"], \"surname\": [\"Fealy\", \"Kenter\", \"Dines\", \"Warren\"], \"given-names\": [\"S\", \"K\", \"JS\", \"RF\"], \"fpage\": [\"0\"], \"volume\": [\"17\"], \"year\": [\"2001\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":16,"string":"16"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50545"},"package_file":{"kind":"string","value":"oa_package/c8/bb/PMC10787846.tar.gz"}}},{"rowIdx":154,"cells":{"accession_id":{"kind":"string","value":"PMC10787847"},"pmid":{"kind":"string","value":"38222263"},"introduction":{"kind":"list like","value":[],"string":"[]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<p>Approved by: Agustin Ibanez, Latin American Brain Health Institute (BrainLat), Chile</p>"],"string":"[\n \"<p>Approved by: Agustin Ibanez, Latin American Brain Health Institute (BrainLat), Chile</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<p>The journal retracts the 18 June 2021 article cited above.</p>","<p>Following publication, the publisher uncovered evidence that false identities were used in the peer-review process. The assignment of fake reviewers was confirmed by an investigation, conducted in accordance with Frontiers' policies and the Committee on Publication Ethics (COPE) guidelines. Given the concerns, the editors no longer have confidence in the findings presented in the article.</p>","<p>This retraction was approved by the Chief Editors of Frontiers in Aging Neuroscience and the Chief Executive Editor of Frontiers. The authors received a communication regarding the retraction and had a chance to respond. This communication has been recorded by the publisher.</p>"],"string":"[\n \"<p>The journal retracts the 18 June 2021 article cited above.</p>\",\n \"<p>Following publication, the publisher uncovered evidence that false identities were used in the peer-review process. The assignment of fake reviewers was confirmed by an investigation, conducted in accordance with Frontiers' policies and the Committee on Publication Ethics (COPE) guidelines. Given the concerns, the editors no longer have confidence in the findings presented in the article.</p>\",\n \"<p>This retraction was approved by the Chief Editors of Frontiers in Aging Neuroscience and the Chief Executive Editor of Frontiers. The authors received a communication regarding the retraction and had a chance to respond. This communication has been recorded by the publisher.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":[],"string":"[]","isConversational":false},"table":{"kind":"list like","value":[],"string":"[]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"graphic":{"kind":"list like","value":[],"string":"[]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":0,"string":"0"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"Front Aging Neurosci. 2023 Dec 28; 15:1358292"},"package_file":{"kind":"string","value":"oa_package/c4/9e/PMC10787847.tar.gz"}}},{"rowIdx":155,"cells":{"accession_id":{"kind":"string","value":"PMC10787848"},"pmid":{"kind":"string","value":"38222136"},"introduction":{"kind":"list like","value":["<title>Introduction and background</title>","<p>The development of more efficient, well-tolerated, and easy combination antiretroviral treatment (cART) has significantly enhanced HIV-positive people's (PLWH) life expectancy, even exceeding that of the general population [##REF##27831953##1##]. Their extended survival, however, has resulted in a shift in their health profile, with a greater frequency of age-related comorbidities, which frequently manifest 5 to 10 years sooner than in the general population [##REF##21998278##2##]. Furthermore, certain PLWH are becoming more susceptible to geriatric disorders such as frailty [##REF##26009828##3##]. The goal of this study is to look into the role of frailty in the holistic treatment of older people with HIV (OPLWH).</p>","<p>Frailty, which was initially used by the insurance industry to assess mortality risks in adults over the age of 65 [##REF##510638##4##], has recently been broadened to include physical, cognitive, social, emotional, and economic components [##REF##28210943##5##]. It denotes vulnerability to unfavorable health impacts as a result of several stresses. While frailty was originally studied in community-living persons over the age of 65, it has subsequently been studied across a wide range of demographics and conditions, indicating prevalence rates ranging from 3% to 5% between the ages of 30 and 60 to 25-30% after the age of 80 in the United Kingdom [##UREF##0##6##]. Frailty has been proven to be a more accurate predictor of survival in COVID-19 individuals than chronological age or comorbidities [##UREF##1##7##]. </p>","<p>Frailty, cognitive decline, and other geriatric disorders are becoming more widespread and emerging sooner as PLWH continues to age, with a median age presently in the mid-50s in high-income countries [##REF##26009828##3##]. Frailty must be identified since it increases the likelihood of acquiring new chronic diseases, such as falling, cognitive impairment, polypharmacy, hospitalization, loss of independence, and mortality. Because aging rates vary, screening for frailty is a critical initial step in the management of OPLWH [##REF##23860844##8##].</p>","<p>As a result of these developments, the approach to caring for OPLWH has moved from immunovirologic treatment to a more multidisciplinary one that places an emphasis on tailored services [##REF##31395144##9##]. Specialized programs have been developed to accommodate the particular needs of OPLWH [##UREF##1##7##]. While frailty assessments are not necessary for every OPLWH, they are crucial in identifying at-risk individuals since chronological age alone is not always a reliable predictor of age-related disorders in geriatrics [##REF##23860844##8##].</p>"],"string":"[\n \"<title>Introduction and background</title>\",\n \"<p>The development of more efficient, well-tolerated, and easy combination antiretroviral treatment (cART) has significantly enhanced HIV-positive people's (PLWH) life expectancy, even exceeding that of the general population [##REF##27831953##1##]. Their extended survival, however, has resulted in a shift in their health profile, with a greater frequency of age-related comorbidities, which frequently manifest 5 to 10 years sooner than in the general population [##REF##21998278##2##]. Furthermore, certain PLWH are becoming more susceptible to geriatric disorders such as frailty [##REF##26009828##3##]. The goal of this study is to look into the role of frailty in the holistic treatment of older people with HIV (OPLWH).</p>\",\n \"<p>Frailty, which was initially used by the insurance industry to assess mortality risks in adults over the age of 65 [##REF##510638##4##], has recently been broadened to include physical, cognitive, social, emotional, and economic components [##REF##28210943##5##]. It denotes vulnerability to unfavorable health impacts as a result of several stresses. While frailty was originally studied in community-living persons over the age of 65, it has subsequently been studied across a wide range of demographics and conditions, indicating prevalence rates ranging from 3% to 5% between the ages of 30 and 60 to 25-30% after the age of 80 in the United Kingdom [##UREF##0##6##]. Frailty has been proven to be a more accurate predictor of survival in COVID-19 individuals than chronological age or comorbidities [##UREF##1##7##]. </p>\",\n \"<p>Frailty, cognitive decline, and other geriatric disorders are becoming more widespread and emerging sooner as PLWH continues to age, with a median age presently in the mid-50s in high-income countries [##REF##26009828##3##]. Frailty must be identified since it increases the likelihood of acquiring new chronic diseases, such as falling, cognitive impairment, polypharmacy, hospitalization, loss of independence, and mortality. Because aging rates vary, screening for frailty is a critical initial step in the management of OPLWH [##REF##23860844##8##].</p>\",\n \"<p>As a result of these developments, the approach to caring for OPLWH has moved from immunovirologic treatment to a more multidisciplinary one that places an emphasis on tailored services [##REF##31395144##9##]. Specialized programs have been developed to accommodate the particular needs of OPLWH [##UREF##1##7##]. While frailty assessments are not necessary for every OPLWH, they are crucial in identifying at-risk individuals since chronological age alone is not always a reliable predictor of age-related disorders in geriatrics [##REF##23860844##8##].</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>As OPLWH lives longer, healthcare for them is changing. However, there are issues with the growth in frailty and other geriatric problems among OPLWH. Once associated with elderly people, fragility is now a critical factor in determining susceptibility to unfavorable health outcomes. Personalized services and an interdisciplinary approach have been developed for OPLWH. Because frailty is associated with mortality and chronic diseases, it is imperative to identify and treat it early. Research on pharmacotherapeutic alternatives and e-health solutions is still ongoing. To offer complete treatment in this evolving healthcare environment, it is vital to steer clear of ageist healthcare practices and give frailty the attention it demands.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>As OPLWH lives longer, healthcare for them is changing. However, there are issues with the growth in frailty and other geriatric problems among OPLWH. Once associated with elderly people, fragility is now a critical factor in determining susceptibility to unfavorable health outcomes. Personalized services and an interdisciplinary approach have been developed for OPLWH. Because frailty is associated with mortality and chronic diseases, it is imperative to identify and treat it early. Research on pharmacotherapeutic alternatives and e-health solutions is still ongoing. To offer complete treatment in this evolving healthcare environment, it is vital to steer clear of ageist healthcare practices and give frailty the attention it demands.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>The life expectancy of people living with HIV (PLWH) has greatly increased due to advancements in combination antiretroviral treatment (cART). However, this longer life has also increased the prevalence of age-related comorbidities, such as frailty, which now manifest sooner in this group. Frailty, a term coined by the insurance industry, has been broadened to include physical, cognitive, and emotional elements and has been recognized as a critical predictor of negative health outcomes. With the median age of PLWH now in the mid-50s, treating frailty is critical given its link to chronic diseases, cognitive decline, and even death. Frailty assessment tools, such as the Frailty Phenotype (FP) and the Frailty Index (FI), are used to identify vulnerable people. Understanding the pathophysiology of frailty in PLWH indicates the role of immunological mechanisms. Frailty screening and management in this group have progressed, with specialized clinics and programs concentrating on multidisciplinary care. Potential pharmacotherapeutic solutions, as well as novel e-health programs and sensors, are in the future of frailty treatment, but it is critical to ensure that frailty evaluation is not exploited to perpetuate ageist healthcare practices. This narrative review investigates the changing healthcare environment for older people living with HIV (OPLWH), notably in high-income countries. It emphasizes the significance of identifying and managing frailty as a crucial feature of OPLWH's holistic care and well-being.</p>"],"string":"[\n \"<p>The life expectancy of people living with HIV (PLWH) has greatly increased due to advancements in combination antiretroviral treatment (cART). However, this longer life has also increased the prevalence of age-related comorbidities, such as frailty, which now manifest sooner in this group. Frailty, a term coined by the insurance industry, has been broadened to include physical, cognitive, and emotional elements and has been recognized as a critical predictor of negative health outcomes. With the median age of PLWH now in the mid-50s, treating frailty is critical given its link to chronic diseases, cognitive decline, and even death. Frailty assessment tools, such as the Frailty Phenotype (FP) and the Frailty Index (FI), are used to identify vulnerable people. Understanding the pathophysiology of frailty in PLWH indicates the role of immunological mechanisms. Frailty screening and management in this group have progressed, with specialized clinics and programs concentrating on multidisciplinary care. Potential pharmacotherapeutic solutions, as well as novel e-health programs and sensors, are in the future of frailty treatment, but it is critical to ensure that frailty evaluation is not exploited to perpetuate ageist healthcare practices. This narrative review investigates the changing healthcare environment for older people living with HIV (OPLWH), notably in high-income countries. It emphasizes the significance of identifying and managing frailty as a crucial feature of OPLWH's holistic care and well-being.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Review</title>","<p>Frailty: concept and evolution</p>","<p>Derived from the Latin word \"fragilis\" (meaning \"breakable\"), the term frailty indicates a condition of increased sensitivity. Fried devised the Frailty Phenotype (FP) and the Frailty Index (FI) to emphasize the complexity of the idea in contrast to impairments or comorbidities [##UREF##2##10##]. Although the fundamental traits and consequences of frailty are well acknowledged, the absence of a clear operational definition makes diagnosis difficult. There are at least 67 metrics that have been developed; the most often used ones in research are the FP and FI [##REF##26674984##11##].</p>","<p>The FP diagnoses frailty when three of the five predefined physical criteria - unintentional weight loss, self-reported fatigue, poor hand grip strength, sluggish walking speed, and low physical activity - are met. One or two conditions indicate pre-frailty, whereas none indicate non-frailty. Based on the accumulation of many age-related health problems, the FI computes frailty. Generally speaking, frailty in older adults who live in communities is indicated by a FI greater than 0.25. A prognosis of poor quality and several acute illnesses is indicated by a FI larger than 0.7 [##UREF##3##12##]. There are 30 prerequisites in all. Even though the FP is simpler to use, it does require specialized assessments and qualified personnel. The FI, on the other hand, appears more complicated but provides superior risk discrimination and may be determined using electronic health information [##REF##26944937##13##].</p>","<p>Models predict comparable results when applied to diverse populations, and they also identify subgroups of frail individuals who do not have impairments or comorbidities. Research on the prevalence of frailty varies, with estimates ranging from 2% to 25% [##REF##31395144##9##]. Other proven methods for diagnosing frailty include the Edmonton Frail Scale (EFS), the Clinical Frailty Scale (CFS), and the Frail questionnaire. Even among those who are not yet diagnosed as frail, a meta-analysis confirmed the association between these markers of frailty and increased rates of death, hospitalization, loss of independence, impairments, falls, fractures, and cognitive decline [##UREF##4##14##].</p>","<p>A gradual loss of normal homeostatic mechanisms brought on by biological and environmental stresses that affect cells, tissues, organs, and the overall health of a person ultimately leads to frailty. A chronic inflammatory state is shaped by immunological traits and CMV seropositivity, and immunosenescence is a major factor in this process. Numerous processes and serum markers are associated with this chronic inflammation, which is sometimes referred to as inflammaging [##REF##20571864##15##]. Hormonal fluctuations, epigenetic alterations, telomere shortening, genetic control, and changes in body composition, such as abdominal fat and muscle loss, can all have an impact on frailty [##REF##30017798##16##].</p>","<p>The CFS is a measure of independence in older PLWH that shows favorable correlations with the FP and FI [##REF##16129869##17##]. A multi-domain evaluation called the EFS has earlier been compared to the FP and FI in PLWH [##REF##16757522##18##]. A simple instrument consisting of five parts is the FRAIL Scale [##REF##22836700##19##]. Originally intended to predict death in PLWH, the Veterans Aging Cohort Study Index (VACS-I) is currently utilized as a frailty screening tool with construct validity and predictive validity [##REF##31565954##20##]. These methods aid in identifying persons who, due to their frailty, may require treatments and care.</p>","<p>Pathophysiology of frailty in PLWH</p>","<p>Frailty in PLWH has molecular underpinnings associated with age-related dysregulation of several physiological systems. Dysregulated systems include those related to the stress response (neuro-immuno-endocrine), metabolism (insulin and mitochondria), and musculoskeletal systems [##REF##27549318##21##]. When dysregulation escalates to a point where negative outcomes are more likely, frailty is categorized as a syndrome. Other variables are involved, even though the majority of evidence indicates that frailty in OPLWH is comparable to physiologic aging [##REF##30885572##22##].</p>","<p>Prolonged low-grade HIV replication triggers persistent immunological activation even in suppressed individuals, which results in immunosenescence and inflammation that invariably produce frailty in clinical trials. Additionally, epigenomic dysregulation is at play. Clinical variables such as co-infections, early suppression of HIV replication, CD4/CD8 ratio, and microbiota translocation facilitate the development of frailty. These traits emphasize the role that immunoinflammatory variables play in the development of frailty [##REF##28203694##23##]. Frailty is a common dysregulated condition of physiological functioning and impaired functional reserve that leads to negative outcomes typical of aging, although it may manifest earlier in PLWH [##REF##28386608##24##].</p>","<p>Studies employing the FP for frailty diagnosis</p>","<p>In the multicenter AIDS Cohort Study (MACS), frailty was measured using an adapted frailty-related phenotype (aFRP) in untreated, seropositive, college-educated, Caucasian males with a mean age of 55. In the same group of males over 65 who tested negative for HIV, frailty was common in 3.4% of cases [##REF##18000149##25##]. Immuno-virologic traits were associated with frailty, and among PLWH who started cART, frailty was associated with a higher risk of developing AIDS or passing away [##REF##21719610##26##]. Risk factors for frailty included old age, non-Hispanic black ethnicity, and potentially controllable conditions such as AIDS, smoking, hepatitis C infection, depression, diabetes, and renal disease. Frailty also happens in the absence of concomitant comorbidities [##REF##18000149##25##].</p>","<p>A 12% frailty prevalence was observed in the AIDS Linked to Intravenous Experience (ALIVE) cohort, which consisted primarily of male African-American injectable drug users (median age 49). Risk factors for this cohort included female sex, advanced HIV disease, lower education, depression, and multimorbidity, as well as older age and HIV infection. Frailty has been associated with all-cause hospitalizations [##UREF##5##27##], overall mortality [##REF##27516622##28##], and chronic illnesses such as lung, heart, and mental disorders.</p>","<p>The HIV Infection, Aging, and Immune Function Long-Term Observational Study (HAILO) cohort examined disability and frailty. The prevalence of pre-frailty and frailty was 37% and 6% of the population, respectively, with little overlap between the two conditions. Premature mortality, type II diabetes, and cardiovascular disease have all been related to frailty [##REF##30590451##29##]. Neurocognitive impairment, obesity, smoking, the first choice of cART (with NNRTI [nucleoside reverse transcriptase inhibitor]-based cART increasing the risk of frailty), and educational level were among the modifiable risk factors. Both moderate alcohol use and physical activity were protective [##REF##28453849##30##].</p>","<p>Additionally, it was demonstrated that more seropositive women than males had frailty, which is in line with the overall population. 10.0% of uninfected women and 17.3% of seropositive women in the Women's Interagency HIV Study (WIHS) were frail [##REF##26980368##31##]. Concerns regarding economic and healthcare delivery challenges as the PLWH population ages are raised by other research, like those carried out in South Africa and Spain, which revealed a high prevalence of frailty in PLWH [##REF##31373215##32##].</p>","<p>Screening of frailty in PLWH</p>","<p>When a senior is diagnosed as frail, it signifies more than simply a condition with a dismal prognosis. For instance, one important risk factor for perioperative issues is frailty. By altering recognized preoperative variables for surgical morbidity, pre-habilitation clinics improve outcomes [##UREF##6##33##]. An integrated geriatric strategy is becoming more and more recommended for certain older PLWH, especially those who have been classified as weak [##REF##28387803##34##].</p>","<p>Other surrogates, besides frailty, can help identify PLWH who would benefit from a geriatric assessment. For instance, polypharmacy is more common in PLWH compared to controls. Similarly, decreased functional status as determined by gait speed or the whole Short Physical Performance Battery (SPPB) assessment tool is also more common [##REF##24576251##35##]. Frailty, functional status, and deficits all interact in PLWH, despite being distinct illnesses [##REF##27715455##36##]. PLWH frequently experience functional impairments and impairments, especially those who also have concurrent geriatric symptoms [##REF##27715455##36##,##REF##24966138##37##]. Strong PLWH in need of geriatric review may also be indicated by a combination of immunological markers (e.g., a low nadir CD4 count of 200, a plateau CD4 level of 500 on suppressive cART, and a CD4/CD8 ratio of one) [##UREF##7##38##].</p>","<p>Weakness is a dynamic state. The majority of non-frail and pre-frail individuals retained their status in a 12-month follow-up analysis involving over 300 treated PLWH, but the majority of frail individuals relapsed to pre-frailty [##REF##25495766##39##]. Pre-frailty, which affects 30-60% of PLWH, has to be identified since it is associated with unfavorable outcomes. We have previously discussed the elements that contribute to the shift of PLWH in the MACS to frailty. The only factor associated with a reversal of frailty was age [##REF##24127428##40##]. Guaraldi spent four years studying the MHMC Cohort's frailty transition determinants. The following factors predicted FI at follow-up: baseline FI, female gender, length of HIV infection and cART usage, and history of smoking [##UREF##8##41##].</p>","<p>There are currently no guidelines for which PLWH to refer, and it is unclear if geriatric referrals are clinically effective. The public should be evaluated for frailty if they are older than 70. It is reasonable to think about screening PLWH above the age of 50 based on data showing PLWH age-advancement. The responsibilities of geriatricians as knowledgeable consultants or involved team members are being established [##REF##23764209##42##].</p>","<p>Management of frailty in PLWH</p>","<p>In high-income nations, the primary healthcare approach for OPLWH is based on specialized community-based primary care or tertiary care that is offered in infectious disease clinics. Specialized geriatric or aging-HIV clinics, HIV-metabolic clinics, and HIV-rehabilitation programs have been established to address the wider range of social, emotional, and health concerns experienced by OPLWH as their healthcare needs have expanded to include non-HIV-related illnesses [##UREF##9##43##].</p>","<p>Numerous of these clinics have adopted care methods designed for senior citizens that are based on geriatric concepts. With an emphasis on modifying the Comprehensive Geriatric Assessment (CGA) to identify the most susceptible OPLWH, these models entail screening for frailty and other geriatric disorders. CGA, a well-researched multidimensional diagnostic procedure, is used to evaluate the functional, psychological, and medical abilities of a subset of elderly people [##UREF##10##44##]. After the age of 70, it is advised to do yearly frailty screenings in the general population [##REF##23764209##42##]. According to recent guidelines, PLWH over 50 should undergo yearly frailty screening using the FRAIL Scale. If the screening findings are positive, the individual should then be sent for a thorough geriatric evaluation [##REF##34652753##45##]. </p>","<p>To test for frailty, HIV clinics that specialize in OPLWH use a variety of measures, such as gait speed, the FRAIL Scale, and the Clinical Frailty Scale [##REF##34652753##45##]. Many clinics have provided insights into how they manage OPLWH who are frail, using multidisciplinary geriatric evaluations to determine who is most in need [##REF##28387803##34##]. For instance, the CFS is used by the Chelsea and Westminster Clinic in London to screen older people with HIV (OPLWH). Individuals with a CFS score of 5 or more, which indicates at least mild frailty, are sent to a specialized geriatric HIV clinic [##REF##34269603##46##]. Clinics may use several measures to screen for frailty. While some use electronic health records to identify the FP, others use the FI. When electronic health data are accessible, the FI may be applied more easily than the FP, which needs specific equipment and training [##REF##26944937##13##, ##REF##30668637##47##].</p>","<p>The goals of therapy for OPLWH who have been classified as frail are to prevent and control impairment and comorbidities. Furthermore, geriatric syndrome assessment and treatment are essential elements of this care approach [##REF##33987636##48##]. To control OPLWH, tactics borrowed from research with frail, uninfected older adults are used. Maintaining the quality of life and possibly averting cognitive decline also depend on acknowledging the increased health risks linked to social isolation. This concept aims to actively involve older people with HIV/AIDS within their social networks and reintegrate HIV care into primary care while facilitating access to community resources [##REF##33128225##49##].</p>","<p>Fundamental care principles for frail older HIV-negative individuals include implementing strength-training exercise programs, managing sarcopenia, assessing for polypharmacy, treating weight loss and undernutrition with protein-calorie supplements, and evaluating and treating reversible causes of fatigue such as anemia, depression, hypothyroidism, and B12 deficiency. Furthermore, vitamin D levels should be monitored, and it should be administered if necessary [##REF##29678160##50##,##REF##31641726##51##].</p>","<p>Programs for preventing frailty that involve many components have shown promise in reducing the pace of frailty development, boosting cognitive performance, and increasing physical function [##UREF##11##52##]. CGA informs care plans that can improve functional abilities, lower the chance of institutionalization, postpone the onset of impairment, cut down on hospital admissions and stays, and increase survival [##UREF##12##53##]. A physiotherapist should ideally be available or included in the multidisciplinary team. Exercise programs of varying intensities have enhanced physical function in OPLWH, while short-duration exercise programs have shown beneficial effects on frail older individuals. Improvements in quality-of-life measures in PLWH have also been associated with participation in yoga programs [##REF##32583707##54##].</p>","<p>While exercise has been shown to be the most effective intervention for frailty, group-based physiotherapy classes have also demonstrated efficacy in protecting against functional decline. On the other hand, addressing the social determinants of frailty is important. This includes, but is not limited to, isolation, loneliness, depression, or any other psychiatric diagnoses. However, this may not always be feasible, as it often depends on the availability of local professional resources and the presence of a community-based support system [##UREF##13##55##].</p>","<p>The New Orleans Alcohol Use in HIV (NOAH) study evaluated the body composition, gait speed, and muscle strength of 341 participants living with HIV. Body composition was found to have a modulatory effect on frailty risk among PLWH, which was statistically significant. That is, while obesity was associated with increased risk, greater muscle mass may have had a protective effect, even among individuals who consume alcohol. These findings emphasize the importance of physical activity and weight control in modulating frailty risk [##REF##36124866##56##].</p>","<p>Furthermore, strict control of the medications PLWH consumes may be beneficial. Polypharmacy may increase the risk of adverse outcomes, and de-prescription is often necessary. For instance, a study demonstrated that frailty was more common in those who used medications with an anticholinergic effect (OR 2.12; 95% CI 0.89-5.0). Therefore, clinicians should be aware of their impact and work to reduce exposure whenever possible [##REF##37644705##57##].</p>","<p>As mentioned earlier, exercise is a well-established preventive intervention for frailty. However, since many of the OPLWH are already in a frail state, high-intensity training and weight-lifting may be too taxing. Therefore, a study investigated a novel game-based training program, referred to as exergame, and its impact on ameliorating frailty among 10 HIV-infected individuals. The exergame program which was conducted twice weekly for six weeks, incorporated activities such as weight shifting, ankle reaching, and obstacle crossing. After the conclusion of the program, changes in balance, gait, and other parameters were assessed, and improvements were seen in balance and mobility [##REF##27768079##58##].</p>","<p>E-health systems are being increasingly utilized to provide health information, personalized recommendations, and smartphone reminders to promote healthy habits and help older people preserve and enhance their functional independence. There is much promise in the continuing evaluation of wearable sensors for frailty identification [##REF##32583707##54##]. The Ecological Momentary Assessment, which gathers real-time patient-reported outcome indicators, can help make it easier to incorporate patient-reported outcomes into innovative healthcare models [##UREF##12##53##]. Nonetheless, it is important to maintain vigilance to prevent the possible ageist approach to healthcare delivery from being supported by frailty [##UREF##11##52##].</p>","<p>Limitations and challenges</p>","<p>While our review provides insight into frailty pathophysiology, screening, detection, and management for PLWH, it also identifies several limitations and research gaps, the most notable of which is the absence of a standardized operational definition for frailty. This presents a challenge not only for consistent diagnosis in the healthcare setting but also for conducting studies with reliable data and results. Therefore, the need to establish a universally accepted definition is paramount. However, more research is needed to explore the most effective and relevant metrics for frailty while considering the diverse impacted populations, including OPLWH.</p>","<p>In a comparable context, the variability of estimates of frailty prevalence in the different studies (ranging from 2% to 25%) highlights the importance of understanding any contributing factors and standardizing assessment methods in future studies. On the other hand, it implies that frailty in this population (PLWH) may manifest differently. Furthermore, while our article touches on the integration of geriatric concepts in HIV care, more research is required to assess the feasibility, effectiveness, and scalability of these approaches. Clear guidelines for identifying PLWH who would benefit from frailty assessments and evaluating the clinical effectiveness of geriatric referrals are lacking, and this is an area we aim to address in our ongoing research.</p>","<p>Lastly, a more thorough exploration of e-health systems and electronic health records used for estimating frailty, especially those employing metrics such as the Frailty Index, would enhance our understanding of technological advancements in frailty assessment and their real-world applicability. Addressing these gaps will contribute to a more comprehensive and nuanced understanding of frailty, improve diagnostic accuracy and inform targeted interventions for vulnerable populations.</p>"],"string":"[\n \"<title>Review</title>\",\n \"<p>Frailty: concept and evolution</p>\",\n \"<p>Derived from the Latin word \\\"fragilis\\\" (meaning \\\"breakable\\\"), the term frailty indicates a condition of increased sensitivity. Fried devised the Frailty Phenotype (FP) and the Frailty Index (FI) to emphasize the complexity of the idea in contrast to impairments or comorbidities [##UREF##2##10##]. Although the fundamental traits and consequences of frailty are well acknowledged, the absence of a clear operational definition makes diagnosis difficult. There are at least 67 metrics that have been developed; the most often used ones in research are the FP and FI [##REF##26674984##11##].</p>\",\n \"<p>The FP diagnoses frailty when three of the five predefined physical criteria - unintentional weight loss, self-reported fatigue, poor hand grip strength, sluggish walking speed, and low physical activity - are met. One or two conditions indicate pre-frailty, whereas none indicate non-frailty. Based on the accumulation of many age-related health problems, the FI computes frailty. Generally speaking, frailty in older adults who live in communities is indicated by a FI greater than 0.25. A prognosis of poor quality and several acute illnesses is indicated by a FI larger than 0.7 [##UREF##3##12##]. There are 30 prerequisites in all. Even though the FP is simpler to use, it does require specialized assessments and qualified personnel. The FI, on the other hand, appears more complicated but provides superior risk discrimination and may be determined using electronic health information [##REF##26944937##13##].</p>\",\n \"<p>Models predict comparable results when applied to diverse populations, and they also identify subgroups of frail individuals who do not have impairments or comorbidities. Research on the prevalence of frailty varies, with estimates ranging from 2% to 25% [##REF##31395144##9##]. Other proven methods for diagnosing frailty include the Edmonton Frail Scale (EFS), the Clinical Frailty Scale (CFS), and the Frail questionnaire. Even among those who are not yet diagnosed as frail, a meta-analysis confirmed the association between these markers of frailty and increased rates of death, hospitalization, loss of independence, impairments, falls, fractures, and cognitive decline [##UREF##4##14##].</p>\",\n \"<p>A gradual loss of normal homeostatic mechanisms brought on by biological and environmental stresses that affect cells, tissues, organs, and the overall health of a person ultimately leads to frailty. A chronic inflammatory state is shaped by immunological traits and CMV seropositivity, and immunosenescence is a major factor in this process. Numerous processes and serum markers are associated with this chronic inflammation, which is sometimes referred to as inflammaging [##REF##20571864##15##]. Hormonal fluctuations, epigenetic alterations, telomere shortening, genetic control, and changes in body composition, such as abdominal fat and muscle loss, can all have an impact on frailty [##REF##30017798##16##].</p>\",\n \"<p>The CFS is a measure of independence in older PLWH that shows favorable correlations with the FP and FI [##REF##16129869##17##]. A multi-domain evaluation called the EFS has earlier been compared to the FP and FI in PLWH [##REF##16757522##18##]. A simple instrument consisting of five parts is the FRAIL Scale [##REF##22836700##19##]. Originally intended to predict death in PLWH, the Veterans Aging Cohort Study Index (VACS-I) is currently utilized as a frailty screening tool with construct validity and predictive validity [##REF##31565954##20##]. These methods aid in identifying persons who, due to their frailty, may require treatments and care.</p>\",\n \"<p>Pathophysiology of frailty in PLWH</p>\",\n \"<p>Frailty in PLWH has molecular underpinnings associated with age-related dysregulation of several physiological systems. Dysregulated systems include those related to the stress response (neuro-immuno-endocrine), metabolism (insulin and mitochondria), and musculoskeletal systems [##REF##27549318##21##]. When dysregulation escalates to a point where negative outcomes are more likely, frailty is categorized as a syndrome. Other variables are involved, even though the majority of evidence indicates that frailty in OPLWH is comparable to physiologic aging [##REF##30885572##22##].</p>\",\n \"<p>Prolonged low-grade HIV replication triggers persistent immunological activation even in suppressed individuals, which results in immunosenescence and inflammation that invariably produce frailty in clinical trials. Additionally, epigenomic dysregulation is at play. Clinical variables such as co-infections, early suppression of HIV replication, CD4/CD8 ratio, and microbiota translocation facilitate the development of frailty. These traits emphasize the role that immunoinflammatory variables play in the development of frailty [##REF##28203694##23##]. Frailty is a common dysregulated condition of physiological functioning and impaired functional reserve that leads to negative outcomes typical of aging, although it may manifest earlier in PLWH [##REF##28386608##24##].</p>\",\n \"<p>Studies employing the FP for frailty diagnosis</p>\",\n \"<p>In the multicenter AIDS Cohort Study (MACS), frailty was measured using an adapted frailty-related phenotype (aFRP) in untreated, seropositive, college-educated, Caucasian males with a mean age of 55. In the same group of males over 65 who tested negative for HIV, frailty was common in 3.4% of cases [##REF##18000149##25##]. Immuno-virologic traits were associated with frailty, and among PLWH who started cART, frailty was associated with a higher risk of developing AIDS or passing away [##REF##21719610##26##]. Risk factors for frailty included old age, non-Hispanic black ethnicity, and potentially controllable conditions such as AIDS, smoking, hepatitis C infection, depression, diabetes, and renal disease. Frailty also happens in the absence of concomitant comorbidities [##REF##18000149##25##].</p>\",\n \"<p>A 12% frailty prevalence was observed in the AIDS Linked to Intravenous Experience (ALIVE) cohort, which consisted primarily of male African-American injectable drug users (median age 49). Risk factors for this cohort included female sex, advanced HIV disease, lower education, depression, and multimorbidity, as well as older age and HIV infection. Frailty has been associated with all-cause hospitalizations [##UREF##5##27##], overall mortality [##REF##27516622##28##], and chronic illnesses such as lung, heart, and mental disorders.</p>\",\n \"<p>The HIV Infection, Aging, and Immune Function Long-Term Observational Study (HAILO) cohort examined disability and frailty. The prevalence of pre-frailty and frailty was 37% and 6% of the population, respectively, with little overlap between the two conditions. Premature mortality, type II diabetes, and cardiovascular disease have all been related to frailty [##REF##30590451##29##]. Neurocognitive impairment, obesity, smoking, the first choice of cART (with NNRTI [nucleoside reverse transcriptase inhibitor]-based cART increasing the risk of frailty), and educational level were among the modifiable risk factors. Both moderate alcohol use and physical activity were protective [##REF##28453849##30##].</p>\",\n \"<p>Additionally, it was demonstrated that more seropositive women than males had frailty, which is in line with the overall population. 10.0% of uninfected women and 17.3% of seropositive women in the Women's Interagency HIV Study (WIHS) were frail [##REF##26980368##31##]. Concerns regarding economic and healthcare delivery challenges as the PLWH population ages are raised by other research, like those carried out in South Africa and Spain, which revealed a high prevalence of frailty in PLWH [##REF##31373215##32##].</p>\",\n \"<p>Screening of frailty in PLWH</p>\",\n \"<p>When a senior is diagnosed as frail, it signifies more than simply a condition with a dismal prognosis. For instance, one important risk factor for perioperative issues is frailty. By altering recognized preoperative variables for surgical morbidity, pre-habilitation clinics improve outcomes [##UREF##6##33##]. An integrated geriatric strategy is becoming more and more recommended for certain older PLWH, especially those who have been classified as weak [##REF##28387803##34##].</p>\",\n \"<p>Other surrogates, besides frailty, can help identify PLWH who would benefit from a geriatric assessment. For instance, polypharmacy is more common in PLWH compared to controls. Similarly, decreased functional status as determined by gait speed or the whole Short Physical Performance Battery (SPPB) assessment tool is also more common [##REF##24576251##35##]. Frailty, functional status, and deficits all interact in PLWH, despite being distinct illnesses [##REF##27715455##36##]. PLWH frequently experience functional impairments and impairments, especially those who also have concurrent geriatric symptoms [##REF##27715455##36##,##REF##24966138##37##]. Strong PLWH in need of geriatric review may also be indicated by a combination of immunological markers (e.g., a low nadir CD4 count of 200, a plateau CD4 level of 500 on suppressive cART, and a CD4/CD8 ratio of one) [##UREF##7##38##].</p>\",\n \"<p>Weakness is a dynamic state. The majority of non-frail and pre-frail individuals retained their status in a 12-month follow-up analysis involving over 300 treated PLWH, but the majority of frail individuals relapsed to pre-frailty [##REF##25495766##39##]. Pre-frailty, which affects 30-60% of PLWH, has to be identified since it is associated with unfavorable outcomes. We have previously discussed the elements that contribute to the shift of PLWH in the MACS to frailty. The only factor associated with a reversal of frailty was age [##REF##24127428##40##]. Guaraldi spent four years studying the MHMC Cohort's frailty transition determinants. The following factors predicted FI at follow-up: baseline FI, female gender, length of HIV infection and cART usage, and history of smoking [##UREF##8##41##].</p>\",\n \"<p>There are currently no guidelines for which PLWH to refer, and it is unclear if geriatric referrals are clinically effective. The public should be evaluated for frailty if they are older than 70. It is reasonable to think about screening PLWH above the age of 50 based on data showing PLWH age-advancement. The responsibilities of geriatricians as knowledgeable consultants or involved team members are being established [##REF##23764209##42##].</p>\",\n \"<p>Management of frailty in PLWH</p>\",\n \"<p>In high-income nations, the primary healthcare approach for OPLWH is based on specialized community-based primary care or tertiary care that is offered in infectious disease clinics. Specialized geriatric or aging-HIV clinics, HIV-metabolic clinics, and HIV-rehabilitation programs have been established to address the wider range of social, emotional, and health concerns experienced by OPLWH as their healthcare needs have expanded to include non-HIV-related illnesses [##UREF##9##43##].</p>\",\n \"<p>Numerous of these clinics have adopted care methods designed for senior citizens that are based on geriatric concepts. With an emphasis on modifying the Comprehensive Geriatric Assessment (CGA) to identify the most susceptible OPLWH, these models entail screening for frailty and other geriatric disorders. CGA, a well-researched multidimensional diagnostic procedure, is used to evaluate the functional, psychological, and medical abilities of a subset of elderly people [##UREF##10##44##]. After the age of 70, it is advised to do yearly frailty screenings in the general population [##REF##23764209##42##]. According to recent guidelines, PLWH over 50 should undergo yearly frailty screening using the FRAIL Scale. If the screening findings are positive, the individual should then be sent for a thorough geriatric evaluation [##REF##34652753##45##]. </p>\",\n \"<p>To test for frailty, HIV clinics that specialize in OPLWH use a variety of measures, such as gait speed, the FRAIL Scale, and the Clinical Frailty Scale [##REF##34652753##45##]. Many clinics have provided insights into how they manage OPLWH who are frail, using multidisciplinary geriatric evaluations to determine who is most in need [##REF##28387803##34##]. For instance, the CFS is used by the Chelsea and Westminster Clinic in London to screen older people with HIV (OPLWH). Individuals with a CFS score of 5 or more, which indicates at least mild frailty, are sent to a specialized geriatric HIV clinic [##REF##34269603##46##]. Clinics may use several measures to screen for frailty. While some use electronic health records to identify the FP, others use the FI. When electronic health data are accessible, the FI may be applied more easily than the FP, which needs specific equipment and training [##REF##26944937##13##, ##REF##30668637##47##].</p>\",\n \"<p>The goals of therapy for OPLWH who have been classified as frail are to prevent and control impairment and comorbidities. Furthermore, geriatric syndrome assessment and treatment are essential elements of this care approach [##REF##33987636##48##]. To control OPLWH, tactics borrowed from research with frail, uninfected older adults are used. Maintaining the quality of life and possibly averting cognitive decline also depend on acknowledging the increased health risks linked to social isolation. This concept aims to actively involve older people with HIV/AIDS within their social networks and reintegrate HIV care into primary care while facilitating access to community resources [##REF##33128225##49##].</p>\",\n \"<p>Fundamental care principles for frail older HIV-negative individuals include implementing strength-training exercise programs, managing sarcopenia, assessing for polypharmacy, treating weight loss and undernutrition with protein-calorie supplements, and evaluating and treating reversible causes of fatigue such as anemia, depression, hypothyroidism, and B12 deficiency. Furthermore, vitamin D levels should be monitored, and it should be administered if necessary [##REF##29678160##50##,##REF##31641726##51##].</p>\",\n \"<p>Programs for preventing frailty that involve many components have shown promise in reducing the pace of frailty development, boosting cognitive performance, and increasing physical function [##UREF##11##52##]. CGA informs care plans that can improve functional abilities, lower the chance of institutionalization, postpone the onset of impairment, cut down on hospital admissions and stays, and increase survival [##UREF##12##53##]. A physiotherapist should ideally be available or included in the multidisciplinary team. Exercise programs of varying intensities have enhanced physical function in OPLWH, while short-duration exercise programs have shown beneficial effects on frail older individuals. Improvements in quality-of-life measures in PLWH have also been associated with participation in yoga programs [##REF##32583707##54##].</p>\",\n \"<p>While exercise has been shown to be the most effective intervention for frailty, group-based physiotherapy classes have also demonstrated efficacy in protecting against functional decline. On the other hand, addressing the social determinants of frailty is important. This includes, but is not limited to, isolation, loneliness, depression, or any other psychiatric diagnoses. However, this may not always be feasible, as it often depends on the availability of local professional resources and the presence of a community-based support system [##UREF##13##55##].</p>\",\n \"<p>The New Orleans Alcohol Use in HIV (NOAH) study evaluated the body composition, gait speed, and muscle strength of 341 participants living with HIV. Body composition was found to have a modulatory effect on frailty risk among PLWH, which was statistically significant. That is, while obesity was associated with increased risk, greater muscle mass may have had a protective effect, even among individuals who consume alcohol. These findings emphasize the importance of physical activity and weight control in modulating frailty risk [##REF##36124866##56##].</p>\",\n \"<p>Furthermore, strict control of the medications PLWH consumes may be beneficial. Polypharmacy may increase the risk of adverse outcomes, and de-prescription is often necessary. For instance, a study demonstrated that frailty was more common in those who used medications with an anticholinergic effect (OR 2.12; 95% CI 0.89-5.0). Therefore, clinicians should be aware of their impact and work to reduce exposure whenever possible [##REF##37644705##57##].</p>\",\n \"<p>As mentioned earlier, exercise is a well-established preventive intervention for frailty. However, since many of the OPLWH are already in a frail state, high-intensity training and weight-lifting may be too taxing. Therefore, a study investigated a novel game-based training program, referred to as exergame, and its impact on ameliorating frailty among 10 HIV-infected individuals. The exergame program which was conducted twice weekly for six weeks, incorporated activities such as weight shifting, ankle reaching, and obstacle crossing. After the conclusion of the program, changes in balance, gait, and other parameters were assessed, and improvements were seen in balance and mobility [##REF##27768079##58##].</p>\",\n \"<p>E-health systems are being increasingly utilized to provide health information, personalized recommendations, and smartphone reminders to promote healthy habits and help older people preserve and enhance their functional independence. There is much promise in the continuing evaluation of wearable sensors for frailty identification [##REF##32583707##54##]. The Ecological Momentary Assessment, which gathers real-time patient-reported outcome indicators, can help make it easier to incorporate patient-reported outcomes into innovative healthcare models [##UREF##12##53##]. Nonetheless, it is important to maintain vigilance to prevent the possible ageist approach to healthcare delivery from being supported by frailty [##UREF##11##52##].</p>\",\n \"<p>Limitations and challenges</p>\",\n \"<p>While our review provides insight into frailty pathophysiology, screening, detection, and management for PLWH, it also identifies several limitations and research gaps, the most notable of which is the absence of a standardized operational definition for frailty. This presents a challenge not only for consistent diagnosis in the healthcare setting but also for conducting studies with reliable data and results. Therefore, the need to establish a universally accepted definition is paramount. However, more research is needed to explore the most effective and relevant metrics for frailty while considering the diverse impacted populations, including OPLWH.</p>\",\n \"<p>In a comparable context, the variability of estimates of frailty prevalence in the different studies (ranging from 2% to 25%) highlights the importance of understanding any contributing factors and standardizing assessment methods in future studies. On the other hand, it implies that frailty in this population (PLWH) may manifest differently. Furthermore, while our article touches on the integration of geriatric concepts in HIV care, more research is required to assess the feasibility, effectiveness, and scalability of these approaches. Clear guidelines for identifying PLWH who would benefit from frailty assessments and evaluating the clinical effectiveness of geriatric referrals are lacking, and this is an area we aim to address in our ongoing research.</p>\",\n \"<p>Lastly, a more thorough exploration of e-health systems and electronic health records used for estimating frailty, especially those employing metrics such as the Frailty Index, would enhance our understanding of technological advancements in frailty assessment and their real-world applicability. Addressing these gaps will contribute to a more comprehensive and nuanced understanding of frailty, improve diagnostic accuracy and inform targeted interventions for vulnerable populations.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":[],"string":"[]","isConversational":false},"table":{"kind":"list like","value":[],"string":"[]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Mohammad Mansour, Monisha Augustine, Mahendra Kumar, Amna Naveed Butt, Thanmai Reddy Thugu, Parvinder Kaur, Nipakumari J. Patel, Ankit Gaudani, M. Bilal Jahania, Elhama Jami, Mouhammad Sharifa, Rohan Raj</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Mohammad Mansour, Monisha Augustine, Mahendra Kumar, Amna Naveed Butt, Thanmai Reddy Thugu, Parvinder Kaur, Nipakumari J. Patel, Ankit Gaudani, M. Bilal Jahania, Elhama Jami, Mouhammad Sharifa, Rohan Raj, Dalia Mehmood</p><p><bold>Drafting of the manuscript:</bold> Mohammad Mansour, Monisha Augustine, Mahendra Kumar, Amna Naveed Butt, Thanmai Reddy Thugu, Parvinder Kaur, Nipakumari J. Patel, Ankit Gaudani, M. Bilal Jahania, Elhama Jami, Mouhammad Sharifa, Rohan Raj</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Mohammad Mansour, Monisha Augustine, Mahendra Kumar, Amna Naveed Butt, Thanmai Reddy Thugu, Parvinder Kaur, Nipakumari J. Patel, Ankit Gaudani, M. Bilal Jahania, Elhama Jami, Mouhammad Sharifa, Rohan Raj, Dalia Mehmood</p><p><bold>Supervision:</bold> Mohammad Mansour</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Concept and design:</bold> Mohammad Mansour, Monisha Augustine, Mahendra Kumar, Amna Naveed Butt, Thanmai Reddy Thugu, Parvinder Kaur, Nipakumari J. Patel, Ankit Gaudani, M. Bilal Jahania, Elhama Jami, Mouhammad Sharifa, Rohan Raj</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Mohammad Mansour, Monisha Augustine, Mahendra Kumar, Amna Naveed Butt, Thanmai Reddy Thugu, Parvinder Kaur, Nipakumari J. Patel, Ankit Gaudani, M. Bilal Jahania, Elhama Jami, Mouhammad Sharifa, Rohan Raj, Dalia Mehmood</p><p><bold>Drafting of the manuscript:</bold> Mohammad Mansour, Monisha Augustine, Mahendra Kumar, Amna Naveed Butt, Thanmai Reddy Thugu, Parvinder Kaur, Nipakumari J. Patel, Ankit Gaudani, M. Bilal Jahania, Elhama Jami, Mouhammad Sharifa, Rohan Raj</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Mohammad Mansour, Monisha Augustine, Mahendra Kumar, Amna Naveed Butt, Thanmai Reddy Thugu, Parvinder Kaur, Nipakumari J. Patel, Ankit Gaudani, M. Bilal Jahania, Elhama Jami, Mouhammad Sharifa, Rohan Raj, Dalia Mehmood</p><p><bold>Supervision:</bold> Mohammad Mansour</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":[],"string":"[]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"6\"], \"article-title\": [\"Frailty and pre-frailty in middle-aged and older adults and its association with multimorbidity and mortality: a prospective analysis of 493\\u2008737 UK Biobank participants\"], \"source\": [\"Lancet Public Health\"], \"person-group\": [\"\\n\"], \"surname\": [\"Hanlon\", \"Nicholl\", \"Jani\", \"Lee\", \"McQueenie\", \"Mair\"], \"given-names\": [\"P\", \"BI\", \"BD\", \"D\", \"R\", \"FS\"], \"fpage\": [\"0\"], \"lpage\": [\"32\"], \"volume\": [\"3\"], \"year\": [\"2018\"]}, {\"label\": [\"7\"], \"article-title\": [\"The effect of frailty on survival in patients with COVID-19 (COPE): a multicentre, European, observational cohort study\"], \"source\": [\"Lancet Public Health\"], \"person-group\": [\"\\n\"], \"surname\": [\"Hewitt\", \"Carter\", \"Vilches-Moraga\"], \"given-names\": [\"J\", \"B\", \"A\"], \"fpage\": [\"0\"], \"lpage\": [\"51\"], \"volume\": [\"5\"], \"year\": [\"2020\"]}, {\"label\": [\"10\"], \"article-title\": [\"Frailty in older adults: evidence for a phenotype\"], \"source\": [\"J Gerontol A Biol Sci Med Sci\"], \"person-group\": [\"\\n\"], \"surname\": [\"Fried\", \"Tangen\", \"Walston\"], \"given-names\": [\"LP\", \"CM\", \"J\"], \"fpage\": [\"0\"], \"lpage\": [\"56\"], \"volume\": [\"56\"], \"year\": [\"2001\"]}, {\"label\": [\"12\"], \"article-title\": [\"Changes in relative fitness and frailty across the adult lifespan: evidence from the Canadian National Population Health Survey\"], \"source\": [\"CMAJ\"], \"person-group\": [\"\\n\"], \"surname\": [\"Rockwood\", \"Song\", \"Mitnitski\"], \"given-names\": [\"K\", \"X\", \"A\"], \"fpage\": [\"0\"], \"lpage\": [\"94\"], \"volume\": [\"183\"], \"year\": [\"2011\"]}, {\"label\": [\"14\"], \"article-title\": [\"Frailty and the prediction of negative health outcomes: a meta-analysis\"], \"source\": [\"J Am Med Dir Assoc\"], \"person-group\": [\"\\n\"], \"surname\": [\"Vermeiren\", \"Vella-Azzopardi\", \"Beckw\\u00e9e\", \"Habbig\", \"Scafoglieri\", \"Jansen\", \"Bautmans\"], \"given-names\": [\"S\", \"R\", \"D\", \"AK\", \"A\", \"B\", \"I\"], \"fpage\": [\"1163\"], \"volume\": [\"17\"], \"year\": [\"2016\"]}, {\"label\": [\"27\"], \"article-title\": [\"Frailty, HIV infection, and mortality in an aging cohort of injection drug users\"], \"source\": [\"PLoS One\"], \"person-group\": [\"\\n\"], \"surname\": [\"Piggott\", \"Muzaale\", \"Mehta\", \"Brown\", \"Patel\", \"Leng\", \"Kirk\"], \"given-names\": [\"DA\", \"AD\", \"SH\", \"TT\", \"KV\", \"SX\", \"GD\"], \"fpage\": [\"0\"], \"volume\": [\"8\"], \"year\": [\"2013\"]}, {\"label\": [\"33\"], \"article-title\": [\"Frailty in surgical preoperative evaluation and postoperative recovery\"], \"source\": [\"Curr Geriatr Rep\"], \"person-group\": [\"\\n\"], \"surname\": [\"Lee\", \"Mak\", \"Tan\"], \"given-names\": [\"DJK\", \"MH\", \"KY\"], \"fpage\": [\"87\"], \"lpage\": [\"96\"], \"volume\": [\"8\"], \"year\": [\"2019\"]}, {\"label\": [\"38\"], \"article-title\": [\"The dynamic association between frailty, CD4 and CD4/CD8 ratio in people aging with HIV\"], \"source\": [\"PLoS One\"], \"person-group\": [\"\\n\"], \"surname\": [\"Guaraldi\", \"Zona\", \"Silva\"], \"given-names\": [\"G\", \"S\", \"AR\"], \"fpage\": [\"0\"], \"volume\": [\"14\"], \"year\": [\"2019\"]}, {\"label\": [\"41\"], \"article-title\": [\"Predictors of transitions in frailty severity and mortality among people aging with HIV\"], \"source\": [\"PLoS One\"], \"person-group\": [\"\\n\"], \"surname\": [\"Brothers\", \"Kirkland\", \"Theou\"], \"given-names\": [\"TD\", \"S\", \"O\"], \"fpage\": [\"0\"], \"volume\": [\"12\"], \"year\": [\"2017\"]}, {\"label\": [\"43\"], \"article-title\": [\"Older people with HIV are an essential part of the continuum of HIV care\"], \"source\": [\"J Int AIDS Soc\"], \"person-group\": [\"\\n\"], \"surname\": [\"Siegler\", \"Burchett\", \"Glesby\"], \"given-names\": [\"EL\", \"CO\", \"MJ\"], \"fpage\": [\"0\"], \"volume\": [\"21\"], \"year\": [\"2018\"]}, {\"label\": [\"44\"], \"article-title\": [\"Frailty and comprehensive geriatric assessment\"], \"source\": [\"J Korean Med Sci\"], \"person-group\": [\"\\n\"], \"surname\": [\"Lee\", \"Lee\", \"Jang\"], \"given-names\": [\"H\", \"E\", \"IY\"], \"fpage\": [\"0\"], \"volume\": [\"35\"], \"year\": [\"2020\"]}, {\"label\": [\"52\"], \"article-title\": [\"Effects of a multicomponent frailty prevention program in prefrail community-dwelling older persons: a 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[\"2022\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":58,"string":"58"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50539"},"package_file":{"kind":"string","value":"oa_package/79/92/PMC10787848.tar.gz"}}},{"rowIdx":156,"cells":{"accession_id":{"kind":"string","value":"PMC10787849"},"pmid":{"kind":"string","value":"38222186"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Autism is a disorder distinguished by significant challenges in social interaction and communication coupled with repetitive and stereotypical patterns of behavior and activities. Deficits in social interaction and language development become apparent before the age of three. In children, this condition is referred to as autism spectrum disorder (ASD). ASD is a disorder that affects a child's neurological system, growth, and general development [##UREF##0##1##]. A child with ASD often has problems communicating; they may have trouble developing social skills. In the Diagnostic and Statistical Manual of Mental Illnesses-5 (DSM-5), ASD is a neurodevelopmental disorder characterized by persistent deficits in social communication and social interaction across multiple contexts, as well as restricted, repetitive patterns of behavior, interests, or activities. The criteria are grouped into two main domains: social communication and behavior. To be diagnosed with ASD, according to the DSM-5, an individual must demonstrate persistent difficulties in social communication and interaction, along with at least two of the four types of restricted, repetitive behaviors. The severity of ASD is specified based on the level of support required: Level 1 (requiring support), Level 2 (requiring substantial support), or Level 3 (requiring very substantial support) [##UREF##1##2##].\nIt is important to note that the field of psychiatry and psychology may evolve, and updates to diagnostic criteria or new editions of diagnostic manuals may occur. Therefore, it is recommended to consult the latest professional literature or experts in the field for the most up-to-date information on the diagnosis of ASD and other mental health conditions. Different approaches, especially psychosocial therapies, are used to address the core symptoms of ASD. These include specific educational programs in special education, speech, occupational, physical, and behavioral analysis, all of which have a favorable impact on the overall efficacy of treatment. Occupational therapy is frequently utilised in managing ASD, although its efficacy can be limited. Consequently, there is considerable anticipation that psychopharmacology may provide additional assistance to these individuals. The CDC states that ASD is a prevalent illness, typically first identified in early childhood [##UREF##2##3##].\nThe signs and symptoms of attention deficit hyperactivity disorder (ADHD), as listed in the DSM-5, include experiencing difficulties with staying focused, paying attention to details, and listening when spoken to directly. Individuals with ADHD often have trouble finishing homework and organizing projects and are reluctant to engage in demanding activities. They regularly lose critical materials needed for assignments, become easily distracted, and are restless or fidgety. Common behaviors include frequently leaving their seat in situations where remaining seated is expected, being unable to play quietly, and exhibiting restlessness as if driven by an inner motor. Additional symptoms are talking excessively, vocalizing thoughts aloud, and showing a lack of self-control or patience [##UREF##3##4##]. The level of impairment in individuals with ASD can vary, affecting perception, cognitive processing, learning, and memory [##UREF##4##5##]. Additionally, genetics plays a significant role in autism, as evidenced by the ASD occurrence rate among twins, which ranges from 36% to 95%. These genetic factors particularly influence the repetitive behavioral aspects associated with ASD [##UREF##5##6##, ####UREF##6##7##, ##UREF##7##8####7##8##].\nClinical assessments using the Indian Scale for Autism Assessment (ISAA) have yielded positive results across the board for symptoms associated with ASD [##UREF##8##9##, ####REF##35996797##10##, ##UREF##9##11####9##11##]. The training intervention was designed to enhance the understanding of managing arousal, preventing annoyance from worsening, and promoting self-regulation skills [##UREF##10##12##]. It is essential to ensure that children on the autism spectrum interventions are supported by research evidence. Given the variety of accessible pharmaceutical therapies, compiling comprehensive research on interventions for children with these conditions is imperative [##REF##36081343##13##]. Physiotherapists are often among the first practitioners to assess children who may be at risk for these disorders [##REF##29971656##14##].\nThe study aims to investigate the efficacy of a multimodal physiotherapy approach in addressing the specific challenges associated with ASD, including speech impairment and attention deficit. The case report will focus on an individual with ASD who presents both speech difficulties and attention deficit symptoms. The multimodal physiotherapy intervention will incorporate a combination of physical therapy techniques, sensory integration strategies, and potentially other therapeutic modalities to enhance both motor and cognitive functions. The study seeks to assess improvements in speech abilities, attention span, and overall functional outcomes in individuals with ASD following the implementation of the multimodal physiotherapy intervention. By exploring the potential benefits of this comprehensive approach, the research aims to contribute valuable insights into the development of effective therapeutic strategies for individuals with ASD who experience speech impairments and attention deficits.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Autism is a disorder distinguished by significant challenges in social interaction and communication coupled with repetitive and stereotypical patterns of behavior and activities. Deficits in social interaction and language development become apparent before the age of three. In children, this condition is referred to as autism spectrum disorder (ASD). ASD is a disorder that affects a child's neurological system, growth, and general development [##UREF##0##1##]. A child with ASD often has problems communicating; they may have trouble developing social skills. In the Diagnostic and Statistical Manual of Mental Illnesses-5 (DSM-5), ASD is a neurodevelopmental disorder characterized by persistent deficits in social communication and social interaction across multiple contexts, as well as restricted, repetitive patterns of behavior, interests, or activities. The criteria are grouped into two main domains: social communication and behavior. To be diagnosed with ASD, according to the DSM-5, an individual must demonstrate persistent difficulties in social communication and interaction, along with at least two of the four types of restricted, repetitive behaviors. The severity of ASD is specified based on the level of support required: Level 1 (requiring support), Level 2 (requiring substantial support), or Level 3 (requiring very substantial support) [##UREF##1##2##].\\nIt is important to note that the field of psychiatry and psychology may evolve, and updates to diagnostic criteria or new editions of diagnostic manuals may occur. Therefore, it is recommended to consult the latest professional literature or experts in the field for the most up-to-date information on the diagnosis of ASD and other mental health conditions. Different approaches, especially psychosocial therapies, are used to address the core symptoms of ASD. These include specific educational programs in special education, speech, occupational, physical, and behavioral analysis, all of which have a favorable impact on the overall efficacy of treatment. Occupational therapy is frequently utilised in managing ASD, although its efficacy can be limited. Consequently, there is considerable anticipation that psychopharmacology may provide additional assistance to these individuals. The CDC states that ASD is a prevalent illness, typically first identified in early childhood [##UREF##2##3##].\\nThe signs and symptoms of attention deficit hyperactivity disorder (ADHD), as listed in the DSM-5, include experiencing difficulties with staying focused, paying attention to details, and listening when spoken to directly. Individuals with ADHD often have trouble finishing homework and organizing projects and are reluctant to engage in demanding activities. They regularly lose critical materials needed for assignments, become easily distracted, and are restless or fidgety. Common behaviors include frequently leaving their seat in situations where remaining seated is expected, being unable to play quietly, and exhibiting restlessness as if driven by an inner motor. Additional symptoms are talking excessively, vocalizing thoughts aloud, and showing a lack of self-control or patience [##UREF##3##4##]. The level of impairment in individuals with ASD can vary, affecting perception, cognitive processing, learning, and memory [##UREF##4##5##]. Additionally, genetics plays a significant role in autism, as evidenced by the ASD occurrence rate among twins, which ranges from 36% to 95%. These genetic factors particularly influence the repetitive behavioral aspects associated with ASD [##UREF##5##6##, ####UREF##6##7##, ##UREF##7##8####7##8##].\\nClinical assessments using the Indian Scale for Autism Assessment (ISAA) have yielded positive results across the board for symptoms associated with ASD [##UREF##8##9##, ####REF##35996797##10##, ##UREF##9##11####9##11##]. The training intervention was designed to enhance the understanding of managing arousal, preventing annoyance from worsening, and promoting self-regulation skills [##UREF##10##12##]. It is essential to ensure that children on the autism spectrum interventions are supported by research evidence. Given the variety of accessible pharmaceutical therapies, compiling comprehensive research on interventions for children with these conditions is imperative [##REF##36081343##13##]. Physiotherapists are often among the first practitioners to assess children who may be at risk for these disorders [##REF##29971656##14##].\\nThe study aims to investigate the efficacy of a multimodal physiotherapy approach in addressing the specific challenges associated with ASD, including speech impairment and attention deficit. The case report will focus on an individual with ASD who presents both speech difficulties and attention deficit symptoms. The multimodal physiotherapy intervention will incorporate a combination of physical therapy techniques, sensory integration strategies, and potentially other therapeutic modalities to enhance both motor and cognitive functions. The study seeks to assess improvements in speech abilities, attention span, and overall functional outcomes in individuals with ASD following the implementation of the multimodal physiotherapy intervention. By exploring the potential benefits of this comprehensive approach, the research aims to contribute valuable insights into the development of effective therapeutic strategies for individuals with ASD who experience speech impairments and attention deficits.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>This study presents intriguing findings that warrant careful consideration and interpretation. The observed improvements in speech abilities and attention deficits following the implementation of the multimodal physiotherapy intervention suggest the potential efficacy of this comprehensive approach. The integration of physical therapy techniques and sensory strategies appears to have a positive impact on both motor and cognitive functions, addressing the unique challenges faced by individuals with ASD who experience concurrent speech impairments and attention deficits. The individualized nature of the treatment plan is a notable strength, emphasizing the importance of tailoring interventions to the specific needs of individuals with ASD.</p>","<p>In the broader context of interventions for ASD, this study contributes to the growing body of literature exploring novel therapeutic approaches. By incorporating physical therapy and sensory strategies into a multimodal intervention, the research underscores the potential benefits of simultaneously addressing both motor and cognitive aspects. This aligns with the evolving understanding of the interconnected nature of sensory-motor functions and cognitive processes in individuals with ASD. The findings may inform clinicians and therapists working with this population, offering insights into developing more comprehensive and tailored interventions. Despite the promising results, it is essential to approach the study's implications cautiously and recognize the need for continued exploration of multimodal approaches within more extensive and diverse samples, allowing for a more robust understanding of their effectiveness across the spectrum of ASD presentations.\nThe physiotherapy interventions presented in this protocol are a comprehensive and multidisciplinary approach to addressing attention deficit and speech impairment in a three-year-old child with developmental anomalies. Each intervention is tailored to target specific aspects of the child's condition, aiming to improve their overall communication and cognitive abilities. Auditory integration training (AIT) involves exposure to specially filtered and modulated music to enhance auditory processing, which may improve sensory integration and attention in individuals with developmental disorders [##REF##7608035##15##]. Sensory integration therapy (SIT) focuses on improving the brain's processing of sensory information, particularly for children with sensory sensitivities often seen in ASD [##REF##10500857##16##]. Holding therapy, based on the belief that autism is linked to a lack of maternal bonding, has its efficacy and safety debated. Facilitated communication involves a skilled facilitator assisting a child with communication challenges by using an output device for spelling words or phrases [##REF##8050988##17##]. Music therapy is recognized for its potential to enhance interaction and communication skills in individuals with ASD [##REF##19535468##18##]. The Picture Exchange Communication System (PECS) is an established method for children with ASD to communicate, offering a structured approach for non-verbal or minimally verbal individuals to express needs and desires using pictures or symbols [##REF##17501728##19##].\nParent-mediated communication is a crucial component of the child's support system, equipping parents to effectively understand and respond to their child's communication. However, the effectiveness of each intervention can vary from one individual to another, and ongoing evaluation and research are needed to determine the long-term impact and effectiveness of these interventions. ADHD is common in children, and the implementation of therapy in mental health, complemented by interdisciplinary collaboration, is crucial [##UREF##11##20##].</p>","<p>The pediatric population benefits from the expertise of physiotherapists who specialize in treating children. These professionals possess a deep understanding of the interplay between early childhood development and the body's systems and functions, as well as typical child growth patterns. Physiotherapists use many advanced devices and tools, as well as generic skills, applying their additional training in development and growth to treat infants through teenagers. Physiotherapists use an extensive span of treatment approaches in the pediatrics field. In each case, careful assessment determines the treatment approach and protocol. However, it is crucial to acknowledge the study's limitations, such as the single-case design, which may restrict the generalizability of the findings. Further research with larger sample sizes and controlled designs is warranted to confirm and extend the current results. </p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>This study presents intriguing findings that warrant careful consideration and interpretation. The observed improvements in speech abilities and attention deficits following the implementation of the multimodal physiotherapy intervention suggest the potential efficacy of this comprehensive approach. The integration of physical therapy techniques and sensory strategies appears to have a positive impact on both motor and cognitive functions, addressing the unique challenges faced by individuals with ASD who experience concurrent speech impairments and attention deficits. The individualized nature of the treatment plan is a notable strength, emphasizing the importance of tailoring interventions to the specific needs of individuals with ASD.</p>\",\n \"<p>In the broader context of interventions for ASD, this study contributes to the growing body of literature exploring novel therapeutic approaches. By incorporating physical therapy and sensory strategies into a multimodal intervention, the research underscores the potential benefits of simultaneously addressing both motor and cognitive aspects. This aligns with the evolving understanding of the interconnected nature of sensory-motor functions and cognitive processes in individuals with ASD. The findings may inform clinicians and therapists working with this population, offering insights into developing more comprehensive and tailored interventions. Despite the promising results, it is essential to approach the study's implications cautiously and recognize the need for continued exploration of multimodal approaches within more extensive and diverse samples, allowing for a more robust understanding of their effectiveness across the spectrum of ASD presentations.\\nThe physiotherapy interventions presented in this protocol are a comprehensive and multidisciplinary approach to addressing attention deficit and speech impairment in a three-year-old child with developmental anomalies. Each intervention is tailored to target specific aspects of the child's condition, aiming to improve their overall communication and cognitive abilities. Auditory integration training (AIT) involves exposure to specially filtered and modulated music to enhance auditory processing, which may improve sensory integration and attention in individuals with developmental disorders [##REF##7608035##15##]. Sensory integration therapy (SIT) focuses on improving the brain's processing of sensory information, particularly for children with sensory sensitivities often seen in ASD [##REF##10500857##16##]. Holding therapy, based on the belief that autism is linked to a lack of maternal bonding, has its efficacy and safety debated. Facilitated communication involves a skilled facilitator assisting a child with communication challenges by using an output device for spelling words or phrases [##REF##8050988##17##]. Music therapy is recognized for its potential to enhance interaction and communication skills in individuals with ASD [##REF##19535468##18##]. The Picture Exchange Communication System (PECS) is an established method for children with ASD to communicate, offering a structured approach for non-verbal or minimally verbal individuals to express needs and desires using pictures or symbols [##REF##17501728##19##].\\nParent-mediated communication is a crucial component of the child's support system, equipping parents to effectively understand and respond to their child's communication. However, the effectiveness of each intervention can vary from one individual to another, and ongoing evaluation and research are needed to determine the long-term impact and effectiveness of these interventions. ADHD is common in children, and the implementation of therapy in mental health, complemented by interdisciplinary collaboration, is crucial [##UREF##11##20##].</p>\",\n \"<p>The pediatric population benefits from the expertise of physiotherapists who specialize in treating children. These professionals possess a deep understanding of the interplay between early childhood development and the body's systems and functions, as well as typical child growth patterns. Physiotherapists use many advanced devices and tools, as well as generic skills, applying their additional training in development and growth to treat infants through teenagers. Physiotherapists use an extensive span of treatment approaches in the pediatrics field. In each case, careful assessment determines the treatment approach and protocol. However, it is crucial to acknowledge the study's limitations, such as the single-case design, which may restrict the generalizability of the findings. Further research with larger sample sizes and controlled designs is warranted to confirm and extend the current results. </p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>In conclusion, this case report provides valuable insights into a holistic intervention strategy for individuals with ASD facing concurrent challenges of speech impairments and attention deficits. The observed improvements in speech abilities and attention following the multimodal physiotherapy intervention suggest the potential effectiveness of integrating physical therapy techniques and sensory strategies. The individualized nature of the treatment plan highlights the importance of tailoring interventions to the specific needs of each individual with ASD.\nWhile the findings are promising, it is crucial to recognize the study's limitations, including its single-case design, which may limit generalizability. Further research with larger and more diverse samples and controlled designs is essential to validate and extend these preliminary results. The study contributes to the broader understanding of therapeutic approaches for ASD by emphasizing the interconnected nature of sensory-motor functions and cognitive processes. The multimodal physiotherapy approach offers a nuanced perspective for clinicians and therapists, suggesting potential benefits in concurrently addressing both motor and cognitive aspects. As we move forward, ongoing research should continue to explore and refine multimodal interventions, considering their applicability across the diverse spectrum of ASD presentations and informing the development of more comprehensive and tailored therapeutic strategies for individuals with ASD experiencing speech impairments and attention deficits.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>In conclusion, this case report provides valuable insights into a holistic intervention strategy for individuals with ASD facing concurrent challenges of speech impairments and attention deficits. The observed improvements in speech abilities and attention following the multimodal physiotherapy intervention suggest the potential effectiveness of integrating physical therapy techniques and sensory strategies. The individualized nature of the treatment plan highlights the importance of tailoring interventions to the specific needs of each individual with ASD.\\nWhile the findings are promising, it is crucial to recognize the study's limitations, including its single-case design, which may limit generalizability. Further research with larger and more diverse samples and controlled designs is essential to validate and extend these preliminary results. The study contributes to the broader understanding of therapeutic approaches for ASD by emphasizing the interconnected nature of sensory-motor functions and cognitive processes. The multimodal physiotherapy approach offers a nuanced perspective for clinicians and therapists, suggesting potential benefits in concurrently addressing both motor and cognitive aspects. As we move forward, ongoing research should continue to explore and refine multimodal interventions, considering their applicability across the diverse spectrum of ASD presentations and informing the development of more comprehensive and tailored therapeutic strategies for individuals with ASD experiencing speech impairments and attention deficits.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Autism is a disorder distinguished by significant challenges in social interaction and communication coupled with repetitive and stereotypical patterns of behavior and activities. Deficits in social interaction and language development become apparent before age three. In children, this condition is referred to as autism spectrum disorder (ASD). Attention Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder characterized by persistent patterns of inattention, hyperactivity, and impulsivity. Individuals with ADHD may struggle with sustaining attention, organizing tasks, and completing assignments. They may exhibit hyperactive behaviors such as fidgeting, difficulty staying seated, and impulsive actions like interrupting others. ADHD can significantly impact daily functioning and is often diagnosed in childhood, with symptoms potentially persisting into adulthood. The disorder has three main subtypes: predominantly inattentive, predominantly hyperactive-impulsive, and combined presentation. Treatment typically involves a combination of behavioral interventions, psychoeducation, and, in some cases, medication, aiming to provide support and strategies for individuals to manage their symptoms effectively in various aspects of life. Cognitive impairment in ASD varies, meaning it could be at the sensory perception level of cognitive processing, learning, and memory. The goal of the training intervention was to control physiological arousal, enhance awareness, keep annoyance from getting worse, and encourage self-regulation abilities. In this case report, we discuss the approach of multimodal physiotherapy for autism with speech impairment and attention deficit. Furthermore, physiotherapy needs to find a position in the new mental health care paradigm in order to contribute to mental health care.</p>"],"string":"[\n \"<p>Autism is a disorder distinguished by significant challenges in social interaction and communication coupled with repetitive and stereotypical patterns of behavior and activities. Deficits in social interaction and language development become apparent before age three. In children, this condition is referred to as autism spectrum disorder (ASD). Attention Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder characterized by persistent patterns of inattention, hyperactivity, and impulsivity. Individuals with ADHD may struggle with sustaining attention, organizing tasks, and completing assignments. They may exhibit hyperactive behaviors such as fidgeting, difficulty staying seated, and impulsive actions like interrupting others. ADHD can significantly impact daily functioning and is often diagnosed in childhood, with symptoms potentially persisting into adulthood. The disorder has three main subtypes: predominantly inattentive, predominantly hyperactive-impulsive, and combined presentation. Treatment typically involves a combination of behavioral interventions, psychoeducation, and, in some cases, medication, aiming to provide support and strategies for individuals to manage their symptoms effectively in various aspects of life. Cognitive impairment in ASD varies, meaning it could be at the sensory perception level of cognitive processing, learning, and memory. The goal of the training intervention was to control physiological arousal, enhance awareness, keep annoyance from getting worse, and encourage self-regulation abilities. In this case report, we discuss the approach of multimodal physiotherapy for autism with speech impairment and attention deficit. Furthermore, physiotherapy needs to find a position in the new mental health care paradigm in order to contribute to mental health care.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Case presentation</title>","<p>Patient information</p>","<p>We present a case involving a three-year-old male child characterized by a spectrum of developmental anomalies. Predominant concerns encompass intellectual disability, hyperactivity, speech dysfluency, and attentional deficits. The patient was delivered at term following an uneventful gestational period of 9 months and 5 days via a lower segment cesarean section (LSCS). Postnatally, delayed initiation of the birth cry necessitated a brief admission to the neonatal intensive care unit (NICU) for observation. Challenges in breastfeeding ensued due to attentional constraints, resulting in a reliance on artificial feeding. \nAdditionally, the patient was identified as having a calcium deficiency. At birth, he weighed 2.4 kilograms and presented with congenital anomalies, which were first observed by his mother at 2.5 years of age. Parental reports at this age indicated deficits in attention, speech fluency, social integration, and communication skills. His vocalizations remained mostly indiscernible babbling until 36 months, when he produced his inaugural linguistically coherent word. He exhibited challenges in apprehending and executing tasks and directives.\nMotor milestones were accomplished within normative time frames. Ambulation with support was initiated at nine months, and independent ambulation was achieved at 10 months. Equilibrium mastery was attained at 14 months. Regarding ADHD, the patient exhibited a diminished attentional span and proclivity for distractibility. Restlessness and loud vocalizations during play were evident, along with difficulty in maintaining a stationary seated position. A sensitive, temperamental disposition was noted. Modest progress was ascertained by both therapeutic interventionists and parents, with circumscribed strides in attentional endurance and communication acumen. The patient remains reliant on external feeding and continues to necessitate diaper use. Proficiency in toilet training remains unattended.</p>","<p>Clinical examination</p>","<p>The child exhibits integration with an examiner but shows less interaction with the surrounding environment. His overall activity level was average, but he exhibited a notable deficit in orientation to time, place, and person. His speech was unimpaired, and his hearing was within normal limits. However, his attention span was observed to be poor, which contributed to difficulties in maintaining focus and engagement. The anthropometric measurements are detailed in Table ##TAB##0##1##.</p>","<p>Investigation</p>","<p>The patient underwent a Brainstem Evoked Response Audiometry (BERA) examination, which revealed normal auditory pathway function, indicating no detectable abnormalities in the auditory system (Table ##TAB##1##2##).</p>","<p>Physiotherapy intervention</p>","<p>The patient underwent physiotherapy treatment for four weeks (Table ##TAB##2##3##). Throughout the treatment, both the patient and his family participated in counseling sessions and followed an exercise regimen.</p>","<p>Figure ##FIG##0##1## shows a child undergoing holding therapy under the supervision of a therapist, and Figure ##FIG##1##2## illustrates the Picture Exchange Communication System (PECS) therapy.</p>","<p>Follow-up and outcome measures</p>","<p>After four weeks of treatment, outcome measures were assessed. Table ##TAB##3##4## summarizes the findings of the scales after the completion of treatment.</p>"],"string":"[\n \"<title>Case presentation</title>\",\n \"<p>Patient information</p>\",\n \"<p>We present a case involving a three-year-old male child characterized by a spectrum of developmental anomalies. Predominant concerns encompass intellectual disability, hyperactivity, speech dysfluency, and attentional deficits. The patient was delivered at term following an uneventful gestational period of 9 months and 5 days via a lower segment cesarean section (LSCS). Postnatally, delayed initiation of the birth cry necessitated a brief admission to the neonatal intensive care unit (NICU) for observation. Challenges in breastfeeding ensued due to attentional constraints, resulting in a reliance on artificial feeding. \\nAdditionally, the patient was identified as having a calcium deficiency. At birth, he weighed 2.4 kilograms and presented with congenital anomalies, which were first observed by his mother at 2.5 years of age. Parental reports at this age indicated deficits in attention, speech fluency, social integration, and communication skills. His vocalizations remained mostly indiscernible babbling until 36 months, when he produced his inaugural linguistically coherent word. He exhibited challenges in apprehending and executing tasks and directives.\\nMotor milestones were accomplished within normative time frames. Ambulation with support was initiated at nine months, and independent ambulation was achieved at 10 months. Equilibrium mastery was attained at 14 months. Regarding ADHD, the patient exhibited a diminished attentional span and proclivity for distractibility. Restlessness and loud vocalizations during play were evident, along with difficulty in maintaining a stationary seated position. A sensitive, temperamental disposition was noted. Modest progress was ascertained by both therapeutic interventionists and parents, with circumscribed strides in attentional endurance and communication acumen. The patient remains reliant on external feeding and continues to necessitate diaper use. Proficiency in toilet training remains unattended.</p>\",\n \"<p>Clinical examination</p>\",\n \"<p>The child exhibits integration with an examiner but shows less interaction with the surrounding environment. His overall activity level was average, but he exhibited a notable deficit in orientation to time, place, and person. His speech was unimpaired, and his hearing was within normal limits. However, his attention span was observed to be poor, which contributed to difficulties in maintaining focus and engagement. The anthropometric measurements are detailed in Table ##TAB##0##1##.</p>\",\n \"<p>Investigation</p>\",\n \"<p>The patient underwent a Brainstem Evoked Response Audiometry (BERA) examination, which revealed normal auditory pathway function, indicating no detectable abnormalities in the auditory system (Table ##TAB##1##2##).</p>\",\n \"<p>Physiotherapy intervention</p>\",\n \"<p>The patient underwent physiotherapy treatment for four weeks (Table ##TAB##2##3##). Throughout the treatment, both the patient and his family participated in counseling sessions and followed an exercise regimen.</p>\",\n \"<p>Figure ##FIG##0##1## shows a child undergoing holding therapy under the supervision of a therapist, and Figure ##FIG##1##2## illustrates the Picture Exchange Communication System (PECS) therapy.</p>\",\n \"<p>Follow-up and outcome measures</p>\",\n \"<p>After four weeks of treatment, outcome measures were assessed. Table ##TAB##3##4## summarizes the findings of the scales after the completion of treatment.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Patient undergoing holding therapy.</title></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Picture Exchange Communication System (PECS).</title></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>Patient undergoing holding therapy.</title></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG2\\\"><label>Figure 2</label><caption><title>Picture Exchange Communication System (PECS).</title></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Physical examination findings.</title><p>cm: Centimeter; kg: Kilogram.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Anthropometric measures</td><td rowspan=\"1\" colspan=\"1\">At birth</td><td rowspan=\"1\" colspan=\"1\">At present</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Length/Height (in cm)</td><td rowspan=\"1\" colspan=\"1\">10 cm</td><td rowspan=\"1\" colspan=\"1\">94cm</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Weight (in kg)</td><td rowspan=\"1\" colspan=\"1\">2.40 kg</td><td rowspan=\"1\" colspan=\"1\">13.5 kg</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Head circumference (in cm)</td><td rowspan=\"1\" colspan=\"1\">35 cm</td><td rowspan=\"1\" colspan=\"1\">46 cm</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Chest circumference (in cm)</td><td rowspan=\"1\" colspan=\"1\">30-33 cm</td><td rowspan=\"1\" colspan=\"1\">49 cm</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>BERA test findings.</title><p>The data has been represented as N.</p><p>EP: Evoked Potentials; SPL: Sound Pressure Level; dB: Decibel; ms: Millisecond; Rec: Recorded; Cz-M: Vertex-Mastoid; R: Right; L: Left; BERA: Brainstem Evoked Response Audiometry; NA: Not Applicable.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Rec.sites</td><td rowspan=\"1\" colspan=\"1\">I Lat., ms</td><td rowspan=\"1\" colspan=\"1\">Ia Lat., ms</td><td rowspan=\"1\" colspan=\"1\">II Lat., ms</td><td rowspan=\"1\" colspan=\"1\">III Lat., ms</td><td rowspan=\"1\" colspan=\"1\">IIIa Lat., ms</td><td rowspan=\"1\" colspan=\"1\">IV Lat., ms</td><td rowspan=\"1\" colspan=\"1\">V Lat., ms</td><td rowspan=\"1\" colspan=\"1\">I-III Lat., ms</td><td rowspan=\"1\" colspan=\"1\">III-V Lat., ms</td><td rowspan=\"1\" colspan=\"1\">I-V Lat., ms</td><td rowspan=\"1\" colspan=\"1\">Stim.side</td><td rowspan=\"1\" colspan=\"1\">Stimulus</td></tr><tr><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">1.43</td><td rowspan=\"1\" colspan=\"1\">1.75</td><td rowspan=\"1\" colspan=\"1\">3.25</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">5.15</td><td rowspan=\"1\" colspan=\"1\">6.63</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">5.2</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">±110 dB SPL</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">1.53</td><td rowspan=\"1\" colspan=\"1\">2.85</td><td rowspan=\"1\" colspan=\"1\">3.35</td><td rowspan=\"1\" colspan=\"1\">4.6</td><td rowspan=\"1\" colspan=\"1\">4.85</td><td rowspan=\"1\" colspan=\"1\">5.25</td><td rowspan=\"1\" colspan=\"1\">6.68</td><td rowspan=\"1\" colspan=\"1\">3.08</td><td rowspan=\"1\" colspan=\"1\">2.08</td><td rowspan=\"1\" colspan=\"1\">5.15</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">±100 dB SPL</td></tr><tr><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">2.38</td><td rowspan=\"1\" colspan=\"1\">2.78</td><td rowspan=\"1\" colspan=\"1\">3.5</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">4.6</td><td rowspan=\"1\" colspan=\"1\">5.4</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">±90 dB SPL</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">2.35</td><td rowspan=\"1\" colspan=\"1\">3.23</td><td rowspan=\"1\" colspan=\"1\">3.55</td><td rowspan=\"1\" colspan=\"1\">3.95</td><td rowspan=\"1\" colspan=\"1\">4.75</td><td rowspan=\"1\" colspan=\"1\">5.5</td><td rowspan=\"1\" colspan=\"1\">5.9</td><td rowspan=\"1\" colspan=\"1\">1.6</td><td rowspan=\"1\" colspan=\"1\">1.95</td><td rowspan=\"1\" colspan=\"1\">3.55</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">±80 dB SPL</td></tr><tr><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">1.73</td><td rowspan=\"1\" colspan=\"1\">2.45</td><td rowspan=\"1\" colspan=\"1\">2.8</td><td rowspan=\"1\" colspan=\"1\">3.75</td><td rowspan=\"1\" colspan=\"1\">4.03</td><td rowspan=\"1\" colspan=\"1\">4.3</td><td rowspan=\"1\" colspan=\"1\">5.83</td><td rowspan=\"1\" colspan=\"1\">2.03</td><td rowspan=\"1\" colspan=\"1\">2.08</td><td rowspan=\"1\" colspan=\"1\">4.1</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">±70 dB SPL</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">1.83</td><td rowspan=\"1\" colspan=\"1\">2.65</td><td rowspan=\"1\" colspan=\"1\">2.88</td><td rowspan=\"1\" colspan=\"1\">3.95</td><td rowspan=\"1\" colspan=\"1\">5.15</td><td rowspan=\"1\" colspan=\"1\">5.35</td><td rowspan=\"1\" colspan=\"1\">5.95</td><td rowspan=\"1\" colspan=\"1\">2.13</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">4.13</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">±60 dB SPL</td></tr><tr><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">2.2</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">3.75</td><td rowspan=\"1\" colspan=\"1\">4.35</td><td rowspan=\"1\" colspan=\"1\">5.68</td><td rowspan=\"1\" colspan=\"1\">6.63</td><td rowspan=\"1\" colspan=\"1\">1.55</td><td rowspan=\"1\" colspan=\"1\">2.88</td><td rowspan=\"1\" colspan=\"1\">4.43</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">±50 dB SPL</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">1.93</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">3.93</td><td rowspan=\"1\" colspan=\"1\">4.65</td><td rowspan=\"1\" colspan=\"1\">4.88</td><td rowspan=\"1\" colspan=\"1\">5.53</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.6</td><td rowspan=\"1\" colspan=\"1\">3.6</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">±40 dB SPL</td></tr><tr><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">1.35</td><td rowspan=\"1\" colspan=\"1\">2.83</td><td rowspan=\"1\" colspan=\"1\">3.3</td><td rowspan=\"1\" colspan=\"1\">4.4</td><td rowspan=\"1\" colspan=\"1\">4.68</td><td rowspan=\"1\" colspan=\"1\">5.25</td><td rowspan=\"1\" colspan=\"1\">6.78</td><td rowspan=\"1\" colspan=\"1\">3.05</td><td rowspan=\"1\" colspan=\"1\">2.38</td><td rowspan=\"1\" colspan=\"1\">5.43</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">±110 dB SPL</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">1.53</td><td rowspan=\"1\" colspan=\"1\">2.9</td><td rowspan=\"1\" colspan=\"1\">3.35</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">5.2</td><td rowspan=\"1\" colspan=\"1\">6.8</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">5.28</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">±100 dB SPL</td></tr><tr><td rowspan=\"1\" colspan=\"1\">11</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">1.6</td><td rowspan=\"1\" colspan=\"1\">2.88</td><td rowspan=\"1\" colspan=\"1\">3.5</td><td rowspan=\"1\" colspan=\"1\">4.65</td><td rowspan=\"1\" colspan=\"1\">4.83</td><td rowspan=\"1\" colspan=\"1\">5.38</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">3.05</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">±90 dB SPL</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">2.15</td><td rowspan=\"1\" colspan=\"1\">3.0</td><td rowspan=\"1\" colspan=\"1\">3.53</td><td rowspan=\"1\" colspan=\"1\">4.6</td><td rowspan=\"1\" colspan=\"1\">4.93</td><td rowspan=\"1\" colspan=\"1\">5.4</td><td rowspan=\"1\" colspan=\"1\">5.73</td><td rowspan=\"1\" colspan=\"1\">2.45</td><td rowspan=\"1\" colspan=\"1\">1.13</td><td rowspan=\"1\" colspan=\"1\">3.58</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">±80 dB SPL</td></tr><tr><td rowspan=\"1\" colspan=\"1\">13</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">2.3</td><td rowspan=\"1\" colspan=\"1\">2.88</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">3.9</td><td rowspan=\"1\" colspan=\"1\">4.8</td><td rowspan=\"1\" colspan=\"1\">5.33</td><td rowspan=\"1\" colspan=\"1\">5.78</td><td rowspan=\"1\" colspan=\"1\">1.6</td><td rowspan=\"1\" colspan=\"1\">1.88</td><td rowspan=\"1\" colspan=\"1\">3.48</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">±60 dB SPL</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">2.08</td><td rowspan=\"1\" colspan=\"1\">3.1</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">4.3</td><td rowspan=\"1\" colspan=\"1\">5.0</td><td rowspan=\"1\" colspan=\"1\">6.05</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">3.98</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">±40 dB SPL</td></tr><tr><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">Cz-M</td><td rowspan=\"1\" colspan=\"1\">1.88</td><td rowspan=\"1\" colspan=\"1\">3.28</td><td rowspan=\"1\" colspan=\"1\">3.6</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">4.63</td><td rowspan=\"1\" colspan=\"1\">6.08</td><td rowspan=\"1\" colspan=\"1\">6.53</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">4.65</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">±50 dB SPL</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB3\"><label>Table 3</label><caption><title>Physiotherapy management for attention deficit and speech impairment.</title><p>ASD: Autism spectrum disorder; AIT: Auditory Integration Training; SIT: Sensory Integration Therapy; PECS: Picture Exchange Communication System; Min: Minute.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Sr. No.</td><td rowspan=\"1\" colspan=\"1\">Treatment</td><td rowspan=\"1\" colspan=\"1\">Description</td><td rowspan=\"1\" colspan=\"1\">Duration</td></tr><tr><td rowspan=\"1\" colspan=\"1\">1)</td><td rowspan=\"1\" colspan=\"1\">Auditory Integration Training (AIT)</td><td rowspan=\"1\" colspan=\"1\">AIT involves experiencing music that has been filtered and modulated, including varied volume levels and pitches.</td><td rowspan=\"1\" colspan=\"1\">10 min 1 session per day/week</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">2)</td><td rowspan=\"1\" colspan=\"1\">Sensory Integration Therapy (SIT)</td><td rowspan=\"1\" colspan=\"1\">SIT aims to enhance brain processing of information, providing a foundation for advanced skill development.</td><td rowspan=\"1\" colspan=\"1\">10 min 1 session per day/week</td></tr><tr><td rowspan=\"1\" colspan=\"1\">3)</td><td rowspan=\"1\" colspan=\"1\">Holding Therapy</td><td rowspan=\"1\" colspan=\"1\">Based on the theory that autism stems from insufficient bonding with the mother.</td><td rowspan=\"1\" colspan=\"1\">10 min 1 session per day/week</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">4)</td><td rowspan=\"1\" colspan=\"1\">Facilitated Communication</td><td rowspan=\"1\" colspan=\"1\">It involves a method in which a skilled facilitator provides assistance to assist a child in using an output device, like a keyboard, typewriter or similar tool to spell.</td><td rowspan=\"1\" colspan=\"1\">15 min 1 session per day/week</td></tr><tr><td rowspan=\"1\" colspan=\"1\">5)</td><td rowspan=\"1\" colspan=\"1\">Music Therapy</td><td rowspan=\"1\" colspan=\"1\">One of the reasons music therapy is believed to be effective for individuals with ASD is because the processes involved in improvisation can potentially aid in the enhancement of interaction and communication skills.</td><td rowspan=\"1\" colspan=\"1\">5 min 1 session per day/week</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">6)</td><td rowspan=\"1\" colspan=\"1\">Picture Exchange Communication System (PECS)</td><td rowspan=\"1\" colspan=\"1\">Its main goal is to help children who have autism learn to start conversations by giving a picture to someone they want to communicate with in order to communicate their needs.</td><td rowspan=\"1\" colspan=\"1\">10 min 1 session per day/week</td></tr><tr><td rowspan=\"1\" colspan=\"1\">7)</td><td rowspan=\"1\" colspan=\"1\">Parent-mediated Communication</td><td rowspan=\"1\" colspan=\"1\">In this approach, the therapist provides training to parents with the goal of enhancing their ability to understand and respond effectively to their child’s communication.</td><td rowspan=\"1\" colspan=\"1\">10 min 1 session per day/week</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB4\"><label>Table 4</label><caption><title>Outcome measures.</title><p>MACS: Manual ability classification system; FIM: Functional independence measures; ACS-C: Attentional control scale.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Sr. Number</td><td rowspan=\"1\" colspan=\"1\">Pediatrics scale</td><td rowspan=\"1\" colspan=\"1\">Before treatment</td><td rowspan=\"1\" colspan=\"1\">After Treatment</td></tr><tr><td rowspan=\"1\" colspan=\"1\">1)</td><td rowspan=\"1\" colspan=\"1\">MACS Scale </td><td rowspan=\"1\" colspan=\"1\">Level 3</td><td rowspan=\"1\" colspan=\"1\">Level 1</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">2)</td><td rowspan=\"1\" colspan=\"1\">WeeFIM Scale</td><td rowspan=\"1\" colspan=\"1\">Score 1</td><td rowspan=\"1\" colspan=\"1\">Score 5</td></tr><tr><td rowspan=\"1\" colspan=\"1\">3)</td><td rowspan=\"1\" colspan=\"1\">Conners Rating Scale</td><td rowspan=\"1\" colspan=\"1\">Score 4</td><td rowspan=\"1\" colspan=\"1\">Score 1</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">4)</td><td rowspan=\"1\" colspan=\"1\">Attentional Control Scale</td><td rowspan=\"1\" colspan=\"1\">Score 1</td><td rowspan=\"1\" colspan=\"1\">Score 4</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB1\\\"><label>Table 1</label><caption><title>Physical examination findings.</title><p>cm: Centimeter; kg: Kilogram.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Anthropometric measures</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">At birth</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">At present</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Length/Height (in cm)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 cm</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">94cm</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Weight (in kg)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.40 kg</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">13.5 kg</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Head circumference (in cm)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">35 cm</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">46 cm</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Chest circumference (in cm)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">30-33 cm</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">49 cm</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB2\\\"><label>Table 2</label><caption><title>BERA test findings.</title><p>The data has been represented as N.</p><p>EP: Evoked Potentials; SPL: Sound Pressure Level; dB: Decibel; ms: Millisecond; Rec: Recorded; Cz-M: Vertex-Mastoid; R: Right; L: Left; BERA: Brainstem Evoked Response Audiometry; NA: Not Applicable.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">N</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Rec.sites</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">I Lat., ms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Ia Lat., ms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">II Lat., ms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">III Lat., ms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">IIIa Lat., ms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">IV Lat., ms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">V Lat., ms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">I-III Lat., ms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">III-V Lat., ms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">I-V Lat., ms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stim.side</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stimulus</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.43</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.75</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.25</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.15</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.63</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±110 dB SPL</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.53</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.85</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.35</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.85</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.25</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.68</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.08</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.08</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.15</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±100 dB SPL</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.38</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.78</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±90 dB SPL</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.35</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.23</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.55</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.95</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.75</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.9</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.95</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.55</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±80 dB SPL</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.73</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.45</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.75</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.03</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.83</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.03</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.08</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±70 dB SPL</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.83</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.65</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.88</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.95</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.15</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.35</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.95</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.13</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.13</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±60 dB SPL</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.75</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.35</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.68</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.63</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.55</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.88</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.43</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±50 dB SPL</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.93</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.93</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.65</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.88</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.53</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±40 dB SPL</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">9</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.35</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.83</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.68</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.25</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.78</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.05</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.38</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.43</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±110 dB SPL</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.53</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.9</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.35</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.28</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±100 dB SPL</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">11</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.88</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.65</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.83</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.38</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.05</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±90 dB SPL</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.15</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.53</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.93</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.73</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.45</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.13</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.58</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±80 dB SPL</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">13</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.88</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.9</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.33</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.78</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.88</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.48</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±60 dB SPL</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.08</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.05</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.98</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±40 dB SPL</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">15</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cz-M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.88</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.28</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.63</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.08</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.53</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.65</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">±50 dB SPL</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB3\\\"><label>Table 3</label><caption><title>Physiotherapy management for attention deficit and speech impairment.</title><p>ASD: Autism spectrum disorder; AIT: Auditory Integration Training; SIT: Sensory Integration Therapy; PECS: Picture Exchange Communication System; Min: Minute.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Sr. No.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Treatment</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Description</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Duration</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Auditory Integration Training (AIT)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">AIT involves experiencing music that has been filtered and modulated, including varied volume levels and pitches.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 min 1 session per day/week</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Sensory Integration Therapy (SIT)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">SIT aims to enhance brain processing of information, providing a foundation for advanced skill development.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 min 1 session per day/week</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Holding Therapy</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Based on the theory that autism stems from insufficient bonding with the mother.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 min 1 session per day/week</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Facilitated Communication</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">It involves a method in which a skilled facilitator provides assistance to assist a child in using an output device, like a keyboard, typewriter or similar tool to spell.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">15 min 1 session per day/week</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Music Therapy</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">One of the reasons music therapy is believed to be effective for individuals with ASD is because the processes involved in improvisation can potentially aid in the enhancement of interaction and communication skills.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5 min 1 session per day/week</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Picture Exchange Communication System (PECS)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Its main goal is to help children who have autism learn to start conversations by giving a picture to someone they want to communicate with in order to communicate their needs.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 min 1 session per day/week</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Parent-mediated Communication</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">In this approach, the therapist provides training to parents with the goal of enhancing their ability to understand and respond effectively to their child’s communication.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 min 1 session per day/week</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB4\\\"><label>Table 4</label><caption><title>Outcome measures.</title><p>MACS: Manual ability classification system; FIM: Functional independence measures; ACS-C: Attentional control scale.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Sr. Number</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Pediatrics scale</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Before treatment</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">After Treatment</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">MACS Scale </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Level 3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Level 1</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">WeeFIM Scale</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Score 1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Score 5</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Conners Rating Scale</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Score 4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Score 1</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Attentional Control Scale</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Score 1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Score 4</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p><p><bold>Drafting of the manuscript:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p><p><bold>Supervision:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Concept and design:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p><p><bold>Drafting of the manuscript:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p><p><bold>Supervision:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0015-00000050547-i01\" position=\"float\"/>","<graphic xlink:href=\"cureus-0015-00000050547-i02\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0015-00000050547-i01\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0015-00000050547-i02\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"1\"], \"article-title\": [\"Addressing health inequities in the united states: a case report on autism spectrum disorder (ASD) and social determinants of health\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Fox\", \"Maribona\", \"Quintero\", \"Lange\", \"Semidey\"], \"given-names\": [\"KE\", \"AS\", \"J\", \"C\", \"K\"], \"fpage\": [\"0\"], \"volume\": [\"15\"], \"year\": [\"2023\"]}, {\"label\": [\"2\"], \"article-title\": [\"Buspirone in autism spectrum disorder: a systematic review\\r\\n\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Gupta\", \"Gupta\", \"Gandhi\"], \"given-names\": [\"N\", \"M\", \"R\"], \"fpage\": [\"0\"], \"volume\": [\"15\"], \"year\": [\"2023\"]}, {\"label\": [\"3\"], \"article-title\": [\"Where is the evidence? A narrative literature review of the treatment modalities for autism spectrum disorders\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Medavarapu\", \"Marella\", \"Sangem\", \"Kairam\"], \"given-names\": [\"S\", \"LL\", \"A\", \"R\"], \"fpage\": [\"0\"], \"volume\": [\"11\"], \"year\": [\"2019\"]}, {\"label\": [\"4\"], \"article-title\": [\"The prevalence and associated factors of attention deficit hyperactivity disorder among primary school children in Amman, Jordan\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Abbasi\", \"Mazzawi\", \"Abasi\", \"Haj Ali\", \"Alqudah\", \"Al-Taiar\"], \"given-names\": [\"LN\", \"T\", \"L\", \"S\", \"A\", \"H\"], \"fpage\": [\"0\"], \"volume\": [\"15\"], \"year\": [\"2023\"], \"uri\": [\"https://www.cureus.com/articles/149695-the-prevalence-and-associated-factors-of-attention-deficit-hyperactivity-disorder-among-primary-school-children-in-amman-jordan\"]}, {\"label\": [\"5\"], \"article-title\": [\"The physiology of cognition in autism spectrum disorder: current and future challenges\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Al-Mazidi\"], \"given-names\": [\"SH\"], \"fpage\": [\"0\"], \"volume\": [\"15\"], \"year\": [\"2023\"]}, {\"label\": [\"6\"], \"article-title\": [\"Clinical discordance in monozygotic twins with autism spectrum disorder\\r\\n\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Ho\", \"Towheed\", \"Luong\", \"Zucker\", \"Fethke\"], \"given-names\": [\"A\", \"A\", \"S\", \"S\", \"E\"], \"fpage\": [\"0\"], \"volume\": [\"14\"], \"year\": [\"2022\"]}, {\"label\": [\"7\"], \"article-title\": [\"Clinical relevance of methylenetetrahydrofolate reductase genetic testing in autism: a case report of successful clinical outcome\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Fadila\", \"Suman\", \"Kumar\", \"Omair\"], \"given-names\": [\"Fadila\", \"P\", \"P\", \"F\"], \"fpage\": [\"0\"], \"volume\": [\"13\"], \"year\": [\"2021\"]}, {\"label\": [\"8\"], \"article-title\": [\"Do parental comorbidities affect the severity of autism spectrum disorder?\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Aldera\", \"Hilabi\", \"Elzahrani\", \"Alhamadh\", \"Alqirnas\", \"Alkahtani\", \"Masuadi\"], \"given-names\": [\"H\", \"A\", \"MR\", \"MS\", \"MQ\", \"R\", \"E\"], \"fpage\": [\"0\"], \"volume\": [\"14\"], \"year\": [\"2022\"]}, {\"label\": [\"9\"], \"article-title\": [\"A comparison of prenatal exposures in children with and without a diagnosis of autism spectrum disorder\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Saunders\", \"Woodland\", \"Gander\"], \"given-names\": [\"A\", \"J\", \"S\"], \"fpage\": [\"0\"], \"volume\": [\"11\"], \"year\": [\"2019\"]}, {\"label\": [\"11\"], \"article-title\": [\"Efficacy of siddha therapeutics on Mantha Sanni (autism spectrum disorder) among pediatric patients: an interventional non-randomized open-label clinical trial\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Elangovan\", \"Ramasamy\", \"Sundaram\", \"Ramasamy\"], \"given-names\": [\"P\", \"G\", \"M\", \"M\"], \"fpage\": [\"0\"], \"volume\": [\"15\"], \"year\": [\"2023\"]}, {\"label\": [\"12\"], \"article-title\": [\"A pediatric patient with autism spectrum disorder and comorbid compulsive behaviors treated with robot-assisted relaxation: a case report\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Nikopoulou\", \"Holeva\", \"Tatsiopoulou\", \"Kaburlasos\", \"Evangeliou\"], \"given-names\": [\"VA\", \"V\", \"P\", \"VG\", \"AE\"], \"fpage\": [\"0\"], \"volume\": [\"14\"], \"year\": [\"2022\"]}, {\"label\": [\"20\"], \"article-title\": [\"Effectiveness of physiotherapy techniques in children with attention deficit disorder/hyperactivity\"], \"source\": [\"Int Arch Med\"], \"person-group\": [\"\\n\"], \"surname\": [\"Daltro\", \"Silva\", \"Dantas\"], \"given-names\": [\"M\", \"J\", \"A\"], \"fpage\": [\"1\"], \"lpage\": [\"6\"], \"volume\": [\"9\"], \"year\": [\"2016\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":20,"string":"20"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50547"},"package_file":{"kind":"string","value":"oa_package/f8/cf/PMC10787849.tar.gz"}}},{"rowIdx":157,"cells":{"accession_id":{"kind":"string","value":"PMC10787871"},"pmid":{"kind":"string","value":"38221988"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Road accidents are a significant public health concern worldwide, with an estimated 1.35 million deaths caused by road traffic accidents each year.\n<sup>\n##UREF##0##1##\n</sup> Developing countries, such as India, are disproportionately affected, with over 150,000 fatalities reported annually.\n<sup>\n##UREF##0##1##\n</sup> Road safety is a major concern in India, with a large number of accidents and fatalities reported each year. According to the Ministry of Road Transport and Highways, there were 449,002 road accidents in India in 2019, resulting in 151,113 deaths and 451,361 injuries.\n<sup>\n##UREF##1##2##\n</sup>\n</p>","<p>Road accidents pose a threat to health, leading to approximately 1.35 million deaths globally each year. Countries such as India are particularly affected, experiencing over 150,000 fatalities annually. This alarming situation emphasizes the importance of comprehending the factors that contribute to these accidents in order to develop prevention measures. Our research is motivated by the urgency to improve road safety in high-risk areas, like India, where the number of accidents and fatalities remains distressingly high.</p>","<p>Our research delves into the modeling of accident severity, a statistical technique in the field of road safety. Traditional statistical models like logit and probit have been used since the 1990s to predict traffic accident severity, but they have limitations, especially when their underlying assumptions are violated.\n<sup>\n##UREF##2##3##\n</sup>\n<sup>,</sup>\n<sup>\n##REF##17920851##4##\n</sup> On the other hand, Artificial Intelligence (AI) models offer adaptability and can handle complex nonlinear relationships without being constrained by such assumptions. We focus on the Random Forest (RF) algorithm, an advanced machine learning model with distinct advantages over other algorithms for predicting accident severity.\n<sup>\n##UREF##3##5##\n</sup> Through tuning parameters and preprocessing techniques, we aim to improve the performance of RF even further, making our academic contribution significant in the pursuit of more accurate and reliable predictions for accident severity.</p>","<p>The modelling process involves analyzing data on past accidents and identifying the factors that contributed to their occurrence and severity.\n<sup>\n##UREF##4##6##\n</sup> These factors can include road conditions, weather, driver behaviour, and vehicle type, among others. The goal of accident severity modelling is to identify the most important factors contributing to accidents and to develop evidence-based strategies to improve road safety and reduce the number and severity of accidents.\n<sup>\n##UREF##5##7##\n</sup>\n<sup>–</sup>\n<sup>\n##UREF##7##9##\n</sup>\n</p>","<p>Statistical models have been widely used for predicting traffic accidents’ severity.\n<sup>\n##UREF##2##3##\n</sup>\n<sup>,</sup>\n<sup>\n##REF##17920851##4##\n</sup> Artificial intelligence models, in contrast, do not make any assumptions and are more adaptable. They can handle intricate nonlinear relationships and generally offer higher predictive accuracy than statistical approaches.\n<sup>\n##UREF##3##5##\n</sup> RF, in particular, has been successfully applied in various contexts, and its performance can be further improved by tuning key parameters and careful data preprocessing.\n<sup>\n##REF##17920851##4##\n</sup>\n<sup>,</sup>\n<sup>\n##UREF##3##5##\n</sup>\n<sup>,</sup>\n<sup>\n##REF##14733994##10##\n</sup>\n<sup>,</sup>\n<sup>\n##UREF##8##11##\n</sup> The performance of the RF algorithm is significantly influenced by the selection of hyperparameters.\n<sup>\n##UREF##9##12##\n</sup> To optimize its performance, identifying the optimal parameter values is crucial.</p>","<p>The main objective of our study is to develop a predictive model for the severity of traffic accidents on Indian highways using RF models due to their accuracy and interpretability.</p>","<p>The findings of our study will be used to develop a predictive model for accident severity that can inform road safety policies and interventions. This model can be used to identify high-risk areas and prioritize resources for accident prevention and mitigation.</p>","<title>Study Areas</title>","<p>The study areas selected are the National Highways two stretches as mentioned below\n<list list-type=\"simple\"><list-item><label>1.</label><p>Pune-Sholapur Section of NH-9 in km.144/400 to Km. 249/000 in the State of Maharashtra (\n##FIG##0##Figure 1##).</p></list-item><list-item><label>2.</label><p>Six-Laning of Barwa-Adda-Panagarh Section of NH-2 from km 398.240 to km 521.120 including Panagarh Bypass in the States of Jharkhand and West Bengal (\n##FIG##1##Figure 2##).</p></list-item></list>\n</p>","<p>The study areas for this research project were selected based on specific criteria. Firstly, the researchers had prior experience of working on one of the stretches, which is the Pune-Sholapur Section of NH-9 in km.144/400 to Km. 249/000 in the State of Maharashtra. This experience could have provided insights and knowledge that could be useful in conducting the study.</p>","<p>Additionally, data was also provided by the same concessionaire as of the previous stretch on request for another stretch, which is the Six-Laning of Barwa-Adda-Panagarh Section of NH-2 from km 398.240 to km 521.120 including Panagarh Bypass in the States of and West Bengal. This data could have been relevant to the research objectives and could have assisted in achieving the desired outcomes.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Road accidents are a significant public health concern worldwide, with an estimated 1.35 million deaths caused by road traffic accidents each year.\\n<sup>\\n##UREF##0##1##\\n</sup> Developing countries, such as India, are disproportionately affected, with over 150,000 fatalities reported annually.\\n<sup>\\n##UREF##0##1##\\n</sup> Road safety is a major concern in India, with a large number of accidents and fatalities reported each year. According to the Ministry of Road Transport and Highways, there were 449,002 road accidents in India in 2019, resulting in 151,113 deaths and 451,361 injuries.\\n<sup>\\n##UREF##1##2##\\n</sup>\\n</p>\",\n \"<p>Road accidents pose a threat to health, leading to approximately 1.35 million deaths globally each year. Countries such as India are particularly affected, experiencing over 150,000 fatalities annually. This alarming situation emphasizes the importance of comprehending the factors that contribute to these accidents in order to develop prevention measures. Our research is motivated by the urgency to improve road safety in high-risk areas, like India, where the number of accidents and fatalities remains distressingly high.</p>\",\n \"<p>Our research delves into the modeling of accident severity, a statistical technique in the field of road safety. Traditional statistical models like logit and probit have been used since the 1990s to predict traffic accident severity, but they have limitations, especially when their underlying assumptions are violated.\\n<sup>\\n##UREF##2##3##\\n</sup>\\n<sup>,</sup>\\n<sup>\\n##REF##17920851##4##\\n</sup> On the other hand, Artificial Intelligence (AI) models offer adaptability and can handle complex nonlinear relationships without being constrained by such assumptions. We focus on the Random Forest (RF) algorithm, an advanced machine learning model with distinct advantages over other algorithms for predicting accident severity.\\n<sup>\\n##UREF##3##5##\\n</sup> Through tuning parameters and preprocessing techniques, we aim to improve the performance of RF even further, making our academic contribution significant in the pursuit of more accurate and reliable predictions for accident severity.</p>\",\n \"<p>The modelling process involves analyzing data on past accidents and identifying the factors that contributed to their occurrence and severity.\\n<sup>\\n##UREF##4##6##\\n</sup> These factors can include road conditions, weather, driver behaviour, and vehicle type, among others. The goal of accident severity modelling is to identify the most important factors contributing to accidents and to develop evidence-based strategies to improve road safety and reduce the number and severity of accidents.\\n<sup>\\n##UREF##5##7##\\n</sup>\\n<sup>–</sup>\\n<sup>\\n##UREF##7##9##\\n</sup>\\n</p>\",\n \"<p>Statistical models have been widely used for predicting traffic accidents’ severity.\\n<sup>\\n##UREF##2##3##\\n</sup>\\n<sup>,</sup>\\n<sup>\\n##REF##17920851##4##\\n</sup> Artificial intelligence models, in contrast, do not make any assumptions and are more adaptable. They can handle intricate nonlinear relationships and generally offer higher predictive accuracy than statistical approaches.\\n<sup>\\n##UREF##3##5##\\n</sup> RF, in particular, has been successfully applied in various contexts, and its performance can be further improved by tuning key parameters and careful data preprocessing.\\n<sup>\\n##REF##17920851##4##\\n</sup>\\n<sup>,</sup>\\n<sup>\\n##UREF##3##5##\\n</sup>\\n<sup>,</sup>\\n<sup>\\n##REF##14733994##10##\\n</sup>\\n<sup>,</sup>\\n<sup>\\n##UREF##8##11##\\n</sup> The performance of the RF algorithm is significantly influenced by the selection of hyperparameters.\\n<sup>\\n##UREF##9##12##\\n</sup> To optimize its performance, identifying the optimal parameter values is crucial.</p>\",\n \"<p>The main objective of our study is to develop a predictive model for the severity of traffic accidents on Indian highways using RF models due to their accuracy and interpretability.</p>\",\n \"<p>The findings of our study will be used to develop a predictive model for accident severity that can inform road safety policies and interventions. This model can be used to identify high-risk areas and prioritize resources for accident prevention and mitigation.</p>\",\n \"<title>Study Areas</title>\",\n \"<p>The study areas selected are the National Highways two stretches as mentioned below\\n<list list-type=\\\"simple\\\"><list-item><label>1.</label><p>Pune-Sholapur Section of NH-9 in km.144/400 to Km. 249/000 in the State of Maharashtra (\\n##FIG##0##Figure 1##).</p></list-item><list-item><label>2.</label><p>Six-Laning of Barwa-Adda-Panagarh Section of NH-2 from km 398.240 to km 521.120 including Panagarh Bypass in the States of Jharkhand and West Bengal (\\n##FIG##1##Figure 2##).</p></list-item></list>\\n</p>\",\n \"<p>The study areas for this research project were selected based on specific criteria. Firstly, the researchers had prior experience of working on one of the stretches, which is the Pune-Sholapur Section of NH-9 in km.144/400 to Km. 249/000 in the State of Maharashtra. This experience could have provided insights and knowledge that could be useful in conducting the study.</p>\",\n \"<p>Additionally, data was also provided by the same concessionaire as of the previous stretch on request for another stretch, which is the Six-Laning of Barwa-Adda-Panagarh Section of NH-2 from km 398.240 to km 521.120 including Panagarh Bypass in the States of and West Bengal. This data could have been relevant to the research objectives and could have assisted in achieving the desired outcomes.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<p>The proposed methodology for this research involves the following steps for implementing a RF model machine learning technique for accident severity prediction.</p>","<p>Data Preparation: The first step in implementing a RF model for accident severity prediction is to collect and prepare data. Raw data on road accidents for the selected stretches of the highway can be obtained from secondary sources such as the Ministry of Road Transport and Highways (MoRTH) and the National Highways Authority of India (NHAI).\n<sup>\n##UREF##1##2##\n</sup> Data wrangling and mining techniques can be used to clean and preprocess the data.</p>","<p>Feature Selection: Once the data is prepared, selecting appropriate features for the model becomes crucial. Feature selection plays a vital role in reducing the dimensionality of the data and enhancing the model’s accuracy. There are several techniques available for feature selection, such as statistical tests, correlation analysis, and principal component analysis (PCA).\n<sup>\n##UREF##10##13##\n</sup>\n</p>","<p>Model Training: In the next step, an RF model can be trained on the preprocessed data. The model can be developed using a machine learning-based framework, as described in Breiman’s work on RF.\n<sup>\n##UREF##11##14##\n</sup> The RF algorithm involves bagging and random feature selection techniques to create multiple decision trees that are aggregated to form a stronger learner.\n<sup>\n##UREF##12##15##\n</sup>\n</p>","<p>RF Algorithm Formulation: The RF algorithm can be represented as:\nwhere X are the input features, B is the number of trees, and T\n<sub>b(X)</sub> is the prediction of the b-th individual decision tree.</p>","<p>Parameter Tuning: To improve the performance of a RF model, it is important to fine-tune its parameters. The three key parameters that significantly impact the tuning performance of the RF model are the total number of trees (n_estimators), the number of features used for each node segmentation (max_feature), and the maximum depth of a tree (max_depth).\n<sup>\n##REF##20159099##16##\n</sup>\n</p>","<p>In the construction of the RF model for predicting accident severity, Gini impurity is employed as a criterion to evaluate the significance of different explanatory variables. Gini impurity, a measure utilized within the framework of decision trees (the base learners in a RF), is crucial for the optimal selection of features at each node split. It offers a quantitative metric to discern the effectiveness of a variable in segregating the target classes.</p>","<p>Mechanism of Gini Impurity: In the context of binary classification, the Gini impurity for a node is calculated as:\nwhere,\n<italic toggle=\"yes\">pk</italic> is the proportion of samples classified to class\n<italic toggle=\"yes\">k</italic> at that node, and the summation operates over all classes. A lower Gini impurity score suggests a higher purity of the node, indicating an enhanced classification.</p>","<p>Gini Importance in RF: In the developed RF model, the Gini impurity plays a dual role:\n<bold>Node Splitting</bold>: It aids in the identification of the most significant variable at each node by evaluating the potential reduction in impurity for each split, and\n<bold>Feature Importance</bold>: Post model training, the average decrease in impurity caused by each feature across all trees is computed, known as Gini importance. This metric offers insights into the relative significance of different features for the prediction task.</p>","<p>Model Evaluation: After training the RF model and optimizing its parameters, it is important to evaluate the model’s performance. Various evaluation metrics can be used, including accuracy, precision, recall, F1 score, and Area Under the Curve - Receiver Operating Characteristics (AUC-ROC) curve.\n<sup>\n##UREF##13##17##\n</sup>\n</p>","<p>Model Implementation: Once the model has been trained and evaluated, it can be deployed for accident severity prediction. The methodology can be designed using Python for building the model and forecasting the severity of road traffic accidents on Indian highways.</p>","<title>Source data</title>","<p>Data on road accidents from selected stretches of highways was obtained from the Concessionaires of the National Highways Authority of India (NHAI) for two projects: Pune-Solapur and Bengal (BAEL) Section. For the Pune-Solapur Section of NH-9, which is located between km. 144/400 and km. 249/000 in the state of Maharashtra, accident dates from 2013 to 2018 were used. For the Six-Laning of Barwa-Adda-Panagarh Section of NH-2, which includes Panagarh Bypass and is located in the States of Jharkhand and West Bengal Stretch, accident dates from 2015 to 2019 were used for the stretch between km 398.240 and km 521.120. The raw data was subject to exploratory data analysis, as detailed in the following section.</p>","<title>Data Preparation</title>","<p>In this stage, data gathering and exploration is performed using secondary source data. The dataset consists of 3257 observations out of which the 1855 observations are of Bengal (BAEL) Section and 1402 observations are of Pune- Solapur and 32 variables, including the target variable “accident severity.” The 32 attributes and their corresponding mappings are presented in\n##TAB##0##Table 1##.</p>","<title>Data Modelling</title>","<p>The RF classification algorithm has been employed in this study to forecast the severity of road traffic accidents in India. This section details the procedure for implementing the model, performance evaluation, and discuss the results obtained. The RF algorithm is written using python programming language.</p>","<p>The target variable for the random RF is selected as the’Accident Severity’ which has classes as Fatal, Grevious Injury, Minor Injury and No Injury and indexed as 1-Fatal, 2-Grevious Injury, 3-Minor Injury, 4-No Injury.</p>","<p>The dataset is partitioned into training and testing sets with a ratio of 80% and 20%, respectively. The hyperparameters’n_estimators’ and’max_depth’ are specified, and a grid search is conducted with cross-validation (cv=5) to identify the optimal hyperparameters. The best parameters and scores are obtained. The best estimator is fit on the training data. Predictions are made on the test data and the accuracy of the model is obtained.</p>","<p>The algorithm and programme for Accident Severity Modelling using RF are written in the Python programming language, and the code is made available to the public for further development. The source code can be\n<ext-link xlink:href=\"http://accessed\" ext-link-type=\"uri\">accessed</ext-link> via the software availability statement.</p>","<p>Accuracy analysis on test data: Three metrics were employed to evaluate the effectiveness of the algorithms: accuracy, precision, and recall. These metrics are defined as follows:</p>","<p>Accuracy: The formula for a metric that measures the proportion of correctly predicted observations to the total number of observations is represented as:\n\n</p>","<p>Precision is a metric that indicates the ratio of correctly predicted positive observations to the total number of predicted positive observations, and is calculated using the formula:\n\n</p>","<p>Recall is a metric that reflects the ratio of correctly predicted positive observations to the total number of actual positive observations, and is determined using the formula:\n\n</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<p>The proposed methodology for this research involves the following steps for implementing a RF model machine learning technique for accident severity prediction.</p>\",\n \"<p>Data Preparation: The first step in implementing a RF model for accident severity prediction is to collect and prepare data. Raw data on road accidents for the selected stretches of the highway can be obtained from secondary sources such as the Ministry of Road Transport and Highways (MoRTH) and the National Highways Authority of India (NHAI).\\n<sup>\\n##UREF##1##2##\\n</sup> Data wrangling and mining techniques can be used to clean and preprocess the data.</p>\",\n \"<p>Feature Selection: Once the data is prepared, selecting appropriate features for the model becomes crucial. Feature selection plays a vital role in reducing the dimensionality of the data and enhancing the model’s accuracy. There are several techniques available for feature selection, such as statistical tests, correlation analysis, and principal component analysis (PCA).\\n<sup>\\n##UREF##10##13##\\n</sup>\\n</p>\",\n \"<p>Model Training: In the next step, an RF model can be trained on the preprocessed data. The model can be developed using a machine learning-based framework, as described in Breiman’s work on RF.\\n<sup>\\n##UREF##11##14##\\n</sup> The RF algorithm involves bagging and random feature selection techniques to create multiple decision trees that are aggregated to form a stronger learner.\\n<sup>\\n##UREF##12##15##\\n</sup>\\n</p>\",\n \"<p>RF Algorithm Formulation: The RF algorithm can be represented as:\\nwhere X are the input features, B is the number of trees, and T\\n<sub>b(X)</sub> is the prediction of the b-th individual decision tree.</p>\",\n \"<p>Parameter Tuning: To improve the performance of a RF model, it is important to fine-tune its parameters. The three key parameters that significantly impact the tuning performance of the RF model are the total number of trees (n_estimators), the number of features used for each node segmentation (max_feature), and the maximum depth of a tree (max_depth).\\n<sup>\\n##REF##20159099##16##\\n</sup>\\n</p>\",\n \"<p>In the construction of the RF model for predicting accident severity, Gini impurity is employed as a criterion to evaluate the significance of different explanatory variables. Gini impurity, a measure utilized within the framework of decision trees (the base learners in a RF), is crucial for the optimal selection of features at each node split. It offers a quantitative metric to discern the effectiveness of a variable in segregating the target classes.</p>\",\n \"<p>Mechanism of Gini Impurity: In the context of binary classification, the Gini impurity for a node is calculated as:\\nwhere,\\n<italic toggle=\\\"yes\\\">pk</italic> is the proportion of samples classified to class\\n<italic toggle=\\\"yes\\\">k</italic> at that node, and the summation operates over all classes. A lower Gini impurity score suggests a higher purity of the node, indicating an enhanced classification.</p>\",\n \"<p>Gini Importance in RF: In the developed RF model, the Gini impurity plays a dual role:\\n<bold>Node Splitting</bold>: It aids in the identification of the most significant variable at each node by evaluating the potential reduction in impurity for each split, and\\n<bold>Feature Importance</bold>: Post model training, the average decrease in impurity caused by each feature across all trees is computed, known as Gini importance. This metric offers insights into the relative significance of different features for the prediction task.</p>\",\n \"<p>Model Evaluation: After training the RF model and optimizing its parameters, it is important to evaluate the model’s performance. Various evaluation metrics can be used, including accuracy, precision, recall, F1 score, and Area Under the Curve - Receiver Operating Characteristics (AUC-ROC) curve.\\n<sup>\\n##UREF##13##17##\\n</sup>\\n</p>\",\n \"<p>Model Implementation: Once the model has been trained and evaluated, it can be deployed for accident severity prediction. The methodology can be designed using Python for building the model and forecasting the severity of road traffic accidents on Indian highways.</p>\",\n \"<title>Source data</title>\",\n \"<p>Data on road accidents from selected stretches of highways was obtained from the Concessionaires of the National Highways Authority of India (NHAI) for two projects: Pune-Solapur and Bengal (BAEL) Section. For the Pune-Solapur Section of NH-9, which is located between km. 144/400 and km. 249/000 in the state of Maharashtra, accident dates from 2013 to 2018 were used. For the Six-Laning of Barwa-Adda-Panagarh Section of NH-2, which includes Panagarh Bypass and is located in the States of Jharkhand and West Bengal Stretch, accident dates from 2015 to 2019 were used for the stretch between km 398.240 and km 521.120. The raw data was subject to exploratory data analysis, as detailed in the following section.</p>\",\n \"<title>Data Preparation</title>\",\n \"<p>In this stage, data gathering and exploration is performed using secondary source data. The dataset consists of 3257 observations out of which the 1855 observations are of Bengal (BAEL) Section and 1402 observations are of Pune- Solapur and 32 variables, including the target variable “accident severity.” The 32 attributes and their corresponding mappings are presented in\\n##TAB##0##Table 1##.</p>\",\n \"<title>Data Modelling</title>\",\n \"<p>The RF classification algorithm has been employed in this study to forecast the severity of road traffic accidents in India. This section details the procedure for implementing the model, performance evaluation, and discuss the results obtained. The RF algorithm is written using python programming language.</p>\",\n \"<p>The target variable for the random RF is selected as the’Accident Severity’ which has classes as Fatal, Grevious Injury, Minor Injury and No Injury and indexed as 1-Fatal, 2-Grevious Injury, 3-Minor Injury, 4-No Injury.</p>\",\n \"<p>The dataset is partitioned into training and testing sets with a ratio of 80% and 20%, respectively. The hyperparameters’n_estimators’ and’max_depth’ are specified, and a grid search is conducted with cross-validation (cv=5) to identify the optimal hyperparameters. The best parameters and scores are obtained. The best estimator is fit on the training data. Predictions are made on the test data and the accuracy of the model is obtained.</p>\",\n \"<p>The algorithm and programme for Accident Severity Modelling using RF are written in the Python programming language, and the code is made available to the public for further development. The source code can be\\n<ext-link xlink:href=\\\"http://accessed\\\" ext-link-type=\\\"uri\\\">accessed</ext-link> via the software availability statement.</p>\",\n \"<p>Accuracy analysis on test data: Three metrics were employed to evaluate the effectiveness of the algorithms: accuracy, precision, and recall. These metrics are defined as follows:</p>\",\n \"<p>Accuracy: The formula for a metric that measures the proportion of correctly predicted observations to the total number of observations is represented as:\\n\\n</p>\",\n \"<p>Precision is a metric that indicates the ratio of correctly predicted positive observations to the total number of predicted positive observations, and is calculated using the formula:\\n\\n</p>\",\n \"<p>Recall is a metric that reflects the ratio of correctly predicted positive observations to the total number of actual positive observations, and is determined using the formula:\\n\\n</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Result and Discussion</title>","<title>Model Performance</title>","<p>The classification model used three hyperparameters -’max_depth’: 10,’max_features’:’sqrt’, and’n_estimators’: 100, and the results generated a confusion matrix for the training set. The matrix indicated the number of correctly and incorrectly classified instances for each class. The classification report provided precision, recall, and f1-score for each class, along with support. The model showed high precision and recall for class 1 but low precision and recall for classes 2, 3, and 4, with an overall accuracy of 67% and a weighted average f1-score of 0.64 on the training set. The macro average f1-score, which assigns equal weight to each class, was 0.53.</p>","<p>The optimal parameters for a RF classifier model were determined through a grid search, with a max depth of 2, n estimators of 5000, and a random state of 0. The model was then applied to the test data, and the predictions were saved in an Excel file called “predicted output3.xls” for further analysis. The accuracy of the model on the test data was determined to be 0.4147, or approximately 41.47%, indicating that it accurately predicted the severity of traffic accidents in about 41.47% of test cases.</p>","<p>\n<bold>Predicted outputs</bold>\n</p>","<p>\n<bold>Comparative analysis of observed and predicted accident severity index against dates</bold>\n</p>","<p>The actual accident severity indices are represented by the observed values, while the predicted values are generated by the RF model using the input features.</p>","<p>The following is a summary (\n##FIG##2##Figure 3##) of the comparison between the observed and predicted values:</p>","<p>On dates such as 25-02-2017, 17-04-2017, and 22-04-2017, the RF model accurately predicts the accident severity index.</p>","<p>In a number of instances, the model predicts a lower accident severity index value than the observed value. 18-02-2017, 23-02-2017, and 27-03-2017, for example.</p>","<p>Occasionally, the model overestimates the accident severity index by predicting a higher value than the observed value, as on 24-05-2017 and 20-10-2017.</p>","<p>In general, the model frequently predicts a severity index of 2 for accidents, even when the observed values are distinct. This may indicate a bias in the model, possibly as a result of an imbalance in the training dataset, in which severity index 2 occurs more frequently than other categories.</p>","<p>\n<bold>Comparative analysis of observed and predicted accident severity index against time</bold>\n</p>","<p>\n##FIG##3##Figure 4## displays the date, day of the week, and time of the accident, as well as the observed and predicted accident severity indices. The plotted for the 165 rows of predicted data doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availbility [i].</p>","<p>The dataset contains accident data from February 18, 2017 to December 31, 2017, as determined by Tableau analysis of the plot generated from the provided Excel table.</p>","<p>The observed accident severity index ranges from 1 to 4, where 1 corresponds to the least severe accident and 4 to the most severe accident.</p>","<p>The observed severity index for the vast majority of accidents in the dataset is 3, followed by 4. 2 indicates a less severe accident, while 4 indicates a more severe accident.</p>","<p>The majority of accidents within the dataset have a predicted severity index of 2, followed by an index of 1.</p>","<p>The analysis of the scatter plot reveals that the predicted severity index is typically lower than the observed severity index. This suggests that the model used to predict the severity of accidents is not always accurate and could be improved.</p>","<title>Comparative analysis of observed and predicted accident severity index against Location and Chainages- RHS</title>","<p>The Tableau plot (\n##FIG##4##Figure 5##) presents a detailed visual analysis of accident data on the right-hand side of the road. The data is organized by date and day of the week, displaying the accident location, observed accident severity index, and predicted accident severity index for each incident. The plot effectively illustrates the spatial distribution of accidents and their severity over time, enabling the identification of patterns and trends. The Tableau plot doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availability [ii].</p>","<p>It is evident from the analysis that the majority of accidents have an observed severity index of 2 or 3, indicating a moderate severity. However, the predicted accident severity index largely remains at 2, indicating that the predictions may be somewhat conservative and do not fully capture the observed severity range.</p>","<p>In addition, there appears to be no correlation between the day of the week and the frequency or severity of accidents across the different days of the week. This may suggest that external factors, such as traffic patterns or weather conditions, have a greater impact on the occurrence and severity of accidents than the day of the week.</p>","<title>Comparative analysis of observed and predicted accident severity index against Location and Chainages- LHS</title>","<p>The graph displays (\n##FIG##5##Figure 6##) the date, day of the week, and accident location on Left Hand Side (LHS) of the road, as well as the observed and predicted accident severity indices. The plotted of predicted data doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availability [iii].</p>","<p>The scatterplot reveals that the majority of accidents on the left side of the road had a severity index of 2 or 3, with only a few instances of severity index 1 and 4. This indicates that the majority of collisions on the left side of the road were of moderate severity.</p>","<p>In the majority of cases, the predicted accident severity index was 2, with only a few instances of values 3 and 4. This suggests that the predictive model may be biased towards predicting less severe accidents.</p>","<p>There was no discernible pattern or trend between the day of the week and the occurrence of accidents. Accidents appeared to occur every day, indicating that the day of the week may not be a significant predictor of accident severity on the left side of the road.</p>","<p>The accident locations, as measured by Accident Location A Chainage km, were scattered along the roadway at various distances. This suggests that there may not be a particular accident hotspot or concentration on the left-hand side of the road.</p>","<title>Data Recording and availability</title>","<p>The recording of road accident data in India must comply with the MoRTH & IRC guidelines, utilizing the Road Accident Recording and Reporting Formats. Despite this, there exists a need for a more advanced data recording system to effectively model road safety. The digital monitoring of road accidents can increase the frequency of data collection and minimize the absence of crucial information. Often, the lack of a system or individual to document the accident leads to the absence of important road accident data. This missing data can be regained through the use of machine learning, thus enhancing the accuracy of road safety modeling.</p>"],"string":"[\n \"<title>Result and Discussion</title>\",\n \"<title>Model Performance</title>\",\n \"<p>The classification model used three hyperparameters -’max_depth’: 10,’max_features’:’sqrt’, and’n_estimators’: 100, and the results generated a confusion matrix for the training set. The matrix indicated the number of correctly and incorrectly classified instances for each class. The classification report provided precision, recall, and f1-score for each class, along with support. The model showed high precision and recall for class 1 but low precision and recall for classes 2, 3, and 4, with an overall accuracy of 67% and a weighted average f1-score of 0.64 on the training set. The macro average f1-score, which assigns equal weight to each class, was 0.53.</p>\",\n \"<p>The optimal parameters for a RF classifier model were determined through a grid search, with a max depth of 2, n estimators of 5000, and a random state of 0. The model was then applied to the test data, and the predictions were saved in an Excel file called “predicted output3.xls” for further analysis. The accuracy of the model on the test data was determined to be 0.4147, or approximately 41.47%, indicating that it accurately predicted the severity of traffic accidents in about 41.47% of test cases.</p>\",\n \"<p>\\n<bold>Predicted outputs</bold>\\n</p>\",\n \"<p>\\n<bold>Comparative analysis of observed and predicted accident severity index against dates</bold>\\n</p>\",\n \"<p>The actual accident severity indices are represented by the observed values, while the predicted values are generated by the RF model using the input features.</p>\",\n \"<p>The following is a summary (\\n##FIG##2##Figure 3##) of the comparison between the observed and predicted values:</p>\",\n \"<p>On dates such as 25-02-2017, 17-04-2017, and 22-04-2017, the RF model accurately predicts the accident severity index.</p>\",\n \"<p>In a number of instances, the model predicts a lower accident severity index value than the observed value. 18-02-2017, 23-02-2017, and 27-03-2017, for example.</p>\",\n \"<p>Occasionally, the model overestimates the accident severity index by predicting a higher value than the observed value, as on 24-05-2017 and 20-10-2017.</p>\",\n \"<p>In general, the model frequently predicts a severity index of 2 for accidents, even when the observed values are distinct. This may indicate a bias in the model, possibly as a result of an imbalance in the training dataset, in which severity index 2 occurs more frequently than other categories.</p>\",\n \"<p>\\n<bold>Comparative analysis of observed and predicted accident severity index against time</bold>\\n</p>\",\n \"<p>\\n##FIG##3##Figure 4## displays the date, day of the week, and time of the accident, as well as the observed and predicted accident severity indices. The plotted for the 165 rows of predicted data doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availbility [i].</p>\",\n \"<p>The dataset contains accident data from February 18, 2017 to December 31, 2017, as determined by Tableau analysis of the plot generated from the provided Excel table.</p>\",\n \"<p>The observed accident severity index ranges from 1 to 4, where 1 corresponds to the least severe accident and 4 to the most severe accident.</p>\",\n \"<p>The observed severity index for the vast majority of accidents in the dataset is 3, followed by 4. 2 indicates a less severe accident, while 4 indicates a more severe accident.</p>\",\n \"<p>The majority of accidents within the dataset have a predicted severity index of 2, followed by an index of 1.</p>\",\n \"<p>The analysis of the scatter plot reveals that the predicted severity index is typically lower than the observed severity index. This suggests that the model used to predict the severity of accidents is not always accurate and could be improved.</p>\",\n \"<title>Comparative analysis of observed and predicted accident severity index against Location and Chainages- RHS</title>\",\n \"<p>The Tableau plot (\\n##FIG##4##Figure 5##) presents a detailed visual analysis of accident data on the right-hand side of the road. The data is organized by date and day of the week, displaying the accident location, observed accident severity index, and predicted accident severity index for each incident. The plot effectively illustrates the spatial distribution of accidents and their severity over time, enabling the identification of patterns and trends. The Tableau plot doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availability [ii].</p>\",\n \"<p>It is evident from the analysis that the majority of accidents have an observed severity index of 2 or 3, indicating a moderate severity. However, the predicted accident severity index largely remains at 2, indicating that the predictions may be somewhat conservative and do not fully capture the observed severity range.</p>\",\n \"<p>In addition, there appears to be no correlation between the day of the week and the frequency or severity of accidents across the different days of the week. This may suggest that external factors, such as traffic patterns or weather conditions, have a greater impact on the occurrence and severity of accidents than the day of the week.</p>\",\n \"<title>Comparative analysis of observed and predicted accident severity index against Location and Chainages- LHS</title>\",\n \"<p>The graph displays (\\n##FIG##5##Figure 6##) the date, day of the week, and accident location on Left Hand Side (LHS) of the road, as well as the observed and predicted accident severity indices. The plotted of predicted data doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availability [iii].</p>\",\n \"<p>The scatterplot reveals that the majority of accidents on the left side of the road had a severity index of 2 or 3, with only a few instances of severity index 1 and 4. This indicates that the majority of collisions on the left side of the road were of moderate severity.</p>\",\n \"<p>In the majority of cases, the predicted accident severity index was 2, with only a few instances of values 3 and 4. This suggests that the predictive model may be biased towards predicting less severe accidents.</p>\",\n \"<p>There was no discernible pattern or trend between the day of the week and the occurrence of accidents. Accidents appeared to occur every day, indicating that the day of the week may not be a significant predictor of accident severity on the left side of the road.</p>\",\n \"<p>The accident locations, as measured by Accident Location A Chainage km, were scattered along the roadway at various distances. This suggests that there may not be a particular accident hotspot or concentration on the left-hand side of the road.</p>\",\n \"<title>Data Recording and availability</title>\",\n \"<p>The recording of road accident data in India must comply with the MoRTH & IRC guidelines, utilizing the Road Accident Recording and Reporting Formats. Despite this, there exists a need for a more advanced data recording system to effectively model road safety. The digital monitoring of road accidents can increase the frequency of data collection and minimize the absence of crucial information. Often, the lack of a system or individual to document the accident leads to the absence of important road accident data. This missing data can be regained through the use of machine learning, thus enhancing the accuracy of road safety modeling.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Result and Discussion</title>","<title>Model Performance</title>","<p>The classification model used three hyperparameters -’max_depth’: 10,’max_features’:’sqrt’, and’n_estimators’: 100, and the results generated a confusion matrix for the training set. The matrix indicated the number of correctly and incorrectly classified instances for each class. The classification report provided precision, recall, and f1-score for each class, along with support. The model showed high precision and recall for class 1 but low precision and recall for classes 2, 3, and 4, with an overall accuracy of 67% and a weighted average f1-score of 0.64 on the training set. The macro average f1-score, which assigns equal weight to each class, was 0.53.</p>","<p>The optimal parameters for a RF classifier model were determined through a grid search, with a max depth of 2, n estimators of 5000, and a random state of 0. The model was then applied to the test data, and the predictions were saved in an Excel file called “predicted output3.xls” for further analysis. The accuracy of the model on the test data was determined to be 0.4147, or approximately 41.47%, indicating that it accurately predicted the severity of traffic accidents in about 41.47% of test cases.</p>","<p>\n<bold>Predicted outputs</bold>\n</p>","<p>\n<bold>Comparative analysis of observed and predicted accident severity index against dates</bold>\n</p>","<p>The actual accident severity indices are represented by the observed values, while the predicted values are generated by the RF model using the input features.</p>","<p>The following is a summary (\n##FIG##2##Figure 3##) of the comparison between the observed and predicted values:</p>","<p>On dates such as 25-02-2017, 17-04-2017, and 22-04-2017, the RF model accurately predicts the accident severity index.</p>","<p>In a number of instances, the model predicts a lower accident severity index value than the observed value. 18-02-2017, 23-02-2017, and 27-03-2017, for example.</p>","<p>Occasionally, the model overestimates the accident severity index by predicting a higher value than the observed value, as on 24-05-2017 and 20-10-2017.</p>","<p>In general, the model frequently predicts a severity index of 2 for accidents, even when the observed values are distinct. This may indicate a bias in the model, possibly as a result of an imbalance in the training dataset, in which severity index 2 occurs more frequently than other categories.</p>","<p>\n<bold>Comparative analysis of observed and predicted accident severity index against time</bold>\n</p>","<p>\n##FIG##3##Figure 4## displays the date, day of the week, and time of the accident, as well as the observed and predicted accident severity indices. The plotted for the 165 rows of predicted data doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availbility [i].</p>","<p>The dataset contains accident data from February 18, 2017 to December 31, 2017, as determined by Tableau analysis of the plot generated from the provided Excel table.</p>","<p>The observed accident severity index ranges from 1 to 4, where 1 corresponds to the least severe accident and 4 to the most severe accident.</p>","<p>The observed severity index for the vast majority of accidents in the dataset is 3, followed by 4. 2 indicates a less severe accident, while 4 indicates a more severe accident.</p>","<p>The majority of accidents within the dataset have a predicted severity index of 2, followed by an index of 1.</p>","<p>The analysis of the scatter plot reveals that the predicted severity index is typically lower than the observed severity index. This suggests that the model used to predict the severity of accidents is not always accurate and could be improved.</p>","<title>Comparative analysis of observed and predicted accident severity index against Location and Chainages- RHS</title>","<p>The Tableau plot (\n##FIG##4##Figure 5##) presents a detailed visual analysis of accident data on the right-hand side of the road. The data is organized by date and day of the week, displaying the accident location, observed accident severity index, and predicted accident severity index for each incident. The plot effectively illustrates the spatial distribution of accidents and their severity over time, enabling the identification of patterns and trends. The Tableau plot doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availability [ii].</p>","<p>It is evident from the analysis that the majority of accidents have an observed severity index of 2 or 3, indicating a moderate severity. However, the predicted accident severity index largely remains at 2, indicating that the predictions may be somewhat conservative and do not fully capture the observed severity range.</p>","<p>In addition, there appears to be no correlation between the day of the week and the frequency or severity of accidents across the different days of the week. This may suggest that external factors, such as traffic patterns or weather conditions, have a greater impact on the occurrence and severity of accidents than the day of the week.</p>","<title>Comparative analysis of observed and predicted accident severity index against Location and Chainages- LHS</title>","<p>The graph displays (\n##FIG##5##Figure 6##) the date, day of the week, and accident location on Left Hand Side (LHS) of the road, as well as the observed and predicted accident severity indices. The plotted of predicted data doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availability [iii].</p>","<p>The scatterplot reveals that the majority of accidents on the left side of the road had a severity index of 2 or 3, with only a few instances of severity index 1 and 4. This indicates that the majority of collisions on the left side of the road were of moderate severity.</p>","<p>In the majority of cases, the predicted accident severity index was 2, with only a few instances of values 3 and 4. This suggests that the predictive model may be biased towards predicting less severe accidents.</p>","<p>There was no discernible pattern or trend between the day of the week and the occurrence of accidents. Accidents appeared to occur every day, indicating that the day of the week may not be a significant predictor of accident severity on the left side of the road.</p>","<p>The accident locations, as measured by Accident Location A Chainage km, were scattered along the roadway at various distances. This suggests that there may not be a particular accident hotspot or concentration on the left-hand side of the road.</p>","<title>Data Recording and availability</title>","<p>The recording of road accident data in India must comply with the MoRTH & IRC guidelines, utilizing the Road Accident Recording and Reporting Formats. Despite this, there exists a need for a more advanced data recording system to effectively model road safety. The digital monitoring of road accidents can increase the frequency of data collection and minimize the absence of crucial information. Often, the lack of a system or individual to document the accident leads to the absence of important road accident data. This missing data can be regained through the use of machine learning, thus enhancing the accuracy of road safety modeling.</p>"],"string":"[\n \"<title>Result and Discussion</title>\",\n \"<title>Model Performance</title>\",\n \"<p>The classification model used three hyperparameters -’max_depth’: 10,’max_features’:’sqrt’, and’n_estimators’: 100, and the results generated a confusion matrix for the training set. The matrix indicated the number of correctly and incorrectly classified instances for each class. The classification report provided precision, recall, and f1-score for each class, along with support. The model showed high precision and recall for class 1 but low precision and recall for classes 2, 3, and 4, with an overall accuracy of 67% and a weighted average f1-score of 0.64 on the training set. The macro average f1-score, which assigns equal weight to each class, was 0.53.</p>\",\n \"<p>The optimal parameters for a RF classifier model were determined through a grid search, with a max depth of 2, n estimators of 5000, and a random state of 0. The model was then applied to the test data, and the predictions were saved in an Excel file called “predicted output3.xls” for further analysis. The accuracy of the model on the test data was determined to be 0.4147, or approximately 41.47%, indicating that it accurately predicted the severity of traffic accidents in about 41.47% of test cases.</p>\",\n \"<p>\\n<bold>Predicted outputs</bold>\\n</p>\",\n \"<p>\\n<bold>Comparative analysis of observed and predicted accident severity index against dates</bold>\\n</p>\",\n \"<p>The actual accident severity indices are represented by the observed values, while the predicted values are generated by the RF model using the input features.</p>\",\n \"<p>The following is a summary (\\n##FIG##2##Figure 3##) of the comparison between the observed and predicted values:</p>\",\n \"<p>On dates such as 25-02-2017, 17-04-2017, and 22-04-2017, the RF model accurately predicts the accident severity index.</p>\",\n \"<p>In a number of instances, the model predicts a lower accident severity index value than the observed value. 18-02-2017, 23-02-2017, and 27-03-2017, for example.</p>\",\n \"<p>Occasionally, the model overestimates the accident severity index by predicting a higher value than the observed value, as on 24-05-2017 and 20-10-2017.</p>\",\n \"<p>In general, the model frequently predicts a severity index of 2 for accidents, even when the observed values are distinct. This may indicate a bias in the model, possibly as a result of an imbalance in the training dataset, in which severity index 2 occurs more frequently than other categories.</p>\",\n \"<p>\\n<bold>Comparative analysis of observed and predicted accident severity index against time</bold>\\n</p>\",\n \"<p>\\n##FIG##3##Figure 4## displays the date, day of the week, and time of the accident, as well as the observed and predicted accident severity indices. The plotted for the 165 rows of predicted data doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availbility [i].</p>\",\n \"<p>The dataset contains accident data from February 18, 2017 to December 31, 2017, as determined by Tableau analysis of the plot generated from the provided Excel table.</p>\",\n \"<p>The observed accident severity index ranges from 1 to 4, where 1 corresponds to the least severe accident and 4 to the most severe accident.</p>\",\n \"<p>The observed severity index for the vast majority of accidents in the dataset is 3, followed by 4. 2 indicates a less severe accident, while 4 indicates a more severe accident.</p>\",\n \"<p>The majority of accidents within the dataset have a predicted severity index of 2, followed by an index of 1.</p>\",\n \"<p>The analysis of the scatter plot reveals that the predicted severity index is typically lower than the observed severity index. This suggests that the model used to predict the severity of accidents is not always accurate and could be improved.</p>\",\n \"<title>Comparative analysis of observed and predicted accident severity index against Location and Chainages- RHS</title>\",\n \"<p>The Tableau plot (\\n##FIG##4##Figure 5##) presents a detailed visual analysis of accident data on the right-hand side of the road. The data is organized by date and day of the week, displaying the accident location, observed accident severity index, and predicted accident severity index for each incident. The plot effectively illustrates the spatial distribution of accidents and their severity over time, enabling the identification of patterns and trends. The Tableau plot doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availability [ii].</p>\",\n \"<p>It is evident from the analysis that the majority of accidents have an observed severity index of 2 or 3, indicating a moderate severity. However, the predicted accident severity index largely remains at 2, indicating that the predictions may be somewhat conservative and do not fully capture the observed severity range.</p>\",\n \"<p>In addition, there appears to be no correlation between the day of the week and the frequency or severity of accidents across the different days of the week. This may suggest that external factors, such as traffic patterns or weather conditions, have a greater impact on the occurrence and severity of accidents than the day of the week.</p>\",\n \"<title>Comparative analysis of observed and predicted accident severity index against Location and Chainages- LHS</title>\",\n \"<p>The graph displays (\\n##FIG##5##Figure 6##) the date, day of the week, and accident location on Left Hand Side (LHS) of the road, as well as the observed and predicted accident severity indices. The plotted of predicted data doesn’t fit in A4 sheet hence the data is published and the link is provided in the Tableau graphs visuals availability [iii].</p>\",\n \"<p>The scatterplot reveals that the majority of accidents on the left side of the road had a severity index of 2 or 3, with only a few instances of severity index 1 and 4. This indicates that the majority of collisions on the left side of the road were of moderate severity.</p>\",\n \"<p>In the majority of cases, the predicted accident severity index was 2, with only a few instances of values 3 and 4. This suggests that the predictive model may be biased towards predicting less severe accidents.</p>\",\n \"<p>There was no discernible pattern or trend between the day of the week and the occurrence of accidents. Accidents appeared to occur every day, indicating that the day of the week may not be a significant predictor of accident severity on the left side of the road.</p>\",\n \"<p>The accident locations, as measured by Accident Location A Chainage km, were scattered along the roadway at various distances. This suggests that there may not be a particular accident hotspot or concentration on the left-hand side of the road.</p>\",\n \"<title>Data Recording and availability</title>\",\n \"<p>The recording of road accident data in India must comply with the MoRTH & IRC guidelines, utilizing the Road Accident Recording and Reporting Formats. Despite this, there exists a need for a more advanced data recording system to effectively model road safety. The digital monitoring of road accidents can increase the frequency of data collection and minimize the absence of crucial information. Often, the lack of a system or individual to document the accident leads to the absence of important road accident data. This missing data can be regained through the use of machine learning, thus enhancing the accuracy of road safety modeling.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p>The RF classifier model predicted the severity of traffic accidents with an overall accuracy of 67% on the training set and approximately 41.47% on the test set. Indicating possible bias or imbalance in the training dataset, the model tended to predict a lower severity index than the observed values. There were no discernible relationships between the day of the week and the occurrence or severity of accidents. The performance of the model can be enhanced by correcting the dataset imbalance and refining the model’s hyperparameters.</p>","<p>The observed and predicted accident severity indices were compared against a number of variables, including dates, times, and locations on both sides of the road. In some instances, the model accurately predicted the accident severity index, but it frequently underestimated accident severity. No discernible patterns or trends were observed in terms of accident location, indicating that external factors may have a greater influence on the occurrence and severity of accidents.</p>","<p>To improve road safety modelling, it is essential to adopt a more sophisticated data recording system consistent with MoRTH and IRC recommendations. Digital monitoring of road accidents can increase the frequency of data collection and reduce the loss of vital information. Integrating machine learning techniques can contribute to more effective interventions and decision-making in the field of traffic accident prevention and mitigation.</p>","<p>In the field of accident severity modeling our research stands out for its contributions. We focus on leveraging Artificial Intelligence (AI) models, which excel at capturing the relationships that traditional statistical methods often overlook. Our, in depth study of the Random Forest (RF) algorithm, combined with careful parameter adjustment and data preprocessing highlights its potential in this area. This research addresses not concerns but also specifically tackles India’s road safety challenges providing insights applicable worldwide as well as tailored solutions for the region. A key aspect of our approach is our unwavering dedication to improving accuracy positioning our work as a standard for precise and reliable accident severity predictions. Overall this study makes a contribution to literature, in this field.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p>The RF classifier model predicted the severity of traffic accidents with an overall accuracy of 67% on the training set and approximately 41.47% on the test set. Indicating possible bias or imbalance in the training dataset, the model tended to predict a lower severity index than the observed values. There were no discernible relationships between the day of the week and the occurrence or severity of accidents. The performance of the model can be enhanced by correcting the dataset imbalance and refining the model’s hyperparameters.</p>\",\n \"<p>The observed and predicted accident severity indices were compared against a number of variables, including dates, times, and locations on both sides of the road. In some instances, the model accurately predicted the accident severity index, but it frequently underestimated accident severity. No discernible patterns or trends were observed in terms of accident location, indicating that external factors may have a greater influence on the occurrence and severity of accidents.</p>\",\n \"<p>To improve road safety modelling, it is essential to adopt a more sophisticated data recording system consistent with MoRTH and IRC recommendations. Digital monitoring of road accidents can increase the frequency of data collection and reduce the loss of vital information. Integrating machine learning techniques can contribute to more effective interventions and decision-making in the field of traffic accident prevention and mitigation.</p>\",\n \"<p>In the field of accident severity modeling our research stands out for its contributions. We focus on leveraging Artificial Intelligence (AI) models, which excel at capturing the relationships that traditional statistical methods often overlook. Our, in depth study of the Random Forest (RF) algorithm, combined with careful parameter adjustment and data preprocessing highlights its potential in this area. This research addresses not concerns but also specifically tackles India’s road safety challenges providing insights applicable worldwide as well as tailored solutions for the region. A key aspect of our approach is our unwavering dedication to improving accuracy positioning our work as a standard for precise and reliable accident severity predictions. Overall this study makes a contribution to literature, in this field.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>No competing interests were disclosed.</p>","<p>\n<bold>Background:</bold> Road accidents claim around 1.35 million lives annually, with countries like India facing a significant impact. In 2019, India reported 449,002 road accidents, causing 151,113 deaths and 451,361 injuries. Accident severity modeling helps understand contributing factors and develop preventive strategies. AI models, such as random forest, offer adaptability and higher predictive accuracy compared to traditional statistical models. This study aims to develop a predictive model for traffic accident severity on Indian highways using the random forest algorithm.</p>","<p>\n<bold>Methods:</bold> A multi-step methodology was employed, involving data collection and preparation, feature selection, training a random forest model, tuning parameters, and evaluating the model using accuracy and F1 score. Data sources included MoRTH and NHAI.</p>","<p>\n<bold>Results:</bold> The classification model had hyperparameters ‘max depth’: 10, ‘max features’: ‘sqrt’, and ‘n estimators’: 100. The model achieved an overall accuracy of 67% and a weighted average F1-score of 0.64 on the training set, with a macro average F1-score of 0.53. Using grid search, a random forest Classifier was fitted with optimal parameters, resulting in 41.47% accuracy on test data.</p>","<p>\n<bold>Conclusions:</bold> The random forest classifier model predicted traffic accident severity with 67% accuracy on the training set and 41.47% on the test set, suggesting possible bias or imbalance in the dataset. No clear patterns were found between the day of the week and accident occurrence or severity. Performance can be improved by addressing dataset imbalance and refining model hyperparameters. The model often underestimated accident severity, highlighting the influence of external factors. Adopting a sophisticated data recording system in line with MoRTH and IRC guidelines and integrating machine learning techniques can enhance road safety modeling, decision-making, and accident prevention efforts.</p>","<title>Amendments from Version 1</title>","<p>We express our gratitude to the reviewers for their constructive comments that greatly enhanced the manuscript's quality. In response to Reviewer-1's insightful observation on the term \"accident\", we have clarified the context and regional usage of the term in the paper, while also acknowledging the global standards. As per suggestions, we've incorporated the Random Forest algorithm formulation in the methods section and discussed the significance of the Gini impurity test in understanding the importance of explanatory variables. Addressing Reviewer-2's feedback, we've emphasized the motivation behind our work and its academic contribution in the Introduction section, ensuring a comprehensive understanding of our study's significance. We revisited and corrected specific literature citations, particularly references 7, 14, and 15. The choice of factors, their contribution, and the transferability of the developed model are now prominently highlighted in a dedicated subsection. Further, to address the concerns about the novelty, we've incorporated a detailed paragraph in the conclusion, elucidating the innovative aspects and unique contributions of our predictive model in comparison to existing literature. We believe these revisions have amplified the clarity, depth, and significance of our research, ensuring alignment with the journal's standards and objectives.</p>"],"string":"[\n \"<p>No competing interests were disclosed.</p>\",\n \"<p>\\n<bold>Background:</bold> Road accidents claim around 1.35 million lives annually, with countries like India facing a significant impact. In 2019, India reported 449,002 road accidents, causing 151,113 deaths and 451,361 injuries. Accident severity modeling helps understand contributing factors and develop preventive strategies. AI models, such as random forest, offer adaptability and higher predictive accuracy compared to traditional statistical models. This study aims to develop a predictive model for traffic accident severity on Indian highways using the random forest algorithm.</p>\",\n \"<p>\\n<bold>Methods:</bold> A multi-step methodology was employed, involving data collection and preparation, feature selection, training a random forest model, tuning parameters, and evaluating the model using accuracy and F1 score. Data sources included MoRTH and NHAI.</p>\",\n \"<p>\\n<bold>Results:</bold> The classification model had hyperparameters ‘max depth’: 10, ‘max features’: ‘sqrt’, and ‘n estimators’: 100. The model achieved an overall accuracy of 67% and a weighted average F1-score of 0.64 on the training set, with a macro average F1-score of 0.53. Using grid search, a random forest Classifier was fitted with optimal parameters, resulting in 41.47% accuracy on test data.</p>\",\n \"<p>\\n<bold>Conclusions:</bold> The random forest classifier model predicted traffic accident severity with 67% accuracy on the training set and 41.47% on the test set, suggesting possible bias or imbalance in the dataset. No clear patterns were found between the day of the week and accident occurrence or severity. Performance can be improved by addressing dataset imbalance and refining model hyperparameters. The model often underestimated accident severity, highlighting the influence of external factors. Adopting a sophisticated data recording system in line with MoRTH and IRC guidelines and integrating machine learning techniques can enhance road safety modeling, decision-making, and accident prevention efforts.</p>\",\n \"<title>Amendments from Version 1</title>\",\n \"<p>We express our gratitude to the reviewers for their constructive comments that greatly enhanced the manuscript's quality. In response to Reviewer-1's insightful observation on the term \\\"accident\\\", we have clarified the context and regional usage of the term in the paper, while also acknowledging the global standards. As per suggestions, we've incorporated the Random Forest algorithm formulation in the methods section and discussed the significance of the Gini impurity test in understanding the importance of explanatory variables. Addressing Reviewer-2's feedback, we've emphasized the motivation behind our work and its academic contribution in the Introduction section, ensuring a comprehensive understanding of our study's significance. We revisited and corrected specific literature citations, particularly references 7, 14, and 15. The choice of factors, their contribution, and the transferability of the developed model are now prominently highlighted in a dedicated subsection. Further, to address the concerns about the novelty, we've incorporated a detailed paragraph in the conclusion, elucidating the innovative aspects and unique contributions of our predictive model in comparison to existing literature. We believe these revisions have amplified the clarity, depth, and significance of our research, ensuring alignment with the journal's standards and objectives.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Future Scope</title>","<p>The study presented provides a good starting point for future research in the field of road safety modeling and accident prevention for Indian highways. However, with the limitations of the present study there opens potential areas for future research as mentioned below which will be taken up in continuation.</p>","<p>Dataset improvement: The study identified the possibility of dataset bias and imbalance affecting model performance. Future research will focus on improving the quality and quantity of data, reducing bias and improving model performance. This will involve exploring alternative data sources, enhancing data collection methods, and addressing data quality issues.</p>","<p>Model improvement: The study used the RF algorithm to develop a predictive model for traffic accident severity. In future research, other machine learning algorithms or ensemble models to improve model performance will be explored. Additionally, refining hyperparameters and addressing dataset imbalance will be done to improve model accuracy.</p>","<p>External factors analysis: The study highlighted the influence of external factors on accident severity prediction. Future research can focus on exploring the impact of external factors such as weather conditions, road infrastructure, and driver behavior on accident severity. This can enhance the accuracy of predictive models and inform decision-making in accident prevention efforts.</p>","<p>Real-time monitoring: The study highlighted the need for a sophisticated data recording system in line with MoRTH and IRC guidelines. Future research can focus on developing a real-time monitoring system that can capture road safety data in real-time and provide insights for accident prevention efforts.</p>"],"string":"[\n \"<title>Future Scope</title>\",\n \"<p>The study presented provides a good starting point for future research in the field of road safety modeling and accident prevention for Indian highways. However, with the limitations of the present study there opens potential areas for future research as mentioned below which will be taken up in continuation.</p>\",\n \"<p>Dataset improvement: The study identified the possibility of dataset bias and imbalance affecting model performance. Future research will focus on improving the quality and quantity of data, reducing bias and improving model performance. This will involve exploring alternative data sources, enhancing data collection methods, and addressing data quality issues.</p>\",\n \"<p>Model improvement: The study used the RF algorithm to develop a predictive model for traffic accident severity. In future research, other machine learning algorithms or ensemble models to improve model performance will be explored. Additionally, refining hyperparameters and addressing dataset imbalance will be done to improve model accuracy.</p>\",\n \"<p>External factors analysis: The study highlighted the influence of external factors on accident severity prediction. Future research can focus on exploring the impact of external factors such as weather conditions, road infrastructure, and driver behavior on accident severity. This can enhance the accuracy of predictive models and inform decision-making in accident prevention efforts.</p>\",\n \"<p>Real-time monitoring: The study highlighted the need for a sophisticated data recording system in line with MoRTH and IRC guidelines. Future research can focus on developing a real-time monitoring system that can capture road safety data in real-time and provide insights for accident prevention efforts.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgement</title>","<p>We are grateful to National Highways Authority of India and IL&FS Engineering and Construction Company for making the raw accident data available.</p>","<title>Data availability</title>","<p>Zenodo. Data for Accident Severity Prediction Modelling for Indian Highways Case Study,\n<ext-link xlink:href=\"https://doi.org/10.5281/zenodo.7773156\" ext-link-type=\"uri\">https://doi.org/10.5281/zenodo.7773156</ext-link>.\n<sup>\n##UREF##14##18##\n</sup>\n</p>","<p>This project contains the following underlying data:\n<list list-type=\"simple\"><list-item><label>•</label><p>\nAccdataset_hk_PS_BAEL_Combined.csv (The dataset consists of 3257 observations out of which the 1855 observations are of Bengal (BAEL) Section and 1402 observations are of Pune- Solapur.)</p></list-item><list-item><label>•</label><p>\npredicted_output_1.xlsx (This is level-2 processed data derived from raw accident data using prediction modeling. The data has been indexed from 1 to 4 for further analysis, and there are a total of 165 rows in the predicted output observations.\n</p></list-item></list>\n</p>","<p>Data are available under the terms of the\n<ext-link xlink:href=\"https://creativecommons.org/licenses/by/4.0/legalcode\" ext-link-type=\"uri\">Creative Commons Attribution 4.0 International license</ext-link> (CC-BY 4.0).</p>","<title>Software availability</title>","<p>\n<list list-type=\"simple\"><list-item><label>•</label><p>Github:\n<ext-link xlink:href=\"https://github.com/humera-k/RF_Accident_Severity\" ext-link-type=\"uri\">https://github.com/humera-k/RF_Accident_Severity\n</ext-link>\n</p></list-item><list-item><label>•</label><p>\n<ext-link xlink:href=\"https://zenodo.org/badge/latestdoi/616376786\" ext-link-type=\"uri\">https://zenodo.org/badge/latestdoi/616376786</ext-link>\n</p></list-item></list>\n</p>","<p>Data are available under the terms of the\n<ext-link xlink:href=\"https://creativecommons.org/licenses/by/4.0/legalcode\" ext-link-type=\"uri\">Creative Commons Attribution 4.0 International license</ext-link> (CC-BY 4.0).</p>","<title>Tableau graphs visuals availability</title>","<p>\n<list list-type=\"simple\"><list-item><label>1.</label><p>\n<ext-link xlink:href=\"https://public.tableau.com/app/profile/humera.khanum/viz/Accidental_Analysis_1/Sheet52\" ext-link-type=\"uri\">\nAccidental_Analysis_1 | Tableau Public</ext-link> (Comparative analysis of observed and predicted accident severity index against time)</p></list-item><list-item><label>2.</label><p>\n<ext-link xlink:href=\"https://public.tableau.com/app/profile/humera.khanum/viz/Accidental_Analysis_1/Sheet3\" ext-link-type=\"uri\">\nAccidental_Analysis_1\n</ext-link> (Comparative analysis of observed and predicted accident severity index against Location and Chainages-Right hand Side (RHS))</p></list-item><list-item><label>3.</label><p>\n<ext-link xlink:href=\"https://public.tableau.com/views/Accidental_Analysis_1/Sheet4?:language=en-US&publish=yes&:display_count=n&:origin=viz_share_link\" ext-link-type=\"uri\">\nAccidental_Analysis_1\n</ext-link> (Comparative analysis of observed and predicted accident severity index against Location and Chainages-Left Hand Side (LHS))\n</p></list-item></list>\n</p>"],"string":"[\n \"<title>Acknowledgement</title>\",\n \"<p>We are grateful to National Highways Authority of India and IL&FS Engineering and Construction Company for making the raw accident data available.</p>\",\n \"<title>Data availability</title>\",\n \"<p>Zenodo. Data for Accident Severity Prediction Modelling for Indian Highways Case Study,\\n<ext-link xlink:href=\\\"https://doi.org/10.5281/zenodo.7773156\\\" ext-link-type=\\\"uri\\\">https://doi.org/10.5281/zenodo.7773156</ext-link>.\\n<sup>\\n##UREF##14##18##\\n</sup>\\n</p>\",\n \"<p>This project contains the following underlying data:\\n<list list-type=\\\"simple\\\"><list-item><label>•</label><p>\\nAccdataset_hk_PS_BAEL_Combined.csv (The dataset consists of 3257 observations out of which the 1855 observations are of Bengal (BAEL) Section and 1402 observations are of Pune- Solapur.)</p></list-item><list-item><label>•</label><p>\\npredicted_output_1.xlsx (This is level-2 processed data derived from raw accident data using prediction modeling. The data has been indexed from 1 to 4 for further analysis, and there are a total of 165 rows in the predicted output observations.\\n</p></list-item></list>\\n</p>\",\n \"<p>Data are available under the terms of the\\n<ext-link xlink:href=\\\"https://creativecommons.org/licenses/by/4.0/legalcode\\\" ext-link-type=\\\"uri\\\">Creative Commons Attribution 4.0 International license</ext-link> (CC-BY 4.0).</p>\",\n \"<title>Software availability</title>\",\n \"<p>\\n<list list-type=\\\"simple\\\"><list-item><label>•</label><p>Github:\\n<ext-link xlink:href=\\\"https://github.com/humera-k/RF_Accident_Severity\\\" ext-link-type=\\\"uri\\\">https://github.com/humera-k/RF_Accident_Severity\\n</ext-link>\\n</p></list-item><list-item><label>•</label><p>\\n<ext-link xlink:href=\\\"https://zenodo.org/badge/latestdoi/616376786\\\" ext-link-type=\\\"uri\\\">https://zenodo.org/badge/latestdoi/616376786</ext-link>\\n</p></list-item></list>\\n</p>\",\n \"<p>Data are available under the terms of the\\n<ext-link xlink:href=\\\"https://creativecommons.org/licenses/by/4.0/legalcode\\\" ext-link-type=\\\"uri\\\">Creative Commons Attribution 4.0 International license</ext-link> (CC-BY 4.0).</p>\",\n \"<title>Tableau graphs visuals availability</title>\",\n \"<p>\\n<list list-type=\\\"simple\\\"><list-item><label>1.</label><p>\\n<ext-link xlink:href=\\\"https://public.tableau.com/app/profile/humera.khanum/viz/Accidental_Analysis_1/Sheet52\\\" ext-link-type=\\\"uri\\\">\\nAccidental_Analysis_1 | Tableau Public</ext-link> (Comparative analysis of observed and predicted accident severity index against time)</p></list-item><list-item><label>2.</label><p>\\n<ext-link xlink:href=\\\"https://public.tableau.com/app/profile/humera.khanum/viz/Accidental_Analysis_1/Sheet3\\\" ext-link-type=\\\"uri\\\">\\nAccidental_Analysis_1\\n</ext-link> (Comparative analysis of observed and predicted accident severity index against Location and Chainages-Right hand Side (RHS))</p></list-item><list-item><label>3.</label><p>\\n<ext-link xlink:href=\\\"https://public.tableau.com/views/Accidental_Analysis_1/Sheet4?:language=en-US&publish=yes&:display_count=n&:origin=viz_share_link\\\" ext-link-type=\\\"uri\\\">\\nAccidental_Analysis_1\\n</ext-link> (Comparative analysis of observed and predicted accident severity index against Location and Chainages-Left Hand Side (LHS))\\n</p></list-item></list>\\n</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"float\" fig-type=\"figure\" id=\"f1\"><label>Figure 1. </label><caption><title>Pune-Sholapur Section of NH-9 in km.144/400 to Km. 249/000 in the State of Maharashtra.</title></caption></fig>","<fig position=\"float\" fig-type=\"figure\" id=\"f2\"><label>Figure 2. </label><caption><title>Six-Laning of Barwa-Adda-Panagarh Section of NH-2 from km 398.240 to km 521.120 including Panagarh Bypass in the States of Jharkhand and West Bengal.</title></caption></fig>","<fig position=\"float\" fig-type=\"figure\" id=\"f3\"><label>Figure 3. </label><caption><title>Comparison between Observed and Predicted Accident Severity Index.</title></caption></fig>","<fig position=\"float\" fig-type=\"figure\" id=\"f4\"><label>Figure 4. </label><caption><title>Comparative analysis of observed and predicted accident severity index against time.</title></caption></fig>","<fig position=\"float\" fig-type=\"figure\" id=\"f5\"><label>Figure 5. </label><caption><title>Comparative analysis of observed and predicted accident severity index against Location and Chainages-RHS.</title></caption></fig>","<fig position=\"float\" fig-type=\"figure\" id=\"f6\"><label>Figure 6. </label><caption><title>Comparative analysis of observed and predicted accident severity index against Location and Chainages-LHS.</title></caption></fig>"],"string":"[\n \"<fig position=\\\"float\\\" fig-type=\\\"figure\\\" id=\\\"f1\\\"><label>Figure 1. </label><caption><title>Pune-Sholapur Section of NH-9 in km.144/400 to Km. 249/000 in the State of Maharashtra.</title></caption></fig>\",\n \"<fig position=\\\"float\\\" fig-type=\\\"figure\\\" id=\\\"f2\\\"><label>Figure 2. </label><caption><title>Six-Laning of Barwa-Adda-Panagarh Section of NH-2 from km 398.240 to km 521.120 including Panagarh Bypass in the States of Jharkhand and West Bengal.</title></caption></fig>\",\n \"<fig position=\\\"float\\\" fig-type=\\\"figure\\\" id=\\\"f3\\\"><label>Figure 3. </label><caption><title>Comparison between Observed and Predicted Accident Severity Index.</title></caption></fig>\",\n \"<fig position=\\\"float\\\" fig-type=\\\"figure\\\" id=\\\"f4\\\"><label>Figure 4. </label><caption><title>Comparative analysis of observed and predicted accident severity index against time.</title></caption></fig>\",\n \"<fig position=\\\"float\\\" fig-type=\\\"figure\\\" id=\\\"f5\\\"><label>Figure 5. </label><caption><title>Comparative analysis of observed and predicted accident severity index against Location and Chainages-RHS.</title></caption></fig>\",\n \"<fig position=\\\"float\\\" fig-type=\\\"figure\\\" id=\\\"f6\\\"><label>Figure 6. </label><caption><title>Comparative analysis of observed and predicted accident severity index against Location and Chainages-LHS.</title></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"T1\"><label>Table 1. </label><caption><title>Dataset Attributes and Parameters Mapping.</title></caption><table frame=\"hsides\" rules=\"groups\" content-type=\"article-table\"><thead><tr><th align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Sl No</th><th align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Attributes</th><th align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Mapping</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Accident Index</td><td colspan=\"1\" rowspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">2</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Date</td><td colspan=\"1\" rowspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">3</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Day of Week</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-Sunday, 2-Monday, 3-Tuesday, 4-Wednesday, 5- Thursday, 6-Friday, 7-Saturday</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">4</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Time of Accident, Accident Location-A</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-Urban, 2-Rural, 3-Unallocated</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">5</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Accident Location-A Chainage-km</td><td colspan=\"1\" rowspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">6</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Accident Location-A Chainage-km-RoadSide</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">LHS, RHS</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">7-9</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Nature of Accident-B1, Nature of Accident- B2, Nature of Accident-B3</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-Overturning, 2-Head on collision, 3-Rear End Collision, 4-Collision Brush/Side Wipe, 5-Right Turn Collision, 6- Skidding, 7a-Others-Hit Cyclist, 7b-Others-Hit Pedestrian, 7c-Others-Hit Parked Vehicle, 7d-Others-Hit Fixed Object, 7e-Others-Wrong Side Driving, 7f-Others-Hit Animal, 7g- Others-Hit Two Wheeler, 7h-Others-Unknown, 7i-Others- Fallen down, 8-Overtaking vehicle, 9-Left Turn Collision</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">10</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Accident Severity -C</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-Fatal, 2-Grevious Injury, 3-Minor Injury, 4-No Injury</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">11-13</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Classification of Accident-C1, Classification of Accident-C2, Classification of Accident-C3</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-Fatal, 2-Grevious Injury, 3-Minor Injury, 4-Non - Injury (Damage only)</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">14-18</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Causes-D1, Causes-D2, Causes-D3, Causes- D4, Causes-D5</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-Drunken, 2-Overspeeding, 3-Vehicle out of control, 4a- Fault of driver of motor vehicle, 4b-Driver of other vehicle, 4c-Cyclist, 4d-Pedestrian, 4e-Passenger, 4f-Animal, 5a- Defect in mechanical condition of motor vehicle, 5b-Road condition</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">19</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Road Feature-E</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-Single lane, 2-Two lanes, 3-Three lanes or more without central divider median, 4-Four lanes or more with central divider alongwith carriageway width</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">20</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Road Condition-F</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-Straight Road, 2-Slight Curve, 3-Sharp Curve, 4-Flat Road, 5-Gentle incline, 6-Steep incline 7-Hump, 8-Dip</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">21</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Intersection Type-G</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-T Junction, 2-’Y Junction, 3-’Four arm junction, 4- Staggered junction, 5-Roundabout, 6-Uncontrolled junction</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">22</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Weather Conditions-H</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-Fine, 2-Mist/Fog\n<break/>3-Cloud, 4-Light Rain, 5-Heavy Rain, 6-Hail/sleet, 7- Snow, 8-Strong Wind, 9-Dust Storm\n<break/>10-Very Hot, 11-Very Cold, 12-Other extraordinary weather condition</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">23-26</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Vehicle Type Involved-J-V1, Vehicle Type Involved-J-V2, Vehicle Type Involved-J-V3, Vehicle Type Involved-J-V4</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">1-Car/Jeep/Van, 2-SUV, 3-Bus, 4-Mini Bus, 5-Truck, 6- Two Wheeler, 7-Three Wheeler, 8-Cycle, 9-Pedestrian, 10- Tractor, 11-Unknown, 12-Animal, 13-Objects, 14-LCV, 15- MAV</td></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">27</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Number of Vehicles</td><td colspan=\"1\" rowspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">28</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Number of Casualties-Fatal</td><td colspan=\"1\" rowspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">29</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Number of Casualties-Grievous Injury</td><td colspan=\"1\" rowspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">30</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Number of Casualties-Minor Injury</td><td colspan=\"1\" rowspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">31</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Number of Casualties-Non Injured</td><td colspan=\"1\" rowspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">32</td><td align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Number of Casualties</td><td colspan=\"1\" rowspan=\"1\"/></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"T1\\\"><label>Table 1. </label><caption><title>Dataset Attributes and Parameters Mapping.</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\" content-type=\\\"article-table\\\"><thead><tr><th align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Sl No</th><th align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Attributes</th><th align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Mapping</th></tr></thead><tbody><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Accident Index</td><td colspan=\\\"1\\\" rowspan=\\\"1\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">2</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Date</td><td colspan=\\\"1\\\" rowspan=\\\"1\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">3</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Day of Week</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-Sunday, 2-Monday, 3-Tuesday, 4-Wednesday, 5- Thursday, 6-Friday, 7-Saturday</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">4</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Time of Accident, Accident Location-A</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-Urban, 2-Rural, 3-Unallocated</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">5</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Accident Location-A Chainage-km</td><td colspan=\\\"1\\\" rowspan=\\\"1\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">6</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Accident Location-A Chainage-km-RoadSide</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">LHS, RHS</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">7-9</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Nature of Accident-B1, Nature of Accident- B2, Nature of Accident-B3</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-Overturning, 2-Head on collision, 3-Rear End Collision, 4-Collision Brush/Side Wipe, 5-Right Turn Collision, 6- Skidding, 7a-Others-Hit Cyclist, 7b-Others-Hit Pedestrian, 7c-Others-Hit Parked Vehicle, 7d-Others-Hit Fixed Object, 7e-Others-Wrong Side Driving, 7f-Others-Hit Animal, 7g- Others-Hit Two Wheeler, 7h-Others-Unknown, 7i-Others- Fallen down, 8-Overtaking vehicle, 9-Left Turn Collision</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">10</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Accident Severity -C</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-Fatal, 2-Grevious Injury, 3-Minor Injury, 4-No Injury</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">11-13</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Classification of Accident-C1, Classification of Accident-C2, Classification of Accident-C3</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-Fatal, 2-Grevious Injury, 3-Minor Injury, 4-Non - Injury (Damage only)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">14-18</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Causes-D1, Causes-D2, Causes-D3, Causes- D4, Causes-D5</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-Drunken, 2-Overspeeding, 3-Vehicle out of control, 4a- Fault of driver of motor vehicle, 4b-Driver of other vehicle, 4c-Cyclist, 4d-Pedestrian, 4e-Passenger, 4f-Animal, 5a- Defect in mechanical condition of motor vehicle, 5b-Road condition</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">19</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Road Feature-E</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-Single lane, 2-Two lanes, 3-Three lanes or more without central divider median, 4-Four lanes or more with central divider alongwith carriageway width</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">20</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Road Condition-F</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-Straight Road, 2-Slight Curve, 3-Sharp Curve, 4-Flat Road, 5-Gentle incline, 6-Steep incline 7-Hump, 8-Dip</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">21</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Intersection Type-G</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-T Junction, 2-’Y Junction, 3-’Four arm junction, 4- Staggered junction, 5-Roundabout, 6-Uncontrolled junction</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">22</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Weather Conditions-H</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-Fine, 2-Mist/Fog\\n<break/>3-Cloud, 4-Light Rain, 5-Heavy Rain, 6-Hail/sleet, 7- Snow, 8-Strong Wind, 9-Dust Storm\\n<break/>10-Very Hot, 11-Very Cold, 12-Other extraordinary weather condition</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">23-26</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Vehicle Type Involved-J-V1, Vehicle Type Involved-J-V2, Vehicle Type Involved-J-V3, Vehicle Type Involved-J-V4</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">1-Car/Jeep/Van, 2-SUV, 3-Bus, 4-Mini Bus, 5-Truck, 6- Two Wheeler, 7-Three Wheeler, 8-Cycle, 9-Pedestrian, 10- Tractor, 11-Unknown, 12-Animal, 13-Objects, 14-LCV, 15- MAV</td></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">27</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Number of Vehicles</td><td colspan=\\\"1\\\" rowspan=\\\"1\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">28</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Number of Casualties-Fatal</td><td colspan=\\\"1\\\" rowspan=\\\"1\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">29</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Number of Casualties-Grievous Injury</td><td colspan=\\\"1\\\" rowspan=\\\"1\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">30</td><td align=\\\"left\\\" colspan=\\\"1\\\" rowspan=\\\"1\\\" valign=\\\"top\\\">Number of Casualties-Minor Injury</td><td colspan=\\\"1\\\" rowspan=\\\"1\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"1\\\" 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In the United States, approximately 5% of patients with breast cancer have de novo metastatic disease at diagnosis, and at least 20% of those initially diagnosed with early-stage breast cancer subsequently develop metastatic disease [##REF##18695137##2##]. Despite advances in therapies, the prognosis of patients with metastatic breast cancer (MBC) remains poor, with a median time to progression on first-line therapy of 9.7 months for triple-negative breast cancer (TNBC) and 25.3 months for HR + , HER2 negative breast cancer and a median overall survival (OS) of 23 months for TNBC and 63.9 months for HR + , HER2 negative breast cancer [##REF##29718092##3##–##UREF##1##5##]. Patients with metastatic disease develop cumulative toxicity from multiple lines of chemotherapy, as well as chemotherapy resistance, which limits efficacy. In order to improve survival while maintaining quality of life, it is important to identify new treatment regimens for patients with MBC. New chemotherapy combinations may improve the duration of response (DOR) and also may provide a superior chemotherapy backbone for the addition of targeted agents in future studies.</p>","<p id=\"Par6\">Eribulin mesylate, a nontaxane microtubule dynamics inhibitor, is a structurally simplified, synthetic analog of the natural product Halichondrin B, isolated from the marine sponge <italic>Halichondria okadai</italic> [##UREF##2##6##, ##UREF##3##7##]. Eribulin suppresses polymerization and sequesters tubulin into nonfunctional aggregates [##UREF##2##6##, ##REF##16020666##8##–##REF##18645010##10##] and has other cytotoxic effects, including vascular remodeling, reversal of the epithelial–mesenchymal transition, induction of the differentiation, and suppression of migration and invasion [##REF##27069131##11##, ##REF##25060424##12##]. Eribulin is administered as a 2- to 5-min IV infusion without the need for premedications, so it is easier to administer than other chemotherapy agents [##UREF##4##13##]. Eribulin was approved as monotherapy for the treatment of taxane and anthracycline-resistant MBC based on results from the EMBRACE trial, which reported a 2.5-month improvement in median OS with eribulin compared to treatment of physicians choice (TPC, 13.1 months vs 10.6 months; HR 0.81, 95% CI 0.66–0.99 p = 0.041) in women with heavily pre-treated MBC [##UREF##5##14##]. Response rate (RR) was significantly longer with eribulin, with a non-significant numerical difference in PFS. In another study, eribulin was compared to capecitabine as an earlier line therapy (up to two lines of chemotherapy) and showed no difference in OS or PFS, but a pooled subset analysis suggested improved OS with eribulin in patients with HER2-negative and TNBC [##UREF##6##15##, ##REF##25381136##16##]. In both studies, treatment-related adverse events (AEs) with eribulin included neuropathy and neutropenia, requiring higher rates of growth factor administration [##REF##26567010##17##–##REF##25381136##19##].</p>","<p id=\"Par7\">Combination chemotherapy with docetaxel and cyclophosphamide (TC) has become a standard treatment option for early-stage lower risk breast cancer based on data from several studies showing improved or similar outcome compared to anthracycline-based regimens [##REF##19204201##20##–##REF##28398846##22##] Compared to doxorubicin and cyclophosphamide (AC), treatment with TC improved OS in patients with up to three positive axillary nodes [##REF##19204201##20##]. Based on encouraging efficacy with TC, we hypothesized that eribulin combined with cyclophosphamide (EC) would be effective in taxane-resistant disease with tolerable toxicity. The aim of this study was to determine the maximum tolerated dose (MTD) of EC, followed by a dose expansion study to estimate the clinical benefit rate (CBR) of EC in patients with advanced breast cancer (ABC). We also performed correlative studies to assess the correlation of circulating tumor cells (CTCs) with response and survival.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">Breast cancer is the most common malignancy affecting women worldwide [##REF##30207593##1##]. In the United States, approximately 5% of patients with breast cancer have de novo metastatic disease at diagnosis, and at least 20% of those initially diagnosed with early-stage breast cancer subsequently develop metastatic disease [##REF##18695137##2##]. Despite advances in therapies, the prognosis of patients with metastatic breast cancer (MBC) remains poor, with a median time to progression on first-line therapy of 9.7 months for triple-negative breast cancer (TNBC) and 25.3 months for HR + , HER2 negative breast cancer and a median overall survival (OS) of 23 months for TNBC and 63.9 months for HR + , HER2 negative breast cancer [##REF##29718092##3##–##UREF##1##5##]. Patients with metastatic disease develop cumulative toxicity from multiple lines of chemotherapy, as well as chemotherapy resistance, which limits efficacy. In order to improve survival while maintaining quality of life, it is important to identify new treatment regimens for patients with MBC. New chemotherapy combinations may improve the duration of response (DOR) and also may provide a superior chemotherapy backbone for the addition of targeted agents in future studies.</p>\",\n \"<p id=\\\"Par6\\\">Eribulin mesylate, a nontaxane microtubule dynamics inhibitor, is a structurally simplified, synthetic analog of the natural product Halichondrin B, isolated from the marine sponge <italic>Halichondria okadai</italic> [##UREF##2##6##, ##UREF##3##7##]. Eribulin suppresses polymerization and sequesters tubulin into nonfunctional aggregates [##UREF##2##6##, ##REF##16020666##8##–##REF##18645010##10##] and has other cytotoxic effects, including vascular remodeling, reversal of the epithelial–mesenchymal transition, induction of the differentiation, and suppression of migration and invasion [##REF##27069131##11##, ##REF##25060424##12##]. Eribulin is administered as a 2- to 5-min IV infusion without the need for premedications, so it is easier to administer than other chemotherapy agents [##UREF##4##13##]. Eribulin was approved as monotherapy for the treatment of taxane and anthracycline-resistant MBC based on results from the EMBRACE trial, which reported a 2.5-month improvement in median OS with eribulin compared to treatment of physicians choice (TPC, 13.1 months vs 10.6 months; HR 0.81, 95% CI 0.66–0.99 p = 0.041) in women with heavily pre-treated MBC [##UREF##5##14##]. Response rate (RR) was significantly longer with eribulin, with a non-significant numerical difference in PFS. In another study, eribulin was compared to capecitabine as an earlier line therapy (up to two lines of chemotherapy) and showed no difference in OS or PFS, but a pooled subset analysis suggested improved OS with eribulin in patients with HER2-negative and TNBC [##UREF##6##15##, ##REF##25381136##16##]. In both studies, treatment-related adverse events (AEs) with eribulin included neuropathy and neutropenia, requiring higher rates of growth factor administration [##REF##26567010##17##–##REF##25381136##19##].</p>\",\n \"<p id=\\\"Par7\\\">Combination chemotherapy with docetaxel and cyclophosphamide (TC) has become a standard treatment option for early-stage lower risk breast cancer based on data from several studies showing improved or similar outcome compared to anthracycline-based regimens [##REF##19204201##20##–##REF##28398846##22##] Compared to doxorubicin and cyclophosphamide (AC), treatment with TC improved OS in patients with up to three positive axillary nodes [##REF##19204201##20##]. Based on encouraging efficacy with TC, we hypothesized that eribulin combined with cyclophosphamide (EC) would be effective in taxane-resistant disease with tolerable toxicity. The aim of this study was to determine the maximum tolerated dose (MTD) of EC, followed by a dose expansion study to estimate the clinical benefit rate (CBR) of EC in patients with advanced breast cancer (ABC). We also performed correlative studies to assess the correlation of circulating tumor cells (CTCs) with response and survival.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<title>Patients</title>","<p id=\"Par8\">Male or female patients ≥ 18 years who had histologically confirmed locally advanced, unresectable or metastatic carcinoma of the breast of all subtypes were eligible to enroll with no limitation on prior lines of therapy. Eligibility included an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0–2, measurable disease, adequate organ and bone marrow functions (neutrophils > 1.0 × 10<sup>9</sup>/L, platelets > 100 × 10<sup>9</sup>/L, hemoglobin > 9 g/dL, total bilirubin < 1.5 × upper limit of normal (ULN), AST and ALT ≤ 3 × ULN or ≤ 5 × ULN in patients with known liver metastasis, creatinine ≤ 1.5 × ULN or ≥ 60 mL/min for patients with creatinine levels > 1.5 × institutional ULN), ≤ grade 1 peripheral neuropathy, and a life expectancy of at least 3 months. Patients with stable treated brain metastases were also eligible to enroll. Exclusion criteria included known active central nervous system (CNS) metastases and/or carcinomatous meningitis, a corrected QT interval (cQT) > 480 ms, or significant cardiovascular disease within the past 6 months. The study was approved by the University of California San Francisco (UCSF) Comprehensive Cancer Center Protocol Review Committee on Human Research, and written informed consent was obtained from all patients prior to trial enrollment. The trial was registered at ClinicalTrials.gov (NCT01554371).</p>","<title>Study design</title>","<p id=\"Par9\">Patients were treated using a 3 + 3 dose confirmation strategy for eribulin with dose expansion at the MTD. Cyclophosphamide was given at a fıxed dose of 600 mg/m<sup>2</sup> on day 1 of a 21-day cycle; eribulin was given day 1 and 8 every of a 21 day cycle, and was escalated from 1.1 mg/m<sup>2</sup> at dose level 0 (DL0) to 1.4 mg/m<sup>2</sup> at dose level 1 (DL1); both drugs were given intravenously. Dose expansion occurred at DL1. The primary objective of dose escalation was to determine the MTD of EC. The highest dose level at which no more than one of six subjects experienced a dose limiting toxicity (DLT) defined the MTD. DLTs were defined as grade 3 or 4 clinically evident non-hematologic toxicity; grade 4 neutropenia, thrombocytopenia lasting > 7 days, febrile neutropenia or any clinically significant toxicity grade 2 or higher that required more than 14 days to resolve occurring within the first 21 days of combination therapy. The primary objective of the dose expansion was CBR at three months; we selected CBR at 3 months rather than 6 months, as these patients were heavily pre-treated so we anticipated shorter responses to therapy in the later line setting. Secondary objectives were RR, DOR, PFS, safety, and correlation of CTCs with clinical benefit.</p>","<p id=\"Par10\">Drug doses were modified for treatment-related toxicity. These toxicities were neutropenia (absolute neutrophil count [ANC] < 1000/μL), thrombocytopenia, rash, GI toxicity, liver abnormalities and neuropathy. If toxicity occurred in a patient, dose reductions were managed as follows. Dose levels -1 and -2 for eribulin were 1.1 mg/m<sup>2</sup> and 0.7 mg/m<sup>2</sup>, for cyclophosphamide one dose reduction was allowed to 500 mg/m<sup>2</sup>. Dose reductions were sequential, with the first dose reduction for cyclophosphamide, followed by eribulin to dose level -1 then -2 for persistent toxicity. If toxicity persisted despite these dose reductions and/or if the participant experienced a cycle delay of three or more weeks, study treatment was discontinued (Supplement 1).</p>","<title>Concomitant medication</title>","<p id=\"Par11\">Patients received prophylactic antiemetics and premedications according to standard institutional guidelines. Colony stimulating growth factor use was allowed at the discretion of the treating physician. Palliative radiotherapy was permitted to control bone pain as long as the irradiated area was limited in extent. Other investigational agents and potent inhibitors or inducers of CYP3A4 were not permitted.</p>","<title>Assessments</title>","<p id=\"Par12\">Baseline evaluations included medical history, a physical examination, Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) tumor imaging with computed tomography (CT), bone scan, laboratory tests (hematology, blood chemistry), a serum pregnancy test for females of child-bearing potential, and an electrocardiogram with QTc measurement. In the dose confirmation cohort (phase Ib), the response to EC was evaluated every 6 weeks until disease progression according to the investigator, based on objective tumor assessments using RECIST version 1.1 criteria. In the dose-expansion cohort (phase II), response to EC was evaluated after study start, then every 9 weeks until end of study therapy.</p>","<title>Safety/tolerability</title>","<p id=\"Par13\">Safety evaluations at baseline and subsequent visits included AEs, clinical laboratory tests, physical examination, and vital signs. AEs were assessed and AE severity was graded in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE version 4.0). Both agents were held for grade 3 or febrile neutropenia (ANC < 1000/μL). Filgrastim or pegylated-filgrastim myeloid growth factor support was encouraged for ANC < 1500/μL and was allowed at the discretion of the treating physician in order to maintain adequate blood counts. Filgrastim was given for ANC < 1000/μL at any time, or as prophylaxis in patients at risk for neutropenia. Neuropathy was assessed using the 10-point Modified Total Neuropathy Score at the start of each cycle and at study termination.</p>","<title>Correlative studies</title>","<p id=\"Par14\">Whole blood samples were obtained in fixative-containing tubes (CellSave tubes, Veridex (currently Menarini)) and processed in the laboratory of Dr. John Park at University of California, San Francisco for CTC identification and enumeration using the CellSearch system. Samples with 5 CTCs per 7.5 mLs of blood were considered CTC-positive.</p>","<title>Statistical analysis</title>","<p id=\"Par15\">The study followed a standard dose-confirmation schema (phase Ib portion) with three patients per cohort (3 + 3 design) for a total of six patients. A two-stage design was performed in the dose-expansion (phase II portion) with a possible total of 40 patients. An overall RR of 25% was considered clinically meaningful. Using a two-stage Simon’s minimax design, the null and alternative hypothesis were H0: p0 < 10% versus Ha: p1 > 25% for the proportion of patients with complete or partial response (PR) by RECIST criteria. Based on the type I error of 5% and the type II error rate of 20%, p0 = 10% and p1 = 25%. Secondary efficacy variables were analyzed using Kaplan–Meier methods, with a corresponding median and a 95% CI.</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<title>Patients</title>\",\n \"<p id=\\\"Par8\\\">Male or female patients ≥ 18 years who had histologically confirmed locally advanced, unresectable or metastatic carcinoma of the breast of all subtypes were eligible to enroll with no limitation on prior lines of therapy. Eligibility included an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0–2, measurable disease, adequate organ and bone marrow functions (neutrophils > 1.0 × 10<sup>9</sup>/L, platelets > 100 × 10<sup>9</sup>/L, hemoglobin > 9 g/dL, total bilirubin < 1.5 × upper limit of normal (ULN), AST and ALT ≤ 3 × ULN or ≤ 5 × ULN in patients with known liver metastasis, creatinine ≤ 1.5 × ULN or ≥ 60 mL/min for patients with creatinine levels > 1.5 × institutional ULN), ≤ grade 1 peripheral neuropathy, and a life expectancy of at least 3 months. Patients with stable treated brain metastases were also eligible to enroll. Exclusion criteria included known active central nervous system (CNS) metastases and/or carcinomatous meningitis, a corrected QT interval (cQT) > 480 ms, or significant cardiovascular disease within the past 6 months. The study was approved by the University of California San Francisco (UCSF) Comprehensive Cancer Center Protocol Review Committee on Human Research, and written informed consent was obtained from all patients prior to trial enrollment. The trial was registered at ClinicalTrials.gov (NCT01554371).</p>\",\n \"<title>Study design</title>\",\n \"<p id=\\\"Par9\\\">Patients were treated using a 3 + 3 dose confirmation strategy for eribulin with dose expansion at the MTD. Cyclophosphamide was given at a fıxed dose of 600 mg/m<sup>2</sup> on day 1 of a 21-day cycle; eribulin was given day 1 and 8 every of a 21 day cycle, and was escalated from 1.1 mg/m<sup>2</sup> at dose level 0 (DL0) to 1.4 mg/m<sup>2</sup> at dose level 1 (DL1); both drugs were given intravenously. Dose expansion occurred at DL1. The primary objective of dose escalation was to determine the MTD of EC. The highest dose level at which no more than one of six subjects experienced a dose limiting toxicity (DLT) defined the MTD. DLTs were defined as grade 3 or 4 clinically evident non-hematologic toxicity; grade 4 neutropenia, thrombocytopenia lasting > 7 days, febrile neutropenia or any clinically significant toxicity grade 2 or higher that required more than 14 days to resolve occurring within the first 21 days of combination therapy. The primary objective of the dose expansion was CBR at three months; we selected CBR at 3 months rather than 6 months, as these patients were heavily pre-treated so we anticipated shorter responses to therapy in the later line setting. Secondary objectives were RR, DOR, PFS, safety, and correlation of CTCs with clinical benefit.</p>\",\n \"<p id=\\\"Par10\\\">Drug doses were modified for treatment-related toxicity. These toxicities were neutropenia (absolute neutrophil count [ANC] < 1000/μL), thrombocytopenia, rash, GI toxicity, liver abnormalities and neuropathy. If toxicity occurred in a patient, dose reductions were managed as follows. Dose levels -1 and -2 for eribulin were 1.1 mg/m<sup>2</sup> and 0.7 mg/m<sup>2</sup>, for cyclophosphamide one dose reduction was allowed to 500 mg/m<sup>2</sup>. Dose reductions were sequential, with the first dose reduction for cyclophosphamide, followed by eribulin to dose level -1 then -2 for persistent toxicity. If toxicity persisted despite these dose reductions and/or if the participant experienced a cycle delay of three or more weeks, study treatment was discontinued (Supplement 1).</p>\",\n \"<title>Concomitant medication</title>\",\n \"<p id=\\\"Par11\\\">Patients received prophylactic antiemetics and premedications according to standard institutional guidelines. Colony stimulating growth factor use was allowed at the discretion of the treating physician. Palliative radiotherapy was permitted to control bone pain as long as the irradiated area was limited in extent. Other investigational agents and potent inhibitors or inducers of CYP3A4 were not permitted.</p>\",\n \"<title>Assessments</title>\",\n \"<p id=\\\"Par12\\\">Baseline evaluations included medical history, a physical examination, Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) tumor imaging with computed tomography (CT), bone scan, laboratory tests (hematology, blood chemistry), a serum pregnancy test for females of child-bearing potential, and an electrocardiogram with QTc measurement. In the dose confirmation cohort (phase Ib), the response to EC was evaluated every 6 weeks until disease progression according to the investigator, based on objective tumor assessments using RECIST version 1.1 criteria. In the dose-expansion cohort (phase II), response to EC was evaluated after study start, then every 9 weeks until end of study therapy.</p>\",\n \"<title>Safety/tolerability</title>\",\n \"<p id=\\\"Par13\\\">Safety evaluations at baseline and subsequent visits included AEs, clinical laboratory tests, physical examination, and vital signs. AEs were assessed and AE severity was graded in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE version 4.0). Both agents were held for grade 3 or febrile neutropenia (ANC < 1000/μL). Filgrastim or pegylated-filgrastim myeloid growth factor support was encouraged for ANC < 1500/μL and was allowed at the discretion of the treating physician in order to maintain adequate blood counts. Filgrastim was given for ANC < 1000/μL at any time, or as prophylaxis in patients at risk for neutropenia. Neuropathy was assessed using the 10-point Modified Total Neuropathy Score at the start of each cycle and at study termination.</p>\",\n \"<title>Correlative studies</title>\",\n \"<p id=\\\"Par14\\\">Whole blood samples were obtained in fixative-containing tubes (CellSave tubes, Veridex (currently Menarini)) and processed in the laboratory of Dr. John Park at University of California, San Francisco for CTC identification and enumeration using the CellSearch system. Samples with 5 CTCs per 7.5 mLs of blood were considered CTC-positive.</p>\",\n \"<title>Statistical analysis</title>\",\n \"<p id=\\\"Par15\\\">The study followed a standard dose-confirmation schema (phase Ib portion) with three patients per cohort (3 + 3 design) for a total of six patients. A two-stage design was performed in the dose-expansion (phase II portion) with a possible total of 40 patients. An overall RR of 25% was considered clinically meaningful. Using a two-stage Simon’s minimax design, the null and alternative hypothesis were H0: p0 < 10% versus Ha: p1 > 25% for the proportion of patients with complete or partial response (PR) by RECIST criteria. Based on the type I error of 5% and the type II error rate of 20%, p0 = 10% and p1 = 25%. Secondary efficacy variables were analyzed using Kaplan–Meier methods, with a corresponding median and a 95% CI.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Patient characteristics</title>","<p id=\"Par16\">Patients with histologically confirmed metastatic or ABC with any number of prior lines of therapy were eligible to enroll in this study, and 44 patients enrolled in total. Baseline patient characteristics are summarized in Table ##TAB##0##1##. The median age was 56 years (range 33–82 years). 31 patients (70.4%) had HR + /HER2- disease, 12 patients (27.3%) had TNBC, and 1 patient (2.3%) had HR + /HER2 + disease. Patients had a median of 1 prior line of hormone therapy (range 0–6) and 2 prior lines of chemotherapy (range 0–7). Most patients (97.7%) had visceral disease. The most common metastatic sites of disease were bone, lymph nodes, liver, and lung (Table ##TAB##0##1##).</p>","<title>Antitumor activity</title>","<p id=\"Par17\">The median duration of treatment was 14.7 weeks (1.8–53.3 weeks). The CBR was 79.5% (35/44; 7 PR, 28 SD). The median PFS was 16.4 weeks (95%CI: 13.8–21.1 weeks). (Figure ##FIG##0##1##a). The median DOR was 16.4 weeks (13.8–21.1 weeks). Clinical response to EC therapy is summarized in Table ##TAB##1##2##. Individual patient characteristics of those who had a PFS 24 weeks on EC are summarized in Table ##TAB##2##3##.</p>","<p id=\"Par18\">The CBR at 3 months in patients with HR + /HER2- disease was 83.9% (n = 31), and 12.9% of patients had a PR. The median PFS was 18.1 weeks. In the 12 patients with TNBC, the CBR was 66.7% with a PR rate of 25%. The median PFS was 10.8 weeks. As expected, PFS was longer in those with HR + disease versus those with TNBC (18.1 vs 10.8 weeks; p = 0.067) (Fig. ##FIG##0##1##b).</p>","<p id=\"Par19\">The CBR among patients who had received (neo)adjuvant treatment with an anthracycline and/or a taxane (A/T) was 81.8% (18/22), similar to that of the overall study population. There was no difference in PFS among patients who had received prior A/T (n = 24) versus those who had treatment without A/T (n = 20) (21.1 vs.15.1 weeks respectively, p = 0.4251). There was no difference in PFS among patients who received 0–2 prior lines of chemotherapy versus who received lines of chemotherapy (18.1 vs. 14.7 weeks respectively, p = 0.6736). In patients with HR + disease, patients who had received 0–2 prior lines of chemotherapy (n = 17) had a median PFS of 21.1 weeks whereas patients who had received lines of chemotherapy (n = 15) had a median PFS of 14.3 weeks. In patients with TNBC, patients who had received 0–2 prior lines of chemotherapy (n = 9) had a median PFS of 9.8 weeks whereas patients who had received lines of chemotherapy (n = 3) had a median PFS of 19.1 weeks.</p>","<title>Drug exposure and safety</title>","<p id=\"Par20\">No DLTs were identified in the dose confirmation (phase Ib) portion of the study. Three patients were treated at DL0 (eribulin 1.1 mg/m<sup>2</sup>) and 3 were treated at DL1 (eribulin 1.4 mg/m<sup>2</sup>), the MTD. Thirty-seven patients (84.1%) completed at least three cycles of treatment and 21 (47.7%) received cycles of treatment. The median number of cycles delivered was 5.8 (1.1–17.8); the median exposure was 3.6 weeks and nine patients received treatment for 6 months or longer (20.5%). Seventeen patients (38.6%) had 1 cyclophosphamide dose reduction and 12 patients (27.3%) had 1 eribulin dose reduction.</p>","<p id=\"Par21\">Adverse events (AEs) are summarized in Table ##TAB##3##4##. Twenty-eight patients (63.6%) experienced a grade 3/4 AE, the most common of which were neutropenia (47.7%, n = 21), fatigue (4.5%, n = 2), dyspnea (4.5%, n = 2), and anemia (2.3%, n = 1). Febrile neutropenia was reported for three patients (6.8%). Most patients (77.3%) received myeloid growth factors. Treatment related AEs led to dose adjustment (interruption/delay or reduction): 26 patients (59.1%) had a dose interruption/delay and 17 patients (38.6%) underwent dose reduction due to a treatment-related AE. Dose reductions due to neutropenia included a decrease of cyclophosphamide to 500 mg/m<sup>2</sup> (n = 17) and of eribulin to 1.1 mg/m<sup>2</sup> (n = 12). Five patients discontinued treatment due to fatigue (n = 3) or neutropenia (n = 2).</p>","<title>Biomarkers</title>","<p id=\"Par22\">CTCs in blood were enumerated at baseline and during treatment to explore the correlation between CTC levels and response to EC. Of the 44 evaluable patients, 26 had baseline CTC data. Of these, 14 were CTC-positive (53.8%). There was no significant difference in the mean CTCs/7.5 mLs of blood at baseline between subtypes (t-test p = 0.6435). There was no significant association between CTC status at baseline and treatment response at first scan (Fisher p = 0.5901). 18 of the 26 patients had paired CTC data at baseline and follow-up, 2 of whom were CTC positive at baseline and turned CTC-negative on treatment. The change in CTC status was not significantly associated with response to treatment (Fisher p = 0.3355).</p>","<p id=\"Par23\">The median PFS was significantly shorter in patients who were CTC-positive at baseline compared to those who were CTC-negative (13.1 vs. 30.6 weeks, p = 0.011). Eighteen patients had on-treatment CTC data available (either during treatment or at the end of study). Of these, nine were CTC-positive (50%). The median PFS was significantly shorter in patients who were CTC-positive during treatment compared to those who were CTC-negative (13.1 weeks vs. 30.6 weeks, p = 0.035).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Patient characteristics</title>\",\n \"<p id=\\\"Par16\\\">Patients with histologically confirmed metastatic or ABC with any number of prior lines of therapy were eligible to enroll in this study, and 44 patients enrolled in total. Baseline patient characteristics are summarized in Table ##TAB##0##1##. The median age was 56 years (range 33–82 years). 31 patients (70.4%) had HR + /HER2- disease, 12 patients (27.3%) had TNBC, and 1 patient (2.3%) had HR + /HER2 + disease. Patients had a median of 1 prior line of hormone therapy (range 0–6) and 2 prior lines of chemotherapy (range 0–7). Most patients (97.7%) had visceral disease. The most common metastatic sites of disease were bone, lymph nodes, liver, and lung (Table ##TAB##0##1##).</p>\",\n \"<title>Antitumor activity</title>\",\n \"<p id=\\\"Par17\\\">The median duration of treatment was 14.7 weeks (1.8–53.3 weeks). The CBR was 79.5% (35/44; 7 PR, 28 SD). The median PFS was 16.4 weeks (95%CI: 13.8–21.1 weeks). (Figure ##FIG##0##1##a). The median DOR was 16.4 weeks (13.8–21.1 weeks). Clinical response to EC therapy is summarized in Table ##TAB##1##2##. Individual patient characteristics of those who had a PFS 24 weeks on EC are summarized in Table ##TAB##2##3##.</p>\",\n \"<p id=\\\"Par18\\\">The CBR at 3 months in patients with HR + /HER2- disease was 83.9% (n = 31), and 12.9% of patients had a PR. The median PFS was 18.1 weeks. In the 12 patients with TNBC, the CBR was 66.7% with a PR rate of 25%. The median PFS was 10.8 weeks. As expected, PFS was longer in those with HR + disease versus those with TNBC (18.1 vs 10.8 weeks; p = 0.067) (Fig. ##FIG##0##1##b).</p>\",\n \"<p id=\\\"Par19\\\">The CBR among patients who had received (neo)adjuvant treatment with an anthracycline and/or a taxane (A/T) was 81.8% (18/22), similar to that of the overall study population. There was no difference in PFS among patients who had received prior A/T (n = 24) versus those who had treatment without A/T (n = 20) (21.1 vs.15.1 weeks respectively, p = 0.4251). There was no difference in PFS among patients who received 0–2 prior lines of chemotherapy versus who received lines of chemotherapy (18.1 vs. 14.7 weeks respectively, p = 0.6736). In patients with HR + disease, patients who had received 0–2 prior lines of chemotherapy (n = 17) had a median PFS of 21.1 weeks whereas patients who had received lines of chemotherapy (n = 15) had a median PFS of 14.3 weeks. In patients with TNBC, patients who had received 0–2 prior lines of chemotherapy (n = 9) had a median PFS of 9.8 weeks whereas patients who had received lines of chemotherapy (n = 3) had a median PFS of 19.1 weeks.</p>\",\n \"<title>Drug exposure and safety</title>\",\n \"<p id=\\\"Par20\\\">No DLTs were identified in the dose confirmation (phase Ib) portion of the study. Three patients were treated at DL0 (eribulin 1.1 mg/m<sup>2</sup>) and 3 were treated at DL1 (eribulin 1.4 mg/m<sup>2</sup>), the MTD. Thirty-seven patients (84.1%) completed at least three cycles of treatment and 21 (47.7%) received cycles of treatment. The median number of cycles delivered was 5.8 (1.1–17.8); the median exposure was 3.6 weeks and nine patients received treatment for 6 months or longer (20.5%). Seventeen patients (38.6%) had 1 cyclophosphamide dose reduction and 12 patients (27.3%) had 1 eribulin dose reduction.</p>\",\n \"<p id=\\\"Par21\\\">Adverse events (AEs) are summarized in Table ##TAB##3##4##. Twenty-eight patients (63.6%) experienced a grade 3/4 AE, the most common of which were neutropenia (47.7%, n = 21), fatigue (4.5%, n = 2), dyspnea (4.5%, n = 2), and anemia (2.3%, n = 1). Febrile neutropenia was reported for three patients (6.8%). Most patients (77.3%) received myeloid growth factors. Treatment related AEs led to dose adjustment (interruption/delay or reduction): 26 patients (59.1%) had a dose interruption/delay and 17 patients (38.6%) underwent dose reduction due to a treatment-related AE. Dose reductions due to neutropenia included a decrease of cyclophosphamide to 500 mg/m<sup>2</sup> (n = 17) and of eribulin to 1.1 mg/m<sup>2</sup> (n = 12). Five patients discontinued treatment due to fatigue (n = 3) or neutropenia (n = 2).</p>\",\n \"<title>Biomarkers</title>\",\n \"<p id=\\\"Par22\\\">CTCs in blood were enumerated at baseline and during treatment to explore the correlation between CTC levels and response to EC. Of the 44 evaluable patients, 26 had baseline CTC data. Of these, 14 were CTC-positive (53.8%). There was no significant difference in the mean CTCs/7.5 mLs of blood at baseline between subtypes (t-test p = 0.6435). There was no significant association between CTC status at baseline and treatment response at first scan (Fisher p = 0.5901). 18 of the 26 patients had paired CTC data at baseline and follow-up, 2 of whom were CTC positive at baseline and turned CTC-negative on treatment. The change in CTC status was not significantly associated with response to treatment (Fisher p = 0.3355).</p>\",\n \"<p id=\\\"Par23\\\">The median PFS was significantly shorter in patients who were CTC-positive at baseline compared to those who were CTC-negative (13.1 vs. 30.6 weeks, p = 0.011). Eighteen patients had on-treatment CTC data available (either during treatment or at the end of study). Of these, nine were CTC-positive (50%). The median PFS was significantly shorter in patients who were CTC-positive during treatment compared to those who were CTC-negative (13.1 weeks vs. 30.6 weeks, p = 0.035).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par24\">This trial was conducted to assess the safety, efficacy, and tolerability of EC for the treatment of patients with metastatic or ABC. CBR and PFS were 79.5% and 16.4 weeks respectively, comparing favorably to historic data of single agent eribulin for ABC (PFS 14.8 weeks) [##UREF##5##14##]. Our data demonstrate that EC has activity in extensively pretreated patients, as 68.2% of patients who enrolled had been treated with three to seven prior lines of chemotherapy. Of note, responses were observed in patients who had received prior anthracycline and taxane therapies.</p>","<p id=\"Par25\">Patients with both HR + and TNBC were enrolled. In particular, metastatic TNBC is an aggressive breast cancer subtype associated with poor clinical outcomes highlighting the importance of identifying novel treatment approaches. Previous phase III studies demonstrated statistically significant improvements in OS in patients with metastatic TNBC treated with eribulin versus treatment of physician choice in both subgroup and pooled analyses. Specifically, in a pooled analysis of the EMBRACE study and Study 301, eribulin significantly improved OS compared with TPC in patients with TNBC (HR: 0.74, p = 0.006) [##REF##25381136##16##, ##REF##27398025##23##]. In our study, 12 patients with metastatic TNBC received EC, with a CBR of 66.7% and a PR rate of 25%. The median PFS was 10.8 weeks with EC treatment. Among patients with metastatic TNBC, the heavily pre-treated patients had longer responses to EC than the less heavily-pretreated patients, although numbers are small; future larger studies can evaluate this further.</p>","<p id=\"Par26\">The adverse events of eribulin in this study is consistent with what has been reported in previous studies [##UREF##5##14##, ##REF##25381136##19##]. In this study, the most frequently reported treatment related AEs were fatigue (68.2%), neutropenia (59.1%), nausea (56.8%), constipation (50%), peripheral neuropathy (47.7%), dyspnea (40.9%), headache (36.4%), and anorexia (36.4%), which reflect the known toxicity profiles of eribulin and cyclophosphamide. The most common grade 3/4 AE was neutropenia (47.7%; 6.8% febrile neutropenia). The incidence of neuropathy was 47.7%, but no patient experienced grade 3/4 neuropathy. Similarly, prior clinical trials also report high incidence of neutropenia and neuropathy with the use of eribulin. In the EMBRACE study, 52% of participants experienced neutropenia (grade 3: 8%, grade 4: 1%) and 35% of participants experienced peripheral neuropathy (grade 3: 8%, grade 4: 0.4%) [##UREF##5##14##]. In real-world studies of patients treated with eribulin grade 3/4 neutropenia occurred in 12% of patients and grade 3/4 neuropathy occurred in 2.6% of patients [##REF##30411979##24##]. Overall, given that the patient population was heavily pre-treated in our study, it was reassuring that the toxicity profile of this combination chemotherapy regimen was similar to those previously reported in single agent studies.</p>","<p id=\"Par27\">In this study, we administered eribulin using a 21-day cycle at a dose of 1.4 mg/m<sup>2</sup> on days 1 and 8 combined with cyclophosphamide at a fıxed dose of 600 mg/m<sup>2</sup> on day 1 of each cycle. Previous studies demonstrated that eribulin was more tolerable when administered on a 21-day schedule compared to a 28-day schedule [##UREF##7##25##]. Alternative schedules of eribulin administration have been investigated to provide better tolerance in patients who experienced myelosuppression. A modified biweekly regimen which provides additional time for bone marrow recovery may potentially improve safety compared with the 21-day dosing regimen [##REF##29435730##26##]. In a prospective phase 2 trial, biweekly eribulin (1.4 mg/m<sup>2</sup> on days 1 and 15 of a 28-day cycle) was tolerable and had comparable antitumor activity in patients who were intolerant of the standard eribulin schedule [##REF##29435730##26##]. Dose reductions due to neutropenia required patients to decrease to 500 mg/m<sup>2</sup> cyclophosphamide (n = 17) and to 1.1 mg/m<sup>2</sup> in eribulin (n = 12), consistent with expected hematologic toxicity in this heavily pre-treated population. Only five patients discontinued treatment due to AEs.</p>","<p id=\"Par28\">Previous studies have suggested that the presence of CTCs in patients with MBC is associated with a worse prognosis [##REF##15317891##27##, ##REF##32770247##28##] and can predict treatment response and progression [##REF##15735118##29##]. Based on these prior work, we performed an exploratory CTC analysis to address whether CTC levels correlate with response to EC. Consistent with previous studies [##REF##15317891##27##, ##REF##32770247##28##], the CTC positivity rate was 53.8% (14 of 26 patients) at baseline. Median PFS was significantly shorter in patients who were CTC-positive at baseline or during treatment compared to those who were CTC-negative.</p>","<p id=\"Par29\">This study has several notable strengths. First, few studies have evaluated combination chemotherapy in patients with MBC who have received multiple lines of prior chemotherapy [##UREF##5##14##, ##REF##32223649##30##]. Inclusion of this patient population in our study suggests that the EC regimen may be more generalizable to real-world treatment scenarios. Second, this trial included patients with multiple breast cancer subtypes: most patients had HR + /HER2- breast cancer, with a smaller number of patients with TNBC, and one patient with HR + /HER2 + disease. The RR to EC was higher in patients with HR + /HER2- disease compared to patients with TNBC, as expected, but our study was not powered to fully detect differences between subtypes. Third, the CTC data provides interesting correlative data, and we found that the median PFS was significantly shorter in patients who were CTC-positive at baseline compared to those who were CTC-negative, providing rationale to continue to study the prognostic and predictive value of CTCs in ABC.</p>","<p id=\"Par30\">This study also has several limitations. First, the primary endpoint of CBR provides important clinical information about response to EC, but this trial is not designed to provide data about OS. Second, since patients were heavily pre-treated, treatment history was fairly heterogenous, which clearly impacts both response to therapy and toxicity. This was a single-arm study so it is not possible to determine the efficacy of this regimen compared to others, and further studies are needed to clarify the efficacy of this regimen. Lastly, our study was conducted before regulatory approval of pembrolizumab plus chemotherapy for PD-L1 + metastatic TNBC, sacituzumab govitecan for metastatic TNBC and heavily pre-treated HR + /HER2- MBC, and trastuzumab deruxtecan for HER2-low MBC. However, as patients being treated with eribulin today will be even more heavily pre-treated than the patients in this trial, the activity of this combination regimen may have implications for current therapeutic options.</p>","<p id=\"Par31\">In conclusion, the results of this trial demonstrate that EC has antitumoral activity in heavily pretreated patients with locally ABC or MBC. Importantly, EC demonstrated a manageable tolerability profile. These results support the additional clinical development of EC as a novel treatment combination for the treatment of ABC.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par24\\\">This trial was conducted to assess the safety, efficacy, and tolerability of EC for the treatment of patients with metastatic or ABC. CBR and PFS were 79.5% and 16.4 weeks respectively, comparing favorably to historic data of single agent eribulin for ABC (PFS 14.8 weeks) [##UREF##5##14##]. Our data demonstrate that EC has activity in extensively pretreated patients, as 68.2% of patients who enrolled had been treated with three to seven prior lines of chemotherapy. Of note, responses were observed in patients who had received prior anthracycline and taxane therapies.</p>\",\n \"<p id=\\\"Par25\\\">Patients with both HR + and TNBC were enrolled. In particular, metastatic TNBC is an aggressive breast cancer subtype associated with poor clinical outcomes highlighting the importance of identifying novel treatment approaches. Previous phase III studies demonstrated statistically significant improvements in OS in patients with metastatic TNBC treated with eribulin versus treatment of physician choice in both subgroup and pooled analyses. Specifically, in a pooled analysis of the EMBRACE study and Study 301, eribulin significantly improved OS compared with TPC in patients with TNBC (HR: 0.74, p = 0.006) [##REF##25381136##16##, ##REF##27398025##23##]. In our study, 12 patients with metastatic TNBC received EC, with a CBR of 66.7% and a PR rate of 25%. The median PFS was 10.8 weeks with EC treatment. Among patients with metastatic TNBC, the heavily pre-treated patients had longer responses to EC than the less heavily-pretreated patients, although numbers are small; future larger studies can evaluate this further.</p>\",\n \"<p id=\\\"Par26\\\">The adverse events of eribulin in this study is consistent with what has been reported in previous studies [##UREF##5##14##, ##REF##25381136##19##]. In this study, the most frequently reported treatment related AEs were fatigue (68.2%), neutropenia (59.1%), nausea (56.8%), constipation (50%), peripheral neuropathy (47.7%), dyspnea (40.9%), headache (36.4%), and anorexia (36.4%), which reflect the known toxicity profiles of eribulin and cyclophosphamide. The most common grade 3/4 AE was neutropenia (47.7%; 6.8% febrile neutropenia). The incidence of neuropathy was 47.7%, but no patient experienced grade 3/4 neuropathy. Similarly, prior clinical trials also report high incidence of neutropenia and neuropathy with the use of eribulin. In the EMBRACE study, 52% of participants experienced neutropenia (grade 3: 8%, grade 4: 1%) and 35% of participants experienced peripheral neuropathy (grade 3: 8%, grade 4: 0.4%) [##UREF##5##14##]. In real-world studies of patients treated with eribulin grade 3/4 neutropenia occurred in 12% of patients and grade 3/4 neuropathy occurred in 2.6% of patients [##REF##30411979##24##]. Overall, given that the patient population was heavily pre-treated in our study, it was reassuring that the toxicity profile of this combination chemotherapy regimen was similar to those previously reported in single agent studies.</p>\",\n \"<p id=\\\"Par27\\\">In this study, we administered eribulin using a 21-day cycle at a dose of 1.4 mg/m<sup>2</sup> on days 1 and 8 combined with cyclophosphamide at a fıxed dose of 600 mg/m<sup>2</sup> on day 1 of each cycle. Previous studies demonstrated that eribulin was more tolerable when administered on a 21-day schedule compared to a 28-day schedule [##UREF##7##25##]. Alternative schedules of eribulin administration have been investigated to provide better tolerance in patients who experienced myelosuppression. A modified biweekly regimen which provides additional time for bone marrow recovery may potentially improve safety compared with the 21-day dosing regimen [##REF##29435730##26##]. In a prospective phase 2 trial, biweekly eribulin (1.4 mg/m<sup>2</sup> on days 1 and 15 of a 28-day cycle) was tolerable and had comparable antitumor activity in patients who were intolerant of the standard eribulin schedule [##REF##29435730##26##]. Dose reductions due to neutropenia required patients to decrease to 500 mg/m<sup>2</sup> cyclophosphamide (n = 17) and to 1.1 mg/m<sup>2</sup> in eribulin (n = 12), consistent with expected hematologic toxicity in this heavily pre-treated population. Only five patients discontinued treatment due to AEs.</p>\",\n \"<p id=\\\"Par28\\\">Previous studies have suggested that the presence of CTCs in patients with MBC is associated with a worse prognosis [##REF##15317891##27##, ##REF##32770247##28##] and can predict treatment response and progression [##REF##15735118##29##]. Based on these prior work, we performed an exploratory CTC analysis to address whether CTC levels correlate with response to EC. Consistent with previous studies [##REF##15317891##27##, ##REF##32770247##28##], the CTC positivity rate was 53.8% (14 of 26 patients) at baseline. Median PFS was significantly shorter in patients who were CTC-positive at baseline or during treatment compared to those who were CTC-negative.</p>\",\n \"<p id=\\\"Par29\\\">This study has several notable strengths. First, few studies have evaluated combination chemotherapy in patients with MBC who have received multiple lines of prior chemotherapy [##UREF##5##14##, ##REF##32223649##30##]. Inclusion of this patient population in our study suggests that the EC regimen may be more generalizable to real-world treatment scenarios. Second, this trial included patients with multiple breast cancer subtypes: most patients had HR + /HER2- breast cancer, with a smaller number of patients with TNBC, and one patient with HR + /HER2 + disease. The RR to EC was higher in patients with HR + /HER2- disease compared to patients with TNBC, as expected, but our study was not powered to fully detect differences between subtypes. Third, the CTC data provides interesting correlative data, and we found that the median PFS was significantly shorter in patients who were CTC-positive at baseline compared to those who were CTC-negative, providing rationale to continue to study the prognostic and predictive value of CTCs in ABC.</p>\",\n \"<p id=\\\"Par30\\\">This study also has several limitations. First, the primary endpoint of CBR provides important clinical information about response to EC, but this trial is not designed to provide data about OS. Second, since patients were heavily pre-treated, treatment history was fairly heterogenous, which clearly impacts both response to therapy and toxicity. This was a single-arm study so it is not possible to determine the efficacy of this regimen compared to others, and further studies are needed to clarify the efficacy of this regimen. Lastly, our study was conducted before regulatory approval of pembrolizumab plus chemotherapy for PD-L1 + metastatic TNBC, sacituzumab govitecan for metastatic TNBC and heavily pre-treated HR + /HER2- MBC, and trastuzumab deruxtecan for HER2-low MBC. However, as patients being treated with eribulin today will be even more heavily pre-treated than the patients in this trial, the activity of this combination regimen may have implications for current therapeutic options.</p>\",\n \"<p id=\\\"Par31\\\">In conclusion, the results of this trial demonstrate that EC has antitumoral activity in heavily pretreated patients with locally ABC or MBC. Importantly, EC demonstrated a manageable tolerability profile. These results support the additional clinical development of EC as a novel treatment combination for the treatment of ABC.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose</title>","<p id=\"Par1\">We hypothesized that eribulin combined with cyclophosphamide (EC) would be an effective combination with tolerable toxicity for the treatment of advanced breast cancer (ABC).</p>","<title>Methods</title>","<p id=\"Par2\">Patients with histologically confirmed metastatic or unresectable ABC with any number of prior lines of therapy were eligible to enroll. In the dose escalation cohort, dose level 0 was defined as eribulin 1.1 mg/m<sup>2</sup> and cyclophosphamide 600 mg/m<sup>2</sup><sub>,</sub> and dose level 1 was defined as eribulin 1.4 mg/m<sup>2</sup> and cyclophosphamide 600 mg/m<sup>2</sup>. Eribulin was given on days 1 and 8 and cyclophosphamide on day 1 of a 21-day cycle. In the dose expansion cohort, enrollment was expanded at dose level 1. The primary objective was clinical benefit rate (CBR), and secondary objectives were response rate (RR), duration of response (DOR), progression-free survival (PFS), and safety.</p>","<title>Results</title>","<p id=\"Par3\">No dose-limiting toxicities were identified in the dose escalation cohort (n = 6). In the dose expansion cohort, an additional 38 patients were enrolled for a total of 44 patients, including 31 patients (70.4%) with hormone receptor-positive (HR +)/HER2- disease, 12 patients (27.3%) with triple-negative breast cancer (TNBC), and 1 patient (2.3%) with HR + /HER2 + disease. Patients had a median age of 56 years (range 33–82 years), 1 prior line of hormone therapy (range 0–6), and 2 prior lines of chemotherapy (range 0–7). CBR was 79.5% (35/44; 7 partial response, 28 stable disease) and the median DOR was 16.4 weeks (range 13.8–21.1 weeks). Median PFS was 16.4 weeks (95% CI: 13.8–21.1 weeks). The most common grade 3/4 adverse event was neutropenia (47.7%, n = 21). Fourteen of 26 patients (53.8%) with circulating tumor cell (CTC) data were CTC-positive ( 5 CTC/7.5 mL) at baseline. Median PFS was shorter in patients who were CTC-positive vs. negative (13.1 vs 30.6 weeks, p = 0.011).</p>","<title>Conclusion</title>","<p id=\"Par4\">In heavily pretreated patients with ABC, treatment with EC resulted in an encouraging CBR of 79.5% and PFS of 16.4 weeks, which compares favorably to single-agent eribulin. Dose reduction and delays were primarily due to neutropenia. The contribution of cyclophosphamide to eribulin remains unclear but warrants further evaluation. NCT01554371.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s10549-023-07073-0.</p>","<title>Keywords</title>"],"string":"[\n \"<title>Purpose</title>\",\n \"<p id=\\\"Par1\\\">We hypothesized that eribulin combined with cyclophosphamide (EC) would be an effective combination with tolerable toxicity for the treatment of advanced breast cancer (ABC).</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">Patients with histologically confirmed metastatic or unresectable ABC with any number of prior lines of therapy were eligible to enroll. In the dose escalation cohort, dose level 0 was defined as eribulin 1.1 mg/m<sup>2</sup> and cyclophosphamide 600 mg/m<sup>2</sup><sub>,</sub> and dose level 1 was defined as eribulin 1.4 mg/m<sup>2</sup> and cyclophosphamide 600 mg/m<sup>2</sup>. Eribulin was given on days 1 and 8 and cyclophosphamide on day 1 of a 21-day cycle. In the dose expansion cohort, enrollment was expanded at dose level 1. The primary objective was clinical benefit rate (CBR), and secondary objectives were response rate (RR), duration of response (DOR), progression-free survival (PFS), and safety.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">No dose-limiting toxicities were identified in the dose escalation cohort (n = 6). In the dose expansion cohort, an additional 38 patients were enrolled for a total of 44 patients, including 31 patients (70.4%) with hormone receptor-positive (HR +)/HER2- disease, 12 patients (27.3%) with triple-negative breast cancer (TNBC), and 1 patient (2.3%) with HR + /HER2 + disease. Patients had a median age of 56 years (range 33–82 years), 1 prior line of hormone therapy (range 0–6), and 2 prior lines of chemotherapy (range 0–7). CBR was 79.5% (35/44; 7 partial response, 28 stable disease) and the median DOR was 16.4 weeks (range 13.8–21.1 weeks). Median PFS was 16.4 weeks (95% CI: 13.8–21.1 weeks). The most common grade 3/4 adverse event was neutropenia (47.7%, n = 21). Fourteen of 26 patients (53.8%) with circulating tumor cell (CTC) data were CTC-positive ( 5 CTC/7.5 mL) at baseline. Median PFS was shorter in patients who were CTC-positive vs. negative (13.1 vs 30.6 weeks, p = 0.011).</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par4\\\">In heavily pretreated patients with ABC, treatment with EC resulted in an encouraging CBR of 79.5% and PFS of 16.4 weeks, which compares favorably to single-agent eribulin. Dose reduction and delays were primarily due to neutropenia. The contribution of cyclophosphamide to eribulin remains unclear but warrants further evaluation. NCT01554371.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s10549-023-07073-0.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Funding</title>","<p>This work was supported in part by funding to the UCSF Regents from Eisai, Inc.</p>","<title>Data availability</title>","<p>Enquiries about data availability should be directed to the authors.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par32\">Amy Jo Chien: Research funding to institution: Merck, Puma, Amgen, Seagen. Michelle E. Melisko: Research funding to institution: Astra Zeneca, Novartis, KCRN Research, Puma, Seattle Genetics. For spouse: Speaker bureau/honoraria: AstraZeneca, Genentech, Gilead. Stock Ownership: Merrimack. John Park: Consulting/honoraria: AstraZenica, Genentech, Diichi, Gilead. Equity/stock ownership: Merrimack Pharma. Hope S. Rugo: Research support for clinical trials through the University of California: Pfizer, Merck, Novartis, Lilly, Roche, Daiichi, Seattle Genetics, Macrogenics, Sermonix, Boehringer Ingelheim, AstraZeneca, Astellas and Gilead. Honoraria from: Puma, Samsung, Mylan, Chugai, Blueprint, and NAPO. The remaining authors, declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>"],"string":"[\n \"<title>Funding</title>\",\n \"<p>This work was supported in part by funding to the UCSF Regents from Eisai, Inc.</p>\",\n \"<title>Data availability</title>\",\n \"<p>Enquiries about data availability should be directed to the authors.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par32\\\">Amy Jo Chien: Research funding to institution: Merck, Puma, Amgen, Seagen. Michelle E. Melisko: Research funding to institution: Astra Zeneca, Novartis, KCRN Research, Puma, Seattle Genetics. For spouse: Speaker bureau/honoraria: AstraZeneca, Genentech, Gilead. Stock Ownership: Merrimack. John Park: Consulting/honoraria: AstraZenica, Genentech, Diichi, Gilead. Equity/stock ownership: Merrimack Pharma. Hope S. Rugo: Research support for clinical trials through the University of California: Pfizer, Merck, Novartis, Lilly, Roche, Daiichi, Seattle Genetics, Macrogenics, Sermonix, Boehringer Ingelheim, AstraZeneca, Astellas and Gilead. Honoraria from: Puma, Samsung, Mylan, Chugai, Blueprint, and NAPO. The remaining authors, declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Kaplan–Meier plot of progression-free survival (PFS). <bold>a</bold> Shown is the Kaplan–Meier plot for PFS for all patients in the study. The median PFS was 16.4 weeks (95% CI: 13.8–21.1 weeks) in all patients. <bold>b</bold> Shown is the Kaplan–Meier plot for PFS for patients with HR + /HER2- disease (red) and TNBC (blue). The median PFS was 18.1 weeks in patients with HR + /HER2- disease and 10.8 weeks in patients with TNBC</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Kaplan–Meier plot of progression-free survival (PFS). <bold>a</bold> Shown is the Kaplan–Meier plot for PFS for all patients in the study. The median PFS was 16.4 weeks (95% CI: 13.8–21.1 weeks) in all patients. <bold>b</bold> Shown is the Kaplan–Meier plot for PFS for patients with HR + /HER2- disease (red) and TNBC (blue). The median PFS was 18.1 weeks in patients with HR + /HER2- disease and 10.8 weeks in patients with TNBC</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Patient characteristics at baseline</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Characteristics</th><th align=\"left\">EC (n = 44)</th><th align=\"left\">%</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"3\">Age, years</td></tr><tr><td align=\"left\"> Median (range, yrs)</td><td align=\"left\">56 (33–82)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"3\">Sex</td></tr><tr><td align=\"left\"> Female</td><td align=\"left\">44</td><td align=\"left\">100</td></tr><tr><td align=\"left\"> Male</td><td align=\"left\">0</td><td align=\"left\">0</td></tr><tr><td align=\"left\" colspan=\"3\">Subtype</td></tr><tr><td align=\"left\"> HR + /HER2-</td><td align=\"left\">31</td><td align=\"left\">70.4</td></tr><tr><td align=\"left\"> TNBC</td><td align=\"left\">12</td><td align=\"left\">27.3</td></tr><tr><td align=\"left\"> HR + /HER2 + </td><td align=\"left\">1</td><td align=\"left\">2.3</td></tr><tr><td align=\"left\" colspan=\"3\">Prior lines of chemotherapy</td></tr><tr><td align=\"left\"> 0</td><td align=\"left\">1</td><td align=\"left\">2.3</td></tr><tr><td align=\"left\"> 1</td><td align=\"left\">15</td><td align=\"left\">34.1</td></tr><tr><td align=\"left\"> 2</td><td align=\"left\">10</td><td align=\"left\">22.7</td></tr><tr><td align=\"left\"> 3</td><td align=\"left\">18</td><td align=\"left\">40.9</td></tr><tr><td align=\"left\" colspan=\"3\">Prior lines of endocrine therapy</td></tr><tr><td align=\"left\"> 0–1</td><td align=\"left\">24</td><td align=\"left\">54.5</td></tr><tr><td align=\"left\"> 2</td><td align=\"left\">8</td><td align=\"left\">18.2</td></tr><tr><td align=\"left\"> 3</td><td align=\"left\">12</td><td align=\"left\">27.3</td></tr><tr><td align=\"left\" colspan=\"3\">Metastatic disease</td></tr><tr><td align=\"left\"> Bone only disease</td><td align=\"left\">1</td><td align=\"left\">2.3</td></tr><tr><td align=\"left\"> Visceral disease</td><td align=\"left\">8</td><td align=\"left\">18.2</td></tr><tr><td align=\"left\"> Bone and visceral disease</td><td align=\"left\">35</td><td align=\"left\">79.5</td></tr><tr><td align=\"left\" colspan=\"3\">Metastatic sites</td></tr><tr><td align=\"left\"> Bone</td><td align=\"left\">34</td><td align=\"left\">77.3</td></tr><tr><td align=\"left\"> Lymph node</td><td align=\"left\">29</td><td align=\"left\">65.9</td></tr><tr><td align=\"left\"> Liver</td><td align=\"left\">29</td><td align=\"left\">65.9</td></tr><tr><td align=\"left\"> Lung</td><td align=\"left\">20</td><td align=\"left\">45.5</td></tr><tr><td align=\"left\"> Soft tissue involvement</td><td align=\"left\">8</td><td align=\"left\">18.2</td></tr><tr><td align=\"left\"> CNS metastasis</td><td align=\"left\">6</td><td align=\"left\">13.6</td></tr><tr><td align=\"left\"> Peritoneum</td><td align=\"left\">6</td><td align=\"left\">13.6</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Clinical response to EC therapy</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Response endpoints</th><th align=\"left\">EC (N = 44)</th></tr></thead><tbody><tr><td align=\"left\">CBR</td><td align=\"left\">35 (79.5)</td></tr><tr><td align=\"left\">CR, n (%)</td><td align=\"left\">0 (0)</td></tr><tr><td align=\"left\">PR, n (%)</td><td align=\"left\">7 (15.9)</td></tr><tr><td align=\"left\">SD, n (%)</td><td align=\"left\">28 (63.6)</td></tr><tr><td align=\"left\">PD, n (%)</td><td align=\"left\">6 (13.6)</td></tr><tr><td align=\"left\">n/a, n (%)</td><td align=\"left\">3 (6.8)</td></tr><tr><td align=\"left\">CBR (CR + PR + SD 6 months), n (%)</td><td align=\"left\">9 (20.5)</td></tr><tr><td align=\"left\">PFS, weeks (median) 95% CI</td><td align=\"left\">16.4 (13.8–21.1)</td></tr><tr><td align=\"left\">PFS, weeks for HR + vs TNBC patients</td><td align=\"left\">18.1 vs 10.8 (p = 0.067)</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Individual patient characteristics whose PFS with EC 24 weeks</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Patient no</th><th align=\"left\">Ph</th><th align=\"left\">Subtype</th><th align=\"left\">Prior lines of ET</th><th align=\"left\">Prior lines of CT</th><th align=\"left\">Metastatic sites</th><th align=\"left\">PFS<break/>(weeks)</th></tr></thead><tbody><tr><td align=\"left\">101</td><td align=\"left\">Ib</td><td align=\"left\">HR + /HER2-</td><td align=\"left\">3</td><td align=\"left\">3</td><td align=\"left\">Bone only</td><td char=\".\" align=\"char\">27.3</td></tr><tr><td align=\"left\">103</td><td align=\"left\">Ib</td><td align=\"left\">HR + /HER2-</td><td align=\"left\">0</td><td align=\"left\">1</td><td align=\"left\">Bone and visceral</td><td char=\".\" align=\"char\">40.7</td></tr><tr><td align=\"left\">107</td><td align=\"left\">Ib</td><td align=\"left\">HR + /HER2-</td><td align=\"left\">2</td><td align=\"left\">1</td><td align=\"left\">Bone and visceral</td><td char=\".\" align=\"char\">34.4</td></tr><tr><td align=\"left\">208</td><td align=\"left\">II</td><td align=\"left\">TNBC</td><td align=\"left\">0</td><td align=\"left\">3</td><td align=\"left\">Bone and visceral</td><td char=\".\" align=\"char\">30.6</td></tr><tr><td align=\"left\">213</td><td align=\"left\">II</td><td align=\"left\">HR + /HER2-</td><td align=\"left\">3</td><td align=\"left\">2</td><td align=\"left\">Bone and visceral</td><td char=\".\" align=\"char\">33.0</td></tr><tr><td align=\"left\">219</td><td align=\"left\">II</td><td align=\"left\">HR + /HER2-</td><td align=\"left\">1</td><td align=\"left\">3</td><td align=\"left\">Bone and visceral</td><td char=\".\" align=\"char\">53.3</td></tr><tr><td align=\"left\">228</td><td align=\"left\">II</td><td align=\"left\">HR + /HER2-</td><td align=\"left\">1</td><td align=\"left\">2</td><td align=\"left\">Bone and visceral</td><td char=\".\" align=\"char\">45.0</td></tr><tr><td align=\"left\">235</td><td align=\"left\">II</td><td align=\"left\">TNBC</td><td align=\"left\">0</td><td align=\"left\">2</td><td align=\"left\">Bone and visceral</td><td char=\".\" align=\"char\">28.6</td></tr><tr><td align=\"left\">240</td><td align=\"left\">II</td><td align=\"left\">HR + /HER2-</td><td align=\"left\">2</td><td align=\"left\">2</td><td align=\"left\">Bone and visceral</td><td char=\".\" align=\"char\">29.4</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Adverse events</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Adverse event</th><th align=\"left\">Any grade, n (%)</th><th align=\"left\">Grade 1–2, n (%)</th><th align=\"left\">Grade 3–4, n (%)</th></tr></thead><tbody><tr><td align=\"left\">Fatigue</td><td char=\"(\" align=\"char\">30 (68.2)</td><td char=\"(\" align=\"char\">28 (63.6)</td><td char=\"(\" align=\"char\">2 (4.5)</td></tr><tr><td align=\"left\">Neutrophil count decrease</td><td char=\"(\" align=\"char\">26 (59.1)</td><td char=\"(\" align=\"char\">5 (11.4)</td><td char=\"(\" align=\"char\">21 (47.7)</td></tr><tr><td align=\"left\">Nausea</td><td char=\"(\" align=\"char\">25 (56.8)</td><td char=\"(\" align=\"char\">25 (56.8)</td><td char=\"(\" align=\"char\">0 (0)</td></tr><tr><td align=\"left\">Constipation</td><td char=\"(\" align=\"char\">22 (50)</td><td char=\"(\" align=\"char\">22 (50)</td><td char=\"(\" align=\"char\">0 (0)</td></tr><tr><td align=\"left\">Peripheral neuropathy</td><td char=\"(\" align=\"char\">21 (47.7)</td><td char=\"(\" align=\"char\">21 (47.7)</td><td char=\"(\" align=\"char\">0 (0)</td></tr><tr><td align=\"left\">Dyspnea</td><td char=\"(\" align=\"char\">18 (40.9)</td><td char=\"(\" align=\"char\">16 (36.4)</td><td char=\"(\" align=\"char\">2 (4.5)</td></tr><tr><td align=\"left\">Headache</td><td char=\"(\" align=\"char\">16 (36.4)</td><td char=\"(\" align=\"char\">16 (36.4)</td><td char=\"(\" align=\"char\">0 (0)</td></tr><tr><td align=\"left\">Anorexia</td><td char=\"(\" align=\"char\">16 (36.4)</td><td char=\"(\" align=\"char\">16 (36.4)</td><td char=\"(\" align=\"char\">0 (0)</td></tr><tr><td align=\"left\">Anemia</td><td char=\"(\" align=\"char\">15 (34.1)</td><td char=\"(\" align=\"char\">14 (31.8)</td><td char=\"(\" align=\"char\">1 (2.3)</td></tr><tr><td align=\"left\">Alopecia</td><td char=\"(\" align=\"char\">14 (31.8)</td><td char=\"(\" align=\"char\">14 (31.8)</td><td char=\"(\" align=\"char\">0 (0)</td></tr><tr><td align=\"left\">Arthralgia</td><td char=\"(\" align=\"char\">14 (31.8)</td><td char=\"(\" align=\"char\">14 (31.8)</td><td char=\"(\" align=\"char\">0 (0)</td></tr><tr><td align=\"left\">Febrile neutropenia</td><td char=\"(\" align=\"char\">3 (6.8)</td><td char=\"(\" align=\"char\">1 (2.3)</td><td char=\"(\" align=\"char\">2 (4.5)</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Patient characteristics at baseline</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Characteristics</th><th align=\\\"left\\\">EC (n = 44)</th><th align=\\\"left\\\">%</th></tr></thead><tbody><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Age, years</td></tr><tr><td align=\\\"left\\\"> Median (range, yrs)</td><td align=\\\"left\\\">56 (33–82)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Sex</td></tr><tr><td align=\\\"left\\\"> Female</td><td align=\\\"left\\\">44</td><td align=\\\"left\\\">100</td></tr><tr><td align=\\\"left\\\"> Male</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">0</td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Subtype</td></tr><tr><td align=\\\"left\\\"> HR + /HER2-</td><td align=\\\"left\\\">31</td><td align=\\\"left\\\">70.4</td></tr><tr><td align=\\\"left\\\"> TNBC</td><td align=\\\"left\\\">12</td><td align=\\\"left\\\">27.3</td></tr><tr><td align=\\\"left\\\"> HR + /HER2 + </td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">2.3</td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Prior lines of chemotherapy</td></tr><tr><td align=\\\"left\\\"> 0</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">2.3</td></tr><tr><td align=\\\"left\\\"> 1</td><td align=\\\"left\\\">15</td><td align=\\\"left\\\">34.1</td></tr><tr><td align=\\\"left\\\"> 2</td><td align=\\\"left\\\">10</td><td align=\\\"left\\\">22.7</td></tr><tr><td align=\\\"left\\\"> 3</td><td align=\\\"left\\\">18</td><td align=\\\"left\\\">40.9</td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Prior lines of endocrine therapy</td></tr><tr><td align=\\\"left\\\"> 0–1</td><td align=\\\"left\\\">24</td><td align=\\\"left\\\">54.5</td></tr><tr><td align=\\\"left\\\"> 2</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">18.2</td></tr><tr><td align=\\\"left\\\"> 3</td><td align=\\\"left\\\">12</td><td align=\\\"left\\\">27.3</td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Metastatic disease</td></tr><tr><td align=\\\"left\\\"> Bone only disease</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">2.3</td></tr><tr><td align=\\\"left\\\"> Visceral disease</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">18.2</td></tr><tr><td align=\\\"left\\\"> Bone and visceral disease</td><td align=\\\"left\\\">35</td><td align=\\\"left\\\">79.5</td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Metastatic sites</td></tr><tr><td align=\\\"left\\\"> Bone</td><td align=\\\"left\\\">34</td><td align=\\\"left\\\">77.3</td></tr><tr><td align=\\\"left\\\"> Lymph node</td><td align=\\\"left\\\">29</td><td align=\\\"left\\\">65.9</td></tr><tr><td align=\\\"left\\\"> Liver</td><td align=\\\"left\\\">29</td><td align=\\\"left\\\">65.9</td></tr><tr><td align=\\\"left\\\"> Lung</td><td align=\\\"left\\\">20</td><td align=\\\"left\\\">45.5</td></tr><tr><td align=\\\"left\\\"> Soft tissue involvement</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">18.2</td></tr><tr><td align=\\\"left\\\"> CNS metastasis</td><td align=\\\"left\\\">6</td><td align=\\\"left\\\">13.6</td></tr><tr><td align=\\\"left\\\"> Peritoneum</td><td align=\\\"left\\\">6</td><td align=\\\"left\\\">13.6</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Clinical response to EC therapy</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Response endpoints</th><th align=\\\"left\\\">EC (N = 44)</th></tr></thead><tbody><tr><td align=\\\"left\\\">CBR</td><td align=\\\"left\\\">35 (79.5)</td></tr><tr><td align=\\\"left\\\">CR, n (%)</td><td align=\\\"left\\\">0 (0)</td></tr><tr><td align=\\\"left\\\">PR, n (%)</td><td align=\\\"left\\\">7 (15.9)</td></tr><tr><td align=\\\"left\\\">SD, n (%)</td><td align=\\\"left\\\">28 (63.6)</td></tr><tr><td align=\\\"left\\\">PD, n (%)</td><td align=\\\"left\\\">6 (13.6)</td></tr><tr><td align=\\\"left\\\">n/a, n (%)</td><td align=\\\"left\\\">3 (6.8)</td></tr><tr><td align=\\\"left\\\">CBR (CR + PR + SD 6 months), n (%)</td><td align=\\\"left\\\">9 (20.5)</td></tr><tr><td align=\\\"left\\\">PFS, weeks (median) 95% CI</td><td align=\\\"left\\\">16.4 (13.8–21.1)</td></tr><tr><td align=\\\"left\\\">PFS, weeks for HR + vs TNBC patients</td><td align=\\\"left\\\">18.1 vs 10.8 (p = 0.067)</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Individual patient characteristics whose PFS with EC 24 weeks</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Patient no</th><th align=\\\"left\\\">Ph</th><th align=\\\"left\\\">Subtype</th><th align=\\\"left\\\">Prior lines of ET</th><th align=\\\"left\\\">Prior lines of CT</th><th align=\\\"left\\\">Metastatic sites</th><th align=\\\"left\\\">PFS<break/>(weeks)</th></tr></thead><tbody><tr><td align=\\\"left\\\">101</td><td align=\\\"left\\\">Ib</td><td align=\\\"left\\\">HR + /HER2-</td><td align=\\\"left\\\">3</td><td align=\\\"left\\\">3</td><td align=\\\"left\\\">Bone only</td><td char=\\\".\\\" align=\\\"char\\\">27.3</td></tr><tr><td align=\\\"left\\\">103</td><td align=\\\"left\\\">Ib</td><td align=\\\"left\\\">HR + /HER2-</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">Bone and visceral</td><td char=\\\".\\\" align=\\\"char\\\">40.7</td></tr><tr><td align=\\\"left\\\">107</td><td align=\\\"left\\\">Ib</td><td align=\\\"left\\\">HR + /HER2-</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">Bone and visceral</td><td char=\\\".\\\" align=\\\"char\\\">34.4</td></tr><tr><td align=\\\"left\\\">208</td><td align=\\\"left\\\">II</td><td align=\\\"left\\\">TNBC</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">3</td><td align=\\\"left\\\">Bone and visceral</td><td char=\\\".\\\" align=\\\"char\\\">30.6</td></tr><tr><td align=\\\"left\\\">213</td><td align=\\\"left\\\">II</td><td align=\\\"left\\\">HR + /HER2-</td><td align=\\\"left\\\">3</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">Bone and visceral</td><td char=\\\".\\\" align=\\\"char\\\">33.0</td></tr><tr><td align=\\\"left\\\">219</td><td align=\\\"left\\\">II</td><td align=\\\"left\\\">HR + /HER2-</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">3</td><td align=\\\"left\\\">Bone and visceral</td><td char=\\\".\\\" align=\\\"char\\\">53.3</td></tr><tr><td align=\\\"left\\\">228</td><td align=\\\"left\\\">II</td><td align=\\\"left\\\">HR + /HER2-</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">Bone and visceral</td><td char=\\\".\\\" align=\\\"char\\\">45.0</td></tr><tr><td align=\\\"left\\\">235</td><td align=\\\"left\\\">II</td><td align=\\\"left\\\">TNBC</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">Bone and visceral</td><td char=\\\".\\\" align=\\\"char\\\">28.6</td></tr><tr><td align=\\\"left\\\">240</td><td align=\\\"left\\\">II</td><td align=\\\"left\\\">HR + /HER2-</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">Bone and visceral</td><td char=\\\".\\\" align=\\\"char\\\">29.4</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab4\\\"><label>Table 4</label><caption><p>Adverse events</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Adverse event</th><th align=\\\"left\\\">Any grade, n (%)</th><th align=\\\"left\\\">Grade 1–2, n (%)</th><th align=\\\"left\\\">Grade 3–4, n (%)</th></tr></thead><tbody><tr><td align=\\\"left\\\">Fatigue</td><td char=\\\"(\\\" align=\\\"char\\\">30 (68.2)</td><td char=\\\"(\\\" align=\\\"char\\\">28 (63.6)</td><td char=\\\"(\\\" align=\\\"char\\\">2 (4.5)</td></tr><tr><td align=\\\"left\\\">Neutrophil count decrease</td><td char=\\\"(\\\" align=\\\"char\\\">26 (59.1)</td><td char=\\\"(\\\" align=\\\"char\\\">5 (11.4)</td><td char=\\\"(\\\" align=\\\"char\\\">21 (47.7)</td></tr><tr><td align=\\\"left\\\">Nausea</td><td char=\\\"(\\\" align=\\\"char\\\">25 (56.8)</td><td char=\\\"(\\\" align=\\\"char\\\">25 (56.8)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0)</td></tr><tr><td align=\\\"left\\\">Constipation</td><td char=\\\"(\\\" align=\\\"char\\\">22 (50)</td><td char=\\\"(\\\" align=\\\"char\\\">22 (50)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0)</td></tr><tr><td align=\\\"left\\\">Peripheral neuropathy</td><td char=\\\"(\\\" align=\\\"char\\\">21 (47.7)</td><td char=\\\"(\\\" align=\\\"char\\\">21 (47.7)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0)</td></tr><tr><td align=\\\"left\\\">Dyspnea</td><td char=\\\"(\\\" align=\\\"char\\\">18 (40.9)</td><td char=\\\"(\\\" align=\\\"char\\\">16 (36.4)</td><td char=\\\"(\\\" align=\\\"char\\\">2 (4.5)</td></tr><tr><td align=\\\"left\\\">Headache</td><td char=\\\"(\\\" align=\\\"char\\\">16 (36.4)</td><td char=\\\"(\\\" align=\\\"char\\\">16 (36.4)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0)</td></tr><tr><td align=\\\"left\\\">Anorexia</td><td char=\\\"(\\\" align=\\\"char\\\">16 (36.4)</td><td char=\\\"(\\\" align=\\\"char\\\">16 (36.4)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0)</td></tr><tr><td align=\\\"left\\\">Anemia</td><td char=\\\"(\\\" align=\\\"char\\\">15 (34.1)</td><td char=\\\"(\\\" align=\\\"char\\\">14 (31.8)</td><td char=\\\"(\\\" align=\\\"char\\\">1 (2.3)</td></tr><tr><td align=\\\"left\\\">Alopecia</td><td char=\\\"(\\\" align=\\\"char\\\">14 (31.8)</td><td char=\\\"(\\\" align=\\\"char\\\">14 (31.8)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0)</td></tr><tr><td align=\\\"left\\\">Arthralgia</td><td char=\\\"(\\\" align=\\\"char\\\">14 (31.8)</td><td char=\\\"(\\\" align=\\\"char\\\">14 (31.8)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0)</td></tr><tr><td align=\\\"left\\\">Febrile neutropenia</td><td char=\\\"(\\\" align=\\\"char\\\">3 (6.8)</td><td char=\\\"(\\\" align=\\\"char\\\">1 (2.3)</td><td char=\\\"(\\\" align=\\\"char\\\">2 (4.5)</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":["<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\ge$$\\end{document}</tex-math><mml:math 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al., ##REF##23544924##2013##; Lengua & Kovacs, ##UREF##10##2005##). However, these studies have focused on global self-reports, which mostly confound consistency of parental reactions across multiple episodes of misbehavior (e.g., punishing misbehavior in one episode, and condoning it in another) with consistency within a single episode of misbehavior (e.g., threatening with punishment, but leaving it in the end). Additionally, observational studies have mostly focused on within-episode consistency (Del Vecchio & O’Leary, ##REF##16597215##2006##; Gardner, ##REF##2745902##1989##). Therefore, it is not known whether within- and across-episode consistency play similar roles in the early development of externalizing problems. In this study, we use a daily diary approach to examine parental consistency both within and across episodes, offering a unique possibility to differentiate these two types of consistency. We examine associations between these aspects of consistency, and how they are associated with child externalizing behavior, both concurrently and longitudinally.</p>","<title>Differentiating Within- from Across Disruptive Behavior Episode Consistency</title>","<p id=\"Par3\">Empirically, studies using questionnaire measures of inconsistent discipline have indeed found it to be associated with more externalizing behavior in children (Barry et al., ##UREF##3##2009##; Gryczkowski et al., ##UREF##8##2010##; Halgunseth et al., ##REF##23544924##2013##; Lengua & Kovacs, ##UREF##10##2005##). However, research to date has not separated within-episode consistency from across-episode consistency. For instance, the frequently used Alabama Parenting Questionnaire (Frick et al., ##UREF##7##1999##) includes items in the inconsistency scale asking parents whether they threaten to punish, but then do not do so in the end, or whether they let their child out of punishment early, which are examples of inconsistent responding <italic>within</italic> a single episode of misbehavior. Additionally, items are included asking parents whether their punishment depends on their mood, which concerns consistency <italic>across</italic> episodes of misbehavior. It is important to note that these types of inconsistency do not necessarily co-occur. Parents may be relatively inconsistent in their responses within a single episode, perhaps not feeling competent enough to follow through with an initial course of action and giving up along the way (Deković et al., ##REF##20101464##2010##), yet they may be consistent in this response style across multiple episodes of misbehavior. Alternatively, parents may be consistent within an episode of misbehavior by providing a negative consequence and sticking to it, but respond in different ways to new episodes, for instance switching to ignoring the misbehavior or trying to redirect attention by making a joke. They may switch approaches because they are in a different mood state themselves (Rueger et al., ##UREF##13##2011##), or because they felt their approach was not effective in a previous instance.</p>","<p id=\"Par4\">Several theoretical frameworks would predict that both types of consistency would be associated with increased externalizing problems. Attachment theory postulates that children form secure attachments to their caregivers if these are consistently responsive to their needs. With consistency, children learn that they can trust their caregiver to provide them with a secure base and a safe haven (Ainsworth et al., ##UREF##1##2015##). With inconsistency in caregiving, in contrast, children learn that their environment is unpredictable and insecure, increasing the risk of attachment problems and problem behavior (Madigan et al., ##REF##26619212##2016##). Supporting this notion, unpredictable behavior from parents has been shown to impact the stress response in both very young children (Noroña-Zhou et al., ##REF##32115696##2020##), as well as older children (Manczak et al., ##REF##28625195##2018##). A dysregulated stress system may in turn result in problems with self-regulation, resulting in heightened disruptive behavior (Wesarg et al., ##UREF##17##2020##). Additionally, unpredictability likely hampers children’s ability to develop a sense of self-efficacy, because it prevents them from developing a sense of control over situations (Bandura, ##UREF##2##1978##; Lippold et al., ##REF##28736495##2016##). Low self-efficacy, in turn, makes it more difficult to regulate anger and frustration, resulting in increased levels of disruptive behavior.</p>","<p id=\"Par5\">Social learning theories emphasize operant conditioning principles which predict that within-episode inconsistency would be associated with increased problematic behavior because failing to follow-through with negative consequences reinforces the child for showing the misbehavior (Patterson, ##UREF##12##1982##). Additionally, consistent negative consequences across different disruptive behavior episodes, or at least a lack of reward, would quickly result in extinction of problematic behavior, whereas inconsistent discipline – with intermittent patterns of positive or negative reinforcement – makes it difficult for children to learn that their behavior is not acceptable. Both social learning and attachment frameworks would thus predict that within- as well as across-episode consistency would play a role in the maintenance of disruptive behavior.</p>","<p id=\"Par6\">Although most questionnaire studies have confounded within- and across-episode consistency, some studies have specifically investigated within-episode consistency. Using observations of parent-child interactions, parents with children who were high on externalizing behavior were less likely to follow-through on an initial demand than parents of children who were low on externalizing behavior (Gardner, ##REF##2745902##1989##). Additionally, mothers of aggressive toddlers have been observed to be more likely to react with both overreactivity and laxness to instances of child aggression than mothers of non-aggressive toddlers (Del Vecchio & O’Leary, ##REF##16597215##2006##). However, with observations it is more difficult to assess how this within-episode consistency relates to across episode consistency. As observations are already so time-consuming, observing (enough) episodes of misbehavior to examine across episode consistency would be especially difficult.</p>","<title>Using Daily Diaries to Assess Parental Consistency</title>","<p id=\"Par7\">Daily diary assessments allow for overcoming the abovementioned limitations of single-time surveys or observations. Single-time self-reports of parental consistency may be biased, as correctly judging how consistent one is in their reactions might be even more difficult than broadly gauging whether one frequently reacts in a certain way (Lippold et al., ##REF##28736495##2016##). A more valid way to assess parental consistency may therefore be to repeatedly ask parents about their actual behavior, and analyze the consistency across their responses. A small scale study examined parents’ daily fluctuations in their overreactive and lax discipline, with lax discipline indicating inconsistency by not following through. Although overreactivity and laxness were positively correlated at the between-person level, indicating that mothers who were generally more overreactive were also more lax, there was a negative association at the within-person level, indicating that when mothers were overreactive in a certain instance, they were less likely to be lax at that time, and vice versa (Passini et al., ##REF##23458698##2013##). These results indicate that mothers can be inconsistent across episodes with regards to their within-episode consistency. However, associations with child externalizing behavior were not examined here. Another diary study did examine associations with externalizing behavior and found that for mothers with 5- 8-year old children maternal consistency across one week was associated with less child externalizing behavior (Villarreal et al., ##UREF##16##2021##). Consistency was operationalized as the within-person fluctuations in destructive conflict characteristics, which included both maternal punitive behavior as well as child and mother negativity, making it unclear whether this was an association with inconsistency in maternal behavior specifically. Yet another study examined daily reports of parents harsh and warm reactions to child misbehavior and found that consistency in the level of warmth was associated with less child ADHD symptoms, whereas consistency in the level of harshness was not (Li & Lansford, ##REF##29608072##2018##). As these studies operationalized inconsistency as fluctuations in mean levels of parenting behaviors, we do not know whether parents exhibited different types of responses in the same instance of misbehavior. For instance, some parents may be more likely to only punish the child by taking away a privilege, whereas other parents are more likely to yell at the child, take away a privilege and also comfort the child, with the latter type of response perhaps especially confusing to the child. A previous observational study has indeed found that some parents were more likely to use both positive and negative discipline strategies within the same episode than others, but did not examine associations between this inconsistency and externalizing behavior (van Zeijl et al., ##UREF##15##2007##).</p>","<title>The Present Study</title>","<p id=\"Par8\">In the present study, we use daily diary data to differentiate consistent responding within a single episode of misbehavior from consistent responding across multiple episodes of misbehavior and examine how they are each associated with the severity of disruptive behavior in children. We assess within-episode consistency as the mean number of different reactions to a specific episode of misbehavior, distinguishing positive attention, positive consequence, negative consequences, negative attention, and ignoring. Across-episode consistency is assessed as the overall dispersion of mothers’ reactions across all possible categories, taking into account the total number of episodes of misbehavior across a week. Parental reactions that were concentrated in fewer reaction categories were indicative of more consistency.</p>","<p id=\"Par9\">We examine associations between the two types of consistency in two independent samples: a community sample (<italic>N</italic> = 134) of mothers of 1.5 to 3.5 year old children who completed a daily diary for 7 days, and an at-risk sample with heightened disruptive behavior (<italic>N</italic> = 149) of 3 to 8 year old children who filled out a daily diary for 14 days. Including these two samples has several benefits. First, we are able to examine whether associations between the two types of consistency conceptually replicate across multiple samples. Second, we can investigate whether both types of consistency are equally associated with disruptive behavior during a developmental stage when disruptive behavior starts to emerge and is relatively more normative, as during a developmental stage when disruptive behavior for most children has started to decline (Tremblay, ##REF##20146751##2010##) as a result of increases in children’s verbal-skills and overall self-regulation (Kuhn et al., ##REF##27101154##2016##). This allows us to investigate whether the two types of consistency play a similar role in the early emergence as in the maintenance of more persistent problem behavior across development. Contemporary accounts of social learning theories would predict that across-episode consistency may be less relevant for problem behavior that persists into preschool age, as repeated coercive cycling in parent-child dyads is thought to result in increasingly rigid, mutually negative interactions over time (Granic & Patterson, ##REF##16478303##2006##).</p>","<p id=\"Par10\">We also examine the added value of computing these types of consistency from daily diary data over a single questionnaire assessment. To this end, we assessed whether it is a better predictor of children’s externalizing behavior one year later than a measure of consistency derived from a general questionnaire as administered in a single – less time-consuming baseline assessment – asking parents to estimate how often in the past month they showed the reactions that we also included in the diary study. Although this ‘general consistency’ measure confounds within- and across-episode consistency, it may still be a better measure of consistency than some of the current measures that are used. Rather than asking parents to report on how consistent they are, we merely asked parents to indicate how often they reacted a certain way, and compute consistency by calculating the dispersion of parents’ responses across the different reaction categories. This approach makes it less likely that this association is for instance explained by parents scoring themselves as inconsistent due to a more negative self-view (Smit et al., ##UREF##14##2021##). This likely plays a role in more traditional questionnaire measures, as most parents will realize that threatening with punishment and then not following through is not an effective parenting strategy. Our approach will allow us to examine whether taking multiple days of measurements to assess consistency is really necessary, or whether we have enough information when we just ask parents how often they react a certain way overall. Additionally, associations between the general consistency and within- and across-episode consistency can be examined to provide an indication of the validity of these measures.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par2\\\">Many studies support the notion that when parents are consistent in their responses to their child’s misbehavior, children show less externalizing behavior (Barry et al., ##UREF##3##2009##; Gardner, ##REF##2745902##1989##; Gryczkowski et al., ##UREF##8##2010##; Halgunseth et al., ##REF##23544924##2013##; Lengua & Kovacs, ##UREF##10##2005##). However, these studies have focused on global self-reports, which mostly confound consistency of parental reactions across multiple episodes of misbehavior (e.g., punishing misbehavior in one episode, and condoning it in another) with consistency within a single episode of misbehavior (e.g., threatening with punishment, but leaving it in the end). Additionally, observational studies have mostly focused on within-episode consistency (Del Vecchio & O’Leary, ##REF##16597215##2006##; Gardner, ##REF##2745902##1989##). Therefore, it is not known whether within- and across-episode consistency play similar roles in the early development of externalizing problems. In this study, we use a daily diary approach to examine parental consistency both within and across episodes, offering a unique possibility to differentiate these two types of consistency. We examine associations between these aspects of consistency, and how they are associated with child externalizing behavior, both concurrently and longitudinally.</p>\",\n \"<title>Differentiating Within- from Across Disruptive Behavior Episode Consistency</title>\",\n \"<p id=\\\"Par3\\\">Empirically, studies using questionnaire measures of inconsistent discipline have indeed found it to be associated with more externalizing behavior in children (Barry et al., ##UREF##3##2009##; Gryczkowski et al., ##UREF##8##2010##; Halgunseth et al., ##REF##23544924##2013##; Lengua & Kovacs, ##UREF##10##2005##). However, research to date has not separated within-episode consistency from across-episode consistency. For instance, the frequently used Alabama Parenting Questionnaire (Frick et al., ##UREF##7##1999##) includes items in the inconsistency scale asking parents whether they threaten to punish, but then do not do so in the end, or whether they let their child out of punishment early, which are examples of inconsistent responding <italic>within</italic> a single episode of misbehavior. Additionally, items are included asking parents whether their punishment depends on their mood, which concerns consistency <italic>across</italic> episodes of misbehavior. It is important to note that these types of inconsistency do not necessarily co-occur. Parents may be relatively inconsistent in their responses within a single episode, perhaps not feeling competent enough to follow through with an initial course of action and giving up along the way (Deković et al., ##REF##20101464##2010##), yet they may be consistent in this response style across multiple episodes of misbehavior. Alternatively, parents may be consistent within an episode of misbehavior by providing a negative consequence and sticking to it, but respond in different ways to new episodes, for instance switching to ignoring the misbehavior or trying to redirect attention by making a joke. They may switch approaches because they are in a different mood state themselves (Rueger et al., ##UREF##13##2011##), or because they felt their approach was not effective in a previous instance.</p>\",\n \"<p id=\\\"Par4\\\">Several theoretical frameworks would predict that both types of consistency would be associated with increased externalizing problems. Attachment theory postulates that children form secure attachments to their caregivers if these are consistently responsive to their needs. With consistency, children learn that they can trust their caregiver to provide them with a secure base and a safe haven (Ainsworth et al., ##UREF##1##2015##). With inconsistency in caregiving, in contrast, children learn that their environment is unpredictable and insecure, increasing the risk of attachment problems and problem behavior (Madigan et al., ##REF##26619212##2016##). Supporting this notion, unpredictable behavior from parents has been shown to impact the stress response in both very young children (Noroña-Zhou et al., ##REF##32115696##2020##), as well as older children (Manczak et al., ##REF##28625195##2018##). A dysregulated stress system may in turn result in problems with self-regulation, resulting in heightened disruptive behavior (Wesarg et al., ##UREF##17##2020##). Additionally, unpredictability likely hampers children’s ability to develop a sense of self-efficacy, because it prevents them from developing a sense of control over situations (Bandura, ##UREF##2##1978##; Lippold et al., ##REF##28736495##2016##). Low self-efficacy, in turn, makes it more difficult to regulate anger and frustration, resulting in increased levels of disruptive behavior.</p>\",\n \"<p id=\\\"Par5\\\">Social learning theories emphasize operant conditioning principles which predict that within-episode inconsistency would be associated with increased problematic behavior because failing to follow-through with negative consequences reinforces the child for showing the misbehavior (Patterson, ##UREF##12##1982##). Additionally, consistent negative consequences across different disruptive behavior episodes, or at least a lack of reward, would quickly result in extinction of problematic behavior, whereas inconsistent discipline – with intermittent patterns of positive or negative reinforcement – makes it difficult for children to learn that their behavior is not acceptable. Both social learning and attachment frameworks would thus predict that within- as well as across-episode consistency would play a role in the maintenance of disruptive behavior.</p>\",\n \"<p id=\\\"Par6\\\">Although most questionnaire studies have confounded within- and across-episode consistency, some studies have specifically investigated within-episode consistency. Using observations of parent-child interactions, parents with children who were high on externalizing behavior were less likely to follow-through on an initial demand than parents of children who were low on externalizing behavior (Gardner, ##REF##2745902##1989##). Additionally, mothers of aggressive toddlers have been observed to be more likely to react with both overreactivity and laxness to instances of child aggression than mothers of non-aggressive toddlers (Del Vecchio & O’Leary, ##REF##16597215##2006##). However, with observations it is more difficult to assess how this within-episode consistency relates to across episode consistency. As observations are already so time-consuming, observing (enough) episodes of misbehavior to examine across episode consistency would be especially difficult.</p>\",\n \"<title>Using Daily Diaries to Assess Parental Consistency</title>\",\n \"<p id=\\\"Par7\\\">Daily diary assessments allow for overcoming the abovementioned limitations of single-time surveys or observations. Single-time self-reports of parental consistency may be biased, as correctly judging how consistent one is in their reactions might be even more difficult than broadly gauging whether one frequently reacts in a certain way (Lippold et al., ##REF##28736495##2016##). A more valid way to assess parental consistency may therefore be to repeatedly ask parents about their actual behavior, and analyze the consistency across their responses. A small scale study examined parents’ daily fluctuations in their overreactive and lax discipline, with lax discipline indicating inconsistency by not following through. Although overreactivity and laxness were positively correlated at the between-person level, indicating that mothers who were generally more overreactive were also more lax, there was a negative association at the within-person level, indicating that when mothers were overreactive in a certain instance, they were less likely to be lax at that time, and vice versa (Passini et al., ##REF##23458698##2013##). These results indicate that mothers can be inconsistent across episodes with regards to their within-episode consistency. However, associations with child externalizing behavior were not examined here. Another diary study did examine associations with externalizing behavior and found that for mothers with 5- 8-year old children maternal consistency across one week was associated with less child externalizing behavior (Villarreal et al., ##UREF##16##2021##). Consistency was operationalized as the within-person fluctuations in destructive conflict characteristics, which included both maternal punitive behavior as well as child and mother negativity, making it unclear whether this was an association with inconsistency in maternal behavior specifically. Yet another study examined daily reports of parents harsh and warm reactions to child misbehavior and found that consistency in the level of warmth was associated with less child ADHD symptoms, whereas consistency in the level of harshness was not (Li & Lansford, ##REF##29608072##2018##). As these studies operationalized inconsistency as fluctuations in mean levels of parenting behaviors, we do not know whether parents exhibited different types of responses in the same instance of misbehavior. For instance, some parents may be more likely to only punish the child by taking away a privilege, whereas other parents are more likely to yell at the child, take away a privilege and also comfort the child, with the latter type of response perhaps especially confusing to the child. A previous observational study has indeed found that some parents were more likely to use both positive and negative discipline strategies within the same episode than others, but did not examine associations between this inconsistency and externalizing behavior (van Zeijl et al., ##UREF##15##2007##).</p>\",\n \"<title>The Present Study</title>\",\n \"<p id=\\\"Par8\\\">In the present study, we use daily diary data to differentiate consistent responding within a single episode of misbehavior from consistent responding across multiple episodes of misbehavior and examine how they are each associated with the severity of disruptive behavior in children. We assess within-episode consistency as the mean number of different reactions to a specific episode of misbehavior, distinguishing positive attention, positive consequence, negative consequences, negative attention, and ignoring. Across-episode consistency is assessed as the overall dispersion of mothers’ reactions across all possible categories, taking into account the total number of episodes of misbehavior across a week. Parental reactions that were concentrated in fewer reaction categories were indicative of more consistency.</p>\",\n \"<p id=\\\"Par9\\\">We examine associations between the two types of consistency in two independent samples: a community sample (<italic>N</italic> = 134) of mothers of 1.5 to 3.5 year old children who completed a daily diary for 7 days, and an at-risk sample with heightened disruptive behavior (<italic>N</italic> = 149) of 3 to 8 year old children who filled out a daily diary for 14 days. Including these two samples has several benefits. First, we are able to examine whether associations between the two types of consistency conceptually replicate across multiple samples. Second, we can investigate whether both types of consistency are equally associated with disruptive behavior during a developmental stage when disruptive behavior starts to emerge and is relatively more normative, as during a developmental stage when disruptive behavior for most children has started to decline (Tremblay, ##REF##20146751##2010##) as a result of increases in children’s verbal-skills and overall self-regulation (Kuhn et al., ##REF##27101154##2016##). This allows us to investigate whether the two types of consistency play a similar role in the early emergence as in the maintenance of more persistent problem behavior across development. Contemporary accounts of social learning theories would predict that across-episode consistency may be less relevant for problem behavior that persists into preschool age, as repeated coercive cycling in parent-child dyads is thought to result in increasingly rigid, mutually negative interactions over time (Granic & Patterson, ##REF##16478303##2006##).</p>\",\n \"<p id=\\\"Par10\\\">We also examine the added value of computing these types of consistency from daily diary data over a single questionnaire assessment. To this end, we assessed whether it is a better predictor of children’s externalizing behavior one year later than a measure of consistency derived from a general questionnaire as administered in a single – less time-consuming baseline assessment – asking parents to estimate how often in the past month they showed the reactions that we also included in the diary study. Although this ‘general consistency’ measure confounds within- and across-episode consistency, it may still be a better measure of consistency than some of the current measures that are used. Rather than asking parents to report on how consistent they are, we merely asked parents to indicate how often they reacted a certain way, and compute consistency by calculating the dispersion of parents’ responses across the different reaction categories. This approach makes it less likely that this association is for instance explained by parents scoring themselves as inconsistent due to a more negative self-view (Smit et al., ##UREF##14##2021##). This likely plays a role in more traditional questionnaire measures, as most parents will realize that threatening with punishment and then not following through is not an effective parenting strategy. Our approach will allow us to examine whether taking multiple days of measurements to assess consistency is really necessary, or whether we have enough information when we just ask parents how often they react a certain way overall. Additionally, associations between the general consistency and within- and across-episode consistency can be examined to provide an indication of the validity of these measures.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Method</title>","<title>Sample</title>","<p id=\"Par11\">We included two samples, to allow for conceptual replication: Sample 1 is a community sample of 134 mothers of 1.5–3.5 year old children (<italic>M</italic> = 30 months, 44.3% girls), who reported on their child’s temper tantrums (frequency and severity) and their responses to these tantrums – in general across the past month and daily for 7 days. Mothers were predominantly, but not exclusively, highly educated (79% higher vocational or university education), and were not selected for experiencing any particular difficulties with their child. Seven percent indicated that they raised their child without a partner. No information regarding ethnicity was collected for this sample.</p>","<p id=\"Par12\">Between February 2016 and June 2017, undergraduate students recruited mothers with children between one and five years old for a research practical. They recruited mothers through online parenting fora and Facebook, and face-to-face outside in Amsterdam. Mothers who participated were also asked to forward the invitation for the study to other mothers. Mothers were informed about the study and gave informed consent in the online study environment. They filled out the general questionnaire regarding: children’s temper tantrums and their own reactions, their personality and sense of parenting competence (<italic>N</italic> = 884). Mothers who indicated that their child was between 1.5 and 3.5 years old were asked if they would like to participate in an additional daily diary study, and <italic>N</italic> = 382 indicated that they would like to receive more information. They were contacted by telephone, with <italic>N</italic> = 220 eventually participating. For this study, we only selected participants if they had participated in at least 4 days of the study (<italic>N</italic> = 185), and who had reported reactions for at least two tantrums, resulting in a final sample of <italic>N</italic> = 134. Participants completed an average of 6.76 days (<italic>SD</italic> = 0.62, range = 4–7 days). For this sample, mothers additionally reported on their child’s externalizing behavior one year later (<italic>n</italic> = 86). Participants who dropped out of the study did not differ significantly from those who participated one year later with regards to age of the mother (<italic>T</italic>(131) = 1.66, <italic>p</italic> = 0.062) or child (<italic>T</italic>(132) = -1.65, <italic>p</italic> = 0.050), the child’s sex (<italic>χ</italic>2(1) = 0.29, <italic>p</italic> = 0.589) or mothers’ educational level (<italic>χ</italic>2(4) = 0.74, <italic>p</italic> = 0.947). Additionally, there were no significant differences in children’s tantrum severity (<italic>T</italic>(132) = 0.90, <italic>p</italic> = 185) and mothers’ within-, across-, or general consistency at T1 (<italic>T</italic>(132) = -0.10,<italic> p</italic> = 0.922; <italic>T</italic>(132) = -0.09, <italic>p</italic> = 0.930; <italic>T</italic>(117) = -0.21, <italic>p</italic> = 0.833, respectively).</p>","<p id=\"Par13\">Mothers who filled out the general questionnaire had a chance of winning a gift certificate of 50 euros. Mothers who participated at least four days of the diary study received a small gift (a small book for their child – 2 euros) by mail. The study was approved by the ethical review board of the Department of Child Development and Education at the University of Amsterdam (#2015-CDE-6367).</p>","<p id=\"Par14\">Sample 2 consists of 149 parents (94% mothers) of 3–8 year old children (<italic>M</italic> = 5.88; 46% girls) oversampled for disruptive behavior – 17% had received parenting support for disruptive child behavior prior to the study; seven percent still received support during the study. Parents reported on how they generally responded to their child’s disruptive behavior as well as their daily responses for 14 days. Parents were predominantly, but not exclusively, highly educated (78% higher vocational or university education). Ten percent indicated that they raised their child without a partner. Culturally, 93% identified as Dutch, of which 19% identified as bicultural (mainly other European cultures or Moroccan). Others self-identified as Moroccan, other European, Asian, Surinamese, or Turkish. This roughly represents the Dutch population where around 25% of families has at least parts of their roots outside the Netherlands, most often in Turkey, Morocco and Surinam (Centraal Bureau voor de Statistiek [CBS], ##UREF##4##2020##).</p>","<p id=\"Par15\">Parents were recruited between March 2020 and June 2021, through social media, primary schools across the Netherlands, and databases from the University of Amsterdam of parents who consented to be contacted for research projects. Children with disruptive behavior problems were oversampled by advertising the study as targeting parents of children with mild to moderate levels of disruptive behavior. Parents who signed up were contacted by phone to explain the study procedures. Parents who agreed to participate signed informed consent, completed a baseline assessment (i.e., demographics and trait measures) with a link to daily online daily questionnaire (<italic>N</italic> = 156). For this study, we only selected participants if they had participated for at least 8 days of the study, and who had reported reactions to at least two disruptive behaviors, resulting in a final sample of <italic>N</italic> = 149. Participants completed an average of 13.22 days (<italic>SD</italic> = 1.34, range = 8–14 days).</p>","<p id=\"Par16\">Parents received €50 for completing the study. Study procedures were approved by the Ethical Review Board of the department of Child Development and Education of the University of Amsterdam (2019-CDE-11055).</p>","<title>Measures</title>","<title>Parental Consistency</title>","<p id=\"Par17\">In Sample 1, parents reported on how they responded to their child’s tantrums both in general over the past month – before they started the diary study, and for each tantrum that took place during the diary study (for a maximum of seven tantrums a day). Parents rated their responses from a list of 11 behaviors. We made a functional classification based on social learning principles (Patterson, ##UREF##12##1982##), differentiating punishment and reward from lack of punishment or reward, and positive and negative attention: negative consequence (2 items: ‘I sent my child to their room/corner/time-out’, ‘I punished my child’), positive consequence (‘I negotiated with my child’, ‘I gave in to my child’), withholding attention (‘I didn’t, I let my child cool off’, ‘I ignored my child’), negative attention (‘I became angry with my child’, ‘I grabbed my child’, ‘I spoke sternly to my child’), positive attention (‘I distracted my child’, ‘I comforted my child’). In the questionnaire about responses to tantrums in general, participants indicated how often they tended to respond that way (1 = <italic>never</italic>; 2 = <italic>almost never</italic>; 3 = < <italic>half the time</italic>; 4 = <italic>about half the time</italic>; 5 = > <italic>half the time</italic>; 6 = <italic>almost always</italic>; 7 = <italic>always</italic>), and we computed a mean score per category. In the daily diaries parents indicated whether or not they responded that way in that particular instance (0 = <italic>no</italic>; 1 = <italic>yes</italic>), allowing for multiple responses. Parents received a score of 1 in a category when answered yes to at least one of the responses in that category.</p>","<p id=\"Par18\">For our measure of across-episode consistency from the daily diary data and the general consistency measure from the baseline questionnaire, we calculated the Index of Qualitative Variation (IQV), which is a measure of variation for nominal variables – where a standard deviation cannot be computed due to qualitative rather than quantitative differences between categories, using the following formula (Frankfort-Nachmias & Leon-Guerrero, ##UREF##6##2018##):</p>","<p id=\"Par19\">For each response category we first computed what proportions of the total number of responses they were for each individual. The squared proportions of each of the categories are summed and then subtracted from 1 and multiplied by K, which is the number of categories (5 in our study). This is then divided by the number of categories minus 1. The resulting value can range from 0 to 1.00, with higher scores indicating greater inconsistency. Therefore, we subtracted this value from 1, so that higher scores indicated greater consistency.</p>","<p id=\"Par20\">From the diary data, we additionally computed a measure of within-episode consistency. For each tantrum, we summed the total number of responses in the different categories (potential range 1–5), and then computed a mean score across all tantrums that were reported during the study. The observed range was 1–3 with higher values indicating less consistency. For ease of interpretation we recoded this variable so that higher values indicated greater consistency by subtracting the values from 3. The final variable thus ranged from 0–2. The intraclass correlation coefficient (ICC) for within-episode consistency was 0.20. This value is similar to ICCs that have previously been reported for parenting variables in diary studies, such as psychological control and autonomy support (Mabbe et al., ##UREF##11##2018##), with somewhat higher levels of around 0.33 also reported for psychological control (Aunola et al., ##REF##23750527##2013##).</p>","<p id=\"Par21\">In Sample 2, parents were asked how they responded to their child’s disruptive behavior in general at the start of the study, and how they responded to their child’s most challenging disruptive behavior that particular day (if any) in the diary study. Parents rated their responses from a list of 13 behaviors, which we grouped into the same five categories as for Sample 1: negative consequence (4 items: ‘I sent my child to their room for at least an hour’, ‘I gave my child a short time-out, away from others’, ‘I took something nice away from my child (e.g., toys or screen time)’, I gave my child extra chores (e.g., set the table)’), positive consequence (2 items: ‘I gave my child his/her way’, ‘I gave in to my child’), withholding attention (2 items: ‘I did nothing’, ‘I talked about it with my child afterwards’), negative attention (3 items: ‘I yelled/swore’, ‘I said things I didn’t mean’, ‘I threatened with punishment, but did not punish’), positive attention (2 items: ‘I begged my child to stop’, ‘I used humor to distract my child’). In the general questionnaire, participants indicated how often they tended to respond that way on a 5-point Likert type scale (1 = <italic>less than once a week,</italic> 2 = <italic>once a week</italic>; 3 = <italic>few times a week</italic>; 4 = <italic>once a day</italic>; 5 = <italic>several times a day</italic>), and we computed a mean score per category. In the daily diaries study parents indicated whether or not they responded that way in that particular instance (0 = <italic>no</italic>, 1 = <italic>yes</italic>), allowing for multiple responses. Parents received a score of 1 in a category when answered yes to at least one of the responses in that category. When parents reported that their child had not shown any disruptive behavior that day, the response category was coded as missing.</p>","<p id=\"Par22\">Like in Sample 1, we computed the IQV as a measure of across-episode consistency from the daily diary data and a measure of general consistency from the baseline questionnaire, and the mean number of selected categories of responses per episode as our measure of within-episode consistency. For within-episode consistency, the ICC was 0.18.</p>","<title>Child Externalizing Behavior</title>","<p id=\"Par23\">In Sample 1, we calculated a measure of severity of the child’s tantrum behavior from the daily diary reports, by summing for each tantrum the total number of aggressive (hitting, kicking, biting, throwing an object, pushing/pulling, spitting, grabbing) and self-injurious behaviors (banging head, holding breath, freezing). A previous study on this sample found that a profile with elevated levels on these behaviors was predictive of both internalizing and externalizing problems above and beyond tantrum frequency and duration (Van den Akker et al., ##REF##35316228##2022##). The ICC for tantrum severity was 0.24.</p>","<p id=\"Par24\">One year later (T2), parents in Sample 1 filled out 24 items of the Externalizing Problem Behavior Scale (the attention problem and aggressive behavior problem subscales, e.g., “My child does not seem to feel guilty after misbehavior”) of the Dutch version of the Child Behavior Checklist (1, 5–5) (Achenbach & Rescorla, ##UREF##0##2000##). Parents were instructed to indicate for the past 2 months how characteristic the item was of their child's behavior, with each item rated as 0 (<italic>not true</italic>), 1 (<italic>sometimes/somewhat true</italic>), or 2 (<italic>often/very true</italic>). Cronbach's alpha for the present sample was 0.89.</p>","<p id=\"Par25\">For Sample 2, rather than indicating how many disruptive behaviors children had displayed, parents rated children’s overall level of disruptive child behavior at T1 each day (i.e., “how disruptive was your child’s behavior today?”) on a 1 − 10 scale. A mean score across the 14 days was computed. The ICC was 0.32.</p>","<title>Analysis Plan</title>","<p id=\"Par26\">Hypotheses and analyses were registered on the Open science Framework (<ext-link ext-link-type=\"uri\" xlink:href=\"https://osf.io/tecr4/?view_only=0c6f1e3d6b2f46e49c5599c4c168be3c\">https://osf.io/tecr4/?view_only=0c6f1e3d6b2f46e49c5599c4c168be3c</ext-link>).</p>","<p id=\"Par27\">We first winsorized outliers (outside 1.5* IQR) to the nearest value if there was a gap in data between that range and the outlier. In Sample 1, for the within-episode consistency measures as derived from the daily diaries we identified two outliers, and for the across-episode consistency derived from the questionnaire asking about tantrums in general, we identified one outlier. For the severity of daily disruptive behavior we identified six outliers, and for externalizing behavior we identified two outliers. In Sample 2, for the within-episode consistency measure as derived from the daily diaries we identified one outlier, and for the across-episode consistency we identified four outlier. For the severity of daily disruptive behavior we identified four outliers. To answer our first research question- whether our measures of within- and across-episode consistency measure different but related aspects of consistency – we computed correlations. Next, we performed regression analyses to predict the severity of daily disruptive behavior from the within- and across-episode consistency measures to see whether they were uniquely associated. In a next step, we examined whether associations were significant above and beyond mean levels of the daily parental reactions. These analyses control for child sex and age and parental educational level and are performed on both Samples 1 and 2. As 11 parents in Sample 2 received parenting support for their child’s behavior, we also controlled for received support in Sample 2. Finally, we performed regression analysis in SPSS (version 28) to examine – in Sample 1 – whether within- and across- episode consistency as derived from the daily diary reports longitudinally predict child externalizing behavior problems one year later (T2), over and above a measure of consistency derived from estimates of parental behavior across the past month, controlling for the severity of temper tantrum behavior as reported in the diary study at T1.</p>"],"string":"[\n \"<title>Method</title>\",\n \"<title>Sample</title>\",\n \"<p id=\\\"Par11\\\">We included two samples, to allow for conceptual replication: Sample 1 is a community sample of 134 mothers of 1.5–3.5 year old children (<italic>M</italic> = 30 months, 44.3% girls), who reported on their child’s temper tantrums (frequency and severity) and their responses to these tantrums – in general across the past month and daily for 7 days. Mothers were predominantly, but not exclusively, highly educated (79% higher vocational or university education), and were not selected for experiencing any particular difficulties with their child. Seven percent indicated that they raised their child without a partner. No information regarding ethnicity was collected for this sample.</p>\",\n \"<p id=\\\"Par12\\\">Between February 2016 and June 2017, undergraduate students recruited mothers with children between one and five years old for a research practical. They recruited mothers through online parenting fora and Facebook, and face-to-face outside in Amsterdam. Mothers who participated were also asked to forward the invitation for the study to other mothers. Mothers were informed about the study and gave informed consent in the online study environment. They filled out the general questionnaire regarding: children’s temper tantrums and their own reactions, their personality and sense of parenting competence (<italic>N</italic> = 884). Mothers who indicated that their child was between 1.5 and 3.5 years old were asked if they would like to participate in an additional daily diary study, and <italic>N</italic> = 382 indicated that they would like to receive more information. They were contacted by telephone, with <italic>N</italic> = 220 eventually participating. For this study, we only selected participants if they had participated in at least 4 days of the study (<italic>N</italic> = 185), and who had reported reactions for at least two tantrums, resulting in a final sample of <italic>N</italic> = 134. Participants completed an average of 6.76 days (<italic>SD</italic> = 0.62, range = 4–7 days). For this sample, mothers additionally reported on their child’s externalizing behavior one year later (<italic>n</italic> = 86). Participants who dropped out of the study did not differ significantly from those who participated one year later with regards to age of the mother (<italic>T</italic>(131) = 1.66, <italic>p</italic> = 0.062) or child (<italic>T</italic>(132) = -1.65, <italic>p</italic> = 0.050), the child’s sex (<italic>χ</italic>2(1) = 0.29, <italic>p</italic> = 0.589) or mothers’ educational level (<italic>χ</italic>2(4) = 0.74, <italic>p</italic> = 0.947). Additionally, there were no significant differences in children’s tantrum severity (<italic>T</italic>(132) = 0.90, <italic>p</italic> = 185) and mothers’ within-, across-, or general consistency at T1 (<italic>T</italic>(132) = -0.10,<italic> p</italic> = 0.922; <italic>T</italic>(132) = -0.09, <italic>p</italic> = 0.930; <italic>T</italic>(117) = -0.21, <italic>p</italic> = 0.833, respectively).</p>\",\n \"<p id=\\\"Par13\\\">Mothers who filled out the general questionnaire had a chance of winning a gift certificate of 50 euros. Mothers who participated at least four days of the diary study received a small gift (a small book for their child – 2 euros) by mail. The study was approved by the ethical review board of the Department of Child Development and Education at the University of Amsterdam (#2015-CDE-6367).</p>\",\n \"<p id=\\\"Par14\\\">Sample 2 consists of 149 parents (94% mothers) of 3–8 year old children (<italic>M</italic> = 5.88; 46% girls) oversampled for disruptive behavior – 17% had received parenting support for disruptive child behavior prior to the study; seven percent still received support during the study. Parents reported on how they generally responded to their child’s disruptive behavior as well as their daily responses for 14 days. Parents were predominantly, but not exclusively, highly educated (78% higher vocational or university education). Ten percent indicated that they raised their child without a partner. Culturally, 93% identified as Dutch, of which 19% identified as bicultural (mainly other European cultures or Moroccan). Others self-identified as Moroccan, other European, Asian, Surinamese, or Turkish. This roughly represents the Dutch population where around 25% of families has at least parts of their roots outside the Netherlands, most often in Turkey, Morocco and Surinam (Centraal Bureau voor de Statistiek [CBS], ##UREF##4##2020##).</p>\",\n \"<p id=\\\"Par15\\\">Parents were recruited between March 2020 and June 2021, through social media, primary schools across the Netherlands, and databases from the University of Amsterdam of parents who consented to be contacted for research projects. Children with disruptive behavior problems were oversampled by advertising the study as targeting parents of children with mild to moderate levels of disruptive behavior. Parents who signed up were contacted by phone to explain the study procedures. Parents who agreed to participate signed informed consent, completed a baseline assessment (i.e., demographics and trait measures) with a link to daily online daily questionnaire (<italic>N</italic> = 156). For this study, we only selected participants if they had participated for at least 8 days of the study, and who had reported reactions to at least two disruptive behaviors, resulting in a final sample of <italic>N</italic> = 149. Participants completed an average of 13.22 days (<italic>SD</italic> = 1.34, range = 8–14 days).</p>\",\n \"<p id=\\\"Par16\\\">Parents received €50 for completing the study. Study procedures were approved by the Ethical Review Board of the department of Child Development and Education of the University of Amsterdam (2019-CDE-11055).</p>\",\n \"<title>Measures</title>\",\n \"<title>Parental Consistency</title>\",\n \"<p id=\\\"Par17\\\">In Sample 1, parents reported on how they responded to their child’s tantrums both in general over the past month – before they started the diary study, and for each tantrum that took place during the diary study (for a maximum of seven tantrums a day). Parents rated their responses from a list of 11 behaviors. We made a functional classification based on social learning principles (Patterson, ##UREF##12##1982##), differentiating punishment and reward from lack of punishment or reward, and positive and negative attention: negative consequence (2 items: ‘I sent my child to their room/corner/time-out’, ‘I punished my child’), positive consequence (‘I negotiated with my child’, ‘I gave in to my child’), withholding attention (‘I didn’t, I let my child cool off’, ‘I ignored my child’), negative attention (‘I became angry with my child’, ‘I grabbed my child’, ‘I spoke sternly to my child’), positive attention (‘I distracted my child’, ‘I comforted my child’). In the questionnaire about responses to tantrums in general, participants indicated how often they tended to respond that way (1 = <italic>never</italic>; 2 = <italic>almost never</italic>; 3 = < <italic>half the time</italic>; 4 = <italic>about half the time</italic>; 5 = > <italic>half the time</italic>; 6 = <italic>almost always</italic>; 7 = <italic>always</italic>), and we computed a mean score per category. In the daily diaries parents indicated whether or not they responded that way in that particular instance (0 = <italic>no</italic>; 1 = <italic>yes</italic>), allowing for multiple responses. Parents received a score of 1 in a category when answered yes to at least one of the responses in that category.</p>\",\n \"<p id=\\\"Par18\\\">For our measure of across-episode consistency from the daily diary data and the general consistency measure from the baseline questionnaire, we calculated the Index of Qualitative Variation (IQV), which is a measure of variation for nominal variables – where a standard deviation cannot be computed due to qualitative rather than quantitative differences between categories, using the following formula (Frankfort-Nachmias & Leon-Guerrero, ##UREF##6##2018##):</p>\",\n \"<p id=\\\"Par19\\\">For each response category we first computed what proportions of the total number of responses they were for each individual. The squared proportions of each of the categories are summed and then subtracted from 1 and multiplied by K, which is the number of categories (5 in our study). This is then divided by the number of categories minus 1. The resulting value can range from 0 to 1.00, with higher scores indicating greater inconsistency. Therefore, we subtracted this value from 1, so that higher scores indicated greater consistency.</p>\",\n \"<p id=\\\"Par20\\\">From the diary data, we additionally computed a measure of within-episode consistency. For each tantrum, we summed the total number of responses in the different categories (potential range 1–5), and then computed a mean score across all tantrums that were reported during the study. The observed range was 1–3 with higher values indicating less consistency. For ease of interpretation we recoded this variable so that higher values indicated greater consistency by subtracting the values from 3. The final variable thus ranged from 0–2. The intraclass correlation coefficient (ICC) for within-episode consistency was 0.20. This value is similar to ICCs that have previously been reported for parenting variables in diary studies, such as psychological control and autonomy support (Mabbe et al., ##UREF##11##2018##), with somewhat higher levels of around 0.33 also reported for psychological control (Aunola et al., ##REF##23750527##2013##).</p>\",\n \"<p id=\\\"Par21\\\">In Sample 2, parents were asked how they responded to their child’s disruptive behavior in general at the start of the study, and how they responded to their child’s most challenging disruptive behavior that particular day (if any) in the diary study. Parents rated their responses from a list of 13 behaviors, which we grouped into the same five categories as for Sample 1: negative consequence (4 items: ‘I sent my child to their room for at least an hour’, ‘I gave my child a short time-out, away from others’, ‘I took something nice away from my child (e.g., toys or screen time)’, I gave my child extra chores (e.g., set the table)’), positive consequence (2 items: ‘I gave my child his/her way’, ‘I gave in to my child’), withholding attention (2 items: ‘I did nothing’, ‘I talked about it with my child afterwards’), negative attention (3 items: ‘I yelled/swore’, ‘I said things I didn’t mean’, ‘I threatened with punishment, but did not punish’), positive attention (2 items: ‘I begged my child to stop’, ‘I used humor to distract my child’). In the general questionnaire, participants indicated how often they tended to respond that way on a 5-point Likert type scale (1 = <italic>less than once a week,</italic> 2 = <italic>once a week</italic>; 3 = <italic>few times a week</italic>; 4 = <italic>once a day</italic>; 5 = <italic>several times a day</italic>), and we computed a mean score per category. In the daily diaries study parents indicated whether or not they responded that way in that particular instance (0 = <italic>no</italic>, 1 = <italic>yes</italic>), allowing for multiple responses. Parents received a score of 1 in a category when answered yes to at least one of the responses in that category. When parents reported that their child had not shown any disruptive behavior that day, the response category was coded as missing.</p>\",\n \"<p id=\\\"Par22\\\">Like in Sample 1, we computed the IQV as a measure of across-episode consistency from the daily diary data and a measure of general consistency from the baseline questionnaire, and the mean number of selected categories of responses per episode as our measure of within-episode consistency. For within-episode consistency, the ICC was 0.18.</p>\",\n \"<title>Child Externalizing Behavior</title>\",\n \"<p id=\\\"Par23\\\">In Sample 1, we calculated a measure of severity of the child’s tantrum behavior from the daily diary reports, by summing for each tantrum the total number of aggressive (hitting, kicking, biting, throwing an object, pushing/pulling, spitting, grabbing) and self-injurious behaviors (banging head, holding breath, freezing). A previous study on this sample found that a profile with elevated levels on these behaviors was predictive of both internalizing and externalizing problems above and beyond tantrum frequency and duration (Van den Akker et al., ##REF##35316228##2022##). The ICC for tantrum severity was 0.24.</p>\",\n \"<p id=\\\"Par24\\\">One year later (T2), parents in Sample 1 filled out 24 items of the Externalizing Problem Behavior Scale (the attention problem and aggressive behavior problem subscales, e.g., “My child does not seem to feel guilty after misbehavior”) of the Dutch version of the Child Behavior Checklist (1, 5–5) (Achenbach & Rescorla, ##UREF##0##2000##). Parents were instructed to indicate for the past 2 months how characteristic the item was of their child's behavior, with each item rated as 0 (<italic>not true</italic>), 1 (<italic>sometimes/somewhat true</italic>), or 2 (<italic>often/very true</italic>). Cronbach's alpha for the present sample was 0.89.</p>\",\n \"<p id=\\\"Par25\\\">For Sample 2, rather than indicating how many disruptive behaviors children had displayed, parents rated children’s overall level of disruptive child behavior at T1 each day (i.e., “how disruptive was your child’s behavior today?”) on a 1 − 10 scale. A mean score across the 14 days was computed. The ICC was 0.32.</p>\",\n \"<title>Analysis Plan</title>\",\n \"<p id=\\\"Par26\\\">Hypotheses and analyses were registered on the Open science Framework (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://osf.io/tecr4/?view_only=0c6f1e3d6b2f46e49c5599c4c168be3c\\\">https://osf.io/tecr4/?view_only=0c6f1e3d6b2f46e49c5599c4c168be3c</ext-link>).</p>\",\n \"<p id=\\\"Par27\\\">We first winsorized outliers (outside 1.5* IQR) to the nearest value if there was a gap in data between that range and the outlier. In Sample 1, for the within-episode consistency measures as derived from the daily diaries we identified two outliers, and for the across-episode consistency derived from the questionnaire asking about tantrums in general, we identified one outlier. For the severity of daily disruptive behavior we identified six outliers, and for externalizing behavior we identified two outliers. In Sample 2, for the within-episode consistency measure as derived from the daily diaries we identified one outlier, and for the across-episode consistency we identified four outlier. For the severity of daily disruptive behavior we identified four outliers. To answer our first research question- whether our measures of within- and across-episode consistency measure different but related aspects of consistency – we computed correlations. Next, we performed regression analyses to predict the severity of daily disruptive behavior from the within- and across-episode consistency measures to see whether they were uniquely associated. In a next step, we examined whether associations were significant above and beyond mean levels of the daily parental reactions. These analyses control for child sex and age and parental educational level and are performed on both Samples 1 and 2. As 11 parents in Sample 2 received parenting support for their child’s behavior, we also controlled for received support in Sample 2. Finally, we performed regression analysis in SPSS (version 28) to examine – in Sample 1 – whether within- and across- episode consistency as derived from the daily diary reports longitudinally predict child externalizing behavior problems one year later (T2), over and above a measure of consistency derived from estimates of parental behavior across the past month, controlling for the severity of temper tantrum behavior as reported in the diary study at T1.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par28\">On average, children in Sample 1 had an average 5.90 tantrums during the 7-day period (<italic>SD</italic> = 4.21, range 2–20), and for children in Sample 2 the mean level of disruptive behavior was rated 3.32 on the 10 point scale across the 14 days (<italic>SD</italic> = 1.21, range 1.14–6.54). Descriptives and intercorrelations for Samples 1 and 2 are provided in Table ##TAB##0##1##. In both samples, within- and across episode consistently were significantly associated. Associations were strong, but not so strong as to indicate that they would actually be measuring the same thing. In Sample 1, only across-episode consistency was negatively associated with disruptive behavior severity; in Sample 2, both within- and across-episode consistency were negatively associated with disruptive behavior severity.</p>","<title>Within- and Across-episode Consistency and Severity of Child Disruptive Behavior</title>","<p id=\"Par29\">To examine whether within- and across-episode consistency were uniquely associated with the severity of daily disruptive behavior, we performed regression analyses. In Sample 1, the first step, controlling for age and sex of the child and educational level of the parent was not significant (<italic>F</italic>(3,130) = 0.47, <italic>p</italic> = 0.707, <italic>R</italic><sup><italic>2</italic></sup> = 0.01). Adding across-episode and within-episode consistency resulted in a significant improvement of the model (Δ<italic>F</italic>(2,128) = 6.95, <italic>p</italic> = 0.001, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.10): when parents were more consistent across disruptive behavior episodes, children displayed less severe disruptive behavior, whereas within-episode consistency was not significantly associated with severity of daily disruptive behavior (Table ##TAB##1##2##).</p>","<p id=\"Par30\">Results of Sample 2 conceptually replicated the findings of Sample 1. The first step, controlling for sex of the child and educational level of the parent was not significant (<italic>F</italic>(3,144) = 0.32, <italic>p</italic> = 0.808, <italic>R</italic><sup><italic>2</italic></sup> = 0.01). Adding across-episode and within-episode consistency resulted in a significant improvement of the model (Δ<italic>F</italic>(2,142) = 9.66, <italic>p</italic> < 0.001, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.12): only across-episode consistency, not within-episode consistency, was significantly associated with severity of daily disruptive behavior (Table ##TAB##2##3##).</p>","<p id=\"Par31\">In a next set of regression analyses, we examined whether within- and across-episode consistency predicted the severity of daily disruptive behavior, above and beyond mean levels of the different response categories. In Sample 1, adding the mean levels of the proportions of the five parental responses across the seven days did not result in a significant improvement over the model including only age and sex of the child and educational level of the parent (Δ<italic>F</italic>(5,125) = 0.47, <italic>p</italic> = 0.801, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.02), indicating that how much parents displayed a certain type of reaction was not predictive of the child’s disruptive behavior. Adding within- and across-episode consistency to the model did result in a significant improvement (Δ<italic>F</italic>(2,123) = 8.63, <italic>p</italic> < 0.001, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.12). Across-episode consistency was predictive of daily disruptive behavior severity, whereas within-episode consistency was not. For model coefficients, see Table ##TAB##1##2##.</p>","<p id=\"Par32\">Different from Sample 1, in Sample 2, adding the mean levels of the proportions of the five parental responses did result in a significant improvement over the model including only sex and educational level of the parent (Δ<italic>F</italic>(5,139) = 6.21, <italic>p</italic> < 0.001, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.18). Providing negative consequences and giving negative attention to disruptive behavior, were each associated with more severe daily disruptive behavior. Here, adding within- and across-episode consistency to the model did not result in a significant improvement (Δ<italic>F</italic>(2,137) = 0.55, <italic>p</italic> = 0.581, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.01), indicating that the association between consistency and child disruptive behavior was explained by the individual negative responses. For model coefficients, see Table ##TAB##2##3##.</p>","<title>Prediction of Externalizing Problems One Year Later</title>","<p id=\"Par33\">In Sample 1, we examined whether the consistency measures predicted externalizing behavior one year later, controlling for the severity of daily disruptive behavior at T1 and for parent-reported consistency as derived from a one-time questionnaire about general responses to tantrums. The first step was significant (<italic>F</italic>(4,73) = 5.01, <italic>p</italic> = 0.001, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.22): more severe daily disruptive behavior was predictive of more externalizing behavior one year later. Above and beyond this effect, less consistency as derived from parents’ reports of how frequently they generally displayed certain responses to their child’s tantrums (i.e. general consistency as computed from the baseline measure), was predictive of more externalizing problems. Importantly however, adding within- and across-episode consistency to the model did not result in a significant improvement (Δ<italic>F</italic>(2,71) = 0.001, <italic>p</italic> = 0.999, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.00). These results indicate that consistency in parental responses as derived from their reports of how often in the last month they displayed certain reactions, was longitudinally predictive of externalizing problems, whereas within- and across-episode consistency as derived from the daily diary measures were not. For model coefficients, see Table ##TAB##3##4##.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par28\\\">On average, children in Sample 1 had an average 5.90 tantrums during the 7-day period (<italic>SD</italic> = 4.21, range 2–20), and for children in Sample 2 the mean level of disruptive behavior was rated 3.32 on the 10 point scale across the 14 days (<italic>SD</italic> = 1.21, range 1.14–6.54). Descriptives and intercorrelations for Samples 1 and 2 are provided in Table ##TAB##0##1##. In both samples, within- and across episode consistently were significantly associated. Associations were strong, but not so strong as to indicate that they would actually be measuring the same thing. In Sample 1, only across-episode consistency was negatively associated with disruptive behavior severity; in Sample 2, both within- and across-episode consistency were negatively associated with disruptive behavior severity.</p>\",\n \"<title>Within- and Across-episode Consistency and Severity of Child Disruptive Behavior</title>\",\n \"<p id=\\\"Par29\\\">To examine whether within- and across-episode consistency were uniquely associated with the severity of daily disruptive behavior, we performed regression analyses. In Sample 1, the first step, controlling for age and sex of the child and educational level of the parent was not significant (<italic>F</italic>(3,130) = 0.47, <italic>p</italic> = 0.707, <italic>R</italic><sup><italic>2</italic></sup> = 0.01). Adding across-episode and within-episode consistency resulted in a significant improvement of the model (Δ<italic>F</italic>(2,128) = 6.95, <italic>p</italic> = 0.001, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.10): when parents were more consistent across disruptive behavior episodes, children displayed less severe disruptive behavior, whereas within-episode consistency was not significantly associated with severity of daily disruptive behavior (Table ##TAB##1##2##).</p>\",\n \"<p id=\\\"Par30\\\">Results of Sample 2 conceptually replicated the findings of Sample 1. The first step, controlling for sex of the child and educational level of the parent was not significant (<italic>F</italic>(3,144) = 0.32, <italic>p</italic> = 0.808, <italic>R</italic><sup><italic>2</italic></sup> = 0.01). Adding across-episode and within-episode consistency resulted in a significant improvement of the model (Δ<italic>F</italic>(2,142) = 9.66, <italic>p</italic> < 0.001, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.12): only across-episode consistency, not within-episode consistency, was significantly associated with severity of daily disruptive behavior (Table ##TAB##2##3##).</p>\",\n \"<p id=\\\"Par31\\\">In a next set of regression analyses, we examined whether within- and across-episode consistency predicted the severity of daily disruptive behavior, above and beyond mean levels of the different response categories. In Sample 1, adding the mean levels of the proportions of the five parental responses across the seven days did not result in a significant improvement over the model including only age and sex of the child and educational level of the parent (Δ<italic>F</italic>(5,125) = 0.47, <italic>p</italic> = 0.801, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.02), indicating that how much parents displayed a certain type of reaction was not predictive of the child’s disruptive behavior. Adding within- and across-episode consistency to the model did result in a significant improvement (Δ<italic>F</italic>(2,123) = 8.63, <italic>p</italic> < 0.001, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.12). Across-episode consistency was predictive of daily disruptive behavior severity, whereas within-episode consistency was not. For model coefficients, see Table ##TAB##1##2##.</p>\",\n \"<p id=\\\"Par32\\\">Different from Sample 1, in Sample 2, adding the mean levels of the proportions of the five parental responses did result in a significant improvement over the model including only sex and educational level of the parent (Δ<italic>F</italic>(5,139) = 6.21, <italic>p</italic> < 0.001, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.18). Providing negative consequences and giving negative attention to disruptive behavior, were each associated with more severe daily disruptive behavior. Here, adding within- and across-episode consistency to the model did not result in a significant improvement (Δ<italic>F</italic>(2,137) = 0.55, <italic>p</italic> = 0.581, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.01), indicating that the association between consistency and child disruptive behavior was explained by the individual negative responses. For model coefficients, see Table ##TAB##2##3##.</p>\",\n \"<title>Prediction of Externalizing Problems One Year Later</title>\",\n \"<p id=\\\"Par33\\\">In Sample 1, we examined whether the consistency measures predicted externalizing behavior one year later, controlling for the severity of daily disruptive behavior at T1 and for parent-reported consistency as derived from a one-time questionnaire about general responses to tantrums. The first step was significant (<italic>F</italic>(4,73) = 5.01, <italic>p</italic> = 0.001, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.22): more severe daily disruptive behavior was predictive of more externalizing behavior one year later. Above and beyond this effect, less consistency as derived from parents’ reports of how frequently they generally displayed certain responses to their child’s tantrums (i.e. general consistency as computed from the baseline measure), was predictive of more externalizing problems. Importantly however, adding within- and across-episode consistency to the model did not result in a significant improvement (Δ<italic>F</italic>(2,71) = 0.001, <italic>p</italic> = 0.999, Δ<italic>R</italic><sup><italic>2</italic></sup> = 0.00). These results indicate that consistency in parental responses as derived from their reports of how often in the last month they displayed certain reactions, was longitudinally predictive of externalizing problems, whereas within- and across-episode consistency as derived from the daily diary measures were not. For model coefficients, see Table ##TAB##3##4##.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par34\">Aim of this study was to investigate how within- and across-episode parental consistency in responding to misbehavior are associated to externalizing problem behavior in children, using a daily diary approach. Within- and across-episode consistency were moderately strongly correlated with each other, but only across-episode consistency was associated with the severity of daily disruptive behavior. In Sample 1, this association was significant above and beyond the content of the parental reactions, whereas in Sample 2, the association was explained by the fact that parents who were more consistent across episodes were less likely to provide negative consequences or negative attention for the disruptive behavior. When we compared the longitudinal predictive value of the measures of consistency derived from the daily diaries to a measure of consistency derived from how often parents indicate they usually react, we found that the measures from the daily diary did not predict externalizing behavior problems one year later, whereas parental consistency as derived from the general questionnaire did.</p>","<title>Within- and Across-episode Consistency</title>","<p id=\"Par35\">In both samples, we found that the two types of consistency were significantly associated with each other, but the associations were not so strong that they indicated that they reflected the same underlying construct. This indicates that some parents were relatively higher on across-episode consistency whereas others were relatively higher on within-episode consistency. These results support the idea that it is relevant to separate the two types of consistency. The correlations between the two types of consistency were quite similar across the two different samples, as were the associations between the two types of consistency as computed from the daily diary data and the ‘trait’ measure of consistency that was computed based on how parents indicated that they generally responded to disruptive behavior in the baseline measure. These associations provide some validation of these measures.</p>","<p id=\"Par36\">Interestingly, despite moderately strong correlations between within- and across episode consistency, when associations between the two types of consistency and the severity of daily disruptive behavior were examined, across-episode consistency was significantly associated with the severity of daily disruptive behavior in both samples, whereas within-episode consistency was not. That within-episode consistency was not associated with disruptive behavior severity is not in line with observational findings that lower within-episode consistency differentiated mother-child dyads with conduct-problems from those without (Gardner, ##REF##2745902##1989##), and mothers of aggressive toddlers from those without (Del Vecchio & O’Leary, ##REF##16597215##2006##). These findings may indicate that with regards to within-episode consistency, the rewarding nature of the final response – when a parent eventually gives in or does not follow through on their initial demand – is more important in explaining this effect of within-episode consistency rather than the mere variation of types of responses as was assessed by our measure. Parents who reward the child for misbehavior are likely to first provide negative attention for instance, scolding the child, and only give in after a sequence of different types of reactions (Gardner, ##REF##2745902##1989##). Alternatively it may mean that across-episode consistency is actually more strongly associated with disruptive behavior. As previous studies examining these rewarding interaction sequences have not controlled for across-episode consistency, more research is necessary to examine whether this association also disappears when across-episode consistency is taken into account.</p>","<p id=\"Par37\">We add to previous findings that inconsistency in responses across episodes of misbehavior may be specifically associated with more severe disruptive behavior, regardless of the variation in types of responses within single episodes. In Sample 1, parents who were less consistent not only varied more within- or across-episode in how they responded to children’s tantrum, but also more frequently used each of the responses, both positive (e.g., positive consequences such as ‘giving in’) and negative (e.g., negative consequences such as ‘punishing’). Importantly, it was the variation between responses rather than the frequency of the individual responses that was associated with child disruptive behavior. This might indicate that parental consistency in responding is more important for lowering child disruptive behavior than how parents respond specifically. Alternatively, it might mean that when children show more disruptive behavior, parents are more likely to try out different ways of responding in an attempt to deal with it. Other studies have found that behavioral or emotional variation is associated with more maladjustment in young children as observed at a more micro time-scale, across real-time interaction. For instance, variability in affective displays has been related to more externalizing problems in mother-toddler dyads (Lunkenheimer et al., ##REF##23786697##2011##), as has behavioral variability (Lunkenheimer et al., ##REF##33180517##2020##).</p>","<p id=\"Par38\">Our findings support the idea that, in a non-clinical sample, predictable parental responses are most important in reducing disruptive behavior. Unpredictable behavior from parents has been shown to impact the stress response in infants, with a blunted cortisol response to a painful stressors for infants of mothers who’s behavior was less predictable (Noroña-Zhou et al., ##REF##32115696##2020##), and variability in the affective quality of mother-child interactions and even in the timing of leisure activities, has been associated with an increased production of proinflammatory cytokines, an index of stress-reactivity, for youth (Manczak et al., ##REF##28625195##2018##). More research is necessary to understand whether these processes play a role in explaining the association between across-episode consistency in parenting behavior and child disruptive behavior.</p>","<p id=\"Par39\">In Sample 2, a sample with older children who were at-risk for problem behavior, across-episode consistency was no longer associated with disruptive behavior above and beyond the individual reactions, whereas the frequency of providing negative consequences and negative attention were associated with more severe disruptive behavior. It seems that, whereas in Sample 1 it did not matter so much what parents did to reduce child disruptive behavior, as long as they did it consistently across episodes, in this sample negative responding was specifically associated with disruptive child behavior. Perhaps for families with older children with elevated levels of disruptive behavior as in Sample 2, parent and child have more strongly established patterns of negative responding to each other (Granic & Patterson, ##REF##16478303##2006##). In support of this idea, in studies of school aged children, affective variability has been associated with less rather than more behavioral problems (Granic et al., ##REF##17549621##2007##; Hollenstein et al., ##REF##15648527##2004##). Settling into a rigid, negative interaction style is a process that takes place in the interaction between parent and child over several years. Heightened variability in other areas may still have negative effects in older children and adolescents. For instance, higher variability in experienced stressors has been associated with worse emotional adjustment in adolescents (Zheng et al., ##REF##36273075##2022##), and higher variability in daily activities is associated with lower psychological well-being in young adults (Lee et al., ##UREF##9##2018##).</p>","<title>Daily Diary Measures</title>","<p id=\"Par40\">In this study, daily disruptive behavior was associated with externalizing problems one year later, and within- and across-episode consistency were associated with our measure of general consistency. Thus, it appears that the daily diary measures were tapping some of the micro-level processes giving rise to increases in problems at a developmental timescale (Granic & Patterson, ##REF##16478303##2006##). However, it also appears that these associations between parenting and child behavior did not cross-over from one level to the other, as the general parental consistency measure was predictive of externalizing problems one year later, whereas the daily measures of consistency (within- and across-episode consistency) were not. Additionally, general consistency was in turn not associated with daily disruptive behavior severity, whereas the daily measure of across-episode consistency was. It thus seems that the parenting and child behavior measures that were measured on a more similar timescale were more likely to be associated with each other. A previous study had similar findings in this regard, with daily measures of parenting variability associated with global parenting measures, but only global measures associated with a measure of the child’s ADHD symptoms (Li & Lansford, ##REF##29608072##2018##). Although there, ADHD symptoms became significantly associated with variability in parental warmth after controlling for parental ADHD symptoms and several types of stress, and daily symptom expression was not assessed. More research is necessary to understand how inconsistency in daily parent-child interactions may eventually increase externalizing problems over months and years.</p>","<p id=\"Par41\">Although daily diary measures are especially helpful in differentiating within- from across-episode consistency, the measure of how often parents indicated to react to their children’s disruptive behavior a certain way over the past month – the general consistency measure – was more predictive longitudinally of externalizing behavior than the daily diary measures. Although this measure again confounds within- and across-episode consistency, it may still be a better measure of consistency than some of the other measures of general consistency. As we asked directly about very specific reactions, our measure is likely a more valid measure of actual consistency in responding (Morsbach & Prinz, ##REF##16636897##2006##). At the same time, we would like to note that the validity of this measure deserves further scrutiny.</p>","<title>Strengths and Limitations</title>","<p id=\"Par42\">This study has several strengths. First, we included two samples of daily diary data that allowed us to differentiate within- from across-episode consistency and examine how our results would replicate across samples. Second, the analyses were registered on the OSF before conducting them. In addition to these strengths, some limitations are also worth mentioning. First, we did not differentiate different types of disruptive behavior episodes. Perhaps some episodes were more similar to each other than others, with a child yelling after not getting what it wanted in separate instances more similar than hitting a sibling in frustration about losing a game. Parents might react differently to different types of misbehavior, but consistently so within the types of misbehavior. Relatedly, in the functionally based categorization of behaviors in this study, certain responses were collapsed into categories as they were highly similar in their function – with these categorizations preregistered. Categories of positive attention, and ‘getting what you want’ were differentiated as they are likely different enough to be inconsistent, as are receiving negative attention or being punished for instance. An even higher level of abstraction could also be chosen, where anything ‘positive’ is contrasted with anything ‘negative’. At present, it is not known how different responses must be to contribute to inconsistency. Relatedly, the categorization of the parental reactions was based on a social learning theory perspective. However, inconsistencies in other aspects of the response might also be relevant. For instance, for several of the reactions that parents could choose from, it would be possible to be quite calm or quite frustrated while doing so, and these differences in affective quality and intensity might also contribute to inconsistency. More research is necessary to investigate whether inconsistency computed from other aspects of parental responses shows similar associations as the inconsistency measures derived from the categorization we made here. Second, both samples consisted of families with mostly highly-educated parents, raising questions about how generalizable these findings are to populations with different educational backgrounds. Additionally, whereas for Sample 2 it was clear that it was representative of ethnicities in the Netherlands, for Sample 1 information about ethnic diversity of the sample was not collected, making it impossible to draw any conclusions about this. Third, although similar measures were available for both samples included in this study, these studies were not designed to be the same, and as a result varied in multiple design aspects, making it impossible to draw any conclusions about why the results may have differed between them. Fourth, there are other aspects of consistency that we have not included in this study. For instance, consistency between different caregivers’ reactions might also play a role adjustment problems (Dwairy, ##UREF##5##2010##).</p>","<title>Conclusion</title>","<p id=\"Par43\">Results of this study show that it is meaningful to separate parental consistency within- from consistency across-episodes of misbehavior as they are correlated, but not strongly so. Furthermore, the aspects of consistency may be differentially important for the severity of child disruptive behavior as it is displayed in daily life, and there is some indication that across-episode consistency might be more important than actual responses, at least in a general population sample of toddlers. However, the actual responses were more important in our sample of early elementary school aged children from an at-risk population. Findings thus suggest that different risk factors (across-episode consistency or negative responding specifically) for disruptive behavior might apply to different subpopulations.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par34\\\">Aim of this study was to investigate how within- and across-episode parental consistency in responding to misbehavior are associated to externalizing problem behavior in children, using a daily diary approach. Within- and across-episode consistency were moderately strongly correlated with each other, but only across-episode consistency was associated with the severity of daily disruptive behavior. In Sample 1, this association was significant above and beyond the content of the parental reactions, whereas in Sample 2, the association was explained by the fact that parents who were more consistent across episodes were less likely to provide negative consequences or negative attention for the disruptive behavior. When we compared the longitudinal predictive value of the measures of consistency derived from the daily diaries to a measure of consistency derived from how often parents indicate they usually react, we found that the measures from the daily diary did not predict externalizing behavior problems one year later, whereas parental consistency as derived from the general questionnaire did.</p>\",\n \"<title>Within- and Across-episode Consistency</title>\",\n \"<p id=\\\"Par35\\\">In both samples, we found that the two types of consistency were significantly associated with each other, but the associations were not so strong that they indicated that they reflected the same underlying construct. This indicates that some parents were relatively higher on across-episode consistency whereas others were relatively higher on within-episode consistency. These results support the idea that it is relevant to separate the two types of consistency. The correlations between the two types of consistency were quite similar across the two different samples, as were the associations between the two types of consistency as computed from the daily diary data and the ‘trait’ measure of consistency that was computed based on how parents indicated that they generally responded to disruptive behavior in the baseline measure. These associations provide some validation of these measures.</p>\",\n \"<p id=\\\"Par36\\\">Interestingly, despite moderately strong correlations between within- and across episode consistency, when associations between the two types of consistency and the severity of daily disruptive behavior were examined, across-episode consistency was significantly associated with the severity of daily disruptive behavior in both samples, whereas within-episode consistency was not. That within-episode consistency was not associated with disruptive behavior severity is not in line with observational findings that lower within-episode consistency differentiated mother-child dyads with conduct-problems from those without (Gardner, ##REF##2745902##1989##), and mothers of aggressive toddlers from those without (Del Vecchio & O’Leary, ##REF##16597215##2006##). These findings may indicate that with regards to within-episode consistency, the rewarding nature of the final response – when a parent eventually gives in or does not follow through on their initial demand – is more important in explaining this effect of within-episode consistency rather than the mere variation of types of responses as was assessed by our measure. Parents who reward the child for misbehavior are likely to first provide negative attention for instance, scolding the child, and only give in after a sequence of different types of reactions (Gardner, ##REF##2745902##1989##). Alternatively it may mean that across-episode consistency is actually more strongly associated with disruptive behavior. As previous studies examining these rewarding interaction sequences have not controlled for across-episode consistency, more research is necessary to examine whether this association also disappears when across-episode consistency is taken into account.</p>\",\n \"<p id=\\\"Par37\\\">We add to previous findings that inconsistency in responses across episodes of misbehavior may be specifically associated with more severe disruptive behavior, regardless of the variation in types of responses within single episodes. In Sample 1, parents who were less consistent not only varied more within- or across-episode in how they responded to children’s tantrum, but also more frequently used each of the responses, both positive (e.g., positive consequences such as ‘giving in’) and negative (e.g., negative consequences such as ‘punishing’). Importantly, it was the variation between responses rather than the frequency of the individual responses that was associated with child disruptive behavior. This might indicate that parental consistency in responding is more important for lowering child disruptive behavior than how parents respond specifically. Alternatively, it might mean that when children show more disruptive behavior, parents are more likely to try out different ways of responding in an attempt to deal with it. Other studies have found that behavioral or emotional variation is associated with more maladjustment in young children as observed at a more micro time-scale, across real-time interaction. For instance, variability in affective displays has been related to more externalizing problems in mother-toddler dyads (Lunkenheimer et al., ##REF##23786697##2011##), as has behavioral variability (Lunkenheimer et al., ##REF##33180517##2020##).</p>\",\n \"<p id=\\\"Par38\\\">Our findings support the idea that, in a non-clinical sample, predictable parental responses are most important in reducing disruptive behavior. Unpredictable behavior from parents has been shown to impact the stress response in infants, with a blunted cortisol response to a painful stressors for infants of mothers who’s behavior was less predictable (Noroña-Zhou et al., ##REF##32115696##2020##), and variability in the affective quality of mother-child interactions and even in the timing of leisure activities, has been associated with an increased production of proinflammatory cytokines, an index of stress-reactivity, for youth (Manczak et al., ##REF##28625195##2018##). More research is necessary to understand whether these processes play a role in explaining the association between across-episode consistency in parenting behavior and child disruptive behavior.</p>\",\n \"<p id=\\\"Par39\\\">In Sample 2, a sample with older children who were at-risk for problem behavior, across-episode consistency was no longer associated with disruptive behavior above and beyond the individual reactions, whereas the frequency of providing negative consequences and negative attention were associated with more severe disruptive behavior. It seems that, whereas in Sample 1 it did not matter so much what parents did to reduce child disruptive behavior, as long as they did it consistently across episodes, in this sample negative responding was specifically associated with disruptive child behavior. Perhaps for families with older children with elevated levels of disruptive behavior as in Sample 2, parent and child have more strongly established patterns of negative responding to each other (Granic & Patterson, ##REF##16478303##2006##). In support of this idea, in studies of school aged children, affective variability has been associated with less rather than more behavioral problems (Granic et al., ##REF##17549621##2007##; Hollenstein et al., ##REF##15648527##2004##). Settling into a rigid, negative interaction style is a process that takes place in the interaction between parent and child over several years. Heightened variability in other areas may still have negative effects in older children and adolescents. For instance, higher variability in experienced stressors has been associated with worse emotional adjustment in adolescents (Zheng et al., ##REF##36273075##2022##), and higher variability in daily activities is associated with lower psychological well-being in young adults (Lee et al., ##UREF##9##2018##).</p>\",\n \"<title>Daily Diary Measures</title>\",\n \"<p id=\\\"Par40\\\">In this study, daily disruptive behavior was associated with externalizing problems one year later, and within- and across-episode consistency were associated with our measure of general consistency. Thus, it appears that the daily diary measures were tapping some of the micro-level processes giving rise to increases in problems at a developmental timescale (Granic & Patterson, ##REF##16478303##2006##). However, it also appears that these associations between parenting and child behavior did not cross-over from one level to the other, as the general parental consistency measure was predictive of externalizing problems one year later, whereas the daily measures of consistency (within- and across-episode consistency) were not. Additionally, general consistency was in turn not associated with daily disruptive behavior severity, whereas the daily measure of across-episode consistency was. It thus seems that the parenting and child behavior measures that were measured on a more similar timescale were more likely to be associated with each other. A previous study had similar findings in this regard, with daily measures of parenting variability associated with global parenting measures, but only global measures associated with a measure of the child’s ADHD symptoms (Li & Lansford, ##REF##29608072##2018##). Although there, ADHD symptoms became significantly associated with variability in parental warmth after controlling for parental ADHD symptoms and several types of stress, and daily symptom expression was not assessed. More research is necessary to understand how inconsistency in daily parent-child interactions may eventually increase externalizing problems over months and years.</p>\",\n \"<p id=\\\"Par41\\\">Although daily diary measures are especially helpful in differentiating within- from across-episode consistency, the measure of how often parents indicated to react to their children’s disruptive behavior a certain way over the past month – the general consistency measure – was more predictive longitudinally of externalizing behavior than the daily diary measures. Although this measure again confounds within- and across-episode consistency, it may still be a better measure of consistency than some of the other measures of general consistency. As we asked directly about very specific reactions, our measure is likely a more valid measure of actual consistency in responding (Morsbach & Prinz, ##REF##16636897##2006##). At the same time, we would like to note that the validity of this measure deserves further scrutiny.</p>\",\n \"<title>Strengths and Limitations</title>\",\n \"<p id=\\\"Par42\\\">This study has several strengths. First, we included two samples of daily diary data that allowed us to differentiate within- from across-episode consistency and examine how our results would replicate across samples. Second, the analyses were registered on the OSF before conducting them. In addition to these strengths, some limitations are also worth mentioning. First, we did not differentiate different types of disruptive behavior episodes. Perhaps some episodes were more similar to each other than others, with a child yelling after not getting what it wanted in separate instances more similar than hitting a sibling in frustration about losing a game. Parents might react differently to different types of misbehavior, but consistently so within the types of misbehavior. Relatedly, in the functionally based categorization of behaviors in this study, certain responses were collapsed into categories as they were highly similar in their function – with these categorizations preregistered. Categories of positive attention, and ‘getting what you want’ were differentiated as they are likely different enough to be inconsistent, as are receiving negative attention or being punished for instance. An even higher level of abstraction could also be chosen, where anything ‘positive’ is contrasted with anything ‘negative’. At present, it is not known how different responses must be to contribute to inconsistency. Relatedly, the categorization of the parental reactions was based on a social learning theory perspective. However, inconsistencies in other aspects of the response might also be relevant. For instance, for several of the reactions that parents could choose from, it would be possible to be quite calm or quite frustrated while doing so, and these differences in affective quality and intensity might also contribute to inconsistency. More research is necessary to investigate whether inconsistency computed from other aspects of parental responses shows similar associations as the inconsistency measures derived from the categorization we made here. Second, both samples consisted of families with mostly highly-educated parents, raising questions about how generalizable these findings are to populations with different educational backgrounds. Additionally, whereas for Sample 2 it was clear that it was representative of ethnicities in the Netherlands, for Sample 1 information about ethnic diversity of the sample was not collected, making it impossible to draw any conclusions about this. Third, although similar measures were available for both samples included in this study, these studies were not designed to be the same, and as a result varied in multiple design aspects, making it impossible to draw any conclusions about why the results may have differed between them. Fourth, there are other aspects of consistency that we have not included in this study. For instance, consistency between different caregivers’ reactions might also play a role adjustment problems (Dwairy, ##UREF##5##2010##).</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par43\\\">Results of this study show that it is meaningful to separate parental consistency within- from consistency across-episodes of misbehavior as they are correlated, but not strongly so. Furthermore, the aspects of consistency may be differentially important for the severity of child disruptive behavior as it is displayed in daily life, and there is some indication that across-episode consistency might be more important than actual responses, at least in a general population sample of toddlers. However, the actual responses were more important in our sample of early elementary school aged children from an at-risk population. Findings thus suggest that different risk factors (across-episode consistency or negative responding specifically) for disruptive behavior might apply to different subpopulations.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par43\">Results of this study show that it is meaningful to separate parental consistency within- from consistency across-episodes of misbehavior as they are correlated, but not strongly so. Furthermore, the aspects of consistency may be differentially important for the severity of child disruptive behavior as it is displayed in daily life, and there is some indication that across-episode consistency might be more important than actual responses, at least in a general population sample of toddlers. However, the actual responses were more important in our sample of early elementary school aged children from an at-risk population. Findings thus suggest that different risk factors (across-episode consistency or negative responding specifically) for disruptive behavior might apply to different subpopulations.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par43\\\">Results of this study show that it is meaningful to separate parental consistency within- from consistency across-episodes of misbehavior as they are correlated, but not strongly so. Furthermore, the aspects of consistency may be differentially important for the severity of child disruptive behavior as it is displayed in daily life, and there is some indication that across-episode consistency might be more important than actual responses, at least in a general population sample of toddlers. However, the actual responses were more important in our sample of early elementary school aged children from an at-risk population. Findings thus suggest that different risk factors (across-episode consistency or negative responding specifically) for disruptive behavior might apply to different subpopulations.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p id=\"Par1\">Consistent discipline is thought to reduce early child externalizing behavior. It is unclear, however, whether consistency is important mainly within episodes of misbehavior (e.g., threatening with discipline but then giving in) or across episodes of misbehavior (e.g., disciplining each instance of misbehavior). Using a daily diary approach, we examine whether these two types of consistency are associated with disruptive child behavior, concurrently and prospectively. We included two samples (Sample 1: <italic>N</italic> = 134, <italic>M</italic><sub>agechild</sub> = 30 months, 44% girls; Sample 2: <italic>N</italic> = 149, <italic>M</italic><sub>agechild</sub> = 5.88 years; 46% girls, at-risk sample) with daily reports of child disruptive behavior and parental responses (Sample 1 = 7 days; Sample 2 = 14 days). Sample 1 parents additionally reported on their reactions over the past month and their child’s externalizing behavior one year later. Within-episode consistency was assessed by the average number of parental reactions per episode; across-episode consistency by the Index of Qualitative Variation; and general consistency by parents’ report of how they had responded to child disruptive behavior in the past month. In both samples correlations between within- and across-episode consistency were significant, but not so strong that they were not differentiated. Again in both samples, regression analyses provided evidence for unique predictive value of across-episode, not within-episode, consistency for daily disruptive behavior. Parental general consistency was longitudinally associated with fewer externalizing problems, whereas within- and across-episode consistency were not. It appears meaningful to differentiate within- from across-episode consistency to better understand the relevance of different aspects of consistency.</p>","<title>Keywords</title>"],"string":"[\n \"<p id=\\\"Par1\\\">Consistent discipline is thought to reduce early child externalizing behavior. It is unclear, however, whether consistency is important mainly within episodes of misbehavior (e.g., threatening with discipline but then giving in) or across episodes of misbehavior (e.g., disciplining each instance of misbehavior). Using a daily diary approach, we examine whether these two types of consistency are associated with disruptive child behavior, concurrently and prospectively. We included two samples (Sample 1: <italic>N</italic> = 134, <italic>M</italic><sub>agechild</sub> = 30 months, 44% girls; Sample 2: <italic>N</italic> = 149, <italic>M</italic><sub>agechild</sub> = 5.88 years; 46% girls, at-risk sample) with daily reports of child disruptive behavior and parental responses (Sample 1 = 7 days; Sample 2 = 14 days). Sample 1 parents additionally reported on their reactions over the past month and their child’s externalizing behavior one year later. Within-episode consistency was assessed by the average number of parental reactions per episode; across-episode consistency by the Index of Qualitative Variation; and general consistency by parents’ report of how they had responded to child disruptive behavior in the past month. In both samples correlations between within- and across-episode consistency were significant, but not so strong that they were not differentiated. Again in both samples, regression analyses provided evidence for unique predictive value of across-episode, not within-episode, consistency for daily disruptive behavior. Parental general consistency was longitudinally associated with fewer externalizing problems, whereas within- and across-episode consistency were not. It appears meaningful to differentiate within- from across-episode consistency to better understand the relevance of different aspects of consistency.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":["<title>Appendix I: Questions as included in the daily diaries</title>","<title>Parental Reactions</title>","<title>Sample 1</title>","<p id=\"Par46\">For each tantrum during that day in daily diary, question:</p>","<title>Sample 2</title>","<p id=\"Par48\">For the most difficult to manage disruptive behavior reported that day, question:</p>","<p id=\"Par50\">For answer options, see Table ##TAB##4##5##. Answer categories for each option in both samples were: check box, coded unchecked = 0, checked = 1. Multiple checks allowed.\n</p>","<title>Child Externalizing Behavior</title>","<title>Sample 1</title>","<p id=\"Par51\">For each tantrum that occurred that day, question:</p>","<p id=\"Par53\">For answer options, see Table ##TAB##5##6##. Answer categories for each option in both samples were: check box, coded unchecked = 0, checked = 1. Multiple checks allowed.\n</p>","<title>Sample 2</title>","<p id=\"Par54\">Question: ‘<italic>How disruptive was your child’s behavior today?’.</italic></p>","<p id=\"Par55\">Answer categories were: slider 1 − 10.</p>","<title>Author Contribution</title>","<p>All authors contributed to the study conception and design. Material preparation and data collection were performed by Alithe Van den akker, Patty Leijten, and Peter Hoffenaar, data analysis was performed by Alithe Van den akker. The first draft of the manuscript was written by Alithe Van den akker and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.</p>","<title>Funding</title>","<p>Study 2 was funded by the Dutch Organization for Health Research and Development (ZonMw) under grant number 636320007 to Patty Leijten. The funder had no role in the design of this protocol, the collection of data, the data analysis, or the interpretation or publication of the study results.</p>","<title>Data Availability</title>","<p>Data are available from the first author upon request.</p>","<title>Compliance with Ethical Standards</title>","<title>Conflict of Interest</title>","<p id=\"Par44\">The authors declare that they have no conflict of interests.</p>","<title>Ethical Approval</title>","<p id=\"Par45\">All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.</p>"],"string":"[\n \"<title>Appendix I: Questions as included in the daily diaries</title>\",\n \"<title>Parental Reactions</title>\",\n \"<title>Sample 1</title>\",\n \"<p id=\\\"Par46\\\">For each tantrum during that day in daily diary, question:</p>\",\n \"<title>Sample 2</title>\",\n \"<p id=\\\"Par48\\\">For the most difficult to manage disruptive behavior reported that day, question:</p>\",\n \"<p id=\\\"Par50\\\">For answer options, see Table ##TAB##4##5##. Answer categories for each option in both samples were: check box, coded unchecked = 0, checked = 1. Multiple checks allowed.\\n</p>\",\n \"<title>Child Externalizing Behavior</title>\",\n \"<title>Sample 1</title>\",\n \"<p id=\\\"Par51\\\">For each tantrum that occurred that day, question:</p>\",\n \"<p id=\\\"Par53\\\">For answer options, see Table ##TAB##5##6##. Answer categories for each option in both samples were: check box, coded unchecked = 0, checked = 1. Multiple checks allowed.\\n</p>\",\n \"<title>Sample 2</title>\",\n \"<p id=\\\"Par54\\\">Question: ‘<italic>How disruptive was your child’s behavior today?’.</italic></p>\",\n \"<p id=\\\"Par55\\\">Answer categories were: slider 1 − 10.</p>\",\n \"<title>Author Contribution</title>\",\n \"<p>All authors contributed to the study conception and design. Material preparation and data collection were performed by Alithe Van den akker, Patty Leijten, and Peter Hoffenaar, data analysis was performed by Alithe Van den akker. The first draft of the manuscript was written by Alithe Van den akker and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Study 2 was funded by the Dutch Organization for Health Research and Development (ZonMw) under grant number 636320007 to Patty Leijten. The funder had no role in the design of this protocol, the collection of data, the data analysis, or the interpretation or publication of the study results.</p>\",\n \"<title>Data Availability</title>\",\n \"<p>Data are available from the first author upon request.</p>\",\n \"<title>Compliance with Ethical Standards</title>\",\n \"<title>Conflict of Interest</title>\",\n \"<p id=\\\"Par44\\\">The authors declare that they have no conflict of interests.</p>\",\n \"<title>Ethical Approval</title>\",\n \"<p id=\\\"Par45\\\">All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":[],"string":"[]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Descriptives and Intercorrelations for the Study Variables</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Measures</th><th align=\"left\">1.</th><th align=\"left\">2.</th><th align=\"left\">3.</th><th align=\"left\">4.</th><th align=\"left\">5.</th><th align=\"left\">6.</th><th align=\"left\">7</th><th align=\"left\">8.</th><th align=\"left\">9.</th><th align=\"left\">10.</th><th align=\"left\"><italic>M(SD)</italic></th></tr></thead><tbody><tr><td align=\"left\">1.Within-episode consistency</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.53**</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">-0.28**</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">-0.34**</td><td char=\".\" align=\"char\">-0.31**</td><td char=\".\" align=\"char\">-0.27**</td><td char=\".\" align=\"char\">-0.50**</td><td char=\".\" align=\"char\">-0.56**</td><td char=\".\" align=\"char\">1.81(0.35)</td></tr><tr><td align=\"left\">2.Across-episode consistency</td><td char=\".\" align=\"char\">0.60**</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">-0.31**</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">-0.40**</td><td char=\".\" align=\"char\">-0.42**</td><td char=\".\" align=\"char\">0.20*</td><td char=\".\" align=\"char\">-0.44**</td><td char=\".\" align=\"char\">-0.38**</td><td char=\".\" align=\"char\">0.25(0.18)</td></tr><tr><td align=\"left\">3.General consistency</td><td char=\".\" align=\"char\">0.40**</td><td char=\".\" align=\"char\">0.49**</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td></tr><tr><td align=\"left\">4.Daily disruptive behavior severity</td><td char=\".\" align=\"char\">-0.11</td><td char=\".\" align=\"char\">-0.29**</td><td char=\".\" align=\"char\">-0.16</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.30**</td><td char=\".\" align=\"char\">0.16</td><td char=\".\" align=\"char\">0.03</td><td char=\".\" align=\"char\">0.30**</td><td char=\".\" align=\"char\">0.19**</td><td char=\".\" align=\"char\">3.32(1.21)</td></tr><tr><td align=\"left\">5.Externalizing problems T2</td><td char=\".\" align=\"char\">-0.08</td><td char=\".\" align=\"char\">-0.22*</td><td char=\".\" align=\"char\">-0.32**</td><td char=\".\" align=\"char\">0.34**</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">–</td></tr><tr><td align=\"left\">6.Negative consequence</td><td char=\".\" align=\"char\">-0.23**</td><td char=\".\" align=\"char\">-0.24**</td><td char=\".\" align=\"char\">-0.31**</td><td char=\".\" align=\"char\">-0.03</td><td char=\".\" align=\"char\">0.10</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.25**</td><td char=\".\" align=\"char\">-0.06</td><td char=\".\" align=\"char\">0.11</td><td char=\".\" align=\"char\">-0.04</td><td char=\".\" align=\"char\">0.27(0.25)</td></tr><tr><td align=\"left\">7.Positive consequence</td><td char=\".\" align=\"char\">-0.34**</td><td char=\".\" align=\"char\">-0.34**</td><td char=\".\" align=\"char\">-0.21*</td><td char=\".\" align=\"char\">-0.03</td><td char=\".\" align=\"char\">-0.03</td><td char=\".\" align=\"char\">0.05</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">-0.05</td><td char=\".\" align=\"char\">0.15</td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">0.08(0.11)</td></tr><tr><td align=\"left\">8.Withhold attention</td><td char=\".\" align=\"char\">-0.20*</td><td char=\".\" align=\"char\">-0.30**</td><td char=\".\" align=\"char\">-0.16</td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">0.04</td><td char=\".\" align=\"char\">-0.09</td><td char=\".\" align=\"char\">-0.24**</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">-0.05</td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">0.51(0.24)</td></tr><tr><td align=\"left\">9.Negative attention</td><td char=\".\" align=\"char\">-0.43**</td><td char=\".\" align=\"char\">-0.29**</td><td char=\".\" align=\"char\">-0.33**</td><td char=\".\" align=\"char\">0.03</td><td char=\".\" align=\"char\">0.20</td><td char=\".\" align=\"char\">0.22*</td><td char=\".\" align=\"char\">-0.00</td><td char=\".\" align=\"char\">0.01</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.17*</td><td char=\".\" align=\"char\">0.18(0.17)</td></tr><tr><td align=\"left\">10.Positive attention</td><td char=\".\" align=\"char\">-0.22*</td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">0.30**</td><td char=\".\" align=\"char\">-0.09</td><td char=\".\" align=\"char\">-0.08</td><td char=\".\" align=\"char\">-0.17</td><td char=\".\" align=\"char\">0.00</td><td char=\".\" align=\"char\">-0.17</td><td char=\".\" align=\"char\">-0.18*</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.20(0.22)</td></tr><tr><td align=\"left\" rowspan=\"2\"><italic>M(SD)</italic></td><td char=\".\" align=\"char\">1.52</td><td char=\".\" align=\"char\">0.40</td><td char=\".\" align=\"char\">0.07</td><td char=\".\" align=\"char\">4.15</td><td char=\".\" align=\"char\">0.60</td><td char=\".\" align=\"char\">0.07</td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">0.30</td><td char=\".\" align=\"char\">0.21</td><td char=\".\" align=\"char\">0.42</td><td char=\".\" align=\"char\">–</td></tr><tr><td char=\".\" align=\"char\">(0.47)</td><td char=\".\" align=\"char\">(0.29)</td><td char=\".\" align=\"char\">(0.05)</td><td char=\".\" align=\"char\">(4.04)</td><td char=\".\" align=\"char\">(0.31)</td><td char=\".\" align=\"char\">(0.16)</td><td char=\".\" align=\"char\">(0.21)</td><td char=\".\" align=\"char\">(0.28)</td><td char=\".\" align=\"char\">(0.26)</td><td char=\".\" align=\"char\">(0.30)</td><td char=\".\" align=\"char\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Results of Regression Analyses Predicting Severity of Daily Disruptive Behavior in Sample 1</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\"/><th align=\"left\"/><th align=\"left\"/><th align=\"left\" colspan=\"4\">Severity of daily disruptive behavior</th></tr><tr><th align=\"left\">variables</th><th align=\"left\"><italic>b(SE)</italic></th><th align=\"left\">β</th><th align=\"left\"><italic>p</italic></th><th align=\"left\">variables</th><th align=\"left\"><italic>b(SE)</italic></th><th align=\"left\">β</th><th align=\"left\"><italic>p</italic></th></tr><tr><th align=\"left\" colspan=\"4\">Step 1</th><th align=\"left\" colspan=\"4\">Step 1</th></tr></thead><tbody><tr><td align=\"left\"> age child</td><td char=\".\" align=\"char\">-0.04(0.04)</td><td char=\".\" align=\"char\">-0.08</td><td char=\".\" align=\"char\">0.370</td><td align=\"left\"> Age child</td><td char=\".\" align=\"char\">-0.04(0.04)</td><td char=\".\" align=\"char\">-0.08</td><td char=\".\" align=\"char\">0.370</td></tr><tr><td align=\"left\"> Sex child</td><td char=\".\" align=\"char\">-0.41(0.72)</td><td char=\".\" align=\"char\">-0.05</td><td char=\".\" align=\"char\">0.572</td><td align=\"left\"> Sex child</td><td char=\".\" align=\"char\">-0.41(0.72)</td><td char=\".\" align=\"char\">-0.05</td><td char=\".\" align=\"char\">0.572</td></tr><tr><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">-0.23(0.38)</td><td char=\".\" align=\"char\">-0.05</td><td char=\".\" align=\"char\">0.553</td><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">-0.23(0.38)</td><td char=\".\" align=\"char\">-0.05</td><td char=\".\" align=\"char\">0.553</td></tr><tr><td align=\"left\" colspan=\"4\">Step 2</td><td align=\"left\" colspan=\"4\">Step 2</td></tr><tr><td align=\"left\"> Age child</td><td char=\".\" align=\"char\">-0.04(0.04)</td><td char=\".\" align=\"char\">-0.12</td><td char=\".\" align=\"char\">0.294</td><td align=\"left\"> Age child</td><td char=\".\" align=\"char\">-0.04(0.04)</td><td char=\".\" align=\"char\">-0.08</td><td char=\".\" align=\"char\">0.385</td></tr><tr><td align=\"left\"> Sex child</td><td char=\".\" align=\"char\">-0.65(0.69)</td><td char=\".\" align=\"char\">-0.07</td><td char=\".\" align=\"char\">0.353</td><td align=\"left\"> Sex child</td><td char=\".\" align=\"char\">-0.30(0.73)</td><td char=\".\" align=\"char\">-0.04</td><td char=\".\" align=\"char\">0.685</td></tr><tr><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">-0.24(0.37)</td><td char=\".\" align=\"char\">-0.05</td><td char=\".\" align=\"char\">0.525</td><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">-0.14(0.40)</td><td char=\".\" align=\"char\">-0.03</td><td char=\".\" align=\"char\">0.725</td></tr><tr><td align=\"left\"> Within-episode consistency</td><td char=\".\" align=\"char\">0.70(0.94)</td><td char=\".\" align=\"char\">0.06</td><td char=\".\" align=\"char\">0.455</td><td align=\"left\"> negative consequence</td><td char=\".\" align=\"char\">-0.77(2.40)</td><td char=\".\" align=\"char\">-0.03</td><td char=\".\" align=\"char\">0.748</td></tr><tr><td align=\"left\"> Across-episode consistency</td><td char=\".\" align=\"char\">-4.92(1.47)</td><td char=\".\" align=\"char\">-0.36</td><td char=\".\" align=\"char\">0.001</td><td align=\"left\"> positive consequence</td><td char=\".\" align=\"char\">0.30(1.79)</td><td char=\".\" align=\"char\">0.02</td><td char=\".\" align=\"char\">0.866</td></tr><tr><td align=\"left\" rowspan=\"14\" colspan=\"4\"/><td align=\"left\"> withholding attention</td><td char=\".\" align=\"char\">1.39(1.33)</td><td char=\".\" align=\"char\">0.10</td><td char=\".\" align=\"char\">0.297</td></tr><tr><td align=\"left\"> negative attention</td><td char=\".\" align=\"char\">0.56(1.47)</td><td char=\".\" align=\"char\">0.04</td><td char=\".\" align=\"char\">0.705</td></tr><tr><td align=\"left\"> positive attention</td><td char=\".\" align=\"char\">-0.96(1.27)</td><td char=\".\" align=\"char\">-0.07</td><td char=\".\" align=\"char\">0.454</td></tr><tr><td align=\"left\" colspan=\"4\">Step 3</td></tr><tr><td align=\"left\"> Age child</td><td char=\".\" align=\"char\">-0.02(0.04)</td><td char=\".\" align=\"char\">-0.04</td><td char=\".\" align=\"char\">0.644</td></tr><tr><td align=\"left\"> Sex child</td><td char=\".\" align=\"char\">-0.80(0.71)</td><td char=\".\" align=\"char\">-0.10</td><td char=\".\" align=\"char\">0.259</td></tr><tr><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">-0.33(0.38)</td><td char=\".\" align=\"char\">-0.08</td><td char=\".\" align=\"char\">0.382</td></tr><tr><td align=\"left\"> negative consequence</td><td char=\".\" align=\"char\">-4.00(2.42)</td><td char=\".\" align=\"char\">-0.16</td><td char=\".\" align=\"char\">0.101</td></tr><tr><td align=\"left\"> positive consequence</td><td char=\".\" align=\"char\">-4.05(2.06)</td><td char=\".\" align=\"char\">-0.21</td><td char=\".\" align=\"char\">0.051</td></tr><tr><td align=\"left\"> withholding attention</td><td char=\".\" align=\"char\">-1.74(1.51)</td><td char=\".\" align=\"char\">-0.12</td><td char=\".\" align=\"char\">0.252</td></tr><tr><td align=\"left\"> negative attention</td><td char=\".\" align=\"char\">-1.78(1.65)</td><td char=\".\" align=\"char\">-0.11</td><td char=\".\" align=\"char\">0.282</td></tr><tr><td align=\"left\"> positive attention</td><td char=\".\" align=\"char\">-1.50(1.38)</td><td char=\".\" align=\"char\">-0.11</td><td char=\".\" align=\"char\">0.282</td></tr><tr><td align=\"left\"> Within-episode consistency</td><td char=\".\" align=\"char\">-0.55(1.17)</td><td char=\".\" align=\"char\">-0.06</td><td char=\".\" align=\"char\">0.638</td></tr><tr><td align=\"left\"> Across-episode consistency</td><td char=\".\" align=\"char\">-6.05(1.67)</td><td char=\".\" align=\"char\">-0.44</td><td char=\".\" align=\"char\">< 0.001</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Results of Regression Analyses Predicting Severity of Daily Disruptive Behavior in Sample 2</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\"/><th align=\"left\"/><th align=\"left\"/><th align=\"left\" colspan=\"4\">Severity of daily disruptive behavior</th></tr><tr><th align=\"left\">variables</th><th align=\"left\"><italic>b(SE)</italic></th><th align=\"left\">β</th><th align=\"left\"><italic>p</italic></th><th align=\"left\">variables</th><th align=\"left\"><italic>b(SE)</italic></th><th align=\"left\">β</th><th align=\"left\"><italic>p</italic></th></tr><tr><th align=\"left\" colspan=\"4\">Step 1</th><th align=\"left\" colspan=\"4\">Step 1</th></tr></thead><tbody><tr><td align=\"left\"> Age child</td><td char=\".\" align=\"char\">-0.00(0.01)</td><td char=\".\" align=\"char\">-0.03</td><td char=\".\" align=\"char\">0.736</td><td align=\"left\"> Age child</td><td char=\".\" align=\"char\">-0.00(0.01)</td><td char=\".\" align=\"char\">-0.01</td><td char=\".\" align=\"char\">0.736</td></tr><tr><td align=\"left\"> Sex child</td><td char=\".\" align=\"char\">-0.12(0.20)</td><td char=\".\" align=\"char\">-0.05</td><td char=\".\" align=\"char\">0.541</td><td align=\"left\"> Sex child</td><td char=\".\" align=\"char\">-0.12(0.20)</td><td char=\".\" align=\"char\">-0.05</td><td char=\".\" align=\"char\">0.541</td></tr><tr><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">0.06(0.10)</td><td char=\".\" align=\"char\">0.05</td><td char=\".\" align=\"char\">0.562</td><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">0.06(0.10)</td><td char=\".\" align=\"char\">0.05</td><td char=\".\" align=\"char\">0.562</td></tr><tr><td align=\"left\"> Received support</td><td char=\".\" align=\"char\">0.71(0.38)</td><td char=\".\" align=\"char\">0.16</td><td char=\".\" align=\"char\">0.062</td><td align=\"left\"> Received support</td><td char=\".\" align=\"char\">0.71(0.38)</td><td char=\".\" align=\"char\">0.16</td><td char=\".\" align=\"char\">0.062</td></tr><tr><td align=\"left\" colspan=\"4\">Step 2</td><td align=\"left\" colspan=\"4\">Step 2</td></tr><tr><td align=\"left\"> Age child</td><td char=\".\" align=\"char\">-0.00(0.01)</td><td char=\".\" align=\"char\">-0.01</td><td char=\".\" align=\"char\">0.988</td><td align=\"left\"> Age child</td><td char=\".\" align=\"char\">0.00(0.01)</td><td char=\".\" align=\"char\">0.02</td><td char=\".\" align=\"char\">0.776</td></tr><tr><td align=\"left\"> Sex child</td><td char=\".\" align=\"char\">-0.09(0.19)</td><td char=\".\" align=\"char\">-0.04</td><td char=\".\" align=\"char\">0.639</td><td align=\"left\"> Sex child</td><td char=\".\" align=\"char\">-0.06(0.19)</td><td char=\".\" align=\"char\">-0.02</td><td char=\".\" align=\"char\">0.770</td></tr><tr><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">0.03(0.10)</td><td char=\".\" align=\"char\">0.02</td><td char=\".\" align=\"char\">0.785</td><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">0.05(0.10)</td><td char=\".\" align=\"char\">0.04</td><td char=\".\" align=\"char\">0.612</td></tr><tr><td align=\"left\"> Received support</td><td char=\".\" align=\"char\">0.83(0.36)</td><td char=\".\" align=\"char\">0.18</td><td char=\".\" align=\"char\">0.021</td><td align=\"left\"> Received support</td><td char=\".\" align=\"char\">0.98(0.36)</td><td char=\".\" align=\"char\">0.21</td><td char=\".\" align=\"char\">0.007</td></tr><tr><td align=\"left\"> Within-episode consistency</td><td char=\".\" align=\"char\">-0.59(0.32)</td><td char=\".\" align=\"char\">-0.17</td><td char=\".\" align=\"char\">0.063</td><td align=\"left\"> negative consequence</td><td char=\".\" align=\"char\">1.45(0.39)</td><td char=\".\" align=\"char\">0.30</td><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> Across-episode consistency</td><td char=\".\" align=\"char\">-1.60(0.61)</td><td char=\".\" align=\"char\">-0.24</td><td char=\".\" align=\"char\">0.010</td><td align=\"left\"> positive consequence</td><td char=\".\" align=\"char\">0.07(0.90)</td><td char=\".\" align=\"char\">0.01</td><td char=\".\" align=\"char\">0.938</td></tr><tr><td align=\"left\" rowspan=\"15\" colspan=\"4\"/><td align=\"left\"> withholding attention</td><td char=\".\" align=\"char\">0.04(0.39)</td><td char=\".\" align=\"char\">0.01</td><td char=\".\" align=\"char\">0.901</td></tr><tr><td align=\"left\"> negative attention</td><td char=\".\" align=\"char\">1.77(0.54)</td><td char=\".\" align=\"char\">0.26</td><td char=\".\" align=\"char\">0.001</td></tr><tr><td align=\"left\"> positive attention</td><td char=\".\" align=\"char\">0.83(0.44)</td><td char=\".\" align=\"char\">0.15</td><td char=\".\" align=\"char\">0.060</td></tr><tr><td align=\"left\" colspan=\"4\">Step 3</td></tr><tr><td align=\"left\"> Age child</td><td char=\".\" align=\"char\">0.00(0.01)</td><td char=\".\" align=\"char\">0.02</td><td char=\".\" align=\"char\">0.785</td></tr><tr><td align=\"left\"> sex</td><td char=\".\" align=\"char\">-0.05(0.19)</td><td char=\".\" align=\"char\">-0.02</td><td char=\".\" align=\"char\">0.778</td></tr><tr><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">0.04(0.10)</td><td char=\".\" align=\"char\">0.03</td><td char=\".\" align=\"char\">0.686</td></tr><tr><td align=\"left\"> Received support</td><td char=\".\" align=\"char\">0.96(0.36)</td><td char=\".\" align=\"char\">0.21</td><td char=\".\" align=\"char\">0.008</td></tr><tr><td align=\"left\"> negative consequence</td><td char=\".\" align=\"char\">1.42(0.46)</td><td char=\".\" align=\"char\">0.29</td><td char=\".\" align=\"char\">0.002</td></tr><tr><td align=\"left\"> positive consequence</td><td char=\".\" align=\"char\">-0.06(0.97)</td><td char=\".\" align=\"char\">-0.01</td><td char=\".\" align=\"char\">0.955</td></tr><tr><td align=\"left\"> withholding attention</td><td char=\".\" align=\"char\">0.22(0.44)</td><td char=\".\" align=\"char\">0.04</td><td char=\".\" align=\"char\">0.624</td></tr><tr><td align=\"left\"> negative attention</td><td char=\".\" align=\"char\">1.77(0.67)</td><td char=\".\" align=\"char\">0.26</td><td char=\".\" align=\"char\">0.009</td></tr><tr><td align=\"left\"> positive attention</td><td char=\".\" align=\"char\">0.86(0.59)</td><td char=\".\" align=\"char\">0.16</td><td char=\".\" align=\"char\">0.142</td></tr><tr><td align=\"left\"> Within-episode consistency</td><td char=\".\" align=\"char\">0.26(0.46)</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">0.578</td></tr><tr><td align=\"left\"> Across-episode consistency</td><td char=\".\" align=\"char\">-0.60(0.74)</td><td char=\".\" align=\"char\">-0.09</td><td char=\".\" align=\"char\">0.421</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Results of Regression Analysis Predicting Externalizing Behavior one year Later</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">variables</th><th align=\"left\"><italic>b(SE)</italic></th><th align=\"left\">β</th><th align=\"left\"><italic>p</italic></th></tr><tr><th align=\"left\" colspan=\"4\">Step 1</th></tr></thead><tbody><tr><td align=\"left\"> Age Child</td><td char=\".\" align=\"char\">-0.01(0.00)</td><td char=\".\" align=\"char\">-0.15</td><td char=\".\" align=\"char\">0.199</td></tr><tr><td align=\"left\"> Sex Child</td><td char=\".\" align=\"char\">0.00(0.07)</td><td char=\".\" align=\"char\">0.01</td><td char=\".\" align=\"char\">0.955</td></tr><tr><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">-0.01(0.03)</td><td char=\".\" align=\"char\">-0.04</td><td char=\".\" align=\"char\">0.676</td></tr><tr><td align=\"left\"> Daily disruptive behavior severity T1</td><td char=\".\" align=\"char\">0.02(0.01)</td><td char=\".\" align=\"char\">0.31</td><td char=\".\" align=\"char\">0.005</td></tr><tr><td align=\"left\"> General consistency</td><td char=\".\" align=\"char\">-1.90(0.68)</td><td char=\".\" align=\"char\">-0.31</td><td char=\".\" align=\"char\">0.006</td></tr><tr><td align=\"left\" colspan=\"4\">Step 2</td></tr><tr><td align=\"left\"> Age Child</td><td char=\".\" align=\"char\">-0.01(0.00)</td><td char=\".\" align=\"char\">-0.13</td><td char=\".\" align=\"char\">0.260</td></tr><tr><td align=\"left\"> Sex Child</td><td char=\".\" align=\"char\">-0.01(0.07)</td><td char=\".\" align=\"char\">-0.01</td><td char=\".\" align=\"char\">0.927</td></tr><tr><td align=\"left\"> Education Level</td><td char=\".\" align=\"char\">-0.01(0.04)</td><td char=\".\" align=\"char\">-0.04</td><td char=\".\" align=\"char\">0.711</td></tr><tr><td align=\"left\"> Daily disruptive behavior severity T1</td><td char=\".\" align=\"char\">0.02(0.01)</td><td char=\".\" align=\"char\">0.30</td><td char=\".\" align=\"char\">0.010</td></tr><tr><td align=\"left\"> General consistency</td><td char=\".\" align=\"char\">-1.76(0.82)</td><td char=\".\" align=\"char\">-0.29</td><td char=\".\" align=\"char\">0.036</td></tr><tr><td align=\"left\"> Within-episode consistency</td><td char=\".\" align=\"char\">0.05(0.09)</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">0.553</td></tr><tr><td align=\"left\"> Across-episode consistency</td><td char=\".\" align=\"char\">-0.12(0.17)</td><td char=\".\" align=\"char\">-0.11</td><td char=\".\" align=\"char\">0.464</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab5\"><label>Table 5</label><caption><p>Answer categories for each parental reaction for the two studies</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"><bold>Study 1</bold></th><th align=\"left\"><bold>Study 2</bold></th></tr></thead><tbody><tr><td align=\"left\" colspan=\"2\"><bold>Negative consequence</bold></td></tr><tr><td align=\"left\">I sent my child to their room/corner/time-out</td><td align=\"left\">I sent my child to their room for at least an hour</td></tr><tr><td align=\"left\">I punished my child</td><td align=\"left\">I gave my child a short time-out, away from others</td></tr><tr><td align=\"left\"/><td align=\"left\">I took something nice away from my child (e.g., toys or screen time)</td></tr><tr><td align=\"left\"/><td align=\"left\">I gave my child extra chores (e.g., set the table)</td></tr><tr><td align=\"left\" colspan=\"2\"><bold>Positive consequence</bold></td></tr><tr><td align=\"left\">I negotiated with my child</td><td align=\"left\">I gave my child his/her way</td></tr><tr><td align=\"left\">I gave in to my child</td><td align=\"left\">I gave in to my child</td></tr><tr><td align=\"left\" colspan=\"2\"><bold>Withholding attention</bold></td></tr><tr><td align=\"left\">I didn’t, I let my child cool off</td><td align=\"left\">I did nothing</td></tr><tr><td align=\"left\">I ignored my child</td><td align=\"left\">I talked about it with my child afterwards</td></tr><tr><td align=\"left\" colspan=\"2\"><bold>Negative attention</bold></td></tr><tr><td align=\"left\">I became angry with my child</td><td align=\"left\">I yelled/swore</td></tr><tr><td align=\"left\">I grabbed my child</td><td align=\"left\">I said things I didn’t mean</td></tr><tr><td align=\"left\">I spoke sternly to my child</td><td align=\"left\">I threatened with punishment, but did not punish</td></tr><tr><td align=\"left\" colspan=\"2\"><bold>Positive attention</bold></td></tr><tr><td align=\"left\">I comforted my child</td><td align=\"left\">I begged my child to stop</td></tr><tr><td align=\"left\">I distracted my child</td><td align=\"left\">I used humor to distract my child</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab6\"><label>Table 6</label><caption><p>Answer categories for the aggressive tantrum behaviors</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\" colspan=\"2\">Hitting</td></tr><tr><td align=\"left\" colspan=\"2\">Kicking</td></tr><tr><td align=\"left\" colspan=\"2\">Biting</td></tr><tr><td align=\"left\" colspan=\"2\">Throwing an object</td></tr><tr><td align=\"left\" colspan=\"2\">Pushing/pulling</td></tr><tr><td align=\"left\" colspan=\"2\">Spitting</td></tr><tr><td align=\"left\" colspan=\"2\">Grabbing</td></tr><tr><td align=\"left\" colspan=\"2\">Banging head</td></tr><tr><td align=\"left\" colspan=\"2\">Holding breath</td></tr><tr><td align=\"left\" colspan=\"2\">freezing</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Descriptives and Intercorrelations for the Study Variables</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Measures</th><th align=\\\"left\\\">1.</th><th align=\\\"left\\\">2.</th><th align=\\\"left\\\">3.</th><th align=\\\"left\\\">4.</th><th align=\\\"left\\\">5.</th><th align=\\\"left\\\">6.</th><th align=\\\"left\\\">7</th><th align=\\\"left\\\">8.</th><th align=\\\"left\\\">9.</th><th align=\\\"left\\\">10.</th><th align=\\\"left\\\"><italic>M(SD)</italic></th></tr></thead><tbody><tr><td align=\\\"left\\\">1.Within-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.53**</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">-0.28**</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">-0.34**</td><td char=\\\".\\\" align=\\\"char\\\">-0.31**</td><td char=\\\".\\\" align=\\\"char\\\">-0.27**</td><td char=\\\".\\\" align=\\\"char\\\">-0.50**</td><td char=\\\".\\\" align=\\\"char\\\">-0.56**</td><td char=\\\".\\\" align=\\\"char\\\">1.81(0.35)</td></tr><tr><td align=\\\"left\\\">2.Across-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">0.60**</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">-0.31**</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">-0.40**</td><td char=\\\".\\\" align=\\\"char\\\">-0.42**</td><td char=\\\".\\\" align=\\\"char\\\">0.20*</td><td char=\\\".\\\" align=\\\"char\\\">-0.44**</td><td char=\\\".\\\" align=\\\"char\\\">-0.38**</td><td char=\\\".\\\" align=\\\"char\\\">0.25(0.18)</td></tr><tr><td align=\\\"left\\\">3.General consistency</td><td char=\\\".\\\" align=\\\"char\\\">0.40**</td><td char=\\\".\\\" align=\\\"char\\\">0.49**</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td></tr><tr><td align=\\\"left\\\">4.Daily disruptive behavior severity</td><td char=\\\".\\\" align=\\\"char\\\">-0.11</td><td char=\\\".\\\" align=\\\"char\\\">-0.29**</td><td char=\\\".\\\" align=\\\"char\\\">-0.16</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.30**</td><td char=\\\".\\\" align=\\\"char\\\">0.16</td><td char=\\\".\\\" align=\\\"char\\\">0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.30**</td><td char=\\\".\\\" align=\\\"char\\\">0.19**</td><td char=\\\".\\\" align=\\\"char\\\">3.32(1.21)</td></tr><tr><td align=\\\"left\\\">5.Externalizing problems T2</td><td char=\\\".\\\" align=\\\"char\\\">-0.08</td><td char=\\\".\\\" align=\\\"char\\\">-0.22*</td><td char=\\\".\\\" align=\\\"char\\\">-0.32**</td><td char=\\\".\\\" align=\\\"char\\\">0.34**</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">–</td></tr><tr><td align=\\\"left\\\">6.Negative consequence</td><td char=\\\".\\\" align=\\\"char\\\">-0.23**</td><td char=\\\".\\\" align=\\\"char\\\">-0.24**</td><td char=\\\".\\\" align=\\\"char\\\">-0.31**</td><td char=\\\".\\\" align=\\\"char\\\">-0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.10</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.25**</td><td char=\\\".\\\" align=\\\"char\\\">-0.06</td><td char=\\\".\\\" align=\\\"char\\\">0.11</td><td char=\\\".\\\" align=\\\"char\\\">-0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.27(0.25)</td></tr><tr><td align=\\\"left\\\">7.Positive consequence</td><td char=\\\".\\\" align=\\\"char\\\">-0.34**</td><td char=\\\".\\\" align=\\\"char\\\">-0.34**</td><td char=\\\".\\\" align=\\\"char\\\">-0.21*</td><td char=\\\".\\\" align=\\\"char\\\">-0.03</td><td char=\\\".\\\" align=\\\"char\\\">-0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.05</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">-0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.15</td><td char=\\\".\\\" align=\\\"char\\\">0.12</td><td char=\\\".\\\" align=\\\"char\\\">0.08(0.11)</td></tr><tr><td align=\\\"left\\\">8.Withhold attention</td><td char=\\\".\\\" align=\\\"char\\\">-0.20*</td><td char=\\\".\\\" align=\\\"char\\\">-0.30**</td><td char=\\\".\\\" align=\\\"char\\\">-0.16</td><td char=\\\".\\\" align=\\\"char\\\">0.12</td><td char=\\\".\\\" align=\\\"char\\\">0.04</td><td char=\\\".\\\" align=\\\"char\\\">-0.09</td><td char=\\\".\\\" align=\\\"char\\\">-0.24**</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">-0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.12</td><td char=\\\".\\\" align=\\\"char\\\">0.51(0.24)</td></tr><tr><td align=\\\"left\\\">9.Negative attention</td><td char=\\\".\\\" align=\\\"char\\\">-0.43**</td><td char=\\\".\\\" align=\\\"char\\\">-0.29**</td><td char=\\\".\\\" align=\\\"char\\\">-0.33**</td><td char=\\\".\\\" align=\\\"char\\\">0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.20</td><td char=\\\".\\\" align=\\\"char\\\">0.22*</td><td char=\\\".\\\" align=\\\"char\\\">-0.00</td><td char=\\\".\\\" align=\\\"char\\\">0.01</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.17*</td><td char=\\\".\\\" align=\\\"char\\\">0.18(0.17)</td></tr><tr><td align=\\\"left\\\">10.Positive attention</td><td char=\\\".\\\" align=\\\"char\\\">-0.22*</td><td char=\\\".\\\" align=\\\"char\\\">0.09</td><td char=\\\".\\\" align=\\\"char\\\">0.30**</td><td char=\\\".\\\" align=\\\"char\\\">-0.09</td><td char=\\\".\\\" align=\\\"char\\\">-0.08</td><td char=\\\".\\\" align=\\\"char\\\">-0.17</td><td char=\\\".\\\" align=\\\"char\\\">0.00</td><td char=\\\".\\\" align=\\\"char\\\">-0.17</td><td char=\\\".\\\" align=\\\"char\\\">-0.18*</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.20(0.22)</td></tr><tr><td align=\\\"left\\\" rowspan=\\\"2\\\"><italic>M(SD)</italic></td><td char=\\\".\\\" align=\\\"char\\\">1.52</td><td char=\\\".\\\" align=\\\"char\\\">0.40</td><td char=\\\".\\\" align=\\\"char\\\">0.07</td><td char=\\\".\\\" align=\\\"char\\\">4.15</td><td char=\\\".\\\" align=\\\"char\\\">0.60</td><td char=\\\".\\\" align=\\\"char\\\">0.07</td><td char=\\\".\\\" align=\\\"char\\\">0.12</td><td char=\\\".\\\" align=\\\"char\\\">0.30</td><td char=\\\".\\\" align=\\\"char\\\">0.21</td><td char=\\\".\\\" align=\\\"char\\\">0.42</td><td char=\\\".\\\" align=\\\"char\\\">–</td></tr><tr><td char=\\\".\\\" align=\\\"char\\\">(0.47)</td><td char=\\\".\\\" align=\\\"char\\\">(0.29)</td><td char=\\\".\\\" align=\\\"char\\\">(0.05)</td><td char=\\\".\\\" align=\\\"char\\\">(4.04)</td><td char=\\\".\\\" align=\\\"char\\\">(0.31)</td><td char=\\\".\\\" align=\\\"char\\\">(0.16)</td><td char=\\\".\\\" align=\\\"char\\\">(0.21)</td><td char=\\\".\\\" align=\\\"char\\\">(0.28)</td><td char=\\\".\\\" align=\\\"char\\\">(0.26)</td><td char=\\\".\\\" align=\\\"char\\\">(0.30)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Results of Regression Analyses Predicting Severity of Daily Disruptive Behavior in Sample 1</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"/><th align=\\\"left\\\"/><th align=\\\"left\\\"/><th align=\\\"left\\\"/><th align=\\\"left\\\" colspan=\\\"4\\\">Severity of daily disruptive behavior</th></tr><tr><th align=\\\"left\\\">variables</th><th align=\\\"left\\\"><italic>b(SE)</italic></th><th align=\\\"left\\\">β</th><th align=\\\"left\\\"><italic>p</italic></th><th align=\\\"left\\\">variables</th><th align=\\\"left\\\"><italic>b(SE)</italic></th><th align=\\\"left\\\">β</th><th align=\\\"left\\\"><italic>p</italic></th></tr><tr><th align=\\\"left\\\" colspan=\\\"4\\\">Step 1</th><th align=\\\"left\\\" colspan=\\\"4\\\">Step 1</th></tr></thead><tbody><tr><td align=\\\"left\\\"> age child</td><td char=\\\".\\\" align=\\\"char\\\">-0.04(0.04)</td><td char=\\\".\\\" align=\\\"char\\\">-0.08</td><td char=\\\".\\\" align=\\\"char\\\">0.370</td><td align=\\\"left\\\"> Age child</td><td char=\\\".\\\" align=\\\"char\\\">-0.04(0.04)</td><td char=\\\".\\\" align=\\\"char\\\">-0.08</td><td char=\\\".\\\" align=\\\"char\\\">0.370</td></tr><tr><td align=\\\"left\\\"> Sex child</td><td char=\\\".\\\" align=\\\"char\\\">-0.41(0.72)</td><td char=\\\".\\\" align=\\\"char\\\">-0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.572</td><td align=\\\"left\\\"> Sex child</td><td char=\\\".\\\" align=\\\"char\\\">-0.41(0.72)</td><td char=\\\".\\\" align=\\\"char\\\">-0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.572</td></tr><tr><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">-0.23(0.38)</td><td char=\\\".\\\" align=\\\"char\\\">-0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.553</td><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">-0.23(0.38)</td><td char=\\\".\\\" align=\\\"char\\\">-0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.553</td></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Step 2</td><td align=\\\"left\\\" colspan=\\\"4\\\">Step 2</td></tr><tr><td align=\\\"left\\\"> Age child</td><td char=\\\".\\\" align=\\\"char\\\">-0.04(0.04)</td><td char=\\\".\\\" align=\\\"char\\\">-0.12</td><td char=\\\".\\\" align=\\\"char\\\">0.294</td><td align=\\\"left\\\"> Age child</td><td char=\\\".\\\" align=\\\"char\\\">-0.04(0.04)</td><td char=\\\".\\\" align=\\\"char\\\">-0.08</td><td char=\\\".\\\" align=\\\"char\\\">0.385</td></tr><tr><td align=\\\"left\\\"> Sex child</td><td char=\\\".\\\" align=\\\"char\\\">-0.65(0.69)</td><td char=\\\".\\\" align=\\\"char\\\">-0.07</td><td char=\\\".\\\" align=\\\"char\\\">0.353</td><td align=\\\"left\\\"> Sex child</td><td char=\\\".\\\" align=\\\"char\\\">-0.30(0.73)</td><td char=\\\".\\\" align=\\\"char\\\">-0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.685</td></tr><tr><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">-0.24(0.37)</td><td char=\\\".\\\" align=\\\"char\\\">-0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.525</td><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">-0.14(0.40)</td><td char=\\\".\\\" align=\\\"char\\\">-0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.725</td></tr><tr><td align=\\\"left\\\"> Within-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">0.70(0.94)</td><td char=\\\".\\\" align=\\\"char\\\">0.06</td><td char=\\\".\\\" align=\\\"char\\\">0.455</td><td align=\\\"left\\\"> negative consequence</td><td char=\\\".\\\" align=\\\"char\\\">-0.77(2.40)</td><td char=\\\".\\\" align=\\\"char\\\">-0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.748</td></tr><tr><td align=\\\"left\\\"> Across-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">-4.92(1.47)</td><td char=\\\".\\\" align=\\\"char\\\">-0.36</td><td char=\\\".\\\" align=\\\"char\\\">0.001</td><td align=\\\"left\\\"> positive consequence</td><td char=\\\".\\\" align=\\\"char\\\">0.30(1.79)</td><td char=\\\".\\\" align=\\\"char\\\">0.02</td><td char=\\\".\\\" align=\\\"char\\\">0.866</td></tr><tr><td align=\\\"left\\\" rowspan=\\\"14\\\" colspan=\\\"4\\\"/><td align=\\\"left\\\"> withholding attention</td><td char=\\\".\\\" align=\\\"char\\\">1.39(1.33)</td><td char=\\\".\\\" align=\\\"char\\\">0.10</td><td char=\\\".\\\" align=\\\"char\\\">0.297</td></tr><tr><td align=\\\"left\\\"> negative attention</td><td char=\\\".\\\" align=\\\"char\\\">0.56(1.47)</td><td char=\\\".\\\" align=\\\"char\\\">0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.705</td></tr><tr><td align=\\\"left\\\"> positive attention</td><td char=\\\".\\\" align=\\\"char\\\">-0.96(1.27)</td><td char=\\\".\\\" align=\\\"char\\\">-0.07</td><td char=\\\".\\\" align=\\\"char\\\">0.454</td></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Step 3</td></tr><tr><td align=\\\"left\\\"> Age child</td><td char=\\\".\\\" align=\\\"char\\\">-0.02(0.04)</td><td char=\\\".\\\" align=\\\"char\\\">-0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.644</td></tr><tr><td align=\\\"left\\\"> Sex child</td><td char=\\\".\\\" align=\\\"char\\\">-0.80(0.71)</td><td char=\\\".\\\" align=\\\"char\\\">-0.10</td><td char=\\\".\\\" align=\\\"char\\\">0.259</td></tr><tr><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">-0.33(0.38)</td><td char=\\\".\\\" align=\\\"char\\\">-0.08</td><td char=\\\".\\\" align=\\\"char\\\">0.382</td></tr><tr><td align=\\\"left\\\"> negative consequence</td><td char=\\\".\\\" align=\\\"char\\\">-4.00(2.42)</td><td char=\\\".\\\" align=\\\"char\\\">-0.16</td><td char=\\\".\\\" align=\\\"char\\\">0.101</td></tr><tr><td align=\\\"left\\\"> positive consequence</td><td char=\\\".\\\" align=\\\"char\\\">-4.05(2.06)</td><td char=\\\".\\\" align=\\\"char\\\">-0.21</td><td char=\\\".\\\" align=\\\"char\\\">0.051</td></tr><tr><td align=\\\"left\\\"> withholding attention</td><td char=\\\".\\\" align=\\\"char\\\">-1.74(1.51)</td><td char=\\\".\\\" align=\\\"char\\\">-0.12</td><td char=\\\".\\\" align=\\\"char\\\">0.252</td></tr><tr><td align=\\\"left\\\"> negative attention</td><td char=\\\".\\\" align=\\\"char\\\">-1.78(1.65)</td><td char=\\\".\\\" align=\\\"char\\\">-0.11</td><td char=\\\".\\\" align=\\\"char\\\">0.282</td></tr><tr><td align=\\\"left\\\"> positive attention</td><td char=\\\".\\\" align=\\\"char\\\">-1.50(1.38)</td><td char=\\\".\\\" align=\\\"char\\\">-0.11</td><td char=\\\".\\\" align=\\\"char\\\">0.282</td></tr><tr><td align=\\\"left\\\"> Within-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">-0.55(1.17)</td><td char=\\\".\\\" align=\\\"char\\\">-0.06</td><td char=\\\".\\\" align=\\\"char\\\">0.638</td></tr><tr><td align=\\\"left\\\"> Across-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">-6.05(1.67)</td><td char=\\\".\\\" align=\\\"char\\\">-0.44</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Results of Regression Analyses Predicting Severity of Daily Disruptive Behavior in Sample 2</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"/><th align=\\\"left\\\"/><th align=\\\"left\\\"/><th align=\\\"left\\\"/><th align=\\\"left\\\" colspan=\\\"4\\\">Severity of daily disruptive behavior</th></tr><tr><th align=\\\"left\\\">variables</th><th align=\\\"left\\\"><italic>b(SE)</italic></th><th align=\\\"left\\\">β</th><th align=\\\"left\\\"><italic>p</italic></th><th align=\\\"left\\\">variables</th><th align=\\\"left\\\"><italic>b(SE)</italic></th><th align=\\\"left\\\">β</th><th align=\\\"left\\\"><italic>p</italic></th></tr><tr><th align=\\\"left\\\" colspan=\\\"4\\\">Step 1</th><th align=\\\"left\\\" colspan=\\\"4\\\">Step 1</th></tr></thead><tbody><tr><td align=\\\"left\\\"> Age child</td><td char=\\\".\\\" align=\\\"char\\\">-0.00(0.01)</td><td char=\\\".\\\" align=\\\"char\\\">-0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.736</td><td align=\\\"left\\\"> Age child</td><td char=\\\".\\\" align=\\\"char\\\">-0.00(0.01)</td><td char=\\\".\\\" align=\\\"char\\\">-0.01</td><td char=\\\".\\\" align=\\\"char\\\">0.736</td></tr><tr><td align=\\\"left\\\"> Sex child</td><td char=\\\".\\\" align=\\\"char\\\">-0.12(0.20)</td><td char=\\\".\\\" align=\\\"char\\\">-0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.541</td><td align=\\\"left\\\"> Sex child</td><td char=\\\".\\\" align=\\\"char\\\">-0.12(0.20)</td><td char=\\\".\\\" align=\\\"char\\\">-0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.541</td></tr><tr><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">0.06(0.10)</td><td char=\\\".\\\" align=\\\"char\\\">0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.562</td><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">0.06(0.10)</td><td char=\\\".\\\" align=\\\"char\\\">0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.562</td></tr><tr><td align=\\\"left\\\"> Received support</td><td char=\\\".\\\" align=\\\"char\\\">0.71(0.38)</td><td char=\\\".\\\" align=\\\"char\\\">0.16</td><td char=\\\".\\\" align=\\\"char\\\">0.062</td><td align=\\\"left\\\"> Received support</td><td char=\\\".\\\" align=\\\"char\\\">0.71(0.38)</td><td char=\\\".\\\" align=\\\"char\\\">0.16</td><td char=\\\".\\\" align=\\\"char\\\">0.062</td></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Step 2</td><td align=\\\"left\\\" colspan=\\\"4\\\">Step 2</td></tr><tr><td align=\\\"left\\\"> Age child</td><td char=\\\".\\\" align=\\\"char\\\">-0.00(0.01)</td><td char=\\\".\\\" align=\\\"char\\\">-0.01</td><td char=\\\".\\\" align=\\\"char\\\">0.988</td><td align=\\\"left\\\"> Age child</td><td char=\\\".\\\" align=\\\"char\\\">0.00(0.01)</td><td char=\\\".\\\" align=\\\"char\\\">0.02</td><td char=\\\".\\\" align=\\\"char\\\">0.776</td></tr><tr><td align=\\\"left\\\"> Sex child</td><td char=\\\".\\\" align=\\\"char\\\">-0.09(0.19)</td><td char=\\\".\\\" align=\\\"char\\\">-0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.639</td><td align=\\\"left\\\"> Sex child</td><td char=\\\".\\\" align=\\\"char\\\">-0.06(0.19)</td><td char=\\\".\\\" align=\\\"char\\\">-0.02</td><td char=\\\".\\\" align=\\\"char\\\">0.770</td></tr><tr><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">0.03(0.10)</td><td char=\\\".\\\" align=\\\"char\\\">0.02</td><td char=\\\".\\\" align=\\\"char\\\">0.785</td><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">0.05(0.10)</td><td char=\\\".\\\" align=\\\"char\\\">0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.612</td></tr><tr><td align=\\\"left\\\"> Received support</td><td char=\\\".\\\" align=\\\"char\\\">0.83(0.36)</td><td char=\\\".\\\" align=\\\"char\\\">0.18</td><td char=\\\".\\\" align=\\\"char\\\">0.021</td><td align=\\\"left\\\"> Received support</td><td char=\\\".\\\" align=\\\"char\\\">0.98(0.36)</td><td char=\\\".\\\" align=\\\"char\\\">0.21</td><td char=\\\".\\\" align=\\\"char\\\">0.007</td></tr><tr><td align=\\\"left\\\"> Within-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">-0.59(0.32)</td><td char=\\\".\\\" align=\\\"char\\\">-0.17</td><td char=\\\".\\\" align=\\\"char\\\">0.063</td><td align=\\\"left\\\"> negative consequence</td><td char=\\\".\\\" align=\\\"char\\\">1.45(0.39)</td><td char=\\\".\\\" align=\\\"char\\\">0.30</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> Across-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">-1.60(0.61)</td><td char=\\\".\\\" align=\\\"char\\\">-0.24</td><td char=\\\".\\\" align=\\\"char\\\">0.010</td><td align=\\\"left\\\"> positive consequence</td><td char=\\\".\\\" align=\\\"char\\\">0.07(0.90)</td><td char=\\\".\\\" align=\\\"char\\\">0.01</td><td char=\\\".\\\" align=\\\"char\\\">0.938</td></tr><tr><td align=\\\"left\\\" rowspan=\\\"15\\\" colspan=\\\"4\\\"/><td align=\\\"left\\\"> withholding attention</td><td char=\\\".\\\" align=\\\"char\\\">0.04(0.39)</td><td char=\\\".\\\" align=\\\"char\\\">0.01</td><td char=\\\".\\\" align=\\\"char\\\">0.901</td></tr><tr><td align=\\\"left\\\"> negative attention</td><td char=\\\".\\\" align=\\\"char\\\">1.77(0.54)</td><td char=\\\".\\\" align=\\\"char\\\">0.26</td><td char=\\\".\\\" align=\\\"char\\\">0.001</td></tr><tr><td align=\\\"left\\\"> positive attention</td><td char=\\\".\\\" align=\\\"char\\\">0.83(0.44)</td><td char=\\\".\\\" align=\\\"char\\\">0.15</td><td char=\\\".\\\" align=\\\"char\\\">0.060</td></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Step 3</td></tr><tr><td align=\\\"left\\\"> Age child</td><td char=\\\".\\\" align=\\\"char\\\">0.00(0.01)</td><td char=\\\".\\\" align=\\\"char\\\">0.02</td><td char=\\\".\\\" align=\\\"char\\\">0.785</td></tr><tr><td align=\\\"left\\\"> sex</td><td char=\\\".\\\" align=\\\"char\\\">-0.05(0.19)</td><td char=\\\".\\\" align=\\\"char\\\">-0.02</td><td char=\\\".\\\" align=\\\"char\\\">0.778</td></tr><tr><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">0.04(0.10)</td><td char=\\\".\\\" align=\\\"char\\\">0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.686</td></tr><tr><td align=\\\"left\\\"> Received support</td><td char=\\\".\\\" align=\\\"char\\\">0.96(0.36)</td><td char=\\\".\\\" align=\\\"char\\\">0.21</td><td char=\\\".\\\" align=\\\"char\\\">0.008</td></tr><tr><td align=\\\"left\\\"> negative consequence</td><td char=\\\".\\\" align=\\\"char\\\">1.42(0.46)</td><td char=\\\".\\\" align=\\\"char\\\">0.29</td><td char=\\\".\\\" align=\\\"char\\\">0.002</td></tr><tr><td align=\\\"left\\\"> positive consequence</td><td char=\\\".\\\" align=\\\"char\\\">-0.06(0.97)</td><td char=\\\".\\\" align=\\\"char\\\">-0.01</td><td char=\\\".\\\" align=\\\"char\\\">0.955</td></tr><tr><td align=\\\"left\\\"> withholding attention</td><td char=\\\".\\\" align=\\\"char\\\">0.22(0.44)</td><td char=\\\".\\\" align=\\\"char\\\">0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.624</td></tr><tr><td align=\\\"left\\\"> negative attention</td><td char=\\\".\\\" align=\\\"char\\\">1.77(0.67)</td><td char=\\\".\\\" align=\\\"char\\\">0.26</td><td char=\\\".\\\" align=\\\"char\\\">0.009</td></tr><tr><td align=\\\"left\\\"> positive attention</td><td char=\\\".\\\" align=\\\"char\\\">0.86(0.59)</td><td char=\\\".\\\" align=\\\"char\\\">0.16</td><td char=\\\".\\\" align=\\\"char\\\">0.142</td></tr><tr><td align=\\\"left\\\"> Within-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">0.26(0.46)</td><td char=\\\".\\\" align=\\\"char\\\">0.08</td><td char=\\\".\\\" align=\\\"char\\\">0.578</td></tr><tr><td align=\\\"left\\\"> Across-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">-0.60(0.74)</td><td char=\\\".\\\" align=\\\"char\\\">-0.09</td><td char=\\\".\\\" align=\\\"char\\\">0.421</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab4\\\"><label>Table 4</label><caption><p>Results of Regression Analysis Predicting Externalizing Behavior one year Later</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">variables</th><th align=\\\"left\\\"><italic>b(SE)</italic></th><th align=\\\"left\\\">β</th><th align=\\\"left\\\"><italic>p</italic></th></tr><tr><th align=\\\"left\\\" colspan=\\\"4\\\">Step 1</th></tr></thead><tbody><tr><td align=\\\"left\\\"> Age Child</td><td char=\\\".\\\" align=\\\"char\\\">-0.01(0.00)</td><td char=\\\".\\\" align=\\\"char\\\">-0.15</td><td char=\\\".\\\" align=\\\"char\\\">0.199</td></tr><tr><td align=\\\"left\\\"> Sex Child</td><td char=\\\".\\\" align=\\\"char\\\">0.00(0.07)</td><td char=\\\".\\\" align=\\\"char\\\">0.01</td><td char=\\\".\\\" align=\\\"char\\\">0.955</td></tr><tr><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">-0.01(0.03)</td><td char=\\\".\\\" align=\\\"char\\\">-0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.676</td></tr><tr><td align=\\\"left\\\"> Daily disruptive behavior severity T1</td><td char=\\\".\\\" align=\\\"char\\\">0.02(0.01)</td><td char=\\\".\\\" align=\\\"char\\\">0.31</td><td char=\\\".\\\" align=\\\"char\\\">0.005</td></tr><tr><td align=\\\"left\\\"> General consistency</td><td char=\\\".\\\" align=\\\"char\\\">-1.90(0.68)</td><td char=\\\".\\\" align=\\\"char\\\">-0.31</td><td char=\\\".\\\" align=\\\"char\\\">0.006</td></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Step 2</td></tr><tr><td align=\\\"left\\\"> Age Child</td><td char=\\\".\\\" align=\\\"char\\\">-0.01(0.00)</td><td char=\\\".\\\" align=\\\"char\\\">-0.13</td><td char=\\\".\\\" align=\\\"char\\\">0.260</td></tr><tr><td align=\\\"left\\\"> Sex Child</td><td char=\\\".\\\" align=\\\"char\\\">-0.01(0.07)</td><td char=\\\".\\\" align=\\\"char\\\">-0.01</td><td char=\\\".\\\" align=\\\"char\\\">0.927</td></tr><tr><td align=\\\"left\\\"> Education Level</td><td char=\\\".\\\" align=\\\"char\\\">-0.01(0.04)</td><td char=\\\".\\\" align=\\\"char\\\">-0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.711</td></tr><tr><td align=\\\"left\\\"> Daily disruptive behavior severity T1</td><td char=\\\".\\\" align=\\\"char\\\">0.02(0.01)</td><td char=\\\".\\\" align=\\\"char\\\">0.30</td><td char=\\\".\\\" align=\\\"char\\\">0.010</td></tr><tr><td align=\\\"left\\\"> General consistency</td><td char=\\\".\\\" align=\\\"char\\\">-1.76(0.82)</td><td char=\\\".\\\" align=\\\"char\\\">-0.29</td><td char=\\\".\\\" align=\\\"char\\\">0.036</td></tr><tr><td align=\\\"left\\\"> Within-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">0.05(0.09)</td><td char=\\\".\\\" align=\\\"char\\\">0.08</td><td char=\\\".\\\" align=\\\"char\\\">0.553</td></tr><tr><td align=\\\"left\\\"> Across-episode consistency</td><td char=\\\".\\\" align=\\\"char\\\">-0.12(0.17)</td><td char=\\\".\\\" align=\\\"char\\\">-0.11</td><td char=\\\".\\\" align=\\\"char\\\">0.464</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab5\\\"><label>Table 5</label><caption><p>Answer categories for each parental reaction for the two studies</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"><bold>Study 1</bold></th><th align=\\\"left\\\"><bold>Study 2</bold></th></tr></thead><tbody><tr><td align=\\\"left\\\" colspan=\\\"2\\\"><bold>Negative consequence</bold></td></tr><tr><td align=\\\"left\\\">I sent my child to their room/corner/time-out</td><td align=\\\"left\\\">I sent my child to their room for at least an hour</td></tr><tr><td align=\\\"left\\\">I punished my child</td><td align=\\\"left\\\">I gave my child a short time-out, away from others</td></tr><tr><td align=\\\"left\\\"/><td align=\\\"left\\\">I took something nice away from my child (e.g., toys or screen time)</td></tr><tr><td align=\\\"left\\\"/><td align=\\\"left\\\">I gave my child extra chores (e.g., set the table)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\"><bold>Positive consequence</bold></td></tr><tr><td align=\\\"left\\\">I negotiated with my child</td><td align=\\\"left\\\">I gave my child his/her way</td></tr><tr><td align=\\\"left\\\">I gave in to my child</td><td align=\\\"left\\\">I gave in to my child</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\"><bold>Withholding attention</bold></td></tr><tr><td align=\\\"left\\\">I didn’t, I let my child cool off</td><td align=\\\"left\\\">I did nothing</td></tr><tr><td align=\\\"left\\\">I ignored my child</td><td align=\\\"left\\\">I talked about it with my child afterwards</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\"><bold>Negative attention</bold></td></tr><tr><td align=\\\"left\\\">I became angry with my child</td><td align=\\\"left\\\">I yelled/swore</td></tr><tr><td align=\\\"left\\\">I grabbed my child</td><td align=\\\"left\\\">I said things I didn’t mean</td></tr><tr><td align=\\\"left\\\">I spoke sternly to my child</td><td align=\\\"left\\\">I threatened with punishment, but did not punish</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\"><bold>Positive attention</bold></td></tr><tr><td align=\\\"left\\\">I comforted my child</td><td align=\\\"left\\\">I begged my child to stop</td></tr><tr><td align=\\\"left\\\">I distracted my child</td><td align=\\\"left\\\">I used humor to distract my child</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab6\\\"><label>Table 6</label><caption><p>Answer categories for the aggressive tantrum behaviors</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Hitting</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Kicking</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Biting</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Throwing an object</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Pushing/pulling</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Spitting</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Grabbing</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Banging head</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Holding breath</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">freezing</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":["<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mathrm{IQV}=\\frac{\\left(\\mathrm{K}\\left(1-\\left(\\sum \\mathrm{Prop}2\\right)\\right)\\right)}{\\left(\\mathrm{K}-1\\right)}$$\\end{document}</tex-math><mml:math id=\"M2\" display=\"block\"><mml:mrow><mml:mi 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0.05, **<italic>p</italic> < 0.01</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"10802_2023_1073_Article_Equa.gif\" position=\"anchor\"/>"],"string":"[\n \"<graphic xlink:href=\\\"10802_2023_1073_Article_Equa.gif\\\" position=\\\"anchor\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"mixed-citation\": [\"Achenbach, T. 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(2020). \"], \"italic\": [\"Jaarrapport integratie 2020\"]}, {\"surname\": [\"Dwairy\"], \"given-names\": [\"M\"], \"article-title\": [\"Parental inconsistency: a third cross-cultural research on parenting and psychological adjustment of children\"], \"source\": [\"Journal of Child and Family Studies\"], \"year\": [\"2010\"], \"volume\": [\"19\"], \"fpage\": [\"23\"], \"lpage\": [\"29\"], \"pub-id\": [\"10.1007/s10826-009-9339-x\"]}, {\"mixed-citation\": [\"Frankfort-Nachmias, C., & Leon-Guerrero, A. (2018). 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T., Ram, N., & Almeida, D. M. (2018). Activity diversity and its associations with psychological well-being across adulthood. \"], \"italic\": [\"The Journals of Gerontology. Series B, Psychological Sciences and Social Sciences\", \"73\"]}, {\"surname\": [\"Lengua\", \"Kovacs\"], \"given-names\": [\"LJ\", \"EA\"], \"article-title\": [\"Bidirectional associations between temperament and parenting and the prediction of adjustment problems in middle childhood\"], \"source\": [\"Journal of Applied Developmental Psychology\"], \"year\": [\"2005\"], \"volume\": [\"26\"], \"issue\": [\"1\"], \"fpage\": [\"21\"], \"lpage\": [\"38\"], \"pub-id\": [\"10.1016/j.appdev.2004.10.001\"]}, {\"mixed-citation\": [\"Mabbe, E., Soenens, B., Vansteenkiste, M., van der Kaap-Deeder, J., & Mouratidis, A. (2018). Day-to-day variation in autonomy-supportive and psychologically controlling parenting: the role of parents\\u2019 daily experiences of need satisfaction and need frustration. \"], \"italic\": [\"Parenting: Science and Practice\", \"18\"]}, {\"mixed-citation\": [\"Patterson, G. R. (1982). \"], \"italic\": [\"Coercive family process\"]}, {\"surname\": [\"Rueger\", \"Katz\", \"Risser\", \"Lovejoy\"], \"given-names\": [\"SY\", \"RL\", \"HJ\", \"MC\"], \"article-title\": [\"Relations between parental affect and parenting behaviors: a meta-analytic review\"], \"source\": [\"Parenting\"], \"year\": [\"2011\"], \"volume\": [\"11\"], \"issue\": [\"1\"], \"fpage\": [\"1\"], \"lpage\": [\"33\"], \"pub-id\": [\"10.1080/15295192.2011.539503\"]}, {\"surname\": [\"Smit\", \"Mikami\", \"Normand\"], \"given-names\": [\"S\", \"AY\", \"S\"], \"article-title\": [\"Parenting children with ADHD: Associations with parental depression, parental ADHD, and child behavior problems\"], \"source\": [\"Journal of Child and Family Studies\"], \"year\": [\"2021\"], \"volume\": [\"30\"], \"issue\": [\"5\"], \"fpage\": [\"1156\"], \"lpage\": [\"1170\"], \"pub-id\": [\"10.1007/s10826-021-01944-0\"]}, {\"mixed-citation\": [\"van Zeijl, J., Mesman, J., Stolk, M. N., Alink, L. R. 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Furthermore, we evaluated the usefulness and the inter-observer variability of the overall LIKERT (subjective operator assessment for the likelihood of a csPCa, from 1 to 5), ECE-LIKERT (subjective operator assessment for the likelihood of ECE, from 1 to 5), PI-RADS v2 and ESUR-ECE, in order to evaluate their performance in predicting the ECE risk in the same cohort of patients.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">Multiparametric MRI (mpMRI) of the prostate is nowadays considered the key tool for the diagnosis of clinically significant prostate cancer (csPCa) [##UREF##0##1##].</p>\",\n \"<p id=\\\"Par6\\\">The quality of MRI has progressively increased in the last years and standardised multiparametric sequences acquisition and reporting have been established, with the development of the Prostate Imaging – Reporting and Data System (PI-RADS) scoring system, first published in 2012 by the European Society of Urogenital Radiology (ESUR) [##REF##22322308##2##].</p>\",\n \"<p id=\\\"Par7\\\">The updated PI-RADS versions 2 and 2.1 are nowadays broadly used in clinical practice, overcoming some ambiguities and limitations related to the overall scoring system of the former version [##REF##26427566##3##, ##REF##30898406##4##].</p>\",\n \"<p id=\\\"Par8\\\">Nevertheless, PI-RADS has not being designed for staging purposes; on this regard, the ESUR developed another score system based on capsular alterations detectable at MRI and related to the likelihood of an extracapsular extension (ECE) of the lesion [##REF##25504428##5##].</p>\",\n \"<p id=\\\"Par9\\\">During treatment planning, the identification of ECE as a strategy for local staging is crucial in order to achieve an adequate balance between cancer control and preservation of potency and continence, ultimately obtaining the best surgical, oncological and functional results.</p>\",\n \"<p id=\\\"Par10\\\">The main objective of this study was to identify clinical, pathological and radiological parameters associated to ECE in a single institution cohort of patients undergoing mpMRI prior to radical prostatectomy (RP), as by whole-mount prostate sections for definitive histological assessment. Furthermore, we evaluated the usefulness and the inter-observer variability of the overall LIKERT (subjective operator assessment for the likelihood of a csPCa, from 1 to 5), ECE-LIKERT (subjective operator assessment for the likelihood of ECE, from 1 to 5), PI-RADS v2 and ESUR-ECE, in order to evaluate their performance in predicting the ECE risk in the same cohort of patients.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Patients and methods</title>","<title>Study design and population</title>","<p id=\"Par11\">This is a retrospective analysis of patients who had undergone mpMRI before RP at Fundació Puigvert – Barcelona (ES), between April 2013 (date of the implementation of mpMRI driven pathway) and December 2017. The mpMRI was requested at urology consultant discretion, either before or after the diagnostic biopsy, as no specific recommendations for requesting an mpMRI were still put in place during the period in observation nor in the international guidelines neither in our internal protocol.</p>","<p id=\"Par12\">Overall, 126 patients met the selection criteria (mpMRI undertaken within 6 months before surgery, either before or after the diagnostic biopsy; availability of full set of data in observation) and their medical records were collected and reviewed. The patients’ data were managed according to our institutional review board protocol, in full compliance with both the principles of the latest version of the Declaration of Helsinki and of the Spanish adaptation of the General Data Protection Regulation (organic law 3/2018, December the 5th 2018).</p>","<title>mpMRI protocol</title>","<p id=\"Par13\">A 3-Tesla mpMRI examination with a pelvic phased-array surface coil was performed for all patients. The mpMRI protocol-included T2-weighted (T2W) sequences in three planes, Diffusion Weighted Imaging (DWI) sequences with high <italic>b</italic>-values (> 1200 s/mm<sup>2</sup>) and apparent diffusion coefficient (ADC) map, and Dynamic Contrast-Enhanced (DCE) sequences with a bolus of gadolinium contrast medium injection. Imaging acquisition protocol was rigorously compliant with the PIRADS v1 guidelines, in force during the period in observation [##REF##22322308##2##]. It is Important to note that these guidelines also included recommendations for waiting a period of 4–6 weeks between a biopsy and the eventual MRI to minimise the effect of eventual haemorrhage, and in case of substantial persisting artifacts to repeat the MRI within further 4 weeks or so. These recommendations were duly followed, and interestingly remained substantially unvaried along the updated v2 and v2.1 versions [##REF##26427566##3##, ##REF##30898406##4##]. All images were retrospectively and independently reassessed by two expert radiologists (L.G. and J.H.) with at least 4 years of experience in prostate mpMRI, both blinded to clinical and histological data; PI-RADS v2 was used to score the MRI explorations, as the updated v2.1 was published posteriorly to the radiological revision of the imaging tests. The maximal index lesion size (ILS), length of capsular involvement (LCI) by tumour, number of lesions and location were recorded whenever visible from the dominant sequence involved. The radiologists also used an overall-LIKERT and ECE-LIKERT scores, as by subjective impression of the likelihood of significant malignancy and extracapsular extension (1 = very unlikely; 5 = very likely), according to the recommendation of the PREDICT (Prostate Diagnostic Imaging Consensus Meeting) panel [##REF##28111879##6##]. Furthermore, the likelihood of ECE was evaluated using the ESUR MRI scoring guidelines of extra-prostatic disease [##REF##25504428##5##].</p>","<title>Reference standard</title>","<p id=\"Par14\">Whole-mount histological sections from the RP specimens were used as the reference standard. The specimens were fixed in 10% buffered formalin, and sectioned into horizontal sections of 3–4 mm. All tissues were paraffin-embedded, and 3–4 microns sections were obtained and stained with hematoxylin–eosin; then, the sections of the tissue were assessed by a single expert uropathologist (F.A.), blinded to mpMRI data. The uropathologist recorded cancer location, size, volume, and Gleason grade group according to the International Society of Urological Pathology (ISUP) consensus conference of 2014 [##REF##26492179##7##].</p>","<title>Outcomes and statistical analysis</title>","<p id=\"Par15\">The primary outcome consisted in identifying the parameters associated to the ECE at the RP specimen. Descriptive data were expressed as median and interquartile range (IQR, 25–75 quartile). Analysis between groups was performed using Student’s <italic>t</italic> test (Mann–Whitney <italic>U</italic> test in variables without normal distribution) for continuous variables, and Chi-square (Fisher’s exact test with observed frequencies < 5) for categorical variables.</p>","<p id=\"Par16\">Quantitative (continuous) variables were transformed to binary (categorical) variables before inclusion in logistic models using the best predictive cut-off point obtained with Receiver Operating Characteristics (ROC) curve analysis.</p>","<p id=\"Par17\">Univariate and multivariate logistic regression models were performed including ECE as a dependent variable. Preoperative clinical, pathological and mpMRI variables with <italic>p</italic> value < 0.2 at the univariate analysis were included as independent variables using the backward stepwise logistic regression analysis. Predictors from the final model were used to calculate the likelihood of ECE according to the following equation: Exp(<italic>β</italic>)/[1 + Exp(<italic>β</italic>)], where <italic>β</italic> = [− 3.00 + <italic>X</italic>*(predictor <italic>A</italic>) + <italic>Y</italic>*(predictor <italic>B</italic>)] for two predictors. The quality of the final model was assessed using the Hosmer–Lemeshow goodness-of-fit-test.</p>","<p id=\"Par18\">Inter-observer agreement between the two radiologists regarding the MRI features/scores was assessed using the intraclass correlation coefficient (ICC) with the 95% confidence interval, applying a two-way ICC with a random rater assumption. The agreement (match) between a PCa lesion detected at mpMRI and an equivalent lesion at whole-mount histological sections from the RP specimens was assessed using Cohen Kappa coefficient, whose results were categorised in standardised ranges < 0.4 poor agreement; 0.4–0.6 moderate agreement; 0.61–0.8 substantial agreement; 0.81–1 as excellent. A <italic>p</italic>-value < 0.05 was considered statistically significant for all cases. Statistical analysis was performed using R studio (V2.5) package.</p>"],"string":"[\n \"<title>Patients and methods</title>\",\n \"<title>Study design and population</title>\",\n \"<p id=\\\"Par11\\\">This is a retrospective analysis of patients who had undergone mpMRI before RP at Fundació Puigvert – Barcelona (ES), between April 2013 (date of the implementation of mpMRI driven pathway) and December 2017. The mpMRI was requested at urology consultant discretion, either before or after the diagnostic biopsy, as no specific recommendations for requesting an mpMRI were still put in place during the period in observation nor in the international guidelines neither in our internal protocol.</p>\",\n \"<p id=\\\"Par12\\\">Overall, 126 patients met the selection criteria (mpMRI undertaken within 6 months before surgery, either before or after the diagnostic biopsy; availability of full set of data in observation) and their medical records were collected and reviewed. The patients’ data were managed according to our institutional review board protocol, in full compliance with both the principles of the latest version of the Declaration of Helsinki and of the Spanish adaptation of the General Data Protection Regulation (organic law 3/2018, December the 5th 2018).</p>\",\n \"<title>mpMRI protocol</title>\",\n \"<p id=\\\"Par13\\\">A 3-Tesla mpMRI examination with a pelvic phased-array surface coil was performed for all patients. The mpMRI protocol-included T2-weighted (T2W) sequences in three planes, Diffusion Weighted Imaging (DWI) sequences with high <italic>b</italic>-values (> 1200 s/mm<sup>2</sup>) and apparent diffusion coefficient (ADC) map, and Dynamic Contrast-Enhanced (DCE) sequences with a bolus of gadolinium contrast medium injection. Imaging acquisition protocol was rigorously compliant with the PIRADS v1 guidelines, in force during the period in observation [##REF##22322308##2##]. It is Important to note that these guidelines also included recommendations for waiting a period of 4–6 weeks between a biopsy and the eventual MRI to minimise the effect of eventual haemorrhage, and in case of substantial persisting artifacts to repeat the MRI within further 4 weeks or so. These recommendations were duly followed, and interestingly remained substantially unvaried along the updated v2 and v2.1 versions [##REF##26427566##3##, ##REF##30898406##4##]. All images were retrospectively and independently reassessed by two expert radiologists (L.G. and J.H.) with at least 4 years of experience in prostate mpMRI, both blinded to clinical and histological data; PI-RADS v2 was used to score the MRI explorations, as the updated v2.1 was published posteriorly to the radiological revision of the imaging tests. The maximal index lesion size (ILS), length of capsular involvement (LCI) by tumour, number of lesions and location were recorded whenever visible from the dominant sequence involved. The radiologists also used an overall-LIKERT and ECE-LIKERT scores, as by subjective impression of the likelihood of significant malignancy and extracapsular extension (1 = very unlikely; 5 = very likely), according to the recommendation of the PREDICT (Prostate Diagnostic Imaging Consensus Meeting) panel [##REF##28111879##6##]. Furthermore, the likelihood of ECE was evaluated using the ESUR MRI scoring guidelines of extra-prostatic disease [##REF##25504428##5##].</p>\",\n \"<title>Reference standard</title>\",\n \"<p id=\\\"Par14\\\">Whole-mount histological sections from the RP specimens were used as the reference standard. The specimens were fixed in 10% buffered formalin, and sectioned into horizontal sections of 3–4 mm. All tissues were paraffin-embedded, and 3–4 microns sections were obtained and stained with hematoxylin–eosin; then, the sections of the tissue were assessed by a single expert uropathologist (F.A.), blinded to mpMRI data. The uropathologist recorded cancer location, size, volume, and Gleason grade group according to the International Society of Urological Pathology (ISUP) consensus conference of 2014 [##REF##26492179##7##].</p>\",\n \"<title>Outcomes and statistical analysis</title>\",\n \"<p id=\\\"Par15\\\">The primary outcome consisted in identifying the parameters associated to the ECE at the RP specimen. Descriptive data were expressed as median and interquartile range (IQR, 25–75 quartile). Analysis between groups was performed using Student’s <italic>t</italic> test (Mann–Whitney <italic>U</italic> test in variables without normal distribution) for continuous variables, and Chi-square (Fisher’s exact test with observed frequencies < 5) for categorical variables.</p>\",\n \"<p id=\\\"Par16\\\">Quantitative (continuous) variables were transformed to binary (categorical) variables before inclusion in logistic models using the best predictive cut-off point obtained with Receiver Operating Characteristics (ROC) curve analysis.</p>\",\n \"<p id=\\\"Par17\\\">Univariate and multivariate logistic regression models were performed including ECE as a dependent variable. Preoperative clinical, pathological and mpMRI variables with <italic>p</italic> value < 0.2 at the univariate analysis were included as independent variables using the backward stepwise logistic regression analysis. Predictors from the final model were used to calculate the likelihood of ECE according to the following equation: Exp(<italic>β</italic>)/[1 + Exp(<italic>β</italic>)], where <italic>β</italic> = [− 3.00 + <italic>X</italic>*(predictor <italic>A</italic>) + <italic>Y</italic>*(predictor <italic>B</italic>)] for two predictors. The quality of the final model was assessed using the Hosmer–Lemeshow goodness-of-fit-test.</p>\",\n \"<p id=\\\"Par18\\\">Inter-observer agreement between the two radiologists regarding the MRI features/scores was assessed using the intraclass correlation coefficient (ICC) with the 95% confidence interval, applying a two-way ICC with a random rater assumption. The agreement (match) between a PCa lesion detected at mpMRI and an equivalent lesion at whole-mount histological sections from the RP specimens was assessed using Cohen Kappa coefficient, whose results were categorised in standardised ranges < 0.4 poor agreement; 0.4–0.6 moderate agreement; 0.61–0.8 substantial agreement; 0.81–1 as excellent. A <italic>p</italic>-value < 0.05 was considered statistically significant for all cases. Statistical analysis was performed using R studio (V2.5) package.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par19\">The clinical, bioptic and MRI features are summarised in Table ##TAB##0##1##. The median age at prostate biopsy, PSA and PSA-density were 66.6 years, 7.2 ng/ml and 0.2 ng/ml<sup>2</sup>, respectively.</p>","<p id=\"Par20\">Overall, 41 patients (32.5%) had T2 or T3 clinical stage. Intraprostatic perineural invasion (IPNI) and ISUP group grade > 3 were observed in 26 (20.6%) and 29 (23%) patients, respectively.</p>","<p id=\"Par21\">The median index lesion size (ILS) and length of capsular involvement (LCI) were 12 and 9 mm (mm), respectively. MRI readings based on PI-RADS v2, ESUR and LIKERT scores are available in the Supplementary material.</p>","<p id=\"Par22\">Pathology data and ECE analysis are summarised in Table ##TAB##1##2## and Supplementary material, respectively. The median ILS of RP specimen was 16 mm. Accordingly, MRI underestimated the ILS by a 25% in comparison to the true specimen size.</p>","<p id=\"Par23\">Overall, ECE was found in 35 (27.8%) patients; definitive ISUP grade > 3 was observed in 40 (31.7%) patients.</p>","<p id=\"Par24\">The following variables were significantly associated to ECE: length of biopsy core (LBC; median: 7 vs 4 mm, <italic>p</italic> < 0.001), intraprostatic perineural invasion (48.6% vs 9.9%, <italic>p</italic> < 0.001), ILS (median: 20 vs 10 mm, <italic>p</italic> < 0.001), LCI (median: 17 vs 7 mm, <italic>p</italic> < 0.001), PSA density (0.24 vs 0.14, <italic>p</italic> = 0.001), and biopsy ISUP grade > 3 (40% vs 16.4%, <italic>p</italic> = 0.003).</p>","<p id=\"Par25\">At ROC Curve analysis, the best cut-off points for LBC, LCI and ILS were identified as 5.5, 9.5 and 11 mm, respectively.</p>","<p id=\"Par26\">Overall, both LCI and IPNI showed statistical significance (<italic>p</italic> < 0.001) at multivariate logistic regression model (Table ##TAB##2##3##). The probability to detect ECE with the generated model was 81.4% by including the two variables (Supplementary material). The model was calibrated with an overall p-value of 0.985 by using the Hosmer–Lemeshow test (<italic>R</italic><sup>2</sup> = 66%), and the predictive accuracy was evaluated through ROC curve analysis, with an area under the curve (AUC) of 0.83 [95% CI (0.76–0.90)], <italic>p</italic> < 0.001 (Fig. ##FIG##0##1##).</p>","<p id=\"Par27\">Univariate and multivariate logistic regression analyses were also conducted to identify predictors in the subgroup of patients with seminal vesicle invasion (SVI), but they were not included in the final model because of the low number of events (<italic>n</italic> = 8). Data analysis and comparison of ROC curve between final model and imaging scores models (PI-RADS v2, LIKERT, ESUR) are available in the Supplementary material.</p>","<p id=\"Par28\">Concordance between the two radiologists was evaluated for ILS, PI-RADS v2, LIKERT and ESUR score, with an overall ICC > 0.6 (<italic>p</italic> < 0.001) in all parameters examined (Supplementary material). Correlation for ILS was 0.66 and 0.62 for L.G. and J.H., respectively (<italic>p</italic> < 0.001).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par19\\\">The clinical, bioptic and MRI features are summarised in Table ##TAB##0##1##. The median age at prostate biopsy, PSA and PSA-density were 66.6 years, 7.2 ng/ml and 0.2 ng/ml<sup>2</sup>, respectively.</p>\",\n \"<p id=\\\"Par20\\\">Overall, 41 patients (32.5%) had T2 or T3 clinical stage. Intraprostatic perineural invasion (IPNI) and ISUP group grade > 3 were observed in 26 (20.6%) and 29 (23%) patients, respectively.</p>\",\n \"<p id=\\\"Par21\\\">The median index lesion size (ILS) and length of capsular involvement (LCI) were 12 and 9 mm (mm), respectively. MRI readings based on PI-RADS v2, ESUR and LIKERT scores are available in the Supplementary material.</p>\",\n \"<p id=\\\"Par22\\\">Pathology data and ECE analysis are summarised in Table ##TAB##1##2## and Supplementary material, respectively. The median ILS of RP specimen was 16 mm. Accordingly, MRI underestimated the ILS by a 25% in comparison to the true specimen size.</p>\",\n \"<p id=\\\"Par23\\\">Overall, ECE was found in 35 (27.8%) patients; definitive ISUP grade > 3 was observed in 40 (31.7%) patients.</p>\",\n \"<p id=\\\"Par24\\\">The following variables were significantly associated to ECE: length of biopsy core (LBC; median: 7 vs 4 mm, <italic>p</italic> < 0.001), intraprostatic perineural invasion (48.6% vs 9.9%, <italic>p</italic> < 0.001), ILS (median: 20 vs 10 mm, <italic>p</italic> < 0.001), LCI (median: 17 vs 7 mm, <italic>p</italic> < 0.001), PSA density (0.24 vs 0.14, <italic>p</italic> = 0.001), and biopsy ISUP grade > 3 (40% vs 16.4%, <italic>p</italic> = 0.003).</p>\",\n \"<p id=\\\"Par25\\\">At ROC Curve analysis, the best cut-off points for LBC, LCI and ILS were identified as 5.5, 9.5 and 11 mm, respectively.</p>\",\n \"<p id=\\\"Par26\\\">Overall, both LCI and IPNI showed statistical significance (<italic>p</italic> < 0.001) at multivariate logistic regression model (Table ##TAB##2##3##). The probability to detect ECE with the generated model was 81.4% by including the two variables (Supplementary material). The model was calibrated with an overall p-value of 0.985 by using the Hosmer–Lemeshow test (<italic>R</italic><sup>2</sup> = 66%), and the predictive accuracy was evaluated through ROC curve analysis, with an area under the curve (AUC) of 0.83 [95% CI (0.76–0.90)], <italic>p</italic> < 0.001 (Fig. ##FIG##0##1##).</p>\",\n \"<p id=\\\"Par27\\\">Univariate and multivariate logistic regression analyses were also conducted to identify predictors in the subgroup of patients with seminal vesicle invasion (SVI), but they were not included in the final model because of the low number of events (<italic>n</italic> = 8). Data analysis and comparison of ROC curve between final model and imaging scores models (PI-RADS v2, LIKERT, ESUR) are available in the Supplementary material.</p>\",\n \"<p id=\\\"Par28\\\">Concordance between the two radiologists was evaluated for ILS, PI-RADS v2, LIKERT and ESUR score, with an overall ICC > 0.6 (<italic>p</italic> < 0.001) in all parameters examined (Supplementary material). Correlation for ILS was 0.66 and 0.62 for L.G. and J.H., respectively (<italic>p</italic> < 0.001).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par29\">The prognosis of PCa is highly related to tumour stage, and ECE is the most common adverse feature found in histopathology after RP [##REF##23083655##8##]. Inadequate selection of patients with ECE to nerve-sparing RP increases the risk of positive surgical margins with subsequent need for either adjuvant or salvage radiotherapy. In order to improve preoperative risk assessment, numerous nomograms have been developed in the last years, mostly based on the association of multiple clinical risk factors (PSA, biopsy ISUP grade, clinical stage, etc.) [##REF##15076291##9##–##REF##16469587##12##].</p>","<p id=\"Par30\">The Partin tables were among the first tools developed for the prediction of pathological stage, and have been widely used for several years for surgical planning [##REF##9145716##10##]. Other predictive tools were subsequently developed using different variables (e.g. percentage of positive biopsy cores), but none of them reached a comparable popularity as for the Partin tables [##REF##15076291##9##, ##REF##16469587##12##]. Nevertheless, they could not distinguish unilateral from bilateral ECE [##REF##11744442##13##].</p>","<p id=\"Par31\">With the advent of mpMRI, further nomograms have been developed in an attempt to improve the accuracy to predict the ECE. Feng et al. first reported that mpMRI could improve the performance of Partin tables and MSKCC nomogram regarding ECE prediction [##REF##26194289##14##].</p>","<p id=\"Par32\">Giganti et al. developed a nomogram exploiting clinical and MRI parameters with strong accuracy for ECE prediction [##UREF##1##15##], subsequently confirmed by an external validation conducted by Alves et al. [##REF##32307562##16##]. One of the most important features of their model was the excellent concordance between MRI-tumour volume and the ADC map of tumour lesion.</p>","<p id=\"Par33\">More recently, Gandaglia et al. developed a model to predict ECE, SVI and stage upgrading in patients diagnosed with MRI-targeted and concomitant systematic biopsies [##REF##31547938##17##], achieving an AUC of 73% (ECE), 81% (SVI) and 73% (upgrading) at internal validation.</p>","<p id=\"Par34\">Nevertheless, a meta-analysis of de Rooij et al. reported high specificity but low sensitivity for mpMRI accuracy in local staging; overall, staging based on MRI alone lacks sensitivity in detecting ECE, especially in case of focal, minimal or microscopic extension because of limitation in spatial resolution [##REF##26215604##18##, ##REF##26260000##19##]. Moreover, the degree of underestimation increases with smaller radiologic tumour size and lower PI-RADS scores [##REF##33026934##20##].</p>","<p id=\"Par35\">The MRI and histology biopsy features have been variably reported in literature in the recent past as predictors of ECE [##REF##33272865##21##].</p>","<p id=\"Par36\">Baco et al. found that MRI-tumour LCI well correlated with ECE, with an AUC that outperformed the Partin tables; they also found higher accuracy for microscopic-ECE detection with a 20 mm threshold [##REF##25150643##22##].</p>","<p id=\"Par37\">Similarly, Kongnyuy et al. identified MRI-LCI as a promising predictor of ECE, positive pathological lymph nodes and biochemical recurrence. The 12.5 mm cut-off showed the highest sensitivity (77%) and specificity (59%) in predicting ECE. The AUC was comparable to that of the Partin tables, outperforming them with LCI and PSA combination [##UREF##2##23##].</p>","<p id=\"Par38\">Moreover, in a recent meta-analysis of Li et al., the LCI showed high diagnostic performance in predicting ECE, with a pooled sensitivity and specificity of 0.79 and 0.77, respectively. When subgroup analysis was performed comparing different threshold values, lower LCI cut-off values yielded slightly better sensitivity and comparable specificity, without substantial differences between sub-groups [##REF##34881183##24##].</p>","<p id=\"Par39\">In addition to LCI, IPNI is another acknowledged parameter often associated with ECE, possibly because in the 85% of cases the ECE goes through neurovascular bundles by dissection of the intraprostatic perineural spaces for tissue planes of least resistance [##REF##17495178##25##]</p>","<p id=\"Par40\">Perineural invasion is defined as the tumour invasion into the perineural sheath, and during the years, it has been associated with tumour progression and prognosis of several malignancies, including prostate cancer [##REF##34994908##26##]. In a recent study on upper urinary tract urothelial carcinomas, Lin et al. found that PNI-positive patients had unfavourable pathological features, including high pathological stage, high tumour grade and lymphovascular invasion, leading to worse progression-free survival (PFS) [##REF##34994908##26##].</p>","<p id=\"Par41\">Algaba et al. found that IPNI is correlated to cancer volume and higher percentage of extraprostatic cancer [##REF##16084008##27##]. Same finding was reported more recently by Leyh-Bannurah et al., being IPNI the only histological parameter found significantly associated to ECE in their nomogram [##REF##32248363##28##] Nevertheless, IPNI has not yet been reported among the most powerful histological features even in the latest version of the EAU guidelines, so that our finding might prompt its inclusion among the reporting recommendations for the prostatic biopsy.</p>","<p id=\"Par42\">In our study, we identified both the LCI and the IPNI as predictors of ECE: the LCI cut-off that best correlated to ECE was 9.5 mm, which was a similar finding reported also by Li et al. [##REF##34881183##24##].</p>","<p id=\"Par43\">Interestingly, in our cohort, the ILS at MRI did not show the same degree of association to the ECE as by the LCI; furthermore, MRI underestimated pathological tumour size by 25%.</p>","<p id=\"Par44\">Overall, these data may have several implications: (1) Prostate mpMRI alone, including singular features and scoring systems, do not adequately predict ECE, except LCI—especially in combination to a histology biopsy feature, as IPNI in our series. (2) A change of PI-RADS score 5 definition should be prompted in the future PI-RADS updated version: score 5 is attributed to ILS ≥ 15 mm, but this threshold was chosen by the PI-RADS steering committee on the basis of old studies of the’90, when csPCa and ECE were found to be correlated to a tumour volume ≥ 0.5 cm<sup>3</sup> (15 mm in major axis) at whole-mount RP specimen [##REF##7506797##29##, ##REF##8709307##30##]. If differences of PI-RADS scores 4 to 5 are to be based on the risk of ECE, LCI should be the preferred MRI feature and with a lower cut-off, as the 15 mm cut-off at MRI might underestimate for a quarter the actual tumour size at specimen. (3) We found excellent or substantial inter-readers agreement for relevant MRI variables, thus strengthening the importance of high-quality imaging acquisition and readers’ skills for their adequate assessment. This latter matter has been popularised with the introduction of a dedicate score (PIQUAL) about the quality of the MRI sequences and the ability to make decision on the basis of it [##REF##32646850##31##].</p>","<p id=\"Par45\">Main limitations of the study includes (1) the retrospective design of our analysis, even though significant efforts have been done in reviewing MRI images by two radiologists blinded to final histology; (2) the number of cases is limited, especially because MRI implementation in the clinical practice has substantially increased in more recent years; (3) the MRI images were reassessed according to the PI-RADS v2, as the version 2.1 became available on a later stage to that phase of our study. Nevertheless, it is very unlikely that the minor changes in score reporting of the latest version would have had an impact on the outcome of our study, as shown in a recent publication comparing the v2.0 vs v2.1 diagnostic performance with no difference in concordance rates between targeted biopsy and radical prostatectomy (doi: 10.2214/AJR.23.29964).</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par29\\\">The prognosis of PCa is highly related to tumour stage, and ECE is the most common adverse feature found in histopathology after RP [##REF##23083655##8##]. Inadequate selection of patients with ECE to nerve-sparing RP increases the risk of positive surgical margins with subsequent need for either adjuvant or salvage radiotherapy. In order to improve preoperative risk assessment, numerous nomograms have been developed in the last years, mostly based on the association of multiple clinical risk factors (PSA, biopsy ISUP grade, clinical stage, etc.) [##REF##15076291##9##–##REF##16469587##12##].</p>\",\n \"<p id=\\\"Par30\\\">The Partin tables were among the first tools developed for the prediction of pathological stage, and have been widely used for several years for surgical planning [##REF##9145716##10##]. Other predictive tools were subsequently developed using different variables (e.g. percentage of positive biopsy cores), but none of them reached a comparable popularity as for the Partin tables [##REF##15076291##9##, ##REF##16469587##12##]. Nevertheless, they could not distinguish unilateral from bilateral ECE [##REF##11744442##13##].</p>\",\n \"<p id=\\\"Par31\\\">With the advent of mpMRI, further nomograms have been developed in an attempt to improve the accuracy to predict the ECE. Feng et al. first reported that mpMRI could improve the performance of Partin tables and MSKCC nomogram regarding ECE prediction [##REF##26194289##14##].</p>\",\n \"<p id=\\\"Par32\\\">Giganti et al. developed a nomogram exploiting clinical and MRI parameters with strong accuracy for ECE prediction [##UREF##1##15##], subsequently confirmed by an external validation conducted by Alves et al. [##REF##32307562##16##]. One of the most important features of their model was the excellent concordance between MRI-tumour volume and the ADC map of tumour lesion.</p>\",\n \"<p id=\\\"Par33\\\">More recently, Gandaglia et al. developed a model to predict ECE, SVI and stage upgrading in patients diagnosed with MRI-targeted and concomitant systematic biopsies [##REF##31547938##17##], achieving an AUC of 73% (ECE), 81% (SVI) and 73% (upgrading) at internal validation.</p>\",\n \"<p id=\\\"Par34\\\">Nevertheless, a meta-analysis of de Rooij et al. reported high specificity but low sensitivity for mpMRI accuracy in local staging; overall, staging based on MRI alone lacks sensitivity in detecting ECE, especially in case of focal, minimal or microscopic extension because of limitation in spatial resolution [##REF##26215604##18##, ##REF##26260000##19##]. Moreover, the degree of underestimation increases with smaller radiologic tumour size and lower PI-RADS scores [##REF##33026934##20##].</p>\",\n \"<p id=\\\"Par35\\\">The MRI and histology biopsy features have been variably reported in literature in the recent past as predictors of ECE [##REF##33272865##21##].</p>\",\n \"<p id=\\\"Par36\\\">Baco et al. found that MRI-tumour LCI well correlated with ECE, with an AUC that outperformed the Partin tables; they also found higher accuracy for microscopic-ECE detection with a 20 mm threshold [##REF##25150643##22##].</p>\",\n \"<p id=\\\"Par37\\\">Similarly, Kongnyuy et al. identified MRI-LCI as a promising predictor of ECE, positive pathological lymph nodes and biochemical recurrence. The 12.5 mm cut-off showed the highest sensitivity (77%) and specificity (59%) in predicting ECE. The AUC was comparable to that of the Partin tables, outperforming them with LCI and PSA combination [##UREF##2##23##].</p>\",\n \"<p id=\\\"Par38\\\">Moreover, in a recent meta-analysis of Li et al., the LCI showed high diagnostic performance in predicting ECE, with a pooled sensitivity and specificity of 0.79 and 0.77, respectively. When subgroup analysis was performed comparing different threshold values, lower LCI cut-off values yielded slightly better sensitivity and comparable specificity, without substantial differences between sub-groups [##REF##34881183##24##].</p>\",\n \"<p id=\\\"Par39\\\">In addition to LCI, IPNI is another acknowledged parameter often associated with ECE, possibly because in the 85% of cases the ECE goes through neurovascular bundles by dissection of the intraprostatic perineural spaces for tissue planes of least resistance [##REF##17495178##25##]</p>\",\n \"<p id=\\\"Par40\\\">Perineural invasion is defined as the tumour invasion into the perineural sheath, and during the years, it has been associated with tumour progression and prognosis of several malignancies, including prostate cancer [##REF##34994908##26##]. In a recent study on upper urinary tract urothelial carcinomas, Lin et al. found that PNI-positive patients had unfavourable pathological features, including high pathological stage, high tumour grade and lymphovascular invasion, leading to worse progression-free survival (PFS) [##REF##34994908##26##].</p>\",\n \"<p id=\\\"Par41\\\">Algaba et al. found that IPNI is correlated to cancer volume and higher percentage of extraprostatic cancer [##REF##16084008##27##]. Same finding was reported more recently by Leyh-Bannurah et al., being IPNI the only histological parameter found significantly associated to ECE in their nomogram [##REF##32248363##28##] Nevertheless, IPNI has not yet been reported among the most powerful histological features even in the latest version of the EAU guidelines, so that our finding might prompt its inclusion among the reporting recommendations for the prostatic biopsy.</p>\",\n \"<p id=\\\"Par42\\\">In our study, we identified both the LCI and the IPNI as predictors of ECE: the LCI cut-off that best correlated to ECE was 9.5 mm, which was a similar finding reported also by Li et al. [##REF##34881183##24##].</p>\",\n \"<p id=\\\"Par43\\\">Interestingly, in our cohort, the ILS at MRI did not show the same degree of association to the ECE as by the LCI; furthermore, MRI underestimated pathological tumour size by 25%.</p>\",\n \"<p id=\\\"Par44\\\">Overall, these data may have several implications: (1) Prostate mpMRI alone, including singular features and scoring systems, do not adequately predict ECE, except LCI—especially in combination to a histology biopsy feature, as IPNI in our series. (2) A change of PI-RADS score 5 definition should be prompted in the future PI-RADS updated version: score 5 is attributed to ILS ≥ 15 mm, but this threshold was chosen by the PI-RADS steering committee on the basis of old studies of the’90, when csPCa and ECE were found to be correlated to a tumour volume ≥ 0.5 cm<sup>3</sup> (15 mm in major axis) at whole-mount RP specimen [##REF##7506797##29##, ##REF##8709307##30##]. If differences of PI-RADS scores 4 to 5 are to be based on the risk of ECE, LCI should be the preferred MRI feature and with a lower cut-off, as the 15 mm cut-off at MRI might underestimate for a quarter the actual tumour size at specimen. (3) We found excellent or substantial inter-readers agreement for relevant MRI variables, thus strengthening the importance of high-quality imaging acquisition and readers’ skills for their adequate assessment. This latter matter has been popularised with the introduction of a dedicate score (PIQUAL) about the quality of the MRI sequences and the ability to make decision on the basis of it [##REF##32646850##31##].</p>\",\n \"<p id=\\\"Par45\\\">Main limitations of the study includes (1) the retrospective design of our analysis, even though significant efforts have been done in reviewing MRI images by two radiologists blinded to final histology; (2) the number of cases is limited, especially because MRI implementation in the clinical practice has substantially increased in more recent years; (3) the MRI images were reassessed according to the PI-RADS v2, as the version 2.1 became available on a later stage to that phase of our study. Nevertheless, it is very unlikely that the minor changes in score reporting of the latest version would have had an impact on the outcome of our study, as shown in a recent publication comparing the v2.0 vs v2.1 diagnostic performance with no difference in concordance rates between targeted biopsy and radical prostatectomy (doi: 10.2214/AJR.23.29964).</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p id=\"Par46\">The mpMRI confirms limitations in predicting the ECE at the RP specimen, even when involving tailored scoring systems. On the other side, MRI-LCI is the most robust factor associated to ECE; in our series, we found a strong predictive accuracy when combined with the IPNI presence. This outcome may prompt a change in the definition of PI-RADS score 5, by reducing IL\nsize cut-off to 10–12mm, or by replacing IL size reporting with LCI (cut-off 9–10mm).</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p id=\\\"Par46\\\">The mpMRI confirms limitations in predicting the ECE at the RP specimen, even when involving tailored scoring systems. On the other side, MRI-LCI is the most robust factor associated to ECE; in our series, we found a strong predictive accuracy when combined with the IPNI presence. This outcome may prompt a change in the definition of PI-RADS score 5, by reducing IL\\nsize cut-off to 10–12mm, or by replacing IL size reporting with LCI (cut-off 9–10mm).</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Objectives</title>","<p id=\"Par1\">To identify the predictive factors of prostate cancer extracapsular extension (ECE) in an institutional cohort of patients who underwent multiparametric MRI of the prostate prior to radical prostatectomy (RP).</p>","<title>Patients and methods</title>","<p id=\"Par2\">Overall, 126 patients met the selection criteria, and their medical records were retrospectively collected and analysed; 2 experienced radiologists reviewed the imaging studies. Logistic regression analysis was conducted to identify the variables associated to ECE at whole-mount histology of RP specimens; according to the statistically significant variables associated, a predictive model was developed and calibrated with the Hosmer–Lomeshow test.</p>","<title>Results</title>","<p id=\"Par3\">The predictive ability to detect ECE with the generated model was 81.4% by including the length of capsular involvement (LCI) and intraprostatic perineural invasion (IPNI). The predictive accuracy of the model at the ROC curve analysis showed an area under the curve (AUC) of 0.83 [95% CI (0.76–0.90)], <italic>p</italic> < 0.001. Concordance between radiologists was substantial in all parameters examined (<italic>p</italic> < 0.001). Limitations include the retrospective design, limited number of cases, and MRI images reassessment according to PI-RADS v2.0.</p>","<title>Conclusion</title>","<p id=\"Par4\">The LCI is the most robust MRI factor associated to ECE; in our series, we found a strong predictive accuracy when combined in a model with the IPNI presence. This outcome may prompt a change in the definition of PI-RADS score 5.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00345-023-04720-5.</p>","<title>Keywords</title>","<p>Open access funding provided by Università degli Studi di Sassari within the CRUI-CARE Agreement.</p>"],"string":"[\n \"<title>Objectives</title>\",\n \"<p id=\\\"Par1\\\">To identify the predictive factors of prostate cancer extracapsular extension (ECE) in an institutional cohort of patients who underwent multiparametric MRI of the prostate prior to radical prostatectomy (RP).</p>\",\n \"<title>Patients and methods</title>\",\n \"<p id=\\\"Par2\\\">Overall, 126 patients met the selection criteria, and their medical records were retrospectively collected and analysed; 2 experienced radiologists reviewed the imaging studies. Logistic regression analysis was conducted to identify the variables associated to ECE at whole-mount histology of RP specimens; according to the statistically significant variables associated, a predictive model was developed and calibrated with the Hosmer–Lomeshow test.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">The predictive ability to detect ECE with the generated model was 81.4% by including the length of capsular involvement (LCI) and intraprostatic perineural invasion (IPNI). The predictive accuracy of the model at the ROC curve analysis showed an area under the curve (AUC) of 0.83 [95% CI (0.76–0.90)], <italic>p</italic> < 0.001. Concordance between radiologists was substantial in all parameters examined (<italic>p</italic> < 0.001). Limitations include the retrospective design, limited number of cases, and MRI images reassessment according to PI-RADS v2.0.</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par4\\\">The LCI is the most robust MRI factor associated to ECE; in our series, we found a strong predictive accuracy when combined in a model with the IPNI presence. This outcome may prompt a change in the definition of PI-RADS score 5.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00345-023-04720-5.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by Università degli Studi di Sassari within the CRUI-CARE Agreement.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>We are thankful to Cristina Esquinas Lopez, Professor of Statistics at at Vall d’Hebron Research Institute, Universitat Autonoma de Barcelona (Spain) for undertaking the statistical analysis on the manuscript. <underline>crise4@hotmail.com</underline></p>","<title>Funding</title>","<p>Open access funding provided by Università degli Studi di Sassari within the CRUI-CARE Agreement. None.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par47\">The authors have nothing to disclose.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>We are thankful to Cristina Esquinas Lopez, Professor of Statistics at at Vall d’Hebron Research Institute, Universitat Autonoma de Barcelona (Spain) for undertaking the statistical analysis on the manuscript. <underline>crise4@hotmail.com</underline></p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by Università degli Studi di Sassari within the CRUI-CARE Agreement. None.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par47\\\">The authors have nothing to disclose.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>ROC curve analysis for predictive model</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>ROC curve analysis for predictive model</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Patients’ clinical data</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Variables</th><th align=\"left\">Median (IQR)</th><th align=\"left\"><italic>N</italic> (%)</th></tr></thead><tbody><tr><td align=\"left\">Age at biopsy (yr)</td><td char=\"(\" align=\"char\">66.6 (61.5–68.9)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\" colspan=\"3\">Clinical stage</td></tr><tr><td align=\"left\"> T1</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">85 (67.5)</td></tr><tr><td align=\"left\"> T2 + T3</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">41 (32.5)</td></tr><tr><td align=\"left\">Baseline PSA at biopsy (ng/ml)</td><td char=\"(\" align=\"char\">7.2 (5.4–10.4)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\">Max length core (mm)</td><td char=\"(\" align=\"char\">5 (3–8)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\" colspan=\"3\">Intraprostatic perineural invasion</td></tr><tr><td align=\"left\"> Yes</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">26 (20.6)</td></tr><tr><td align=\"left\"> No</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">100 (79.4)</td></tr><tr><td align=\"left\">Prostatic volume Observer #1 (cc)</td><td char=\"(\" align=\"char\">45.5 (32–65.2)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\">Prostatic volume Observer #2 (cc)</td><td char=\"(\" align=\"char\">50 (35–74)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\">PSA-density at biopsy (ng/cc)</td><td char=\"(\" align=\"char\">0.17 (0.11–0.26)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\">Number of targeted biopsies (n. 49, 38.89%)</td><td char=\"(\" align=\"char\">3 (3–4)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\">Number of positive targeted biopsy (n. 49, 38.89%)</td><td char=\"(\" align=\"char\">2 (0–3)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\" colspan=\"3\">ISUP group at biopsy</td></tr><tr><td align=\"left\"> 1</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">41 (32.5)</td></tr><tr><td align=\"left\"> 2</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">47 (37.3)</td></tr><tr><td align=\"left\"> 3</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">9 (7.1)</td></tr><tr><td align=\"left\"> 4</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">18 (14.3)</td></tr><tr><td align=\"left\"> 5</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">11 (8.8)</td></tr><tr><td align=\"left\" colspan=\"3\">Type of MRI</td></tr><tr><td align=\"left\"> T2w + DWI + DCE</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">123 (97.6)</td></tr><tr><td align=\"left\"> T2w + DWI</td><td char=\"(\" align=\"char\"/><td char=\"(\" align=\"char\">3 (2.4)</td></tr><tr><td align=\"left\"><p>Index Lesion size (mm), Observer #1</p><p>Median (IQR)</p></td><td char=\"(\" align=\"char\">12 (8–19)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\"><p>Index Lesion size (mm), Observer #2</p><p>Median (IQR)</p></td><td char=\"(\" align=\"char\">12 (8–18)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\"><p>Length of capsular involvement (mm)</p><p>Median (IQR)</p></td><td char=\"(\" align=\"char\">9 (3.7–15.2)</td><td char=\"(\" align=\"char\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Patients’ pathology data (<italic>N</italic> = 126 patients)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Variables</th><th align=\"left\">Median (IQR)</th><th align=\"left\"><italic>N</italic> (%)</th></tr></thead><tbody><tr><td align=\"left\">Index Lesion size (mm)</td><td char=\"–\" align=\"char\">16 (10–21)</td><td char=\"(\" align=\"char\"/></tr><tr><td align=\"left\" colspan=\"3\">Histology</td></tr><tr><td align=\"left\"> pT0</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">2 (1.6)</td></tr><tr><td align=\"left\"> pT2a</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">25 (19.9)</td></tr><tr><td align=\"left\"> pT2b</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">8 (6.3)</td></tr><tr><td align=\"left\"> pT2c</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">56 (44.4)</td></tr><tr><td align=\"left\"> pT3a</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">27 (21.4)</td></tr><tr><td align=\"left\"> pT3b</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">8 (6.4)</td></tr><tr><td align=\"left\" colspan=\"3\">ISUP grade group</td></tr><tr><td align=\"left\"> 0</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">2 (1.6)</td></tr><tr><td align=\"left\"> 1</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">15 (11.9)</td></tr><tr><td align=\"left\"> 2</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">51 (40.5)</td></tr><tr><td align=\"left\"> 3</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">18 (14.4)</td></tr><tr><td align=\"left\"> 4</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">23 (18.2)</td></tr><tr><td align=\"left\"> 5</td><td char=\".\" align=\"char\"/><td char=\"(\" align=\"char\">17 (13.4)</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Logistic Regression for ECE</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"3\">Univariate</th><th align=\"left\" colspan=\"3\">Multivariate</th><th align=\"left\" colspan=\"3\">Multivariate*</th></tr><tr><th align=\"left\">OR</th><th align=\"left\">95% CI</th><th align=\"left\"><italic>p</italic> value</th><th align=\"left\">OR</th><th align=\"left\">95% CI</th><th align=\"left\"><italic>p</italic> value</th><th align=\"left\">OR</th><th align=\"left\">95% CI</th><th align=\"left\"><italic>p</italic> value</th></tr></thead><tbody><tr><td align=\"left\">Clinical stage</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\" colspan=\"3\"/></tr><tr><td align=\"left\"> T1</td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><bold><italic>0.002</italic></bold></td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><italic>0.971</italic></td><td align=\"left\" rowspan=\"8\" colspan=\"3\"/></tr><tr><td align=\"left\"> T2 + T3</td><td align=\"left\">3.72</td><td align=\"left\">1.65–8.58</td><td align=\"left\">1.02</td><td align=\"left\">0.31–3.22</td></tr><tr><td align=\"left\">PSA density (ng/ml<sup>2</sup>)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> ≤ 0.15</td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><bold><italic>0.003</italic></bold></td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><italic>0.406</italic></td></tr><tr><td align=\"left\"> > 0.15</td><td align=\"left\">4.16</td><td align=\"left\">1.71–11.29</td><td align=\"left\">1.75</td><td align=\"left\">0.48–6.97</td></tr><tr><td align=\"left\">Max length core (mm)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> ≤ 5.5</td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><bold> < </bold><bold><italic>0.001</italic></bold></td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><italic>0.096</italic></td></tr><tr><td align=\"left\"> > 5.5</td><td align=\"left\">5.93</td><td align=\"left\">2.57–14.55</td><td align=\"left\">2.85</td><td align=\"left\">0.85–10.36</td></tr><tr><td align=\"left\">IPNI</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> No</td><td align=\"left\">Ref</td><td align=\"left\">-</td><td align=\"left\" rowspan=\"2\"><bold> < </bold><bold><italic>0.001</italic></bold></td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><bold><italic>0.001</italic></bold></td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><bold> < </bold><bold><italic>0.001</italic></bold></td></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">8.61</td><td align=\"left\">3.31–22.37</td><td align=\"left\">10.24</td><td align=\"left\">2.57–40.85</td><td align=\"left\">8.17</td><td align=\"left\">2.78–26.32</td></tr><tr><td align=\"left\">ISUP grade at biopsy</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\" colspan=\"3\"/></tr><tr><td align=\"left\"> ≤ 3</td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><bold> < </bold><bold><italic>0.001</italic></bold></td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><italic>0.078</italic></td><td align=\"left\" rowspan=\"2\" colspan=\"3\"/></tr><tr><td align=\"left\"> > 3</td><td align=\"left\">4.39</td><td align=\"left\">1.93–10.66</td><td align=\"left\">2.81</td><td align=\"left\">0.91–9.37</td></tr><tr><td align=\"left\">LCI (mm)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> ≤ 9.5</td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><bold> < </bold><bold><italic>0.001</italic></bold></td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><bold><italic>0.002</italic></bold></td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><bold> < </bold><bold><italic>0.001</italic></bold></td></tr><tr><td align=\"left\"> > 9.5</td><td align=\"left\">11.61</td><td align=\"left\">4.41–36.77</td><td align=\"left\">14.34</td><td align=\"left\">2.87–95.10</td><td align=\"left\">11.09</td><td align=\"left\">3.91–38.18</td></tr><tr><td align=\"left\">ILS at MRI (mm)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\" colspan=\"3\"/></tr><tr><td align=\"left\"> ≤ 11</td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><bold> < </bold><bold><italic>0.001</italic></bold></td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\" rowspan=\"2\"><italic>0.673</italic></td><td align=\"left\" rowspan=\"2\" colspan=\"3\"/></tr><tr><td align=\"left\"> > 11</td><td align=\"left\">8.86</td><td align=\"left\">3.38–27.96</td><td align=\"left\">0.69</td><td align=\"left\">0.12–3.81</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Patients’ clinical data</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Variables</th><th align=\\\"left\\\">Median (IQR)</th><th align=\\\"left\\\"><italic>N</italic> (%)</th></tr></thead><tbody><tr><td align=\\\"left\\\">Age at biopsy (yr)</td><td char=\\\"(\\\" align=\\\"char\\\">66.6 (61.5–68.9)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Clinical stage</td></tr><tr><td align=\\\"left\\\"> T1</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">85 (67.5)</td></tr><tr><td align=\\\"left\\\"> T2 + T3</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">41 (32.5)</td></tr><tr><td align=\\\"left\\\">Baseline PSA at biopsy (ng/ml)</td><td char=\\\"(\\\" align=\\\"char\\\">7.2 (5.4–10.4)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Max length core (mm)</td><td char=\\\"(\\\" align=\\\"char\\\">5 (3–8)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Intraprostatic perineural invasion</td></tr><tr><td align=\\\"left\\\"> Yes</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">26 (20.6)</td></tr><tr><td align=\\\"left\\\"> No</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">100 (79.4)</td></tr><tr><td align=\\\"left\\\">Prostatic volume Observer #1 (cc)</td><td char=\\\"(\\\" align=\\\"char\\\">45.5 (32–65.2)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Prostatic volume Observer #2 (cc)</td><td char=\\\"(\\\" align=\\\"char\\\">50 (35–74)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">PSA-density at biopsy (ng/cc)</td><td char=\\\"(\\\" align=\\\"char\\\">0.17 (0.11–0.26)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Number of targeted biopsies (n. 49, 38.89%)</td><td char=\\\"(\\\" align=\\\"char\\\">3 (3–4)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Number of positive targeted biopsy (n. 49, 38.89%)</td><td char=\\\"(\\\" align=\\\"char\\\">2 (0–3)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">ISUP group at biopsy</td></tr><tr><td align=\\\"left\\\"> 1</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">41 (32.5)</td></tr><tr><td align=\\\"left\\\"> 2</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">47 (37.3)</td></tr><tr><td align=\\\"left\\\"> 3</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">9 (7.1)</td></tr><tr><td align=\\\"left\\\"> 4</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">18 (14.3)</td></tr><tr><td align=\\\"left\\\"> 5</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">11 (8.8)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Type of MRI</td></tr><tr><td align=\\\"left\\\"> T2w + DWI + DCE</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">123 (97.6)</td></tr><tr><td align=\\\"left\\\"> T2w + DWI</td><td char=\\\"(\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">3 (2.4)</td></tr><tr><td align=\\\"left\\\"><p>Index Lesion size (mm), Observer #1</p><p>Median (IQR)</p></td><td char=\\\"(\\\" align=\\\"char\\\">12 (8–19)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"><p>Index Lesion size (mm), Observer #2</p><p>Median (IQR)</p></td><td char=\\\"(\\\" align=\\\"char\\\">12 (8–18)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"><p>Length of capsular involvement (mm)</p><p>Median (IQR)</p></td><td char=\\\"(\\\" align=\\\"char\\\">9 (3.7–15.2)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Patients’ pathology data (<italic>N</italic> = 126 patients)</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Variables</th><th align=\\\"left\\\">Median (IQR)</th><th align=\\\"left\\\"><italic>N</italic> (%)</th></tr></thead><tbody><tr><td align=\\\"left\\\">Index Lesion size (mm)</td><td char=\\\"–\\\" align=\\\"char\\\">16 (10–21)</td><td char=\\\"(\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Histology</td></tr><tr><td align=\\\"left\\\"> pT0</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">2 (1.6)</td></tr><tr><td align=\\\"left\\\"> pT2a</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">25 (19.9)</td></tr><tr><td align=\\\"left\\\"> pT2b</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">8 (6.3)</td></tr><tr><td align=\\\"left\\\"> pT2c</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">56 (44.4)</td></tr><tr><td align=\\\"left\\\"> pT3a</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">27 (21.4)</td></tr><tr><td align=\\\"left\\\"> pT3b</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">8 (6.4)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">ISUP grade group</td></tr><tr><td align=\\\"left\\\"> 0</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">2 (1.6)</td></tr><tr><td align=\\\"left\\\"> 1</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">15 (11.9)</td></tr><tr><td align=\\\"left\\\"> 2</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">51 (40.5)</td></tr><tr><td align=\\\"left\\\"> 3</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">18 (14.4)</td></tr><tr><td align=\\\"left\\\"> 4</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">23 (18.2)</td></tr><tr><td align=\\\"left\\\"> 5</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\"(\\\" align=\\\"char\\\">17 (13.4)</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Logistic Regression for ECE</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" rowspan=\\\"2\\\"/><th align=\\\"left\\\" colspan=\\\"3\\\">Univariate</th><th align=\\\"left\\\" colspan=\\\"3\\\">Multivariate</th><th align=\\\"left\\\" colspan=\\\"3\\\">Multivariate*</th></tr><tr><th align=\\\"left\\\">OR</th><th align=\\\"left\\\">95% CI</th><th align=\\\"left\\\"><italic>p</italic> value</th><th align=\\\"left\\\">OR</th><th align=\\\"left\\\">95% CI</th><th align=\\\"left\\\"><italic>p</italic> value</th><th align=\\\"left\\\">OR</th><th align=\\\"left\\\">95% CI</th><th align=\\\"left\\\"><italic>p</italic> value</th></tr></thead><tbody><tr><td align=\\\"left\\\">Clinical stage</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\" colspan=\\\"3\\\"/></tr><tr><td align=\\\"left\\\"> T1</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold><italic>0.002</italic></bold></td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><italic>0.971</italic></td><td align=\\\"left\\\" rowspan=\\\"8\\\" colspan=\\\"3\\\"/></tr><tr><td align=\\\"left\\\"> T2 + T3</td><td align=\\\"left\\\">3.72</td><td align=\\\"left\\\">1.65–8.58</td><td align=\\\"left\\\">1.02</td><td align=\\\"left\\\">0.31–3.22</td></tr><tr><td align=\\\"left\\\">PSA density (ng/ml<sup>2</sup>)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ≤ 0.15</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold><italic>0.003</italic></bold></td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><italic>0.406</italic></td></tr><tr><td align=\\\"left\\\"> > 0.15</td><td align=\\\"left\\\">4.16</td><td align=\\\"left\\\">1.71–11.29</td><td align=\\\"left\\\">1.75</td><td align=\\\"left\\\">0.48–6.97</td></tr><tr><td align=\\\"left\\\">Max length core (mm)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ≤ 5.5</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold> < </bold><bold><italic>0.001</italic></bold></td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><italic>0.096</italic></td></tr><tr><td align=\\\"left\\\"> > 5.5</td><td align=\\\"left\\\">5.93</td><td align=\\\"left\\\">2.57–14.55</td><td align=\\\"left\\\">2.85</td><td align=\\\"left\\\">0.85–10.36</td></tr><tr><td align=\\\"left\\\">IPNI</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold> < </bold><bold><italic>0.001</italic></bold></td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold><italic>0.001</italic></bold></td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold> < </bold><bold><italic>0.001</italic></bold></td></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">8.61</td><td align=\\\"left\\\">3.31–22.37</td><td align=\\\"left\\\">10.24</td><td align=\\\"left\\\">2.57–40.85</td><td align=\\\"left\\\">8.17</td><td align=\\\"left\\\">2.78–26.32</td></tr><tr><td align=\\\"left\\\">ISUP grade at biopsy</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\" colspan=\\\"3\\\"/></tr><tr><td align=\\\"left\\\"> ≤ 3</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold> < </bold><bold><italic>0.001</italic></bold></td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><italic>0.078</italic></td><td align=\\\"left\\\" rowspan=\\\"2\\\" colspan=\\\"3\\\"/></tr><tr><td align=\\\"left\\\"> > 3</td><td align=\\\"left\\\">4.39</td><td align=\\\"left\\\">1.93–10.66</td><td align=\\\"left\\\">2.81</td><td align=\\\"left\\\">0.91–9.37</td></tr><tr><td align=\\\"left\\\">LCI (mm)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ≤ 9.5</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold> < </bold><bold><italic>0.001</italic></bold></td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold><italic>0.002</italic></bold></td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold> < </bold><bold><italic>0.001</italic></bold></td></tr><tr><td align=\\\"left\\\"> > 9.5</td><td align=\\\"left\\\">11.61</td><td align=\\\"left\\\">4.41–36.77</td><td align=\\\"left\\\">14.34</td><td align=\\\"left\\\">2.87–95.10</td><td align=\\\"left\\\">11.09</td><td align=\\\"left\\\">3.91–38.18</td></tr><tr><td align=\\\"left\\\">ILS at MRI (mm)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\" colspan=\\\"3\\\"/></tr><tr><td align=\\\"left\\\"> ≤ 11</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><bold> < </bold><bold><italic>0.001</italic></bold></td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\" rowspan=\\\"2\\\"><italic>0.673</italic></td><td align=\\\"left\\\" rowspan=\\\"2\\\" colspan=\\\"3\\\"/></tr><tr><td align=\\\"left\\\"> > 11</td><td align=\\\"left\\\">8.86</td><td align=\\\"left\\\">3.38–27.96</td><td align=\\\"left\\\">0.69</td><td align=\\\"left\\\">0.12–3.81</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p><italic>yr</italic> years, <italic>ng</italic> nanograms, <italic>ml</italic> millilitres, <italic>mm</italic> millimetres, <italic>cc</italic> cubic centimetres</p></table-wrap-foot>","<table-wrap-foot><p><italic>mm</italic> millimetres</p></table-wrap-foot>","<table-wrap-foot><p><bold>Multivariate</bold>: Analysis conducted considering all parameters; <bold>Multivariate*</bold>: Analysis conducted considering all parameters with stepwise method for statistical significance (<italic>p</italic> < 0.05)</p><p><italic>ECE</italic> extracapsular extension, <italic>OR</italic> odds-ratio, <italic>IPNI</italic> intraprostatic perineural invasion, <italic>LCI</italic> length of capsular involvement, <italic>ILS</italic> index lesion size</p><p>Bold Italic are reported the statistically significant <italic>p</italic>-values</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p><italic>yr</italic> years, <italic>ng</italic> nanograms, <italic>ml</italic> millilitres, <italic>mm</italic> millimetres, <italic>cc</italic> cubic centimetres</p></table-wrap-foot>\",\n \"<table-wrap-foot><p><italic>mm</italic> millimetres</p></table-wrap-foot>\",\n \"<table-wrap-foot><p><bold>Multivariate</bold>: Analysis conducted considering all parameters; <bold>Multivariate*</bold>: Analysis conducted considering all parameters with stepwise method for statistical significance (<italic>p</italic> < 0.05)</p><p><italic>ECE</italic> extracapsular extension, <italic>OR</italic> odds-ratio, <italic>IPNI</italic> intraprostatic perineural invasion, <italic>LCI</italic> length of capsular involvement, <italic>ILS</italic> index lesion size</p><p>Bold Italic are reported the statistically significant <italic>p</italic>-values</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"345_2023_4720_Fig1_HTML\" id=\"MO1\"/>"],"string":"[\n \"<graphic xlink:href=\\\"345_2023_4720_Fig1_HTML\\\" id=\\\"MO1\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"345_2023_4720_MOESM1_ESM.docx\"><caption><p>Supplementary file1 (DOCX 129 KB)</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"345_2023_4720_MOESM1_ESM.docx\\\"><caption><p>Supplementary file1 (DOCX 129 KB)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"1.\"], \"mixed-citation\": [\"Mottet N, B J, Briers E, Bolla M, Bourke L, Cornford P, De Santis M, Henry A, Joniau S, Lam T, Mason MD, Van den Poel H, Van den Kwast TH, Rouvi\\u00e8re O, Wiegel T, members of the EAU \\u2013 ESTRO \\u2013 ESUR \\u2013SIOG Prostate Cancer Guidelines Panel (2021) EAU \\u2013 ESTRO \\u2013 ESUR \\u2013 SIOG Guidelines on Prostate Cancer. Edn. presented at the EAU Annual Congress Milan 2021. 978\\u201394\\u201392671\\u201313\\u20134., T.N. Publisher: EAU Guidelines Office. Place published: Arnhem, Editor\"]}, {\"label\": [\"15.\"], \"surname\": [\"Giganti\"], \"given-names\": [\"F\"], \"article-title\": [\"Apparent diffusion coefficient in the evaluation of side-specific extracapsular extension in prostate cancer: development and external validation of a nomogram of clinical use\"], \"source\": [\"Urol Oncol\"], \"year\": [\"2016\"], \"volume\": [\"34\"], \"issue\": [\"7\"], \"fpage\": [\"291\"], \"pub-id\": [\"10.1016/j.urolonc.2016.02.015\"]}, {\"label\": [\"23.\"], \"surname\": [\"Kongnyuy\"], \"given-names\": [\"M\"], \"article-title\": [\"Tumor contact with prostate capsule on magnetic resonance imaging: a potential biomarker for staging and prognosis\"], \"source\": [\"Urol Oncol\"], \"year\": [\"2017\"], \"volume\": [\"35\"], \"issue\": [\"1\"], \"fpage\": [\"30e1\"], \"lpage\": [\"30e8\"], \"pub-id\": [\"10.1016/j.urolonc.2016.07.013\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":31,"string":"31"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"World J Urol. 2024 Jan 13; 42(1):37"},"package_file":{"kind":"string","value":"oa_package/5e/6f/PMC10787875.tar.gz"}}},{"rowIdx":161,"cells":{"accession_id":{"kind":"string","value":"PMC10787876"},"pmid":{"kind":"string","value":"37833450"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par16\">Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer after invasive ductal carcinoma (IDC), representing 10–15% of all cases [##REF##25848941##1##]. In the metastatic setting, ILC differs in its pattern of metastatic sites, often involving the bone, and gastrointestinal tract [##REF##28757653##2##–##REF##37020372##4##]. Additionally, several studies demonstrate worse overall survival (OS) in metastatic ILC compared to IDC, even when evaluating patients with similar receptor subtypes [##REF##37020372##4##–##REF##18704988##6##].</p>","<p id=\"Par17\">While investigators have evaluated surgical outcomes by histologic subtype in early-stage disease, there are scant data evaluating the use of primary-site surgery in the metastatic setting in those with ILC versus IDC. The current recommended therapy for metastatic breast cancer is systemic therapy, with local therapy reserved for palliation of symptoms [##REF##17891444##7##]. While retrospective studies and institutional series have found associations between primary tumor resection and longer survival in those with metastatic breast cancer [##REF##17522944##8##], most randomized control trials have not demonstrated such a survival advantage [##REF##34995128##9##–##REF##26363985##11##]. A previous study found that ILC patients with bone-only metastases had longer OS than those with visceral metastases when given a combination of chemotherapy and surgery, but it is unclear whether this reflects the more indolent course of osseous metastases, or an ILC specific effect of treatment [##REF##37020372##4##, ##REF##17687611##12##].</p>","<p id=\"Par18\">As such, whether histologic subtype should factor into patient selection for primary-site surgery is unknown. Prior analyses have suggested that if surgery of the primary tumor is associated with improved survival, this may be more likely in those with hormone receptor (HR) positive disease, or bone-only metastases [##REF##29777404##10##, ##REF##20101736##13##]. Given that ILC is largely HR-positive, HER2-negative, and has a propensity for bone metastases, we wondered if primary-site surgery use differed in patients with metastatic ILC compared to IDC.</p>","<p id=\"Par19\">We used the National Cancer Database (NCDB) to evaluate differences in practice patterns and management of patients with metastatic ILC compared to metastatic IDC. Specifically, we investigated the following questions: whether rates of primary-site surgery differ by histologic subtype and whether selection factors associated with undergoing primary-site surgery differ by histologic subtype. As secondary endpoints, we evaluated the use of chemotherapy and radiotherapy relative to surgery by histologic subtype, and the association between primary-site surgery and OS in ILC and IDC cohorts in both unmatched and propensity score matched multivariable models.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par16\\\">Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer after invasive ductal carcinoma (IDC), representing 10–15% of all cases [##REF##25848941##1##]. In the metastatic setting, ILC differs in its pattern of metastatic sites, often involving the bone, and gastrointestinal tract [##REF##28757653##2##–##REF##37020372##4##]. Additionally, several studies demonstrate worse overall survival (OS) in metastatic ILC compared to IDC, even when evaluating patients with similar receptor subtypes [##REF##37020372##4##–##REF##18704988##6##].</p>\",\n \"<p id=\\\"Par17\\\">While investigators have evaluated surgical outcomes by histologic subtype in early-stage disease, there are scant data evaluating the use of primary-site surgery in the metastatic setting in those with ILC versus IDC. The current recommended therapy for metastatic breast cancer is systemic therapy, with local therapy reserved for palliation of symptoms [##REF##17891444##7##]. While retrospective studies and institutional series have found associations between primary tumor resection and longer survival in those with metastatic breast cancer [##REF##17522944##8##], most randomized control trials have not demonstrated such a survival advantage [##REF##34995128##9##–##REF##26363985##11##]. A previous study found that ILC patients with bone-only metastases had longer OS than those with visceral metastases when given a combination of chemotherapy and surgery, but it is unclear whether this reflects the more indolent course of osseous metastases, or an ILC specific effect of treatment [##REF##37020372##4##, ##REF##17687611##12##].</p>\",\n \"<p id=\\\"Par18\\\">As such, whether histologic subtype should factor into patient selection for primary-site surgery is unknown. Prior analyses have suggested that if surgery of the primary tumor is associated with improved survival, this may be more likely in those with hormone receptor (HR) positive disease, or bone-only metastases [##REF##29777404##10##, ##REF##20101736##13##]. Given that ILC is largely HR-positive, HER2-negative, and has a propensity for bone metastases, we wondered if primary-site surgery use differed in patients with metastatic ILC compared to IDC.</p>\",\n \"<p id=\\\"Par19\\\">We used the National Cancer Database (NCDB) to evaluate differences in practice patterns and management of patients with metastatic ILC compared to metastatic IDC. Specifically, we investigated the following questions: whether rates of primary-site surgery differ by histologic subtype and whether selection factors associated with undergoing primary-site surgery differ by histologic subtype. As secondary endpoints, we evaluated the use of chemotherapy and radiotherapy relative to surgery by histologic subtype, and the association between primary-site surgery and OS in ILC and IDC cohorts in both unmatched and propensity score matched multivariable models.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<title>Data source and study cohort</title>","<p id=\"Par20\">The NCDB is a national comprehensive clinical surveillance resource representing over 70% of all newly diagnosed cancer cases in the United States and includes patient demographics, clinical information, and survival outcomes [##REF##19636004##14##, ##REF##28241198##15##]. Participants User Files from 2010 to 2016 were used. Due to the de-identified nature of the public-access user files, the study was exempted from institutional review board approval.</p>","<p id=\"Par21\">Since most ILC tumors are HR-positive and HER2-negative, we limited analysis to tumors with this receptor subtype. Tumors that were estrogen receptor (ER) and/or progesterone receptor (PR) positive were considered HR-positive. Histology codes were used to identify cohorts, with the ILC cohort comprising those with codes for ILC or mixed ILC/IDC (histology codes 8520, 8522, and 8524 if invasive behavior), and the IDC cohort comprising codes for IDC or invasive mammary carcinoma not otherwise specified (histology codes 8500, 8501, 8502, 8503, and 8523 if invasive behavior). We excluded patients with stage I-III disease, histologic subtypes other than IDC or ILC, individuals who died within 6 months of their diagnosis, and those missing critical clinical information including disease stage, HR-status, HER2-status, or treatment type.</p>","<title>Clinical measures</title>","<p id=\"Par22\">Charlson-Deyo Co-Morbidity Index (CDCI) was recorded as a measure of severity of co-morbid conditions. Age at diagnosis was subdivided into under 50 years and over 50 years to estimate pre- and post-menopausal status, respectively. Metastatic disease sites were categorized as bone-only, visceral-only, bone and visceral, or unknown [##REF##37020372##4##]. We utilized data on treatment facility type, insurance status, and median income in univariate and multivariate analyses.</p>","<title>Statistical methods</title>","<p id=\"Par23\">We compared clinicopathologic and demographic features between the ILC and IDC cohorts using chi-square tests for categorical variables and t-tests for continuous variables. We investigated factors associated with receiving surgery for the primary tumor, radiotherapy, chemotherapy, and timing of chemotherapy relative to surgery by histologic subtype. For univariate analyses, we used Kaplan-Meier plots and log-rank tests to assess associations between receipt of surgery and OS by histologic subtype, and by timing of chemotherapy and receipt of radiotherapy. We also evaluated treatment facility type, insurance type, and median income quartiles by surgery and histologic subtype.</p>","<p id=\"Par24\">For multivariate analysis, we used Cox proportional hazards models to account for confounders with OS. The multivariable model included age, CDCI (0/1+), metastatic site (bone-only versus all other), and receptor subtype (ER-positive, PR-positive, HER2-negative versus ER-positive, PR-negative, HER2-negative).</p>","<p id=\"Par25\">Finally, we performed propensity score matching including age, tumor grade, receptor subtype, site of metastatic disease, CDCI (0/1+), and treating facility variables to account for likelihood of having primary-site surgery to determine the association between primary-site surgery and OS. Within each histology category among those who had survival data available patients who had surgery (ILC n = 1,444; IDC n = 4,924) were matched to patients who did not have surgery (ILC n = 3,553; IDC n = 10,894) using the greedy nearest neighbor matching algorithm. Matching was restricted to observations that had propensity scores in the extended common support region (ILC 0.05–0.71; IDC 0.06–0.66), which extends the common support region by 0.25 times a pooled estimate of the common standard deviation of the logit of the propensity score. The PSMATCH procedure in SAS version 9.4 was used to perform matching. To account for the matched nature of the sample, log-rank tests and Cox models were stratified on the matched pairs.</p>","<p id=\"Par26\">Hypothesis tests were two-sided, and the significance threshold was set to 0.05. Statistical analyses were performed using Stata 16 and SAS version 9.4.</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<title>Data source and study cohort</title>\",\n \"<p id=\\\"Par20\\\">The NCDB is a national comprehensive clinical surveillance resource representing over 70% of all newly diagnosed cancer cases in the United States and includes patient demographics, clinical information, and survival outcomes [##REF##19636004##14##, ##REF##28241198##15##]. Participants User Files from 2010 to 2016 were used. Due to the de-identified nature of the public-access user files, the study was exempted from institutional review board approval.</p>\",\n \"<p id=\\\"Par21\\\">Since most ILC tumors are HR-positive and HER2-negative, we limited analysis to tumors with this receptor subtype. Tumors that were estrogen receptor (ER) and/or progesterone receptor (PR) positive were considered HR-positive. Histology codes were used to identify cohorts, with the ILC cohort comprising those with codes for ILC or mixed ILC/IDC (histology codes 8520, 8522, and 8524 if invasive behavior), and the IDC cohort comprising codes for IDC or invasive mammary carcinoma not otherwise specified (histology codes 8500, 8501, 8502, 8503, and 8523 if invasive behavior). We excluded patients with stage I-III disease, histologic subtypes other than IDC or ILC, individuals who died within 6 months of their diagnosis, and those missing critical clinical information including disease stage, HR-status, HER2-status, or treatment type.</p>\",\n \"<title>Clinical measures</title>\",\n \"<p id=\\\"Par22\\\">Charlson-Deyo Co-Morbidity Index (CDCI) was recorded as a measure of severity of co-morbid conditions. Age at diagnosis was subdivided into under 50 years and over 50 years to estimate pre- and post-menopausal status, respectively. Metastatic disease sites were categorized as bone-only, visceral-only, bone and visceral, or unknown [##REF##37020372##4##]. We utilized data on treatment facility type, insurance status, and median income in univariate and multivariate analyses.</p>\",\n \"<title>Statistical methods</title>\",\n \"<p id=\\\"Par23\\\">We compared clinicopathologic and demographic features between the ILC and IDC cohorts using chi-square tests for categorical variables and t-tests for continuous variables. We investigated factors associated with receiving surgery for the primary tumor, radiotherapy, chemotherapy, and timing of chemotherapy relative to surgery by histologic subtype. For univariate analyses, we used Kaplan-Meier plots and log-rank tests to assess associations between receipt of surgery and OS by histologic subtype, and by timing of chemotherapy and receipt of radiotherapy. We also evaluated treatment facility type, insurance type, and median income quartiles by surgery and histologic subtype.</p>\",\n \"<p id=\\\"Par24\\\">For multivariate analysis, we used Cox proportional hazards models to account for confounders with OS. The multivariable model included age, CDCI (0/1+), metastatic site (bone-only versus all other), and receptor subtype (ER-positive, PR-positive, HER2-negative versus ER-positive, PR-negative, HER2-negative).</p>\",\n \"<p id=\\\"Par25\\\">Finally, we performed propensity score matching including age, tumor grade, receptor subtype, site of metastatic disease, CDCI (0/1+), and treating facility variables to account for likelihood of having primary-site surgery to determine the association between primary-site surgery and OS. Within each histology category among those who had survival data available patients who had surgery (ILC n = 1,444; IDC n = 4,924) were matched to patients who did not have surgery (ILC n = 3,553; IDC n = 10,894) using the greedy nearest neighbor matching algorithm. Matching was restricted to observations that had propensity scores in the extended common support region (ILC 0.05–0.71; IDC 0.06–0.66), which extends the common support region by 0.25 times a pooled estimate of the common standard deviation of the logit of the propensity score. The PSMATCH procedure in SAS version 9.4 was used to perform matching. To account for the matched nature of the sample, log-rank tests and Cox models were stratified on the matched pairs.</p>\",\n \"<p id=\\\"Par26\\\">Hypothesis tests were two-sided, and the significance threshold was set to 0.05. Statistical analyses were performed using Stata 16 and SAS version 9.4.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Study cohort</title>","<p id=\"Par27\">There were 100,147 patients with stage IV breast cancer, of whom 25,294 had HR-positive, HER2-negative invasive lobular or ductal histology, and met study criteria (Fig. ##FIG##0##1##). Of these patients, 19,171 (75.8%) had IDC and 6,123 (24.2%) had ILC. Within the ILC cohort, 4,484 (73.2%) had pure ILC, with the remaining having mixed ILC/IDC. Median follow-up time of the ILC cohort was 27.2 months (IQ range 14.7–41.5 months), which was similar to the median follow-up time of 26.8 months (IQ range 14.6–42.6 months) for the IDC cohort (Table ##TAB##0##1##). Patients with ILC were slightly older than those with IDC (mean age 64 years versus 61 years, <italic>p</italic> < 0.001) and differed significantly by race (<italic>p</italic> < 0.001), with a higher proportion of White patients (79.4% versus 74.2%, <italic>p</italic> < 0.0001) (Table ##TAB##0##1##). Additionally, ILC and IDC patients differed by tumor grade (<italic>p</italic> < 0.001), with ILC patients having a higher proportion of grade 1 tumors (22.1% versus 9.48%, p < 0.0001) and more bone-only metastases than those with IDC (60.8% versus 45.2%, <italic>p</italic> < 0.001).</p>","<p id=\"Par28\">\n\n</p>","<p id=\"Par53\">\n\n</p>","<p id=\"Par30\">While overall most patients were treated at community cancer centers, slightly more ILC patients than IDC patients were treated at academic centers (ILC 36.0% versus IDC 33.5%, <italic>p</italic> = 0.001) (Table ##TAB##0##1##). ILC patients were significantly more likely to have public insurance (ILC 54.7% versus IDC 51.6%) and less likely to be uninsured (ILC 3.82% versus IDC 5.15%, <italic>p</italic> < 0.0001). Patients with ILC had higher median income, with 39.7% in the highest quartile compared to 35.7% of IDC patients in the highest median income quartile (<italic>p</italic> < 0.0001).</p>","<title>Primary-site surgery by histologic subtype</title>","<p id=\"Par31\">In the overall study population, 7,158 (28.3%) underwent primary-site surgery. Although the absolute difference is small, primary-site surgery was performed less often in those with metastatic ILC than those with metastatic IDC (n = 1,644 [26.8%] versus n = 5,514 [28.8%] respectively, <italic>p</italic> = 0.004) (Table ##TAB##1##2##). This difference was more pronounced when restricting the analysis to patients with pure ILC, where only 23.8% underwent primary-site surgery. Additionally, the slightly lower rate of primary-site surgery among patients with ILC compared to IDC was observed among those with bone-only or unknown site of metastases, but not among those with visceral metastases (Fig. ##FIG##1##2##). Among those who had primary-site surgery in both histologic cohorts, the site of metastatic disease was significantly more likely to be bone-only compared to other sites (ILC 63.5%; IDC 48.5%, <italic>p</italic> < 0.001) (Table ##TAB##2##3##).</p>","<p id=\"Par54\">\n\n</p>","<p id=\"Par32\">\n\n</p>","<p id=\"Par33\">For both cohorts, patients with private insurance were significantly more likely to receive primary-site surgery (ILC 46.4%; IDC 49.4%, <italic>p</italic> < 0.0001) compared to patients with public insurance (Table ##TAB##2##3##). Both ILC and IDC patients who received surgery were equally likely to have been treated at an academic treating facility (ILC n = 415 [26.3%]; IDC n = 1,318 [26.2%]) compared to a community setting (ILC n = 1,164 [73.7%]; IDC n = 3,714 [73.8%]).</p>","<p id=\"Par55\">\n\n</p>","<p id=\"Par34\">Among those who underwent primary-site surgery (n = 7,158), the differences by histology reflected those seen in the overall study population. Those with ILC were older (ILC mean age 61.7 years versus IDC 58.1 years, <italic>p</italic> < 0.001), had more T3 tumors (ILC 30.8% versus IDC 15.5%, <italic>p</italic> < 0.001), had more N3 nodal status (ILC 35.3% versus IDC 20.5%, <italic>p</italic> < 0.001), and had more grade 2 disease (ILC 61.4% versus IDC 45.9%, <italic>p</italic> < 0.001) (Table ##TAB##0##1##). There was no significant difference in mastectomy rate between the two cohorts (Table ##TAB##1##2##). Positive surgical margins were significantly more common in those with ILC who underwent lumpectomy compared to IDC (15.7% versus 11.2%, <italic>p =</italic> 0.025) (Table ##TAB##0##1##).</p>","<p id=\"Par35\">The factors associated with undergoing primary-site surgery were similar in the ILC and IDC cohorts. In both groups, primary-site surgery was less common in older patients, and more common in those with larger tumors (except T4) and higher N category (Table ##TAB##3##4##). The odds of undergoing primary-site surgery were highest for ILC patients with pathologic stage T3 disease versus T1 (OR 2.65, 95% CI 1.17–3.51, <italic>p</italic> = 0.002; Table ##TAB##3##4##) whereas the odds of surgery for patients with IDC were highest in pathologic stage T2 disease versus T1 (OR 1.71, 95% CI 1.30–2.25, p < 0.001). In both groups, those with T4 disease had significantly lower odds of primary-site surgery compared to T1 (Table ##TAB##3##4##). Over time, the odds of undergoing primary-site surgery decreased. Specifically, the odds of surgery decreased by 16% per each additional year of diagnosis (OR 0.84 and <italic>p</italic> < 0.001 for both groups; 95% CI 0.82–0.87 in ILC group; 95% CI 0.83–0.85 in IDC group, Table ##TAB##3##4##).</p>","<p id=\"Par56\">\n\n</p>","<title>Radiotherapy by histologic subtype</title>","<p id=\"Par36\">The use of radiotherapy overall was lower for patients with ILC than IDC (29.1% versus 37.9%, <italic>p <</italic> 0.001) (Table ##TAB##1##2##). In IDC patients who had surgery, 51.5% also had radiation, while in ILC patients who had surgery, 42.5% also had radiation (Table ##TAB##2##3##). Among those who received radiotherapy, there was no difference in the rate of radiation to local versus distant sites by histologic subtype (<italic>p</italic> = 0.55).</p>","<title>Use and timing of chemotherapy</title>","<p id=\"Par37\">More IDC patients received chemotherapy (41.3% ILC versus 47.4% IDC, <italic>p</italic> < 0.001), while those with ILC were more likely to receive endocrine therapy (83.5% ILC versus 78.6% IDC, <italic>p</italic> < 0.001). For those who had primary-site surgery, the sequence of chemotherapy and surgery differed by histologic subtype; while 40.5% of patients with IDC had chemotherapy prior to surgery, only 29.0% of patients with ILC had chemotherapy prior to surgery (<italic>p</italic> < 0.001).</p>","<title>Survival analyses in unmatched cohorts</title>","<p id=\"Par38\">Overall, patients with ILC had slightly but significantly shorter OS than those with IDC (median 38 months ILC versus 40 months IDC, <italic>p</italic> = 0.006). In both cohorts, primary-site surgery was associated with significantly improved OS (Table ##TAB##0##1##). In the ILC cohort, undergoing primary-site surgery was associated with 35% lower risk of death compared to those who did not undergo surgery (HR 0.65, 95% CI 0.57–0.68, <italic>p</italic> < 0.001) (Fig. ##FIG##2##3##). This association persisted when controlling for age, CDCI (0/1+), metastatic site, and receptor subtype (HR 0.64, 95% CI 0.58–0.70, <italic>p</italic> < 0.001). The timing of surgery (before or after systemic chemotherapy) was not significantly associated with OS among those with ILC in unadjusted analysis nor after controlling for age, CDCI (0/1+), metastatic site, and receptor subtype.</p>","<p id=\"Par39\">\n\n</p>","<p id=\"Par40\">Similarly, patients in the IDC cohort who underwent primary-site surgery had an associated 40% lower risk of death compared to those without surgery (HR 0.60, 95% CI 0.57–0.63, <italic>p</italic> < 0.001) (Fig. ##FIG##2##3##). This remained true after controlling for age, CDCI (0/1+), metastatic site, and receptor subtype (HR 0.61, 95% CI 0.58–0.64, <italic>p</italic> < 0.001). Unlike for those with ILC, timing of surgery was significantly associated with OS. Patients with IDC who had chemotherapy before surgery had 24% less risk of death compared to those who had surgery prior to chemotherapy (HR 0.76, 95% CI 0.70–0.84, <italic>p</italic> < 0.001). This association persisted after controlling for age, CDCI (0/1+), site of metastasis, and receptor subtype (HR 0.83, 95% CI 0.76–0.92, <italic>p</italic> < 0.001).</p>","<p id=\"Par41\">The type of surgery (mastectomy versus lumpectomy) was not significantly associated with different OS in either group. However, we found a significant statistical interaction between having surgery for the primary tumor and the site of radiation. Primary-site surgery was associated with a greater reduction in risk of death among those who had local radiation compared to those who had distant radiation (HR 0.35, 95% CI 0.3–0.4 versus HR 0.67, 95% CI 0.61–0.74 respectively, test of interaction <italic>p</italic> < 0.001). This interaction between primary-site surgery and site of radiation was similar in both the ILC and IDC cohorts separately.</p>","<title>Survival analyses in propensity score matched cohorts</title>","<p id=\"Par42\">The propensity score model included age, tumor grade, receptor subtype, metastatic site, CDCI (0/1+), and treating facility variables, with 3,089 ILC patients (991 with surgery, 2,098 without surgery) and 11,216 IDC patients (3,429 with surgery, 7,787 without surgery) in each cohort with available data for matching. In both the ILC and IDC matched samples there was still a significant association between having surgery for the primary tumor and improved OS (ILC HR 0.71, 95% CI 0.59–0.85, <italic>p</italic> < 0.001; IDC HR 0.67, 95% CI 0.61–0.74, <italic>p</italic> < 0.001).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Study cohort</title>\",\n \"<p id=\\\"Par27\\\">There were 100,147 patients with stage IV breast cancer, of whom 25,294 had HR-positive, HER2-negative invasive lobular or ductal histology, and met study criteria (Fig. ##FIG##0##1##). Of these patients, 19,171 (75.8%) had IDC and 6,123 (24.2%) had ILC. Within the ILC cohort, 4,484 (73.2%) had pure ILC, with the remaining having mixed ILC/IDC. Median follow-up time of the ILC cohort was 27.2 months (IQ range 14.7–41.5 months), which was similar to the median follow-up time of 26.8 months (IQ range 14.6–42.6 months) for the IDC cohort (Table ##TAB##0##1##). Patients with ILC were slightly older than those with IDC (mean age 64 years versus 61 years, <italic>p</italic> < 0.001) and differed significantly by race (<italic>p</italic> < 0.001), with a higher proportion of White patients (79.4% versus 74.2%, <italic>p</italic> < 0.0001) (Table ##TAB##0##1##). Additionally, ILC and IDC patients differed by tumor grade (<italic>p</italic> < 0.001), with ILC patients having a higher proportion of grade 1 tumors (22.1% versus 9.48%, p < 0.0001) and more bone-only metastases than those with IDC (60.8% versus 45.2%, <italic>p</italic> < 0.001).</p>\",\n \"<p id=\\\"Par28\\\">\\n\\n</p>\",\n \"<p id=\\\"Par53\\\">\\n\\n</p>\",\n \"<p id=\\\"Par30\\\">While overall most patients were treated at community cancer centers, slightly more ILC patients than IDC patients were treated at academic centers (ILC 36.0% versus IDC 33.5%, <italic>p</italic> = 0.001) (Table ##TAB##0##1##). ILC patients were significantly more likely to have public insurance (ILC 54.7% versus IDC 51.6%) and less likely to be uninsured (ILC 3.82% versus IDC 5.15%, <italic>p</italic> < 0.0001). Patients with ILC had higher median income, with 39.7% in the highest quartile compared to 35.7% of IDC patients in the highest median income quartile (<italic>p</italic> < 0.0001).</p>\",\n \"<title>Primary-site surgery by histologic subtype</title>\",\n \"<p id=\\\"Par31\\\">In the overall study population, 7,158 (28.3%) underwent primary-site surgery. Although the absolute difference is small, primary-site surgery was performed less often in those with metastatic ILC than those with metastatic IDC (n = 1,644 [26.8%] versus n = 5,514 [28.8%] respectively, <italic>p</italic> = 0.004) (Table ##TAB##1##2##). This difference was more pronounced when restricting the analysis to patients with pure ILC, where only 23.8% underwent primary-site surgery. Additionally, the slightly lower rate of primary-site surgery among patients with ILC compared to IDC was observed among those with bone-only or unknown site of metastases, but not among those with visceral metastases (Fig. ##FIG##1##2##). Among those who had primary-site surgery in both histologic cohorts, the site of metastatic disease was significantly more likely to be bone-only compared to other sites (ILC 63.5%; IDC 48.5%, <italic>p</italic> < 0.001) (Table ##TAB##2##3##).</p>\",\n \"<p id=\\\"Par54\\\">\\n\\n</p>\",\n \"<p id=\\\"Par32\\\">\\n\\n</p>\",\n \"<p id=\\\"Par33\\\">For both cohorts, patients with private insurance were significantly more likely to receive primary-site surgery (ILC 46.4%; IDC 49.4%, <italic>p</italic> < 0.0001) compared to patients with public insurance (Table ##TAB##2##3##). Both ILC and IDC patients who received surgery were equally likely to have been treated at an academic treating facility (ILC n = 415 [26.3%]; IDC n = 1,318 [26.2%]) compared to a community setting (ILC n = 1,164 [73.7%]; IDC n = 3,714 [73.8%]).</p>\",\n \"<p id=\\\"Par55\\\">\\n\\n</p>\",\n \"<p id=\\\"Par34\\\">Among those who underwent primary-site surgery (n = 7,158), the differences by histology reflected those seen in the overall study population. Those with ILC were older (ILC mean age 61.7 years versus IDC 58.1 years, <italic>p</italic> < 0.001), had more T3 tumors (ILC 30.8% versus IDC 15.5%, <italic>p</italic> < 0.001), had more N3 nodal status (ILC 35.3% versus IDC 20.5%, <italic>p</italic> < 0.001), and had more grade 2 disease (ILC 61.4% versus IDC 45.9%, <italic>p</italic> < 0.001) (Table ##TAB##0##1##). There was no significant difference in mastectomy rate between the two cohorts (Table ##TAB##1##2##). Positive surgical margins were significantly more common in those with ILC who underwent lumpectomy compared to IDC (15.7% versus 11.2%, <italic>p =</italic> 0.025) (Table ##TAB##0##1##).</p>\",\n \"<p id=\\\"Par35\\\">The factors associated with undergoing primary-site surgery were similar in the ILC and IDC cohorts. In both groups, primary-site surgery was less common in older patients, and more common in those with larger tumors (except T4) and higher N category (Table ##TAB##3##4##). The odds of undergoing primary-site surgery were highest for ILC patients with pathologic stage T3 disease versus T1 (OR 2.65, 95% CI 1.17–3.51, <italic>p</italic> = 0.002; Table ##TAB##3##4##) whereas the odds of surgery for patients with IDC were highest in pathologic stage T2 disease versus T1 (OR 1.71, 95% CI 1.30–2.25, p < 0.001). In both groups, those with T4 disease had significantly lower odds of primary-site surgery compared to T1 (Table ##TAB##3##4##). Over time, the odds of undergoing primary-site surgery decreased. Specifically, the odds of surgery decreased by 16% per each additional year of diagnosis (OR 0.84 and <italic>p</italic> < 0.001 for both groups; 95% CI 0.82–0.87 in ILC group; 95% CI 0.83–0.85 in IDC group, Table ##TAB##3##4##).</p>\",\n \"<p id=\\\"Par56\\\">\\n\\n</p>\",\n \"<title>Radiotherapy by histologic subtype</title>\",\n \"<p id=\\\"Par36\\\">The use of radiotherapy overall was lower for patients with ILC than IDC (29.1% versus 37.9%, <italic>p <</italic> 0.001) (Table ##TAB##1##2##). In IDC patients who had surgery, 51.5% also had radiation, while in ILC patients who had surgery, 42.5% also had radiation (Table ##TAB##2##3##). Among those who received radiotherapy, there was no difference in the rate of radiation to local versus distant sites by histologic subtype (<italic>p</italic> = 0.55).</p>\",\n \"<title>Use and timing of chemotherapy</title>\",\n \"<p id=\\\"Par37\\\">More IDC patients received chemotherapy (41.3% ILC versus 47.4% IDC, <italic>p</italic> < 0.001), while those with ILC were more likely to receive endocrine therapy (83.5% ILC versus 78.6% IDC, <italic>p</italic> < 0.001). For those who had primary-site surgery, the sequence of chemotherapy and surgery differed by histologic subtype; while 40.5% of patients with IDC had chemotherapy prior to surgery, only 29.0% of patients with ILC had chemotherapy prior to surgery (<italic>p</italic> < 0.001).</p>\",\n \"<title>Survival analyses in unmatched cohorts</title>\",\n \"<p id=\\\"Par38\\\">Overall, patients with ILC had slightly but significantly shorter OS than those with IDC (median 38 months ILC versus 40 months IDC, <italic>p</italic> = 0.006). In both cohorts, primary-site surgery was associated with significantly improved OS (Table ##TAB##0##1##). In the ILC cohort, undergoing primary-site surgery was associated with 35% lower risk of death compared to those who did not undergo surgery (HR 0.65, 95% CI 0.57–0.68, <italic>p</italic> < 0.001) (Fig. ##FIG##2##3##). This association persisted when controlling for age, CDCI (0/1+), metastatic site, and receptor subtype (HR 0.64, 95% CI 0.58–0.70, <italic>p</italic> < 0.001). The timing of surgery (before or after systemic chemotherapy) was not significantly associated with OS among those with ILC in unadjusted analysis nor after controlling for age, CDCI (0/1+), metastatic site, and receptor subtype.</p>\",\n \"<p id=\\\"Par39\\\">\\n\\n</p>\",\n \"<p id=\\\"Par40\\\">Similarly, patients in the IDC cohort who underwent primary-site surgery had an associated 40% lower risk of death compared to those without surgery (HR 0.60, 95% CI 0.57–0.63, <italic>p</italic> < 0.001) (Fig. ##FIG##2##3##). This remained true after controlling for age, CDCI (0/1+), metastatic site, and receptor subtype (HR 0.61, 95% CI 0.58–0.64, <italic>p</italic> < 0.001). Unlike for those with ILC, timing of surgery was significantly associated with OS. Patients with IDC who had chemotherapy before surgery had 24% less risk of death compared to those who had surgery prior to chemotherapy (HR 0.76, 95% CI 0.70–0.84, <italic>p</italic> < 0.001). This association persisted after controlling for age, CDCI (0/1+), site of metastasis, and receptor subtype (HR 0.83, 95% CI 0.76–0.92, <italic>p</italic> < 0.001).</p>\",\n \"<p id=\\\"Par41\\\">The type of surgery (mastectomy versus lumpectomy) was not significantly associated with different OS in either group. However, we found a significant statistical interaction between having surgery for the primary tumor and the site of radiation. Primary-site surgery was associated with a greater reduction in risk of death among those who had local radiation compared to those who had distant radiation (HR 0.35, 95% CI 0.3–0.4 versus HR 0.67, 95% CI 0.61–0.74 respectively, test of interaction <italic>p</italic> < 0.001). This interaction between primary-site surgery and site of radiation was similar in both the ILC and IDC cohorts separately.</p>\",\n \"<title>Survival analyses in propensity score matched cohorts</title>\",\n \"<p id=\\\"Par42\\\">The propensity score model included age, tumor grade, receptor subtype, metastatic site, CDCI (0/1+), and treating facility variables, with 3,089 ILC patients (991 with surgery, 2,098 without surgery) and 11,216 IDC patients (3,429 with surgery, 7,787 without surgery) in each cohort with available data for matching. In both the ILC and IDC matched samples there was still a significant association between having surgery for the primary tumor and improved OS (ILC HR 0.71, 95% CI 0.59–0.85, <italic>p</italic> < 0.001; IDC HR 0.67, 95% CI 0.61–0.74, <italic>p</italic> < 0.001).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par43\">Recent randomized trial data suggest that the role of primary-site surgery in the management of patients with metastatic breast cancer is limited to local control in select cases, with no evidence of impact on OS [##REF##34995128##9##–##REF##26363985##11##]. However, optimal selection criteria for primary-site surgery are unknown, with decisions being made on an individualized basis in clinical practice [##REF##20101736##13##]. Given the known differences in surgical management in the early stage setting and disease patterns in the metastatic setting between ILC and IDC, we explored whether the use of primary-site surgery differs in HR-positive HER2-negative metastatic lobular versus ductal breast cancer.</p>","<p id=\"Par44\">In this cohort of 25,294 patients from the NCDB, we found that a high proportion of patients overall underwent primary-site surgery in the setting of metastatic breast cancer (28.3%). It is important to note that these data represent patients diagnosed between 2010 and 2016, prior to the publication of randomized trials demonstrating the lack of OS benefit from primary-site surgery [##REF##34995128##9##–##REF##26363985##11##]. Indeed, we found that the odds of undergoing primary-site surgery significantly decreased over time. While primary-site surgery was more common in those with bone-only metastases, and those with ILC were more likely to have such a disease pattern, the overall usage of primary-site surgery in the ILC cohort was slightly but significantly lower than in the IDC cohort. Although a slightly smaller proportion of patients with ILC had primary-site surgery, the majority of factors associated with receiving surgery did not differ between the lobular and ductal groups; in both groups, primary-site surgery was more common among younger patients, those with T2 or T3 tumors, more nodal disease, and private insurance.</p>","<p id=\"Par45\">Interestingly, while patients with ILC had larger tumors than those with IDC, there was no difference in the rate of mastectomy by histologic subtype. This differs from the early-stage setting, where lobular histology is associated with higher mastectomy rates. Similar to the early-stage setting, however, those with metastatic ILC who had primary-site surgery experienced significantly higher positive margin rates after lumpectomy than those with metastatic IDC. This suggests that the local control benefit of primary-site surgery might be attenuated in those with ILC, who may require more extensive surgery to achieve negative margins. We did find an association between local radiotherapy and improved OS in this cohort; whether this association reflects a relationship between improved local control and survival outcomes versus improved outcomes in those selected to have radiation is unknown. Of note, patients with ILC were significantly less likely to receive radiation than those with IDC, which is consistent with other studies [##REF##35949419##16##, ##REF##33415073##17##].</p>","<p id=\"Par46\">Interestingly, we found significantly lower odds of primary-site surgery in patients with T4 tumors in both lobular and ductal groups. Since palliation is the most accepted purpose of primary-site surgery in the stage IV setting, we would have expected higher rates of surgery in those with T4 tumors. Alternatively, these tumors may have been deemed unresectable; one of the challenges of analyzing this retrospective dataset is the inability to discern the reasons for performing primary-site surgery.</p>","<p id=\"Par47\">This limitation likely impacts the strong association between primary-site surgery and improved OS that we found in both groups. For example, in both the ILC and IDC cohorts, patients who had private insurance were more likely to have surgery compared to patients who had public insurance. The improved outcomes associated with primary-site surgery may reflect improved access to care as opposed to a biologic effect of surgery. This is consistent with prior data; retrospective series tend to show a survival advantage associated with primary-site surgery [##REF##37020372##4##, ##REF##17687611##12##, ##REF##20101736##13##], whereas recent randomized trial data do not [##REF##34995128##9##, ##REF##26363985##11##]. Such discrepancies suggest selection bias in which patients undergo primary-site surgery. We are likely unable to account for the many factors that influence why surgery would be used in some patients versus others despite using propensity score matched models.</p>","<p id=\"Par48\">Of more interest, perhaps, is the finding that the use of pre-operative systemic therapy was associated with improved OS in the IDC cohort, but not in the ILC cohort. We suspect that pre-operative systemic therapy in the IDC cohort may have helped to select patients who would have more durable response to therapy, and therefore have improved OS. In contrast, response to therapy in those with ILC may be more difficult to ascertain, or less likely to be associated with outcomes.</p>","<p id=\"Par49\">For systemic therapy, those with ILC were significantly more likely to receive endocrine therapy than those with IDC, despite all studied cases being HR-positive. Likewise, those with IDC were more likely to receive chemotherapy. This treatment pattern has been observed in previous literature and may point to the notion that early-stage ILC has reduced sensitivity to chemotherapies, or perceived as such, and is therefore utilized less frequently [##REF##35347549##18##, ##REF##27482285##19##]. However, more recent studies show that in the metastatic setting, response to eribulin and CDK4/6 inhibitors may be similar between ILC and IDC [##REF##30578311##20##, ##REF##31859246##21##]. These findings highlight the need to identify lobular specific therapies for those with metastatic disease.</p>","<p id=\"Par50\">As a secondary endpoint, we looked at OS by histology. Similar to our findings, worse OS in those with metastatic ILC has been shown in other studies as well [##REF##28757653##2##, ##REF##15084238##3##, ##REF##18458044##22##]. While the underlying reason for this difference is unclear, it suggests that ILC is indeed biologically different than IDC, given differential outcomes despite restricting the study population to those with HR-positive, HER2-negative tumor types, and ILC tumors being of lower grade than IDC tumors. One potential explanation could be that those with metastatic ILC may have an overall higher burden of disease than is typically detected on standard imaging modalities [##REF##33520704##23##].</p>","<p id=\"Par51\">To our knowledge, this is the largest reported study evaluating primary-site surgery by histologic subtype in metastatic breast cancer. However, this study is subject to several limitations, including selection bias, lack of detailed systemic therapy information, radiation field data, and the absence of local recurrence events as an endpoint. However, the findings reflect real-world management patterns which appear to differ by histologic subtype.</p>","<p id=\"Par52\">While ILC has long been regarded as a less aggressive tumor type, our findings from this large NCDB study are consistent with others showing worse outcomes in ILC than IDC. The differences between the IDC and ILC groups in this study were relatively small, however, it is interesting to note that histology appears to be influencing management. The use of primary-site surgery was slightly lower, and the use of both radiotherapy and chemotherapy were much lower in those with metastatic ILC compared to metastatic IDC. It is unclear what is driving the lower usage of chemotherapy and radiotherapy in ILC cases; this may reflect an underlying bias that lobular tumors are more indolent and slow growing. Coupled with shorter OS in the ILC cohort, these findings reinforce the need for further study to determine histologic subtype-specific management options. In regard to surgical management, the significantly larger tumor size and higher positive margin rates in the ILC cohort suggest that if primary-site surgery is to be utilized, one should consider a larger excision and likely incorporate radiotherapy to maximize potential benefit of locoregional intervention. Further work is needed to improve management outcomes for those with metastatic ILC.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par43\\\">Recent randomized trial data suggest that the role of primary-site surgery in the management of patients with metastatic breast cancer is limited to local control in select cases, with no evidence of impact on OS [##REF##34995128##9##–##REF##26363985##11##]. However, optimal selection criteria for primary-site surgery are unknown, with decisions being made on an individualized basis in clinical practice [##REF##20101736##13##]. Given the known differences in surgical management in the early stage setting and disease patterns in the metastatic setting between ILC and IDC, we explored whether the use of primary-site surgery differs in HR-positive HER2-negative metastatic lobular versus ductal breast cancer.</p>\",\n \"<p id=\\\"Par44\\\">In this cohort of 25,294 patients from the NCDB, we found that a high proportion of patients overall underwent primary-site surgery in the setting of metastatic breast cancer (28.3%). It is important to note that these data represent patients diagnosed between 2010 and 2016, prior to the publication of randomized trials demonstrating the lack of OS benefit from primary-site surgery [##REF##34995128##9##–##REF##26363985##11##]. Indeed, we found that the odds of undergoing primary-site surgery significantly decreased over time. While primary-site surgery was more common in those with bone-only metastases, and those with ILC were more likely to have such a disease pattern, the overall usage of primary-site surgery in the ILC cohort was slightly but significantly lower than in the IDC cohort. Although a slightly smaller proportion of patients with ILC had primary-site surgery, the majority of factors associated with receiving surgery did not differ between the lobular and ductal groups; in both groups, primary-site surgery was more common among younger patients, those with T2 or T3 tumors, more nodal disease, and private insurance.</p>\",\n \"<p id=\\\"Par45\\\">Interestingly, while patients with ILC had larger tumors than those with IDC, there was no difference in the rate of mastectomy by histologic subtype. This differs from the early-stage setting, where lobular histology is associated with higher mastectomy rates. Similar to the early-stage setting, however, those with metastatic ILC who had primary-site surgery experienced significantly higher positive margin rates after lumpectomy than those with metastatic IDC. This suggests that the local control benefit of primary-site surgery might be attenuated in those with ILC, who may require more extensive surgery to achieve negative margins. We did find an association between local radiotherapy and improved OS in this cohort; whether this association reflects a relationship between improved local control and survival outcomes versus improved outcomes in those selected to have radiation is unknown. Of note, patients with ILC were significantly less likely to receive radiation than those with IDC, which is consistent with other studies [##REF##35949419##16##, ##REF##33415073##17##].</p>\",\n \"<p id=\\\"Par46\\\">Interestingly, we found significantly lower odds of primary-site surgery in patients with T4 tumors in both lobular and ductal groups. Since palliation is the most accepted purpose of primary-site surgery in the stage IV setting, we would have expected higher rates of surgery in those with T4 tumors. Alternatively, these tumors may have been deemed unresectable; one of the challenges of analyzing this retrospective dataset is the inability to discern the reasons for performing primary-site surgery.</p>\",\n \"<p id=\\\"Par47\\\">This limitation likely impacts the strong association between primary-site surgery and improved OS that we found in both groups. For example, in both the ILC and IDC cohorts, patients who had private insurance were more likely to have surgery compared to patients who had public insurance. The improved outcomes associated with primary-site surgery may reflect improved access to care as opposed to a biologic effect of surgery. This is consistent with prior data; retrospective series tend to show a survival advantage associated with primary-site surgery [##REF##37020372##4##, ##REF##17687611##12##, ##REF##20101736##13##], whereas recent randomized trial data do not [##REF##34995128##9##, ##REF##26363985##11##]. Such discrepancies suggest selection bias in which patients undergo primary-site surgery. We are likely unable to account for the many factors that influence why surgery would be used in some patients versus others despite using propensity score matched models.</p>\",\n \"<p id=\\\"Par48\\\">Of more interest, perhaps, is the finding that the use of pre-operative systemic therapy was associated with improved OS in the IDC cohort, but not in the ILC cohort. We suspect that pre-operative systemic therapy in the IDC cohort may have helped to select patients who would have more durable response to therapy, and therefore have improved OS. In contrast, response to therapy in those with ILC may be more difficult to ascertain, or less likely to be associated with outcomes.</p>\",\n \"<p id=\\\"Par49\\\">For systemic therapy, those with ILC were significantly more likely to receive endocrine therapy than those with IDC, despite all studied cases being HR-positive. Likewise, those with IDC were more likely to receive chemotherapy. This treatment pattern has been observed in previous literature and may point to the notion that early-stage ILC has reduced sensitivity to chemotherapies, or perceived as such, and is therefore utilized less frequently [##REF##35347549##18##, ##REF##27482285##19##]. However, more recent studies show that in the metastatic setting, response to eribulin and CDK4/6 inhibitors may be similar between ILC and IDC [##REF##30578311##20##, ##REF##31859246##21##]. These findings highlight the need to identify lobular specific therapies for those with metastatic disease.</p>\",\n \"<p id=\\\"Par50\\\">As a secondary endpoint, we looked at OS by histology. Similar to our findings, worse OS in those with metastatic ILC has been shown in other studies as well [##REF##28757653##2##, ##REF##15084238##3##, ##REF##18458044##22##]. While the underlying reason for this difference is unclear, it suggests that ILC is indeed biologically different than IDC, given differential outcomes despite restricting the study population to those with HR-positive, HER2-negative tumor types, and ILC tumors being of lower grade than IDC tumors. One potential explanation could be that those with metastatic ILC may have an overall higher burden of disease than is typically detected on standard imaging modalities [##REF##33520704##23##].</p>\",\n \"<p id=\\\"Par51\\\">To our knowledge, this is the largest reported study evaluating primary-site surgery by histologic subtype in metastatic breast cancer. However, this study is subject to several limitations, including selection bias, lack of detailed systemic therapy information, radiation field data, and the absence of local recurrence events as an endpoint. However, the findings reflect real-world management patterns which appear to differ by histologic subtype.</p>\",\n \"<p id=\\\"Par52\\\">While ILC has long been regarded as a less aggressive tumor type, our findings from this large NCDB study are consistent with others showing worse outcomes in ILC than IDC. The differences between the IDC and ILC groups in this study were relatively small, however, it is interesting to note that histology appears to be influencing management. The use of primary-site surgery was slightly lower, and the use of both radiotherapy and chemotherapy were much lower in those with metastatic ILC compared to metastatic IDC. It is unclear what is driving the lower usage of chemotherapy and radiotherapy in ILC cases; this may reflect an underlying bias that lobular tumors are more indolent and slow growing. Coupled with shorter OS in the ILC cohort, these findings reinforce the need for further study to determine histologic subtype-specific management options. In regard to surgical management, the significantly larger tumor size and higher positive margin rates in the ILC cohort suggest that if primary-site surgery is to be utilized, one should consider a larger excision and likely incorporate radiotherapy to maximize potential benefit of locoregional intervention. Further work is needed to improve management outcomes for those with metastatic ILC.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose</title>","<p id=\"Par1\">Primary site surgery for metastatic breast cancer improves local control but does not impact overall survival. Whether histologic subtype influences patient selection for surgery is unknown. Given differences in surgical management between early-stage lobular versus ductal disease, we evaluated the impact of histology on primary site surgery in patients with metastatic breast cancer.</p>","<title>Methods</title>","<p id=\"Par2\">The National Cancer Database (NCDB, 2010–2016) was queried for patients with stage IV HR-positive, HER2-negative invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC). We compared clinicopathologic features, primary site surgery rates, and outcomes by histologic subtype. Multivariable Cox proportional hazard models with and without propensity score matching were used for overall survival (OS) analyses.</p>","<title>Results</title>","<p id=\"Par3\">In 25,294 patients, primary site surgery was slightly but significantly less common in the 6,123 patients with ILC compared to the 19,171 patients with IDC (26.9% versus 28.8%, <italic>p</italic> = 0.004). Those with ILC were less likely to receive chemotherapy (41.3% versus 47.4%, <italic>p</italic> < 0.0001) or radiotherapy (29.1% versus 37.9%, <italic>p <</italic> 0.0001), and had shorter OS. While mastectomy rates were similar, those with ILC who underwent lumpectomy had significantly higher positive margin rates (ILC 15.7% versus IDC 11.2%, <italic>p =</italic> 0.025). In both groups, the odds of undergoing surgery decreased over time, and were higher in younger patients with T2/T3 tumors and higher nodal burden.</p>","<title>Conclusion</title>","<p id=\"Par4\">Lobular histology is associated with less primary site surgery, higher positive margin rates, less radiotherapy and chemotherapy, and shorter OS compared to those with HR-positive HER2-negative IDC. These findings support the need for ILC-specific data and treatment approaches in the setting of metastatic disease.</p>","<title>Keywords</title>"],"string":"[\n \"<title>Purpose</title>\",\n \"<p id=\\\"Par1\\\">Primary site surgery for metastatic breast cancer improves local control but does not impact overall survival. Whether histologic subtype influences patient selection for surgery is unknown. Given differences in surgical management between early-stage lobular versus ductal disease, we evaluated the impact of histology on primary site surgery in patients with metastatic breast cancer.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">The National Cancer Database (NCDB, 2010–2016) was queried for patients with stage IV HR-positive, HER2-negative invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC). We compared clinicopathologic features, primary site surgery rates, and outcomes by histologic subtype. Multivariable Cox proportional hazard models with and without propensity score matching were used for overall survival (OS) analyses.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">In 25,294 patients, primary site surgery was slightly but significantly less common in the 6,123 patients with ILC compared to the 19,171 patients with IDC (26.9% versus 28.8%, <italic>p</italic> = 0.004). Those with ILC were less likely to receive chemotherapy (41.3% versus 47.4%, <italic>p</italic> < 0.0001) or radiotherapy (29.1% versus 37.9%, <italic>p <</italic> 0.0001), and had shorter OS. While mastectomy rates were similar, those with ILC who underwent lumpectomy had significantly higher positive margin rates (ILC 15.7% versus IDC 11.2%, <italic>p =</italic> 0.025). In both groups, the odds of undergoing surgery decreased over time, and were higher in younger patients with T2/T3 tumors and higher nodal burden.</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par4\\\">Lobular histology is associated with less primary site surgery, higher positive margin rates, less radiotherapy and chemotherapy, and shorter OS compared to those with HR-positive HER2-negative IDC. These findings support the need for ILC-specific data and treatment approaches in the setting of metastatic disease.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":["<title>Author contributions</title>","<p>Supervision, study concept and design, and interpretation of data were provided by Rita A Mukhtar. The first draft of the manuscript was written by Harriet Rothschild and all authors commented on previous versions of the manuscript. Amy Shui performed statistical analysis and data interpretation. All authors read and approved the final manuscript.</p>","<title>Funding</title>","<p>Author HTR was supported by the National Center for Advancing Translational Sciences, National Institutes of Health (NIH) (Grant number TL1 TR 001871). The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Author AMS is part of the Biostatistics Core that is generously supported by the University of California, San Francisco Department of Surgery. Author RAM is supported by National Cancer Institute Award K08CA256047. For the remaining authors no sources of funding were declared.</p>","<title>Data Availability</title>","<p>The datasets analyzed during the current study are available in the National Cancer Database, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.facs.org/quality-programs/cancer-programs/national-cancer-database/\">https://www.facs.org/quality-programs/cancer-programs/national-cancer-database/</ext-link>.</p>","<title>Declarations</title>","<title>Ethics approval</title>","<p id=\"Par73\">Due to the de-identified nature of the public-access user files in the National Cancer Database, the study was deemed exempt from institutional review board approval.</p>","<title>Competing interests</title>","<p id=\"Par310\">The authors have no relevant financial or non-financial interests to disclose.</p>"],"string":"[\n \"<title>Author contributions</title>\",\n \"<p>Supervision, study concept and design, and interpretation of data were provided by Rita A Mukhtar. The first draft of the manuscript was written by Harriet Rothschild and all authors commented on previous versions of the manuscript. Amy Shui performed statistical analysis and data interpretation. All authors read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Author HTR was supported by the National Center for Advancing Translational Sciences, National Institutes of Health (NIH) (Grant number TL1 TR 001871). The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Author AMS is part of the Biostatistics Core that is generously supported by the University of California, San Francisco Department of Surgery. Author RAM is supported by National Cancer Institute Award K08CA256047. For the remaining authors no sources of funding were declared.</p>\",\n \"<title>Data Availability</title>\",\n \"<p>The datasets analyzed during the current study are available in the National Cancer Database, <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://www.facs.org/quality-programs/cancer-programs/national-cancer-database/\\\">https://www.facs.org/quality-programs/cancer-programs/national-cancer-database/</ext-link>.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethics approval</title>\",\n \"<p id=\\\"Par73\\\">Due to the de-identified nature of the public-access user files in the National Cancer Database, the study was deemed exempt from institutional review board approval.</p>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par310\\\">The authors have no relevant financial or non-financial interests to disclose.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>CONSORT flow diagram for study population of stage IV, hormone receptor positive, HER2-negative breast cancer patients from the National Cancer Database (PUF 2010–2016). <italic>ER</italic>, estrogen receptor; <italic>PR</italic>, progesterone receptor; <italic>HER2</italic>, human epidermal growth factor receptor 2; <italic>IDC</italic>, invasive ductal carcinoma; <italic>ILC</italic>, invasive lobular carcinoma; <italic>PUF</italic>, Participant User Files</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Proportion of patients undergoing primary-site surgery by histologic subtype grouped by site of metastatic disease. For patients with bone metastasis only or unknown metastatic site, use of primary-site surgery was significantly less common in those with ILC compared to IDC</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Kaplan-Meier survival curves based on primary-site surgery and histology for stage IV hormone receptor positive, HER2-negative patients in the National Cancer Database (PUF 2010–2016). Estimated overall survival in patients with invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) with or without surgery (unmatched, unadjusted cohorts)</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>CONSORT flow diagram for study population of stage IV, hormone receptor positive, HER2-negative breast cancer patients from the National Cancer Database (PUF 2010–2016). <italic>ER</italic>, estrogen receptor; <italic>PR</italic>, progesterone receptor; <italic>HER2</italic>, human epidermal growth factor receptor 2; <italic>IDC</italic>, invasive ductal carcinoma; <italic>ILC</italic>, invasive lobular carcinoma; <italic>PUF</italic>, Participant User Files</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Proportion of patients undergoing primary-site surgery by histologic subtype grouped by site of metastatic disease. For patients with bone metastasis only or unknown metastatic site, use of primary-site surgery was significantly less common in those with ILC compared to IDC</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Kaplan-Meier survival curves based on primary-site surgery and histology for stage IV hormone receptor positive, HER2-negative patients in the National Cancer Database (PUF 2010–2016). Estimated overall survival in patients with invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) with or without surgery (unmatched, unadjusted cohorts)</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparison of patient characteristics between invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) cohorts in unmatched population</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Patient Characteristics</th><th align=\"left\">IDC, n (%)<break/>Total n = 19,171</th><th align=\"left\">ILC, n (%)<break/>Total n = 6,123</th><th align=\"left\"><italic>P-</italic>value</th></tr></thead><tbody><tr><td align=\"left\"><bold>Mean Age</bold> [S.D.]</td><td align=\"left\">61.3 [± 13.9]</td><td align=\"left\">63.6 [± 12.6]</td><td char=\".\" align=\"char\">< 0.0001</td></tr><tr><td align=\"left\"> Age < 50</td><td align=\"left\">3,861 (20.1%)</td><td align=\"left\">836 (13.7%)</td><td char=\".\" align=\"char\">< 0.0001</td></tr><tr><td align=\"left\"> Age ≥ 50</td><td align=\"left\">15,310 (79.8%)</td><td align=\"left\">5,287 (86.3%)</td><td align=\"left\"/></tr><tr><td align=\"left\"><bold>Median Follow up time</bold>, months [IQ range]</td><td align=\"left\">26.8 [14.6–42.6]</td><td align=\"left\">27.2 [17.7–41.5]</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Race</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> White</td><td align=\"left\">14,116 (74.2%)</td><td align=\"left\">4,825 (79.4%)</td><td char=\".\" align=\"char\">< 0.0001</td></tr><tr><td align=\"left\"> African American</td><td align=\"left\">3,010 (15.8%)</td><td align=\"left\">753 (12.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> East Asian</td><td align=\"left\">130 (0.68%)</td><td align=\"left\">27 (0.44%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Other</td><td align=\"left\">1,763 (9.27%)</td><td align=\"left\">470 (7.74%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Hispanic</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">0.19</td></tr><tr><td align=\"left\"> Hispanic</td><td align=\"left\">1,081 (5.82%)</td><td align=\"left\">319 (5.36%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Non-Hispanic</td><td align=\"left\">17,508 (94.2%)</td><td align=\"left\">5,629 (94.6%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Treatment Facility Type</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">0.001</td></tr><tr><td align=\"left\"> Academic</td><td align=\"left\">6,003 (33.5%)</td><td align=\"left\">2146 (36%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Community<sup>a</sup></td><td align=\"left\">11,927 (66.5%)</td><td align=\"left\">3810 (64%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Primary Payer</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.0001</td></tr><tr><td align=\"left\"> No insurance</td><td align=\"left\">987 (5.15%)</td><td align=\"left\">234 (3.82%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Private insurance</td><td align=\"left\">7,990 (41.7%)</td><td align=\"left\">2,475 (40.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Public insurance</td><td align=\"left\">9,890 (51.6%)</td><td align=\"left\">3,348 (54.7%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Unknown insurance</td><td align=\"left\">304 (1.59%)</td><td align=\"left\">66 (1.08%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Median Income Quartiles</bold>\n<sup><bold>b</bold></sup>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.0001</td></tr><tr><td align=\"left\"> <$40,227</td><td align=\"left\">3,724 (19.7%)</td><td align=\"left\">1,030 (17.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> $40,227–50,353</td><td align=\"left\">4,053 (21.4%)</td><td align=\"left\">1,212 (20.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> $50,354–63,332</td><td align=\"left\">4,389 (23.2%)</td><td align=\"left\">1,398 (23.2%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> ≥$63,333</td><td align=\"left\">6,739 (35.6%)</td><td align=\"left\">2,394 (39.7%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Receptor Status</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">0.017</td></tr><tr><td align=\"left\"> ER positive / PR positive</td><td align=\"left\">16,170 (84.3%)</td><td align=\"left\">5,086 (83.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> ER positive / PR negative</td><td align=\"left\">3,001 (15.7%)</td><td align=\"left\">1,037 (16.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Grade</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.0001</td></tr><tr><td align=\"left\"> 1</td><td align=\"left\">1,580 (9.48%)</td><td align=\"left\">1,020 (22.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> 2</td><td align=\"left\">8,822 (52.9%)</td><td align=\"left\">2,861 (62%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> 3</td><td align=\"left\">6,272 (37.6%)</td><td align=\"left\">732 (15.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>N Stage</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> 1</td><td align=\"left\">1,932 (47.6%)</td><td align=\"left\">480 (36.5%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> 2</td><td align=\"left\">1,295 (31.9%)</td><td align=\"left\">372 (28.3%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> 3</td><td align=\"left\">831 (20.5%)</td><td align=\"left\">465 (35.3%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>T Stage</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> 1</td><td align=\"left\">1,217 (23.4%)</td><td align=\"left\">328 (21.0%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> 2</td><td align=\"left\">2,056 (39.6%)</td><td align=\"left\">570 (36.5%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> 3</td><td align=\"left\">803 (15.5%)</td><td align=\"left\">481 (30.8%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> 4</td><td align=\"left\">1,118 (21.5%)</td><td align=\"left\">182 (11.7%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Charlson-Deyo Score</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">0.007</td></tr><tr><td align=\"left\"> 0</td><td align=\"left\">15,894 (82.9%)</td><td align=\"left\">4,984 (81.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> ≥1</td><td align=\"left\">3,277 (17.1%)</td><td align=\"left\">1,139 (18.6%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Positive Surgical Margins</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Lumpectomy</td><td align=\"left\">182/1621 (11.2%)</td><td align=\"left\">70/445 (15.7%)</td><td char=\".\" align=\"char\">0.025</td></tr><tr><td align=\"left\"> Mastectomy</td><td align=\"left\">262/3893 (6.7%)</td><td align=\"left\">99/1199 (8.2%)</td><td char=\".\" align=\"char\">0.14</td></tr><tr><td align=\"left\">\n<bold>Metastasis site</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.0001</td></tr><tr><td align=\"left\"> Bone-only</td><td align=\"left\">8,510 (45.2%)</td><td align=\"left\">3,641 (60.8%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> All other</td><td align=\"left\">10,334 (54.8%)</td><td align=\"left\">2,346 (39.2%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Overall Survival in months (Median (95% CI))</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> All patients</td><td align=\"left\">39.7 (39.0-40.6)</td><td align=\"left\">38.4 (47.2–39.7)</td><td char=\".\" align=\"char\">0.006</td></tr><tr><td align=\"left\"> With surgery</td><td align=\"left\">50.9 (49.1–52.9)</td><td align=\"left\">47.4 (44.9–50.6)</td><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> Without surgery</td><td align=\"left\">35.3 (34.4–36.0)</td><td align=\"left\">34.7 (33.5–35.9)</td><td char=\".\" align=\"char\">< 0.001</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Comparison of treatment patterns by histology. <italic>IDC</italic>, invasive ductal carcinoma; <italic>ILC</italic>, invasive lobular carcinoma; <italic>CNS</italic>, central nervous system</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Treatment</th><th align=\"left\">IDC, n (%)<break/>Total n = 19,171</th><th align=\"left\">ILC, n (%)<break/>Total n = 6,123</th><th align=\"left\"><italic>p-</italic>value</th></tr></thead><tbody><tr><td align=\"left\">\n<bold>Any surgery</bold>\n</td><td align=\"left\">5,514 (28.8%)</td><td align=\"left\">1,644 (26.8%)</td><td char=\".\" align=\"char\">0.004</td></tr><tr><td align=\"left\">\n<bold>Surgery type</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">0.19</td></tr><tr><td align=\"left\"> Lumpectomy</td><td align=\"left\">1,621 (27.5%)</td><td align=\"left\">445 (27.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Mastectomy</td><td align=\"left\">3,788 (64.2%)</td><td align=\"left\">1,166 (70.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Radical mastectomy</td><td align=\"left\">105 (8.38%)</td><td align=\"left\">33 (2.01%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Chemotherapy</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> No chemotherapy</td><td align=\"left\">9,592 (50%)</td><td align=\"left\">3,406 (55.6%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Yes chemotherapy</td><td align=\"left\">9,084 (47.4%)</td><td align=\"left\">2,529 (41.3%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Unknown chemotherapy</td><td align=\"left\">495 (2.58%)</td><td align=\"left\">188 (3.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Chemotherapy timing</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> Preoperative chemotherapy</td><td align=\"left\">2,231 (40.5%)</td><td align=\"left\">477 (29%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Postoperative chemotherapy</td><td align=\"left\">3,283 (59.5%)</td><td align=\"left\">1,167 (71%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Endocrine therapy</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> No endocrine therapy</td><td align=\"left\">3,520 (18.4%)</td><td align=\"left\">869 (14.2%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Yes endocrine therapy</td><td align=\"left\">15,075 (78.6%)</td><td align=\"left\">5,112 (83.5%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Unknown endocrine therapy</td><td align=\"left\">576 (3%)</td><td align=\"left\">142 (2.32%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Any radiation therapy</bold>\n</td><td align=\"left\">7,260 (37.9%)</td><td align=\"left\">1,781 (29.1%)</td><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\">\n<bold>Radiation location</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">0.55</td></tr><tr><td align=\"left\"> Local radiation</td><td align=\"left\">2,365 (35.9%)</td><td align=\"left\">564 (35.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Distant radiation</td><td align=\"left\">4,220 (64.1%)</td><td align=\"left\">1,042 (64.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Detailed radiation location</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> CNS/head</td><td align=\"left\">600 (8.26%)</td><td align=\"left\">168 (9.43%)</td><td char=\".\" align=\"char\">0.02</td></tr><tr><td align=\"left\"> Viscera</td><td align=\"left\">86 (1.18%)</td><td align=\"left\">28 (1.57%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Breast</td><td align=\"left\">2,365 (3.25%)</td><td align=\"left\">564 (31.7%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Bone</td><td align=\"left\">3,534 (48.7%)</td><td align=\"left\">846 (47.5%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Other</td><td align=\"left\">383 (5.28%)</td><td align=\"left\">79 (4.43%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Unknown</td><td align=\"left\">292 (4.02%)</td><td align=\"left\">96 (5.4%)</td><td align=\"left\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Sociodemographic and treatment patterns broken down by histology and surgery status. Total number of patients, (n= ), unless otherwise stated. <italic>IDC</italic>, invasive ductal carcinoma; <italic>ILC</italic>, invasive lobular carcinoma; <italic>CNS</italic>, central nervous system</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"4\">Invasive Ductal Carcinoma</th><th align=\"left\"/><th align=\"left\" colspan=\"4\">Invasive Lobular Carcinoma</th></tr><tr><th align=\"left\">All IDC (n = 19,171)</th><th align=\"left\">IDC w/surgery<break/>(n = 5,514)</th><th align=\"left\">IDC w/out surgery (n = 13,657)</th><th align=\"left\"><italic>P-</italic>value</th><th align=\"left\"/><th align=\"left\">All ILC (n = 6,123)</th><th align=\"left\">ILC w/surgery<break/>(n = 1,644)</th><th align=\"left\">ILC w/out surgery (n = 4,479)</th><th align=\"left\"><italic>P-</italic>value</th></tr></thead><tbody><tr><td align=\"left\">\n<bold>Treatment Facility</bold>\n<sup><bold>a</bold></sup>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.0001</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.0001</td></tr><tr><td align=\"left\"> Academic</td><td char=\".\" align=\"char\">6,003 (33.5%)</td><td char=\".\" align=\"char\">1,318 (26.2%)</td><td align=\"left\">4,685 (36.3%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">2,146 (36%)</td><td char=\".\" align=\"char\">415 (26.3%)</td><td align=\"left\">1,731 (39.6%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Community</td><td char=\".\" align=\"char\">11,927 (66.5%)</td><td char=\".\" align=\"char\">3,714 (73.8%)</td><td align=\"left\">8,213 (63.7%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">3,810 (64%)</td><td char=\".\" align=\"char\">1,164 (73.7%)</td><td align=\"left\">2,646 (60.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Primary Payer</bold>\n<sup><bold>b</bold></sup>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> No insurance</td><td char=\".\" align=\"char\">987 (5.15%)</td><td char=\".\" align=\"char\">207 (3.75%)</td><td align=\"left\">780 (5.71%)</td><td char=\".\" align=\"char\">< 0.0001</td><td align=\"left\"/><td align=\"left\">234 (3.82%)</td><td char=\".\" align=\"char\">41 (2.49%)</td><td align=\"left\">193 (4.31%)</td><td char=\".\" align=\"char\">< 0.0001</td></tr><tr><td align=\"left\"> Private insurance</td><td char=\".\" align=\"char\">7,990 (41.7%)</td><td char=\".\" align=\"char\">2,723 (49.4%)</td><td align=\"left\">5,267 (38.6%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">2,475 (40.4%)</td><td char=\".\" align=\"char\">763 (46.4%)</td><td align=\"left\">1,712 (38.2%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Public insurance</td><td char=\".\" align=\"char\">9,890 (51.6%)</td><td char=\".\" align=\"char\">2,509 (45.5%)</td><td align=\"left\">7,381 (54.1%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">3,348 (54.7%)</td><td char=\".\" align=\"char\">824 (50.1%)</td><td align=\"left\">2,524 (56.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Unknown</td><td char=\".\" align=\"char\">304 (1.59%)</td><td char=\".\" align=\"char\">75 (1.36%)</td><td align=\"left\">229 (1.68%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">66 (1.08%)</td><td char=\".\" align=\"char\">16 (1.12%)</td><td align=\"left\">50 (1.12%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Median Income Quartile</bold>\n<sup><bold>c</bold></sup>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.0001</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.0001</td></tr><tr><td align=\"left\"> <$40,227</td><td char=\".\" align=\"char\">3,724 (19.7%)</td><td char=\".\" align=\"char\">1,084 (20.0%)</td><td align=\"left\">2,640 (19.6%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">1,030 (17.1%)</td><td char=\".\" align=\"char\">260 (16.1%)</td><td align=\"left\">770 (17.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> $40,227–50,353</td><td char=\".\" align=\"char\">4,053 (21.4%)</td><td char=\".\" align=\"char\">1,175 (21.6%)</td><td align=\"left\">2,878 (21.4%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">1,212 (20.1%)</td><td char=\".\" align=\"char\">351 (21.7%)</td><td align=\"left\">861 (19.5%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> $50,354–63,332</td><td char=\".\" align=\"char\">4,389 (23.2%)</td><td char=\".\" align=\"char\">1,261 (23.2%)</td><td align=\"left\">3,128 (23.2%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">1,398 (23.2%)</td><td char=\".\" align=\"char\">365 (22.6%)</td><td align=\"left\">1,033 (23.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> ≥$63,333</td><td char=\".\" align=\"char\">6,739 (35.7%)</td><td char=\".\" align=\"char\">1,911 (35.2%)</td><td align=\"left\">4,828 (35.8%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">2,394 (39.7%)</td><td char=\".\" align=\"char\">641 (39.6%)</td><td align=\"left\">1,753 (39.7%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Metastatic Site</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\"> Bone metastasis only</td><td char=\".\" align=\"char\">8510 (44.4%)</td><td char=\".\" align=\"char\">2,677 (48.5%)</td><td align=\"left\">5833 (42.7%)</td><td char=\".\" align=\"char\">< 0.001</td><td align=\"left\"/><td align=\"left\">3641 (59.5%)</td><td char=\".\" align=\"char\">1044 (63.5%)</td><td align=\"left\">2597 (58%)</td><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> Visceral metastasis only</td><td char=\".\" align=\"char\">3226 (16.8%)</td><td char=\".\" align=\"char\">1,059 (19.2%)</td><td align=\"left\">2167 (15.9%)</td><td char=\".\" align=\"char\">< 0.001</td><td align=\"left\"/><td align=\"left\">515 (8.4%)</td><td char=\".\" align=\"char\">150 (9.12%)</td><td align=\"left\">365 (8.15%)</td><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> Bone and visceral metastases</td><td char=\".\" align=\"char\">5424 (28.3%)</td><td char=\".\" align=\"char\">873 (15.8%)</td><td align=\"left\">4551 (33.3%)</td><td char=\".\" align=\"char\">< 0.001</td><td align=\"left\"/><td align=\"left\">1167 (19.0%)</td><td char=\".\" align=\"char\">163 (9.91%)</td><td align=\"left\">1004 (22.4%)</td><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> Unknown metastatic site</td><td char=\".\" align=\"char\">2011 (10.5%)</td><td char=\".\" align=\"char\">905 (16.4%)</td><td align=\"left\">1106 (8.1%)</td><td char=\".\" align=\"char\">< 0.001</td><td align=\"left\"/><td align=\"left\">800 (13.1%)</td><td char=\".\" align=\"char\">287 (17.5%)</td><td align=\"left\">513 (11.5%)</td><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\">\n<bold>Radiation frequency</bold>\n</td><td char=\".\" align=\"char\">7260 (37.9%)</td><td char=\".\" align=\"char\">2,842 (51.5%)</td><td align=\"left\">4418 (32.3%)</td><td char=\".\" align=\"char\">< 0.001</td><td align=\"left\"/><td align=\"left\">1781 (29.1%)</td><td char=\".\" align=\"char\">698 (42.5%)</td><td align=\"left\">1083 (24.2%)</td><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\">\n<bold>Radiation binary</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.001</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> Local</td><td char=\".\" align=\"char\">2,365 (35.9%)</td><td char=\".\" align=\"char\">1835 (69.2%)</td><td align=\"left\">530 (13.5%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">564 (35.1%)</td><td char=\".\" align=\"char\">458 (69.5%)</td><td align=\"left\">106 (11.2%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Distant</td><td char=\".\" align=\"char\">4,220 (64.1%)</td><td char=\".\" align=\"char\">817 (30.8%)</td><td align=\"left\">3,403 (86.5%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">1042 (64.8%)</td><td char=\".\" align=\"char\">201 (30.5%)</td><td align=\"left\">841 (88.8%)</td><td align=\"left\"/></tr><tr><td align=\"left\">\n<bold>Radiation detailed</bold>\n</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.001</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\">< 0.001</td></tr><tr><td align=\"left\"> CNS/head</td><td char=\".\" align=\"char\">600 (8.26%)</td><td char=\".\" align=\"char\">87 (3.06%)</td><td align=\"left\">513 (11.6%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">168 (9.43%)</td><td char=\".\" align=\"char\">18 (2.58%)</td><td align=\"left\">150 (13.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Viscera</td><td char=\".\" align=\"char\">86 (1.18%)</td><td char=\".\" align=\"char\">39 (1.37%)</td><td align=\"left\">47 (1.06%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">28 (1.57%)</td><td char=\".\" align=\"char\">8 (1.15%)</td><td align=\"left\">20 (1.84%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Breast</td><td char=\".\" align=\"char\">2365 (32.6%)</td><td char=\".\" align=\"char\">1,835 (64.6%)</td><td align=\"left\">530 (12%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">564 (31.7%)</td><td char=\".\" align=\"char\">458 (65.6%)</td><td align=\"left\">106 (9.79%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Bone</td><td char=\".\" align=\"char\">3534 (48.7%)</td><td char=\".\" align=\"char\">691 (24.3%)</td><td align=\"left\">2843 (64.4%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">846 (47.5%)</td><td char=\".\" align=\"char\">175 (25.1%)</td><td align=\"left\">671 (62%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Other</td><td char=\".\" align=\"char\">383 (5.28%)</td><td char=\".\" align=\"char\">100 (3.52%)</td><td align=\"left\">283 (6.41%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">79 (4.44%)</td><td char=\".\" align=\"char\">18 (2.58%)</td><td align=\"left\">61 (5.63%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Unknown</td><td char=\".\" align=\"char\">292 (4.02%)</td><td char=\".\" align=\"char\">90 (3.17%)</td><td align=\"left\">202 (4.57%)</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">96 (5.39%)</td><td char=\".\" align=\"char\">21 (3.01%)</td><td align=\"left\">75 (6.93%)</td><td align=\"left\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Factors associated with receiving primary-site surgery in those with metastatic invasive ductal carcinoma and those with metastatic invasive lobular carcinoma. <italic>OR odds ratio; CI confidence interval</italic></p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Unadjusted OR</th><th align=\"left\">95% CI</th><th align=\"left\" colspan=\"2\"><italic>p</italic>-value</th></tr></thead><tbody><tr><td align=\"left\">\n<bold>Invasive Ductal Carcinoma</bold>\n</td><td align=\"left\"/><td align=\"left\" colspan=\"2\"/><td align=\"left\"/></tr><tr><td align=\"left\">Age<sup>a</sup></td><td align=\"left\">0.83</td><td align=\"left\" colspan=\"2\">(0.81–0.85)</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">Stage 0 vs. T1</td><td align=\"left\">0.50</td><td align=\"left\" colspan=\"2\">(0.31–0.81)</td><td align=\"left\">0.0045</td></tr><tr><td align=\"left\">Stage T2 vs. T1</td><td align=\"left\">1.71</td><td align=\"left\" colspan=\"2\">(1.30–2.25)</td><td align=\"left\">0.0001</td></tr><tr><td align=\"left\">Stage T3 vs. T1</td><td align=\"left\">1.68</td><td align=\"left\" colspan=\"2\">(1.17–2.41)</td><td align=\"left\">0.0050</td></tr><tr><td align=\"left\">Stage T4 vs. T1</td><td align=\"left\">0.63</td><td align=\"left\" colspan=\"2\">(0.49–0.82)</td><td align=\"left\">0.0006</td></tr><tr><td align=\"left\">Node N1 vs. N0</td><td align=\"left\">0.73</td><td align=\"left\" colspan=\"2\">(0.60–0.88)</td><td align=\"left\">0.0008</td></tr><tr><td align=\"left\">Node N2 vs. N0</td><td align=\"left\">4.21</td><td align=\"left\" colspan=\"2\">(3.18–5.58)</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">Node N3 vs. N0</td><td align=\"left\">2.91</td><td align=\"left\" colspan=\"2\">(2.17–3.91)</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">Year of diagnosis</td><td align=\"left\">0.84</td><td align=\"left\" colspan=\"2\">(0.83–0.85)</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">\n<bold>Invasive Lobular Carcinoma</bold>\n</td><td align=\"left\"/><td align=\"left\" colspan=\"2\"/><td align=\"left\"/></tr><tr><td align=\"left\">Age<sup>a</sup></td><td align=\"left\">0.85</td><td align=\"left\" colspan=\"2\">(0.81–0.89)</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">Stage 0 vs. T1</td><td align=\"left\">0.08</td><td align=\"left\" colspan=\"2\">(0.04–0.20)</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">Stage T2 vs. T1</td><td align=\"left\">2.03</td><td align=\"left\" colspan=\"2\">(1.17–3.51)</td><td align=\"left\">0.0113</td></tr><tr><td align=\"left\">Stage T3 vs. T1</td><td align=\"left\">2.65</td><td align=\"left\" colspan=\"2\">(1.43–4.90)</td><td align=\"left\">0.0020</td></tr><tr><td align=\"left\">Stage T4 vs. T1</td><td align=\"left\">0.45</td><td align=\"left\" colspan=\"2\">(0.27–0.78)</td><td align=\"left\">0.0042</td></tr><tr><td align=\"left\">Node N1 vs. N0</td><td align=\"left\">0.79</td><td align=\"left\" colspan=\"2\">(0.57–1.09)</td><td align=\"left\">0.1529</td></tr><tr><td align=\"left\">Node N2 vs. N0</td><td align=\"left\">19.65</td><td align=\"left\" colspan=\"2\">(9.28–41.61)</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">Node N3 vs. N0</td><td align=\"left\">12.12</td><td align=\"left\" colspan=\"2\">(6.88–21.35)</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">Year of diagnosis</td><td align=\"left\">0.84</td><td align=\"left\" colspan=\"2\">(0.82–0.87)</td><td align=\"left\">< 0.001</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Comparison of patient characteristics between invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) cohorts in unmatched population</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Patient Characteristics</th><th align=\\\"left\\\">IDC, n (%)<break/>Total n = 19,171</th><th align=\\\"left\\\">ILC, n (%)<break/>Total n = 6,123</th><th align=\\\"left\\\"><italic>P-</italic>value</th></tr></thead><tbody><tr><td align=\\\"left\\\"><bold>Mean Age</bold> [S.D.]</td><td align=\\\"left\\\">61.3 [± 13.9]</td><td align=\\\"left\\\">63.6 [± 12.6]</td><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td></tr><tr><td align=\\\"left\\\"> Age < 50</td><td align=\\\"left\\\">3,861 (20.1%)</td><td align=\\\"left\\\">836 (13.7%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td></tr><tr><td align=\\\"left\\\"> Age ≥ 50</td><td align=\\\"left\\\">15,310 (79.8%)</td><td align=\\\"left\\\">5,287 (86.3%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><bold>Median Follow up time</bold>, months [IQ range]</td><td align=\\\"left\\\">26.8 [14.6–42.6]</td><td align=\\\"left\\\">27.2 [17.7–41.5]</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Race</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> White</td><td align=\\\"left\\\">14,116 (74.2%)</td><td align=\\\"left\\\">4,825 (79.4%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td></tr><tr><td align=\\\"left\\\"> African American</td><td align=\\\"left\\\">3,010 (15.8%)</td><td align=\\\"left\\\">753 (12.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> East Asian</td><td align=\\\"left\\\">130 (0.68%)</td><td align=\\\"left\\\">27 (0.44%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Other</td><td align=\\\"left\\\">1,763 (9.27%)</td><td align=\\\"left\\\">470 (7.74%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Hispanic</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">0.19</td></tr><tr><td align=\\\"left\\\"> Hispanic</td><td align=\\\"left\\\">1,081 (5.82%)</td><td align=\\\"left\\\">319 (5.36%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Non-Hispanic</td><td align=\\\"left\\\">17,508 (94.2%)</td><td align=\\\"left\\\">5,629 (94.6%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Treatment Facility Type</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">0.001</td></tr><tr><td align=\\\"left\\\"> Academic</td><td align=\\\"left\\\">6,003 (33.5%)</td><td align=\\\"left\\\">2146 (36%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Community<sup>a</sup></td><td align=\\\"left\\\">11,927 (66.5%)</td><td align=\\\"left\\\">3810 (64%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Primary Payer</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td></tr><tr><td align=\\\"left\\\"> No insurance</td><td align=\\\"left\\\">987 (5.15%)</td><td align=\\\"left\\\">234 (3.82%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Private insurance</td><td align=\\\"left\\\">7,990 (41.7%)</td><td align=\\\"left\\\">2,475 (40.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Public insurance</td><td align=\\\"left\\\">9,890 (51.6%)</td><td align=\\\"left\\\">3,348 (54.7%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Unknown insurance</td><td align=\\\"left\\\">304 (1.59%)</td><td align=\\\"left\\\">66 (1.08%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Median Income Quartiles</bold>\\n<sup><bold>b</bold></sup>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td></tr><tr><td align=\\\"left\\\"> <$40,227</td><td align=\\\"left\\\">3,724 (19.7%)</td><td align=\\\"left\\\">1,030 (17.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> $40,227–50,353</td><td align=\\\"left\\\">4,053 (21.4%)</td><td align=\\\"left\\\">1,212 (20.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> $50,354–63,332</td><td align=\\\"left\\\">4,389 (23.2%)</td><td align=\\\"left\\\">1,398 (23.2%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ≥$63,333</td><td align=\\\"left\\\">6,739 (35.6%)</td><td align=\\\"left\\\">2,394 (39.7%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Receptor Status</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">0.017</td></tr><tr><td align=\\\"left\\\"> ER positive / PR positive</td><td align=\\\"left\\\">16,170 (84.3%)</td><td align=\\\"left\\\">5,086 (83.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ER positive / PR negative</td><td align=\\\"left\\\">3,001 (15.7%)</td><td align=\\\"left\\\">1,037 (16.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Grade</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td></tr><tr><td align=\\\"left\\\"> 1</td><td align=\\\"left\\\">1,580 (9.48%)</td><td align=\\\"left\\\">1,020 (22.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> 2</td><td align=\\\"left\\\">8,822 (52.9%)</td><td align=\\\"left\\\">2,861 (62%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> 3</td><td align=\\\"left\\\">6,272 (37.6%)</td><td align=\\\"left\\\">732 (15.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>N Stage</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> 1</td><td align=\\\"left\\\">1,932 (47.6%)</td><td align=\\\"left\\\">480 (36.5%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> 2</td><td align=\\\"left\\\">1,295 (31.9%)</td><td align=\\\"left\\\">372 (28.3%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> 3</td><td align=\\\"left\\\">831 (20.5%)</td><td align=\\\"left\\\">465 (35.3%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>T Stage</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> 1</td><td align=\\\"left\\\">1,217 (23.4%)</td><td align=\\\"left\\\">328 (21.0%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> 2</td><td align=\\\"left\\\">2,056 (39.6%)</td><td align=\\\"left\\\">570 (36.5%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> 3</td><td align=\\\"left\\\">803 (15.5%)</td><td align=\\\"left\\\">481 (30.8%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> 4</td><td align=\\\"left\\\">1,118 (21.5%)</td><td align=\\\"left\\\">182 (11.7%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Charlson-Deyo Score</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">0.007</td></tr><tr><td align=\\\"left\\\"> 0</td><td align=\\\"left\\\">15,894 (82.9%)</td><td align=\\\"left\\\">4,984 (81.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ≥1</td><td align=\\\"left\\\">3,277 (17.1%)</td><td align=\\\"left\\\">1,139 (18.6%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Positive Surgical Margins</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Lumpectomy</td><td align=\\\"left\\\">182/1621 (11.2%)</td><td align=\\\"left\\\">70/445 (15.7%)</td><td char=\\\".\\\" align=\\\"char\\\">0.025</td></tr><tr><td align=\\\"left\\\"> Mastectomy</td><td align=\\\"left\\\">262/3893 (6.7%)</td><td align=\\\"left\\\">99/1199 (8.2%)</td><td char=\\\".\\\" align=\\\"char\\\">0.14</td></tr><tr><td align=\\\"left\\\">\\n<bold>Metastasis site</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td></tr><tr><td align=\\\"left\\\"> Bone-only</td><td align=\\\"left\\\">8,510 (45.2%)</td><td align=\\\"left\\\">3,641 (60.8%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> All other</td><td align=\\\"left\\\">10,334 (54.8%)</td><td align=\\\"left\\\">2,346 (39.2%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Overall Survival in months (Median (95% CI))</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> All patients</td><td align=\\\"left\\\">39.7 (39.0-40.6)</td><td align=\\\"left\\\">38.4 (47.2–39.7)</td><td char=\\\".\\\" align=\\\"char\\\">0.006</td></tr><tr><td align=\\\"left\\\"> With surgery</td><td align=\\\"left\\\">50.9 (49.1–52.9)</td><td align=\\\"left\\\">47.4 (44.9–50.6)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> Without surgery</td><td align=\\\"left\\\">35.3 (34.4–36.0)</td><td align=\\\"left\\\">34.7 (33.5–35.9)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Comparison of treatment patterns by histology. <italic>IDC</italic>, invasive ductal carcinoma; <italic>ILC</italic>, invasive lobular carcinoma; <italic>CNS</italic>, central nervous system</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Treatment</th><th align=\\\"left\\\">IDC, n (%)<break/>Total n = 19,171</th><th align=\\\"left\\\">ILC, n (%)<break/>Total n = 6,123</th><th align=\\\"left\\\"><italic>p-</italic>value</th></tr></thead><tbody><tr><td align=\\\"left\\\">\\n<bold>Any surgery</bold>\\n</td><td align=\\\"left\\\">5,514 (28.8%)</td><td align=\\\"left\\\">1,644 (26.8%)</td><td char=\\\".\\\" align=\\\"char\\\">0.004</td></tr><tr><td align=\\\"left\\\">\\n<bold>Surgery type</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">0.19</td></tr><tr><td align=\\\"left\\\"> Lumpectomy</td><td align=\\\"left\\\">1,621 (27.5%)</td><td align=\\\"left\\\">445 (27.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Mastectomy</td><td align=\\\"left\\\">3,788 (64.2%)</td><td align=\\\"left\\\">1,166 (70.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Radical mastectomy</td><td align=\\\"left\\\">105 (8.38%)</td><td align=\\\"left\\\">33 (2.01%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Chemotherapy</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> No chemotherapy</td><td align=\\\"left\\\">9,592 (50%)</td><td align=\\\"left\\\">3,406 (55.6%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Yes chemotherapy</td><td align=\\\"left\\\">9,084 (47.4%)</td><td align=\\\"left\\\">2,529 (41.3%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Unknown chemotherapy</td><td align=\\\"left\\\">495 (2.58%)</td><td align=\\\"left\\\">188 (3.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Chemotherapy timing</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> Preoperative chemotherapy</td><td align=\\\"left\\\">2,231 (40.5%)</td><td align=\\\"left\\\">477 (29%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Postoperative chemotherapy</td><td align=\\\"left\\\">3,283 (59.5%)</td><td align=\\\"left\\\">1,167 (71%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Endocrine therapy</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> No endocrine therapy</td><td align=\\\"left\\\">3,520 (18.4%)</td><td align=\\\"left\\\">869 (14.2%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Yes endocrine therapy</td><td align=\\\"left\\\">15,075 (78.6%)</td><td align=\\\"left\\\">5,112 (83.5%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Unknown endocrine therapy</td><td align=\\\"left\\\">576 (3%)</td><td align=\\\"left\\\">142 (2.32%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Any radiation therapy</bold>\\n</td><td align=\\\"left\\\">7,260 (37.9%)</td><td align=\\\"left\\\">1,781 (29.1%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">\\n<bold>Radiation location</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">0.55</td></tr><tr><td align=\\\"left\\\"> Local radiation</td><td align=\\\"left\\\">2,365 (35.9%)</td><td align=\\\"left\\\">564 (35.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Distant radiation</td><td align=\\\"left\\\">4,220 (64.1%)</td><td align=\\\"left\\\">1,042 (64.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Detailed radiation location</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> CNS/head</td><td align=\\\"left\\\">600 (8.26%)</td><td align=\\\"left\\\">168 (9.43%)</td><td char=\\\".\\\" align=\\\"char\\\">0.02</td></tr><tr><td align=\\\"left\\\"> Viscera</td><td align=\\\"left\\\">86 (1.18%)</td><td align=\\\"left\\\">28 (1.57%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Breast</td><td align=\\\"left\\\">2,365 (3.25%)</td><td align=\\\"left\\\">564 (31.7%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Bone</td><td align=\\\"left\\\">3,534 (48.7%)</td><td align=\\\"left\\\">846 (47.5%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Other</td><td align=\\\"left\\\">383 (5.28%)</td><td align=\\\"left\\\">79 (4.43%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Unknown</td><td align=\\\"left\\\">292 (4.02%)</td><td align=\\\"left\\\">96 (5.4%)</td><td align=\\\"left\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Sociodemographic and treatment patterns broken down by histology and surgery status. Total number of patients, (n= ), unless otherwise stated. <italic>IDC</italic>, invasive ductal carcinoma; <italic>ILC</italic>, invasive lobular carcinoma; <italic>CNS</italic>, central nervous system</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" rowspan=\\\"2\\\"/><th align=\\\"left\\\" colspan=\\\"4\\\">Invasive Ductal Carcinoma</th><th align=\\\"left\\\"/><th align=\\\"left\\\" colspan=\\\"4\\\">Invasive Lobular Carcinoma</th></tr><tr><th align=\\\"left\\\">All IDC (n = 19,171)</th><th align=\\\"left\\\">IDC w/surgery<break/>(n = 5,514)</th><th align=\\\"left\\\">IDC w/out surgery (n = 13,657)</th><th align=\\\"left\\\"><italic>P-</italic>value</th><th align=\\\"left\\\"/><th align=\\\"left\\\">All ILC (n = 6,123)</th><th align=\\\"left\\\">ILC w/surgery<break/>(n = 1,644)</th><th align=\\\"left\\\">ILC w/out surgery (n = 4,479)</th><th align=\\\"left\\\"><italic>P-</italic>value</th></tr></thead><tbody><tr><td align=\\\"left\\\">\\n<bold>Treatment Facility</bold>\\n<sup><bold>a</bold></sup>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td></tr><tr><td align=\\\"left\\\"> Academic</td><td char=\\\".\\\" align=\\\"char\\\">6,003 (33.5%)</td><td char=\\\".\\\" align=\\\"char\\\">1,318 (26.2%)</td><td align=\\\"left\\\">4,685 (36.3%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">2,146 (36%)</td><td char=\\\".\\\" align=\\\"char\\\">415 (26.3%)</td><td align=\\\"left\\\">1,731 (39.6%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Community</td><td char=\\\".\\\" align=\\\"char\\\">11,927 (66.5%)</td><td char=\\\".\\\" align=\\\"char\\\">3,714 (73.8%)</td><td align=\\\"left\\\">8,213 (63.7%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">3,810 (64%)</td><td char=\\\".\\\" align=\\\"char\\\">1,164 (73.7%)</td><td align=\\\"left\\\">2,646 (60.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Primary Payer</bold>\\n<sup><bold>b</bold></sup>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> No insurance</td><td char=\\\".\\\" align=\\\"char\\\">987 (5.15%)</td><td char=\\\".\\\" align=\\\"char\\\">207 (3.75%)</td><td align=\\\"left\\\">780 (5.71%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td><td align=\\\"left\\\"/><td align=\\\"left\\\">234 (3.82%)</td><td char=\\\".\\\" align=\\\"char\\\">41 (2.49%)</td><td align=\\\"left\\\">193 (4.31%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td></tr><tr><td align=\\\"left\\\"> Private insurance</td><td char=\\\".\\\" align=\\\"char\\\">7,990 (41.7%)</td><td char=\\\".\\\" align=\\\"char\\\">2,723 (49.4%)</td><td align=\\\"left\\\">5,267 (38.6%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">2,475 (40.4%)</td><td char=\\\".\\\" align=\\\"char\\\">763 (46.4%)</td><td align=\\\"left\\\">1,712 (38.2%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Public insurance</td><td char=\\\".\\\" align=\\\"char\\\">9,890 (51.6%)</td><td char=\\\".\\\" align=\\\"char\\\">2,509 (45.5%)</td><td align=\\\"left\\\">7,381 (54.1%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">3,348 (54.7%)</td><td char=\\\".\\\" align=\\\"char\\\">824 (50.1%)</td><td align=\\\"left\\\">2,524 (56.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Unknown</td><td char=\\\".\\\" align=\\\"char\\\">304 (1.59%)</td><td char=\\\".\\\" align=\\\"char\\\">75 (1.36%)</td><td align=\\\"left\\\">229 (1.68%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">66 (1.08%)</td><td char=\\\".\\\" align=\\\"char\\\">16 (1.12%)</td><td align=\\\"left\\\">50 (1.12%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Median Income Quartile</bold>\\n<sup><bold>c</bold></sup>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.0001</td></tr><tr><td align=\\\"left\\\"> <$40,227</td><td char=\\\".\\\" align=\\\"char\\\">3,724 (19.7%)</td><td char=\\\".\\\" align=\\\"char\\\">1,084 (20.0%)</td><td align=\\\"left\\\">2,640 (19.6%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">1,030 (17.1%)</td><td char=\\\".\\\" align=\\\"char\\\">260 (16.1%)</td><td align=\\\"left\\\">770 (17.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> $40,227–50,353</td><td char=\\\".\\\" align=\\\"char\\\">4,053 (21.4%)</td><td char=\\\".\\\" align=\\\"char\\\">1,175 (21.6%)</td><td align=\\\"left\\\">2,878 (21.4%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">1,212 (20.1%)</td><td char=\\\".\\\" align=\\\"char\\\">351 (21.7%)</td><td align=\\\"left\\\">861 (19.5%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> $50,354–63,332</td><td char=\\\".\\\" align=\\\"char\\\">4,389 (23.2%)</td><td char=\\\".\\\" align=\\\"char\\\">1,261 (23.2%)</td><td align=\\\"left\\\">3,128 (23.2%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">1,398 (23.2%)</td><td char=\\\".\\\" align=\\\"char\\\">365 (22.6%)</td><td align=\\\"left\\\">1,033 (23.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ≥$63,333</td><td char=\\\".\\\" align=\\\"char\\\">6,739 (35.7%)</td><td char=\\\".\\\" align=\\\"char\\\">1,911 (35.2%)</td><td align=\\\"left\\\">4,828 (35.8%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">2,394 (39.7%)</td><td char=\\\".\\\" align=\\\"char\\\">641 (39.6%)</td><td align=\\\"left\\\">1,753 (39.7%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Metastatic Site</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Bone metastasis only</td><td char=\\\".\\\" align=\\\"char\\\">8510 (44.4%)</td><td char=\\\".\\\" align=\\\"char\\\">2,677 (48.5%)</td><td align=\\\"left\\\">5833 (42.7%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td><td align=\\\"left\\\"/><td align=\\\"left\\\">3641 (59.5%)</td><td char=\\\".\\\" align=\\\"char\\\">1044 (63.5%)</td><td align=\\\"left\\\">2597 (58%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> Visceral metastasis only</td><td char=\\\".\\\" align=\\\"char\\\">3226 (16.8%)</td><td char=\\\".\\\" align=\\\"char\\\">1,059 (19.2%)</td><td align=\\\"left\\\">2167 (15.9%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td><td align=\\\"left\\\"/><td align=\\\"left\\\">515 (8.4%)</td><td char=\\\".\\\" align=\\\"char\\\">150 (9.12%)</td><td align=\\\"left\\\">365 (8.15%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> Bone and visceral metastases</td><td char=\\\".\\\" align=\\\"char\\\">5424 (28.3%)</td><td char=\\\".\\\" align=\\\"char\\\">873 (15.8%)</td><td align=\\\"left\\\">4551 (33.3%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td><td align=\\\"left\\\"/><td align=\\\"left\\\">1167 (19.0%)</td><td char=\\\".\\\" align=\\\"char\\\">163 (9.91%)</td><td align=\\\"left\\\">1004 (22.4%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> Unknown metastatic site</td><td char=\\\".\\\" align=\\\"char\\\">2011 (10.5%)</td><td char=\\\".\\\" align=\\\"char\\\">905 (16.4%)</td><td align=\\\"left\\\">1106 (8.1%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td><td align=\\\"left\\\"/><td align=\\\"left\\\">800 (13.1%)</td><td char=\\\".\\\" align=\\\"char\\\">287 (17.5%)</td><td align=\\\"left\\\">513 (11.5%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">\\n<bold>Radiation frequency</bold>\\n</td><td char=\\\".\\\" align=\\\"char\\\">7260 (37.9%)</td><td char=\\\".\\\" align=\\\"char\\\">2,842 (51.5%)</td><td align=\\\"left\\\">4418 (32.3%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td><td align=\\\"left\\\"/><td align=\\\"left\\\">1781 (29.1%)</td><td char=\\\".\\\" align=\\\"char\\\">698 (42.5%)</td><td align=\\\"left\\\">1083 (24.2%)</td><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">\\n<bold>Radiation binary</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> Local</td><td char=\\\".\\\" align=\\\"char\\\">2,365 (35.9%)</td><td char=\\\".\\\" align=\\\"char\\\">1835 (69.2%)</td><td align=\\\"left\\\">530 (13.5%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">564 (35.1%)</td><td char=\\\".\\\" align=\\\"char\\\">458 (69.5%)</td><td align=\\\"left\\\">106 (11.2%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Distant</td><td char=\\\".\\\" align=\\\"char\\\">4,220 (64.1%)</td><td char=\\\".\\\" align=\\\"char\\\">817 (30.8%)</td><td align=\\\"left\\\">3,403 (86.5%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">1042 (64.8%)</td><td char=\\\".\\\" align=\\\"char\\\">201 (30.5%)</td><td align=\\\"left\\\">841 (88.8%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">\\n<bold>Radiation detailed</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\">< 0.001</td></tr><tr><td align=\\\"left\\\"> CNS/head</td><td char=\\\".\\\" align=\\\"char\\\">600 (8.26%)</td><td char=\\\".\\\" align=\\\"char\\\">87 (3.06%)</td><td align=\\\"left\\\">513 (11.6%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">168 (9.43%)</td><td char=\\\".\\\" align=\\\"char\\\">18 (2.58%)</td><td align=\\\"left\\\">150 (13.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Viscera</td><td char=\\\".\\\" align=\\\"char\\\">86 (1.18%)</td><td char=\\\".\\\" align=\\\"char\\\">39 (1.37%)</td><td align=\\\"left\\\">47 (1.06%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">28 (1.57%)</td><td char=\\\".\\\" align=\\\"char\\\">8 (1.15%)</td><td align=\\\"left\\\">20 (1.84%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Breast</td><td char=\\\".\\\" align=\\\"char\\\">2365 (32.6%)</td><td char=\\\".\\\" align=\\\"char\\\">1,835 (64.6%)</td><td align=\\\"left\\\">530 (12%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">564 (31.7%)</td><td char=\\\".\\\" align=\\\"char\\\">458 (65.6%)</td><td align=\\\"left\\\">106 (9.79%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Bone</td><td char=\\\".\\\" align=\\\"char\\\">3534 (48.7%)</td><td char=\\\".\\\" align=\\\"char\\\">691 (24.3%)</td><td align=\\\"left\\\">2843 (64.4%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">846 (47.5%)</td><td char=\\\".\\\" align=\\\"char\\\">175 (25.1%)</td><td align=\\\"left\\\">671 (62%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Other</td><td char=\\\".\\\" align=\\\"char\\\">383 (5.28%)</td><td char=\\\".\\\" align=\\\"char\\\">100 (3.52%)</td><td align=\\\"left\\\">283 (6.41%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">79 (4.44%)</td><td char=\\\".\\\" align=\\\"char\\\">18 (2.58%)</td><td align=\\\"left\\\">61 (5.63%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Unknown</td><td char=\\\".\\\" align=\\\"char\\\">292 (4.02%)</td><td char=\\\".\\\" align=\\\"char\\\">90 (3.17%)</td><td align=\\\"left\\\">202 (4.57%)</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">96 (5.39%)</td><td char=\\\".\\\" align=\\\"char\\\">21 (3.01%)</td><td align=\\\"left\\\">75 (6.93%)</td><td align=\\\"left\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab4\\\"><label>Table 4</label><caption><p>Factors associated with receiving primary-site surgery in those with metastatic invasive ductal carcinoma and those with metastatic invasive lobular carcinoma. <italic>OR odds ratio; CI confidence interval</italic></p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"/><th align=\\\"left\\\">Unadjusted OR</th><th align=\\\"left\\\">95% CI</th><th align=\\\"left\\\" colspan=\\\"2\\\"><italic>p</italic>-value</th></tr></thead><tbody><tr><td align=\\\"left\\\">\\n<bold>Invasive Ductal Carcinoma</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\" colspan=\\\"2\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">Age<sup>a</sup></td><td align=\\\"left\\\">0.83</td><td align=\\\"left\\\" colspan=\\\"2\\\">(0.81–0.85)</td><td align=\\\"left\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">Stage 0 vs. T1</td><td align=\\\"left\\\">0.50</td><td align=\\\"left\\\" colspan=\\\"2\\\">(0.31–0.81)</td><td align=\\\"left\\\">0.0045</td></tr><tr><td align=\\\"left\\\">Stage T2 vs. T1</td><td align=\\\"left\\\">1.71</td><td align=\\\"left\\\" colspan=\\\"2\\\">(1.30–2.25)</td><td align=\\\"left\\\">0.0001</td></tr><tr><td align=\\\"left\\\">Stage T3 vs. T1</td><td align=\\\"left\\\">1.68</td><td align=\\\"left\\\" colspan=\\\"2\\\">(1.17–2.41)</td><td align=\\\"left\\\">0.0050</td></tr><tr><td align=\\\"left\\\">Stage T4 vs. T1</td><td align=\\\"left\\\">0.63</td><td align=\\\"left\\\" colspan=\\\"2\\\">(0.49–0.82)</td><td align=\\\"left\\\">0.0006</td></tr><tr><td align=\\\"left\\\">Node N1 vs. N0</td><td align=\\\"left\\\">0.73</td><td align=\\\"left\\\" colspan=\\\"2\\\">(0.60–0.88)</td><td align=\\\"left\\\">0.0008</td></tr><tr><td align=\\\"left\\\">Node N2 vs. N0</td><td align=\\\"left\\\">4.21</td><td align=\\\"left\\\" colspan=\\\"2\\\">(3.18–5.58)</td><td align=\\\"left\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">Node N3 vs. N0</td><td align=\\\"left\\\">2.91</td><td align=\\\"left\\\" colspan=\\\"2\\\">(2.17–3.91)</td><td align=\\\"left\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">Year of diagnosis</td><td align=\\\"left\\\">0.84</td><td align=\\\"left\\\" colspan=\\\"2\\\">(0.83–0.85)</td><td align=\\\"left\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">\\n<bold>Invasive Lobular Carcinoma</bold>\\n</td><td align=\\\"left\\\"/><td align=\\\"left\\\" colspan=\\\"2\\\"/><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">Age<sup>a</sup></td><td align=\\\"left\\\">0.85</td><td align=\\\"left\\\" colspan=\\\"2\\\">(0.81–0.89)</td><td align=\\\"left\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">Stage 0 vs. T1</td><td align=\\\"left\\\">0.08</td><td align=\\\"left\\\" colspan=\\\"2\\\">(0.04–0.20)</td><td align=\\\"left\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">Stage T2 vs. T1</td><td align=\\\"left\\\">2.03</td><td align=\\\"left\\\" colspan=\\\"2\\\">(1.17–3.51)</td><td align=\\\"left\\\">0.0113</td></tr><tr><td align=\\\"left\\\">Stage T3 vs. T1</td><td align=\\\"left\\\">2.65</td><td align=\\\"left\\\" colspan=\\\"2\\\">(1.43–4.90)</td><td align=\\\"left\\\">0.0020</td></tr><tr><td align=\\\"left\\\">Stage T4 vs. T1</td><td align=\\\"left\\\">0.45</td><td align=\\\"left\\\" colspan=\\\"2\\\">(0.27–0.78)</td><td align=\\\"left\\\">0.0042</td></tr><tr><td align=\\\"left\\\">Node N1 vs. N0</td><td align=\\\"left\\\">0.79</td><td align=\\\"left\\\" colspan=\\\"2\\\">(0.57–1.09)</td><td align=\\\"left\\\">0.1529</td></tr><tr><td align=\\\"left\\\">Node N2 vs. N0</td><td align=\\\"left\\\">19.65</td><td align=\\\"left\\\" colspan=\\\"2\\\">(9.28–41.61)</td><td align=\\\"left\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">Node N3 vs. N0</td><td align=\\\"left\\\">12.12</td><td align=\\\"left\\\" colspan=\\\"2\\\">(6.88–21.35)</td><td align=\\\"left\\\">< 0.001</td></tr><tr><td align=\\\"left\\\">Year of diagnosis</td><td align=\\\"left\\\">0.84</td><td align=\\\"left\\\" colspan=\\\"2\\\">(0.82–0.87)</td><td align=\\\"left\\\">< 0.001</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p><sup>a</sup>Community treatment facility includes Community Cancer Programs, Comprehensive Community Cancer Programs, and Integrated Network Cancer Programs</p><p><sup>b</sup>Median Income Quartiles from 2012–2016</p></table-wrap-foot>","<table-wrap-foot><p><sup>a</sup> Treatment facility data available for 17,930 IDC and 5,956 ILC patients</p><p><sup>b</sup> Primary payer data available for 19,171 IDC and 6,123 ILC patients</p><p><sup>c</sup> Median income quartile data available for 18,905 IDC and 6,034 ILC patients</p></table-wrap-foot>","<table-wrap-foot><p><sup>a</sup> In this analysis, patient age at diagnosis is scaled to every 10 years</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p><sup>a</sup>Community treatment facility includes Community Cancer Programs, Comprehensive Community Cancer Programs, and Integrated Network Cancer Programs</p><p><sup>b</sup>Median Income Quartiles from 2012–2016</p></table-wrap-foot>\",\n \"<table-wrap-foot><p><sup>a</sup> Treatment facility data available for 17,930 IDC and 5,956 ILC patients</p><p><sup>b</sup> Primary payer data available for 19,171 IDC and 6,123 ILC patients</p><p><sup>c</sup> Median income quartile data available for 18,905 IDC and 6,034 ILC patients</p></table-wrap-foot>\",\n \"<table-wrap-foot><p><sup>a</sup> In this analysis, patient age at diagnosis is scaled to every 10 years</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"10549_2023_7125_Fig1_HTML\" id=\"d32e462\"/>","<graphic xlink:href=\"10549_2023_7125_Fig2_HTML\" id=\"d32e1318\"/>","<graphic xlink:href=\"10549_2023_7125_Fig3_HTML\" id=\"d32e2276\"/>"],"string":"[\n \"<graphic xlink:href=\\\"10549_2023_7125_Fig1_HTML\\\" id=\\\"d32e462\\\"/>\",\n \"<graphic xlink:href=\\\"10549_2023_7125_Fig2_HTML\\\" id=\\\"d32e1318\\\"/>\",\n \"<graphic xlink:href=\\\"10549_2023_7125_Fig3_HTML\\\" id=\\\"d32e2276\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [\n \"CDCI\",\n \"ER\",\n \"HER2\",\n \"HR\",\n \"IDC\",\n \"ILC\",\n \"NCDB\",\n \"OS\",\n \"PR\"\n ],\n \"definition\": [\n \"Charlson-Deyo Co-Morbidity Index\",\n \"Estrogen receptor\",\n \"Human epidermal growth factor receptor 2\",\n \"Hormone receptor\",\n \"Invasive ductal carcinoma\",\n \"Invasive lobular carcinoma\",\n \"National Cancer Database\",\n \"Overall survival\",\n \"Progesterone receptor\"\n ]\n}"},"n_references":{"kind":"number","value":23,"string":"23"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"Breast Cancer Res Treat. 2024 Oct 13; 203(2):245-256"},"package_file":{"kind":"string","value":"oa_package/92/6e/PMC10787876.tar.gz"}}},{"rowIdx":162,"cells":{"accession_id":{"kind":"string","value":"PMC10787878"},"pmid":{"kind":"string","value":"0"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par5\">Terpenoids are a large group of natural compounds constructed from isoprene units (C5). Many different terpenoids are used in the food, beverage, cosmetic, and pharmaceutical industries (Tetali ##REF##30467631##2019##). The current industrial supply of terpenoids relies mainly on chemical synthesis or extraction from plants. The chemical synthesis of terpenoids harboring complex structures has been actively researched (Phil ##REF##29635919##2018##). However, the multi-stereo center in the complex terpenoids requires diastereoselective synthesis, resulting in multi-step total synthesis with high costs and low yield (Santosh et al. ##UREF##2##2020##; Le et al. ##REF##36296479##2022##). Therefore, the chemical synthesis of terpenoids harboring complex structures is expensive and requires fine-controlled and complex processes. Plant extraction has been also used to supply terpenoids; however, it is limited by the presence of various compounds harboring similar physicochemical properties in plants and the difficulty of separating these compounds (Rodger et al. ##REF##21855882##2011##). In addition, natural plant supplies are limited owing to their finite amount (Nur and Henrik ##REF##29187859##2017##). Therefore, the engineering of microorganisms to produce terpenoids has emerged as a promising approach.</p>","<p id=\"Par6\">Research on terpenoid production using microorganisms has rapidly increased in the twenty-first century. <italic>Saccharomyces cerevisiae</italic> and <italic>Escherichia coli</italic> have been used to produce plant terpenoids by heterologous expression of their genes because they have advantages for production, such as the presence of a native terpenoid synthetic pathway and facile genetic manipulation (Navale et al. ##REF##33394155##2021##; Sun et al. ##REF##33616900##2020##). <italic>Saccharomyces cerevisiae</italic> offers several advantages for terpenoid production, including a high sugar catabolic rate, a relatively fast growth rate, and its GRAS status (Claudia et al. ##REF##28623722##2017##). In addition, <italic>saccharomyces cerevisiae</italic> has an inherent terpenoid production pathway, the mevalonate (MVA) pathway, in which isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP), essential intermediates of sterols and ubiquinones, are synthesized from acetyl-CoA (Guo et al. ##REF##32786348##2020##). Metabolic reactions in the MVA pathway proceed in the cytosol, which, in turn, are used for terpenoid production. Recently, terpenoid production using organelles has attracted attention owing to their ability to produce acetyl-CoA.</p>","<p id=\"Par7\">Peroxisomes are considered potential acetyl-CoA pools because β-oxidation of fatty acids proceeds in them (Hammer and Avalos ##REF##28853733##2017##). In fact, a terpenoid, β-amyrin, was produced at high level (57.8 mg/g dry cell weight (DCW)) by introducing two enzymes of the MVA pathway into peroxisomes and four downstream enzymes into the cytosol or peroxisomes (Du et al. ##REF##34955018##2022##). The concentration of acetyl-CoA in the mitochondria is as much as 20–30 times higher than that in the cytosol because of the high enzymatic activity of the mitochondrial pyruvate dehydrogenase complex (Weinert et al. ##UREF##3##2014##). Thus, mitochondria are considered suitable organelles for the introduction of the MVA pathway for terpenoid production (Duran et al. ##REF##32592388##2020##). For example, patchoulol, a terpenoid, was produced at a high level (19.24 mg/L) by introducing all the enzymes of the MVA pathway into the mitochondria and a fusion protein of the downstream enzymes into the cytosol (Tao et al. ##REF##35254611##2022##). In another example, amorphadiene was produced at 400 mg/L by introducing several enzymes of the MVA pathway into the mitochondria and two downstream enzymes into the cytosol (Yuan and Ching ##REF##27471067##2016##).</p>","<p id=\"Par8\">Although introducing the MVA pathway into the mitochondria is attractive for producing terpenoids in yeast, there may be a limit because the mitochondria comprise a small percentage of the entire cell (Uchida et al. ##REF##21360734##2011##). We hypothesized that terpenoid production will increase by introducing the MVA pathway into the mitochondria when mitochondrial volume increases. In the present study, we constructed yeast strains to analyze squalene production or β-carotene production and investigated the relationship between mitochondria-mediated terpenoid (squalene or β-carotene) production and mitochondrial volume.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">Terpenoids are a large group of natural compounds constructed from isoprene units (C5). Many different terpenoids are used in the food, beverage, cosmetic, and pharmaceutical industries (Tetali ##REF##30467631##2019##). The current industrial supply of terpenoids relies mainly on chemical synthesis or extraction from plants. The chemical synthesis of terpenoids harboring complex structures has been actively researched (Phil ##REF##29635919##2018##). However, the multi-stereo center in the complex terpenoids requires diastereoselective synthesis, resulting in multi-step total synthesis with high costs and low yield (Santosh et al. ##UREF##2##2020##; Le et al. ##REF##36296479##2022##). Therefore, the chemical synthesis of terpenoids harboring complex structures is expensive and requires fine-controlled and complex processes. Plant extraction has been also used to supply terpenoids; however, it is limited by the presence of various compounds harboring similar physicochemical properties in plants and the difficulty of separating these compounds (Rodger et al. ##REF##21855882##2011##). In addition, natural plant supplies are limited owing to their finite amount (Nur and Henrik ##REF##29187859##2017##). Therefore, the engineering of microorganisms to produce terpenoids has emerged as a promising approach.</p>\",\n \"<p id=\\\"Par6\\\">Research on terpenoid production using microorganisms has rapidly increased in the twenty-first century. <italic>Saccharomyces cerevisiae</italic> and <italic>Escherichia coli</italic> have been used to produce plant terpenoids by heterologous expression of their genes because they have advantages for production, such as the presence of a native terpenoid synthetic pathway and facile genetic manipulation (Navale et al. ##REF##33394155##2021##; Sun et al. ##REF##33616900##2020##). <italic>Saccharomyces cerevisiae</italic> offers several advantages for terpenoid production, including a high sugar catabolic rate, a relatively fast growth rate, and its GRAS status (Claudia et al. ##REF##28623722##2017##). In addition, <italic>saccharomyces cerevisiae</italic> has an inherent terpenoid production pathway, the mevalonate (MVA) pathway, in which isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP), essential intermediates of sterols and ubiquinones, are synthesized from acetyl-CoA (Guo et al. ##REF##32786348##2020##). Metabolic reactions in the MVA pathway proceed in the cytosol, which, in turn, are used for terpenoid production. Recently, terpenoid production using organelles has attracted attention owing to their ability to produce acetyl-CoA.</p>\",\n \"<p id=\\\"Par7\\\">Peroxisomes are considered potential acetyl-CoA pools because β-oxidation of fatty acids proceeds in them (Hammer and Avalos ##REF##28853733##2017##). In fact, a terpenoid, β-amyrin, was produced at high level (57.8 mg/g dry cell weight (DCW)) by introducing two enzymes of the MVA pathway into peroxisomes and four downstream enzymes into the cytosol or peroxisomes (Du et al. ##REF##34955018##2022##). The concentration of acetyl-CoA in the mitochondria is as much as 20–30 times higher than that in the cytosol because of the high enzymatic activity of the mitochondrial pyruvate dehydrogenase complex (Weinert et al. ##UREF##3##2014##). Thus, mitochondria are considered suitable organelles for the introduction of the MVA pathway for terpenoid production (Duran et al. ##REF##32592388##2020##). For example, patchoulol, a terpenoid, was produced at a high level (19.24 mg/L) by introducing all the enzymes of the MVA pathway into the mitochondria and a fusion protein of the downstream enzymes into the cytosol (Tao et al. ##REF##35254611##2022##). In another example, amorphadiene was produced at 400 mg/L by introducing several enzymes of the MVA pathway into the mitochondria and two downstream enzymes into the cytosol (Yuan and Ching ##REF##27471067##2016##).</p>\",\n \"<p id=\\\"Par8\\\">Although introducing the MVA pathway into the mitochondria is attractive for producing terpenoids in yeast, there may be a limit because the mitochondria comprise a small percentage of the entire cell (Uchida et al. ##REF##21360734##2011##). We hypothesized that terpenoid production will increase by introducing the MVA pathway into the mitochondria when mitochondrial volume increases. In the present study, we constructed yeast strains to analyze squalene production or β-carotene production and investigated the relationship between mitochondria-mediated terpenoid (squalene or β-carotene) production and mitochondrial volume.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<title>Genes for yeast strain construction</title>","<p id=\"Par9\">The genes used for the construction of the yeast strains were prepared as described in the Supplementary Information. The plasmids, primers, and DNA fragments used are listed in Tables ##SUPPL##0##S1##, ##SUPPL##0##S2##, and ##SUPPL##0##S3##, respectively. <italic>Escherichia coli</italic> NovaBlue (Merck Millipore, Darmstadt, Germany) was used for plasmid preparation using the Orthodox method.</p>","<title>Yeast strains</title>","<p id=\"Par10\"><italic>Saccharomyces cerevisiae</italic> BY4741 was used as the host strain. The strains used in this study are listed in Table ##TAB##0##1##. The methods used for strain construction are described in the Supplementary Information.</p>","<title>Mitochondrial volume analysis</title>","<p id=\"Par11\">Mutants with defective mitochondrial morphology were selected from the Yeast Knockout Collection (Open Biosystems, Huntsville, AL, USA) database. The total mitochondrial volume per cell was quantified using a previously described method (Viana et al. ##REF##25640425##2015##). The mutant strains were transformed with pGK426-MLS-GFP and cultured in 5 mL of synthetic complete media without uracil (2% glucose, 1.46 g/L Yeast Synthetic Drop-out Medium Supplements; Merck, Darmstadt, Germany) overnight at 30 °C with shaking at 200 rpm. The strains were transferred to YPD media (2% glucose, 2% peptone, and 1% yeast extract) at 0.1–0.4 OD<sub>600</sub> and cultured further for 4 h under the same conditions. When the OD<sub>600</sub> reached 0.5–1.0, the cultures were diluted 8–16-fold with YPD, transferred to a 96-well glass plate coated with concanavalin-A, and incubated at 30 °C for 20 min. The supernatant was removed and fresh YPD was added to each well. MLS-GFP signals in mutants were detected using an Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan). Z-stack images of MLS-GFP were constructed and mitochondrial volumes were calculated using ImageJ and MitoGraph software. The mitochondrial volume in one cell was calculated as the average of at least 10 cells per strain.</p>","<title>Analyses for IPP/DMAPP, squalene, and β-carotene contents</title>","<p id=\"Par12\">For squalene production, strains, pre-incubated in 5 mL of YPD at 30 °C overnight, were inoculated into 30 mL of YPD at 0.01 OD<sub>600</sub> and incubated at 30 °C for 24 h in 250-mL baffled flask with shaking at 200 rpm. For β-carotene production, YPD supplemented with 200 μg/mL hygromycin (Nacalai Tesque, Japan) was used, and sampling was performed 48 h after inoculation. DCW was determined using the equation, DCW/OD<sub>600</sub> = 0.561 derived beforehand.</p>","<p id=\"Par13\">Intracellular IPP/DMAPP were extracted as previously described (Luo et al. ##REF##32758537##2020##). Briefly, 2 mL of each culture was centrifuged at 5,000 × <italic>g</italic> for 2 min, and the supernatant was removed. Cells were lysed in an acetonitrile/methanol/water (40:40:20) solution. The solution was chilled at –20 °C for 20 min, followed by centrifugation at 16,000 × <italic>g</italic> for 10 min. The supernatant was collected, dried, and resuspended in 50 μL of water. IPP/DMAPP were analyzed using liquid chromatography-tandem mass spectrometry (LC–MS/MS) (Agilent Nexera 1260 series high-performance liquid chromatography system and 6460 Triple Quad LC/MS; Agilent Technologies, Santa Clara, CA, USA). Separation, detection, and quantification of IPP /DMAPP were performed as previously described (Kato et al. ##REF##22280965##2012##); however, IPP and DMAPP could not be separated from each other. Thus, in this study, the contents are presented as the sum of IPP and DMAPP (referred to as IPP/DMAPP).</p>","<p id=\"Par14\">Intracellular squalene was extracted as previously described (Zhu et al. ##REF##34710614##2021##). Briefly, 600 μL of each culture was centrifuged at 5,000 × <italic>g</italic> for 2 min and the supernatant was removed. 600 μL of ethyl acetate and approximately 200 μL of grass beads (diameter of 0.5 mm) were added to the cells, which were then lysed by shaking at 1,500 rpm for 10 min using a grinder (shake Master NEO BMS, Japan) with a pre-cooled sample holder at 4 °C. The cell lysate was centrifuged at 21,880 × <italic>g</italic> for 10 min and the upper organic solvent was collected. A GCMS-QP2010 (Shimadzu, Japan) equipped with a DB-5MS column (15 m × 0.25 mm, 0.25 μm film thickness; Agilent Technologies) was used to analyze squalene. The oven temperature was maintained at 100 °C for 2 min, gradually increased to 250 °C at a rate of 20 °C/min, maintained for 2 min, then increased to 325 °C at a rate of 50 °C/min and maintained for 2 min.</p>","<p id=\"Par15\">Intracellular β-carotene was extracted as previously described (Ma et al. ##REF##35102143##2022##). Briefly, 100 μL of each culture was centrifuged at 5,000 × <italic>g</italic> for 2 min and the supernatant was removed. A total of 900 μL of dimethyl sulfoxide was added to the cells, and β-carotene was extracted after incubation at 50 °C for 1 h. After the incubation, 450 μL of methanol was added and the mixtures were centrifuged at 21,880 × <italic>g</italic> for 5 min. The supernatants were collected. A high-performance liquid chromatography (HPLC) (Shimadzu, Japan) equipped with a BDS Hypersil C18 column (4.6 × 150 mm<sup>2</sup>, 5 μm particle size; Thermo Fisher Scientific, Waltham, MA, USA) and a SPD-20A UV–VIS detector (Shimadzu) was used to analyze β-carotene. The oven temperature was maintained at 25 °C. Methanol and acetonitrile were used as mobile phases at a flow rate of 0.28 mL/min and 0.52 mL/min. β-carotene was detected by absorbance at 450 nm.</p>","<p id=\"Par16\">The representative chromatograms of IPP/DMAPP, squalene, and β-carotene were shown in Figs, ##SUPPL##0##S1##, ##SUPPL##0##S2##, and ##SUPPL##0##S3##.</p>","<title>Measurement of intracellular oxidation levels</title>","<p id=\"Par17\">The reactive oxygen species (ROS) level was analyzed using ROS Assay Kit -Photo-oxidation Resistant DCFH-DA- (Dojindo, kumamoto, Japan). Briefly, strains, pre-incubated in 5 mL of YPD at 30 °C overnight, were inoculated into 5 mL of fresh YPD at 1.0 OD<sub>600</sub> and incubated at 30 °C for 4 h in test tubes with shaking at 200 rpm. Then, 1 mL of each culture was centrifuged at 5,000 × <italic>g</italic> for 2 min, and the supernatant was removed. The cells were treated according to the manufacturer’s protocol and the fluorescence was measured with λ<sub>ex</sub> = 500 nm and λ<sub>em</sub> = 540 nm using a plate-reader (INFINITE M NANO + ; Tercan, Männedorf, Switzerland).</p>","<title>Spot assay using dithiothreitol (DTT)</title>","<p id=\"Par18\">The effect of DTT addition to media on growth was analyzed as previously described (Bode et al. ##REF##23198688##2013##). Briefly, strains, pre-incubated in 5 mL of YPD at 30 °C overnight, were inoculated at 1.0 OD<sub>600</sub> into 5 mL of fresh YPD and incubated at 30 °C for 4 h in a test tube with shaking at 200 rpm. OD<sub>600</sub> was adjusted to 1, and tenfold serial dilutions were spotted at 3μL onto YPD, YPD + DTT 0.5 mM, or YPD + DTT 5 mM plates. The plates were incubated at 30 °C for 24 h.</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<title>Genes for yeast strain construction</title>\",\n \"<p id=\\\"Par9\\\">The genes used for the construction of the yeast strains were prepared as described in the Supplementary Information. The plasmids, primers, and DNA fragments used are listed in Tables ##SUPPL##0##S1##, ##SUPPL##0##S2##, and ##SUPPL##0##S3##, respectively. <italic>Escherichia coli</italic> NovaBlue (Merck Millipore, Darmstadt, Germany) was used for plasmid preparation using the Orthodox method.</p>\",\n \"<title>Yeast strains</title>\",\n \"<p id=\\\"Par10\\\"><italic>Saccharomyces cerevisiae</italic> BY4741 was used as the host strain. The strains used in this study are listed in Table ##TAB##0##1##. The methods used for strain construction are described in the Supplementary Information.</p>\",\n \"<title>Mitochondrial volume analysis</title>\",\n \"<p id=\\\"Par11\\\">Mutants with defective mitochondrial morphology were selected from the Yeast Knockout Collection (Open Biosystems, Huntsville, AL, USA) database. The total mitochondrial volume per cell was quantified using a previously described method (Viana et al. ##REF##25640425##2015##). The mutant strains were transformed with pGK426-MLS-GFP and cultured in 5 mL of synthetic complete media without uracil (2% glucose, 1.46 g/L Yeast Synthetic Drop-out Medium Supplements; Merck, Darmstadt, Germany) overnight at 30 °C with shaking at 200 rpm. The strains were transferred to YPD media (2% glucose, 2% peptone, and 1% yeast extract) at 0.1–0.4 OD<sub>600</sub> and cultured further for 4 h under the same conditions. When the OD<sub>600</sub> reached 0.5–1.0, the cultures were diluted 8–16-fold with YPD, transferred to a 96-well glass plate coated with concanavalin-A, and incubated at 30 °C for 20 min. The supernatant was removed and fresh YPD was added to each well. MLS-GFP signals in mutants were detected using an Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan). Z-stack images of MLS-GFP were constructed and mitochondrial volumes were calculated using ImageJ and MitoGraph software. The mitochondrial volume in one cell was calculated as the average of at least 10 cells per strain.</p>\",\n \"<title>Analyses for IPP/DMAPP, squalene, and β-carotene contents</title>\",\n \"<p id=\\\"Par12\\\">For squalene production, strains, pre-incubated in 5 mL of YPD at 30 °C overnight, were inoculated into 30 mL of YPD at 0.01 OD<sub>600</sub> and incubated at 30 °C for 24 h in 250-mL baffled flask with shaking at 200 rpm. For β-carotene production, YPD supplemented with 200 μg/mL hygromycin (Nacalai Tesque, Japan) was used, and sampling was performed 48 h after inoculation. DCW was determined using the equation, DCW/OD<sub>600</sub> = 0.561 derived beforehand.</p>\",\n \"<p id=\\\"Par13\\\">Intracellular IPP/DMAPP were extracted as previously described (Luo et al. ##REF##32758537##2020##). Briefly, 2 mL of each culture was centrifuged at 5,000 × <italic>g</italic> for 2 min, and the supernatant was removed. Cells were lysed in an acetonitrile/methanol/water (40:40:20) solution. The solution was chilled at –20 °C for 20 min, followed by centrifugation at 16,000 × <italic>g</italic> for 10 min. The supernatant was collected, dried, and resuspended in 50 μL of water. IPP/DMAPP were analyzed using liquid chromatography-tandem mass spectrometry (LC–MS/MS) (Agilent Nexera 1260 series high-performance liquid chromatography system and 6460 Triple Quad LC/MS; Agilent Technologies, Santa Clara, CA, USA). Separation, detection, and quantification of IPP /DMAPP were performed as previously described (Kato et al. ##REF##22280965##2012##); however, IPP and DMAPP could not be separated from each other. Thus, in this study, the contents are presented as the sum of IPP and DMAPP (referred to as IPP/DMAPP).</p>\",\n \"<p id=\\\"Par14\\\">Intracellular squalene was extracted as previously described (Zhu et al. ##REF##34710614##2021##). Briefly, 600 μL of each culture was centrifuged at 5,000 × <italic>g</italic> for 2 min and the supernatant was removed. 600 μL of ethyl acetate and approximately 200 μL of grass beads (diameter of 0.5 mm) were added to the cells, which were then lysed by shaking at 1,500 rpm for 10 min using a grinder (shake Master NEO BMS, Japan) with a pre-cooled sample holder at 4 °C. The cell lysate was centrifuged at 21,880 × <italic>g</italic> for 10 min and the upper organic solvent was collected. A GCMS-QP2010 (Shimadzu, Japan) equipped with a DB-5MS column (15 m × 0.25 mm, 0.25 μm film thickness; Agilent Technologies) was used to analyze squalene. The oven temperature was maintained at 100 °C for 2 min, gradually increased to 250 °C at a rate of 20 °C/min, maintained for 2 min, then increased to 325 °C at a rate of 50 °C/min and maintained for 2 min.</p>\",\n \"<p id=\\\"Par15\\\">Intracellular β-carotene was extracted as previously described (Ma et al. ##REF##35102143##2022##). Briefly, 100 μL of each culture was centrifuged at 5,000 × <italic>g</italic> for 2 min and the supernatant was removed. A total of 900 μL of dimethyl sulfoxide was added to the cells, and β-carotene was extracted after incubation at 50 °C for 1 h. After the incubation, 450 μL of methanol was added and the mixtures were centrifuged at 21,880 × <italic>g</italic> for 5 min. The supernatants were collected. A high-performance liquid chromatography (HPLC) (Shimadzu, Japan) equipped with a BDS Hypersil C18 column (4.6 × 150 mm<sup>2</sup>, 5 μm particle size; Thermo Fisher Scientific, Waltham, MA, USA) and a SPD-20A UV–VIS detector (Shimadzu) was used to analyze β-carotene. The oven temperature was maintained at 25 °C. Methanol and acetonitrile were used as mobile phases at a flow rate of 0.28 mL/min and 0.52 mL/min. β-carotene was detected by absorbance at 450 nm.</p>\",\n \"<p id=\\\"Par16\\\">The representative chromatograms of IPP/DMAPP, squalene, and β-carotene were shown in Figs, ##SUPPL##0##S1##, ##SUPPL##0##S2##, and ##SUPPL##0##S3##.</p>\",\n \"<title>Measurement of intracellular oxidation levels</title>\",\n \"<p id=\\\"Par17\\\">The reactive oxygen species (ROS) level was analyzed using ROS Assay Kit -Photo-oxidation Resistant DCFH-DA- (Dojindo, kumamoto, Japan). Briefly, strains, pre-incubated in 5 mL of YPD at 30 °C overnight, were inoculated into 5 mL of fresh YPD at 1.0 OD<sub>600</sub> and incubated at 30 °C for 4 h in test tubes with shaking at 200 rpm. Then, 1 mL of each culture was centrifuged at 5,000 × <italic>g</italic> for 2 min, and the supernatant was removed. The cells were treated according to the manufacturer’s protocol and the fluorescence was measured with λ<sub>ex</sub> = 500 nm and λ<sub>em</sub> = 540 nm using a plate-reader (INFINITE M NANO + ; Tercan, Männedorf, Switzerland).</p>\",\n \"<title>Spot assay using dithiothreitol (DTT)</title>\",\n \"<p id=\\\"Par18\\\">The effect of DTT addition to media on growth was analyzed as previously described (Bode et al. ##REF##23198688##2013##). Briefly, strains, pre-incubated in 5 mL of YPD at 30 °C overnight, were inoculated at 1.0 OD<sub>600</sub> into 5 mL of fresh YPD and incubated at 30 °C for 4 h in a test tube with shaking at 200 rpm. OD<sub>600</sub> was adjusted to 1, and tenfold serial dilutions were spotted at 3μL onto YPD, YPD + DTT 0.5 mM, or YPD + DTT 5 mM plates. The plates were incubated at 30 °C for 24 h.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>The strain expressing the MVA pathway in mitochondria</title>","<p id=\"Par19\">Proteins can be delivered to the mitochondria by fusion with a mitochondrial localization signal (MLS); however, their enzymatic activities are sometimes interrupted in the mitochondria if the MLS remains fused to them (Duran et al. ##REF##32592388##2020##). To introduce the entire MVA pathway into the mitochondria and activate it, we constructed a BY4741 background strain, SSY1, expressing all the enzymes of the MVA pathway fused with the MLS of CoxIV (Fig. ##FIG##0##1##a), because it is cleaved away in mitochondria. The MLS of CoxIV consists of 26 amino acids; the first 25 amino acids and last glutamine are required for translocation and cleavage, respectively. To ascertain the action of MLS, we constructed a strain, expressing a GFP fused with MLS, and examined the localization of GFP as compared with MitoTracker. As shown in Fig. ##FIG##0##1##b, the signal of the MLS-fused GFP coincided with that of MitoTracker, confirming that the MLS of CoxIV worked effectively in translocation to the mitochondria. Thus, we hypothesized that all enzymes of the MVA pathway fused with MLS were also translocated to the mitochondria. Next, we examined whether the MVA pathway introduced into the mitochondria functioned correctly by measuring the amount of IPP/DMAPP in SSY1 compared to that in BY4741. The IPP/DMAPP content in SSY1 was 164 nmol/g DCW, which was 15-fold higher than that in BY4741, 11 nmol/g DCW (Fig. ##FIG##0##1##c). This result indicates that the MVA pathway introduced into the mitochondria functioned and was beneficial for terpenoid production, as previously reported (Lv et al. ##UREF##0##2016##).</p>","<title>The total mitochondrial volumes in mutants with defective mitochondrial morphology</title>","<p id=\"Par20\">Mitochondrial volume has been reported to be decreased in a deletion mutant of <italic>MGM1</italic>, a mitochondrial morphology-related gene (Bernhardt et al. ##REF##25601284##2015##). We hypothesized that mutants with defective mitochondrial morphology could be used to examine the effect of total mitochondrial volume on terpenoid production in yeast expressing the MVA pathway in their mitochondria. We selected 13 genes, <italic>FZO1</italic>, <italic>MGM1</italic>, <italic>UGO1</italic>, <italic>DNM1</italic>, <italic>FIS1</italic>, <italic>MDV1</italic>, <italic>CAF4</italic>, <italic>MMM1</italic>, <italic>MDM10</italic>, <italic>MDM12</italic>, <italic>MDM31</italic>, <italic>MDM32</italic>, and <italic>MDM33</italic> (Ohsumi and Shimoda ##UREF##1##2007##). GFP fused with the MLS of CoxIV was expressed in BY4741 and the deletion mutants, and mitochondrial volumes were calculated from the fluorescent signals of GFP using the confocal microscopic Z-stack method (Fig. ##FIG##1##2##). As expected, we found differences in mitochondrial volume among them. The only strain with a higher volume than that in BY4741 was Δ<italic>mdm32</italic> (2.2 m<sup>3</sup>/cell), which was 1.8-fold higher than that in BY4741 (1.2 μm<sup>3</sup>/cell). The strains with volumes lower than those in BY4741 were Δ<italic>mdm10</italic>, Δ<italic>mdm12</italic>, Δ<italic>fzo1</italic>, Δ<italic>mmm1,</italic> Δ<italic>mdm31,</italic> Δ<italic>mdm33,</italic> Δ<italic>mgm1</italic>, and Δ<italic>ugo1</italic>. The strain with the lowest volume was Δ<italic>ugo1</italic> (0.4 μm<sup>3</sup>/cell), which was threefold lower than that in BY4741.</p>","<title>The effect of mitochondrial volume on mitochondria-mediated terpenoid production</title>","<p id=\"Par21\">We hypothesized that an increase in mitochondrial volume may augment the terpenoid production in yeast expressing the MVA pathway in their mitochondria. To examine the effect of mitochondrial volume on terpenoid production, we used mutants with defective mitochondrial morphology, Δ<italic>mdm32</italic>, Δ<italic>fzo1</italic>, Δ<italic>mgm1</italic>, and Δ<italic>ugo1</italic>. Four mutants were constructed from SSY1, and the relationship between mitochondrial volume and IPP/DMAPP or squalene content was examined. In SSY2 (Δ<italic>mdm32</italic>) harboring a large mitochondrial volume, both the contents of IPP/DMAPP (25 nmol/g DCW) and squalene (707 nmol/ g DCW) increased 1.3- and 2.8-fold higher than that in SSY1 (19 nmol/g DCW for IPP/DMAPP, 256 nmol/ g DCW for squalene) (Fig. ##FIG##2##3##a and b). In contrast, in SSY3 (Δ<italic>fzo1</italic>), SSY4 (Δ<italic>mgm1</italic>), and SSY5 (Δ<italic>ugo1</italic>) harboring a low of mitochondrial volume, the contents of IPP/DMAPP and squalene decreased compared with that in SSY1, with the exception of squalene in SSY3. The contents of IPP/DMAPP in SSY3, 4, and 5 were 4.65, 1.29, and 1.32 nmol/g DCW, which were 4.2-, 15.1-, and 14.7-fold less than those in SSY1. The concentrations of squalene in SSY3, 4, and 5 were 233, 14, and 25 nmol/ g DCW, which were 1.1-, 18.2-, and 10.1-fold less than those in SSY1. Furthermore, we found that the contents of both IPP/DMAPP and squalene were clearly correlated with the mitochondrial volume (Fig. ##FIG##2##3##c and d).</p>","<p id=\"Par22\">In addition, we investigated the effect of the mitochondrial volume on β-carotene production to evaluate the effectiveness of our approach on other terpenoid production. In SSY7 (Δ<italic>mdm32</italic>) harboring a large mitochondrial volume, the contents of IPP/DMAPP (15 nmol/g DCW) and β-carotene (1609 nmol/ g DCW) were 1.2- and 1.4-fold higher than those in SSY6 (12.4 nmol/g DCW for IPP/DMAPP, 1132 nmol/ g DCW for β-carotene) (Fig. ##FIG##3##4##a and b). In contrast, the contents of IPP/DMAPP in SSY8 (Δ<italic>fzo1</italic>), 9 (Δ<italic>mgm1</italic>), and 10 (Δ<italic>ugo1</italic>) were 3.0-, 2.7-, and 12.4-fold lower than those in SSY6 at 4.1, 4.6, and 1.0 nmol/g DCW, respectively. The concentrations of β-carotene in SSY8, 9, and 10 were 1030, 491, and 678 nmol/ g DCW, 1.1-, 2.3-, and 1.7-fold lower than those in SSY6, respectively. Furthermore, the contents of both IPP/DMAPP and β-carotene were correlated with the mitochondrial volume (Fig. ##FIG##3##4##c and d). These data indicate that the increase in mitochondrial volume augmented terpenoid production in yeast expressing the MVA pathway in the mitochondria.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>The strain expressing the MVA pathway in mitochondria</title>\",\n \"<p id=\\\"Par19\\\">Proteins can be delivered to the mitochondria by fusion with a mitochondrial localization signal (MLS); however, their enzymatic activities are sometimes interrupted in the mitochondria if the MLS remains fused to them (Duran et al. ##REF##32592388##2020##). To introduce the entire MVA pathway into the mitochondria and activate it, we constructed a BY4741 background strain, SSY1, expressing all the enzymes of the MVA pathway fused with the MLS of CoxIV (Fig. ##FIG##0##1##a), because it is cleaved away in mitochondria. The MLS of CoxIV consists of 26 amino acids; the first 25 amino acids and last glutamine are required for translocation and cleavage, respectively. To ascertain the action of MLS, we constructed a strain, expressing a GFP fused with MLS, and examined the localization of GFP as compared with MitoTracker. As shown in Fig. ##FIG##0##1##b, the signal of the MLS-fused GFP coincided with that of MitoTracker, confirming that the MLS of CoxIV worked effectively in translocation to the mitochondria. Thus, we hypothesized that all enzymes of the MVA pathway fused with MLS were also translocated to the mitochondria. Next, we examined whether the MVA pathway introduced into the mitochondria functioned correctly by measuring the amount of IPP/DMAPP in SSY1 compared to that in BY4741. The IPP/DMAPP content in SSY1 was 164 nmol/g DCW, which was 15-fold higher than that in BY4741, 11 nmol/g DCW (Fig. ##FIG##0##1##c). This result indicates that the MVA pathway introduced into the mitochondria functioned and was beneficial for terpenoid production, as previously reported (Lv et al. ##UREF##0##2016##).</p>\",\n \"<title>The total mitochondrial volumes in mutants with defective mitochondrial morphology</title>\",\n \"<p id=\\\"Par20\\\">Mitochondrial volume has been reported to be decreased in a deletion mutant of <italic>MGM1</italic>, a mitochondrial morphology-related gene (Bernhardt et al. ##REF##25601284##2015##). We hypothesized that mutants with defective mitochondrial morphology could be used to examine the effect of total mitochondrial volume on terpenoid production in yeast expressing the MVA pathway in their mitochondria. We selected 13 genes, <italic>FZO1</italic>, <italic>MGM1</italic>, <italic>UGO1</italic>, <italic>DNM1</italic>, <italic>FIS1</italic>, <italic>MDV1</italic>, <italic>CAF4</italic>, <italic>MMM1</italic>, <italic>MDM10</italic>, <italic>MDM12</italic>, <italic>MDM31</italic>, <italic>MDM32</italic>, and <italic>MDM33</italic> (Ohsumi and Shimoda ##UREF##1##2007##). GFP fused with the MLS of CoxIV was expressed in BY4741 and the deletion mutants, and mitochondrial volumes were calculated from the fluorescent signals of GFP using the confocal microscopic Z-stack method (Fig. ##FIG##1##2##). As expected, we found differences in mitochondrial volume among them. The only strain with a higher volume than that in BY4741 was Δ<italic>mdm32</italic> (2.2 m<sup>3</sup>/cell), which was 1.8-fold higher than that in BY4741 (1.2 μm<sup>3</sup>/cell). The strains with volumes lower than those in BY4741 were Δ<italic>mdm10</italic>, Δ<italic>mdm12</italic>, Δ<italic>fzo1</italic>, Δ<italic>mmm1,</italic> Δ<italic>mdm31,</italic> Δ<italic>mdm33,</italic> Δ<italic>mgm1</italic>, and Δ<italic>ugo1</italic>. The strain with the lowest volume was Δ<italic>ugo1</italic> (0.4 μm<sup>3</sup>/cell), which was threefold lower than that in BY4741.</p>\",\n \"<title>The effect of mitochondrial volume on mitochondria-mediated terpenoid production</title>\",\n \"<p id=\\\"Par21\\\">We hypothesized that an increase in mitochondrial volume may augment the terpenoid production in yeast expressing the MVA pathway in their mitochondria. To examine the effect of mitochondrial volume on terpenoid production, we used mutants with defective mitochondrial morphology, Δ<italic>mdm32</italic>, Δ<italic>fzo1</italic>, Δ<italic>mgm1</italic>, and Δ<italic>ugo1</italic>. Four mutants were constructed from SSY1, and the relationship between mitochondrial volume and IPP/DMAPP or squalene content was examined. In SSY2 (Δ<italic>mdm32</italic>) harboring a large mitochondrial volume, both the contents of IPP/DMAPP (25 nmol/g DCW) and squalene (707 nmol/ g DCW) increased 1.3- and 2.8-fold higher than that in SSY1 (19 nmol/g DCW for IPP/DMAPP, 256 nmol/ g DCW for squalene) (Fig. ##FIG##2##3##a and b). In contrast, in SSY3 (Δ<italic>fzo1</italic>), SSY4 (Δ<italic>mgm1</italic>), and SSY5 (Δ<italic>ugo1</italic>) harboring a low of mitochondrial volume, the contents of IPP/DMAPP and squalene decreased compared with that in SSY1, with the exception of squalene in SSY3. The contents of IPP/DMAPP in SSY3, 4, and 5 were 4.65, 1.29, and 1.32 nmol/g DCW, which were 4.2-, 15.1-, and 14.7-fold less than those in SSY1. The concentrations of squalene in SSY3, 4, and 5 were 233, 14, and 25 nmol/ g DCW, which were 1.1-, 18.2-, and 10.1-fold less than those in SSY1. Furthermore, we found that the contents of both IPP/DMAPP and squalene were clearly correlated with the mitochondrial volume (Fig. ##FIG##2##3##c and d).</p>\",\n \"<p id=\\\"Par22\\\">In addition, we investigated the effect of the mitochondrial volume on β-carotene production to evaluate the effectiveness of our approach on other terpenoid production. In SSY7 (Δ<italic>mdm32</italic>) harboring a large mitochondrial volume, the contents of IPP/DMAPP (15 nmol/g DCW) and β-carotene (1609 nmol/ g DCW) were 1.2- and 1.4-fold higher than those in SSY6 (12.4 nmol/g DCW for IPP/DMAPP, 1132 nmol/ g DCW for β-carotene) (Fig. ##FIG##3##4##a and b). In contrast, the contents of IPP/DMAPP in SSY8 (Δ<italic>fzo1</italic>), 9 (Δ<italic>mgm1</italic>), and 10 (Δ<italic>ugo1</italic>) were 3.0-, 2.7-, and 12.4-fold lower than those in SSY6 at 4.1, 4.6, and 1.0 nmol/g DCW, respectively. The concentrations of β-carotene in SSY8, 9, and 10 were 1030, 491, and 678 nmol/ g DCW, 1.1-, 2.3-, and 1.7-fold lower than those in SSY6, respectively. Furthermore, the contents of both IPP/DMAPP and β-carotene were correlated with the mitochondrial volume (Fig. ##FIG##3##4##c and d). These data indicate that the increase in mitochondrial volume augmented terpenoid production in yeast expressing the MVA pathway in the mitochondria.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par23\">In this study, we demonstrated the beneficial effect of the MVA pathway introduced into the mitochondria for terpenoid production by measuring IPP/DMAPP, an essential precursor of terpenoids. Furthermore, using mutants with defective mitochondrial morphology, we found that the contents of IPP/DMAPP and terpenoids (squalene or β-carotene) were positively correlated with the mitochondrial volume and showed differences in the mitochondrial volume. These results not only support the advantages of introducing the MVA pathway into mitochondria for terpenoid production but also suggest that increasing the mitochondrial volume might enhance the production.</p>","<p id=\"Par24\">Both IPP/DMAPP and terpenoids contents were positively correlated with the mitochondrial volume. However, notably, there was a difference in the degree of increase between IPP/DMAPP and terpenoids in SSY2 (Δ<italic>mdm32</italic>) and SSY7 (Δ<italic>mdm32</italic>) (1.3-fold for IPP/DMAPP and 2.8-fold for squalene in squalene production, 1.2-fold for IPP/DMAPP and 1.4-fold for in β-carotene production). IDI1 was not introduced into the mitochondria of any of the strains used in this study. Thus, DMAPP was not synthesized in the mitochondria. The growth defect compared to the wild-type strain is believed to be caused by the accumulation of the pyrophosphate compounds, mevalonate-5-PP, IPP, and DMAPP in the mitochondria (Zhu et al. ##REF##34710614##2021##), which was also observed in this study (Fig. ##FIG##0##1## C). Pyrophosphate compounds can react with adenosine monophosphate-amino acids through aminoacyl tRNA synthetases and convert them to toxic ATP analogs, which inhibit mitochondrial adenine nucleotide translocase and F1-ATPase (Mönkkönen et al. ##REF##16402039##2006##, Mookerjee-Basu et al. ##REF##20483757##2010##). However, we first speculated that the defective mitochondrial morphology or the introduction of MVA pathway enzymes into the mitochondria might cause mitochondrial dysfunction and induce oxidative stress. Therefore, we measured the ROS levels and growth of BY4741, <italic>mdm32</italic> deletion strain, SSY1, and SSY2 (Δ<italic>mdm32</italic>) and found that the ROS levels in SSY2 (Δ<italic>mdm32</italic>) tended to be slightly higher compared to those in BY4741 (Fig. ##SUPPL##0##S4##). Moreover, the addition of DTT as a reducing agent to media improved the growth of the strain, which was exposed to oxidative stress (Bode et al. ##REF##23198688##2013##). Thus, a DTT assay was performed using the SSY2 (Δ<italic>mdm32</italic>) strain, which revealed that DDT addition to YPD media did not improve the growth of SSY2 (Δ<italic>mdm32</italic>) (Fig. ##SUPPL##0##S5##). Therefore, the slight increase in ROS levels in SSY2 (Δ<italic>mdm32</italic>) was not the cause of the growth defect of SSY2 (Δ<italic>mdm32</italic>), supporting the theory that growth defects are attributed to the accumulation of the pyrophosphate compounds mentioned in the previous study (Zhu et al. ##REF##34710614##2021##).</p>","<p id=\"Par25\">Thus, it is plausible that IPP/DMAPP mainly contains IPP accumulated in the mitochondria. After production in the mitochondrial matrix, IPP is exported to the cytosol, where squalene production proceeds (Zhu et al. ##REF##34710614##2021##). During export, IPP must pass through the inner and outer mitochondrial membranes. Several transporters selectively pass through small molecules in the inner membranes (Duran et al ##REF##32592388##2020##). Porin is present in the outer membrane and allows small molecules (< 5 kDa) to pass freely between the cytosol and intermembrane space. Thus, passage through the inner membrane is thought to be critical for IPP export from the mitochondria to the cytosol. Mdm32 is a protein present in the mitochondrial inner membrane, and loss of Mdm32 leads to defects in mitochondrial morphology (Okamoto and Shaw ##REF##16285870##2005##). Although the mechanism underlying the IPP export remains to be elucidated, structural changes in the inner mitochondrial membrane may enhance IPP export or diffusion.</p>","<p id=\"Par26\">Regarding the Δ<italic>fzo1</italic> strains (SSY3 (Δ<italic>fzo1</italic>) and SSY8 (Δ<italic>fzo1</italic>)), although both IPP and terpenoid decreased in the Δ<italic>fzo1</italic> strains compared to the normal strains (SSY1 and SSY6), we also observed a difference in the degree of decrease between IPP and squalene content: 76% for IPP and 9% for squalene (Fig. ##FIG##2##3##a and c) and that between IPP and β-carotene content: 68% for IPP and 9% for β-carotene (Fig. ##FIG##3##4##a and c). These differences seem to arise from the balance between IPP synthesis in the mitochondria and IPP export. Due to the low mitochondrial volume, a sufficient amount of enzymes in the MVA pathway could not be transported to the mitochondria; thus, the rate of IPP synthesis decreased. The IPP produced via the mitochondria in the Δ<italic>fzo1</italic> strains seem to be exported smoothly to the cytosol, because of its low synthetic activity, and is subsequently metabolized to squalene or β-carotene. Thus, although the terpenoid level in the Δ<italic>fzo1</italic> strains was slightly lower than that in the normal strains, the difference in terpenoid levels between the normal strains and the Δ<italic>fzo1</italic> strains was not as drastic as that in IPP. In contrast, the IPP/DMAPP levels in the Δ<italic>fzo1</italic> strains decreased drastically compared with those in the normal strains (Figs. ##FIG##2##3##a and ##FIG##3##4## a). The Fzo1 protein resides in the outer mitochondrial membrane, and the loss of <italic>Fzo1</italic> may not affect IPP export or diffusion due to the presence of porins in the outer membrane, suggesting that there may be no difference in the export ability of mitochondria. There may be a limit to export, and the IPP in the normal strains probably accumulates in the mitochondrial matrix.</p>","<p id=\"Par27\">In the present study, SSY1 to 10 showed growth defects compared to the wild-type strain, BY4741. The defects were severe in the Δ<italic>mdm32</italic> strains (SSY2 and SSY7). The deletion of <italic>FZO1</italic>, <italic>MGM1</italic>, <italic>UGO1</italic>, or <italic>MDM32</italic> leads growth defects by dysfunction of mitochondria (Okamoto and Shaw ##REF##16285870##2005##). In the normal and Δ<italic>mdm32</italic> strains, the IPP/DMAPP levels were high (Figs. ##FIG##2##3##a and ##FIG##3##4##a), indicating that pyrophosphate compounds may accumulate in the mitochondria. Therefore, the severe growth defects in the Δ<italic>mdm32</italic> strains is probably due to both mitochondrial dysfunction and pyrophosphate compound accumulation in the mitochondria. As the normal strains had no mitochondrial morphological defects. Thus, the growth defect in the normal strains may only be due to the accumulation of pyrophosphate compounds in the mitochondria. In contrast, the growth defects in Δ<italic>fzo1</italic> strains, Δ<italic>mgm1</italic> strains (SSY4 and SSY9), and Δ<italic>ugo1</italic> strains (SSY5 and SSY10) may be due to mitochondrial dysfunction because their IPP levels, that is, their activities in the MVA pathway introduced in the mitochondria, were considerably low. Further approaches to expand the volume of mitochondria without growth defects by mitochondrial dysfunction and enhance the export of IPP, inhibiting pyrophosphate compound accumulation, would be required to increase terpenoid production using our proposed mitochondria-based strategy.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par23\\\">In this study, we demonstrated the beneficial effect of the MVA pathway introduced into the mitochondria for terpenoid production by measuring IPP/DMAPP, an essential precursor of terpenoids. Furthermore, using mutants with defective mitochondrial morphology, we found that the contents of IPP/DMAPP and terpenoids (squalene or β-carotene) were positively correlated with the mitochondrial volume and showed differences in the mitochondrial volume. These results not only support the advantages of introducing the MVA pathway into mitochondria for terpenoid production but also suggest that increasing the mitochondrial volume might enhance the production.</p>\",\n \"<p id=\\\"Par24\\\">Both IPP/DMAPP and terpenoids contents were positively correlated with the mitochondrial volume. However, notably, there was a difference in the degree of increase between IPP/DMAPP and terpenoids in SSY2 (Δ<italic>mdm32</italic>) and SSY7 (Δ<italic>mdm32</italic>) (1.3-fold for IPP/DMAPP and 2.8-fold for squalene in squalene production, 1.2-fold for IPP/DMAPP and 1.4-fold for in β-carotene production). IDI1 was not introduced into the mitochondria of any of the strains used in this study. Thus, DMAPP was not synthesized in the mitochondria. The growth defect compared to the wild-type strain is believed to be caused by the accumulation of the pyrophosphate compounds, mevalonate-5-PP, IPP, and DMAPP in the mitochondria (Zhu et al. ##REF##34710614##2021##), which was also observed in this study (Fig. ##FIG##0##1## C). Pyrophosphate compounds can react with adenosine monophosphate-amino acids through aminoacyl tRNA synthetases and convert them to toxic ATP analogs, which inhibit mitochondrial adenine nucleotide translocase and F1-ATPase (Mönkkönen et al. ##REF##16402039##2006##, Mookerjee-Basu et al. ##REF##20483757##2010##). However, we first speculated that the defective mitochondrial morphology or the introduction of MVA pathway enzymes into the mitochondria might cause mitochondrial dysfunction and induce oxidative stress. Therefore, we measured the ROS levels and growth of BY4741, <italic>mdm32</italic> deletion strain, SSY1, and SSY2 (Δ<italic>mdm32</italic>) and found that the ROS levels in SSY2 (Δ<italic>mdm32</italic>) tended to be slightly higher compared to those in BY4741 (Fig. ##SUPPL##0##S4##). Moreover, the addition of DTT as a reducing agent to media improved the growth of the strain, which was exposed to oxidative stress (Bode et al. ##REF##23198688##2013##). Thus, a DTT assay was performed using the SSY2 (Δ<italic>mdm32</italic>) strain, which revealed that DDT addition to YPD media did not improve the growth of SSY2 (Δ<italic>mdm32</italic>) (Fig. ##SUPPL##0##S5##). Therefore, the slight increase in ROS levels in SSY2 (Δ<italic>mdm32</italic>) was not the cause of the growth defect of SSY2 (Δ<italic>mdm32</italic>), supporting the theory that growth defects are attributed to the accumulation of the pyrophosphate compounds mentioned in the previous study (Zhu et al. ##REF##34710614##2021##).</p>\",\n \"<p id=\\\"Par25\\\">Thus, it is plausible that IPP/DMAPP mainly contains IPP accumulated in the mitochondria. After production in the mitochondrial matrix, IPP is exported to the cytosol, where squalene production proceeds (Zhu et al. ##REF##34710614##2021##). During export, IPP must pass through the inner and outer mitochondrial membranes. Several transporters selectively pass through small molecules in the inner membranes (Duran et al ##REF##32592388##2020##). Porin is present in the outer membrane and allows small molecules (< 5 kDa) to pass freely between the cytosol and intermembrane space. Thus, passage through the inner membrane is thought to be critical for IPP export from the mitochondria to the cytosol. Mdm32 is a protein present in the mitochondrial inner membrane, and loss of Mdm32 leads to defects in mitochondrial morphology (Okamoto and Shaw ##REF##16285870##2005##). Although the mechanism underlying the IPP export remains to be elucidated, structural changes in the inner mitochondrial membrane may enhance IPP export or diffusion.</p>\",\n \"<p id=\\\"Par26\\\">Regarding the Δ<italic>fzo1</italic> strains (SSY3 (Δ<italic>fzo1</italic>) and SSY8 (Δ<italic>fzo1</italic>)), although both IPP and terpenoid decreased in the Δ<italic>fzo1</italic> strains compared to the normal strains (SSY1 and SSY6), we also observed a difference in the degree of decrease between IPP and squalene content: 76% for IPP and 9% for squalene (Fig. ##FIG##2##3##a and c) and that between IPP and β-carotene content: 68% for IPP and 9% for β-carotene (Fig. ##FIG##3##4##a and c). These differences seem to arise from the balance between IPP synthesis in the mitochondria and IPP export. Due to the low mitochondrial volume, a sufficient amount of enzymes in the MVA pathway could not be transported to the mitochondria; thus, the rate of IPP synthesis decreased. The IPP produced via the mitochondria in the Δ<italic>fzo1</italic> strains seem to be exported smoothly to the cytosol, because of its low synthetic activity, and is subsequently metabolized to squalene or β-carotene. Thus, although the terpenoid level in the Δ<italic>fzo1</italic> strains was slightly lower than that in the normal strains, the difference in terpenoid levels between the normal strains and the Δ<italic>fzo1</italic> strains was not as drastic as that in IPP. In contrast, the IPP/DMAPP levels in the Δ<italic>fzo1</italic> strains decreased drastically compared with those in the normal strains (Figs. ##FIG##2##3##a and ##FIG##3##4## a). The Fzo1 protein resides in the outer mitochondrial membrane, and the loss of <italic>Fzo1</italic> may not affect IPP export or diffusion due to the presence of porins in the outer membrane, suggesting that there may be no difference in the export ability of mitochondria. There may be a limit to export, and the IPP in the normal strains probably accumulates in the mitochondrial matrix.</p>\",\n \"<p id=\\\"Par27\\\">In the present study, SSY1 to 10 showed growth defects compared to the wild-type strain, BY4741. The defects were severe in the Δ<italic>mdm32</italic> strains (SSY2 and SSY7). The deletion of <italic>FZO1</italic>, <italic>MGM1</italic>, <italic>UGO1</italic>, or <italic>MDM32</italic> leads growth defects by dysfunction of mitochondria (Okamoto and Shaw ##REF##16285870##2005##). In the normal and Δ<italic>mdm32</italic> strains, the IPP/DMAPP levels were high (Figs. ##FIG##2##3##a and ##FIG##3##4##a), indicating that pyrophosphate compounds may accumulate in the mitochondria. Therefore, the severe growth defects in the Δ<italic>mdm32</italic> strains is probably due to both mitochondrial dysfunction and pyrophosphate compound accumulation in the mitochondria. As the normal strains had no mitochondrial morphological defects. Thus, the growth defect in the normal strains may only be due to the accumulation of pyrophosphate compounds in the mitochondria. In contrast, the growth defects in Δ<italic>fzo1</italic> strains, Δ<italic>mgm1</italic> strains (SSY4 and SSY9), and Δ<italic>ugo1</italic> strains (SSY5 and SSY10) may be due to mitochondrial dysfunction because their IPP levels, that is, their activities in the MVA pathway introduced in the mitochondria, were considerably low. Further approaches to expand the volume of mitochondria without growth defects by mitochondrial dysfunction and enhance the export of IPP, inhibiting pyrophosphate compound accumulation, would be required to increase terpenoid production using our proposed mitochondria-based strategy.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<title>Abstract</title>","<p id=\"Par1\">Terpenoids are widely used in the food, beverage, cosmetics, and pharmaceutical industries. Microorganisms have been extensively studied for terpenoid production. In yeast, the introduction of the mevalonate (MVA) pathway in organelles in addition to the augmentation of its own MVA pathway have been challenging. Introduction of the MVA pathway into mitochondria is considered a promising approach for terpenoid production because acetyl-CoA, the starting molecule of the MVA pathway, is abundant in mitochondria. However, mitochondria comprise only a small percentage of the entire cell. Therefore, we hypothesized that increasing the total mitochondrial volume per cell would increase terpenoid production. First, we ascertained that the amounts of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the final molecules of the MVA pathway, were 15-fold higher of the strain expressing the MVA pathway in mitochondria than in the wild-type yeast strain. Second, we found that different deletion mutants induced different mitochondrial volumes by measuring the mitochondrial volume in various deletion mutants affecting mitochondrial morphology; for example,Δ<italic>mdm32</italic> increased mitochondrial volume, and Δ<italic>fzo1</italic> decreased it. Finally, the effects of mitochondrial volume on amounts of IPP/DMAPP and terpenoids (squalene or β-carotene) were investigated using mutants harboring large or small mitochondria expressing the MVA pathway in mitochondria. Amounts of IPP/DMAPP and terpenoids (squalene or β-carotene) increased when the mitochondrial volume expanded. Introducing the MVA pathway into mitochondria for terpenoid production in yeast may become more attractive by enlarging the mitochondrial volume.</p>","<title>Key points</title>","<p id=\"Par2\">\n<italic>• IPP/DMAPP content increased in the strain expressing the MVA pathway in mitochondria</italic>\n</p>","<p id=\"Par3\">\n<italic>• IPP/DMAPP and terpenoid contents are positively correlated with mitochondrial volume</italic>\n</p>","<p id=\"Par4\">\n<italic>• Enlarging the mitochondria may improve mitochondria-mediated terpenoid production</italic>\n</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00253-023-12922-5.</p>","<title>Keywords</title>","<p>Open Access funding provided by Kobe University.</p>"],"string":"[\n \"<title>Abstract</title>\",\n \"<p id=\\\"Par1\\\">Terpenoids are widely used in the food, beverage, cosmetics, and pharmaceutical industries. Microorganisms have been extensively studied for terpenoid production. In yeast, the introduction of the mevalonate (MVA) pathway in organelles in addition to the augmentation of its own MVA pathway have been challenging. Introduction of the MVA pathway into mitochondria is considered a promising approach for terpenoid production because acetyl-CoA, the starting molecule of the MVA pathway, is abundant in mitochondria. However, mitochondria comprise only a small percentage of the entire cell. Therefore, we hypothesized that increasing the total mitochondrial volume per cell would increase terpenoid production. First, we ascertained that the amounts of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the final molecules of the MVA pathway, were 15-fold higher of the strain expressing the MVA pathway in mitochondria than in the wild-type yeast strain. Second, we found that different deletion mutants induced different mitochondrial volumes by measuring the mitochondrial volume in various deletion mutants affecting mitochondrial morphology; for example,Δ<italic>mdm32</italic> increased mitochondrial volume, and Δ<italic>fzo1</italic> decreased it. Finally, the effects of mitochondrial volume on amounts of IPP/DMAPP and terpenoids (squalene or β-carotene) were investigated using mutants harboring large or small mitochondria expressing the MVA pathway in mitochondria. Amounts of IPP/DMAPP and terpenoids (squalene or β-carotene) increased when the mitochondrial volume expanded. Introducing the MVA pathway into mitochondria for terpenoid production in yeast may become more attractive by enlarging the mitochondrial volume.</p>\",\n \"<title>Key points</title>\",\n \"<p id=\\\"Par2\\\">\\n<italic>• IPP/DMAPP content increased in the strain expressing the MVA pathway in mitochondria</italic>\\n</p>\",\n \"<p id=\\\"Par3\\\">\\n<italic>• IPP/DMAPP and terpenoid contents are positively correlated with mitochondrial volume</italic>\\n</p>\",\n \"<p id=\\\"Par4\\\">\\n<italic>• Enlarging the mitochondria may improve mitochondria-mediated terpenoid production</italic>\\n</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00253-023-12922-5.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open Access funding provided by Kobe University.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>The authors are grateful for the support from NEDO Projects No. P16009 (Development of production techniques for highly functional biomaterials using plant and other organism smart cells).</p>","<title>Author contribution</title>","<p>SY designed and performed the experiments, interpreted the results, and helped write the manuscript. TB was involved in planning and performing the experiments. TK performed an interpretation of the results and helped write the manuscript. AK reviewed the research plan and manuscript. TH was the supervisor in research planning, experiments, interpretation of the results, and writing of the manuscript. All authors read and approved the final manuscript.</p>","<title>Funding</title>","<p>Open Access funding provided by Kobe University.</p>","<title>Data availability</title>","<p>The data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.</p>","<title>Declarations</title>","<title>Ethical approval</title>","<p id=\"Par28\">This article does not contain any studies with human participants or animals performed by any of the authors.</p>","<title>Competing interests</title>","<p id=\"Par29\">The authors declare no competing interests.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>The authors are grateful for the support from NEDO Projects No. P16009 (Development of production techniques for highly functional biomaterials using plant and other organism smart cells).</p>\",\n \"<title>Author contribution</title>\",\n \"<p>SY designed and performed the experiments, interpreted the results, and helped write the manuscript. TB was involved in planning and performing the experiments. TK performed an interpretation of the results and helped write the manuscript. AK reviewed the research plan and manuscript. TH was the supervisor in research planning, experiments, interpretation of the results, and writing of the manuscript. All authors read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open Access funding provided by Kobe University.</p>\",\n \"<title>Data availability</title>\",\n \"<p>The data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethical approval</title>\",\n \"<p id=\\\"Par28\\\">This article does not contain any studies with human participants or animals performed by any of the authors.</p>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par29\\\">The authors declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Introduction of the MVA pathway into mitochondria. <bold>a</bold> Introduction of the MVA pathway into mitochondria. Native and engineered pathways are shown in black and blue, respectively. <bold>b</bold> The localization of the MLS-fused GFP. The left and right panels show the fluorescent images of the MLS-fused GFP and MitoTracker. IPP production in BY4741 and SSY1. <bold>c</bold> The amount of IPP/DMAPP (columns) and OD<sub>600</sub> (dots) were measured 24 h after inoculation. Data are presented as the mean ± standard deviation (SD). P-values were determined using two-tailed Student’s <italic>t</italic>-tests (* <italic>P</italic> < 0.05). Biological replication was achieved by three individual cultures</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Mitochondrial volume in the mutants with defective mitochondrial morphology. All strains expressed MLS-fused GFP. Mitochondrial volume was calculated from the fluorescent signals of GFP using the confocal microscopic Z-stack method. Data are presented as the mean ± SD. <italic>P</italic>-values were determined using two-tailed Student’s <italic>t</italic>-tests (* <italic>P</italic> < 0.05) compared with BY4741 cells. Biological replication was achieved using at least ten individual cells</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Effect of mitochondrial volume on squalene production. <bold>a</bold> Effect of mitochondrial volume on IPP/DMAPP level. The amounts of IPP/DMAPP (columns) and OD<sub>600</sub> (dots) were measured 24 h after inoculation. Biological replication was achieved using three individual cultures. <italic>P</italic>-values were determined using two-tailed Student’s <italic>t</italic>-tests (* <italic>P</italic> < 0.05) compared with SSY1. <bold>b</bold> Squalene production; the amount of squalene (columns) and OD<sub>600</sub> (dots) were measured 24 h after inoculation. Biological replication and P-values were the same in (<bold>a</bold>). <bold>c</bold> The relationship between the amount of IPP/DMAPP and mitochondrial volume. <bold>d</bold> The relationship between squalene production and mitochondrial volume</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Effect of mitochondrial volume on β-carotene production. <bold>a</bold> Effect of mitochondrial volume on IPP/DMAPP level. The amounts of IPP/DMAPP (columns) and OD<sub>600</sub> (dots) were measured 48 h after inoculation. Biological replication was achieved using three individual cultures. P-values were determined using two-tailed Student’s <italic>t</italic>-tests (* P < 0.05) compared with SSY6. <bold>b</bold> β-carotene production; the amount of β-carotene (columns) and OD<sub>600</sub> (dots) were measured 48 h after inoculation. Biological replication and P-values were the same in (<bold>a</bold>). <bold>c</bold> The relationship between the amount of IPP/DMAPP and mitochondrial volume. <bold>d</bold> The relationship between β-carotene production and mitochondrial volume</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Introduction of the MVA pathway into mitochondria. <bold>a</bold> Introduction of the MVA pathway into mitochondria. Native and engineered pathways are shown in black and blue, respectively. <bold>b</bold> The localization of the MLS-fused GFP. The left and right panels show the fluorescent images of the MLS-fused GFP and MitoTracker. IPP production in BY4741 and SSY1. <bold>c</bold> The amount of IPP/DMAPP (columns) and OD<sub>600</sub> (dots) were measured 24 h after inoculation. Data are presented as the mean ± standard deviation (SD). P-values were determined using two-tailed Student’s <italic>t</italic>-tests (* <italic>P</italic> < 0.05). Biological replication was achieved by three individual cultures</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Mitochondrial volume in the mutants with defective mitochondrial morphology. All strains expressed MLS-fused GFP. Mitochondrial volume was calculated from the fluorescent signals of GFP using the confocal microscopic Z-stack method. Data are presented as the mean ± SD. <italic>P</italic>-values were determined using two-tailed Student’s <italic>t</italic>-tests (* <italic>P</italic> < 0.05) compared with BY4741 cells. Biological replication was achieved using at least ten individual cells</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Effect of mitochondrial volume on squalene production. <bold>a</bold> Effect of mitochondrial volume on IPP/DMAPP level. The amounts of IPP/DMAPP (columns) and OD<sub>600</sub> (dots) were measured 24 h after inoculation. Biological replication was achieved using three individual cultures. <italic>P</italic>-values were determined using two-tailed Student’s <italic>t</italic>-tests (* <italic>P</italic> < 0.05) compared with SSY1. <bold>b</bold> Squalene production; the amount of squalene (columns) and OD<sub>600</sub> (dots) were measured 24 h after inoculation. Biological replication and P-values were the same in (<bold>a</bold>). <bold>c</bold> The relationship between the amount of IPP/DMAPP and mitochondrial volume. <bold>d</bold> The relationship between squalene production and mitochondrial volume</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Effect of mitochondrial volume on β-carotene production. <bold>a</bold> Effect of mitochondrial volume on IPP/DMAPP level. The amounts of IPP/DMAPP (columns) and OD<sub>600</sub> (dots) were measured 48 h after inoculation. Biological replication was achieved using three individual cultures. P-values were determined using two-tailed Student’s <italic>t</italic>-tests (* P < 0.05) compared with SSY6. <bold>b</bold> β-carotene production; the amount of β-carotene (columns) and OD<sub>600</sub> (dots) were measured 48 h after inoculation. Biological replication and P-values were the same in (<bold>a</bold>). <bold>c</bold> The relationship between the amount of IPP/DMAPP and mitochondrial volume. <bold>d</bold> The relationship between β-carotene production and mitochondrial volume</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Strains used or constructed in this study</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Strain</th><th align=\"left\">Genotype</th></tr></thead><tbody><tr><td align=\"left\">BY4741</td><td align=\"left\">MATa, <italic>his3</italic>Δ<italic>1</italic>, <italic>leu2</italic>Δ<italic>0</italic>, <italic>met15</italic>Δ<italic>0</italic>, <italic>ura3</italic>Δ<italic>0</italic></td></tr><tr><td align=\"left\">SSY1</td><td align=\"left\">BY4741, ARS208::T<sub>TDH3</sub>-<italic>ERG10</italic>-MLS-P<sub>TDH3</sub>-P<sub>ADH1</sub>-MLS-<italic>ERG13</italic>-T<sub>ADH1</sub>; ARS308::T<sub>TDH3</sub>-<italic>tHMG1</italic>-MLS-P<sub>TDH3</sub>-P<sub>ADH1</sub>-MLS-<italic>ERG12</italic>-T<sub>ADH1</sub>; ARS416::T<sub>TDH3</sub>-<italic>ERG19</italic>-MLS-P<sub>TDH3</sub>-P<sub>ADH1</sub>-MLS-<italic>ERG8</italic>-T<sub>ADH1</sub></td></tr><tr><td align=\"left\">SSY2</td><td align=\"left\">SSY1, Δ<italic>mdm32</italic></td></tr><tr><td align=\"left\">SSY3</td><td align=\"left\">SSY1, Δ<italic>fzo1</italic></td></tr><tr><td align=\"left\">SSY4</td><td align=\"left\">SSY1, Δ<italic>mgm1</italic></td></tr><tr><td align=\"left\">SSY5</td><td align=\"left\">SSY1, Δ<italic>ugo1</italic></td></tr><tr><td align=\"left\">SSY6</td><td align=\"left\">SSY1, pCrtYBI-BTS1</td></tr><tr><td align=\"left\">SSY7</td><td align=\"left\">SSY1, Δ<italic>mdm32</italic>, pCrtYBI-BTS1</td></tr><tr><td align=\"left\">SSY8</td><td align=\"left\">SSY1, Δ<italic>fzo1</italic>, pCrtYBI-BTS1</td></tr><tr><td align=\"left\">SSY9</td><td align=\"left\">SSY1, Δ<italic>mgm</italic>, pCrtYBI-BTS1</td></tr><tr><td align=\"left\">SSY10</td><td align=\"left\">SSY1, Δ<italic>ugo1</italic>, pCrtYBI-BTS1</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Strains used or constructed in this study</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Strain</th><th align=\\\"left\\\">Genotype</th></tr></thead><tbody><tr><td align=\\\"left\\\">BY4741</td><td align=\\\"left\\\">MATa, <italic>his3</italic>Δ<italic>1</italic>, <italic>leu2</italic>Δ<italic>0</italic>, <italic>met15</italic>Δ<italic>0</italic>, <italic>ura3</italic>Δ<italic>0</italic></td></tr><tr><td align=\\\"left\\\">SSY1</td><td align=\\\"left\\\">BY4741, ARS208::T<sub>TDH3</sub>-<italic>ERG10</italic>-MLS-P<sub>TDH3</sub>-P<sub>ADH1</sub>-MLS-<italic>ERG13</italic>-T<sub>ADH1</sub>; ARS308::T<sub>TDH3</sub>-<italic>tHMG1</italic>-MLS-P<sub>TDH3</sub>-P<sub>ADH1</sub>-MLS-<italic>ERG12</italic>-T<sub>ADH1</sub>; ARS416::T<sub>TDH3</sub>-<italic>ERG19</italic>-MLS-P<sub>TDH3</sub>-P<sub>ADH1</sub>-MLS-<italic>ERG8</italic>-T<sub>ADH1</sub></td></tr><tr><td align=\\\"left\\\">SSY2</td><td align=\\\"left\\\">SSY1, Δ<italic>mdm32</italic></td></tr><tr><td align=\\\"left\\\">SSY3</td><td align=\\\"left\\\">SSY1, Δ<italic>fzo1</italic></td></tr><tr><td align=\\\"left\\\">SSY4</td><td align=\\\"left\\\">SSY1, Δ<italic>mgm1</italic></td></tr><tr><td align=\\\"left\\\">SSY5</td><td align=\\\"left\\\">SSY1, Δ<italic>ugo1</italic></td></tr><tr><td align=\\\"left\\\">SSY6</td><td align=\\\"left\\\">SSY1, pCrtYBI-BTS1</td></tr><tr><td align=\\\"left\\\">SSY7</td><td align=\\\"left\\\">SSY1, Δ<italic>mdm32</italic>, pCrtYBI-BTS1</td></tr><tr><td align=\\\"left\\\">SSY8</td><td align=\\\"left\\\">SSY1, Δ<italic>fzo1</italic>, pCrtYBI-BTS1</td></tr><tr><td align=\\\"left\\\">SSY9</td><td align=\\\"left\\\">SSY1, Δ<italic>mgm</italic>, pCrtYBI-BTS1</td></tr><tr><td align=\\\"left\\\">SSY10</td><td align=\\\"left\\\">SSY1, Δ<italic>ugo1</italic>, pCrtYBI-BTS1</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"253_2023_12922_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"253_2023_12922_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"253_2023_12922_Fig3_HTML\" id=\"MO3\"/>","<graphic xlink:href=\"253_2023_12922_Fig4_HTML\" id=\"MO4\"/>"],"string":"[\n \"<graphic xlink:href=\\\"253_2023_12922_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12922_Fig2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12922_Fig3_HTML\\\" id=\\\"MO3\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12922_Fig4_HTML\\\" id=\\\"MO4\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"253_2023_12922_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 1399 KB)</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"253_2023_12922_MOESM1_ESM.pdf\\\"><caption><p>Supplementary file1 (PDF 1399 KB)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"mixed-citation\": [\"Lv X, Wang F, Zhou P, Ye L, Xie W, Xu H, Yu H (2016) Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in \"], \"italic\": [\"Saccharomyces cerevisiae\"]}, {\"mixed-citation\": [\"Ohsumi Y, Shimoda T (2007) \\u201cAll about yeast\\u201d [Translated from Japanese]. \\u201cKobo no subete \\u201d [In Japanese]. Springer Japan. ISBN978\\u20134\\u2013431\\u201371308\\u20131\"]}, {\"surname\": [\"Santosh\", \"Paradyota\", \"Prabhupada\", \"Subhalaxmi\", \"Laxmidhar\"], \"given-names\": [\"KS\", \"KB\", \"C\", \"P\", \"R\"], \"article-title\": [\"Strategy and Problems for Synthesis of Antimalaria Artemisinin (Qinghaosu)\"], \"source\": [\"ChemSelect\"], \"year\": [\"2020\"], \"volume\": [\"5\"], \"fpage\": [\"12333\"], \"lpage\": [\"12344\"], \"pub-id\": [\"10.1002/slct.202002885\"]}, {\"surname\": [\"Weinert\", \"Iesmantavicius\", \"Moustafa\", \"Sch\\u00f6lz\", \"Wagner\", \"Magnes\", \"Zechner\", \"Choudhary\"], \"given-names\": [\"BT\", \"V\", \"T\", \"C\", \"SA\", \"C\", \"R\", \"C\"], \"article-title\": [\"Acetylation dynamics and stoichiometry in \"], \"italic\": [\"Saccharomyces cerevisiae\"], \"source\": [\"Mol Sys Biol\"], \"year\": [\"2014\"], \"volume\": [\"10\"], \"fpage\": [\"716\"], \"pub-id\": [\"10.1002/msb.134766\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":29,"string":"29"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"Appl Microbiol Biotechnol. 2024 Jan 13; 108(1):1-9"},"package_file":{"kind":"string","value":"oa_package/7d/53/PMC10787878.tar.gz"}}},{"rowIdx":163,"cells":{"accession_id":{"kind":"string","value":"PMC10787880"},"pmid":{"kind":"string","value":"0"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par6\">The asymmetric reduction of C = C double bonds is widely applied in the pharmaceutical, fine chemical, and agrochemical industries, as up to two stereogenic centers can be created in those processes. Unfortunately, the chemo-catalyzed reduction of olefins using hydrogen gas and transition metal catalysts with expensive chiral ligands suffers from poor environmental sustainability, narrow substrate range, or low enantioselectivity (Hollmann et al. ##UREF##2##2021##). The biocatalyzed reduction provides an alternative green synthetic route with a broad substrate range and excellent enantioselectivity. Ene-reductases (ERs), NAD(P)H-dependent flavoproteins from the old yellow enzyme (OYEs) family, are powerful biocatalysts for the asymmetric reduction of activated alkenes, generating the products up to two new stereogenic centers with excellent enantioselectivities (Hall and Bommarius ##REF##21692484##2011##; Toogood and Scrutton ##REF##31157123##2018##).</p>","<p id=\"Par7\">Since the first OYE was isolated from yeast, many OYEs have been identified from bacteria, yeasts, fungi, plants, and algae (Parmeggiani et al. ##REF##34586700##2022##). The OYEs could be used in the reduction of C = C double bonds bearing an electron-withdrawing group, such as nitro, aldehyde, ketone, carboxylic acid, ester, imide, or nitrile moiety. The enzymes of the OYE family were proposed to be divided into five subclasses based on the phylogenetic and biochemical analysis, called class I (classical OYEs), class II, class III (thermophilic-like OYEs), class IV, and class IV (fungal OYEs). Most of the known OYEs belong to the classical OYE subgroup, which contains OYE1-like OYEs from fungi, OYE3 from <italic>Saccharomyces</italic> sp<italic>.</italic>, PETNR-like OYEs from bacteria, and OPR-like OYEs from plants (Peters et al. ##REF##30758121##2019##). These enzymes have been used in the synthesis of various industrial interested building blocks, such as (<italic>R</italic>)-citronellal (Zheng et al. ##UREF##11##2018##), amino acid derivatives (Winkler et al. ##REF##22498437##2012##), and (<italic>R</italic>)-3-Hydroxy-2-methylpropanoate (Stueckler et al. ##UREF##9##2010##).</p>","<p id=\"Par8\">Thermophilic enzymes have great potential for industrial use as they have high resistance to temperature and organic solvents. Since the identification of the first thermophilic-like OYE YqjM from <italic>Bacillus subtilis</italic> (Kitzing et al. ##REF##15890652##2005##), another 10 thermophilic-like OYEs belonging to class III have been isolated and characterized, including XenA from <italic>Pseudomonas putida</italic> (Griese et al. ##REF##16822524##2006##), <italic>Ts</italic>OYE from <italic>Thermus scotoductus</italic> SA-01 (Opperman et al. ##REF##18263719##2008##), TOYE from <italic>Thermoanaerobacter pseudethanolicus</italic> (Adalbjornsson et al. ##REF##19943268##2010##), <italic>Gk</italic>OYE from <italic>Geobacillus kaustophilus</italic> (Schittmayer et al. ##UREF##6##2011##), <italic>Chr</italic>-OYE3 from <italic>Chryseobacterium</italic> sp. CA49 (Xu et al. ##UREF##10##2014##), <italic>Geo</italic>ER from <italic>Geobacillus</italic> sp. 30 (Tsuji et al. ##REF##24927695##2014##), OYERo2 from <italic>Rhodococcus opacus</italic> 1CP (Riedel et al. ##REF##26483784##2015##), <italic>F</italic>OYE-1 from <italic>Ferrovum</italic> sp. JA12 (Scholtissek et al. ##REF##27542380##2017##), <italic>Ca</italic>OYE from <italic>Chloroflexus aggregans</italic> (Robescu et al. ##REF##33925162##2021##), and <italic>Pf</italic>ER2 from <italic>Pseudomonas fluorescens</italic> (Shi et al. ##REF##34489033##2021##). Moreover, four thermal-tolerant OYEs were also identified from other subclasses, including <italic>Bc</italic>OYE (class IV) from <italic>Bacillus coagulans</italic> (Zhou et al. ##UREF##12##2014##), YqiG (class IV) from <italic>B. subtilis</italic> (Sheng et al. ##UREF##7##2016##), <italic>Ct</italic>OYE (class I) from <italic>Chroococcidiopsis thermalis</italic>, and <italic>Gs</italic>OYE (class I) from <italic>Galdieria sulphuraria</italic> (Robescu et al. ##REF##31930452##2020##). All the thermophilic-like OYEs showed activity optima around 40–55 °C exhibited high thermostability and high resistance toward organic solvents, so they have huge potential for industrial application.</p>","<p id=\"Par9\">Several thermophilic-like OYE structures have been revealed, including YqjM (PDB: 1Z41) (Kitzing et al. ##REF##15890652##2005##), XenA (PDB: 2H8X) (Griese et al. ##REF##16822524##2006##), CrS (PDB: 3HF3) (Opperman et al. ##REF##20138824##2010##), TOYE (PDB: 3KRU) (Adalbjornsson et al. ##REF##19943268##2010##), <italic>Gk</italic>OYE (PDB: 3GR7) (Schittmayer et al. ##UREF##6##2011##), <italic>Rm</italic>ER (PDB: 5OCS) (Opperman ##REF##28833623##2017##), and <italic>Ca</italic>OYE (PDB: 7O0T) (Robescu et al. ##REF##33925162##2021##), which showed that OYE homologs are highly conserved. The general catalytic mechanism of OYE-mediated reduction is well understood. The electron-withdrawing group moiety forms an H-bond interaction with two donor residues, usually histidine and asparagine, and then the addition of hydride from the reduced flavin to the Cβ of the activated alkene, followed by proton transfer to the Cα of the substrate from a tyrosine or cysteine residue.</p>","<p id=\"Par10\">Although many OYEs have been identified over the last years, the demand for new OYEs keeps growing to overcome application limitations, such as low catalytic activity and poor stability in harsh reaction conditions. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. <italic>Aspergillus</italic> sp<italic>.</italic> produces many secondary metabolites, and it has been proposed that ene-reductases participate in the synthetic pathways. For example, the <italic>Aspergillus fumigatus</italic> old yellow enzyme EasA reduces chanoclavine-I aldehyde to dihydrochanoclavine aldehyde in the ergot alkaloid pathways (Cheng et al. ##REF##20102147##2010##). More recently, four OYEs were identified from <italic>Aspergillus niger</italic> and <italic>Botryotinia fuckeliana</italic> (Robescu et al. ##UREF##5##2022a##, ##REF##35328465##2022b##). Herein, we mined the OYEs in the filamentous fungus <italic>Aspergillus flavus</italic> to expand the ene-reductase toolbox. A new thermophilic-like OYE (<italic>Af</italic>OYE1) was identified and carefully characterized, which exhibited broad substrate scope and excellent enantioselectivity.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par6\\\">The asymmetric reduction of C = C double bonds is widely applied in the pharmaceutical, fine chemical, and agrochemical industries, as up to two stereogenic centers can be created in those processes. Unfortunately, the chemo-catalyzed reduction of olefins using hydrogen gas and transition metal catalysts with expensive chiral ligands suffers from poor environmental sustainability, narrow substrate range, or low enantioselectivity (Hollmann et al. ##UREF##2##2021##). The biocatalyzed reduction provides an alternative green synthetic route with a broad substrate range and excellent enantioselectivity. Ene-reductases (ERs), NAD(P)H-dependent flavoproteins from the old yellow enzyme (OYEs) family, are powerful biocatalysts for the asymmetric reduction of activated alkenes, generating the products up to two new stereogenic centers with excellent enantioselectivities (Hall and Bommarius ##REF##21692484##2011##; Toogood and Scrutton ##REF##31157123##2018##).</p>\",\n \"<p id=\\\"Par7\\\">Since the first OYE was isolated from yeast, many OYEs have been identified from bacteria, yeasts, fungi, plants, and algae (Parmeggiani et al. ##REF##34586700##2022##). The OYEs could be used in the reduction of C = C double bonds bearing an electron-withdrawing group, such as nitro, aldehyde, ketone, carboxylic acid, ester, imide, or nitrile moiety. The enzymes of the OYE family were proposed to be divided into five subclasses based on the phylogenetic and biochemical analysis, called class I (classical OYEs), class II, class III (thermophilic-like OYEs), class IV, and class IV (fungal OYEs). Most of the known OYEs belong to the classical OYE subgroup, which contains OYE1-like OYEs from fungi, OYE3 from <italic>Saccharomyces</italic> sp<italic>.</italic>, PETNR-like OYEs from bacteria, and OPR-like OYEs from plants (Peters et al. ##REF##30758121##2019##). These enzymes have been used in the synthesis of various industrial interested building blocks, such as (<italic>R</italic>)-citronellal (Zheng et al. ##UREF##11##2018##), amino acid derivatives (Winkler et al. ##REF##22498437##2012##), and (<italic>R</italic>)-3-Hydroxy-2-methylpropanoate (Stueckler et al. ##UREF##9##2010##).</p>\",\n \"<p id=\\\"Par8\\\">Thermophilic enzymes have great potential for industrial use as they have high resistance to temperature and organic solvents. Since the identification of the first thermophilic-like OYE YqjM from <italic>Bacillus subtilis</italic> (Kitzing et al. ##REF##15890652##2005##), another 10 thermophilic-like OYEs belonging to class III have been isolated and characterized, including XenA from <italic>Pseudomonas putida</italic> (Griese et al. ##REF##16822524##2006##), <italic>Ts</italic>OYE from <italic>Thermus scotoductus</italic> SA-01 (Opperman et al. ##REF##18263719##2008##), TOYE from <italic>Thermoanaerobacter pseudethanolicus</italic> (Adalbjornsson et al. ##REF##19943268##2010##), <italic>Gk</italic>OYE from <italic>Geobacillus kaustophilus</italic> (Schittmayer et al. ##UREF##6##2011##), <italic>Chr</italic>-OYE3 from <italic>Chryseobacterium</italic> sp. CA49 (Xu et al. ##UREF##10##2014##), <italic>Geo</italic>ER from <italic>Geobacillus</italic> sp. 30 (Tsuji et al. ##REF##24927695##2014##), OYERo2 from <italic>Rhodococcus opacus</italic> 1CP (Riedel et al. ##REF##26483784##2015##), <italic>F</italic>OYE-1 from <italic>Ferrovum</italic> sp. JA12 (Scholtissek et al. ##REF##27542380##2017##), <italic>Ca</italic>OYE from <italic>Chloroflexus aggregans</italic> (Robescu et al. ##REF##33925162##2021##), and <italic>Pf</italic>ER2 from <italic>Pseudomonas fluorescens</italic> (Shi et al. ##REF##34489033##2021##). Moreover, four thermal-tolerant OYEs were also identified from other subclasses, including <italic>Bc</italic>OYE (class IV) from <italic>Bacillus coagulans</italic> (Zhou et al. ##UREF##12##2014##), YqiG (class IV) from <italic>B. subtilis</italic> (Sheng et al. ##UREF##7##2016##), <italic>Ct</italic>OYE (class I) from <italic>Chroococcidiopsis thermalis</italic>, and <italic>Gs</italic>OYE (class I) from <italic>Galdieria sulphuraria</italic> (Robescu et al. ##REF##31930452##2020##). All the thermophilic-like OYEs showed activity optima around 40–55 °C exhibited high thermostability and high resistance toward organic solvents, so they have huge potential for industrial application.</p>\",\n \"<p id=\\\"Par9\\\">Several thermophilic-like OYE structures have been revealed, including YqjM (PDB: 1Z41) (Kitzing et al. ##REF##15890652##2005##), XenA (PDB: 2H8X) (Griese et al. ##REF##16822524##2006##), CrS (PDB: 3HF3) (Opperman et al. ##REF##20138824##2010##), TOYE (PDB: 3KRU) (Adalbjornsson et al. ##REF##19943268##2010##), <italic>Gk</italic>OYE (PDB: 3GR7) (Schittmayer et al. ##UREF##6##2011##), <italic>Rm</italic>ER (PDB: 5OCS) (Opperman ##REF##28833623##2017##), and <italic>Ca</italic>OYE (PDB: 7O0T) (Robescu et al. ##REF##33925162##2021##), which showed that OYE homologs are highly conserved. The general catalytic mechanism of OYE-mediated reduction is well understood. The electron-withdrawing group moiety forms an H-bond interaction with two donor residues, usually histidine and asparagine, and then the addition of hydride from the reduced flavin to the Cβ of the activated alkene, followed by proton transfer to the Cα of the substrate from a tyrosine or cysteine residue.</p>\",\n \"<p id=\\\"Par10\\\">Although many OYEs have been identified over the last years, the demand for new OYEs keeps growing to overcome application limitations, such as low catalytic activity and poor stability in harsh reaction conditions. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. <italic>Aspergillus</italic> sp<italic>.</italic> produces many secondary metabolites, and it has been proposed that ene-reductases participate in the synthetic pathways. For example, the <italic>Aspergillus fumigatus</italic> old yellow enzyme EasA reduces chanoclavine-I aldehyde to dihydrochanoclavine aldehyde in the ergot alkaloid pathways (Cheng et al. ##REF##20102147##2010##). More recently, four OYEs were identified from <italic>Aspergillus niger</italic> and <italic>Botryotinia fuckeliana</italic> (Robescu et al. ##UREF##5##2022a##, ##REF##35328465##2022b##). Herein, we mined the OYEs in the filamentous fungus <italic>Aspergillus flavus</italic> to expand the ene-reductase toolbox. A new thermophilic-like OYE (<italic>Af</italic>OYE1) was identified and carefully characterized, which exhibited broad substrate scope and excellent enantioselectivity.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<title>Reagents</title>","<p id=\"Par11\">Alkene substrates and racemic reduction products were purchased from MilliporeSigma (MA, USA), Aladdin (Shanghai, China), or TCI (Shanghai, China). Glucose dehydrogenase from <italic>Bacillus</italic> was purchased from Aladdin (G139687, Shanghai, China), and NADH disodium salt hydrate was purchased from TCI (Shanghai, China). All protein commercial crystallization kits were purchased from Hampton Research (CA, USA), MiTeGen LLC (NY, USA), Qiagen (Hilden, Germany), or Molecular Dimensions (OH, USA). All other reagents and solvents were commercially available and were used without further purification.</p>","<title>General analysis methods</title>","<p id=\"Par12\"><sup>1</sup>H NMR spectra were acquired on a Bruker-400 spectrometer in CDCl<sub>3</sub>, and all signals were reported in parts per million (ppm) downfield relative to tetramethylsilane. Optical rotations were measured with a Perkin Elmer 341 polarimeter. Gas chromatographic analyses were performed on a Thermo Scientific TRACE 1300 gas chromatograph equipped with a CHIRASIL-DEX CB column (Agilent Technologies, USA), and using a flame ionization detector, nitrogen was used as the carrier gas at 5 mL/min, the split ratio was 1:20 (v/v), and the column temperature was programmed as being kept at 50 °C for 1 min and then upgraded to 150 °C at the rate of 3 °C/min. The protein purities were analyzed using SDS-PAGE using 4–12% polyacrylamide gradient gel (SurePAGE, Genscript, Nanjing, China) running in 50 mM MOPS, 50 mM Tris base, 0.1% SDS, and 1 mM EDTA, at pH 7.7, and stained with Coomassie Blue. Protein concentrations were measured using the Bradford assay (Coomassie Brilliant Blue G-250) and bovine serum albumin as the protein standard.</p>","<title>Mining OYE from Aspergillus flavus and plasmid construction</title>","<p id=\"Par13\">The thermophilic-like old yellow enzyme YqjM was submitted to a BLASTp program against the <italic>A. flavus</italic> strain NRRL3357 proteins in UniProtKB/Swiss-Prot database, which returned an NADPH dehydrogenase afvA (UniProt: B8N8Q9, GenBank: XP_041144250.1) with 41.7% identities and an NADP-dependent oxidoreductase lnbE (UniProt: B8NWW6) with 34% identities. Here, the NADPH dehydrogenase afvA was selected, and its gene sequence with an N-terminal His6-tag followed by a tobacco etch virus (TEV) protease cleavage site was codon-optimized by OPTIMIZER (<ext-link ext-link-type=\"uri\" xlink:href=\"http://genomes.urv.es/OPTIMIZER/\">http://genomes.urv.es/OPTIMIZER/</ext-link>, See Supplementary Information for nucleotide sequence) (Puigbò et al. ##REF##17439967##2007##) for the expression in <italic>E. coli</italic> and synthesized by GENEWIZ (Nanjing, China). The synthesized gene was inserted into the pET28b vector between the <italic>Nco</italic>I and <italic>Xho</italic>I enzyme sites, creating the plasmid pET28b-<italic>afvA</italic>, and the sequence was confirmed via sequencing (Shenggong, Shanghai, China).</p>","<title>Protein production and purification</title>","<p id=\"Par14\">The plasmid pET28b-<italic>afvA</italic> was transferred into <italic>E. coli</italic> BL21 (DE3) for protein production. LB medium containing kanamycin (50 μg/mL) was inoculated with <italic>E. coli</italic> BL21 (pET28b-<italic>afvA</italic>), and the culture was incubated at 37 °C and 220 rpm. Then, isopropyl-β-D-thio-galactoside (IPTG) was added into the culture at the final concentration of 1 mM when the OD<sub>600</sub> reached about 0.5. The culture was further cultured at 16 °C for 18 h for protein expression, and then the cells were harvested by centrifugation (5000 g, 4 °C) for 6 min.</p>","<p id=\"Par15\">The cell pellets were re-suspended in HEPES buffer (25 mM, pH 7.5, 10 M NaOH solution was used to adjust pH) containing 300 mM NaCl, 20 mM imidazole, and 1 mM tris(2-carboxyethyl)phosphine (TCEP), and then the cells were lysed by low-temperature ultra-high‐pressure homogenizer (JN-mini, JNBio, China) at 1100 bar and 4 °C. The lysis was centrifuged at 18,000 g and 4 °C for 1 h to remove the cell debris. The clear lysate was filtered through a surfactant-free cellulose acetate membrane with a 0.45 μm pore size and loaded on the His Trap™ HP column, which was pre-equilibrated with HEPES buffer (25 mM, pH 7.5) containing 300 mM NaCl, 20 mM imidazole, and 1 mM TCEP. Proteins were eluted by HEPES buffer (25 mM, pH 7.5) containing 300 mM NaCl and 1 mM TCEP with a linear imidazole concentration gradient from 20 to 500 mM. The peak fractions were collected and followed by His6-TEV protease cleavage and dialysis against HEPES buffer (25 mM, pH 7.5) containing 300 mM NaCl and 1 mM TCEP overnight. The dialysate was loaded onto a His Trap™ HP column again, which was pre-equilibrated with HEPES buffer (25 mM, pH 7.5) containing 300 mM NaCl and 1 mM TCEP. Flow through and wash fractions that contain the desired purified protein were collected and checked by SDS-PAGE. The fractions containing desired purified protein were pooled and concentrated by Amicon® Ultra-15 Centrifugal Filter Unit (10 K, Merck Millipore) and used as the catalyst.</p>","<p id=\"Par16\">To prepare protein samples for crystallization, the purified protein was further purified using gel filtration (HiLoad 16/60 Superdex pg 200, GE Healthcare) with HEPES buffer (25 mM, pH 7.5) containing 100 mM NaCl and 1 mM TCEP. The peak fractions were concentrated and used for crystallization.</p>","<title>General procedures of AfOYE1-catalyzed reductions</title>","<p id=\"Par17\">The purified <italic>Af</italic>OYE1 was used in all the measurements. All the reaction mixtures contained 5 mL potassium phosphate buffer (100 mM, pH 7.0), 0.1 μM purified <italic>Af</italic>OYE1, 5 U glucose dehydrogenase from <italic>Bacillus</italic>, 50 mM glucose, 20 mM NADH, and 8 mM substrate, and then the reactions were conducted at 45 °C for 10 min in triplicates. Then, the mixtures were extracted with ethyl acetate (1 mL), and then the yields were determined by GC with an external standard calibration curve. One unit of enzyme activity was defined as the amount of enzyme producing 1 μmol product per minute under the assay condition.</p>","<p id=\"Par18\">The irreversible thermal inactivation (<italic>T</italic><sub>1/2</sub>) of <italic>Af</italic>OYE1 was analyzed by heating the purified <italic>Af</italic>OYE1 in potassium phosphate buffer (pH 7.0) at different temperatures (30–70 °C) for 5 min, chilled on ice. The residual activities on 2-cyclopentenone were then measured under standard assay conditions.</p>","<p id=\"Par19\">To determine the optimal reaction conditions, 8 mM 2-cyclopentenone was used as a substrate, and the reactions were initiated by the addition of purified <italic>Af</italic>OYE1. The reaction mixtures in 5 mL potassium phosphate buffer (100 mM, pH 7.0), were conducted at 25–60 °C for 10 min, or in 5 mL potassium phosphate buffer (100 mM, pH 5.5–8.0) were conducted at 45 °C for 10 min. Then, the mixtures were extracted with ethyl acetate (1 mL), and the yield of cyclopentanone was determined by GC with an external standard calibration curve.</p>","<p id=\"Par20\">Preparative biotransformation was carried out in 50 mL potassium phosphate buffer (100 mM, pH 7.0), 0.5 μM purified <italic>Af</italic>OYE1, 20 U glucose dehydrogenase from <italic>Bacillus</italic>, 200 mM glucose, 20 mM NADH, and 50 mM substrate, and then the reactions were conducted at 45 °C for 10 h. Then, the mixtures were extracted with ethyl acetate (3 × 50 mL), the organic phase was combined and dried by anhydrous NaSO<sub>4</sub>, the solvents were removed under certain reduced pressure, and then the final product was purified with column chromatography and subjected to NMR and optical rotation analysis.</p>","<p id=\"Par21\">The kinetic parameters <italic>K</italic><sub><italic>m</italic></sub> and <italic>k</italic><sub>cat</sub> of <italic>Af</italic>OYE1 for 2-cyclopentenone were determined by measuring the production of cyclopentanone. The reaction mixture containing varying concentrations of 2-cyclopentenone in 5 mL potassium phosphate buffer (100 mM, pH 7.0) was incubated under the optimal reaction conditions (pH 7.0, and 45 °C) at 220 rpm for 2.0 min. The mixtures were extracted with ethyl acetate (1 mL), and then the yield of cyclopentanone was determined by GC. The parameters were calculated using the Prism program (GraphPad, San Diego, CA, USA).</p>","<title>Determination of the flavin cofactor</title>","<p id=\"Par22\">The flavin content of <italic>Af</italic>OYE1 was determined spectrophotometrically from absorption scans (350–750 nm) using a SpectraMax® M2e (Molecular Devices, USA) microplate reader. Free flavin was released from <italic>Af</italic>OYE1 by the protein denaturation through incubating in a boiling water bath for 10 min; then, the samples were centrifuged at 15,000 g for 10 min. The supernatant, <italic>Af</italic>OYE1 solution, and standard flavin mononucleotide (FMN) solution were analyzed by microplate readers to determine flavin content.</p>","<title>Crystallization</title>","<p id=\"Par23\">Initial crystallization screening of <italic>Af</italic>OYE1 (20 mg/mL) was carried out at 18 °C with high-throughput sparse matrix crystallization trails using a Gryphon crystallization robot (Art Robbins Instruments). MRC 96-well two-drop standard plates were adopted with the sitting-drop vapor diffusion method by mixing 0.5 μL protein and 0.5 μL reservoir solution in the initial screening. A 24-well plate was used with the hanging-drop vapor diffusion method by mixing 1 μL protein and 1 μL reservoir solution in manual optimization steps. Well-diffracted crystals were achieved in the condition containing 0.1 M Bis–Tris propane, pH 6.5, 15% (w/v) PEG 3350, and 0.2 M sodium nitrate, after seeding with smaller crystals initially obtained in the same condition. Crystals were cryoprotected in mother liquor supplemented with 25% (v/v) ethylene glycerol and snap-frozen in liquid nitrogen for data collection.</p>","<title>Data collection and structural determination</title>","<p id=\"Par24\">The X-ray diffraction data were collected at 100 K Crystallography Beamline 18U1 (BL18U1) using a Pilatus 3 S 6 M detector at Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China) and at the wavelength of 0.979 Å. Datasets were processed using XDS (Kabsch ##UREF##3##2010##). The structure was solved by molecular replacement using a modified monomer model of ene-reductase (PDB code: 5OCS, sharing 42% identity with <italic>Af</italic>OYE1) (Opperman ##REF##28833623##2017##) with all amino acids mutating to alanine in coot as a search model. An <italic>Af</italic>OYE1 dimer was positioned in the asymmetric unit using PHENIX Phaser-MR (McCoy et al. ##REF##19461840##2007##). All refinement and model-building procedures were carried out by PHENIX.refine (Adams et al. ##UREF##0##2010##) and COOT (Emsley and Cowtan ##UREF##1##2004##). FMN was automatically imported from the Coot dictionary. The final parameters obtained for the best models reached a Rfactor/Rfree of 0.144/0.167 at 1.55 Å (Table ##SUPPL##0##S1##). All the structure figures were prepared by PyMOL.</p>","<title>Phylogenetic analysis</title>","<p id=\"Par25\">The sequence alignment of 83 known OYEs (Table ##SUPPL##0##S2##) and <italic>Af</italic>OYE1 was done by ClustalW, and then phylogenetic analysis was conducted using the Maximum Likelihood statistical method based on the Jones-Taylar-Thornton (JTT) model in MEGAX. The initial tree for the heuristic search was obtained by applying NJ/BioNJ algorithms to a matrix of pairwise distance that was estimated by a <italic>p</italic>-distance and then selecting the topology with superior log likelihood value (− 28,266.79). The visualizing phylogenetic tree was produced by an online tree viewer iTOL Version 6.5.8, and the assignment of the subclasses was based on a previous evolutionary study.</p>","<title>Construction of point mutations</title>","<p id=\"Par26\">The mutants were constructed by site-directed mutagenesis using primers listed in Table ##SUPPL##0##S3## and confirmed by sequencing (Shenggong, Shanghai, China). The correct plasmid was transformed into <italic>E. coli</italic> BL21 for protein production.</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<title>Reagents</title>\",\n \"<p id=\\\"Par11\\\">Alkene substrates and racemic reduction products were purchased from MilliporeSigma (MA, USA), Aladdin (Shanghai, China), or TCI (Shanghai, China). Glucose dehydrogenase from <italic>Bacillus</italic> was purchased from Aladdin (G139687, Shanghai, China), and NADH disodium salt hydrate was purchased from TCI (Shanghai, China). All protein commercial crystallization kits were purchased from Hampton Research (CA, USA), MiTeGen LLC (NY, USA), Qiagen (Hilden, Germany), or Molecular Dimensions (OH, USA). All other reagents and solvents were commercially available and were used without further purification.</p>\",\n \"<title>General analysis methods</title>\",\n \"<p id=\\\"Par12\\\"><sup>1</sup>H NMR spectra were acquired on a Bruker-400 spectrometer in CDCl<sub>3</sub>, and all signals were reported in parts per million (ppm) downfield relative to tetramethylsilane. Optical rotations were measured with a Perkin Elmer 341 polarimeter. Gas chromatographic analyses were performed on a Thermo Scientific TRACE 1300 gas chromatograph equipped with a CHIRASIL-DEX CB column (Agilent Technologies, USA), and using a flame ionization detector, nitrogen was used as the carrier gas at 5 mL/min, the split ratio was 1:20 (v/v), and the column temperature was programmed as being kept at 50 °C for 1 min and then upgraded to 150 °C at the rate of 3 °C/min. The protein purities were analyzed using SDS-PAGE using 4–12% polyacrylamide gradient gel (SurePAGE, Genscript, Nanjing, China) running in 50 mM MOPS, 50 mM Tris base, 0.1% SDS, and 1 mM EDTA, at pH 7.7, and stained with Coomassie Blue. Protein concentrations were measured using the Bradford assay (Coomassie Brilliant Blue G-250) and bovine serum albumin as the protein standard.</p>\",\n \"<title>Mining OYE from Aspergillus flavus and plasmid construction</title>\",\n \"<p id=\\\"Par13\\\">The thermophilic-like old yellow enzyme YqjM was submitted to a BLASTp program against the <italic>A. flavus</italic> strain NRRL3357 proteins in UniProtKB/Swiss-Prot database, which returned an NADPH dehydrogenase afvA (UniProt: B8N8Q9, GenBank: XP_041144250.1) with 41.7% identities and an NADP-dependent oxidoreductase lnbE (UniProt: B8NWW6) with 34% identities. Here, the NADPH dehydrogenase afvA was selected, and its gene sequence with an N-terminal His6-tag followed by a tobacco etch virus (TEV) protease cleavage site was codon-optimized by OPTIMIZER (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"http://genomes.urv.es/OPTIMIZER/\\\">http://genomes.urv.es/OPTIMIZER/</ext-link>, See Supplementary Information for nucleotide sequence) (Puigbò et al. ##REF##17439967##2007##) for the expression in <italic>E. coli</italic> and synthesized by GENEWIZ (Nanjing, China). The synthesized gene was inserted into the pET28b vector between the <italic>Nco</italic>I and <italic>Xho</italic>I enzyme sites, creating the plasmid pET28b-<italic>afvA</italic>, and the sequence was confirmed via sequencing (Shenggong, Shanghai, China).</p>\",\n \"<title>Protein production and purification</title>\",\n \"<p id=\\\"Par14\\\">The plasmid pET28b-<italic>afvA</italic> was transferred into <italic>E. coli</italic> BL21 (DE3) for protein production. LB medium containing kanamycin (50 μg/mL) was inoculated with <italic>E. coli</italic> BL21 (pET28b-<italic>afvA</italic>), and the culture was incubated at 37 °C and 220 rpm. Then, isopropyl-β-D-thio-galactoside (IPTG) was added into the culture at the final concentration of 1 mM when the OD<sub>600</sub> reached about 0.5. The culture was further cultured at 16 °C for 18 h for protein expression, and then the cells were harvested by centrifugation (5000 g, 4 °C) for 6 min.</p>\",\n \"<p id=\\\"Par15\\\">The cell pellets were re-suspended in HEPES buffer (25 mM, pH 7.5, 10 M NaOH solution was used to adjust pH) containing 300 mM NaCl, 20 mM imidazole, and 1 mM tris(2-carboxyethyl)phosphine (TCEP), and then the cells were lysed by low-temperature ultra-high‐pressure homogenizer (JN-mini, JNBio, China) at 1100 bar and 4 °C. The lysis was centrifuged at 18,000 g and 4 °C for 1 h to remove the cell debris. The clear lysate was filtered through a surfactant-free cellulose acetate membrane with a 0.45 μm pore size and loaded on the His Trap™ HP column, which was pre-equilibrated with HEPES buffer (25 mM, pH 7.5) containing 300 mM NaCl, 20 mM imidazole, and 1 mM TCEP. Proteins were eluted by HEPES buffer (25 mM, pH 7.5) containing 300 mM NaCl and 1 mM TCEP with a linear imidazole concentration gradient from 20 to 500 mM. The peak fractions were collected and followed by His6-TEV protease cleavage and dialysis against HEPES buffer (25 mM, pH 7.5) containing 300 mM NaCl and 1 mM TCEP overnight. The dialysate was loaded onto a His Trap™ HP column again, which was pre-equilibrated with HEPES buffer (25 mM, pH 7.5) containing 300 mM NaCl and 1 mM TCEP. Flow through and wash fractions that contain the desired purified protein were collected and checked by SDS-PAGE. The fractions containing desired purified protein were pooled and concentrated by Amicon® Ultra-15 Centrifugal Filter Unit (10 K, Merck Millipore) and used as the catalyst.</p>\",\n \"<p id=\\\"Par16\\\">To prepare protein samples for crystallization, the purified protein was further purified using gel filtration (HiLoad 16/60 Superdex pg 200, GE Healthcare) with HEPES buffer (25 mM, pH 7.5) containing 100 mM NaCl and 1 mM TCEP. The peak fractions were concentrated and used for crystallization.</p>\",\n \"<title>General procedures of AfOYE1-catalyzed reductions</title>\",\n \"<p id=\\\"Par17\\\">The purified <italic>Af</italic>OYE1 was used in all the measurements. All the reaction mixtures contained 5 mL potassium phosphate buffer (100 mM, pH 7.0), 0.1 μM purified <italic>Af</italic>OYE1, 5 U glucose dehydrogenase from <italic>Bacillus</italic>, 50 mM glucose, 20 mM NADH, and 8 mM substrate, and then the reactions were conducted at 45 °C for 10 min in triplicates. Then, the mixtures were extracted with ethyl acetate (1 mL), and then the yields were determined by GC with an external standard calibration curve. One unit of enzyme activity was defined as the amount of enzyme producing 1 μmol product per minute under the assay condition.</p>\",\n \"<p id=\\\"Par18\\\">The irreversible thermal inactivation (<italic>T</italic><sub>1/2</sub>) of <italic>Af</italic>OYE1 was analyzed by heating the purified <italic>Af</italic>OYE1 in potassium phosphate buffer (pH 7.0) at different temperatures (30–70 °C) for 5 min, chilled on ice. The residual activities on 2-cyclopentenone were then measured under standard assay conditions.</p>\",\n \"<p id=\\\"Par19\\\">To determine the optimal reaction conditions, 8 mM 2-cyclopentenone was used as a substrate, and the reactions were initiated by the addition of purified <italic>Af</italic>OYE1. The reaction mixtures in 5 mL potassium phosphate buffer (100 mM, pH 7.0), were conducted at 25–60 °C for 10 min, or in 5 mL potassium phosphate buffer (100 mM, pH 5.5–8.0) were conducted at 45 °C for 10 min. Then, the mixtures were extracted with ethyl acetate (1 mL), and the yield of cyclopentanone was determined by GC with an external standard calibration curve.</p>\",\n \"<p id=\\\"Par20\\\">Preparative biotransformation was carried out in 50 mL potassium phosphate buffer (100 mM, pH 7.0), 0.5 μM purified <italic>Af</italic>OYE1, 20 U glucose dehydrogenase from <italic>Bacillus</italic>, 200 mM glucose, 20 mM NADH, and 50 mM substrate, and then the reactions were conducted at 45 °C for 10 h. Then, the mixtures were extracted with ethyl acetate (3 × 50 mL), the organic phase was combined and dried by anhydrous NaSO<sub>4</sub>, the solvents were removed under certain reduced pressure, and then the final product was purified with column chromatography and subjected to NMR and optical rotation analysis.</p>\",\n \"<p id=\\\"Par21\\\">The kinetic parameters <italic>K</italic><sub><italic>m</italic></sub> and <italic>k</italic><sub>cat</sub> of <italic>Af</italic>OYE1 for 2-cyclopentenone were determined by measuring the production of cyclopentanone. The reaction mixture containing varying concentrations of 2-cyclopentenone in 5 mL potassium phosphate buffer (100 mM, pH 7.0) was incubated under the optimal reaction conditions (pH 7.0, and 45 °C) at 220 rpm for 2.0 min. The mixtures were extracted with ethyl acetate (1 mL), and then the yield of cyclopentanone was determined by GC. The parameters were calculated using the Prism program (GraphPad, San Diego, CA, USA).</p>\",\n \"<title>Determination of the flavin cofactor</title>\",\n \"<p id=\\\"Par22\\\">The flavin content of <italic>Af</italic>OYE1 was determined spectrophotometrically from absorption scans (350–750 nm) using a SpectraMax® M2e (Molecular Devices, USA) microplate reader. Free flavin was released from <italic>Af</italic>OYE1 by the protein denaturation through incubating in a boiling water bath for 10 min; then, the samples were centrifuged at 15,000 g for 10 min. The supernatant, <italic>Af</italic>OYE1 solution, and standard flavin mononucleotide (FMN) solution were analyzed by microplate readers to determine flavin content.</p>\",\n \"<title>Crystallization</title>\",\n \"<p id=\\\"Par23\\\">Initial crystallization screening of <italic>Af</italic>OYE1 (20 mg/mL) was carried out at 18 °C with high-throughput sparse matrix crystallization trails using a Gryphon crystallization robot (Art Robbins Instruments). MRC 96-well two-drop standard plates were adopted with the sitting-drop vapor diffusion method by mixing 0.5 μL protein and 0.5 μL reservoir solution in the initial screening. A 24-well plate was used with the hanging-drop vapor diffusion method by mixing 1 μL protein and 1 μL reservoir solution in manual optimization steps. Well-diffracted crystals were achieved in the condition containing 0.1 M Bis–Tris propane, pH 6.5, 15% (w/v) PEG 3350, and 0.2 M sodium nitrate, after seeding with smaller crystals initially obtained in the same condition. Crystals were cryoprotected in mother liquor supplemented with 25% (v/v) ethylene glycerol and snap-frozen in liquid nitrogen for data collection.</p>\",\n \"<title>Data collection and structural determination</title>\",\n \"<p id=\\\"Par24\\\">The X-ray diffraction data were collected at 100 K Crystallography Beamline 18U1 (BL18U1) using a Pilatus 3 S 6 M detector at Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China) and at the wavelength of 0.979 Å. Datasets were processed using XDS (Kabsch ##UREF##3##2010##). The structure was solved by molecular replacement using a modified monomer model of ene-reductase (PDB code: 5OCS, sharing 42% identity with <italic>Af</italic>OYE1) (Opperman ##REF##28833623##2017##) with all amino acids mutating to alanine in coot as a search model. An <italic>Af</italic>OYE1 dimer was positioned in the asymmetric unit using PHENIX Phaser-MR (McCoy et al. ##REF##19461840##2007##). All refinement and model-building procedures were carried out by PHENIX.refine (Adams et al. ##UREF##0##2010##) and COOT (Emsley and Cowtan ##UREF##1##2004##). FMN was automatically imported from the Coot dictionary. The final parameters obtained for the best models reached a Rfactor/Rfree of 0.144/0.167 at 1.55 Å (Table ##SUPPL##0##S1##). All the structure figures were prepared by PyMOL.</p>\",\n \"<title>Phylogenetic analysis</title>\",\n \"<p id=\\\"Par25\\\">The sequence alignment of 83 known OYEs (Table ##SUPPL##0##S2##) and <italic>Af</italic>OYE1 was done by ClustalW, and then phylogenetic analysis was conducted using the Maximum Likelihood statistical method based on the Jones-Taylar-Thornton (JTT) model in MEGAX. The initial tree for the heuristic search was obtained by applying NJ/BioNJ algorithms to a matrix of pairwise distance that was estimated by a <italic>p</italic>-distance and then selecting the topology with superior log likelihood value (− 28,266.79). The visualizing phylogenetic tree was produced by an online tree viewer iTOL Version 6.5.8, and the assignment of the subclasses was based on a previous evolutionary study.</p>\",\n \"<title>Construction of point mutations</title>\",\n \"<p id=\\\"Par26\\\">The mutants were constructed by site-directed mutagenesis using primers listed in Table ##SUPPL##0##S3## and confirmed by sequencing (Shenggong, Shanghai, China). The correct plasmid was transformed into <italic>E. coli</italic> BL21 for protein production.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Mining OYEs from A. flavus and sequence analysis</title>","<p id=\"Par27\">To identify new OYEs from <italic>A. flavus</italic>, the thermophilic-like OYE YqjM from <italic>Bacillus subtilis</italic> was used as a reference to search the <italic>A. flavus</italic> proteins in UniProtKB/Swiss-Prot database. The returned results showed that <italic>A. flavus</italic> contains two OYEs, including a putative NADPH dehydrogenase afvA (UniProt: B8N8Q9) and an NADP-dependent oxidoreductase lnbE (UniProt: B8NWW6). The afvA and lnbE have 41.7% and 34% identities with YqjM, respectively. Here, the afvA was selected since it has higher identities with the known YqjM, and then it was overexpressed in <italic>E. coli</italic> for further analysis. Functional analysis showed that afvA has the ability to reduce 2-cyclopentenone into 2-cyclopentanone. The <italic>Af</italic>OYE1 was previously named afvA, which was annotated as an NADPH dehydrogenase. afvA was proposed to catalyze the hydroxylation of siderin and 7-demethyl siderin to the corresponding hydroxylsiderin and hydroxydemethylsiderin (Uka et al. ##REF##33337039##2020##). However, we found that afvA could not hydroxylate the siderin analogs, such as 6-hydroxy-4-methylcoumarin, 6-methylcoumarin, and 7-methylcoumarin, which indicated that afvA might not be able to catalyze the hydroxylation. Here, we annotated afvA as <italic>Af</italic>OYE1 based on the functional analysis.</p>","<p id=\"Par28\">The phylogenetic analysis of <italic>Af</italic>OYE1 and 83 known OYEs was conducted, and the results showed that <italic>Af</italic>OYE1 belongs to the group of Class III OYEs (previous thermophilic-like OYEs group) (Fig. ##FIG##0##1##) (Peters et al. ##REF##30758121##2019##). The sequence alignments showed that <italic>Af</italic>OYE1 conserved the two fingerprint motifs of the thermophilic-like OYE homologs and the catalytic residues of OYEs, including His211, His214, and Tyr216 (Fig. ##SUPPL##0##S1##) (Kitzing et al. ##REF##15890652##2005##). <italic>Af</italic>OYE1 has the highest similarities to the OYEs from <italic>Botryotinia fuckeliana</italic> (<italic>Bf</italic>OYE4) and <italic>Aspergillus niger</italic> (<italic>An</italic>OYE8), with 53.6% and 51.7% identities, respectively (Robescu et al. ##UREF##5##2022a##). The phylogenetic analysis and sequence properties also indicated that <italic>Af</italic>OYE1 is an ene-reductase other than a hydroxylase.</p>","<title>Expression and characterization of the recombinant</title>","<p id=\"Par29\">The heterologous expression of <italic>Af</italic>OYE1 with an N-terminal His6-tag was conducted in the host <italic>E. coli</italic> BL21 (DE3) at 16 °C. SDS-PAGE analysis showed that <italic>Af</italic>OYE1 was produced with a molecular mass of around 43 kDa (<bold>Fig. S2</bold>), which was in accordance with the predicted molecular mass derived from the amino acid sequence.</p>","<p id=\"Par30\">The UV–VIS absorption spectra of the purified <italic>Af</italic>OYE1, released flavin, and standard FMN suggested that <italic>Af</italic>OYE1 contained an FMN molecule as its prosthetic group and the FMN was non-covalently bound to the protein (Fig. ##FIG##1##2##). The supernatant after boiling of <italic>Af</italic>OYE1 and centrifugation turned into bright yellow, and both the released flavin and standard FMN had identical spectra, which had maxima absorbance at about 370 and 450 nm. The flavin contained <italic>Af</italic>OYE1 exhibited maxima absorbance at 370 and 460 nm.</p>","<title>Evaluation of the thermostability of AfOYE1 and optimal reaction conditions</title>","<p id=\"Par31\">The <italic>Af</italic>OYE1 thermal stability was evaluated by detecting the midpoint for thermal inactivation (<italic>T</italic><sub>1/2</sub>). The irreversible thermal deactivation curve showed that the <italic>T</italic><sub>1/2</sub> of <italic>Af</italic>OYE1 was about 60 °C (Fig. ##FIG##2##3##A). Moreover, the catalytic activity of <italic>Af</italic>OYE1 decreased slowly after 5 min heat treatment at the temperature below 55 °C, and then a drastic decline was observed at the temperature above 55 °C.</p>","<p id=\"Par32\">To further characterize <italic>Af</italic>OYE1, the effects of temperature and pH on its catalytic activity with 2-cyclopentenone were investigated carefully. <italic>Af</italic>OYE1 exhibited high catalytic activities at temperatures 40–50 °C and observed maximum activity (0.85 U/mg) at 45 °C (Fig. ##FIG##2##3##B). The data showed that <italic>Af</italic>OYE1 still conserved 70% of the maximal activity at 60 °C (0.6 U/mg) and over 60% of the maximal activity when the temperature was down to 20 °C (0.52 U/mg). Moreover, <italic>Af</italic>OYE1 had high catalytic activity in a wide range of pH (pH 5.5–8.0) (Fig. ##FIG##2##3##C). The highest catalytic activity (1.06 U/mg) of <italic>Af</italic>OYE1 was observed at pH 7.0, and it displayed over 85% of the maximal activity at pH 5.5 (0.91 U/mg) and pH 8.0 (0.94 U/mg).</p>","<title>Evaluation of the catalytic activity of AfOYE1 toward activated alkenes and kinetic analysis</title>","<p id=\"Par33\">The <italic>Af</italic>OYE1-catalyzed bioreduction system was further applied to different kinds of activated α, β-unsaturated alkenes. The results showed that cyclic enones (<bold>1</bold>, <bold>2</bold>, <bold>3</bold>, and <bold>4</bold>), acrylamide (<bold>5</bold>), nitroalkenes (<bold>6</bold> and <bold>7</bold>), and α, β-unsaturated aldehydes (<bold>8</bold>) could be reduced by <italic>Af</italic>OYE1 (Fig. ##FIG##3##4##). However, cyclic imide (<bold>9</bold>) and α, β-unsaturated esters (<bold>12</bold> and <bold>13</bold>) could not be accepted as substrates for <italic>Af</italic>OYE1. <italic>Af</italic>OYE1 displayed the highest activity toward 2-cyclohexenone with TTN of 2050. For the <italic>Af</italic>OYE1-catalyzed bioreduction of cyclic enones, it had higher activity toward cyclohexenones than cyclopentenones (<bold>3</bold> vs. <bold>1</bold>, <bold>4</bold> vs. <bold>2</bold>). Similar to other OYEs in the thermophilic-like group, <italic>Af</italic>OYE1 was shown to have a restricted substrate spectrum (Amato and Stewart ##REF##25940546##2015##).</p>","<p id=\"Par34\">The results showed that the substituent on the cyclic moiety had an important effect on the catalytic activity. <italic>Af</italic>OYE1 could catalyze the reduction of α-methyl substituted cyclopentenone (<bold>2</bold>) and cyclohexenone (<bold>4</bold>). However, it could not convert the β-methyl substituted cyclopentenone (<bold>10</bold>) and cyclohexenone (<bold>11</bold>) into the corresponding product. Moreover, the <italic>Af</italic>OYE1-catalyzed reduction had excellent enantioselectivities toward the prochiral alkenes. The bioreduction of 2-methylcyclopentenone (<bold>2</bold>) and 2-methylcyclohexenone (<bold>4</bold>) yielded the corresponding (<italic>S</italic>)-2-methylcyclopentanone ([α]<sup>25</sup><sub>D</sub> = + 16.5° (c 0.4, CHCl<sub>3</sub>) ((<italic>S</italic>), [α]<sup>25</sup><sub>D</sub> = + 114.9° (c 0.52, CHCl<sub>3</sub>) (Shimoda et al. ##UREF##8##2004##)) and (<italic>S</italic>)-2-methylcyclohexanone with > 99% ee.</p>","<p id=\"Par35\">The kinetic parameters <italic>K</italic><sub><italic>m</italic></sub> and <italic>k</italic><sub>cat</sub> of <italic>Af</italic>OYE1 were investigated with varying concentrations of 2-cyclopentenone (Fig. ##SUPPL##0##S3##). The <italic>K</italic><sub><italic>m</italic></sub> value determined by non-linear regression was 0.061 ± 0.005 mM<sup>−1</sup>, which is significantly smaller than those of most of the known OYEs (Robescu et al. ##REF##35328465##2022b##, ##REF##31930452##2020##; Xu et al. ##UREF##10##2014##). The apparent <italic>k</italic><sub>cat</sub> of the <italic>Af</italic>OYE1 on 2-cyclopentenone was 34.12 ± 0.74 min<sup>−1</sup>, resulting in catalytic efficiencies of 9.32 mM<sup>−1</sup> s<sup>−1</sup>.</p>","<title>Crystal structure of AfOYE1</title>","<p id=\"Par36\">The structure of <italic>Af</italic>OYE1 (PDB: 8J59) was determined to have a maximum resolution of 1.55 Å in the P1211 space group with an elongated dimer per asymmetric unit (Table ##SUPPL##0##S2##). The overall structure of <italic>Af</italic>OYE1 showed an expected eight-stranded (α, β)-barrel fold of triosephosphate isomerase (TIM), which is highly conserved in the thermophilic-like OYEs (Fig. ##FIG##4##5##A). On the top of the β-barrel core of each monomer encircles one FMN cofactor, which points toward the C-terminal side and is buried within the active site cavity. Similar to other thermophilic-like OYEs, the PDBePISA analysis (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver\">https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver</ext-link>) of the <italic>Af</italic>OYE1 structure showed that the inverse dimeric architecture with an interacting surface of 492 Å<sup>2</sup> is contributed by α1-helix, the peculiar long N-terminal loops, and the C-terminal helix of each protomer, which contains 7 hydrogen bonds and 6 salt bridges among the 41 interfacing residues (Fig. ##FIG##4##5##B and Table ##SUPPL##0##S4##) (Krissinel and Henrick ##REF##17681537##2007##). Meanwhile, <italic>Af</italic>OYE1 has a slightly negative surface but a highly positive inner surface at the entrance of the active site, which endows its ability to bind the negatively charged FMN cofactor (Fig. ##SUPPL##0##S4##).</p>","<p id=\"Par37\">The DoGSiteScorer analysis (<ext-link ext-link-type=\"uri\" xlink:href=\"https://proteins.plus\">https://proteins.plus</ext-link>) of the structure showed that the catalytic cavity of <italic>Af</italic>OYE1 is quite deep, and the FMN cofactor is heavily buried at the bottom of the catalytic hole with a depth of 24.23 Å (Table ##SUPPL##0##S4##). As shown by the difference electron density map <italic>F</italic><sub><italic>o</italic></sub>-<italic>F</italic><sub><italic>c</italic></sub>, the <italic>si</italic>-face of the FMN faced the solvent, while the <italic>re</italic>-side was in contact with the protein backbone tightly via the extensive hydrogen bonding and hydrophobic interactions with the side chain and main chain elements (Fig. ##FIG##4##5##C and Fig. ##SUPPL##0##S5##) (Robescu et al. ##REF##35328465##2022b##). Meanwhile, the histidine pair (His211/His214) together with the highly conserved proton donor Tyr216 interacts with a buffer-sourced chloride anion and is positioned on the <italic>si</italic>-face of the isoalloxazine ring of FMN. It indicated that His/His together with Tyr 216 participated in the binding and correct orientation of substrate (Fig. ##FIG##4##5##C) (Robescu et al. ##REF##35328465##2022b##, ##REF##31930452##2020##, ##REF##33925162##2021##).</p>","<p id=\"Par38\">Until now, two OYE structures from filamentous fungi, including OYE4 from <italic>B. fuckeliana</italic> (<italic>Bf</italic>OYE4, PDB: 7BLF) (Robescu et al. ##UREF##5##2022a##) and OYE from <italic>A. niger</italic> (<italic>An</italic>OYE8, PDB: 7QFX) (Robescu et al. ##REF##35328465##2022b##), have been reported. The superposition structures of <italic>Af</italic>OYE1 with <italic>Bf</italic>OYE4 and <italic>An</italic>OYE8, with an r.m.s.d. of 0.694 Å and 0.588 Å, respectively, showed that they have similar monomer folding, except the α6-helix for the residues (from Phe271 to Glu280), the loop region (from Ser316 to Gly325), and the cap subdomain region (from Ala132 to Thr182) (Fig. ##SUPPL##0##S6##). The protruding α6-helix and the long N-terminal loops of <italic>Af</italic>OYE1 work dimerization function in multi-asymmetry units (Fig. ##SUPPL##0##S7##). However, the protruding α6-helix shows random loops in other OYEs (Fig. ##SUPPL##0##S6##). The loop region from Ser316 to Gly325 runs on the enzyme surface and reaches the active site entrance, which contributes to the size and feature of the catalytic cavity (Fig. ##SUPPL##0##S8##A). The residues Ile318 and Ile320 in the loop have a distance to the FMN flavin group of 5.1 Å and 4.1 Å, respectively. Meanwhile, the residue Ile320 points its side chain toward the top of the catalytic cavity, which is stabilized by a hydrogen bond with Arg371 at the C-terminal α-helix (from Arg371 to Gln375) (Fig. ##SUPPL##0##S8##B). The cap region of <italic>Af</italic>OYE1 with large unstructured turns is located on the top of the entrance of the active site (from Ala132 to Thr182), while the <italic>Bf</italic>OYE4 shows α-helices and the <italic>Gs</italic>OYE (PDB: 6S0G) shows β-hairpins (Robescu et al. ##REF##31930452##2020##) (Fig. ##SUPPL##0##S8##C), which may involve interacting with NADH/NADPH and adjusting the selectivity toward FMN cofactor (Adalbjornsson et al. ##REF##19943268##2010##; Knaus et al. ##REF##26727612##2016##; Pompeu et al. ##UREF##4##2012##; Pudney et al. ##REF##17939663##2007##). The C-terminal α helix of each monomer (from Thr395 to Phe401) points to the active sites of the adjacent monomer, and the “finger” residue Trp399 interacts with the respective FMN flavin group at a distance of 3.8 Å (Fig. ##SUPPL##0##S8##B). This may play an important role in enzyme assembling, TIM barrel catalytic domain surface stabling, and two protomers dimerization (Fig. ##SUPPL##0##S8##B).</p>","<title>Analysis of the catalytic residues of AfOYE1</title>","<p id=\"Par39\">Both the sequence alignment and structure analysis indicated that <italic>Af</italic>OYE1 highly conserved the catalytic residues, including His211, His214, and Tyr216. Here, the function of those residues was investigated. Both His211 and His214 were mutated into leucine, creating the variants H211L and H214L. The catalytic activity analysis showed that these two variants still conserved about 30% of catalytic activity of the wild type <italic>Af</italic>OYE1 (Fig. ##FIG##5##6##). Previously, the histidine pair has been considered as the binding motifs of OYE homologs, such as His164 and His167 in YqjM (Shi et al. ##REF##32830294##2020##), His172 and His175 in Crs (Opperman et al. ##REF##20138824##2010##), and His182 and Asn185 in “thermophilic-like” OYE homologs (Riedel et al. ##REF##26483784##2015##). The variants H211L and H214L had only 30% catalytic activity of the wild type, which indicated that the histidine residues in the catalytic center were important for substrate binding. Furthermore, the tyrosine residue in the catalytic center of OYE was proposed as a proton donor of OYE, which contributed to the transfer of proton to substrate at the α-position (Kitzing et al. ##REF##15890652##2005##). Therefore, Tyr216 was mutated to phenylalanine (Y216F) to investigate whether Tyr216 serves as the proton donor in <italic>Af</italic>OYE1. The results showed that the variant Y216F conserved 30% catalytic activity of the wild type (Fig. ##FIG##5##6##), which indicated that Tyr216 did act as a proton donor in <italic>Af</italic>OYE1. However, alternative proton donors might exist, because the variant Y216F was still able to reduce 2-cyclopentenone.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Mining OYEs from A. flavus and sequence analysis</title>\",\n \"<p id=\\\"Par27\\\">To identify new OYEs from <italic>A. flavus</italic>, the thermophilic-like OYE YqjM from <italic>Bacillus subtilis</italic> was used as a reference to search the <italic>A. flavus</italic> proteins in UniProtKB/Swiss-Prot database. The returned results showed that <italic>A. flavus</italic> contains two OYEs, including a putative NADPH dehydrogenase afvA (UniProt: B8N8Q9) and an NADP-dependent oxidoreductase lnbE (UniProt: B8NWW6). The afvA and lnbE have 41.7% and 34% identities with YqjM, respectively. Here, the afvA was selected since it has higher identities with the known YqjM, and then it was overexpressed in <italic>E. coli</italic> for further analysis. Functional analysis showed that afvA has the ability to reduce 2-cyclopentenone into 2-cyclopentanone. The <italic>Af</italic>OYE1 was previously named afvA, which was annotated as an NADPH dehydrogenase. afvA was proposed to catalyze the hydroxylation of siderin and 7-demethyl siderin to the corresponding hydroxylsiderin and hydroxydemethylsiderin (Uka et al. ##REF##33337039##2020##). However, we found that afvA could not hydroxylate the siderin analogs, such as 6-hydroxy-4-methylcoumarin, 6-methylcoumarin, and 7-methylcoumarin, which indicated that afvA might not be able to catalyze the hydroxylation. Here, we annotated afvA as <italic>Af</italic>OYE1 based on the functional analysis.</p>\",\n \"<p id=\\\"Par28\\\">The phylogenetic analysis of <italic>Af</italic>OYE1 and 83 known OYEs was conducted, and the results showed that <italic>Af</italic>OYE1 belongs to the group of Class III OYEs (previous thermophilic-like OYEs group) (Fig. ##FIG##0##1##) (Peters et al. ##REF##30758121##2019##). The sequence alignments showed that <italic>Af</italic>OYE1 conserved the two fingerprint motifs of the thermophilic-like OYE homologs and the catalytic residues of OYEs, including His211, His214, and Tyr216 (Fig. ##SUPPL##0##S1##) (Kitzing et al. ##REF##15890652##2005##). <italic>Af</italic>OYE1 has the highest similarities to the OYEs from <italic>Botryotinia fuckeliana</italic> (<italic>Bf</italic>OYE4) and <italic>Aspergillus niger</italic> (<italic>An</italic>OYE8), with 53.6% and 51.7% identities, respectively (Robescu et al. ##UREF##5##2022a##). The phylogenetic analysis and sequence properties also indicated that <italic>Af</italic>OYE1 is an ene-reductase other than a hydroxylase.</p>\",\n \"<title>Expression and characterization of the recombinant</title>\",\n \"<p id=\\\"Par29\\\">The heterologous expression of <italic>Af</italic>OYE1 with an N-terminal His6-tag was conducted in the host <italic>E. coli</italic> BL21 (DE3) at 16 °C. SDS-PAGE analysis showed that <italic>Af</italic>OYE1 was produced with a molecular mass of around 43 kDa (<bold>Fig. S2</bold>), which was in accordance with the predicted molecular mass derived from the amino acid sequence.</p>\",\n \"<p id=\\\"Par30\\\">The UV–VIS absorption spectra of the purified <italic>Af</italic>OYE1, released flavin, and standard FMN suggested that <italic>Af</italic>OYE1 contained an FMN molecule as its prosthetic group and the FMN was non-covalently bound to the protein (Fig. ##FIG##1##2##). The supernatant after boiling of <italic>Af</italic>OYE1 and centrifugation turned into bright yellow, and both the released flavin and standard FMN had identical spectra, which had maxima absorbance at about 370 and 450 nm. The flavin contained <italic>Af</italic>OYE1 exhibited maxima absorbance at 370 and 460 nm.</p>\",\n \"<title>Evaluation of the thermostability of AfOYE1 and optimal reaction conditions</title>\",\n \"<p id=\\\"Par31\\\">The <italic>Af</italic>OYE1 thermal stability was evaluated by detecting the midpoint for thermal inactivation (<italic>T</italic><sub>1/2</sub>). The irreversible thermal deactivation curve showed that the <italic>T</italic><sub>1/2</sub> of <italic>Af</italic>OYE1 was about 60 °C (Fig. ##FIG##2##3##A). Moreover, the catalytic activity of <italic>Af</italic>OYE1 decreased slowly after 5 min heat treatment at the temperature below 55 °C, and then a drastic decline was observed at the temperature above 55 °C.</p>\",\n \"<p id=\\\"Par32\\\">To further characterize <italic>Af</italic>OYE1, the effects of temperature and pH on its catalytic activity with 2-cyclopentenone were investigated carefully. <italic>Af</italic>OYE1 exhibited high catalytic activities at temperatures 40–50 °C and observed maximum activity (0.85 U/mg) at 45 °C (Fig. ##FIG##2##3##B). The data showed that <italic>Af</italic>OYE1 still conserved 70% of the maximal activity at 60 °C (0.6 U/mg) and over 60% of the maximal activity when the temperature was down to 20 °C (0.52 U/mg). Moreover, <italic>Af</italic>OYE1 had high catalytic activity in a wide range of pH (pH 5.5–8.0) (Fig. ##FIG##2##3##C). The highest catalytic activity (1.06 U/mg) of <italic>Af</italic>OYE1 was observed at pH 7.0, and it displayed over 85% of the maximal activity at pH 5.5 (0.91 U/mg) and pH 8.0 (0.94 U/mg).</p>\",\n \"<title>Evaluation of the catalytic activity of AfOYE1 toward activated alkenes and kinetic analysis</title>\",\n \"<p id=\\\"Par33\\\">The <italic>Af</italic>OYE1-catalyzed bioreduction system was further applied to different kinds of activated α, β-unsaturated alkenes. The results showed that cyclic enones (<bold>1</bold>, <bold>2</bold>, <bold>3</bold>, and <bold>4</bold>), acrylamide (<bold>5</bold>), nitroalkenes (<bold>6</bold> and <bold>7</bold>), and α, β-unsaturated aldehydes (<bold>8</bold>) could be reduced by <italic>Af</italic>OYE1 (Fig. ##FIG##3##4##). However, cyclic imide (<bold>9</bold>) and α, β-unsaturated esters (<bold>12</bold> and <bold>13</bold>) could not be accepted as substrates for <italic>Af</italic>OYE1. <italic>Af</italic>OYE1 displayed the highest activity toward 2-cyclohexenone with TTN of 2050. For the <italic>Af</italic>OYE1-catalyzed bioreduction of cyclic enones, it had higher activity toward cyclohexenones than cyclopentenones (<bold>3</bold> vs. <bold>1</bold>, <bold>4</bold> vs. <bold>2</bold>). Similar to other OYEs in the thermophilic-like group, <italic>Af</italic>OYE1 was shown to have a restricted substrate spectrum (Amato and Stewart ##REF##25940546##2015##).</p>\",\n \"<p id=\\\"Par34\\\">The results showed that the substituent on the cyclic moiety had an important effect on the catalytic activity. <italic>Af</italic>OYE1 could catalyze the reduction of α-methyl substituted cyclopentenone (<bold>2</bold>) and cyclohexenone (<bold>4</bold>). However, it could not convert the β-methyl substituted cyclopentenone (<bold>10</bold>) and cyclohexenone (<bold>11</bold>) into the corresponding product. Moreover, the <italic>Af</italic>OYE1-catalyzed reduction had excellent enantioselectivities toward the prochiral alkenes. The bioreduction of 2-methylcyclopentenone (<bold>2</bold>) and 2-methylcyclohexenone (<bold>4</bold>) yielded the corresponding (<italic>S</italic>)-2-methylcyclopentanone ([α]<sup>25</sup><sub>D</sub> = + 16.5° (c 0.4, CHCl<sub>3</sub>) ((<italic>S</italic>), [α]<sup>25</sup><sub>D</sub> = + 114.9° (c 0.52, CHCl<sub>3</sub>) (Shimoda et al. ##UREF##8##2004##)) and (<italic>S</italic>)-2-methylcyclohexanone with > 99% ee.</p>\",\n \"<p id=\\\"Par35\\\">The kinetic parameters <italic>K</italic><sub><italic>m</italic></sub> and <italic>k</italic><sub>cat</sub> of <italic>Af</italic>OYE1 were investigated with varying concentrations of 2-cyclopentenone (Fig. ##SUPPL##0##S3##). The <italic>K</italic><sub><italic>m</italic></sub> value determined by non-linear regression was 0.061 ± 0.005 mM<sup>−1</sup>, which is significantly smaller than those of most of the known OYEs (Robescu et al. ##REF##35328465##2022b##, ##REF##31930452##2020##; Xu et al. ##UREF##10##2014##). The apparent <italic>k</italic><sub>cat</sub> of the <italic>Af</italic>OYE1 on 2-cyclopentenone was 34.12 ± 0.74 min<sup>−1</sup>, resulting in catalytic efficiencies of 9.32 mM<sup>−1</sup> s<sup>−1</sup>.</p>\",\n \"<title>Crystal structure of AfOYE1</title>\",\n \"<p id=\\\"Par36\\\">The structure of <italic>Af</italic>OYE1 (PDB: 8J59) was determined to have a maximum resolution of 1.55 Å in the P1211 space group with an elongated dimer per asymmetric unit (Table ##SUPPL##0##S2##). The overall structure of <italic>Af</italic>OYE1 showed an expected eight-stranded (α, β)-barrel fold of triosephosphate isomerase (TIM), which is highly conserved in the thermophilic-like OYEs (Fig. ##FIG##4##5##A). On the top of the β-barrel core of each monomer encircles one FMN cofactor, which points toward the C-terminal side and is buried within the active site cavity. Similar to other thermophilic-like OYEs, the PDBePISA analysis (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver\\\">https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver</ext-link>) of the <italic>Af</italic>OYE1 structure showed that the inverse dimeric architecture with an interacting surface of 492 Å<sup>2</sup> is contributed by α1-helix, the peculiar long N-terminal loops, and the C-terminal helix of each protomer, which contains 7 hydrogen bonds and 6 salt bridges among the 41 interfacing residues (Fig. ##FIG##4##5##B and Table ##SUPPL##0##S4##) (Krissinel and Henrick ##REF##17681537##2007##). Meanwhile, <italic>Af</italic>OYE1 has a slightly negative surface but a highly positive inner surface at the entrance of the active site, which endows its ability to bind the negatively charged FMN cofactor (Fig. ##SUPPL##0##S4##).</p>\",\n \"<p id=\\\"Par37\\\">The DoGSiteScorer analysis (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://proteins.plus\\\">https://proteins.plus</ext-link>) of the structure showed that the catalytic cavity of <italic>Af</italic>OYE1 is quite deep, and the FMN cofactor is heavily buried at the bottom of the catalytic hole with a depth of 24.23 Å (Table ##SUPPL##0##S4##). As shown by the difference electron density map <italic>F</italic><sub><italic>o</italic></sub>-<italic>F</italic><sub><italic>c</italic></sub>, the <italic>si</italic>-face of the FMN faced the solvent, while the <italic>re</italic>-side was in contact with the protein backbone tightly via the extensive hydrogen bonding and hydrophobic interactions with the side chain and main chain elements (Fig. ##FIG##4##5##C and Fig. ##SUPPL##0##S5##) (Robescu et al. ##REF##35328465##2022b##). Meanwhile, the histidine pair (His211/His214) together with the highly conserved proton donor Tyr216 interacts with a buffer-sourced chloride anion and is positioned on the <italic>si</italic>-face of the isoalloxazine ring of FMN. It indicated that His/His together with Tyr 216 participated in the binding and correct orientation of substrate (Fig. ##FIG##4##5##C) (Robescu et al. ##REF##35328465##2022b##, ##REF##31930452##2020##, ##REF##33925162##2021##).</p>\",\n \"<p id=\\\"Par38\\\">Until now, two OYE structures from filamentous fungi, including OYE4 from <italic>B. fuckeliana</italic> (<italic>Bf</italic>OYE4, PDB: 7BLF) (Robescu et al. ##UREF##5##2022a##) and OYE from <italic>A. niger</italic> (<italic>An</italic>OYE8, PDB: 7QFX) (Robescu et al. ##REF##35328465##2022b##), have been reported. The superposition structures of <italic>Af</italic>OYE1 with <italic>Bf</italic>OYE4 and <italic>An</italic>OYE8, with an r.m.s.d. of 0.694 Å and 0.588 Å, respectively, showed that they have similar monomer folding, except the α6-helix for the residues (from Phe271 to Glu280), the loop region (from Ser316 to Gly325), and the cap subdomain region (from Ala132 to Thr182) (Fig. ##SUPPL##0##S6##). The protruding α6-helix and the long N-terminal loops of <italic>Af</italic>OYE1 work dimerization function in multi-asymmetry units (Fig. ##SUPPL##0##S7##). However, the protruding α6-helix shows random loops in other OYEs (Fig. ##SUPPL##0##S6##). The loop region from Ser316 to Gly325 runs on the enzyme surface and reaches the active site entrance, which contributes to the size and feature of the catalytic cavity (Fig. ##SUPPL##0##S8##A). The residues Ile318 and Ile320 in the loop have a distance to the FMN flavin group of 5.1 Å and 4.1 Å, respectively. Meanwhile, the residue Ile320 points its side chain toward the top of the catalytic cavity, which is stabilized by a hydrogen bond with Arg371 at the C-terminal α-helix (from Arg371 to Gln375) (Fig. ##SUPPL##0##S8##B). The cap region of <italic>Af</italic>OYE1 with large unstructured turns is located on the top of the entrance of the active site (from Ala132 to Thr182), while the <italic>Bf</italic>OYE4 shows α-helices and the <italic>Gs</italic>OYE (PDB: 6S0G) shows β-hairpins (Robescu et al. ##REF##31930452##2020##) (Fig. ##SUPPL##0##S8##C), which may involve interacting with NADH/NADPH and adjusting the selectivity toward FMN cofactor (Adalbjornsson et al. ##REF##19943268##2010##; Knaus et al. ##REF##26727612##2016##; Pompeu et al. ##UREF##4##2012##; Pudney et al. ##REF##17939663##2007##). The C-terminal α helix of each monomer (from Thr395 to Phe401) points to the active sites of the adjacent monomer, and the “finger” residue Trp399 interacts with the respective FMN flavin group at a distance of 3.8 Å (Fig. ##SUPPL##0##S8##B). This may play an important role in enzyme assembling, TIM barrel catalytic domain surface stabling, and two protomers dimerization (Fig. ##SUPPL##0##S8##B).</p>\",\n \"<title>Analysis of the catalytic residues of AfOYE1</title>\",\n \"<p id=\\\"Par39\\\">Both the sequence alignment and structure analysis indicated that <italic>Af</italic>OYE1 highly conserved the catalytic residues, including His211, His214, and Tyr216. Here, the function of those residues was investigated. Both His211 and His214 were mutated into leucine, creating the variants H211L and H214L. The catalytic activity analysis showed that these two variants still conserved about 30% of catalytic activity of the wild type <italic>Af</italic>OYE1 (Fig. ##FIG##5##6##). Previously, the histidine pair has been considered as the binding motifs of OYE homologs, such as His164 and His167 in YqjM (Shi et al. ##REF##32830294##2020##), His172 and His175 in Crs (Opperman et al. ##REF##20138824##2010##), and His182 and Asn185 in “thermophilic-like” OYE homologs (Riedel et al. ##REF##26483784##2015##). The variants H211L and H214L had only 30% catalytic activity of the wild type, which indicated that the histidine residues in the catalytic center were important for substrate binding. Furthermore, the tyrosine residue in the catalytic center of OYE was proposed as a proton donor of OYE, which contributed to the transfer of proton to substrate at the α-position (Kitzing et al. ##REF##15890652##2005##). Therefore, Tyr216 was mutated to phenylalanine (Y216F) to investigate whether Tyr216 serves as the proton donor in <italic>Af</italic>OYE1. The results showed that the variant Y216F conserved 30% catalytic activity of the wild type (Fig. ##FIG##5##6##), which indicated that Tyr216 did act as a proton donor in <italic>Af</italic>OYE1. However, alternative proton donors might exist, because the variant Y216F was still able to reduce 2-cyclopentenone.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par40\">The <italic>Af</italic>OYE1 was previously named afvA, which was annotated as an NADPH dehydrogenase. The proposed function of afvA was to hydroxylate siderin and 7-demethyl siderin in the biosynthesis of aflavarin (Cary et al. ##REF##26209694##2015##). Here, the recombinant <italic>Af</italic>OYE1 was shown to have the ability to reduce the activated alkenes. However, it could not catalyze the hydroxylation of the siderin analogs, such as 6-hydroxy-4-methylcoumarin, 6-methylcoumarin, and 7-methylcoumarin. These data indicated that <italic>Af</italic>OYE1 might not play as a hydroxylase in the biosynthesis of aflavarin. Similar to other OYEs in the thermophilic-like group, the recombinant <italic>Af</italic>OYE1 was shown to have a restricted substrate spectrum (Amato and Stewart ##REF##25940546##2015##). It has the catalytic activity toward cyclic enones, acrylamide, nitroalkenes, and α, β-unsaturated aldehydes, but the cyclic imide and α, β-unsaturated esters could not be accepted as substrate.</p>","<p id=\"Par41\">Until now, over twenty OYE homologs from Class III OYEs have been identified, including <italic>Bf</italic>OYE4 from filamentous fungus <italic>B. fuckeliana</italic> and <italic>An</italic>OYE8 from filamentous fungus <italic>A. niger</italic>. Although <italic>Bf</italic>OYE4 and <italic>An</italic>OYE8 were phylogenetically clustered in the group of Class III OYEs, the thermostability of these two filamentous fungi source OYEs is similar to those of non-thermostable OYEs (Robescu et al. ##REF##35328465##2022b##). This newly identified <italic>Af</italic>OYE1 from filamentous fungus <italic>A. flavus</italic> had high catalytic activity at 40–50 °C and with the optima temperature at 45 °C. Moreover, the thermal stability analysis showed that the midpoint for thermal inactivation (<italic>T</italic><sub>1/2</sub>) of <italic>Af</italic>OYE1 was 60 °C. All the data indicated that <italic>Af</italic>OYE1 is a thermostable OYE. Therefore, the first thermostable OYE is identified from <italic>A. flavus</italic>, which provides a new optional thermostable OYE in asymmetric reduction of activated alkenes.</p>","<p id=\"Par42\"><italic>Af</italic>OYE1 catalyzed the bioreduction of 2-methylcyclopentenone and 2-methylcyclohexenone with excellent (<italic>S</italic>)-enantioselectivity, producing the corresponding (<italic>S</italic>)-2-methylcyclopentanone and (<italic>S</italic>)-2-methylcyclohexanone with > 99% ee. Until now, most of the known OYEs are (<italic>R</italic>)-stereoselectivity, and only 4 OYEs, including KYE1 from <italic>Kluyveromyces lactis</italic> (Yanto et al. ##REF##21510626##2011##), YersER from <italic>Yersinia bercovieri</italic> (Yanto et al. ##REF##21510626##2011##), <italic>Ct</italic>OYE from <italic>Chroococcidiopsis thermalis</italic> (Yanto et al. ##REF##21510626##2011##), and <italic>Gs</italic>OYE from <italic>Galdieria sulphuraria</italic> (Robescu et al. ##REF##31930452##2020##), were (<italic>S</italic>)-stereoselectivity in the reduction of 2-methylcyclohexenone to 2-methylcyclohexanone. However, <italic>Af</italic>OYE1 only shared 23–33% of identities with those four OYEs. The identification of a new thermostable <italic>Af</italic>OYE1 would provide a robust enzyme for (<italic>S</italic>)-enantioselective bioreductions.</p>","<p id=\"Par43\">The novel structure of the loop of Ser316 to Gly325 narrows the entrance of the catalytic pocket. This 10aa-long loop of <italic>Af</italic>OYE1 spans the entrance of the catalytic pocket and bumps close to the entrance of the catalytic pocket, resulting in a narrow entrance of the catalytic center (Fig. ##SUPPL##0##S9##A). However, the corresponding loop in <italic>Ca</italic>OYE (Ser265 to Try280), <italic>An</italic>OYE8 (Ser308 to Phe323), <italic>Bf</italic>OYE4 (Ser324 to Tyr339), YqjM (His 255 to Pro262), <italic>Ts</italic>OYE (Ser260 to Val277), and <italic>Gk</italic>OYE (Ser250 to Gln265) was away from the entrance of the catalytic pocket (Fig. ##SUPPL##0##S9##A), resulting in an open catalytic pocket (Robescu et al. ##REF##35328465##2022b##, ##REF##33925162##2021##). Meanwhile, similar to the residue Arg325 of <italic>Ca</italic>OYE and Phe368 of <italic>Rm</italic>ER, the “finger” residue Trp399 of <italic>Af</italic>OYE1, which is located in the C-terminal α helix of each monomer and points to the FMN flavin group, and together with the cap region (residues Ala132 to Thr182) may interact with NADH/NADPH and adjust the selectivity of FMN cofactor (Opperman ##REF##28833623##2017##).</p>","<p id=\"Par44\">The functional analysis of the conserved catalytic residues His211, His214, and Tyr216 of <italic>Af</italic>OYE1 showed that all three residues were important to its catalytic activity. However, the mutagenesis of those three residues did not eliminate their catalytic activity. The histidine pair has been considered as the binding motifs of OYE homologs, such as His164 and His167 in YqjM (Shi et al. ##REF##32830294##2020##), His172 and His175 in Crs (Opperman et al. ##REF##20138824##2010##), and His182 and Asn185 in “thermophilic-like” OYE homologs (Riedel et al. ##REF##26483784##2015##). The <italic>Af</italic>OYE1 variants H211L and H214L conserved 30% catalytic activity of the wild type, which showed that those two histidine residues played an important role in the substrate binding. Meanwhile, the variants H211L and H214L still exhibited activity, which indicated that other residues in the catalytic center of <italic>Af</italic>OYE1 might enable the enzyme to bind the substrate when the histidine was replaced. The replacement of Tyr216 by phenylalanine resulted in a dramatic decrease in catalytic activity, indicating Tyr216 acts as a proton donor in the <italic>Af</italic>OYE1 (Kitzing et al. ##REF##15890652##2005##). Thus, a common catalytic mechanism can be anticipated for <italic>Af</italic>OYE1 and other OYEs. On the other hand, the variant Y216F could still reduce 2-cyclopentenone into 2-cyclopentanone, which suggested that the proton transfer process could be compensated. Previously, it has also been shown that the replacement of Tyr177 in CrS resulted in the variant only harboring 20% of the catalytic efficiency (Opperman et al. ##REF##20138824##2010##).</p>","<p id=\"Par45\">In summary, a new thermophilic-like OYE <italic>Af</italic>OYE1 was identified from <italic>A. flavus</italic>. It could reduce a spectrum of activated alkenes, had an optimal temperature of 45 °C, and exhibited high reduction activity in a wide pH range (pH 5.5–8.0). Unlike most of the known OYEs, the <italic>Af</italic>OYE1-catalyzed asymmetric reduction had (<italic>S</italic>)-selective with excellent enantioselectivity, which expands the toolbox of thermostable (<italic>S</italic>)-selective OYEs. The crystal structure of <italic>Af</italic>OYE1 revealed that it had a special catalytic cavity, which may be relevant to its enantioselectivity. Our results indicate that fungi are good sources for the identification of new OYEs. We believe the newly identified <italic>Af</italic>OYE1 will give an alternative for asymmetric alkene hydrogenation in industrial processes.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par40\\\">The <italic>Af</italic>OYE1 was previously named afvA, which was annotated as an NADPH dehydrogenase. The proposed function of afvA was to hydroxylate siderin and 7-demethyl siderin in the biosynthesis of aflavarin (Cary et al. ##REF##26209694##2015##). Here, the recombinant <italic>Af</italic>OYE1 was shown to have the ability to reduce the activated alkenes. However, it could not catalyze the hydroxylation of the siderin analogs, such as 6-hydroxy-4-methylcoumarin, 6-methylcoumarin, and 7-methylcoumarin. These data indicated that <italic>Af</italic>OYE1 might not play as a hydroxylase in the biosynthesis of aflavarin. Similar to other OYEs in the thermophilic-like group, the recombinant <italic>Af</italic>OYE1 was shown to have a restricted substrate spectrum (Amato and Stewart ##REF##25940546##2015##). It has the catalytic activity toward cyclic enones, acrylamide, nitroalkenes, and α, β-unsaturated aldehydes, but the cyclic imide and α, β-unsaturated esters could not be accepted as substrate.</p>\",\n \"<p id=\\\"Par41\\\">Until now, over twenty OYE homologs from Class III OYEs have been identified, including <italic>Bf</italic>OYE4 from filamentous fungus <italic>B. fuckeliana</italic> and <italic>An</italic>OYE8 from filamentous fungus <italic>A. niger</italic>. Although <italic>Bf</italic>OYE4 and <italic>An</italic>OYE8 were phylogenetically clustered in the group of Class III OYEs, the thermostability of these two filamentous fungi source OYEs is similar to those of non-thermostable OYEs (Robescu et al. ##REF##35328465##2022b##). This newly identified <italic>Af</italic>OYE1 from filamentous fungus <italic>A. flavus</italic> had high catalytic activity at 40–50 °C and with the optima temperature at 45 °C. Moreover, the thermal stability analysis showed that the midpoint for thermal inactivation (<italic>T</italic><sub>1/2</sub>) of <italic>Af</italic>OYE1 was 60 °C. All the data indicated that <italic>Af</italic>OYE1 is a thermostable OYE. Therefore, the first thermostable OYE is identified from <italic>A. flavus</italic>, which provides a new optional thermostable OYE in asymmetric reduction of activated alkenes.</p>\",\n \"<p id=\\\"Par42\\\"><italic>Af</italic>OYE1 catalyzed the bioreduction of 2-methylcyclopentenone and 2-methylcyclohexenone with excellent (<italic>S</italic>)-enantioselectivity, producing the corresponding (<italic>S</italic>)-2-methylcyclopentanone and (<italic>S</italic>)-2-methylcyclohexanone with > 99% ee. Until now, most of the known OYEs are (<italic>R</italic>)-stereoselectivity, and only 4 OYEs, including KYE1 from <italic>Kluyveromyces lactis</italic> (Yanto et al. ##REF##21510626##2011##), YersER from <italic>Yersinia bercovieri</italic> (Yanto et al. ##REF##21510626##2011##), <italic>Ct</italic>OYE from <italic>Chroococcidiopsis thermalis</italic> (Yanto et al. ##REF##21510626##2011##), and <italic>Gs</italic>OYE from <italic>Galdieria sulphuraria</italic> (Robescu et al. ##REF##31930452##2020##), were (<italic>S</italic>)-stereoselectivity in the reduction of 2-methylcyclohexenone to 2-methylcyclohexanone. However, <italic>Af</italic>OYE1 only shared 23–33% of identities with those four OYEs. The identification of a new thermostable <italic>Af</italic>OYE1 would provide a robust enzyme for (<italic>S</italic>)-enantioselective bioreductions.</p>\",\n \"<p id=\\\"Par43\\\">The novel structure of the loop of Ser316 to Gly325 narrows the entrance of the catalytic pocket. This 10aa-long loop of <italic>Af</italic>OYE1 spans the entrance of the catalytic pocket and bumps close to the entrance of the catalytic pocket, resulting in a narrow entrance of the catalytic center (Fig. ##SUPPL##0##S9##A). However, the corresponding loop in <italic>Ca</italic>OYE (Ser265 to Try280), <italic>An</italic>OYE8 (Ser308 to Phe323), <italic>Bf</italic>OYE4 (Ser324 to Tyr339), YqjM (His 255 to Pro262), <italic>Ts</italic>OYE (Ser260 to Val277), and <italic>Gk</italic>OYE (Ser250 to Gln265) was away from the entrance of the catalytic pocket (Fig. ##SUPPL##0##S9##A), resulting in an open catalytic pocket (Robescu et al. ##REF##35328465##2022b##, ##REF##33925162##2021##). Meanwhile, similar to the residue Arg325 of <italic>Ca</italic>OYE and Phe368 of <italic>Rm</italic>ER, the “finger” residue Trp399 of <italic>Af</italic>OYE1, which is located in the C-terminal α helix of each monomer and points to the FMN flavin group, and together with the cap region (residues Ala132 to Thr182) may interact with NADH/NADPH and adjust the selectivity of FMN cofactor (Opperman ##REF##28833623##2017##).</p>\",\n \"<p id=\\\"Par44\\\">The functional analysis of the conserved catalytic residues His211, His214, and Tyr216 of <italic>Af</italic>OYE1 showed that all three residues were important to its catalytic activity. However, the mutagenesis of those three residues did not eliminate their catalytic activity. The histidine pair has been considered as the binding motifs of OYE homologs, such as His164 and His167 in YqjM (Shi et al. ##REF##32830294##2020##), His172 and His175 in Crs (Opperman et al. ##REF##20138824##2010##), and His182 and Asn185 in “thermophilic-like” OYE homologs (Riedel et al. ##REF##26483784##2015##). The <italic>Af</italic>OYE1 variants H211L and H214L conserved 30% catalytic activity of the wild type, which showed that those two histidine residues played an important role in the substrate binding. Meanwhile, the variants H211L and H214L still exhibited activity, which indicated that other residues in the catalytic center of <italic>Af</italic>OYE1 might enable the enzyme to bind the substrate when the histidine was replaced. The replacement of Tyr216 by phenylalanine resulted in a dramatic decrease in catalytic activity, indicating Tyr216 acts as a proton donor in the <italic>Af</italic>OYE1 (Kitzing et al. ##REF##15890652##2005##). Thus, a common catalytic mechanism can be anticipated for <italic>Af</italic>OYE1 and other OYEs. On the other hand, the variant Y216F could still reduce 2-cyclopentenone into 2-cyclopentanone, which suggested that the proton transfer process could be compensated. Previously, it has also been shown that the replacement of Tyr177 in CrS resulted in the variant only harboring 20% of the catalytic efficiency (Opperman et al. ##REF##20138824##2010##).</p>\",\n \"<p id=\\\"Par45\\\">In summary, a new thermophilic-like OYE <italic>Af</italic>OYE1 was identified from <italic>A. flavus</italic>. It could reduce a spectrum of activated alkenes, had an optimal temperature of 45 °C, and exhibited high reduction activity in a wide pH range (pH 5.5–8.0). Unlike most of the known OYEs, the <italic>Af</italic>OYE1-catalyzed asymmetric reduction had (<italic>S</italic>)-selective with excellent enantioselectivity, which expands the toolbox of thermostable (<italic>S</italic>)-selective OYEs. The crystal structure of <italic>Af</italic>OYE1 revealed that it had a special catalytic cavity, which may be relevant to its enantioselectivity. Our results indicate that fungi are good sources for the identification of new OYEs. We believe the newly identified <italic>Af</italic>OYE1 will give an alternative for asymmetric alkene hydrogenation in industrial processes.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<title>Abstract</title>","<p id=\"Par1\">Old yellow enzymes (OYEs) have been proven as powerful biocatalysts for the asymmetric reduction of activated alkenes. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. To expand the OYEs toolbox, a new thermophilic-like OYE (<italic>Af</italic>OYE1) was identified from <italic>Aspergillus flavus</italic> strain NRRL3357. The thermal stability analysis showed that the <italic>T</italic><sub>1/2</sub> of <italic>Af</italic>OYE1 was 60 °C, and it had the optimal temperature at 45 °C. Moreover, <italic>Af</italic>OYE1 exhibited high reduction activity in a wide pH range (pH 5.5–8.0). <italic>Af</italic>OYE1 could accept cyclic enones, acrylamide, nitroalkenes, and α, β-unsaturated aldehydes as substrates and had excellent enantioselectivity toward prochiral alkenes (> 99% ee). Interestingly, an unexpected (<italic>S</italic>)-stereoselectivity bioreduction toward 2-methylcyclohexenone was observed. The further crystal structure of <italic>Af</italic>OYE1 revealed that the “cap” region from Ala132 to Thr182, the loop of Ser316 to Gly325, α short helix of Arg371 to Gln375, and the C-terminal “finger” structure endow the catalytic cavity of <italic>Af</italic>OYE1 quite deep and narrow, and flavin mononucleotide (FMN) heavily buried at the bottom of the active site tunnel. Furthermore, the catalytic mechanism of <italic>Af</italic>OYE1 was also investigated, and the results confirmed that the residues His211, His214, and Tyr216 compose its catalytic triad. This newly identified thermophilic-like OYE would thus be valuable for asymmetric alkene hydrogenation in industrial processes.</p>","<title>Key points</title>","<p id=\"Par2\">\n<list list-type=\"bullet\"><list-item><p id=\"Par3\"><italic>A new thermophilic-like OYE AfOYE1 was identified from Aspergillus flavus, and the T</italic><sub>1/2</sub>\n<italic>of AfOYE1 was 60 °C</italic></p></list-item><list-item><p id=\"Par4\"><italic>AfOYE1 catalyzed the reduction of 2-methylcyclohexenone with (S)-stereoselectivity</italic></p></list-item><list-item><p id=\"Par5\"><italic>The crystal structure of AfOYE1 was revealedv</italic></p></list-item></list></p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00253-023-12963-w.</p>","<title>Keywords</title>"],"string":"[\n \"<title>Abstract</title>\",\n \"<p id=\\\"Par1\\\">Old yellow enzymes (OYEs) have been proven as powerful biocatalysts for the asymmetric reduction of activated alkenes. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. To expand the OYEs toolbox, a new thermophilic-like OYE (<italic>Af</italic>OYE1) was identified from <italic>Aspergillus flavus</italic> strain NRRL3357. The thermal stability analysis showed that the <italic>T</italic><sub>1/2</sub> of <italic>Af</italic>OYE1 was 60 °C, and it had the optimal temperature at 45 °C. Moreover, <italic>Af</italic>OYE1 exhibited high reduction activity in a wide pH range (pH 5.5–8.0). <italic>Af</italic>OYE1 could accept cyclic enones, acrylamide, nitroalkenes, and α, β-unsaturated aldehydes as substrates and had excellent enantioselectivity toward prochiral alkenes (> 99% ee). Interestingly, an unexpected (<italic>S</italic>)-stereoselectivity bioreduction toward 2-methylcyclohexenone was observed. The further crystal structure of <italic>Af</italic>OYE1 revealed that the “cap” region from Ala132 to Thr182, the loop of Ser316 to Gly325, α short helix of Arg371 to Gln375, and the C-terminal “finger” structure endow the catalytic cavity of <italic>Af</italic>OYE1 quite deep and narrow, and flavin mononucleotide (FMN) heavily buried at the bottom of the active site tunnel. Furthermore, the catalytic mechanism of <italic>Af</italic>OYE1 was also investigated, and the results confirmed that the residues His211, His214, and Tyr216 compose its catalytic triad. This newly identified thermophilic-like OYE would thus be valuable for asymmetric alkene hydrogenation in industrial processes.</p>\",\n \"<title>Key points</title>\",\n \"<p id=\\\"Par2\\\">\\n<list list-type=\\\"bullet\\\"><list-item><p id=\\\"Par3\\\"><italic>A new thermophilic-like OYE AfOYE1 was identified from Aspergillus flavus, and the T</italic><sub>1/2</sub>\\n<italic>of AfOYE1 was 60 °C</italic></p></list-item><list-item><p id=\\\"Par4\\\"><italic>AfOYE1 catalyzed the reduction of 2-methylcyclohexenone with (S)-stereoselectivity</italic></p></list-item><list-item><p id=\\\"Par5\\\"><italic>The crystal structure of AfOYE1 was revealedv</italic></p></list-item></list></p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00253-023-12963-w.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>We thank SSRF 18U1 beamline scientists for their help with data collection and the NMR center in the College of Science at Henan Agricultural University for taking NMR spectra. We acknowledge English editorial assistance by Maria Ajmal (Henan Agricultural University, China).</p>","<title>Author contribution</title>","<p>NL, YW, and YM carried out the experimental work and drafted the manuscript; YL, SZ, SW, and PM analyzed the data and revised the manuscript; NL, YH, and HL designed the experiments and revised the manuscript. All authors have read and approved the final manuscript.</p>","<title>Funding</title>","<p>This study was funded by the National Natural Science Foundation of China (Nos. 32171472 and 31900876), the Key Scientific Research Projects for Higher Education of Henan Province (No. 22A180013), the Key Scientific and Technological Project of Henan Province (No. 232102311151), and High-level Talent Scientific Research Startup Fund Program of Henan University of Technology (2019BS020).</p>","<title>Data availability</title>","<p>All data generated or analyzed during this study are included in this published article (and its supplementary information files).</p>","<title>Declarations</title>","<title>Ethics approval</title>","<p id=\"Par46\">This article does not contain any studies with human participants or animals performed by any of the authors.</p>","<title>Conflict of interest</title>","<p id=\"Par47\">The authors declare no competing interests.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>We thank SSRF 18U1 beamline scientists for their help with data collection and the NMR center in the College of Science at Henan Agricultural University for taking NMR spectra. We acknowledge English editorial assistance by Maria Ajmal (Henan Agricultural University, China).</p>\",\n \"<title>Author contribution</title>\",\n \"<p>NL, YW, and YM carried out the experimental work and drafted the manuscript; YL, SZ, SW, and PM analyzed the data and revised the manuscript; NL, YH, and HL designed the experiments and revised the manuscript. All authors have read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>This study was funded by the National Natural Science Foundation of China (Nos. 32171472 and 31900876), the Key Scientific Research Projects for Higher Education of Henan Province (No. 22A180013), the Key Scientific and Technological Project of Henan Province (No. 232102311151), and High-level Talent Scientific Research Startup Fund Program of Henan University of Technology (2019BS020).</p>\",\n \"<title>Data availability</title>\",\n \"<p>All data generated or analyzed during this study are included in this published article (and its supplementary information files).</p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethics approval</title>\",\n \"<p id=\\\"Par46\\\">This article does not contain any studies with human participants or animals performed by any of the authors.</p>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par47\\\">The authors declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Phylogenetic analysis of <italic>Af</italic>OYE1 and previously described OYEs. The phylogenetic analysis was conducted using the Maximum Likelihood statistical method based on the Jones-Taylar-Thornton (JTT) model in MEGAX. The corresponding alignment was done by ClustalW. The accession numbers of the previously described OYEs are listed in Table ##SUPPL##0##S2##</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>UV–visible absorption spectra of purified <italic>Af</italic>OYE1 (solid line), released flavin after thermal denaturation (100 °C for 10 min, dotted line), and standard FMN (dash line)</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Thermostability <bold>(A)</bold>, temperature <bold>(B)</bold>, and pH <bold>(C)</bold> optima of <italic>Af</italic>OYE1. The standard deviations of triplicates are represented by error bars</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p><italic>Af</italic>OYE1-catalyzed reduction of activated α, β-unsaturated alkenes. Reaction conditions: 5 mL PBS (100 mM, pH 7.0), 0.1 μM <italic>Af</italic>OYE1, 5 U glucose dehydrogenase, 50 mM glucose, 20 mM NADH, and 8 mM substrate, at 45 °C for 10 min. TTN (total turnover number): the total moles of product yield divided by the moles of <italic>Af</italic>OYE1</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Crystal structure of <italic>Af</italic>OYE1. <bold>A</bold> Overall cartoon structure of <italic>Af</italic>OYE1. The two protomers of <italic>Af</italic>OYE1 are colored green and cyan, with FMN (yellow) and Cl (magenta) buried in each protomer’s active pocket. <bold>B</bold> Surface representation showing the two protein chains forming the dimeric architecture contributed deeply by the interaction of α1-helix, N-terminal loops, and C-terminal helix of each protomer. <bold>C</bold> Details of <italic>Af</italic>OYE1 catalytic cavity. The omit |<italic>F</italic><sub><italic>o</italic></sub>|-|<italic>F</italic><sub><italic>c</italic></sub>| map is shown in gray mesh contoured at 2σ level indicating locations of FMN and ethylene glycol (EDO). FMN and EDO bound in the active site are shown with C atoms in cyan and Cl in the green sphere together with H<sub>2</sub>O in the red dot. Relevant residues of the catalytic cavity are shown with green C atoms, Red O, and Blue N. Hydrogen bonds are shown as yellow dashed lines</p></caption></fig>","<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>The catalytic activity of the variants and wild type <italic>Af</italic>OYE1 toward 2-cyclopentenone</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Phylogenetic analysis of <italic>Af</italic>OYE1 and previously described OYEs. The phylogenetic analysis was conducted using the Maximum Likelihood statistical method based on the Jones-Taylar-Thornton (JTT) model in MEGAX. The corresponding alignment was done by ClustalW. The accession numbers of the previously described OYEs are listed in Table ##SUPPL##0##S2##</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>UV–visible absorption spectra of purified <italic>Af</italic>OYE1 (solid line), released flavin after thermal denaturation (100 °C for 10 min, dotted line), and standard FMN (dash line)</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Thermostability <bold>(A)</bold>, temperature <bold>(B)</bold>, and pH <bold>(C)</bold> optima of <italic>Af</italic>OYE1. The standard deviations of triplicates are represented by error bars</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p><italic>Af</italic>OYE1-catalyzed reduction of activated α, β-unsaturated alkenes. Reaction conditions: 5 mL PBS (100 mM, pH 7.0), 0.1 μM <italic>Af</italic>OYE1, 5 U glucose dehydrogenase, 50 mM glucose, 20 mM NADH, and 8 mM substrate, at 45 °C for 10 min. TTN (total turnover number): the total moles of product yield divided by the moles of <italic>Af</italic>OYE1</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><p>Crystal structure of <italic>Af</italic>OYE1. <bold>A</bold> Overall cartoon structure of <italic>Af</italic>OYE1. The two protomers of <italic>Af</italic>OYE1 are colored green and cyan, with FMN (yellow) and Cl (magenta) buried in each protomer’s active pocket. <bold>B</bold> Surface representation showing the two protein chains forming the dimeric architecture contributed deeply by the interaction of α1-helix, N-terminal loops, and C-terminal helix of each protomer. <bold>C</bold> Details of <italic>Af</italic>OYE1 catalytic cavity. The omit |<italic>F</italic><sub><italic>o</italic></sub>|-|<italic>F</italic><sub><italic>c</italic></sub>| map is shown in gray mesh contoured at 2σ level indicating locations of FMN and ethylene glycol (EDO). FMN and EDO bound in the active site are shown with C atoms in cyan and Cl in the green sphere together with H<sub>2</sub>O in the red dot. Relevant residues of the catalytic cavity are shown with green C atoms, Red O, and Blue N. Hydrogen bonds are shown as yellow dashed lines</p></caption></fig>\",\n \"<fig id=\\\"Fig6\\\"><label>Fig. 6</label><caption><p>The catalytic activity of the variants and wild type <italic>Af</italic>OYE1 toward 2-cyclopentenone</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":[],"string":"[]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>Na Li and Yuan Wang contributed equally to the work.</p></fn></fn-group>"],"string":"[\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>Na Li and Yuan Wang contributed equally to the work.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"253_2023_12963_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"253_2023_12963_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"253_2023_12963_Fig3_HTML\" id=\"MO3\"/>","<graphic xlink:href=\"253_2023_12963_Fig4_HTML\" id=\"MO4\"/>","<graphic xlink:href=\"253_2023_12963_Fig5_HTML\" id=\"MO5\"/>","<graphic xlink:href=\"253_2023_12963_Fig6_HTML\" id=\"MO6\"/>"],"string":"[\n \"<graphic xlink:href=\\\"253_2023_12963_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12963_Fig2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12963_Fig3_HTML\\\" id=\\\"MO3\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12963_Fig4_HTML\\\" id=\\\"MO4\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12963_Fig5_HTML\\\" id=\\\"MO5\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12963_Fig6_HTML\\\" id=\\\"MO6\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"253_2023_12963_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 22613 KB)</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"253_2023_12963_MOESM1_ESM.pdf\\\"><caption><p>Supplementary file1 (PDF 22613 KB)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"surname\": [\"Adams\", \"Afonine\", \"Bunkoczi\", \"Chen\", \"Davis\", \"Echols\", \"Headd\", \"Hung\", \"Kapral\", \"Grosse-Kunstleve\", \"McCoy\", \"Moriarty\", \"Oeffner\", \"Read\", \"Richardson\", \"Richardson\", \"Terwilliger\", \"Zwart\"], \"given-names\": [\"PD\", \"PV\", \"G\", \"VB\", \"IW\", \"N\", \"JJ\", \"L-W\", \"GJ\", \"RW\", \"AJ\", \"NW\", \"R\", \"RJ\", \"DC\", \"JS\", \"TC\", \"PH\"], \"article-title\": [\"PHENIX: a comprehensive Python-based system for macromolecular structure solution\"], \"source\": [\"Acta Crystallogr, Sec D: Biol Crystallogr\"], \"year\": [\"2010\"], \"volume\": [\"66\"], \"fpage\": [\"213\"], \"lpage\": [\"221\"], \"pub-id\": [\"10.1107/S0907444909052925\"]}, {\"surname\": [\"Emsley\", \"Cowtan\"], \"given-names\": [\"P\", \"K\"], \"article-title\": [\"Coot: model-building tools for molecular graphics\"], \"source\": [\"Acta Crystallogr, Sec D: Biol Crystallogr\"], \"year\": [\"2004\"], \"volume\": [\"60\"], \"fpage\": [\"2126\"], \"lpage\": [\"2132\"], \"pub-id\": [\"10.1107/S0907444904019158\"]}, {\"surname\": [\"Hollmann\", \"Opperman\", \"Paul\"], \"given-names\": [\"F\", \"DJ\", \"CE\"], \"article-title\": [\"Biocatalytic reduction reactions from a chemist\\u2019s perspective\"], \"source\": [\"Angew Chem Int Ed\"], \"year\": [\"2021\"], \"volume\": [\"60\"], \"fpage\": [\"5644\"], \"lpage\": [\"5665\"], \"pub-id\": [\"10.1002/anie.202001876\"]}, {\"mixed-citation\": [\"Kabsch W (2010) XDS. 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108(1):1-11"},"package_file":{"kind":"string","value":"oa_package/54/2b/PMC10787880.tar.gz"}}},{"rowIdx":164,"cells":{"accession_id":{"kind":"string","value":"PMC10787881"},"pmid":{"kind":"string","value":"0"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par5\">The model yeast species <italic>Saccharomyces cerevisiae</italic> is used in many fundamental and applied research applications, such as production of many industrially relevant compounds (Krivoruchko and Nielsen ##REF##25544013##2015##) and as biosensors to various substances, for example, heavy metals or estrogens (Martin-Yken ##REF##32413968##2020##). The compounds produced in yeast include D-lactic acid (DLA) (Baek et al. ##REF##26596574##2016##), which is used for production of stereocomplex type poly-lactic acid, a promising biodegradable polymer (de Albuquerque et al. ##REF##34273343##2021##). Genetically engineered yeast strains produce up to 112 g/L (1.24 M) of DLA in neutralizing conditions or over 53.2 g/L (0.59 M) of this substance without neutralizing agents (Ishida et al. ##REF##16569615##2006##, ##UREF##3##2011##; Baek et al. ##REF##26596574##2016##; Yamada et al. ##REF##28475210##2017##; Mitsui et al. ##REF##32960291##2020##). Efficient sensing systems for DLA would also be relevant in medicine, as D-lactic acidosis is a rare but serious neurologic condition specific to individuals with short bowel syndrome (Kowlgi and Chhabra ##UREF##4##2015##; Petersen ##REF##16306301##2005##).</p>","<p id=\"Par6\">However, a systematic evaluation of yeast cell response is scarce not only for DLA but even for its incomparably more common enantiomer L-lactic acid (LLA). The response to LLA has been studied in several yeast strains on the levels of viability or growth rate reduction, as well as transcription. Specifically, it was found that lactic acid (LA) in industrially relevant concentrations of 90 or even 280 mM LA (presumably LLA) had a rather limited effect on the metabolism of <italic>S. cerevisiae</italic> in thermostat cultures but did affect the energy status of the cell by provoking a reduction in the ATP content (Thomsson and Larsson ##REF##16317544##2006##). Another study estimated the minimal inhibitory concentration of LA (presumably LLA) as 2.5% w/v (278 mM), while 0.2% began to stress the cells (Narendranath et al. ##REF##11420658##2001##). There are also two studies on the transcriptional effect of LLA. One of them dealt with LLA in comparison with acetic and hydrochloric acids with DNA microarrays in shake flask cultures. The authors found that these organic acids triggered relatively similar gene expression perturbations and affected cell wall and metal metabolism; the latter was intermediated by the Atp1p transcription factor (Kawahata et al. ##REF##16911514##2006##). Another study, using chemostat cultures, also found iron metabolism remodeling; it was very pronounced at pH 5 and 500 mM LLA and much less severe at pH 3 and 900 mM LLA (Abbott et al. ##REF##18676708##2008##). To the best of our knowledge, there is no published data on the effect of DLA on yeast transcriptome or proteome.</p>","<p id=\"Par7\">In this study, we evaluated the transcriptional response of <italic>S. cerevisiae</italic> to varying concentrations of DLA (from 0.05 mM to 45 mM) and 45 mM of LLA in order to check if this model species possessed any promoters that would quantitatively respond to DLA but not LLA and could thus be promising for designing a yeast-based stereo-specific biosensor to lactic acid. Such transcriptome-based approach has already been successfully applied to find a 1-butanol sensing promoter in <italic>S. cerevisiae</italic> (Shi et al. ##REF##28712783##2017##). In addition, we aimed at enriching the data on transcriptional response to DLA in comparison to LLA. We found that the concentrations of DLA of 0.05 or 0.5 mM did not trigger any changes in gene expression compared to the control samples. The genes activated in response to 5 mM DLA were enriched in those controlling cell wall organization, while the genes upregulated upon 45 mM DLA treatment included several genes functioning in lactate metabolism and iron uptake. Finally, the genes responding to LLA contained many genes known to respond to this and other weak acids.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">The model yeast species <italic>Saccharomyces cerevisiae</italic> is used in many fundamental and applied research applications, such as production of many industrially relevant compounds (Krivoruchko and Nielsen ##REF##25544013##2015##) and as biosensors to various substances, for example, heavy metals or estrogens (Martin-Yken ##REF##32413968##2020##). The compounds produced in yeast include D-lactic acid (DLA) (Baek et al. ##REF##26596574##2016##), which is used for production of stereocomplex type poly-lactic acid, a promising biodegradable polymer (de Albuquerque et al. ##REF##34273343##2021##). Genetically engineered yeast strains produce up to 112 g/L (1.24 M) of DLA in neutralizing conditions or over 53.2 g/L (0.59 M) of this substance without neutralizing agents (Ishida et al. ##REF##16569615##2006##, ##UREF##3##2011##; Baek et al. ##REF##26596574##2016##; Yamada et al. ##REF##28475210##2017##; Mitsui et al. ##REF##32960291##2020##). Efficient sensing systems for DLA would also be relevant in medicine, as D-lactic acidosis is a rare but serious neurologic condition specific to individuals with short bowel syndrome (Kowlgi and Chhabra ##UREF##4##2015##; Petersen ##REF##16306301##2005##).</p>\",\n \"<p id=\\\"Par6\\\">However, a systematic evaluation of yeast cell response is scarce not only for DLA but even for its incomparably more common enantiomer L-lactic acid (LLA). The response to LLA has been studied in several yeast strains on the levels of viability or growth rate reduction, as well as transcription. Specifically, it was found that lactic acid (LA) in industrially relevant concentrations of 90 or even 280 mM LA (presumably LLA) had a rather limited effect on the metabolism of <italic>S. cerevisiae</italic> in thermostat cultures but did affect the energy status of the cell by provoking a reduction in the ATP content (Thomsson and Larsson ##REF##16317544##2006##). Another study estimated the minimal inhibitory concentration of LA (presumably LLA) as 2.5% w/v (278 mM), while 0.2% began to stress the cells (Narendranath et al. ##REF##11420658##2001##). There are also two studies on the transcriptional effect of LLA. One of them dealt with LLA in comparison with acetic and hydrochloric acids with DNA microarrays in shake flask cultures. The authors found that these organic acids triggered relatively similar gene expression perturbations and affected cell wall and metal metabolism; the latter was intermediated by the Atp1p transcription factor (Kawahata et al. ##REF##16911514##2006##). Another study, using chemostat cultures, also found iron metabolism remodeling; it was very pronounced at pH 5 and 500 mM LLA and much less severe at pH 3 and 900 mM LLA (Abbott et al. ##REF##18676708##2008##). To the best of our knowledge, there is no published data on the effect of DLA on yeast transcriptome or proteome.</p>\",\n \"<p id=\\\"Par7\\\">In this study, we evaluated the transcriptional response of <italic>S. cerevisiae</italic> to varying concentrations of DLA (from 0.05 mM to 45 mM) and 45 mM of LLA in order to check if this model species possessed any promoters that would quantitatively respond to DLA but not LLA and could thus be promising for designing a yeast-based stereo-specific biosensor to lactic acid. Such transcriptome-based approach has already been successfully applied to find a 1-butanol sensing promoter in <italic>S. cerevisiae</italic> (Shi et al. ##REF##28712783##2017##). In addition, we aimed at enriching the data on transcriptional response to DLA in comparison to LLA. We found that the concentrations of DLA of 0.05 or 0.5 mM did not trigger any changes in gene expression compared to the control samples. The genes activated in response to 5 mM DLA were enriched in those controlling cell wall organization, while the genes upregulated upon 45 mM DLA treatment included several genes functioning in lactate metabolism and iron uptake. Finally, the genes responding to LLA contained many genes known to respond to this and other weak acids.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<title>Yeast cultivation</title>","<p id=\"Par8\">The yeast strain used for this work was BY4742 (<italic>MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0</italic>; Baker Brachmann et al. ##REF##9483801##1998##). For the experimental exposures, overnight suspension cultures were inoculated from an independent BY4742 colony on solid YPD medium (1% yeast extract, 2% peptone, 2% D-glucose, and 2% agar) and grown in synthetic medium containing 0.67% (w/v) yeast nitrogen base, 2% (w/v) glucose, 150 mM NaCl, 20 mg/L L-histidine HCl, 100 mg/L L-leucine, 30 mg/L L-lysine HCl, and 20 mg/L uracil at 28 °C with orbital shaking (120 rpm). The addition of NaCl had the purpose of mimicking the conditions in the blood plasma for further use in sensor applications. The next day, approximately 1.5 optical density units of each culture were collected by centrifugation (1700 × g for 5 min at room temperature) and resuspended in 3 ml of the same media (control) or media with lactic acid. OD600 was recorded at the beginning of the exposure and in 3–4 h. After this time, the experimental cultures were collected by centrifugation and frozen at -80 °C for RNA extraction. The relative growth rate was calculated as the difference between logarithmic (base 2) final OD600 and initial OD600 divided over incubation time in hours. This experiment was performed in total seven times with independent suspension cultures with 0.05, 0.5, 5, or 45 mM DLA, or 45 mM LLA and a control medium; three of these replicates were used for the RNA sequencing. Moreover, similar exposures were performed with eight independent suspension cultures with 45 mM DLA, 45 mM sodium D-lactate (DLS), and a control medium; five of these replicates were used for qPCR testing.</p>","<title>RNA extraction, sequencing, and quantitative PCR (qPCR)</title>","<p id=\"Par9\">RNA extraction, library construction, and sequencing were performed by the CeGaT company (Tübingen, Germany). RNA isolation was performed with the RNeasy kit (Qiagen, Hilden, Germany) according to manual (RNeasy Mini Handbook) with slight modifications. Cells were homogenized by mechanical disruption. After the addition of RLT buffer (Qiagen, Hilden, Germany) and glass beads, the samples were vortexed three times for 3 min. After each vortexing step, the samples were cooled on ice. Then, the lysate was centrifuged for 2 min at maximum speed, and the supernatant was transferred to new microcentrifuge tubes, combined with the same volume of 70% ethanol and mixed by pipetting. Sequencing libraries were prepared with the TruSeq Stranded mRNA kit (Illumina Inc., CA, USA) and sequenced at 2 × 100 bp with a NovaSeq 6000 (Illumina Inc., USA). Demultiplexing of the sequencing reads was performed with Illumina bcl2fastq v2.20, and adapters were trimmed with Skewer v 0.2.2 (Jiang et al. ##REF##24925680##2014##). For each sample, between 2.8 and 7.4 Gb were sequenced.</p>","<p id=\"Par10\">RNA extraction for qPCR-based gene expression analysis was performed with the RNASwift method according to the original protocol (Nwokeoji et al. ##REF##27495141##2016##) using GeneJET spin columns (Thermo Scientific, Waltham, MA, USA) and buffers provided with the RNeasy kit (Qiagen, Hilden, Germany) at the last step. RNA purification was performed according to the recommendation of the buffer manufacturer. Then, RNA was treated with RapidOut DNA removal kit (Thermo Scientific, Waltham, MA, USA) to remove residual genomic DNA. Then, RNA concentration was measured with the Nano-300 (Allsheng, Hangzhou, China) micro-spectrophotometer, and approximately 60–70 ng of DNA-free RNA was used for reverse transcription, which was performed with RevertAid reverse transcriptase and the corresponding buffer (Thermo Scientific, Waltham, MA, USA), RiboLock RNase inhibitor (Thermo Scientific, Waltham, MA, USA), dNTPs and Oligo(dT)18 primers (Thermo Scientific, Waltham, MA, USA) according to the recommendation of the enzyme manufacturer. Then, 1 μL of the resulting cDNA of the resulting solution was used for 10-μL qPCR. The amplification was performed using a StepOne Plus instrument (Thermo Scientific, Massachusetts, USA) with the 5X qPCRmix-HS SYBR Hi-Rox (Evrogen, Moscow, Russia) and primers (5 pmol each) specific for the following genes: <italic>ACT1</italic> and <italic>CDC19</italic> used as reference genes; <italic>AQR1</italic>; <italic>DLD3</italic>; <italic>FIT2</italic>; and <italic>YPS3</italic>. Primer sequences (Supplemental Table ##SUPPL##0##S1##) for <italic>ACT1</italic> and <italic>CDC19</italic> were taken from the work by Cankorur-Cetinkaya et al. (##REF##22675547##2012##); the other primer pairs were designed with NCBI Primer Blast (Ye et al. ##UREF##10##2012##). Amplification efficiency was tested for each primer pair with serial dilutions of a control cDNA sample and lied in the range of 88–100% (Supplemental Table ##SUPPL##0##S1##).</p>","<title>Data analysis and availability</title>","<p id=\"Par11\">Quality control of raw data was performed with FastQC v0.11.9 and summarized with MultiQC v1.13 (Ewels et al. ##REF##27312411##2016##). The R64-1–1 release of <italic>S. cerevisiae</italic> strain S288C genome (Engel et al. ##UREF##2##2014##) was downloaded from Ensembl (Cunningham et al. ##REF##34791404##2022##) release 108 and used as a reference. The reads were aligned to the genome with hisat2 (Kim et al. ##REF##31375807##2019##) v2.2.1, sorted with samtools (Li et al. ##REF##19505943##2009##) v1.9 and quantified with featureCounts (Liao et al. ##REF##24227677##2014##) from subread v2.0.4.</p>","<p id=\"Par12\">The resulting count table was further processed with the DESeq2 (Love et al. ##REF##25516281##2014##) v1.34.0 for R (R Core Team ##UREF##7##2022##) v4.1.2 to compare expression levels. The figures were prepared using the ggplot2 (Wickham ##UREF##8##2016##) v3.4.2, enhancedVolcano (Blighe et al. ##UREF##0##2023##) v1.12.0 and DEGReport (Pantano et al. ##UREF##6##2023##) v1.30.3 packages for R. The data from the Abbott et al. (##REF##18676708##2008##) manuscript, which were used for comparison, were downloaded from the NCBI GEO database (accession number GSE10066) with the script generated by the GEO2R service (Edgar et al. ##REF##11752295##2002##), which utilizes the GEOquery (Davis and Meltzer ##REF##17496320##2007##) 2.62.2, limma (Ritchie et al. ##REF##25605792##2015##) 3.50.3, and DESeq2 packages for R. Gene ontology (GO) term and publication enrichment analyses were performed with YeastMine (Balakrishnan et al. ##REF##22434830##2012##; Cherry et al. ##REF##22110037##2012##) using the database of 1 Apr 2023.</p>","<p id=\"Par13\">All the code used is available at GitHub (Drozdova ##UREF##1##2023##). The raw and processed RNA sequencing data are also available from the NCBI GEO repository under the accession number GSE231937.</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<title>Yeast cultivation</title>\",\n \"<p id=\\\"Par8\\\">The yeast strain used for this work was BY4742 (<italic>MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0</italic>; Baker Brachmann et al. ##REF##9483801##1998##). For the experimental exposures, overnight suspension cultures were inoculated from an independent BY4742 colony on solid YPD medium (1% yeast extract, 2% peptone, 2% D-glucose, and 2% agar) and grown in synthetic medium containing 0.67% (w/v) yeast nitrogen base, 2% (w/v) glucose, 150 mM NaCl, 20 mg/L L-histidine HCl, 100 mg/L L-leucine, 30 mg/L L-lysine HCl, and 20 mg/L uracil at 28 °C with orbital shaking (120 rpm). The addition of NaCl had the purpose of mimicking the conditions in the blood plasma for further use in sensor applications. The next day, approximately 1.5 optical density units of each culture were collected by centrifugation (1700 × g for 5 min at room temperature) and resuspended in 3 ml of the same media (control) or media with lactic acid. OD600 was recorded at the beginning of the exposure and in 3–4 h. After this time, the experimental cultures were collected by centrifugation and frozen at -80 °C for RNA extraction. The relative growth rate was calculated as the difference between logarithmic (base 2) final OD600 and initial OD600 divided over incubation time in hours. This experiment was performed in total seven times with independent suspension cultures with 0.05, 0.5, 5, or 45 mM DLA, or 45 mM LLA and a control medium; three of these replicates were used for the RNA sequencing. Moreover, similar exposures were performed with eight independent suspension cultures with 45 mM DLA, 45 mM sodium D-lactate (DLS), and a control medium; five of these replicates were used for qPCR testing.</p>\",\n \"<title>RNA extraction, sequencing, and quantitative PCR (qPCR)</title>\",\n \"<p id=\\\"Par9\\\">RNA extraction, library construction, and sequencing were performed by the CeGaT company (Tübingen, Germany). RNA isolation was performed with the RNeasy kit (Qiagen, Hilden, Germany) according to manual (RNeasy Mini Handbook) with slight modifications. Cells were homogenized by mechanical disruption. After the addition of RLT buffer (Qiagen, Hilden, Germany) and glass beads, the samples were vortexed three times for 3 min. After each vortexing step, the samples were cooled on ice. Then, the lysate was centrifuged for 2 min at maximum speed, and the supernatant was transferred to new microcentrifuge tubes, combined with the same volume of 70% ethanol and mixed by pipetting. Sequencing libraries were prepared with the TruSeq Stranded mRNA kit (Illumina Inc., CA, USA) and sequenced at 2 × 100 bp with a NovaSeq 6000 (Illumina Inc., USA). Demultiplexing of the sequencing reads was performed with Illumina bcl2fastq v2.20, and adapters were trimmed with Skewer v 0.2.2 (Jiang et al. ##REF##24925680##2014##). For each sample, between 2.8 and 7.4 Gb were sequenced.</p>\",\n \"<p id=\\\"Par10\\\">RNA extraction for qPCR-based gene expression analysis was performed with the RNASwift method according to the original protocol (Nwokeoji et al. ##REF##27495141##2016##) using GeneJET spin columns (Thermo Scientific, Waltham, MA, USA) and buffers provided with the RNeasy kit (Qiagen, Hilden, Germany) at the last step. RNA purification was performed according to the recommendation of the buffer manufacturer. Then, RNA was treated with RapidOut DNA removal kit (Thermo Scientific, Waltham, MA, USA) to remove residual genomic DNA. Then, RNA concentration was measured with the Nano-300 (Allsheng, Hangzhou, China) micro-spectrophotometer, and approximately 60–70 ng of DNA-free RNA was used for reverse transcription, which was performed with RevertAid reverse transcriptase and the corresponding buffer (Thermo Scientific, Waltham, MA, USA), RiboLock RNase inhibitor (Thermo Scientific, Waltham, MA, USA), dNTPs and Oligo(dT)18 primers (Thermo Scientific, Waltham, MA, USA) according to the recommendation of the enzyme manufacturer. Then, 1 μL of the resulting cDNA of the resulting solution was used for 10-μL qPCR. The amplification was performed using a StepOne Plus instrument (Thermo Scientific, Massachusetts, USA) with the 5X qPCRmix-HS SYBR Hi-Rox (Evrogen, Moscow, Russia) and primers (5 pmol each) specific for the following genes: <italic>ACT1</italic> and <italic>CDC19</italic> used as reference genes; <italic>AQR1</italic>; <italic>DLD3</italic>; <italic>FIT2</italic>; and <italic>YPS3</italic>. Primer sequences (Supplemental Table ##SUPPL##0##S1##) for <italic>ACT1</italic> and <italic>CDC19</italic> were taken from the work by Cankorur-Cetinkaya et al. (##REF##22675547##2012##); the other primer pairs were designed with NCBI Primer Blast (Ye et al. ##UREF##10##2012##). Amplification efficiency was tested for each primer pair with serial dilutions of a control cDNA sample and lied in the range of 88–100% (Supplemental Table ##SUPPL##0##S1##).</p>\",\n \"<title>Data analysis and availability</title>\",\n \"<p id=\\\"Par11\\\">Quality control of raw data was performed with FastQC v0.11.9 and summarized with MultiQC v1.13 (Ewels et al. ##REF##27312411##2016##). The R64-1–1 release of <italic>S. cerevisiae</italic> strain S288C genome (Engel et al. ##UREF##2##2014##) was downloaded from Ensembl (Cunningham et al. ##REF##34791404##2022##) release 108 and used as a reference. The reads were aligned to the genome with hisat2 (Kim et al. ##REF##31375807##2019##) v2.2.1, sorted with samtools (Li et al. ##REF##19505943##2009##) v1.9 and quantified with featureCounts (Liao et al. ##REF##24227677##2014##) from subread v2.0.4.</p>\",\n \"<p id=\\\"Par12\\\">The resulting count table was further processed with the DESeq2 (Love et al. ##REF##25516281##2014##) v1.34.0 for R (R Core Team ##UREF##7##2022##) v4.1.2 to compare expression levels. The figures were prepared using the ggplot2 (Wickham ##UREF##8##2016##) v3.4.2, enhancedVolcano (Blighe et al. ##UREF##0##2023##) v1.12.0 and DEGReport (Pantano et al. ##UREF##6##2023##) v1.30.3 packages for R. The data from the Abbott et al. (##REF##18676708##2008##) manuscript, which were used for comparison, were downloaded from the NCBI GEO database (accession number GSE10066) with the script generated by the GEO2R service (Edgar et al. ##REF##11752295##2002##), which utilizes the GEOquery (Davis and Meltzer ##REF##17496320##2007##) 2.62.2, limma (Ritchie et al. ##REF##25605792##2015##) 3.50.3, and DESeq2 packages for R. Gene ontology (GO) term and publication enrichment analyses were performed with YeastMine (Balakrishnan et al. ##REF##22434830##2012##; Cherry et al. ##REF##22110037##2012##) using the database of 1 Apr 2023.</p>\",\n \"<p id=\\\"Par13\\\">All the code used is available at GitHub (Drozdova ##UREF##1##2023##). The raw and processed RNA sequencing data are also available from the NCBI GEO repository under the accession number GSE231937.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Overview of transcriptional response to lactic acid enantiomers</title>","<p id=\"Par14\">The performed analysis revealed differentially expressed genes (hereafter DEGs; absolute log2 fold change > 1 and adjusted <italic>p</italic>-value < 0.05) only in the case of the two highest DLA concentrations (5 mM and 45 mM), as well as in the case of 45 mM LLA (Fig. ##FIG##0##1##a–c). The concentrations of 0.5 mM DLA and below did not produce any significant transcriptional response (Fig. ##FIG##0##1##d, e). Overall, the presence/absence of DEGs correlated with the growth inhibition: whenever growth was inhibited, we recorded differential expression (Supplemental Fig. ##SUPPL##0##S1##; Supplemental Table ##SUPPL##1##S2##). Finally, there were 10 genes that were differentially expressed between the maximal concentration of DLA and the same concentration of LLA (see below in the section “Transcriptional response to LLA and search for DLA-specific genes”).</p>","<p id=\"Par15\">Furthermore, we functionally characterized DEG lists with gene ontology terms using YeastMine (Table ##TAB##0##1##). We found that the genes upregulated in response to 45 mM were enriched with those participating in lactate biosynthesis and metabolism (these genes will be characterized in detail below) and siderophore transport. The genes upregulated in response to a lower concentration of DLA (5 mM) were connected to the cell wall, while those downregulated in these conditions contained three genes regulating leucine biosynthesis. In the case of 45 mM LLA, we only found one very general enriched GO term for downregulated genes, generation of precursor metabolites, and energy.\n</p>","<title>Transcriptional response to DLA</title>","<p id=\"Par16\">In the case of the highest concentration of DLA, we found significant enrichment of two GO terms connected to lactate (Table ##TAB##0##1##). The DEGs annotated with the terms “lactate metabolic process” (GO:0006089) and “lactate biosynthetic process” (GO:0019249) largely overlapped and contained <italic>DLD1</italic> (YDL174C), <italic>DLD3</italic> (YEL071W), <italic>SNO4</italic> (YMR322C), and <italic>HSP32</italic> (YPL280W). The former two genes indeed encode D-lactate dehydrogenases, mitochondrial Dld1 and cytoplasmic Dld3 (Pallotta ##REF##22460278##2012##); according to the literature, Dld3 can also oxidize D-2-hydroxyglutarate to α-ketoglutarate (Becker-Kettern et al. ##REF##26774271##2016##). The latter two are specific small chaperones (Gong et al. ##REF##19536198##2009##). Unfortunately, none of these four genes was both DLA-specific and quantitatively responding to DLA (Fig. ##FIG##1##2##a).</p>","<p id=\"Par17\">In the case of 5 mM DLA, the main groups of upregulated genes were those associated with cell wall biogenesis (Table ##TAB##0##1##; Supplemental Table ##SUPPL##0##S4##). This effect is not probably specific for DLA, as a similar effect was found for different organic acids (Kawahata et al. ##REF##16911514##2006##). These genes also reacted to 45 mM DLA, even though less strongly (Supplemental Fig. ##SUPPL##0##S2##). Overall, the transcriptional responses to 5 mM DLA and 45 mM DLA correlated quite well (Fig. ##FIG##1##2##b, c).</p>","<title>Transcriptional response to LLA and search for DLA-specific genes</title>","<p id=\"Par18\">We found fewer DEGs in response to LLA in comparison to DLA; moreover, there were no overrepresented GO terms for upregulated genes and only a rather vague term “generation of precursor metabolites and energy” in the case of downregulated genes. However, the lists of genes differentially expressed in response to LLA were associated with many (over 20) publications enriched in some of these genes (Supplemental Table ##SUPPL##0##S4##). Impressively, four of the six manuscripts enriched in LLA-upregulated genes dealt with the Haa1 transcription factor, which was indeed shown to mediate the adaptation of yeast cells to lactic and other weak acids (Fernandes et al. ##REF##16176797##2005##; Mira et al. ##UREF##5##2010##, ##REF##21586585##2011##; Sugiyama et al. ##REF##24682296##2014##).</p>","<p id=\"Par19\">Generally, the changes triggered by LLA correlated well to the changes observed in response to the same concentration of DLA (Fig. ##FIG##2##3##a, b). In order to reveal if there were any genes that quantitatively responded to DLA and did not respond to LLA, we performed a clustering analysis of 214 genes that were differentially expressed in at least one condition. Within the obtained six clusters, none had the desired pattern for a DLA sensor, i.e., monotonous increase or decrease in response to DLA and absence or very slight response to LLA (Fig. ##FIG##2##3##c). It is worth mentioning that the first cluster featured genes which were seemingly affected more by the high concentrations of DLA than by LLA, but in fact, the changes were very subtle (Supplemental Fig. ##SUPPL##0##S3##).</p>","<p id=\"Par20\">In addition, we analyzed the genes differentially expressed between 45 mM DLA and 45 mM LLA (Fig. ##FIG##0##1##f). There were ten such genes, YHL028W (<italic>WSC4</italic>), YLR054C (<italic>OSW2</italic>), YLR121C (<italic>YPS3</italic>), YGR189C (<italic>CRH1</italic>), YGR146C (<italic>ECL1</italic>), YGL255W (<italic>ZRT1</italic>), YHR209W (<italic>CRG1</italic>), YLR205C (<italic>HMX1</italic>), YKR091W (<italic>SRL3</italic>), and YCR005C (<italic>CIT2</italic>). Some of these genes (<italic>WSC4</italic>, <italic>YPS3</italic>, <italic>CHR1</italic>, and <italic>OSW2</italic>) regulate cell wall assembly. Wsc4, Yps3, and Crh1 have functions in maintaining cell wall integrity (Verna et al. ##REF##9391108##1997##; Krysan et al. ##REF##16087741##2005##; Cabib et al. ##REF##17302808##2007##). Osw2 is a protein of unknown function, which is putatively involved in spore wall assembly (Coluccio et al. ##REF##15590821##2004##). Several genes (<italic>ZRT1</italic>, <italic>ECL1</italic>, and <italic>HMX1</italic>) are implicated in metal transport. Zrt1 is a zinc transporter (Zhao and Eide ##REF##8637895##1996##). Ecl1 is a protein of unknown function upregulated by overexpression of the other iron deprivation-responding transcription factor, Aft2 (Rutherford et al. ##REF##12756250##2003##). <italic>HMX1</italic> encodes a heme oxygenase, and expression of this gene is regulated by the iron deprivation-responding transcription factor Aft1 (Protchenko and Philpott ##REF##12840010##2003##). Finally, the link between some of the genes and LA stress was unclear. Crg1 is a small molecule methyltransferase regulating lipid homeostasis in response to a drug cantharidin (Lissina et al. ##REF##22028670##2011##), Cit2 is a citrate synthase (Kim et al. ##REF##3023912##1986##), and Srl3 (or Whi7) participates in cell cycle regulation (Gomar-Alba et al. ##REF##28839131##2017##). Of these genes, five reacted to both 5 mM and 45 mM DLA but not to 45 mM LLA (Fig. ##FIG##2##3##d). These could be candidates for qualitative sensors for DLA but required further exploration.</p>","<title>Neutralization compensates for the effect of the DLA on growth rate and transcription of selected genes</title>","<p id=\"Par21\">During all previous analyses, we found several groups of genes that reacted to one (45 mM) or two (5 and 45 mM) concentrations of DLA, and we also found that these treatments slowed down yeast growth (Supplemental Fig. ##SUPPL##0##S1##). In order to check if the slow growth was caused by the lower pH values, we performed the same treatment but with 45 mM sodium D-lactate (DLS) obtained with addition of the same amount of NaOH (the concentration of additional NaCl in the media was adjusted to sum up to 150 mM) and indeed observed compensation of the growth defect (Fig. ##FIG##3##4##a). Thus, the observed slow growth in yeast treated with 45 mM DLA is explained by pH shift and not by the influence of the D-lactate ion.</p>","<p id=\"Par22\">Moreover, we checked if the expression of several genes that we found to be DLA-responsive changed in response to DLS. For this analysis, we chose the <italic>AQR1</italic>, <italic>DLD3</italic>, and <italic>FIT2</italic> genes, which were activated to 45 mM DLA but did not respond to 5 mM DLA, as well as the <italic>YPS3</italic> gene that responded to both concentrations of DLA. These genes belong to different functional groups. <italic>AQR1</italic> (YNL065W) encodes a membrane protein from the major facilitator superfamily, which provides the cells with resistance to short-chain monocarboxylic acids (Tenreiro et al. ##REF##11922628##2002##). <italic>FI</italic><italic>T2</italic> (YOR382W) is a cell wall mannoprotein involved in the siderophore transport (Protchenko et al. ##REF##11673473##2001##), and we chose it as a representative of a larger group of iron uptake-related proteins (Table ##TAB##0##1##). <italic>DLD3</italic> (YEL071W) codes for a protein with D-lactate dehydrogenase activity in vitro, but there is evidence that in vivo it contributes to D-lactate synthesis (Chelstowska et al. ##UREF##11##1999##; Becker-Kettern et al. ##REF##26774271##2016##). Finally, <italic>YPS3</italic> (YLR121C) encodes an aspartic protease required for cell wall integrity (Olsen et al. ##REF##10191273##1999##), and we chose it as a representative of the cell wall integrity-related genes and also because it had strong changes in expression in response to both DLA concentrations but not to LLA (Fig. ##FIG##2##3##d) and thus could act as qualitative DLA sensor. However, we found that the relative expression levels of all of these genes were very similar in the control samples and in those treated with 45 mM DLS. Taken together, our data suggest that the expression changes in response to DLA were mostly triggered by the change in the pH value.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Overview of transcriptional response to lactic acid enantiomers</title>\",\n \"<p id=\\\"Par14\\\">The performed analysis revealed differentially expressed genes (hereafter DEGs; absolute log2 fold change > 1 and adjusted <italic>p</italic>-value < 0.05) only in the case of the two highest DLA concentrations (5 mM and 45 mM), as well as in the case of 45 mM LLA (Fig. ##FIG##0##1##a–c). The concentrations of 0.5 mM DLA and below did not produce any significant transcriptional response (Fig. ##FIG##0##1##d, e). Overall, the presence/absence of DEGs correlated with the growth inhibition: whenever growth was inhibited, we recorded differential expression (Supplemental Fig. ##SUPPL##0##S1##; Supplemental Table ##SUPPL##1##S2##). Finally, there were 10 genes that were differentially expressed between the maximal concentration of DLA and the same concentration of LLA (see below in the section “Transcriptional response to LLA and search for DLA-specific genes”).</p>\",\n \"<p id=\\\"Par15\\\">Furthermore, we functionally characterized DEG lists with gene ontology terms using YeastMine (Table ##TAB##0##1##). We found that the genes upregulated in response to 45 mM were enriched with those participating in lactate biosynthesis and metabolism (these genes will be characterized in detail below) and siderophore transport. The genes upregulated in response to a lower concentration of DLA (5 mM) were connected to the cell wall, while those downregulated in these conditions contained three genes regulating leucine biosynthesis. In the case of 45 mM LLA, we only found one very general enriched GO term for downregulated genes, generation of precursor metabolites, and energy.\\n</p>\",\n \"<title>Transcriptional response to DLA</title>\",\n \"<p id=\\\"Par16\\\">In the case of the highest concentration of DLA, we found significant enrichment of two GO terms connected to lactate (Table ##TAB##0##1##). The DEGs annotated with the terms “lactate metabolic process” (GO:0006089) and “lactate biosynthetic process” (GO:0019249) largely overlapped and contained <italic>DLD1</italic> (YDL174C), <italic>DLD3</italic> (YEL071W), <italic>SNO4</italic> (YMR322C), and <italic>HSP32</italic> (YPL280W). The former two genes indeed encode D-lactate dehydrogenases, mitochondrial Dld1 and cytoplasmic Dld3 (Pallotta ##REF##22460278##2012##); according to the literature, Dld3 can also oxidize D-2-hydroxyglutarate to α-ketoglutarate (Becker-Kettern et al. ##REF##26774271##2016##). The latter two are specific small chaperones (Gong et al. ##REF##19536198##2009##). Unfortunately, none of these four genes was both DLA-specific and quantitatively responding to DLA (Fig. ##FIG##1##2##a).</p>\",\n \"<p id=\\\"Par17\\\">In the case of 5 mM DLA, the main groups of upregulated genes were those associated with cell wall biogenesis (Table ##TAB##0##1##; Supplemental Table ##SUPPL##0##S4##). This effect is not probably specific for DLA, as a similar effect was found for different organic acids (Kawahata et al. ##REF##16911514##2006##). These genes also reacted to 45 mM DLA, even though less strongly (Supplemental Fig. ##SUPPL##0##S2##). Overall, the transcriptional responses to 5 mM DLA and 45 mM DLA correlated quite well (Fig. ##FIG##1##2##b, c).</p>\",\n \"<title>Transcriptional response to LLA and search for DLA-specific genes</title>\",\n \"<p id=\\\"Par18\\\">We found fewer DEGs in response to LLA in comparison to DLA; moreover, there were no overrepresented GO terms for upregulated genes and only a rather vague term “generation of precursor metabolites and energy” in the case of downregulated genes. However, the lists of genes differentially expressed in response to LLA were associated with many (over 20) publications enriched in some of these genes (Supplemental Table ##SUPPL##0##S4##). Impressively, four of the six manuscripts enriched in LLA-upregulated genes dealt with the Haa1 transcription factor, which was indeed shown to mediate the adaptation of yeast cells to lactic and other weak acids (Fernandes et al. ##REF##16176797##2005##; Mira et al. ##UREF##5##2010##, ##REF##21586585##2011##; Sugiyama et al. ##REF##24682296##2014##).</p>\",\n \"<p id=\\\"Par19\\\">Generally, the changes triggered by LLA correlated well to the changes observed in response to the same concentration of DLA (Fig. ##FIG##2##3##a, b). In order to reveal if there were any genes that quantitatively responded to DLA and did not respond to LLA, we performed a clustering analysis of 214 genes that were differentially expressed in at least one condition. Within the obtained six clusters, none had the desired pattern for a DLA sensor, i.e., monotonous increase or decrease in response to DLA and absence or very slight response to LLA (Fig. ##FIG##2##3##c). It is worth mentioning that the first cluster featured genes which were seemingly affected more by the high concentrations of DLA than by LLA, but in fact, the changes were very subtle (Supplemental Fig. ##SUPPL##0##S3##).</p>\",\n \"<p id=\\\"Par20\\\">In addition, we analyzed the genes differentially expressed between 45 mM DLA and 45 mM LLA (Fig. ##FIG##0##1##f). There were ten such genes, YHL028W (<italic>WSC4</italic>), YLR054C (<italic>OSW2</italic>), YLR121C (<italic>YPS3</italic>), YGR189C (<italic>CRH1</italic>), YGR146C (<italic>ECL1</italic>), YGL255W (<italic>ZRT1</italic>), YHR209W (<italic>CRG1</italic>), YLR205C (<italic>HMX1</italic>), YKR091W (<italic>SRL3</italic>), and YCR005C (<italic>CIT2</italic>). Some of these genes (<italic>WSC4</italic>, <italic>YPS3</italic>, <italic>CHR1</italic>, and <italic>OSW2</italic>) regulate cell wall assembly. Wsc4, Yps3, and Crh1 have functions in maintaining cell wall integrity (Verna et al. ##REF##9391108##1997##; Krysan et al. ##REF##16087741##2005##; Cabib et al. ##REF##17302808##2007##). Osw2 is a protein of unknown function, which is putatively involved in spore wall assembly (Coluccio et al. ##REF##15590821##2004##). Several genes (<italic>ZRT1</italic>, <italic>ECL1</italic>, and <italic>HMX1</italic>) are implicated in metal transport. Zrt1 is a zinc transporter (Zhao and Eide ##REF##8637895##1996##). Ecl1 is a protein of unknown function upregulated by overexpression of the other iron deprivation-responding transcription factor, Aft2 (Rutherford et al. ##REF##12756250##2003##). <italic>HMX1</italic> encodes a heme oxygenase, and expression of this gene is regulated by the iron deprivation-responding transcription factor Aft1 (Protchenko and Philpott ##REF##12840010##2003##). Finally, the link between some of the genes and LA stress was unclear. Crg1 is a small molecule methyltransferase regulating lipid homeostasis in response to a drug cantharidin (Lissina et al. ##REF##22028670##2011##), Cit2 is a citrate synthase (Kim et al. ##REF##3023912##1986##), and Srl3 (or Whi7) participates in cell cycle regulation (Gomar-Alba et al. ##REF##28839131##2017##). Of these genes, five reacted to both 5 mM and 45 mM DLA but not to 45 mM LLA (Fig. ##FIG##2##3##d). These could be candidates for qualitative sensors for DLA but required further exploration.</p>\",\n \"<title>Neutralization compensates for the effect of the DLA on growth rate and transcription of selected genes</title>\",\n \"<p id=\\\"Par21\\\">During all previous analyses, we found several groups of genes that reacted to one (45 mM) or two (5 and 45 mM) concentrations of DLA, and we also found that these treatments slowed down yeast growth (Supplemental Fig. ##SUPPL##0##S1##). In order to check if the slow growth was caused by the lower pH values, we performed the same treatment but with 45 mM sodium D-lactate (DLS) obtained with addition of the same amount of NaOH (the concentration of additional NaCl in the media was adjusted to sum up to 150 mM) and indeed observed compensation of the growth defect (Fig. ##FIG##3##4##a). Thus, the observed slow growth in yeast treated with 45 mM DLA is explained by pH shift and not by the influence of the D-lactate ion.</p>\",\n \"<p id=\\\"Par22\\\">Moreover, we checked if the expression of several genes that we found to be DLA-responsive changed in response to DLS. For this analysis, we chose the <italic>AQR1</italic>, <italic>DLD3</italic>, and <italic>FIT2</italic> genes, which were activated to 45 mM DLA but did not respond to 5 mM DLA, as well as the <italic>YPS3</italic> gene that responded to both concentrations of DLA. These genes belong to different functional groups. <italic>AQR1</italic> (YNL065W) encodes a membrane protein from the major facilitator superfamily, which provides the cells with resistance to short-chain monocarboxylic acids (Tenreiro et al. ##REF##11922628##2002##). <italic>FI</italic><italic>T2</italic> (YOR382W) is a cell wall mannoprotein involved in the siderophore transport (Protchenko et al. ##REF##11673473##2001##), and we chose it as a representative of a larger group of iron uptake-related proteins (Table ##TAB##0##1##). <italic>DLD3</italic> (YEL071W) codes for a protein with D-lactate dehydrogenase activity in vitro, but there is evidence that in vivo it contributes to D-lactate synthesis (Chelstowska et al. ##UREF##11##1999##; Becker-Kettern et al. ##REF##26774271##2016##). Finally, <italic>YPS3</italic> (YLR121C) encodes an aspartic protease required for cell wall integrity (Olsen et al. ##REF##10191273##1999##), and we chose it as a representative of the cell wall integrity-related genes and also because it had strong changes in expression in response to both DLA concentrations but not to LLA (Fig. ##FIG##2##3##d) and thus could act as qualitative DLA sensor. However, we found that the relative expression levels of all of these genes were very similar in the control samples and in those treated with 45 mM DLS. Taken together, our data suggest that the expression changes in response to DLA were mostly triggered by the change in the pH value.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par23\">In this study, we explored the transcriptional response of <italic>S. cerevisiae</italic> to LA enantiomers. One of the goals of this work was to find genes that would specifically and quantitatively respond to DLA but not to LLA. We did not find such genes. In general, we found that the response to high concentration of DLA and LLA was quite similar and even more pronounced to DLA than to LLA. It is possible that the reason for this difference is that LLA is metabolized faster. There was no response to the concentrations of DLA of 0.5 mM and below. It is possible that higher DLA concentrations (> 50 mM) or longer exposures (about a day) could have made the effect more pronounced and reveal differentially expressed genes. However, higher concentrations would be outside the range of DLA concentrations typically found in biological fluids. While normal levels of LLA in mammal blood plasma are about 1–2 mM, the levels of DLA are about two orders of magnitude lower, 0.01–0.07 mM (Ewaschuk et al. ##REF##15987839##2005##). In general, the levels of < 0.2 mM are considered normal (Zhang et al. ##REF##12612331##2003##). The most well-known condition leading to D-lactic acidosis in human is short bowel syndrome, during which plasma DLA levels may reach millimolar concentrations (Zhang et al. ##REF##12612331##2003##; Yilmaz et al. ##REF##30089656##2018##). Similar symptoms in ruminants appear upon elevated carbohydrate level in their diet (Lorenz and Gentile ##REF##24980724##2014##) and may lead to DLA plasma levels as high as about 25 mM (Ewaschuk et al. ##REF##15987839##2005##). Thus, an ideal biosensor for DLA should be sensitive at 0.1–5 mM concentration range. Similarly, the need to use longer exposures of sensor yeast would also hinder its usage in biosensor applications. So, we find it unlikely that a quantitative yeast sensor to DLA may be constructed based on the native yeast transcriptional networks, but the possibility of integrating a heterologous cassette remains open. Such a cassette has been described for <italic>Pseudomonas</italic>, but its sensitivity starts from about 20 mM DLA (Singh et al. ##REF##31517075##2019##), which is also far from ideal for monitoring DLA levels in biological fluids.</p>","<p id=\"Par24\">Importantly, we present the first dataset on yeast transcriptional response to DLA. Generally, we found that if the particular concentration did not inhibit growth, we did not see a transcriptional response either. This result is very similar to the study, in which the authors compared the transcriptome-wide responses to different alcohols in search for 1-butanol sensor and found that the samples treated with ethanol, which did not cause significant growth inhibition, clustered with the control samples, while samples treated with 1-butanol and 1-propanol, which were much more toxic, clustered separately (Shi et al. ##REF##28712783##2017##). Intriguingly, the response to 5 mM and 45 mM was seemingly different if judging by enriched GO terms (5 mM DLA caused overexpression of cell wall-related genes, while 45 mM led to increased expression of lactate metabolism and siderophore transport genes), but the transcriptional profiles in response to 5 mM and 45 mM DLA were highly correlated (Fig. ##FIG##1##2##c), and more than half of the DEGs were shared between the two comparisons (Fig. ##FIG##0##1##g, h).</p>","<p id=\"Par25\">In general, the groups of cell-wall related genes and genes of iron/siderophore uptake have already been described to respond to weak acid stress (Kawahata et al. ##REF##16911514##2006##; Abbott et al. ##REF##17484738##2007##, ##REF##18676708##2008##), so our findings fully corroborate the previously published data but at the same time suggest that this response might be pH-dependent rather than specific for the lactic acid. However, comparison of four weak acids, benzoate, sorbate, acetate, and propionate, showed that the transcriptional responses were largely specific (Abbott et al. ##REF##17484738##2007##), proving that the response to pH was not the only reason for gene expression changes. We have checked this hypothesis for the <italic>FIT2</italic> gene, which codes for cell wall mannoprotein involved in the siderophore transport (Protchenko et al. ##REF##11673473##2001##) as a representative of the iron/siderophore uptake functional group and <italic>YPS3</italic>, the gene encoding an aspartic protease required for cell wall integrity (Olsen et al. ##REF##10191273##1999##), as a representative of the cell wall genes group. Both genes did not respond to D-lactate treatment if pH was compensated (Fig. ##FIG##3##4##b). Moreover, in this experiment we also measured the expression levels of the <italic>AQR1</italic> gene. It encodes a membrane protein from the major facilitator superfamily, which provides the cells with resistance to short-chain monocarboxylic acids (Tenreiro et al. ##REF##11922628##2002##). We found that <italic>AQR1</italic> also only responded to DLA at low pH. Interestingly, the authors of the original manuscript noted that the expression of this gene was not stimulated by weak acid stress (Tenreiro et al. ##REF##11922628##2002##). We found that it was upregulated in response to 45 mM DLA, while Abbott et al. (##REF##17484738##2007##) found it as a common gene downregulated in response to the four weak organic acids they tested. Aqr1 has not been shown to have a role in lactic acid transport, but it acts as a lactic acid exporter and has been shown to be important for yeast co-cultivation with lactic acid bacteria (Velasco et al. ##REF##15590823##2004##; Kapetanakis et al. ##REF##34887841##2021##).</p>","<p id=\"Par26\">While the transcriptional response to DLA has not been explored before, there are studies on transcriptional changes in response to LLA (Kawahata et al. ##REF##16911514##2006##; Abbott et al. ##REF##18676708##2008##). We have compared the changes in the genes differentially expressed in response to 45 mM LLA according to our data and each of the two studies and found substantial positive correlation (Supplemental Fig. ##SUPPL##0##S4##), even though all experimental designs were different.</p>","<p id=\"Par27\">In the case of the work by Kawahata et al. (##REF##16911514##2006##), experimental exposures were performed in shake flask cultures with 0.3% LLA (about 33 mM), and the S288C strain (parental to BY472) was used. Two experimental designs were used. First, acid shock was performed by pre-growing the cultures to the optical density at 660 nm (OD660) of 1.0 and exposing them to LLA for 30 min. The second design, acid adaptation, involved diluting overnight cultures to the OD660 = 0.1 in the media with LLA and growing until OD660 reached 1. The number of overlapping DEGs was quite low in both cases (five genes), but the changes in the transcription of these genes were mostly similar to our results (Supplemental Fig. ##SUPPL##0##S4##a, b).</p>","<p id=\"Par28\">In the study by Abbott et al. (##REF##18676708##2008##), chemostat cultures of the CEN.PK 113-7D strain (not closely related to S288C and BY4742) were subjected to quite high concentrations of LLA, namely, 500 mM LLA at pH 3 and 900 mM LLA at pH 5. The lists of DEGs shared in our results and these data were larger (over 30 genes in each case), and the correlation of our 45 mM-LLA exposure was much higher with 500 mM LLA than with 900 mM LLA.</p>","<p id=\"Par29\">We were particularly interested in the D-lactate metabolism genes <italic>DLD1</italic> and <italic>DLD3</italic>. Originally, both of these genes were shown to code for a mitochondrial and cytoplasmic D-lactate dehydrogenases, respectively (Lodi and Ferrero ##REF##8492799##1993##; Chelstowska et al. ##UREF##11##1999##). According to our results (Fig. ##FIG##1##2##a; Supplemental Table ##SUPPL##2##S3##), <italic>DLD1</italic> was upregulated in response to 5 mM DLA (fold change = 1.92 and adjusted <italic>p</italic> = 0.04), as well as in response to 45 mM DLA (fold change = 2.76 and adjusted <italic>p</italic> = 0.0001) and had a similar trend in response to 45 mM LLA, even though the difference did not reach statistical significance (fold change = 1.85 and adjusted <italic>p</italic> = 0.06). Interestingly, Abbott et al. (##REF##18676708##2008##) also found upregulation of <italic>DLD1</italic> to the highest LLA concentration used in their experimental design, 900 mM (fold change = 4 and adjusted <italic>p</italic> = 0.0002). This non-stereo-specific regulation could be interesting to explore further.</p>","<p id=\"Par30\">While the Dld1 enzyme is the major D-lactate dehydrogenase, Dld3 is a minor D-lactate dehydrogenase and mostly acts as a transhydrogenase coupling D-2-hydroxyglutarate degradation to DLA synthesis (Lodi and Ferrero ##REF##8492799##1993##; Chestowska et al. 1999; Becker-Kettern et al. ##REF##26774271##2016##). In our experiment, <italic>DLD3</italic> was only upregulated in response to 45 mM DLA but not 5 mM DLA or lower concentrations and did not react to DLS, also corroborating the idea of its very minor role as a D-lactate dehygrogenase. It is possible that this protein acts at high DLA concentrations to prevent cell damage by low pH.</p>","<p id=\"Par31\">In general, our data enrich our understanding of the yeast transcriptome-wide response to LLA and provide the first description of the response to DLA. We found that even though the response to different stereoisomers of lactic acid had quite significant similarities to the response to other weak acids tested previously and largely dependent on pH, there are large differences between DLA and LLA responses, which probably reflect the difference in their role in yeast biology. The role of pH in the DLA response highlights the importance of controlling and optimizing lactic acid production in yeast under neutralizing and non-neutralizing conditions separately, which is also corroborated by a recent work on a recombinant yeast strain with improved lactic acid tolerance and lactic acid yield under non-neutralizing conditions (Yamada et al. ##UREF##9##2021##).</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par23\\\">In this study, we explored the transcriptional response of <italic>S. cerevisiae</italic> to LA enantiomers. One of the goals of this work was to find genes that would specifically and quantitatively respond to DLA but not to LLA. We did not find such genes. In general, we found that the response to high concentration of DLA and LLA was quite similar and even more pronounced to DLA than to LLA. It is possible that the reason for this difference is that LLA is metabolized faster. There was no response to the concentrations of DLA of 0.5 mM and below. It is possible that higher DLA concentrations (> 50 mM) or longer exposures (about a day) could have made the effect more pronounced and reveal differentially expressed genes. However, higher concentrations would be outside the range of DLA concentrations typically found in biological fluids. While normal levels of LLA in mammal blood plasma are about 1–2 mM, the levels of DLA are about two orders of magnitude lower, 0.01–0.07 mM (Ewaschuk et al. ##REF##15987839##2005##). In general, the levels of < 0.2 mM are considered normal (Zhang et al. ##REF##12612331##2003##). The most well-known condition leading to D-lactic acidosis in human is short bowel syndrome, during which plasma DLA levels may reach millimolar concentrations (Zhang et al. ##REF##12612331##2003##; Yilmaz et al. ##REF##30089656##2018##). Similar symptoms in ruminants appear upon elevated carbohydrate level in their diet (Lorenz and Gentile ##REF##24980724##2014##) and may lead to DLA plasma levels as high as about 25 mM (Ewaschuk et al. ##REF##15987839##2005##). Thus, an ideal biosensor for DLA should be sensitive at 0.1–5 mM concentration range. Similarly, the need to use longer exposures of sensor yeast would also hinder its usage in biosensor applications. So, we find it unlikely that a quantitative yeast sensor to DLA may be constructed based on the native yeast transcriptional networks, but the possibility of integrating a heterologous cassette remains open. Such a cassette has been described for <italic>Pseudomonas</italic>, but its sensitivity starts from about 20 mM DLA (Singh et al. ##REF##31517075##2019##), which is also far from ideal for monitoring DLA levels in biological fluids.</p>\",\n \"<p id=\\\"Par24\\\">Importantly, we present the first dataset on yeast transcriptional response to DLA. Generally, we found that if the particular concentration did not inhibit growth, we did not see a transcriptional response either. This result is very similar to the study, in which the authors compared the transcriptome-wide responses to different alcohols in search for 1-butanol sensor and found that the samples treated with ethanol, which did not cause significant growth inhibition, clustered with the control samples, while samples treated with 1-butanol and 1-propanol, which were much more toxic, clustered separately (Shi et al. ##REF##28712783##2017##). Intriguingly, the response to 5 mM and 45 mM was seemingly different if judging by enriched GO terms (5 mM DLA caused overexpression of cell wall-related genes, while 45 mM led to increased expression of lactate metabolism and siderophore transport genes), but the transcriptional profiles in response to 5 mM and 45 mM DLA were highly correlated (Fig. ##FIG##1##2##c), and more than half of the DEGs were shared between the two comparisons (Fig. ##FIG##0##1##g, h).</p>\",\n \"<p id=\\\"Par25\\\">In general, the groups of cell-wall related genes and genes of iron/siderophore uptake have already been described to respond to weak acid stress (Kawahata et al. ##REF##16911514##2006##; Abbott et al. ##REF##17484738##2007##, ##REF##18676708##2008##), so our findings fully corroborate the previously published data but at the same time suggest that this response might be pH-dependent rather than specific for the lactic acid. However, comparison of four weak acids, benzoate, sorbate, acetate, and propionate, showed that the transcriptional responses were largely specific (Abbott et al. ##REF##17484738##2007##), proving that the response to pH was not the only reason for gene expression changes. We have checked this hypothesis for the <italic>FIT2</italic> gene, which codes for cell wall mannoprotein involved in the siderophore transport (Protchenko et al. ##REF##11673473##2001##) as a representative of the iron/siderophore uptake functional group and <italic>YPS3</italic>, the gene encoding an aspartic protease required for cell wall integrity (Olsen et al. ##REF##10191273##1999##), as a representative of the cell wall genes group. Both genes did not respond to D-lactate treatment if pH was compensated (Fig. ##FIG##3##4##b). Moreover, in this experiment we also measured the expression levels of the <italic>AQR1</italic> gene. It encodes a membrane protein from the major facilitator superfamily, which provides the cells with resistance to short-chain monocarboxylic acids (Tenreiro et al. ##REF##11922628##2002##). We found that <italic>AQR1</italic> also only responded to DLA at low pH. Interestingly, the authors of the original manuscript noted that the expression of this gene was not stimulated by weak acid stress (Tenreiro et al. ##REF##11922628##2002##). We found that it was upregulated in response to 45 mM DLA, while Abbott et al. (##REF##17484738##2007##) found it as a common gene downregulated in response to the four weak organic acids they tested. Aqr1 has not been shown to have a role in lactic acid transport, but it acts as a lactic acid exporter and has been shown to be important for yeast co-cultivation with lactic acid bacteria (Velasco et al. ##REF##15590823##2004##; Kapetanakis et al. ##REF##34887841##2021##).</p>\",\n \"<p id=\\\"Par26\\\">While the transcriptional response to DLA has not been explored before, there are studies on transcriptional changes in response to LLA (Kawahata et al. ##REF##16911514##2006##; Abbott et al. ##REF##18676708##2008##). We have compared the changes in the genes differentially expressed in response to 45 mM LLA according to our data and each of the two studies and found substantial positive correlation (Supplemental Fig. ##SUPPL##0##S4##), even though all experimental designs were different.</p>\",\n \"<p id=\\\"Par27\\\">In the case of the work by Kawahata et al. (##REF##16911514##2006##), experimental exposures were performed in shake flask cultures with 0.3% LLA (about 33 mM), and the S288C strain (parental to BY472) was used. Two experimental designs were used. First, acid shock was performed by pre-growing the cultures to the optical density at 660 nm (OD660) of 1.0 and exposing them to LLA for 30 min. The second design, acid adaptation, involved diluting overnight cultures to the OD660 = 0.1 in the media with LLA and growing until OD660 reached 1. The number of overlapping DEGs was quite low in both cases (five genes), but the changes in the transcription of these genes were mostly similar to our results (Supplemental Fig. ##SUPPL##0##S4##a, b).</p>\",\n \"<p id=\\\"Par28\\\">In the study by Abbott et al. (##REF##18676708##2008##), chemostat cultures of the CEN.PK 113-7D strain (not closely related to S288C and BY4742) were subjected to quite high concentrations of LLA, namely, 500 mM LLA at pH 3 and 900 mM LLA at pH 5. The lists of DEGs shared in our results and these data were larger (over 30 genes in each case), and the correlation of our 45 mM-LLA exposure was much higher with 500 mM LLA than with 900 mM LLA.</p>\",\n \"<p id=\\\"Par29\\\">We were particularly interested in the D-lactate metabolism genes <italic>DLD1</italic> and <italic>DLD3</italic>. Originally, both of these genes were shown to code for a mitochondrial and cytoplasmic D-lactate dehydrogenases, respectively (Lodi and Ferrero ##REF##8492799##1993##; Chelstowska et al. ##UREF##11##1999##). According to our results (Fig. ##FIG##1##2##a; Supplemental Table ##SUPPL##2##S3##), <italic>DLD1</italic> was upregulated in response to 5 mM DLA (fold change = 1.92 and adjusted <italic>p</italic> = 0.04), as well as in response to 45 mM DLA (fold change = 2.76 and adjusted <italic>p</italic> = 0.0001) and had a similar trend in response to 45 mM LLA, even though the difference did not reach statistical significance (fold change = 1.85 and adjusted <italic>p</italic> = 0.06). Interestingly, Abbott et al. (##REF##18676708##2008##) also found upregulation of <italic>DLD1</italic> to the highest LLA concentration used in their experimental design, 900 mM (fold change = 4 and adjusted <italic>p</italic> = 0.0002). This non-stereo-specific regulation could be interesting to explore further.</p>\",\n \"<p id=\\\"Par30\\\">While the Dld1 enzyme is the major D-lactate dehydrogenase, Dld3 is a minor D-lactate dehydrogenase and mostly acts as a transhydrogenase coupling D-2-hydroxyglutarate degradation to DLA synthesis (Lodi and Ferrero ##REF##8492799##1993##; Chestowska et al. 1999; Becker-Kettern et al. ##REF##26774271##2016##). In our experiment, <italic>DLD3</italic> was only upregulated in response to 45 mM DLA but not 5 mM DLA or lower concentrations and did not react to DLS, also corroborating the idea of its very minor role as a D-lactate dehygrogenase. It is possible that this protein acts at high DLA concentrations to prevent cell damage by low pH.</p>\",\n \"<p id=\\\"Par31\\\">In general, our data enrich our understanding of the yeast transcriptome-wide response to LLA and provide the first description of the response to DLA. We found that even though the response to different stereoisomers of lactic acid had quite significant similarities to the response to other weak acids tested previously and largely dependent on pH, there are large differences between DLA and LLA responses, which probably reflect the difference in their role in yeast biology. The role of pH in the DLA response highlights the importance of controlling and optimizing lactic acid production in yeast under neutralizing and non-neutralizing conditions separately, which is also corroborated by a recent work on a recombinant yeast strain with improved lactic acid tolerance and lactic acid yield under non-neutralizing conditions (Yamada et al. ##UREF##9##2021##).</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<title>Abstract</title>","<p id=\"Par1\">The model yeast, <italic>Saccharomyces cerevisiae</italic>, is a popular object for both fundamental and applied research, including the development of biosensors and industrial production of pharmaceutical compounds. However, despite multiple studies exploring <italic>S. cerevisiae</italic> transcriptional response to various substances, this response is unknown for some substances produced in yeast, such as D-lactic acid (DLA). Here, we explore the transcriptional response of the BY4742 strain to a wide range of DLA concentrations (from 0.05 to 45 mM), and compare it to the response to 45 mM L-lactic acid (LLA). We recorded a response to 5 and 45 mM DLA (125 and 113 differentially expressed genes (DEGs), respectively; > 50% shared) and a less pronounced response to 45 mM LLA (63 DEGs; > 30% shared with at least one DLA treatment). Our data did not reveal natural yeast promoters quantitatively sensing DLA but provide the first description of the transcriptome-wide response to DLA and enrich our understanding of the LLA response. Some DLA-activated genes were indeed related to lactate metabolism, as well as iron uptake and cell wall structure. Additional analyses showed that at least some of these genes were activated only by acidic form of DLA but not its salt, revealing the role of pH. The list of LLA-responsive genes was similar to those published previously and also included iron uptake and cell wall genes, as well as genes responding to other weak acids. These data might be instrumental for optimization of lactate production in yeast and yeast co-cultivation with lactic acid bacteria.</p>","<title>Key points</title>","<p id=\"Par2\">\n<italic>• We present the first dataset on yeast transcriptional response to DLA.</italic>\n</p>","<p id=\"Par3\">\n<italic>• Differential gene expression was correlated with yeast growth inhibition.</italic>\n</p>","<p id=\"Par4\">\n<italic>• The transcriptome response to DLA was richer in comparison to LLA.</italic>\n</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00253-023-12863-z.</p>","<title>Keywords</title>"],"string":"[\n \"<title>Abstract</title>\",\n \"<p id=\\\"Par1\\\">The model yeast, <italic>Saccharomyces cerevisiae</italic>, is a popular object for both fundamental and applied research, including the development of biosensors and industrial production of pharmaceutical compounds. However, despite multiple studies exploring <italic>S. cerevisiae</italic> transcriptional response to various substances, this response is unknown for some substances produced in yeast, such as D-lactic acid (DLA). Here, we explore the transcriptional response of the BY4742 strain to a wide range of DLA concentrations (from 0.05 to 45 mM), and compare it to the response to 45 mM L-lactic acid (LLA). We recorded a response to 5 and 45 mM DLA (125 and 113 differentially expressed genes (DEGs), respectively; > 50% shared) and a less pronounced response to 45 mM LLA (63 DEGs; > 30% shared with at least one DLA treatment). Our data did not reveal natural yeast promoters quantitatively sensing DLA but provide the first description of the transcriptome-wide response to DLA and enrich our understanding of the LLA response. Some DLA-activated genes were indeed related to lactate metabolism, as well as iron uptake and cell wall structure. Additional analyses showed that at least some of these genes were activated only by acidic form of DLA but not its salt, revealing the role of pH. The list of LLA-responsive genes was similar to those published previously and also included iron uptake and cell wall genes, as well as genes responding to other weak acids. These data might be instrumental for optimization of lactate production in yeast and yeast co-cultivation with lactic acid bacteria.</p>\",\n \"<title>Key points</title>\",\n \"<p id=\\\"Par2\\\">\\n<italic>• We present the first dataset on yeast transcriptional response to DLA.</italic>\\n</p>\",\n \"<p id=\\\"Par3\\\">\\n<italic>• Differential gene expression was correlated with yeast growth inhibition.</italic>\\n</p>\",\n \"<p id=\\\"Par4\\\">\\n<italic>• The transcriptome response to DLA was richer in comparison to LLA.</italic>\\n</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00253-023-12863-z.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Author contribution</title>","<p>PD, AG, MT, and EB designed the research. PD, AS, AV, and EI performed the experiments. PD, AG, AV, and EZ analyzed the data and prepared text and figures. MT and EB supervised the project. All authors read and approved the final manuscript.</p>","<title>Funding</title>","<p>The work was supported by the Russian Science Foundation (interdisciplinary project 20–64-47011 performed in association with the Petrozavodsk State University, Petrozavodsk, Russia; link to information about the project: <ext-link ext-link-type=\"uri\" xlink:href=\"https://rscf.ru/en/project/20-64-47011/\">https://rscf.ru/en/project/20-64-47011/</ext-link>).</p>","<title>Data availability</title>","<p>The datasets generated during and analyzed during the current study are available in the GitHub repository, <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/drozdovapb/S_cerevisiae_lactate_transcriptome\">https://github.com/drozdovapb/S_cerevisiae_lactate_transcriptome</ext-link> and the NCBI GEO repository under the accession number GSE231937. </p>","<title>Declarations</title>","<title>Ethics approval</title>","<p id=\"Par32\">This article does not contain any studies with human participants or animals performed by any of the authors.</p>","<title>Competing interests</title>","<p id=\"Par33\">The authors declare no competing interests.</p>"],"string":"[\n \"<title>Author contribution</title>\",\n \"<p>PD, AG, MT, and EB designed the research. PD, AS, AV, and EI performed the experiments. PD, AG, AV, and EZ analyzed the data and prepared text and figures. MT and EB supervised the project. All authors read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>The work was supported by the Russian Science Foundation (interdisciplinary project 20–64-47011 performed in association with the Petrozavodsk State University, Petrozavodsk, Russia; link to information about the project: <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://rscf.ru/en/project/20-64-47011/\\\">https://rscf.ru/en/project/20-64-47011/</ext-link>).</p>\",\n \"<title>Data availability</title>\",\n \"<p>The datasets generated during and analyzed during the current study are available in the GitHub repository, <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://github.com/drozdovapb/S_cerevisiae_lactate_transcriptome\\\">https://github.com/drozdovapb/S_cerevisiae_lactate_transcriptome</ext-link> and the NCBI GEO repository under the accession number GSE231937. </p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethics approval</title>\",\n \"<p id=\\\"Par32\\\">This article does not contain any studies with human participants or animals performed by any of the authors.</p>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par33\\\">The authors declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Overview of gene expression changes in all comparisons. <bold>a</bold>–<bold>f</bold> show volcano plots for major comparisons with differentially expressed genes (absolute log<sub>2</sub> fold change > 1 and adjusted <italic>p</italic>-value < 0.05) are indicated by red dots (each dot corresponds to one gene). <bold>g</bold>, <bold>h</bold> show intersections of the lists of genes upregulated and downregulated, respectively, in response to different treatments, represented as Venn diagrams. Full expression data are available in Supplemental Table ##SUPPL##2##S3## and from the GEO database (GSE231937)</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Overview of transcriptional response to DLA. Shown are <bold>a</bold> logarithmic (base 2) normalized expression counts of the genes DE in response to 45 mM DLA and annotated with the GO terms “lactate metabolic process” or “lactate biosynthesis process” (ORF (open reading frame) symbols and gene names shown in the titles of the panels), as well as the correlation between expression changes in response to 5/45 mM DLA of shared DEGs (<bold>b</bold>) and all genes (<bold>c</bold>). Pearson’s correlation coefficient = 0.99 for (<bold>b</bold>) and 0.72 for (<bold>c</bold>)</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Comparison of transcriptional responses to LLA and DLA shows significant similarities and reveals several candidate qualitative DLA sensor genes. Correlation between expression changes in response to 45 mM DLA or LLA of shared DEGs (<bold>a</bold>) and all genes (<bold>b</bold>). Pearson’s correlation coefficient = 0.98 for <bold>a</bold> and 0.65 for <bold>b</bold>. <bold>c</bold> Clustering of expression profiles does not reveal any groups with quantitative response to DLA. <bold>d</bold> Expression profiles of potential qualitative sensors, genes responding to DLA but not LLA. The vertical axis shows logarithmic (base 2) normalized expression counts of the genes</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Addition of 45 mM sodium D-lactate compensates for the growth defect caused by 45 mM DLA treatment (<bold>a</bold>) and does not trigger changes in the expression of selective DLA-responsive genes (<bold>b</bold>). <bold>a</bold> Relative growth rate (the difference between logarithmic (base 2) final OD600 and initial OD600 divided over incubation time in hours) of cultures incubated for 3 h in the media with 45 mM DLA or DLS. *<italic>p</italic> < 0.05; ns, not significant (paired pairwise Wilcoxon rank sum test). <bold>b</bold> Expression levels of the <italic>AQR1</italic>, <italic>DLD3</italic>, <italic>FIT2</italic>, and <italic>YPS3</italic> genes relative to the geometric mean of the reference genes <italic>ACT1</italic> and <italic>CDC19</italic> measured with quantitative PCR. Raw qPCR data are presented in Supplemental Table ##SUPPL##3##S5##</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Overview of gene expression changes in all comparisons. <bold>a</bold>–<bold>f</bold> show volcano plots for major comparisons with differentially expressed genes (absolute log<sub>2</sub> fold change > 1 and adjusted <italic>p</italic>-value < 0.05) are indicated by red dots (each dot corresponds to one gene). <bold>g</bold>, <bold>h</bold> show intersections of the lists of genes upregulated and downregulated, respectively, in response to different treatments, represented as Venn diagrams. Full expression data are available in Supplemental Table ##SUPPL##2##S3## and from the GEO database (GSE231937)</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Overview of transcriptional response to DLA. Shown are <bold>a</bold> logarithmic (base 2) normalized expression counts of the genes DE in response to 45 mM DLA and annotated with the GO terms “lactate metabolic process” or “lactate biosynthesis process” (ORF (open reading frame) symbols and gene names shown in the titles of the panels), as well as the correlation between expression changes in response to 5/45 mM DLA of shared DEGs (<bold>b</bold>) and all genes (<bold>c</bold>). Pearson’s correlation coefficient = 0.99 for (<bold>b</bold>) and 0.72 for (<bold>c</bold>)</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Comparison of transcriptional responses to LLA and DLA shows significant similarities and reveals several candidate qualitative DLA sensor genes. Correlation between expression changes in response to 45 mM DLA or LLA of shared DEGs (<bold>a</bold>) and all genes (<bold>b</bold>). Pearson’s correlation coefficient = 0.98 for <bold>a</bold> and 0.65 for <bold>b</bold>. <bold>c</bold> Clustering of expression profiles does not reveal any groups with quantitative response to DLA. <bold>d</bold> Expression profiles of potential qualitative sensors, genes responding to DLA but not LLA. The vertical axis shows logarithmic (base 2) normalized expression counts of the genes</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Addition of 45 mM sodium D-lactate compensates for the growth defect caused by 45 mM DLA treatment (<bold>a</bold>) and does not trigger changes in the expression of selective DLA-responsive genes (<bold>b</bold>). <bold>a</bold> Relative growth rate (the difference between logarithmic (base 2) final OD600 and initial OD600 divided over incubation time in hours) of cultures incubated for 3 h in the media with 45 mM DLA or DLS. *<italic>p</italic> < 0.05; ns, not significant (paired pairwise Wilcoxon rank sum test). <bold>b</bold> Expression levels of the <italic>AQR1</italic>, <italic>DLD3</italic>, <italic>FIT2</italic>, and <italic>YPS3</italic> genes relative to the geometric mean of the reference genes <italic>ACT1</italic> and <italic>CDC19</italic> measured with quantitative PCR. Raw qPCR data are presented in Supplemental Table ##SUPPL##3##S5##</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Significantly (<italic>p</italic> < 0.05) overrepresented GO terms</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\" colspan=\"2\">45 mM DLA vs. control</td></tr><tr><td align=\"left\">Upregulated (82 genes)</td><td align=\"left\">Downregulated (31 genes)</td></tr><tr><td align=\"left\">Lactate metabolic process (GO:0006089)</td><td align=\"left\">No enrichment found</td></tr><tr><td align=\"left\" colspan=\"2\">Siderophore transport (GO:0015891)</td></tr><tr><td align=\"left\" colspan=\"2\">Lactate biosynthetic process (GO:0019249)</td></tr><tr><td align=\"left\" colspan=\"2\">5 mM DLA vs. control</td></tr><tr><td align=\"left\">Upregulated (74 genes)</td><td align=\"left\">Downregulated (51 genes)</td></tr><tr><td align=\"left\">Cell wall organization or biogenesis (GO:0071554)</td><td align=\"left\">Leucine biosynthetic process (GO:0009098)</td></tr><tr><td align=\"left\" colspan=\"2\">External encapsulating structure organization (GO:0045229)</td></tr><tr><td align=\"left\" colspan=\"2\">Cell wall organization (GO:0071555)</td></tr><tr><td align=\"left\" colspan=\"2\">Fungal-type cell wall organization or biogenesis (GO:0071852)</td></tr><tr><td align=\"left\" colspan=\"2\">Fungal-type cell wall organization (GO:0031505)</td></tr><tr><td align=\"left\" colspan=\"2\">Cell wall biogenesis (GO:0042546)</td></tr><tr><td align=\"left\" colspan=\"2\">45 mM LLA vs. control</td></tr><tr><td align=\"left\">Upregulated (17 genes)</td><td align=\"left\">Downregulated (45 genes)</td></tr><tr><td align=\"left\">No enrichment found</td><td align=\"left\">Generation of precursor metabolites and energy (GO:0006091)</td></tr><tr><td align=\"left\" colspan=\"2\">45 mM DLA vs. 45 mM LLA</td></tr><tr><td align=\"left\">Upregulated (8 genes)</td><td align=\"left\">Downregulated (2 genes)</td></tr><tr><td align=\"left\">No enrichment found</td><td align=\"left\">No enrichment found</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Significantly (<italic>p</italic> < 0.05) overrepresented GO terms</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr><td align=\\\"left\\\" colspan=\\\"2\\\">45 mM DLA vs. control</td></tr><tr><td align=\\\"left\\\">Upregulated (82 genes)</td><td align=\\\"left\\\">Downregulated (31 genes)</td></tr><tr><td align=\\\"left\\\">Lactate metabolic process (GO:0006089)</td><td align=\\\"left\\\">No enrichment found</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Siderophore transport (GO:0015891)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Lactate biosynthetic process (GO:0019249)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">5 mM DLA vs. control</td></tr><tr><td align=\\\"left\\\">Upregulated (74 genes)</td><td align=\\\"left\\\">Downregulated (51 genes)</td></tr><tr><td align=\\\"left\\\">Cell wall organization or biogenesis (GO:0071554)</td><td align=\\\"left\\\">Leucine biosynthetic process (GO:0009098)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">External encapsulating structure organization (GO:0045229)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Cell wall organization (GO:0071555)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Fungal-type cell wall organization or biogenesis (GO:0071852)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Fungal-type cell wall organization (GO:0031505)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Cell wall biogenesis (GO:0042546)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">45 mM LLA vs. control</td></tr><tr><td align=\\\"left\\\">Upregulated (17 genes)</td><td align=\\\"left\\\">Downregulated (45 genes)</td></tr><tr><td align=\\\"left\\\">No enrichment found</td><td align=\\\"left\\\">Generation of precursor metabolites and energy (GO:0006091)</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">45 mM DLA vs. 45 mM LLA</td></tr><tr><td align=\\\"left\\\">Upregulated (8 genes)</td><td align=\\\"left\\\">Downregulated (2 genes)</td></tr><tr><td align=\\\"left\\\">No enrichment found</td><td align=\\\"left\\\">No enrichment found</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" 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affiliations.</p></fn></fn-group>"],"string":"[\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"253_2023_12863_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"253_2023_12863_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"253_2023_12863_Fig3_HTML\" id=\"MO3\"/>","<graphic xlink:href=\"253_2023_12863_Fig4_HTML\" id=\"MO4\"/>"],"string":"[\n \"<graphic xlink:href=\\\"253_2023_12863_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12863_Fig2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12863_Fig3_HTML\\\" id=\\\"MO3\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12863_Fig4_HTML\\\" id=\\\"MO4\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media 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Approximately, 1 in 300 to 500 people in the population will carry a PV in either <italic>BRCA1</italic> or <italic>BRCA2</italic> (<italic>BRCA</italic>; the genes most commonly associated with hereditary breast and ovarian cancer (HBOC)) [##REF##11044354##3##–##REF##10359546##6##], and 1 in 370 individuals will carry a pathogenic variant (PV) in one of the Lynch syndrome genes (the genes most commonly associated with hereditary colorectal cancer) [##REF##35177335##7##]. Early identification of a PV in a cancer susceptibility gene provides individuals with the opportunity for enhanced surveillance and risk-reducing interventions which can significantly reduce the morbidity and mortality of these cancers [##REF##20810374##8##–##REF##21858794##13##]. Identification of carrier status also provides the opportunity for cascade testing in relatives [##REF##31857708##14##–##REF##35115620##16##].</p>","<p id=\"Par6\">Genetic testing for cancer susceptibility genes is currently offered predominantly to individuals who are considered high-risk for either <italic>BRCA</italic> or Lynch syndrome based on clinical and family history criteria. However, only around half of the carriers of PVs in <italic>BRCA</italic> and Lynch syndrome genes will meet clinical and family history criteria for genetic testing [##REF##29361001##5##, ##REF##31406321##17##, ##REF##24493722##18##]. Additionally, current approaches to testing for hereditary cancer genes are associated with inequities in referral and access to testing [##REF##32720237##19##, ##REF##35380723##20##].</p>","<p id=\"Par7\">An alternative approach is population-based hereditary cancer genetic testing which may provide a more clinically effective strategy for early identification of high-risk individuals [##REF##29361001##5##, ##REF##26483301##21##]. To date, population-based testing has been primarily studied in the context of <italic>BRCA</italic> testing in the Ashkenazi Jewish population [##REF##25435541##22##, ##REF##20008623##23##], and data are limited in the general population, especially among under-represented racial and ethnic groups. The objective of this study was to determine the yield of hereditary cancer gene PVs among unselected diverse women attending breast imaging centers, as a potential strategy for more complete identification of high-risk individuals who could benefit from enhanced surveillance and/or risk reduction interventions.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">A substantial proportion of cancers are associated with pathogenic variants (PV) in hereditary cancer genes [##REF##35971132##1##, ##REF##27978560##2##], including an estimated 10% of breast, 10% of colon, and 20% of ovarian cancers. Approximately, 1 in 300 to 500 people in the population will carry a PV in either <italic>BRCA1</italic> or <italic>BRCA2</italic> (<italic>BRCA</italic>; the genes most commonly associated with hereditary breast and ovarian cancer (HBOC)) [##REF##11044354##3##–##REF##10359546##6##], and 1 in 370 individuals will carry a pathogenic variant (PV) in one of the Lynch syndrome genes (the genes most commonly associated with hereditary colorectal cancer) [##REF##35177335##7##]. Early identification of a PV in a cancer susceptibility gene provides individuals with the opportunity for enhanced surveillance and risk-reducing interventions which can significantly reduce the morbidity and mortality of these cancers [##REF##20810374##8##–##REF##21858794##13##]. Identification of carrier status also provides the opportunity for cascade testing in relatives [##REF##31857708##14##–##REF##35115620##16##].</p>\",\n \"<p id=\\\"Par6\\\">Genetic testing for cancer susceptibility genes is currently offered predominantly to individuals who are considered high-risk for either <italic>BRCA</italic> or Lynch syndrome based on clinical and family history criteria. However, only around half of the carriers of PVs in <italic>BRCA</italic> and Lynch syndrome genes will meet clinical and family history criteria for genetic testing [##REF##29361001##5##, ##REF##31406321##17##, ##REF##24493722##18##]. Additionally, current approaches to testing for hereditary cancer genes are associated with inequities in referral and access to testing [##REF##32720237##19##, ##REF##35380723##20##].</p>\",\n \"<p id=\\\"Par7\\\">An alternative approach is population-based hereditary cancer genetic testing which may provide a more clinically effective strategy for early identification of high-risk individuals [##REF##29361001##5##, ##REF##26483301##21##]. To date, population-based testing has been primarily studied in the context of <italic>BRCA</italic> testing in the Ashkenazi Jewish population [##REF##25435541##22##, ##REF##20008623##23##], and data are limited in the general population, especially among under-represented racial and ethnic groups. The objective of this study was to determine the yield of hereditary cancer gene PVs among unselected diverse women attending breast imaging centers, as a potential strategy for more complete identification of high-risk individuals who could benefit from enhanced surveillance and/or risk reduction interventions.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<title>Study population</title>","<p id=\"Par8\">This retrospective cohort study included unselected female patients who were offered and underwent genetic testing at the time of breast imaging at three imaging centers (Memorial MRI and Diagnostics, Texas) from November 2020 through March 2022. All patients arriving at the imaging centers were given a written flier with an invitation to undergo genetic testing for a panel of hereditary cancer genes. Patients were also offered the option of an online genetic information session with a board-certified genetic counselor prior to testing. The lead clinician investigator (DM) served as the ordering clinician for the testing at all three centers. A limited number of providers using the imaging centers opted out of having their patients participate. Only patients (including those with a previous history of breast cancer) undergoing routine breast imaging, either by mammogram or ultrasound, were included. Patients undergoing imaging for newly diagnosed breast cancer were excluded.</p>","<p id=\"Par9\">Clinical, demographic, and family cancer history information were ascertained through test requisition forms and family cancer history questionnaires completed by the patients. Patient questionnaires included clinical questions needed for Tyrer–Cuzick breast cancer risk assessment. Race/ethnicity (ancestry) was self-reported. National Comprehensive Cancer Network (NCCN) guidelines for Genetic/Familial High-Risk Assessment: Breast, Ovarian, and Pancreatic Cancer [##UREF##0##24##] and Lynch Syndrome (LS) were reviewed to determine if genetic testing would have been guideline indicated for a particular patient [##UREF##1##25##]. For the patients unaffected with breast cancer who completed the clinical portion of their questionnaire for breast cancer risk assessment, a Tyrer–Cuzick breast cancer risk 5 years and lifetime risk score was calculated [##REF##14627668##26##]. This score was only reported back to patients if their genetic testing (described below) was negative/uninformative for PVs associated with increased breast cancer risk. It was not available if enough information about personal/family history was not provided to compute a Tyrer–Cuzick score or the patient had a personal history of breast cancer.</p>","<p id=\"Par10\">If the patients’ clinical and/or family history met NCCN guidelines for hereditary cancer testing, they were given the option to request insurance coverage for testing or self-pay. Patients who did not meet guidelines were offered the option to self-pay and financial assistance options were available to eligible patients, based on their income and family size. Genetic information sessions performed by board-certified genetic counselors (Natera, Inc.) were available to patients on a pre- and post- test basis. All patients with a PV in a breast cancer-associated predisposition gene were offered in person risk counseling by the lead clinician investigator (DM).</p>","<p id=\"Par11\">This study was granted a waiver of consent process under 45 CFR 46.116(d), a waiver of the requirement for documentation of informed consent according to 45 CFR 46.117(c)(2), and a waiver from the HIPAA Authorization Requirement according to 45 CFR 46.164.512(i) (Salus IRB, ID# 21204—01A).</p>","<title>Hereditary cancer testing</title>","<p id=\"Par12\">Next-generation sequencing (NGS)-based hereditary cancer risk assessment was carried out utilizing a multiplex gene panel (40 or 53 genes) testing (Empower™, Natera, Inc. in collaboration with Baylor Genetics). The targeted regions of the genes associated with hereditary cancer syndromes are enriched using a capture-based method and sequenced by next-generation sequencing (NGS) using the Illumina platform. The variants detected in exons and within 20 bp of the exon/intron boundary are reported, unless otherwise specified. Read depth analysis is used to detect copy number variation (CNV) for genes. Positive sequencing results from certain genes or regions with highly homologous sequences in the genome are confirmed by gene-specific long-range PCR and Sanger sequencing. Multiplex ligation-dependent probe amplification (MLPA), PCR-based methods, and/or array comparative genomic hybridization (aCGH) are used to confirm copy number changes involving the genes in the test.</p>","<p id=\"Par13\">All patients underwent testing for at least 25 clinically actionable genes (Table ##TAB##0##1##). Genes were considered clinically actionable based on the presence of established NCCN and/or peer-reviewed consensus management recommendations for enhanced surveillance, or risk-reducing interventions, and family cascade testing if a PV was detected. Clinically actionable genes were categorized as “high risk” or “moderate risk” based on their reported relative risk for cancer, relative risk of > 4, and relative risk of 2–4, respectively (Table ##TAB##0##1##). Testing included both HBOC and Lynch syndrome (LS) genes (NCCN HBOC, NCCN Colorectal Cancer (CRC)). Variants were classified consistent with guidelines from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology as previously described [##REF##14627668##26##–##REF##30964716##28##]. Only likely pathogenic and pathogenic variants (PVs) were considered in the analysis: benign and likely benign variants, and variants of unknown significance were not considered.</p>","<p id=\"Par14\">For the purposes of the current analysis, variants were not considered clinically actionable (i.e., had no potential impact on patient care) and were not included if they:<list list-type=\"order\"><list-item><p id=\"Par15\">Were in genes only associated with autosomal recessive disease association (e.g., monoallelic <italic>MUTYH</italic> carriers which may conflate estimates of pathogenic variant prevalence) or</p></list-item><list-item><p id=\"Par16\">Were low penetrance variants in clinically actionable genes, such as <italic>CHEK2</italic> c.470 T > C [p.Ile157Thr].</p></list-item></list></p>","<title>Analysis</title>","<p id=\"Par17\">The sociodemographic characteristics and personal and family history of cancer of the study population were explored. Patients’ characteristics were stratified based on the presence and type of PV. The prevalence of P/LP variants was calculated. For patients with a P/LP variant, the proportion who did and did not meet NCCN guidelines for genetic testing was evaluated. We also evaluated the proportion of patients with PV who would have qualified for additional screening based on the empiric risk model (i.e., Tyrer–Cuzick score > = 20%).</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<title>Study population</title>\",\n \"<p id=\\\"Par8\\\">This retrospective cohort study included unselected female patients who were offered and underwent genetic testing at the time of breast imaging at three imaging centers (Memorial MRI and Diagnostics, Texas) from November 2020 through March 2022. All patients arriving at the imaging centers were given a written flier with an invitation to undergo genetic testing for a panel of hereditary cancer genes. Patients were also offered the option of an online genetic information session with a board-certified genetic counselor prior to testing. The lead clinician investigator (DM) served as the ordering clinician for the testing at all three centers. A limited number of providers using the imaging centers opted out of having their patients participate. Only patients (including those with a previous history of breast cancer) undergoing routine breast imaging, either by mammogram or ultrasound, were included. Patients undergoing imaging for newly diagnosed breast cancer were excluded.</p>\",\n \"<p id=\\\"Par9\\\">Clinical, demographic, and family cancer history information were ascertained through test requisition forms and family cancer history questionnaires completed by the patients. Patient questionnaires included clinical questions needed for Tyrer–Cuzick breast cancer risk assessment. Race/ethnicity (ancestry) was self-reported. National Comprehensive Cancer Network (NCCN) guidelines for Genetic/Familial High-Risk Assessment: Breast, Ovarian, and Pancreatic Cancer [##UREF##0##24##] and Lynch Syndrome (LS) were reviewed to determine if genetic testing would have been guideline indicated for a particular patient [##UREF##1##25##]. For the patients unaffected with breast cancer who completed the clinical portion of their questionnaire for breast cancer risk assessment, a Tyrer–Cuzick breast cancer risk 5 years and lifetime risk score was calculated [##REF##14627668##26##]. This score was only reported back to patients if their genetic testing (described below) was negative/uninformative for PVs associated with increased breast cancer risk. It was not available if enough information about personal/family history was not provided to compute a Tyrer–Cuzick score or the patient had a personal history of breast cancer.</p>\",\n \"<p id=\\\"Par10\\\">If the patients’ clinical and/or family history met NCCN guidelines for hereditary cancer testing, they were given the option to request insurance coverage for testing or self-pay. Patients who did not meet guidelines were offered the option to self-pay and financial assistance options were available to eligible patients, based on their income and family size. Genetic information sessions performed by board-certified genetic counselors (Natera, Inc.) were available to patients on a pre- and post- test basis. All patients with a PV in a breast cancer-associated predisposition gene were offered in person risk counseling by the lead clinician investigator (DM).</p>\",\n \"<p id=\\\"Par11\\\">This study was granted a waiver of consent process under 45 CFR 46.116(d), a waiver of the requirement for documentation of informed consent according to 45 CFR 46.117(c)(2), and a waiver from the HIPAA Authorization Requirement according to 45 CFR 46.164.512(i) (Salus IRB, ID# 21204—01A).</p>\",\n \"<title>Hereditary cancer testing</title>\",\n \"<p id=\\\"Par12\\\">Next-generation sequencing (NGS)-based hereditary cancer risk assessment was carried out utilizing a multiplex gene panel (40 or 53 genes) testing (Empower™, Natera, Inc. in collaboration with Baylor Genetics). The targeted regions of the genes associated with hereditary cancer syndromes are enriched using a capture-based method and sequenced by next-generation sequencing (NGS) using the Illumina platform. The variants detected in exons and within 20 bp of the exon/intron boundary are reported, unless otherwise specified. Read depth analysis is used to detect copy number variation (CNV) for genes. Positive sequencing results from certain genes or regions with highly homologous sequences in the genome are confirmed by gene-specific long-range PCR and Sanger sequencing. Multiplex ligation-dependent probe amplification (MLPA), PCR-based methods, and/or array comparative genomic hybridization (aCGH) are used to confirm copy number changes involving the genes in the test.</p>\",\n \"<p id=\\\"Par13\\\">All patients underwent testing for at least 25 clinically actionable genes (Table ##TAB##0##1##). Genes were considered clinically actionable based on the presence of established NCCN and/or peer-reviewed consensus management recommendations for enhanced surveillance, or risk-reducing interventions, and family cascade testing if a PV was detected. Clinically actionable genes were categorized as “high risk” or “moderate risk” based on their reported relative risk for cancer, relative risk of > 4, and relative risk of 2–4, respectively (Table ##TAB##0##1##). Testing included both HBOC and Lynch syndrome (LS) genes (NCCN HBOC, NCCN Colorectal Cancer (CRC)). Variants were classified consistent with guidelines from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology as previously described [##REF##14627668##26##–##REF##30964716##28##]. Only likely pathogenic and pathogenic variants (PVs) were considered in the analysis: benign and likely benign variants, and variants of unknown significance were not considered.</p>\",\n \"<p id=\\\"Par14\\\">For the purposes of the current analysis, variants were not considered clinically actionable (i.e., had no potential impact on patient care) and were not included if they:<list list-type=\\\"order\\\"><list-item><p id=\\\"Par15\\\">Were in genes only associated with autosomal recessive disease association (e.g., monoallelic <italic>MUTYH</italic> carriers which may conflate estimates of pathogenic variant prevalence) or</p></list-item><list-item><p id=\\\"Par16\\\">Were low penetrance variants in clinically actionable genes, such as <italic>CHEK2</italic> c.470 T > C [p.Ile157Thr].</p></list-item></list></p>\",\n \"<title>Analysis</title>\",\n \"<p id=\\\"Par17\\\">The sociodemographic characteristics and personal and family history of cancer of the study population were explored. Patients’ characteristics were stratified based on the presence and type of PV. The prevalence of P/LP variants was calculated. For patients with a P/LP variant, the proportion who did and did not meet NCCN guidelines for genetic testing was evaluated. We also evaluated the proportion of patients with PV who would have qualified for additional screening based on the empiric risk model (i.e., Tyrer–Cuzick score > = 20%).</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Study population</title>","<p id=\"Par18\">A total of 1,943 women undergoing breast imaging elected to have hereditary cancer genetic testing during the study period (Table ##TAB##1##2##). Median age was 66 years (range 18–89 years). Self-reported race and ethnicity were Asian 5.0% (<italic>N</italic> = 85); Black 20% (<italic>N</italic> = 339); White 38% (<italic>N</italic> = 650); and Hispanic 32% (<italic>N</italic> = 534).</p>","<p id=\"Par19\">A personal history of cancer was documented for 7.5% (<italic>N</italic> = 146) (Fig. ##FIG##0##1##), a family history of cancer in 42.3% (<italic>N</italic> = 822), and no personal or family history of cancer in 50.2% (<italic>N</italic> = 975). A personal history of breast or ovarian-related cancers was recorded in 4% (<italic>N</italic> = 80), endometrial cancer in 0.8% (<italic>N</italic> = 15), and colorectal cancer in 0.5% (<italic>N</italic> = 10).</p>","<p id=\"Par20\">Overall, 18.2% (354/1943) of patients met current NCCN guidelines for hereditary breast and ovarian cancer (HBOC) gene testing, 3.7% (71/1943) met NCCN guidelines for Lynch syndrome genetic testing, and 1.0% (19/1943) met both HBOC and Lynch syndrome guidelines for testing (Table ##TAB##2##3##).</p>","<title>Prevalence of pathogenic variants</title>","<p id=\"Par21\">Among 1943 patients who received genetic testing, 39 (2%) were identified as carriers of a PV in an autosomal dominant clinically actionable HBOC-related or LS gene (Table ##TAB##2##3##). Of the 39 PVs identified, 84.6% (<italic>N</italic> = 33) were in HBOC-related genes which corresponds to a prevalence of 1.7% (33/1943) in the total cohort. The remaining 15.4% (<italic>N</italic> = 6) PVs were in LS genes which corresponds to a prevalence of 0.3% (6/1943) in the total cohort. The most common PVs were in <italic>CHEK2</italic> (10/39; 25.6%); <italic>PALB2</italic> (8/39; 20.5%); <italic>BRCA2</italic> (6/39; 15.4%); and <italic>PMS2</italic> (5/39; 12.8%) (Fig. ##FIG##1##2##). Of the 34 PVs where race/ethnicity were known, 47% were detected among non-White patients (Table ##TAB##1##2##). The PV prevalence (%) was distributed across the respective ancestral groups as follows: Black 3/339 (0.89%); White 16/650 (2.4%); Asian 3/85 (3.5%), and Hispanic 9/534 (1.7%).</p>","<p id=\"Par22\">Patients with a PV were over 50 years of age at the time of their testing in 82.1% (32/39) of cases. This is similar to the 1587/1943 (81.7%) of patients aged 51 or older who were tested in the total cohort and consistent with National screening guidelines for women at average risk [##REF##26757170##27##] (Table ##TAB##1##2##).</p>","<title>Guideline eligibility for genetic testing or enhanced surveillance breast cancer</title>","<p id=\"Par23\">Only 38.5% (15/39) of PV carriers met either NCCN guidelines for HBOC testing or LS testing prior to genetic testing. Of these, 25.6% (10/39) met HBOC criteria for genetic testing with PVs in HBOC-related genes and 7.7% (3/39) had a PV in an LS gene, and 5.1% (2/39) met criteria for LS testing with PV in HBOC-related genes.</p>","<p id=\"Par24\">Notably, 5 out of 6 patients with a <italic>BRCA2</italic> PV and 5 out of 8 patients with a <italic>PALB2</italic> PV did not meet criteria for HBOC testing. The frequencies of clinically actionable PVs identified in high- or moderate-risk genes in patients who either met or did not meet NCCN guidelines for inclusion of the genes are shown in Fig. ##FIG##1##2##.</p>","<p id=\"Par25\">Data allowing calculation of the patient’s Tyrer–Cuzick score were available for 64.1% (25/39) of patients with a PV in an autosomal dominant clinically actionable HBOC (Table ##TAB##2##3##). Of these, only 8% (2/25) had a Tyrer–Cuzick score ≥ 20 which would have triggered health insurance coverage for increased surveillance for breast cancer, absent in the PV finding (Table ##TAB##2##3##).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Study population</title>\",\n \"<p id=\\\"Par18\\\">A total of 1,943 women undergoing breast imaging elected to have hereditary cancer genetic testing during the study period (Table ##TAB##1##2##). Median age was 66 years (range 18–89 years). Self-reported race and ethnicity were Asian 5.0% (<italic>N</italic> = 85); Black 20% (<italic>N</italic> = 339); White 38% (<italic>N</italic> = 650); and Hispanic 32% (<italic>N</italic> = 534).</p>\",\n \"<p id=\\\"Par19\\\">A personal history of cancer was documented for 7.5% (<italic>N</italic> = 146) (Fig. ##FIG##0##1##), a family history of cancer in 42.3% (<italic>N</italic> = 822), and no personal or family history of cancer in 50.2% (<italic>N</italic> = 975). A personal history of breast or ovarian-related cancers was recorded in 4% (<italic>N</italic> = 80), endometrial cancer in 0.8% (<italic>N</italic> = 15), and colorectal cancer in 0.5% (<italic>N</italic> = 10).</p>\",\n \"<p id=\\\"Par20\\\">Overall, 18.2% (354/1943) of patients met current NCCN guidelines for hereditary breast and ovarian cancer (HBOC) gene testing, 3.7% (71/1943) met NCCN guidelines for Lynch syndrome genetic testing, and 1.0% (19/1943) met both HBOC and Lynch syndrome guidelines for testing (Table ##TAB##2##3##).</p>\",\n \"<title>Prevalence of pathogenic variants</title>\",\n \"<p id=\\\"Par21\\\">Among 1943 patients who received genetic testing, 39 (2%) were identified as carriers of a PV in an autosomal dominant clinically actionable HBOC-related or LS gene (Table ##TAB##2##3##). Of the 39 PVs identified, 84.6% (<italic>N</italic> = 33) were in HBOC-related genes which corresponds to a prevalence of 1.7% (33/1943) in the total cohort. The remaining 15.4% (<italic>N</italic> = 6) PVs were in LS genes which corresponds to a prevalence of 0.3% (6/1943) in the total cohort. The most common PVs were in <italic>CHEK2</italic> (10/39; 25.6%); <italic>PALB2</italic> (8/39; 20.5%); <italic>BRCA2</italic> (6/39; 15.4%); and <italic>PMS2</italic> (5/39; 12.8%) (Fig. ##FIG##1##2##). Of the 34 PVs where race/ethnicity were known, 47% were detected among non-White patients (Table ##TAB##1##2##). The PV prevalence (%) was distributed across the respective ancestral groups as follows: Black 3/339 (0.89%); White 16/650 (2.4%); Asian 3/85 (3.5%), and Hispanic 9/534 (1.7%).</p>\",\n \"<p id=\\\"Par22\\\">Patients with a PV were over 50 years of age at the time of their testing in 82.1% (32/39) of cases. This is similar to the 1587/1943 (81.7%) of patients aged 51 or older who were tested in the total cohort and consistent with National screening guidelines for women at average risk [##REF##26757170##27##] (Table ##TAB##1##2##).</p>\",\n \"<title>Guideline eligibility for genetic testing or enhanced surveillance breast cancer</title>\",\n \"<p id=\\\"Par23\\\">Only 38.5% (15/39) of PV carriers met either NCCN guidelines for HBOC testing or LS testing prior to genetic testing. Of these, 25.6% (10/39) met HBOC criteria for genetic testing with PVs in HBOC-related genes and 7.7% (3/39) had a PV in an LS gene, and 5.1% (2/39) met criteria for LS testing with PV in HBOC-related genes.</p>\",\n \"<p id=\\\"Par24\\\">Notably, 5 out of 6 patients with a <italic>BRCA2</italic> PV and 5 out of 8 patients with a <italic>PALB2</italic> PV did not meet criteria for HBOC testing. The frequencies of clinically actionable PVs identified in high- or moderate-risk genes in patients who either met or did not meet NCCN guidelines for inclusion of the genes are shown in Fig. ##FIG##1##2##.</p>\",\n \"<p id=\\\"Par25\\\">Data allowing calculation of the patient’s Tyrer–Cuzick score were available for 64.1% (25/39) of patients with a PV in an autosomal dominant clinically actionable HBOC (Table ##TAB##2##3##). Of these, only 8% (2/25) had a Tyrer–Cuzick score ≥ 20 which would have triggered health insurance coverage for increased surveillance for breast cancer, absent in the PV finding (Table ##TAB##2##3##).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par26\">We found that 2% (39/1943) of women undergoing hereditary cancer gene testing as part of their clinical care at the time of breast imaging had PVs in hereditary cancer genes that had implications for cancer surveillance and clinical management. Importantly, the population undergoing testing was racially and ethnically diverse compared to previous studies [##REF##35380723##20##, ##REF##30964716##28##], predominantly over the age of 50 and without a personal history of cancer (92.5%). This was similar or more diverse compared to CDC population data on race and ethnicity for females aged 55 to 74 years who were resident in Texas in 2021 (4.8% Asian, 12.5% Black, 80.7% White, and 28.1% Hispanic) [##UREF##2##29##].</p>","<p id=\"Par27\">Among women who had a PV in either a HBOC or LS gene, only 38.5% met NCCN guidelines for testing of either of these conditions. Of those who had a PV in an HBOC gene and had data needed for a Tyrer–Cuzick score, only 8% had an estimated lifetime score of ≥ 20%. Therefore, while testing as part of clinical care at the time of breast imaging identified some women who were eligible for hereditary cancer gene testing based on NCCN guidelines, it predominantly identified women with PVs who neither met NCCN guidelines for testing nor the Tyrer–Cuzick threshold for increased surveillance for breast cancer [##REF##15057881##30##]. Consequently, without this opportunity for genetic testing, women with PVs may not have accessed risk-appropriate enhanced surveillance.</p>","<p id=\"Par28\">Identification of individuals with PVs in HBOC and LS genes provides opportunities for cancer prevention and earlier detection [##REF##21858794##13##, ##REF##32245629##31##]. For people with PVs in HBOC genes, there are recommendations and options for earlier mammography, screening breast MRI, chemoprophylaxis, and risk-reducing surgeries, such as mastectomy and bilateral salpingo-oophorectomy [##REF##20810374##8##, ##REF##18268356##10##, ##REF##28383664##11##, ##REF##21858794##13##]. Knowledge of specific PVs can also impact decisions about surgery and adjuvant therapies [##REF##35585432##32##–##REF##34081848##34##]. The median age of the population in the current cohort was 66 years and previous research has suggested that HBOC gene testing in younger women would be the most cost-effective approach to testing [##REF##33758026##35##]. Nonetheless, a study of the remaining lifetime risk for the subset of women who were older than 65 years in the population-based CARRIERS project [##REF##33471974##36##] indicated that <italic>BRCA1</italic>, <italic>BRCA2</italic>, and <italic>PALB2</italic> PVs were associated with enough breast cancer risk to warrant high-risk screening [##REF##34292776##37##]. For people who have PVs in LS genes, there is also compelling evidence of the benefit of colonoscopy in reducing mortality from CRC [##REF##19720893##9##, ##REF##33915171##38##]. Additionally, in women with PVs in LS genes, the lifetime risk of endometrial cancer is similar to that of CRC, which can largely be prevented by hysterectomy [##REF##16421367##39##]. For women with PVs who chose to share this information with family members, it can provide more accurate risk assessment and cascade testing [##REF##31857708##14##–##REF##35115620##16##]. The yield of PVs is typically higher (~ 10%) [##REF##21858794##13##] when we use the guideline criteria to screen the eligibility for genetic testing. However, this study demonstrated a meaningful yield (~ 2%) of clinically actionable PVs among participants who did not meet any guideline. Amplifying this point, 5 out of 6 <italic>BRCA2</italic> carriers identified by this unselected approach did not meet any guideline and there is ample evidence of reduction in cancer-specific and all-cause mortality by standard of care gene-specific clinical management [##REF##20810374##8##, ##REF##28914396##40##, ##REF##27087880##41##]. Thus, the universal testing approach and increasingly cost-effective genetic testing are likely to have a high impact despite a modest yield.</p>","<p id=\"Par29\">Perhaps one of the most striking findings in this clinical cohort was the diversity of the population that accessed the testing. Later stages of cancer at the time of diagnosis and lower cancer survival rates are clearly documented in Black people in the USA [##REF##33603954##42##]. There are data indicating that people of color or Hispanic ancestry are less likely to be referred for genetic counseling or testing and are less likely to take up genetic testing in the absence of a personal history of cancer [##REF##35380723##20##, ##REF##33603954##42##, ##REF##31078448##43##]. As demonstrated in our study, increasing access and convenience of testing may help overcome barriers to more equitable testing [##REF##31078448##43##, ##REF##31239069##44##].</p>","<p id=\"Par30\">Limitations of this study include that women under 40 years of age without documented increased risk are unlikely to have routine mammography and therefore, may not be represented in this cohort. Thus, <italic>BRCA</italic> carriers may be under-represented. Nonetheless, several <italic>BRCA2</italic> carriers, the majority of which did not meet any testing guidelines, were identified representing a critical opportunity for screening and prevention. Another limitation of the current cohort is that the number of patients who declined hereditary cancer testing or who may have had genetic testing prior to this study was unknown. Given the uncertainty about the total number of women who received the invitation to have genetic testing, the potential benefit of our strategy with regard to the yield of actionable PVs in the imaging center population could be over- or underestimated. Finally, though the racial and ethnic composition of the study participants were exceptionally diverse, we do not know if there were significant differences in the uptake among the respective groups [##REF##36370464##45##]. Nonetheless, (as cited above) the population who underwent testing is diverse and generally representative of the ethnic makeup of the state of Texas. Further, we do not have qualitative or quantitative data on how the patients made their decision to participate and receive testing as it was beyond the scope of our study. However, we believe that this will be important for future research in the context of population health implementation. Finally, the presence of a PV or elevated empiric risk (e.g., > 20%) does not guarantee insurance coverage, and access has been problematic across different healthcare systems and among the underinsured. Nonetheless, we believe that this report provides additional evidence supporting access to risk-appropriate care. Without granular insurance data, we note that all of the individuals in the study at the least had access to the imaging centers.</p>","<p id=\"Par31\">Additionally, while genetic information sessions both before and after testing were available to all patients who underwent testing, formal pre-test genetic counseling was not required. Requiring pre-test genetic counseling may itself present a barrier to testing [##REF##30526229##46##], so there may be a trade-off between the benchmark of full-genetic counseling and the use of abbreviated genetic information sessions to improve access to potentially life-saving information, especially in population health settings. Additional research is needed to establish what constitutes optimal pre- and post-test counseling and informed consent for patients receiving genetic testing in non-genetic/population health settings. However, in the meantime, the results of this and similar studies suggest that testing in a diverse imaging center population can extend the reach of genetic cancer risk assessment, has a clinically meaningful and actionable yield of cancer-associated PVs, and can help address inequity in access to testing and risk-appropriate screening and prevention.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par26\\\">We found that 2% (39/1943) of women undergoing hereditary cancer gene testing as part of their clinical care at the time of breast imaging had PVs in hereditary cancer genes that had implications for cancer surveillance and clinical management. Importantly, the population undergoing testing was racially and ethnically diverse compared to previous studies [##REF##35380723##20##, ##REF##30964716##28##], predominantly over the age of 50 and without a personal history of cancer (92.5%). This was similar or more diverse compared to CDC population data on race and ethnicity for females aged 55 to 74 years who were resident in Texas in 2021 (4.8% Asian, 12.5% Black, 80.7% White, and 28.1% Hispanic) [##UREF##2##29##].</p>\",\n \"<p id=\\\"Par27\\\">Among women who had a PV in either a HBOC or LS gene, only 38.5% met NCCN guidelines for testing of either of these conditions. Of those who had a PV in an HBOC gene and had data needed for a Tyrer–Cuzick score, only 8% had an estimated lifetime score of ≥ 20%. Therefore, while testing as part of clinical care at the time of breast imaging identified some women who were eligible for hereditary cancer gene testing based on NCCN guidelines, it predominantly identified women with PVs who neither met NCCN guidelines for testing nor the Tyrer–Cuzick threshold for increased surveillance for breast cancer [##REF##15057881##30##]. Consequently, without this opportunity for genetic testing, women with PVs may not have accessed risk-appropriate enhanced surveillance.</p>\",\n \"<p id=\\\"Par28\\\">Identification of individuals with PVs in HBOC and LS genes provides opportunities for cancer prevention and earlier detection [##REF##21858794##13##, ##REF##32245629##31##]. For people with PVs in HBOC genes, there are recommendations and options for earlier mammography, screening breast MRI, chemoprophylaxis, and risk-reducing surgeries, such as mastectomy and bilateral salpingo-oophorectomy [##REF##20810374##8##, ##REF##18268356##10##, ##REF##28383664##11##, ##REF##21858794##13##]. Knowledge of specific PVs can also impact decisions about surgery and adjuvant therapies [##REF##35585432##32##–##REF##34081848##34##]. The median age of the population in the current cohort was 66 years and previous research has suggested that HBOC gene testing in younger women would be the most cost-effective approach to testing [##REF##33758026##35##]. Nonetheless, a study of the remaining lifetime risk for the subset of women who were older than 65 years in the population-based CARRIERS project [##REF##33471974##36##] indicated that <italic>BRCA1</italic>, <italic>BRCA2</italic>, and <italic>PALB2</italic> PVs were associated with enough breast cancer risk to warrant high-risk screening [##REF##34292776##37##]. For people who have PVs in LS genes, there is also compelling evidence of the benefit of colonoscopy in reducing mortality from CRC [##REF##19720893##9##, ##REF##33915171##38##]. Additionally, in women with PVs in LS genes, the lifetime risk of endometrial cancer is similar to that of CRC, which can largely be prevented by hysterectomy [##REF##16421367##39##]. For women with PVs who chose to share this information with family members, it can provide more accurate risk assessment and cascade testing [##REF##31857708##14##–##REF##35115620##16##]. The yield of PVs is typically higher (~ 10%) [##REF##21858794##13##] when we use the guideline criteria to screen the eligibility for genetic testing. However, this study demonstrated a meaningful yield (~ 2%) of clinically actionable PVs among participants who did not meet any guideline. Amplifying this point, 5 out of 6 <italic>BRCA2</italic> carriers identified by this unselected approach did not meet any guideline and there is ample evidence of reduction in cancer-specific and all-cause mortality by standard of care gene-specific clinical management [##REF##20810374##8##, ##REF##28914396##40##, ##REF##27087880##41##]. Thus, the universal testing approach and increasingly cost-effective genetic testing are likely to have a high impact despite a modest yield.</p>\",\n \"<p id=\\\"Par29\\\">Perhaps one of the most striking findings in this clinical cohort was the diversity of the population that accessed the testing. Later stages of cancer at the time of diagnosis and lower cancer survival rates are clearly documented in Black people in the USA [##REF##33603954##42##]. There are data indicating that people of color or Hispanic ancestry are less likely to be referred for genetic counseling or testing and are less likely to take up genetic testing in the absence of a personal history of cancer [##REF##35380723##20##, ##REF##33603954##42##, ##REF##31078448##43##]. As demonstrated in our study, increasing access and convenience of testing may help overcome barriers to more equitable testing [##REF##31078448##43##, ##REF##31239069##44##].</p>\",\n \"<p id=\\\"Par30\\\">Limitations of this study include that women under 40 years of age without documented increased risk are unlikely to have routine mammography and therefore, may not be represented in this cohort. Thus, <italic>BRCA</italic> carriers may be under-represented. Nonetheless, several <italic>BRCA2</italic> carriers, the majority of which did not meet any testing guidelines, were identified representing a critical opportunity for screening and prevention. Another limitation of the current cohort is that the number of patients who declined hereditary cancer testing or who may have had genetic testing prior to this study was unknown. Given the uncertainty about the total number of women who received the invitation to have genetic testing, the potential benefit of our strategy with regard to the yield of actionable PVs in the imaging center population could be over- or underestimated. Finally, though the racial and ethnic composition of the study participants were exceptionally diverse, we do not know if there were significant differences in the uptake among the respective groups [##REF##36370464##45##]. Nonetheless, (as cited above) the population who underwent testing is diverse and generally representative of the ethnic makeup of the state of Texas. Further, we do not have qualitative or quantitative data on how the patients made their decision to participate and receive testing as it was beyond the scope of our study. However, we believe that this will be important for future research in the context of population health implementation. Finally, the presence of a PV or elevated empiric risk (e.g., > 20%) does not guarantee insurance coverage, and access has been problematic across different healthcare systems and among the underinsured. Nonetheless, we believe that this report provides additional evidence supporting access to risk-appropriate care. Without granular insurance data, we note that all of the individuals in the study at the least had access to the imaging centers.</p>\",\n \"<p id=\\\"Par31\\\">Additionally, while genetic information sessions both before and after testing were available to all patients who underwent testing, formal pre-test genetic counseling was not required. Requiring pre-test genetic counseling may itself present a barrier to testing [##REF##30526229##46##], so there may be a trade-off between the benchmark of full-genetic counseling and the use of abbreviated genetic information sessions to improve access to potentially life-saving information, especially in population health settings. Additional research is needed to establish what constitutes optimal pre- and post-test counseling and informed consent for patients receiving genetic testing in non-genetic/population health settings. However, in the meantime, the results of this and similar studies suggest that testing in a diverse imaging center population can extend the reach of genetic cancer risk assessment, has a clinically meaningful and actionable yield of cancer-associated PVs, and can help address inequity in access to testing and risk-appropriate screening and prevention.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose</title>","<p id=\"Par1\">Up to 10% of all breast cancers (BC) are attributed to inherited pathogenic variants (PV) in BC susceptibility genes; however, most carriers of PVs remain unidentified. Here, we sought to determine the yield of hereditary cancer gene PVs among diverse women attending breast imaging centers, who could benefit from enhanced surveillance and/or risk reduction interventions.</p>","<title>Methods</title>","<p id=\"Par2\">This cross-sectional retrospective cohort study included consecutive women, unselected for personal or family cancer history, who were offered genetic testing for hereditary cancer genes at the time of breast imaging at three centers (November 2020–March 2022).</p>","<title>Results</title>","<p id=\"Par3\">Among 1943 patients (median age: 66 years), self-reported race/ethnicity was White (34.5%), Hispanic (27.7%), African American (17.9%), Asian (4.5%), Ashkenazi Jewish (0.6%), Other (3.5%), and missing (13.0%). Thirty-nine patients (2%) were identified as carriers of a PV in an autosomal dominant clinically actionable hereditary breast and ovarian cancer (HBOC)-related or Lynch syndrome gene, most frequently, <italic>BRCA2</italic> (6/39; 15.4%), <italic>PALB2</italic> (8/39; 20.5%), <italic>CHEK2</italic> (10/39; 25.6%), and <italic>PMS2</italic> (5/39; 12.8%). Of the 34 PVs with known race/ethnicity, 47% were detected among non-White patients. Overall, 354/1,943 (18.2%) of patients met NCCN guidelines for HBOC gene testing and only 15/39 (38.5%) patients with an autosomal dominant clinically actionable PV met guidelines.</p>","<title>Conclusion</title>","<p id=\"Par4\">This population health approach extended the reach of genetic cancer risk assessment in a diverse population and highlighted the limits of a guideline-based approach. This may help address inequity in access to risk-appropriate screening and cancer prevention.</p>","<title>Keywords</title>"],"string":"[\n \"<title>Purpose</title>\",\n \"<p id=\\\"Par1\\\">Up to 10% of all breast cancers (BC) are attributed to inherited pathogenic variants (PV) in BC susceptibility genes; however, most carriers of PVs remain unidentified. Here, we sought to determine the yield of hereditary cancer gene PVs among diverse women attending breast imaging centers, who could benefit from enhanced surveillance and/or risk reduction interventions.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">This cross-sectional retrospective cohort study included consecutive women, unselected for personal or family cancer history, who were offered genetic testing for hereditary cancer genes at the time of breast imaging at three centers (November 2020–March 2022).</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">Among 1943 patients (median age: 66 years), self-reported race/ethnicity was White (34.5%), Hispanic (27.7%), African American (17.9%), Asian (4.5%), Ashkenazi Jewish (0.6%), Other (3.5%), and missing (13.0%). Thirty-nine patients (2%) were identified as carriers of a PV in an autosomal dominant clinically actionable hereditary breast and ovarian cancer (HBOC)-related or Lynch syndrome gene, most frequently, <italic>BRCA2</italic> (6/39; 15.4%), <italic>PALB2</italic> (8/39; 20.5%), <italic>CHEK2</italic> (10/39; 25.6%), and <italic>PMS2</italic> (5/39; 12.8%). Of the 34 PVs with known race/ethnicity, 47% were detected among non-White patients. Overall, 354/1,943 (18.2%) of patients met NCCN guidelines for HBOC gene testing and only 15/39 (38.5%) patients with an autosomal dominant clinically actionable PV met guidelines.</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par4\\\">This population health approach extended the reach of genetic cancer risk assessment in a diverse population and highlighted the limits of a guideline-based approach. This may help address inequity in access to risk-appropriate screening and cancer prevention.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>We would like to acknowledge Sofia Hurtado, BS and Mayra Rodas, BS from Natera, Inc. for data acquisition and Urmi Sengupta, PhD, from Natera, Inc., for assistance with the development of the manuscript.</p>","<title>Author contributions</title>","<p>LW, VS, and JW have contributed to the study concept and design. Data acquisition, analysis, and interpretation were performed by LW, DM, MM, KH, MY, and NK. LW, DM, VS, AR, MM, KH, YS, MY, NK, and JW were involved in the administrative process. LW, VS, and JW supervised the study. Original drafting of the manuscript was performed by LW, VS, and MY. Critical revision and editing of the manuscript were performed by LW, DM, VS, AR, MM, KH, YS, MY, and JW. All authors read and approved the final version of the manuscript for submission.</p>","<title>Funding</title>","<p>Not applicable.</p>","<title>Data availability</title>","<p>This is descriptive summary data on genetic testing outcome. The specific variant level data are contributed to ClinVar by Baylor Genetics, Houston, Texas.</p>","<title>Declarations</title>","<title>Competing interests</title>","<p id=\"Par32\">VS, MM, KH, YS, MY, RG, and WS are employees of Natera, Inc. with stocks or options to own stocks. LW, DM, and NK declare no conflict of interest.</p>","<title>Ethical approval</title>","<p id=\"Par33\">This study was granted a waiver of consent process under 45 CFR 46.116(d), a waiver of the requirement for documentation of informed consent according to 45 CFR 46.117(c)(2), and a waiver from the HIPAA Authorization Requirement according to 45 CFR 46.164.512(i) (Salus IRB, ID# 21204—01A).</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>We would like to acknowledge Sofia Hurtado, BS and Mayra Rodas, BS from Natera, Inc. for data acquisition and Urmi Sengupta, PhD, from Natera, Inc., for assistance with the development of the manuscript.</p>\",\n \"<title>Author contributions</title>\",\n \"<p>LW, VS, and JW have contributed to the study concept and design. Data acquisition, analysis, and interpretation were performed by LW, DM, MM, KH, MY, and NK. LW, DM, VS, AR, MM, KH, YS, MY, NK, and JW were involved in the administrative process. LW, VS, and JW supervised the study. Original drafting of the manuscript was performed by LW, VS, and MY. Critical revision and editing of the manuscript were performed by LW, DM, VS, AR, MM, KH, YS, MY, and JW. All authors read and approved the final version of the manuscript for submission.</p>\",\n \"<title>Funding</title>\",\n \"<p>Not applicable.</p>\",\n \"<title>Data availability</title>\",\n \"<p>This is descriptive summary data on genetic testing outcome. The specific variant level data are contributed to ClinVar by Baylor Genetics, Houston, Texas.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par32\\\">VS, MM, KH, YS, MY, RG, and WS are employees of Natera, Inc. with stocks or options to own stocks. LW, DM, and NK declare no conflict of interest.</p>\",\n \"<title>Ethical approval</title>\",\n \"<p id=\\\"Par33\\\">This study was granted a waiver of consent process under 45 CFR 46.116(d), a waiver of the requirement for documentation of informed consent according to 45 CFR 46.117(c)(2), and a waiver from the HIPAA Authorization Requirement according to 45 CFR 46.164.512(i) (Salus IRB, ID# 21204—01A).</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Personal cancer history in the full cohort (<italic>N</italic> = 1943)</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Number of clinically actionable PVs identified in high- or moderate-risk genes in patients who either met or did not meet NCCN guidelines for testing of the indicated gene</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Personal cancer history in the full cohort (<italic>N</italic> = 1943)</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Number of clinically actionable PVs identified in high- or moderate-risk genes in patients who either met or did not meet NCCN guidelines for testing of the indicated gene</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>List of 25 actionable genes reported for all patients</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">High-risk Genes</th><th align=\"left\">Moderate-risk Genes</th></tr></thead><tbody><tr><td align=\"left\"><italic>APC</italic></td><td align=\"left\"><italic>ATM</italic></td></tr><tr><td align=\"left\"><italic>BRCA1</italic></td><td align=\"left\"><italic>BARD1</italic></td></tr><tr><td align=\"left\"><italic>BRCA2</italic></td><td align=\"left\"><italic>BRIP1</italic></td></tr><tr><td align=\"left\"><italic>BMPR1A</italic></td><td align=\"left\"><italic>CHEK2</italic></td></tr><tr><td align=\"left\"><italic>CDH1</italic></td><td align=\"left\"><italic>NF1</italic></td></tr><tr><td align=\"left\"><italic>EPCAM</italic></td><td align=\"left\"><italic>RAD51C</italic></td></tr><tr><td align=\"left\"><italic>MEN1</italic></td><td align=\"left\"><italic>RAD51D</italic></td></tr><tr><td align=\"left\"><italic>MLH1</italic></td><td align=\"left\"/></tr><tr><td align=\"left\"><italic>MSH2</italic></td><td align=\"left\"/></tr><tr><td align=\"left\"><italic>MSH6</italic></td><td align=\"left\"/></tr><tr><td align=\"left\"><italic>MUTYH (Biallelic)</italic></td><td align=\"left\"/></tr><tr><td align=\"left\"><italic>PALB2</italic></td><td align=\"left\"/></tr><tr><td align=\"left\"><italic>PMS2</italic></td><td align=\"left\"/></tr><tr><td align=\"left\"><italic>PTEN</italic></td><td align=\"left\"/></tr><tr><td align=\"left\"><italic>SMAD4</italic></td><td align=\"left\"/></tr><tr><td align=\"left\"><italic>STK11</italic></td><td align=\"left\"/></tr><tr><td align=\"left\"><italic>TP53</italic></td><td align=\"left\"/></tr><tr><td align=\"left\"><italic>VHL</italic></td><td align=\"left\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Characteristics of individuals who underwent genetic testing (<italic>N</italic> = 1943)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Characteristics</th><th align=\"left\">Patients with a PV in HBOC gene<break/> N= 33</th><th align=\"left\">Patients with a PV in a LS gene<break/> N= 6</th><th align=\"left\">Patients without a PV<break/> N= 1904</th><th align=\"left\">All<break/> N= 1943</th></tr></thead><tbody><tr><td align=\"left\">Median age in years (Range)</td><td char=\"(\" align=\"char\">61 (36–78)</td><td char=\"(\" align=\"char\">67 (19–73)</td><td char=\"(\" align=\"char\">65 (18–89)</td><td char=\"(\" align=\"char\">66 (18–89</td></tr><tr><td align=\"left\">< 50 years, n (%)</td><td char=\"(\" align=\"char\">5 (15.2)</td><td char=\"(\" align=\"char\">2 (33.3)</td><td char=\"(\" align=\"char\">349 (18.3)</td><td char=\"(\" align=\"char\">356 (18.3)</td></tr><tr><td align=\"left\"> ≥ 50 years, n (%)</td><td char=\"(\" align=\"char\">28 (84.8)</td><td char=\"(\" align=\"char\">4 (66.7)</td><td char=\"(\" align=\"char\">1555 (81.7)</td><td char=\"(\" align=\"char\">1587 (81.7)</td></tr><tr><td align=\"left\">Race and Ethnicity, n (%)</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Black</td><td char=\"(\" align=\"char\">2 (6.1)</td><td char=\"(\" align=\"char\">1 (16.7)</td><td char=\"(\" align=\"char\">336 (20)</td><td char=\"(\" align=\"char\">339 (20)</td></tr><tr><td align=\"left\">White</td><td char=\"(\" align=\"char\">14 (42.4)</td><td char=\"(\" align=\"char\">2 (33.3)</td><td char=\"(\" align=\"char\">634 (38.0)</td><td char=\"(\" align=\"char\">650 (38)</td></tr><tr><td align=\"left\">Asian</td><td char=\"(\" align=\"char\">3 (9.1)</td><td char=\"(\" align=\"char\">0 (0.0)</td><td char=\"(\" align=\"char\">82 (5.0)</td><td char=\"(\" align=\"char\">85 (5.0)</td></tr><tr><td align=\"left\">Hispanic</td><td char=\"(\" align=\"char\">8 (24.2)</td><td char=\"(\" align=\"char\">1 (16.7)</td><td char=\"(\" align=\"char\">525 (32)</td><td char=\"(\" align=\"char\">534 (32)</td></tr><tr><td align=\"left\">Multiple races selected</td><td char=\"(\" align=\"char\">1 (3.0)</td><td char=\"(\" align=\"char\">0 (0.0)</td><td char=\"(\" align=\"char\">23 (1.4)</td><td char=\"(\" align=\"char\">24 (1.4)</td></tr><tr><td align=\"left\">Other</td><td char=\"(\" align=\"char\">1 (3.0)</td><td char=\"(\" align=\"char\">1 (16.7)</td><td char=\"(\" align=\"char\">56 (3.4)</td><td char=\"(\" align=\"char\">58 (3.4)</td></tr><tr><td align=\"left\">Missing</td><td char=\"(\" align=\"char\">4 (12.1)</td><td char=\"(\" align=\"char\">1 (16.7)</td><td char=\"(\" align=\"char\">248 (13.0)</td><td char=\"(\" align=\"char\">253 (13.0)</td></tr><tr><td align=\"left\">Personal history of cancer (any type)</td><td char=\"(\" align=\"char\">1 (3.0)</td><td char=\"(\" align=\"char\">0 (0.0)</td><td char=\"(\" align=\"char\">145 (7.6)</td><td char=\"(\" align=\"char\">146 (7.5)</td></tr><tr><td align=\"left\">Family history of cancer (in patients with no personal cancer history)</td><td char=\"(\" align=\"char\">25 (75.8)</td><td char=\"(\" align=\"char\">6 (100.0)</td><td char=\"(\" align=\"char\">791 (41.5)</td><td char=\"(\" align=\"char\">822 (42.3)</td></tr><tr><td align=\"left\">No known personal or family history of cancer</td><td char=\"(\" align=\"char\">7 (21.2)</td><td char=\"(\" align=\"char\">0 (0.0)</td><td char=\"(\" align=\"char\">968 (50.8)</td><td char=\"(\" align=\"char\">975 (50.2)</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Number (percentage) of patients who met NCCN criteria for hereditary cancer testing based on personal and/or family history</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Characteristics</th><th align=\"left\">Patients with a PV in HBOC gene<break/> N= 33 (%)</th><th align=\"left\">Patients with a PV in a LS gene<break/> N= 6 (%)</th><th align=\"left\">Patients without a PV<break/> N= 1904 (%)</th><th align=\"left\">All<break/> N= 1943 (%)</th></tr></thead><tbody><tr><td align=\"left\">HBOC</td><td char=\"(\" align=\"char\">10 (30.3)</td><td char=\"(\" align=\"char\">3 (50.0)</td><td char=\"(\" align=\"char\">341 (17.9)</td><td char=\"(\" align=\"char\">354 (18.2)</td></tr><tr><td align=\"left\">Lynch</td><td char=\"(\" align=\"char\">1 (3.0)</td><td char=\"(\" align=\"char\">0 (0.0)</td><td char=\"(\" align=\"char\">70 (3.7)</td><td char=\"(\" align=\"char\">71 (3.7)</td></tr><tr><td align=\"left\">Both HBOC and Lynch</td><td char=\"(\" align=\"char\">1 (3.0)</td><td char=\"(\" align=\"char\">0 (0.0)</td><td char=\"(\" align=\"char\">18 (0.9)</td><td char=\"(\" align=\"char\">19 (1.0)</td></tr><tr><td align=\"left\"> Pre-test Tyrer-Cuzick* risk score calculated, n (%)</td><td char=\"(\" align=\"char\">25 (75.8)</td><td char=\"(\" align=\"char\">4(66.6)</td><td char=\"(\" align=\"char\">1405 (73.8)</td><td char=\"(\" align=\"char\">1434 (73.8)</td></tr><tr><td align=\"left\"> Pre-test Tyrer-Cuzick* risk >20%</td><td char=\"(\" align=\"char\">2 (8.0) *</td><td char=\"(\" align=\"char\">1 (16.7)</td><td char=\"(\" align=\"char\">66 (6.0)</td><td char=\"(\" align=\"char\">67 (4.7)</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>List of 25 actionable genes reported for all patients</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">High-risk Genes</th><th align=\\\"left\\\">Moderate-risk Genes</th></tr></thead><tbody><tr><td align=\\\"left\\\"><italic>APC</italic></td><td align=\\\"left\\\"><italic>ATM</italic></td></tr><tr><td align=\\\"left\\\"><italic>BRCA1</italic></td><td align=\\\"left\\\"><italic>BARD1</italic></td></tr><tr><td align=\\\"left\\\"><italic>BRCA2</italic></td><td align=\\\"left\\\"><italic>BRIP1</italic></td></tr><tr><td align=\\\"left\\\"><italic>BMPR1A</italic></td><td align=\\\"left\\\"><italic>CHEK2</italic></td></tr><tr><td align=\\\"left\\\"><italic>CDH1</italic></td><td align=\\\"left\\\"><italic>NF1</italic></td></tr><tr><td align=\\\"left\\\"><italic>EPCAM</italic></td><td align=\\\"left\\\"><italic>RAD51C</italic></td></tr><tr><td align=\\\"left\\\"><italic>MEN1</italic></td><td align=\\\"left\\\"><italic>RAD51D</italic></td></tr><tr><td align=\\\"left\\\"><italic>MLH1</italic></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><italic>MSH2</italic></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><italic>MSH6</italic></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><italic>MUTYH (Biallelic)</italic></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><italic>PALB2</italic></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><italic>PMS2</italic></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><italic>PTEN</italic></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><italic>SMAD4</italic></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><italic>STK11</italic></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><italic>TP53</italic></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"><italic>VHL</italic></td><td align=\\\"left\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Characteristics of individuals who underwent genetic testing (<italic>N</italic> = 1943)</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Characteristics</th><th align=\\\"left\\\">Patients with a PV in HBOC gene<break/> N= 33</th><th align=\\\"left\\\">Patients with a PV in a LS gene<break/> N= 6</th><th align=\\\"left\\\">Patients without a PV<break/> N= 1904</th><th align=\\\"left\\\">All<break/> N= 1943</th></tr></thead><tbody><tr><td align=\\\"left\\\">Median age in years (Range)</td><td char=\\\"(\\\" align=\\\"char\\\">61 (36–78)</td><td char=\\\"(\\\" align=\\\"char\\\">67 (19–73)</td><td char=\\\"(\\\" align=\\\"char\\\">65 (18–89)</td><td char=\\\"(\\\" align=\\\"char\\\">66 (18–89</td></tr><tr><td align=\\\"left\\\">< 50 years, n (%)</td><td char=\\\"(\\\" align=\\\"char\\\">5 (15.2)</td><td char=\\\"(\\\" align=\\\"char\\\">2 (33.3)</td><td char=\\\"(\\\" align=\\\"char\\\">349 (18.3)</td><td char=\\\"(\\\" align=\\\"char\\\">356 (18.3)</td></tr><tr><td align=\\\"left\\\"> ≥ 50 years, n (%)</td><td char=\\\"(\\\" align=\\\"char\\\">28 (84.8)</td><td char=\\\"(\\\" align=\\\"char\\\">4 (66.7)</td><td char=\\\"(\\\" align=\\\"char\\\">1555 (81.7)</td><td char=\\\"(\\\" align=\\\"char\\\">1587 (81.7)</td></tr><tr><td align=\\\"left\\\">Race and Ethnicity, n (%)</td><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\".\\\" align=\\\"char\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Black</td><td char=\\\"(\\\" align=\\\"char\\\">2 (6.1)</td><td char=\\\"(\\\" align=\\\"char\\\">1 (16.7)</td><td char=\\\"(\\\" align=\\\"char\\\">336 (20)</td><td char=\\\"(\\\" align=\\\"char\\\">339 (20)</td></tr><tr><td align=\\\"left\\\">White</td><td char=\\\"(\\\" align=\\\"char\\\">14 (42.4)</td><td char=\\\"(\\\" align=\\\"char\\\">2 (33.3)</td><td char=\\\"(\\\" align=\\\"char\\\">634 (38.0)</td><td char=\\\"(\\\" align=\\\"char\\\">650 (38)</td></tr><tr><td align=\\\"left\\\">Asian</td><td char=\\\"(\\\" align=\\\"char\\\">3 (9.1)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0.0)</td><td char=\\\"(\\\" align=\\\"char\\\">82 (5.0)</td><td char=\\\"(\\\" align=\\\"char\\\">85 (5.0)</td></tr><tr><td align=\\\"left\\\">Hispanic</td><td char=\\\"(\\\" align=\\\"char\\\">8 (24.2)</td><td char=\\\"(\\\" align=\\\"char\\\">1 (16.7)</td><td char=\\\"(\\\" align=\\\"char\\\">525 (32)</td><td char=\\\"(\\\" align=\\\"char\\\">534 (32)</td></tr><tr><td align=\\\"left\\\">Multiple races selected</td><td char=\\\"(\\\" align=\\\"char\\\">1 (3.0)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0.0)</td><td char=\\\"(\\\" align=\\\"char\\\">23 (1.4)</td><td char=\\\"(\\\" align=\\\"char\\\">24 (1.4)</td></tr><tr><td align=\\\"left\\\">Other</td><td char=\\\"(\\\" align=\\\"char\\\">1 (3.0)</td><td char=\\\"(\\\" align=\\\"char\\\">1 (16.7)</td><td char=\\\"(\\\" align=\\\"char\\\">56 (3.4)</td><td char=\\\"(\\\" align=\\\"char\\\">58 (3.4)</td></tr><tr><td align=\\\"left\\\">Missing</td><td char=\\\"(\\\" align=\\\"char\\\">4 (12.1)</td><td char=\\\"(\\\" align=\\\"char\\\">1 (16.7)</td><td char=\\\"(\\\" align=\\\"char\\\">248 (13.0)</td><td char=\\\"(\\\" align=\\\"char\\\">253 (13.0)</td></tr><tr><td align=\\\"left\\\">Personal history of cancer (any type)</td><td char=\\\"(\\\" align=\\\"char\\\">1 (3.0)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0.0)</td><td char=\\\"(\\\" align=\\\"char\\\">145 (7.6)</td><td char=\\\"(\\\" align=\\\"char\\\">146 (7.5)</td></tr><tr><td align=\\\"left\\\">Family history of cancer (in patients with no personal cancer history)</td><td char=\\\"(\\\" align=\\\"char\\\">25 (75.8)</td><td char=\\\"(\\\" align=\\\"char\\\">6 (100.0)</td><td char=\\\"(\\\" align=\\\"char\\\">791 (41.5)</td><td char=\\\"(\\\" align=\\\"char\\\">822 (42.3)</td></tr><tr><td align=\\\"left\\\">No known personal or family history of cancer</td><td char=\\\"(\\\" align=\\\"char\\\">7 (21.2)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0.0)</td><td char=\\\"(\\\" align=\\\"char\\\">968 (50.8)</td><td char=\\\"(\\\" align=\\\"char\\\">975 (50.2)</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Number (percentage) of patients who met NCCN criteria for hereditary cancer testing based on personal and/or family history</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Characteristics</th><th align=\\\"left\\\">Patients with a PV in HBOC gene<break/> N= 33 (%)</th><th align=\\\"left\\\">Patients with a PV in a LS gene<break/> N= 6 (%)</th><th align=\\\"left\\\">Patients without a PV<break/> N= 1904 (%)</th><th align=\\\"left\\\">All<break/> N= 1943 (%)</th></tr></thead><tbody><tr><td align=\\\"left\\\">HBOC</td><td char=\\\"(\\\" align=\\\"char\\\">10 (30.3)</td><td char=\\\"(\\\" align=\\\"char\\\">3 (50.0)</td><td char=\\\"(\\\" align=\\\"char\\\">341 (17.9)</td><td char=\\\"(\\\" align=\\\"char\\\">354 (18.2)</td></tr><tr><td align=\\\"left\\\">Lynch</td><td char=\\\"(\\\" align=\\\"char\\\">1 (3.0)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0.0)</td><td char=\\\"(\\\" align=\\\"char\\\">70 (3.7)</td><td char=\\\"(\\\" align=\\\"char\\\">71 (3.7)</td></tr><tr><td align=\\\"left\\\">Both HBOC and Lynch</td><td char=\\\"(\\\" align=\\\"char\\\">1 (3.0)</td><td char=\\\"(\\\" align=\\\"char\\\">0 (0.0)</td><td char=\\\"(\\\" align=\\\"char\\\">18 (0.9)</td><td char=\\\"(\\\" align=\\\"char\\\">19 (1.0)</td></tr><tr><td align=\\\"left\\\"> Pre-test Tyrer-Cuzick* risk score calculated, n (%)</td><td char=\\\"(\\\" align=\\\"char\\\">25 (75.8)</td><td char=\\\"(\\\" align=\\\"char\\\">4(66.6)</td><td char=\\\"(\\\" align=\\\"char\\\">1405 (73.8)</td><td char=\\\"(\\\" align=\\\"char\\\">1434 (73.8)</td></tr><tr><td align=\\\"left\\\"> Pre-test Tyrer-Cuzick* risk >20%</td><td char=\\\"(\\\" align=\\\"char\\\">2 (8.0) *</td><td char=\\\"(\\\" align=\\\"char\\\">1 (16.7)</td><td char=\\\"(\\\" align=\\\"char\\\">66 (6.0)</td><td char=\\\"(\\\" align=\\\"char\\\">67 (4.7)</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p>HBOC, Hereditary Breast and Ovarian Cancer-related genes <italic>(BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, NF1, BRIP1)</italic></p><p>LS, Lynch syndrome genes <italic>(MSH2, PMS2)</italic></p></table-wrap-foot>","<table-wrap-foot><p>*Pre-test Tyrer–Cuzick score was calculated for the patients with no prior history of breast cancer</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>HBOC, Hereditary Breast and Ovarian Cancer-related genes <italic>(BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, NF1, BRIP1)</italic></p><p>LS, Lynch syndrome genes <italic>(MSH2, PMS2)</italic></p></table-wrap-foot>\",\n \"<table-wrap-foot><p>*Pre-test Tyrer–Cuzick score was calculated for the patients with no prior history of breast cancer</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"10549_2023_7137_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"10549_2023_7137_Fig2_HTML\" id=\"MO2\"/>"],"string":"[\n \"<graphic xlink:href=\\\"10549_2023_7137_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"10549_2023_7137_Fig2_HTML\\\" id=\\\"MO2\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"24.\"], \"mixed-citation\": [\"Network NCC (2023) Genetic/familial high-risk assessment: breast, ovarian, and pancreatic. \"], \"ext-link\": [\"https://www.nccn.org/professionals/physician_gls/pdf/genetics_bop.pdf\"]}, {\"label\": [\"25.\"], \"mixed-citation\": [\"NCCN.org. (2023). \"], \"ext-link\": [\"https://www.nccn.org/professionals/physician_gls/pdf/genetics_colon.pdf\"]}, {\"label\": [\"29.\"], \"mixed-citation\": [\"Prevention CfDCa: CDC WONDER. \"], \"ext-link\": [\"https://wonder.cdc.gov/\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":46,"string":"46"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"Breast Cancer Res Treat. 2024 Oct 20; 203(2):365-372"},"package_file":{"kind":"string","value":"oa_package/86/b6/PMC10787882.tar.gz"}}},{"rowIdx":166,"cells":{"accession_id":{"kind":"string","value":"PMC10787883"},"pmid":{"kind":"string","value":"38217724"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par5\">Robotic surgery has revolutionized the treatment of several diseases given the improved clinical outcomes, such as less blood loss, shorter length of hospital stay and fewer complications compared with laparoscopy or open approach. The main limitation has been the high cost due to purchase and maintenance of the da Vinci™ robotic surgical system (Intuitive Surgical, Sunnyvale, CA, USA) that represented the only alternative of the global market so far. After the Intuitive’s patent expiry, novel robotic platforms became available, including the Revo-I, the Senhance, the Versius, Avatera, Hinotori, and Hugo RAS. Some of these new systems share features differing from those of the da Vinci, both in terms of console, robotic units, and arm; thus, the introduction of novel platforms poses specific challenges to be addressed by the whole operating team; information and training about indications, setup, and outcomes are mandatory.</p>","<p id=\"Par6\">The Versius CMR is one of those new systems that has been approved in the UK in 2018. It displays an open surgical console with hand controllers and a head-up display (HUD). Versius is a multi-modular system with independent bedside unit (BSU), one dedicated to visualization with an endoscopic camera. The HUD provides the surgeon with a three-dimensional, high-definition visualization. The system carries the potentials of versatility, due to the open console design—improving communication in the OR—and adaptability with up to 4 BSU located around the operating table with multiple allowed configurations. The hand controllers are ergonomically designed and, unlike the Da Vinci and other new systems, accommodate all functions—including camera movement and energy delivery—without pedal control [##REF##37325289##1##–##REF##35047780##3##].</p>","<p id=\"Par7\">After strong evidence of feasibility developed in the pre-clinical setting [##REF##35807035##4##], clinical outcomes from the official “Versius Robotic Surgical System Registry” are yet to come; a low rate of conversion to alternative technique, serious adverse events, and 90-day mortality is anticipated [##REF##37036097##5##].</p>","<p id=\"Par8\">However, even if recognized viable from wide series, the description of each individual Versius procedures with specific challenges is still crucial.</p>","<p id=\"Par9\">The aim of the article is to report a comprehensive analysis of the Versius system for pelvic surgery, by describing indications, setup, and early outcomes in a multicentric study.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">Robotic surgery has revolutionized the treatment of several diseases given the improved clinical outcomes, such as less blood loss, shorter length of hospital stay and fewer complications compared with laparoscopy or open approach. The main limitation has been the high cost due to purchase and maintenance of the da Vinci™ robotic surgical system (Intuitive Surgical, Sunnyvale, CA, USA) that represented the only alternative of the global market so far. After the Intuitive’s patent expiry, novel robotic platforms became available, including the Revo-I, the Senhance, the Versius, Avatera, Hinotori, and Hugo RAS. Some of these new systems share features differing from those of the da Vinci, both in terms of console, robotic units, and arm; thus, the introduction of novel platforms poses specific challenges to be addressed by the whole operating team; information and training about indications, setup, and outcomes are mandatory.</p>\",\n \"<p id=\\\"Par6\\\">The Versius CMR is one of those new systems that has been approved in the UK in 2018. It displays an open surgical console with hand controllers and a head-up display (HUD). Versius is a multi-modular system with independent bedside unit (BSU), one dedicated to visualization with an endoscopic camera. The HUD provides the surgeon with a three-dimensional, high-definition visualization. The system carries the potentials of versatility, due to the open console design—improving communication in the OR—and adaptability with up to 4 BSU located around the operating table with multiple allowed configurations. The hand controllers are ergonomically designed and, unlike the Da Vinci and other new systems, accommodate all functions—including camera movement and energy delivery—without pedal control [##REF##37325289##1##–##REF##35047780##3##].</p>\",\n \"<p id=\\\"Par7\\\">After strong evidence of feasibility developed in the pre-clinical setting [##REF##35807035##4##], clinical outcomes from the official “Versius Robotic Surgical System Registry” are yet to come; a low rate of conversion to alternative technique, serious adverse events, and 90-day mortality is anticipated [##REF##37036097##5##].</p>\",\n \"<p id=\\\"Par8\\\">However, even if recognized viable from wide series, the description of each individual Versius procedures with specific challenges is still crucial.</p>\",\n \"<p id=\\\"Par9\\\">The aim of the article is to report a comprehensive analysis of the Versius system for pelvic surgery, by describing indications, setup, and early outcomes in a multicentric study.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<p id=\"Par10\">This is a retrospective observational study involving two Institutions, ASST Santi Paolo and Carlo, Milan, and at the Apuane Hospital, Massa, Italy.</p>","<p id=\"Par11\">The Versius CMR was installed by September, 2022, at the former institution, and by 2021 in the latter. The console surgeons involved were urologists (BR and DM), gynecologists (GG, MF), and general surgeons (AP). All except two were prior Da Vinci users; the latter (AP an MF) had an extensive laparoscopic background.</p>","<p id=\"Par12\">All interventions performed in the pelvic area with the Versius were included in the analysis. Data about indications, intra-, and post-operative course were prospectively collected.</p>","<title>Training</title>","<p id=\"Par13\">The training involved the whole OR Team (console surgeon, bedside assistant, scrub nurse, circulating nurse). For surgeons, the Versius training package consisted of a 3.5-day program following 10 h of online didactic training; it includes dry box exercises and wet lab sessions simulated in an operating room using cadaveric models.</p>","<title>Interventions</title>","<p id=\"Par14\">Urologic pelvic procedures, gynecological procedures, pelvic procedures from general surgeons were considered. Colectomy and hernia repair were excluded. Interventions could be either performed for oncological or non-oncological purposes; reconstructive interventions with the use of devices (i.e., mesh) were included as well.</p>","<title>Data collection and analysis</title>","<p id=\"Par15\">The following variables were collected:<list list-type=\"bullet\"><list-item><p id=\"Par16\">Demographics: age, gender;</p></list-item><list-item><p id=\"Par17\">Surgical indications</p></list-item><list-item><p id=\"Par18\">Intra-operative data: OR setup, number of ports, number of robotic arms, console time, estimated blood loss (EBL), conversion to alternative approaches, complications invoking a change in surgical strategy; use of additional devices for repair (i.e., mesh); malfunctioning of the system requiring a re-start</p></list-item><list-item><p id=\"Par19\">Post-operative data: complications classified by Clavien– Dindo, transfusion rate, length of stay (LOS), 30- and 90-day re-admission</p></list-item></list></p>","<p id=\"Par20\">Data were inserted in a dedicated data base (Excel, Microsoft) and analyzed by an external reviewer (ST) with formal habilitation as a coordinator with the Versius and the Da Vinci systems. A descriptive analysis of all variables was performed. Console Time, EBL, and LOS were provided as mean value and range.</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<p id=\\\"Par10\\\">This is a retrospective observational study involving two Institutions, ASST Santi Paolo and Carlo, Milan, and at the Apuane Hospital, Massa, Italy.</p>\",\n \"<p id=\\\"Par11\\\">The Versius CMR was installed by September, 2022, at the former institution, and by 2021 in the latter. The console surgeons involved were urologists (BR and DM), gynecologists (GG, MF), and general surgeons (AP). All except two were prior Da Vinci users; the latter (AP an MF) had an extensive laparoscopic background.</p>\",\n \"<p id=\\\"Par12\\\">All interventions performed in the pelvic area with the Versius were included in the analysis. Data about indications, intra-, and post-operative course were prospectively collected.</p>\",\n \"<title>Training</title>\",\n \"<p id=\\\"Par13\\\">The training involved the whole OR Team (console surgeon, bedside assistant, scrub nurse, circulating nurse). For surgeons, the Versius training package consisted of a 3.5-day program following 10 h of online didactic training; it includes dry box exercises and wet lab sessions simulated in an operating room using cadaveric models.</p>\",\n \"<title>Interventions</title>\",\n \"<p id=\\\"Par14\\\">Urologic pelvic procedures, gynecological procedures, pelvic procedures from general surgeons were considered. Colectomy and hernia repair were excluded. Interventions could be either performed for oncological or non-oncological purposes; reconstructive interventions with the use of devices (i.e., mesh) were included as well.</p>\",\n \"<title>Data collection and analysis</title>\",\n \"<p id=\\\"Par15\\\">The following variables were collected:<list list-type=\\\"bullet\\\"><list-item><p id=\\\"Par16\\\">Demographics: age, gender;</p></list-item><list-item><p id=\\\"Par17\\\">Surgical indications</p></list-item><list-item><p id=\\\"Par18\\\">Intra-operative data: OR setup, number of ports, number of robotic arms, console time, estimated blood loss (EBL), conversion to alternative approaches, complications invoking a change in surgical strategy; use of additional devices for repair (i.e., mesh); malfunctioning of the system requiring a re-start</p></list-item><list-item><p id=\\\"Par19\\\">Post-operative data: complications classified by Clavien– Dindo, transfusion rate, length of stay (LOS), 30- and 90-day re-admission</p></list-item></list></p>\",\n \"<p id=\\\"Par20\\\">Data were inserted in a dedicated data base (Excel, Microsoft) and analyzed by an external reviewer (ST) with formal habilitation as a coordinator with the Versius and the Da Vinci systems. A descriptive analysis of all variables was performed. Console Time, EBL, and LOS were provided as mean value and range.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par21\">Overall, a total of 171 interventions were performed with the Versius CMR at the ASST Santi Paolo and Carlo (120) and at the Apuane Hospital (51). Forty-two of them involved pelvic procedures. A full list of interventions stratified by center and specialty is reported in Table ##TAB##0##1##. Twenty-two interventions had an oncological indication (localized prostate cancer) whereas the remaining ones had a non-oncological or functional purpose. The mostly performed procedures were radical prostatectomy (22) and annexectomy (9). The OR setup for urological, gynecological and general surgical procedure (rectopexy and sigmoidectomy) is depicted in Fig. ##FIG##0##1##a–d, respectively. No intra-operative complication invoking a change in surgical strategy occurred nor conversion to open or laparoscopic surgery. Two Clavien II and one IIIb complications were evident (pelvic hematoma requiring transfusion, a urinary tract infection treated with antibiotic therapy and a bowel obstruction due to port-site herniation). Table ##TAB##1##2## reports console time, EBL, complications, and LOS stratified by procedure. Malfunctioning/alarms requiring the whole system re-activation occurred in 2 different cases. An adjustment in trocar placement according to patients’ height was required in 2 patients undergoing RALP, in which the trocar was moved caudally. In two cases, a pelvic organ prolapse (POP) was repaired concomitant with other gynecological procedures: an hysterectomy, bilateral salpingo-oophorectomy, and lateral suspension with a titanium-coated polypropylene mesh, according to Dubuisson technique. A single case, a 53-year-old woman, underwent a ventral rectopexy for full-thickness rectal external prolapse.</p>","<p id=\"Par22\">Positive margin (PM) rate can be evaluated for radical prostatectomies. The outcomes are variable, one series showed 83.3% of PM rate on 18 cases (11 pT3, published data) [##REF##37604773##6##] and in the other one (4 pT2c cases), no PM were found. At a follow-up ranging from 9 to 11 months, a single case of biochemical recurrence was noticed. One re-admission at 30 days (herniation) was recorded, and none at 90 days.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par21\\\">Overall, a total of 171 interventions were performed with the Versius CMR at the ASST Santi Paolo and Carlo (120) and at the Apuane Hospital (51). Forty-two of them involved pelvic procedures. A full list of interventions stratified by center and specialty is reported in Table ##TAB##0##1##. Twenty-two interventions had an oncological indication (localized prostate cancer) whereas the remaining ones had a non-oncological or functional purpose. The mostly performed procedures were radical prostatectomy (22) and annexectomy (9). The OR setup for urological, gynecological and general surgical procedure (rectopexy and sigmoidectomy) is depicted in Fig. ##FIG##0##1##a–d, respectively. No intra-operative complication invoking a change in surgical strategy occurred nor conversion to open or laparoscopic surgery. Two Clavien II and one IIIb complications were evident (pelvic hematoma requiring transfusion, a urinary tract infection treated with antibiotic therapy and a bowel obstruction due to port-site herniation). Table ##TAB##1##2## reports console time, EBL, complications, and LOS stratified by procedure. Malfunctioning/alarms requiring the whole system re-activation occurred in 2 different cases. An adjustment in trocar placement according to patients’ height was required in 2 patients undergoing RALP, in which the trocar was moved caudally. In two cases, a pelvic organ prolapse (POP) was repaired concomitant with other gynecological procedures: an hysterectomy, bilateral salpingo-oophorectomy, and lateral suspension with a titanium-coated polypropylene mesh, according to Dubuisson technique. A single case, a 53-year-old woman, underwent a ventral rectopexy for full-thickness rectal external prolapse.</p>\",\n \"<p id=\\\"Par22\\\">Positive margin (PM) rate can be evaluated for radical prostatectomies. The outcomes are variable, one series showed 83.3% of PM rate on 18 cases (11 pT3, published data) [##REF##37604773##6##] and in the other one (4 pT2c cases), no PM were found. At a follow-up ranging from 9 to 11 months, a single case of biochemical recurrence was noticed. One re-admission at 30 days (herniation) was recorded, and none at 90 days.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par23\">The current series represents the first one completely focused on pelvic surgery with the new Versius surgical system.</p>","<p id=\"Par24\">Sparse reports evaluating single indications with Versius [##REF##37221826##7##–##UREF##0##9##] are available yet; similarly, some series reported on the use of Versius for abdominal surgery, the majority including preliminary procedures—hernia repair, cholecystectomy—to become familiar with the system [##REF##36002682##10##, ##REF##32989548##11##]. In the present series, we intentionally addressed the pelvic area, to evaluate if robotic surgery with a novel platform may address the challenges posed by such interventions, often made up by complex tasks (dissection and reconstruction) in small spaces with limited accessibility and range of movements. A comprehensive analysis of pelvic approach with Versius has been already reported in the pre-clinical setting: Vasdess et al. [##REF##32989548##11##] described the possible setup for prostate surgery on cadaveric models, exploring multiple options of trocar placement with either 3- and 4-arm configurations. To note, authors evaluated also the feasibility of radical cystectomy (even if in vivo cystectomy has not yet performed so far with the Versius).</p>","<p id=\"Par25\">Our series confirms the feasibility of Versius pelvic surgery in different settings without major complication; actually, we did not face the need for conversion to other approaches. In preliminary series, conversion has been reported in up to 4–6% of Versius cases [##REF##37334028##12##, ##REF##35752748##13##] together with a certain variable degree of adverse events [##REF##35752748##13##–##REF##35861102##15##]. Herein, a single Clavien IIIb complication was reported (bowel herniation through abdominal wall requiring surgical repair); the occurrence is seemingly unrelated to the use of a novel robotic system. As far as surgical margin status is concerned, radical prostatectomy is the only oncological intervention we included. In this setting, PM rate from a single institution series appears of importance (83.3%); the small sample size and a high rate of pT3 could have accounted for the occurrence [##REF##37604773##6##]. Moreover, PM is counterbalanced by low BCR, (a single case showing a raise in post-operative PSA out of 22 RALP), provided long-term PSA is still awaited.</p>","<p id=\"Par26\">Unlike other series dealing with the Versius, some complex cases have been performed as well in the present article: this is the case of surgery for endometriosis and of a case of post-radiation sigmoidectomy.</p>","<p id=\"Par27\">Some specifics to pelvic surgery may apply to the Versius system.</p>","<p id=\"Par28\">Surgical instruments are shorter than those of the Da Vinci (30 cm): the issue should be considered and an estimation of the distance to the target area (and to other areas to be reached, i.e., pelvic nodal dissection) should be pre-planned. Port placement can be, therefore, moved caudally than the conventional Da Vinci configuration, especially considering patient’s height. In two RALP cases, we had to move one port more caudally to make the instrument reach the target area (urethral stump). The BMI of the patient is another issue that needs to be taken into account: in the current series, extreme BMIs have been successfully managed with a prior accurate planning of port and BSU placement. This was the case of gynecological surgery, which encompassed a range of BMI from 16 to 43 (non-published data).</p>","<p id=\"Par29\">One of the major concerns of multi-modular robotic system is the likelihood of external clashing between arms, a point somehow counterbalancing the versatility invoked by new systems with independent units [##REF##35637355##16##]. Overall, whereas the Da Vinci Xi allows for a linear trocar placement with a standardized docking, such a step with the Versius should be accurately planned to minimize clashing or limitation in instruments motion [##REF##35637355##16##]. Another feature typical of the Versius system is the docking of the robotic arm to the instrument and not to the trocar, as occurs with the Da Vinci or Hugo RAS; if the use of a non-dedicated trocar may represent an advantage, on the other side collisions between the robotic arm and the skin of the patient are recognized by the system and given as an alarm. The issue should be taken into account as well during the setup, especially in pelvic surgery in which a Trendelenburg of the patient is required and angles with the trocar could be relevant.</p>","<p id=\"Par30\">Beyond technical details, some general considerations may arise from the current experience.</p>","<p id=\"Par31\">Versius can be used by surgeons from different disciplines and it heavily comprises diverse surgical specialties; the same occurs with the Da Vinci, even if major users have initially been urologists [##REF##37325289##1##]. It is representative that in a center owning three robotic systems such as the ASST Santi Paolo and Carlo (Da Vinci, Versius CMR and Hugo RAS), the majority of Versius cases have been accomplished by gynecologists and general surgeons.</p>","<p id=\"Par32\">In line with these findings, laparoscopists seem to be those surgeons mostly advantaging from the introduction of Versius. The instruments are designed to mimic the articulation of the human arm and the wristed joints with seven degrees of freedom overcome the difficulties of laparoscopic surgery. The use of laparoscopic trocars—not robotic dedicated—raises opportunity for a hybrid procedure; thus, laparoscopists appreciate Versius features designed to simplify laparoscopic surgery.</p>","<p id=\"Par33\">Severe malfunctioning of the Versius has been reported in few cases and has been fixed with the re-start of the system (2 cases). If compared to the Da Vinci, it should be recognized that the latter has reached its fourth generation, whereas the Versius system is at its very first one; technological improvement and optimization are awaited. Noticeably, similar considerations may apply also to other novel robotic platforms that entered the market within the last two years.</p>","<p id=\"Par34\">As far as the Versius is concerned, technological updates are already developing: an improvement in arm clash recovery has been recently released—i.e., maintenance of arm engagement from the surgeon in case of clash—and other enhancements are yet to come, such as improvement of bipolar devices and the implementation of an energy sealer device [##REF##37334028##12##].</p>","<p id=\"Par35\">The article is not devoid of limitations.</p>","<p id=\"Par36\">First, the small sample size and short follow-up preclude any conclusion about mid- and long-term outcomes. The different kind of surgeries included make outcome assumptions weak; however, the report of long-term clinical follow-up was beyond the purpose of the paper, that merely aimed to address the feasibility of Versius surgery in the pelvic area.</p>","<p id=\"Par37\">Second, no matched comparison with the Da Vinci nor a subjective comparison is herein provided. Some console surgeons (BR, DM, GG) and assistants (MCS, FT, MS) are currently operating on multiple platforms, but a subjective perception about systems is not herein provided. However, an objective comparison of the first RALP cases with the Versius and Hugo RAS has been described by our group throughout the metrics developed by the ERUS working group for radical prostatectomy [##UREF##1##17##]. Further comparative analysis of robotic systems is expected in the very next future, to highlight differences and peculiarity of each platform trying to draw definite outcomes.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par23\\\">The current series represents the first one completely focused on pelvic surgery with the new Versius surgical system.</p>\",\n \"<p id=\\\"Par24\\\">Sparse reports evaluating single indications with Versius [##REF##37221826##7##–##UREF##0##9##] are available yet; similarly, some series reported on the use of Versius for abdominal surgery, the majority including preliminary procedures—hernia repair, cholecystectomy—to become familiar with the system [##REF##36002682##10##, ##REF##32989548##11##]. In the present series, we intentionally addressed the pelvic area, to evaluate if robotic surgery with a novel platform may address the challenges posed by such interventions, often made up by complex tasks (dissection and reconstruction) in small spaces with limited accessibility and range of movements. A comprehensive analysis of pelvic approach with Versius has been already reported in the pre-clinical setting: Vasdess et al. [##REF##32989548##11##] described the possible setup for prostate surgery on cadaveric models, exploring multiple options of trocar placement with either 3- and 4-arm configurations. To note, authors evaluated also the feasibility of radical cystectomy (even if in vivo cystectomy has not yet performed so far with the Versius).</p>\",\n \"<p id=\\\"Par25\\\">Our series confirms the feasibility of Versius pelvic surgery in different settings without major complication; actually, we did not face the need for conversion to other approaches. In preliminary series, conversion has been reported in up to 4–6% of Versius cases [##REF##37334028##12##, ##REF##35752748##13##] together with a certain variable degree of adverse events [##REF##35752748##13##–##REF##35861102##15##]. Herein, a single Clavien IIIb complication was reported (bowel herniation through abdominal wall requiring surgical repair); the occurrence is seemingly unrelated to the use of a novel robotic system. As far as surgical margin status is concerned, radical prostatectomy is the only oncological intervention we included. In this setting, PM rate from a single institution series appears of importance (83.3%); the small sample size and a high rate of pT3 could have accounted for the occurrence [##REF##37604773##6##]. Moreover, PM is counterbalanced by low BCR, (a single case showing a raise in post-operative PSA out of 22 RALP), provided long-term PSA is still awaited.</p>\",\n \"<p id=\\\"Par26\\\">Unlike other series dealing with the Versius, some complex cases have been performed as well in the present article: this is the case of surgery for endometriosis and of a case of post-radiation sigmoidectomy.</p>\",\n \"<p id=\\\"Par27\\\">Some specifics to pelvic surgery may apply to the Versius system.</p>\",\n \"<p id=\\\"Par28\\\">Surgical instruments are shorter than those of the Da Vinci (30 cm): the issue should be considered and an estimation of the distance to the target area (and to other areas to be reached, i.e., pelvic nodal dissection) should be pre-planned. Port placement can be, therefore, moved caudally than the conventional Da Vinci configuration, especially considering patient’s height. In two RALP cases, we had to move one port more caudally to make the instrument reach the target area (urethral stump). The BMI of the patient is another issue that needs to be taken into account: in the current series, extreme BMIs have been successfully managed with a prior accurate planning of port and BSU placement. This was the case of gynecological surgery, which encompassed a range of BMI from 16 to 43 (non-published data).</p>\",\n \"<p id=\\\"Par29\\\">One of the major concerns of multi-modular robotic system is the likelihood of external clashing between arms, a point somehow counterbalancing the versatility invoked by new systems with independent units [##REF##35637355##16##]. Overall, whereas the Da Vinci Xi allows for a linear trocar placement with a standardized docking, such a step with the Versius should be accurately planned to minimize clashing or limitation in instruments motion [##REF##35637355##16##]. Another feature typical of the Versius system is the docking of the robotic arm to the instrument and not to the trocar, as occurs with the Da Vinci or Hugo RAS; if the use of a non-dedicated trocar may represent an advantage, on the other side collisions between the robotic arm and the skin of the patient are recognized by the system and given as an alarm. The issue should be taken into account as well during the setup, especially in pelvic surgery in which a Trendelenburg of the patient is required and angles with the trocar could be relevant.</p>\",\n \"<p id=\\\"Par30\\\">Beyond technical details, some general considerations may arise from the current experience.</p>\",\n \"<p id=\\\"Par31\\\">Versius can be used by surgeons from different disciplines and it heavily comprises diverse surgical specialties; the same occurs with the Da Vinci, even if major users have initially been urologists [##REF##37325289##1##]. It is representative that in a center owning three robotic systems such as the ASST Santi Paolo and Carlo (Da Vinci, Versius CMR and Hugo RAS), the majority of Versius cases have been accomplished by gynecologists and general surgeons.</p>\",\n \"<p id=\\\"Par32\\\">In line with these findings, laparoscopists seem to be those surgeons mostly advantaging from the introduction of Versius. The instruments are designed to mimic the articulation of the human arm and the wristed joints with seven degrees of freedom overcome the difficulties of laparoscopic surgery. The use of laparoscopic trocars—not robotic dedicated—raises opportunity for a hybrid procedure; thus, laparoscopists appreciate Versius features designed to simplify laparoscopic surgery.</p>\",\n \"<p id=\\\"Par33\\\">Severe malfunctioning of the Versius has been reported in few cases and has been fixed with the re-start of the system (2 cases). If compared to the Da Vinci, it should be recognized that the latter has reached its fourth generation, whereas the Versius system is at its very first one; technological improvement and optimization are awaited. Noticeably, similar considerations may apply also to other novel robotic platforms that entered the market within the last two years.</p>\",\n \"<p id=\\\"Par34\\\">As far as the Versius is concerned, technological updates are already developing: an improvement in arm clash recovery has been recently released—i.e., maintenance of arm engagement from the surgeon in case of clash—and other enhancements are yet to come, such as improvement of bipolar devices and the implementation of an energy sealer device [##REF##37334028##12##].</p>\",\n \"<p id=\\\"Par35\\\">The article is not devoid of limitations.</p>\",\n \"<p id=\\\"Par36\\\">First, the small sample size and short follow-up preclude any conclusion about mid- and long-term outcomes. The different kind of surgeries included make outcome assumptions weak; however, the report of long-term clinical follow-up was beyond the purpose of the paper, that merely aimed to address the feasibility of Versius surgery in the pelvic area.</p>\",\n \"<p id=\\\"Par37\\\">Second, no matched comparison with the Da Vinci nor a subjective comparison is herein provided. Some console surgeons (BR, DM, GG) and assistants (MCS, FT, MS) are currently operating on multiple platforms, but a subjective perception about systems is not herein provided. However, an objective comparison of the first RALP cases with the Versius and Hugo RAS has been described by our group throughout the metrics developed by the ERUS working group for radical prostatectomy [##UREF##1##17##]. Further comparative analysis of robotic systems is expected in the very next future, to highlight differences and peculiarity of each platform trying to draw definite outcomes.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p id=\"Par38\">From the present series, pelvic surgery with the Versius system is feasible without severe intra- or peri-operative complications; long-term oncological and functional outcomes are yet to be defined. All steps of pelvic surgery are reproducible with the Versius, provided a proper surgical setup and trocar placement are pursued. Versius can be easily adopted by surgeons of different disciplines and background within the same institution; a further multi-specialty implementation is expected, and a cost analysis is awaited to highlight its future role into healthcare systems.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p id=\\\"Par38\\\">From the present series, pelvic surgery with the Versius system is feasible without severe intra- or peri-operative complications; long-term oncological and functional outcomes are yet to be defined. All steps of pelvic surgery are reproducible with the Versius, provided a proper surgical setup and trocar placement are pursued. Versius can be easily adopted by surgeons of different disciplines and background within the same institution; a further multi-specialty implementation is expected, and a cost analysis is awaited to highlight its future role into healthcare systems.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par1\">Versius CMR is a novel robotic system characterized by an open surgical console and independent bedside units. The system has potentials of flexibility and versatility, and has been used in urological, gynecological, and general surgical procedure. The aim is to depict a comprehensive analysis of the Versius system for pelvic surgery.</p>","<title>Methods</title>","<p id=\"Par2\">This is a study involving two Institutions, ASST Santi Paolo and Carlo, Milan, and Apuane Hospital, Massa, Italy. All interventions performed in the pelvic area with the Versius were included. Data about indications, intra-, and post-operative course were prospectively collected and analyzed.</p>","<title>Results</title>","<p id=\"Par3\">A total of 171 interventions were performed with the Versius. Forty-two of them involved pelvic procedures. Twenty-two had an oncological indication (localized prostate cancer), the remaining had a non-oncological or functional purpose. The mostly performed pelvic procedure was radical prostatectomy (22) followed by annexectomy (9). No intra-operative complication nor conversion to other approaches occurred. A Clavien II complication and one Clavien IIIb were reported. Malfunctioning/alarms requiring a power cycle of the system occurred in 2 different cases. An adjustment in trocar placement according to patients’ height was required in 2 patients undergoing prostatectomy, in which the trocar was moved caudally. In two cases, a pelvic prolapse was repaired concomitant with other gynecological procedures.</p>","<title>Conclusions</title>","<p id=\"Par4\">Pelvic surgery with the Versius is feasible without major complications; either dissection and reconstructive steps could be accomplished, provided a proper OR setup and trocar placement are pursued. Versius can be easily adopted by surgeons of different disciplines and backgrounds; a further multi-specialty implementation is presumed and long-term oncological and functional outcomes are awaited.</p>","<title>Keywords</title>","<p>Open access funding provided by Università degli Studi di Milano within the CRUI-CARE Agreement.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par1\\\">Versius CMR is a novel robotic system characterized by an open surgical console and independent bedside units. The system has potentials of flexibility and versatility, and has been used in urological, gynecological, and general surgical procedure. The aim is to depict a comprehensive analysis of the Versius system for pelvic surgery.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">This is a study involving two Institutions, ASST Santi Paolo and Carlo, Milan, and Apuane Hospital, Massa, Italy. All interventions performed in the pelvic area with the Versius were included. Data about indications, intra-, and post-operative course were prospectively collected and analyzed.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">A total of 171 interventions were performed with the Versius. Forty-two of them involved pelvic procedures. Twenty-two had an oncological indication (localized prostate cancer), the remaining had a non-oncological or functional purpose. The mostly performed pelvic procedure was radical prostatectomy (22) followed by annexectomy (9). No intra-operative complication nor conversion to other approaches occurred. A Clavien II complication and one Clavien IIIb were reported. Malfunctioning/alarms requiring a power cycle of the system occurred in 2 different cases. An adjustment in trocar placement according to patients’ height was required in 2 patients undergoing prostatectomy, in which the trocar was moved caudally. In two cases, a pelvic prolapse was repaired concomitant with other gynecological procedures.</p>\",\n \"<title>Conclusions</title>\",\n \"<p id=\\\"Par4\\\">Pelvic surgery with the Versius is feasible without major complications; either dissection and reconstructive steps could be accomplished, provided a proper OR setup and trocar placement are pursued. Versius can be easily adopted by surgeons of different disciplines and backgrounds; a further multi-specialty implementation is presumed and long-term oncological and functional outcomes are awaited.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by Università degli Studi di Milano within the CRUI-CARE Agreement.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":["<title>Author contributions</title>","<p>MCS: protocol and project development, manuscript writing. MDM: project development, data management. JM: data collection, data analysis. MF: project development, data management. APC: project development, data management, manuscript editing. LM: data management. CC: data management. AM: data collection, data analysis. TC: data collection, data analysis. EP: data collection, data analysis. MS: data management. FT: data management. ST: data analysis, manuscript editing. SA: data collection. LS: data collection. MA: data collection. AM: project development. PPB: project development, supervision. SM: supervision. BR: project development, supervision. GG: project development, data management, manuscript editing.</p>","<title>Funding</title>","<p>Open access funding provided by Università degli Studi di Milano within the CRUI-CARE Agreement.</p>","<title>Data availability</title>","<p>Data will be shared upon appropriate request.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par39\">Authors have no potential conflict of interest to disclose.</p>","<title>The research involves human participants</title>","<p id=\"Par40\">All patients signed an informed consent to the procedure they underwent; all interventions were planned and performed according to the current international guidelines and treatment recommendation.</p>"],"string":"[\n \"<title>Author contributions</title>\",\n \"<p>MCS: protocol and project development, manuscript writing. MDM: project development, data management. JM: data collection, data analysis. MF: project development, data management. APC: project development, data management, manuscript editing. LM: data management. CC: data management. AM: data collection, data analysis. TC: data collection, data analysis. EP: data collection, data analysis. MS: data management. FT: data management. ST: data analysis, manuscript editing. SA: data collection. LS: data collection. MA: data collection. AM: project development. PPB: project development, supervision. SM: supervision. BR: project development, supervision. GG: project development, data management, manuscript editing.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by Università degli Studi di Milano within the CRUI-CARE Agreement.</p>\",\n \"<title>Data availability</title>\",\n \"<p>Data will be shared upon appropriate request.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par39\\\">Authors have no potential conflict of interest to disclose.</p>\",\n \"<title>The research involves human participants</title>\",\n \"<p id=\\\"Par40\\\">All patients signed an informed consent to the procedure they underwent; all interventions were planned and performed according to the current international guidelines and treatment recommendation.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>OR setup for prostatectomy (<bold>a</bold>), gynecologic surgery (<bold>b</bold>), rectopexy (<bold>c</bold>), signoidectomy (<bold>d</bold>)</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>OR setup for prostatectomy (<bold>a</bold>), gynecologic surgery (<bold>b</bold>), rectopexy (<bold>c</bold>), signoidectomy (<bold>d</bold>)</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Procedures stratified by Center</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Procedure</th><th align=\"left\">Number</th><th align=\"left\">Center</th><th align=\"left\">Details</th></tr></thead><tbody><tr><td align=\"left\">Radical prostatectomy</td><td char=\".\" align=\"char\">22</td><td align=\"left\"><p>ASST Santi Paolo and Carlo (4)</p><p>Apuane Hospital (18)</p></td><td align=\"left\"/></tr><tr><td align=\"left\">Annexectomy</td><td char=\".\" align=\"char\">9</td><td align=\"left\">ASST Santi Paolo and Carlo</td><td align=\"left\">Bilateral in 8 cases</td></tr><tr><td align=\"left\">Surgery for Endometriosis</td><td char=\".\" align=\"char\">2</td><td align=\"left\">ASST Santi Paolo and Carlo</td><td align=\"left\"/></tr><tr><td align=\"left\">Hysterectomy</td><td char=\".\" align=\"char\">3</td><td align=\"left\">ASST Santi Paolo and Carlo</td><td align=\"left\">Concomitant POP repair in 1 case</td></tr><tr><td align=\"left\">Ovarian cyst enucleation</td><td char=\".\" align=\"char\">4</td><td align=\"left\">ASST Santi Paolo and Carlo</td><td align=\"left\">Bilateral in 2 cases</td></tr><tr><td align=\"left\">Dubuisson repair</td><td char=\".\" align=\"char\">1</td><td align=\"left\">ASST Santi Paolo and Carlo</td><td align=\"left\"/></tr><tr><td align=\"left\">Ventral rectopexy</td><td char=\".\" align=\"char\">1</td><td align=\"left\">ASST Santi Paolo and Carlo</td><td align=\"left\"/></tr><tr><td align=\"left\">Sigmoidectomy</td><td char=\".\" align=\"char\">1</td><td align=\"left\">ASST Santi Paolo and Carlo</td><td align=\"left\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Peri-operative outcomes stratified by procedure</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Procedure</th><th align=\"left\">N° robotic arms</th><th align=\"left\">Console time</th><th align=\"left\">EBL</th><th align=\"left\">Complications</th><th align=\"left\">LOS</th></tr></thead><tbody><tr><td align=\"left\">Radical prostatectomy</td><td align=\"left\">4</td><td align=\"left\">201 (130–242)</td><td align=\"left\">140 (100–550)</td><td align=\"left\"><p>1 pelvic hematoma</p><p>1 UTI</p><p>1 port-site herniation</p></td><td align=\"left\">4 (3.75–7)</td></tr><tr><td align=\"left\">Annexectomy</td><td align=\"left\">4</td><td align=\"left\">58 (31–77)</td><td align=\"left\">0 (0–0)</td><td align=\"left\">No</td><td align=\"left\">2</td></tr><tr><td align=\"left\">Hysterectomy</td><td align=\"left\">4</td><td align=\"left\">142 (122–176)</td><td align=\"left\">133 (50–200)</td><td align=\"left\">No</td><td align=\"left\">2</td></tr><tr><td align=\"left\">Ovarian cyst enucleation</td><td align=\"left\">3 in 2 cases, 4 in 1</td><td align=\"left\">115 (76–147)</td><td align=\"left\">37 (0–62)</td><td align=\"left\">No</td><td align=\"left\">2</td></tr><tr><td align=\"left\">Surgery for Endometriosis</td><td align=\"left\">4</td><td align=\"left\">217 (210–225)</td><td align=\"left\">50 (0–100)</td><td align=\"left\">No</td><td align=\"left\">2</td></tr><tr><td align=\"left\">Dubuisson Lateral suspension</td><td align=\"left\">4</td><td align=\"left\">170</td><td align=\"left\">0</td><td align=\"left\">No</td><td align=\"left\">3</td></tr><tr><td align=\"left\">Ventral rectopexy</td><td align=\"left\">4</td><td align=\"left\">94</td><td align=\"left\">100</td><td align=\"left\">No</td><td align=\"left\">3</td></tr><tr><td align=\"left\">Sigmoidectomy</td><td align=\"left\">4</td><td align=\"left\">80</td><td align=\"left\">100</td><td align=\"left\">No</td><td align=\"left\">5</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Procedures stratified by Center</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Procedure</th><th align=\\\"left\\\">Number</th><th align=\\\"left\\\">Center</th><th align=\\\"left\\\">Details</th></tr></thead><tbody><tr><td align=\\\"left\\\">Radical prostatectomy</td><td char=\\\".\\\" align=\\\"char\\\">22</td><td align=\\\"left\\\"><p>ASST Santi Paolo and Carlo (4)</p><p>Apuane Hospital (18)</p></td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">Annexectomy</td><td char=\\\".\\\" align=\\\"char\\\">9</td><td align=\\\"left\\\">ASST Santi Paolo and Carlo</td><td align=\\\"left\\\">Bilateral in 8 cases</td></tr><tr><td align=\\\"left\\\">Surgery for Endometriosis</td><td char=\\\".\\\" align=\\\"char\\\">2</td><td align=\\\"left\\\">ASST Santi Paolo and Carlo</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">Hysterectomy</td><td char=\\\".\\\" align=\\\"char\\\">3</td><td align=\\\"left\\\">ASST Santi Paolo and Carlo</td><td align=\\\"left\\\">Concomitant POP repair in 1 case</td></tr><tr><td align=\\\"left\\\">Ovarian cyst enucleation</td><td char=\\\".\\\" align=\\\"char\\\">4</td><td align=\\\"left\\\">ASST Santi Paolo and Carlo</td><td align=\\\"left\\\">Bilateral in 2 cases</td></tr><tr><td align=\\\"left\\\">Dubuisson repair</td><td char=\\\".\\\" align=\\\"char\\\">1</td><td align=\\\"left\\\">ASST Santi Paolo and Carlo</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">Ventral rectopexy</td><td char=\\\".\\\" align=\\\"char\\\">1</td><td align=\\\"left\\\">ASST Santi Paolo and Carlo</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\">Sigmoidectomy</td><td char=\\\".\\\" align=\\\"char\\\">1</td><td align=\\\"left\\\">ASST Santi Paolo and Carlo</td><td align=\\\"left\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Peri-operative outcomes stratified by procedure</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Procedure</th><th align=\\\"left\\\">N° robotic arms</th><th align=\\\"left\\\">Console time</th><th align=\\\"left\\\">EBL</th><th align=\\\"left\\\">Complications</th><th align=\\\"left\\\">LOS</th></tr></thead><tbody><tr><td align=\\\"left\\\">Radical prostatectomy</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">201 (130–242)</td><td align=\\\"left\\\">140 (100–550)</td><td align=\\\"left\\\"><p>1 pelvic hematoma</p><p>1 UTI</p><p>1 port-site herniation</p></td><td align=\\\"left\\\">4 (3.75–7)</td></tr><tr><td align=\\\"left\\\">Annexectomy</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">58 (31–77)</td><td align=\\\"left\\\">0 (0–0)</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">2</td></tr><tr><td align=\\\"left\\\">Hysterectomy</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">142 (122–176)</td><td align=\\\"left\\\">133 (50–200)</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">2</td></tr><tr><td align=\\\"left\\\">Ovarian cyst enucleation</td><td align=\\\"left\\\">3 in 2 cases, 4 in 1</td><td align=\\\"left\\\">115 (76–147)</td><td align=\\\"left\\\">37 (0–62)</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">2</td></tr><tr><td align=\\\"left\\\">Surgery for Endometriosis</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">217 (210–225)</td><td align=\\\"left\\\">50 (0–100)</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">2</td></tr><tr><td align=\\\"left\\\">Dubuisson Lateral suspension</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">170</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">3</td></tr><tr><td align=\\\"left\\\">Ventral rectopexy</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">94</td><td align=\\\"left\\\">100</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">3</td></tr><tr><td align=\\\"left\\\">Sigmoidectomy</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">80</td><td align=\\\"left\\\">100</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">5</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>Maria Chiara Sighinolfi and Maurizio De Maria shared the first-authorship.</p></fn></fn-group>"],"string":"[\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>Maria Chiara Sighinolfi and Maurizio De Maria shared the first-authorship.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"345_2023_4730_Fig1_HTML\" id=\"MO1\"/>"],"string":"[\n \"<graphic xlink:href=\\\"345_2023_4730_Fig1_HTML\\\" id=\\\"MO1\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"9.\"], \"surname\": [\"El Dahdah\", \"Halabi\", \"Kamal\", \"Zenilman\", \"Moussa\"], \"given-names\": [\"J\", \"M\", \"J\", \"ME\", \"HJ\"], \"article-title\": [\"Initial experience with a novel robotic surgical system in abdominal surgery\"], \"source\": [\"Robot Surg\"], \"year\": [\"2023\"], \"volume\": [\"17\"], \"issue\": [\"3\"], \"fpage\": [\"841\"], \"lpage\": [\"846\"], \"pub-id\": [\"10.1007/s11701-022-01471-0\"]}, {\"label\": [\"17.\"], \"surname\": [\"Rocco\", \"Turri\", \"Sangalli\", \"Centanni\", \"Stocco\", \"Chiumello\", \"Assumma\", \"Coelho\", \"Sarchi\", \"Grasso\", \"Piacentini\", \"Dellrto\", \"Calcagnile\", \"Sighinolfi\"], \"given-names\": [\"B\", \"F\", \"M\", \"S\", \"M\", \"D\", \"S\", \"RF\", \"L\", \"A\", \"I\", \"P\", \"T\", \"MC\"], \"article-title\": [\"Concomitant introduction of new robotic systems (Hugo RAS and Versius) at a single center: analysis of first clinical cases of radical prostatectomy by a single surgeon\"], \"source\": [\"JU Open Plus.\"], \"year\": [\"2023\"], \"volume\": [\"1\"], \"issue\": [\"6\"], \"fpage\": [\"e00025\"], \"pub-id\": [\"10.1097/JU9.0000000000000020\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":17,"string":"17"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"World J Urol. 2024 Jan 13; 42(1):31"},"package_file":{"kind":"string","value":"oa_package/36/d9/PMC10787883.tar.gz"}}},{"rowIdx":167,"cells":{"accession_id":{"kind":"string","value":"PMC10787884"},"pmid":{"kind":"string","value":"37812362"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par5\">Breast cancer in men (male breast cancer; MBC) is a rare entity accounting for 0.5–1% of all breast cancer diagnoses and an estimated lifetime risk of 1:1000 [##REF##30620402##1##]. Due to its rarity and lack of prospective dedicated MBC trials, the treatment guidelines are mainly based on extrapolation from randomized evidence from trials including mainly women with breast cancer (female breast cancer; FBC) [##REF##32058842##2##].</p>","<p id=\"Par6\">The utilization of neoadjuvant chemotherapy (NAC) has steadily increased over the years due to its potential to de-escalate breast and axillary surgery as well as to provide an opportunity for response-based tailored adjuvant therapy [##REF##33507815##3##]. There is solid evidence supporting the use of NAC in FBC, while the utilization and effectiveness of NAC in MBC is less studied [##REF##33507815##3##, ##REF##34933868##4##]. In fact, two retrospective studies have shown conflicting results regarding potential sex disparities on the effectiveness of NAC [##REF##33997921##5##, ##REF##36069365##6##], whereas one of them also showed a lower utilization of NAC in MBC compared to FBC [##REF##33997921##5##].</p>","<p id=\"Par7\">Considering the limited and conflicting evidence on the role of NAC in MBC, the aims of the present nationwide, register-based, retrospective cohort study were to investigate the utilization of NAC in men and women with early breast cancer, and to compare the effectiveness of NAC between men and women in terms of pathologic complete response (pCR).</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">Breast cancer in men (male breast cancer; MBC) is a rare entity accounting for 0.5–1% of all breast cancer diagnoses and an estimated lifetime risk of 1:1000 [##REF##30620402##1##]. Due to its rarity and lack of prospective dedicated MBC trials, the treatment guidelines are mainly based on extrapolation from randomized evidence from trials including mainly women with breast cancer (female breast cancer; FBC) [##REF##32058842##2##].</p>\",\n \"<p id=\\\"Par6\\\">The utilization of neoadjuvant chemotherapy (NAC) has steadily increased over the years due to its potential to de-escalate breast and axillary surgery as well as to provide an opportunity for response-based tailored adjuvant therapy [##REF##33507815##3##]. There is solid evidence supporting the use of NAC in FBC, while the utilization and effectiveness of NAC in MBC is less studied [##REF##33507815##3##, ##REF##34933868##4##]. In fact, two retrospective studies have shown conflicting results regarding potential sex disparities on the effectiveness of NAC [##REF##33997921##5##, ##REF##36069365##6##], whereas one of them also showed a lower utilization of NAC in MBC compared to FBC [##REF##33997921##5##].</p>\",\n \"<p id=\\\"Par7\\\">Considering the limited and conflicting evidence on the role of NAC in MBC, the aims of the present nationwide, register-based, retrospective cohort study were to investigate the utilization of NAC in men and women with early breast cancer, and to compare the effectiveness of NAC between men and women in terms of pathologic complete response (pCR).</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Patients and methods</title>","<title>Study design, data sources, participants and data collection</title>","<p id=\"Par8\">For this nationwide, register-based retrospective <italic>cohort study</italic>, all patients with stage I–III invasive breast cancer diagnosed in Sweden between January 1, 2008 and December 31, 2019 were identified through the National Quality Registry for breast cancer (Nationellt Kvalitetsregister för bröstcancer; NKBC). NKBC has a high coverage (99.8%) and data completeness ensuring the validity of data and the generalizability of the study results [##REF##31046737##7##]. Using the ten-digit personal identity number, data from NKBC was linked to other national databases of interest to build the research database BCBaSe 3.0 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://cancercentrum.se/samverkan/regional-cancer-centres/research-and-innovation/register-based-research-databases/)\">https://cancercentrum.se/samverkan/regional-cancer-centres/research-and-innovation/register-based-research-databases/)</ext-link>. The study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Regional Ethics Committee, Stockholm (Approval number: 2019–02610).</p>","<p id=\"Par9\">All patients with stage I-III breast cancer in NKBC were included except those with lacking information on estrogen-receptor (ER) status, progesterone-receptor (PgR) status, HER2-status or information on preoperative or postoperative TNM stage. Patients who did not undergo surgery were also excluded. The number of patients treated with NAC but not underwent surgery were 91 females (1.4% among females with NAC) and 1 male (4.0% among males with NAC). The decision of no surgery has been considered unrelated to disease progression since all patients had a registered adjuvant therapy and none of the patients was registered with metastatic disease during the first three months from diagnosis (Fig. ##FIG##0##1##).</p>","<p id=\"Par10\">Patient demographics (age, educational level, household income, health care region at diagnosis), tumor characteristics (tumor size, histological grade, clinical stage, pathological stage, morphological type and surrogate molecular subtypes based on immunohistochemistry (IHC) status) and treatment characteristics (data on breast and axillary surgery, chemotherapy, radiotherapy and endocrine therapy) were collected.</p>","<title>Outcomes and definitions</title>","<p id=\"Par11\">IHC-subtyping was used to classify tumors into three surrogate subtypes, namely luminal (ER or PgR-status ≥ 10%, HER2-negative), HER2-positive (any ER and PgR status, HER2-positive), and triple negative (ER and PgR status < 10% and HER2-negative) breast cancer.</p>","<p id=\"Par12\">For the research question on NAC utilization, patients were classified as treated with NAC if there was a treatment strategy including NAC, irrespective of chemotherapeutic agent used.</p>","<p id=\"Par13\">For the research question on pCR, we defined pCR as the absence of invasive breast cancer in the surgical specimens from breast and axillary lymph nodes.</p>","<title>Statistical analysis</title>","<p id=\"Par14\">Data were summarized using descriptive statistics including numbers and percentages for categorical variables and median with range for continuous variables. Comparisons of patient characteristics between males and females were made by Pearson’s Chi-squared test, Fisher´s exact test, or Mann–Whitney test as appropriate. For the first research question on NAC utilization, a multivariable logistic regression analysis was performed to analyze the association between sex and the likelihood of receiving NAC while adjusting for prespecified patient- and tumor characteristics including age at diagnosis, educational level, household income, health care region at diagnosis, clinical T and N stage, morphological type, histological grade, and IHC-based subtype. For the second research question on pCR, a multivariable logistic regression analysis was conducted to evaluate the impact of sex on the odds of pCR, adjusted for prespecified parameters including age at diagnosis, clinical T and N stage, histological grade, and IHC-subtype. All <italic>p</italic> values reported were two-sided, and <italic>p</italic> values of < 0.05 were considered statistically significant. All statistical analyses were performed using the SPSS statistical package (IBM Corp. Released 2021. IBM SPSS Statistics for Windows, Version 28.0. Armonk, NY: IBM Corp).</p>"],"string":"[\n \"<title>Patients and methods</title>\",\n \"<title>Study design, data sources, participants and data collection</title>\",\n \"<p id=\\\"Par8\\\">For this nationwide, register-based retrospective <italic>cohort study</italic>, all patients with stage I–III invasive breast cancer diagnosed in Sweden between January 1, 2008 and December 31, 2019 were identified through the National Quality Registry for breast cancer (Nationellt Kvalitetsregister för bröstcancer; NKBC). NKBC has a high coverage (99.8%) and data completeness ensuring the validity of data and the generalizability of the study results [##REF##31046737##7##]. Using the ten-digit personal identity number, data from NKBC was linked to other national databases of interest to build the research database BCBaSe 3.0 (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://cancercentrum.se/samverkan/regional-cancer-centres/research-and-innovation/register-based-research-databases/)\\\">https://cancercentrum.se/samverkan/regional-cancer-centres/research-and-innovation/register-based-research-databases/)</ext-link>. The study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Regional Ethics Committee, Stockholm (Approval number: 2019–02610).</p>\",\n \"<p id=\\\"Par9\\\">All patients with stage I-III breast cancer in NKBC were included except those with lacking information on estrogen-receptor (ER) status, progesterone-receptor (PgR) status, HER2-status or information on preoperative or postoperative TNM stage. Patients who did not undergo surgery were also excluded. The number of patients treated with NAC but not underwent surgery were 91 females (1.4% among females with NAC) and 1 male (4.0% among males with NAC). The decision of no surgery has been considered unrelated to disease progression since all patients had a registered adjuvant therapy and none of the patients was registered with metastatic disease during the first three months from diagnosis (Fig. ##FIG##0##1##).</p>\",\n \"<p id=\\\"Par10\\\">Patient demographics (age, educational level, household income, health care region at diagnosis), tumor characteristics (tumor size, histological grade, clinical stage, pathological stage, morphological type and surrogate molecular subtypes based on immunohistochemistry (IHC) status) and treatment characteristics (data on breast and axillary surgery, chemotherapy, radiotherapy and endocrine therapy) were collected.</p>\",\n \"<title>Outcomes and definitions</title>\",\n \"<p id=\\\"Par11\\\">IHC-subtyping was used to classify tumors into three surrogate subtypes, namely luminal (ER or PgR-status ≥ 10%, HER2-negative), HER2-positive (any ER and PgR status, HER2-positive), and triple negative (ER and PgR status < 10% and HER2-negative) breast cancer.</p>\",\n \"<p id=\\\"Par12\\\">For the research question on NAC utilization, patients were classified as treated with NAC if there was a treatment strategy including NAC, irrespective of chemotherapeutic agent used.</p>\",\n \"<p id=\\\"Par13\\\">For the research question on pCR, we defined pCR as the absence of invasive breast cancer in the surgical specimens from breast and axillary lymph nodes.</p>\",\n \"<title>Statistical analysis</title>\",\n \"<p id=\\\"Par14\\\">Data were summarized using descriptive statistics including numbers and percentages for categorical variables and median with range for continuous variables. Comparisons of patient characteristics between males and females were made by Pearson’s Chi-squared test, Fisher´s exact test, or Mann–Whitney test as appropriate. For the first research question on NAC utilization, a multivariable logistic regression analysis was performed to analyze the association between sex and the likelihood of receiving NAC while adjusting for prespecified patient- and tumor characteristics including age at diagnosis, educational level, household income, health care region at diagnosis, clinical T and N stage, morphological type, histological grade, and IHC-based subtype. For the second research question on pCR, a multivariable logistic regression analysis was conducted to evaluate the impact of sex on the odds of pCR, adjusted for prespecified parameters including age at diagnosis, clinical T and N stage, histological grade, and IHC-subtype. All <italic>p</italic> values reported were two-sided, and <italic>p</italic> values of < 0.05 were considered statistically significant. All statistical analyses were performed using the SPSS statistical package (IBM Corp. Released 2021. IBM SPSS Statistics for Windows, Version 28.0. Armonk, NY: IBM Corp).</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Characteristics of study cohort</title>","<p id=\"Par15\">In total, 114,290 patients with breast cancer were registered in NKBC between 2008 and 2020. Applying the inclusion and exclusion criteria, 82,888 patients (82,401 women and 428 men) with stage I-III breast cancer who underwent surgery and had adequate information for IHC-subtyping were identified. This cohort comprised the NAC utilization cohort. When the cohort was restricted only to patients who received NAC, 6487 breast cancer patients (6463 women and 24 men) were available for analyses related to the effectiveness of NAC. A flowchart diagram of patients’ selection process is shown in Fig. ##FIG##0##1##.</p>","<p id=\"Par16\">Table ##TAB##0##1## summarizes patient, tumor and treatment characteristics in the NAC utilization cohort, by sex. Men with breast cancer were older, had a lower educational level, more advanced anatomical stage at diagnosis (both T and N stage), higher histological grade, fewer lobular carcinomas (1.5% vs. 13.8% in women) and a different IHC-subtype distribution (luminal 86.9% vs. 77.3%; HER2-positive 12.5% vs. 13.4%, triple negative 0.6% vs. 9.2%, respectively). Treatment patterns, including breast and axillary surgery as well as adjuvant therapeutic approaches followed the statistically significant differences in anatomical staging and IHC-subtype. Regarding NAC effectiveness cohort, 6463 women (7.8%) and 24 men (4.9%) received neoadjuvant chemotherapy, respectively. When comparing baseline characteristics of women and men treated with NAC, statistically significant differences regarding educational level, IHC-subtype, use of adjuvant endocrine therapy, as well as type of breast surgery were seen (Table ##TAB##1##2##). The utilization of NAC seems to be steadily increased over time for both males and females in Sweden as shown in Table ##TAB##1##2##.</p>","<title>Factors associated with NAC utilization patterns</title>","<p id=\"Par17\">Using multivariate logistic regression model, using the complete case analysis method, no statistically significant difference in NAC utilization between women and men was observed (Odds Ratio (OR): 1.135; 95% Confidence Interval (CI): 0.606–2.128). The total number of patients included in this model was 78,760. Factors associated with higher likelihood of NAC were: young age, high educational level, high household income, treatment in certain healthcare regions Stockholm/Gotland, South, or Southeast), high clinical T and N stage, high histological grade, HER2-positive and triple negative IHC-subtype and ductal histology (Table ##TAB##2##3##).</p>","<title>Factors associated with pCR after NAC</title>","<p id=\"Par18\">No statistically significant difference in pCR rates were observed between women and men in the multivariate logistic regression analysis, using the complete case analysis method (OR: 1.141; 95% CI 0.141–9.238). The total number of patients included in this model was 6215. Factors associated with higher pCR rates were young age, high histologic grade, and HER2-positive IHC-subtype (Table ##TAB##3##4##).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Characteristics of study cohort</title>\",\n \"<p id=\\\"Par15\\\">In total, 114,290 patients with breast cancer were registered in NKBC between 2008 and 2020. Applying the inclusion and exclusion criteria, 82,888 patients (82,401 women and 428 men) with stage I-III breast cancer who underwent surgery and had adequate information for IHC-subtyping were identified. This cohort comprised the NAC utilization cohort. When the cohort was restricted only to patients who received NAC, 6487 breast cancer patients (6463 women and 24 men) were available for analyses related to the effectiveness of NAC. A flowchart diagram of patients’ selection process is shown in Fig. ##FIG##0##1##.</p>\",\n \"<p id=\\\"Par16\\\">Table ##TAB##0##1## summarizes patient, tumor and treatment characteristics in the NAC utilization cohort, by sex. Men with breast cancer were older, had a lower educational level, more advanced anatomical stage at diagnosis (both T and N stage), higher histological grade, fewer lobular carcinomas (1.5% vs. 13.8% in women) and a different IHC-subtype distribution (luminal 86.9% vs. 77.3%; HER2-positive 12.5% vs. 13.4%, triple negative 0.6% vs. 9.2%, respectively). Treatment patterns, including breast and axillary surgery as well as adjuvant therapeutic approaches followed the statistically significant differences in anatomical staging and IHC-subtype. Regarding NAC effectiveness cohort, 6463 women (7.8%) and 24 men (4.9%) received neoadjuvant chemotherapy, respectively. When comparing baseline characteristics of women and men treated with NAC, statistically significant differences regarding educational level, IHC-subtype, use of adjuvant endocrine therapy, as well as type of breast surgery were seen (Table ##TAB##1##2##). The utilization of NAC seems to be steadily increased over time for both males and females in Sweden as shown in Table ##TAB##1##2##.</p>\",\n \"<title>Factors associated with NAC utilization patterns</title>\",\n \"<p id=\\\"Par17\\\">Using multivariate logistic regression model, using the complete case analysis method, no statistically significant difference in NAC utilization between women and men was observed (Odds Ratio (OR): 1.135; 95% Confidence Interval (CI): 0.606–2.128). The total number of patients included in this model was 78,760. Factors associated with higher likelihood of NAC were: young age, high educational level, high household income, treatment in certain healthcare regions Stockholm/Gotland, South, or Southeast), high clinical T and N stage, high histological grade, HER2-positive and triple negative IHC-subtype and ductal histology (Table ##TAB##2##3##).</p>\",\n \"<title>Factors associated with pCR after NAC</title>\",\n \"<p id=\\\"Par18\\\">No statistically significant difference in pCR rates were observed between women and men in the multivariate logistic regression analysis, using the complete case analysis method (OR: 1.141; 95% CI 0.141–9.238). The total number of patients included in this model was 6215. Factors associated with higher pCR rates were young age, high histologic grade, and HER2-positive IHC-subtype (Table ##TAB##3##4##).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par19\">Using nationwide, register-based data from Sweden, we found no evidence of sex disparities regarding utilization of NAC in breast cancer patients when analyses were adjusted for patient- and tumor characteristics. In addition, the effectiveness of NAC in terms of pCR seems to be similar between men and women with breast cancer, supporting the current recommendations on treating men with breast cancer with the same principles as women with regard to NAC indications.</p>","<p id=\"Par20\">Due to the rarity of MBC and subsequently the lack of prospective trials, potential differences in utilization and effectiveness of NAC between men and women have been investigated only through register-based studies.</p>","<p id=\"Par21\">Regarding utilization of NAC in men with breast cancer, Cao et al. analyzed data from the United States National Cancer Database (NCDB) between 2004 and 2016, and found that men with node positive (N +) disease were less likely to be treated with NAC when compared to women. Interestingly, Cao et al. found an underutilization of oncological treatment in men with breast cancer in general, a pattern not seen in the present study cohort. Breast cancer treatment practices may vary between countries, and may also have changed over time. Our study cohort included patients diagnosed during more recent years, when sex disparities in breast cancer treatment strategies have been acknowledged [##REF##19996029##8##], thus leading to efforts to mitigate these disparities. Differences between study cohorts with regard to age (a higher proportion of older adults in our cohort) and stage (only patients with N + disease in NCDB cohort) could also explain the partly conflicting study results.</p>","<p id=\"Par22\">Also, with regard to NAC effectiveness in men compared to women with breast cancer, the current literature shows somewhat conflicting results. Cao et al. found similar pCR rate between men and women treated with neoadjuvant chemotherapy, as well as a comparable overall survival. Leone et al. found the odds for pCR in women compared to men to be nearly twice as high when studying patients diagnosed 2010 to 2016 from the same database as Cao. Interestingly, the difference in pCR rates observed in the latter study was mostly driven by differences in pCR within the luminal HER2-negative and luminal HER2-positive subgroups. Our results are in accordance with Cao et al., but differ from Leone et al. Although the number of included patients in certain subgroups in our study cohort was not large enough to enable subgroup analyses, we included this potential confounding factor into the multivariate model when the impact of sex on NAC effectiveness was analyzed. The lack of difference in pCR rates between men and women with breast cancer, in spite of the higher percentage of luminal breast cancer in men, could possibly be explained by different distribution of Luminal A/B tumors between men and women, i.e. a higher proportion of more high risk Luminal B tumors in men [##REF##32579768##9##, ##REF##28984296##10##]. Luminal B breast cancer is associated with higher pCR rates than Luminal A [##REF##22508812##11##], possibly balancing the chance of pCR between the two cohorts. Another potential explanation of similar NAC effectiveness between men and women despite the dominance of luminal tumors in men could be a higher presence of an immunological-enriched tumor microenvironment in male luminal tumors [##REF##36614261##12##], a condition that has been associated with improved pCR rates in all breast cancer subtypes [##REF##29233559##13##].</p>","<p id=\"Par23\">The comparison of patient- and tumor-related characteristics between men and women with breast cancer confirmed some well-established differences between the sexes; older age at diagnosis, more advanced N-stage at diagnosis, the dominance of luminal subtype and the rarity of TNBC and lobular histology in men with breast cancer.</p>","<p id=\"Par24\">Considering NAC utilization in general, some study results deserve attention. Our study results confirm a tumor-driven approach regarding NAC utilization with a higher use in more advanced and biologically more aggressive disease, which is in line with the current evidence and clinical practice. On the other hand, our study results imply some socioeconomic and geographic inequalities with lower odds to receive NAC among patients with lower income, lower education and among those from specific regions. Although similar inequalities have been reported previously [##REF##35658495##14##, ##REF##30232683##15##], such disparities are not acceptable and a deeper understanding is necessary in an effort to eliminate healthcare-related inequalities.</p>","<p id=\"Par25\">An interesting finding in terms of factors associated with higher pCR rates was the association between young age and a higher pCR rate. This finding is in accordance with a pooled analysis from eight randomized trials, where younger patients had higher odds of pCR, thus supporting the notion that tumor biology in younger patients might be more aggressive also within subtypes and therefore more susceptible to chemotherapy [##REF##29632634##16##].</p>","<p id=\"Par26\">The study has several limitations that need to be addressed. First, data on planned treatment was used rather than actual treatment given, as NKBC data on planned treatment have a higher validity than given treatment for the studied time period. As a result, however, a risk of misclassification between NAC and primary surgery for some patients does exist. Second, the duration of planned NAC is lacking. However, the Swedish guidelines have steadily and throughout the years, recommended the use of six cycles (q3w) of chemotherapy, similar to the recommendation for adjuvant chemotherapy. In our study cohort, we lacked information about dose-dense chemotherapeutic regimens. However, this strategy has been rather uncommon in Sweden during the study period and there is no reason to believe that there would be any sex disparity in using dose dense regimens that could impact the prognosis. The limited sample size for men with breast cancer in some specific subtypes is also a limitation as it precludes from relevant subgroup analysis. To mitigate this source of bias, we tried to adjust the multivariate analyses using parameters of potential interest as breast cancer subtype. One could argue that the exclusion of patients who did not undergo surgery can result in immortal-time bias if disease progression during NAC is the reason for no surgery. However, within the group of patients treated with NAC, the proportion of patients who did not undergo surgery was extremely low in both sexes and was considered unrelated to disease progression, thus eliminating the risk for immortal-time bias. Finally, the nature of collected data for the present study does not allow any information about the role of patient or clinician in treatment decision regarding the type and sequence of treatment strategy between sexes.</p>","<p id=\"Par27\">Acknowledging the relative limited number of men with breast cancer included in the analyses, the current study did not find any sex disparities either in the NAC utilization or effectiveness supporting the current recommendations on treating men with breast cancer similar to women with regard to indications for NAC. The observed socioeconomical and geographical disparities in NAC utilization deserves a deeper understanding before designing strategies to eliminate these inequalities towards an equitable access to breast cancer care.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par19\\\">Using nationwide, register-based data from Sweden, we found no evidence of sex disparities regarding utilization of NAC in breast cancer patients when analyses were adjusted for patient- and tumor characteristics. In addition, the effectiveness of NAC in terms of pCR seems to be similar between men and women with breast cancer, supporting the current recommendations on treating men with breast cancer with the same principles as women with regard to NAC indications.</p>\",\n \"<p id=\\\"Par20\\\">Due to the rarity of MBC and subsequently the lack of prospective trials, potential differences in utilization and effectiveness of NAC between men and women have been investigated only through register-based studies.</p>\",\n \"<p id=\\\"Par21\\\">Regarding utilization of NAC in men with breast cancer, Cao et al. analyzed data from the United States National Cancer Database (NCDB) between 2004 and 2016, and found that men with node positive (N +) disease were less likely to be treated with NAC when compared to women. Interestingly, Cao et al. found an underutilization of oncological treatment in men with breast cancer in general, a pattern not seen in the present study cohort. Breast cancer treatment practices may vary between countries, and may also have changed over time. Our study cohort included patients diagnosed during more recent years, when sex disparities in breast cancer treatment strategies have been acknowledged [##REF##19996029##8##], thus leading to efforts to mitigate these disparities. Differences between study cohorts with regard to age (a higher proportion of older adults in our cohort) and stage (only patients with N + disease in NCDB cohort) could also explain the partly conflicting study results.</p>\",\n \"<p id=\\\"Par22\\\">Also, with regard to NAC effectiveness in men compared to women with breast cancer, the current literature shows somewhat conflicting results. Cao et al. found similar pCR rate between men and women treated with neoadjuvant chemotherapy, as well as a comparable overall survival. Leone et al. found the odds for pCR in women compared to men to be nearly twice as high when studying patients diagnosed 2010 to 2016 from the same database as Cao. Interestingly, the difference in pCR rates observed in the latter study was mostly driven by differences in pCR within the luminal HER2-negative and luminal HER2-positive subgroups. Our results are in accordance with Cao et al., but differ from Leone et al. Although the number of included patients in certain subgroups in our study cohort was not large enough to enable subgroup analyses, we included this potential confounding factor into the multivariate model when the impact of sex on NAC effectiveness was analyzed. The lack of difference in pCR rates between men and women with breast cancer, in spite of the higher percentage of luminal breast cancer in men, could possibly be explained by different distribution of Luminal A/B tumors between men and women, i.e. a higher proportion of more high risk Luminal B tumors in men [##REF##32579768##9##, ##REF##28984296##10##]. Luminal B breast cancer is associated with higher pCR rates than Luminal A [##REF##22508812##11##], possibly balancing the chance of pCR between the two cohorts. Another potential explanation of similar NAC effectiveness between men and women despite the dominance of luminal tumors in men could be a higher presence of an immunological-enriched tumor microenvironment in male luminal tumors [##REF##36614261##12##], a condition that has been associated with improved pCR rates in all breast cancer subtypes [##REF##29233559##13##].</p>\",\n \"<p id=\\\"Par23\\\">The comparison of patient- and tumor-related characteristics between men and women with breast cancer confirmed some well-established differences between the sexes; older age at diagnosis, more advanced N-stage at diagnosis, the dominance of luminal subtype and the rarity of TNBC and lobular histology in men with breast cancer.</p>\",\n \"<p id=\\\"Par24\\\">Considering NAC utilization in general, some study results deserve attention. Our study results confirm a tumor-driven approach regarding NAC utilization with a higher use in more advanced and biologically more aggressive disease, which is in line with the current evidence and clinical practice. On the other hand, our study results imply some socioeconomic and geographic inequalities with lower odds to receive NAC among patients with lower income, lower education and among those from specific regions. Although similar inequalities have been reported previously [##REF##35658495##14##, ##REF##30232683##15##], such disparities are not acceptable and a deeper understanding is necessary in an effort to eliminate healthcare-related inequalities.</p>\",\n \"<p id=\\\"Par25\\\">An interesting finding in terms of factors associated with higher pCR rates was the association between young age and a higher pCR rate. This finding is in accordance with a pooled analysis from eight randomized trials, where younger patients had higher odds of pCR, thus supporting the notion that tumor biology in younger patients might be more aggressive also within subtypes and therefore more susceptible to chemotherapy [##REF##29632634##16##].</p>\",\n \"<p id=\\\"Par26\\\">The study has several limitations that need to be addressed. First, data on planned treatment was used rather than actual treatment given, as NKBC data on planned treatment have a higher validity than given treatment for the studied time period. As a result, however, a risk of misclassification between NAC and primary surgery for some patients does exist. Second, the duration of planned NAC is lacking. However, the Swedish guidelines have steadily and throughout the years, recommended the use of six cycles (q3w) of chemotherapy, similar to the recommendation for adjuvant chemotherapy. In our study cohort, we lacked information about dose-dense chemotherapeutic regimens. However, this strategy has been rather uncommon in Sweden during the study period and there is no reason to believe that there would be any sex disparity in using dose dense regimens that could impact the prognosis. The limited sample size for men with breast cancer in some specific subtypes is also a limitation as it precludes from relevant subgroup analysis. To mitigate this source of bias, we tried to adjust the multivariate analyses using parameters of potential interest as breast cancer subtype. One could argue that the exclusion of patients who did not undergo surgery can result in immortal-time bias if disease progression during NAC is the reason for no surgery. However, within the group of patients treated with NAC, the proportion of patients who did not undergo surgery was extremely low in both sexes and was considered unrelated to disease progression, thus eliminating the risk for immortal-time bias. Finally, the nature of collected data for the present study does not allow any information about the role of patient or clinician in treatment decision regarding the type and sequence of treatment strategy between sexes.</p>\",\n \"<p id=\\\"Par27\\\">Acknowledging the relative limited number of men with breast cancer included in the analyses, the current study did not find any sex disparities either in the NAC utilization or effectiveness supporting the current recommendations on treating men with breast cancer similar to women with regard to indications for NAC. The observed socioeconomical and geographical disparities in NAC utilization deserves a deeper understanding before designing strategies to eliminate these inequalities towards an equitable access to breast cancer care.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose</title>","<p id=\"Par1\">Evidence supporting the use of neoadjuvant chemotherapy (NAC) in early breast cancer is based on studies mainly including women, whereas the utilization and effectiveness of NAC in men is less studied. The present study aimed to investigate the utilization and effectiveness of NAC in men and women with early breast cancer.</p>","<title>Methods</title>","<p id=\"Par2\">Eligible patients were identified through the Swedish National Breast Cancer Quality Register, that includes all newly diagnosed breast cancer cases in Sweden from 2008 and onwards. For the treatment utilization analysis, all patients with stage I–III between 2008 and 2020 were included (n = 82,888), whereas for the effectiveness analysis the cohort was restricted to patients receiving NAC (n = 6487). For both analyses, multivariate logistic regression models were applied to investigate potential sex disparities in NAC utilization and effectiveness, adjusted for patient- and tumor characteristics.</p>","<title>Results</title>","<p id=\"Par3\">In the NAC utilization analysis, 487 men and 82,401 women with stage I–III were included. No statistically significant difference between sexes in terms of NAC utilization was observed (adjusted Odds Ratio (adjOR): 1.135; 95% Confidence Interval (CI) 0.606–2.128) with an overall utilization rate of 4.9% in men compared to 7.8% in women. Among the 24 men and 6463 women who received NAC, the pathologic complete response (pCR) rates were 16.7% and 21.2%, respectively (adjOR: 1.141; 95% CI 0.141–9.238).</p>","<title>Conclusion</title>","<p id=\"Par4\">The present study did not find any sex disparities in NAC utilization or effectiveness in terms of pCR. This supports the current recommendations of treating men with breast cancer with the same indications for NAC as women.</p>","<title>Keywords</title>","<p>Open access funding provided by Uppsala University.</p>"],"string":"[\n \"<title>Purpose</title>\",\n \"<p id=\\\"Par1\\\">Evidence supporting the use of neoadjuvant chemotherapy (NAC) in early breast cancer is based on studies mainly including women, whereas the utilization and effectiveness of NAC in men is less studied. The present study aimed to investigate the utilization and effectiveness of NAC in men and women with early breast cancer.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">Eligible patients were identified through the Swedish National Breast Cancer Quality Register, that includes all newly diagnosed breast cancer cases in Sweden from 2008 and onwards. For the treatment utilization analysis, all patients with stage I–III between 2008 and 2020 were included (n = 82,888), whereas for the effectiveness analysis the cohort was restricted to patients receiving NAC (n = 6487). For both analyses, multivariate logistic regression models were applied to investigate potential sex disparities in NAC utilization and effectiveness, adjusted for patient- and tumor characteristics.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">In the NAC utilization analysis, 487 men and 82,401 women with stage I–III were included. No statistically significant difference between sexes in terms of NAC utilization was observed (adjusted Odds Ratio (adjOR): 1.135; 95% Confidence Interval (CI) 0.606–2.128) with an overall utilization rate of 4.9% in men compared to 7.8% in women. Among the 24 men and 6463 women who received NAC, the pathologic complete response (pCR) rates were 16.7% and 21.2%, respectively (adjOR: 1.141; 95% CI 0.141–9.238).</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par4\\\">The present study did not find any sex disparities in NAC utilization or effectiveness in terms of pCR. This supports the current recommendations of treating men with breast cancer with the same indications for NAC as women.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by Uppsala University.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":["<title>Author contributions</title>","<p>AV conceived the original idea about the study and study’s design. Material preparation, data collection and analysis were performed by AV. The first draft of the manuscript was written by AS and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.</p>","<title>Funding</title>","<p>Open access funding provided by Uppsala University. This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.</p>","<title>Data availability</title>","<p>Data are available from register holders (Statistics Sweden, Swedish National Board of Health and Welfare, the Regional Cancer Center Stockholm Gotland) for researchers with relevant ethical approvals and who meet the criteria for access to confidential data. The data are not publicly available due to restrictions by Swedish and European law, in order to protect patient privacy.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par28\">AS, MS and AV have no competing interests. IF has received institutional research grants from MSD unrelated to the current work.</p>"],"string":"[\n \"<title>Author contributions</title>\",\n \"<p>AV conceived the original idea about the study and study’s design. Material preparation, data collection and analysis were performed by AV. The first draft of the manuscript was written by AS and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by Uppsala University. This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.</p>\",\n \"<title>Data availability</title>\",\n \"<p>Data are available from register holders (Statistics Sweden, Swedish National Board of Health and Welfare, the Regional Cancer Center Stockholm Gotland) for researchers with relevant ethical approvals and who meet the criteria for access to confidential data. The data are not publicly available due to restrictions by Swedish and European law, in order to protect patient privacy.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par28\\\">AS, MS and AV have no competing interests. IF has received institutional research grants from MSD unrelated to the current work.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>CONSORT diagram of study population. Cohort I: All patients registered in NKBC with stage I-III breast cancer who underwent surgery and had adequate information for IHC-subtyping. Cohort II: restricted only to patients who received neoadjuvant chemotherapy (NAC) (BcBASE 3.0, NKBC, 2008-2019).</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>CONSORT diagram of study population. Cohort I: All patients registered in NKBC with stage I-III breast cancer who underwent surgery and had adequate information for IHC-subtyping. Cohort II: restricted only to patients who received neoadjuvant chemotherapy (NAC) (BcBASE 3.0, NKBC, 2008-2019).</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Summary of demographics, tumor-related variables, and treatment patterns for the utilization cohort; women and men with stage I-III breast cancer who underwent surgery and had adequate information for IHC-subtyping (BCBaSe 3.0/NKBC, 2008–2019)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Factors</th><th align=\"left\">Female (N = 82,401), n (%)</th><th align=\"left\">Male (N = 487), n (%)</th><th align=\"left\">p value</th></tr></thead><tbody><tr><td align=\"left\">Age in yrs, median (range)</td><td align=\"left\">64 (19–99)</td><td align=\"left\">69 (29–94)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\">Calendar year at diagnosis</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> 2008–2011</td><td align=\"left\">22,001 (26.7)</td><td align=\"left\">108 (22.2)</td><td char=\".\" align=\"char\"><bold>0.026</bold></td></tr><tr><td align=\"left\"> 2012–2015</td><td align=\"left\">26,780 (32.5)</td><td align=\"left\">154 (21.6)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> 2016–2020</td><td align=\"left\">33,620 (40.8)</td><td align=\"left\">225 (46.2)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Education Level</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Low ≦9 years</td><td align=\"left\">17,330 (21.3)</td><td align=\"left\">149 (30.8)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Intermediate 10–12 years</td><td align=\"left\">34,310 (42.1)</td><td align=\"left\">200 (41.3)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> High ≧13 years</td><td align=\"left\">29,790 (36.6)</td><td align=\"left\">135 (27.9)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Household Income</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Q1</td><td align=\"left\">20,166 (24.6)</td><td align=\"left\">114 (23.5)</td><td char=\".\" align=\"char\">0.602</td></tr><tr><td align=\"left\"> Q2</td><td align=\"left\">20,792 (25.4)</td><td align=\"left\">127 (26.2)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Q3</td><td align=\"left\">20,545 (25.1)</td><td align=\"left\">132 (27.2)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Q4</td><td align=\"left\">20,383 (24.9)</td><td align=\"left\">112 (23.1)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Regions</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Northern</td><td align=\"left\">6822 (8.3)</td><td align=\"left\">47 (9.7)</td><td char=\".\" align=\"char\">0.519</td></tr><tr><td align=\"left\"> Stockholm-Gotland</td><td align=\"left\">19,185 (23.4)</td><td align=\"left\">99 (20.4)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Uppsala-Örebro</td><td align=\"left\">17,143 (20.9)</td><td align=\"left\">102 (21.0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> South</td><td align=\"left\">12,965 (15.8)</td><td align=\"left\">79 (16.3)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Southeast</td><td align=\"left\">8803 (10.7)</td><td align=\"left\">48 (9.9)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Western (Halland)</td><td align=\"left\">17,142 (20.9)</td><td align=\"left\">11 (22.8)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Clinical T stage</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> T 0–1</td><td align=\"left\">54,155 (65.7)</td><td align=\"left\">263 (54.0)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> T 2–4</td><td align=\"left\">27,893 (33.9)</td><td align=\"left\">221 (45.4)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Clinical N stage</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> cN + </td><td align=\"left\">10,246 (12.4)</td><td align=\"left\">108 (22.2)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> cN-</td><td align=\"left\">71,881 (87.2)</td><td align=\"left\">378 (77.6)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Histological grade</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Well differentiated (G1)</td><td align=\"left\">16,013 (19.4)</td><td align=\"left\">48 (9.9)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Moderately differentiated (G2)</td><td align=\"left\">40,137 (48.7)</td><td align=\"left\">246 (50.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Poorly differentiated (G3)</td><td align=\"left\">21,346 (25.9)</td><td align=\"left\">171 (35.1)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Subtype according to IHC</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Luminal</td><td align=\"left\">63,728 (77.3)</td><td align=\"left\">423 (86.9)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Her2 positive</td><td align=\"left\">11,059 (13.4)</td><td align=\"left\">61 (12.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> TNBC</td><td align=\"left\">7614 (9.2)</td><td align=\"left\">3 (0.6)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Morphological subtype</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Ductal</td><td align=\"left\">61,414 (79.1)</td><td align=\"left\">429 (91.9)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Lobular</td><td align=\"left\">10,719 (13.8)</td><td align=\"left\">7 (1.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Other</td><td align=\"left\">5503 (7.1)</td><td align=\"left\">31 (7.0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Breast surgery</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Breast conserving surgery</td><td align=\"left\">49,891 (60.5)</td><td align=\"left\">480 (98.6)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Mastectomy</td><td align=\"left\">32,510 (39.5)</td><td align=\"left\">7 (1.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Axillary surgery</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Sentinel lymph node dissection (SLND)</td><td align=\"left\">55,566 (70.2)</td><td align=\"left\">265 (57.7)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Axillary lymph node dissection (ALND)</td><td align=\"left\">12,253 (15.5)</td><td align=\"left\">113 (24.6)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> SLND = > ALND</td><td align=\"left\">11,363 (14.4)</td><td align=\"left\">81 (17.6)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Adjuvant chemotherapy</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">23,029 (27.9)</td><td align=\"left\">163 (33.5)</td><td char=\".\" align=\"char\"><bold>0.007</bold></td></tr><tr><td align=\"left\"> No</td><td align=\"left\">59,372 (72.1)</td><td align=\"left\">324 (66.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Adjuvant radiοtherapy</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">50,513 (61.3)</td><td align=\"left\">156 (32.0)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> No</td><td align=\"left\">31,888 (38.7)</td><td align=\"left\">331 (68.0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Adjuvant endocrine therapy</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">53,368 (64.8)</td><td align=\"left\">383 (78.6)</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> No</td><td align=\"left\">29,033 (35.2)</td><td align=\"left\">104 (21.4)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Neoadjuvant chemotherapy</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">6463 (7.8)</td><td align=\"left\">24 (4.9)</td><td char=\".\" align=\"char\"><bold>0.017</bold></td></tr><tr><td align=\"left\"> No</td><td align=\"left\">75,938 (92.2)</td><td align=\"left\">463 (95.1)</td><td char=\".\" align=\"char\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Summary of demographic and clinical variables as well as treatment modalities for female and male patients with breast cancer who received NAC (BCBaSe 3.0/NKBC, 2008–2019)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Factors</th><th align=\"left\">Female (N = 6463), n (%)</th><th align=\"left\">Male (N = 24), n (%)</th><th align=\"left\">p value</th></tr></thead><tbody><tr><td align=\"left\">Age in yrs, median (range)</td><td align=\"left\">54 (21–83)</td><td align=\"left\">64.5 (41–84)</td><td char=\".\" align=\"char\">0.054</td></tr><tr><td align=\"left\">Calendar year at diagnosis</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> 2008–2011</td><td align=\"left\">866 (13.4)</td><td align=\"left\">3 (12.5)</td><td char=\".\" align=\"char\">0.462</td></tr><tr><td align=\"left\"> 2012–2015</td><td align=\"left\">1706 (26.4)</td><td align=\"left\">9 (37.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> 2016–2020</td><td align=\"left\">3891 (60.2)</td><td align=\"left\">12 (50.0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Education Level</td><td align=\"left\">N = 6344</td><td align=\"left\">N = 23</td><td char=\".\" align=\"char\"><bold>0.001</bold></td></tr><tr><td align=\"left\"> Low ≦9 years</td><td align=\"left\">965 (15.2)</td><td align=\"left\">10 (43.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Intermediate 10–12 years</td><td align=\"left\">2576 (40.6)</td><td align=\"left\">8 (34.8)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> High ≧13 years</td><td align=\"left\">2803 (44.2)</td><td align=\"left\">5 (21.7)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Household Income</td><td align=\"left\">N = 6401</td><td align=\"left\">N = 24</td><td char=\".\" align=\"char\">0.405</td></tr><tr><td align=\"left\"> Q4</td><td align=\"left\">2162 (33.8)</td><td align=\"left\">9 (37.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Q3</td><td align=\"left\">1611 (25.2)</td><td align=\"left\">8 (33.3)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Q2</td><td align=\"left\">1435 (22.4)</td><td align=\"left\">2 (8.3)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Q1</td><td align=\"left\">1193 (18.6)</td><td align=\"left\">5 (20.8)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Regions</td><td align=\"left\">N = 6420</td><td align=\"left\">N = 24</td><td char=\".\" align=\"char\">0.715</td></tr><tr><td align=\"left\"> Northern</td><td align=\"left\">408 (6.4)</td><td align=\"left\">1 (4.2)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Stockholm-Gotland</td><td align=\"left\">2476 (38.6)</td><td align=\"left\">10 (41.7)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Uppsala-Örebro</td><td align=\"left\">794 (12.4)</td><td align=\"left\">1 (4.2)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> South</td><td align=\"left\">1224 (19.1)</td><td align=\"left\">7 (29.2)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Southeast</td><td align=\"left\">615 (9.6)</td><td align=\"left\">2 (8.3)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Western (Halland)</td><td align=\"left\">903 (14.1)</td><td align=\"left\">3 (12.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Clinical T stage</td><td align=\"left\">N = 6463</td><td align=\"left\">N = 24</td><td char=\".\" align=\"char\">0.951</td></tr><tr><td align=\"left\"> T 0–1</td><td align=\"left\">1066 (16.5)</td><td align=\"left\">4 (16.7)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> T 2–4</td><td align=\"left\">5370 (83.1)</td><td align=\"left\">20 (83.3)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Clinical N stage</td><td align=\"left\">N = 6463</td><td align=\"left\">N = 24</td><td char=\".\" align=\"char\">0.407</td></tr><tr><td align=\"left\"> cN + </td><td align=\"left\">3444 (53.3)</td><td align=\"left\">16 (66.7)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> cN-</td><td align=\"left\">2978 (46.1)</td><td align=\"left\">8 (33.3)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Histological grade</td><td align=\"left\">N = 2394</td><td align=\"left\">N = 11</td><td char=\".\" align=\"char\">0.054</td></tr><tr><td align=\"left\"> Well differentiated (G1)</td><td align=\"left\">266 (11.1)</td><td align=\"left\">1 (9.1)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Moderately differentiated (G2)</td><td align=\"left\">1357 (56.7)</td><td align=\"left\">10 (90.9)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Poorly differentiated (G3)</td><td align=\"left\">771 (32.2)</td><td align=\"left\">0 (0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Subtype according to IHC</td><td align=\"left\">N = 6463</td><td align=\"left\">N = 24</td><td char=\".\" align=\"char\"><bold>0.001</bold></td></tr><tr><td align=\"left\"> Luminal</td><td align=\"left\">2942 (45.4)</td><td align=\"left\">20 (83.3)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Her2 positive</td><td align=\"left\">2147 (33.2)</td><td align=\"left\">4 (16.7)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> TNBC</td><td align=\"left\">1374 (21.3)</td><td align=\"left\">0 (0.0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Morphological</td><td align=\"left\">N = 2337</td><td align=\"left\">N = 11</td><td char=\".\" align=\"char\">0.349</td></tr><tr><td align=\"left\"> Ductal</td><td align=\"left\">1961 (83.9)</td><td align=\"left\">11 (100)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Lobular</td><td align=\"left\">262 (11.2)</td><td align=\"left\">0 (0.0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Other</td><td align=\"left\">114 (4.9)</td><td align=\"left\">0 (0.0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Breast surgery</td><td align=\"left\">N = 6463</td><td align=\"left\">N = 24</td><td char=\".\" align=\"char\"><bold>0.001</bold></td></tr><tr><td align=\"left\"> Breast conserving surgery</td><td align=\"left\">2132 (33)</td><td align=\"left\">0 (0.0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Mastectomy</td><td align=\"left\">4331 (67)</td><td align=\"left\">24 (100)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Axillary surgery</td><td align=\"left\">N = 6271</td><td align=\"left\">N = 23</td><td char=\".\" align=\"char\">0.756</td></tr><tr><td align=\"left\"> Sentinel lymph node dissection (SLND)</td><td align=\"left\">1575 (25.1)</td><td align=\"left\">5 (21.7)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Axillary lymph node dissection (ALND)</td><td align=\"left\">3912 (62.4)</td><td align=\"left\">16 (69.6)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> SLND = > ALND</td><td align=\"left\">784 (12.5)</td><td align=\"left\">2 (8.7)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Type of chemotherapy</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Anthracycline- and taxane-based</td><td align=\"left\">4858 (75.2)</td><td align=\"left\">18 (75.0)</td><td char=\".\" align=\"char\">0.817</td></tr><tr><td align=\"left\"> Anthracycline-based</td><td align=\"left\">1004 (15.5)</td><td align=\"left\">3 (12.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Taxane-based</td><td align=\"left\">601 (9.3)</td><td align=\"left\">3 (12.5)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Anti-HER2 treatment</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Trastuzumab</td><td align=\"left\">1917 (29.7)</td><td align=\"left\">4 (16.7)</td><td char=\".\" align=\"char\">0.101</td></tr><tr><td align=\"left\">Adjuvant radiotherapy</td><td align=\"left\">N = 6463</td><td align=\"left\">N = 24</td><td char=\".\" align=\"char\">0.629</td></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">4854 (75.1)</td><td align=\"left\">17 (70.8)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> No</td><td align=\"left\">1609 (24.9)</td><td align=\"left\">7 (29.2)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Adjuvant endocrine therapy</td><td align=\"left\">N = 6463</td><td align=\"left\">N = 24</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">3853 (55.4)</td><td align=\"left\">22 (91.7)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> No</td><td align=\"left\">2880 (44.6)</td><td align=\"left\">2 (8.3)</td><td char=\".\" align=\"char\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>In utilization cohort: multivariable logistic regression analyzing the association between receipt of NAC and clinical factors among patients with breast cancer (BCBaSe 3.0/NKBC, 2008–2019)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">OR</th><th align=\"left\">95% CI</th><th align=\"left\">95% CI</th><th align=\"left\">P-value</th></tr><tr><th align=\"left\"/><th align=\"left\"/><th align=\"left\">Low</th><th align=\"left\">High</th><th align=\"left\"/></tr></thead><tbody><tr><td align=\"left\">Female versus male</td><td align=\"left\">1.135</td><td align=\"left\">0.606</td><td align=\"left\">2.128</td><td char=\".\" align=\"char\">0.692</td></tr><tr><td align=\"left\">Age</td><td align=\"left\">0.967</td><td align=\"left\">0.963</td><td align=\"left\">0.970</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\">Educational Level</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> High (≧13 years)</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Intermediate (10–12 years)</td><td align=\"left\">0.732</td><td align=\"left\">0.628</td><td align=\"left\">0.853</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Low (≦9 years)</td><td align=\"left\">0.890</td><td align=\"left\">0.801</td><td align=\"left\">0.989</td><td char=\".\" align=\"char\"><bold>0.030</bold></td></tr><tr><td align=\"left\">Household Income</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Q4 (High)</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Q3</td><td align=\"left\">0.708</td><td align=\"left\">0.624</td><td align=\"left\">0.803</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Q2</td><td align=\"left\">0.676</td><td align=\"left\">0.591</td><td align=\"left\">0.773</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Q1 (Low)</td><td align=\"left\">0.585</td><td align=\"left\">0.504</td><td align=\"left\">0.680</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\">Healtcare regions</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Northern</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Stockholm Gotland</td><td align=\"left\">2.707</td><td align=\"left\">2.197</td><td align=\"left\">3.335</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Uppsala-Örebro</td><td align=\"left\">0.826</td><td align=\"left\">0.657</td><td align=\"left\">1.038</td><td char=\".\" align=\"char\">0.101</td></tr><tr><td align=\"left\"> South</td><td align=\"left\">2.119</td><td align=\"left\">1.698</td><td align=\"left\">2.645</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Southeast</td><td align=\"left\">1.664</td><td align=\"left\">1.313</td><td align=\"left\">2.108</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Western</td><td align=\"left\">0.813</td><td align=\"left\">0.647</td><td align=\"left\">1.023</td><td char=\".\" align=\"char\">0.077</td></tr><tr><td align=\"left\">Clinical T stage</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> T1-2</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> T3-4</td><td align=\"left\">4.151</td><td align=\"left\">1.782</td><td align=\"left\">9.667</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\">Clinical N stage</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> cN-</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> cN + </td><td align=\"left\">1.888</td><td align=\"left\">1.039</td><td align=\"left\">3.432</td><td char=\".\" align=\"char\"><bold>0.037</bold></td></tr><tr><td align=\"left\">Histological grade</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> I</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> II</td><td align=\"left\">4.305</td><td align=\"left\">3.587</td><td align=\"left\">5.167</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> III</td><td align=\"left\">4.049</td><td align=\"left\">3.575</td><td align=\"left\">4.586</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\">Subtype according to IHC</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Luminal</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> HER2-positive</td><td align=\"left\">3.077</td><td align=\"left\">2.714</td><td align=\"left\">3.489</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Triple-negative</td><td align=\"left\">5.876</td><td align=\"left\">5.077</td><td align=\"left\">6.802</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\">Morphological type</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Ductal</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Lobular</td><td align=\"left\">0.778</td><td align=\"left\">0.669</td><td align=\"left\">0.906</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Other</td><td align=\"left\">0.667</td><td align=\"left\">0.530</td><td align=\"left\">0.838</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Multivariable logistic regression analyzing factors associated with complete pathologic response, among patients who received NAC for breast cancer (NKBC, 2008–2019)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">OR</th><th align=\"left\">95% CI</th><th align=\"left\">95% CI</th><th align=\"left\">P-value</th></tr><tr><th align=\"left\"/><th align=\"left\"/><th align=\"left\">Low</th><th align=\"left\">High</th><th align=\"left\"/></tr></thead><tbody><tr><td align=\"left\">Female versus male</td><td align=\"left\">1.141</td><td align=\"left\">0.141</td><td align=\"left\">9.238</td><td char=\".\" align=\"char\">0.902</td></tr><tr><td align=\"left\">Age</td><td align=\"left\">0.989</td><td align=\"left\">0.981</td><td align=\"left\">0.997</td><td char=\".\" align=\"char\"><bold>0.010</bold></td></tr><tr><td align=\"left\">Clinical T stage</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> T1-2</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> T3-4</td><td align=\"left\">1.227</td><td align=\"left\">0.142</td><td align=\"left\">10.612</td><td char=\".\" align=\"char\">0.853</td></tr><tr><td align=\"left\">Clinical N stage</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> cN− </td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> cN + </td><td align=\"left\">0.693</td><td align=\"left\">0.180</td><td align=\"left\">2.675</td><td char=\".\" align=\"char\">0.595</td></tr><tr><td align=\"left\">Histological grade</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> I</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> II</td><td align=\"left\">1.989</td><td align=\"left\">1.191</td><td align=\"left\">3.320</td><td char=\".\" align=\"char\"><bold>0.009</bold></td></tr><tr><td align=\"left\"> III</td><td align=\"left\">4.623</td><td align=\"left\">2.743</td><td align=\"left\">7.793</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\">Subtype according to IHC</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> Luminal</td><td align=\"left\">Ref</td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"> HER2-positive</td><td align=\"left\">2.774</td><td align=\"left\">2.134</td><td align=\"left\">3.607</td><td char=\".\" align=\"char\"><bold> < 0.001</bold></td></tr><tr><td align=\"left\"> Triple-negative</td><td align=\"left\">1.046</td><td align=\"left\">0.753</td><td align=\"left\">1.451</td><td char=\".\" align=\"char\">0.790</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Summary of demographics, tumor-related variables, and treatment patterns for the utilization cohort; women and men with stage I-III breast cancer who underwent surgery and had adequate information for IHC-subtyping (BCBaSe 3.0/NKBC, 2008–2019)</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Factors</th><th align=\\\"left\\\">Female (N = 82,401), n (%)</th><th align=\\\"left\\\">Male (N = 487), n (%)</th><th align=\\\"left\\\">p value</th></tr></thead><tbody><tr><td align=\\\"left\\\">Age in yrs, median (range)</td><td align=\\\"left\\\">64 (19–99)</td><td align=\\\"left\\\">69 (29–94)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\">Calendar year at diagnosis</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> 2008–2011</td><td align=\\\"left\\\">22,001 (26.7)</td><td align=\\\"left\\\">108 (22.2)</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.026</bold></td></tr><tr><td align=\\\"left\\\"> 2012–2015</td><td align=\\\"left\\\">26,780 (32.5)</td><td align=\\\"left\\\">154 (21.6)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> 2016–2020</td><td align=\\\"left\\\">33,620 (40.8)</td><td align=\\\"left\\\">225 (46.2)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Education Level</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Low ≦9 years</td><td align=\\\"left\\\">17,330 (21.3)</td><td align=\\\"left\\\">149 (30.8)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Intermediate 10–12 years</td><td align=\\\"left\\\">34,310 (42.1)</td><td align=\\\"left\\\">200 (41.3)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> High ≧13 years</td><td align=\\\"left\\\">29,790 (36.6)</td><td align=\\\"left\\\">135 (27.9)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Household Income</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Q1</td><td align=\\\"left\\\">20,166 (24.6)</td><td align=\\\"left\\\">114 (23.5)</td><td char=\\\".\\\" align=\\\"char\\\">0.602</td></tr><tr><td align=\\\"left\\\"> Q2</td><td align=\\\"left\\\">20,792 (25.4)</td><td align=\\\"left\\\">127 (26.2)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Q3</td><td align=\\\"left\\\">20,545 (25.1)</td><td align=\\\"left\\\">132 (27.2)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Q4</td><td align=\\\"left\\\">20,383 (24.9)</td><td align=\\\"left\\\">112 (23.1)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Regions</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Northern</td><td align=\\\"left\\\">6822 (8.3)</td><td align=\\\"left\\\">47 (9.7)</td><td char=\\\".\\\" align=\\\"char\\\">0.519</td></tr><tr><td align=\\\"left\\\"> Stockholm-Gotland</td><td align=\\\"left\\\">19,185 (23.4)</td><td align=\\\"left\\\">99 (20.4)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Uppsala-Örebro</td><td align=\\\"left\\\">17,143 (20.9)</td><td align=\\\"left\\\">102 (21.0)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> South</td><td align=\\\"left\\\">12,965 (15.8)</td><td align=\\\"left\\\">79 (16.3)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Southeast</td><td align=\\\"left\\\">8803 (10.7)</td><td align=\\\"left\\\">48 (9.9)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Western (Halland)</td><td align=\\\"left\\\">17,142 (20.9)</td><td align=\\\"left\\\">11 (22.8)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Clinical T stage</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> T 0–1</td><td align=\\\"left\\\">54,155 (65.7)</td><td align=\\\"left\\\">263 (54.0)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> T 2–4</td><td align=\\\"left\\\">27,893 (33.9)</td><td align=\\\"left\\\">221 (45.4)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Clinical N stage</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> cN + </td><td align=\\\"left\\\">10,246 (12.4)</td><td align=\\\"left\\\">108 (22.2)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> cN-</td><td align=\\\"left\\\">71,881 (87.2)</td><td align=\\\"left\\\">378 (77.6)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Histological grade</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Well differentiated (G1)</td><td align=\\\"left\\\">16,013 (19.4)</td><td align=\\\"left\\\">48 (9.9)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Moderately differentiated (G2)</td><td align=\\\"left\\\">40,137 (48.7)</td><td align=\\\"left\\\">246 (50.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Poorly differentiated (G3)</td><td align=\\\"left\\\">21,346 (25.9)</td><td align=\\\"left\\\">171 (35.1)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Subtype according to IHC</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Luminal</td><td align=\\\"left\\\">63,728 (77.3)</td><td align=\\\"left\\\">423 (86.9)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Her2 positive</td><td align=\\\"left\\\">11,059 (13.4)</td><td align=\\\"left\\\">61 (12.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> TNBC</td><td align=\\\"left\\\">7614 (9.2)</td><td align=\\\"left\\\">3 (0.6)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Morphological subtype</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Ductal</td><td align=\\\"left\\\">61,414 (79.1)</td><td align=\\\"left\\\">429 (91.9)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Lobular</td><td align=\\\"left\\\">10,719 (13.8)</td><td align=\\\"left\\\">7 (1.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Other</td><td align=\\\"left\\\">5503 (7.1)</td><td align=\\\"left\\\">31 (7.0)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Breast surgery</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Breast conserving surgery</td><td align=\\\"left\\\">49,891 (60.5)</td><td align=\\\"left\\\">480 (98.6)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Mastectomy</td><td align=\\\"left\\\">32,510 (39.5)</td><td align=\\\"left\\\">7 (1.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Axillary surgery</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Sentinel lymph node dissection (SLND)</td><td align=\\\"left\\\">55,566 (70.2)</td><td align=\\\"left\\\">265 (57.7)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Axillary lymph node dissection (ALND)</td><td align=\\\"left\\\">12,253 (15.5)</td><td align=\\\"left\\\">113 (24.6)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> SLND = > ALND</td><td align=\\\"left\\\">11,363 (14.4)</td><td align=\\\"left\\\">81 (17.6)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Adjuvant chemotherapy</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">23,029 (27.9)</td><td align=\\\"left\\\">163 (33.5)</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.007</bold></td></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\">59,372 (72.1)</td><td align=\\\"left\\\">324 (66.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Adjuvant radiοtherapy</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">50,513 (61.3)</td><td align=\\\"left\\\">156 (32.0)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\">31,888 (38.7)</td><td align=\\\"left\\\">331 (68.0)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Adjuvant endocrine therapy</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">53,368 (64.8)</td><td align=\\\"left\\\">383 (78.6)</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\">29,033 (35.2)</td><td align=\\\"left\\\">104 (21.4)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Neoadjuvant chemotherapy</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">6463 (7.8)</td><td align=\\\"left\\\">24 (4.9)</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.017</bold></td></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\">75,938 (92.2)</td><td align=\\\"left\\\">463 (95.1)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Summary of demographic and clinical variables as well as treatment modalities for female and male patients with breast cancer who received NAC (BCBaSe 3.0/NKBC, 2008–2019)</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Factors</th><th align=\\\"left\\\">Female (N = 6463), n (%)</th><th align=\\\"left\\\">Male (N = 24), n (%)</th><th align=\\\"left\\\">p value</th></tr></thead><tbody><tr><td align=\\\"left\\\">Age in yrs, median (range)</td><td align=\\\"left\\\">54 (21–83)</td><td align=\\\"left\\\">64.5 (41–84)</td><td char=\\\".\\\" align=\\\"char\\\">0.054</td></tr><tr><td align=\\\"left\\\">Calendar year at diagnosis</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> 2008–2011</td><td align=\\\"left\\\">866 (13.4)</td><td align=\\\"left\\\">3 (12.5)</td><td char=\\\".\\\" align=\\\"char\\\">0.462</td></tr><tr><td align=\\\"left\\\"> 2012–2015</td><td align=\\\"left\\\">1706 (26.4)</td><td align=\\\"left\\\">9 (37.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> 2016–2020</td><td align=\\\"left\\\">3891 (60.2)</td><td align=\\\"left\\\">12 (50.0)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Education Level</td><td align=\\\"left\\\">N = 6344</td><td align=\\\"left\\\">N = 23</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.001</bold></td></tr><tr><td align=\\\"left\\\"> Low ≦9 years</td><td align=\\\"left\\\">965 (15.2)</td><td align=\\\"left\\\">10 (43.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Intermediate 10–12 years</td><td align=\\\"left\\\">2576 (40.6)</td><td align=\\\"left\\\">8 (34.8)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> High ≧13 years</td><td align=\\\"left\\\">2803 (44.2)</td><td align=\\\"left\\\">5 (21.7)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Household Income</td><td align=\\\"left\\\">N = 6401</td><td align=\\\"left\\\">N = 24</td><td char=\\\".\\\" align=\\\"char\\\">0.405</td></tr><tr><td align=\\\"left\\\"> Q4</td><td align=\\\"left\\\">2162 (33.8)</td><td align=\\\"left\\\">9 (37.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Q3</td><td align=\\\"left\\\">1611 (25.2)</td><td align=\\\"left\\\">8 (33.3)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Q2</td><td align=\\\"left\\\">1435 (22.4)</td><td align=\\\"left\\\">2 (8.3)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Q1</td><td align=\\\"left\\\">1193 (18.6)</td><td align=\\\"left\\\">5 (20.8)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Regions</td><td align=\\\"left\\\">N = 6420</td><td align=\\\"left\\\">N = 24</td><td char=\\\".\\\" align=\\\"char\\\">0.715</td></tr><tr><td align=\\\"left\\\"> Northern</td><td align=\\\"left\\\">408 (6.4)</td><td align=\\\"left\\\">1 (4.2)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Stockholm-Gotland</td><td align=\\\"left\\\">2476 (38.6)</td><td align=\\\"left\\\">10 (41.7)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Uppsala-Örebro</td><td align=\\\"left\\\">794 (12.4)</td><td align=\\\"left\\\">1 (4.2)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> South</td><td align=\\\"left\\\">1224 (19.1)</td><td align=\\\"left\\\">7 (29.2)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Southeast</td><td align=\\\"left\\\">615 (9.6)</td><td align=\\\"left\\\">2 (8.3)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Western (Halland)</td><td align=\\\"left\\\">903 (14.1)</td><td align=\\\"left\\\">3 (12.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Clinical T stage</td><td align=\\\"left\\\">N = 6463</td><td align=\\\"left\\\">N = 24</td><td char=\\\".\\\" align=\\\"char\\\">0.951</td></tr><tr><td align=\\\"left\\\"> T 0–1</td><td align=\\\"left\\\">1066 (16.5)</td><td align=\\\"left\\\">4 (16.7)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> T 2–4</td><td align=\\\"left\\\">5370 (83.1)</td><td align=\\\"left\\\">20 (83.3)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Clinical N stage</td><td align=\\\"left\\\">N = 6463</td><td align=\\\"left\\\">N = 24</td><td char=\\\".\\\" align=\\\"char\\\">0.407</td></tr><tr><td align=\\\"left\\\"> cN + </td><td align=\\\"left\\\">3444 (53.3)</td><td align=\\\"left\\\">16 (66.7)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> cN-</td><td align=\\\"left\\\">2978 (46.1)</td><td align=\\\"left\\\">8 (33.3)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Histological grade</td><td align=\\\"left\\\">N = 2394</td><td align=\\\"left\\\">N = 11</td><td char=\\\".\\\" align=\\\"char\\\">0.054</td></tr><tr><td align=\\\"left\\\"> Well differentiated (G1)</td><td align=\\\"left\\\">266 (11.1)</td><td align=\\\"left\\\">1 (9.1)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Moderately differentiated (G2)</td><td align=\\\"left\\\">1357 (56.7)</td><td align=\\\"left\\\">10 (90.9)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Poorly differentiated (G3)</td><td align=\\\"left\\\">771 (32.2)</td><td align=\\\"left\\\">0 (0)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Subtype according to IHC</td><td align=\\\"left\\\">N = 6463</td><td align=\\\"left\\\">N = 24</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.001</bold></td></tr><tr><td align=\\\"left\\\"> Luminal</td><td align=\\\"left\\\">2942 (45.4)</td><td align=\\\"left\\\">20 (83.3)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Her2 positive</td><td align=\\\"left\\\">2147 (33.2)</td><td align=\\\"left\\\">4 (16.7)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> TNBC</td><td align=\\\"left\\\">1374 (21.3)</td><td align=\\\"left\\\">0 (0.0)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Morphological</td><td align=\\\"left\\\">N = 2337</td><td align=\\\"left\\\">N = 11</td><td char=\\\".\\\" align=\\\"char\\\">0.349</td></tr><tr><td align=\\\"left\\\"> Ductal</td><td align=\\\"left\\\">1961 (83.9)</td><td align=\\\"left\\\">11 (100)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Lobular</td><td align=\\\"left\\\">262 (11.2)</td><td align=\\\"left\\\">0 (0.0)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Other</td><td align=\\\"left\\\">114 (4.9)</td><td align=\\\"left\\\">0 (0.0)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Breast surgery</td><td align=\\\"left\\\">N = 6463</td><td align=\\\"left\\\">N = 24</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.001</bold></td></tr><tr><td align=\\\"left\\\"> Breast conserving surgery</td><td align=\\\"left\\\">2132 (33)</td><td align=\\\"left\\\">0 (0.0)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Mastectomy</td><td align=\\\"left\\\">4331 (67)</td><td align=\\\"left\\\">24 (100)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Axillary surgery</td><td align=\\\"left\\\">N = 6271</td><td align=\\\"left\\\">N = 23</td><td char=\\\".\\\" align=\\\"char\\\">0.756</td></tr><tr><td align=\\\"left\\\"> Sentinel lymph node dissection (SLND)</td><td align=\\\"left\\\">1575 (25.1)</td><td align=\\\"left\\\">5 (21.7)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Axillary lymph node dissection (ALND)</td><td align=\\\"left\\\">3912 (62.4)</td><td align=\\\"left\\\">16 (69.6)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> SLND = > ALND</td><td align=\\\"left\\\">784 (12.5)</td><td align=\\\"left\\\">2 (8.7)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Type of chemotherapy</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Anthracycline- and taxane-based</td><td align=\\\"left\\\">4858 (75.2)</td><td align=\\\"left\\\">18 (75.0)</td><td char=\\\".\\\" align=\\\"char\\\">0.817</td></tr><tr><td align=\\\"left\\\"> Anthracycline-based</td><td align=\\\"left\\\">1004 (15.5)</td><td align=\\\"left\\\">3 (12.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Taxane-based</td><td align=\\\"left\\\">601 (9.3)</td><td align=\\\"left\\\">3 (12.5)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Anti-HER2 treatment</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Trastuzumab</td><td align=\\\"left\\\">1917 (29.7)</td><td align=\\\"left\\\">4 (16.7)</td><td char=\\\".\\\" align=\\\"char\\\">0.101</td></tr><tr><td align=\\\"left\\\">Adjuvant radiotherapy</td><td align=\\\"left\\\">N = 6463</td><td align=\\\"left\\\">N = 24</td><td char=\\\".\\\" align=\\\"char\\\">0.629</td></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">4854 (75.1)</td><td align=\\\"left\\\">17 (70.8)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\">1609 (24.9)</td><td align=\\\"left\\\">7 (29.2)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\">Adjuvant endocrine therapy</td><td align=\\\"left\\\">N = 6463</td><td align=\\\"left\\\">N = 24</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">3853 (55.4)</td><td align=\\\"left\\\">22 (91.7)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\">2880 (44.6)</td><td align=\\\"left\\\">2 (8.3)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>In utilization cohort: multivariable logistic regression analyzing the association between receipt of NAC and clinical factors among patients with breast cancer (BCBaSe 3.0/NKBC, 2008–2019)</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"/><th align=\\\"left\\\">OR</th><th align=\\\"left\\\">95% CI</th><th align=\\\"left\\\">95% CI</th><th align=\\\"left\\\">P-value</th></tr><tr><th align=\\\"left\\\"/><th align=\\\"left\\\"/><th align=\\\"left\\\">Low</th><th align=\\\"left\\\">High</th><th align=\\\"left\\\"/></tr></thead><tbody><tr><td align=\\\"left\\\">Female versus male</td><td align=\\\"left\\\">1.135</td><td align=\\\"left\\\">0.606</td><td align=\\\"left\\\">2.128</td><td char=\\\".\\\" align=\\\"char\\\">0.692</td></tr><tr><td align=\\\"left\\\">Age</td><td align=\\\"left\\\">0.967</td><td align=\\\"left\\\">0.963</td><td align=\\\"left\\\">0.970</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\">Educational Level</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> High (≧13 years)</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Intermediate (10–12 years)</td><td align=\\\"left\\\">0.732</td><td align=\\\"left\\\">0.628</td><td align=\\\"left\\\">0.853</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Low (≦9 years)</td><td align=\\\"left\\\">0.890</td><td align=\\\"left\\\">0.801</td><td align=\\\"left\\\">0.989</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.030</bold></td></tr><tr><td align=\\\"left\\\">Household Income</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Q4 (High)</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Q3</td><td align=\\\"left\\\">0.708</td><td align=\\\"left\\\">0.624</td><td align=\\\"left\\\">0.803</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Q2</td><td align=\\\"left\\\">0.676</td><td align=\\\"left\\\">0.591</td><td align=\\\"left\\\">0.773</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Q1 (Low)</td><td align=\\\"left\\\">0.585</td><td align=\\\"left\\\">0.504</td><td align=\\\"left\\\">0.680</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\">Healtcare regions</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Northern</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Stockholm Gotland</td><td align=\\\"left\\\">2.707</td><td align=\\\"left\\\">2.197</td><td align=\\\"left\\\">3.335</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Uppsala-Örebro</td><td align=\\\"left\\\">0.826</td><td align=\\\"left\\\">0.657</td><td align=\\\"left\\\">1.038</td><td char=\\\".\\\" align=\\\"char\\\">0.101</td></tr><tr><td align=\\\"left\\\"> South</td><td align=\\\"left\\\">2.119</td><td align=\\\"left\\\">1.698</td><td align=\\\"left\\\">2.645</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Southeast</td><td align=\\\"left\\\">1.664</td><td align=\\\"left\\\">1.313</td><td align=\\\"left\\\">2.108</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Western</td><td align=\\\"left\\\">0.813</td><td align=\\\"left\\\">0.647</td><td align=\\\"left\\\">1.023</td><td char=\\\".\\\" align=\\\"char\\\">0.077</td></tr><tr><td align=\\\"left\\\">Clinical T stage</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> T1-2</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> T3-4</td><td align=\\\"left\\\">4.151</td><td align=\\\"left\\\">1.782</td><td align=\\\"left\\\">9.667</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\">Clinical N stage</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> cN-</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> cN + </td><td align=\\\"left\\\">1.888</td><td align=\\\"left\\\">1.039</td><td align=\\\"left\\\">3.432</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.037</bold></td></tr><tr><td align=\\\"left\\\">Histological grade</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> I</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> II</td><td align=\\\"left\\\">4.305</td><td align=\\\"left\\\">3.587</td><td align=\\\"left\\\">5.167</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> III</td><td align=\\\"left\\\">4.049</td><td align=\\\"left\\\">3.575</td><td align=\\\"left\\\">4.586</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\">Subtype according to IHC</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Luminal</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> HER2-positive</td><td align=\\\"left\\\">3.077</td><td align=\\\"left\\\">2.714</td><td align=\\\"left\\\">3.489</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Triple-negative</td><td align=\\\"left\\\">5.876</td><td align=\\\"left\\\">5.077</td><td align=\\\"left\\\">6.802</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\">Morphological type</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Ductal</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Lobular</td><td align=\\\"left\\\">0.778</td><td align=\\\"left\\\">0.669</td><td align=\\\"left\\\">0.906</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Other</td><td align=\\\"left\\\">0.667</td><td align=\\\"left\\\">0.530</td><td align=\\\"left\\\">0.838</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab4\\\"><label>Table 4</label><caption><p>Multivariable logistic regression analyzing factors associated with complete pathologic response, among patients who received NAC for breast cancer (NKBC, 2008–2019)</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"/><th align=\\\"left\\\">OR</th><th align=\\\"left\\\">95% CI</th><th align=\\\"left\\\">95% CI</th><th align=\\\"left\\\">P-value</th></tr><tr><th align=\\\"left\\\"/><th align=\\\"left\\\"/><th align=\\\"left\\\">Low</th><th align=\\\"left\\\">High</th><th align=\\\"left\\\"/></tr></thead><tbody><tr><td align=\\\"left\\\">Female versus male</td><td align=\\\"left\\\">1.141</td><td align=\\\"left\\\">0.141</td><td align=\\\"left\\\">9.238</td><td char=\\\".\\\" align=\\\"char\\\">0.902</td></tr><tr><td align=\\\"left\\\">Age</td><td align=\\\"left\\\">0.989</td><td align=\\\"left\\\">0.981</td><td align=\\\"left\\\">0.997</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.010</bold></td></tr><tr><td align=\\\"left\\\">Clinical T stage</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> T1-2</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> T3-4</td><td align=\\\"left\\\">1.227</td><td align=\\\"left\\\">0.142</td><td align=\\\"left\\\">10.612</td><td char=\\\".\\\" align=\\\"char\\\">0.853</td></tr><tr><td align=\\\"left\\\">Clinical N stage</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> cN− </td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> cN + </td><td align=\\\"left\\\">0.693</td><td align=\\\"left\\\">0.180</td><td align=\\\"left\\\">2.675</td><td char=\\\".\\\" align=\\\"char\\\">0.595</td></tr><tr><td align=\\\"left\\\">Histological grade</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> I</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> II</td><td align=\\\"left\\\">1.989</td><td align=\\\"left\\\">1.191</td><td align=\\\"left\\\">3.320</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.009</bold></td></tr><tr><td align=\\\"left\\\"> III</td><td align=\\\"left\\\">4.623</td><td align=\\\"left\\\">2.743</td><td align=\\\"left\\\">7.793</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\">Subtype according to IHC</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> Luminal</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\"> HER2-positive</td><td align=\\\"left\\\">2.774</td><td align=\\\"left\\\">2.134</td><td align=\\\"left\\\">3.607</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001</bold></td></tr><tr><td align=\\\"left\\\"> Triple-negative</td><td align=\\\"left\\\">1.046</td><td align=\\\"left\\\">0.753</td><td align=\\\"left\\\">1.451</td><td char=\\\".\\\" align=\\\"char\\\">0.790</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list 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complete case analyses</p><p>Bold text indicates a statistically significant difference with a p-value less than 0.05</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>Bold text indicates a statistically significant difference with a p-value less than 0.05</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>Bold text indicates a statistically difference with a p-value less than 0.05</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>The multivariate models were complete case analyses</p><p>Bold text indicates a statistically significant difference with a p-value less than 0.05</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>The multivariate models were complete case analyses</p><p>Bold text indicates a statistically significant difference with a p-value less than 0.05</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"10549_2023_7129_Fig1_HTML\" id=\"MO1\"/>"],"string":"[\n \"<graphic xlink:href=\\\"10549_2023_7129_Fig1_HTML\\\" id=\\\"MO1\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":16,"string":"16"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"Breast Cancer Res Treat. 2024 Oct 9; 203(2):235-243"},"package_file":{"kind":"string","value":"oa_package/e1/ed/PMC10787884.tar.gz"}}},{"rowIdx":168,"cells":{"accession_id":{"kind":"string","value":"PMC10787885"},"pmid":{"kind":"string","value":"38217834"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par2\">Robotic-assisted surgery (RAS) has gained widespread diffusion over the last years, demonstrating the ability to overcome the technical limitations of conventional laparoscopy. Enhancements provided by robotic assistance include three-dimensional view and magnification, increased dexterity with 7-degrees of freedom of robotic instruments, tremor filtering, and improved surgeons’ ergonomics [##REF##14685095##1##, ##REF##21815795##2##]. Some major drawbacks must be considered before using RAS in children: anesthesia, placement of trocars, and technical difficulties related to small space [##REF##28889938##3##]. Nevertheless, RAS has been described as safe and feasible option for a wide range of surgical indications in children, including urological, oncological, and gastrointestinal pathologies [##UREF##0##4##–##REF##36865692##8##]. Several reports have investigated safety and feasibility of RAS in pediatric population, compared with different approaches (open or laparoscopic) [##REF##29530407##9##–##REF##31555861##12##].</p>","<p id=\"Par3\">To the current state, the field of pediatric gynecology remains the least explored, with only few pediatric reports of application of RAS for gynecological indications [##REF##23158752##13##–##REF##15313068##16##].</p>","<p id=\"Par4\">This descriptive, retrospective study aimed to report a multicenter experience regarding the application of RAS for gynecological indications in pediatric patients.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par2\\\">Robotic-assisted surgery (RAS) has gained widespread diffusion over the last years, demonstrating the ability to overcome the technical limitations of conventional laparoscopy. Enhancements provided by robotic assistance include three-dimensional view and magnification, increased dexterity with 7-degrees of freedom of robotic instruments, tremor filtering, and improved surgeons’ ergonomics [##REF##14685095##1##, ##REF##21815795##2##]. Some major drawbacks must be considered before using RAS in children: anesthesia, placement of trocars, and technical difficulties related to small space [##REF##28889938##3##]. Nevertheless, RAS has been described as safe and feasible option for a wide range of surgical indications in children, including urological, oncological, and gastrointestinal pathologies [##UREF##0##4##–##REF##36865692##8##]. Several reports have investigated safety and feasibility of RAS in pediatric population, compared with different approaches (open or laparoscopic) [##REF##29530407##9##–##REF##31555861##12##].</p>\",\n \"<p id=\\\"Par3\\\">To the current state, the field of pediatric gynecology remains the least explored, with only few pediatric reports of application of RAS for gynecological indications [##REF##23158752##13##–##REF##15313068##16##].</p>\",\n \"<p id=\\\"Par4\\\">This descriptive, retrospective study aimed to report a multicenter experience regarding the application of RAS for gynecological indications in pediatric patients.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<p id=\"Par5\">All children and adolescents up to 18 years of age, operated using RAS for gynecological indications in 4 different institutions over a 3-year period, were included. The exclusion criteria were patients over 18 years old as well as all gynecological surgical conditions not treated with RAS.</p>","<p id=\"Par6\">The surgical centers were contacted via mail and those accepting to participate to the study, were required to fill a study form, with requested information, for each enrolled patient.</p>","<p id=\"Par7\">Patient baseline, including age at time of surgery, weight, possible comorbidities, clinical presentation, pre-operative diagnosis, and side of pathology, were reported in the first section.</p>","<p id=\"Par8\">Details of operative technique, such as type of procedure, number of robotic and/or accessory ports, use of sealing device, method for specimen extraction, were reported in the second section.</p>","<p id=\"Par9\">Operative results, including robot docking time, total operative time, length of stay (LOS), requirement time of pain medication, complication rate, conversion rate, pathology, and follow-up results, were analyzed in the third section.</p>","<p id=\"Par10\">All data were elaborated using the statistical software Microsoft Excel, Windows vers.11. Descriptive statistics were used to present findings, and quantitative variables were expressed as median (range) to report the data.</p>","<p id=\"Par11\">The study received appropriate Institute Review Board (IRB) approval.</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<p id=\\\"Par5\\\">All children and adolescents up to 18 years of age, operated using RAS for gynecological indications in 4 different institutions over a 3-year period, were included. The exclusion criteria were patients over 18 years old as well as all gynecological surgical conditions not treated with RAS.</p>\",\n \"<p id=\\\"Par6\\\">The surgical centers were contacted via mail and those accepting to participate to the study, were required to fill a study form, with requested information, for each enrolled patient.</p>\",\n \"<p id=\\\"Par7\\\">Patient baseline, including age at time of surgery, weight, possible comorbidities, clinical presentation, pre-operative diagnosis, and side of pathology, were reported in the first section.</p>\",\n \"<p id=\\\"Par8\\\">Details of operative technique, such as type of procedure, number of robotic and/or accessory ports, use of sealing device, method for specimen extraction, were reported in the second section.</p>\",\n \"<p id=\\\"Par9\\\">Operative results, including robot docking time, total operative time, length of stay (LOS), requirement time of pain medication, complication rate, conversion rate, pathology, and follow-up results, were analyzed in the third section.</p>\",\n \"<p id=\\\"Par10\\\">All data were elaborated using the statistical software Microsoft Excel, Windows vers.11. Descriptive statistics were used to present findings, and quantitative variables were expressed as median (range) to report the data.</p>\",\n \"<p id=\\\"Par11\\\">The study received appropriate Institute Review Board (IRB) approval.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Patient baseline</title>","<p id=\"Par12\">Twenty-three girls, with median age at surgery of 12.3 years (range 0.6–17.8) and median weight of 47.2 kg (range 9–73), received RAS for gynecological indication in the study period and were included. Associated comorbidities were reported in 5/23 (21.7%). Most patients (16/23, 69.5%) were symptomatic at time of diagnosis, with non-specific abdominal/pelvic pain being the most frequent presentation. Pre-operative work-up included abdominal ultrasonography (US), pelvic computed tomography (CT) and/or magnetic resonance imaging (MRI), and voiding cystourethrogram (VCUG) in selected cases. Serum tumor markers, such as beta human chorionic gonadotropin (β-HCG), alfa-fetoprotein (α-FP), Cancer Antigen 125 (Ca125), lactic dehydrogenase (LDH), and human epididymis secretory protein 4 (HE-4), were performed in all patients with adnexal mass. Pre-operative diagnosis was ovarian cyst (<italic>n</italic> = 4), ovarian “complex” mass (<italic>n</italic> = 12), fallopian tube lesion (<italic>n</italic> = 3), uterine cyst (<italic>n</italic> = 1), gonadal dysgenesis (<italic>n</italic> = 1), pelvic paravaginal mass (<italic>n</italic> = 1), cloaca malformation (<italic>n</italic> = 1), and high-confluence urogenital sinus (UGS) (<italic>n</italic> = 1). One patient (4.3%) presented concomitant ovarian “complex” mass and paratubal cyst.</p>","<title>Installation and operative technique</title>","<p id=\"Par13\">All RAS procedures were carried out using the da Vinci Xi Surgical System (Intuitive Surgical, Sunnyvale, CA, USA). The patient was placed supine on the operative table and appropriate age-sized Foley catheter was inserted using sterile precautions pre-operatively.</p>","<p id=\"Par14\">Three robotic arms, one 12-mm with 12–8 mm reducer, for the 3D, 0-degree, robotic optic, and two 8-mm ports to accommodate the robotic instruments, were placed on the umbilical line in all procedures. A fourth 5-mm accessory port was also placed. The robot was finally docked over the patient’s feet. Robotic vessel sealer was adopted in all procedures. Indocyanine green (ICG) near-infrared fluorescence (NIRF) was adopted in ovarian mass to check the resection margins and guide intra-operative decision making and in paratubal lesion to check the vascular permeability of the fallopian tube following the removal of the lesion (Fig. ##FIG##0##1##). A 10-mm bag-retrieval, introduced through the umbilical port, was adopted for specimen extraction.</p>","<p id=\"Par15\">Video ##MEDIA##0##1## reproduces the technique of robotic-assisted resection of ovarian mass using ICG-NIRF.</p>","<p>\n</p>","<title>Operative results</title>","<p id=\"Par16\">The RAS procedures included: ovarian cystectomy (<italic>n</italic> = 10), salpingo-oophorectomy (<italic>n</italic> = 6), bilateral gonadectomy (<italic>n</italic> = 1), salpingectomy (<italic>n</italic> = 1), paratubal cyst excision (<italic>n</italic> = 1), Gartner cyst excision (<italic>n</italic> = 1), paravaginal ganglioneuroma resection (<italic>n</italic> = 1), fistula closure in UGS (<italic>n</italic> = 1), and vaginoplasty using ileal flap in cloaca malformation (<italic>n</italic> = 1). Median operative time was 144.9 min (range 64–360), and median docking time was 17.3 min (range 7–50). Conversion to open or laparoscopy was not necessary in any case. Median LOS was 2.1 days (range 1–7), and median analgesic requirement was 2.2 days (range 1–6). One patient (4.3%) needed redo-surgery for recurrent Gartner cyst (Clavien 3b).</p>","<p id=\"Par17\">The histopathology confirmed diagnosis of ovarian serous cystadenoma (<italic>n</italic> = 2), ovarian functional follicular cyst (<italic>n</italic> = 2), mature cystic teratoma (<italic>n</italic> = 6), immature teratoma (<italic>n</italic> = 4) (Fig. ##FIG##1##2##), ovotestis (<italic>n</italic> = 2), streak gonads in Turner syndrome SRY + (<italic>n</italic> = 1), paratubal cystadenoma (<italic>n</italic> = 2), Gartner cyst (<italic>n</italic> = 1), and ganglioneuroblastoma (<italic>n</italic> = 1).</p>","<p id=\"Par18\">The median length of follow-up was 2.2 years (range 0.5–4.5). No patients required adjuvant chemotherapy following surgery and none reported recurrence of tumoral pathology.</p>","<p id=\"Par19\">All results are summarized in Table ##TAB##0##1##.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Patient baseline</title>\",\n \"<p id=\\\"Par12\\\">Twenty-three girls, with median age at surgery of 12.3 years (range 0.6–17.8) and median weight of 47.2 kg (range 9–73), received RAS for gynecological indication in the study period and were included. Associated comorbidities were reported in 5/23 (21.7%). Most patients (16/23, 69.5%) were symptomatic at time of diagnosis, with non-specific abdominal/pelvic pain being the most frequent presentation. Pre-operative work-up included abdominal ultrasonography (US), pelvic computed tomography (CT) and/or magnetic resonance imaging (MRI), and voiding cystourethrogram (VCUG) in selected cases. Serum tumor markers, such as beta human chorionic gonadotropin (β-HCG), alfa-fetoprotein (α-FP), Cancer Antigen 125 (Ca125), lactic dehydrogenase (LDH), and human epididymis secretory protein 4 (HE-4), were performed in all patients with adnexal mass. Pre-operative diagnosis was ovarian cyst (<italic>n</italic> = 4), ovarian “complex” mass (<italic>n</italic> = 12), fallopian tube lesion (<italic>n</italic> = 3), uterine cyst (<italic>n</italic> = 1), gonadal dysgenesis (<italic>n</italic> = 1), pelvic paravaginal mass (<italic>n</italic> = 1), cloaca malformation (<italic>n</italic> = 1), and high-confluence urogenital sinus (UGS) (<italic>n</italic> = 1). One patient (4.3%) presented concomitant ovarian “complex” mass and paratubal cyst.</p>\",\n \"<title>Installation and operative technique</title>\",\n \"<p id=\\\"Par13\\\">All RAS procedures were carried out using the da Vinci Xi Surgical System (Intuitive Surgical, Sunnyvale, CA, USA). The patient was placed supine on the operative table and appropriate age-sized Foley catheter was inserted using sterile precautions pre-operatively.</p>\",\n \"<p id=\\\"Par14\\\">Three robotic arms, one 12-mm with 12–8 mm reducer, for the 3D, 0-degree, robotic optic, and two 8-mm ports to accommodate the robotic instruments, were placed on the umbilical line in all procedures. A fourth 5-mm accessory port was also placed. The robot was finally docked over the patient’s feet. Robotic vessel sealer was adopted in all procedures. Indocyanine green (ICG) near-infrared fluorescence (NIRF) was adopted in ovarian mass to check the resection margins and guide intra-operative decision making and in paratubal lesion to check the vascular permeability of the fallopian tube following the removal of the lesion (Fig. ##FIG##0##1##). A 10-mm bag-retrieval, introduced through the umbilical port, was adopted for specimen extraction.</p>\",\n \"<p id=\\\"Par15\\\">Video ##MEDIA##0##1## reproduces the technique of robotic-assisted resection of ovarian mass using ICG-NIRF.</p>\",\n \"<p>\\n</p>\",\n \"<title>Operative results</title>\",\n \"<p id=\\\"Par16\\\">The RAS procedures included: ovarian cystectomy (<italic>n</italic> = 10), salpingo-oophorectomy (<italic>n</italic> = 6), bilateral gonadectomy (<italic>n</italic> = 1), salpingectomy (<italic>n</italic> = 1), paratubal cyst excision (<italic>n</italic> = 1), Gartner cyst excision (<italic>n</italic> = 1), paravaginal ganglioneuroma resection (<italic>n</italic> = 1), fistula closure in UGS (<italic>n</italic> = 1), and vaginoplasty using ileal flap in cloaca malformation (<italic>n</italic> = 1). Median operative time was 144.9 min (range 64–360), and median docking time was 17.3 min (range 7–50). Conversion to open or laparoscopy was not necessary in any case. Median LOS was 2.1 days (range 1–7), and median analgesic requirement was 2.2 days (range 1–6). One patient (4.3%) needed redo-surgery for recurrent Gartner cyst (Clavien 3b).</p>\",\n \"<p id=\\\"Par17\\\">The histopathology confirmed diagnosis of ovarian serous cystadenoma (<italic>n</italic> = 2), ovarian functional follicular cyst (<italic>n</italic> = 2), mature cystic teratoma (<italic>n</italic> = 6), immature teratoma (<italic>n</italic> = 4) (Fig. ##FIG##1##2##), ovotestis (<italic>n</italic> = 2), streak gonads in Turner syndrome SRY + (<italic>n</italic> = 1), paratubal cystadenoma (<italic>n</italic> = 2), Gartner cyst (<italic>n</italic> = 1), and ganglioneuroblastoma (<italic>n</italic> = 1).</p>\",\n \"<p id=\\\"Par18\\\">The median length of follow-up was 2.2 years (range 0.5–4.5). No patients required adjuvant chemotherapy following surgery and none reported recurrence of tumoral pathology.</p>\",\n \"<p id=\\\"Par19\\\">All results are summarized in Table ##TAB##0##1##.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par20\">Despite the growing number of indications in pediatric urology, the application of RAS remains still limited in other fields of pediatric surgery. Analyzing the pediatric literature, very few reports on RAS application in gynecology are available, with limited case series or single-case observations [##REF##23158752##13##–##REF##36061048##15##].</p>","<p id=\"Par21\">Our study collected the number of 4 pediatric surgery units with high volume robotic activity and 24 patients, operated over a 3-year period, were enrolled. Despite the small number of patients included, our preliminary results were promising and showed that RAS may be fully applicable even to gynecological indications in pediatric patients. Improved dexterity, coordination, and visualization were provided by robot assistance. The absence of intra- and post-operative complications also confirmed the safety and feasibility of this approach in children.</p>","<p id=\"Par22\">Moreover, our study added new elements to the current knowledge. First, the previous reports have described benign ovarian pathology as main indication to RAS [##REF##23158752##13##–##REF##36061048##15##]. Our study introduced further undescribed indications, such as tubal, uterine, and vaginal malformations, and demonstrated the feasibility of complex reconstructive procedures of internal genitalia such as vaginoplasty using ileal flap using robotic approach.</p>","<p id=\"Par23\">Based on our experience, we believe that the key-points for an optimal management of such pathology using RAS are correct trocar placement, use of sealing device and ICG-NIRF technology, use of endobag for specimen extraction, and teamwork. The most critical step is the placement of trocars, especially in children [##REF##24548088##17##]. Improper placement of the trocars would limit robotic manipulation in the abdominal cavity and increase the chances of instrument conflicts due to the small surface area of the abdominal wall of children and the relatively small space in the abdominal cavity. As described by Xie et al. [##REF##31559290##14##], we always adopted three robotic arms and a fourth accessory laparoscopic port for the bedside surgeon. We placed the robotic ports on the umbilical line to keep proper distance from the pelvic area and have enough working space for manipulation of giant masses or insertion of retrieval bag. Our standard operation order was to insert the trocar for the scope first and then insert the trocars for the robotic arms. Furthermore, if use of specimen retrieval bag is planned, our suggestion is to place 12-mm umbilical robotic port with 12–8 mm reducer to use the 8-mm robotic scope. At time of specimen extraction, the optic is moved to one working arm and the 10-mm retrieval bag is inserted into the abdominal cavity through the umbilical robotic port and the specimen extraction is finally done under direct vision.</p>","<p id=\"Par24\">Use of robotic vessel sealer is very helpful to perform a bloodless dissection of anatomic structures or resection of giant tumors. In some indications, such as ovarian tumors, use of ICG-NIRF was very helpful to visualize the resection margins of the mass and help guide intra-operative decision between salpingo-oophorectomy and ovarian-sparing surgery. This technology required an intra-operative administration of ICG (0.5 mg/kg) via intravenous route and in a matter of 60 s, fluorescence appeared in the target organs, allowing to identify the resection margins and the vascularization of the mass [##REF##32626676##18##–##REF##34616696##20##]. Recently, ICG-NIRF was also adopted during removal of paratubal lesion, to check the vascular permeability of the fallopian tube following the resection of the lesion.</p>","<p id=\"Par25\">Use of endobag is needed for extraction of resected tumors with high suspicion of malignancy. We suggest adopting large endobags (volume up to 1000 mL) and extract the specimen by enlarging the umbilical incision and, whenever possible, aspirating the liquid content of cystic masses before extraction, to avoid additional large Pfannestiel incision.</p>","<p id=\"Par26\">Finally, the teamwork is essential to perform a smooth operation, shorten the learning curve for docking, and ultimately reduce total operative time and anesthetic times.</p>","<p id=\"Par27\">Limitations of the presented study are the small number series, the limited follow-up period, and the multi-institutional participation, that made the data hardly comparable. The number of accumulated procedures in children is difficult to compare with that in adults. Thus, it was necessary collect the data of different pediatric surgery units to collect more conspicuous evidence.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par20\\\">Despite the growing number of indications in pediatric urology, the application of RAS remains still limited in other fields of pediatric surgery. Analyzing the pediatric literature, very few reports on RAS application in gynecology are available, with limited case series or single-case observations [##REF##23158752##13##–##REF##36061048##15##].</p>\",\n \"<p id=\\\"Par21\\\">Our study collected the number of 4 pediatric surgery units with high volume robotic activity and 24 patients, operated over a 3-year period, were enrolled. Despite the small number of patients included, our preliminary results were promising and showed that RAS may be fully applicable even to gynecological indications in pediatric patients. Improved dexterity, coordination, and visualization were provided by robot assistance. The absence of intra- and post-operative complications also confirmed the safety and feasibility of this approach in children.</p>\",\n \"<p id=\\\"Par22\\\">Moreover, our study added new elements to the current knowledge. First, the previous reports have described benign ovarian pathology as main indication to RAS [##REF##23158752##13##–##REF##36061048##15##]. Our study introduced further undescribed indications, such as tubal, uterine, and vaginal malformations, and demonstrated the feasibility of complex reconstructive procedures of internal genitalia such as vaginoplasty using ileal flap using robotic approach.</p>\",\n \"<p id=\\\"Par23\\\">Based on our experience, we believe that the key-points for an optimal management of such pathology using RAS are correct trocar placement, use of sealing device and ICG-NIRF technology, use of endobag for specimen extraction, and teamwork. The most critical step is the placement of trocars, especially in children [##REF##24548088##17##]. Improper placement of the trocars would limit robotic manipulation in the abdominal cavity and increase the chances of instrument conflicts due to the small surface area of the abdominal wall of children and the relatively small space in the abdominal cavity. As described by Xie et al. [##REF##31559290##14##], we always adopted three robotic arms and a fourth accessory laparoscopic port for the bedside surgeon. We placed the robotic ports on the umbilical line to keep proper distance from the pelvic area and have enough working space for manipulation of giant masses or insertion of retrieval bag. Our standard operation order was to insert the trocar for the scope first and then insert the trocars for the robotic arms. Furthermore, if use of specimen retrieval bag is planned, our suggestion is to place 12-mm umbilical robotic port with 12–8 mm reducer to use the 8-mm robotic scope. At time of specimen extraction, the optic is moved to one working arm and the 10-mm retrieval bag is inserted into the abdominal cavity through the umbilical robotic port and the specimen extraction is finally done under direct vision.</p>\",\n \"<p id=\\\"Par24\\\">Use of robotic vessel sealer is very helpful to perform a bloodless dissection of anatomic structures or resection of giant tumors. In some indications, such as ovarian tumors, use of ICG-NIRF was very helpful to visualize the resection margins of the mass and help guide intra-operative decision between salpingo-oophorectomy and ovarian-sparing surgery. This technology required an intra-operative administration of ICG (0.5 mg/kg) via intravenous route and in a matter of 60 s, fluorescence appeared in the target organs, allowing to identify the resection margins and the vascularization of the mass [##REF##32626676##18##–##REF##34616696##20##]. Recently, ICG-NIRF was also adopted during removal of paratubal lesion, to check the vascular permeability of the fallopian tube following the resection of the lesion.</p>\",\n \"<p id=\\\"Par25\\\">Use of endobag is needed for extraction of resected tumors with high suspicion of malignancy. We suggest adopting large endobags (volume up to 1000 mL) and extract the specimen by enlarging the umbilical incision and, whenever possible, aspirating the liquid content of cystic masses before extraction, to avoid additional large Pfannestiel incision.</p>\",\n \"<p id=\\\"Par26\\\">Finally, the teamwork is essential to perform a smooth operation, shorten the learning curve for docking, and ultimately reduce total operative time and anesthetic times.</p>\",\n \"<p id=\\\"Par27\\\">Limitations of the presented study are the small number series, the limited follow-up period, and the multi-institutional participation, that made the data hardly comparable. The number of accumulated procedures in children is difficult to compare with that in adults. Thus, it was necessary collect the data of different pediatric surgery units to collect more conspicuous evidence.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par28\">This preliminary experience showed that RAS is safe and feasible for surgical treatment of pediatric gynecological pathology, although no conclusive data are available to confirm its superiority over traditional laparoscopy. Case–control and comparative prospective studies will help to delineate better the advantages of this new technology as well as its optimal use in pediatrics. The primary focus for future studies should therefore be on quality management, optimization of patient outcomes for the largest number of patients, and surgical and team training.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par28\\\">This preliminary experience showed that RAS is safe and feasible for surgical treatment of pediatric gynecological pathology, although no conclusive data are available to confirm its superiority over traditional laparoscopy. Case–control and comparative prospective studies will help to delineate better the advantages of this new technology as well as its optimal use in pediatrics. The primary focus for future studies should therefore be on quality management, optimization of patient outcomes for the largest number of patients, and surgical and team training.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p id=\"Par1\">Robotic-assisted surgery (RAS) is increasingly adopted in the pediatric population. This retrospective multicenter study aimed to report application of RAS for gynecological indications in pediatric patients. The medical records of all girls with gynecological pathology, operated in 4 different institutions over a 3-year period, were retrospectively collected. Robot docking time, total operative time, length of stay (LOS), requirement time of pain medication, complication rate, conversion rate, and pathology were analyzed. Twenty-three girls, with median age of 12.3 years (range 0.6–17.8) and median weight of 47.2 kg (range 9–73), received the following RAS procedures: ovarian cystectomy for ovarian cyst/mass (<italic>n</italic> = 10), salpingo-oophorectomy for ovarian complex mass (<italic>n</italic> = 6), bilateral gonadectomy for Turner syndrome SRY + (<italic>n</italic> = 1), salpingectomy for fallopian tube lesion (<italic>n</italic> = 1), paratubal cyst excision (<italic>n</italic> = 1), Gartner cyst excision (<italic>n</italic> = 1), paravaginal ganglioneuroma resection (<italic>n</italic> = 1), fistula closure in urogenital sinus (<italic>n</italic> = 1), and vaginoplasty using ileal flap in cloaca malformation (<italic>n</italic> = 1). Median operative time was 144.9 min (range 64–360), and median docking time was 17.3 min (range 7–50). Conversion to open or laparoscopy was not necessary in any case. Median LOS was 2.1 days (range 1–7), and median analgesic requirement was 2.2 days (range 1–6). One patient (4.3%) needed redo-surgery for recurrent Gartner cyst (Clavien 3b). This preliminary experience showed that RAS is safe and feasible for surgical treatment of gynecological pathology in pediatric patients, although no conclusive data are available to confirm its superiority over traditional laparoscopy. Randomized, prospective, comparative studies are needed to identify the gold standard approach for such indication.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s11701-023-01767-9.</p>","<title>Keywords</title>","<p>Open access funding provided by Università degli Studi di Napoli Federico II within the CRUI-CARE Agreement.</p>"],"string":"[\n \"<p id=\\\"Par1\\\">Robotic-assisted surgery (RAS) is increasingly adopted in the pediatric population. This retrospective multicenter study aimed to report application of RAS for gynecological indications in pediatric patients. The medical records of all girls with gynecological pathology, operated in 4 different institutions over a 3-year period, were retrospectively collected. Robot docking time, total operative time, length of stay (LOS), requirement time of pain medication, complication rate, conversion rate, and pathology were analyzed. Twenty-three girls, with median age of 12.3 years (range 0.6–17.8) and median weight of 47.2 kg (range 9–73), received the following RAS procedures: ovarian cystectomy for ovarian cyst/mass (<italic>n</italic> = 10), salpingo-oophorectomy for ovarian complex mass (<italic>n</italic> = 6), bilateral gonadectomy for Turner syndrome SRY + (<italic>n</italic> = 1), salpingectomy for fallopian tube lesion (<italic>n</italic> = 1), paratubal cyst excision (<italic>n</italic> = 1), Gartner cyst excision (<italic>n</italic> = 1), paravaginal ganglioneuroma resection (<italic>n</italic> = 1), fistula closure in urogenital sinus (<italic>n</italic> = 1), and vaginoplasty using ileal flap in cloaca malformation (<italic>n</italic> = 1). Median operative time was 144.9 min (range 64–360), and median docking time was 17.3 min (range 7–50). Conversion to open or laparoscopy was not necessary in any case. Median LOS was 2.1 days (range 1–7), and median analgesic requirement was 2.2 days (range 1–6). One patient (4.3%) needed redo-surgery for recurrent Gartner cyst (Clavien 3b). This preliminary experience showed that RAS is safe and feasible for surgical treatment of gynecological pathology in pediatric patients, although no conclusive data are available to confirm its superiority over traditional laparoscopy. Randomized, prospective, comparative studies are needed to identify the gold standard approach for such indication.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s11701-023-01767-9.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by Università degli Studi di Napoli Federico II within the CRUI-CARE Agreement.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":["<title>Author contributions</title>","<p>All authors contributed to the study conception and design. All authors performed material preparation, data collection, and data analysis. M.E. and C.E. wrote the first draft of the manuscript. G.E. and C.D.M. prepared Fig. ##FIG##0##1##. A.C. and M.E. prepared video ##MEDIA##0##1##. All authors read and approved the final manuscript.</p>","<title>Funding</title>","<p>Open access funding provided by Università degli Studi di Napoli Federico II within the CRUI-CARE Agreement. The authors declare that no funds, grants, or other supports were received during the preparation of this manuscript.</p>","<title>Data availability</title>","<p>All data will be available on request.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par29\">The authors have no relevant financial or non-financial interests to disclose.</p>","<title>Ethics approval</title>","<p id=\"Par30\">This is a retrospective study. No ethical approval is required.</p>","<title>Consent to participate</title>","<p id=\"Par31\">Written informed consent was obtained from all individual participants included in the study and their parents.</p>"],"string":"[\n \"<title>Author contributions</title>\",\n \"<p>All authors contributed to the study conception and design. All authors performed material preparation, data collection, and data analysis. M.E. and C.E. wrote the first draft of the manuscript. G.E. and C.D.M. prepared Fig. ##FIG##0##1##. A.C. and M.E. prepared video ##MEDIA##0##1##. All authors read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by Università degli Studi di Napoli Federico II within the CRUI-CARE Agreement. The authors declare that no funds, grants, or other supports were received during the preparation of this manuscript.</p>\",\n \"<title>Data availability</title>\",\n \"<p>All data will be available on request.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par29\\\">The authors have no relevant financial or non-financial interests to disclose.</p>\",\n \"<title>Ethics approval</title>\",\n \"<p id=\\\"Par30\\\">This is a retrospective study. No ethical approval is required.</p>\",\n \"<title>Consent to participate</title>\",\n \"<p id=\\\"Par31\\\">Written informed consent was obtained from all individual participants included in the study and their parents.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>After removal of giant paratubal cyst (<bold>a</bold>, <bold>b</bold>), ICG-guided fluorescence was helpful to check the vascular permeability of fallopian tube (<bold>c</bold>)</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Pre-operative MRI and specimen of right ovarian immature teratoma</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>After removal of giant paratubal cyst (<bold>a</bold>, <bold>b</bold>), ICG-guided fluorescence was helpful to check the vascular permeability of fallopian tube (<bold>c</bold>)</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Pre-operative MRI and specimen of right ovarian immature teratoma</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Outcomes of RAS in pediatric gynecological indications</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Patient</th><th align=\"left\">Age (years)</th><th align=\"left\">Side</th><th align=\"left\">Comorbidity</th><th align=\"left\">Pre-operative diagnosis</th><th align=\"left\">Surgical procedure</th><th align=\"left\">OT (min)</th><th align=\"left\">LOS (days)</th><th align=\"left\">Post-operative complications</th><th align=\"left\">Pathology results</th></tr></thead><tbody><tr><td align=\"left\">1</td><td align=\"left\">10</td><td align=\"left\">Right</td><td align=\"left\">No</td><td align=\"left\">Ovarian mass</td><td align=\"left\">Salpingo-oophorectomy</td><td char=\".\" align=\"char\">85</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">Mature cystic teratoma</td></tr><tr><td align=\"left\">2</td><td align=\"left\">16</td><td align=\"left\">Left</td><td align=\"left\">No</td><td align=\"left\">Giant ovarian cyst (12 cm)</td><td align=\"left\">Ovarian cystectomy</td><td char=\".\" align=\"char\">72</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">Serous cystadenoma</td></tr><tr><td align=\"left\">3</td><td align=\"left\">17</td><td align=\"left\">Right</td><td align=\"left\">No</td><td align=\"left\">Giant ovarian cyst (10 cm)</td><td align=\"left\">Ovarian cystectomy</td><td char=\".\" align=\"char\">75</td><td char=\".\" align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">Serous cystadenoma</td></tr><tr><td align=\"left\">4</td><td align=\"left\">12</td><td align=\"left\">Right</td><td align=\"left\">Type I neurofibromatosis</td><td align=\"left\">Ovarian mass</td><td align=\"left\">Salpingo-oophorectomy</td><td char=\".\" align=\"char\">124</td><td char=\".\" align=\"left\">3</td><td align=\"left\">0</td><td align=\"left\">Immature teratoma</td></tr><tr><td align=\"left\">5</td><td align=\"left\">12</td><td align=\"left\">Right</td><td align=\"left\">Rheumatoid arthritis</td><td align=\"left\">Ovarian mass</td><td align=\"left\">Salpingo-oophorectomy</td><td char=\".\" align=\"char\">95</td><td char=\".\" align=\"left\">2</td><td align=\"left\">0</td><td align=\"left\">Immature teratoma</td></tr><tr><td align=\"left\">6</td><td align=\"left\">13</td><td align=\"left\">Left</td><td align=\"left\">No</td><td align=\"left\">Ovarian mass + paratubal cyst</td><td align=\"left\">Salpingo-oophorectomy</td><td char=\".\" align=\"char\">78</td><td align=\"left\">2</td><td align=\"left\">0</td><td align=\"left\">Immature teratoma + paratubal cyst</td></tr><tr><td align=\"left\">7</td><td align=\"left\">3</td><td align=\"left\">Bilateral</td><td align=\"left\">Turner syndrome SRY + </td><td align=\"left\">Gonadal dysgenesis</td><td align=\"left\">Bilateral gonadectomy</td><td char=\".\" align=\"char\">64</td><td char=\".\" align=\"left\">2</td><td align=\"left\">0</td><td align=\"left\">Streak gonads</td></tr><tr><td align=\"left\">8</td><td align=\"left\">13</td><td align=\"left\">Right</td><td align=\"left\">No</td><td align=\"left\">Fallopian tube lesion</td><td align=\"left\">Salpingectomy</td><td char=\".\" align=\"char\">96</td><td align=\"left\">2</td><td align=\"left\">0</td><td align=\"left\">Tubal cystadenoma</td></tr><tr><td align=\"left\">9</td><td align=\"left\">17</td><td align=\"left\">N/A</td><td align=\"left\">No</td><td align=\"left\">Uterine cyst</td><td align=\"left\">Excision</td><td char=\".\" align=\"char\">200</td><td char=\".\" align=\"left\">3</td><td align=\"left\">Cyst recurrence (Clavien 3b)</td><td align=\"left\">Gartner cyst</td></tr><tr><td align=\"left\">10</td><td align=\"left\">7</td><td align=\"left\">Right</td><td align=\"left\">No</td><td align=\"left\"><p>Ovarian</p><p>Mass</p></td><td align=\"left\">Ovarian cystectomy</td><td char=\".\" align=\"char\">155</td><td align=\"left\">2</td><td align=\"left\">0</td><td align=\"left\">Mature cystic teratoma</td></tr><tr><td align=\"left\">11</td><td align=\"left\">14</td><td align=\"left\">Left</td><td align=\"left\">No</td><td align=\"left\">Pelvic paravaginal mass</td><td align=\"left\">Excision</td><td char=\".\" align=\"char\">198</td><td char=\".\" align=\"left\">2</td><td align=\"left\">0</td><td align=\"left\">Ganglioneuroblastoma</td></tr><tr><td align=\"left\">12</td><td align=\"left\">10</td><td align=\"left\">N/A</td><td align=\"left\">No</td><td align=\"left\">Cloaca</td><td align=\"left\">Vaginoplasty using ileal flap</td><td char=\".\" align=\"char\">360</td><td char=\".\" align=\"left\">7</td><td align=\"left\">0</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\">13</td><td align=\"left\">0.6</td><td align=\"left\">N/A</td><td align=\"left\">No</td><td align=\"left\">Urogenital sinus</td><td align=\"left\">Fistula closure</td><td char=\".\" align=\"char\">155</td><td char=\".\" align=\"left\">3</td><td align=\"left\">0</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\">14</td><td align=\"left\">17</td><td align=\"left\">Right</td><td align=\"left\">DSD</td><td align=\"left\">Ovarian mass</td><td align=\"left\">Ovarian cystectomy</td><td char=\".\" align=\"char\">145</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">Ovotestis</td></tr><tr><td align=\"left\">15</td><td align=\"left\">13</td><td align=\"left\">Left</td><td align=\"left\">DSD</td><td align=\"left\">Ovarian mass</td><td align=\"left\">Salpingo-oophorectomy</td><td char=\".\" align=\"char\">120</td><td char=\".\" align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">Ovotestis</td></tr><tr><td align=\"left\">16</td><td align=\"left\">13</td><td align=\"left\">Right</td><td align=\"left\">Epilepsy, mild cystic fibrosis</td><td align=\"left\">Ovarian mass</td><td align=\"left\">Salpingo-oophorectomy</td><td char=\".\" align=\"char\">80</td><td char=\".\" align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">Immature teratoma</td></tr><tr><td align=\"left\">17</td><td align=\"left\">17.8</td><td align=\"left\">Right</td><td align=\"left\">Epilepsy, Hashimoto thyroiditis</td><td align=\"left\">Paratubal cyst</td><td align=\"left\">Paratubal cystectomy</td><td char=\".\" align=\"char\">115</td><td char=\".\" align=\"left\">2</td><td align=\"left\">0</td><td align=\"left\">Serous papillary cystadenoma</td></tr><tr><td align=\"left\">18</td><td align=\"left\">13.2</td><td align=\"left\">Left</td><td align=\"left\">No</td><td align=\"left\">Ovarian mass</td><td align=\"left\">Ovarian cystectomy</td><td char=\".\" align=\"char\">228</td><td align=\"left\">3</td><td align=\"left\">0</td><td align=\"left\">Mature cystic teratoma</td></tr><tr><td align=\"left\">19</td><td align=\"left\">12.9</td><td align=\"left\">Right</td><td align=\"left\">No</td><td align=\"left\">Ovarian mass</td><td align=\"left\">Ovarian cystectomy</td><td char=\".\" align=\"char\">218</td><td align=\"left\">2.1</td><td align=\"left\">0</td><td align=\"left\">Mature cystic teratoma</td></tr><tr><td align=\"left\">20</td><td align=\"left\">8</td><td align=\"left\">Right</td><td align=\"left\">No</td><td align=\"left\">Ovarian mass</td><td align=\"left\">Ovarian cystectomy</td><td char=\".\" align=\"char\">185</td><td align=\"left\">2.7</td><td align=\"left\">0</td><td align=\"left\">Mature cystic teratoma</td></tr><tr><td align=\"left\">21</td><td align=\"left\">14.8</td><td align=\"left\">Right</td><td align=\"left\">No</td><td align=\"left\">Ovarian mass</td><td align=\"left\">Ovarian cystectomy</td><td char=\".\" align=\"char\">198</td><td align=\"left\">2.3</td><td align=\"left\">0</td><td align=\"left\">Mature cystic teratoma</td></tr><tr><td align=\"left\">22</td><td align=\"left\">14.7</td><td align=\"left\">Bilateral</td><td align=\"left\">No</td><td align=\"left\">Ovarian cyst (6 cm)</td><td align=\"left\">Ovarian cystectomy</td><td char=\".\" align=\"char\">155</td><td align=\"left\">1.5</td><td align=\"left\">0</td><td align=\"left\">Functional follicular cyst</td></tr><tr><td align=\"left\">23</td><td align=\"left\">15</td><td align=\"left\">Left</td><td align=\"left\">No</td><td align=\"left\"><p>Ovarian cyst</p><p>(7 cm)</p></td><td align=\"left\">Ovarian cystectomy</td><td char=\".\" align=\"char\">132</td><td align=\"left\">1</td><td align=\"left\">0</td><td align=\"left\">Functional follicular cyst</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Outcomes of RAS in pediatric gynecological indications</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Patient</th><th align=\\\"left\\\">Age (years)</th><th align=\\\"left\\\">Side</th><th align=\\\"left\\\">Comorbidity</th><th align=\\\"left\\\">Pre-operative diagnosis</th><th align=\\\"left\\\">Surgical procedure</th><th align=\\\"left\\\">OT (min)</th><th align=\\\"left\\\">LOS (days)</th><th align=\\\"left\\\">Post-operative complications</th><th align=\\\"left\\\">Pathology results</th></tr></thead><tbody><tr><td align=\\\"left\\\">1</td><td align=\\\"left\\\">10</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Ovarian mass</td><td align=\\\"left\\\">Salpingo-oophorectomy</td><td char=\\\".\\\" align=\\\"char\\\">85</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Mature cystic teratoma</td></tr><tr><td align=\\\"left\\\">2</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">Left</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Giant ovarian cyst (12 cm)</td><td align=\\\"left\\\">Ovarian cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">72</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Serous cystadenoma</td></tr><tr><td align=\\\"left\\\">3</td><td align=\\\"left\\\">17</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Giant ovarian cyst (10 cm)</td><td align=\\\"left\\\">Ovarian cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">75</td><td char=\\\".\\\" align=\\\"left\\\">1</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Serous cystadenoma</td></tr><tr><td align=\\\"left\\\">4</td><td align=\\\"left\\\">12</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">Type I neurofibromatosis</td><td align=\\\"left\\\">Ovarian mass</td><td align=\\\"left\\\">Salpingo-oophorectomy</td><td char=\\\".\\\" align=\\\"char\\\">124</td><td char=\\\".\\\" align=\\\"left\\\">3</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Immature teratoma</td></tr><tr><td align=\\\"left\\\">5</td><td align=\\\"left\\\">12</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">Rheumatoid arthritis</td><td align=\\\"left\\\">Ovarian mass</td><td align=\\\"left\\\">Salpingo-oophorectomy</td><td char=\\\".\\\" align=\\\"char\\\">95</td><td char=\\\".\\\" align=\\\"left\\\">2</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Immature teratoma</td></tr><tr><td align=\\\"left\\\">6</td><td align=\\\"left\\\">13</td><td align=\\\"left\\\">Left</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Ovarian mass + paratubal cyst</td><td align=\\\"left\\\">Salpingo-oophorectomy</td><td char=\\\".\\\" align=\\\"char\\\">78</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Immature teratoma + paratubal cyst</td></tr><tr><td align=\\\"left\\\">7</td><td align=\\\"left\\\">3</td><td align=\\\"left\\\">Bilateral</td><td align=\\\"left\\\">Turner syndrome SRY + </td><td align=\\\"left\\\">Gonadal dysgenesis</td><td align=\\\"left\\\">Bilateral gonadectomy</td><td char=\\\".\\\" align=\\\"char\\\">64</td><td char=\\\".\\\" align=\\\"left\\\">2</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Streak gonads</td></tr><tr><td align=\\\"left\\\">8</td><td align=\\\"left\\\">13</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Fallopian tube lesion</td><td align=\\\"left\\\">Salpingectomy</td><td char=\\\".\\\" align=\\\"char\\\">96</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Tubal cystadenoma</td></tr><tr><td align=\\\"left\\\">9</td><td align=\\\"left\\\">17</td><td align=\\\"left\\\">N/A</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Uterine cyst</td><td align=\\\"left\\\">Excision</td><td char=\\\".\\\" align=\\\"char\\\">200</td><td char=\\\".\\\" align=\\\"left\\\">3</td><td align=\\\"left\\\">Cyst recurrence (Clavien 3b)</td><td align=\\\"left\\\">Gartner cyst</td></tr><tr><td align=\\\"left\\\">10</td><td align=\\\"left\\\">7</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\"><p>Ovarian</p><p>Mass</p></td><td align=\\\"left\\\">Ovarian cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">155</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Mature cystic teratoma</td></tr><tr><td align=\\\"left\\\">11</td><td align=\\\"left\\\">14</td><td align=\\\"left\\\">Left</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Pelvic paravaginal mass</td><td align=\\\"left\\\">Excision</td><td char=\\\".\\\" align=\\\"char\\\">198</td><td char=\\\".\\\" align=\\\"left\\\">2</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Ganglioneuroblastoma</td></tr><tr><td align=\\\"left\\\">12</td><td align=\\\"left\\\">10</td><td align=\\\"left\\\">N/A</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Cloaca</td><td align=\\\"left\\\">Vaginoplasty using ileal flap</td><td char=\\\".\\\" align=\\\"char\\\">360</td><td char=\\\".\\\" align=\\\"left\\\">7</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">N/A</td></tr><tr><td align=\\\"left\\\">13</td><td align=\\\"left\\\">0.6</td><td align=\\\"left\\\">N/A</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Urogenital sinus</td><td align=\\\"left\\\">Fistula closure</td><td char=\\\".\\\" align=\\\"char\\\">155</td><td char=\\\".\\\" align=\\\"left\\\">3</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">N/A</td></tr><tr><td align=\\\"left\\\">14</td><td align=\\\"left\\\">17</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">DSD</td><td align=\\\"left\\\">Ovarian mass</td><td align=\\\"left\\\">Ovarian cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">145</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Ovotestis</td></tr><tr><td align=\\\"left\\\">15</td><td align=\\\"left\\\">13</td><td align=\\\"left\\\">Left</td><td align=\\\"left\\\">DSD</td><td align=\\\"left\\\">Ovarian mass</td><td align=\\\"left\\\">Salpingo-oophorectomy</td><td char=\\\".\\\" align=\\\"char\\\">120</td><td char=\\\".\\\" align=\\\"left\\\">1</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Ovotestis</td></tr><tr><td align=\\\"left\\\">16</td><td align=\\\"left\\\">13</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">Epilepsy, mild cystic fibrosis</td><td align=\\\"left\\\">Ovarian mass</td><td align=\\\"left\\\">Salpingo-oophorectomy</td><td char=\\\".\\\" align=\\\"char\\\">80</td><td char=\\\".\\\" align=\\\"left\\\">1</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Immature teratoma</td></tr><tr><td align=\\\"left\\\">17</td><td align=\\\"left\\\">17.8</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">Epilepsy, Hashimoto thyroiditis</td><td align=\\\"left\\\">Paratubal cyst</td><td align=\\\"left\\\">Paratubal cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">115</td><td char=\\\".\\\" align=\\\"left\\\">2</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Serous papillary cystadenoma</td></tr><tr><td align=\\\"left\\\">18</td><td align=\\\"left\\\">13.2</td><td align=\\\"left\\\">Left</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Ovarian mass</td><td align=\\\"left\\\">Ovarian cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">228</td><td align=\\\"left\\\">3</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Mature cystic teratoma</td></tr><tr><td align=\\\"left\\\">19</td><td align=\\\"left\\\">12.9</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Ovarian mass</td><td align=\\\"left\\\">Ovarian cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">218</td><td align=\\\"left\\\">2.1</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Mature cystic teratoma</td></tr><tr><td align=\\\"left\\\">20</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Ovarian mass</td><td align=\\\"left\\\">Ovarian cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">185</td><td align=\\\"left\\\">2.7</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Mature cystic teratoma</td></tr><tr><td align=\\\"left\\\">21</td><td align=\\\"left\\\">14.8</td><td align=\\\"left\\\">Right</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Ovarian mass</td><td align=\\\"left\\\">Ovarian cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">198</td><td align=\\\"left\\\">2.3</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Mature cystic teratoma</td></tr><tr><td align=\\\"left\\\">22</td><td align=\\\"left\\\">14.7</td><td align=\\\"left\\\">Bilateral</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">Ovarian cyst (6 cm)</td><td align=\\\"left\\\">Ovarian cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">155</td><td align=\\\"left\\\">1.5</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Functional follicular cyst</td></tr><tr><td align=\\\"left\\\">23</td><td align=\\\"left\\\">15</td><td align=\\\"left\\\">Left</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\"><p>Ovarian cyst</p><p>(7 cm)</p></td><td align=\\\"left\\\">Ovarian cystectomy</td><td char=\\\".\\\" align=\\\"char\\\">132</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">0</td><td align=\\\"left\\\">Functional follicular cyst</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p><italic>RAS</italic> robotic-assisted surgery, <italic>OT</italic> operative time, <italic>LOS</italic> length of stay, <italic>DSD</italic> disorder of sex development</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p><italic>RAS</italic> robotic-assisted surgery, <italic>OT</italic> operative time, <italic>LOS</italic> length of stay, <italic>DSD</italic> disorder of sex development</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"11701_2023_1767_Fig1_HTML\" id=\"MO1\"/>","<graphic 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\"fpage\": [\"159\"], \"lpage\": [\"166\"], \"pub-id\": [\"10.1089/lap.2018.0745\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":20,"string":"20"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:01"},"citation":{"kind":"string","value":"J Robot Surg. 2024 Jan 13; 18(1):20"},"package_file":{"kind":"string","value":"oa_package/d2/98/PMC10787885.tar.gz"}}},{"rowIdx":169,"cells":{"accession_id":{"kind":"string","value":"PMC10787886"},"pmid":{"kind":"string","value":"37249705"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par2\">In the DSM-5, a “with Limited Prosocial Emotions” specifier was added to the Conduct Disorder diagnostic criteria to differentiate those children and adolescents who also display a callous absence of empathy and remorse, shallow or narrow affect, and deficient concern regarding performance in activities (American Psychiatric Association, ##UREF##0##2013##). This addition was based on research extending the affective dimension of the adult psychopathy personality construct downward to advance developmental theories (Frick, ##REF##35878505##2022##; Frick et al., ##REF##24117854##2014##). Greater knowledge on how these traits manifest earlier in development, when they may be more malleable, can inform clinical prevention and intervention efforts. In research settings, this specifier is commonly referred to as callous-unemotional (CU) traits and, like psychopathy, these traits are associated with several unique behavioral, emotional, and cognitive correlates relative to other externalizing and antisocial constructs (Frick et al., ##REF##24117854##2014##). However, empirical work on CU traits has relied on panel (or cross-sectional) designs that primarily investigate within-person change. Such panel designs do not sufficiently assess within-person fluctuations and have comparatively low ecological validity–two important limitations of this line of research that can be circumvented through intensive longitudinal methodologies.</p>","<title>Correlates of Callous-unemotional Traits</title>","<p id=\"Par3\">Studies have pinpointed several distinguishing correlates of CU traits. Individuals with conduct problems and normative CU traits are often characterized by a defiant or difficult temperament with heightened levels of emotion dysregulation, negative affect, and impulsive aggression to provocation (Frick & Viding, ##REF##19825260##2009##). In contrast, those with elevated conduct problems <italic>and</italic> CU traits do not tend to show the same types of emotional reactivity and are instead discerned by a fearless disposition reflected as emotional hypoactivity, reduced sensitivity to punishment, and often greater aggression severity (Fanti et al., ##REF##25916948##2016##; McMahon et al., ##REF##20939651##2010##). Despite the name, CU traits do not always confer risk for blunted emotional responsivity. For example, although differentiated on fear processing, one meta-analytic review (<italic>k</italic> = 20) found no differences across CU traits and control groups in processing angry, disgusted, or happy cues (Marsh & Blair, ##REF##17915324##2008##). In addition to dysfunctional parenting practices, it is this fearlessness that may explain why individuals with CU traits have been less successfully served by treatment efforts (Hawes et al., ##REF##24748077##2014a##; McMahon et al., ##REF##33787407##2021##). Distinctions have also been made regarding the structure of CU traits, with studies having identified three (callousness, uncaring, unemotional; Ray & Frick, ##REF##30142284##2020##) or two (callousness, uncaring; Hawes et al., ##REF##24188153##2014b##; Zheng et al., ##REF##34928648##2021##) dimensions. Callousness tends to capture deficient empathy and guilt, uncaring reflects an aloof attitude regarding others, and unemotionality is defined as impoverished emotional experiences. Regardless of the structure, CU traits discriminates an etiologically and clinically atypical group (Frick et al., ##REF##24117854##2014##).</p>","<title>Traits and Symptoms in Adolescence</title>","<p id=\"Par4\">Adolescence is a sensitive developmental period marked by profound cognitive, neurobiological, and interpersonal change (Dahl et al., ##REF##29469094##2018##). In a large meta-analysis including 192 epidemiological studies, Solmi et al. (##REF##34079068##2022##) found that the peak age of onset of any mental disorder was 14.5 years. Therefore, adolescence represents a prime time for understanding vulnerabilities and the development of psychopathological symptoms. Personality and psychopathology are typically considered distinct domains sometimes differentiated based on temporal stability. Specifically, some researchers have suggested that personality traits reflect long-term dispositions and that psychopathological symptoms are state-like instances of an experience or behavior (DeYoung et al., ##REF##32978977##2022##). In childhood and adolescence, however, personality traits may also change over time (Soto et al., ##REF##21171787##2011##). Indeed, some research has found little differences between mean-level change of several personality facets and internalizing symptoms across five 9-month follow-ups in a sample of female adolescents (Goldstein et al., ##REF##35241862##2022##). Other evidence has shown that personality pathology tends to decline across adolescence (Álvarez-Tomás et al., ##REF##30599336##2019##). In the CU traits literature, many panel studies have examined the long-term stability of CU traits with one review suggesting that CU traits show substantial rank-order stability and modest mean-level stability during childhood, adolescence, and into early adulthood (Frick et al., ##REF##24117854##2014##). However, what cannot be ascertained from this work is whether adolescents with CU traits are consistent in their manifestation of CU traits. In other words, do CU traits fluctuate day-to-day (or moment-by-moment)?</p>","<title>Intensive Longitudinal Methods</title>","<p id=\"Par5\">Innovations in technology (e.g., smart phones and wearables) have generated a surge in studies investigating dynamic processes on micro timescales using a range of intensive longitudinal methods (for recent reviews, see Russell & Gajos, ##REF##31997358##2020##; Urben et al., ##UREF##10##2022##). Broadly, intensive longitudinal methods, such as daily diary, experience sampling, ecological momentary assessment, and other ambulatory assessment designs, are characterized by many measurement occasions over a brief observation window. These methods focus on within-person variability, such that the measured variables may show deviations or fluctuations relative to the individual mean at specific observation occasions, but the individual mean is relatively stable over time compared to developmental growth or decrease in conventional longitudinal designs. Major advantages of intensive longitudinal methods also include the capability to assess real-time experiences and memory compared to traditional longitudinal designs, which can result in retrospective and recall biases (Russell & Gajos, ##REF##31997358##2020##). These methods also enhance ecological validity, such that assessments are conducted when participants are in real-world settings relative to artificial laboratory experiments. Hence, intensive longitudinal data provide a high level of temporal granularity and inform our understanding of real-life short-term within-person dynamics as opposed to long-term developmental change.</p>","<title>Testing Personality and Psychopathology with Intensive Longitudinal Methods</title>","<p id=\"Par6\">Studies are beginning to apply intensive longitudinal designs to elucidate the dynamics of personality and psychopathology. For example, across three samples of adults, Edershile and Wright (##REF##33090821##2021##) tested whether narcissism reflects a dynamic process and found variability in narcissistic states at a moment-to-moment level. Among 91 adults with a personality disorder, daily assessments of personality pathology including negative affect, detachment, impulsivity, and hostility predicted clinical characterizations of personality disorders and daily stress levels (Dotterer et al., ##REF##31597579##2019##). In a sample of adolescent girls, daily negative affect, detachment, disinhibition, and psychoticism were associated with daily socio-affective processes (Kaurin et al., ##UREF##3##2022##). Such studies enhanced our understanding of state-level fluctuations in personality and psychopathology. Yet, no research to date has investigated whether CU traits also vary day-to-day or moment-to-moment. Only two intensive longitudinal studies have included a measure of CU traits using justice-involved adolescent samples; however, both studies only assessed CU traits as a total score at a single timepoint (De Ridder et al., ##UREF##2##2016##; Suter et al., ##UREF##8##2017##). Another study examined callousness in the context of interpersonal antagonism in samples of undergraduates, community adults, and psychiatric outpatients, but, similarly, callousness was only assessed at one timepoint as a predictor (Vize et al., ##REF##35402088##2022##). Across these studies, higher levels of CU traits were associated with higher levels of perceived angry affect and self-reported antisocial behavior (De Ridder et al., ##UREF##2##2016##; Suter et al., ##UREF##8##2017##), and callousness was negatively associated with positive affect and empathy (Vize et al., ##REF##35402088##2022##).</p>","<p id=\"Par7\">In line with other personality constructs (Wright & Kaurin, ##REF##32375147##2020##), much of our current understanding of CU traits is founded on structural or trait-based models (Frick et al., ##REF##24117854##2014##). Research on dynamic modeling at micro timescales may help further inform functional narratives. As the affective domain of psychopathy, CU traits may demonstrate meaningful within-person fluctuations. Notably, the links between CU traits and conduct problems at the daily within-person level could possibly be instantiated by their intermediate and differential links involving daily positive and negative affect. Although most research places negatively valenced emotion processes at the center of personality and psychopathology, some research has implicated positive affect in the development of aggressive behaviors (Toro et al., ##REF##32952964##2020##). In addition, affect and emotion are best conceptualized as dynamic processes underpinning a range of psychopathological outcomes (Houben et al., ##REF##25822133##2015##). Other theoretical accounts suggest that certain forms of personality are derived and maintained by state-level fluctuations of the traits themselves (e.g., grandiosity and vulnerability in narcissism; Edershile & Wright, ##REF##33090821##2021##). It may be the case that daily fluctuations in callousness and uncaring serves to perpetuate CU traits and associated conduct problems. Overall, state-level personality may be more malleable than traits, and thus, studying phenomena at the state-level can illuminate more effective treatment targets for this typically treatment-resistant group.</p>","<title>The Present Study</title>","<p id=\"Par8\">There is a lack of knowledge on CU traits on a micro timescale. In this exploratory work with data collected from a sample of adolescents via an intensive 30-day daily diary design, our aims were twofold. First, we aimed to explore whether CU traits (at the item level) demonstrated daily within-person fluctuations. In addition to the potential novel knowledge gained from investigating CU traits using intensive longitudinal designs, examining CU traits at the item level is congruent with recent research initiatives (e.g., Hierarchical Taxonomy of Psychopathology; Kotov et al., ##REF##33577350##2021##) that emphasize fine-grained trait or symptom level analyses to advance mental health science. Second, we aimed to investigate whether daily CU traits (including callousness and uncaring) were associated with daily positive and negative affect, as well as emotional and conduct problems. To address these aims, we applied dynamic structural equation modeling (DSEM) to parse both <italic>within-person</italic> state-level fluctuations including autoregressive and cross-lagged associations (i.e., whether participants’ level at time <italic>t</italic> was significantly predicted by their level of the same or different construct, respectively, at time <italic>t</italic>-1) from stable <italic>between-person</italic> trait-like differences (Asparouhov et al., ##UREF##1##2018##; Hamaker et al., ##REF##29624092##2018##). Given the exploratory nature of this study, we pose no specific a priori hypotheses.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par2\\\">In the DSM-5, a “with Limited Prosocial Emotions” specifier was added to the Conduct Disorder diagnostic criteria to differentiate those children and adolescents who also display a callous absence of empathy and remorse, shallow or narrow affect, and deficient concern regarding performance in activities (American Psychiatric Association, ##UREF##0##2013##). This addition was based on research extending the affective dimension of the adult psychopathy personality construct downward to advance developmental theories (Frick, ##REF##35878505##2022##; Frick et al., ##REF##24117854##2014##). Greater knowledge on how these traits manifest earlier in development, when they may be more malleable, can inform clinical prevention and intervention efforts. In research settings, this specifier is commonly referred to as callous-unemotional (CU) traits and, like psychopathy, these traits are associated with several unique behavioral, emotional, and cognitive correlates relative to other externalizing and antisocial constructs (Frick et al., ##REF##24117854##2014##). However, empirical work on CU traits has relied on panel (or cross-sectional) designs that primarily investigate within-person change. Such panel designs do not sufficiently assess within-person fluctuations and have comparatively low ecological validity–two important limitations of this line of research that can be circumvented through intensive longitudinal methodologies.</p>\",\n \"<title>Correlates of Callous-unemotional Traits</title>\",\n \"<p id=\\\"Par3\\\">Studies have pinpointed several distinguishing correlates of CU traits. Individuals with conduct problems and normative CU traits are often characterized by a defiant or difficult temperament with heightened levels of emotion dysregulation, negative affect, and impulsive aggression to provocation (Frick & Viding, ##REF##19825260##2009##). In contrast, those with elevated conduct problems <italic>and</italic> CU traits do not tend to show the same types of emotional reactivity and are instead discerned by a fearless disposition reflected as emotional hypoactivity, reduced sensitivity to punishment, and often greater aggression severity (Fanti et al., ##REF##25916948##2016##; McMahon et al., ##REF##20939651##2010##). Despite the name, CU traits do not always confer risk for blunted emotional responsivity. For example, although differentiated on fear processing, one meta-analytic review (<italic>k</italic> = 20) found no differences across CU traits and control groups in processing angry, disgusted, or happy cues (Marsh & Blair, ##REF##17915324##2008##). In addition to dysfunctional parenting practices, it is this fearlessness that may explain why individuals with CU traits have been less successfully served by treatment efforts (Hawes et al., ##REF##24748077##2014a##; McMahon et al., ##REF##33787407##2021##). Distinctions have also been made regarding the structure of CU traits, with studies having identified three (callousness, uncaring, unemotional; Ray & Frick, ##REF##30142284##2020##) or two (callousness, uncaring; Hawes et al., ##REF##24188153##2014b##; Zheng et al., ##REF##34928648##2021##) dimensions. Callousness tends to capture deficient empathy and guilt, uncaring reflects an aloof attitude regarding others, and unemotionality is defined as impoverished emotional experiences. Regardless of the structure, CU traits discriminates an etiologically and clinically atypical group (Frick et al., ##REF##24117854##2014##).</p>\",\n \"<title>Traits and Symptoms in Adolescence</title>\",\n \"<p id=\\\"Par4\\\">Adolescence is a sensitive developmental period marked by profound cognitive, neurobiological, and interpersonal change (Dahl et al., ##REF##29469094##2018##). In a large meta-analysis including 192 epidemiological studies, Solmi et al. (##REF##34079068##2022##) found that the peak age of onset of any mental disorder was 14.5 years. Therefore, adolescence represents a prime time for understanding vulnerabilities and the development of psychopathological symptoms. Personality and psychopathology are typically considered distinct domains sometimes differentiated based on temporal stability. Specifically, some researchers have suggested that personality traits reflect long-term dispositions and that psychopathological symptoms are state-like instances of an experience or behavior (DeYoung et al., ##REF##32978977##2022##). In childhood and adolescence, however, personality traits may also change over time (Soto et al., ##REF##21171787##2011##). Indeed, some research has found little differences between mean-level change of several personality facets and internalizing symptoms across five 9-month follow-ups in a sample of female adolescents (Goldstein et al., ##REF##35241862##2022##). Other evidence has shown that personality pathology tends to decline across adolescence (Álvarez-Tomás et al., ##REF##30599336##2019##). In the CU traits literature, many panel studies have examined the long-term stability of CU traits with one review suggesting that CU traits show substantial rank-order stability and modest mean-level stability during childhood, adolescence, and into early adulthood (Frick et al., ##REF##24117854##2014##). However, what cannot be ascertained from this work is whether adolescents with CU traits are consistent in their manifestation of CU traits. In other words, do CU traits fluctuate day-to-day (or moment-by-moment)?</p>\",\n \"<title>Intensive Longitudinal Methods</title>\",\n \"<p id=\\\"Par5\\\">Innovations in technology (e.g., smart phones and wearables) have generated a surge in studies investigating dynamic processes on micro timescales using a range of intensive longitudinal methods (for recent reviews, see Russell & Gajos, ##REF##31997358##2020##; Urben et al., ##UREF##10##2022##). Broadly, intensive longitudinal methods, such as daily diary, experience sampling, ecological momentary assessment, and other ambulatory assessment designs, are characterized by many measurement occasions over a brief observation window. These methods focus on within-person variability, such that the measured variables may show deviations or fluctuations relative to the individual mean at specific observation occasions, but the individual mean is relatively stable over time compared to developmental growth or decrease in conventional longitudinal designs. Major advantages of intensive longitudinal methods also include the capability to assess real-time experiences and memory compared to traditional longitudinal designs, which can result in retrospective and recall biases (Russell & Gajos, ##REF##31997358##2020##). These methods also enhance ecological validity, such that assessments are conducted when participants are in real-world settings relative to artificial laboratory experiments. Hence, intensive longitudinal data provide a high level of temporal granularity and inform our understanding of real-life short-term within-person dynamics as opposed to long-term developmental change.</p>\",\n \"<title>Testing Personality and Psychopathology with Intensive Longitudinal Methods</title>\",\n \"<p id=\\\"Par6\\\">Studies are beginning to apply intensive longitudinal designs to elucidate the dynamics of personality and psychopathology. For example, across three samples of adults, Edershile and Wright (##REF##33090821##2021##) tested whether narcissism reflects a dynamic process and found variability in narcissistic states at a moment-to-moment level. Among 91 adults with a personality disorder, daily assessments of personality pathology including negative affect, detachment, impulsivity, and hostility predicted clinical characterizations of personality disorders and daily stress levels (Dotterer et al., ##REF##31597579##2019##). In a sample of adolescent girls, daily negative affect, detachment, disinhibition, and psychoticism were associated with daily socio-affective processes (Kaurin et al., ##UREF##3##2022##). Such studies enhanced our understanding of state-level fluctuations in personality and psychopathology. Yet, no research to date has investigated whether CU traits also vary day-to-day or moment-to-moment. Only two intensive longitudinal studies have included a measure of CU traits using justice-involved adolescent samples; however, both studies only assessed CU traits as a total score at a single timepoint (De Ridder et al., ##UREF##2##2016##; Suter et al., ##UREF##8##2017##). Another study examined callousness in the context of interpersonal antagonism in samples of undergraduates, community adults, and psychiatric outpatients, but, similarly, callousness was only assessed at one timepoint as a predictor (Vize et al., ##REF##35402088##2022##). Across these studies, higher levels of CU traits were associated with higher levels of perceived angry affect and self-reported antisocial behavior (De Ridder et al., ##UREF##2##2016##; Suter et al., ##UREF##8##2017##), and callousness was negatively associated with positive affect and empathy (Vize et al., ##REF##35402088##2022##).</p>\",\n \"<p id=\\\"Par7\\\">In line with other personality constructs (Wright & Kaurin, ##REF##32375147##2020##), much of our current understanding of CU traits is founded on structural or trait-based models (Frick et al., ##REF##24117854##2014##). Research on dynamic modeling at micro timescales may help further inform functional narratives. As the affective domain of psychopathy, CU traits may demonstrate meaningful within-person fluctuations. Notably, the links between CU traits and conduct problems at the daily within-person level could possibly be instantiated by their intermediate and differential links involving daily positive and negative affect. Although most research places negatively valenced emotion processes at the center of personality and psychopathology, some research has implicated positive affect in the development of aggressive behaviors (Toro et al., ##REF##32952964##2020##). In addition, affect and emotion are best conceptualized as dynamic processes underpinning a range of psychopathological outcomes (Houben et al., ##REF##25822133##2015##). Other theoretical accounts suggest that certain forms of personality are derived and maintained by state-level fluctuations of the traits themselves (e.g., grandiosity and vulnerability in narcissism; Edershile & Wright, ##REF##33090821##2021##). It may be the case that daily fluctuations in callousness and uncaring serves to perpetuate CU traits and associated conduct problems. Overall, state-level personality may be more malleable than traits, and thus, studying phenomena at the state-level can illuminate more effective treatment targets for this typically treatment-resistant group.</p>\",\n \"<title>The Present Study</title>\",\n \"<p id=\\\"Par8\\\">There is a lack of knowledge on CU traits on a micro timescale. In this exploratory work with data collected from a sample of adolescents via an intensive 30-day daily diary design, our aims were twofold. First, we aimed to explore whether CU traits (at the item level) demonstrated daily within-person fluctuations. In addition to the potential novel knowledge gained from investigating CU traits using intensive longitudinal designs, examining CU traits at the item level is congruent with recent research initiatives (e.g., Hierarchical Taxonomy of Psychopathology; Kotov et al., ##REF##33577350##2021##) that emphasize fine-grained trait or symptom level analyses to advance mental health science. Second, we aimed to investigate whether daily CU traits (including callousness and uncaring) were associated with daily positive and negative affect, as well as emotional and conduct problems. To address these aims, we applied dynamic structural equation modeling (DSEM) to parse both <italic>within-person</italic> state-level fluctuations including autoregressive and cross-lagged associations (i.e., whether participants’ level at time <italic>t</italic> was significantly predicted by their level of the same or different construct, respectively, at time <italic>t</italic>-1) from stable <italic>between-person</italic> trait-like differences (Asparouhov et al., ##UREF##1##2018##; Hamaker et al., ##REF##29624092##2018##). Given the exploratory nature of this study, we pose no specific a priori hypotheses.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Method</title>","<title>Participants and Procedure</title>","<p id=\"Par9\">Adolescents living in a western Canadian province were recruited between April 2019 and October 2020 to participate in an online study examining their psychological and behavioral adjustment (see Xu & Zheng, ##REF##35583795##2022##). A total of 99 participants (12–17 years old, <italic>M</italic> = 14.60, <italic>SD</italic> = 1.76, 55.8% female, 51.5% white, 23.2% Asian, 8.1% multiracial, 4.0% Hispanic/Latinx, 2.0% Black, 6.1% Other) completed a baseline survey and at least one day of the 30-day daily diary surveys (2,108 total observations, <italic>M</italic> = 21.72 days, <italic>Range</italic> = 1–30, <italic>SD</italic> = 7.80). Among the adolescents, 81.1% reported living with both biological parents, 17.8% reported living with one biological parent, and 1.1% reported living with someone other than a biological parent. Most participants’ parents reported a personal annual income within the average (CAD $65,700) and median ($51,600) personal total incomes for groups of 25–54-year-olds in this Canadian province (17.2% had a personal annual income below $35,000, 9.1% were between $35,000–$45,000, 12.1% were between $45,000–$55,000, 17.2% were between $55,000–$65,000, and 38.4% had an annual income above $65,000).</p>","<p id=\"Par10\">The research ethics committee at the University of Alberta approved the procedure and instruments for this study. Survey instruments were developed and administered via email through RedCap. Participants were recruited through newsletters, social media, and flyers posted or circulated in the western Canadian province. All adolescents were eligible for inclusion in the study. Interested participants were asked to contact the research team and were provided with information for the study. Participants received an online baseline survey (~45 min to complete) following the obtainment of assent online. Participants’ parents provided consent online for their children to participate in this study. Daily surveys (~10 min to complete) were sent out five days after completion of the baseline survey for 30 consecutive days. The daily survey was sent out at 5 pm each day and adolescents were asked to fill out the survey before going to sleep that night. Participants received a $45 e-gift card of their choosing as compensation for their participation in the baseline and daily surveys.</p>","<title>Measures</title>","<title>Callous-unemotional Traits</title>","<p id=\"Par11\">Daily callous-unemotional traits were assessed with a shortened 12-item version of the 24-item Inventory of Callous Unemotional Traits (ICU; Hawes et al., ##REF##24188153##2014b##; Zheng et al., ##REF##34928648##2021##; see Table ##TAB##0##1##). Each item is scored on a 4-point Likert scale (0 = <italic>not at all true</italic>, 1 = <italic>somewhat true</italic>, 2 = <italic>mostly true</italic>, 3 = <italic>definitely true</italic>) in response to the stem “indicate how well the following statements described you today.” This ICU scale includes two subscales: callousness (6 items; ordinal ω<sub>w</sub> = 0.48, ordinal ω<sub>b</sub> = 0.91) and uncaring (5 items; ordinal ω<sub>w</sub> = 0.71, ordinal ω<sub>b</sub> = 0.91). Items that needed to be reverse-coded were done so prior to the analysis. Multilevel Confirmatory Factor Analyses (MLCFA) with the weighted least squares means and variances adjusted estimator demonstrated that the 2-factor model provided satisfactory and better model fit at within- and between-levels (CFI = 0.97, TLI = 0.97, RMSEA = 0.02, SRMR<sub>within(w)</sub> = 0.08, SRMR<sub>between(b)</sub> = 0.04), compared to the 1-factor model at both levels (CFI = 0.87, TLI = 0.83, RMSEA = 0.04, SRMR<sub>w</sub> = 0.15, SRMR<sub>b</sub> = 0.07). The estimated averaged standardized factor loading for the callousness factor was 0.60 and 0.84 at within- and between-levels, and for the uncaring factor was 0.70 and 0.80 at within- and between-levels. MLCFA using the maximum likelihood estimator with robust standard errors (MLR) demonstrated that the 2-factor model at within- and between-levels provided better model fit (Δ scaling correction = 3.68, Δχ<sup>2</sup> = 163.03, Δdfs = 2.00, <italic>p</italic> < 0.001) compared to the 1-factor model at both levels. For the first aim, all 12-items were used. However, in line with several studies that have found that item 3 (<italic>does not show emotions</italic>), the only item left from the original unemotional subscale, exhibits low factor loading (Colins et al., ##REF##26493393##2016##; Zheng et al., ##REF##34928648##2021##), this item was not included in calculations of subscales for the second aim.</p>","<title>Positive and Negative Affect</title>","<p id=\"Par12\">Positive (5 items; e.g., <italic>active</italic>, <italic>attentive</italic>) and negative (5 items; e.g., <italic>ashamed</italic>, <italic>nervous</italic>) affect were assessed daily (i.e., “indicate to what extent you have felt the way as described by each of the following words today”) with the short-form Positive and Negative Affect Schedule (PANAS-SF; Cooke et al., ##REF##36174167##2022##; Thompson, ##UREF##9##2007##) on a 5-point Likert scale (1 = <italic>very slightly or not at all</italic> 2 = <italic>a little</italic>, 3 = <italic>moderately</italic>, 4 = <italic>quite a bit</italic>, 5 = <italic>extremely</italic>). MLCFA model fit for the 2-factor model are as follows: CFI = 0.91, TLI = 0.88, RMSEA = 0.04, SRMR<sub>w</sub> = 0.13, SRMR<sub>b</sub> = 0.14. The estimated averaged standardized factor loading for the positive affect factor was 0.58 and 0.71 at within- and between-levels, and for the negative affect factor was 0.70 and 0.90 at within- and between-levels. Items were averaged within days with higher scores indicative of higher levels of positive (ω<sub>w</sub> = 0.63, ω<sub>b</sub> = 0.88, intra-class correlation [ICC] = 0.71) and negative (ω<sub>w</sub> = 0.74, ω<sub>b</sub> = 0.94, ICC = 0.61) affect.</p>","<title>Emotional and Conduct Problems</title>","<p id=\"Par13\">Participants reported their daily emotional and conduct problems using 5 items each from the emotional problems (e.g., <italic>I worry a lot</italic>) and conduct problems (e.g., <italic>I take things that are not mine from home, school, or elsewhere</italic>) subscales of the Strengths and Difficulties Questionnaire (Goodman et al., ##REF##9826298##1998##). Items were rated on a 3-point Likert scale (0 = <italic>not true</italic>, 1 = <italic>somewhat true</italic>, 2 = <italic>certainly true</italic>) and averaged within days. MLCFA model fit for the 2-factor model are as follows: CFI = 0.98, TLI = 0.98, RMSEA = 0.01, SRMR<sub>w</sub> = 0.10, SRMR<sub>b</sub> = 0.05. The estimated averaged standardized factor loading for the emotional problems factor was 0.57 and 0.88 at within- and between-levels, and for the conduct problems factor was 0.48 and 0.84 at within- and between-levels. Higher scores represented more emotional (ordinal ω<sub>w</sub> = 0.70, ordinal ω<sub>b</sub> = 0.94, ICC = 0.76) and conduct (ordinal ω<sub>w</sub> = 0.63, ordinal ω<sub>b</sub> = 0.92, ICC = 0.60) problems that day (i.e., “mark how the following items described you on the basis of how things have been for you today”).</p>","<title>Analytic Approach</title>","<p id=\"Par14\">In M<italic>plus</italic> version 8.6 (Muthén & Muthén, ##UREF##5##2021##), DSEM (Asparouhov et al., ##UREF##1##2018##) was conducted to examine both research aims (see Fig. ##FIG##0##1## for DSEM schematic of aim 2). Combining time-series and structural equation modeling, this multilevel modeling approach tests temporal relations between measured variables by decomposing the data into within-person (Level 1) and between-person (Level 2) components (Asparouhov et al., ##UREF##1##2018##; Hamaker et al., ##REF##29624092##2018##). At the within-person level, <italic>inertia</italic>, otherwise known as autoregression or carryover, is an estimate of the extent to which scores return to equilibrium (or persist) over measurement occasions, with higher scores indicating greater resistance to change (Kuppens et al., ##REF##20501521##2010##). Regarding cross-lagged associations, <italic>augmentation</italic> describes positive cross-lagged associations, such that an increase in the level of one construct predicts an increase in another construct at a successive time point (Pe & Kuppens, ##REF##22642355##2012##). By contrast, <italic>blunting</italic> characterizes negative cross-lagged associations, in which an increase in the level of one construct predicts a decrease in another construct at the next time point. In other words, these within-person components assess whether participants’ level at time <italic>t</italic> were significantly predicted by their level of the same or different construct, respectively, at time <italic>t</italic>-1. Contemporaneously, <italic>covariation</italic> specifies whether constructs at the same time point are associated with one another (Krone et al., ##REF##29265839##2018##). Accordingly, at the within-person level (person-level centered), both autoregressive and cross-lagged parameters are estimated. Covariances among residuals between constructs within the same day after controlling for the previous day’s effect (i.e., autoregression and cross-lagged effects) were also estimated. At the between-person level, DSEM models means and (co)variances to examine between-person differences aggregated over the 30-day survey period (i.e., random intercepts). Bayesian Markov Chain Monte Carlo (MCMC) is used for estimation resulting in an entire distribution of possible values for each unknown parameter. Models were estimated using default priors and convergence was established by examining several diagnostic criteria including the Potential Scale Reduction (PSR) statistic, autocorrelation plots, and trace plots. Significance is determined by whether the 95% credible interval (CI) contains value 0 for each parameter. M<italic>plus</italic> code and output are supplied on the Open Science Framework: <ext-link ext-link-type=\"uri\" xlink:href=\"https://osf.io/n2rdf/?view_only=357ec650bfac4a63905e7ed34efd9255\">https://osf.io/n2rdf/?view_only=357ec650bfac4a63905e7ed34efd9255</ext-link>.</p>"],"string":"[\n \"<title>Method</title>\",\n \"<title>Participants and Procedure</title>\",\n \"<p id=\\\"Par9\\\">Adolescents living in a western Canadian province were recruited between April 2019 and October 2020 to participate in an online study examining their psychological and behavioral adjustment (see Xu & Zheng, ##REF##35583795##2022##). A total of 99 participants (12–17 years old, <italic>M</italic> = 14.60, <italic>SD</italic> = 1.76, 55.8% female, 51.5% white, 23.2% Asian, 8.1% multiracial, 4.0% Hispanic/Latinx, 2.0% Black, 6.1% Other) completed a baseline survey and at least one day of the 30-day daily diary surveys (2,108 total observations, <italic>M</italic> = 21.72 days, <italic>Range</italic> = 1–30, <italic>SD</italic> = 7.80). Among the adolescents, 81.1% reported living with both biological parents, 17.8% reported living with one biological parent, and 1.1% reported living with someone other than a biological parent. Most participants’ parents reported a personal annual income within the average (CAD $65,700) and median ($51,600) personal total incomes for groups of 25–54-year-olds in this Canadian province (17.2% had a personal annual income below $35,000, 9.1% were between $35,000–$45,000, 12.1% were between $45,000–$55,000, 17.2% were between $55,000–$65,000, and 38.4% had an annual income above $65,000).</p>\",\n \"<p id=\\\"Par10\\\">The research ethics committee at the University of Alberta approved the procedure and instruments for this study. Survey instruments were developed and administered via email through RedCap. Participants were recruited through newsletters, social media, and flyers posted or circulated in the western Canadian province. All adolescents were eligible for inclusion in the study. Interested participants were asked to contact the research team and were provided with information for the study. Participants received an online baseline survey (~45 min to complete) following the obtainment of assent online. Participants’ parents provided consent online for their children to participate in this study. Daily surveys (~10 min to complete) were sent out five days after completion of the baseline survey for 30 consecutive days. The daily survey was sent out at 5 pm each day and adolescents were asked to fill out the survey before going to sleep that night. Participants received a $45 e-gift card of their choosing as compensation for their participation in the baseline and daily surveys.</p>\",\n \"<title>Measures</title>\",\n \"<title>Callous-unemotional Traits</title>\",\n \"<p id=\\\"Par11\\\">Daily callous-unemotional traits were assessed with a shortened 12-item version of the 24-item Inventory of Callous Unemotional Traits (ICU; Hawes et al., ##REF##24188153##2014b##; Zheng et al., ##REF##34928648##2021##; see Table ##TAB##0##1##). Each item is scored on a 4-point Likert scale (0 = <italic>not at all true</italic>, 1 = <italic>somewhat true</italic>, 2 = <italic>mostly true</italic>, 3 = <italic>definitely true</italic>) in response to the stem “indicate how well the following statements described you today.” This ICU scale includes two subscales: callousness (6 items; ordinal ω<sub>w</sub> = 0.48, ordinal ω<sub>b</sub> = 0.91) and uncaring (5 items; ordinal ω<sub>w</sub> = 0.71, ordinal ω<sub>b</sub> = 0.91). Items that needed to be reverse-coded were done so prior to the analysis. Multilevel Confirmatory Factor Analyses (MLCFA) with the weighted least squares means and variances adjusted estimator demonstrated that the 2-factor model provided satisfactory and better model fit at within- and between-levels (CFI = 0.97, TLI = 0.97, RMSEA = 0.02, SRMR<sub>within(w)</sub> = 0.08, SRMR<sub>between(b)</sub> = 0.04), compared to the 1-factor model at both levels (CFI = 0.87, TLI = 0.83, RMSEA = 0.04, SRMR<sub>w</sub> = 0.15, SRMR<sub>b</sub> = 0.07). The estimated averaged standardized factor loading for the callousness factor was 0.60 and 0.84 at within- and between-levels, and for the uncaring factor was 0.70 and 0.80 at within- and between-levels. MLCFA using the maximum likelihood estimator with robust standard errors (MLR) demonstrated that the 2-factor model at within- and between-levels provided better model fit (Δ scaling correction = 3.68, Δχ<sup>2</sup> = 163.03, Δdfs = 2.00, <italic>p</italic> < 0.001) compared to the 1-factor model at both levels. For the first aim, all 12-items were used. However, in line with several studies that have found that item 3 (<italic>does not show emotions</italic>), the only item left from the original unemotional subscale, exhibits low factor loading (Colins et al., ##REF##26493393##2016##; Zheng et al., ##REF##34928648##2021##), this item was not included in calculations of subscales for the second aim.</p>\",\n \"<title>Positive and Negative Affect</title>\",\n \"<p id=\\\"Par12\\\">Positive (5 items; e.g., <italic>active</italic>, <italic>attentive</italic>) and negative (5 items; e.g., <italic>ashamed</italic>, <italic>nervous</italic>) affect were assessed daily (i.e., “indicate to what extent you have felt the way as described by each of the following words today”) with the short-form Positive and Negative Affect Schedule (PANAS-SF; Cooke et al., ##REF##36174167##2022##; Thompson, ##UREF##9##2007##) on a 5-point Likert scale (1 = <italic>very slightly or not at all</italic> 2 = <italic>a little</italic>, 3 = <italic>moderately</italic>, 4 = <italic>quite a bit</italic>, 5 = <italic>extremely</italic>). MLCFA model fit for the 2-factor model are as follows: CFI = 0.91, TLI = 0.88, RMSEA = 0.04, SRMR<sub>w</sub> = 0.13, SRMR<sub>b</sub> = 0.14. The estimated averaged standardized factor loading for the positive affect factor was 0.58 and 0.71 at within- and between-levels, and for the negative affect factor was 0.70 and 0.90 at within- and between-levels. Items were averaged within days with higher scores indicative of higher levels of positive (ω<sub>w</sub> = 0.63, ω<sub>b</sub> = 0.88, intra-class correlation [ICC] = 0.71) and negative (ω<sub>w</sub> = 0.74, ω<sub>b</sub> = 0.94, ICC = 0.61) affect.</p>\",\n \"<title>Emotional and Conduct Problems</title>\",\n \"<p id=\\\"Par13\\\">Participants reported their daily emotional and conduct problems using 5 items each from the emotional problems (e.g., <italic>I worry a lot</italic>) and conduct problems (e.g., <italic>I take things that are not mine from home, school, or elsewhere</italic>) subscales of the Strengths and Difficulties Questionnaire (Goodman et al., ##REF##9826298##1998##). Items were rated on a 3-point Likert scale (0 = <italic>not true</italic>, 1 = <italic>somewhat true</italic>, 2 = <italic>certainly true</italic>) and averaged within days. MLCFA model fit for the 2-factor model are as follows: CFI = 0.98, TLI = 0.98, RMSEA = 0.01, SRMR<sub>w</sub> = 0.10, SRMR<sub>b</sub> = 0.05. The estimated averaged standardized factor loading for the emotional problems factor was 0.57 and 0.88 at within- and between-levels, and for the conduct problems factor was 0.48 and 0.84 at within- and between-levels. Higher scores represented more emotional (ordinal ω<sub>w</sub> = 0.70, ordinal ω<sub>b</sub> = 0.94, ICC = 0.76) and conduct (ordinal ω<sub>w</sub> = 0.63, ordinal ω<sub>b</sub> = 0.92, ICC = 0.60) problems that day (i.e., “mark how the following items described you on the basis of how things have been for you today”).</p>\",\n \"<title>Analytic Approach</title>\",\n \"<p id=\\\"Par14\\\">In M<italic>plus</italic> version 8.6 (Muthén & Muthén, ##UREF##5##2021##), DSEM (Asparouhov et al., ##UREF##1##2018##) was conducted to examine both research aims (see Fig. ##FIG##0##1## for DSEM schematic of aim 2). Combining time-series and structural equation modeling, this multilevel modeling approach tests temporal relations between measured variables by decomposing the data into within-person (Level 1) and between-person (Level 2) components (Asparouhov et al., ##UREF##1##2018##; Hamaker et al., ##REF##29624092##2018##). At the within-person level, <italic>inertia</italic>, otherwise known as autoregression or carryover, is an estimate of the extent to which scores return to equilibrium (or persist) over measurement occasions, with higher scores indicating greater resistance to change (Kuppens et al., ##REF##20501521##2010##). Regarding cross-lagged associations, <italic>augmentation</italic> describes positive cross-lagged associations, such that an increase in the level of one construct predicts an increase in another construct at a successive time point (Pe & Kuppens, ##REF##22642355##2012##). By contrast, <italic>blunting</italic> characterizes negative cross-lagged associations, in which an increase in the level of one construct predicts a decrease in another construct at the next time point. In other words, these within-person components assess whether participants’ level at time <italic>t</italic> were significantly predicted by their level of the same or different construct, respectively, at time <italic>t</italic>-1. Contemporaneously, <italic>covariation</italic> specifies whether constructs at the same time point are associated with one another (Krone et al., ##REF##29265839##2018##). Accordingly, at the within-person level (person-level centered), both autoregressive and cross-lagged parameters are estimated. Covariances among residuals between constructs within the same day after controlling for the previous day’s effect (i.e., autoregression and cross-lagged effects) were also estimated. At the between-person level, DSEM models means and (co)variances to examine between-person differences aggregated over the 30-day survey period (i.e., random intercepts). Bayesian Markov Chain Monte Carlo (MCMC) is used for estimation resulting in an entire distribution of possible values for each unknown parameter. Models were estimated using default priors and convergence was established by examining several diagnostic criteria including the Potential Scale Reduction (PSR) statistic, autocorrelation plots, and trace plots. Significance is determined by whether the 95% credible interval (CI) contains value 0 for each parameter. M<italic>plus</italic> code and output are supplied on the Open Science Framework: <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://osf.io/n2rdf/?view_only=357ec650bfac4a63905e7ed34efd9255\\\">https://osf.io/n2rdf/?view_only=357ec650bfac4a63905e7ed34efd9255</ext-link>.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Callous-unemotional Traits at the Item Level</title>","<p id=\"Par15\">Descriptive statistics, ICCs, and within-level and between-level CU item correlations are shown in ##SUPPL##0##Supplementary Table S1##. ICC is the proportion of the total variance that is accounted for by stable or trait-like between-person differences plus measurement error (Hamaker et al., ##REF##29624092##2018##). Accordingly, lower ICCs would indicate more within-person fluctuations. At the within-level, almost all items were significantly positively correlated with each other. At the between-level, items 2 (<italic>feels bad or guilty when I have done something wrong</italic>) and 3 (<italic>does not show emotions</italic>) showed few significant associations with other items and the highest ICC values (0.77 and 0.78, respectively).</p>","<p id=\"Par16\">After controlling for the previous day’s autoregression and cross-lagged effects, item-level within-day residual correlations are shown in Table ##TAB##1##2##. Similar to the within-level item correlations presented above, the majority of items were still positively correlated with each other (although magnitudes were smaller) after controlling for the previous day’s effect.</p>","<p id=\"Par17\">Significant item-level autoregressive effects are shown in Fig. ##FIG##1##2## (as indicated by curved arrows) and all item-level autoregressive estimates are described in ##SUPPL##0##Supplementary Table S2##. Except for items 6 (<italic>does not care about doing things well</italic>) and 10 (<italic>shows no remorse when I have done something wrong</italic>), all items showed significant autoregressive effects (βs = 0.08–0.23; 95% CI [0.02–0.18, 0.13–0.28]), suggesting that adolescents experiencing high levels of CU traits on the previous day were more likely to experience high levels the next day compared to their average level. More specifically, items 1 (<italic>does not care who I hurt to get what I want</italic>; β = 0.08; 95% CI [0.02, 0.14]), 4 (<italic>concerned about the feelings of others</italic>; β = 0.08; 95% CI [0.02, 0.15]), 8 (<italic>apologizes to persons I have hurt</italic>; β = 0.08; 95% CI [0.02, 0.14]), and 11 (<italic>feelings of others are unimportant</italic>; β = 0.08; 95% CI [0.02, 0.13]) showed the lowest levels of inertia and item 3 (<italic>does not show emotions</italic>; β = 0.23; 95% CI [0.18, 0.28]) revealed the largest inertia.</p>","<p id=\"Par18\">Significant item-level cross-lagged effects are shown in Fig. ##FIG##1##2## (as indicated by straight arrows) and all item-level cross-lagged estimates are described in ##SUPPL##0##Supplementary Table S3##. All items, except for item 10, demonstrated cross-lagged effects with at least one other item. However, it is notable that most cross-lagged associations involved only four items as outcomes: items 2 (<italic>feels bad or guilty when I have done something wrong</italic>; βs = -0.07–0.06 95% CIs [-0.12–0.01, -0.02–0.12]), 7 (<italic>seems very cold and uncaring;</italic> βs = 0.06–0.10; 95% CIs [0.01–0.05, 0.11–0.14]), 9 (<italic>tries not to hurt others’ feelings</italic>; βs = -0.06–0.10; 95% CIs [-0.12–0.05, -0.00–0.14]), and 12 (<italic>does things to make others feel good</italic>; βs = 0.06–0.13; 95% CIs [0.00–0.07, 0.12–0.18]). These findings highlight several CU traits that adolescents reported as endorsing higher levels the next day compared to their average level. In addition, most items only showed two significant cross-lagged associations, with items 4, 8, and 9 displaying three cross-lagged effects. All significant cross-lagged associations were augmented, apart from four effects (i.e., 3 → 9, 7 → 2, 9 → 6, and 12 → 6), which revealed blunting effects.</p>","<p id=\"Par19\">Between-level item correlations, means, and variances are shown in Table ##TAB##1##2##. Overall, consistent with the between-level descriptive statistics, all items displayed significant positive associations with almost all other items, except items 2 (<italic>feels bad or guilty when I have done something wrong</italic>) and 3 (<italic>does not show emotions</italic>), which showed few significant associations.</p>","<title>Callous-unemotional Traits and Associations with Subscales</title>","<p id=\"Par20\">Descriptive statistics, ICCs, and within-level and between-level subscale correlations are shown in ##SUPPL##0##Supplementary Table S4##. Notably, at the within-level, callousness was significantly positively associated with all variables except positive affect, whereas uncaring was positively associated with negative affect and conduct problems, and negatively with positive affect. At the between-level, callousness was significantly positively associated with all variables and negatively associated with positive affect; uncaring was positively associated with conduct problems and negatively with positive affect. Also, emotional problems displayed the highest ICC (0.76) and uncaring had the lowest ICC (0.60). Table ##TAB##2##3## shows within-day residual correlations after controlling for the previous day’s effects. Findings were similar to the within-level item correlations presented above.</p>","<p id=\"Par21\">Significant autoregressive (curved arrows) and cross-lagged (straight arrows) effects are displayed in Fig. ##FIG##2##3## (all estimates are shown in ##SUPPL##0##Supplementary Tables S5 and S6##, respectively). All variables showed significant autoregressive effects (βs = 0.13–0.35; 95% CIs [0.08–0.29, 0.18–0.41]). Thus, adolescents experiencing high levels of each variable on the previous day were more likely to experience high levels of each variable, respectively, the next day compared to their average level. Regarding cross-lagged effects, all significant associations were positive (i.e., augmentation). Specifically, adolescents who reported higher than their average levels of callousness also reported higher than their average levels of uncaring the next day (β = 0.08; 95% CI [0.03, 0.13]). Similar cross-day associations were observed between conduct problems and uncaring (β = 0.05; 95% CI [0.00, 0.10]), positive affect and callousness (β = 0.05; 95% CI [0.00, 0.10]), negative affect and emotional problems (β = 0.07; 95% CI [0.01, 0.13]), and, finally, emotional problems and negative affect (β = 0.09; 95% CI [0.03, 0.15]). Notably, uncaring did not show any significant cross-lagged effects.</p>","<p id=\"Par22\">Between-level correlations, means, and variances are shown in Table ##TAB##2##3##. Notably, adolescents with higher levels of callousness, on average, tended to also have higher average levels of uncaring, negative affect, conduct problems, and emotional problems and lower average levels of positive affect over the month. Those with higher average levels of uncaring also had higher average levels of conduct problems and lower average levels of positive affect over the month.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Callous-unemotional Traits at the Item Level</title>\",\n \"<p id=\\\"Par15\\\">Descriptive statistics, ICCs, and within-level and between-level CU item correlations are shown in ##SUPPL##0##Supplementary Table S1##. ICC is the proportion of the total variance that is accounted for by stable or trait-like between-person differences plus measurement error (Hamaker et al., ##REF##29624092##2018##). Accordingly, lower ICCs would indicate more within-person fluctuations. At the within-level, almost all items were significantly positively correlated with each other. At the between-level, items 2 (<italic>feels bad or guilty when I have done something wrong</italic>) and 3 (<italic>does not show emotions</italic>) showed few significant associations with other items and the highest ICC values (0.77 and 0.78, respectively).</p>\",\n \"<p id=\\\"Par16\\\">After controlling for the previous day’s autoregression and cross-lagged effects, item-level within-day residual correlations are shown in Table ##TAB##1##2##. Similar to the within-level item correlations presented above, the majority of items were still positively correlated with each other (although magnitudes were smaller) after controlling for the previous day’s effect.</p>\",\n \"<p id=\\\"Par17\\\">Significant item-level autoregressive effects are shown in Fig. ##FIG##1##2## (as indicated by curved arrows) and all item-level autoregressive estimates are described in ##SUPPL##0##Supplementary Table S2##. Except for items 6 (<italic>does not care about doing things well</italic>) and 10 (<italic>shows no remorse when I have done something wrong</italic>), all items showed significant autoregressive effects (βs = 0.08–0.23; 95% CI [0.02–0.18, 0.13–0.28]), suggesting that adolescents experiencing high levels of CU traits on the previous day were more likely to experience high levels the next day compared to their average level. More specifically, items 1 (<italic>does not care who I hurt to get what I want</italic>; β = 0.08; 95% CI [0.02, 0.14]), 4 (<italic>concerned about the feelings of others</italic>; β = 0.08; 95% CI [0.02, 0.15]), 8 (<italic>apologizes to persons I have hurt</italic>; β = 0.08; 95% CI [0.02, 0.14]), and 11 (<italic>feelings of others are unimportant</italic>; β = 0.08; 95% CI [0.02, 0.13]) showed the lowest levels of inertia and item 3 (<italic>does not show emotions</italic>; β = 0.23; 95% CI [0.18, 0.28]) revealed the largest inertia.</p>\",\n \"<p id=\\\"Par18\\\">Significant item-level cross-lagged effects are shown in Fig. ##FIG##1##2## (as indicated by straight arrows) and all item-level cross-lagged estimates are described in ##SUPPL##0##Supplementary Table S3##. All items, except for item 10, demonstrated cross-lagged effects with at least one other item. However, it is notable that most cross-lagged associations involved only four items as outcomes: items 2 (<italic>feels bad or guilty when I have done something wrong</italic>; βs = -0.07–0.06 95% CIs [-0.12–0.01, -0.02–0.12]), 7 (<italic>seems very cold and uncaring;</italic> βs = 0.06–0.10; 95% CIs [0.01–0.05, 0.11–0.14]), 9 (<italic>tries not to hurt others’ feelings</italic>; βs = -0.06–0.10; 95% CIs [-0.12–0.05, -0.00–0.14]), and 12 (<italic>does things to make others feel good</italic>; βs = 0.06–0.13; 95% CIs [0.00–0.07, 0.12–0.18]). These findings highlight several CU traits that adolescents reported as endorsing higher levels the next day compared to their average level. In addition, most items only showed two significant cross-lagged associations, with items 4, 8, and 9 displaying three cross-lagged effects. All significant cross-lagged associations were augmented, apart from four effects (i.e., 3 → 9, 7 → 2, 9 → 6, and 12 → 6), which revealed blunting effects.</p>\",\n \"<p id=\\\"Par19\\\">Between-level item correlations, means, and variances are shown in Table ##TAB##1##2##. Overall, consistent with the between-level descriptive statistics, all items displayed significant positive associations with almost all other items, except items 2 (<italic>feels bad or guilty when I have done something wrong</italic>) and 3 (<italic>does not show emotions</italic>), which showed few significant associations.</p>\",\n \"<title>Callous-unemotional Traits and Associations with Subscales</title>\",\n \"<p id=\\\"Par20\\\">Descriptive statistics, ICCs, and within-level and between-level subscale correlations are shown in ##SUPPL##0##Supplementary Table S4##. Notably, at the within-level, callousness was significantly positively associated with all variables except positive affect, whereas uncaring was positively associated with negative affect and conduct problems, and negatively with positive affect. At the between-level, callousness was significantly positively associated with all variables and negatively associated with positive affect; uncaring was positively associated with conduct problems and negatively with positive affect. Also, emotional problems displayed the highest ICC (0.76) and uncaring had the lowest ICC (0.60). Table ##TAB##2##3## shows within-day residual correlations after controlling for the previous day’s effects. Findings were similar to the within-level item correlations presented above.</p>\",\n \"<p id=\\\"Par21\\\">Significant autoregressive (curved arrows) and cross-lagged (straight arrows) effects are displayed in Fig. ##FIG##2##3## (all estimates are shown in ##SUPPL##0##Supplementary Tables S5 and S6##, respectively). All variables showed significant autoregressive effects (βs = 0.13–0.35; 95% CIs [0.08–0.29, 0.18–0.41]). Thus, adolescents experiencing high levels of each variable on the previous day were more likely to experience high levels of each variable, respectively, the next day compared to their average level. Regarding cross-lagged effects, all significant associations were positive (i.e., augmentation). Specifically, adolescents who reported higher than their average levels of callousness also reported higher than their average levels of uncaring the next day (β = 0.08; 95% CI [0.03, 0.13]). Similar cross-day associations were observed between conduct problems and uncaring (β = 0.05; 95% CI [0.00, 0.10]), positive affect and callousness (β = 0.05; 95% CI [0.00, 0.10]), negative affect and emotional problems (β = 0.07; 95% CI [0.01, 0.13]), and, finally, emotional problems and negative affect (β = 0.09; 95% CI [0.03, 0.15]). Notably, uncaring did not show any significant cross-lagged effects.</p>\",\n \"<p id=\\\"Par22\\\">Between-level correlations, means, and variances are shown in Table ##TAB##2##3##. Notably, adolescents with higher levels of callousness, on average, tended to also have higher average levels of uncaring, negative affect, conduct problems, and emotional problems and lower average levels of positive affect over the month. Those with higher average levels of uncaring also had higher average levels of conduct problems and lower average levels of positive affect over the month.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par23\">By collecting intensive longitudinal data and applying analytic approaches that elucidate temporal dynamics, we can expand our understanding of CU traits in a more ecologically valid way. Accordingly, this exploratory study examined whether adolescent CU traits manifest fluctuations across a 1-month observation window. Many CU traits items showed within-person autoregressive and cross-lagged links over the timeframe (i.e., participants’ levels at time <italic>t</italic> were significantly predicted by their levels at time <italic>t</italic>-1), and we described these effects using terminology from the affective dynamics literature. We also illustrated how daily CU traits were associated with daily affect and emotional and conduct problems. Overall, we observed several within- and between-person effects that serve as a starting point for further understanding of temporal dynamics and functional accounts of CU traits and related emotional and behavioral functioning.</p>","<title>Callous-unemotional Traits in Daily Life</title>","<p id=\"Par24\">In line with recent research calling for lower-order precision level analyses (Goulter et al., ##REF##36074612##2022##; Kotov et al., ##REF##33577350##2021##), we assessed CU traits at the item-level. Almost all items displayed within-person autoregressive effects (inertia). Of those items, two items that typically load onto callousness (<italic>does not care who I hurt to get what I want</italic>, <italic>feelings of others are unimportant</italic>) and two items that represent uncaring (<italic>concerned about the feelings of others</italic>, <italic>apologizes to persons I have hurt</italic>) had lower inertia and ICC scores (i.e., greater within-person fluctuations) relative to other items. Notably, these items all reflect CU traits related to behaviors directed toward others or others’ emotions or feelings. These items demonstrate greater variability likely because they tap interpersonal interactions with others, which could be less salient and stable relative to items regarding one’s own motivations and behaviors (e.g., <italic>does not care if I am in trouble</italic>, <italic>does not care about doing things well</italic>). Conceptualizations of CU traits point to deficits in both affiliative capacity and emotional responding, and intensive longitudinal designs may prove especially useful in informing socio-emotional accounts of CU traits.</p>","<p id=\"Par25\">Conversely, <italic>does not show emotions</italic> showed the largest inertia, ICC (i.e., the least within-person fluctuations), and highest between-level mean. This is a notable finding because this item is the only item retained from the 24-item ICU to the brief 12-item version presumably loading onto the unemotional subscale. Although this 12-item adaptation has been well-validated in child, adolescent, and young adult samples (e.g., Hawes et al., ##REF##24188153##2014b##; Zheng et al., ##REF##34928648##2021##), others have emphasized the importance of unemotional items for understanding the broader CU construct (Ray & Frick, ##REF##30142284##2020##). For example, one meta-analytic study found that although the unemotional subscale was only modestly associated with aggression (<italic>r</italic> = 0.06), it was more highly associated with lower empathy (<italic>r</italic> = -0.22; Cardinale & Marsh, ##REF##29239206##2020##). These findings suggest that the unemotional scale may be more consequential for delineating “trait-like” impoverished emotion and empathy aspects of CU traits (Ray & Frick, ##REF##30142284##2020##). Furthermore, both <italic>does not care about doing things well</italic> and <italic>shows no remorse when I have done something wrong</italic> did not show significant autoregressive effects. These results are somewhat expected as it is unlikely that our community sample committed transgressions every day, thus necessitating further research with higher-risk and clinical samples.</p>","<p id=\"Par26\">A greater understanding of the short-term fluctuations in CU traits can also help inform ethical considerations in this field regarding labelling, stigmatization, and nomenclature. As a construct, CU traits was initially developed by downwardly extending the affective component of adult psychopathy to children and adolescents. Although definitions of psychopathy typically also comprise interpersonal and impulsive/antisocial dimensions, an extensive child and adult literature has revealed that the affective or CU dimension tends to distinguish those individuals with distinct neurocognitive correlates, as well as heightened risk for persistent antisocial behavior (Frick et al., ##REF##24117854##2014##). However, there are reasonable concerns regarding the stigmatization of labelling children and adolescents with the term CU “traits” (Prasad & Kimonis, ##UREF##6##2018##). Several scholars have accordingly advocated for alternative language, such as CU <italic>behaviors</italic>, <italic>features</italic>, or <italic>symptoms</italic>, particularly in younger children when these characteristics may be less stable (e.g., Schuberth et al., ##REF##30155686##2019##; Waller & Hyde, ##REF##28824706##2017##). Others have made clear at the outset that although they use the term CU “traits” in young samples, they are not suggesting that these indicators are immutable (e.g., Fleming et al., ##REF##36229121##2022##). As well-stated by Kaurin et al. (##UREF##3##2022##, p. 23) “A stricter focus on dysregulated interpersonal and affective processes in daily life [therefore] may also reform the professional and public view on personality pathology, which is dominated by perceptions of destiny rather than manageable risk.” Overall, our item-level findings provide preliminary granular and temporal evidence of within-person “state” fluctuations of CU traits in real-life.</p>","<title>Daily Callous-unemotional Traits and Emotional and Behavioral Functioning</title>","<p id=\"Par27\">We also evaluated whether daily CU traits, including callousness and uncaring, were associated with several indicators of emotional and behavioral functioning. Both callousness and conduct problems augmented uncaring over time. One explanation for these findings may relate to how adolescents with elevated callousness appraise certain situations. It may be the case that adolescents high on callousness with a cognitive predisposition underpinned by fearlessness and deficits in emotional processing interpret certain situational features (e.g., negative interactions with parents or peers) in a way that results in higher levels of uncaring. Also noteworthy, uncaring did not predict any other construct. Structural differences in the ICU have been suggested to be a spurious by-product of method variance (Ray & Frick, ##REF##30142284##2020##); however, our findings contribute some evidence pointing to meaningful differences between CU subscales. Taken together, these findings suggest that uncaring may be better conceptualized as an outcome rather than an antecedent, and callousness could represent a critical treatment target.</p>","<p id=\"Par28\">An interesting finding was that positive affect was linked with callousness. Previous research found that callousness at baseline negatively predicted positive affect assessed six times per day across a 1-week period (Vize et al., ##REF##35402088##2022##); however, callousness was assessed with a personality measure only at baseline. One explanation for our findings may be due to the specific measure of positive affect assessing high valence constructs, including being active, inspired, and determined (Cooke et al., ##REF##36174167##2022##; Thompson, ##UREF##9##2007##). It may be the case that callousness also designates resolute individuals characterized by ambition. This notion goes against the DSM-5 “with Limited Prosocial Emotions” criterion specifying an absence of interest in school or work performance (American Psychiatric Association, ##UREF##0##2013##). However, the current uncaring subscale from the 12-item ICU is operationalized as a lack of concern for others (not activities). In addition, echoing Vize et al. (##REF##35402088##2022##), such traits may be strongly associated with extroversion, which is linked to heightened positive affect (Watson et al., ##REF##25751628##2015##)–although these findings are derived from adult samples. Future studies should consider a broader range of positive affect items to replicate and extend current findings, as well as further research on personality in adolescent samples.</p>","<title>Strengths and Limitations</title>","<p id=\"Par29\">Notable strengths of the present study include the naturalistic 30-day diary design, which advances understanding of CU traits under higher ecological validity relative to traditional longitudinal studies. In addition, we applied an analytic approach that disentangles within-person fluctuations from between-person differences. We also evaluated whether daily CU traits were associated with multiple forms of daily emotional and behavioral functioning. However, current findings should be interpreted within the context of some limitations. First, the current sample was primarily comprised of community adolescents from families with higher annual income than the provincial median income. Participants also endorsed lower levels of CU traits (<italic>M</italic> = 6.88; <italic>SD</italic> = 4.26) relative to other studies using the 12-item ICU scale. For example, Colins et al. (##REF##26493393##2016##) found higher levels of CU traits in a sample of justice-involved adolescent girls (<italic>M</italic> = 9.14; <italic>SD</italic> = 5.88). Thus, our results may not generalize to other populations, and as highlighted, it would be particularly important to examine daily CU traits in higher-risk or clinical samples.</p>","<p id=\"Par30\">Second, all data are solely based on adolescent self-reports–we did not collect data from other informants (e.g., parents) on adolescent daily CU traits. Past research has revealed distinct associations between CU traits and conduct problems across adolescent- and parent-reports (e.g., Goulter & Moretti, ##REF##33825099##2021##). Meta-analytic work has also shown less discrepancies in cross-informant correspondence when informants are reporting on observable behaviors (e.g., externalizing vs. internalizing) or when the behaviors are measured within the same context (De Los Reyes et al., ##REF##25915035##2015##). Regarding CU traits, it may be more difficult for other informants to report on CU traits given these traits are not necessarily readily observable. </p>","<p id=\"Par31\">Third, our sample was relatively small, although simulation studies have shown that sufficient power can be achieved for DSEM with a sample size of 100 and a minimum of 25 measured occasions (e.g., Schultzberg & Muthén, ##UREF##7##2018##). In particular, power at Level 2 is relatively small compared to conventional longitudinal studies, and thus, these between-person effects should be interpreted with caution. However, with over 2,000 observations at the daily level, power at Level 1 is sufficient for the current approach. Because of our sample size, we were not able to optimally investigate gender differences–an important direction for future research given most studies examining CU traits have relied predominantly on male samples.</p>","<title>Future Directions and Implications</title>","<p id=\"Par32\">By decomposing “trait” and “state”-like aspects of CU traits, current findings inform understanding of fluctuations in CU traits and lay a foundation for future intensive longitudinal designs to test critical questions in the field. For instance, adolescence represents a sensitive period marked by distinct change including within neural systems involved in social, emotional, and motivational processes (Dahl et al., ##REF##29469094##2018##). Because of this, studies testing daily dynamics among adolescents may be particularly well-suited to reveal important information regarding socio-behavioral functioning. Atypical levels of CU traits have also been observed in samples as young as preschool age (Kimonis et al., ##REF##26344015##2016##), and developmental models describe early childhood as the period in which the emergence of conscience and empathy typically occurs (Kochanska, ##UREF##4##1993##). Thus, future research should also investigate daily CU traits in younger samples. Ideally, CU traits would be examined at multiple developmental stages across multiple timescales, and measurement burst designs that combine micro and macro longitudinal methods could identify changes in short-term CU dynamics and their relations with long-term outcomes. Future research should also apply multiple measurement approaches (e.g., self-report, observational, psychophysiological) to better characterize the emotional components of daily experiences–a particularly important avenue in CU traits research given the distinct psychophysiological profiles among these samples (Fanti et al., ##REF##30797946##2019##).</p>","<p id=\"Par33\">It will also be crucial to investigate the role of specific situational features in CU traits fluctuations and employ different intensive longitudinal methods to test these relations. For example, the present study employed a time-based sampling scheme prompting participants at 5 pm each day. By contrast, other research may use a variable-interval scheme generating prompts at random throughout the day to further enhance ecological validity (Russell & Gajos, ##REF##31997358##2020##). Future studies may also want to consider event contingent designs, which could be useful for examining negative interactions with parents, peers, or the broader environment. Theoretical and empirical works highlight the role of warm and supportive parent–child relationships in promoting empathy development and eliciting positive affect (Kochanska, ##UREF##4##1993##). Intensive longitudinal methods with parent–child dyads (e.g., Xu & Zheng, ##REF##35583795##2022##) have the potential to further advance understanding of familial relations in the development of CU traits. Particularly, parenting behaviors, including parental warmth and harsh discipline, have been linked to child CU traits (Goulter et al., ##REF##31166154##2020##; Waller et al., ##REF##23583974##2013##; Zheng et al., ##REF##27179538##2017##), and recent daily diary studies have demonstrated substantial daily fluctuations in parental warmth (Xu & Zheng, ##REF##36273075##2023##). Thus, examining how daily parenting behaviors are associated with CU traits may provide modifiable targets in daily family processes for future treatment efforts. In adolescence, research may want to examine the role of peer affiliation and interactions in daily CU traits fluctuations. Although the present study assessed negative affect, a greater understanding of the dynamics of stress and associated stressful experiences would also be particularly informative.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par23\\\">By collecting intensive longitudinal data and applying analytic approaches that elucidate temporal dynamics, we can expand our understanding of CU traits in a more ecologically valid way. Accordingly, this exploratory study examined whether adolescent CU traits manifest fluctuations across a 1-month observation window. Many CU traits items showed within-person autoregressive and cross-lagged links over the timeframe (i.e., participants’ levels at time <italic>t</italic> were significantly predicted by their levels at time <italic>t</italic>-1), and we described these effects using terminology from the affective dynamics literature. We also illustrated how daily CU traits were associated with daily affect and emotional and conduct problems. Overall, we observed several within- and between-person effects that serve as a starting point for further understanding of temporal dynamics and functional accounts of CU traits and related emotional and behavioral functioning.</p>\",\n \"<title>Callous-unemotional Traits in Daily Life</title>\",\n \"<p id=\\\"Par24\\\">In line with recent research calling for lower-order precision level analyses (Goulter et al., ##REF##36074612##2022##; Kotov et al., ##REF##33577350##2021##), we assessed CU traits at the item-level. Almost all items displayed within-person autoregressive effects (inertia). Of those items, two items that typically load onto callousness (<italic>does not care who I hurt to get what I want</italic>, <italic>feelings of others are unimportant</italic>) and two items that represent uncaring (<italic>concerned about the feelings of others</italic>, <italic>apologizes to persons I have hurt</italic>) had lower inertia and ICC scores (i.e., greater within-person fluctuations) relative to other items. Notably, these items all reflect CU traits related to behaviors directed toward others or others’ emotions or feelings. These items demonstrate greater variability likely because they tap interpersonal interactions with others, which could be less salient and stable relative to items regarding one’s own motivations and behaviors (e.g., <italic>does not care if I am in trouble</italic>, <italic>does not care about doing things well</italic>). Conceptualizations of CU traits point to deficits in both affiliative capacity and emotional responding, and intensive longitudinal designs may prove especially useful in informing socio-emotional accounts of CU traits.</p>\",\n \"<p id=\\\"Par25\\\">Conversely, <italic>does not show emotions</italic> showed the largest inertia, ICC (i.e., the least within-person fluctuations), and highest between-level mean. This is a notable finding because this item is the only item retained from the 24-item ICU to the brief 12-item version presumably loading onto the unemotional subscale. Although this 12-item adaptation has been well-validated in child, adolescent, and young adult samples (e.g., Hawes et al., ##REF##24188153##2014b##; Zheng et al., ##REF##34928648##2021##), others have emphasized the importance of unemotional items for understanding the broader CU construct (Ray & Frick, ##REF##30142284##2020##). For example, one meta-analytic study found that although the unemotional subscale was only modestly associated with aggression (<italic>r</italic> = 0.06), it was more highly associated with lower empathy (<italic>r</italic> = -0.22; Cardinale & Marsh, ##REF##29239206##2020##). These findings suggest that the unemotional scale may be more consequential for delineating “trait-like” impoverished emotion and empathy aspects of CU traits (Ray & Frick, ##REF##30142284##2020##). Furthermore, both <italic>does not care about doing things well</italic> and <italic>shows no remorse when I have done something wrong</italic> did not show significant autoregressive effects. These results are somewhat expected as it is unlikely that our community sample committed transgressions every day, thus necessitating further research with higher-risk and clinical samples.</p>\",\n \"<p id=\\\"Par26\\\">A greater understanding of the short-term fluctuations in CU traits can also help inform ethical considerations in this field regarding labelling, stigmatization, and nomenclature. As a construct, CU traits was initially developed by downwardly extending the affective component of adult psychopathy to children and adolescents. Although definitions of psychopathy typically also comprise interpersonal and impulsive/antisocial dimensions, an extensive child and adult literature has revealed that the affective or CU dimension tends to distinguish those individuals with distinct neurocognitive correlates, as well as heightened risk for persistent antisocial behavior (Frick et al., ##REF##24117854##2014##). However, there are reasonable concerns regarding the stigmatization of labelling children and adolescents with the term CU “traits” (Prasad & Kimonis, ##UREF##6##2018##). Several scholars have accordingly advocated for alternative language, such as CU <italic>behaviors</italic>, <italic>features</italic>, or <italic>symptoms</italic>, particularly in younger children when these characteristics may be less stable (e.g., Schuberth et al., ##REF##30155686##2019##; Waller & Hyde, ##REF##28824706##2017##). Others have made clear at the outset that although they use the term CU “traits” in young samples, they are not suggesting that these indicators are immutable (e.g., Fleming et al., ##REF##36229121##2022##). As well-stated by Kaurin et al. (##UREF##3##2022##, p. 23) “A stricter focus on dysregulated interpersonal and affective processes in daily life [therefore] may also reform the professional and public view on personality pathology, which is dominated by perceptions of destiny rather than manageable risk.” Overall, our item-level findings provide preliminary granular and temporal evidence of within-person “state” fluctuations of CU traits in real-life.</p>\",\n \"<title>Daily Callous-unemotional Traits and Emotional and Behavioral Functioning</title>\",\n \"<p id=\\\"Par27\\\">We also evaluated whether daily CU traits, including callousness and uncaring, were associated with several indicators of emotional and behavioral functioning. Both callousness and conduct problems augmented uncaring over time. One explanation for these findings may relate to how adolescents with elevated callousness appraise certain situations. It may be the case that adolescents high on callousness with a cognitive predisposition underpinned by fearlessness and deficits in emotional processing interpret certain situational features (e.g., negative interactions with parents or peers) in a way that results in higher levels of uncaring. Also noteworthy, uncaring did not predict any other construct. Structural differences in the ICU have been suggested to be a spurious by-product of method variance (Ray & Frick, ##REF##30142284##2020##); however, our findings contribute some evidence pointing to meaningful differences between CU subscales. Taken together, these findings suggest that uncaring may be better conceptualized as an outcome rather than an antecedent, and callousness could represent a critical treatment target.</p>\",\n \"<p id=\\\"Par28\\\">An interesting finding was that positive affect was linked with callousness. Previous research found that callousness at baseline negatively predicted positive affect assessed six times per day across a 1-week period (Vize et al., ##REF##35402088##2022##); however, callousness was assessed with a personality measure only at baseline. One explanation for our findings may be due to the specific measure of positive affect assessing high valence constructs, including being active, inspired, and determined (Cooke et al., ##REF##36174167##2022##; Thompson, ##UREF##9##2007##). It may be the case that callousness also designates resolute individuals characterized by ambition. This notion goes against the DSM-5 “with Limited Prosocial Emotions” criterion specifying an absence of interest in school or work performance (American Psychiatric Association, ##UREF##0##2013##). However, the current uncaring subscale from the 12-item ICU is operationalized as a lack of concern for others (not activities). In addition, echoing Vize et al. (##REF##35402088##2022##), such traits may be strongly associated with extroversion, which is linked to heightened positive affect (Watson et al., ##REF##25751628##2015##)–although these findings are derived from adult samples. Future studies should consider a broader range of positive affect items to replicate and extend current findings, as well as further research on personality in adolescent samples.</p>\",\n \"<title>Strengths and Limitations</title>\",\n \"<p id=\\\"Par29\\\">Notable strengths of the present study include the naturalistic 30-day diary design, which advances understanding of CU traits under higher ecological validity relative to traditional longitudinal studies. In addition, we applied an analytic approach that disentangles within-person fluctuations from between-person differences. We also evaluated whether daily CU traits were associated with multiple forms of daily emotional and behavioral functioning. However, current findings should be interpreted within the context of some limitations. First, the current sample was primarily comprised of community adolescents from families with higher annual income than the provincial median income. Participants also endorsed lower levels of CU traits (<italic>M</italic> = 6.88; <italic>SD</italic> = 4.26) relative to other studies using the 12-item ICU scale. For example, Colins et al. (##REF##26493393##2016##) found higher levels of CU traits in a sample of justice-involved adolescent girls (<italic>M</italic> = 9.14; <italic>SD</italic> = 5.88). Thus, our results may not generalize to other populations, and as highlighted, it would be particularly important to examine daily CU traits in higher-risk or clinical samples.</p>\",\n \"<p id=\\\"Par30\\\">Second, all data are solely based on adolescent self-reports–we did not collect data from other informants (e.g., parents) on adolescent daily CU traits. Past research has revealed distinct associations between CU traits and conduct problems across adolescent- and parent-reports (e.g., Goulter & Moretti, ##REF##33825099##2021##). Meta-analytic work has also shown less discrepancies in cross-informant correspondence when informants are reporting on observable behaviors (e.g., externalizing vs. internalizing) or when the behaviors are measured within the same context (De Los Reyes et al., ##REF##25915035##2015##). Regarding CU traits, it may be more difficult for other informants to report on CU traits given these traits are not necessarily readily observable. </p>\",\n \"<p id=\\\"Par31\\\">Third, our sample was relatively small, although simulation studies have shown that sufficient power can be achieved for DSEM with a sample size of 100 and a minimum of 25 measured occasions (e.g., Schultzberg & Muthén, ##UREF##7##2018##). In particular, power at Level 2 is relatively small compared to conventional longitudinal studies, and thus, these between-person effects should be interpreted with caution. However, with over 2,000 observations at the daily level, power at Level 1 is sufficient for the current approach. Because of our sample size, we were not able to optimally investigate gender differences–an important direction for future research given most studies examining CU traits have relied predominantly on male samples.</p>\",\n \"<title>Future Directions and Implications</title>\",\n \"<p id=\\\"Par32\\\">By decomposing “trait” and “state”-like aspects of CU traits, current findings inform understanding of fluctuations in CU traits and lay a foundation for future intensive longitudinal designs to test critical questions in the field. For instance, adolescence represents a sensitive period marked by distinct change including within neural systems involved in social, emotional, and motivational processes (Dahl et al., ##REF##29469094##2018##). Because of this, studies testing daily dynamics among adolescents may be particularly well-suited to reveal important information regarding socio-behavioral functioning. Atypical levels of CU traits have also been observed in samples as young as preschool age (Kimonis et al., ##REF##26344015##2016##), and developmental models describe early childhood as the period in which the emergence of conscience and empathy typically occurs (Kochanska, ##UREF##4##1993##). Thus, future research should also investigate daily CU traits in younger samples. Ideally, CU traits would be examined at multiple developmental stages across multiple timescales, and measurement burst designs that combine micro and macro longitudinal methods could identify changes in short-term CU dynamics and their relations with long-term outcomes. Future research should also apply multiple measurement approaches (e.g., self-report, observational, psychophysiological) to better characterize the emotional components of daily experiences–a particularly important avenue in CU traits research given the distinct psychophysiological profiles among these samples (Fanti et al., ##REF##30797946##2019##).</p>\",\n \"<p id=\\\"Par33\\\">It will also be crucial to investigate the role of specific situational features in CU traits fluctuations and employ different intensive longitudinal methods to test these relations. For example, the present study employed a time-based sampling scheme prompting participants at 5 pm each day. By contrast, other research may use a variable-interval scheme generating prompts at random throughout the day to further enhance ecological validity (Russell & Gajos, ##REF##31997358##2020##). Future studies may also want to consider event contingent designs, which could be useful for examining negative interactions with parents, peers, or the broader environment. Theoretical and empirical works highlight the role of warm and supportive parent–child relationships in promoting empathy development and eliciting positive affect (Kochanska, ##UREF##4##1993##). Intensive longitudinal methods with parent–child dyads (e.g., Xu & Zheng, ##REF##35583795##2022##) have the potential to further advance understanding of familial relations in the development of CU traits. Particularly, parenting behaviors, including parental warmth and harsh discipline, have been linked to child CU traits (Goulter et al., ##REF##31166154##2020##; Waller et al., ##REF##23583974##2013##; Zheng et al., ##REF##27179538##2017##), and recent daily diary studies have demonstrated substantial daily fluctuations in parental warmth (Xu & Zheng, ##REF##36273075##2023##). Thus, examining how daily parenting behaviors are associated with CU traits may provide modifiable targets in daily family processes for future treatment efforts. In adolescence, research may want to examine the role of peer affiliation and interactions in daily CU traits fluctuations. Although the present study assessed negative affect, a greater understanding of the dynamics of stress and associated stressful experiences would also be particularly informative.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par34\">In this exploratory study, we observed meaningful fluctuations of CU traits at both item- and subscale-levels. These findings suggest that CU traits may be more susceptible to change and malleable than previously thought. In addition, callousness and uncaring subscales demonstrated distinct cross-day dynamics in relation to emotional and behavioral problems. Here, we have laid a foundation for future research focus applying intensive longitudinal methods to advance understanding of CU traits in daily life. Further functional information gained from these methods can potentially inform intervention efforts by offering modifiable targets situated in daily lives to promote personalized and just-in-time interventions. By conducting sampling in real-time in real-world situations, we can further inform personalized models of treatment targeting daily processes.\n</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par34\\\">In this exploratory study, we observed meaningful fluctuations of CU traits at both item- and subscale-levels. These findings suggest that CU traits may be more susceptible to change and malleable than previously thought. In addition, callousness and uncaring subscales demonstrated distinct cross-day dynamics in relation to emotional and behavioral problems. Here, we have laid a foundation for future research focus applying intensive longitudinal methods to advance understanding of CU traits in daily life. Further functional information gained from these methods can potentially inform intervention efforts by offering modifiable targets situated in daily lives to promote personalized and just-in-time interventions. By conducting sampling in real-time in real-world situations, we can further inform personalized models of treatment targeting daily processes.\\n</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p id=\"Par1\">Intensive longitudinal methods (e.g., daily diary) inform understanding of dynamic processes by parsing <italic>within-person</italic> state-like fluctuations from stable <italic>between-person</italic> trait-like differences. In this exploratory study, we investigated whether self-reported callous-unemotional (CU) traits (callousness, uncaring) demonstrated daily fluctuations, as well as whether daily CU traits were associated with multiple forms of daily emotional and behavioral functioning. A sample of 99 adolescents (55.8% female; <italic>M</italic><sub>age</sub> = 14.60 years) provided baseline information and completed a naturalistic 30-day diary reporting on CU traits, positive and negative affect, and emotional and conduct problems in their daily lives. Dynamic structural equation modeling revealed that many CU traits items showed within-person autoregressive and cross-lagged links; however, there was substantial between-person variation in within-person fluctuations across items. At the subscale level, cross-day associations were observed between callousness and uncaring, conduct problems and uncaring, positive affect and callousness, negative affect and emotional problems, and emotional problems and negative affect. By harnessing intensive longitudinal data, our findings provide preliminary state-level evidence of CU traits, as well as functional information with regards to CU traits and emotional and behavioral problems in daily life. We consider the implications of our findings in terms of informing future CU traits intensive longitudinal evaluations.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s10802-023-01077-6.</p>","<title>Keywords</title>"],"string":"[\n \"<p id=\\\"Par1\\\">Intensive longitudinal methods (e.g., daily diary) inform understanding of dynamic processes by parsing <italic>within-person</italic> state-like fluctuations from stable <italic>between-person</italic> trait-like differences. In this exploratory study, we investigated whether self-reported callous-unemotional (CU) traits (callousness, uncaring) demonstrated daily fluctuations, as well as whether daily CU traits were associated with multiple forms of daily emotional and behavioral functioning. A sample of 99 adolescents (55.8% female; <italic>M</italic><sub>age</sub> = 14.60 years) provided baseline information and completed a naturalistic 30-day diary reporting on CU traits, positive and negative affect, and emotional and conduct problems in their daily lives. Dynamic structural equation modeling revealed that many CU traits items showed within-person autoregressive and cross-lagged links; however, there was substantial between-person variation in within-person fluctuations across items. At the subscale level, cross-day associations were observed between callousness and uncaring, conduct problems and uncaring, positive affect and callousness, negative affect and emotional problems, and emotional problems and negative affect. By harnessing intensive longitudinal data, our findings provide preliminary state-level evidence of CU traits, as well as functional information with regards to CU traits and emotional and behavioral problems in daily life. We consider the implications of our findings in terms of informing future CU traits intensive longitudinal evaluations.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s10802-023-01077-6.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>We thank all the participating parents, adolescents, Elk Island and St. Albert public schools, and our research assistants. Study data were collected and managed using REDCap electronic data capture tools hosted and supported by the Women and Children’s Health Research Institute at the University of Alberta.</p>","<title>Author Contribution</title>","<p>Goulter, N: Conceptualization, Writing-Original Draft, Writing-Review/Editing; Cooke, EM: Formal Analysis, Writing-Original Draft, Writing-Review/Editing; Zheng, Y: Conceptualization, Funding Acquisition, Methodology, Writing-Review/Editing.</p>","<title>Funding</title>","<p>This research was supported partly with funding from the Social Sciences and Humanities Research Council (IDG 430-2018-00317 and 409-2020-00080) and Natural Sciences and Engineering Research Council (RGPIN-2020-04458 and DGECR-2020-00077) of Canada. EC was supported by a Mitacs Accelerate grant (IT18227) awarded to YZ.</p>","<title>Data Availability</title>","<p>Data are available by emailing the last author. Code and output are available on the Open Science Framework: <ext-link ext-link-type=\"uri\" xlink:href=\"https://osf.io/n2rdf/?view_only=357ec650bfac4a63905e7ed34efd9255\">https://osf.io/n2rdf/?view_only=357ec650bfac4a63905e7ed34efd9255</ext-link>.</p>","<title>Compliance with Ethical Standards</title>","<title>Ethical Approval</title>","<p id=\"Par35\">The study was approved by the institutional research ethics committee and was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.</p>","<title>Informed Consent</title>","<p id=\"Par36\">Informed consent and assent for study participation and research publication was obtained from all individual participants included in the study.</p>","<title>Conflicts of Interest</title>","<p id=\"Par37\">The authors have no relevant or non-financial interests to disclose.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>We thank all the participating parents, adolescents, Elk Island and St. Albert public schools, and our research assistants. Study data were collected and managed using REDCap electronic data capture tools hosted and supported by the Women and Children’s Health Research Institute at the University of Alberta.</p>\",\n \"<title>Author Contribution</title>\",\n \"<p>Goulter, N: Conceptualization, Writing-Original Draft, Writing-Review/Editing; Cooke, EM: Formal Analysis, Writing-Original Draft, Writing-Review/Editing; Zheng, Y: Conceptualization, Funding Acquisition, Methodology, Writing-Review/Editing.</p>\",\n \"<title>Funding</title>\",\n \"<p>This research was supported partly with funding from the Social Sciences and Humanities Research Council (IDG 430-2018-00317 and 409-2020-00080) and Natural Sciences and Engineering Research Council (RGPIN-2020-04458 and DGECR-2020-00077) of Canada. EC was supported by a Mitacs Accelerate grant (IT18227) awarded to YZ.</p>\",\n \"<title>Data Availability</title>\",\n \"<p>Data are available by emailing the last author. Code and output are available on the Open Science Framework: <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://osf.io/n2rdf/?view_only=357ec650bfac4a63905e7ed34efd9255\\\">https://osf.io/n2rdf/?view_only=357ec650bfac4a63905e7ed34efd9255</ext-link>.</p>\",\n \"<title>Compliance with Ethical Standards</title>\",\n \"<title>Ethical Approval</title>\",\n \"<p id=\\\"Par35\\\">The study was approved by the institutional research ethics committee and was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.</p>\",\n \"<title>Informed Consent</title>\",\n \"<p id=\\\"Par36\\\">Informed consent and assent for study participation and research publication was obtained from all individual participants included in the study.</p>\",\n \"<title>Conflicts of Interest</title>\",\n \"<p id=\\\"Par37\\\">The authors have no relevant or non-financial interests to disclose.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Schematic of DSEM. Note. Observed items are decomposed into time-varying within-level (w) and time-invariant between-level (b) components. CAL = callousness, UNC = uncaring, POS = positive affect, NEG = negative affect, CON = conduct problems, EMO = emotional problems</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>DSEM Standardized Estimates for Callous-Unemotional Traits Items Within-Person Autoregressive (curved arrows) and Cross-Lagged Effects (straight arrows). Note. Only significant effects based on 95% credible intervals are shown. CU = callous-unemotional</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>DSEM Standardized Estimates for Callous-Unemotional Traits Subscales, Positive Affect, Negative Affect, Conduct Problems, and Emotional Problems Within-Person Autoregressive (curved arrows) and Cross-Lagged Effects (straight arrows). Note. Only significant effects based on 95% credible intervals are shown. CAL = callousness, UNC = uncaring, POS = positive affect, NEG = negative affect, CON = conduct problems, EMO = emotional problems</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Schematic of DSEM. Note. Observed items are decomposed into time-varying within-level (w) and time-invariant between-level (b) components. CAL = callousness, UNC = uncaring, POS = positive affect, NEG = negative affect, CON = conduct problems, EMO = emotional problems</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>DSEM Standardized Estimates for Callous-Unemotional Traits Items Within-Person Autoregressive (curved arrows) and Cross-Lagged Effects (straight arrows). Note. Only significant effects based on 95% credible intervals are shown. CU = callous-unemotional</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>DSEM Standardized Estimates for Callous-Unemotional Traits Subscales, Positive Affect, Negative Affect, Conduct Problems, and Emotional Problems Within-Person Autoregressive (curved arrows) and Cross-Lagged Effects (straight arrows). Note. Only significant effects based on 95% credible intervals are shown. CAL = callousness, UNC = uncaring, POS = positive affect, NEG = negative affect, CON = conduct problems, EMO = emotional problems</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Callous-Unemotional Traits Items and Subscales</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"><italic>Item Description</italic></th><th align=\"left\"><italic>Subscale</italic></th></tr></thead><tbody><tr><td align=\"left\">CU1: Does not care who I hurt to get what I want</td><td align=\"left\">Callousness</td></tr><tr><td align=\"left\">CU2: Feels bad or guilty when I have done something wrong (r)</td><td align=\"left\">Uncaring</td></tr><tr><td align=\"left\">CU3: Does not show emotions</td><td align=\"left\">Unemotional</td></tr><tr><td align=\"left\">CU4: Concerned about the feelings of others (r)</td><td align=\"left\">Uncaring</td></tr><tr><td align=\"left\">CU5: Does not care if I am in trouble</td><td align=\"left\">Callousness</td></tr><tr><td align=\"left\">CU6: Does not care about doing things well</td><td align=\"left\">Callousness</td></tr><tr><td align=\"left\">CU7: Seems very cold and uncaring</td><td align=\"left\">Callousness</td></tr><tr><td align=\"left\">CU8: Apologizes to persons I have hurt (r)</td><td align=\"left\">Uncaring</td></tr><tr><td align=\"left\">CU9: Tries not to hurt others’ feelings (r)</td><td align=\"left\">Uncaring</td></tr><tr><td align=\"left\">CU10: Shows no remorse when I have done something wrong</td><td align=\"left\">Callousness</td></tr><tr><td align=\"left\">CU11: Feelings of others are unimportant</td><td align=\"left\">Callousness</td></tr><tr><td align=\"left\">CU12: Does things to make others feel good (r)</td><td align=\"left\">Uncaring</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Within-Person Same Day Residual Correlations and Between-Person Correlations for Callous-Unemotional Traits Items</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"><bold>Item</bold></th><th align=\"left\"><bold>CU1</bold></th><th align=\"left\"><bold>CU2</bold></th><th align=\"left\"><bold>CU3</bold></th><th align=\"left\"><bold>CU4</bold></th><th align=\"left\"><bold>CU5</bold></th><th align=\"left\"><bold>CU6</bold></th><th align=\"left\"><bold>CU7</bold></th><th align=\"left\"><bold>CU8</bold></th><th align=\"left\"><bold>CU9</bold></th><th align=\"left\"><bold>CU10</bold></th><th align=\"left\"><bold>CU11</bold></th><th align=\"left\"><bold>CU12</bold></th></tr></thead><tbody><tr><td align=\"left\">CU1</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.01</td><td char=\".\" align=\"char\">0.03</td><td char=\".\" align=\"char\">0.13<sup>***</sup></td><td char=\".\" align=\"char\">0.14<sup>***</sup></td><td char=\".\" align=\"char\">0.21<sup>***</sup></td><td char=\".\" align=\"char\">0.12<sup>***</sup></td><td char=\".\" align=\"char\">0.08<sup>***</sup></td><td char=\".\" align=\"char\">0.09<sup>***</sup></td><td char=\".\" align=\"char\">0.10<sup>***</sup></td><td char=\".\" align=\"char\">0.07<sup>***</sup></td><td char=\".\" align=\"char\">0.12<sup>***</sup></td></tr><tr><td align=\"left\">CU2</td><td char=\".\" align=\"char\">0.22<sup>*</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">-0.08<sup>***</sup></td><td char=\".\" align=\"char\">0.24<sup>***</sup></td><td char=\".\" align=\"char\">0.12<sup>***</sup></td><td char=\".\" align=\"char\">0.04</td><td char=\".\" align=\"char\">-0.04<sup>*</sup></td><td char=\".\" align=\"char\">0.17<sup>***</sup></td><td char=\".\" align=\"char\">0.18<sup>***</sup></td><td char=\".\" align=\"char\">0.02</td><td char=\".\" align=\"char\">0.06<sup>**</sup></td><td char=\".\" align=\"char\">0.15<sup>***</sup></td></tr><tr><td align=\"left\">CU3</td><td char=\".\" align=\"char\">-0.01</td><td char=\".\" align=\"char\">0.46<sup>***</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">-0.20<sup>***</sup></td><td char=\".\" align=\"char\">0.04<sup>*</sup></td><td char=\".\" align=\"char\">0.05<sup>*</sup></td><td char=\".\" align=\"char\">0.10<sup>***</sup></td><td char=\".\" align=\"char\">-0.12<sup>***</sup></td><td char=\".\" align=\"char\">-0.11<sup>***</sup></td><td char=\".\" align=\"char\">0.04<sup>*</sup></td><td char=\".\" align=\"char\">0.03</td><td char=\".\" align=\"char\">-0.06<sup>**</sup></td></tr><tr><td align=\"left\">CU4</td><td char=\".\" align=\"char\">0.52<sup>***</sup></td><td char=\".\" align=\"char\">0.23<sup>*</sup></td><td char=\".\" align=\"char\">-0.28<sup>*</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.07<sup>**</sup></td><td char=\".\" align=\"char\">0.07<sup>**</sup></td><td char=\".\" align=\"char\">0.08<sup>***</sup></td><td char=\".\" align=\"char\">0.37<sup>***</sup></td><td char=\".\" align=\"char\">0.38<sup>***</sup></td><td char=\".\" align=\"char\">0.04</td><td char=\".\" align=\"char\">0.10<sup>***</sup></td><td char=\".\" align=\"char\">0.34<sup>***</sup></td></tr><tr><td align=\"left\">CU5</td><td char=\".\" align=\"char\">0.75<sup>***</sup></td><td char=\".\" align=\"char\">0.17</td><td char=\".\" align=\"char\">-0.04</td><td char=\".\" align=\"char\">0.57<sup>***</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.31<sup>***</sup></td><td char=\".\" align=\"char\">0.09<sup>***</sup></td><td char=\".\" align=\"char\">0.04<sup>*</sup></td><td char=\".\" align=\"char\">0.06<sup>**</sup></td><td char=\".\" align=\"char\">0.09<sup>***</sup></td><td char=\".\" align=\"char\">0.07<sup>**</sup></td><td char=\".\" align=\"char\">0.05<sup>*</sup></td></tr><tr><td align=\"left\">CU6</td><td char=\".\" align=\"char\">0.73<sup>***</sup></td><td char=\".\" align=\"char\">0.13</td><td char=\".\" align=\"char\">0.03</td><td char=\".\" align=\"char\">0.49<sup>***</sup></td><td char=\".\" align=\"char\">0.87<sup>***</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.19<sup>***</sup></td><td char=\".\" align=\"char\">0.07<sup>**</sup></td><td char=\".\" align=\"char\">0.05<sup>*</sup></td><td char=\".\" align=\"char\">0.11<sup>***</sup></td><td char=\".\" align=\"char\">0.10<sup>***</sup></td><td char=\".\" align=\"char\">0.09<sup>***</sup></td></tr><tr><td align=\"left\">CU7</td><td char=\".\" align=\"char\">0.35<sup>**</sup></td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">0.34<sup>**</sup></td><td char=\".\" align=\"char\">0.40<sup>**</sup></td><td char=\".\" align=\"char\">0.42<sup>***</sup></td><td char=\".\" align=\"char\">0.61<sup>***</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.05<sup>*</sup></td><td char=\".\" align=\"char\">0.07<sup>**</sup></td><td char=\".\" align=\"char\">0.11<sup>***</sup></td><td char=\".\" align=\"char\">0.09<sup>***</sup></td><td char=\".\" align=\"char\">0.10<sup>***</sup></td></tr><tr><td align=\"left\">CU8</td><td char=\".\" align=\"char\">0.50<sup>***</sup></td><td char=\".\" align=\"char\">0.18</td><td char=\".\" align=\"char\">-0.23<sup>*</sup></td><td char=\".\" align=\"char\">0.84<sup>***</sup></td><td char=\".\" align=\"char\">0.56<sup>***</sup></td><td char=\".\" align=\"char\">0.48<sup>***</sup></td><td char=\".\" align=\"char\">0.34<sup>**</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.54<sup>***</sup></td><td char=\".\" align=\"char\">0.07<sup>**</sup></td><td char=\".\" align=\"char\">0.08<sup>***</sup></td><td char=\".\" align=\"char\">0.34<sup>***</sup></td></tr><tr><td align=\"left\">CU9</td><td char=\".\" align=\"char\">0.53<sup>***</sup></td><td char=\".\" align=\"char\">0.28<sup>*</sup></td><td char=\".\" align=\"char\">-0.19</td><td char=\".\" align=\"char\">0.93<sup>***</sup></td><td char=\".\" align=\"char\">0.56<sup>***</sup></td><td char=\".\" align=\"char\">0.51<sup>***</sup></td><td char=\".\" align=\"char\">0.40<sup>***</sup></td><td char=\".\" align=\"char\">0.82<sup>***</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.00</td><td char=\".\" align=\"char\">0.08<sup>***</sup></td><td char=\".\" align=\"char\">0.34<sup>***</sup></td></tr><tr><td align=\"left\">CU10</td><td char=\".\" align=\"char\">0.67<sup>***</sup></td><td char=\".\" align=\"char\">0.19</td><td char=\".\" align=\"char\">-0.05</td><td char=\".\" align=\"char\">0.61<sup>***</sup></td><td char=\".\" align=\"char\">0.72<sup>***</sup></td><td char=\".\" align=\"char\">0.75<sup>***</sup></td><td char=\".\" align=\"char\">0.48<sup>***</sup></td><td char=\".\" align=\"char\">0.48<sup>***</sup></td><td char=\".\" align=\"char\">0.62<sup>***</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.11<sup>***</sup></td><td char=\".\" align=\"char\">0.05<sup>*</sup></td></tr><tr><td align=\"left\">CU11</td><td char=\".\" align=\"char\">0.76<sup>***</sup></td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">0.01</td><td char=\".\" align=\"char\">0.60<sup>***</sup></td><td char=\".\" align=\"char\">0.64<sup>***</sup></td><td char=\".\" align=\"char\">0.73<sup>***</sup></td><td char=\".\" align=\"char\">0.54<sup>***</sup></td><td char=\".\" align=\"char\">0.47<sup>***</sup></td><td char=\".\" align=\"char\">0.54<sup>***</sup></td><td char=\".\" align=\"char\">0.81<sup>***</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.08<sup>**</sup></td></tr><tr><td align=\"left\">CU12</td><td char=\".\" align=\"char\">0.39<sup>**</sup></td><td char=\".\" align=\"char\">0.15</td><td char=\".\" align=\"char\">-0.29<sup>**</sup></td><td char=\".\" align=\"char\">0.90<sup>***</sup></td><td char=\".\" align=\"char\">0.44<sup>***</sup></td><td char=\".\" align=\"char\">0.41<sup>***</sup></td><td char=\".\" align=\"char\">0.36<sup>**</sup></td><td char=\".\" align=\"char\">0.79<sup>***</sup></td><td char=\".\" align=\"char\">0.84<sup>***</sup></td><td char=\".\" align=\"char\">0.49<sup>***</sup></td><td char=\".\" align=\"char\">0.40<sup>***</sup></td><td char=\".\" align=\"char\">–</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\"><bold><italic>M</italic></bold></td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">1.00</td><td char=\".\" align=\"char\">1.50</td><td char=\".\" align=\"char\">0.53</td><td char=\".\" align=\"char\">0.29</td><td char=\".\" align=\"char\">0.24</td><td char=\".\" align=\"char\">0.42</td><td char=\".\" align=\"char\">0.63</td><td char=\".\" align=\"char\">0.51</td><td char=\".\" align=\"char\">0.25</td><td char=\".\" align=\"char\">0.17</td><td char=\".\" align=\"char\">0.66</td></tr><tr><td align=\"left\"><bold><italic>σ</italic></bold><sup><bold>2</bold></sup></td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">1.19</td><td char=\".\" align=\"char\">1.26</td><td char=\".\" align=\"char\">0.37</td><td char=\".\" align=\"char\">0.26</td><td char=\".\" align=\"char\">0.21</td><td char=\".\" align=\"char\">0.36</td><td char=\".\" align=\"char\">0.46</td><td char=\".\" align=\"char\">0.38</td><td char=\".\" align=\"char\">0.17</td><td char=\".\" align=\"char\">0.13</td><td char=\".\" align=\"char\">0.52</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Within-Person Same Day Residual Correlations and Between-Person Correlations for Callous-Unemotional Traits Subscales, Positive Affect, Negative Affect, Conduct Problems, and Emotional Problems</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"><bold>Item</bold></th><th align=\"left\"><bold>1</bold></th><th align=\"left\"><bold>2</bold></th><th align=\"left\"><bold>3</bold></th><th align=\"left\"><bold>4</bold></th><th align=\"left\"><bold>5</bold></th><th align=\"left\"><bold>6</bold></th></tr></thead><tbody><tr><td align=\"left\">1. Callousness</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.17<sup>***</sup></td><td char=\".\" align=\"char\">-0.03</td><td char=\".\" align=\"char\">0.21<sup>***</sup></td><td char=\".\" align=\"char\">0.22<sup>***</sup></td><td char=\".\" align=\"char\">0.13<sup>***</sup></td></tr><tr><td align=\"left\">2. Uncaring</td><td char=\".\" align=\"char\">0.58<sup>***</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">-0.10<sup>***</sup></td><td char=\".\" align=\"char\">0.09<sup>***</sup></td><td char=\".\" align=\"char\">0.21<sup>***</sup></td><td char=\".\" align=\"char\">0.03</td></tr><tr><td align=\"left\">3. Positive Affect</td><td char=\".\" align=\"char\">-0.24<sup>*</sup></td><td char=\".\" align=\"char\">-0.36<sup>**</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.06<sup>**</sup></td><td char=\".\" align=\"char\">-0.06<sup>**</sup></td><td char=\".\" align=\"char\">-0.09<sup>***</sup></td></tr><tr><td align=\"left\">4. Negative Affect</td><td char=\".\" align=\"char\">0.52<sup>***</sup></td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\">-0.16</td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.28<sup>***</sup></td><td char=\".\" align=\"char\">0.48<sup>***</sup></td></tr><tr><td align=\"left\">5. Conduct Problems</td><td char=\".\" align=\"char\">0.72<sup>***</sup></td><td char=\".\" align=\"char\">0.59<sup>***</sup></td><td char=\".\" align=\"char\">-0.29<sup>**</sup></td><td char=\".\" align=\"char\">0.52<sup>***</sup></td><td char=\".\" align=\"char\">–</td><td char=\".\" align=\"char\">0.17<sup>***</sup></td></tr><tr><td align=\"left\">6. Emotional Problems</td><td char=\".\" align=\"char\">0.41<sup>***</sup></td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">-0.38<sup>***</sup></td><td char=\".\" align=\"char\">0.88<sup>***</sup></td><td char=\".\" align=\"char\">0.47<sup>***</sup></td><td char=\".\" align=\"char\">–</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\"><bold><italic>M</italic></bold></td><td char=\".\" align=\"char\">0.25</td><td char=\".\" align=\"char\">0.66</td><td char=\".\" align=\"char\">2.67</td><td char=\".\" align=\"char\">1.61</td><td char=\".\" align=\"char\">0.18</td><td char=\".\" align=\"char\">0.46</td></tr><tr><td align=\"left\"><bold><italic>σ</italic></bold><sup><bold>2</bold></sup></td><td char=\".\" align=\"char\">0.12</td><td char=\".\" align=\"char\">0.29</td><td char=\".\" align=\"char\">0.74</td><td char=\".\" align=\"char\">0.46</td><td char=\".\" align=\"char\">0.05</td><td char=\".\" align=\"char\">0.24</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Callous-Unemotional Traits Items and Subscales</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"><italic>Item Description</italic></th><th align=\\\"left\\\"><italic>Subscale</italic></th></tr></thead><tbody><tr><td align=\\\"left\\\">CU1: Does not care who I hurt to get what I want</td><td align=\\\"left\\\">Callousness</td></tr><tr><td align=\\\"left\\\">CU2: Feels bad or guilty when I have done something wrong (r)</td><td align=\\\"left\\\">Uncaring</td></tr><tr><td align=\\\"left\\\">CU3: Does not show emotions</td><td align=\\\"left\\\">Unemotional</td></tr><tr><td align=\\\"left\\\">CU4: Concerned about the feelings of others (r)</td><td align=\\\"left\\\">Uncaring</td></tr><tr><td align=\\\"left\\\">CU5: Does not care if I am in trouble</td><td align=\\\"left\\\">Callousness</td></tr><tr><td align=\\\"left\\\">CU6: Does not care about doing things well</td><td align=\\\"left\\\">Callousness</td></tr><tr><td align=\\\"left\\\">CU7: Seems very cold and uncaring</td><td align=\\\"left\\\">Callousness</td></tr><tr><td align=\\\"left\\\">CU8: Apologizes to persons I have hurt (r)</td><td align=\\\"left\\\">Uncaring</td></tr><tr><td align=\\\"left\\\">CU9: Tries not to hurt others’ feelings (r)</td><td align=\\\"left\\\">Uncaring</td></tr><tr><td align=\\\"left\\\">CU10: Shows no remorse when I have done something wrong</td><td align=\\\"left\\\">Callousness</td></tr><tr><td align=\\\"left\\\">CU11: Feelings of others are unimportant</td><td align=\\\"left\\\">Callousness</td></tr><tr><td align=\\\"left\\\">CU12: Does things to make others feel good (r)</td><td align=\\\"left\\\">Uncaring</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Within-Person Same Day Residual Correlations and Between-Person Correlations for Callous-Unemotional Traits Items</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"><bold>Item</bold></th><th align=\\\"left\\\"><bold>CU1</bold></th><th align=\\\"left\\\"><bold>CU2</bold></th><th align=\\\"left\\\"><bold>CU3</bold></th><th align=\\\"left\\\"><bold>CU4</bold></th><th align=\\\"left\\\"><bold>CU5</bold></th><th align=\\\"left\\\"><bold>CU6</bold></th><th align=\\\"left\\\"><bold>CU7</bold></th><th align=\\\"left\\\"><bold>CU8</bold></th><th align=\\\"left\\\"><bold>CU9</bold></th><th align=\\\"left\\\"><bold>CU10</bold></th><th align=\\\"left\\\"><bold>CU11</bold></th><th align=\\\"left\\\"><bold>CU12</bold></th></tr></thead><tbody><tr><td align=\\\"left\\\">CU1</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.01</td><td char=\\\".\\\" align=\\\"char\\\">0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.13<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.14<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.21<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.12<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.08<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.09<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.10<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.07<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.12<sup>***</sup></td></tr><tr><td align=\\\"left\\\">CU2</td><td char=\\\".\\\" align=\\\"char\\\">0.22<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">-0.08<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.24<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.12<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.04</td><td char=\\\".\\\" align=\\\"char\\\">-0.04<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.17<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.18<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.02</td><td char=\\\".\\\" align=\\\"char\\\">0.06<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.15<sup>***</sup></td></tr><tr><td align=\\\"left\\\">CU3</td><td char=\\\".\\\" align=\\\"char\\\">-0.01</td><td char=\\\".\\\" align=\\\"char\\\">0.46<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">-0.20<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.04<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.05<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.10<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">-0.12<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">-0.11<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.04<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.03</td><td char=\\\".\\\" align=\\\"char\\\">-0.06<sup>**</sup></td></tr><tr><td align=\\\"left\\\">CU4</td><td char=\\\".\\\" align=\\\"char\\\">0.52<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.23<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">-0.28<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.07<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.07<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.08<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.37<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.38<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.10<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.34<sup>***</sup></td></tr><tr><td align=\\\"left\\\">CU5</td><td char=\\\".\\\" align=\\\"char\\\">0.75<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.17</td><td char=\\\".\\\" align=\\\"char\\\">-0.04</td><td char=\\\".\\\" align=\\\"char\\\">0.57<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.31<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.09<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.04<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.06<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.09<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.07<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.05<sup>*</sup></td></tr><tr><td align=\\\"left\\\">CU6</td><td char=\\\".\\\" align=\\\"char\\\">0.73<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.13</td><td char=\\\".\\\" align=\\\"char\\\">0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.49<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.87<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.19<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.07<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.05<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.11<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.10<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.09<sup>***</sup></td></tr><tr><td align=\\\"left\\\">CU7</td><td char=\\\".\\\" align=\\\"char\\\">0.35<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.09</td><td char=\\\".\\\" align=\\\"char\\\">0.34<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.40<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.42<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.61<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.05<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.07<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.11<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.09<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.10<sup>***</sup></td></tr><tr><td align=\\\"left\\\">CU8</td><td char=\\\".\\\" align=\\\"char\\\">0.50<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.18</td><td char=\\\".\\\" align=\\\"char\\\">-0.23<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.84<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.56<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.48<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.34<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.54<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.07<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.08<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.34<sup>***</sup></td></tr><tr><td align=\\\"left\\\">CU9</td><td char=\\\".\\\" align=\\\"char\\\">0.53<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.28<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">-0.19</td><td char=\\\".\\\" align=\\\"char\\\">0.93<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.56<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.51<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.40<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.82<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.00</td><td char=\\\".\\\" align=\\\"char\\\">0.08<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.34<sup>***</sup></td></tr><tr><td align=\\\"left\\\">CU10</td><td char=\\\".\\\" align=\\\"char\\\">0.67<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.19</td><td char=\\\".\\\" align=\\\"char\\\">-0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.61<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.72<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.75<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.48<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.48<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.62<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.11<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.05<sup>*</sup></td></tr><tr><td align=\\\"left\\\">CU11</td><td char=\\\".\\\" align=\\\"char\\\">0.76<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.12</td><td char=\\\".\\\" align=\\\"char\\\">0.01</td><td char=\\\".\\\" align=\\\"char\\\">0.60<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.64<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.73<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.54<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.47<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.54<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.81<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.08<sup>**</sup></td></tr><tr><td align=\\\"left\\\">CU12</td><td char=\\\".\\\" align=\\\"char\\\">0.39<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.15</td><td char=\\\".\\\" align=\\\"char\\\">-0.29<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.90<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.44<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.41<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.36<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.79<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.84<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.49<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.40<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td></tr></tbody></table><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr><td align=\\\"left\\\"><bold><italic>M</italic></bold></td><td char=\\\".\\\" align=\\\"char\\\">0.12</td><td char=\\\".\\\" align=\\\"char\\\">1.00</td><td char=\\\".\\\" align=\\\"char\\\">1.50</td><td char=\\\".\\\" align=\\\"char\\\">0.53</td><td char=\\\".\\\" align=\\\"char\\\">0.29</td><td char=\\\".\\\" align=\\\"char\\\">0.24</td><td char=\\\".\\\" align=\\\"char\\\">0.42</td><td char=\\\".\\\" align=\\\"char\\\">0.63</td><td char=\\\".\\\" align=\\\"char\\\">0.51</td><td char=\\\".\\\" align=\\\"char\\\">0.25</td><td char=\\\".\\\" align=\\\"char\\\">0.17</td><td char=\\\".\\\" align=\\\"char\\\">0.66</td></tr><tr><td align=\\\"left\\\"><bold><italic>σ</italic></bold><sup><bold>2</bold></sup></td><td char=\\\".\\\" align=\\\"char\\\">0.09</td><td char=\\\".\\\" align=\\\"char\\\">1.19</td><td char=\\\".\\\" align=\\\"char\\\">1.26</td><td char=\\\".\\\" align=\\\"char\\\">0.37</td><td char=\\\".\\\" align=\\\"char\\\">0.26</td><td char=\\\".\\\" align=\\\"char\\\">0.21</td><td char=\\\".\\\" align=\\\"char\\\">0.36</td><td char=\\\".\\\" align=\\\"char\\\">0.46</td><td char=\\\".\\\" align=\\\"char\\\">0.38</td><td char=\\\".\\\" align=\\\"char\\\">0.17</td><td char=\\\".\\\" align=\\\"char\\\">0.13</td><td char=\\\".\\\" align=\\\"char\\\">0.52</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Within-Person Same Day Residual Correlations and Between-Person Correlations for Callous-Unemotional Traits Subscales, Positive Affect, Negative Affect, Conduct Problems, and Emotional Problems</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"><bold>Item</bold></th><th align=\\\"left\\\"><bold>1</bold></th><th align=\\\"left\\\"><bold>2</bold></th><th align=\\\"left\\\"><bold>3</bold></th><th align=\\\"left\\\"><bold>4</bold></th><th align=\\\"left\\\"><bold>5</bold></th><th align=\\\"left\\\"><bold>6</bold></th></tr></thead><tbody><tr><td align=\\\"left\\\">1. Callousness</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.17<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">-0.03</td><td char=\\\".\\\" align=\\\"char\\\">0.21<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.22<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.13<sup>***</sup></td></tr><tr><td align=\\\"left\\\">2. Uncaring</td><td char=\\\".\\\" align=\\\"char\\\">0.58<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">-0.10<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.09<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.21<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.03</td></tr><tr><td align=\\\"left\\\">3. Positive Affect</td><td char=\\\".\\\" align=\\\"char\\\">-0.24<sup>*</sup></td><td char=\\\".\\\" align=\\\"char\\\">-0.36<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.06<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">-0.06<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">-0.09<sup>***</sup></td></tr><tr><td align=\\\"left\\\">4. Negative Affect</td><td char=\\\".\\\" align=\\\"char\\\">0.52<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.08</td><td char=\\\".\\\" align=\\\"char\\\">-0.16</td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.28<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.48<sup>***</sup></td></tr><tr><td align=\\\"left\\\">5. Conduct Problems</td><td char=\\\".\\\" align=\\\"char\\\">0.72<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.59<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">-0.29<sup>**</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.52<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td><td char=\\\".\\\" align=\\\"char\\\">0.17<sup>***</sup></td></tr><tr><td align=\\\"left\\\">6. Emotional Problems</td><td char=\\\".\\\" align=\\\"char\\\">0.41<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.09</td><td char=\\\".\\\" align=\\\"char\\\">-0.38<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.88<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">0.47<sup>***</sup></td><td char=\\\".\\\" align=\\\"char\\\">–</td></tr></tbody></table><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr><td align=\\\"left\\\"><bold><italic>M</italic></bold></td><td char=\\\".\\\" align=\\\"char\\\">0.25</td><td char=\\\".\\\" align=\\\"char\\\">0.66</td><td char=\\\".\\\" align=\\\"char\\\">2.67</td><td char=\\\".\\\" align=\\\"char\\\">1.61</td><td char=\\\".\\\" align=\\\"char\\\">0.18</td><td char=\\\".\\\" align=\\\"char\\\">0.46</td></tr><tr><td align=\\\"left\\\"><bold><italic>σ</italic></bold><sup><bold>2</bold></sup></td><td char=\\\".\\\" align=\\\"char\\\">0.12</td><td char=\\\".\\\" align=\\\"char\\\">0.29</td><td char=\\\".\\\" align=\\\"char\\\">0.74</td><td char=\\\".\\\" align=\\\"char\\\">0.46</td><td char=\\\".\\\" align=\\\"char\\\">0.05</td><td char=\\\".\\\" align=\\\"char\\\">0.24</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p><italic>r</italic> reverse coded items</p></table-wrap-foot>","<table-wrap-foot><p>Correlations estimated using the Bayes estimator. Within-day residual correlations are shown above the diagonal. Between-level correlations, means (<italic>M</italic>), and variances (σ<sup>2</sup><bold>)</bold> are shown below the diagonal. Items 2, 4, 8, 9, 12 are reverse coded</p><p>*<italic>p</italic> ≤ 0.05, **<italic>p</italic> ≤ 0.01, ***<italic>p</italic> ≤ 0.001</p></table-wrap-foot>","<table-wrap-foot><p>Correlations estimated using the Bayes estimator. Within-day residual correlations are shown above the diagonal. Between-level correlations, means (<italic>M</italic>), and variances (σ<sup>2</sup><bold>)</bold> are shown below the diagonal</p><p>*<italic>p</italic> ≤ 0.05, **<italic>p</italic> ≤ 0.01, ***<italic>p</italic> ≤ 0.001</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p><italic>r</italic> reverse coded items</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>Correlations estimated using the Bayes estimator. Within-day residual correlations are shown above the diagonal. Between-level correlations, means (<italic>M</italic>), and variances (σ<sup>2</sup><bold>)</bold> are shown below the diagonal. Items 2, 4, 8, 9, 12 are reverse coded</p><p>*<italic>p</italic> ≤ 0.05, **<italic>p</italic> ≤ 0.01, ***<italic>p</italic> ≤ 0.001</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>Correlations estimated using the Bayes estimator. Within-day residual correlations are shown above the diagonal. Between-level correlations, means (<italic>M</italic>), and variances (σ<sup>2</sup><bold>)</bold> are shown below the diagonal</p><p>*<italic>p</italic> ≤ 0.05, **<italic>p</italic> ≤ 0.01, ***<italic>p</italic> ≤ 0.001</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"10802_2023_1077_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"10802_2023_1077_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"10802_2023_1077_Fig3_HTML\" id=\"MO3\"/>"],"string":"[\n \"<graphic xlink:href=\\\"10802_2023_1077_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"10802_2023_1077_Fig2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic xlink:href=\\\"10802_2023_1077_Fig3_HTML\\\" id=\\\"MO3\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"10802_2023_1077_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 306 KB)</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"10802_2023_1077_MOESM1_ESM.pdf\\\"><caption><p>Supplementary file1 (PDF 306 KB)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"collab\": [\"American Psychiatric Association\"], \"source\": [\"Diagnostic and statistical manual of mental disorders\"], \"year\": [\"2013\"], \"edition\": [\"5\"], \"publisher-name\": [\"American Psychiatric Publishing\"]}, {\"surname\": [\"Asparouhov\", \"Hamaker\", \"Muth\\u00e9n\"], \"given-names\": [\"T\", \"EL\", \"B\"], \"article-title\": [\"Dynamic structural equation models\"], \"source\": [\"Structural Equation Modeling: A Multidisciplinary Journal\"], \"year\": [\"2018\"], \"volume\": [\"25\"], \"issue\": [\"3\"], \"fpage\": [\"359\"], \"lpage\": [\"388\"], \"pub-id\": [\"10.1080/10705511.2017.1406803\"]}, {\"surname\": [\"De Ridder\", \"Pihet\", \"Suter\", \"Caldara\"], \"given-names\": [\"J\", \"S\", \"M\", \"R\"], \"article-title\": [\"Empathy in institutionalized adolescents with callous-unemotional traits: an ecological momentary assessment study of emotion recognition\"], \"source\": [\"Criminal Justice and Behavior\"], \"year\": [\"2016\"], \"volume\": [\"43\"], \"issue\": [\"5\"], \"fpage\": [\"653\"], \"lpage\": [\"669\"], \"pub-id\": [\"10.1177/0093854815618431\"]}, {\"mixed-citation\": [\"Kaurin, A., Do, Q., Ladouceur, C. D., Silk, J., & Wright, A. G. (2022). Daily interpersonal and affective manifestations of maladaptive personality in adolescent girls. \"], \"italic\": [\"PsyArXiv,\"]}, {\"surname\": [\"Kochanska\"], \"given-names\": [\"G\"], \"article-title\": [\"Toward a synthesis of parental socialization and child temperament in early development of conscience\"], \"source\": [\"Child Development\"], \"year\": [\"1993\"], \"volume\": [\"64\"], \"issue\": [\"2\"], \"fpage\": [\"325\"], \"lpage\": [\"347\"], \"pub-id\": [\"10.1111/j.1467-8624.1993.tb02913.x\"]}, {\"surname\": [\"Muth\\u00e9n\", \"Muth\\u00e9n\"], \"given-names\": [\"BO\", \"LK\"], \"source\": [\"Mplus version 8.6: user\\u2019s guide\"], \"year\": [\"2021\"], \"publisher-name\": [\"Authors\"]}, {\"surname\": [\"Prasad\", \"Kimonis\"], \"given-names\": [\"AH\", \"ER\"], \"article-title\": [\"Effects of the \\u201climited prosocial emotions\\u201d specifier for conduct disorder on juror perceptions of juvenile offenders\"], \"source\": [\"Criminal Justice and Behavior\"], \"year\": [\"2018\"], \"volume\": [\"45\"], \"issue\": [\"10\"], \"fpage\": [\"1547\"], \"lpage\": [\"1564\"], \"pub-id\": [\"10.1177/0093854818774381\"]}, {\"surname\": [\"Schultzberg\", \"Muth\\u00e9n\"], \"given-names\": [\"M\", \"B\"], \"article-title\": [\"Number of subjects and time points needed for multilevel time-series analysis: a simulation study of dynamic structural equation modeling\"], \"source\": [\"Structural Equation Modeling: A Multidisciplinary Journal\"], \"year\": [\"2018\"], \"volume\": [\"25\"], \"issue\": [\"4\"], \"fpage\": [\"495\"], \"lpage\": [\"515\"], \"pub-id\": [\"10.1080/10705511.2017.1392862\"]}, {\"surname\": [\"Suter\", \"Pihet\", \"Zimmermann\", \"de Ridder\", \"Urben\", \"Stephan\"], \"given-names\": [\"M\", \"S\", \"G\", \"J\", \"S\", \"P\"], \"article-title\": [\"Predicting daily-life antisocial behaviour in institutionalized adolescents with transgression-related implicit association tests\"], \"source\": [\"Journal of Forensic Psychiatry & Psychology\"], \"year\": [\"2017\"], \"volume\": [\"28\"], \"issue\": [\"6\"], \"fpage\": [\"881\"], \"lpage\": [\"900\"], \"pub-id\": [\"10.1080/14789949.2017.1332772\"]}, {\"surname\": [\"Thompson\"], \"given-names\": [\"ER\"], \"article-title\": [\"Development and validation of an internationally reliable short-form of the Positive and Negative Affect Schedule (PANAS)\"], \"source\": [\"Journal of Cross-Cultural Psychology\"], \"year\": [\"2007\"], \"volume\": [\"38\"], \"issue\": [\"2\"], \"fpage\": [\"227\"], \"lpage\": [\"242\"], \"pub-id\": [\"10.1177/0022022106297301\"]}, {\"mixed-citation\": [\"Urben, S., Constanty, L., Lepage, C., Rosselet Amoussou, J., Durussel, J., Turri, F., Wouters, E., M\\u00fcrner-Lavanchy, I., & Plessen, K. J. (2022). The added value of a micro-level ecological approach when mapping self-regulatory control processes and externalizing symptoms during adolescence: a systematic review. \"], \"italic\": [\"European Child & Adolescent Psychiatry,\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":58,"string":"58"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Res Child Adolesc Psychopathol. 2024 May 30; 52(1):51-63"},"package_file":{"kind":"string","value":"oa_package/3c/1a/PMC10787886.tar.gz"}}},{"rowIdx":170,"cells":{"accession_id":{"kind":"string","value":"PMC10787887"},"pmid":{"kind":"string","value":"0"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par3\">Social learning can be defined “as a change in behavior that follows the observation of another (typically a conspecific) perform a similar behavior, the products of the behavior, or even the products alone” (Zentall ##REF##21895354##2012##: 114). Which type of information is acquired through observation may differ strongly and is reflected in so-called social learning mechanisms. Motivational factors such as social facilitation (Zajonc ##REF##14300526##1965##) promote the acquisition of information or change in behaviour by the observer via the mere presence of a demonstrator (Zentall ##UREF##29##2001##). Perceptual factors such as local (Roberts ##UREF##21##1941##) or stimulus enhancement (Galef ##UREF##8##1988##) can facilitate information acquisition by drawing the attention of the observer to either the location or stimulus of importance (Zentall ##UREF##29##2001##). In emulation (Tomasello ##UREF##24##1998##) the results of a demonstrated behaviour affect the observer who may strive to generate the same effect on the environment/objects as the demonstration did, without necessarily understanding the actions or reproducing the behaviour. Imitation is a specific learning mechanism, defined by a high degree of copying fidelity/response matching (Whiten and Ham ##UREF##27##1992##; Whiten et al. ##UREF##28##2009##), and cannot be explained by motivational, perceptual, or attentional factors alone (Zentall ##REF##21895354##2012##), for a comprehensive overview of social learning mechanisms see Hoppitt and Laland (##UREF##11##2013##). Social learning is taxonomically widespread, ranging from insects to birds and mammals, possibly because it is a cost-effective way of acquiring information. Yet, social learning is not always advantageous (Giraldeau et al. ##UREF##9##2002##; Garcia-Nisa et al. ##REF##36670123##2023##) and different social learning mechanisms may have different thresholds in this respect.</p>","<p id=\"Par4\">Great apes, for instance, seem to be less prone to show imitation than other forms of social learning like emulation (Horner and Whiten ##REF##15549502##2005##; Tennie et al. ##UREF##23##2006##; Clay and Tennie ##REF##28741660##2018##). On the other hand, Marmosets (<italic>Callithrix jacchus</italic>) (Bugnyar and Huber ##REF##9344436##1997##; Voelkl and Huber ##REF##10973721##2000##, ##REF##17622356##2007##) and dogs (<italic>Canis familiaris</italic>) (Huber et al. ##UREF##14##2009##, ##REF##29980941##2018##, ##REF##31975325##2020##) show high-fidelity imitation despite not being closely related to humans (<italic>Homo imitans</italic> as proposed by Meltzoff ##UREF##16##1988##). This suggests that high-fidelity imitation may be driven by natural ecology and social structure rather than phylogenetic relatedness. In fact, experimental evidence has illustrated that various avian species, namely budgerigars (<italic>Melopsittacus undulates</italic>) (Dawson and Foss ##REF##5882805##1965##; Heyes and Saggerson ##UREF##10##2002##), European starlings (<italic>Sturnus vulgaris</italic>) (Fawcett et al. ##UREF##5##2002##), Japanese Quail (<italic>Coturnix japonica</italic>) (Akins and Zentall ##UREF##0##1998##; Akins et al. ##REF##12391793##2002##), Common Ravens (<italic>Corvus corax</italic>) (Loretto et al. ##UREF##15##2020##) and Pigeons (<italic>Columba livia</italic>) (Nguyen et al. ##REF##16235638##2005##) show (simple forms) of motor imitation.</p>","<p id=\"Par5\">Parrots are renowned for their technical intelligence, vocal mimicry and social learning capacities (Pepperberg and Funk ##UREF##20##1990##; Huber et al. ##UREF##13##2001##; Funk ##REF##12357289##2002##; Huber and Gajdon ##REF##16909237##2006##; Werdenich and Huber ##UREF##25##2006##; Auersperg et al. ##REF##19411271##2009##, ##REF##21687666##2011##, ##REF##23137681##2012##, ##UREF##1##2014##; Miyata et al. ##REF##20640911##2011##; Goodman et al. ##REF##30224791##2018##; Klump et al. ##REF##34437121##2021##; Smith et al. ##REF##36258015##2022##). Yet surprisingly few parrot species have been tested on their motor imitation skills (budgerigars: Dawson and Foss ##REF##5882805##1965##; Galef et al. ##UREF##7##1986##; Heyes and Saggerson ##UREF##10##2002##; grey parrots (<italic>Psittacus erithacus</italic>): Moore ##UREF##18##1992##; kea (<italic>Nestor notabilis</italic>): Huber et al. ##UREF##13##2001##; Suwandschieff et al. ##REF##37261570##2023##; Goffin cockatoos (<italic>Cacatua goffiniana</italic>): Auersperg et al. ##REF##23137681##2012##), revealing mixed results. Whereas most studies find evidence for motor imitation, the studies on kea remained inconclusive.</p>","<p id=\"Par6\">Kea (<italic>Nestor notabilis</italic>) possess well-developed technical skills (Huber and Gajdon ##REF##16909237##2006##), have long lifespans with multiple reproductive cycles, extended juvenile periods accompanied by considerable in-group tolerance, are highly neophilic and exploratory (Diamond and Bond ##UREF##4##1999##). They also have a very large number of documented food sources (Brejaart ##UREF##2##1988##; Clarke ##UREF##3##1971##; O’Donnell and Dilks ##UREF##19##1994##) many of which need to be extracted, which strongly suggest transfer of knowledge between individuals. All these characteristics facilitate the development of social learning (Gajdon et al. ##UREF##6##2004##), yet experimental evidence for imitation in this species is still missing. Therefore, we tested kea, for their social learning skills in a demonstrated sequence task. Specifically, we aimed at exploring kea’s imitative social learning capacities. We hypothesised that when confronted with a relatively complex two-step task, kea would pay attention to, and copy the behaviour of, a skilled conspecific. We thus predicted that observers would preferentially use the demonstrated opening side, sequence and colour whereas non-observing control individuals would apply trial-and-error learning to solve the task.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par3\\\">Social learning can be defined “as a change in behavior that follows the observation of another (typically a conspecific) perform a similar behavior, the products of the behavior, or even the products alone” (Zentall ##REF##21895354##2012##: 114). Which type of information is acquired through observation may differ strongly and is reflected in so-called social learning mechanisms. Motivational factors such as social facilitation (Zajonc ##REF##14300526##1965##) promote the acquisition of information or change in behaviour by the observer via the mere presence of a demonstrator (Zentall ##UREF##29##2001##). Perceptual factors such as local (Roberts ##UREF##21##1941##) or stimulus enhancement (Galef ##UREF##8##1988##) can facilitate information acquisition by drawing the attention of the observer to either the location or stimulus of importance (Zentall ##UREF##29##2001##). In emulation (Tomasello ##UREF##24##1998##) the results of a demonstrated behaviour affect the observer who may strive to generate the same effect on the environment/objects as the demonstration did, without necessarily understanding the actions or reproducing the behaviour. Imitation is a specific learning mechanism, defined by a high degree of copying fidelity/response matching (Whiten and Ham ##UREF##27##1992##; Whiten et al. ##UREF##28##2009##), and cannot be explained by motivational, perceptual, or attentional factors alone (Zentall ##REF##21895354##2012##), for a comprehensive overview of social learning mechanisms see Hoppitt and Laland (##UREF##11##2013##). Social learning is taxonomically widespread, ranging from insects to birds and mammals, possibly because it is a cost-effective way of acquiring information. Yet, social learning is not always advantageous (Giraldeau et al. ##UREF##9##2002##; Garcia-Nisa et al. ##REF##36670123##2023##) and different social learning mechanisms may have different thresholds in this respect.</p>\",\n \"<p id=\\\"Par4\\\">Great apes, for instance, seem to be less prone to show imitation than other forms of social learning like emulation (Horner and Whiten ##REF##15549502##2005##; Tennie et al. ##UREF##23##2006##; Clay and Tennie ##REF##28741660##2018##). On the other hand, Marmosets (<italic>Callithrix jacchus</italic>) (Bugnyar and Huber ##REF##9344436##1997##; Voelkl and Huber ##REF##10973721##2000##, ##REF##17622356##2007##) and dogs (<italic>Canis familiaris</italic>) (Huber et al. ##UREF##14##2009##, ##REF##29980941##2018##, ##REF##31975325##2020##) show high-fidelity imitation despite not being closely related to humans (<italic>Homo imitans</italic> as proposed by Meltzoff ##UREF##16##1988##). This suggests that high-fidelity imitation may be driven by natural ecology and social structure rather than phylogenetic relatedness. In fact, experimental evidence has illustrated that various avian species, namely budgerigars (<italic>Melopsittacus undulates</italic>) (Dawson and Foss ##REF##5882805##1965##; Heyes and Saggerson ##UREF##10##2002##), European starlings (<italic>Sturnus vulgaris</italic>) (Fawcett et al. ##UREF##5##2002##), Japanese Quail (<italic>Coturnix japonica</italic>) (Akins and Zentall ##UREF##0##1998##; Akins et al. ##REF##12391793##2002##), Common Ravens (<italic>Corvus corax</italic>) (Loretto et al. ##UREF##15##2020##) and Pigeons (<italic>Columba livia</italic>) (Nguyen et al. ##REF##16235638##2005##) show (simple forms) of motor imitation.</p>\",\n \"<p id=\\\"Par5\\\">Parrots are renowned for their technical intelligence, vocal mimicry and social learning capacities (Pepperberg and Funk ##UREF##20##1990##; Huber et al. ##UREF##13##2001##; Funk ##REF##12357289##2002##; Huber and Gajdon ##REF##16909237##2006##; Werdenich and Huber ##UREF##25##2006##; Auersperg et al. ##REF##19411271##2009##, ##REF##21687666##2011##, ##REF##23137681##2012##, ##UREF##1##2014##; Miyata et al. ##REF##20640911##2011##; Goodman et al. ##REF##30224791##2018##; Klump et al. ##REF##34437121##2021##; Smith et al. ##REF##36258015##2022##). Yet surprisingly few parrot species have been tested on their motor imitation skills (budgerigars: Dawson and Foss ##REF##5882805##1965##; Galef et al. ##UREF##7##1986##; Heyes and Saggerson ##UREF##10##2002##; grey parrots (<italic>Psittacus erithacus</italic>): Moore ##UREF##18##1992##; kea (<italic>Nestor notabilis</italic>): Huber et al. ##UREF##13##2001##; Suwandschieff et al. ##REF##37261570##2023##; Goffin cockatoos (<italic>Cacatua goffiniana</italic>): Auersperg et al. ##REF##23137681##2012##), revealing mixed results. Whereas most studies find evidence for motor imitation, the studies on kea remained inconclusive.</p>\",\n \"<p id=\\\"Par6\\\">Kea (<italic>Nestor notabilis</italic>) possess well-developed technical skills (Huber and Gajdon ##REF##16909237##2006##), have long lifespans with multiple reproductive cycles, extended juvenile periods accompanied by considerable in-group tolerance, are highly neophilic and exploratory (Diamond and Bond ##UREF##4##1999##). They also have a very large number of documented food sources (Brejaart ##UREF##2##1988##; Clarke ##UREF##3##1971##; O’Donnell and Dilks ##UREF##19##1994##) many of which need to be extracted, which strongly suggest transfer of knowledge between individuals. All these characteristics facilitate the development of social learning (Gajdon et al. ##UREF##6##2004##), yet experimental evidence for imitation in this species is still missing. Therefore, we tested kea, for their social learning skills in a demonstrated sequence task. Specifically, we aimed at exploring kea’s imitative social learning capacities. We hypothesised that when confronted with a relatively complex two-step task, kea would pay attention to, and copy the behaviour of, a skilled conspecific. We thus predicted that observers would preferentially use the demonstrated opening side, sequence and colour whereas non-observing control individuals would apply trial-and-error learning to solve the task.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Method</title>","<title>Subjects</title>","<p id=\"Par7\">Eighteen kea from the Haidlhof Research Station (Bad Vöslau) participated in this study. All individuals were group-housed in an outdoor aviary equipped with perches, nesting areas, ponds, and various enrichment. All birds were fed three times a day, had access to water ad libitum and were not food deprived for testing. All individuals had prior experience with experimental testing and participated on a voluntary basis in the task. The testing compartment at the Haidlhof Research Station can be visually separated from the rest of the aviary and can be further divided into two different areas. The individuals were assigned to test groups of three and five individuals and two control groups of five individuals each. The distribution was sex and age balanced, for details see Table 1 of the supplementary material.\n</p>","<title>Apparatus</title>","<p id=\"Par8\">A rectangular box (44 × 18 × 18 cm) with two aluminium sliding lids, two pins, two strings and two rings served as the test box, see Fig. ##FIG##0##1##. The test box was designed to provide a sequence function, requiring the subjects to pull a pin on top of the box, to then be able to open the opposite sliding lid by pulling a ring attached at the side of the box. The adjacent sliding lid remained locked. Hence, the only solving sequences for the test box were left pin–right ring or right pin–left ring. Electronics were added inside the box to provide the sequence function. If no pin or both pins were pulled the mechanism locked both lids and no rewards could be retrieved. Manual locking keys were added to provide a full and partial locking function for demonstrator training and demonstration sessions. The box was divided into equally sized reward sections underneath the sliding lids and a GoPro fixture was attached at the base.</p>","<p id=\"Par9\">The pins were coloured in red and yellow, which is on the preferred spectrum for kea, and the strings and rings in green and blue (less preferred spectrum) respectively (Weser and Ross ##UREF##26##2013##). Each side and sequence had one preferred and one less preferred colour pairing, minimising the potential for a side bias based on colour preference alone. To increase salience, only those parts that needed direct manipulation were coloured, i.e., the pins, strings, and rings.</p>","<title>Procedure</title>","<p id=\"Par10\">To minimize the effects of individual learning over social learning, all test sessions consisted of one trial only. Each session/trial was terminated either after successful reward retrieval or a maximum of two minutes of exploration—i.e. pulling and touching different ring-pin combinations without opening success. A stopwatch was used to track the two minutes and all sessions were terminated by removing the individuals from the test compartment once either criterion (removal response or time-out) was met. Subjects from test groups received two experimental phases, whereas subjects from control groups received three phases (see below). In all phases, both sides of the box were baited and each sequence was rewarded. However, removing a pin and pulling on the wrong ring, pulling both pins or pulling on the rings without having removed the respective pin did not lead to any reward (as the doors were locked). Altogether four sessions (of one trial each) were tested on three consecutive days. On the first day, phase one (consisting of only one session) was tested in the morning and the first session of phase two (consisting of three sessions) in the afternoon. On the second and third day, each individual only received a morning test session of session two and three of phase two respectively.</p>","<p id=\"Par11\">The experimenter wore mirrored sunglasses (as has been applied before by Bastos and Taylor ##REF##31911652##2020##; Suwandschieff et al. ##REF##37261570##2023##) and remained silent during testing (excluding direct commands such as “enter”, “exit” etc.) to avoid unintentional cueing.</p>","<p><bold>Phase 1: Forced failure task to all birds</bold> Pins were removed on this occasion and the test box could not be opened. All individuals (of all experimental groups) received access to the test box and were allowed to try and open it for two minutes. As the pins were missing all individuals were forced to fail this task. After two minutes passed the individual exited the testing compartment and the session was completed. This phase was introduced to prime individuals on the non-functioning of the task, focusing the attention towards the essential pins and increasing the motivation to follow a demonstration.</p>","<p id=\"Par13\"><bold>Phase 2: Non-demonstrated task Control Group (CG)</bold> Control group individuals were allowed to try and solve the task by trial-and-error for three sessions (on three consecutive days).</p>","<p id=\"Par14\"><bold>Phase 2: Demonstrated task Test Groups (TG)</bold> Test group individuals received three demonstration sessions of three trials each followed by three test sessions of one trial each (test following demonstration session in direct succession) on three consecutive days. Side/sequence and demonstrator assignment were counterbalanced across the two groups (see Table 1 of the supplementary material).</p>","<p id=\"Par15\"><bold>Phase 3: Demonstrated task Control Test (CGTest)</bold> In the third phase, former control group birds (minus the two demonstrator birds) were randomly assigned to the two demonstrators and were tested again, this time as observer individuals. The test setup was identical to the phase two demonstrated task of the test group.</p>","<title>Data scoring and analysis</title>","<p id=\"Par16\">All experiments were videotaped from two sides, behind the observation compartment and directly above the test box (GoPro) within the test compartment, for the exact setup see Fig. ##FIG##0##1## of the supplementary material. All GoPro videos of the test sessions were scored/coded with Solomon Coder (version beta 19.08.02) and one independent rater (blind to the study) scored 10% of all videos. Interobserver reliability was tested with Cohen’s Kappa for the categorical (<italic>k</italic> = 0.93) and Intraclass Correlation Coefficient for the numerical data (ICC = 0.711, approach duration; ICC = 0.999, response duration; ICC = 1, solving latency), for more information see the supplementary material.</p>","<p id=\"Par17\">A total of 74 sessions were analysed. Two individuals did not participate in two sessions and one session respectively, all other individuals completed all three of their test sessions. A binomial test was applied to compare the success rate of the test versus the control group, using the proportion of successful control birds as the baseline chance level of solving the setup spontaneously without demonstration. All other analyses were strictly descriptive.</p>"],"string":"[\n \"<title>Method</title>\",\n \"<title>Subjects</title>\",\n \"<p id=\\\"Par7\\\">Eighteen kea from the Haidlhof Research Station (Bad Vöslau) participated in this study. All individuals were group-housed in an outdoor aviary equipped with perches, nesting areas, ponds, and various enrichment. All birds were fed three times a day, had access to water ad libitum and were not food deprived for testing. All individuals had prior experience with experimental testing and participated on a voluntary basis in the task. The testing compartment at the Haidlhof Research Station can be visually separated from the rest of the aviary and can be further divided into two different areas. The individuals were assigned to test groups of three and five individuals and two control groups of five individuals each. The distribution was sex and age balanced, for details see Table 1 of the supplementary material.\\n</p>\",\n \"<title>Apparatus</title>\",\n \"<p id=\\\"Par8\\\">A rectangular box (44 × 18 × 18 cm) with two aluminium sliding lids, two pins, two strings and two rings served as the test box, see Fig. ##FIG##0##1##. The test box was designed to provide a sequence function, requiring the subjects to pull a pin on top of the box, to then be able to open the opposite sliding lid by pulling a ring attached at the side of the box. The adjacent sliding lid remained locked. Hence, the only solving sequences for the test box were left pin–right ring or right pin–left ring. Electronics were added inside the box to provide the sequence function. If no pin or both pins were pulled the mechanism locked both lids and no rewards could be retrieved. Manual locking keys were added to provide a full and partial locking function for demonstrator training and demonstration sessions. The box was divided into equally sized reward sections underneath the sliding lids and a GoPro fixture was attached at the base.</p>\",\n \"<p id=\\\"Par9\\\">The pins were coloured in red and yellow, which is on the preferred spectrum for kea, and the strings and rings in green and blue (less preferred spectrum) respectively (Weser and Ross ##UREF##26##2013##). Each side and sequence had one preferred and one less preferred colour pairing, minimising the potential for a side bias based on colour preference alone. To increase salience, only those parts that needed direct manipulation were coloured, i.e., the pins, strings, and rings.</p>\",\n \"<title>Procedure</title>\",\n \"<p id=\\\"Par10\\\">To minimize the effects of individual learning over social learning, all test sessions consisted of one trial only. Each session/trial was terminated either after successful reward retrieval or a maximum of two minutes of exploration—i.e. pulling and touching different ring-pin combinations without opening success. A stopwatch was used to track the two minutes and all sessions were terminated by removing the individuals from the test compartment once either criterion (removal response or time-out) was met. Subjects from test groups received two experimental phases, whereas subjects from control groups received three phases (see below). In all phases, both sides of the box were baited and each sequence was rewarded. However, removing a pin and pulling on the wrong ring, pulling both pins or pulling on the rings without having removed the respective pin did not lead to any reward (as the doors were locked). Altogether four sessions (of one trial each) were tested on three consecutive days. On the first day, phase one (consisting of only one session) was tested in the morning and the first session of phase two (consisting of three sessions) in the afternoon. On the second and third day, each individual only received a morning test session of session two and three of phase two respectively.</p>\",\n \"<p id=\\\"Par11\\\">The experimenter wore mirrored sunglasses (as has been applied before by Bastos and Taylor ##REF##31911652##2020##; Suwandschieff et al. ##REF##37261570##2023##) and remained silent during testing (excluding direct commands such as “enter”, “exit” etc.) to avoid unintentional cueing.</p>\",\n \"<p><bold>Phase 1: Forced failure task to all birds</bold> Pins were removed on this occasion and the test box could not be opened. All individuals (of all experimental groups) received access to the test box and were allowed to try and open it for two minutes. As the pins were missing all individuals were forced to fail this task. After two minutes passed the individual exited the testing compartment and the session was completed. This phase was introduced to prime individuals on the non-functioning of the task, focusing the attention towards the essential pins and increasing the motivation to follow a demonstration.</p>\",\n \"<p id=\\\"Par13\\\"><bold>Phase 2: Non-demonstrated task Control Group (CG)</bold> Control group individuals were allowed to try and solve the task by trial-and-error for three sessions (on three consecutive days).</p>\",\n \"<p id=\\\"Par14\\\"><bold>Phase 2: Demonstrated task Test Groups (TG)</bold> Test group individuals received three demonstration sessions of three trials each followed by three test sessions of one trial each (test following demonstration session in direct succession) on three consecutive days. Side/sequence and demonstrator assignment were counterbalanced across the two groups (see Table 1 of the supplementary material).</p>\",\n \"<p id=\\\"Par15\\\"><bold>Phase 3: Demonstrated task Control Test (CGTest)</bold> In the third phase, former control group birds (minus the two demonstrator birds) were randomly assigned to the two demonstrators and were tested again, this time as observer individuals. The test setup was identical to the phase two demonstrated task of the test group.</p>\",\n \"<title>Data scoring and analysis</title>\",\n \"<p id=\\\"Par16\\\">All experiments were videotaped from two sides, behind the observation compartment and directly above the test box (GoPro) within the test compartment, for the exact setup see Fig. ##FIG##0##1## of the supplementary material. All GoPro videos of the test sessions were scored/coded with Solomon Coder (version beta 19.08.02) and one independent rater (blind to the study) scored 10% of all videos. Interobserver reliability was tested with Cohen’s Kappa for the categorical (<italic>k</italic> = 0.93) and Intraclass Correlation Coefficient for the numerical data (ICC = 0.711, approach duration; ICC = 0.999, response duration; ICC = 1, solving latency), for more information see the supplementary material.</p>\",\n \"<p id=\\\"Par17\\\">A total of 74 sessions were analysed. Two individuals did not participate in two sessions and one session respectively, all other individuals completed all three of their test sessions. A binomial test was applied to compare the success rate of the test versus the control group, using the proportion of successful control birds as the baseline chance level of solving the setup spontaneously without demonstration. All other analyses were strictly descriptive.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par18\">After all subjects had experienced a non-functional apparatus (phase 1), they were allowed to engage in the task with or without prior demonstration of a skilled conspecific (phase 2). In sum, four individuals from the test group (out of a total of 8) successfully solved the task and two individuals from the control group (out of a total of 10). The exact binomial test resulted in a nearly significant trend between the test and control group (<italic>x</italic> = 4, <italic>n</italic> = 8, <italic>p</italic> = 2/10; <italic>p-value</italic> = 0.056).</p>","<p id=\"Par19\">The control group individuals participated in an additional round of testing (phase 3), in which they received a demonstration prior to getting access to the task again. Four individuals who had failed to solve the task without receiving a demonstration successfully solved the task after receiving a demonstration. In contrast, one of the individuals from the control test group, who had solved the task without receiving a demonstration (in the control group round), did not solve the task after receiving a demonstration (in the control test group round). Assessing the birds’ performance after receiving a demonstration (test group plus control test group) <italic>versus</italic> without a demonstration (control group), the exact binomial test revealed a significant difference between the groups with the test groups performing much better on average than the control group (<italic>x</italic> = 8, <italic>n</italic> = 16, <italic>p</italic> = 2/10; <italic>p-value</italic> = 0.007). When comparing the within-subject design of the control group <italic>versus</italic> the control test group (repeated measure) twice as many individuals successfully solved the task after having seen a demonstration (<italic>x</italic> = 4, <italic>n</italic> = 8, <italic>p</italic> = 2/10; <italic>p-value</italic> = 0.056). Half of the successful individuals (4/8) that saw a demonstration solved the task via the opposite (non-demonstrated) sequence and later reversed to the demonstrated sequence in subsequent sessions, see Table ##TAB##0##1##.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par18\\\">After all subjects had experienced a non-functional apparatus (phase 1), they were allowed to engage in the task with or without prior demonstration of a skilled conspecific (phase 2). In sum, four individuals from the test group (out of a total of 8) successfully solved the task and two individuals from the control group (out of a total of 10). The exact binomial test resulted in a nearly significant trend between the test and control group (<italic>x</italic> = 4, <italic>n</italic> = 8, <italic>p</italic> = 2/10; <italic>p-value</italic> = 0.056).</p>\",\n \"<p id=\\\"Par19\\\">The control group individuals participated in an additional round of testing (phase 3), in which they received a demonstration prior to getting access to the task again. Four individuals who had failed to solve the task without receiving a demonstration successfully solved the task after receiving a demonstration. In contrast, one of the individuals from the control test group, who had solved the task without receiving a demonstration (in the control group round), did not solve the task after receiving a demonstration (in the control test group round). Assessing the birds’ performance after receiving a demonstration (test group plus control test group) <italic>versus</italic> without a demonstration (control group), the exact binomial test revealed a significant difference between the groups with the test groups performing much better on average than the control group (<italic>x</italic> = 8, <italic>n</italic> = 16, <italic>p</italic> = 2/10; <italic>p-value</italic> = 0.007). When comparing the within-subject design of the control group <italic>versus</italic> the control test group (repeated measure) twice as many individuals successfully solved the task after having seen a demonstration (<italic>x</italic> = 4, <italic>n</italic> = 8, <italic>p</italic> = 2/10; <italic>p-value</italic> = 0.056). Half of the successful individuals (4/8) that saw a demonstration solved the task via the opposite (non-demonstrated) sequence and later reversed to the demonstrated sequence in subsequent sessions, see Table ##TAB##0##1##.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par20\">We show that kea were able to solve a rather difficult two-step sequence task and that receiving a demonstration of a skilled conspecific had a positive effect on solving success. However, successful birds showed a high variation in their response topography and often abandoned faithfully copying the task in favour of exploration. This is particularly interesting in the case of John, who followed the demonstrated sequence twice and then generalised in the other direction.</p>","<p id=\"Par21\">The effect of demonstration becomes particularly clear when combining the results from phase two and three. Eight out of 10 subjects that came to solve the task did so after a demonstration. That kea can profit from social learning is in line with previous studies (Huber et al. ##UREF##13##2001##; Huber ##UREF##12##2002##; Suwandschieff et al. ##REF##37261570##2023##). However, the current study provides little evidence for motor imitation, despite various methodological differences from the other studies. For instance, previous experiments illustrated that the motivation to follow a demonstration was low. It was theorized that the complexity of the demonstrated actions could have contributed, as they likely were too simple to require a demonstration to solve the task (Suwandschieff et al. ##REF##37261570##2023##), or too complex to follow the demonstration from afar (Huber et al. ##UREF##13##2001##) and reproduce with high fidelity. Therefore, the task was made more difficult than the basic two-choice task, while avoiding the complexity of the multi-lock box, and the forced failure in phase one was introduced to prime individuals on the task difficulty and to increase their motivation to follow a demonstration. Yet, these measures did not result in a higher copying fidelity than the other studies. On the one hand, half of the solvers used the opposite sequence to the demonstrators. On the other hand, solving success appeared not to reference solving consistency, as successful birds continued to explore the solving potential by applying different opening methods (see supplementary material for details). This variation in solving behaviour unfortunately made a statistical comparison not possible in terms of actual mechanisms, or quantifiable behavioural differences with previous studies.</p>","<p id=\"Par22\">As all individuals, regardless of the experimental group, participated in the forced failure task, it is improbable that our results can be solely explained by social facilitation or local enhancement. Although we cannot rule out the possibility that the mere presence of a demonstrator motivated individuals to engage in the task, we have established that all individuals will do so even in the absence of a conspecific. Therefore, the presence of another individual does not appear to be the primary factor explaining our results. Additionally, since the location of the test box remained constant throughout the different phases, no additional information could have been gained from the demonstration. Consequently, this does not explain the discrepancy between the experimental groups. While the success rate of observer birds and the varied response patterns exhibited by successful individuals indicate emulation, we cannot dismiss the potential influence of stimulus enhancement. A test setup that clearly distinguishes these two mechanisms would have to be devised, i.e., one including a ghost control (Whiten and Ham ##UREF##27##1992##). Consistent with previous findings kea show high behavioural flexibility (Werdenich and Huber ##UREF##25##2006##; Auersperg et al. ##REF##21687666##2011##; Laschober et al. ##REF##34110523##2021##) and preferentially engage in exploratory behaviour, being more interested in potential affordances than feeding success (Diamond and Bond ##UREF##4##1999##; Huber et al. ##UREF##13##2001##; Smith et al. ##REF##36258015##2022##; Suwandschieff et al. ##REF##37261570##2023##). These results are in accordance with kea’s natural feeding strategies, as opportunistic group foragers, with kea paying close attention to what others feed on while engaging in individual manipulation strategies to obtain the resources (Diamond and Bond ##UREF##4##1999##). In addition, it corresponds with the characteristics of island-dwelling parrots, as described by Mettke-Hofmann and colleagues (##UREF##17##2002##) who found that island species spend significantly more time on exploratory behaviour especially in areas of seasonally fluctuating food availability.</p>","<p id=\"Par23\">In conclusion, we find strong evidence that observing a conspecific opening an apparatus via two steps affected the solving success of observer kea. They thus profit from social learning, which aligns well with other studies on parrots and songbirds showing social information transmission and the spread of novel foraging techniques within captive groups and wild populations (e.g. Slagsvold and Wiebe ##UREF##22##2011##; Auersperg et al. ##UREF##1##2014##; Aplin et al. ##REF##25470065##2015##; Klump et al. ##REF##34437121##2021##). However, in our study, the response topography of solvers was variable and the copying fidelity was at a very low level, providing no indication of motor imitation over emulation or stimulus enhancement in kea. Therefore, our findings corroborate that kea display strong behavioural variability when attempting to solve a complex motor task. They also keep on exploring options after a successful solution and may rapidly shift solving strategies. Taken together, this makes kea a great model system to study behavioural flexibility but not so much for imitation.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par20\\\">We show that kea were able to solve a rather difficult two-step sequence task and that receiving a demonstration of a skilled conspecific had a positive effect on solving success. However, successful birds showed a high variation in their response topography and often abandoned faithfully copying the task in favour of exploration. This is particularly interesting in the case of John, who followed the demonstrated sequence twice and then generalised in the other direction.</p>\",\n \"<p id=\\\"Par21\\\">The effect of demonstration becomes particularly clear when combining the results from phase two and three. Eight out of 10 subjects that came to solve the task did so after a demonstration. That kea can profit from social learning is in line with previous studies (Huber et al. ##UREF##13##2001##; Huber ##UREF##12##2002##; Suwandschieff et al. ##REF##37261570##2023##). However, the current study provides little evidence for motor imitation, despite various methodological differences from the other studies. For instance, previous experiments illustrated that the motivation to follow a demonstration was low. It was theorized that the complexity of the demonstrated actions could have contributed, as they likely were too simple to require a demonstration to solve the task (Suwandschieff et al. ##REF##37261570##2023##), or too complex to follow the demonstration from afar (Huber et al. ##UREF##13##2001##) and reproduce with high fidelity. Therefore, the task was made more difficult than the basic two-choice task, while avoiding the complexity of the multi-lock box, and the forced failure in phase one was introduced to prime individuals on the task difficulty and to increase their motivation to follow a demonstration. Yet, these measures did not result in a higher copying fidelity than the other studies. On the one hand, half of the solvers used the opposite sequence to the demonstrators. On the other hand, solving success appeared not to reference solving consistency, as successful birds continued to explore the solving potential by applying different opening methods (see supplementary material for details). This variation in solving behaviour unfortunately made a statistical comparison not possible in terms of actual mechanisms, or quantifiable behavioural differences with previous studies.</p>\",\n \"<p id=\\\"Par22\\\">As all individuals, regardless of the experimental group, participated in the forced failure task, it is improbable that our results can be solely explained by social facilitation or local enhancement. Although we cannot rule out the possibility that the mere presence of a demonstrator motivated individuals to engage in the task, we have established that all individuals will do so even in the absence of a conspecific. Therefore, the presence of another individual does not appear to be the primary factor explaining our results. Additionally, since the location of the test box remained constant throughout the different phases, no additional information could have been gained from the demonstration. Consequently, this does not explain the discrepancy between the experimental groups. While the success rate of observer birds and the varied response patterns exhibited by successful individuals indicate emulation, we cannot dismiss the potential influence of stimulus enhancement. A test setup that clearly distinguishes these two mechanisms would have to be devised, i.e., one including a ghost control (Whiten and Ham ##UREF##27##1992##). Consistent with previous findings kea show high behavioural flexibility (Werdenich and Huber ##UREF##25##2006##; Auersperg et al. ##REF##21687666##2011##; Laschober et al. ##REF##34110523##2021##) and preferentially engage in exploratory behaviour, being more interested in potential affordances than feeding success (Diamond and Bond ##UREF##4##1999##; Huber et al. ##UREF##13##2001##; Smith et al. ##REF##36258015##2022##; Suwandschieff et al. ##REF##37261570##2023##). These results are in accordance with kea’s natural feeding strategies, as opportunistic group foragers, with kea paying close attention to what others feed on while engaging in individual manipulation strategies to obtain the resources (Diamond and Bond ##UREF##4##1999##). In addition, it corresponds with the characteristics of island-dwelling parrots, as described by Mettke-Hofmann and colleagues (##UREF##17##2002##) who found that island species spend significantly more time on exploratory behaviour especially in areas of seasonally fluctuating food availability.</p>\",\n \"<p id=\\\"Par23\\\">In conclusion, we find strong evidence that observing a conspecific opening an apparatus via two steps affected the solving success of observer kea. They thus profit from social learning, which aligns well with other studies on parrots and songbirds showing social information transmission and the spread of novel foraging techniques within captive groups and wild populations (e.g. Slagsvold and Wiebe ##UREF##22##2011##; Auersperg et al. ##UREF##1##2014##; Aplin et al. ##REF##25470065##2015##; Klump et al. ##REF##34437121##2021##). However, in our study, the response topography of solvers was variable and the copying fidelity was at a very low level, providing no indication of motor imitation over emulation or stimulus enhancement in kea. Therefore, our findings corroborate that kea display strong behavioural variability when attempting to solve a complex motor task. They also keep on exploring options after a successful solution and may rapidly shift solving strategies. Taken together, this makes kea a great model system to study behavioural flexibility but not so much for imitation.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<p>Communicated by F. Bairlein.</p>","<p id=\"Par1\">Social learning is an important aspect of dealing with the complexity of life. The transmission of information via the observation of other individuals is a cost-effective way of acquiring information. It is widespread within the animal kingdom but may differ strongly in the social learning mechanisms applied by the divergent species. Here we tested eighteen Kea (<italic>Nestor notabilis</italic>) parrots on their propensity to socially learn, and imitate, a demonstrated sequence of steps necessary to open an apparatus containing food. The demonstration by a conspecific led to more successful openings by observer birds, than control birds without a demonstration. However, all successful individuals showed great variation in their response topography and abandoned faithfully copying the task in favour of exploration. While the results provide little evidence for motor imitation they do provide further evidence for kea’s propensity towards exploration and rapidly shifting solving strategies, indicative of behavioural flexibility.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s10336-023-02127-y.</p>","<p id=\"Par2\"><bold>Keas, Vögel der Vielseitigkeit. Kea-Papageien (</bold><bold><italic>Nestor notabilis</italic></bold><bold>) zeigen eine hohe Verhaltensflexibilität bei der Lösung einer demonstrierten Sequenzaufgabe.</bold>\n</p>","<p>Soziales Lernen ist ein wichtiger Aspekt im Umgang mit der Komplexität des Lebens. Das Erlangen von Informationen durch die Beobachtung Anderer ist eine effiziente Möglichkeit der Informationsbeschaffung. Diese Art des Lernens ist im Tierreich weit verbreitet, kann sich jedoch hinsichtlich der unterschiedlichen sozialen Lernmechanismen, welcher sich die verschiedenen Arten bedienen, stark unterscheiden. Achtzehn Kea-Papageien (<italic>Nestor notabilis</italic>) wurden auf soziales Lernen, im Speziellen auf imitatives Lernen einer demonstrierten Abfolge von Schritten zum Öffnen eines mit Futter gefüllten Apparats, getestet. Die Resultate ergaben, dass eine Demonstration durch einen Artgenossen bei Beobachtervögeln zu erfolgreicheren Lösungsansätzen führten als bei Kontrollvögeln welche keine Demonstration erhielten. Allerdings zeigten alle erfolgreichen Individuen große Unterschiede in der Topographie ihrer Reaktion, der genauen Abfolge an Bewegungen. Ebenfalls gaben sie das getreue Kopieren der Aufgabe zugunsten von Erkundungs- und/oder individuellen Lösungsstrategien auf. Insgesamt zeigten unsere Ergebnisse, dass Keas starke Verhaltensvariabilität und Flexibilität zeigen, wenn sie eine komplexe motorische Aufgabe lösen. Damit stellen sie ein großartiges Modellsystem zur Untersuchung der Verhaltensflexibilität dar.</p>","<title>Keywords</title>","<p>Open access funding provided by Austrian Science Fund (FWF).</p>"],"string":"[\n \"<p>Communicated by F. Bairlein.</p>\",\n \"<p id=\\\"Par1\\\">Social learning is an important aspect of dealing with the complexity of life. The transmission of information via the observation of other individuals is a cost-effective way of acquiring information. It is widespread within the animal kingdom but may differ strongly in the social learning mechanisms applied by the divergent species. Here we tested eighteen Kea (<italic>Nestor notabilis</italic>) parrots on their propensity to socially learn, and imitate, a demonstrated sequence of steps necessary to open an apparatus containing food. The demonstration by a conspecific led to more successful openings by observer birds, than control birds without a demonstration. However, all successful individuals showed great variation in their response topography and abandoned faithfully copying the task in favour of exploration. While the results provide little evidence for motor imitation they do provide further evidence for kea’s propensity towards exploration and rapidly shifting solving strategies, indicative of behavioural flexibility.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s10336-023-02127-y.</p>\",\n \"<p id=\\\"Par2\\\"><bold>Keas, Vögel der Vielseitigkeit. Kea-Papageien (</bold><bold><italic>Nestor notabilis</italic></bold><bold>) zeigen eine hohe Verhaltensflexibilität bei der Lösung einer demonstrierten Sequenzaufgabe.</bold>\\n</p>\",\n \"<p>Soziales Lernen ist ein wichtiger Aspekt im Umgang mit der Komplexität des Lebens. Das Erlangen von Informationen durch die Beobachtung Anderer ist eine effiziente Möglichkeit der Informationsbeschaffung. Diese Art des Lernens ist im Tierreich weit verbreitet, kann sich jedoch hinsichtlich der unterschiedlichen sozialen Lernmechanismen, welcher sich die verschiedenen Arten bedienen, stark unterscheiden. Achtzehn Kea-Papageien (<italic>Nestor notabilis</italic>) wurden auf soziales Lernen, im Speziellen auf imitatives Lernen einer demonstrierten Abfolge von Schritten zum Öffnen eines mit Futter gefüllten Apparats, getestet. Die Resultate ergaben, dass eine Demonstration durch einen Artgenossen bei Beobachtervögeln zu erfolgreicheren Lösungsansätzen führten als bei Kontrollvögeln welche keine Demonstration erhielten. Allerdings zeigten alle erfolgreichen Individuen große Unterschiede in der Topographie ihrer Reaktion, der genauen Abfolge an Bewegungen. Ebenfalls gaben sie das getreue Kopieren der Aufgabe zugunsten von Erkundungs- und/oder individuellen Lösungsstrategien auf. Insgesamt zeigten unsere Ergebnisse, dass Keas starke Verhaltensvariabilität und Flexibilität zeigen, wenn sie eine komplexe motorische Aufgabe lösen. Damit stellen sie ein großartiges Modellsystem zur Untersuchung der Verhaltensflexibilität dar.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by Austrian Science Fund (FWF).</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>We are thankful to Andràs Pèter for his help with devising the test box, Louise Mackie for her help with interobserver reliability check, Remco Folkertsma for his help with data analysis, the Austrian Science Fund (FWF) for funding the project (P 33507-B) and to the entire staff at the research station Haidlhof for their hard work and ongoing support.</p>","<title>Author contributions</title>","<p>ES: design, data collection, data curation and analysis, manuscript original draft, revisions, final manuscript; LH: conceptualisation, design, supervision, manuscript review; TB: conceptualisation, design, supervision, manuscript review; RS: conceptualisation, funding acquisition, design, supervision, manuscript review and editing. All authors read and approved the final manuscript.</p>","<title>Funding</title>","<p>Open access funding provided by Austrian Science Fund (FWF). This research was funded in whole, or in part, by the Austrian Science Fund (FWF) [P 33507-B]. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.</p>","<title>Data availability</title>","<p>All data generated or analysed during this study are included in this published article [supplementary material file: raw data].</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par24\">All authors have read the submitted version of this manuscript and declare no conflicts of interest. The authors have no relevant non-financial interests to disclose.</p>","<title>Ethical approval</title>","<p id=\"Par25\">The study is in accordance with the Good Scientific Practice guidelines and national legislation (ETK-10/11/2016) and has been approved by the institutional ethics and animal welfare committee at the University of Veterinary Medicine, Vienna.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>We are thankful to Andràs Pèter for his help with devising the test box, Louise Mackie for her help with interobserver reliability check, Remco Folkertsma for his help with data analysis, the Austrian Science Fund (FWF) for funding the project (P 33507-B) and to the entire staff at the research station Haidlhof for their hard work and ongoing support.</p>\",\n \"<title>Author contributions</title>\",\n \"<p>ES: design, data collection, data curation and analysis, manuscript original draft, revisions, final manuscript; LH: conceptualisation, design, supervision, manuscript review; TB: conceptualisation, design, supervision, manuscript review; RS: conceptualisation, funding acquisition, design, supervision, manuscript review and editing. All authors read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by Austrian Science Fund (FWF). This research was funded in whole, or in part, by the Austrian Science Fund (FWF) [P 33507-B]. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.</p>\",\n \"<title>Data availability</title>\",\n \"<p>All data generated or analysed during this study are included in this published article [supplementary material file: raw data].</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par24\\\">All authors have read the submitted version of this manuscript and declare no conflicts of interest. The authors have no relevant non-financial interests to disclose.</p>\",\n \"<title>Ethical approval</title>\",\n \"<p id=\\\"Par25\\\">The study is in accordance with the Good Scientific Practice guidelines and national legislation (ETK-10/11/2016) and has been approved by the institutional ethics and animal welfare committee at the University of Veterinary Medicine, Vienna.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Test box <bold>a</bold> front view of two pins <bold>b</bold> side view of green and blue ring respectively</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Test box <bold>a</bold> front view of two pins <bold>b</bold> side view of green and blue ring respectively</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Individuals that successfully solved the task per experimental group</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Name</th><th align=\"left\">Sex</th><th align=\"left\">Age</th><th align=\"left\">Raised</th><th align=\"left\">Group</th><th align=\"left\">No of successful sessions (out of 3)</th><th align=\"left\">Demonstrator</th><th align=\"left\">Applied demonstrated sequence</th><th align=\"left\">Applied non demonstrated sequence</th><th align=\"left\">Stayed consistent for no of sessions</th><th align=\"left\">Switched in session no</th></tr></thead><tbody><tr><td align=\"left\">Frowin</td><td align=\"left\">Male</td><td align=\"left\">17</td><td align=\"left\">Parent</td><td align=\"left\">Control</td><td align=\"left\">3</td><td align=\"left\">NA</td><td align=\"left\">NA</td><td align=\"left\">NA</td><td align=\"left\">3/3</td><td align=\"left\">NA</td></tr><tr><td align=\"left\">Papu</td><td align=\"left\">Female</td><td align=\"left\">8</td><td align=\"left\">Hand</td><td align=\"left\">Control</td><td align=\"left\">1</td><td align=\"left\">NA</td><td align=\"left\">NA</td><td align=\"left\">NA</td><td align=\"left\">1/1</td><td align=\"left\">NA</td></tr><tr><td align=\"left\">John*</td><td align=\"left\">Male</td><td align=\"left\">22</td><td align=\"left\">Parent</td><td align=\"left\">Test</td><td align=\"left\">3</td><td align=\"left\">Frowin</td><td align=\"left\">in Session 1 and 2</td><td align=\"left\">in Session 3</td><td align=\"left\">2/3</td><td align=\"left\">3</td></tr><tr><td align=\"left\">Tai*</td><td align=\"left\">Female</td><td align=\"left\">3</td><td align=\"left\">Parent</td><td align=\"left\">Test</td><td align=\"left\">2</td><td align=\"left\">Frowin</td><td align=\"left\">in Session 1 and 3</td><td align=\"left\">Never</td><td align=\"left\">2/2</td><td align=\"left\">NA</td></tr><tr><td align=\"left\">Coco</td><td align=\"left\">Female</td><td align=\"left\">14</td><td align=\"left\">Hand</td><td align=\"left\">Test</td><td align=\"left\">2</td><td align=\"left\">Paul</td><td align=\"left\">in Session 2</td><td align=\"left\">In Session 1</td><td align=\"left\">1/2</td><td align=\"left\">2</td></tr><tr><td align=\"left\">Pick</td><td align=\"left\">Male</td><td align=\"left\">17</td><td align=\"left\">Hand</td><td align=\"left\">Test</td><td align=\"left\">2</td><td align=\"left\">Paul</td><td align=\"left\">Never</td><td align=\"left\">In Session 1 and 2</td><td align=\"left\">2/2</td><td align=\"left\">NA</td></tr><tr><td align=\"left\">Sunny</td><td align=\"left\">Female</td><td align=\"left\">14</td><td align=\"left\">Hand</td><td align=\"left\">Control Test</td><td align=\"left\">1</td><td align=\"left\">Paul</td><td align=\"left\">Never</td><td align=\"left\">In Session 1</td><td align=\"left\">1/1</td><td align=\"left\">NA</td></tr><tr><td align=\"left\">Skipper</td><td align=\"left\">Male</td><td align=\"left\">4</td><td align=\"left\">Hand</td><td align=\"left\">Control Test</td><td align=\"left\">2</td><td align=\"left\">Paul</td><td align=\"left\">in Session 2 and 3</td><td align=\"left\">Never</td><td align=\"left\">2/2</td><td align=\"left\">NA</td></tr><tr><td align=\"left\">Fay</td><td align=\"left\">Female</td><td align=\"left\">5</td><td align=\"left\">Parent</td><td align=\"left\">Control Test</td><td align=\"left\">1</td><td align=\"left\">Frowin</td><td align=\"left\">in Session 1</td><td align=\"left\">Never</td><td align=\"left\">1/1</td><td align=\"left\">NA</td></tr><tr><td align=\"left\">Plume</td><td align=\"left\">Female</td><td align=\"left\">14</td><td align=\"left\">Hand</td><td align=\"left\">Control Test</td><td align=\"left\">2</td><td align=\"left\">Frowin</td><td align=\"left\">in Session 3</td><td align=\"left\">In Session 1</td><td align=\"left\">1/2</td><td align=\"left\">3</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Individuals that successfully solved the task per experimental group</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Name</th><th align=\\\"left\\\">Sex</th><th align=\\\"left\\\">Age</th><th align=\\\"left\\\">Raised</th><th align=\\\"left\\\">Group</th><th align=\\\"left\\\">No of successful sessions (out of 3)</th><th align=\\\"left\\\">Demonstrator</th><th align=\\\"left\\\">Applied demonstrated sequence</th><th align=\\\"left\\\">Applied non demonstrated sequence</th><th align=\\\"left\\\">Stayed consistent for no of sessions</th><th align=\\\"left\\\">Switched in session no</th></tr></thead><tbody><tr><td align=\\\"left\\\">Frowin</td><td align=\\\"left\\\">Male</td><td align=\\\"left\\\">17</td><td align=\\\"left\\\">Parent</td><td align=\\\"left\\\">Control</td><td align=\\\"left\\\">3</td><td align=\\\"left\\\">NA</td><td align=\\\"left\\\">NA</td><td align=\\\"left\\\">NA</td><td align=\\\"left\\\">3/3</td><td align=\\\"left\\\">NA</td></tr><tr><td align=\\\"left\\\">Papu</td><td align=\\\"left\\\">Female</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">Hand</td><td align=\\\"left\\\">Control</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">NA</td><td align=\\\"left\\\">NA</td><td align=\\\"left\\\">NA</td><td align=\\\"left\\\">1/1</td><td align=\\\"left\\\">NA</td></tr><tr><td align=\\\"left\\\">John*</td><td align=\\\"left\\\">Male</td><td align=\\\"left\\\">22</td><td align=\\\"left\\\">Parent</td><td align=\\\"left\\\">Test</td><td align=\\\"left\\\">3</td><td align=\\\"left\\\">Frowin</td><td align=\\\"left\\\">in Session 1 and 2</td><td align=\\\"left\\\">in Session 3</td><td align=\\\"left\\\">2/3</td><td align=\\\"left\\\">3</td></tr><tr><td align=\\\"left\\\">Tai*</td><td align=\\\"left\\\">Female</td><td align=\\\"left\\\">3</td><td align=\\\"left\\\">Parent</td><td align=\\\"left\\\">Test</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">Frowin</td><td align=\\\"left\\\">in Session 1 and 3</td><td align=\\\"left\\\">Never</td><td align=\\\"left\\\">2/2</td><td align=\\\"left\\\">NA</td></tr><tr><td align=\\\"left\\\">Coco</td><td align=\\\"left\\\">Female</td><td align=\\\"left\\\">14</td><td align=\\\"left\\\">Hand</td><td align=\\\"left\\\">Test</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">Paul</td><td align=\\\"left\\\">in Session 2</td><td align=\\\"left\\\">In Session 1</td><td align=\\\"left\\\">1/2</td><td align=\\\"left\\\">2</td></tr><tr><td align=\\\"left\\\">Pick</td><td align=\\\"left\\\">Male</td><td align=\\\"left\\\">17</td><td align=\\\"left\\\">Hand</td><td align=\\\"left\\\">Test</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">Paul</td><td align=\\\"left\\\">Never</td><td align=\\\"left\\\">In Session 1 and 2</td><td align=\\\"left\\\">2/2</td><td align=\\\"left\\\">NA</td></tr><tr><td align=\\\"left\\\">Sunny</td><td align=\\\"left\\\">Female</td><td align=\\\"left\\\">14</td><td align=\\\"left\\\">Hand</td><td align=\\\"left\\\">Control Test</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">Paul</td><td align=\\\"left\\\">Never</td><td align=\\\"left\\\">In Session 1</td><td align=\\\"left\\\">1/1</td><td align=\\\"left\\\">NA</td></tr><tr><td align=\\\"left\\\">Skipper</td><td align=\\\"left\\\">Male</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">Hand</td><td align=\\\"left\\\">Control Test</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">Paul</td><td align=\\\"left\\\">in Session 2 and 3</td><td align=\\\"left\\\">Never</td><td align=\\\"left\\\">2/2</td><td align=\\\"left\\\">NA</td></tr><tr><td align=\\\"left\\\">Fay</td><td align=\\\"left\\\">Female</td><td align=\\\"left\\\">5</td><td align=\\\"left\\\">Parent</td><td align=\\\"left\\\">Control Test</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">Frowin</td><td align=\\\"left\\\">in Session 1</td><td align=\\\"left\\\">Never</td><td align=\\\"left\\\">1/1</td><td align=\\\"left\\\">NA</td></tr><tr><td align=\\\"left\\\">Plume</td><td align=\\\"left\\\">Female</td><td align=\\\"left\\\">14</td><td align=\\\"left\\\">Hand</td><td align=\\\"left\\\">Control Test</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">Frowin</td><td align=\\\"left\\\">in Session 3</td><td align=\\\"left\\\">In Session 1</td><td align=\\\"left\\\">1/2</td><td align=\\\"left\\\">3</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>","<supplementary-material 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Number of successful sessions out of three test sessions, assigned demonstrator, application of demonstrated sequence and according session information, application of non-demonstrated sequence and according session information, count of consistent opening sequence out of three test sessions, and number of switches in opening sequence out of three test sessions</p><p>*Received two additional trials</p></table-wrap-foot>","<fn-group><fn><p>This article is a contribution to the Topical Collection ‘50 years anniversary of the Nobel Prize in Physiology or Medicine to Karl von Frisch, Konrad Lorenz and Niko Tinbergen in 1973’.</p></fn><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>Table indicates name, sex, age, rearing, and experimental group. Number of successful sessions out of three test sessions, assigned demonstrator, application of demonstrated sequence and according session information, application of non-demonstrated sequence and according session information, count of consistent opening sequence out of three test sessions, and number of switches in opening sequence out of three test sessions</p><p>*Received two additional trials</p></table-wrap-foot>\",\n \"<fn-group><fn><p>This article is a contribution to the Topical Collection ‘50 years anniversary of the Nobel Prize in Physiology or Medicine to Karl von Frisch, Konrad Lorenz and Niko Tinbergen in 1973’.</p></fn><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"10336_2023_2127_Fig1_HTML\" id=\"MO1\"/>"],"string":"[\n \"<graphic xlink:href=\\\"10336_2023_2127_Fig1_HTML\\\" id=\\\"MO1\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"10336_2023_2127_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 574 KB)</p></caption></media>","<media xlink:href=\"10336_2023_2127_MOESM2_ESM.xlsx\"><caption><p>Supplementary file2 (XLSX 60 KB)</p></caption></media>","<media xlink:href=\"10336_2023_2127_MOESM3_ESM.mov\"><caption><p>Supplementary file3 (MOV 212979 KB)</p></caption></media>","<media xlink:href=\"10336_2023_2127_MOESM4_ESM.mov\"><caption><p>Supplementary file4 (MOV 115808 KB)</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"10336_2023_2127_MOESM1_ESM.pdf\\\"><caption><p>Supplementary file1 (PDF 574 KB)</p></caption></media>\",\n \"<media xlink:href=\\\"10336_2023_2127_MOESM2_ESM.xlsx\\\"><caption><p>Supplementary file2 (XLSX 60 KB)</p></caption></media>\",\n \"<media xlink:href=\\\"10336_2023_2127_MOESM3_ESM.mov\\\"><caption><p>Supplementary file3 (MOV 212979 KB)</p></caption></media>\",\n \"<media xlink:href=\\\"10336_2023_2127_MOESM4_ESM.mov\\\"><caption><p>Supplementary file4 (MOV 115808 KB)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"surname\": [\"Akins\", \"Zentall\"], \"given-names\": [\"CK\", \"TR\"], \"article-title\": [\"Imitation in Japanese quail: the role of reinforcement of demonstrator responding\"], \"source\": [\"Psychon Bull Rev\"], \"year\": [\"1998\"], \"volume\": [\"5\"], \"fpage\": [\"694\"], \"lpage\": [\"697\"], \"pub-id\": [\"10.3758/BF03208847\"]}, {\"surname\": [\"Auersperg\", \"von Bayern\", \"Weber\", \"Szabadvari\", \"Bugnyar\", \"Kacelnik\"], \"given-names\": [\"AMI\", \"AMP\", \"S\", \"A\", \"T\", \"A\"], \"article-title\": [\"Social transmission of tool use and tool manufacture in Goffin cockatoos (\"], \"italic\": [\"Cacatua goffini)\"], \"source\": [\"Proc Royal Soc B Biol Sci\"], \"year\": [\"2014\"], \"volume\": [\"281\"], \"fpage\": [\"20140972\"], \"pub-id\": [\"10.1098/rspb.2014.0972\"]}, {\"mixed-citation\": [\"Brejaart R (1988) Diet and feeding behaviour of the kea (\"], \"italic\": [\"Nestor notabilis\"], \"ext-link\": [\"https://hdl.handle.net/10182/5018\"]}, {\"mixed-citation\": [\"Clarke CMH (1971) Observations on population, movements and food of the Kea (\"], \"italic\": [\"Nestor Notabilis).\"]}, {\"surname\": [\"Diamond\", \"Bond\"], \"given-names\": [\"J\", \"A\"], \"source\": [\"Kea, bird of paradox: the evolution and behavior of a New Zealand parrot\"], \"year\": [\"1999\"], \"publisher-name\": [\"University of California Press\"]}, {\"surname\": [\"Fawcett\", \"Skinner\", \"Goldsmith\"], \"given-names\": [\"TW\", \"AM\", \"AR\"], \"article-title\": [\"A test of imitative learning in starlings using a two-action method with an enhanced ghost control\"], \"source\": [\"Anim Behav\"], \"year\": [\"2002\"], \"volume\": [\"64\"], \"fpage\": [\"547\"], \"lpage\": [\"556\"], \"pub-id\": [\"10.1006/anbe.2002.3092\"]}, {\"surname\": [\"Gajdon\", \"Fijn\", \"Huber\"], \"given-names\": [\"GK\", \"N\", \"L\"], \"article-title\": [\"Testing social learning in a wild mountain parrot, the kea (\"], \"italic\": [\"Nestor notabilis\"], \"source\": [\"Anim Learn Behav\"], \"year\": [\"2004\"], \"volume\": [\"32\"], \"fpage\": [\"62\"], \"lpage\": [\"71\"], \"pub-id\": [\"10.3758/BF03196007\"]}, {\"surname\": [\"Galef\", \"Manzig\", \"Field\"], \"given-names\": [\"BG\", \"LA\", \"RM\"], \"article-title\": [\"Imitation learning in budgerigars: Dawson and Foss (1965) revisited\"], \"source\": [\"Behav Proc\"], \"year\": [\"1986\"], \"volume\": [\"13\"], \"fpage\": [\"191\"], \"lpage\": [\"202\"], \"pub-id\": [\"10.1016/0376-6357(86)90025-2\"]}, {\"mixed-citation\": [\"Galef BG (1988) Imitation in animals: History, definition, and interpretation of data from the psychological laboratory. In: Zentall TR, Galef BG (Eds) (1988) Social learning: Psychological and biological perspectives. Psychology Press. 10.4324/9781315801889\"]}, {\"surname\": [\"Giraldeau\", \"Valone\", \"Templeton\"], \"given-names\": [\"LA\", \"TJ\", \"JJ\"], \"article-title\": [\"Potential disadvantages of using socially acquired information\"], \"source\": [\"Phil Trans Royal Soc Lond\"], \"year\": [\"2002\"], \"volume\": [\"B357\"], \"fpage\": [\"1559\"], \"lpage\": [\"1566\"], \"pub-id\": [\"10.1098/rstb.2002.1065\"]}, {\"surname\": [\"Heyes\", \"Saggerson\"], \"given-names\": [\"C\", \"A\"], \"article-title\": [\"Testing for imitative and nonimitative social learning in the budgerigar using a two-object/two-action test\"], \"source\": [\"Anim Behav\"], \"year\": [\"2002\"], \"volume\": [\"64\"], \"fpage\": [\"851\"], \"lpage\": [\"859\"], \"pub-id\": [\"10.1006/anbe.2003.2002\"]}, {\"surname\": [\"Hoppitt\", \"Laland\"], \"given-names\": [\"W\", \"KN\"], \"source\": [\"Social learning: an introduction to mechanisms, methods, and models\"], \"year\": [\"2013\"], \"publisher-name\": [\"Princeton University Press\"]}, {\"surname\": [\"Huber\"], \"given-names\": [\"L\"], \"article-title\": [\"Clever birds: Keas learn through observation\"], \"source\": [\"Interpretive Birding Bulletin\"], \"year\": [\"2002\"], \"volume\": [\"3\"], \"fpage\": [\"57\"], \"lpage\": [\"59\"]}, {\"surname\": [\"Huber\", \"Rechberger\", \"Taborsky\"], \"given-names\": [\"L\", \"S\", \"M\"], \"article-title\": [\"Social learning affects object exploration and manipulation in keas, \"], \"italic\": [\"Nestor notabilis\"], \"source\": [\"Anim Behav\"], \"year\": [\"2001\"], \"volume\": [\"62\"], \"fpage\": [\"945\"], \"lpage\": [\"954\"], \"pub-id\": [\"10.1006/anbe.2001.1822\"]}, {\"surname\": [\"Huber\", \"Range\", \"Voelkl\", \"Szucsich\", \"Viranyi\", \"Miklosi\"], \"given-names\": [\"L\", \"F\", \"B\", \"A\", \"Z\", \"A\"], \"article-title\": [\"The evolution of imitation: what do the capacities of non-human animals tell us about the mechanisms of imitation?\"], \"source\": [\"Philosoph Transact Royal Soc B Biol Sci\"], \"year\": [\"2009\"], \"volume\": [\"364\"], \"fpage\": [\"2299\"], \"lpage\": [\"2309\"], \"pub-id\": [\"10.1098/rstb.2009.0060\"]}, {\"surname\": [\"Loretto\", \"Schuster\", \"Federspiel\", \"Heinrich\", \"Bugnyar\"], \"given-names\": [\"MC\", \"R\", \"IG\", \"B\", \"T\"], \"article-title\": [\"Contextual imitation in juvenile common ravens, \"], \"italic\": [\"Corvus corax\"], \"source\": [\"Anim Behav\"], \"year\": [\"2020\"], \"volume\": [\"163\"], \"fpage\": [\"127\"], \"lpage\": [\"134\"], \"pub-id\": [\"10.1016/j.anbehav.2020.03.007\"]}, {\"surname\": [\"Meltzoff\", \"Zentall\", \"Galef\"], \"given-names\": [\"AN\", \"TR\", \"BG\"], \"article-title\": [\"The human infant as \"], \"italic\": [\"Homo imitans\"], \"source\": [\"Social learning: psychological and biological perspectives\"], \"year\": [\"1988\"], \"publisher-loc\": [\"Hillsdale\"], \"publisher-name\": [\"Erlbaum\"], \"fpage\": [\"319\"], \"lpage\": [\"341\"]}, {\"surname\": [\"Mettke-Hofmann\", \"Winkler\", \"Leisler\"], \"given-names\": [\"C\", \"H\", \"B\"], \"article-title\": [\"The significance of ecological factors for exploration and neophobia in parrots\"], \"source\": [\"Ethology\"], \"year\": [\"2002\"], \"volume\": [\"108\"], \"fpage\": [\"249\"], \"lpage\": [\"272\"], \"pub-id\": [\"10.1046/j.1439-0310.2002.00773.x\"]}, {\"surname\": [\"Moore\"], \"given-names\": [\"BR\"], \"article-title\": [\"Avian movement imitation and a new form of mimicry: tracing the evolution of a complex form of learning\"], \"source\": [\"Behaviour\"], \"year\": [\"1992\"], \"volume\": [\"122\"], \"fpage\": [\"231\"], \"lpage\": [\"263\"], \"pub-id\": [\"10.1163/156853992X00525\"]}, {\"mixed-citation\": [\"O'Donnell CF, Dilks PJ (1994) Foods and foraging of forest birds in temperate rainforest, South Westland, New Zealand. NZd J Ecol 18:87\\u2013107. \"], \"ext-link\": [\"http://www.jstor.org/stable/24066768\"]}, {\"surname\": [\"Pepperberg\", \"Funk\"], \"given-names\": [\"IM\", \"MS\"], \"article-title\": [\"Object permanence in four species of psittacine birds: An African Grey parrot (\"], \"italic\": [\"Psittacus erithacus\", \"Ara maracana\", \"Melopsittacus undulatus\", \"Nymphicus hollandicus\"], \"source\": [\"Anim Learn Behav\"], \"year\": [\"1990\"], \"volume\": [\"18\"], \"fpage\": [\"97\"], \"lpage\": [\"108\"], \"pub-id\": [\"10.3758/BF03205244\"]}, {\"surname\": [\"Roberts\"], \"given-names\": [\"D\"], \"article-title\": [\"Imitation and suggestion in animals\"], \"source\": [\"Bull Anim Behav\"], \"year\": [\"1941\"], \"volume\": [\"1\"], \"fpage\": [\"11\"], \"lpage\": [\"19\"]}, {\"surname\": [\"Slagsvold\", \"Wiebe\"], \"given-names\": [\"T\", \"KL\"], \"article-title\": [\"Social learning in birds and its role in shaping a foraging niche\"], \"source\": [\"Philosoph Transact Royal Soc B Biol Sci\"], \"year\": [\"2011\"], \"volume\": [\"366\"], \"fpage\": [\"969\"], \"lpage\": [\"977\"], \"pub-id\": [\"10.1098/rstb.2010.0343\"]}, {\"surname\": [\"Tennie\", \"Call\", \"Tomasello\"], \"given-names\": [\"C\", \"J\", \"M\"], \"article-title\": [\"Push or pull: Imitation vs. emulation in great apes and human children\"], \"source\": [\"Ethology\"], \"year\": [\"2006\"], \"volume\": [\"112\"], \"fpage\": [\"1159\"], \"lpage\": [\"1169\"], \"pub-id\": [\"10.1111/j.1439-0310.2006.01269.x\"]}, {\"surname\": [\"Tomasello\"], \"given-names\": [\"M\"], \"article-title\": [\"Emulation learning and cultural learning\"], \"source\": [\"Behav Brain Sci\"], \"year\": [\"1998\"], \"volume\": [\"21\"], \"fpage\": [\"703\"], \"lpage\": [\"704\"], \"pub-id\": [\"10.1017/S0140525X98441748\"]}, {\"surname\": [\"Werdenich\", \"Huber\"], \"given-names\": [\"D\", \"L\"], \"article-title\": [\"A case of quick problem solving in birds: string pulling in keas, \"], \"italic\": [\"Nestor notabilis\"], \"source\": [\"Anim Behav\"], \"year\": [\"2006\"], \"volume\": [\"71\"], \"fpage\": [\"855\"], \"lpage\": [\"863\"], \"pub-id\": [\"10.1016/j.anbehav.2005.06.018\"]}, {\"surname\": [\"Weser\", \"Ross\"], \"given-names\": [\"C\", \"JG\"], \"article-title\": [\"The effect of colour on bait consumption of kea (\"], \"italic\": [\"Nestor notabilis\"], \"source\": [\"NZd J Zool\"], \"year\": [\"2013\"], \"volume\": [\"40\"], \"fpage\": [\"137\"], \"lpage\": [\"144\"], \"pub-id\": [\"10.1080/03014223.2012.710639\"]}, {\"surname\": [\"Whiten\", \"Ham\"], \"given-names\": [\"A\", \"R\"], \"article-title\": [\"On the Nature and evolution of imitation in the animal kingdom: reappraisal of a century of research\"], \"source\": [\"Adv Study of Behav\"], \"year\": [\"1992\"], \"volume\": [\"21\"], \"fpage\": [\"239\"], \"lpage\": [\"283\"], \"pub-id\": [\"10.1016/S0065-3454(08)60146-1\"]}, {\"surname\": [\"Whiten\", \"McGuigan\", \"Marshall-Pescini\", \"Hopper\"], \"given-names\": [\"A\", \"N\", \"S\", \"LM\"], \"article-title\": [\"Emulation, imitation, over-imitation and the scope of culture for child and chimpanzee\"], \"source\": [\"Philosoph Transact Royal Soc B Biol Sci\"], \"year\": [\"2009\"], \"volume\": [\"364\"], \"fpage\": [\"2417\"], \"lpage\": [\"2428\"], \"pub-id\": [\"10.1098/rstb.2009.0069\"]}, {\"surname\": [\"Zentall\"], \"given-names\": [\"TR\"], \"article-title\": [\"Imitation in animals: evidence, function, and mechanisms\"], \"source\": [\"Cybern Syst\"], \"year\": [\"2001\"], \"volume\": [\"32\"], \"fpage\": [\"53\"], \"lpage\": [\"96\"], \"pub-id\": [\"10.1080/019697201300001812\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":56,"string":"56"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"J Ornithol. 2024 Dec 16; 165(1):49-55"},"package_file":{"kind":"string","value":"oa_package/79/d5/PMC10787887.tar.gz"}}},{"rowIdx":171,"cells":{"accession_id":{"kind":"string","value":"PMC10787888"},"pmid":{"kind":"string","value":"38150097"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par4\">Glucuronoyl esterases cleave ester-linked lignin-carbohydrate complexes (LCCs) in lignocellulosic biomass (Mosbech et al. ##REF##29321810##2018##). In this way, their catalytic action assists in decomposing the plant cell wall matrix by removing some of the covalent interpolymeric linkages that sustain lignocellulose recalcitrance.</p>","<p id=\"Par5\">As literature sees it today, ester-linked LCCs exist in vivo between the α-1,2-linked-D-glucuronoyl substitutions of glucuronoxylan and the aliphatic hydroxyl groups (Balakshin et al. ##REF##21298285##2011##; Yuan et al. ##REF##21879769##2011##). An important additional feature observed in many wood and cereal types of biomass, is the 4-<italic>O</italic>-methylation on said glucuronoyl, which creates a unique plant-based trademark for these aldouronic acids. We commonly agree on the carbohydrate side of the LCC, but the exact nature of the aliphatic alcohol on lignin remains a matter of debate (Giummarella et al. ##REF##31294799##2019##; Sapouna & Lawoko ##UREF##3##2021##). In the most abundant lignin-substructures, two alcohol moieties present themselves as possible esterification points, namely the α- and the γ-positioned ones (Fig. ##FIG##0##1##, structure 1 vs. structure 2), and whichever is most dominant in vivo is difficult to verify, because the occurrence is low, and because they prove difficult to isolate and annotate exactly. Ester bound acids tend to migrate (Puchart et al. ##REF##31952591##2020##) and extensive sample handling in order to enrich these linkages may introduce artifacts. Hence, de novo ester synthesis in the plant cell may start in one position and migrate to a different one, as the cell wall matures (Li & Helm ##UREF##1##1995##).</p>","<p id=\"Par6\">Glucuronoyl esterases of both fungal and bacterial origin has received much attention in research during the past ten to fifteen years since their discovery (Agger et al. ##REF##37256329##2023##; Biely ##UREF##0##2016##; Larsbrink and Lo Leggio ##REF##36651189##2023##). Activity has been demonstrated widely on both synthetic and natural substrates, and several crystal structures reveal the catalytic mechanism of these canonical α/β-hydrolases of the serine-esterase type (Baath et al. ##REF##30814248##2019##; Charavgi et al. ##REF##23275164##2013##; Ernst et al. ##REF##31911652##2020##; Mazurkewich et al. ##UREF##2##2019##; Topakas et al. ##REF##20473662##2010##). We know that fungal variants are generally more restricted in their substrate preferences than the bacterial enzymes, because most of the fungal ones are dependent on the 4-<italic>O</italic>-methyl-modification on the glucuronoyl moiety for recognition. Bacterial enzymes are broader in their substrate preferences in terms of the sugar-moiety, but it is still uncertain for all types of glucuronoyl esterases, if they have specificity towards the configuration of the lignin-alcohol and if so, which of the two possible variants they prefer.</p>","<p id=\"Par7\">We have investigated the substrate specificity of a fungal glucuronoyl esterase from <italic>Cerrena unicolor</italic> (<italic>Cu</italic>GE) by exploiting the fact that esterases often display transesterification capacities under the right reaction conditions. We hypothesize that the enzyme will preferably perform transesterification with the type of alcohol (α- or γ-positioned) that fits its substrate specificities best. We performed transesterifications with either methyl-glucuronate or methyl-4-<italic>O</italic>-methyl-glucuronate as donor substrates, and benzyl-alcohol or 3-phenyl-1-propanol as the alcohol-acceptors according to the four reactions outlined in Fig. ##FIG##0##1##.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par4\\\">Glucuronoyl esterases cleave ester-linked lignin-carbohydrate complexes (LCCs) in lignocellulosic biomass (Mosbech et al. ##REF##29321810##2018##). In this way, their catalytic action assists in decomposing the plant cell wall matrix by removing some of the covalent interpolymeric linkages that sustain lignocellulose recalcitrance.</p>\",\n \"<p id=\\\"Par5\\\">As literature sees it today, ester-linked LCCs exist in vivo between the α-1,2-linked-D-glucuronoyl substitutions of glucuronoxylan and the aliphatic hydroxyl groups (Balakshin et al. ##REF##21298285##2011##; Yuan et al. ##REF##21879769##2011##). An important additional feature observed in many wood and cereal types of biomass, is the 4-<italic>O</italic>-methylation on said glucuronoyl, which creates a unique plant-based trademark for these aldouronic acids. We commonly agree on the carbohydrate side of the LCC, but the exact nature of the aliphatic alcohol on lignin remains a matter of debate (Giummarella et al. ##REF##31294799##2019##; Sapouna & Lawoko ##UREF##3##2021##). In the most abundant lignin-substructures, two alcohol moieties present themselves as possible esterification points, namely the α- and the γ-positioned ones (Fig. ##FIG##0##1##, structure 1 vs. structure 2), and whichever is most dominant in vivo is difficult to verify, because the occurrence is low, and because they prove difficult to isolate and annotate exactly. Ester bound acids tend to migrate (Puchart et al. ##REF##31952591##2020##) and extensive sample handling in order to enrich these linkages may introduce artifacts. Hence, de novo ester synthesis in the plant cell may start in one position and migrate to a different one, as the cell wall matures (Li & Helm ##UREF##1##1995##).</p>\",\n \"<p id=\\\"Par6\\\">Glucuronoyl esterases of both fungal and bacterial origin has received much attention in research during the past ten to fifteen years since their discovery (Agger et al. ##REF##37256329##2023##; Biely ##UREF##0##2016##; Larsbrink and Lo Leggio ##REF##36651189##2023##). Activity has been demonstrated widely on both synthetic and natural substrates, and several crystal structures reveal the catalytic mechanism of these canonical α/β-hydrolases of the serine-esterase type (Baath et al. ##REF##30814248##2019##; Charavgi et al. ##REF##23275164##2013##; Ernst et al. ##REF##31911652##2020##; Mazurkewich et al. ##UREF##2##2019##; Topakas et al. ##REF##20473662##2010##). We know that fungal variants are generally more restricted in their substrate preferences than the bacterial enzymes, because most of the fungal ones are dependent on the 4-<italic>O</italic>-methyl-modification on the glucuronoyl moiety for recognition. Bacterial enzymes are broader in their substrate preferences in terms of the sugar-moiety, but it is still uncertain for all types of glucuronoyl esterases, if they have specificity towards the configuration of the lignin-alcohol and if so, which of the two possible variants they prefer.</p>\",\n \"<p id=\\\"Par7\\\">We have investigated the substrate specificity of a fungal glucuronoyl esterase from <italic>Cerrena unicolor</italic> (<italic>Cu</italic>GE) by exploiting the fact that esterases often display transesterification capacities under the right reaction conditions. We hypothesize that the enzyme will preferably perform transesterification with the type of alcohol (α- or γ-positioned) that fits its substrate specificities best. We performed transesterifications with either methyl-glucuronate or methyl-4-<italic>O</italic>-methyl-glucuronate as donor substrates, and benzyl-alcohol or 3-phenyl-1-propanol as the alcohol-acceptors according to the four reactions outlined in Fig. ##FIG##0##1##.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>LC–MS method</title>","<p id=\"Par17\">Reaction product profiles were analyzed by LC–MS. 5 µL of reaction mixture was injected onto a Hypercarb column (150 mm × 2.1 mm; 3μm, Thermo Fischer Scientific, Sunnyvale, CA, USA). The chromatography was performed on a Dionex UltiMate 3000 UPLC (Thermo Fischer Scientific, Sunnyvale, CA, USA) at 0.4 mL min<sup>−1</sup> and 70 °C with a two-eluent system consisting of eluent A (acetonitrile) and eluent B (water). The elution was performed as follows (time indicated in min): 0–3, 0% A 100% B; 3–10, isocratic 40% A 60% B; 10–19, isocratic 0% A 100% B. Water was used as eluent B to avoid excessive spontaneous hydrolysis of either donor substrates or products on the LC-column.</p>","<p id=\"Par18\">For the analysis of the transesterification of PhPrOH and Me-4-<italic>O</italic>-MeGlcA the chromatography was performed on the same system and condition of flow, temperature and eluent system but the elution was performed as follows (time indicated in min): 0–3, 0% A 100% B; 3–10, isocratic 70% A 30% B; 10–19, isocratic 0% A 100% B. The differences in elution system for this particular reaction was in order to elute the stronger retained transesterification product of this reaction compared to the other reactions within the same period.</p>","<p id=\"Par19\">The HPLC was connected to an ESI-iontrap (model Amazon SL from Bruker Daltonics, Bremen, Germany) and the electrospray was operated in positive ultra scan mode using a target mass of 300 m/z. A scan range from 100 to 2000 m/z was selected and capillary voltage was set to 4.5 kV, end plate offset 0.5 kV, nebulizer pressure at 3.0 bar, dry gas flow at 12.0 L min<sup>−1</sup>, and dry gas temperature at 280 °C.</p>"],"string":"[\n \"<title>LC–MS method</title>\",\n \"<p id=\\\"Par17\\\">Reaction product profiles were analyzed by LC–MS. 5 µL of reaction mixture was injected onto a Hypercarb column (150 mm × 2.1 mm; 3μm, Thermo Fischer Scientific, Sunnyvale, CA, USA). The chromatography was performed on a Dionex UltiMate 3000 UPLC (Thermo Fischer Scientific, Sunnyvale, CA, USA) at 0.4 mL min<sup>−1</sup> and 70 °C with a two-eluent system consisting of eluent A (acetonitrile) and eluent B (water). The elution was performed as follows (time indicated in min): 0–3, 0% A 100% B; 3–10, isocratic 40% A 60% B; 10–19, isocratic 0% A 100% B. Water was used as eluent B to avoid excessive spontaneous hydrolysis of either donor substrates or products on the LC-column.</p>\",\n \"<p id=\\\"Par18\\\">For the analysis of the transesterification of PhPrOH and Me-4-<italic>O</italic>-MeGlcA the chromatography was performed on the same system and condition of flow, temperature and eluent system but the elution was performed as follows (time indicated in min): 0–3, 0% A 100% B; 3–10, isocratic 70% A 30% B; 10–19, isocratic 0% A 100% B. The differences in elution system for this particular reaction was in order to elute the stronger retained transesterification product of this reaction compared to the other reactions within the same period.</p>\",\n \"<p id=\\\"Par19\\\">The HPLC was connected to an ESI-iontrap (model Amazon SL from Bruker Daltonics, Bremen, Germany) and the electrospray was operated in positive ultra scan mode using a target mass of 300 m/z. A scan range from 100 to 2000 m/z was selected and capillary voltage was set to 4.5 kV, end plate offset 0.5 kV, nebulizer pressure at 3.0 bar, dry gas flow at 12.0 L min<sup>−1</sup>, and dry gas temperature at 280 °C.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results and discussion</title>","<p id=\"Par26\">Initial hydrolytic experiments with either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA as substrate and in the absence of alcohol acceptors show substrate conversion as expected (Fig. ##FIG##1##2##), with a significantly higher level of conversion for the 4-<italic>O</italic>-methylated ester compared to the non-derivatized glucuronate ester within the first 100 min of reaction (Table ##TAB##0##1##). Enzyme concentrations were dosed to allow similar rates of catalysis in all experiments, independent of substrate, and consequently <italic>Cu</italic>GE was dosed 50 times less in the experiments where Me-4-<italic>O</italic>-MeGlcA was the substrate compared to the experiments with Me-GlcA as substrate. The fact that <italic>Cu</italic>GE prefers its substrates to have the 4-<italic>O</italic>-methyl-modification on the glucuronoyl is well-establised (d’Errico et al. ##REF##25425346##2015##; Ernst et al. ##REF##31911652##2020##; Monrad et al. ##REF##29739247##2018##). As the 100 min reaction time progresses, it becomes evident that the hydrolytic reaction slows down for both substrates (Table ##TAB##0##1##), which again is not surprising as the affinity (K<sub>M</sub>) for these synthetic model substrates is known to be relatively high (d’Errico et al. ##REF##25425346##2015##).</p>","<p id=\"Par27\">It is known, that glucuronoyl esterases prefer bulky alcohols and in that respect, the methyl-esters are poor substrates. Experiments with either benzyl-alcohol or 3-phenyl-1-propanol as the primary solvent and thereby dominating acceptor molecule (14% v/v water), show that the transesterification reaction occurring between Me-4-<italic>O</italic>-MeGlcA and PhPrOH (Fig. ##FIG##0##1##, reaction 1) is significantly higher than any of the other combinations (Fig. ##FIG##2##3##). The reaction between Me-4-<italic>O</italic>-MeGlcA and BnzOH is second best (Fig. ##FIG##0##1##, reaction 2) with about 3–4 fold less product compared to the PhPr-GlcA ester. Quantifications of the ester products are done relative to the standard of Bnz-GlcA dissolved either in BnzOH or PhPrOH depending on the relevant acceptor alcohol, and the calibrations indicate a certain level of ion suppression when PhPrOH is the solvent (Supplementary standards curves), hence leading to a potential underestimation in that respect. PhPrOH has similar retention time as the transesterification products. At the same time, compounds carrying the 4-<italic>O</italic>-methylation tends to ionize better, and therefore give stronger responses. In the reactions performed here, enzyme loadings vary according to formation of transesterification products, as the goal is to quantify transesterification products. Hence, at low enzyme loadings, hydrolysis is not observed for reactions containing Me-4-<italic>O</italic>-MeGlcA as donor (and relatively low water concentration), whereas increasing the enzyme concentration yields hydrolytic reactions in parallel to the transesterification (data not shown). Ultimately, the extent of hydrolysis is a competition for acceptor substrate, which appears favored in the case of the alternative alcohol when enzyme concentrations are low. Hydrolytic reactions are certainly present but becomes un-quantifiable at conditions where transesterification is dominating.</p>","<p id=\"Par28\">Interestingly, the 4-<italic>O</italic>-methylation is a more important feature for catalysis than the nature of the acceptor-alcohol, since the Bnz-4-<italic>O</italic>-MeGlcA-ester forms faster than the PhPr-GlcA ester. A cautious estimation of the effect of the donor versus the effect of the acceptor demonstrates that the ratio between concentrations of products formed during the reaction time is higher when the donor is changed, compared to when the acceptor is changed (Fig. ##FIG##3##4##).</p>","<p id=\"Par29\">Hence, the effect of changing the donor substrate between the 4-<italic>O</italic>-methylated and non-methylated counterparts is larger than the effect of changing the alcohol acceptor. This observation may not be valid for all glucuronoyl esterases, as it is common knowledge that not all GEs favor the 4-<italic>O</italic>-methylation.</p>","<p id=\"Par30\">Recent QM/MM studies of the fungal <italic>Thermothelomyces thermophila</italic> glucuronoyl esterase (<italic>Tt</italic>GE) show that the acylation step is the most energy demanding and hence rate-limiting step (Viegas et al. ##REF##35925549##2022##), whereas similar studies with a bacterial <italic>Ot</italic>CE15A from <italic>Opitutus terrae</italic> show that the deacylation-step is rate-limiting (Zong et al. ##REF##34983933##2022##). Without having investigated the energetic landscape of catalysis by <italic>Cu</italic>GE, the transesterification reactions we observe support the observation that the acylation-step is determining for reaction speed. However, it is well-known that the differences between fungal and bacterial GEs are quite large in terms of overall structure (Larsbrink and Lo Leggio ##REF##36651189##2023##), and transesterification reactions of the kind presented here, may turn out different for bacterial GEs with respect to energetic fingerprints.</p>","<p id=\"Par31\">The aim of this study is to investigate the preference for either an α- or a γ-positioned alcohol, and the results show that the γ-positioned alcohol is most favorable, yet the enzyme can perform the reaction with the α-alcohol. There are currently no univocal structural explanations for how the GEs interact with the alcohol moiety of the substrate. However, we have recently performed docking simulations with these exact two esters; Bnz-4-<italic>O</italic>-MeGlcA and PhPr-4-<italic>O</italic>-MeGlcA as ligands in <italic>Cu</italic>GE and guided by the sugar-moiety, which show steric obstacles when the α-benzyl constitutes the alcohol part of the ligand in contrary to the γ-linked ester (Agger et al. ##REF##37256329##2023##).</p>","<p id=\"Par32\">Exploiting the transesterification capacities of these enzymes potentially opens for other biotechnological applications than biomass deconstruction, such as functionalization of hemicellulose or smaller aldouronic acids with alcohols of different properties (hydrophobicity, other functional groups etc.). Future studies may explore the field of potential acceptors more broadly than here, and thereby determine the diversity of alcohols and properties that could be relevant to investigate further.</p>"],"string":"[\n \"<title>Results and discussion</title>\",\n \"<p id=\\\"Par26\\\">Initial hydrolytic experiments with either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA as substrate and in the absence of alcohol acceptors show substrate conversion as expected (Fig. ##FIG##1##2##), with a significantly higher level of conversion for the 4-<italic>O</italic>-methylated ester compared to the non-derivatized glucuronate ester within the first 100 min of reaction (Table ##TAB##0##1##). Enzyme concentrations were dosed to allow similar rates of catalysis in all experiments, independent of substrate, and consequently <italic>Cu</italic>GE was dosed 50 times less in the experiments where Me-4-<italic>O</italic>-MeGlcA was the substrate compared to the experiments with Me-GlcA as substrate. The fact that <italic>Cu</italic>GE prefers its substrates to have the 4-<italic>O</italic>-methyl-modification on the glucuronoyl is well-establised (d’Errico et al. ##REF##25425346##2015##; Ernst et al. ##REF##31911652##2020##; Monrad et al. ##REF##29739247##2018##). As the 100 min reaction time progresses, it becomes evident that the hydrolytic reaction slows down for both substrates (Table ##TAB##0##1##), which again is not surprising as the affinity (K<sub>M</sub>) for these synthetic model substrates is known to be relatively high (d’Errico et al. ##REF##25425346##2015##).</p>\",\n \"<p id=\\\"Par27\\\">It is known, that glucuronoyl esterases prefer bulky alcohols and in that respect, the methyl-esters are poor substrates. Experiments with either benzyl-alcohol or 3-phenyl-1-propanol as the primary solvent and thereby dominating acceptor molecule (14% v/v water), show that the transesterification reaction occurring between Me-4-<italic>O</italic>-MeGlcA and PhPrOH (Fig. ##FIG##0##1##, reaction 1) is significantly higher than any of the other combinations (Fig. ##FIG##2##3##). The reaction between Me-4-<italic>O</italic>-MeGlcA and BnzOH is second best (Fig. ##FIG##0##1##, reaction 2) with about 3–4 fold less product compared to the PhPr-GlcA ester. Quantifications of the ester products are done relative to the standard of Bnz-GlcA dissolved either in BnzOH or PhPrOH depending on the relevant acceptor alcohol, and the calibrations indicate a certain level of ion suppression when PhPrOH is the solvent (Supplementary standards curves), hence leading to a potential underestimation in that respect. PhPrOH has similar retention time as the transesterification products. At the same time, compounds carrying the 4-<italic>O</italic>-methylation tends to ionize better, and therefore give stronger responses. In the reactions performed here, enzyme loadings vary according to formation of transesterification products, as the goal is to quantify transesterification products. Hence, at low enzyme loadings, hydrolysis is not observed for reactions containing Me-4-<italic>O</italic>-MeGlcA as donor (and relatively low water concentration), whereas increasing the enzyme concentration yields hydrolytic reactions in parallel to the transesterification (data not shown). Ultimately, the extent of hydrolysis is a competition for acceptor substrate, which appears favored in the case of the alternative alcohol when enzyme concentrations are low. Hydrolytic reactions are certainly present but becomes un-quantifiable at conditions where transesterification is dominating.</p>\",\n \"<p id=\\\"Par28\\\">Interestingly, the 4-<italic>O</italic>-methylation is a more important feature for catalysis than the nature of the acceptor-alcohol, since the Bnz-4-<italic>O</italic>-MeGlcA-ester forms faster than the PhPr-GlcA ester. A cautious estimation of the effect of the donor versus the effect of the acceptor demonstrates that the ratio between concentrations of products formed during the reaction time is higher when the donor is changed, compared to when the acceptor is changed (Fig. ##FIG##3##4##).</p>\",\n \"<p id=\\\"Par29\\\">Hence, the effect of changing the donor substrate between the 4-<italic>O</italic>-methylated and non-methylated counterparts is larger than the effect of changing the alcohol acceptor. This observation may not be valid for all glucuronoyl esterases, as it is common knowledge that not all GEs favor the 4-<italic>O</italic>-methylation.</p>\",\n \"<p id=\\\"Par30\\\">Recent QM/MM studies of the fungal <italic>Thermothelomyces thermophila</italic> glucuronoyl esterase (<italic>Tt</italic>GE) show that the acylation step is the most energy demanding and hence rate-limiting step (Viegas et al. ##REF##35925549##2022##), whereas similar studies with a bacterial <italic>Ot</italic>CE15A from <italic>Opitutus terrae</italic> show that the deacylation-step is rate-limiting (Zong et al. ##REF##34983933##2022##). Without having investigated the energetic landscape of catalysis by <italic>Cu</italic>GE, the transesterification reactions we observe support the observation that the acylation-step is determining for reaction speed. However, it is well-known that the differences between fungal and bacterial GEs are quite large in terms of overall structure (Larsbrink and Lo Leggio ##REF##36651189##2023##), and transesterification reactions of the kind presented here, may turn out different for bacterial GEs with respect to energetic fingerprints.</p>\",\n \"<p id=\\\"Par31\\\">The aim of this study is to investigate the preference for either an α- or a γ-positioned alcohol, and the results show that the γ-positioned alcohol is most favorable, yet the enzyme can perform the reaction with the α-alcohol. There are currently no univocal structural explanations for how the GEs interact with the alcohol moiety of the substrate. However, we have recently performed docking simulations with these exact two esters; Bnz-4-<italic>O</italic>-MeGlcA and PhPr-4-<italic>O</italic>-MeGlcA as ligands in <italic>Cu</italic>GE and guided by the sugar-moiety, which show steric obstacles when the α-benzyl constitutes the alcohol part of the ligand in contrary to the γ-linked ester (Agger et al. ##REF##37256329##2023##).</p>\",\n \"<p id=\\\"Par32\\\">Exploiting the transesterification capacities of these enzymes potentially opens for other biotechnological applications than biomass deconstruction, such as functionalization of hemicellulose or smaller aldouronic acids with alcohols of different properties (hydrophobicity, other functional groups etc.). Future studies may explore the field of potential acceptors more broadly than here, and thereby determine the diversity of alcohols and properties that could be relevant to investigate further.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Results and discussion</title>","<p id=\"Par26\">Initial hydrolytic experiments with either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA as substrate and in the absence of alcohol acceptors show substrate conversion as expected (Fig. ##FIG##1##2##), with a significantly higher level of conversion for the 4-<italic>O</italic>-methylated ester compared to the non-derivatized glucuronate ester within the first 100 min of reaction (Table ##TAB##0##1##). Enzyme concentrations were dosed to allow similar rates of catalysis in all experiments, independent of substrate, and consequently <italic>Cu</italic>GE was dosed 50 times less in the experiments where Me-4-<italic>O</italic>-MeGlcA was the substrate compared to the experiments with Me-GlcA as substrate. The fact that <italic>Cu</italic>GE prefers its substrates to have the 4-<italic>O</italic>-methyl-modification on the glucuronoyl is well-establised (d’Errico et al. ##REF##25425346##2015##; Ernst et al. ##REF##31911652##2020##; Monrad et al. ##REF##29739247##2018##). As the 100 min reaction time progresses, it becomes evident that the hydrolytic reaction slows down for both substrates (Table ##TAB##0##1##), which again is not surprising as the affinity (K<sub>M</sub>) for these synthetic model substrates is known to be relatively high (d’Errico et al. ##REF##25425346##2015##).</p>","<p id=\"Par27\">It is known, that glucuronoyl esterases prefer bulky alcohols and in that respect, the methyl-esters are poor substrates. Experiments with either benzyl-alcohol or 3-phenyl-1-propanol as the primary solvent and thereby dominating acceptor molecule (14% v/v water), show that the transesterification reaction occurring between Me-4-<italic>O</italic>-MeGlcA and PhPrOH (Fig. ##FIG##0##1##, reaction 1) is significantly higher than any of the other combinations (Fig. ##FIG##2##3##). The reaction between Me-4-<italic>O</italic>-MeGlcA and BnzOH is second best (Fig. ##FIG##0##1##, reaction 2) with about 3–4 fold less product compared to the PhPr-GlcA ester. Quantifications of the ester products are done relative to the standard of Bnz-GlcA dissolved either in BnzOH or PhPrOH depending on the relevant acceptor alcohol, and the calibrations indicate a certain level of ion suppression when PhPrOH is the solvent (Supplementary standards curves), hence leading to a potential underestimation in that respect. PhPrOH has similar retention time as the transesterification products. At the same time, compounds carrying the 4-<italic>O</italic>-methylation tends to ionize better, and therefore give stronger responses. In the reactions performed here, enzyme loadings vary according to formation of transesterification products, as the goal is to quantify transesterification products. Hence, at low enzyme loadings, hydrolysis is not observed for reactions containing Me-4-<italic>O</italic>-MeGlcA as donor (and relatively low water concentration), whereas increasing the enzyme concentration yields hydrolytic reactions in parallel to the transesterification (data not shown). Ultimately, the extent of hydrolysis is a competition for acceptor substrate, which appears favored in the case of the alternative alcohol when enzyme concentrations are low. Hydrolytic reactions are certainly present but becomes un-quantifiable at conditions where transesterification is dominating.</p>","<p id=\"Par28\">Interestingly, the 4-<italic>O</italic>-methylation is a more important feature for catalysis than the nature of the acceptor-alcohol, since the Bnz-4-<italic>O</italic>-MeGlcA-ester forms faster than the PhPr-GlcA ester. A cautious estimation of the effect of the donor versus the effect of the acceptor demonstrates that the ratio between concentrations of products formed during the reaction time is higher when the donor is changed, compared to when the acceptor is changed (Fig. ##FIG##3##4##).</p>","<p id=\"Par29\">Hence, the effect of changing the donor substrate between the 4-<italic>O</italic>-methylated and non-methylated counterparts is larger than the effect of changing the alcohol acceptor. This observation may not be valid for all glucuronoyl esterases, as it is common knowledge that not all GEs favor the 4-<italic>O</italic>-methylation.</p>","<p id=\"Par30\">Recent QM/MM studies of the fungal <italic>Thermothelomyces thermophila</italic> glucuronoyl esterase (<italic>Tt</italic>GE) show that the acylation step is the most energy demanding and hence rate-limiting step (Viegas et al. ##REF##35925549##2022##), whereas similar studies with a bacterial <italic>Ot</italic>CE15A from <italic>Opitutus terrae</italic> show that the deacylation-step is rate-limiting (Zong et al. ##REF##34983933##2022##). Without having investigated the energetic landscape of catalysis by <italic>Cu</italic>GE, the transesterification reactions we observe support the observation that the acylation-step is determining for reaction speed. However, it is well-known that the differences between fungal and bacterial GEs are quite large in terms of overall structure (Larsbrink and Lo Leggio ##REF##36651189##2023##), and transesterification reactions of the kind presented here, may turn out different for bacterial GEs with respect to energetic fingerprints.</p>","<p id=\"Par31\">The aim of this study is to investigate the preference for either an α- or a γ-positioned alcohol, and the results show that the γ-positioned alcohol is most favorable, yet the enzyme can perform the reaction with the α-alcohol. There are currently no univocal structural explanations for how the GEs interact with the alcohol moiety of the substrate. However, we have recently performed docking simulations with these exact two esters; Bnz-4-<italic>O</italic>-MeGlcA and PhPr-4-<italic>O</italic>-MeGlcA as ligands in <italic>Cu</italic>GE and guided by the sugar-moiety, which show steric obstacles when the α-benzyl constitutes the alcohol part of the ligand in contrary to the γ-linked ester (Agger et al. ##REF##37256329##2023##).</p>","<p id=\"Par32\">Exploiting the transesterification capacities of these enzymes potentially opens for other biotechnological applications than biomass deconstruction, such as functionalization of hemicellulose or smaller aldouronic acids with alcohols of different properties (hydrophobicity, other functional groups etc.). Future studies may explore the field of potential acceptors more broadly than here, and thereby determine the diversity of alcohols and properties that could be relevant to investigate further.</p>"],"string":"[\n \"<title>Results and discussion</title>\",\n \"<p id=\\\"Par26\\\">Initial hydrolytic experiments with either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA as substrate and in the absence of alcohol acceptors show substrate conversion as expected (Fig. ##FIG##1##2##), with a significantly higher level of conversion for the 4-<italic>O</italic>-methylated ester compared to the non-derivatized glucuronate ester within the first 100 min of reaction (Table ##TAB##0##1##). Enzyme concentrations were dosed to allow similar rates of catalysis in all experiments, independent of substrate, and consequently <italic>Cu</italic>GE was dosed 50 times less in the experiments where Me-4-<italic>O</italic>-MeGlcA was the substrate compared to the experiments with Me-GlcA as substrate. The fact that <italic>Cu</italic>GE prefers its substrates to have the 4-<italic>O</italic>-methyl-modification on the glucuronoyl is well-establised (d’Errico et al. ##REF##25425346##2015##; Ernst et al. ##REF##31911652##2020##; Monrad et al. ##REF##29739247##2018##). As the 100 min reaction time progresses, it becomes evident that the hydrolytic reaction slows down for both substrates (Table ##TAB##0##1##), which again is not surprising as the affinity (K<sub>M</sub>) for these synthetic model substrates is known to be relatively high (d’Errico et al. ##REF##25425346##2015##).</p>\",\n \"<p id=\\\"Par27\\\">It is known, that glucuronoyl esterases prefer bulky alcohols and in that respect, the methyl-esters are poor substrates. Experiments with either benzyl-alcohol or 3-phenyl-1-propanol as the primary solvent and thereby dominating acceptor molecule (14% v/v water), show that the transesterification reaction occurring between Me-4-<italic>O</italic>-MeGlcA and PhPrOH (Fig. ##FIG##0##1##, reaction 1) is significantly higher than any of the other combinations (Fig. ##FIG##2##3##). The reaction between Me-4-<italic>O</italic>-MeGlcA and BnzOH is second best (Fig. ##FIG##0##1##, reaction 2) with about 3–4 fold less product compared to the PhPr-GlcA ester. Quantifications of the ester products are done relative to the standard of Bnz-GlcA dissolved either in BnzOH or PhPrOH depending on the relevant acceptor alcohol, and the calibrations indicate a certain level of ion suppression when PhPrOH is the solvent (Supplementary standards curves), hence leading to a potential underestimation in that respect. PhPrOH has similar retention time as the transesterification products. At the same time, compounds carrying the 4-<italic>O</italic>-methylation tends to ionize better, and therefore give stronger responses. In the reactions performed here, enzyme loadings vary according to formation of transesterification products, as the goal is to quantify transesterification products. Hence, at low enzyme loadings, hydrolysis is not observed for reactions containing Me-4-<italic>O</italic>-MeGlcA as donor (and relatively low water concentration), whereas increasing the enzyme concentration yields hydrolytic reactions in parallel to the transesterification (data not shown). Ultimately, the extent of hydrolysis is a competition for acceptor substrate, which appears favored in the case of the alternative alcohol when enzyme concentrations are low. Hydrolytic reactions are certainly present but becomes un-quantifiable at conditions where transesterification is dominating.</p>\",\n \"<p id=\\\"Par28\\\">Interestingly, the 4-<italic>O</italic>-methylation is a more important feature for catalysis than the nature of the acceptor-alcohol, since the Bnz-4-<italic>O</italic>-MeGlcA-ester forms faster than the PhPr-GlcA ester. A cautious estimation of the effect of the donor versus the effect of the acceptor demonstrates that the ratio between concentrations of products formed during the reaction time is higher when the donor is changed, compared to when the acceptor is changed (Fig. ##FIG##3##4##).</p>\",\n \"<p id=\\\"Par29\\\">Hence, the effect of changing the donor substrate between the 4-<italic>O</italic>-methylated and non-methylated counterparts is larger than the effect of changing the alcohol acceptor. This observation may not be valid for all glucuronoyl esterases, as it is common knowledge that not all GEs favor the 4-<italic>O</italic>-methylation.</p>\",\n \"<p id=\\\"Par30\\\">Recent QM/MM studies of the fungal <italic>Thermothelomyces thermophila</italic> glucuronoyl esterase (<italic>Tt</italic>GE) show that the acylation step is the most energy demanding and hence rate-limiting step (Viegas et al. ##REF##35925549##2022##), whereas similar studies with a bacterial <italic>Ot</italic>CE15A from <italic>Opitutus terrae</italic> show that the deacylation-step is rate-limiting (Zong et al. ##REF##34983933##2022##). Without having investigated the energetic landscape of catalysis by <italic>Cu</italic>GE, the transesterification reactions we observe support the observation that the acylation-step is determining for reaction speed. However, it is well-known that the differences between fungal and bacterial GEs are quite large in terms of overall structure (Larsbrink and Lo Leggio ##REF##36651189##2023##), and transesterification reactions of the kind presented here, may turn out different for bacterial GEs with respect to energetic fingerprints.</p>\",\n \"<p id=\\\"Par31\\\">The aim of this study is to investigate the preference for either an α- or a γ-positioned alcohol, and the results show that the γ-positioned alcohol is most favorable, yet the enzyme can perform the reaction with the α-alcohol. There are currently no univocal structural explanations for how the GEs interact with the alcohol moiety of the substrate. However, we have recently performed docking simulations with these exact two esters; Bnz-4-<italic>O</italic>-MeGlcA and PhPr-4-<italic>O</italic>-MeGlcA as ligands in <italic>Cu</italic>GE and guided by the sugar-moiety, which show steric obstacles when the α-benzyl constitutes the alcohol part of the ligand in contrary to the γ-linked ester (Agger et al. ##REF##37256329##2023##).</p>\",\n \"<p id=\\\"Par32\\\">Exploiting the transesterification capacities of these enzymes potentially opens for other biotechnological applications than biomass deconstruction, such as functionalization of hemicellulose or smaller aldouronic acids with alcohols of different properties (hydrophobicity, other functional groups etc.). Future studies may explore the field of potential acceptors more broadly than here, and thereby determine the diversity of alcohols and properties that could be relevant to investigate further.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par33\"><italic>Cu</italic>GE prefers methyl-4-<italic>O</italic>-methyl-glucuronate as donor substrate and the γ-positioned acceptor according to the experiments conducted here, and evaluated based on yield and rate of product formation. It is important to emphasize that the results reported here illustrate the substrate preferences of <italic>Cu</italic>GE, which is not to be generalized for all GEs. These results exemplify a methodology for investigating substrate preferences, and future studies may include reverse hydrolytic reactions.</p>","<p id=\"Par34\">We observe large differences in product formation rates during the first 100 min of reaction with the formation of the 4-<italic>O</italic>-methyl-glucuronoyl-3-propane-phenyl as fastest, and the results clearly indicate that <italic>Cu</italic>GE prefers the γ-positioned alcohol during transesterification. The most important factor for reaction though, continues to be the presence of the 4-<italic>O</italic>-methylation derivatization of the glucuronoyl moiety.</p>","<p id=\"Par35\">These experiments demonstrate a methodology where the enzyme reveals its substrate preferences immediately by favoring product formation from the most suitable substrates. These results do not inform about the prevalence of either α- or γ-LCC esters in lignocellulosic biomass, but it certainly informs about the preferences of this particular enzyme. Given that enzymes evolve to tackle the linkages present in biomass, it is tempting to speculate that the γ-esters LCC is the more prevalent of the two types in biomass.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par33\\\"><italic>Cu</italic>GE prefers methyl-4-<italic>O</italic>-methyl-glucuronate as donor substrate and the γ-positioned acceptor according to the experiments conducted here, and evaluated based on yield and rate of product formation. It is important to emphasize that the results reported here illustrate the substrate preferences of <italic>Cu</italic>GE, which is not to be generalized for all GEs. These results exemplify a methodology for investigating substrate preferences, and future studies may include reverse hydrolytic reactions.</p>\",\n \"<p id=\\\"Par34\\\">We observe large differences in product formation rates during the first 100 min of reaction with the formation of the 4-<italic>O</italic>-methyl-glucuronoyl-3-propane-phenyl as fastest, and the results clearly indicate that <italic>Cu</italic>GE prefers the γ-positioned alcohol during transesterification. The most important factor for reaction though, continues to be the presence of the 4-<italic>O</italic>-methylation derivatization of the glucuronoyl moiety.</p>\",\n \"<p id=\\\"Par35\\\">These experiments demonstrate a methodology where the enzyme reveals its substrate preferences immediately by favoring product formation from the most suitable substrates. These results do not inform about the prevalence of either α- or γ-LCC esters in lignocellulosic biomass, but it certainly informs about the preferences of this particular enzyme. Given that enzymes evolve to tackle the linkages present in biomass, it is tempting to speculate that the γ-esters LCC is the more prevalent of the two types in biomass.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose</title>","<p id=\"Par1\">Glucuronoyl esterases (GE, family CE15) catalyse the cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), and this study demonstrate how transesterification reactions with a fungal GE from <italic>Cerrena unicolor</italic> (<italic>Cu</italic>GE) can reveal the enzyme’s preference for the alcohol-part of the ester-bond.</p>","<title>Methods</title>","<p id=\"Par2\">This alcohol-preference relates to where the ester-LCCs are located on the lignin molecule, and has consequences for how the enzymes potentially interact with lignin. It is unknown exactly what the enzymes prefer; either the α-benzyl or the γ-benzyl position. By providing the enzyme with a donor substrate (the methyl ester of either glucuronate or 4-<italic>O</italic>-methyl-glucuronate) and either one of two acceptor molecules (benzyl alcohol or 3-phenyl-1-propanol) we demonstrate that the enzyme can perform transesterification and it serves as a method for assessing the enzyme’s alcohol preferences.</p>","<title>Conclusion</title>","<p id=\"Par3\"><italic>Cu</italic>GE preferentially forms the γ-ester from the methyl ester of 4-<italic>O</italic>-methyl-glucuronate and 3-phenyl-1-propanol and the enzyme’s substrate preferences are primarily dictated by the presence of the 4-<italic>O</italic>-methylation on the glucuronoyl donor, and secondly on the type of alcohol.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s10529-023-03456-x.</p>","<title>Keywords</title>","<p>Open access funding provided by Technical University of Denmark</p>"],"string":"[\n \"<title>Purpose</title>\",\n \"<p id=\\\"Par1\\\">Glucuronoyl esterases (GE, family CE15) catalyse the cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), and this study demonstrate how transesterification reactions with a fungal GE from <italic>Cerrena unicolor</italic> (<italic>Cu</italic>GE) can reveal the enzyme’s preference for the alcohol-part of the ester-bond.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">This alcohol-preference relates to where the ester-LCCs are located on the lignin molecule, and has consequences for how the enzymes potentially interact with lignin. It is unknown exactly what the enzymes prefer; either the α-benzyl or the γ-benzyl position. By providing the enzyme with a donor substrate (the methyl ester of either glucuronate or 4-<italic>O</italic>-methyl-glucuronate) and either one of two acceptor molecules (benzyl alcohol or 3-phenyl-1-propanol) we demonstrate that the enzyme can perform transesterification and it serves as a method for assessing the enzyme’s alcohol preferences.</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par3\\\"><italic>Cu</italic>GE preferentially forms the γ-ester from the methyl ester of 4-<italic>O</italic>-methyl-glucuronate and 3-phenyl-1-propanol and the enzyme’s substrate preferences are primarily dictated by the presence of the 4-<italic>O</italic>-methylation on the glucuronoyl donor, and secondly on the type of alcohol.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s10529-023-03456-x.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by Technical University of Denmark</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Materials and enzyme preparation</title>","<p id=\"Par8\">Methyl 4-<italic>O</italic>-methyl-D-glucopyranosyluronate (Me-4-O-MeGlcA) was purchased from Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia. Glucuronic acid methyl ester (Me-GlcA) and benzyl D-glucuronate (Bnz-GlcA) were purchased from Carbosynth. Benzyl alcohol (BnzOH), 3-phenyl-1-propanol (PhPrOH) and all other chemicals were purchased from Sigma.</p>","<p id=\"Par9\">The construct containing the gene encoding for the CE15 glucuronoyl esterase from <italic>Cerrena unicolor</italic> (<italic>Cu</italic>GE) was produced in <italic>P. pastoris</italic> as previously described (Mosbech et al. ##REF##29321810##2018##). After fermentation the cells were separated by centrifugation and the fermentation broth (3 L) was sterile filtered and concentrated by ultrafiltration on a 10 kDa cut-off membrane to a final volume of approx. 80 mL. <italic>Cu</italic>GE was purified by affinity chromatography on an IMAC-column (HisTrap HP 5 mL column, GE Healthcare) using an Äkta Purifier 100 (GE Healthcare, Uppsala Sweden).</p>","<title>Enzyme reactions</title>","<p id=\"Par10\">The transesterification activity of <italic>Cu</italic>GE was tested in four different experimental setups using either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA as donor substrates and benzyl alcohol (BnzOH) or 3-phenyl-1-propanol (PhPrOH) as acceptor substrates, allowing the assessment of the transesterification ability and preferences of <italic>Cu</italic>GE in either the α and γ position (Fig. ##FIG##0##1##).</p>","<p id=\"Par11\">Transesterification with PhPrOH as acceptor molecule was performed in a 100 µL reaction mixture containing 0.45 mM of Me-4-<italic>O</italic>-MeGlcA or 0.48 mM of Me-GlcA (dissolved in 10 mM Na acetate buffer pH 6) and 6.32 M of PhPrOH in order to have an acceptor–donor ratio of approx. 14,000 and of 13,000 with Me-4-<italic>O</italic>-MeGlcA and Me-GlcA, respectively. The reaction was started in a HPLC vial by addition of 0.02 µM or 0.87 µM of <italic>Cu</italic>GE for the reaction with Me-4-<italic>O</italic>-MeGlcA and Me-GlcA, respectively.</p>","<p id=\"Par12\">Transesterification with BnzOH as acceptor molecule was performed in a 100 µL reaction mixture containing 0.45 mM of Me-4-<italic>O</italic>-MeGlcA or 0.48 mM of Me-GlcA (dissolved in 10 mM Na acetate buffer pH 6) and 8.31 M of BnzOH in order to have an acceptor donor ratio of approx.. 18,500 and of 17,500 with Me-4-<italic>O</italic>-MeGlcA and Me-GlcA, respectively. The reaction was started in a HPLC vial by addition of 0.11 µM or 4.33 µM of <italic>Cu</italic>GE for the reaction with Me-4-<italic>O</italic>-MeGlcA and Me-GlcA, respectively. All transesterification reactions were run in triplicates.</p>","<p id=\"Par13\">The reaction samples were placed in the UHPLC’s autosampler at 40 °C and the reaction evolution was followed directly on LC–MS by injecting 5 µL of the reaction mixture onto the column every 20 min followed by the LC–MS method described below.</p>","<p id=\"Par14\">Control experiments containing 0.45 mM of Me-4-<italic>O</italic>-MeGlcA or 0.48 mM of Me-GlcA (dissolved in 10 mM Na acetate buffer pH 6) and 6.32 M of PhPrOH or 8.31 M of BnzOH and replacing the enzyme volume with an equal amount of 10 mM Na acetate buffer pH 6 were run to assess auto-hydrolysis during the reaction time.</p>","<p id=\"Par15\">Chromatograms of all transesterification reactions and auto-hydrolytic control experiments are found in Supplementary Figs. S1-S8.</p>","<p id=\"Par16\">Control experiments showing <italic>Cu</italic>GE’s hydrolytic activity were performed in 100 µL reaction mixture containing 0.45 mM of Me-4-<italic>O</italic>-MeGlcA or 0.48 mM of Me-GlcA dissolved in 10 mM Na acetate buffer pH 6. The reactions were started in the HPLC vial by the addition of 0.08 µM or 4.33 µM of <italic>Cu</italic>GE for the reaction with Me-4-<italic>O</italic>-MeGlcA and Me-GlcA, respectively. The hydrolytic reactions were performed in triplicates. Direct quantification results of substrate depletion are provided in Supplementary Table S1.</p>","<title>Quantification method</title>","<p id=\"Par20\">Quantification of all precursor ions was performed using Bruker TASQ software (Bruker Daltonics, Bremen, Germany). All ions were observed as [M + Na]<sup>+</sup>.</p>","<p id=\"Par21\">Quantification of the Me-4-<italic>O</italic>-MeGlcA hydrolysis by <italic>Cu</italic>GE was performed by defining an Extracted Ion Chromatogram (EIC) of <italic>m/z</italic> 244.95 and <italic>m/z</italic> 467.03 (pseudo-double ion with one sodium), with a width of ± 0.5 and retention time 6.5 min ± 0.2 min, and reaction extend was quantified as substrate depletion relative to calibration with Me-4-<italic>O</italic>-MeGlcA.</p>","<p id=\"Par22\">Quantification of the Me-GlcA hydrolysis by <italic>Cu</italic>GE was performed by defining an EIC of <italic>m/z</italic> 230.92 and <italic>m/z</italic> 439.07 (pseudo-double ion with one sodium), with a width of ± 0.5, retention time 3.0 min ± 0.2 min, and reaction extent was quantified as substrate depletion relative to calibration with Me-GlcA.</p>","<p id=\"Par23\">Quantification of product formations of both Bnz-4-<italic>O</italic>-MeGlcA (EIC on <italic>m/z</italic> 321.08 with a width of ± 0.5, retention time 9.8 min) and Bnz-GlcA (EIC on <italic>m/z</italic> 307.04 and <italic>m/z</italic> 591.01 with a width of ± 0.5, retention time 8.0 min), were performed relative to a Bnz-GlcA standard dissolved in BnzOH (EIC on <italic>m/z</italic> 307.04 and <italic>m/z</italic> 591.01 with a width of ± 0.5 and retention time 8.0 min with a window of 0.2 min).</p>","<p id=\"Par24\">Estimations of PhPr-4-<italic>O</italic>-MeGlcA (EIC on <italic>m/z</italic> 349.14, retention time 9.3 min) and PhPr-GlcA (EIC on <italic>m/z</italic> 335.12, retention time 7.7 min); products of <italic>Cu</italic>GE transesterification of PhPrOH with Me-4-<italic>O</italic>-MeGlcA or Me-GlcA, respectively, were performed using Bnz-GlcA diluted in PhPrOH as standard.</p>","<p id=\"Par25\">Calibration curves was performed using 6–8 levels of concentrations and fitting the data with a quadratic curve for the Me-4-<italic>O</italic>-MeGlcA and Me-GlcA calibrations and with a linear curve for the Bnz-GlcA (Supplementary Figure S9).</p>","<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Materials and enzyme preparation</title>\",\n \"<p id=\\\"Par8\\\">Methyl 4-<italic>O</italic>-methyl-D-glucopyranosyluronate (Me-4-O-MeGlcA) was purchased from Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia. Glucuronic acid methyl ester (Me-GlcA) and benzyl D-glucuronate (Bnz-GlcA) were purchased from Carbosynth. Benzyl alcohol (BnzOH), 3-phenyl-1-propanol (PhPrOH) and all other chemicals were purchased from Sigma.</p>\",\n \"<p id=\\\"Par9\\\">The construct containing the gene encoding for the CE15 glucuronoyl esterase from <italic>Cerrena unicolor</italic> (<italic>Cu</italic>GE) was produced in <italic>P. pastoris</italic> as previously described (Mosbech et al. ##REF##29321810##2018##). After fermentation the cells were separated by centrifugation and the fermentation broth (3 L) was sterile filtered and concentrated by ultrafiltration on a 10 kDa cut-off membrane to a final volume of approx. 80 mL. <italic>Cu</italic>GE was purified by affinity chromatography on an IMAC-column (HisTrap HP 5 mL column, GE Healthcare) using an Äkta Purifier 100 (GE Healthcare, Uppsala Sweden).</p>\",\n \"<title>Enzyme reactions</title>\",\n \"<p id=\\\"Par10\\\">The transesterification activity of <italic>Cu</italic>GE was tested in four different experimental setups using either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA as donor substrates and benzyl alcohol (BnzOH) or 3-phenyl-1-propanol (PhPrOH) as acceptor substrates, allowing the assessment of the transesterification ability and preferences of <italic>Cu</italic>GE in either the α and γ position (Fig. ##FIG##0##1##).</p>\",\n \"<p id=\\\"Par11\\\">Transesterification with PhPrOH as acceptor molecule was performed in a 100 µL reaction mixture containing 0.45 mM of Me-4-<italic>O</italic>-MeGlcA or 0.48 mM of Me-GlcA (dissolved in 10 mM Na acetate buffer pH 6) and 6.32 M of PhPrOH in order to have an acceptor–donor ratio of approx. 14,000 and of 13,000 with Me-4-<italic>O</italic>-MeGlcA and Me-GlcA, respectively. The reaction was started in a HPLC vial by addition of 0.02 µM or 0.87 µM of <italic>Cu</italic>GE for the reaction with Me-4-<italic>O</italic>-MeGlcA and Me-GlcA, respectively.</p>\",\n \"<p id=\\\"Par12\\\">Transesterification with BnzOH as acceptor molecule was performed in a 100 µL reaction mixture containing 0.45 mM of Me-4-<italic>O</italic>-MeGlcA or 0.48 mM of Me-GlcA (dissolved in 10 mM Na acetate buffer pH 6) and 8.31 M of BnzOH in order to have an acceptor donor ratio of approx.. 18,500 and of 17,500 with Me-4-<italic>O</italic>-MeGlcA and Me-GlcA, respectively. The reaction was started in a HPLC vial by addition of 0.11 µM or 4.33 µM of <italic>Cu</italic>GE for the reaction with Me-4-<italic>O</italic>-MeGlcA and Me-GlcA, respectively. All transesterification reactions were run in triplicates.</p>\",\n \"<p id=\\\"Par13\\\">The reaction samples were placed in the UHPLC’s autosampler at 40 °C and the reaction evolution was followed directly on LC–MS by injecting 5 µL of the reaction mixture onto the column every 20 min followed by the LC–MS method described below.</p>\",\n \"<p id=\\\"Par14\\\">Control experiments containing 0.45 mM of Me-4-<italic>O</italic>-MeGlcA or 0.48 mM of Me-GlcA (dissolved in 10 mM Na acetate buffer pH 6) and 6.32 M of PhPrOH or 8.31 M of BnzOH and replacing the enzyme volume with an equal amount of 10 mM Na acetate buffer pH 6 were run to assess auto-hydrolysis during the reaction time.</p>\",\n \"<p id=\\\"Par15\\\">Chromatograms of all transesterification reactions and auto-hydrolytic control experiments are found in Supplementary Figs. S1-S8.</p>\",\n \"<p id=\\\"Par16\\\">Control experiments showing <italic>Cu</italic>GE’s hydrolytic activity were performed in 100 µL reaction mixture containing 0.45 mM of Me-4-<italic>O</italic>-MeGlcA or 0.48 mM of Me-GlcA dissolved in 10 mM Na acetate buffer pH 6. The reactions were started in the HPLC vial by the addition of 0.08 µM or 4.33 µM of <italic>Cu</italic>GE for the reaction with Me-4-<italic>O</italic>-MeGlcA and Me-GlcA, respectively. The hydrolytic reactions were performed in triplicates. Direct quantification results of substrate depletion are provided in Supplementary Table S1.</p>\",\n \"<title>Quantification method</title>\",\n \"<p id=\\\"Par20\\\">Quantification of all precursor ions was performed using Bruker TASQ software (Bruker Daltonics, Bremen, Germany). All ions were observed as [M + Na]<sup>+</sup>.</p>\",\n \"<p id=\\\"Par21\\\">Quantification of the Me-4-<italic>O</italic>-MeGlcA hydrolysis by <italic>Cu</italic>GE was performed by defining an Extracted Ion Chromatogram (EIC) of <italic>m/z</italic> 244.95 and <italic>m/z</italic> 467.03 (pseudo-double ion with one sodium), with a width of ± 0.5 and retention time 6.5 min ± 0.2 min, and reaction extend was quantified as substrate depletion relative to calibration with Me-4-<italic>O</italic>-MeGlcA.</p>\",\n \"<p id=\\\"Par22\\\">Quantification of the Me-GlcA hydrolysis by <italic>Cu</italic>GE was performed by defining an EIC of <italic>m/z</italic> 230.92 and <italic>m/z</italic> 439.07 (pseudo-double ion with one sodium), with a width of ± 0.5, retention time 3.0 min ± 0.2 min, and reaction extent was quantified as substrate depletion relative to calibration with Me-GlcA.</p>\",\n \"<p id=\\\"Par23\\\">Quantification of product formations of both Bnz-4-<italic>O</italic>-MeGlcA (EIC on <italic>m/z</italic> 321.08 with a width of ± 0.5, retention time 9.8 min) and Bnz-GlcA (EIC on <italic>m/z</italic> 307.04 and <italic>m/z</italic> 591.01 with a width of ± 0.5, retention time 8.0 min), were performed relative to a Bnz-GlcA standard dissolved in BnzOH (EIC on <italic>m/z</italic> 307.04 and <italic>m/z</italic> 591.01 with a width of ± 0.5 and retention time 8.0 min with a window of 0.2 min).</p>\",\n \"<p id=\\\"Par24\\\">Estimations of PhPr-4-<italic>O</italic>-MeGlcA (EIC on <italic>m/z</italic> 349.14, retention time 9.3 min) and PhPr-GlcA (EIC on <italic>m/z</italic> 335.12, retention time 7.7 min); products of <italic>Cu</italic>GE transesterification of PhPrOH with Me-4-<italic>O</italic>-MeGlcA or Me-GlcA, respectively, were performed using Bnz-GlcA diluted in PhPrOH as standard.</p>\",\n \"<p id=\\\"Par25\\\">Calibration curves was performed using 6–8 levels of concentrations and fitting the data with a quadratic curve for the Me-4-<italic>O</italic>-MeGlcA and Me-GlcA calibrations and with a linear curve for the Bnz-GlcA (Supplementary Figure S9).</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Author contributions</title>","<p>Author VP performed the experiments and wrote parts of the paper. Author JWA designed the experiments and wrote the paper.</p>","<title>Funding</title>","<p>Open access funding provided by Technical University of Denmark. The work was funded by the Novo Nordisk Foundation “Emerging Investigator 2022 – Research within Industrial Biotechnology and Environmental Biotechnology” (grant number NNF22OC0074634) Lignin for the Future granted to JWA.</p>","<title>Declarations</title>","<title>Competing interests</title>","<p id=\"Par36\">The author’s declare no competing interests.</p>"],"string":"[\n \"<title>Author contributions</title>\",\n \"<p>Author VP performed the experiments and wrote parts of the paper. Author JWA designed the experiments and wrote the paper.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by Technical University of Denmark. The work was funded by the Novo Nordisk Foundation “Emerging Investigator 2022 – Research within Industrial Biotechnology and Environmental Biotechnology” (grant number NNF22OC0074634) Lignin for the Future granted to JWA.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par36\\\">The author’s declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Four transesterification reactions catalyzed by <italic>Cu</italic>GE performed by combining either one of the two different donor substrates, methyl-4-<italic>O</italic>-methyl-glucuronate (1 and 2) and methyl-glucuronate (3 and 4) with either 3-phenyl-1-propanol (PhPrOH, reaction 1 and 3) or benzyl alcohol (BnzOH, reaction 2 and 4) as acceptor substrates</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Comparison between the hydrolysis of Me-4-<italic>O</italic>-MeGlcA and Me-GlcA by <italic>Cu</italic>GE. Reaction evolution over 100 min of the Me-GlcA hydrolysis by (4.3 µM) <italic>Cu</italic>GE is showed in violet at retention time (RT) 3.0 min. MS2 spectrum of Me-GlcA m/z 230.92 is reported in the upper right corner (violet). Reaction evolution over 100 min of the Me-4-<italic>O</italic>-MeGlcA hydrolysis by (0.087 µM) <italic>Cu</italic>GE is showed in blue at RT 6.5 min. MS2 spectrum of Me-4-<italic>O</italic>-MeGlcA m/z 244.95 is reported in the lower right corner (blue). All ions are shown as [M + Na]<sup>+</sup>. The two hydrolysis were run separately</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Product formation after transesterification reactions catalyzed by <italic>Cu</italic>GE. Reactions are combinations of Me-GlcA or Me-4-<italic>O</italic>-MeGlcA with either benzyl-alcohol or 3-phenyl-1-propanol according to reactions in Fig. ##FIG##0##1##. Blue circle: PhPr-4-<italic>O</italic>-MeGlcA ester (product reaction 1, Fig. ##FIG##0##1##). <italic>Cu</italic>GE dosage; 0.02 µM. Green triangle: Bnz-4-<italic>O</italic>-MeGlcA ester (product reaction 2, Fig. ##FIG##0##1##). <italic>Cu</italic>GE dosage; 0.11 µM. Red square: PhPr-GlcA ester (product reaction 3, Fig. ##FIG##0##1##). <italic>Cu</italic>GE dosage; 0.87 µM. Grey diamond: Bnz-GlcA ester (product reaction 4, Fig. ##FIG##0##1##). <italic>Cu</italic>GE dosage; 4.3 µM. Individual plots of each reaction is found in Supplementary Figure S10</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Ratios between concentrations of product formations, which illustrates the effect of changing the donor (left hand side), or changing the acceptor (right hand side). The ratios are calculated based on product concentrations at 100 min reaction time. On the left hand side, the donor substrate is changed, and the acceptor is kept constant. In the right hand side, the acceptor is changed and the donor is kept constant</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Four transesterification reactions catalyzed by <italic>Cu</italic>GE performed by combining either one of the two different donor substrates, methyl-4-<italic>O</italic>-methyl-glucuronate (1 and 2) and methyl-glucuronate (3 and 4) with either 3-phenyl-1-propanol (PhPrOH, reaction 1 and 3) or benzyl alcohol (BnzOH, reaction 2 and 4) as acceptor substrates</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Comparison between the hydrolysis of Me-4-<italic>O</italic>-MeGlcA and Me-GlcA by <italic>Cu</italic>GE. Reaction evolution over 100 min of the Me-GlcA hydrolysis by (4.3 µM) <italic>Cu</italic>GE is showed in violet at retention time (RT) 3.0 min. MS2 spectrum of Me-GlcA m/z 230.92 is reported in the upper right corner (violet). Reaction evolution over 100 min of the Me-4-<italic>O</italic>-MeGlcA hydrolysis by (0.087 µM) <italic>Cu</italic>GE is showed in blue at RT 6.5 min. MS2 spectrum of Me-4-<italic>O</italic>-MeGlcA m/z 244.95 is reported in the lower right corner (blue). All ions are shown as [M + Na]<sup>+</sup>. The two hydrolysis were run separately</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Product formation after transesterification reactions catalyzed by <italic>Cu</italic>GE. Reactions are combinations of Me-GlcA or Me-4-<italic>O</italic>-MeGlcA with either benzyl-alcohol or 3-phenyl-1-propanol according to reactions in Fig. ##FIG##0##1##. Blue circle: PhPr-4-<italic>O</italic>-MeGlcA ester (product reaction 1, Fig. ##FIG##0##1##). <italic>Cu</italic>GE dosage; 0.02 µM. Green triangle: Bnz-4-<italic>O</italic>-MeGlcA ester (product reaction 2, Fig. ##FIG##0##1##). <italic>Cu</italic>GE dosage; 0.11 µM. Red square: PhPr-GlcA ester (product reaction 3, Fig. ##FIG##0##1##). <italic>Cu</italic>GE dosage; 0.87 µM. Grey diamond: Bnz-GlcA ester (product reaction 4, Fig. ##FIG##0##1##). <italic>Cu</italic>GE dosage; 4.3 µM. Individual plots of each reaction is found in Supplementary Figure S10</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Ratios between concentrations of product formations, which illustrates the effect of changing the donor (left hand side), or changing the acceptor (right hand side). The ratios are calculated based on product concentrations at 100 min reaction time. On the left hand side, the donor substrate is changed, and the acceptor is kept constant. In the right hand side, the acceptor is changed and the donor is kept constant</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparison of substrate conversion of either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA relative to enzyme dosage within 100 min reaction time</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Reaction time</th><th align=\"left\" colspan=\"2\">Accumulated substrate conversion</th><th align=\"left\" colspan=\"2\">Substrate conversion within each time interval</th></tr><tr><th align=\"left\">Me-4-<italic>O</italic>-MeGlcA</th><th align=\"left\">Me-GlcA</th><th align=\"left\">Me-4-<italic>O</italic>-MeGlcA</th><th align=\"left\">Me-GlcA</th></tr><tr><th align=\"left\">min</th><th align=\"left\">mmol/µmol <italic>Cu</italic>GE</th><th align=\"left\">mmol/µmol <italic>Cu</italic>GE</th><th align=\"left\">mmol/µmol <italic>Cu</italic>GE</th><th align=\"left\">mmol/µmol <italic>Cu</italic>GE</th></tr></thead><tbody><tr><td align=\"left\">20</td><td align=\"left\">2.3 ± 0.1</td><td align=\"left\">0.05 ± 0.00</td><td align=\"left\">2.31 ± 0.1</td><td align=\"left\">0.05 ± 0.00</td></tr><tr><td align=\"left\">40</td><td align=\"left\">3.0 ± 0.08</td><td align=\"left\">0.07 ± 0.00</td><td align=\"left\">0.91 ± 0.03</td><td align=\"left\">0.02 ± 0.00</td></tr><tr><td align=\"left\">60</td><td align=\"left\">3.2 ± 0.03</td><td align=\"left\">0.08 ± 0.00</td><td align=\"left\">0.91 ± 0.05</td><td align=\"left\">0.02 ± 0.01</td></tr><tr><td align=\"left\">80</td><td align=\"left\">3.6 ± 0.13</td><td align=\"left\">0.09 ± 0.01</td><td align=\"left\">0.52 ± 0.00</td><td align=\"left\">0.02 ± 0.01</td></tr><tr><td align=\"left\">100</td><td align=\"left\">3.6 ± 0.1</td><td align=\"left\">0.10 ± 0.01</td><td align=\"left\">0.43 ± 0.12</td><td align=\"left\">0.01 ± 0.00</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Comparison of substrate conversion of either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA relative to enzyme dosage within 100 min reaction time</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" rowspan=\\\"2\\\">Reaction time</th><th align=\\\"left\\\" colspan=\\\"2\\\">Accumulated substrate conversion</th><th align=\\\"left\\\" colspan=\\\"2\\\">Substrate conversion within each time interval</th></tr><tr><th align=\\\"left\\\">Me-4-<italic>O</italic>-MeGlcA</th><th align=\\\"left\\\">Me-GlcA</th><th align=\\\"left\\\">Me-4-<italic>O</italic>-MeGlcA</th><th align=\\\"left\\\">Me-GlcA</th></tr><tr><th align=\\\"left\\\">min</th><th align=\\\"left\\\">mmol/µmol <italic>Cu</italic>GE</th><th align=\\\"left\\\">mmol/µmol <italic>Cu</italic>GE</th><th align=\\\"left\\\">mmol/µmol <italic>Cu</italic>GE</th><th align=\\\"left\\\">mmol/µmol <italic>Cu</italic>GE</th></tr></thead><tbody><tr><td align=\\\"left\\\">20</td><td align=\\\"left\\\">2.3 ± 0.1</td><td align=\\\"left\\\">0.05 ± 0.00</td><td align=\\\"left\\\">2.31 ± 0.1</td><td align=\\\"left\\\">0.05 ± 0.00</td></tr><tr><td align=\\\"left\\\">40</td><td align=\\\"left\\\">3.0 ± 0.08</td><td align=\\\"left\\\">0.07 ± 0.00</td><td align=\\\"left\\\">0.91 ± 0.03</td><td align=\\\"left\\\">0.02 ± 0.00</td></tr><tr><td align=\\\"left\\\">60</td><td align=\\\"left\\\">3.2 ± 0.03</td><td align=\\\"left\\\">0.08 ± 0.00</td><td align=\\\"left\\\">0.91 ± 0.05</td><td align=\\\"left\\\">0.02 ± 0.01</td></tr><tr><td align=\\\"left\\\">80</td><td align=\\\"left\\\">3.6 ± 0.13</td><td align=\\\"left\\\">0.09 ± 0.01</td><td align=\\\"left\\\">0.52 ± 0.00</td><td align=\\\"left\\\">0.02 ± 0.01</td></tr><tr><td align=\\\"left\\\">100</td><td align=\\\"left\\\">3.6 ± 0.1</td><td align=\\\"left\\\">0.10 ± 0.01</td><td align=\\\"left\\\">0.43 ± 0.12</td><td align=\\\"left\\\">0.01 ± 0.00</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p>Accumulated substrate conversion signifies how much substrate is converted at a given time point relative to the enzyme concentration. Substrate conversion within each time interval describes how much additional substrate is converted at a given time point since the previous, relative to the enzyme concentration</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>Accumulated substrate conversion signifies how much substrate is converted at a given time point relative to the enzyme concentration. Substrate conversion within each time interval describes how much additional substrate is converted at a given time point since the previous, relative to the enzyme concentration</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"10529_2023_3456_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"10529_2023_3456_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"10529_2023_3456_Fig3_HTML\" id=\"MO3\"/>","<graphic xlink:href=\"10529_2023_3456_Fig4_HTML\" id=\"MO4\"/>"],"string":"[\n \"<graphic xlink:href=\\\"10529_2023_3456_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"10529_2023_3456_Fig2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic xlink:href=\\\"10529_2023_3456_Fig3_HTML\\\" id=\\\"MO3\\\"/>\",\n \"<graphic xlink:href=\\\"10529_2023_3456_Fig4_HTML\\\" id=\\\"MO4\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"10529_2023_3456_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 3147 KB)</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"10529_2023_3456_MOESM1_ESM.pdf\\\"><caption><p>Supplementary file1 (PDF 3147 KB)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"surname\": [\"Biely\"], \"given-names\": [\"P\"], \"article-title\": [\"Microbial glucuronoyl esterases \\u2013 ten years after discovery\"], \"source\": [\"Appl\\u00a0environ Microbiol\"], \"year\": [\"2016\"], \"volume\": [\"82\"], \"issue\": [\"24\"], \"fpage\": [\"02396\"], \"lpage\": [\"2416\"], \"pub-id\": [\"10.1128/AEM.02396-16\"]}, {\"surname\": [\"Li\", \"Helm\"], \"given-names\": [\"K\", \"RF\"], \"article-title\": [\"Synthesis and rearrangement reactions of ester-linked lignin-carbohydrate model compounds\"], \"source\": [\"J Agric Food Chem\"], \"year\": [\"1995\"], \"volume\": [\"43\"], \"issue\": [\"8\"], \"fpage\": [\"2098\"], \"lpage\": [\"2103\"], \"pub-id\": [\"10.1021/jf00056a026\"]}, {\"surname\": [\"Mazurkewich\", \"Poulsen\", \"Lo Leggio\", \"Larsbrink\"], \"given-names\": [\"S\", \"J-CN\", \"L\", \"J\"], \"article-title\": [\"Structural and biochemical studies of the glucuronoyl esterase \"], \"italic\": [\"Ot\"], \"source\": [\"J Biol Chem\"], \"year\": [\"2019\"], \"volume\": [\"1\"], \"issue\": [\"1\"], \"fpage\": [\"1\"], \"lpage\": [\"13\"], \"pub-id\": [\"10.1017/CBO9781107415324.004\"]}, {\"surname\": [\"Sapouna\", \"Lawoko\"], \"given-names\": [\"I\", \"M\"], \"article-title\": [\"Deciphering lignin heterogeneity in ball milled softwood: unravelling the synergy between the supramolecular cell wall structure and molecular events\"], \"source\": [\"Green Chem\"], \"year\": [\"2021\"], \"volume\": [\"23\"], \"issue\": [\"9\"], \"fpage\": [\"3348\"], \"lpage\": [\"3364\"], \"pub-id\": [\"10.1039/D0GC04319B\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":19,"string":"19"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Biotechnol Lett. 2024 Dec 27; 46(1):107-114"},"package_file":{"kind":"string","value":"oa_package/ad/28/PMC10787888.tar.gz"}}},{"rowIdx":172,"cells":{"accession_id":{"kind":"string","value":"PMC10787889"},"pmid":{"kind":"string","value":"38217712"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par5\">Adolescent and young adult cancer survivors (AYAs), those aged 15–39 years at initial cancer diagnosis, are considered a unique group with age-specific challenges, from cancer diagnosis until end of life [##REF##25228567##1##, ##UREF##0##2##]. This age range is, however, flexibly applied depending on the research question of interest, country, and health care system [##REF##26849003##3##]. In the Netherlands, pediatric oncology is centralized and includes patients until 18 years of age at diagnosis. The Dutch AYA definition applies to all cancer patients initially diagnosed between 18 and 39 years. As the overall cancer incidence of AYAs has increased over the last decades and the 5-year relative survival is now exceeding 80%, a large part of this growing population will eventually become long-term survivors [##REF##33218178##4##]. As a result of the cancer diagnosis and treatment, these AYAs are at increased risk of long-term (e.g., infertility) and late effects (e.g., secondary malignancies), and experience unmet (age-specific) needs related to finances and mental health for example [##REF##34638332##5##–##UREF##1##7##]. AYAs are in a particularly exposed position for these risk factors due to their often invasive and long-lasting treatments [##UREF##2##8##], and being diagnosed during a complex phase of life, including many physical, emotional, and social transitions [##REF##17613877##9##].</p>","<p id=\"Par6\">AYAs can have a long life ahead in which suffering from these long-term and late effects can have a significant impact on their health-related quality of life (HRQoL). HRQoL is defined by the survivor’s own perception of one’s health or well-being, including physical, mental, and social aspects [##UREF##3##10##]. Several studies have focused on the HRQoL issues among AYAs: literature shows that AYAs are at increased risk of fatigue [##REF##28917270##11##], cognitive impairment [##REF##34118000##12##], work and financial problems [##REF##34118000##12##, ##REF##35772237##13##], psychological distress [##REF##30636177##14##], and body image issues [##REF##33275802##15##], which can result in a diminished HRQoL. In addition, studies described impacted physical health and functioning [##UREF##4##16##–##REF##29572734##19##], and lower mental health [##REF##33332603##17##–##REF##29572734##19##] in AYAs compared to the general population/older cancer survivors [##REF##29572734##19##]. In line, the systematic review of Quinn showed that AYAs are more likely to have impaired HRQoL compared to the general population, although QoL was difficult to measure due to their age-specific needs [##UREF##5##20##].</p>","<p id=\"Par7\">Many factors can independently impact HRQoL, yet they often co-occur in cancer patients [##REF##26187660##21##–##REF##26645947##25##]. To study the interconnectedness between these factors, a network analysis can be performed. It provides insight into the relationships among symptoms, risk factors, and protective factors. Network approaches involve the identification of symptoms and factors (network nodes) and the relations among them (positive or negative associations between nodes) [##UREF##6##26##]. Taking into account the dependence of factors, this type of analysis is more likely reflecting reality compared to focusing on these factors independently [##REF##31435892##27##].</p>","<p id=\"Par8\">Although network analyses have been performed previously among cancer survivors in general [##REF##31435892##27##–##REF##26370099##29##], they are lacking among AYAs, especially when focusing on HRQoL outcomes. Gaining these insights is of importance to optimize supportive care and provide targeted interventions for this unique long-term surviving population. Within a large population-based sample of long-term AYAs, using an exploratory approach, we want to (1) assess HRQoL in long-term AYAs, (2) identify the most central nodes in a HRQoL network, and (3) determine how these nodes are linked to one another ((strengths of) interconnectedness).</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">Adolescent and young adult cancer survivors (AYAs), those aged 15–39 years at initial cancer diagnosis, are considered a unique group with age-specific challenges, from cancer diagnosis until end of life [##REF##25228567##1##, ##UREF##0##2##]. This age range is, however, flexibly applied depending on the research question of interest, country, and health care system [##REF##26849003##3##]. In the Netherlands, pediatric oncology is centralized and includes patients until 18 years of age at diagnosis. The Dutch AYA definition applies to all cancer patients initially diagnosed between 18 and 39 years. As the overall cancer incidence of AYAs has increased over the last decades and the 5-year relative survival is now exceeding 80%, a large part of this growing population will eventually become long-term survivors [##REF##33218178##4##]. As a result of the cancer diagnosis and treatment, these AYAs are at increased risk of long-term (e.g., infertility) and late effects (e.g., secondary malignancies), and experience unmet (age-specific) needs related to finances and mental health for example [##REF##34638332##5##–##UREF##1##7##]. AYAs are in a particularly exposed position for these risk factors due to their often invasive and long-lasting treatments [##UREF##2##8##], and being diagnosed during a complex phase of life, including many physical, emotional, and social transitions [##REF##17613877##9##].</p>\",\n \"<p id=\\\"Par6\\\">AYAs can have a long life ahead in which suffering from these long-term and late effects can have a significant impact on their health-related quality of life (HRQoL). HRQoL is defined by the survivor’s own perception of one’s health or well-being, including physical, mental, and social aspects [##UREF##3##10##]. Several studies have focused on the HRQoL issues among AYAs: literature shows that AYAs are at increased risk of fatigue [##REF##28917270##11##], cognitive impairment [##REF##34118000##12##], work and financial problems [##REF##34118000##12##, ##REF##35772237##13##], psychological distress [##REF##30636177##14##], and body image issues [##REF##33275802##15##], which can result in a diminished HRQoL. In addition, studies described impacted physical health and functioning [##UREF##4##16##–##REF##29572734##19##], and lower mental health [##REF##33332603##17##–##REF##29572734##19##] in AYAs compared to the general population/older cancer survivors [##REF##29572734##19##]. In line, the systematic review of Quinn showed that AYAs are more likely to have impaired HRQoL compared to the general population, although QoL was difficult to measure due to their age-specific needs [##UREF##5##20##].</p>\",\n \"<p id=\\\"Par7\\\">Many factors can independently impact HRQoL, yet they often co-occur in cancer patients [##REF##26187660##21##–##REF##26645947##25##]. To study the interconnectedness between these factors, a network analysis can be performed. It provides insight into the relationships among symptoms, risk factors, and protective factors. Network approaches involve the identification of symptoms and factors (network nodes) and the relations among them (positive or negative associations between nodes) [##UREF##6##26##]. Taking into account the dependence of factors, this type of analysis is more likely reflecting reality compared to focusing on these factors independently [##REF##31435892##27##].</p>\",\n \"<p id=\\\"Par8\\\">Although network analyses have been performed previously among cancer survivors in general [##REF##31435892##27##–##REF##26370099##29##], they are lacking among AYAs, especially when focusing on HRQoL outcomes. Gaining these insights is of importance to optimize supportive care and provide targeted interventions for this unique long-term surviving population. Within a large population-based sample of long-term AYAs, using an exploratory approach, we want to (1) assess HRQoL in long-term AYAs, (2) identify the most central nodes in a HRQoL network, and (3) determine how these nodes are linked to one another ((strengths of) interconnectedness).</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<title>Study population and data collection</title>","<p id=\"Par9\">Data of the population-based, cross-sectional SURVAYA study was used, which was approved by the Institutional Review Board (IRBd18122) and registered within clinical trial registration (NCT05379387). The study population is extensively described previously [##REF##36005166##30##]. In short, the SURVAYA study was performed among AYAs (18–39 years old at time of initial cancer diagnosis) diagnosed with cancer between 1999 and 2015 (5–20 years post diagnosis at study invitation), treated at the Netherlands Cancer Institute or one of the University Medical Centers in the Netherlands, and registered within the Netherlands Cancer Registry (NCR). Survivors were invited to complete a one-time questionnaire within PROFILES (Patient Reported Outcomes Following Initial treatment and Long-term Evaluation of Survivorship) [##REF##21621408##31##].</p>","<title>Measures</title>","<p id=\"Par10\">Sociodemographic characteristics were obtained through a one-time questionnaire including age at time of questionnaire, sex at birth, marital status, and educational level. Clinical characteristics, obtained by the NCR, include tumor type, stage, primary treatment received, and time since diagnosis. Tumor type was classified according to the third International Classification of Diseases for Oncology (ICDO-3) [##UREF##7##32##]. Cancer stage was classified according to TNM or Ann Arbor Code (Hodgkin lymphoma and Non-Hodgkin lymphoma) [##UREF##8##33##].</p>","<p id=\"Par11\">The EORTC QLQ-SURV111 [##UREF##9##34##], a cancer core survivorship questionnaire that is currently being developed by the European Organization for Research and Treatment of Cancer (EORTC), was used for our network analysis. This questionnaire assesses long-term HRQoL outcomes, including physical, mental, and social HRQoL issues specifically relevant to cancer survivors. We selected 8 functioning scales (physical functioning, cognitive functioning, emotional functioning, role functioning, body image, symptom awareness, sexual functioning, and overall quality of life), 9 symptoms scales (fatigue, sleep problems, pain, social interference, health distress, negative health outlook, social isolation, symptom checklist, and sexual problems), and 3 single items (financial difficulties, worry cancer risk family, and treated differently). Scales and items measuring positive factors of HRQoL and items that were not applicable for all participants were excluded from the analysis. The rationale for this selection is that including these positive scales/items and optional items would result in difficulties with respectively interpreting network associations to intervene on and the development of a network structure. Participants scored the items on a 4-point Likert scale from 1 (not at all) to 4 (very much). Overall quality of life scores ranged from 1 (very poor) to 7 (excellent). All scales and single items were linearly transformed to a “0–100” scale [##UREF##10##35##]. A higher score on the functioning scales indicates better HRQoL/functioning, while a higher score for symptoms indicates more complaints. For our analysis, we transformed the scores of the symptom scales once again, so a higher score on the symptom scale indicates fewer complaints. Now, a higher score on all scales corresponds with better functioning, fewer complaints, and a better overall quality of life.</p>","<title>Data analysis</title>","<p id=\"Par12\">Data were analyzed using SPSS Statistics (IBM Corporation, version 26.0, Armonk, NY, USA) and R version 4.2.1 packages MVN, huge, qgraph, and bootnet. Descriptive statistics were calculated and presented as frequencies, percentages, means, and standard deviations.</p>","<p id=\"Par13\">We used listwise deletion to exclude participants with missing items on the EORTC QLQ-SURV111 questionnaire, except for the items related to sexuality. This is because sexual functioning and problems can be an important aspect of HRQoL, and missing responses to sensitive questions like sexuality are common [##REF##21134739##36##]. If half of the items from the two sexuality scales were answered, we assumed that the missing item had a value equal to the item that was present for that respondent according to the QLQ-SURV111 scoring manual. In case both items for the scale were missing, we used a copy mean imputation of the study population.</p>","<p id=\"Par14\">We assessed the assumption of multivariate normality with Mardia’s test [##UREF##11##37##], which needs to be fulfilled prior to estimating the network [##REF##28342071##38##]. Mardia’s multivariate skewness and kurtosis coefficients of the numeric scales were calculated [##UREF##11##37##]. In the case of multivariate normality, both <italic>p</italic>-values of skewness and kurtosis should be greater than 0.05 [##UREF##11##37##]. As the data were not multivariate normally distributed according to Mardia’s test (<italic>p</italic><0.05), a nonparanormal transformation was applied to relax the normality assumption [##UREF##12##39##].</p>","<p id=\"Par15\">In our network model, HRQoL is conceptualized as a network of mutually interrelated factors. Because data are continuous scales or items, we used Guassian Graphical model (GGM) [##UREF##6##26##, ##REF##29595293##40##]. Nodes represent the selected HRQoL scales and items, and edges (links connecting two nodes) represent the regularized partial correlation coefficients after controlling for all other nodes. The thickness of the edge visualizes the strength, and the color a positive (red) or negative (blue) partial correlation. The partial correlation is indicated as very small (<italic>r</italic><0.1), small (0.1≤<italic>r</italic><0.3), moderate (0.3≤<italic>r</italic><0.5), and large (<italic>r</italic>>0.5) [##UREF##13##41##].</p>","<p id=\"Par16\">We applied graphical lasso tuned with the Extended Bayesian Information Criterion (EBIC) [##REF##28342071##38##, ##UREF##14##42##]. The EBIC hyperparameter, used to set the preferred simplicity of the model, was set to 0.5 to minimize spurious connections [##REF##28342071##38##]. Graphical lasso is a form of lasso regularization to prevent that edges between two nodes are spurious because of other nodes (i.e., conditional independence association) and small edges were shrinked to zero by dropping them from the model [##UREF##14##42##]. In this way, the estimated network is not over fitted and interpretable.</p>","<p id=\"Par17\">We estimated node strength (i.e., number and strength of edges between nodes), betweenness (i.e., how often a node lies in shortest path between any combination of two nodes), and closeness (i.e., average distance from one node to all other nodes, which indicates how fast a node can be reached), which are indices of node centrality [##REF##28342071##38##, ##UREF##15##43##].</p>","<p id=\"Par18\">Bootstrapping was performed to explore the accuracy and stability of the network [##REF##28342071##38##]. To estimate the accuracy of edge weights, 95% bootstrapped confidence intervals (CIs) around each edge in the network were calculated. Non-parametric bootstrapping (1000 bootstrap samples) was used to construct CIs. To estimate the stability of node centrality, we applied case-dropping bootstrap (1000 bootstrap samples) to calculate the correlational stability coefficient (CS coefficient) [##REF##28342071##38##]. This coefficient represents the maximum proportion of participants that can be dropped from the analysis with the correlation between the original centrality indices and the subset centrality indices of at least 0.7 with 95% probability [##UREF##16##44##]. The CS coefficients of at least above 0.25, but preferable above 0.5, are considered stable [##UREF##16##44##]. Additionally, the bootstrapped values were used to test the significance of edge weights and node strength [##REF##28342071##38##]. These bootstrapped difference tests indicate the difference between two different edge weights or node strengths. A bootstrapped CI around these difference scores was calculated [##REF##28342071##38##].</p>","<p id=\"Par19\">Detection of communities was performed using the Louvain clustering method, a hierarchical clustering method based on multi-level modularity optimization algorithm [##UREF##17##45##].</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<title>Study population and data collection</title>\",\n \"<p id=\\\"Par9\\\">Data of the population-based, cross-sectional SURVAYA study was used, which was approved by the Institutional Review Board (IRBd18122) and registered within clinical trial registration (NCT05379387). The study population is extensively described previously [##REF##36005166##30##]. In short, the SURVAYA study was performed among AYAs (18–39 years old at time of initial cancer diagnosis) diagnosed with cancer between 1999 and 2015 (5–20 years post diagnosis at study invitation), treated at the Netherlands Cancer Institute or one of the University Medical Centers in the Netherlands, and registered within the Netherlands Cancer Registry (NCR). Survivors were invited to complete a one-time questionnaire within PROFILES (Patient Reported Outcomes Following Initial treatment and Long-term Evaluation of Survivorship) [##REF##21621408##31##].</p>\",\n \"<title>Measures</title>\",\n \"<p id=\\\"Par10\\\">Sociodemographic characteristics were obtained through a one-time questionnaire including age at time of questionnaire, sex at birth, marital status, and educational level. Clinical characteristics, obtained by the NCR, include tumor type, stage, primary treatment received, and time since diagnosis. Tumor type was classified according to the third International Classification of Diseases for Oncology (ICDO-3) [##UREF##7##32##]. Cancer stage was classified according to TNM or Ann Arbor Code (Hodgkin lymphoma and Non-Hodgkin lymphoma) [##UREF##8##33##].</p>\",\n \"<p id=\\\"Par11\\\">The EORTC QLQ-SURV111 [##UREF##9##34##], a cancer core survivorship questionnaire that is currently being developed by the European Organization for Research and Treatment of Cancer (EORTC), was used for our network analysis. This questionnaire assesses long-term HRQoL outcomes, including physical, mental, and social HRQoL issues specifically relevant to cancer survivors. We selected 8 functioning scales (physical functioning, cognitive functioning, emotional functioning, role functioning, body image, symptom awareness, sexual functioning, and overall quality of life), 9 symptoms scales (fatigue, sleep problems, pain, social interference, health distress, negative health outlook, social isolation, symptom checklist, and sexual problems), and 3 single items (financial difficulties, worry cancer risk family, and treated differently). Scales and items measuring positive factors of HRQoL and items that were not applicable for all participants were excluded from the analysis. The rationale for this selection is that including these positive scales/items and optional items would result in difficulties with respectively interpreting network associations to intervene on and the development of a network structure. Participants scored the items on a 4-point Likert scale from 1 (not at all) to 4 (very much). Overall quality of life scores ranged from 1 (very poor) to 7 (excellent). All scales and single items were linearly transformed to a “0–100” scale [##UREF##10##35##]. A higher score on the functioning scales indicates better HRQoL/functioning, while a higher score for symptoms indicates more complaints. For our analysis, we transformed the scores of the symptom scales once again, so a higher score on the symptom scale indicates fewer complaints. Now, a higher score on all scales corresponds with better functioning, fewer complaints, and a better overall quality of life.</p>\",\n \"<title>Data analysis</title>\",\n \"<p id=\\\"Par12\\\">Data were analyzed using SPSS Statistics (IBM Corporation, version 26.0, Armonk, NY, USA) and R version 4.2.1 packages MVN, huge, qgraph, and bootnet. Descriptive statistics were calculated and presented as frequencies, percentages, means, and standard deviations.</p>\",\n \"<p id=\\\"Par13\\\">We used listwise deletion to exclude participants with missing items on the EORTC QLQ-SURV111 questionnaire, except for the items related to sexuality. This is because sexual functioning and problems can be an important aspect of HRQoL, and missing responses to sensitive questions like sexuality are common [##REF##21134739##36##]. If half of the items from the two sexuality scales were answered, we assumed that the missing item had a value equal to the item that was present for that respondent according to the QLQ-SURV111 scoring manual. In case both items for the scale were missing, we used a copy mean imputation of the study population.</p>\",\n \"<p id=\\\"Par14\\\">We assessed the assumption of multivariate normality with Mardia’s test [##UREF##11##37##], which needs to be fulfilled prior to estimating the network [##REF##28342071##38##]. Mardia’s multivariate skewness and kurtosis coefficients of the numeric scales were calculated [##UREF##11##37##]. In the case of multivariate normality, both <italic>p</italic>-values of skewness and kurtosis should be greater than 0.05 [##UREF##11##37##]. As the data were not multivariate normally distributed according to Mardia’s test (<italic>p</italic><0.05), a nonparanormal transformation was applied to relax the normality assumption [##UREF##12##39##].</p>\",\n \"<p id=\\\"Par15\\\">In our network model, HRQoL is conceptualized as a network of mutually interrelated factors. Because data are continuous scales or items, we used Guassian Graphical model (GGM) [##UREF##6##26##, ##REF##29595293##40##]. Nodes represent the selected HRQoL scales and items, and edges (links connecting two nodes) represent the regularized partial correlation coefficients after controlling for all other nodes. The thickness of the edge visualizes the strength, and the color a positive (red) or negative (blue) partial correlation. The partial correlation is indicated as very small (<italic>r</italic><0.1), small (0.1≤<italic>r</italic><0.3), moderate (0.3≤<italic>r</italic><0.5), and large (<italic>r</italic>>0.5) [##UREF##13##41##].</p>\",\n \"<p id=\\\"Par16\\\">We applied graphical lasso tuned with the Extended Bayesian Information Criterion (EBIC) [##REF##28342071##38##, ##UREF##14##42##]. The EBIC hyperparameter, used to set the preferred simplicity of the model, was set to 0.5 to minimize spurious connections [##REF##28342071##38##]. Graphical lasso is a form of lasso regularization to prevent that edges between two nodes are spurious because of other nodes (i.e., conditional independence association) and small edges were shrinked to zero by dropping them from the model [##UREF##14##42##]. In this way, the estimated network is not over fitted and interpretable.</p>\",\n \"<p id=\\\"Par17\\\">We estimated node strength (i.e., number and strength of edges between nodes), betweenness (i.e., how often a node lies in shortest path between any combination of two nodes), and closeness (i.e., average distance from one node to all other nodes, which indicates how fast a node can be reached), which are indices of node centrality [##REF##28342071##38##, ##UREF##15##43##].</p>\",\n \"<p id=\\\"Par18\\\">Bootstrapping was performed to explore the accuracy and stability of the network [##REF##28342071##38##]. To estimate the accuracy of edge weights, 95% bootstrapped confidence intervals (CIs) around each edge in the network were calculated. Non-parametric bootstrapping (1000 bootstrap samples) was used to construct CIs. To estimate the stability of node centrality, we applied case-dropping bootstrap (1000 bootstrap samples) to calculate the correlational stability coefficient (CS coefficient) [##REF##28342071##38##]. This coefficient represents the maximum proportion of participants that can be dropped from the analysis with the correlation between the original centrality indices and the subset centrality indices of at least 0.7 with 95% probability [##UREF##16##44##]. The CS coefficients of at least above 0.25, but preferable above 0.5, are considered stable [##UREF##16##44##]. Additionally, the bootstrapped values were used to test the significance of edge weights and node strength [##REF##28342071##38##]. These bootstrapped difference tests indicate the difference between two different edge weights or node strengths. A bootstrapped CI around these difference scores was calculated [##REF##28342071##38##].</p>\",\n \"<p id=\\\"Par19\\\">Detection of communities was performed using the Louvain clustering method, a hierarchical clustering method based on multi-level modularity optimization algorithm [##UREF##17##45##].</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Characteristics AYA cancer survivors</title>","<p id=\"Par20\">In total, 11296 AYAs were invited to participate in the study, of whom 4010 (36%) responded. After excluding 414 records with missing data, we included 3596 AYAs in our final analysis; their sociodemographic and clinical characteristics are described in Table ##TAB##0##1##. AYAs were on average 31.5 years old at diagnosis and mostly female (61%). The average time since diagnosis was 12.4 years and the most common cancer types were breast cancer (24%), germ cell tumors (18%), lymphoid hematological malignancies (15%), and tumors of female genitalia (11%).\n</p>","<p id=\"Par21\">The mean scores of the functioning and symptom scales of the QLQ-SURV111 are shown in Table ##TAB##1##2##. The overall global quality of life score of AYAs was on average 77.3 (SD 18.7). The functioning scale with the highest score was physical functioning (mean 91.7; SD 13.9), whereas sexual functioning was the scale with the lowest score (mean 43.7; SD 25.5). AYAs scored the lowest on the symptom scales social isolation (mean 69.1; SD 30.7), fatigue (mean 70.3; SD 26.0), and negative health outlook (mean 74.2; SD 20.0).\n</p>","<title>Network analysis</title>","<title>Overall network</title>","<p id=\"Par22\">The partial correlation network model is shown in Fig. ##FIG##0##1##. In our sample, health distress had a strong partial correlation with negative health outlook (<italic>r</italic> = 0.71) and moderate partial correlation with worries about family getting cancer (<italic>r</italic> =0.48). Symptom checklist had a strong partial correlation with pain (<italic>r</italic> =0.67). Role functioning was strongly partially correlated to physical functioning (<italic>r</italic> =0.70) and to social interference (<italic>r</italic> =0.72), and there was a moderate partial correlation between sexual functioning and sexual problems (<italic>r</italic> =0.43).</p>","<p id=\"Par23\">The bootstrapped confidence intervals of estimated edge weights (Fig. ##FIG##1##2##) show that the previously described strong/moderate correlations in our network are robust. The negative correlations in our network, on the other hand, are not reliable. To test if a correlation between two nodes was significantly different from other correlations, we used the edge difference test (Supplementary material Figure ##SUPPL##0##S1##). This plot showed that the correlation between health distress and negative health outlook was significantly different from all other correlations.</p>","<title>Cluster analysis</title>","<p id=\"Par24\">Within our network model, we identified four clusters (Fig. ##FIG##0##1##); (1) the worriment cluster (orange), (2) the daily functioning cluster (yellow), (3) the psychological cluster (pink), and (4) sexual cluster (green). The worriment cluster consists of the nodes: health distress, negative health outlook, symptom awareness, social isolation, worried about family getting cancer, and people treating you differently. Role functioning, physical functioning, pain, symptom checklist, social interference, and financial difficulties were all part of the daily functioning cluster. Emotional functioning, fatigue, sleep problems, cognitive functioning, and overall quality of life formed the psychological cluster. The sexual cluster consists of sexual functioning, sexual problems, and body image.</p>","<title>Network stability and centrality</title>","<p id=\"Par25\">The CS coefficient for strength, closeness, and betweenness were 0.75, 0.75, and 0.75 respectively, indicating a stable and reliable network (Fig. ##FIG##2##3##). Regarding centrality, the nodes with the highest strength are the most central, and therefore the most important nodes of the network model. In our model, nodes with the highest strength were negative health outlook (standardized centrality estimates (SCE) = 1.60), role functioning (SCE = 1.40), health distress (SCE = 1.40), and emotional functioning (SCE = 1.30). In addition, health distress and negative health outlook had the highest betweenness, and emotional functioning and health distress had the highest closeness (Fig. ##FIG##3##4##). The centrality difference plot (Supplementary material Figure ##SUPPL##0##S2##) demonstrates that the strength of the node negative health outlook significantly differed from the other nodes in the network model.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Characteristics AYA cancer survivors</title>\",\n \"<p id=\\\"Par20\\\">In total, 11296 AYAs were invited to participate in the study, of whom 4010 (36%) responded. After excluding 414 records with missing data, we included 3596 AYAs in our final analysis; their sociodemographic and clinical characteristics are described in Table ##TAB##0##1##. AYAs were on average 31.5 years old at diagnosis and mostly female (61%). The average time since diagnosis was 12.4 years and the most common cancer types were breast cancer (24%), germ cell tumors (18%), lymphoid hematological malignancies (15%), and tumors of female genitalia (11%).\\n</p>\",\n \"<p id=\\\"Par21\\\">The mean scores of the functioning and symptom scales of the QLQ-SURV111 are shown in Table ##TAB##1##2##. The overall global quality of life score of AYAs was on average 77.3 (SD 18.7). The functioning scale with the highest score was physical functioning (mean 91.7; SD 13.9), whereas sexual functioning was the scale with the lowest score (mean 43.7; SD 25.5). AYAs scored the lowest on the symptom scales social isolation (mean 69.1; SD 30.7), fatigue (mean 70.3; SD 26.0), and negative health outlook (mean 74.2; SD 20.0).\\n</p>\",\n \"<title>Network analysis</title>\",\n \"<title>Overall network</title>\",\n \"<p id=\\\"Par22\\\">The partial correlation network model is shown in Fig. ##FIG##0##1##. In our sample, health distress had a strong partial correlation with negative health outlook (<italic>r</italic> = 0.71) and moderate partial correlation with worries about family getting cancer (<italic>r</italic> =0.48). Symptom checklist had a strong partial correlation with pain (<italic>r</italic> =0.67). Role functioning was strongly partially correlated to physical functioning (<italic>r</italic> =0.70) and to social interference (<italic>r</italic> =0.72), and there was a moderate partial correlation between sexual functioning and sexual problems (<italic>r</italic> =0.43).</p>\",\n \"<p id=\\\"Par23\\\">The bootstrapped confidence intervals of estimated edge weights (Fig. ##FIG##1##2##) show that the previously described strong/moderate correlations in our network are robust. The negative correlations in our network, on the other hand, are not reliable. To test if a correlation between two nodes was significantly different from other correlations, we used the edge difference test (Supplementary material Figure ##SUPPL##0##S1##). This plot showed that the correlation between health distress and negative health outlook was significantly different from all other correlations.</p>\",\n \"<title>Cluster analysis</title>\",\n \"<p id=\\\"Par24\\\">Within our network model, we identified four clusters (Fig. ##FIG##0##1##); (1) the worriment cluster (orange), (2) the daily functioning cluster (yellow), (3) the psychological cluster (pink), and (4) sexual cluster (green). The worriment cluster consists of the nodes: health distress, negative health outlook, symptom awareness, social isolation, worried about family getting cancer, and people treating you differently. Role functioning, physical functioning, pain, symptom checklist, social interference, and financial difficulties were all part of the daily functioning cluster. Emotional functioning, fatigue, sleep problems, cognitive functioning, and overall quality of life formed the psychological cluster. The sexual cluster consists of sexual functioning, sexual problems, and body image.</p>\",\n \"<title>Network stability and centrality</title>\",\n \"<p id=\\\"Par25\\\">The CS coefficient for strength, closeness, and betweenness were 0.75, 0.75, and 0.75 respectively, indicating a stable and reliable network (Fig. ##FIG##2##3##). Regarding centrality, the nodes with the highest strength are the most central, and therefore the most important nodes of the network model. In our model, nodes with the highest strength were negative health outlook (standardized centrality estimates (SCE) = 1.60), role functioning (SCE = 1.40), health distress (SCE = 1.40), and emotional functioning (SCE = 1.30). In addition, health distress and negative health outlook had the highest betweenness, and emotional functioning and health distress had the highest closeness (Fig. ##FIG##3##4##). The centrality difference plot (Supplementary material Figure ##SUPPL##0##S2##) demonstrates that the strength of the node negative health outlook significantly differed from the other nodes in the network model.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par26\">This study shows the results of a stable and reliable network analysis based on HRQoL data of 3596 long-term AYAs, as the first to our knowledge. Although the overall global quality of life score was 77.3 on average, the lowest functioning scale score was sexual functioning and the lowest symptom scale scores were social isolation, fatigue, and negative health outlook. This network showed several strong/moderate partial correlations, with the partial correlation between health distress and negative health outlook being the only one significantly different from all other. Also, the strength of the node negative health outlook was significantly different from all other nodes. In total, four clusters of negative symptom and functioning HRQoL scales were identified, including a worriment cluster, daily functioning cluster, psychological cluster, and sexual cluster.</p>","<p id=\"Par27\">As this study is the first to apply a network analysis based on a wide range of HRQoL data of AYAs using the relatively new QLQ-SURV111 questionnaire, findings are difficult to compare with other studies. Although previous studies had similar aims, they mostly used the EORTC QLQ-C30 to assess HRQoL and studied adult cancer survivors [##REF##34387856##28##, ##REF##35472477##46##]. This was also the case in the network analysis conducted by Rooij et al. in a heterogeneous sample of adult cancer survivors where the EORTC QLQ-C30 was used [##REF##34387856##28##]. In their analysis, fatigue was consistently central and had moderate direct relationships with emotional symptoms, cognitive symptoms, appetite loss, dyspnea, and pain. Fatigue being the most central symptom was in contrast with our results, which might be explained by the much younger population in our study (31.5 years vs 61 years) and the difference in time since diagnosis, which was considerably longer in our study (12.4 years vs 4.2 years). In our network, negative health outlook was the node with the highest strength, which had a strong correlation with health distress within the worriment cluster. This suggests that psychological and emotional issues remain more of relevance to AYA cancer survivors also after long-term follow-up, and are better picked up by the QLQ-SURV111 questionnaire that covers a more complete range of relevant survivorship issues.</p>","<p id=\"Par28\">Other network studies focused specifically on a construct (e.g., fear of recurrence) or predefined clusters of symptoms, for example, the network analysis on fear of cancer recurrence, anxiety, and depression in breast cancer patients of Yang et al. [##REF##35472477##46##]. In their network, “having trouble relaxing” was the most central node, anxiety and depression were well-connected, and fear of cancer recurrence formed a distinct cluster. The use of the broad range of survivorship issues of the QLQ-SURV111, including psychological, social, physical symptoms and functioning, allowed us to explore clusters within our network as well. An interesting cluster that emerged was the worriment cluster, which consisted of negative health outlook, health distress, symptom awareness, social isolation, worried about family getting cancer, and people treating you differently. Although our questions regarding worries were different, one other network analysis on AYAs focused on the construct of fear of cancer recurrence [##REF##35565220##47##]. Here, the researchers found fear of serious medical interventions as the most central symptom in their network, with the highest node strengths for fear of pain, fear of relying on strangers for activities of daily living, and fear of severe medical treatments. Based on their results, which emphasize the centrality of emotional issues among AYA patients, they stress the importance of prioritizing these symptoms for interventions. However, as stated previously, comparing the results of these studies with our results should be done cautiously as study populations, study designs, aims, and used questionnaires differ. The lack of studies among AYAs to compare our findings with stresses the need for more AYA-specialized research.</p>","<p id=\"Par29\">The results of a network analysis can provide more insight in the HRQoL of AYAs, in which symptoms and functioning can influence each other, instead of perceiving them as individual factors [##UREF##18##48##]. Identifying the most important factors in a network can help to address these problems with targeted interventions and healthcare, and lead to novel research ideas. First, we recommend to replicate network analysis studies in other groups of AYAs to be able to make comparisons between studies with similar study populations and draw conclusions with more certainty—changing the exploratory approach into a confirmative approach. Ideally, longitudinal data needs to be collected to draw conclusions over time and adapt healthcare to these time-related changes where needed. This could even be specific to longitudinal ecological momentary assessment (EMA) in which participants are asked to complete (parts of) the expected momentary dynamic items of the HRQoL questionnaire multiple times a day during a study period. In this way, variations over time can be taken into account to a more detailed and individual level. Inter- and intra-individual differences over time might result in changes in prevalence (scores of the scales or items), partial correlations of nodes, centrality, and clusters formed.</p>","<p id=\"Par30\">In addition, subgroup analysis should be performed to make healthcare interventions even more tailored. AYAs subgroup analysis could focus on age, gender, stage, treatment, and type of cancer. In order to establish tailored care, it is important to make the subgroups as specific as possible while remaining a stable and reliable network. However, it should also be noted that these results represent means and thus differences may exist on an individual level in clinical practice.</p>","<p id=\"Par31\">Future research can lead to and optimize supportive care and targeted interventions, like psycho-oncological care and psychosocial, behavioral, and supportive interventions [##REF##33675909##49##]. For example, the strong significant correlation between health distress and negative health outlook, which is part of the worriment cluster in this study, might be intervened on by distress screening and renewed/tailored, age-specific psycho-oncological aftercare [##REF##35602702##50##]. For healthcare providers (HCPs) involved in AYA healthcare, and in general, visualization of the nodes, correlations, and clusters may help to understand the cohesion between different factors/symptoms and the influence they might have on each other, and more important, how a targeted intervention can influence several HRQoL outcomes simultaneously. This advocates for holistic and age-specific psycho-oncological aftercare in which multiple factors are targeted simultaneously by a multidisciplinary team of HCPs, to be as efficient and effective as possible.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par26\\\">This study shows the results of a stable and reliable network analysis based on HRQoL data of 3596 long-term AYAs, as the first to our knowledge. Although the overall global quality of life score was 77.3 on average, the lowest functioning scale score was sexual functioning and the lowest symptom scale scores were social isolation, fatigue, and negative health outlook. This network showed several strong/moderate partial correlations, with the partial correlation between health distress and negative health outlook being the only one significantly different from all other. Also, the strength of the node negative health outlook was significantly different from all other nodes. In total, four clusters of negative symptom and functioning HRQoL scales were identified, including a worriment cluster, daily functioning cluster, psychological cluster, and sexual cluster.</p>\",\n \"<p id=\\\"Par27\\\">As this study is the first to apply a network analysis based on a wide range of HRQoL data of AYAs using the relatively new QLQ-SURV111 questionnaire, findings are difficult to compare with other studies. Although previous studies had similar aims, they mostly used the EORTC QLQ-C30 to assess HRQoL and studied adult cancer survivors [##REF##34387856##28##, ##REF##35472477##46##]. This was also the case in the network analysis conducted by Rooij et al. in a heterogeneous sample of adult cancer survivors where the EORTC QLQ-C30 was used [##REF##34387856##28##]. In their analysis, fatigue was consistently central and had moderate direct relationships with emotional symptoms, cognitive symptoms, appetite loss, dyspnea, and pain. Fatigue being the most central symptom was in contrast with our results, which might be explained by the much younger population in our study (31.5 years vs 61 years) and the difference in time since diagnosis, which was considerably longer in our study (12.4 years vs 4.2 years). In our network, negative health outlook was the node with the highest strength, which had a strong correlation with health distress within the worriment cluster. This suggests that psychological and emotional issues remain more of relevance to AYA cancer survivors also after long-term follow-up, and are better picked up by the QLQ-SURV111 questionnaire that covers a more complete range of relevant survivorship issues.</p>\",\n \"<p id=\\\"Par28\\\">Other network studies focused specifically on a construct (e.g., fear of recurrence) or predefined clusters of symptoms, for example, the network analysis on fear of cancer recurrence, anxiety, and depression in breast cancer patients of Yang et al. [##REF##35472477##46##]. In their network, “having trouble relaxing” was the most central node, anxiety and depression were well-connected, and fear of cancer recurrence formed a distinct cluster. The use of the broad range of survivorship issues of the QLQ-SURV111, including psychological, social, physical symptoms and functioning, allowed us to explore clusters within our network as well. An interesting cluster that emerged was the worriment cluster, which consisted of negative health outlook, health distress, symptom awareness, social isolation, worried about family getting cancer, and people treating you differently. Although our questions regarding worries were different, one other network analysis on AYAs focused on the construct of fear of cancer recurrence [##REF##35565220##47##]. Here, the researchers found fear of serious medical interventions as the most central symptom in their network, with the highest node strengths for fear of pain, fear of relying on strangers for activities of daily living, and fear of severe medical treatments. Based on their results, which emphasize the centrality of emotional issues among AYA patients, they stress the importance of prioritizing these symptoms for interventions. However, as stated previously, comparing the results of these studies with our results should be done cautiously as study populations, study designs, aims, and used questionnaires differ. The lack of studies among AYAs to compare our findings with stresses the need for more AYA-specialized research.</p>\",\n \"<p id=\\\"Par29\\\">The results of a network analysis can provide more insight in the HRQoL of AYAs, in which symptoms and functioning can influence each other, instead of perceiving them as individual factors [##UREF##18##48##]. Identifying the most important factors in a network can help to address these problems with targeted interventions and healthcare, and lead to novel research ideas. First, we recommend to replicate network analysis studies in other groups of AYAs to be able to make comparisons between studies with similar study populations and draw conclusions with more certainty—changing the exploratory approach into a confirmative approach. Ideally, longitudinal data needs to be collected to draw conclusions over time and adapt healthcare to these time-related changes where needed. This could even be specific to longitudinal ecological momentary assessment (EMA) in which participants are asked to complete (parts of) the expected momentary dynamic items of the HRQoL questionnaire multiple times a day during a study period. In this way, variations over time can be taken into account to a more detailed and individual level. Inter- and intra-individual differences over time might result in changes in prevalence (scores of the scales or items), partial correlations of nodes, centrality, and clusters formed.</p>\",\n \"<p id=\\\"Par30\\\">In addition, subgroup analysis should be performed to make healthcare interventions even more tailored. AYAs subgroup analysis could focus on age, gender, stage, treatment, and type of cancer. In order to establish tailored care, it is important to make the subgroups as specific as possible while remaining a stable and reliable network. However, it should also be noted that these results represent means and thus differences may exist on an individual level in clinical practice.</p>\",\n \"<p id=\\\"Par31\\\">Future research can lead to and optimize supportive care and targeted interventions, like psycho-oncological care and psychosocial, behavioral, and supportive interventions [##REF##33675909##49##]. For example, the strong significant correlation between health distress and negative health outlook, which is part of the worriment cluster in this study, might be intervened on by distress screening and renewed/tailored, age-specific psycho-oncological aftercare [##REF##35602702##50##]. For healthcare providers (HCPs) involved in AYA healthcare, and in general, visualization of the nodes, correlations, and clusters may help to understand the cohesion between different factors/symptoms and the influence they might have on each other, and more important, how a targeted intervention can influence several HRQoL outcomes simultaneously. This advocates for holistic and age-specific psycho-oncological aftercare in which multiple factors are targeted simultaneously by a multidisciplinary team of HCPs, to be as efficient and effective as possible.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p id=\"Par33\">This innovative network analysis provides insight in the nodes, correlations, and clusters that could be targeted to improve the HRQoL outcomes of AYAs. Future studies with longitudinal data and subgroup analyses can tailor the interventions and provided healthcare even more, specifically for those at risk of poor HRQoL outcomes. With these insights, more targeted interventions and healthcare can be provided and developed.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p id=\\\"Par33\\\">This innovative network analysis provides insight in the nodes, correlations, and clusters that could be targeted to improve the HRQoL outcomes of AYAs. Future studies with longitudinal data and subgroup analyses can tailor the interventions and provided healthcare even more, specifically for those at risk of poor HRQoL outcomes. With these insights, more targeted interventions and healthcare can be provided and developed.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose</title>","<p id=\"Par1\">Adolescent and young adult cancer survivors (AYAs) are at increased risk of long-term and late effects, and experience unmet needs, impacting their health-related quality of life (HRQoL). In order to provide and optimize supportive care and targeted interventions for this unique population, it is important to study HRQoL factors’ interconnectedness on a population level. Therefore, this network analysis was performed with the aim to explore the interconnectedness between HRQoL factors, in the analysis described as nodes, among long-term AYAs.</p>","<title>Methods</title>","<p id=\"Par2\">This population-based cohort study used cross-sectional survey data of long-term AYAs, who were identified by the Netherlands Cancer Registry (NCR). Participants completed a one-time survey (SURVAYA study), including the EORTC survivorship questionnaire (QLQ-SURV111) to assess their long-term HRQoL outcomes and sociodemographic characteristics. The NCR provided the clinical data. Descriptive statistics and a network analysis, including network clustering, were performed.</p>","<title>Results</title>","<p id=\"Par3\">In total, 3596 AYAs (on average 12.4 years post diagnosis) were included in our network analysis. The network was proven stable and reliable and, in total, four clusters were identified, including a worriment, daily functioning, psychological, and sexual cluster. Negative health outlook, part of the worriment cluster, was the node with the highest strength and its partial correlation with health distress was significantly different from all other partial correlations.</p>","<title>Conclusion</title>","<p id=\"Par4\">This study shows the results of a stable and reliable network analysis based on HRQoL data of long-term AYAs, and identified nodes, correlations, and clusters that could be intervened on to improve the HRQoL outcomes of AYAs.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00520-023-08295-0.</p>","<title>Keywords</title>"],"string":"[\n \"<title>Purpose</title>\",\n \"<p id=\\\"Par1\\\">Adolescent and young adult cancer survivors (AYAs) are at increased risk of long-term and late effects, and experience unmet needs, impacting their health-related quality of life (HRQoL). In order to provide and optimize supportive care and targeted interventions for this unique population, it is important to study HRQoL factors’ interconnectedness on a population level. Therefore, this network analysis was performed with the aim to explore the interconnectedness between HRQoL factors, in the analysis described as nodes, among long-term AYAs.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">This population-based cohort study used cross-sectional survey data of long-term AYAs, who were identified by the Netherlands Cancer Registry (NCR). Participants completed a one-time survey (SURVAYA study), including the EORTC survivorship questionnaire (QLQ-SURV111) to assess their long-term HRQoL outcomes and sociodemographic characteristics. The NCR provided the clinical data. Descriptive statistics and a network analysis, including network clustering, were performed.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">In total, 3596 AYAs (on average 12.4 years post diagnosis) were included in our network analysis. The network was proven stable and reliable and, in total, four clusters were identified, including a worriment, daily functioning, psychological, and sexual cluster. Negative health outlook, part of the worriment cluster, was the node with the highest strength and its partial correlation with health distress was significantly different from all other partial correlations.</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par4\\\">This study shows the results of a stable and reliable network analysis based on HRQoL data of long-term AYAs, and identified nodes, correlations, and clusters that could be intervened on to improve the HRQoL outcomes of AYAs.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00520-023-08295-0.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Strengths and limitations</title>","<p id=\"Par32\">This explorative study represents the very first network analysis using data on a range of HRQoL outcomes of long-term AYAs to our knowledge. Strengths include the large sample size, the establishment of a stable and reliable network, and the inclusion of a wide range of survivorship issues. However, the results should be interpreted with caution as the EORTC QLQ-SURV111 is not yet finalized and validated. In addition, our study included mostly females and over 40% was diagnosed with a stage I tumor. With a response rate of 36%, there are several subgroups (males, AYAs with a more aggressive disease, and AYAs diagnosed at the age of 18–24) underrepresented in this analysis who might have different HRQoL outcomes [##REF##36005166##30##]. Results may therefore not be generalizable to the total AYAs population. Also, we have not taken a closer look at the outcomes of specific subgroups (between groups), as the study group as a whole was analyzed. As mentioned previously, the subgroup analyses should be part of future research to tailor interventions with a risk-based approach. In line with this, due to the methodology of this study, i.e., a cross-sectional questionnaire study, no causal pathways or changes in HRQoL factors over time can be assessed. In the future, this might be tackled by using longitudinal data instead of cross-sectional data.</p>","<title>Supplementary information</title>","<p>\n</p>"],"string":"[\n \"<title>Strengths and limitations</title>\",\n \"<p id=\\\"Par32\\\">This explorative study represents the very first network analysis using data on a range of HRQoL outcomes of long-term AYAs to our knowledge. Strengths include the large sample size, the establishment of a stable and reliable network, and the inclusion of a wide range of survivorship issues. However, the results should be interpreted with caution as the EORTC QLQ-SURV111 is not yet finalized and validated. In addition, our study included mostly females and over 40% was diagnosed with a stage I tumor. With a response rate of 36%, there are several subgroups (males, AYAs with a more aggressive disease, and AYAs diagnosed at the age of 18–24) underrepresented in this analysis who might have different HRQoL outcomes [##REF##36005166##30##]. Results may therefore not be generalizable to the total AYAs population. Also, we have not taken a closer look at the outcomes of specific subgroups (between groups), as the study group as a whole was analyzed. As mentioned previously, the subgroup analyses should be part of future research to tailor interventions with a risk-based approach. In line with this, due to the methodology of this study, i.e., a cross-sectional questionnaire study, no causal pathways or changes in HRQoL factors over time can be assessed. In the future, this might be tackled by using longitudinal data instead of cross-sectional data.</p>\",\n \"<title>Supplementary information</title>\",\n \"<p>\\n</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>The authors wish to thank all the patients for their participation in the study and the registration team of the Netherlands Comprehensive Cancer Organisation (IKNL) for the collection of data for the Netherlands Cancer Registry.</p>","<title>Author contribution</title>","<p>Conceptualization: C.V., S.H.M.J., T.I.B., D.W., D.C.R., C.D., and O.H.; data curation: C.V. and T.I.B.; formal analysis: T.I.B., D.W., and C.D.; funding acquisition: W.T.A.G. and O.H.; investigation: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; methodology: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; project administration: C.V.; resources: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; software: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; supervision: W.T.A.G. and O.H.; validation: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; visualization: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; writing—original draft preparation: C.V., D.C.R., and S.H.M.J.; writing—review and editing: T.I.B., D.W., C.V., D.C.R., C.D., R.T., R.B., S.E.J.K., J.M.K., J.M.T., M.E.M.M.B., T.H., R.I.L., J.N., M.C.M.K., W.T.A.G., S.H.M.J., and O.H.; all authors have read and approved the final manuscript.</p>","<title>Funding</title>","<p>This study was funded by a VIDI grant (VIDI198.007) and an investment grant (#480-08-009) from the Netherlands Organization for Scientific Research. This research was also supported by an institutional grant of the Dutch Cancer Society and of the Dutch Ministry of Health, Welfare and Sport.</p>","<title>Data availability</title>","<p>The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy issues.</p>","<title>Declarations</title>","<title>Ethics approval</title>","<p id=\"Par34\">The study was conducted in accordance with the Declaration of Helsinki, and approved by the Netherlands Cancer Institute Institutional Review Board (IRBd18122) on February 6, 2019.</p>","<title>Consent to participate</title>","<p id=\"Par35\">Informed consent was obtained from all subjects (responders) involved in the study.</p>","<title>Competing interests</title>","<p id=\"Par36\">The authors have no relevant financial or non-financial interests to disclose.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>The authors wish to thank all the patients for their participation in the study and the registration team of the Netherlands Comprehensive Cancer Organisation (IKNL) for the collection of data for the Netherlands Cancer Registry.</p>\",\n \"<title>Author contribution</title>\",\n \"<p>Conceptualization: C.V., S.H.M.J., T.I.B., D.W., D.C.R., C.D., and O.H.; data curation: C.V. and T.I.B.; formal analysis: T.I.B., D.W., and C.D.; funding acquisition: W.T.A.G. and O.H.; investigation: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; methodology: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; project administration: C.V.; resources: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; software: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; supervision: W.T.A.G. and O.H.; validation: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; visualization: T.I.B., D.W., C.V., D.C.R., C.D., and S.H.M.J.; writing—original draft preparation: C.V., D.C.R., and S.H.M.J.; writing—review and editing: T.I.B., D.W., C.V., D.C.R., C.D., R.T., R.B., S.E.J.K., J.M.K., J.M.T., M.E.M.M.B., T.H., R.I.L., J.N., M.C.M.K., W.T.A.G., S.H.M.J., and O.H.; all authors have read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>This study was funded by a VIDI grant (VIDI198.007) and an investment grant (#480-08-009) from the Netherlands Organization for Scientific Research. This research was also supported by an institutional grant of the Dutch Cancer Society and of the Dutch Ministry of Health, Welfare and Sport.</p>\",\n \"<title>Data availability</title>\",\n \"<p>The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy issues.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethics approval</title>\",\n \"<p id=\\\"Par34\\\">The study was conducted in accordance with the Declaration of Helsinki, and approved by the Netherlands Cancer Institute Institutional Review Board (IRBd18122) on February 6, 2019.</p>\",\n \"<title>Consent to participate</title>\",\n \"<p id=\\\"Par35\\\">Informed consent was obtained from all subjects (responders) involved in the study.</p>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par36\\\">The authors have no relevant financial or non-financial interests to disclose.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>The (cluster) network of HRQoL outcomes of long-term AYA cancer survivors. In this partial correlation network model, the nodes (PF: physical functioning, CF: cognitive functioning, EF: emotional functioning, RF: role functioning, BI: body image, SA: symptom awareness, SF: sexual functioning, FA: fatigue, SL: sleep problems, PA: pain, Sif: social interference, HD: health distress, NHO: negative health outlook, SI: social isolation, SC: symptom checklist, SP: sexual problems, FD: financial difficulties, WF: worried about family getting cancer, TD: people treating you differently, QL: overall quality of life) represent all the HRQoL scales of the QLQ-SURV111 and the edges (links connecting two nodes) represent the regularized partial correlation coefficients after controlling for all other nodes. The blue color indicates a positive partial correlation and a red color a negative partial correlation between two nodes. The thickness of the edge visualizes the strength of the partial correlation between two nodes. The four clusters that we identified within our network model include in orange the worriment cluster, in yellow the daily functioning cluster, in pink the psychological cluster, and in green the sexual cluster</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Bootstrapped confidence intervals of estimated edge weights. Each horizontal line represents one edge of the network, ordered from the edge with the highest edge weight to the edge with the lowest edge weight. The red line indicates the sample values and the gray lines are the bootstrapped CIs. The larger the gray line the less certain the edge value is</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Correlational stability plot of centrality indices by case-dropping subset bootstrap. Correlations between centrality indices of network sampled with persons dropped and the original sample. Lines indicate the means and areas indicate the range from the 2.5th quantile to the 97.5th quantile</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Central indices of the network. Centrality indices are shown as standardized <italic>z</italic>-scores. PF: physical functioning, CF: cognitive functioning, EF: emotional functioning, RF: role functioning, BI: body image, SA: symptom awareness, SF: sexual functioning, FA: fatigue, SL: sleep problems, PA: pain, Sif: social interference, HD: health distress, NHO: negative health outlook, SI: social isolation, SC: symptom checklist, SP: sexual problems, FD: financial difficulties, WF: worried about family getting cancer, TD: people treating you differently, QL: overall quality of life</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>The (cluster) network of HRQoL outcomes of long-term AYA cancer survivors. In this partial correlation network model, the nodes (PF: physical functioning, CF: cognitive functioning, EF: emotional functioning, RF: role functioning, BI: body image, SA: symptom awareness, SF: sexual functioning, FA: fatigue, SL: sleep problems, PA: pain, Sif: social interference, HD: health distress, NHO: negative health outlook, SI: social isolation, SC: symptom checklist, SP: sexual problems, FD: financial difficulties, WF: worried about family getting cancer, TD: people treating you differently, QL: overall quality of life) represent all the HRQoL scales of the QLQ-SURV111 and the edges (links connecting two nodes) represent the regularized partial correlation coefficients after controlling for all other nodes. The blue color indicates a positive partial correlation and a red color a negative partial correlation between two nodes. The thickness of the edge visualizes the strength of the partial correlation between two nodes. The four clusters that we identified within our network model include in orange the worriment cluster, in yellow the daily functioning cluster, in pink the psychological cluster, and in green the sexual cluster</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Bootstrapped confidence intervals of estimated edge weights. Each horizontal line represents one edge of the network, ordered from the edge with the highest edge weight to the edge with the lowest edge weight. The red line indicates the sample values and the gray lines are the bootstrapped CIs. The larger the gray line the less certain the edge value is</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Correlational stability plot of centrality indices by case-dropping subset bootstrap. Correlations between centrality indices of network sampled with persons dropped and the original sample. Lines indicate the means and areas indicate the range from the 2.5th quantile to the 97.5th quantile</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Central indices of the network. Centrality indices are shown as standardized <italic>z</italic>-scores. PF: physical functioning, CF: cognitive functioning, EF: emotional functioning, RF: role functioning, BI: body image, SA: symptom awareness, SF: sexual functioning, FA: fatigue, SL: sleep problems, PA: pain, Sif: social interference, HD: health distress, NHO: negative health outlook, SI: social isolation, SC: symptom checklist, SP: sexual problems, FD: financial difficulties, WF: worried about family getting cancer, TD: people treating you differently, QL: overall quality of life</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Sociodemographic and clinical characteristics of the study cohort</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" colspan=\"2\"/><th colspan=\"2\">Respondents N = 3596</th></tr><tr><th><italic>n</italic></th><th>%</th></tr></thead><tbody><tr><td rowspan=\"2\">Gender</td><td>Male</td><td>1420</td><td>39</td></tr><tr><td>Female</td><td>2176</td><td>61</td></tr><tr><td rowspan=\"4\">Age at diagnosis mean (SD)</td><td/><td colspan=\"2\">31.5 (5.9)</td></tr><tr><td>18–24 years</td><td>569</td><td>16</td></tr><tr><td>25–34 years</td><td>1584</td><td>44</td></tr><tr><td>35–39 years</td><td>1443</td><td>40</td></tr><tr><td>Age at completing questionnaire mean (SD)</td><td/><td colspan=\"2\">44.5 (7.5)</td></tr><tr><td rowspan=\"4\">Time since diagnosis</td><td/><td colspan=\"2\">12.4 (4.5)</td></tr><tr><td>5–10 years</td><td>1452</td><td>40</td></tr><tr><td>11–15 years</td><td>1247</td><td>35</td></tr><tr><td>16–20 years</td><td>897</td><td>25</td></tr><tr><td rowspan=\"12\">Type of cancer</td><td>Breast cancer</td><td>846</td><td>23.5</td></tr><tr><td>Germ cell tumors</td><td>637</td><td>17.7</td></tr><tr><td>Lymphoid hematological malignancies</td><td>540</td><td>15.0</td></tr><tr><td>Female genitalia tumors</td><td>383</td><td>10.7</td></tr><tr><td>Melanoma</td><td>252</td><td>7.0</td></tr><tr><td>Thyroid cancer</td><td>224</td><td>6.2</td></tr><tr><td>Bone or soft tissue sarcoma</td><td>161</td><td>4.5</td></tr><tr><td>Myeloid hematological malignancies</td><td>136</td><td>3.8</td></tr><tr><td>Central nervous system tumors</td><td>131</td><td>3.6</td></tr><tr><td>Head and neck cancer</td><td>107</td><td>3.0</td></tr><tr><td>Digestive tract tumors</td><td>104</td><td>2.9</td></tr><tr><td>Other*</td><td>75</td><td>2.1</td></tr><tr><td rowspan=\"5\">Tumor stage</td><td>I</td><td>1537</td><td>42.7</td></tr><tr><td>II</td><td>957</td><td>26.6</td></tr><tr><td>III</td><td>515</td><td>14.3</td></tr><tr><td>IV</td><td>165</td><td>4.6</td></tr><tr><td>Missing</td><td>422</td><td>11.7</td></tr><tr><td rowspan=\"6\">Primary treatment modality</td><td>Surgery</td><td>2799</td><td>77.8</td></tr><tr><td>Chemotherapy</td><td>2030</td><td>56.5</td></tr><tr><td>Radiotherapy</td><td>1716</td><td>47.7</td></tr><tr><td>Hormonal therapy</td><td>435</td><td>12.1</td></tr><tr><td>Targeted therapy</td><td>282</td><td>7.8</td></tr><tr><td>Stem cell therapy</td><td>130</td><td>3.6</td></tr><tr><td>Marital status (at time of questionnaire)</td><td>In a relation</td><td>3001</td><td>83.5</td></tr><tr><td rowspan=\"4\">Educational level</td><td>Primary school or equivalent</td><td>16</td><td>0.4</td></tr><tr><td>Secondary school or equivalent</td><td>1514</td><td>42.1</td></tr><tr><td>College/university or equivalent</td><td>2060</td><td>57.3</td></tr><tr><td>Missing</td><td>6</td><td>0.2</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>The long-term HRQoL outcomes from the cancer survivorship core questionnaire (QLQ-SURV111) of the AYA cancer survivors, arranged by scale and total score mean</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Scales</th><th>Short names</th><th>Functional/symptom</th><th>Number of items (<italic>n</italic>)</th><th>Item numbers</th><th>Raw scores<break/>mean (SD)</th><th>Total scores<break/>mean (SD)</th></tr></thead><tbody><tr><td>Physical functioning</td><td>PF</td><td>Functional</td><td>5</td><td>1–5</td><td>1.2 (0.4)</td><td>91.7 (13.9)</td></tr><tr><td>Role functioning</td><td>RF</td><td>Functional</td><td>3</td><td>69–71</td><td>1.5 (0.7)</td><td>83.7 (24.5)</td></tr><tr><td>Emotional functioning</td><td>EF</td><td>Functional</td><td>7</td><td>52–58</td><td>1.6 (0.6)</td><td>81.0 (19.9)</td></tr><tr><td>Cognitive functioning</td><td>CF</td><td>Functional</td><td>4</td><td>47, 48, 50, 51</td><td>1.6 (0.7)</td><td>79.6 (22.6)</td></tr><tr><td>Body image</td><td>BI</td><td>Functional</td><td>2</td><td>40, 41</td><td>1.7 (0.7)</td><td>77.8 (24.6)</td></tr><tr><td>Overall quality of life</td><td>QL</td><td>Functional</td><td>1</td><td>121</td><td>5.6 (1.1)</td><td>77.3 (18.7)</td></tr><tr><td>Symptom awareness</td><td>SA</td><td>Functional</td><td>2</td><td>76, 77</td><td>2.2 (0.8)</td><td>60.9 (25.4)</td></tr><tr><td>Sexual functioning</td><td>SF</td><td>Functional</td><td>2</td><td>111, 112</td><td>2.3 (0.8)</td><td>43.7 (25.5)</td></tr><tr><td>Financial difficulties</td><td>FD</td><td>Symptom</td><td>1</td><td>68</td><td>1.3 (0.7)</td><td>89.4 (23.8)</td></tr><tr><td>Social interference</td><td>Sif</td><td>Symptom</td><td>2</td><td>73, 74</td><td>1.4 (0.7)</td><td>88.1 (22.0)</td></tr><tr><td>Worry cancer risk family</td><td>WF</td><td>Symptom</td><td>1</td><td>61</td><td>1.4 (0.7)</td><td>86.6 (23.1)</td></tr><tr><td>Symptom checklist</td><td>SC</td><td>Symptom</td><td>17</td><td>20, 21, 23–26, 28, 30–38, 75</td><td>1.4 (0.4)</td><td>85.8 (13.5)</td></tr><tr><td>Treated differently</td><td>TD</td><td>Symptom</td><td>1</td><td>107</td><td>1.4 (0.7)</td><td>85.2 (22.6)</td></tr><tr><td>Pain</td><td>PA</td><td>Symptom</td><td>2</td><td>22, 72</td><td>1.5 (0.7)</td><td>84.2 (23.0)</td></tr><tr><td>Sexual problems</td><td>SP</td><td>Symptom</td><td>2</td><td>113, 117</td><td>1.5 (0.8)</td><td>82.8 (26.3)</td></tr><tr><td>Health distress</td><td>HD</td><td>Symptom</td><td>3</td><td>63–65</td><td>1.6 (0.7)</td><td>79.0 (22.3)</td></tr><tr><td>Sleep problems</td><td>SL</td><td>Symptom</td><td>4</td><td>16–19</td><td>1.8 (0.7)</td><td>74.5 (23.6)</td></tr><tr><td>Negative health outlook</td><td>NHO</td><td>Symptom</td><td>7</td><td>60, 62, 64, 81, 82, 98, 99</td><td>1.8 (0.6)</td><td>74.2 (20.0)</td></tr><tr><td>Fatigue</td><td>FA</td><td>Symptom</td><td>4</td><td>6–9</td><td>1.9 (0.8)</td><td>70.3 (26.0)</td></tr><tr><td>Social isolation</td><td>SI</td><td>Symptom</td><td>2</td><td>89, 90</td><td>1.9 (0.9)</td><td>69.1 (30.7)</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Sociodemographic and clinical characteristics of the study cohort</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th rowspan=\\\"2\\\" colspan=\\\"2\\\"/><th colspan=\\\"2\\\">Respondents N = 3596</th></tr><tr><th><italic>n</italic></th><th>%</th></tr></thead><tbody><tr><td rowspan=\\\"2\\\">Gender</td><td>Male</td><td>1420</td><td>39</td></tr><tr><td>Female</td><td>2176</td><td>61</td></tr><tr><td rowspan=\\\"4\\\">Age at diagnosis mean (SD)</td><td/><td colspan=\\\"2\\\">31.5 (5.9)</td></tr><tr><td>18–24 years</td><td>569</td><td>16</td></tr><tr><td>25–34 years</td><td>1584</td><td>44</td></tr><tr><td>35–39 years</td><td>1443</td><td>40</td></tr><tr><td>Age at completing questionnaire mean (SD)</td><td/><td colspan=\\\"2\\\">44.5 (7.5)</td></tr><tr><td rowspan=\\\"4\\\">Time since diagnosis</td><td/><td colspan=\\\"2\\\">12.4 (4.5)</td></tr><tr><td>5–10 years</td><td>1452</td><td>40</td></tr><tr><td>11–15 years</td><td>1247</td><td>35</td></tr><tr><td>16–20 years</td><td>897</td><td>25</td></tr><tr><td rowspan=\\\"12\\\">Type of cancer</td><td>Breast cancer</td><td>846</td><td>23.5</td></tr><tr><td>Germ cell tumors</td><td>637</td><td>17.7</td></tr><tr><td>Lymphoid hematological malignancies</td><td>540</td><td>15.0</td></tr><tr><td>Female genitalia tumors</td><td>383</td><td>10.7</td></tr><tr><td>Melanoma</td><td>252</td><td>7.0</td></tr><tr><td>Thyroid cancer</td><td>224</td><td>6.2</td></tr><tr><td>Bone or soft tissue sarcoma</td><td>161</td><td>4.5</td></tr><tr><td>Myeloid hematological malignancies</td><td>136</td><td>3.8</td></tr><tr><td>Central nervous system tumors</td><td>131</td><td>3.6</td></tr><tr><td>Head and neck cancer</td><td>107</td><td>3.0</td></tr><tr><td>Digestive tract tumors</td><td>104</td><td>2.9</td></tr><tr><td>Other*</td><td>75</td><td>2.1</td></tr><tr><td rowspan=\\\"5\\\">Tumor stage</td><td>I</td><td>1537</td><td>42.7</td></tr><tr><td>II</td><td>957</td><td>26.6</td></tr><tr><td>III</td><td>515</td><td>14.3</td></tr><tr><td>IV</td><td>165</td><td>4.6</td></tr><tr><td>Missing</td><td>422</td><td>11.7</td></tr><tr><td rowspan=\\\"6\\\">Primary treatment modality</td><td>Surgery</td><td>2799</td><td>77.8</td></tr><tr><td>Chemotherapy</td><td>2030</td><td>56.5</td></tr><tr><td>Radiotherapy</td><td>1716</td><td>47.7</td></tr><tr><td>Hormonal therapy</td><td>435</td><td>12.1</td></tr><tr><td>Targeted therapy</td><td>282</td><td>7.8</td></tr><tr><td>Stem cell therapy</td><td>130</td><td>3.6</td></tr><tr><td>Marital status (at time of questionnaire)</td><td>In a relation</td><td>3001</td><td>83.5</td></tr><tr><td rowspan=\\\"4\\\">Educational level</td><td>Primary school or equivalent</td><td>16</td><td>0.4</td></tr><tr><td>Secondary school or equivalent</td><td>1514</td><td>42.1</td></tr><tr><td>College/university or equivalent</td><td>2060</td><td>57.3</td></tr><tr><td>Missing</td><td>6</td><td>0.2</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>The long-term HRQoL outcomes from the cancer survivorship core questionnaire (QLQ-SURV111) of the AYA cancer survivors, arranged by scale and total score mean</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th>Scales</th><th>Short names</th><th>Functional/symptom</th><th>Number of items (<italic>n</italic>)</th><th>Item numbers</th><th>Raw scores<break/>mean (SD)</th><th>Total scores<break/>mean (SD)</th></tr></thead><tbody><tr><td>Physical functioning</td><td>PF</td><td>Functional</td><td>5</td><td>1–5</td><td>1.2 (0.4)</td><td>91.7 (13.9)</td></tr><tr><td>Role functioning</td><td>RF</td><td>Functional</td><td>3</td><td>69–71</td><td>1.5 (0.7)</td><td>83.7 (24.5)</td></tr><tr><td>Emotional functioning</td><td>EF</td><td>Functional</td><td>7</td><td>52–58</td><td>1.6 (0.6)</td><td>81.0 (19.9)</td></tr><tr><td>Cognitive functioning</td><td>CF</td><td>Functional</td><td>4</td><td>47, 48, 50, 51</td><td>1.6 (0.7)</td><td>79.6 (22.6)</td></tr><tr><td>Body image</td><td>BI</td><td>Functional</td><td>2</td><td>40, 41</td><td>1.7 (0.7)</td><td>77.8 (24.6)</td></tr><tr><td>Overall quality of life</td><td>QL</td><td>Functional</td><td>1</td><td>121</td><td>5.6 (1.1)</td><td>77.3 (18.7)</td></tr><tr><td>Symptom awareness</td><td>SA</td><td>Functional</td><td>2</td><td>76, 77</td><td>2.2 (0.8)</td><td>60.9 (25.4)</td></tr><tr><td>Sexual functioning</td><td>SF</td><td>Functional</td><td>2</td><td>111, 112</td><td>2.3 (0.8)</td><td>43.7 (25.5)</td></tr><tr><td>Financial difficulties</td><td>FD</td><td>Symptom</td><td>1</td><td>68</td><td>1.3 (0.7)</td><td>89.4 (23.8)</td></tr><tr><td>Social interference</td><td>Sif</td><td>Symptom</td><td>2</td><td>73, 74</td><td>1.4 (0.7)</td><td>88.1 (22.0)</td></tr><tr><td>Worry cancer risk family</td><td>WF</td><td>Symptom</td><td>1</td><td>61</td><td>1.4 (0.7)</td><td>86.6 (23.1)</td></tr><tr><td>Symptom checklist</td><td>SC</td><td>Symptom</td><td>17</td><td>20, 21, 23–26, 28, 30–38, 75</td><td>1.4 (0.4)</td><td>85.8 (13.5)</td></tr><tr><td>Treated differently</td><td>TD</td><td>Symptom</td><td>1</td><td>107</td><td>1.4 (0.7)</td><td>85.2 (22.6)</td></tr><tr><td>Pain</td><td>PA</td><td>Symptom</td><td>2</td><td>22, 72</td><td>1.5 (0.7)</td><td>84.2 (23.0)</td></tr><tr><td>Sexual problems</td><td>SP</td><td>Symptom</td><td>2</td><td>113, 117</td><td>1.5 (0.8)</td><td>82.8 (26.3)</td></tr><tr><td>Health distress</td><td>HD</td><td>Symptom</td><td>3</td><td>63–65</td><td>1.6 (0.7)</td><td>79.0 (22.3)</td></tr><tr><td>Sleep problems</td><td>SL</td><td>Symptom</td><td>4</td><td>16–19</td><td>1.8 (0.7)</td><td>74.5 (23.6)</td></tr><tr><td>Negative health outlook</td><td>NHO</td><td>Symptom</td><td>7</td><td>60, 62, 64, 81, 82, 98, 99</td><td>1.8 (0.6)</td><td>74.2 (20.0)</td></tr><tr><td>Fatigue</td><td>FA</td><td>Symptom</td><td>4</td><td>6–9</td><td>1.9 (0.8)</td><td>70.3 (26.0)</td></tr><tr><td>Social isolation</td><td>SI</td><td>Symptom</td><td>2</td><td>89, 90</td><td>1.9 (0.9)</td><td>69.1 (30.7)</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" 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Bootsma and Deborah van de Wal share first authorship.</p></fn><fn><p><bold>Publisher’s note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>*Other includes urinary tract, respiratory tract, male genitalia, neuroblastoma, adrenal, paraganglioma, and eyes</p></table-wrap-foot>\",\n \"<fn-group><fn><p>Tom I. 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In particular, the homeostasis of the sphingolipids ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate (S1P) has been described to regulate proliferation, differentiation, cell growth and inflammation in the cells of the central nervous system (CNS) [##REF##24459205##1##]. S1P is produced from ceramide by ceramidase followed by phosphorylation by sphingosine kinase (SphK1/2) [##REF##18216770##2##]. S1P is abundantly produced by erythrocytes, platelets and endothelial cells [##REF##19268560##3##, ##REF##7795224##4##]. Although, S1P synthesis in the CNS has also been reported, namely by astrocytes after fibroblast growth factor stimulation [##REF##16470810##5##].</p>","<p id=\"Par12\">S1P in the extracellular space binds to its five specific G-coupled receptors S1PR1-5, which signal through diverse downstream pathways [##REF##24459205##1##]. All S1P receptors are expressed in the CNS [##REF##35781853##6##]. They participate in multiple important functions during CNS development, and further contribute to the development and/or resolution of neurodegeneration in pathological conditions such as ischemic stroke [##REF##31165772##7##], multiple sclerosis [##REF##27825807##8##], hearing loss [##REF##27080739##9##] and seizures [##UREF##0##10##, ##REF##20930159##11##]. S1P receptors have received attention in the field of multiple sclerosis, for which immunomodulation is achieved by the oral drug Fingolimod (FTY720) that activates S1PR1,3–5 [##REF##20061941##12##]. S1PR1 is the most studied S1P receptor with important roles in angiogenesis and neurogenesis [##REF##16314531##13##]. S1PR2 is a modulator of neuronal excitability during neuronal development [##REF##11553273##14##] and controls spontaneous activity of cultured neurons [##REF##35781853##6##]. S1PR3 receptor controls activity of microglia and their participation in neuroinflammation [##REF##23813380##15##–##REF##27098703##17##], and is highly expressed in astrocytes, where it regulates astrogliosis via activation of the small GTPase RhoA [##REF##28577576##18##]. Until recently, S1PR4 has received little attention in the CNS [##REF##25309325##19##]. Our recent work proposed S1PR4 presence in synapses, and a role in neuronal activity control [##REF##35781853##6##]. Furthermore, previous studies have demonstrated loss of sphingosine kinase activity and lowering of brain region-specific S1P levels early in the progression of Alzheimer’s disease [##REF##24456642##20##, ##REF##29615132##21##].</p>","<p id=\"Par13\">Recent evidence also suggests an important role for S1P and its generating enzymes SphK1/2 in the development of diabetes [##REF##18458870##22##] through adverse effects on endothelial function [##REF##27098703##17##, ##REF##16179586##23##], hepatic insulin sensitivity and secretion [##REF##30448236##24##], regulating insulin secretion in pancreatic β-cell [##REF##22389505##25##], and contributing to diabetes-associated inflammation [##REF##16179586##23##].</p>","<p id=\"Par14\">The sphingosine rheostat has been extensively studied in diabetes and insulin resistance, with special focus on ceramide and ceramide-1-phosphate [##REF##18458870##22##]. Because S1P is involved in a plethora of cellular functions, a tight regulation of S1P homeostasis seems important for proper brain functioning, which would be disrupted in metabolic syndrome and diabetes. Diabetes impacts synapses with negative consequences for brain function, including memory performance [##REF##32265637##26##]. Furthermore, rodent models of diabetes show altered neuromodulation systems operated by adenosine [##REF##16256246##27##–##REF##30686981##29##], ATP [##REF##17869435##30##], or endocannabinoids [##REF##17222407##31##, ##REF##32428627##32##], which control synaptic activity and brain energy metabolism, and might interact with brain insulin signaling [##REF##36629507##33##]. These systems can also afford neuroprotection. For example, pharmacologically targeting the altered adenosinergic system components confers neuroprotection and improves memory performance in diabetes models [##REF##22514596##28##, ##REF##30686981##29##, ##REF##19694901##34##]. It is hitherto unknown whether the neuromodulation system operated by S1P in the CNS is altered in diabetes and metabolic syndrome.</p>","<p id=\"Par15\">In this study, we set out to test the hypothesis that T2D impacts the neuromodulation system operated by S1P in the CNS. Since S1PRs are present in nerve terminals of the cortex [##REF##35781853##6##], we determined changes induced by T2D in the density of S1PR1-4 in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and from diet-induced obese mice, which are models with well-established brain dysfunction (e.g., [##REF##30686981##29##, ##REF##36222315##35##, ##REF##30670942##36##]).</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par11\\\">Bioactive lipids are important for brain function. In particular, the homeostasis of the sphingolipids ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate (S1P) has been described to regulate proliferation, differentiation, cell growth and inflammation in the cells of the central nervous system (CNS) [##REF##24459205##1##]. S1P is produced from ceramide by ceramidase followed by phosphorylation by sphingosine kinase (SphK1/2) [##REF##18216770##2##]. S1P is abundantly produced by erythrocytes, platelets and endothelial cells [##REF##19268560##3##, ##REF##7795224##4##]. Although, S1P synthesis in the CNS has also been reported, namely by astrocytes after fibroblast growth factor stimulation [##REF##16470810##5##].</p>\",\n \"<p id=\\\"Par12\\\">S1P in the extracellular space binds to its five specific G-coupled receptors S1PR1-5, which signal through diverse downstream pathways [##REF##24459205##1##]. All S1P receptors are expressed in the CNS [##REF##35781853##6##]. They participate in multiple important functions during CNS development, and further contribute to the development and/or resolution of neurodegeneration in pathological conditions such as ischemic stroke [##REF##31165772##7##], multiple sclerosis [##REF##27825807##8##], hearing loss [##REF##27080739##9##] and seizures [##UREF##0##10##, ##REF##20930159##11##]. S1P receptors have received attention in the field of multiple sclerosis, for which immunomodulation is achieved by the oral drug Fingolimod (FTY720) that activates S1PR1,3–5 [##REF##20061941##12##]. S1PR1 is the most studied S1P receptor with important roles in angiogenesis and neurogenesis [##REF##16314531##13##]. S1PR2 is a modulator of neuronal excitability during neuronal development [##REF##11553273##14##] and controls spontaneous activity of cultured neurons [##REF##35781853##6##]. S1PR3 receptor controls activity of microglia and their participation in neuroinflammation [##REF##23813380##15##–##REF##27098703##17##], and is highly expressed in astrocytes, where it regulates astrogliosis via activation of the small GTPase RhoA [##REF##28577576##18##]. Until recently, S1PR4 has received little attention in the CNS [##REF##25309325##19##]. Our recent work proposed S1PR4 presence in synapses, and a role in neuronal activity control [##REF##35781853##6##]. Furthermore, previous studies have demonstrated loss of sphingosine kinase activity and lowering of brain region-specific S1P levels early in the progression of Alzheimer’s disease [##REF##24456642##20##, ##REF##29615132##21##].</p>\",\n \"<p id=\\\"Par13\\\">Recent evidence also suggests an important role for S1P and its generating enzymes SphK1/2 in the development of diabetes [##REF##18458870##22##] through adverse effects on endothelial function [##REF##27098703##17##, ##REF##16179586##23##], hepatic insulin sensitivity and secretion [##REF##30448236##24##], regulating insulin secretion in pancreatic β-cell [##REF##22389505##25##], and contributing to diabetes-associated inflammation [##REF##16179586##23##].</p>\",\n \"<p id=\\\"Par14\\\">The sphingosine rheostat has been extensively studied in diabetes and insulin resistance, with special focus on ceramide and ceramide-1-phosphate [##REF##18458870##22##]. Because S1P is involved in a plethora of cellular functions, a tight regulation of S1P homeostasis seems important for proper brain functioning, which would be disrupted in metabolic syndrome and diabetes. Diabetes impacts synapses with negative consequences for brain function, including memory performance [##REF##32265637##26##]. Furthermore, rodent models of diabetes show altered neuromodulation systems operated by adenosine [##REF##16256246##27##–##REF##30686981##29##], ATP [##REF##17869435##30##], or endocannabinoids [##REF##17222407##31##, ##REF##32428627##32##], which control synaptic activity and brain energy metabolism, and might interact with brain insulin signaling [##REF##36629507##33##]. These systems can also afford neuroprotection. For example, pharmacologically targeting the altered adenosinergic system components confers neuroprotection and improves memory performance in diabetes models [##REF##22514596##28##, ##REF##30686981##29##, ##REF##19694901##34##]. It is hitherto unknown whether the neuromodulation system operated by S1P in the CNS is altered in diabetes and metabolic syndrome.</p>\",\n \"<p id=\\\"Par15\\\">In this study, we set out to test the hypothesis that T2D impacts the neuromodulation system operated by S1P in the CNS. Since S1PRs are present in nerve terminals of the cortex [##REF##35781853##6##], we determined changes induced by T2D in the density of S1PR1-4 in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and from diet-induced obese mice, which are models with well-established brain dysfunction (e.g., [##REF##30686981##29##, ##REF##36222315##35##, ##REF##30670942##36##]).</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<title>Animals</title>","<p id=\"Par16\">Experiments were performed according to EU Directive 2010/63/EU under approval of the Malmö/Lund Committee for Animal Experiment Ethics (permit numbers 994/2018 and 9987/2020) and are reported following the ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments, NC3Rs initiative, UK). Male and female GK rats were obtained from a local colony, and male and female age-matched Wistar rats from Janvier (Saint Berthevin, France) were used as controls [##REF##30686981##29##]. Wistar rats were housed in the facility for at least a month before experimentation. Eight weeks-old C57BL/6 J mice were purchased from Taconic (Ry, Denmark). Only male mice were used due to sex differences in responses to diet-induced obesity (see [##REF##36222315##35##], and references therein). Animals were housed in ventilated cages enriched with a cylinder, wood toys and nesting material, at controlled temperature of 22 °C, 50–60% humidity and a 12:12-h light–dark cycle. Food and water were provided ad libitum. Rats were kept on a regular chow and were euthanized at 6 months of age (Fig. ##FIG##0##1##A). Mice were fed a lard-based high-fat diet (HFD; 60% calories from fat) or a control diet (CD, 10% calories from fat) from Research diets (New Brunswick, NJ-USA), as previously described [##REF##36222315##35##]. Mice were held on the diet for 1 week, 1 or 2 months, starting from 9 weeks of age (Fig. ##FIG##0##1##B).</p>","<title>Glucose Tolerance Test and Insulin Determination</title>","<p id=\"Par17\">A glucose tolerance test (GTT) was performed 1–3 days before sacrifice. Food was removed at 08:00 for 6 h, and mice were put into clean cages to avoid coprophagy before and during the test. Before each test, a blood sample was collected from vena saphena into a heparinized tube for determination of plasma insulin concentration. Glycemia was measured from tail tip blood with the Accu-Chek Aviva glucometer (Roche, Solna, Sweden). Mice were then administered 2 g/kg glucose i.p. from a 20%(w/v) solution in saline, followed by determination of glucose levels after 15, 30, 60, 90 and 120 min.</p>","<p id=\"Par18\">Plasma insulin was measured with ELISA kits from Mercodia (Uppsala, Sweden; #10-1250-01 for rats; #10-1247-10 for mice).</p>","<title>Preparation of Nerve Terminal-Enriched Membranes</title>","<p id=\"Par19\">After weighing and measuring tail-tip blood glucose, animals were anesthetized with isoflurane and quickly decapitated. Trunk blood was collected, and plasma was stored at − 80 °C for insulin and S1P determination. Brains were quickly dissected, frozen in N<sub>2</sub> (l) and stored at − 80 °C until further experiments. Synaptosomes were prepared as described previously [##REF##30670942##36##]. Briefly, cortical tissue was homogenized (12 strokes at 800 rotations/min) with a glass-teflon Potter–Elvehjem homogenizer (rotor head InterMed/STIR20) in Sucrose-HEPES buffer (0.32 mol/L sucrose, 1 mmol/L EDTA, 10 mmol/L HEPES, 1 mg/mL bovine serum albumin, pH 7.4) at 4 °C. Homogenates were centrifuged at 3000 <italic>g</italic> for 10 min at 4 °C (Beckman Coulter/Avanti J-20 XP). After, supernatants were centrifuged at 14,000 <italic>g</italic> for 12 min at 4 °C. The pellet was re-suspended in 1 mL of 45% (v/v) Percoll (GE Healthcare, Uppsala, Sweden) solution prepared in Krebs-HEPES buffer (in mmol/L: 140 NaCl, 5 KCl, 10 HEPES, 1 EDTA, 5 glucose, pH 7.4), and then centrifuged at 21,000 <italic>g</italic> for 2 min at 4 °C. The top layer (rich in synaptosomes) was washed by re-suspending in Krebs-HEPES buffer and centrifuging again. The resulting pellets were re-suspended in Krebs-HEPES solution containing protease inhibitors (cOmplete cocktail from Roche, Mannheim, Germany), and stored at − 80 °C until immunoblotting.</p>","<title>Immunoblotting</title>","<p id=\"Par20\">Total protein content of the samples was measured with the bicinchoninic acid assay (kit from Pierce, ThermoFisher Scientific, Uppsala, Sweden). Then, Western blotting was carried out as detailed by Lizarbe et al. [##REF##30670942##36##]. Briefly, samples were dissolved in sample buffer (#NP0007, Invitrogen, ThermoFisher), boiled at 95 °C for 5 min, and then separated by SDS-PAGE in 4–12% Bis–Tris gradient gels (#NP0336, Invitrogen), followed by transfer onto nitrocellulose membranes of 0.45-μm pore size (#GE10600002, GE Healthcare). The membranes were blocked for 1–2 h in Tris-buffered saline (in mmol/L: 20 Tris, 150 NaCl, pH 7.6) containing 5% (w/v) skim milk, 1% (v/v) Tween-20, and then sequentially incubated with primary and secondary antibodies (Table ##TAB##0##1##) diluted in this blocking solution. Immunoblots were developed with the chemiluminescence Super-Signal kit (#34,580, ThermoFisher). Whenever necessary, sensitivity was enhanced with the biotin-streptavidin kit VectaStain ABC-HRP according to manufacturer’s instructions (#PK-4000, Vectorlabs, CA-USA). Luminescence was detected using the Chemidoc XRS + interfaced to Image Lab 5.2.1 (Biorad, Stockholm, Sweden).</p>","<title>Mass Spectrometry</title>","<p id=\"Par21\">Plasma samples obtained from trunk blood, and cortical homogenates in phosphate-buffered saline (PBS; in mmol/L: 137 NaCl, 2.7 KCl, 1.5 KH<sub>2</sub>PO<sub>4</sub>, 8.1 Na<sub>2</sub>HPO<sub>4</sub>, pH 7.4) were spiked with deuterated S1P as internal standard (S1P-D7 > 99% deuterated; Avanti Polar Lipids/Merck, Darmstadt, Germany), and S1P was extracted as described in [##REF##22528437##37##]. Extracts were dried under a nitrogen stream, dissolved in methanol, and subjected to liquid chromatography-coupled tandem mass spectrometry as previously described [##REF##33993723##38##, ##REF##35055052##39##].</p>","<title>Statistics</title>","<p id=\"Par22\">Results are shown as mean ± SD, and were analyzed with Prism 9.4.1 (GraphPad Software, San Diego, CA). Normal distribution was assessed with the Kolmogorov–Smirnov test. In the presence of normality deviations, results were analyzed using Mann–Whitney test or Kruskal–Wallis test followed by Dunn’s multiple comparisons. Normally distributed data was analyzed with unpaired, two-tailed Student <italic>t</italic>-test or ANOVA followed by independent comparisons with the Fisher’s least significant difference (LSD) test. Significance was accepted for <italic>P</italic> < 0.05.</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<title>Animals</title>\",\n \"<p id=\\\"Par16\\\">Experiments were performed according to EU Directive 2010/63/EU under approval of the Malmö/Lund Committee for Animal Experiment Ethics (permit numbers 994/2018 and 9987/2020) and are reported following the ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments, NC3Rs initiative, UK). Male and female GK rats were obtained from a local colony, and male and female age-matched Wistar rats from Janvier (Saint Berthevin, France) were used as controls [##REF##30686981##29##]. Wistar rats were housed in the facility for at least a month before experimentation. Eight weeks-old C57BL/6 J mice were purchased from Taconic (Ry, Denmark). Only male mice were used due to sex differences in responses to diet-induced obesity (see [##REF##36222315##35##], and references therein). Animals were housed in ventilated cages enriched with a cylinder, wood toys and nesting material, at controlled temperature of 22 °C, 50–60% humidity and a 12:12-h light–dark cycle. Food and water were provided ad libitum. Rats were kept on a regular chow and were euthanized at 6 months of age (Fig. ##FIG##0##1##A). Mice were fed a lard-based high-fat diet (HFD; 60% calories from fat) or a control diet (CD, 10% calories from fat) from Research diets (New Brunswick, NJ-USA), as previously described [##REF##36222315##35##]. Mice were held on the diet for 1 week, 1 or 2 months, starting from 9 weeks of age (Fig. ##FIG##0##1##B).</p>\",\n \"<title>Glucose Tolerance Test and Insulin Determination</title>\",\n \"<p id=\\\"Par17\\\">A glucose tolerance test (GTT) was performed 1–3 days before sacrifice. Food was removed at 08:00 for 6 h, and mice were put into clean cages to avoid coprophagy before and during the test. Before each test, a blood sample was collected from vena saphena into a heparinized tube for determination of plasma insulin concentration. Glycemia was measured from tail tip blood with the Accu-Chek Aviva glucometer (Roche, Solna, Sweden). Mice were then administered 2 g/kg glucose i.p. from a 20%(w/v) solution in saline, followed by determination of glucose levels after 15, 30, 60, 90 and 120 min.</p>\",\n \"<p id=\\\"Par18\\\">Plasma insulin was measured with ELISA kits from Mercodia (Uppsala, Sweden; #10-1250-01 for rats; #10-1247-10 for mice).</p>\",\n \"<title>Preparation of Nerve Terminal-Enriched Membranes</title>\",\n \"<p id=\\\"Par19\\\">After weighing and measuring tail-tip blood glucose, animals were anesthetized with isoflurane and quickly decapitated. Trunk blood was collected, and plasma was stored at − 80 °C for insulin and S1P determination. Brains were quickly dissected, frozen in N<sub>2</sub> (l) and stored at − 80 °C until further experiments. Synaptosomes were prepared as described previously [##REF##30670942##36##]. Briefly, cortical tissue was homogenized (12 strokes at 800 rotations/min) with a glass-teflon Potter–Elvehjem homogenizer (rotor head InterMed/STIR20) in Sucrose-HEPES buffer (0.32 mol/L sucrose, 1 mmol/L EDTA, 10 mmol/L HEPES, 1 mg/mL bovine serum albumin, pH 7.4) at 4 °C. Homogenates were centrifuged at 3000 <italic>g</italic> for 10 min at 4 °C (Beckman Coulter/Avanti J-20 XP). After, supernatants were centrifuged at 14,000 <italic>g</italic> for 12 min at 4 °C. The pellet was re-suspended in 1 mL of 45% (v/v) Percoll (GE Healthcare, Uppsala, Sweden) solution prepared in Krebs-HEPES buffer (in mmol/L: 140 NaCl, 5 KCl, 10 HEPES, 1 EDTA, 5 glucose, pH 7.4), and then centrifuged at 21,000 <italic>g</italic> for 2 min at 4 °C. The top layer (rich in synaptosomes) was washed by re-suspending in Krebs-HEPES buffer and centrifuging again. The resulting pellets were re-suspended in Krebs-HEPES solution containing protease inhibitors (cOmplete cocktail from Roche, Mannheim, Germany), and stored at − 80 °C until immunoblotting.</p>\",\n \"<title>Immunoblotting</title>\",\n \"<p id=\\\"Par20\\\">Total protein content of the samples was measured with the bicinchoninic acid assay (kit from Pierce, ThermoFisher Scientific, Uppsala, Sweden). Then, Western blotting was carried out as detailed by Lizarbe et al. [##REF##30670942##36##]. Briefly, samples were dissolved in sample buffer (#NP0007, Invitrogen, ThermoFisher), boiled at 95 °C for 5 min, and then separated by SDS-PAGE in 4–12% Bis–Tris gradient gels (#NP0336, Invitrogen), followed by transfer onto nitrocellulose membranes of 0.45-μm pore size (#GE10600002, GE Healthcare). The membranes were blocked for 1–2 h in Tris-buffered saline (in mmol/L: 20 Tris, 150 NaCl, pH 7.6) containing 5% (w/v) skim milk, 1% (v/v) Tween-20, and then sequentially incubated with primary and secondary antibodies (Table ##TAB##0##1##) diluted in this blocking solution. Immunoblots were developed with the chemiluminescence Super-Signal kit (#34,580, ThermoFisher). Whenever necessary, sensitivity was enhanced with the biotin-streptavidin kit VectaStain ABC-HRP according to manufacturer’s instructions (#PK-4000, Vectorlabs, CA-USA). Luminescence was detected using the Chemidoc XRS + interfaced to Image Lab 5.2.1 (Biorad, Stockholm, Sweden).</p>\",\n \"<title>Mass Spectrometry</title>\",\n \"<p id=\\\"Par21\\\">Plasma samples obtained from trunk blood, and cortical homogenates in phosphate-buffered saline (PBS; in mmol/L: 137 NaCl, 2.7 KCl, 1.5 KH<sub>2</sub>PO<sub>4</sub>, 8.1 Na<sub>2</sub>HPO<sub>4</sub>, pH 7.4) were spiked with deuterated S1P as internal standard (S1P-D7 > 99% deuterated; Avanti Polar Lipids/Merck, Darmstadt, Germany), and S1P was extracted as described in [##REF##22528437##37##]. Extracts were dried under a nitrogen stream, dissolved in methanol, and subjected to liquid chromatography-coupled tandem mass spectrometry as previously described [##REF##33993723##38##, ##REF##35055052##39##].</p>\",\n \"<title>Statistics</title>\",\n \"<p id=\\\"Par22\\\">Results are shown as mean ± SD, and were analyzed with Prism 9.4.1 (GraphPad Software, San Diego, CA). Normal distribution was assessed with the Kolmogorov–Smirnov test. In the presence of normality deviations, results were analyzed using Mann–Whitney test or Kruskal–Wallis test followed by Dunn’s multiple comparisons. Normally distributed data was analyzed with unpaired, two-tailed Student <italic>t</italic>-test or ANOVA followed by independent comparisons with the Fisher’s least significant difference (LSD) test. Significance was accepted for <italic>P</italic> < 0.05.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par23\">We have previously detailed metabolic phenotypes of insulin-resistant GK rats [##REF##30686981##29##] and HFD-induced obese mice [##REF##29027041##40##]. In the present study, we have confirmed that GK rats displayed lower body weight, increased glycemia, and similar fed plasma insulin concentration, when compared to controls (Table ##TAB##1##2##). HFD-fed mice showed increased body weight, a tendency for increased fasting glycemia and plasma insulin concentrations, reduced glucose tolerance, and increased insulin resistance, when compared to controls (Table ##TAB##2##3##).</p>","<p id=\"Par24\">Plasma concentrations of S1P were higher in GK than Wistar rats (+ 60%, <italic>P</italic> = 0.008, Fig. ##FIG##0##1##A). However, S1P levels in homogenates from the cortex of GK rats were not significantly different from those in Wistar rats (<italic>P</italic> > 0.05, Fig. ##FIG##1##2##B). Nerve terminal-enriched membranes from GK rats showed lower immunoreactivity of S1PR1 (− 21%, <italic>P</italic> = 0.004), S1PR2 (− 26%, <italic>P</italic> = 0.006) and S1PR4 (− 40%, <italic>P</italic> = 0.046,) than those from control Wistar rats (Fig. ##FIG##1##2##C–D). S1PR3 immunoreactivity was similar between the groups (<italic>P</italic> > 0.05).</p>","<p id=\"Par25\">Plasma concentrations of S1P were increased by both HFD exposure and age (interaction F(2,64) = 1.4, <italic>P</italic> > 0.05; diet F(1,64) = 7.2, <italic>P</italic> = 0.009; time F(2,64) = 17, <italic>P</italic> < 0.001). In particular, HFD-feeding increased plasma S1P levels by 11–16%, most prominently at 1 month after diet intervention (Fig. ##FIG##2##3##A). In the cortex, S1P levels were also increased with HFD, and showed a variation with age that was unrelated to plasma S1P levels (interaction F(2,27) = 0.12, <italic>P</italic> > 0.05; diet F(1,27) = 5.5, <italic>P</italic> = 0.026; time F(2,27) = 4.4, <italic>P</italic> = 0.022, Fig. ##FIG##2##3##B). The density of S1PRs was analyzed in nerve-terminal membranes from the cortex of mice fed a CD or HFD for 1 week, 1 month, and 2 months (Fig. ##FIG##2##3##C–E). SNAP25 analyzed as constitutive protein showed no HFD-induced changes (Fig. ##FIG##2##3##F). Relative to CD, HFD exposure reduced S1PR1 immunoreactivity (interaction F(2,30) = 0.64, <italic>P</italic> > 0.05; diet F(1,30) = 13, <italic>P</italic> = 0.001; time F(2,30) = 0.64, <italic>P</italic> > 0.05; Fig. ##FIG##2##3##G), which is particularly prominent at 1 week (− 52%, <italic>P</italic> = 0.011) and 2 months of HFD feeding (− 50%, <italic>P</italic> = 0.014). The density of S1PR2 and S1PR3 was not modified by HFD feeding (Fig. ##FIG##2##3##G). S1PR4 immunoreactivity in cortical nerve terminal membranes was lower in HFD-fed mice than controls (interaction F(2,31) = 1.6, <italic>P</italic> > 0.05; diet F(1,31) = 7.4, <italic>P</italic> = 0.011; time F(2,31) = 1.6, <italic>P</italic> > 0.05; Fig. ##FIG##2##3##G), most prominently after 1 week of HFD feeding (− 54%, <italic>P</italic> = 0.005), and recovering to control levels at 2 months of HFD.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par23\\\">We have previously detailed metabolic phenotypes of insulin-resistant GK rats [##REF##30686981##29##] and HFD-induced obese mice [##REF##29027041##40##]. In the present study, we have confirmed that GK rats displayed lower body weight, increased glycemia, and similar fed plasma insulin concentration, when compared to controls (Table ##TAB##1##2##). HFD-fed mice showed increased body weight, a tendency for increased fasting glycemia and plasma insulin concentrations, reduced glucose tolerance, and increased insulin resistance, when compared to controls (Table ##TAB##2##3##).</p>\",\n \"<p id=\\\"Par24\\\">Plasma concentrations of S1P were higher in GK than Wistar rats (+ 60%, <italic>P</italic> = 0.008, Fig. ##FIG##0##1##A). However, S1P levels in homogenates from the cortex of GK rats were not significantly different from those in Wistar rats (<italic>P</italic> > 0.05, Fig. ##FIG##1##2##B). Nerve terminal-enriched membranes from GK rats showed lower immunoreactivity of S1PR1 (− 21%, <italic>P</italic> = 0.004), S1PR2 (− 26%, <italic>P</italic> = 0.006) and S1PR4 (− 40%, <italic>P</italic> = 0.046,) than those from control Wistar rats (Fig. ##FIG##1##2##C–D). S1PR3 immunoreactivity was similar between the groups (<italic>P</italic> > 0.05).</p>\",\n \"<p id=\\\"Par25\\\">Plasma concentrations of S1P were increased by both HFD exposure and age (interaction F(2,64) = 1.4, <italic>P</italic> > 0.05; diet F(1,64) = 7.2, <italic>P</italic> = 0.009; time F(2,64) = 17, <italic>P</italic> < 0.001). In particular, HFD-feeding increased plasma S1P levels by 11–16%, most prominently at 1 month after diet intervention (Fig. ##FIG##2##3##A). In the cortex, S1P levels were also increased with HFD, and showed a variation with age that was unrelated to plasma S1P levels (interaction F(2,27) = 0.12, <italic>P</italic> > 0.05; diet F(1,27) = 5.5, <italic>P</italic> = 0.026; time F(2,27) = 4.4, <italic>P</italic> = 0.022, Fig. ##FIG##2##3##B). The density of S1PRs was analyzed in nerve-terminal membranes from the cortex of mice fed a CD or HFD for 1 week, 1 month, and 2 months (Fig. ##FIG##2##3##C–E). SNAP25 analyzed as constitutive protein showed no HFD-induced changes (Fig. ##FIG##2##3##F). Relative to CD, HFD exposure reduced S1PR1 immunoreactivity (interaction F(2,30) = 0.64, <italic>P</italic> > 0.05; diet F(1,30) = 13, <italic>P</italic> = 0.001; time F(2,30) = 0.64, <italic>P</italic> > 0.05; Fig. ##FIG##2##3##G), which is particularly prominent at 1 week (− 52%, <italic>P</italic> = 0.011) and 2 months of HFD feeding (− 50%, <italic>P</italic> = 0.014). The density of S1PR2 and S1PR3 was not modified by HFD feeding (Fig. ##FIG##2##3##G). S1PR4 immunoreactivity in cortical nerve terminal membranes was lower in HFD-fed mice than controls (interaction F(2,31) = 1.6, <italic>P</italic> > 0.05; diet F(1,31) = 7.4, <italic>P</italic> = 0.011; time F(2,31) = 1.6, <italic>P</italic> > 0.05; Fig. ##FIG##2##3##G), most prominently after 1 week of HFD feeding (− 54%, <italic>P</italic> = 0.005), and recovering to control levels at 2 months of HFD.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par26\">This study demonstrates for the first time that S1PR density in cortical nerve terminals is reduced in lean and obese models of insulin resistance, along with possible increases of S1P concentrations in the cortex, implying a role for S1PR signaling in the neuropathological process that occurs in T2D. Since S1PR activation generally attenuates neuronal activity [##REF##35781853##6##], lower S1PR density might lead to overall cortical hyper-excitability.</p>","<p id=\"Par27\">From those analyzed, S1PR1 is the receptor with the highest expression level in the CNS. Its density was decreased in cortical synapses of both insulin-resistant GK rats and HFD-fed mice, relative to their respective controls. Previous studies have reported similar S1PR1 level reductions in the hypothalamus of HFD-fed rats or mice, as well as in the leptin deficient ob/ob mice [##REF##25255053##41##]. Silva et al. proposed that S1P is an appetite inhibitor through S1PR1 signaling, and reduced S1PR1 levels in the hypothalamus of obese models were associated with increased food intake. S1PR1 activation inhibits neuronal activity [##REF##35781853##6##] and, therefore, the S1PR1 reduction observed in cortical synapses is likely to result in loss of S1P-dependent synaptic control. However, S1PR1 is ubiquitously expressed, and exert a multitude of regulatory actions, not being specific to synapses. Thus, a potential approach of pharmacologically targeting S1PR1 for synaptic modulation might also trigger a plethora of non-synaptic actions. Adding to the complexity of S1PR1 signaling, others have reported that specific S1PR1 activation increases excitability of some sensory neurons in the rat dorsal root ganglia [##REF##20844107##42##]. Given the ubiquitous actions of S1P, any systemic interventions on S1PR1 should be taken with caution.</p>","<p id=\"Par28\">The density of both S1PR2 and S1PR4 was decreased in cortical synapses from GK rats compared to controls. Considering the role of S1PR2 as neuromodulator in excitatory neurons [##REF##11553273##14##], and its preferential localization at the pre-synaptic level in the cortex [##REF##35781853##6##], dampening of S1PR2 signaling in GK rats is likely to contribute to the synaptic dysfunction and impaired synaptic plasticity [##REF##30686981##29##]. Our previous study [##REF##35781853##6##] showed that specifically S1PR2 and S1PR4 activation results in dampening of spontaneous spiking frequency of cultured primary neurons. A reduction in levels of synaptic S1PR2/4 could thus result in uncontrolled, excessive synaptic activity that might lead to excitotoxicity and synaptic damage [##REF##32265637##26##]. We thus speculate that S1PR2/4 activation might allow for synaptic protection in diabetic conditions.</p>","<p id=\"Par29\">HFD-fed mice did not reproduce the T2D-induced S1PR2 alterations observed in GK rats. Moreover, S1PR4 density was reduced in short- but not long-term HFD, when compared to the respective controls. Besides obesity, a key difference between the two models is the hyperglycemia in GK rats [##REF##23855509##43##, ##REF##29220408##44##] that is negligible in HFD-fed mice [##REF##32265637##26##, ##REF##30670942##36##]. On the other hand, both models become insulin resistant. In this regard, it needs to be noted that S1P signaling through S1PR2 has been shown to interact with insulin signaling, and might participate in the development of insulin resistance in peripheral cells [##REF##26943364##45##]. Given the ability of S1PR2 to inhibit insulin-mediated signals, it is plausible that reduced S1PR2 in synaptic membranes results in enhanced insulin signaling. Thus, S1PR2 might contribute to the development of central insulin resistance. In turn, putatively increased S1PR2 signaling due to higher S1P concentrations in HFD-fed mice could also dampen synaptic insulin signaling. Loss of tonic insulin receptor signaling in nerve terminals is believed to contribute to memory impairment [##REF##36629507##33##, ##REF##27881773##46##].</p>","<p id=\"Par30\">Although S1PR5 is present in the CNS, its relevance for neuromodulation at the synaptic level remains to be established ([##REF##35781853##6##], and references therein). Therefore, we have not investigated diabetes-induced alterations of S1PR5 density in the present study.</p>","<p id=\"Par31\">Concentrations of S1P measured by ELISA were found to increase in the liver of patients with T2D as well as in streptozotocin-induced T2D rats [##REF##30972941##47##]. Plasma S1P concentration also increases in the ob/ob mouse model, mice exposed to HFD for 6 weeks, and in a population of young obese humans [##REF##24039766##48##]. Interestingly, plasma S1P in humans was found to be associated with body fat, insulin levels, insulin resistance (by HOMA-IR), as well with cholesterol [##REF##24039766##48##]. The same study reported an increase in concentrations of S1P in plasma after 12 h of fasting, suggesting a relation between S1P levels and increased inter-organ lipid flux. In our study, plasma samples for S1P determination were collected under fed state, which we therefore expect to depict diabetes-induced S1P changes rather than acute fasting-induced mobilization of lipid stores.</p>","<p id=\"Par32\">While plasma S1P levels increase in non-diabetic young adults with obesity (< 30 years old; body mass index ~ 37 kg/m<sup>2</sup>) relative to lean controls [##REF##24039766##48##], others have reported that T2D is associated with a decrease of plasma S1P concentrations [##REF##29543843##49##, ##REF##31602237##50##]. Namely, these studies found lower plasma S1P concentrations in individuals with T2D relative to healthy controls matched for age and body mass index (average: ~ 60 years old and ~ 29 kg/m<sup>2</sup> in Vaisar et al. [##REF##29543843##49##]; 44–49 years old and 25–26 kg/m<sup>2</sup> in Sui et al. [##REF##31602237##50##]). While reported effects of obesity and T2D on plasma S1P levels are opposite, these studies differ in the studied populations, age of the subjects, and methods for S1P extraction and detection.</p>","<p id=\"Par33\">S1P levels in the cortex are reported to be lower than in other brain regions [##REF##30805275##51##], and we now found S1P concentrations increased in the cortex of HFD-fed mice. To our knowledge, this is the first study investigating S1P concentration in the cerebral cortex of T2D/pre-diabetic animal models. We report an HFD-induced increase of S1P in cortical tissue that seems to be independent of HFD-induced increases in circulating S1P levels. In turn, in GK rats, the large variance of S1P concentrations measured in the cortex precluded determining the expected diabetes-induced increase in S1P levels. This contrasts the concurrent increases of brain and plasma S1P that have previously been reported in a mouse model of hypertension [##REF##29856066##52##].</p>","<p id=\"Par34\">The assessment of S1PR density in mouse cortical synapses across the HFD treatment is limited to the effects of the exposure to the diet, and not the effect of age. This is due to the semi-quantitative nature of immunoblotting methods, and the fact that we have decided to analyze all age-matched samples in parallel. Nevertheless, mice were treated for a relatively short period of time (only 2 months), at an age range from about 2 to 4 months, during which changes of S1PR density might not be as important as effects of the diet. Another limitation of this study is that despite including both sexes in the GK rat part of the study, analysis of plasma S1P levels was only available for males. We are not able to determine sex effects on plasma S1P concentrations, although they were not apparent in cortical levels of S1P. Finally, another important limitation of this study is that the nerve-terminal enriched membranes obtained with our protocol also contain non-synaptic components, such as the perisynaptic astrocytic processes. In particular, S1PR1 is abundant in astrocytes, and the observed S1PR1 reduction could be due to alterations in astrocytic rather than synaptic compartments. However, in total membranes obtained as previously described [##REF##16256246##27##], we have not observed any S1PR1 reduction in GK vs Wistar rats, or HFD-fed mice vs controls (data not shown). Thus, S1PR density alterations in the present study most likely take place within the synaptic membranes.</p>","<p id=\"Par35\">In conclusion, we have observed a decrease in the protein levels of three S1P receptors in the cortex of the GK model that were likely unrelated to cortical concentrations of S1P. Two of the altered receptors, namely S1PR2 and S1PR4, have been proposed to control neuronal activity and synaptic transmission ([##REF##35781853##6##], and references therein). Furthermore, S1PR4 levels were also decreased in the cortex of a diet-induced obese mouse model with metabolic syndrome, along with increased cortical levels of S1P. Altogether, these results point towards T2D-induced alterations of the neuromodulation system to which S1P signaling contributes. Our results open the door for testing brain-specific S1P-S1PR modulation as a potential neuroprotection strategy in diabetes.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par26\\\">This study demonstrates for the first time that S1PR density in cortical nerve terminals is reduced in lean and obese models of insulin resistance, along with possible increases of S1P concentrations in the cortex, implying a role for S1PR signaling in the neuropathological process that occurs in T2D. Since S1PR activation generally attenuates neuronal activity [##REF##35781853##6##], lower S1PR density might lead to overall cortical hyper-excitability.</p>\",\n \"<p id=\\\"Par27\\\">From those analyzed, S1PR1 is the receptor with the highest expression level in the CNS. Its density was decreased in cortical synapses of both insulin-resistant GK rats and HFD-fed mice, relative to their respective controls. Previous studies have reported similar S1PR1 level reductions in the hypothalamus of HFD-fed rats or mice, as well as in the leptin deficient ob/ob mice [##REF##25255053##41##]. Silva et al. proposed that S1P is an appetite inhibitor through S1PR1 signaling, and reduced S1PR1 levels in the hypothalamus of obese models were associated with increased food intake. S1PR1 activation inhibits neuronal activity [##REF##35781853##6##] and, therefore, the S1PR1 reduction observed in cortical synapses is likely to result in loss of S1P-dependent synaptic control. However, S1PR1 is ubiquitously expressed, and exert a multitude of regulatory actions, not being specific to synapses. Thus, a potential approach of pharmacologically targeting S1PR1 for synaptic modulation might also trigger a plethora of non-synaptic actions. Adding to the complexity of S1PR1 signaling, others have reported that specific S1PR1 activation increases excitability of some sensory neurons in the rat dorsal root ganglia [##REF##20844107##42##]. Given the ubiquitous actions of S1P, any systemic interventions on S1PR1 should be taken with caution.</p>\",\n \"<p id=\\\"Par28\\\">The density of both S1PR2 and S1PR4 was decreased in cortical synapses from GK rats compared to controls. Considering the role of S1PR2 as neuromodulator in excitatory neurons [##REF##11553273##14##], and its preferential localization at the pre-synaptic level in the cortex [##REF##35781853##6##], dampening of S1PR2 signaling in GK rats is likely to contribute to the synaptic dysfunction and impaired synaptic plasticity [##REF##30686981##29##]. Our previous study [##REF##35781853##6##] showed that specifically S1PR2 and S1PR4 activation results in dampening of spontaneous spiking frequency of cultured primary neurons. A reduction in levels of synaptic S1PR2/4 could thus result in uncontrolled, excessive synaptic activity that might lead to excitotoxicity and synaptic damage [##REF##32265637##26##]. We thus speculate that S1PR2/4 activation might allow for synaptic protection in diabetic conditions.</p>\",\n \"<p id=\\\"Par29\\\">HFD-fed mice did not reproduce the T2D-induced S1PR2 alterations observed in GK rats. Moreover, S1PR4 density was reduced in short- but not long-term HFD, when compared to the respective controls. Besides obesity, a key difference between the two models is the hyperglycemia in GK rats [##REF##23855509##43##, ##REF##29220408##44##] that is negligible in HFD-fed mice [##REF##32265637##26##, ##REF##30670942##36##]. On the other hand, both models become insulin resistant. In this regard, it needs to be noted that S1P signaling through S1PR2 has been shown to interact with insulin signaling, and might participate in the development of insulin resistance in peripheral cells [##REF##26943364##45##]. Given the ability of S1PR2 to inhibit insulin-mediated signals, it is plausible that reduced S1PR2 in synaptic membranes results in enhanced insulin signaling. Thus, S1PR2 might contribute to the development of central insulin resistance. In turn, putatively increased S1PR2 signaling due to higher S1P concentrations in HFD-fed mice could also dampen synaptic insulin signaling. Loss of tonic insulin receptor signaling in nerve terminals is believed to contribute to memory impairment [##REF##36629507##33##, ##REF##27881773##46##].</p>\",\n \"<p id=\\\"Par30\\\">Although S1PR5 is present in the CNS, its relevance for neuromodulation at the synaptic level remains to be established ([##REF##35781853##6##], and references therein). Therefore, we have not investigated diabetes-induced alterations of S1PR5 density in the present study.</p>\",\n \"<p id=\\\"Par31\\\">Concentrations of S1P measured by ELISA were found to increase in the liver of patients with T2D as well as in streptozotocin-induced T2D rats [##REF##30972941##47##]. Plasma S1P concentration also increases in the ob/ob mouse model, mice exposed to HFD for 6 weeks, and in a population of young obese humans [##REF##24039766##48##]. Interestingly, plasma S1P in humans was found to be associated with body fat, insulin levels, insulin resistance (by HOMA-IR), as well with cholesterol [##REF##24039766##48##]. The same study reported an increase in concentrations of S1P in plasma after 12 h of fasting, suggesting a relation between S1P levels and increased inter-organ lipid flux. In our study, plasma samples for S1P determination were collected under fed state, which we therefore expect to depict diabetes-induced S1P changes rather than acute fasting-induced mobilization of lipid stores.</p>\",\n \"<p id=\\\"Par32\\\">While plasma S1P levels increase in non-diabetic young adults with obesity (< 30 years old; body mass index ~ 37 kg/m<sup>2</sup>) relative to lean controls [##REF##24039766##48##], others have reported that T2D is associated with a decrease of plasma S1P concentrations [##REF##29543843##49##, ##REF##31602237##50##]. Namely, these studies found lower plasma S1P concentrations in individuals with T2D relative to healthy controls matched for age and body mass index (average: ~ 60 years old and ~ 29 kg/m<sup>2</sup> in Vaisar et al. [##REF##29543843##49##]; 44–49 years old and 25–26 kg/m<sup>2</sup> in Sui et al. [##REF##31602237##50##]). While reported effects of obesity and T2D on plasma S1P levels are opposite, these studies differ in the studied populations, age of the subjects, and methods for S1P extraction and detection.</p>\",\n \"<p id=\\\"Par33\\\">S1P levels in the cortex are reported to be lower than in other brain regions [##REF##30805275##51##], and we now found S1P concentrations increased in the cortex of HFD-fed mice. To our knowledge, this is the first study investigating S1P concentration in the cerebral cortex of T2D/pre-diabetic animal models. We report an HFD-induced increase of S1P in cortical tissue that seems to be independent of HFD-induced increases in circulating S1P levels. In turn, in GK rats, the large variance of S1P concentrations measured in the cortex precluded determining the expected diabetes-induced increase in S1P levels. This contrasts the concurrent increases of brain and plasma S1P that have previously been reported in a mouse model of hypertension [##REF##29856066##52##].</p>\",\n \"<p id=\\\"Par34\\\">The assessment of S1PR density in mouse cortical synapses across the HFD treatment is limited to the effects of the exposure to the diet, and not the effect of age. This is due to the semi-quantitative nature of immunoblotting methods, and the fact that we have decided to analyze all age-matched samples in parallel. Nevertheless, mice were treated for a relatively short period of time (only 2 months), at an age range from about 2 to 4 months, during which changes of S1PR density might not be as important as effects of the diet. Another limitation of this study is that despite including both sexes in the GK rat part of the study, analysis of plasma S1P levels was only available for males. We are not able to determine sex effects on plasma S1P concentrations, although they were not apparent in cortical levels of S1P. Finally, another important limitation of this study is that the nerve-terminal enriched membranes obtained with our protocol also contain non-synaptic components, such as the perisynaptic astrocytic processes. In particular, S1PR1 is abundant in astrocytes, and the observed S1PR1 reduction could be due to alterations in astrocytic rather than synaptic compartments. However, in total membranes obtained as previously described [##REF##16256246##27##], we have not observed any S1PR1 reduction in GK vs Wistar rats, or HFD-fed mice vs controls (data not shown). Thus, S1PR density alterations in the present study most likely take place within the synaptic membranes.</p>\",\n \"<p id=\\\"Par35\\\">In conclusion, we have observed a decrease in the protein levels of three S1P receptors in the cortex of the GK model that were likely unrelated to cortical concentrations of S1P. Two of the altered receptors, namely S1PR2 and S1PR4, have been proposed to control neuronal activity and synaptic transmission ([##REF##35781853##6##], and references therein). Furthermore, S1PR4 levels were also decreased in the cortex of a diet-induced obese mouse model with metabolic syndrome, along with increased cortical levels of S1P. Altogether, these results point towards T2D-induced alterations of the neuromodulation system to which S1P signaling contributes. Our results open the door for testing brain-specific S1P-S1PR modulation as a potential neuroprotection strategy in diabetes.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<p id=\"Par1\">Sphingosine-1-phosphate (S1P) is a phosphosphingolipid with pleiotropic biological functions. S1P acts as an intracellular second messenger, as well as extracellular ligand to five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation, apoptosis, synaptic activity and neuroglia activation. Moreover, S1P metabolism alterations have been reported in neurodegenerative disorders. We have previously reported that S1PRs are present in nerve terminals, exhibiting distinct sub-synaptic localization and neuromodulation actions. Since type 2 diabetes (T2D) causes synaptic dysfunction, we hypothesized that S1P signaling is modified in nerve terminals. In this study, we determined the density of S1PRs in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and Wistar controls, and from mice fed a high-fat diet (HFD) and low-fat-fed controls. Relative to their controls, GK rats showed similar cortical S1P concentration despite higher S1P levels in plasma, yet lower density of S1PR1, S1PR2 and S1PR4 in nerve-terminal-enriched membranes. HFD-fed mice exhibited increased plasma and cortical concentrations of S1P, and decreased density of S1PR1 and S1PR4. These findings point towards altered S1P signaling in synapses of insulin resistance and diet-induced obesity models, suggesting a role of S1P signaling in T2D-associated synaptic dysfunction.</p>","<title>Keywords</title>","<p>Open access funding provided by Lund University.</p>"],"string":"[\n \"<p id=\\\"Par1\\\">Sphingosine-1-phosphate (S1P) is a phosphosphingolipid with pleiotropic biological functions. S1P acts as an intracellular second messenger, as well as extracellular ligand to five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation, apoptosis, synaptic activity and neuroglia activation. Moreover, S1P metabolism alterations have been reported in neurodegenerative disorders. We have previously reported that S1PRs are present in nerve terminals, exhibiting distinct sub-synaptic localization and neuromodulation actions. Since type 2 diabetes (T2D) causes synaptic dysfunction, we hypothesized that S1P signaling is modified in nerve terminals. In this study, we determined the density of S1PRs in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and Wistar controls, and from mice fed a high-fat diet (HFD) and low-fat-fed controls. Relative to their controls, GK rats showed similar cortical S1P concentration despite higher S1P levels in plasma, yet lower density of S1PR1, S1PR2 and S1PR4 in nerve-terminal-enriched membranes. HFD-fed mice exhibited increased plasma and cortical concentrations of S1P, and decreased density of S1PR1 and S1PR4. These findings point towards altered S1P signaling in synapses of insulin resistance and diet-induced obesity models, suggesting a role of S1P signaling in T2D-associated synaptic dysfunction.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by Lund University.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>The authors thank Nadiia Kravchenko for technical assistance with insulin determination, and Eugenia Cordero Concha for handling the GK rats.</p>","<title>Author Contributions</title>","<p>AM, JMND designed the study. CS, HE, LV, JPPV, FM conducted experiments, CS, JMND analyzed data. CS wrote the manuscript. AM, LE and JMND revised the manuscript. CS and JMND verified all data. All authors approved the final version of the manuscript.</p>","<title>Funding</title>","<p>Open access funding provided by Lund University. This work was supported by the Swedish foundation for International Cooperation in Research and Higher education (#BR2019- 8508), Swedish Research Council (#2019-01130 #2019-01406), Diabetesfonden (#Dia2019-440, #Dia2021-637, #Dia2022-723), Dementiafonden, and Direktör Albert Påhlssons Foundation. The authors acknowledge support from the Lund University Diabetes Center, which is funded by the Swedish Research Council (Strategic Research Area EXODIAB; #2009-1039) and the Swedish Foundation for Strategic Research (#IRC15-0067).</p>","<title>Data Availability</title>","<p>The datasets from the current study are available from the corresponding author on reasonable request.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par37\">The authors declare no competing interests.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>The authors thank Nadiia Kravchenko for technical assistance with insulin determination, and Eugenia Cordero Concha for handling the GK rats.</p>\",\n \"<title>Author Contributions</title>\",\n \"<p>AM, JMND designed the study. CS, HE, LV, JPPV, FM conducted experiments, CS, JMND analyzed data. CS wrote the manuscript. AM, LE and JMND revised the manuscript. CS and JMND verified all data. All authors approved the final version of the manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by Lund University. This work was supported by the Swedish foundation for International Cooperation in Research and Higher education (#BR2019- 8508), Swedish Research Council (#2019-01130 #2019-01406), Diabetesfonden (#Dia2019-440, #Dia2021-637, #Dia2022-723), Dementiafonden, and Direktör Albert Påhlssons Foundation. The authors acknowledge support from the Lund University Diabetes Center, which is funded by the Swedish Research Council (Strategic Research Area EXODIAB; #2009-1039) and the Swedish Foundation for Strategic Research (#IRC15-0067).</p>\",\n \"<title>Data Availability</title>\",\n \"<p>The datasets from the current study are available from the corresponding author on reasonable request.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par37\\\">The authors declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Study design. Samples from Wistar and GK rats were collected at 6 months of age (<bold>A</bold>). Mice were kept on CD or HFD for 1, 4 or 8 weeks before tissue sampling (<bold>B</bold>)</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>S1P concentrations and cortical nerve-terminal density of S1PRs of GK and Wistar rats. S1P concentration in <bold>A</bold> plasma and <bold>B</bold> cortical homogenates. <bold>C</bold> Immunoblots for S1PR1-4 and β-actin (protein loading control) after SDS-PAGE separation of 15 µg of protein from nerve terminal-enriched cortical membranes. <bold>D</bold> relative immunoreactivity estimated from the relative signal intensity in the immunoblots. Data shown as mean ± SD overlaid on individual data points (<italic>n</italic> = 3–7; circles = male rats, triangles = female rats). Symbols over data-points indicate significant differences between Wistar and GK rats (*<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01) based on Mann–Whitney (for cortical S1P concentration and S1PR1 immunoreactivity) or Student t-tests (remaining comparisons)</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>S1P concentrations and cortical nerve-terminal density of S1PRs of mice fed HFD and CD. S1P concentration in <bold>A</bold> plasma and <bold>B</bold> cortical homogenates. <bold>C</bold> Immunoblots for S1PR1-4 and SNAP25 (protein loading control) after SDS-PAGE separation of 15 µg of protein from nerve terminal-enriched cortical membranes. <bold>D–G</bold> Scatter plots show relative immunoreactivity estimated from the signal intensity in the immunoblots after normalization to mean of CD samples. Data shown as mean ± SD overlaid on individual data points (<italic>n</italic> = 5–20). Circles, triangles, and squares represent 1 week, 1 month, and 2 months of diet intervention, respectively. Symbols over data-points (*<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01) indicate significant mean differences from post-tests comparing CD vs HFD within each age, or age within each diet, using Kruskal–Wallis test followed by Dunn’s multiple comparisons (in panel B), or ANOVA followed by independent comparisons with the Fisher’s least significant difference (LSD) test (in panels A, F–G). Samples indicated by <italic>ex</italic> were excluded from analysis</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Study design. Samples from Wistar and GK rats were collected at 6 months of age (<bold>A</bold>). Mice were kept on CD or HFD for 1, 4 or 8 weeks before tissue sampling (<bold>B</bold>)</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>S1P concentrations and cortical nerve-terminal density of S1PRs of GK and Wistar rats. S1P concentration in <bold>A</bold> plasma and <bold>B</bold> cortical homogenates. <bold>C</bold> Immunoblots for S1PR1-4 and β-actin (protein loading control) after SDS-PAGE separation of 15 µg of protein from nerve terminal-enriched cortical membranes. <bold>D</bold> relative immunoreactivity estimated from the relative signal intensity in the immunoblots. Data shown as mean ± SD overlaid on individual data points (<italic>n</italic> = 3–7; circles = male rats, triangles = female rats). Symbols over data-points indicate significant differences between Wistar and GK rats (*<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01) based on Mann–Whitney (for cortical S1P concentration and S1PR1 immunoreactivity) or Student t-tests (remaining comparisons)</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>S1P concentrations and cortical nerve-terminal density of S1PRs of mice fed HFD and CD. S1P concentration in <bold>A</bold> plasma and <bold>B</bold> cortical homogenates. <bold>C</bold> Immunoblots for S1PR1-4 and SNAP25 (protein loading control) after SDS-PAGE separation of 15 µg of protein from nerve terminal-enriched cortical membranes. <bold>D–G</bold> Scatter plots show relative immunoreactivity estimated from the signal intensity in the immunoblots after normalization to mean of CD samples. Data shown as mean ± SD overlaid on individual data points (<italic>n</italic> = 5–20). Circles, triangles, and squares represent 1 week, 1 month, and 2 months of diet intervention, respectively. Symbols over data-points (*<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01) indicate significant mean differences from post-tests comparing CD vs HFD within each age, or age within each diet, using Kruskal–Wallis test followed by Dunn’s multiple comparisons (in panel B), or ANOVA followed by independent comparisons with the Fisher’s least significant difference (LSD) test (in panels A, F–G). Samples indicated by <italic>ex</italic> were excluded from analysis</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Antibodies used for Western blot (WB) and molecular weight of the analyzed band</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Antibody</th><th align=\"left\">Dilution</th><th align=\"left\">Source</th><th align=\"left\">Molecular weight (kDa)</th></tr></thead><tbody><tr><td align=\"left\">Rabbit anti-S1PR1</td><td align=\"left\">1:1,000</td><td align=\"left\">ThermoFisher (PA1-1040)</td><td align=\"left\">47 kDa</td></tr><tr><td align=\"left\">Rabbit anti-S1PR2</td><td align=\"left\">1:500</td><td align=\"left\">Origene (AP01311PU-N)</td><td align=\"left\">42 kDa</td></tr><tr><td align=\"left\">Rabbit anti-S1PR3</td><td align=\"left\">1:1,000</td><td align=\"left\">Origene (TA329055)</td><td align=\"left\">50 kDa</td></tr><tr><td align=\"left\">Rabbit anti-S1PR4</td><td align=\"left\">1:1,000</td><td align=\"left\">Novus (NBP2-24,500)</td><td align=\"left\">39 kDa</td></tr><tr><td align=\"left\">Rabbit anti-SNAP25</td><td align=\"left\">1:5,000</td><td align=\"left\">Abcam (ab109105)</td><td align=\"left\">25 kDa</td></tr><tr><td align=\"left\">HRP-tagged anti-β-actin</td><td align=\"left\">1:10,000</td><td align=\"left\">Sigma-Aldrich (A3854)</td><td align=\"left\">42 kDa</td></tr><tr><td align=\"left\">HRP-tagged anti-rabbit IgG</td><td align=\"left\">1:5,000</td><td align=\"left\">Abcam (ab6802)</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Biotinylated anti-rabbit IgG</td><td align=\"left\">1:5,000</td><td align=\"left\">Vectorlabs (BA-1000)</td><td align=\"left\">–</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Metabolic parameters of GK and Wistar rats</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Wistar (<italic>n</italic> = 3)</th><th align=\"left\" colspan=\"2\">GK (<italic>n</italic> = 3)</th><th align=\"left\">F(DFn,DFd), <italic>P</italic>-value</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"4\">Body weight (g)</td><td align=\"left\">Interaction F(1,8) = 38, <italic>P</italic> < 0.001</td></tr><tr><td align=\"left\"> Male</td><td char=\"±\" align=\"char\">582 ± 37</td><td char=\"±\" align=\"char\">353 ± 44</td><td align=\"left\"><italic>P</italic> < 0.001</td><td align=\"left\">Diabetes F(1,8) = 47, <italic>P</italic> < 0.001</td></tr><tr><td align=\"left\"> Female</td><td char=\"±\" align=\"char\">279 ± 7</td><td char=\"±\" align=\"char\">266 ± 20</td><td align=\"left\">n.s.</td><td align=\"left\">Sex F(1,8) = 122, <italic>P</italic> < 0.001</td></tr><tr><td align=\"left\" colspan=\"4\">Glycemia (mmol/L)</td><td align=\"left\">Interaction F(1,8) = 18, <italic>P</italic> = 0.003</td></tr><tr><td align=\"left\"> Male</td><td char=\"±\" align=\"char\">6.5 ± 0.8</td><td char=\"±\" align=\"char\">18.2 ± 3.4</td><td align=\"left\"><italic>P</italic> < 0.001</td><td align=\"left\">Diabetes F(1,8) = 39, <italic>P</italic> < 0.001</td></tr><tr><td align=\"left\"> Female</td><td char=\"±\" align=\"char\">6.3 ± 0.6</td><td char=\"±\" align=\"char\">8.7 ± 1.1</td><td align=\"left\"><italic>P</italic> = 0.035</td><td align=\"left\">Sex F(1,8) = 19, <italic>P</italic> = 0.002</td></tr><tr><td align=\"left\" colspan=\"4\">Plasma insulin (µg/L)</td><td align=\"left\">Interaction F(1,8) = 0.096, n.s.</td></tr><tr><td align=\"left\"> Male</td><td char=\"±\" align=\"char\">2.0 ± 0.6</td><td char=\"±\" align=\"char\">2.8 ± 1.1</td><td align=\"left\">n.s.</td><td align=\"left\">Diabetes F(1,8) = 3.6, n.s.</td></tr><tr><td align=\"left\"> Female</td><td char=\"±\" align=\"char\">0.9 ± 0.3</td><td char=\"±\" align=\"char\">2.0 ± 1.1</td><td align=\"left\">n.s.</td><td align=\"left\">Sex F(1,8) = 4.0, n.s.</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Metabolic parameters of HFD-fed mice compared to CD-fed mice</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">CD (<italic>n</italic> = 9–16)</th><th align=\"left\" colspan=\"2\">HFD (<italic>n</italic> = 10–16)</th><th align=\"left\">F(DFn,DFd), <italic>P</italic>-value</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"5\">Body weight (g)</td></tr><tr><td align=\"left\"> 1 week</td><td char=\".\" align=\"char\">23.6 ± 1.4</td><td char=\".\" align=\"char\">26.0 ± 1.8</td><td align=\"left\"><italic>P</italic> < 0.001</td><td align=\"left\">Interaction F(2,70) = 2.2, n.s.</td></tr><tr><td align=\"left\"> 1 month</td><td char=\".\" align=\"char\">28.3 ± 1.5</td><td char=\".\" align=\"char\">31.9 ± 2.1</td><td align=\"left\"><italic>P</italic> < 0.001</td><td align=\"left\">Diet F(1,70) = 50, <italic>P</italic> < 0.001</td></tr><tr><td align=\"left\"> 2 months</td><td char=\".\" align=\"char\">30.6 ± 2.3</td><td char=\".\" align=\"char\">35.2 ± 3.4</td><td align=\"left\"><italic>P</italic> = 0.017</td><td align=\"left\">Time F(2,70) = 91, <italic>P</italic> < 0.001</td></tr><tr><td align=\"left\" colspan=\"5\">Fasting glycemia (mmol/L)</td></tr><tr><td align=\"left\"> 1 week</td><td char=\".\" align=\"char\">8.6 ± 1.3</td><td char=\".\" align=\"char\">10.0 ± 0.9</td><td align=\"left\">n.s.</td><td align=\"left\">Interaction F(2,70) = 1.67, n.s.</td></tr><tr><td align=\"left\"> 1 month</td><td char=\".\" align=\"char\">8.2 ± 1.5</td><td char=\".\" align=\"char\">11.0 ± 3.0</td><td align=\"left\"><italic>P</italic> < 0.001</td><td align=\"left\">Diet F(1,70) = 17, <italic>P</italic> < 0.001</td></tr><tr><td align=\"left\"> 2 months</td><td char=\".\" align=\"char\">8.4 ± 1.1</td><td char=\".\" align=\"char\">9.2 ± 1.1</td><td align=\"left\">n.s.</td><td align=\"left\">Time F(2,70) = 1.9, n.s.</td></tr><tr><td align=\"left\" colspan=\"5\">Fasting plasma insulin (µg/L)</td></tr><tr><td align=\"left\"> 1 week</td><td char=\".\" align=\"char\">1.7 ± 1.1</td><td char=\".\" align=\"char\">1.8 ± 0.8</td><td align=\"left\">n.s.</td><td align=\"left\">Interaction F(2,62) = 0.85, n.s.</td></tr><tr><td align=\"left\"> 1 month</td><td char=\".\" align=\"char\">0.6 ± 0.3</td><td char=\".\" align=\"char\">1.0 ± 0.6</td><td align=\"left\">n.s.</td><td align=\"left\">Diet F(1,70) = 95, <italic>P</italic> = 0.023</td></tr><tr><td align=\"left\"> 2 months</td><td char=\".\" align=\"char\">0.8 ± 0.7</td><td char=\".\" align=\"char\">1.5 ± 0.8</td><td align=\"left\"><italic>P</italic> = 0.026</td><td align=\"left\">Time F(2,70) = 10, <italic>P</italic> < 0.001</td></tr><tr><td align=\"left\" colspan=\"5\">2-h glycemia in GTT (mmol/L)</td></tr><tr><td align=\"left\"> 1 week</td><td char=\".\" align=\"char\">8.3 ± 1.5</td><td char=\".\" align=\"char\">12.0 ± 1.8</td><td align=\"left\"><italic>P</italic> < 0.001</td><td align=\"left\">Interaction F(2,70) = 0.66, n.s.</td></tr><tr><td align=\"left\"> 1 month</td><td char=\".\" align=\"char\">7.7 ± 1.0</td><td char=\".\" align=\"char\">11.5 ± 2.2</td><td align=\"left\"><italic>P</italic> < 0.001</td><td align=\"left\">Diet F(1,70) = 95, <italic>P</italic> < 0.001</td></tr><tr><td align=\"left\"> 2 months</td><td char=\".\" align=\"char\">7.3 ± 1.1</td><td char=\".\" align=\"char\">12.1 ± 2.5</td><td align=\"left\"><italic>P</italic> < 0.001</td><td align=\"left\">Time F(2,70) = 0.76, n.s.</td></tr><tr><td align=\"left\" colspan=\"5\">HOMA-IR*</td></tr><tr><td align=\"left\"> 1 week</td><td char=\".\" align=\"char\">13.8 ± 9.2</td><td char=\".\" align=\"char\">17.7 ± 7.3</td><td align=\"left\">n.s.</td><td align=\"left\">Interaction F(2,62) = 0.42, n.s.</td></tr><tr><td align=\"left\"> 1 month</td><td char=\".\" align=\"char\">4.7 ± 2.4</td><td char=\".\" align=\"char\">11.2 ± 7.4</td><td align=\"left\"><italic>P</italic> = 0.011</td><td align=\"left\">Diet F(1,62) = 12, <italic>P</italic> < 0.001</td></tr><tr><td align=\"left\"> 2 months</td><td char=\".\" align=\"char\">6.7 ± 6.2</td><td char=\".\" align=\"char\">14.8 ± 9.1</td><td align=\"left\"><italic>P</italic> = 0.011</td><td align=\"left\">Time F(2,62) = 6.8, <italic>P</italic> = 0.002</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Antibodies used for Western blot (WB) and molecular weight of the analyzed band</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Antibody</th><th align=\\\"left\\\">Dilution</th><th align=\\\"left\\\">Source</th><th align=\\\"left\\\">Molecular weight (kDa)</th></tr></thead><tbody><tr><td align=\\\"left\\\">Rabbit anti-S1PR1</td><td align=\\\"left\\\">1:1,000</td><td align=\\\"left\\\">ThermoFisher (PA1-1040)</td><td align=\\\"left\\\">47 kDa</td></tr><tr><td align=\\\"left\\\">Rabbit anti-S1PR2</td><td align=\\\"left\\\">1:500</td><td align=\\\"left\\\">Origene (AP01311PU-N)</td><td align=\\\"left\\\">42 kDa</td></tr><tr><td align=\\\"left\\\">Rabbit anti-S1PR3</td><td align=\\\"left\\\">1:1,000</td><td align=\\\"left\\\">Origene (TA329055)</td><td align=\\\"left\\\">50 kDa</td></tr><tr><td align=\\\"left\\\">Rabbit anti-S1PR4</td><td align=\\\"left\\\">1:1,000</td><td align=\\\"left\\\">Novus (NBP2-24,500)</td><td align=\\\"left\\\">39 kDa</td></tr><tr><td align=\\\"left\\\">Rabbit anti-SNAP25</td><td align=\\\"left\\\">1:5,000</td><td align=\\\"left\\\">Abcam (ab109105)</td><td align=\\\"left\\\">25 kDa</td></tr><tr><td align=\\\"left\\\">HRP-tagged anti-β-actin</td><td align=\\\"left\\\">1:10,000</td><td align=\\\"left\\\">Sigma-Aldrich (A3854)</td><td align=\\\"left\\\">42 kDa</td></tr><tr><td align=\\\"left\\\">HRP-tagged anti-rabbit IgG</td><td align=\\\"left\\\">1:5,000</td><td align=\\\"left\\\">Abcam (ab6802)</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Biotinylated anti-rabbit IgG</td><td align=\\\"left\\\">1:5,000</td><td align=\\\"left\\\">Vectorlabs (BA-1000)</td><td align=\\\"left\\\">–</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Metabolic parameters of GK and Wistar rats</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"/><th align=\\\"left\\\">Wistar (<italic>n</italic> = 3)</th><th align=\\\"left\\\" colspan=\\\"2\\\">GK (<italic>n</italic> = 3)</th><th align=\\\"left\\\">F(DFn,DFd), <italic>P</italic>-value</th></tr></thead><tbody><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Body weight (g)</td><td align=\\\"left\\\">Interaction F(1,8) = 38, <italic>P</italic> < 0.001</td></tr><tr><td align=\\\"left\\\"> Male</td><td char=\\\"±\\\" align=\\\"char\\\">582 ± 37</td><td char=\\\"±\\\" align=\\\"char\\\">353 ± 44</td><td align=\\\"left\\\"><italic>P</italic> < 0.001</td><td align=\\\"left\\\">Diabetes F(1,8) = 47, <italic>P</italic> < 0.001</td></tr><tr><td align=\\\"left\\\"> Female</td><td char=\\\"±\\\" align=\\\"char\\\">279 ± 7</td><td char=\\\"±\\\" align=\\\"char\\\">266 ± 20</td><td align=\\\"left\\\">n.s.</td><td align=\\\"left\\\">Sex F(1,8) = 122, <italic>P</italic> < 0.001</td></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Glycemia (mmol/L)</td><td align=\\\"left\\\">Interaction F(1,8) = 18, <italic>P</italic> = 0.003</td></tr><tr><td align=\\\"left\\\"> Male</td><td char=\\\"±\\\" align=\\\"char\\\">6.5 ± 0.8</td><td char=\\\"±\\\" align=\\\"char\\\">18.2 ± 3.4</td><td align=\\\"left\\\"><italic>P</italic> < 0.001</td><td align=\\\"left\\\">Diabetes F(1,8) = 39, <italic>P</italic> < 0.001</td></tr><tr><td align=\\\"left\\\"> Female</td><td char=\\\"±\\\" align=\\\"char\\\">6.3 ± 0.6</td><td char=\\\"±\\\" align=\\\"char\\\">8.7 ± 1.1</td><td align=\\\"left\\\"><italic>P</italic> = 0.035</td><td align=\\\"left\\\">Sex F(1,8) = 19, <italic>P</italic> = 0.002</td></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Plasma insulin (µg/L)</td><td align=\\\"left\\\">Interaction F(1,8) = 0.096, n.s.</td></tr><tr><td align=\\\"left\\\"> Male</td><td char=\\\"±\\\" align=\\\"char\\\">2.0 ± 0.6</td><td char=\\\"±\\\" align=\\\"char\\\">2.8 ± 1.1</td><td align=\\\"left\\\">n.s.</td><td align=\\\"left\\\">Diabetes F(1,8) = 3.6, n.s.</td></tr><tr><td align=\\\"left\\\"> Female</td><td char=\\\"±\\\" align=\\\"char\\\">0.9 ± 0.3</td><td char=\\\"±\\\" align=\\\"char\\\">2.0 ± 1.1</td><td align=\\\"left\\\">n.s.</td><td align=\\\"left\\\">Sex F(1,8) = 4.0, n.s.</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Metabolic parameters of HFD-fed mice compared to CD-fed mice</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"/><th align=\\\"left\\\">CD (<italic>n</italic> = 9–16)</th><th align=\\\"left\\\" colspan=\\\"2\\\">HFD (<italic>n</italic> = 10–16)</th><th align=\\\"left\\\">F(DFn,DFd), <italic>P</italic>-value</th></tr></thead><tbody><tr><td align=\\\"left\\\" colspan=\\\"5\\\">Body weight (g)</td></tr><tr><td align=\\\"left\\\"> 1 week</td><td char=\\\".\\\" align=\\\"char\\\">23.6 ± 1.4</td><td char=\\\".\\\" align=\\\"char\\\">26.0 ± 1.8</td><td align=\\\"left\\\"><italic>P</italic> < 0.001</td><td align=\\\"left\\\">Interaction F(2,70) = 2.2, n.s.</td></tr><tr><td align=\\\"left\\\"> 1 month</td><td char=\\\".\\\" align=\\\"char\\\">28.3 ± 1.5</td><td char=\\\".\\\" align=\\\"char\\\">31.9 ± 2.1</td><td align=\\\"left\\\"><italic>P</italic> < 0.001</td><td align=\\\"left\\\">Diet F(1,70) = 50, <italic>P</italic> < 0.001</td></tr><tr><td align=\\\"left\\\"> 2 months</td><td char=\\\".\\\" align=\\\"char\\\">30.6 ± 2.3</td><td char=\\\".\\\" align=\\\"char\\\">35.2 ± 3.4</td><td align=\\\"left\\\"><italic>P</italic> = 0.017</td><td align=\\\"left\\\">Time F(2,70) = 91, <italic>P</italic> < 0.001</td></tr><tr><td align=\\\"left\\\" colspan=\\\"5\\\">Fasting glycemia (mmol/L)</td></tr><tr><td align=\\\"left\\\"> 1 week</td><td char=\\\".\\\" align=\\\"char\\\">8.6 ± 1.3</td><td char=\\\".\\\" align=\\\"char\\\">10.0 ± 0.9</td><td align=\\\"left\\\">n.s.</td><td align=\\\"left\\\">Interaction F(2,70) = 1.67, n.s.</td></tr><tr><td align=\\\"left\\\"> 1 month</td><td char=\\\".\\\" align=\\\"char\\\">8.2 ± 1.5</td><td char=\\\".\\\" align=\\\"char\\\">11.0 ± 3.0</td><td align=\\\"left\\\"><italic>P</italic> < 0.001</td><td align=\\\"left\\\">Diet F(1,70) = 17, <italic>P</italic> < 0.001</td></tr><tr><td align=\\\"left\\\"> 2 months</td><td char=\\\".\\\" align=\\\"char\\\">8.4 ± 1.1</td><td char=\\\".\\\" align=\\\"char\\\">9.2 ± 1.1</td><td align=\\\"left\\\">n.s.</td><td align=\\\"left\\\">Time F(2,70) = 1.9, n.s.</td></tr><tr><td align=\\\"left\\\" colspan=\\\"5\\\">Fasting plasma insulin (µg/L)</td></tr><tr><td align=\\\"left\\\"> 1 week</td><td char=\\\".\\\" align=\\\"char\\\">1.7 ± 1.1</td><td char=\\\".\\\" align=\\\"char\\\">1.8 ± 0.8</td><td align=\\\"left\\\">n.s.</td><td align=\\\"left\\\">Interaction F(2,62) = 0.85, n.s.</td></tr><tr><td align=\\\"left\\\"> 1 month</td><td char=\\\".\\\" align=\\\"char\\\">0.6 ± 0.3</td><td char=\\\".\\\" align=\\\"char\\\">1.0 ± 0.6</td><td align=\\\"left\\\">n.s.</td><td align=\\\"left\\\">Diet F(1,70) = 95, <italic>P</italic> = 0.023</td></tr><tr><td align=\\\"left\\\"> 2 months</td><td char=\\\".\\\" align=\\\"char\\\">0.8 ± 0.7</td><td char=\\\".\\\" align=\\\"char\\\">1.5 ± 0.8</td><td align=\\\"left\\\"><italic>P</italic> = 0.026</td><td align=\\\"left\\\">Time F(2,70) = 10, <italic>P</italic> < 0.001</td></tr><tr><td align=\\\"left\\\" colspan=\\\"5\\\">2-h glycemia in GTT (mmol/L)</td></tr><tr><td align=\\\"left\\\"> 1 week</td><td char=\\\".\\\" align=\\\"char\\\">8.3 ± 1.5</td><td char=\\\".\\\" align=\\\"char\\\">12.0 ± 1.8</td><td align=\\\"left\\\"><italic>P</italic> < 0.001</td><td align=\\\"left\\\">Interaction F(2,70) = 0.66, n.s.</td></tr><tr><td align=\\\"left\\\"> 1 month</td><td char=\\\".\\\" align=\\\"char\\\">7.7 ± 1.0</td><td char=\\\".\\\" align=\\\"char\\\">11.5 ± 2.2</td><td align=\\\"left\\\"><italic>P</italic> < 0.001</td><td align=\\\"left\\\">Diet F(1,70) = 95, <italic>P</italic> < 0.001</td></tr><tr><td align=\\\"left\\\"> 2 months</td><td char=\\\".\\\" align=\\\"char\\\">7.3 ± 1.1</td><td char=\\\".\\\" align=\\\"char\\\">12.1 ± 2.5</td><td align=\\\"left\\\"><italic>P</italic> < 0.001</td><td align=\\\"left\\\">Time F(2,70) = 0.76, n.s.</td></tr><tr><td align=\\\"left\\\" colspan=\\\"5\\\">HOMA-IR*</td></tr><tr><td align=\\\"left\\\"> 1 week</td><td char=\\\".\\\" align=\\\"char\\\">13.8 ± 9.2</td><td char=\\\".\\\" align=\\\"char\\\">17.7 ± 7.3</td><td align=\\\"left\\\">n.s.</td><td align=\\\"left\\\">Interaction F(2,62) = 0.42, n.s.</td></tr><tr><td align=\\\"left\\\"> 1 month</td><td char=\\\".\\\" align=\\\"char\\\">4.7 ± 2.4</td><td char=\\\".\\\" align=\\\"char\\\">11.2 ± 7.4</td><td align=\\\"left\\\"><italic>P</italic> = 0.011</td><td align=\\\"left\\\">Diet F(1,62) = 12, <italic>P</italic> < 0.001</td></tr><tr><td align=\\\"left\\\"> 2 months</td><td char=\\\".\\\" align=\\\"char\\\">6.7 ± 6.2</td><td char=\\\".\\\" align=\\\"char\\\">14.8 ± 9.1</td><td align=\\\"left\\\"><italic>P</italic> = 0.011</td><td align=\\\"left\\\">Time F(2,62) = 6.8, <italic>P</italic> = 0.002</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p>Data is mean ± SD (<italic>n.s.</italic> non-significant difference, <italic>P</italic> > 0.05)</p></table-wrap-foot>","<table-wrap-foot><p>Data is mean ± SD (<italic>n.s.</italic> non-significant difference, <italic>P</italic> > 0.05)</p><p>*Homeostatic model assessment for insulin resistance calculated from fasting insulinemia and glycemia</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>Data is mean ± SD (<italic>n.s.</italic> non-significant difference, <italic>P</italic> > 0.05)</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>Data is mean ± SD (<italic>n.s.</italic> non-significant difference, <italic>P</italic> > 0.05)</p><p>*Homeostatic model assessment for insulin resistance calculated from fasting insulinemia and glycemia</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"11064_2023_4033_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"11064_2023_4033_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"11064_2023_4033_Fig3_HTML\" id=\"MO3\"/>"],"string":"[\n \"<graphic xlink:href=\\\"11064_2023_4033_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"11064_2023_4033_Fig2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic xlink:href=\\\"11064_2023_4033_Fig3_HTML\\\" id=\\\"MO3\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"10.\"], \"surname\": [\"Choi\", \"Chun\"], \"given-names\": [\"JW\", \"J\"], \"article-title\": [\"Lysophospholipids and their receptors in the central nervous system\"], \"source\": [\"Biochim Biophys Acta\"], \"year\": [\"1831\"], \"volume\": [\"1\"], \"fpage\": [\"20\"], \"lpage\": [\"32\"], \"pub-id\": [\"10.1016/j.bbalip.2012.07.015\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [\n \"CNS\",\n \"HFD\",\n \"GK\",\n \"GTT\",\n \"S1P\",\n \"S1PR\",\n \"Sph\",\n \"SphK\",\n \"T2D\"\n ],\n \"definition\": [\n \"Central nervous system\",\n \"High-fat diet\",\n \"Goto-Kakizaki\",\n \"Glucose tolerance test\",\n \"Sphingosine 1-phosphate\",\n \"Sphingosine-1-phosphate receptors\",\n \"Sphingosine\",\n \"Sphingosine kinase\",\n \"Type 2 diabetes\"\n ]\n}"},"n_references":{"kind":"number","value":53,"string":"53"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Neurochem Res. 2024 Oct 4; 49(2):338-347"},"package_file":{"kind":"string","value":"oa_package/c1/70/PMC10787890.tar.gz"}}},{"rowIdx":174,"cells":{"accession_id":{"kind":"string","value":"PMC10787891"},"pmid":{"kind":"string","value":"0"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par5\">Antibiotic resistance, a globalized public health peril in this new era of modern medicine, prevailed from the pathogenic microbial development of novel resistance mechanisms of genetic and epigenetic origins against the available antibiotics by circumventing the therapeutic actions of these drugs, leading to the failure of antimicrobial drugs curbing the menace of infection (Dar et al. ##UREF##3##2017##; Soares et al. ##UREF##9##2021##). The discovery, research, and clinical applications of immuno-peptides known as antimicrobial peptides (AMPs), such as defensins, LL-37, gramicidins D, histatins, Renalexin, and others with antibacterial properties, have been considered and proven as alternative antimicrobial agents to antibiotics (Perez-Perez et al. ##REF##34680851##2021##; Peters et al. ##REF##21060861##2010##; Zhang et al. ##REF##34496967##2021##). Antimicrobial peptides are mostly cationic, amphipathic, and composed of 10–100 amino acid residues. These biological agents have shown novel therapeutic potencies against resistant bacterial pathogens elicited via the disruption of the cell wall and plasma membrane, inducing pore formation, upon the establishment of electrostatic interactions with the negatively charged membrane phospholipids, or inhibiting DNA replication and protein synthesis, thereby exhibiting a broad spectrum of therapeutic activities (Jindal et al. ##REF##26046345##2015##; Ołdak and Zielińska ##UREF##8##2017##). Many AMPs possess a net charge of +2 to +9, confirming strong polarity to bacterial cell membrane surface structures, thereby conceding antibacterial activity against bacterial pathogens that pose global public health threats (Wei and Zhang ##UREF##10##2022##). AMPs are known for modulation and orchestration and as indispensable components of the innate immune system functioning as the first line of defense against bacterial attack in eukaryotes and often synthesized as a competitive strategy in prokaryotes to limit and outcompete the cellular growth of competitive microbes (Seyedjavadi et al. ##REF##37744143##2021##). The mechanisms of therapeutic action of AMPs, including antibacterial peptides, have been extensively exploited over the years. Studies in both in vitro, in vivo, and model plasma membranes have confirmed novel and distinct modes of action compared to clinically prescribed antibiotics by extensively provoking plasma membrane incision and permeability, leading to membrane disruption and cell death (Erdem Büyükkiraz and Kesmen ##REF##34606679##2022##; Wei and Zhang ##UREF##10##2022##).</p>","<p id=\"Par6\">The recent applications of AMPs as disease control therapeutics, coupled with their growing interest as antimicrobial agents produced via recombinant expression systems, have provided a promising and safer platform for the expression and the clinical applications of AMPs <italic>(</italic>Jindal et al. ##REF##26046345##2015##; Montfort-Gardeazabal et al. ##REF##33129981##2021##; Nuti et al. ##REF##28814242##2017##). This study focused on two antimicrobial peptides, LL-37 and Renalexin. LL−37 is the only α-helix cathelicidin-based AMP with therapeutic action against bacterial, viral, and fungal infections (Kang et al. ##REF##31170191##2019##). The biosynthesis of LL−37 occurs in most immune cells, including the mast cell, neutrophils, mucosal epithelial cell, keratinocytes, adipocytes, and the T and B lymphocytes. Cathelicidins (hCAP18) are distinct mammalian immune proteins that act as precursor molecules that undergo proteolytic cleavage at the C-terminal to release a short peptide with antimicrobial and immune-modulatory activity commonly known as LL-37 (Erdem Büyükkiraz and Kesmen ##REF##34606679##2022##). The antibacterial activity of naturally and recombinantly purified LL−37 against multi-drug resistant (MDR) bacteria pathogens has been elucidated and published <italic>(</italic>Dürr et al. ##UREF##4##2006##; Erdem Büyükkiraz and Kesmen ##REF##34606679##2022##; Perez-Perez et al. ##REF##34680851##2021##; Scott et al. ##REF##12244186##2002##). Antimicrobial activity of single-peptide LL-37 showed promising results against <italic>Staphylococcus aureus</italic> (69% sensitivity) and <italic>Escherichia coli</italic> (64% sensitivity) at 50 μM peptide concentration (Perez-Perez et al. ##REF##34680851##2021##). A study by Douglas Clark and colleagues in early 1994 led to the discovery of a novel antimicrobial peptide Renalexin (sometimes referred to as Ranalexin), isolated from the skin of the American Bullfrog <italic>Rana catesbeiana</italic>. It contains a single intramolecular disulfide bond between two cysteine amino acids at positions 14 and 20, which forms a heptapeptide ring within the molecule similar to that seen in the antibiotic Polymyxin B. Renalexin is initially synthesized as a precursor peptide with a putative signal sequence and an acidic amino acid–rich region at its N-terminal <italic>(</italic>Aleinein et al. ##REF##23053091##2013##; Clark et al. ##REF##8144672##1994##). Renalexin has shown therapeutic actions against both gram-positive <italic>S. aureus</italic> and gram-negative <italic>E. coli</italic> bacteria at concentrations ranging from 50 μM and above by interacting with phospholipid membrane via electrostatic binding; in cases where the peptide traverses the membrane, it inhibits protein expression which leads to cell death (Dar et al. ##UREF##3##2017##; Nuti et al. ##REF##28814242##2017##).</p>","<p id=\"Par7\">Here, we report on an effective and reliable design and expression of soluble hybrid antimicrobial peptide LL-37_Renalexin in <italic>Escherichia coli</italic>. We started with synthetic DNA encoding for the target peptide and using CusF3H+ and SmbP as carrier proteins. The recombinant LL-37_Renalexin gene was cloned into the expression vector pET30a+. The fusion peptides CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin were expressed in <italic>E. coli</italic> with isopropyl β-D-1-thiogalactopyranoside induction under optimized conditions. The recombinant tag-free LL-37_Renalexin was purified via immobilized metal affinity chromatography after being released from the carrier proteins by enterokinase treatment. The in vitro antibacterial activities of the hybrid peptide were ascertained and evaluated.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">Antibiotic resistance, a globalized public health peril in this new era of modern medicine, prevailed from the pathogenic microbial development of novel resistance mechanisms of genetic and epigenetic origins against the available antibiotics by circumventing the therapeutic actions of these drugs, leading to the failure of antimicrobial drugs curbing the menace of infection (Dar et al. ##UREF##3##2017##; Soares et al. ##UREF##9##2021##). The discovery, research, and clinical applications of immuno-peptides known as antimicrobial peptides (AMPs), such as defensins, LL-37, gramicidins D, histatins, Renalexin, and others with antibacterial properties, have been considered and proven as alternative antimicrobial agents to antibiotics (Perez-Perez et al. ##REF##34680851##2021##; Peters et al. ##REF##21060861##2010##; Zhang et al. ##REF##34496967##2021##). Antimicrobial peptides are mostly cationic, amphipathic, and composed of 10–100 amino acid residues. These biological agents have shown novel therapeutic potencies against resistant bacterial pathogens elicited via the disruption of the cell wall and plasma membrane, inducing pore formation, upon the establishment of electrostatic interactions with the negatively charged membrane phospholipids, or inhibiting DNA replication and protein synthesis, thereby exhibiting a broad spectrum of therapeutic activities (Jindal et al. ##REF##26046345##2015##; Ołdak and Zielińska ##UREF##8##2017##). Many AMPs possess a net charge of +2 to +9, confirming strong polarity to bacterial cell membrane surface structures, thereby conceding antibacterial activity against bacterial pathogens that pose global public health threats (Wei and Zhang ##UREF##10##2022##). AMPs are known for modulation and orchestration and as indispensable components of the innate immune system functioning as the first line of defense against bacterial attack in eukaryotes and often synthesized as a competitive strategy in prokaryotes to limit and outcompete the cellular growth of competitive microbes (Seyedjavadi et al. ##REF##37744143##2021##). The mechanisms of therapeutic action of AMPs, including antibacterial peptides, have been extensively exploited over the years. Studies in both in vitro, in vivo, and model plasma membranes have confirmed novel and distinct modes of action compared to clinically prescribed antibiotics by extensively provoking plasma membrane incision and permeability, leading to membrane disruption and cell death (Erdem Büyükkiraz and Kesmen ##REF##34606679##2022##; Wei and Zhang ##UREF##10##2022##).</p>\",\n \"<p id=\\\"Par6\\\">The recent applications of AMPs as disease control therapeutics, coupled with their growing interest as antimicrobial agents produced via recombinant expression systems, have provided a promising and safer platform for the expression and the clinical applications of AMPs <italic>(</italic>Jindal et al. ##REF##26046345##2015##; Montfort-Gardeazabal et al. ##REF##33129981##2021##; Nuti et al. ##REF##28814242##2017##). This study focused on two antimicrobial peptides, LL-37 and Renalexin. LL−37 is the only α-helix cathelicidin-based AMP with therapeutic action against bacterial, viral, and fungal infections (Kang et al. ##REF##31170191##2019##). The biosynthesis of LL−37 occurs in most immune cells, including the mast cell, neutrophils, mucosal epithelial cell, keratinocytes, adipocytes, and the T and B lymphocytes. Cathelicidins (hCAP18) are distinct mammalian immune proteins that act as precursor molecules that undergo proteolytic cleavage at the C-terminal to release a short peptide with antimicrobial and immune-modulatory activity commonly known as LL-37 (Erdem Büyükkiraz and Kesmen ##REF##34606679##2022##). The antibacterial activity of naturally and recombinantly purified LL−37 against multi-drug resistant (MDR) bacteria pathogens has been elucidated and published <italic>(</italic>Dürr et al. ##UREF##4##2006##; Erdem Büyükkiraz and Kesmen ##REF##34606679##2022##; Perez-Perez et al. ##REF##34680851##2021##; Scott et al. ##REF##12244186##2002##). Antimicrobial activity of single-peptide LL-37 showed promising results against <italic>Staphylococcus aureus</italic> (69% sensitivity) and <italic>Escherichia coli</italic> (64% sensitivity) at 50 μM peptide concentration (Perez-Perez et al. ##REF##34680851##2021##). A study by Douglas Clark and colleagues in early 1994 led to the discovery of a novel antimicrobial peptide Renalexin (sometimes referred to as Ranalexin), isolated from the skin of the American Bullfrog <italic>Rana catesbeiana</italic>. It contains a single intramolecular disulfide bond between two cysteine amino acids at positions 14 and 20, which forms a heptapeptide ring within the molecule similar to that seen in the antibiotic Polymyxin B. Renalexin is initially synthesized as a precursor peptide with a putative signal sequence and an acidic amino acid–rich region at its N-terminal <italic>(</italic>Aleinein et al. ##REF##23053091##2013##; Clark et al. ##REF##8144672##1994##). Renalexin has shown therapeutic actions against both gram-positive <italic>S. aureus</italic> and gram-negative <italic>E. coli</italic> bacteria at concentrations ranging from 50 μM and above by interacting with phospholipid membrane via electrostatic binding; in cases where the peptide traverses the membrane, it inhibits protein expression which leads to cell death (Dar et al. ##UREF##3##2017##; Nuti et al. ##REF##28814242##2017##).</p>\",\n \"<p id=\\\"Par7\\\">Here, we report on an effective and reliable design and expression of soluble hybrid antimicrobial peptide LL-37_Renalexin in <italic>Escherichia coli</italic>. We started with synthetic DNA encoding for the target peptide and using CusF3H+ and SmbP as carrier proteins. The recombinant LL-37_Renalexin gene was cloned into the expression vector pET30a+. The fusion peptides CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin were expressed in <italic>E. coli</italic> with isopropyl β-D-1-thiogalactopyranoside induction under optimized conditions. The recombinant tag-free LL-37_Renalexin was purified via immobilized metal affinity chromatography after being released from the carrier proteins by enterokinase treatment. The in vitro antibacterial activities of the hybrid peptide were ascertained and evaluated.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<title>Reagents, plasmid, enzymes, and bacteria strains</title>","<p id=\"Par8\">\n<italic>Escherichia coli</italic> DH5α cells employed for routine plasmid propagation and subcloning experiments were supplied by New England Biolabs (NEB) (Ipswich MA, USA). Protease-deficient bacteria strains <italic>Escherichia coli</italic> BL21(DE3) and <italic>Escherichia coli</italic> SHuffle T7(DE3) also supplied by NEB were used as microbial expression hosts. The plasmid pET30a+ purchased from EMD Biosciences (Darmstadt, Germany) was chosen for the design and construction of plasmid expression vectors. The MEGAquick-spin DNA and plasmid purification kits were bought from iNtRON Biotechnology (Seoul, South Korea). Restriction enzymes <italic>Nde</italic>I and <italic>Xho</italic>I were purchased from NEB. T4 DNA ligase, <italic>Vent</italic>, and <italic>Taq</italic> DNA polymerases used for molecular cloning and amplification were provided from NEB as well. The synthetic DNA encoding for the hybrid peptide and the protease enterokinase were purchased from GenScript Inc. (Centennial, Piscataway, USA). Isopropyl-β-D-1-thiogalactopyranoside (IPTG) used for induction of expression was purchased from A.G. Scientific Inc. (San Diego, CA, USA). Kanamycin employed as a selective marker for transformed cells and as a positive control was supplied by Sigma-Aldrich (Darmstadt, Germany). Standard protein markers used for peptide size characterization were purchased from NEB and Bio Basic Inc. (Amherst, NY, USA). All culture media including Luria-Bertani, Tryptic Soy Broth, bacteriological agar, and Mueller-Hinton agar used for microbial cultivation, expression, enrichment, and antimicrobial activity were provided by Sigma-Aldrich (Darmstadt, Germany), and Legacy Biologicals (Mount Prospect, IL, USA). All clinical bacteria pathogens including <italic>S. aureus</italic> and <italic>E. coli</italic> used for the antimicrobial activities were supplied from the University Hospital, Department of Pathology, Clinical Microbiology Laboratory (UANL, Monterrey, Mexico).</p>","<title>Design of the hybrid AMP LL-37_Renalexin</title>","<p id=\"Par9\">The mature amino acid sequences of the single antimicrobial peptide LL-37 and Renalexin were retrieved from the AMP database (<ext-link ext-link-type=\"uri\" xlink:href=\"https://APD3.unmc.edu/structure\">https://APD3.unmc.edu/structure</ext-link>) with the accession number AP0030/2K60 and AP00513/P39084, respectively, and were used for the design of a novel hybrid peptide LL-37_Renalexin via the application of our newly designed GS peptide linker and the protein tags CusF3H+ and SmbP (Fig. ##FIG##0##1##) that allow for expression and immobilized metal-affinity chromatography (IMAC) purification. The designed hybrid peptide amino acid sequence was flanked at respective positions with the amino acid sequences of protein tags SmbP or CusF3H+, enterokinase site, GS linker, <italic>Nde</italic>I, <italic>Kpn</italic>I, and <italic>Xho</italic>I restriction enzyme sites. The entire amino acid sequence of the designed complete hybrid peptide was optimized (<ext-link ext-link-type=\"uri\" xlink:href=\"http://genomes.urv.es/CAIcal\">http://genomes.urv.es/CAIcal</ext-link>) for efficient expression based on codon usage in <italic>E. coli</italic> as the microbial expression host. The biochemical properties and molecular structure of the designed hybrid peptide were elucidated and analyzed using Expasy and I-TASSER tools (<ext-link ext-link-type=\"uri\" xlink:href=\"https://web.expasy.org/cgi-bin/protparam/protparam\">https://web.expasy.org/cgi-bin/protparam/protparam</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://zhanggroup.org/I-TASSER/\">https://zhanggroup.org/I-TASSER/</ext-link>) to access the efficiency and the reliability of production in microbial systems.</p>","<title>Construction of pET30a+ expression vectors</title>","<p id=\"Par10\">A 492- and 480-mer oligonucleotide DNA (gene) sequences (Fig. ##SUPPL##0##S1##) encoding for SmbP_LL-37_Renalexin and CusF3H+_LL-37_Renalexin with the accession numbers OR356112 and OR356113, respectively, (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/Genbank.html\">https://www.ncbi.nlm.nih.gov/Genbank.html</ext-link>) were synthesized based on the optimized hybrid peptide sequence with reference to codon usage in <italic>E. coli</italic>. The gene was cloned into pUC57 and supplied to the protein expression and purification laboratory, UANL, Mexico. Both the pUC57 and pET30a+ plasmid vectors were digested with <italic>Nde</italic>I and <italic>Xho</italic>I restriction enzymes (Fig. ##SUPPL##0##S2##). The restriction digestion products were visualized on a 1% agarose gel stained with 0.5 μg/ml ethidium bromide. The target DNA fragment was cut from the gel and purified using the MEGAquick-spin DNA purification kit. Purified DNA fragments were ligated with the T4 DNA ligase for the design of two plasmid expression vectors pET30a+_CusF3H+_LL-37_Renalexin and pET30a+_SmbP_LL-37_Renalexin. The resulting plasmid construct was transformed for propagation into <italic>E. coli</italic> DH5α by heat shock at 42 °C for 45 s. Transformant cells were selected on Luria-Bertani–kanamycin (30 μg/ml) agar plates. To confirm the presence of the gene in the designed plasmid constructs, the 5′-T7 promoter forward primer (5′-TAATACGACTCACTATAGGG-3′) and the 3′-T7 terminator reserve primer (3′-GCTAGTTATTGCTCACGG-5′) pairs were used for polymerase chain reaction (DNase-free water, 5 mM dNTPs, 10 ng DNA template, 0.5 μM primer, 5U <italic>Taq</italic> polymerase) under the following conditions: initial denaturation at 95 °C for 1 min, denaturation at 95 °C for 30 s, primer annealing at 55 °C for 45 s, elongation at 72 °C for 50 s, and final elongations at 72 °C for 5 min, 32 reaction cycles of amplification. The PCR amplicons were analyzed on a 1% agarose gel and visualized under a UV transilluminator (Fig. ##SUPPL##0##S3##). The plasmid constructs were sequenced by STARSEQ GmbH (Instituto de Biotecnologia, UNAM, Mexico). FinchTV version 1.4 was employed to analyze and confirm the sequenced nucleotides using EMBOSS (data not shown) (Fig. ##SUPPL##0##S4##).</p>","<title>Expression and purification of recombinant fusion peptides CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin</title>","<p id=\"Par11\">For expression, the designed recombinant plasmid DNA construct was transformed by heat shock at 42 °C for 45 s into <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) calcium competent cells. An inoculum of 5 ml LB broth (30 μg/ml kanamycin) was made, inoculated with a fresh single colony of transformed cells, and incubated at 37 °C overnight with shaking at 220 rpm. Overnight cultures confirming cell viability and plasmid stability were used to inoculate 1000 ml LB broth (30 μg/ml kanamycin) in a baffled flask and incubated at 37 °C, 220 rpm for about 3–4 h until optical cell density (OD<sub>600nm</sub>) of 0.4–0.6 was obtained. Recombinant fusion peptide expression was induced with 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. Induced culture flasks were incubated under the optimized condition of 25 °C, 220 rpm for 16 h. Cell pellets harboring the expressed recombinant fusion peptide were harvested by centrifugation at 8500 × g, 4 °C for 15 min into sterilized 50-ml Eppendorf tubes. Collected pellets were resuspended in ice-cold lysis buffer (500 mM NaCl, 50 mM Tris-HCl pH 8.0) and lysed by vortexing on ice with 0.1 mm glass beads. Clear soluble cell lysate was obtained after centrifugation at 8500 × g, 4 °C for 15 min. Purification of recombinant fusion peptide was performed via IMAC using the ÄKTA Prime Plus System (GE Healthcare) for fast protein liquid chromatography (FPLC). Briefly, a 1-ml HisTrap FF agarose-resin Ni(II) charged column was employed for the IMAC purification. The column was equilibrated with 5 column volumes (CV) of equilibrating buffer (500 mM NaCl, 50 mM Tris-HCl pH 8.0). The clarified soluble lysate was loaded onto the column under the conditions of 0.5 MPa pressure and 0.5 ml/min flow rate. Subsequently, the column was washed with 3 CVs of washing buffer (500 mM NaCl, 2.5 mM Imidazole, 50 mM Tris-HCl pH 8.0). The recombinant fusion peptide was eluted by gradient elution with elution buffer (500 mM NaCl, 200 mM Imidazole, 50 mM Tris-HCl pH 8.0). A 10 μl cell lysate, column flow-through, and elution fractions were analyzed on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the purity of protein bands (Tab. ##SUPPL##0##S1##) was measured by densitometry using ImageJ software (v.2.0) (Adamíková et al. ##REF##30810853##2019##).</p>","<title>Enterokinase cleavage and purification of LL-37_Renalexin</title>","<p id=\"Par12\">To induce enterokinase treatment, the purified fusion peptide elution fractions were pooled together in a 6-cm dialysis membrane and dialyzed against dialysis buffer (1× PBS pH 7.2) to desalt the purified peptide and exchange the elution buffer. In brief, the dialysis setup was incubated at room temperature for 1 h with gentle shaking on a magnetic stirrer, followed by overnight incubation at 4 °C. The concentration of the dialyzed fusion peptide was estimated by Bradford analysis using the Bovine Serum Albumin (BSA) (Tab. ##SUPPL##0##S2##, Fig. ##SUPPL##0##S5## and Tab. ##SUPPL##0##S3##, Fig. ##SUPPL##0##S6##) as standard protein (Colyer and Walker ##UREF##2##1996##). A 1 mg purified fusion peptide was exposed to 20 U enterokinase cleavage (5 U/μl) for the release of hybrid peptide LL-37_Renalexin (tag-free). The cleavage reaction was incubated at room temperature for 16 h, followed by enzyme inactivation at −20 °C for 3 h. 10 μl of cleavage mixture was analyzed on a 15% and 18% Tricine SDS-PAGE.</p>","<p id=\"Par13\">In purifying the tag-free hybrid peptide by IMAC, a 1.0 × 10-cm chromatographic syringe column was loaded with 1 ml of agarose resins charged with 1 M Ni(II) ions. The column was equilibrated with 10 CV of 1× PBS pH 7.2, and the enterokinase cleavage mixture was loaded into the column, mixed, and incubated at 4 °C for 1.5 h for efficient binding of protein tags to the affinity column. Column flow-through was collected as tag-free hybrid AMP LL-37_Renalexin. The protein tags were released with elution buffer (500 mM NaCl, 200 mM Imidazole, 50 mM Tris-HCl pH 8.0). The purified tag-free hybrid peptide was characterized on a 15% Tricine SDS-PAGE, and the concentration was estimated by Bradford analysis and Nanodrop absorbance readings at 280 nm (Tab. ##SUPPL##0##S4##, Fig. ##SUPPL##0##S7## and Tab. ##SUPPL##0##S5##, Fig ##SUPPL##0##S8##).</p>","<title>Antimicrobial activity assay of recombinant hybrid peptide LL-37_Renalexin</title>","<title>Culture media</title>","<p id=\"Par14\">Tryptic soy broth (TSB): tryptone (pancreatic digest of casein) 17.0 g, soytone (peptic digest of soybean) 3.0 g, glucose (dextrose) 2.5 g, sodium chloride 5.0 g, and dipotassium phosphate 2.5 g, pH 7.3. Bacteriological agar: Mueller–Hinton agar (MHA), beef extract 2.0 g, acid hydrolysate of casein 17.5 g, starch 1.5 g, agar 17.0 g, pH 7.3. Phosphate-buffered saline (PBS); sodium chloride 8 g, potassium chloride 0.2 g, sodium phosphate dibasic 1.44 g, potassium phosphate monobasic 0.245 g, pH 7.4. All culture media and buffers were used for microbial cultivation of bacteria clinical isolates, inoculum suspension preparations, and antimicrobial activity tests.</p>","<title>Inoculum preparation</title>","<p id=\"Par15\">Single colonies of test bacteria clinical isolates were cultured in 5 ml TSB and incubated at 37 °C, 220 rpm for 16 h. A 20 μl overnight culture was inoculated into 5 ml TSB and incubated at 37 °C with shaking until an optical cell density (OD<sub>600nm</sub>) of 0.8–1.0 was obtained (mid-logarithmic growth). Cells at the log growth phase were serially diluted in 1× PBS pH 7.2 buffer and the dilution suspensions at 1 × 10<sup>5</sup> CFU/ml equivalent to 0.5 McFarland turbidity standard were employed for antimicrobial assay.</p>","<title>Antimicrobial activity</title>","<title>Dose-response assay: minimum inhibition concentration (MIC) determination</title>","<p id=\"Par16\">The antimicrobial activity of the hybrid peptide was evaluated against gram-positive and gram-negative bacteria clinical isolates of <italic>Staphylococcus aureus</italic>, <italic>Escherichia coli</italic>, methicillin-resistant <italic>Staphylococcus aureus</italic>, and <italic>Klebsiella pneumoniae</italic> from the University Hospital. The minimum inhibition concentrations against the test pathogens were ascertained and estimated in accordance with the modification of the National Committee for Clinical Laboratory Standards (NCCLS) for the broth microdilution method (CLS ##UREF##1##2022##). Briefly, bacteria cell cultures in the mid-log growth phase in TSB were serially diluted in 1× PBS pH 7.2 buffer to 1 × 10<sup>5</sup> CFU/ml. A 2-fold broth microdilution assay was performed in a 96-well microtiter plate with a 200 μl assay volume per well composed of 100 μl diluted peptide at concentrations ranging from 0.5–33 μM, 80 μl bacteria suspensions, and 20 μl TSB medium. The plate was incubated at 37°C for 3 h with shaking. After 3 h incubation, 0.1 ml aliquot was taken per well and a 10-fold dilution was made from which a 100 μl aliquot was spread on tryptic soy agar (TSA). Inoculated plates were incubated at 37 °C for 20 h and the remaining colony-forming units were evaluated (Tab. ##SUPPL##0##S6##, ##SUPPL##0##S7##, ##SUPPL##0##S8##, ##SUPPL##0##S9##, ##SUPPL##0##S14##, and ##SUPPL##0##S15##), and MICs were calculated as the lowest peptide concentration that obviate visible turbidity using a modified B. Gompertz function for the line of best fit in dose-response analysis (Lambert and Pearson ##REF##10792538##2000##).</p>","<title>Time-killing assay</title>","<p id=\"Par17\">The antibacterial killing kinetics of the hybrid peptide was evaluated against two bacterial isolates by ascertaining the time course to kill test bacteria isolates suspension of <italic>S. aureus</italic> (gram+), and <italic>E. coli</italic> (gram−). In brief, bacterial cultures in the mid-logarithmic growth phase were incubated as described previously with the peptide LL-37_Renalexin at a concentration of approximately 2× MIC in a TSB medium. The 10 μl aliquot suspensions were taken at every 20 min interval until a period of 3 h incubation was observed. A 10-fold dilution in 1× PBS pH 7.2 was made, and 0.1 ml aliquots were inoculated on TSA medium. Inoculated plates were incubated at 37 °C for 20 h. Log remaining CFU/ml of test pathogens were taken and plotted against time (Tab. ##SUPPL##0##S10##, and ##SUPPL##0##S11##). Two control samples were made, positive control (bacterial suspension and kanamycin at the same peptide concentration) and negative control (bacterial suspension and 1X PBS buffer). The average of the total remaining CFU/ml from each treatment was evaluated (Tab. ##SUPPL##0##S12## and ##SUPPL##0##S13##) and analyzed via one-way analysis of variance (ANOVA). The hybrid peptide with a single disulfide linkage (S–S bond) expressed in <italic>E. coli</italic> SHuffle T7(DE3) showing relatively lower minimum inhibitory concentrations (MICs) was employed for time-killing kinetic assay.</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<title>Reagents, plasmid, enzymes, and bacteria strains</title>\",\n \"<p id=\\\"Par8\\\">\\n<italic>Escherichia coli</italic> DH5α cells employed for routine plasmid propagation and subcloning experiments were supplied by New England Biolabs (NEB) (Ipswich MA, USA). Protease-deficient bacteria strains <italic>Escherichia coli</italic> BL21(DE3) and <italic>Escherichia coli</italic> SHuffle T7(DE3) also supplied by NEB were used as microbial expression hosts. The plasmid pET30a+ purchased from EMD Biosciences (Darmstadt, Germany) was chosen for the design and construction of plasmid expression vectors. The MEGAquick-spin DNA and plasmid purification kits were bought from iNtRON Biotechnology (Seoul, South Korea). Restriction enzymes <italic>Nde</italic>I and <italic>Xho</italic>I were purchased from NEB. T4 DNA ligase, <italic>Vent</italic>, and <italic>Taq</italic> DNA polymerases used for molecular cloning and amplification were provided from NEB as well. The synthetic DNA encoding for the hybrid peptide and the protease enterokinase were purchased from GenScript Inc. (Centennial, Piscataway, USA). Isopropyl-β-D-1-thiogalactopyranoside (IPTG) used for induction of expression was purchased from A.G. Scientific Inc. (San Diego, CA, USA). Kanamycin employed as a selective marker for transformed cells and as a positive control was supplied by Sigma-Aldrich (Darmstadt, Germany). Standard protein markers used for peptide size characterization were purchased from NEB and Bio Basic Inc. (Amherst, NY, USA). All culture media including Luria-Bertani, Tryptic Soy Broth, bacteriological agar, and Mueller-Hinton agar used for microbial cultivation, expression, enrichment, and antimicrobial activity were provided by Sigma-Aldrich (Darmstadt, Germany), and Legacy Biologicals (Mount Prospect, IL, USA). All clinical bacteria pathogens including <italic>S. aureus</italic> and <italic>E. coli</italic> used for the antimicrobial activities were supplied from the University Hospital, Department of Pathology, Clinical Microbiology Laboratory (UANL, Monterrey, Mexico).</p>\",\n \"<title>Design of the hybrid AMP LL-37_Renalexin</title>\",\n \"<p id=\\\"Par9\\\">The mature amino acid sequences of the single antimicrobial peptide LL-37 and Renalexin were retrieved from the AMP database (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://APD3.unmc.edu/structure\\\">https://APD3.unmc.edu/structure</ext-link>) with the accession number AP0030/2K60 and AP00513/P39084, respectively, and were used for the design of a novel hybrid peptide LL-37_Renalexin via the application of our newly designed GS peptide linker and the protein tags CusF3H+ and SmbP (Fig. ##FIG##0##1##) that allow for expression and immobilized metal-affinity chromatography (IMAC) purification. The designed hybrid peptide amino acid sequence was flanked at respective positions with the amino acid sequences of protein tags SmbP or CusF3H+, enterokinase site, GS linker, <italic>Nde</italic>I, <italic>Kpn</italic>I, and <italic>Xho</italic>I restriction enzyme sites. The entire amino acid sequence of the designed complete hybrid peptide was optimized (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"http://genomes.urv.es/CAIcal\\\">http://genomes.urv.es/CAIcal</ext-link>) for efficient expression based on codon usage in <italic>E. coli</italic> as the microbial expression host. The biochemical properties and molecular structure of the designed hybrid peptide were elucidated and analyzed using Expasy and I-TASSER tools (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://web.expasy.org/cgi-bin/protparam/protparam\\\">https://web.expasy.org/cgi-bin/protparam/protparam</ext-link>, <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://zhanggroup.org/I-TASSER/\\\">https://zhanggroup.org/I-TASSER/</ext-link>) to access the efficiency and the reliability of production in microbial systems.</p>\",\n \"<title>Construction of pET30a+ expression vectors</title>\",\n \"<p id=\\\"Par10\\\">A 492- and 480-mer oligonucleotide DNA (gene) sequences (Fig. ##SUPPL##0##S1##) encoding for SmbP_LL-37_Renalexin and CusF3H+_LL-37_Renalexin with the accession numbers OR356112 and OR356113, respectively, (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://www.ncbi.nlm.nih.gov/Genbank.html\\\">https://www.ncbi.nlm.nih.gov/Genbank.html</ext-link>) were synthesized based on the optimized hybrid peptide sequence with reference to codon usage in <italic>E. coli</italic>. The gene was cloned into pUC57 and supplied to the protein expression and purification laboratory, UANL, Mexico. Both the pUC57 and pET30a+ plasmid vectors were digested with <italic>Nde</italic>I and <italic>Xho</italic>I restriction enzymes (Fig. ##SUPPL##0##S2##). The restriction digestion products were visualized on a 1% agarose gel stained with 0.5 μg/ml ethidium bromide. The target DNA fragment was cut from the gel and purified using the MEGAquick-spin DNA purification kit. Purified DNA fragments were ligated with the T4 DNA ligase for the design of two plasmid expression vectors pET30a+_CusF3H+_LL-37_Renalexin and pET30a+_SmbP_LL-37_Renalexin. The resulting plasmid construct was transformed for propagation into <italic>E. coli</italic> DH5α by heat shock at 42 °C for 45 s. Transformant cells were selected on Luria-Bertani–kanamycin (30 μg/ml) agar plates. To confirm the presence of the gene in the designed plasmid constructs, the 5′-T7 promoter forward primer (5′-TAATACGACTCACTATAGGG-3′) and the 3′-T7 terminator reserve primer (3′-GCTAGTTATTGCTCACGG-5′) pairs were used for polymerase chain reaction (DNase-free water, 5 mM dNTPs, 10 ng DNA template, 0.5 μM primer, 5U <italic>Taq</italic> polymerase) under the following conditions: initial denaturation at 95 °C for 1 min, denaturation at 95 °C for 30 s, primer annealing at 55 °C for 45 s, elongation at 72 °C for 50 s, and final elongations at 72 °C for 5 min, 32 reaction cycles of amplification. The PCR amplicons were analyzed on a 1% agarose gel and visualized under a UV transilluminator (Fig. ##SUPPL##0##S3##). The plasmid constructs were sequenced by STARSEQ GmbH (Instituto de Biotecnologia, UNAM, Mexico). FinchTV version 1.4 was employed to analyze and confirm the sequenced nucleotides using EMBOSS (data not shown) (Fig. ##SUPPL##0##S4##).</p>\",\n \"<title>Expression and purification of recombinant fusion peptides CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin</title>\",\n \"<p id=\\\"Par11\\\">For expression, the designed recombinant plasmid DNA construct was transformed by heat shock at 42 °C for 45 s into <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) calcium competent cells. An inoculum of 5 ml LB broth (30 μg/ml kanamycin) was made, inoculated with a fresh single colony of transformed cells, and incubated at 37 °C overnight with shaking at 220 rpm. Overnight cultures confirming cell viability and plasmid stability were used to inoculate 1000 ml LB broth (30 μg/ml kanamycin) in a baffled flask and incubated at 37 °C, 220 rpm for about 3–4 h until optical cell density (OD<sub>600nm</sub>) of 0.4–0.6 was obtained. Recombinant fusion peptide expression was induced with 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. Induced culture flasks were incubated under the optimized condition of 25 °C, 220 rpm for 16 h. Cell pellets harboring the expressed recombinant fusion peptide were harvested by centrifugation at 8500 × g, 4 °C for 15 min into sterilized 50-ml Eppendorf tubes. Collected pellets were resuspended in ice-cold lysis buffer (500 mM NaCl, 50 mM Tris-HCl pH 8.0) and lysed by vortexing on ice with 0.1 mm glass beads. Clear soluble cell lysate was obtained after centrifugation at 8500 × g, 4 °C for 15 min. Purification of recombinant fusion peptide was performed via IMAC using the ÄKTA Prime Plus System (GE Healthcare) for fast protein liquid chromatography (FPLC). Briefly, a 1-ml HisTrap FF agarose-resin Ni(II) charged column was employed for the IMAC purification. The column was equilibrated with 5 column volumes (CV) of equilibrating buffer (500 mM NaCl, 50 mM Tris-HCl pH 8.0). The clarified soluble lysate was loaded onto the column under the conditions of 0.5 MPa pressure and 0.5 ml/min flow rate. Subsequently, the column was washed with 3 CVs of washing buffer (500 mM NaCl, 2.5 mM Imidazole, 50 mM Tris-HCl pH 8.0). The recombinant fusion peptide was eluted by gradient elution with elution buffer (500 mM NaCl, 200 mM Imidazole, 50 mM Tris-HCl pH 8.0). A 10 μl cell lysate, column flow-through, and elution fractions were analyzed on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the purity of protein bands (Tab. ##SUPPL##0##S1##) was measured by densitometry using ImageJ software (v.2.0) (Adamíková et al. ##REF##30810853##2019##).</p>\",\n \"<title>Enterokinase cleavage and purification of LL-37_Renalexin</title>\",\n \"<p id=\\\"Par12\\\">To induce enterokinase treatment, the purified fusion peptide elution fractions were pooled together in a 6-cm dialysis membrane and dialyzed against dialysis buffer (1× PBS pH 7.2) to desalt the purified peptide and exchange the elution buffer. In brief, the dialysis setup was incubated at room temperature for 1 h with gentle shaking on a magnetic stirrer, followed by overnight incubation at 4 °C. The concentration of the dialyzed fusion peptide was estimated by Bradford analysis using the Bovine Serum Albumin (BSA) (Tab. ##SUPPL##0##S2##, Fig. ##SUPPL##0##S5## and Tab. ##SUPPL##0##S3##, Fig. ##SUPPL##0##S6##) as standard protein (Colyer and Walker ##UREF##2##1996##). A 1 mg purified fusion peptide was exposed to 20 U enterokinase cleavage (5 U/μl) for the release of hybrid peptide LL-37_Renalexin (tag-free). The cleavage reaction was incubated at room temperature for 16 h, followed by enzyme inactivation at −20 °C for 3 h. 10 μl of cleavage mixture was analyzed on a 15% and 18% Tricine SDS-PAGE.</p>\",\n \"<p id=\\\"Par13\\\">In purifying the tag-free hybrid peptide by IMAC, a 1.0 × 10-cm chromatographic syringe column was loaded with 1 ml of agarose resins charged with 1 M Ni(II) ions. The column was equilibrated with 10 CV of 1× PBS pH 7.2, and the enterokinase cleavage mixture was loaded into the column, mixed, and incubated at 4 °C for 1.5 h for efficient binding of protein tags to the affinity column. Column flow-through was collected as tag-free hybrid AMP LL-37_Renalexin. The protein tags were released with elution buffer (500 mM NaCl, 200 mM Imidazole, 50 mM Tris-HCl pH 8.0). The purified tag-free hybrid peptide was characterized on a 15% Tricine SDS-PAGE, and the concentration was estimated by Bradford analysis and Nanodrop absorbance readings at 280 nm (Tab. ##SUPPL##0##S4##, Fig. ##SUPPL##0##S7## and Tab. ##SUPPL##0##S5##, Fig ##SUPPL##0##S8##).</p>\",\n \"<title>Antimicrobial activity assay of recombinant hybrid peptide LL-37_Renalexin</title>\",\n \"<title>Culture media</title>\",\n \"<p id=\\\"Par14\\\">Tryptic soy broth (TSB): tryptone (pancreatic digest of casein) 17.0 g, soytone (peptic digest of soybean) 3.0 g, glucose (dextrose) 2.5 g, sodium chloride 5.0 g, and dipotassium phosphate 2.5 g, pH 7.3. Bacteriological agar: Mueller–Hinton agar (MHA), beef extract 2.0 g, acid hydrolysate of casein 17.5 g, starch 1.5 g, agar 17.0 g, pH 7.3. Phosphate-buffered saline (PBS); sodium chloride 8 g, potassium chloride 0.2 g, sodium phosphate dibasic 1.44 g, potassium phosphate monobasic 0.245 g, pH 7.4. All culture media and buffers were used for microbial cultivation of bacteria clinical isolates, inoculum suspension preparations, and antimicrobial activity tests.</p>\",\n \"<title>Inoculum preparation</title>\",\n \"<p id=\\\"Par15\\\">Single colonies of test bacteria clinical isolates were cultured in 5 ml TSB and incubated at 37 °C, 220 rpm for 16 h. A 20 μl overnight culture was inoculated into 5 ml TSB and incubated at 37 °C with shaking until an optical cell density (OD<sub>600nm</sub>) of 0.8–1.0 was obtained (mid-logarithmic growth). Cells at the log growth phase were serially diluted in 1× PBS pH 7.2 buffer and the dilution suspensions at 1 × 10<sup>5</sup> CFU/ml equivalent to 0.5 McFarland turbidity standard were employed for antimicrobial assay.</p>\",\n \"<title>Antimicrobial activity</title>\",\n \"<title>Dose-response assay: minimum inhibition concentration (MIC) determination</title>\",\n \"<p id=\\\"Par16\\\">The antimicrobial activity of the hybrid peptide was evaluated against gram-positive and gram-negative bacteria clinical isolates of <italic>Staphylococcus aureus</italic>, <italic>Escherichia coli</italic>, methicillin-resistant <italic>Staphylococcus aureus</italic>, and <italic>Klebsiella pneumoniae</italic> from the University Hospital. The minimum inhibition concentrations against the test pathogens were ascertained and estimated in accordance with the modification of the National Committee for Clinical Laboratory Standards (NCCLS) for the broth microdilution method (CLS ##UREF##1##2022##). Briefly, bacteria cell cultures in the mid-log growth phase in TSB were serially diluted in 1× PBS pH 7.2 buffer to 1 × 10<sup>5</sup> CFU/ml. A 2-fold broth microdilution assay was performed in a 96-well microtiter plate with a 200 μl assay volume per well composed of 100 μl diluted peptide at concentrations ranging from 0.5–33 μM, 80 μl bacteria suspensions, and 20 μl TSB medium. The plate was incubated at 37°C for 3 h with shaking. After 3 h incubation, 0.1 ml aliquot was taken per well and a 10-fold dilution was made from which a 100 μl aliquot was spread on tryptic soy agar (TSA). Inoculated plates were incubated at 37 °C for 20 h and the remaining colony-forming units were evaluated (Tab. ##SUPPL##0##S6##, ##SUPPL##0##S7##, ##SUPPL##0##S8##, ##SUPPL##0##S9##, ##SUPPL##0##S14##, and ##SUPPL##0##S15##), and MICs were calculated as the lowest peptide concentration that obviate visible turbidity using a modified B. Gompertz function for the line of best fit in dose-response analysis (Lambert and Pearson ##REF##10792538##2000##).</p>\",\n \"<title>Time-killing assay</title>\",\n \"<p id=\\\"Par17\\\">The antibacterial killing kinetics of the hybrid peptide was evaluated against two bacterial isolates by ascertaining the time course to kill test bacteria isolates suspension of <italic>S. aureus</italic> (gram+), and <italic>E. coli</italic> (gram−). In brief, bacterial cultures in the mid-logarithmic growth phase were incubated as described previously with the peptide LL-37_Renalexin at a concentration of approximately 2× MIC in a TSB medium. The 10 μl aliquot suspensions were taken at every 20 min interval until a period of 3 h incubation was observed. A 10-fold dilution in 1× PBS pH 7.2 was made, and 0.1 ml aliquots were inoculated on TSA medium. Inoculated plates were incubated at 37 °C for 20 h. Log remaining CFU/ml of test pathogens were taken and plotted against time (Tab. ##SUPPL##0##S10##, and ##SUPPL##0##S11##). Two control samples were made, positive control (bacterial suspension and kanamycin at the same peptide concentration) and negative control (bacterial suspension and 1X PBS buffer). The average of the total remaining CFU/ml from each treatment was evaluated (Tab. ##SUPPL##0##S12## and ##SUPPL##0##S13##) and analyzed via one-way analysis of variance (ANOVA). The hybrid peptide with a single disulfide linkage (S–S bond) expressed in <italic>E. coli</italic> SHuffle T7(DE3) showing relatively lower minimum inhibitory concentrations (MICs) was employed for time-killing kinetic assay.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Design of hybrid peptide and construction of recombinant plasmids</title>","<p id=\"Par18\">In designing the hybrid peptide with the molecular gene map shown (Fig. ##FIG##1##2##A), we employed the mature amino acid sequences that encode for peptides LL-37 and Renalexin retrieved from the antimicrobial peptide database <ext-link ext-link-type=\"uri\" xlink:href=\"http://aps.unmc.edu/AP/prediction/\">http://aps.unmc.edu/AP/prediction/</ext-link> with the accession number AP00310/2K60 and AP00513/P39084, respectively. The amino acid GS (glycine and serine) between LL-37 and Renalexin in the gene construct was employed as a novel, simple, flexible peptide linker allowing for the construction of the hybrid peptide LL-37_Renalexin. DDDDK amino acid sequence at the N-terminal of the hybrid peptide serves as an enterokinase site that allows for the molecular cleavage of the protein tags SmbP and CusF3H+ yielding a tag-free hybrid AMP LL-37_Renalexin. For efficient production and affinity purification, the carrier proteins CusF3H+ and SmbP were inserted between the LL-37_Renalexin gene sequence and the start codon. The molecular Riben structure (Fig. ##FIG##1##2##B) of the designed hybrid peptide predicted in i-TASSER (<ext-link ext-link-type=\"uri\" xlink:href=\"https://zhanggroup.org/I-TASSER/\">https://zhanggroup.org/I-TASSER/</ext-link>) suggests a secondary structural motif with efficient production in <italic>E. coli</italic> (Kesidis et al. ##REF##32534131##2020##). The structure predicted and modeled in i-Tasser was confirmed in the Phyre2 server (Fig. ##SUPPL##0##S9##) (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.sbg.bio.ic.ac.uk/phyre2\">http://www.sbg.bio.ic.ac.uk/phyre2</ext-link>) with 99.9–99.3% confidence level under the model template identifier (i.d) c2K6oA (LL-37) and c2fcgF (Renalexin) (Kelley et al. ##UREF##6##2016##). The application of GS peptide linker facilitates the design of a novel hybrid peptide by maintaining the molecular α-helix structure in LL-37 and the heptapeptide ring of Renalexin (<ext-link ext-link-type=\"uri\" xlink:href=\"https://APD3.unmc.edu/structure\">https://APD3.unmc.edu/structure</ext-link>). The target hybrid peptide has the following biochemical properties: 59 amino acid length, +9 net charge, pI of 10.3, 44% hydrophobicity and 56% hydrophilicity, GRAVY index of 0.00, instability index of 21.3, aliphatic index of 102.37, and a molecular weight of 6.740 kDa theoretically predicted in <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.expasy.org/tools/proparameter\">http://www.expasy.org/tools/proparameter</ext-link>. The physicochemical properties of the hybrid peptide suggest strong electrostatic cationic polarity, hydropathicity, stability, and good isoelectric potentials which provide theoretical evidence of strong antimicrobial activity of this hybrid peptide at a varying pH range compared to its counterpart single peptides. Cellular toxicity of the hybrid peptide against healthy human cells was ascertained using ToxIBTL advanced bioinformatic online server (<ext-link ext-link-type=\"uri\" xlink:href=\"https://server.wei-group.net/ToxIBTL/server.html\">https://server.wei-group.net/ToxIBTL/server.html</ext-link>). ToxIBTL is an online in silico peptide and protein toxicity prediction tool that operates using evolutionary information and the physicochemical properties of peptide sequence via the integration of bottleneck principle to predict peptide or protein toxicity level. Our in silico cytotoxicity analysis (Fig. ##SUPPL##0##S10##) showed that the hybrid peptide has no toxic effect (zero toxicity) on normal human cells at the niche of infection at a toxicity score of 3.7139784e-05 (0.0000371) which is by far below the standard threshold of 0.5.</p>","<p id=\"Par19\">The DNA nucleotide (gene) of the coding sequence (CDS) was chemically synthesized by GenScript based on the optimized amino acid sequence that encodes for the recombinant fusion proteins CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin. The gene map of the synthetic DNA constituted restriction sites for <italic>Nde</italic>I, <italic>Kpn</italic>I, <italic>Xho</italic>I, and enterokinase, a protein tag site at the N-terminal, with the hybrid peptide site between the protein tag site and a stop codon. The 492-bp (SmbP tag construct) and 480-bp (CusF3H+ tag construct) synthetic DNAs were molecularly cloned into pET30a+ at <italic>Nde</italic>I and <italic>Xho</italic>I restriction sites under the control of T7 promoter using the T4-DNA ligase for the design of two plasmid expression vectors pET30a+_CusF3H+_LL-37_Renalexin and pET30a+_SmbP_LL-37_Renalexin. The plasmid construct was confirmed by colony-based PCR screening using the T7 promoter and terminator-specific primers. After colony-based PCR screening, the correct recombinant plasmid sequence was further confirmed by DNA sequencing (data not shown).</p>","<title>Expression and IMAC purification of recombinant fusion protein</title>","<p id=\"Par20\">The protease-deficient bacterial strains <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) competent cells used as expression hosts encode for the T7 RNA polymerase allowing for the expression of the recombinant fusion peptides CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin under the influence of T7 promoter. IPTG induction and expression of fusion peptides successfully showed an efficient insertion of DNA under the T7 promoter in the designed plasmid constructs mentioned above. The carrier proteins CusF3H+ and SmbP show significant advantages in the production and purification of the recombinant fusion peptides expressed as soluble proteins with no formation of inclusion bodies (Perez-Perez et al. ##REF##34680851##2021##; Vargas-Cortez et al. ##REF##26494603##2016##). In evaluating small-scale expression of recombinant fusion proteins CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin (17 kDa) in <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3), soluble cell lysates were prepared by lysing cell pellets collected from 2 ml of 1 mM IPTG-induced overnight cultures to access the presence of the target protein in soluble cell lysate. Analysis of 5 μl aliquot of clear lysate on a 15% SDS-PAGE showed successful evidence of expression of the target fusion proteins with 17–18 kDa-expected protein band compared to non-induced cells as negative control samples (Fig. ##FIG##2##3##). For the large-scale production of recombinant fusion proteins, <italic>E. coli</italic> strains <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) were used as expression hosts. From a 1-L expression volume, cell pellets were collected from 1 mM IPTG-induced overnight cultures and lysed by mechanical vortexing on ice with 0.1 mm glass beads. After cell lysis, clear soluble lysate was collected and employed as a protein source for the immobilized metal affinity chromatography.</p>","<p id=\"Par21\">For the purification of fusion protein SmbP_LL-37_Renalexin and CusF3H+_LL-37_Renalexin, the ÄKTA Prime Plus system (GE Healthcare Systems) was employed for fast protein liquid chromatography (FPLC) by metal affinity chromatography. A 1-ml HisTrap FF column charged with Ni(II) was used to isolate the target recombinant peptide from the pool of cellular proteins present in the soluble lysate fractions collected. Analysis of IMAC purification fractions of the recombinant fusion protein on a 15% SDS PAGE (Fig. ##FIG##3##4##) showed evidence of target peptide in the elution fractions (200 mM imidazole) without any trace of peptide indications in the column flow-through. This result confirms the high affinity of CusF3H+ and SmbP to agarose-resin Ni(II)-charged column that facilitates the binding of the target proteins unto the column while the untargeted cellular proteins exit the column as flow-through (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##; Vargas-Cortez et al. ##REF##28087367##2017##). Our Bradford quantification analysis using standard Bovine Serum Albumin (BSA) calibration equations (data not shown) indicated a peptide concentration of 3.136 mg/L for CusF3H+_LL-37_Renalexin produced in <italic>E. coli</italic> SHuffle T7(DE3) and 1.523 mg/L for SmbP_LL-37_Renalexin produced in <italic>E. coli</italic> BL21(DE3) with a purity of 90–95% matching the purity standard of commercially available synthetic therapeutic peptides. We observed a 2-fold higher recombinant fusion peptide yield in <italic>E. coli</italic> SHuffle T7(DE3) compared to production in <italic>E. coli</italic> BL21(DE3).</p>","<title>Enterokinase cleavage and purification recombinant LL-37_Renalexin</title>","<p id=\"Par22\">Our previous studies (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##) reported the abrogative effect on the bioactivity of recombinant antimicrobial peptides with attached protein tags. We employed the restriction enzyme enterokinase for the selective cleavage of protein tags CusF3H+ and SmbP from the above-purified fusion peptides. Electrophoretic analysis of inactivated enterokinase cleavage mixture revealed three protein bands of size 18 kDa (uncleaved fusion peptide), ≈13 kDa (CusF3H+ or SmbP), and ≈10 kDa (LL-37_Renalexin, tag-free) on Tricine SDS-PAGE (Fig. ##FIG##4##5##). The uncleaved fusion peptide detected was due to an incomplete enzymatic cleavage reaction. Finally, the tag-free LL-37_Renalexin was purified via a one-step IMAC purification using an agarose resin Ni(II)-charged syringe column unto which the uncleaved fusion peptide and the carrier proteins CusF3H+ and SmbP remain bounded, allowing for the elution of the target hybrid peptide LL-37_Renalexin as column flowthrough. A 2.16 mg/L for tag-free LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3) and 0.72 mg/L expressed in <italic>E. coli</italic> BL21(DE3) were obtained after Bradford and Nanodrop spectrometry (A280nm) quantification analysis.</p>","<title>Antimicrobial activity of recombinant LL-37_Renalexin</title>","<p id=\"Par23\">The antimicrobial potency of the purified hybrid peptide against clinical isolates of <italic>S. aureus</italic>, <italic>E. coli</italic>, MRSA, and <italic>K. pneumoniae</italic> was evaluated, and the minimum inhibitory concentrations (MICs) were determined via the broth microdilution assay as described by NCCLSI (Lacy et al. ##UREF##7##2004##). Data on remaining colony-forming units (CFU/ml) of test pathogens were taken and analyzed after overnight culture with the peptide. The MIC, defined as the minimum peptide concentration that prevented visible turbidity in the test pathogen, was calculated using a modified Benjamin Gompertz sigmoid function (Lambert and Pearson ##REF##10792538##2000##) from the plot of peptide concentrations against the remaining CFU/ml (dose-response plot). The dose-response plots (Fig. ##FIG##5##6##) show the antibacterial activity of the hybrid AMP LL-37_Renalexin at MIC levels of 10–27 μM, much lower than the reported MICs of the single-peptide LL-37 and Renalexin (50–100 μM) (Aleinein et al. ##REF##23053091##2013##; Kang et al. ##REF##31170191##2019##; Perez-Perez et al. ##REF##34680851##2021##). The MIC result we observed in this study affirms with data reported on related hybrid and dimeric peptides tested against the same bacterial pathogens (Cheng et al. ##REF##34885732##2021##; Dürr et al. ##UREF##4##2006##; Kang et al. ##REF##31170191##2019##; Wei and Zhang ##UREF##10##2022##; Seyedjavadi et al. ##REF##37744143##2021##). The dose-response antimicrobial activity results indicated that the test bacterial pathogens were sensitive to the recombinant hybrid peptide at active peptide concentrations as low as 10 μM and 33 μM. Interestingly, we evaluated the bioactivity of the hybrid peptide without disulfide-linkage expressed in <italic>E. coli</italic> BL21(DE3); the results (Table ##TAB##0##1##) suggested no significant difference in the MICs compared to the hybrid peptide with disulfide-linkage expressed in <italic>E. coli</italic> SHuffle T7(DE3).</p>","<title>Time-kill kinetics analysis</title>","<p id=\"Par24\">The time-kill kinetic assay (Fig. ##FIG##6##7##) disclosed that the hybrid peptide shows a multifunctional antibacterial activity within 1.5 h via disruption of membrane integrity and membrane traversing, demonstrating a strong but relatively slow antibacterial potency against all investigated gram-positive and gram-negative bacterial pathogens as compared to the classical antibiotic kanamycin that exhibited its antibacterial activity within less than an hour. We observed that the antibacterial activity of the hybrid peptide in comparison to the known antibiotic kanamycin analyzed against total remaining CFU/ml showed a significant difference at <italic>p</italic>-values < 0.05 (Fig. ##FIG##7##8##), with the hybrid peptide showing relatively similar antibacterial actions as exhibited by kanamycin (Aleinein et al. ##REF##23053091##2013##; Hanafiah et al. ##REF##33281340##2020##; Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Design of hybrid peptide and construction of recombinant plasmids</title>\",\n \"<p id=\\\"Par18\\\">In designing the hybrid peptide with the molecular gene map shown (Fig. ##FIG##1##2##A), we employed the mature amino acid sequences that encode for peptides LL-37 and Renalexin retrieved from the antimicrobial peptide database <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"http://aps.unmc.edu/AP/prediction/\\\">http://aps.unmc.edu/AP/prediction/</ext-link> with the accession number AP00310/2K60 and AP00513/P39084, respectively. The amino acid GS (glycine and serine) between LL-37 and Renalexin in the gene construct was employed as a novel, simple, flexible peptide linker allowing for the construction of the hybrid peptide LL-37_Renalexin. DDDDK amino acid sequence at the N-terminal of the hybrid peptide serves as an enterokinase site that allows for the molecular cleavage of the protein tags SmbP and CusF3H+ yielding a tag-free hybrid AMP LL-37_Renalexin. For efficient production and affinity purification, the carrier proteins CusF3H+ and SmbP were inserted between the LL-37_Renalexin gene sequence and the start codon. The molecular Riben structure (Fig. ##FIG##1##2##B) of the designed hybrid peptide predicted in i-TASSER (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://zhanggroup.org/I-TASSER/\\\">https://zhanggroup.org/I-TASSER/</ext-link>) suggests a secondary structural motif with efficient production in <italic>E. coli</italic> (Kesidis et al. ##REF##32534131##2020##). The structure predicted and modeled in i-Tasser was confirmed in the Phyre2 server (Fig. ##SUPPL##0##S9##) (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"http://www.sbg.bio.ic.ac.uk/phyre2\\\">http://www.sbg.bio.ic.ac.uk/phyre2</ext-link>) with 99.9–99.3% confidence level under the model template identifier (i.d) c2K6oA (LL-37) and c2fcgF (Renalexin) (Kelley et al. ##UREF##6##2016##). The application of GS peptide linker facilitates the design of a novel hybrid peptide by maintaining the molecular α-helix structure in LL-37 and the heptapeptide ring of Renalexin (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://APD3.unmc.edu/structure\\\">https://APD3.unmc.edu/structure</ext-link>). The target hybrid peptide has the following biochemical properties: 59 amino acid length, +9 net charge, pI of 10.3, 44% hydrophobicity and 56% hydrophilicity, GRAVY index of 0.00, instability index of 21.3, aliphatic index of 102.37, and a molecular weight of 6.740 kDa theoretically predicted in <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"http://www.expasy.org/tools/proparameter\\\">http://www.expasy.org/tools/proparameter</ext-link>. The physicochemical properties of the hybrid peptide suggest strong electrostatic cationic polarity, hydropathicity, stability, and good isoelectric potentials which provide theoretical evidence of strong antimicrobial activity of this hybrid peptide at a varying pH range compared to its counterpart single peptides. Cellular toxicity of the hybrid peptide against healthy human cells was ascertained using ToxIBTL advanced bioinformatic online server (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://server.wei-group.net/ToxIBTL/server.html\\\">https://server.wei-group.net/ToxIBTL/server.html</ext-link>). ToxIBTL is an online in silico peptide and protein toxicity prediction tool that operates using evolutionary information and the physicochemical properties of peptide sequence via the integration of bottleneck principle to predict peptide or protein toxicity level. Our in silico cytotoxicity analysis (Fig. ##SUPPL##0##S10##) showed that the hybrid peptide has no toxic effect (zero toxicity) on normal human cells at the niche of infection at a toxicity score of 3.7139784e-05 (0.0000371) which is by far below the standard threshold of 0.5.</p>\",\n \"<p id=\\\"Par19\\\">The DNA nucleotide (gene) of the coding sequence (CDS) was chemically synthesized by GenScript based on the optimized amino acid sequence that encodes for the recombinant fusion proteins CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin. The gene map of the synthetic DNA constituted restriction sites for <italic>Nde</italic>I, <italic>Kpn</italic>I, <italic>Xho</italic>I, and enterokinase, a protein tag site at the N-terminal, with the hybrid peptide site between the protein tag site and a stop codon. The 492-bp (SmbP tag construct) and 480-bp (CusF3H+ tag construct) synthetic DNAs were molecularly cloned into pET30a+ at <italic>Nde</italic>I and <italic>Xho</italic>I restriction sites under the control of T7 promoter using the T4-DNA ligase for the design of two plasmid expression vectors pET30a+_CusF3H+_LL-37_Renalexin and pET30a+_SmbP_LL-37_Renalexin. The plasmid construct was confirmed by colony-based PCR screening using the T7 promoter and terminator-specific primers. After colony-based PCR screening, the correct recombinant plasmid sequence was further confirmed by DNA sequencing (data not shown).</p>\",\n \"<title>Expression and IMAC purification of recombinant fusion protein</title>\",\n \"<p id=\\\"Par20\\\">The protease-deficient bacterial strains <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) competent cells used as expression hosts encode for the T7 RNA polymerase allowing for the expression of the recombinant fusion peptides CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin under the influence of T7 promoter. IPTG induction and expression of fusion peptides successfully showed an efficient insertion of DNA under the T7 promoter in the designed plasmid constructs mentioned above. The carrier proteins CusF3H+ and SmbP show significant advantages in the production and purification of the recombinant fusion peptides expressed as soluble proteins with no formation of inclusion bodies (Perez-Perez et al. ##REF##34680851##2021##; Vargas-Cortez et al. ##REF##26494603##2016##). In evaluating small-scale expression of recombinant fusion proteins CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin (17 kDa) in <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3), soluble cell lysates were prepared by lysing cell pellets collected from 2 ml of 1 mM IPTG-induced overnight cultures to access the presence of the target protein in soluble cell lysate. Analysis of 5 μl aliquot of clear lysate on a 15% SDS-PAGE showed successful evidence of expression of the target fusion proteins with 17–18 kDa-expected protein band compared to non-induced cells as negative control samples (Fig. ##FIG##2##3##). For the large-scale production of recombinant fusion proteins, <italic>E. coli</italic> strains <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) were used as expression hosts. From a 1-L expression volume, cell pellets were collected from 1 mM IPTG-induced overnight cultures and lysed by mechanical vortexing on ice with 0.1 mm glass beads. After cell lysis, clear soluble lysate was collected and employed as a protein source for the immobilized metal affinity chromatography.</p>\",\n \"<p id=\\\"Par21\\\">For the purification of fusion protein SmbP_LL-37_Renalexin and CusF3H+_LL-37_Renalexin, the ÄKTA Prime Plus system (GE Healthcare Systems) was employed for fast protein liquid chromatography (FPLC) by metal affinity chromatography. A 1-ml HisTrap FF column charged with Ni(II) was used to isolate the target recombinant peptide from the pool of cellular proteins present in the soluble lysate fractions collected. Analysis of IMAC purification fractions of the recombinant fusion protein on a 15% SDS PAGE (Fig. ##FIG##3##4##) showed evidence of target peptide in the elution fractions (200 mM imidazole) without any trace of peptide indications in the column flow-through. This result confirms the high affinity of CusF3H+ and SmbP to agarose-resin Ni(II)-charged column that facilitates the binding of the target proteins unto the column while the untargeted cellular proteins exit the column as flow-through (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##; Vargas-Cortez et al. ##REF##28087367##2017##). Our Bradford quantification analysis using standard Bovine Serum Albumin (BSA) calibration equations (data not shown) indicated a peptide concentration of 3.136 mg/L for CusF3H+_LL-37_Renalexin produced in <italic>E. coli</italic> SHuffle T7(DE3) and 1.523 mg/L for SmbP_LL-37_Renalexin produced in <italic>E. coli</italic> BL21(DE3) with a purity of 90–95% matching the purity standard of commercially available synthetic therapeutic peptides. We observed a 2-fold higher recombinant fusion peptide yield in <italic>E. coli</italic> SHuffle T7(DE3) compared to production in <italic>E. coli</italic> BL21(DE3).</p>\",\n \"<title>Enterokinase cleavage and purification recombinant LL-37_Renalexin</title>\",\n \"<p id=\\\"Par22\\\">Our previous studies (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##) reported the abrogative effect on the bioactivity of recombinant antimicrobial peptides with attached protein tags. We employed the restriction enzyme enterokinase for the selective cleavage of protein tags CusF3H+ and SmbP from the above-purified fusion peptides. Electrophoretic analysis of inactivated enterokinase cleavage mixture revealed three protein bands of size 18 kDa (uncleaved fusion peptide), ≈13 kDa (CusF3H+ or SmbP), and ≈10 kDa (LL-37_Renalexin, tag-free) on Tricine SDS-PAGE (Fig. ##FIG##4##5##). The uncleaved fusion peptide detected was due to an incomplete enzymatic cleavage reaction. Finally, the tag-free LL-37_Renalexin was purified via a one-step IMAC purification using an agarose resin Ni(II)-charged syringe column unto which the uncleaved fusion peptide and the carrier proteins CusF3H+ and SmbP remain bounded, allowing for the elution of the target hybrid peptide LL-37_Renalexin as column flowthrough. A 2.16 mg/L for tag-free LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3) and 0.72 mg/L expressed in <italic>E. coli</italic> BL21(DE3) were obtained after Bradford and Nanodrop spectrometry (A280nm) quantification analysis.</p>\",\n \"<title>Antimicrobial activity of recombinant LL-37_Renalexin</title>\",\n \"<p id=\\\"Par23\\\">The antimicrobial potency of the purified hybrid peptide against clinical isolates of <italic>S. aureus</italic>, <italic>E. coli</italic>, MRSA, and <italic>K. pneumoniae</italic> was evaluated, and the minimum inhibitory concentrations (MICs) were determined via the broth microdilution assay as described by NCCLSI (Lacy et al. ##UREF##7##2004##). Data on remaining colony-forming units (CFU/ml) of test pathogens were taken and analyzed after overnight culture with the peptide. The MIC, defined as the minimum peptide concentration that prevented visible turbidity in the test pathogen, was calculated using a modified Benjamin Gompertz sigmoid function (Lambert and Pearson ##REF##10792538##2000##) from the plot of peptide concentrations against the remaining CFU/ml (dose-response plot). The dose-response plots (Fig. ##FIG##5##6##) show the antibacterial activity of the hybrid AMP LL-37_Renalexin at MIC levels of 10–27 μM, much lower than the reported MICs of the single-peptide LL-37 and Renalexin (50–100 μM) (Aleinein et al. ##REF##23053091##2013##; Kang et al. ##REF##31170191##2019##; Perez-Perez et al. ##REF##34680851##2021##). The MIC result we observed in this study affirms with data reported on related hybrid and dimeric peptides tested against the same bacterial pathogens (Cheng et al. ##REF##34885732##2021##; Dürr et al. ##UREF##4##2006##; Kang et al. ##REF##31170191##2019##; Wei and Zhang ##UREF##10##2022##; Seyedjavadi et al. ##REF##37744143##2021##). The dose-response antimicrobial activity results indicated that the test bacterial pathogens were sensitive to the recombinant hybrid peptide at active peptide concentrations as low as 10 μM and 33 μM. Interestingly, we evaluated the bioactivity of the hybrid peptide without disulfide-linkage expressed in <italic>E. coli</italic> BL21(DE3); the results (Table ##TAB##0##1##) suggested no significant difference in the MICs compared to the hybrid peptide with disulfide-linkage expressed in <italic>E. coli</italic> SHuffle T7(DE3).</p>\",\n \"<title>Time-kill kinetics analysis</title>\",\n \"<p id=\\\"Par24\\\">The time-kill kinetic assay (Fig. ##FIG##6##7##) disclosed that the hybrid peptide shows a multifunctional antibacterial activity within 1.5 h via disruption of membrane integrity and membrane traversing, demonstrating a strong but relatively slow antibacterial potency against all investigated gram-positive and gram-negative bacterial pathogens as compared to the classical antibiotic kanamycin that exhibited its antibacterial activity within less than an hour. We observed that the antibacterial activity of the hybrid peptide in comparison to the known antibiotic kanamycin analyzed against total remaining CFU/ml showed a significant difference at <italic>p</italic>-values < 0.05 (Fig. ##FIG##7##8##), with the hybrid peptide showing relatively similar antibacterial actions as exhibited by kanamycin (Aleinein et al. ##REF##23053091##2013##; Hanafiah et al. ##REF##33281340##2020##; Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par25\">Antimicrobial peptides (AMPs) as drug candidates are being considered the new hope for the biomedical and pharmaceutical industries in conjunction with their multifunctional antibacterial pharmacological actions against infectious agents (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Moretta et al. ##REF##34195099##2021##; Nuti et al. ##REF##28814242##2017##). The production of AMPs as therapeutic peptides via the applications of recombinant DNA technology (rDNA) and the use of cost-effective microbial expression systems have facilitated the large-scale acquisition of bioactive AMPs, enhancing clinical and research applications (Akhtar et al. ##REF##22675369##2012##; Dar et al. ##UREF##3##2017##). In addition to the successful application of rDNA, the efficient, fast, and easy growth of microbial expression hosts like <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) has allowed for commercial production of bioactive cationic AMPs independent of posttranslational modifications (Aleinein et al. ##REF##23053091##2013##; Klubthawee et al. ##REF##32499514##2020##; Montfort-Gardeazabal et al. ##REF##33129981##2021##). Novel AMPs like Brevinin-2R, Renalexin, Cecropin A, and LL-37 as single peptides have been produced as recombinant protein-based drug candidates in microbial systems and have shown a promising antimicrobial effect (Aleinein et al. ##REF##23053091##2013##; Chhetri et al. ##REF##26629417##2015##; Zhang et al. ##UREF##11##2018##). The thriving commercial production of AMPs in <italic>E. coli</italic> largely depends on the design and use of protein tags, including but not limited to poly-histidine, maltose-binding protein (MBP), thioredoxin protein (THX), and glutathione S-transferase (GST), that allow for the expression of recombinant proteins as fusion proteins in benign forms (Akhtar et al. ##REF##22675369##2012##; El-Gayar ##UREF##5##2015##; Gddoa Al-sahlany et al. ##REF##32168785##2020##; Mo et al. ##REF##30267582##2018##; Riguero et al. ##REF##32861092##2020##). Recently, our newly designed small metal-binding proteins SmbP and CusF3H+ have been exploited as protein tags which aided in the production and purification of AMPs like LL-37 and Bin1b, and green fluorescence protein (GFP) in different <italic>E. coli</italic> strains (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##; Vargas-Cortez et al. ##REF##26494603##2016##; Vargas-Cortez et al. ##REF##28087367##2017##). Our previous study has unraveled the capabilities of these protein tags that facilitate the secretion, folding, and purification of expressed recombinant LL-37 and Bin1b as single peptides with intact bioactivity at purity above 80% (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##; Santos et al. ##REF##30997666##2019##). Peptide hybridization has been considered an advanced technique for the design of novel AMPs having reliable peptide stability, long half-life with intact therapeutic activity with hybrid peptides like Cecropin A_Thanatin, and Indolicidin_Renalexin showing broad-spectrum antimicrobial activity against multidrug-resistant bacterial pathogens (Bayarbat et al. ##UREF##0##2016##; Seyedjavadi et al. ##REF##37744143##2021##; Wade et al. ##REF##31067436##2019##).</p>","<p id=\"Par26\">The broad-spectrum antibacterial activity of LL-37 as a single peptide although at a higher peptide concentration has led us to design a recombinant hybrid peptide production strategy via the application of GS flexible peptide linker and a carrier proteins CusF3H+ and SmbP. The simple GS peptide linker enhances the expression of the hybrid peptide LL-37_Renalexin and maintains the spatial configuration within the hybrid peptide with intact and advanced bioactivity. This data strongly suggests that the GS peptide linker can be employed as a reliable, simple, flexible linker for the design and expression of recombinant therapeutic peptides as compared to the RGGPDGSGPDESGPDE flexible linker employed in the design of hybrid and dimeric peptides with primary structural modifications (Klubthawee et al. ##REF##32499514##2020##; Seyedjavadi et al. ##REF##37744143##2021##).</p>","<p id=\"Par27\">In this study, we have efficiently employed the mature amino acids of LL-37 and Renalexin for the design of a novel hybrid peptide LL-37_Renalexin with zero cytotoxicity against healthy human cells. We reliably cloned the cDNA that encodes for the target peptide into pET30a+ under the T7 promoter and terminator regions and obtained a successful expression in <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) under the condition of 25 °C, for 16 h with 1 mM IPTG. In other studies, the microbial strains, induction, and temperature conditions demonstrated profound effect on the expression of both single, hybrid, and dimeric peptides with inclusion bodies indications (Chhetri et al. ##REF##26629417##2015##; Montfort-Gardeazabal et al. ##REF##33129981##2021##; Seyedjavadi et al. ##REF##37744143##2021##; Shang et al. ##REF##33042031##2020##; Wade et al. ##REF##31067436##2019##; Xu et al. ##REF##24945359##2014##). The expression condition and peptide isolation protocols observed in this study made it possible for efficient production coupled with higher yield.</p>","<p id=\"Par28\">Recombinant fusion peptides CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin expression level and its presence in soluble cell lysate provide an evidential advantage of protein tag CusF3H+ and SmbP over others like glutathione S-transferase, maltose-binding protein, amyloid-β peptide, and thioredoxin tag (Aleinein et al. ##REF##23053091##2013##; Chhetri et al. ##REF##26629417##2015##; Zhang et al. ##UREF##11##2018##) yielding up to 95% peptide purity which matched the purity standard of commercially available synthetic therapeutic peptides (Zhongxuan et al. ##REF##33255863##2020##). In this present study, Bradford quantification of purified and PBS-dialyzed protein elution fractions revealed a total recombinant peptide yield of 1.5–3.1 mg/L fusion proteins SmbP_LL-37_Renalexin and CusF3H+_LL-37_Renalexin expressed in BL21(DE3) and SHuffle T7(DE3), respectively. We observed a 2-fold higher peptide yield in <italic>E. coli</italic> SHuffle T7(DE3) for both SmbP and CusF3H+ tagged fusion proteins than in <italic>E. coli</italic> BL21(DE3), providing relevant supportive data on the usage of <italic>E. coli</italic> SHuffle T7(DE3) as microbial host for the production of either single or hybrid recombinant hybrid peptides with or without disulfide bonds (Montfort-Gardeazabal et al. ##REF##33129981##2021##). Our result showed a higher expression level in recombinant hybrid peptide as soluble protein than reported from other studies, 0.9 mg/L by Seyedjavadi et al. (##REF##37744143##2021##); 900 μg/L by Clement et al. (##REF##25587248##2015##), and 0.3 mg/L by Cheng et al. (##REF##34885732##2021##).</p>","<p id=\"Par29\">We employed the enzyme enterokinase for the selective cleavage of protein tags CusF3H+ and SmbP from the purified fusion peptides due to the presence of an enterokinase site between the hybrid peptide and the protein tag. Analysis of inactivated enterokinase cleavage mixture revealed three protein bands of an approximate molecular size of 18 kDa (uncleaved fusion peptide), 13 kDa (CusF3H+ or SmbP), and 10 kDa (LL-37_Renalexin, tag-free) on Tricine SDS-PAGE with tag-free recombinant hybrid peptide showing slightly higher band size than the theoretically expected size (6.740 kDa). This result can be attributed to the inefficient enterokinase cleavage observed, which may be associated with the presence of phenylalanine, leucine, and isoleucine residues and heptapeptide motif (Rana box) in Renalexin that forms a cyclic disulfide bond aiding a molecular structural loop formation folded unto the N-terminal that is known to influence enzymatic cleavage and peptide reduction. Also, the high hydrophobicity of the peptide which prevents complete reduction with SDS and mercaptoethanol reagents, and the peptide molecular folding in aqueous systems all of which influence poor peptide mobility. These findings agree with previous studies reported where the single peptides LL-37 and Renalexin show higher protein band sizes than the theoretically determined sizes (Aleinein et al. ##REF##23053091##2013##; Perez-Perez et al. ##REF##34680851##2021##). A 0.7–2.1 mg/L LL-37_Renalexin (tag-free) peptide was obtained upon spectroscopic and Bradford quantification of the second IMAC purification elution fractions (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##; Vargas-Cortez et al. ##REF##28087367##2017##).</p>","<p id=\"Par30\">The relatively slow induction of the antibacterial activity of LL-37_Renalexin compared to the known antibiotic kanamycin we observed in the time-killing kinetic assay results can be related to the high hydrophobicity of the hybrid peptide which may cause partial exposure of hydrophilic regions to bacterial membrane (Wei and Zhang ##UREF##10##2022##) coupled with the presence of monovalent Na(I) and K(I) cations in the assay medium that are known to cause shielding effects between the cationic peptides and anionic bacterial membrane surface, hence, the delay in eliciting antibacterial activity in the case of the hybrid peptide as compared to kanamycin (Huan et al. ##REF##33178164##2020##; Nuti et al. ##REF##28814242##2017##). Our findings show that the hybrid peptide LL-37_Renalexin with 44% hydrophobicity and 56% hydrophilicity elicited near-microbicidal activity against all tested pathogens with above 85% reduction in bacteria colony-forming units at 33 μM peptide concentration. The reduction in CFU/ml observed suggests a bactericidal activity since the level of reduction (<italic>R</italic><sub>L</sub>) in CFU/ml is more significant than three times the logarithm CFU/ml of bacteria (Klubthawee et al. ##REF##32499514##2020##; Zhang et al. ##UREF##11##2018##). All <italic>S. aureus</italic>, <italic>E. coli</italic>, MRSA, and <italic>K. pneumoniae</italic> clinical isolates showed 85% sensitivity at 33 μM as minimum peptide bactericidal concentration with about 25% increment in sensitivity indicative of higher antibacterial potency of this novel hybrid peptide compared to its counterpart single-peptide LL-37 as reported from our previous study with 64% sensitivity against <italic>E. coli</italic> and 69% against <italic>S. aureus</italic> (Perez-Perez et al. ##REF##34680851##2021##). We also observed approximately a 2-fold reduction with respect to the minimum inhibitory hybrid peptide concentration required to inhibit bacterial growth as compared to its single-peptide LL-37. We envisioned that the hybrid peptide’s antimicrobial effects are brought about through its ability to disrupt cell membranes, thanks to its pronounced cationic polarity which enables it to create a strong electrostatic bond with the negatively charged bacteria membrane. Additionally, it is thought that the hybrid peptide’s v-shaped structure, made possible by the presence of the GS flexible linker, allows it to effectively engage with bacterial chromosomal DNA. This interaction can lead to the formation of supercoils, ultimately hindering DNA replication and transcription (Zhang et al. ##REF##34496967##2021##).</p>","<p id=\"Par31\">In this study, we have designed, produced, and purified a novel multifunctional recombinant hybrid peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker and a characterized carrier proteins SmbP and CusF3H+. The small metal-binding protein tags SmbP and CusF3H+ provide an evidential advantage in cytoplasmic production and purification of the novel hybrid AMP LL-37_Renalexin with intact biochemical properties and can be applied as a new avenue to produce recombinant peptides and proteins. The purified tag-free hybrid peptide LL-37_Renalexin exhibited above 85% reduction in bacteria CFU/ml against <italic>S. aureus</italic>, <italic>E. coli</italic>, MRSA, and <italic>K. pneumoniae</italic> clinical isolates at lower minimum inhibition concentration levels of 10–33 μM as compared to its counterpart single-AMPs LL-37 and Renalexin of 50–100 μM reported (Aleinein et al. ##REF##23053091##2013##; Kang et al. ##REF##31170191##2019##; Perez-Perez et al. ##REF##34680851##2021##), making it a competitive antimicrobial agent.</p>","<p id=\"Par32\">From our antibacterial bioassay findings, we proposed that the newly designed hybrid peptide LL-37_Renalexin can be classified as an antibacterial peptide that may have no toxic effects against normal cells at the niche of infection as theoretically predicted. Previous investigations (Aleinein et al. ##REF##23053091##2013##; Jindal et al. ##REF##26046345##2015##; Kang et al. ##REF##31170191##2019##; Perez-Perez et al. ##REF##34680851##2021##) have confirmed the exclusive antibacterial effect of recombinant single-peptides LL-37 and Renalexin, respectively, showing no toxic effects against normal human cells. Notably, we successfully express the hybrid peptide in <italic>E. coli</italic> with intact bioactivity conferred by the conserved secondary α-helical structures as seen in the single peptides supporting the predicted secondary structure of LL-37_Renalexin clearly showing the conserved helical domains of LL-37 and Renalexin. It is imperative to note, however, that this study lacks empirical data from wet laboratory circular dichroism (CD) spectrometry and 3-(4,5-dimthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to support the theoretically predicted structure and toxicity. The recombinant DNA strategies used in the design, production, and purification of recombinant fusion proteins provide a reliable platform and protocol for the expression and purification of therapeutic recombinant proteins in <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) as microbial expression hosts.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par25\\\">Antimicrobial peptides (AMPs) as drug candidates are being considered the new hope for the biomedical and pharmaceutical industries in conjunction with their multifunctional antibacterial pharmacological actions against infectious agents (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Moretta et al. ##REF##34195099##2021##; Nuti et al. ##REF##28814242##2017##). The production of AMPs as therapeutic peptides via the applications of recombinant DNA technology (rDNA) and the use of cost-effective microbial expression systems have facilitated the large-scale acquisition of bioactive AMPs, enhancing clinical and research applications (Akhtar et al. ##REF##22675369##2012##; Dar et al. ##UREF##3##2017##). In addition to the successful application of rDNA, the efficient, fast, and easy growth of microbial expression hosts like <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) has allowed for commercial production of bioactive cationic AMPs independent of posttranslational modifications (Aleinein et al. ##REF##23053091##2013##; Klubthawee et al. ##REF##32499514##2020##; Montfort-Gardeazabal et al. ##REF##33129981##2021##). Novel AMPs like Brevinin-2R, Renalexin, Cecropin A, and LL-37 as single peptides have been produced as recombinant protein-based drug candidates in microbial systems and have shown a promising antimicrobial effect (Aleinein et al. ##REF##23053091##2013##; Chhetri et al. ##REF##26629417##2015##; Zhang et al. ##UREF##11##2018##). The thriving commercial production of AMPs in <italic>E. coli</italic> largely depends on the design and use of protein tags, including but not limited to poly-histidine, maltose-binding protein (MBP), thioredoxin protein (THX), and glutathione S-transferase (GST), that allow for the expression of recombinant proteins as fusion proteins in benign forms (Akhtar et al. ##REF##22675369##2012##; El-Gayar ##UREF##5##2015##; Gddoa Al-sahlany et al. ##REF##32168785##2020##; Mo et al. ##REF##30267582##2018##; Riguero et al. ##REF##32861092##2020##). Recently, our newly designed small metal-binding proteins SmbP and CusF3H+ have been exploited as protein tags which aided in the production and purification of AMPs like LL-37 and Bin1b, and green fluorescence protein (GFP) in different <italic>E. coli</italic> strains (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##; Vargas-Cortez et al. ##REF##26494603##2016##; Vargas-Cortez et al. ##REF##28087367##2017##). Our previous study has unraveled the capabilities of these protein tags that facilitate the secretion, folding, and purification of expressed recombinant LL-37 and Bin1b as single peptides with intact bioactivity at purity above 80% (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##; Santos et al. ##REF##30997666##2019##). Peptide hybridization has been considered an advanced technique for the design of novel AMPs having reliable peptide stability, long half-life with intact therapeutic activity with hybrid peptides like Cecropin A_Thanatin, and Indolicidin_Renalexin showing broad-spectrum antimicrobial activity against multidrug-resistant bacterial pathogens (Bayarbat et al. ##UREF##0##2016##; Seyedjavadi et al. ##REF##37744143##2021##; Wade et al. ##REF##31067436##2019##).</p>\",\n \"<p id=\\\"Par26\\\">The broad-spectrum antibacterial activity of LL-37 as a single peptide although at a higher peptide concentration has led us to design a recombinant hybrid peptide production strategy via the application of GS flexible peptide linker and a carrier proteins CusF3H+ and SmbP. The simple GS peptide linker enhances the expression of the hybrid peptide LL-37_Renalexin and maintains the spatial configuration within the hybrid peptide with intact and advanced bioactivity. This data strongly suggests that the GS peptide linker can be employed as a reliable, simple, flexible linker for the design and expression of recombinant therapeutic peptides as compared to the RGGPDGSGPDESGPDE flexible linker employed in the design of hybrid and dimeric peptides with primary structural modifications (Klubthawee et al. ##REF##32499514##2020##; Seyedjavadi et al. ##REF##37744143##2021##).</p>\",\n \"<p id=\\\"Par27\\\">In this study, we have efficiently employed the mature amino acids of LL-37 and Renalexin for the design of a novel hybrid peptide LL-37_Renalexin with zero cytotoxicity against healthy human cells. We reliably cloned the cDNA that encodes for the target peptide into pET30a+ under the T7 promoter and terminator regions and obtained a successful expression in <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) under the condition of 25 °C, for 16 h with 1 mM IPTG. In other studies, the microbial strains, induction, and temperature conditions demonstrated profound effect on the expression of both single, hybrid, and dimeric peptides with inclusion bodies indications (Chhetri et al. ##REF##26629417##2015##; Montfort-Gardeazabal et al. ##REF##33129981##2021##; Seyedjavadi et al. ##REF##37744143##2021##; Shang et al. ##REF##33042031##2020##; Wade et al. ##REF##31067436##2019##; Xu et al. ##REF##24945359##2014##). The expression condition and peptide isolation protocols observed in this study made it possible for efficient production coupled with higher yield.</p>\",\n \"<p id=\\\"Par28\\\">Recombinant fusion peptides CusF3H+_LL-37_Renalexin and SmbP_LL-37_Renalexin expression level and its presence in soluble cell lysate provide an evidential advantage of protein tag CusF3H+ and SmbP over others like glutathione S-transferase, maltose-binding protein, amyloid-β peptide, and thioredoxin tag (Aleinein et al. ##REF##23053091##2013##; Chhetri et al. ##REF##26629417##2015##; Zhang et al. ##UREF##11##2018##) yielding up to 95% peptide purity which matched the purity standard of commercially available synthetic therapeutic peptides (Zhongxuan et al. ##REF##33255863##2020##). In this present study, Bradford quantification of purified and PBS-dialyzed protein elution fractions revealed a total recombinant peptide yield of 1.5–3.1 mg/L fusion proteins SmbP_LL-37_Renalexin and CusF3H+_LL-37_Renalexin expressed in BL21(DE3) and SHuffle T7(DE3), respectively. We observed a 2-fold higher peptide yield in <italic>E. coli</italic> SHuffle T7(DE3) for both SmbP and CusF3H+ tagged fusion proteins than in <italic>E. coli</italic> BL21(DE3), providing relevant supportive data on the usage of <italic>E. coli</italic> SHuffle T7(DE3) as microbial host for the production of either single or hybrid recombinant hybrid peptides with or without disulfide bonds (Montfort-Gardeazabal et al. ##REF##33129981##2021##). Our result showed a higher expression level in recombinant hybrid peptide as soluble protein than reported from other studies, 0.9 mg/L by Seyedjavadi et al. (##REF##37744143##2021##); 900 μg/L by Clement et al. (##REF##25587248##2015##), and 0.3 mg/L by Cheng et al. (##REF##34885732##2021##).</p>\",\n \"<p id=\\\"Par29\\\">We employed the enzyme enterokinase for the selective cleavage of protein tags CusF3H+ and SmbP from the purified fusion peptides due to the presence of an enterokinase site between the hybrid peptide and the protein tag. Analysis of inactivated enterokinase cleavage mixture revealed three protein bands of an approximate molecular size of 18 kDa (uncleaved fusion peptide), 13 kDa (CusF3H+ or SmbP), and 10 kDa (LL-37_Renalexin, tag-free) on Tricine SDS-PAGE with tag-free recombinant hybrid peptide showing slightly higher band size than the theoretically expected size (6.740 kDa). This result can be attributed to the inefficient enterokinase cleavage observed, which may be associated with the presence of phenylalanine, leucine, and isoleucine residues and heptapeptide motif (Rana box) in Renalexin that forms a cyclic disulfide bond aiding a molecular structural loop formation folded unto the N-terminal that is known to influence enzymatic cleavage and peptide reduction. Also, the high hydrophobicity of the peptide which prevents complete reduction with SDS and mercaptoethanol reagents, and the peptide molecular folding in aqueous systems all of which influence poor peptide mobility. These findings agree with previous studies reported where the single peptides LL-37 and Renalexin show higher protein band sizes than the theoretically determined sizes (Aleinein et al. ##REF##23053091##2013##; Perez-Perez et al. ##REF##34680851##2021##). A 0.7–2.1 mg/L LL-37_Renalexin (tag-free) peptide was obtained upon spectroscopic and Bradford quantification of the second IMAC purification elution fractions (Montfort-Gardeazabal et al. ##REF##33129981##2021##; Perez-Perez et al. ##REF##34680851##2021##; Vargas-Cortez et al. ##REF##28087367##2017##).</p>\",\n \"<p id=\\\"Par30\\\">The relatively slow induction of the antibacterial activity of LL-37_Renalexin compared to the known antibiotic kanamycin we observed in the time-killing kinetic assay results can be related to the high hydrophobicity of the hybrid peptide which may cause partial exposure of hydrophilic regions to bacterial membrane (Wei and Zhang ##UREF##10##2022##) coupled with the presence of monovalent Na(I) and K(I) cations in the assay medium that are known to cause shielding effects between the cationic peptides and anionic bacterial membrane surface, hence, the delay in eliciting antibacterial activity in the case of the hybrid peptide as compared to kanamycin (Huan et al. ##REF##33178164##2020##; Nuti et al. ##REF##28814242##2017##). Our findings show that the hybrid peptide LL-37_Renalexin with 44% hydrophobicity and 56% hydrophilicity elicited near-microbicidal activity against all tested pathogens with above 85% reduction in bacteria colony-forming units at 33 μM peptide concentration. The reduction in CFU/ml observed suggests a bactericidal activity since the level of reduction (<italic>R</italic><sub>L</sub>) in CFU/ml is more significant than three times the logarithm CFU/ml of bacteria (Klubthawee et al. ##REF##32499514##2020##; Zhang et al. ##UREF##11##2018##). All <italic>S. aureus</italic>, <italic>E. coli</italic>, MRSA, and <italic>K. pneumoniae</italic> clinical isolates showed 85% sensitivity at 33 μM as minimum peptide bactericidal concentration with about 25% increment in sensitivity indicative of higher antibacterial potency of this novel hybrid peptide compared to its counterpart single-peptide LL-37 as reported from our previous study with 64% sensitivity against <italic>E. coli</italic> and 69% against <italic>S. aureus</italic> (Perez-Perez et al. ##REF##34680851##2021##). We also observed approximately a 2-fold reduction with respect to the minimum inhibitory hybrid peptide concentration required to inhibit bacterial growth as compared to its single-peptide LL-37. We envisioned that the hybrid peptide’s antimicrobial effects are brought about through its ability to disrupt cell membranes, thanks to its pronounced cationic polarity which enables it to create a strong electrostatic bond with the negatively charged bacteria membrane. Additionally, it is thought that the hybrid peptide’s v-shaped structure, made possible by the presence of the GS flexible linker, allows it to effectively engage with bacterial chromosomal DNA. This interaction can lead to the formation of supercoils, ultimately hindering DNA replication and transcription (Zhang et al. ##REF##34496967##2021##).</p>\",\n \"<p id=\\\"Par31\\\">In this study, we have designed, produced, and purified a novel multifunctional recombinant hybrid peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker and a characterized carrier proteins SmbP and CusF3H+. The small metal-binding protein tags SmbP and CusF3H+ provide an evidential advantage in cytoplasmic production and purification of the novel hybrid AMP LL-37_Renalexin with intact biochemical properties and can be applied as a new avenue to produce recombinant peptides and proteins. The purified tag-free hybrid peptide LL-37_Renalexin exhibited above 85% reduction in bacteria CFU/ml against <italic>S. aureus</italic>, <italic>E. coli</italic>, MRSA, and <italic>K. pneumoniae</italic> clinical isolates at lower minimum inhibition concentration levels of 10–33 μM as compared to its counterpart single-AMPs LL-37 and Renalexin of 50–100 μM reported (Aleinein et al. ##REF##23053091##2013##; Kang et al. ##REF##31170191##2019##; Perez-Perez et al. ##REF##34680851##2021##), making it a competitive antimicrobial agent.</p>\",\n \"<p id=\\\"Par32\\\">From our antibacterial bioassay findings, we proposed that the newly designed hybrid peptide LL-37_Renalexin can be classified as an antibacterial peptide that may have no toxic effects against normal cells at the niche of infection as theoretically predicted. Previous investigations (Aleinein et al. ##REF##23053091##2013##; Jindal et al. ##REF##26046345##2015##; Kang et al. ##REF##31170191##2019##; Perez-Perez et al. ##REF##34680851##2021##) have confirmed the exclusive antibacterial effect of recombinant single-peptides LL-37 and Renalexin, respectively, showing no toxic effects against normal human cells. Notably, we successfully express the hybrid peptide in <italic>E. coli</italic> with intact bioactivity conferred by the conserved secondary α-helical structures as seen in the single peptides supporting the predicted secondary structure of LL-37_Renalexin clearly showing the conserved helical domains of LL-37 and Renalexin. It is imperative to note, however, that this study lacks empirical data from wet laboratory circular dichroism (CD) spectrometry and 3-(4,5-dimthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to support the theoretically predicted structure and toxicity. The recombinant DNA strategies used in the design, production, and purification of recombinant fusion proteins provide a reliable platform and protocol for the expression and purification of therapeutic recombinant proteins in <italic>E. coli</italic> BL21(DE3) and <italic>E. coli</italic> SHuffle T7(DE3) as microbial expression hosts.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<title>Abstract</title>","<p id=\"Par1\">An alarming global public health and economic peril has been the emergence of antibiotic resistance resulting from clinically relevant bacteria pathogens, including <italic>Enterococcus faecium</italic>, <italic>Staphylococcus aureus</italic>, <italic>Klebsiella pneumonia</italic>, <italic>Acinetobacter baumannii</italic>, <italic>Pseudomonas aeruginosa</italic>, and <italic>Enterobacter</italic> species constantly exhibiting intrinsic and extrinsic resistance mechanisms against last-resort antibiotics like gentamycin, ciprofloxacin, tetracycline, colistin, and standard ampicillin prescription in clinical practices. The discovery and applications of antimicrobial peptides (AMPs) with antibacterial properties have been considered and proven as alternative antimicrobial agents to antibiotics. In this study, we have designed, produced, and purified a recombinant novel multifunctional hybrid antimicrobial peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker coupled with the use of our previously characterized small metal-binding proteins SmbP and CusF3H+ as carrier proteins that allow for an enhanced bacterial expression, using BL21(DE3) and SHuffle T7(DE3) <italic>Escherichia coli</italic> strains, and purification of the hybrid peptide via immobilized metal affinity chromatography. The purified tag-free LL-37_Renalexin hybrid peptide exhibited above 85% reduction in bacteria colony-forming units and broad-spectrum antimicrobial effects against <italic>Staphylococcus aureus</italic>, <italic>Escherichia coli</italic>, Methicillin-resistant <italic>Staphylococcus aureus</italic> (MRSA), and <italic>Klebsiella pneumoniae</italic> bacteria clinical isolates at a lower minimum inhibition concentration level (10–33 μM) as compared to its counterpart single-AMPs LL-37 and Renalexin (50–100 μM).</p>","<title>Key points</title>","<p id=\"Par2\">• <italic>The hybrid antimicrobial peptide LL-37_Renalexin has been designed using a GS linker.</italic></p>","<p id=\"Par3\">• <italic>The peptide was expressed with the carrier proteins SmbP and CusF3H+.</italic></p>","<p id=\"Par4\">• <italic>The hybrid peptide shows antibacterial potency against clinical bacterial isolates.</italic></p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00253-023-12887-5.</p>","<title>Keywords</title>"],"string":"[\n \"<title>Abstract</title>\",\n \"<p id=\\\"Par1\\\">An alarming global public health and economic peril has been the emergence of antibiotic resistance resulting from clinically relevant bacteria pathogens, including <italic>Enterococcus faecium</italic>, <italic>Staphylococcus aureus</italic>, <italic>Klebsiella pneumonia</italic>, <italic>Acinetobacter baumannii</italic>, <italic>Pseudomonas aeruginosa</italic>, and <italic>Enterobacter</italic> species constantly exhibiting intrinsic and extrinsic resistance mechanisms against last-resort antibiotics like gentamycin, ciprofloxacin, tetracycline, colistin, and standard ampicillin prescription in clinical practices. The discovery and applications of antimicrobial peptides (AMPs) with antibacterial properties have been considered and proven as alternative antimicrobial agents to antibiotics. In this study, we have designed, produced, and purified a recombinant novel multifunctional hybrid antimicrobial peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker coupled with the use of our previously characterized small metal-binding proteins SmbP and CusF3H+ as carrier proteins that allow for an enhanced bacterial expression, using BL21(DE3) and SHuffle T7(DE3) <italic>Escherichia coli</italic> strains, and purification of the hybrid peptide via immobilized metal affinity chromatography. The purified tag-free LL-37_Renalexin hybrid peptide exhibited above 85% reduction in bacteria colony-forming units and broad-spectrum antimicrobial effects against <italic>Staphylococcus aureus</italic>, <italic>Escherichia coli</italic>, Methicillin-resistant <italic>Staphylococcus aureus</italic> (MRSA), and <italic>Klebsiella pneumoniae</italic> bacteria clinical isolates at a lower minimum inhibition concentration level (10–33 μM) as compared to its counterpart single-AMPs LL-37 and Renalexin (50–100 μM).</p>\",\n \"<title>Key points</title>\",\n \"<p id=\\\"Par2\\\">• <italic>The hybrid antimicrobial peptide LL-37_Renalexin has been designed using a GS linker.</italic></p>\",\n \"<p id=\\\"Par3\\\">• <italic>The peptide was expressed with the carrier proteins SmbP and CusF3H+.</italic></p>\",\n \"<p id=\\\"Par4\\\">• <italic>The hybrid peptide shows antibacterial potency against clinical bacterial isolates.</italic></p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00253-023-12887-5.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>\n</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>\\n</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>We thank Mexico’s Consejo Nacional de Humanidades Ciencias y Tecnologias (CONAHCYT) for the financial support to the graduate student JKN.</p>","<title>Author contribution</title>","<p>JKN made the DNA constructs, performed the experiments, and wrote the manuscript. NGC-V provided the fully characterized <italic>S. aureus</italic>, <italic>E. coli</italic>, MRSA, and <italic>K. pneumoniae</italic> strains from his laboratory at the University Hospital and assisted in the evaluation of the antimicrobial assays for MRSA and <italic>K. pneumoniae</italic>. XZ edited the manuscript, designed the experiments, and proposed the research as Principal Investigator. All authors read and approved the final manuscript.</p>","<title>Funding</title>","<p>This work was funded by grant UANL-PAICYT-330-CN-2022 awarded to XZ.</p>","<title>Data availability</title>","<p>All data supporting the findings of this study are available within the paper and its supplementary information.</p>","<title>Declarations</title>","<title>Ethics approval and consent to participate</title>","<p id=\"Par33\">This in vitro research study does not involve human or animal models; hence, no ethical consent and approval were required.</p>","<title>Conflict of interest</title>","<p id=\"Par34\">The authors declare no competing interest.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>We thank Mexico’s Consejo Nacional de Humanidades Ciencias y Tecnologias (CONAHCYT) for the financial support to the graduate student JKN.</p>\",\n \"<title>Author contribution</title>\",\n \"<p>JKN made the DNA constructs, performed the experiments, and wrote the manuscript. NGC-V provided the fully characterized <italic>S. aureus</italic>, <italic>E. coli</italic>, MRSA, and <italic>K. pneumoniae</italic> strains from his laboratory at the University Hospital and assisted in the evaluation of the antimicrobial assays for MRSA and <italic>K. pneumoniae</italic>. XZ edited the manuscript, designed the experiments, and proposed the research as Principal Investigator. All authors read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>This work was funded by grant UANL-PAICYT-330-CN-2022 awarded to XZ.</p>\",\n \"<title>Data availability</title>\",\n \"<p>All data supporting the findings of this study are available within the paper and its supplementary information.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethics approval and consent to participate</title>\",\n \"<p id=\\\"Par33\\\">This in vitro research study does not involve human or animal models; hence, no ethical consent and approval were required.</p>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par34\\\">The authors declare no competing interest.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Amino acid sequence flowchart representation of the expression cassette encoding for the recombinant fusion peptides. <bold>A</bold> Expression cassette encoding for CusF3H+_LL-37_Renalexin. <bold>B</bold> Expression cassette encoding for SmbP_LL-37_Renalexin</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Design of the hybrid antimicrobial peptide LL-37_Renalexin. <bold>A</bold> Representation of expression cassette gene map for LL-37_Renalexin expression in <italic>E. coli</italic>. <bold>B</bold> 3D Riben molecular secondary structure predicted for the hybrid peptide LL-37_Renalexin showing the components including LL-37 (long peptide on left), the flexible GS peptide linker (midway), and Renalexin (short peptide on far right). The peptide structure was predicted in i-Tasser server and confirmed in Phyre2 server with good positive <italic>z</italic>-score (1.00) and <italic>c</italic>-score (−2.09) suggesting an efficient sequence alignment with good, modelled confidence level supporting the predicted structure</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Small-scale expression of recombinant fusion peptides in <italic>E. coli</italic>. <bold>A</bold> 15% SDS-PAGE analysis of CusF3H+_LL-37_Renalexin expressed in <italic>E. coli</italic> BL21(DE3): Lane 1—protein ladder; Lanes 2 and 3—soluble (SF) and insoluble (IF) fractions of untransformed control cells; Lanes 4 and 5—SF and IF of uninduced transformed cells; Lanes 6, 7, and 8—SF of induced transformed cells. <bold>B</bold> 15% SDS PAGE analysis of SmbP_LL-37_Renalexin expressed in <italic>E. coli</italic> BL21(DE3): Lane 1—protein ladder; Lanes 2 and 3—SF and IF of untransformed control cells; Lanes 4 and 5—SF and IF of uninduced transformed cells; Lane 6, 7, and 8—SF of induced transformed cells. <bold>C</bold> 15% SDS-PAGE analysis of CusF3H+_LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3): Lane 1—protein ladder; Lane 2—soluble fraction of uninduced transformed cells (control); Lanes 3, 4, and 5—SF of induced transformed cells</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Large-scale expression and IMAC purification of recombinant fusion peptides. <bold>A</bold> IMAC purification of SmbP_LL-37_Renalexin (17 kDa) expressed in <italic>E. coli</italic> BL21(DE3). A 15% SDS-PAGE analysis of elution fractions. Lane 1, protein marker; Lane 2, cell lysate; Lane 3, column flow-through; Lane 4–10, elution fractions. <bold>B</bold> IMAC purification of CusF3H+_LL-37_Renalexin (17 kDa) expressed in <italic>E. coli</italic> SHuffle T7. A 15% SDS PAGE analysis of the elution fractions. Lane 1, protein marker; Lane 2, cell lysate; Lane 3, column flow-through; Lane 4-10, elution fractions</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Enterokinase cleavage, tag removal, and second IMAC purification of tag-free LL-37_Renalexin analyzed on Tricine SDS-PAGE. <bold>A</bold> 18% Tricine gel—Lane 1, protein ladder; Lane 2, CusF3H+_LL-37_Renalexin (uncut) expressed in <italic>E. coli</italic> SHuffle T7(DE3); Lane 3, cut CusF3H+_LL-37_Renalexin (enterokinase mix). <bold>B</bold> 18% Tricine gel—Lane 1, protein ladder; Lane 2, SmbP_LL-37_Renalexin (uncut) expressed in <italic>E. coli</italic> BL21(DE3); Lane 3, cut SmbP_LL-37_Renalexin (enterokinase mix). <bold>C</bold> 15% Tricine gel—Lane 1, fusion peptide CusF3H+_LL-37_Renalexin (uncut); Lane 2, enerokinase mix (Protein tag and tag-free LL-37_Renalexin); Lane 3, second IMAC purified hybrid peptide LL-37_Renlexin (tag-free)</p></caption></fig>","<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Antimicrobial activity of purified recombinant hybrid AMP LL-37_Renalexin (tag-free) against 1 × 10<sup>5</sup> CFU/ml of bacteria pathogens. <bold>A</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3) against CFU/ml of <italic>S. aureus</italic>. <bold>B</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3) against CFU/ml of <italic>E. coli</italic>. <bold>C</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3) against CFU/ml of MRSA. <bold>D</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3) against CFU/ml of <italic>K</italic>. <italic>pneumoniae</italic>. <bold>E</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> BL21(DE3) against CFU/ml of <italic>S. aureus</italic>. <bold>F</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> BL21(DE3) against CFU/ml of <italic>E. coli</italic>. The data points represent the mean remaining CFU/ml of the test pathogen from three replica plates, and the error bar represents the standard deviation of the mean. Statistical analysis was performed using Gompertz sigmoid function for non-linear regression between the peptide concentration and the CFU/ml of test pathogen</p></caption></fig>","<fig id=\"Fig7\"><label>Fig. 7</label><caption><p>Time-killing kinetics of LL-37_Renalexin (tag-free) expressed in <italic>E. coli</italic> SHuffle T7(DE3) at 2X MIC against the log 1 × 10<sup>5</sup> CFU/ml of the test pathogens within 3 h time interval of treatment. <bold>A</bold> Time-kill assay of the hybrid peptide against <italic>S. aureus.</italic>\n<bold>B</bold> Time-kill assay of the hybrid peptide against <italic>E. coli</italic>. A 1 × PBS buffer (pH 7.2) and suspensions of bacteria inoculum were used as the negative control. Antibiotic kanamycin was employed as positive control. The data points represent the mean of log remaining CFU/ml of the test pathogens from three replica plates, and the error bars represent the standard deviation (SD) of the mean CFUs</p></caption></fig>","<fig id=\"Fig8\"><label>Fig. 8</label><caption><p>Antimicrobial activity of the hybrid peptide LL-37_Renalexin (tag-free) expressed in <italic>E. coli</italic> SHuffle T7(DE3) against 1 × 10<sup>5</sup> CFU/ml of test pathogens analyzed by one-way analysis of variance (ANOVA). <bold>A</bold> Antibacterial activity against the CFU/ml of <italic>S. aureus</italic>, <bold>B</bold> Antibacterial activity against the CFU/ml of <italic>E. coli</italic>. A 1 × PBS buffer (pH 7.2) and suspension of bacteria inoculum were used as negative control. The bars represent the mean remaining CFU/ml of the test pathogens from three replica plates and the error bars represent the standard deviation (SD) of the means. Asterisks indicate the statistical significance difference (all <italic>P</italic>-values < 0.05) between the peptide, the negative control, and kanamycin (positive control)</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Amino acid sequence flowchart representation of the expression cassette encoding for the recombinant fusion peptides. <bold>A</bold> Expression cassette encoding for CusF3H+_LL-37_Renalexin. <bold>B</bold> Expression cassette encoding for SmbP_LL-37_Renalexin</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Design of the hybrid antimicrobial peptide LL-37_Renalexin. <bold>A</bold> Representation of expression cassette gene map for LL-37_Renalexin expression in <italic>E. coli</italic>. <bold>B</bold> 3D Riben molecular secondary structure predicted for the hybrid peptide LL-37_Renalexin showing the components including LL-37 (long peptide on left), the flexible GS peptide linker (midway), and Renalexin (short peptide on far right). The peptide structure was predicted in i-Tasser server and confirmed in Phyre2 server with good positive <italic>z</italic>-score (1.00) and <italic>c</italic>-score (−2.09) suggesting an efficient sequence alignment with good, modelled confidence level supporting the predicted structure</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Small-scale expression of recombinant fusion peptides in <italic>E. coli</italic>. <bold>A</bold> 15% SDS-PAGE analysis of CusF3H+_LL-37_Renalexin expressed in <italic>E. coli</italic> BL21(DE3): Lane 1—protein ladder; Lanes 2 and 3—soluble (SF) and insoluble (IF) fractions of untransformed control cells; Lanes 4 and 5—SF and IF of uninduced transformed cells; Lanes 6, 7, and 8—SF of induced transformed cells. <bold>B</bold> 15% SDS PAGE analysis of SmbP_LL-37_Renalexin expressed in <italic>E. coli</italic> BL21(DE3): Lane 1—protein ladder; Lanes 2 and 3—SF and IF of untransformed control cells; Lanes 4 and 5—SF and IF of uninduced transformed cells; Lane 6, 7, and 8—SF of induced transformed cells. <bold>C</bold> 15% SDS-PAGE analysis of CusF3H+_LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3): Lane 1—protein ladder; Lane 2—soluble fraction of uninduced transformed cells (control); Lanes 3, 4, and 5—SF of induced transformed cells</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Large-scale expression and IMAC purification of recombinant fusion peptides. <bold>A</bold> IMAC purification of SmbP_LL-37_Renalexin (17 kDa) expressed in <italic>E. coli</italic> BL21(DE3). A 15% SDS-PAGE analysis of elution fractions. Lane 1, protein marker; Lane 2, cell lysate; Lane 3, column flow-through; Lane 4–10, elution fractions. <bold>B</bold> IMAC purification of CusF3H+_LL-37_Renalexin (17 kDa) expressed in <italic>E. coli</italic> SHuffle T7. A 15% SDS PAGE analysis of the elution fractions. Lane 1, protein marker; Lane 2, cell lysate; Lane 3, column flow-through; Lane 4-10, elution fractions</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><p>Enterokinase cleavage, tag removal, and second IMAC purification of tag-free LL-37_Renalexin analyzed on Tricine SDS-PAGE. <bold>A</bold> 18% Tricine gel—Lane 1, protein ladder; Lane 2, CusF3H+_LL-37_Renalexin (uncut) expressed in <italic>E. coli</italic> SHuffle T7(DE3); Lane 3, cut CusF3H+_LL-37_Renalexin (enterokinase mix). <bold>B</bold> 18% Tricine gel—Lane 1, protein ladder; Lane 2, SmbP_LL-37_Renalexin (uncut) expressed in <italic>E. coli</italic> BL21(DE3); Lane 3, cut SmbP_LL-37_Renalexin (enterokinase mix). <bold>C</bold> 15% Tricine gel—Lane 1, fusion peptide CusF3H+_LL-37_Renalexin (uncut); Lane 2, enerokinase mix (Protein tag and tag-free LL-37_Renalexin); Lane 3, second IMAC purified hybrid peptide LL-37_Renlexin (tag-free)</p></caption></fig>\",\n \"<fig id=\\\"Fig6\\\"><label>Fig. 6</label><caption><p>Antimicrobial activity of purified recombinant hybrid AMP LL-37_Renalexin (tag-free) against 1 × 10<sup>5</sup> CFU/ml of bacteria pathogens. <bold>A</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3) against CFU/ml of <italic>S. aureus</italic>. <bold>B</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3) against CFU/ml of <italic>E. coli</italic>. <bold>C</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3) against CFU/ml of MRSA. <bold>D</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> SHuffle T7(DE3) against CFU/ml of <italic>K</italic>. <italic>pneumoniae</italic>. <bold>E</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> BL21(DE3) against CFU/ml of <italic>S. aureus</italic>. <bold>F</bold> Dose-response activity of LL-37_Renalexin expressed in <italic>E. coli</italic> BL21(DE3) against CFU/ml of <italic>E. coli</italic>. The data points represent the mean remaining CFU/ml of the test pathogen from three replica plates, and the error bar represents the standard deviation of the mean. Statistical analysis was performed using Gompertz sigmoid function for non-linear regression between the peptide concentration and the CFU/ml of test pathogen</p></caption></fig>\",\n \"<fig id=\\\"Fig7\\\"><label>Fig. 7</label><caption><p>Time-killing kinetics of LL-37_Renalexin (tag-free) expressed in <italic>E. coli</italic> SHuffle T7(DE3) at 2X MIC against the log 1 × 10<sup>5</sup> CFU/ml of the test pathogens within 3 h time interval of treatment. <bold>A</bold> Time-kill assay of the hybrid peptide against <italic>S. aureus.</italic>\\n<bold>B</bold> Time-kill assay of the hybrid peptide against <italic>E. coli</italic>. A 1 × PBS buffer (pH 7.2) and suspensions of bacteria inoculum were used as the negative control. Antibiotic kanamycin was employed as positive control. The data points represent the mean of log remaining CFU/ml of the test pathogens from three replica plates, and the error bars represent the standard deviation (SD) of the mean CFUs</p></caption></fig>\",\n \"<fig id=\\\"Fig8\\\"><label>Fig. 8</label><caption><p>Antimicrobial activity of the hybrid peptide LL-37_Renalexin (tag-free) expressed in <italic>E. coli</italic> SHuffle T7(DE3) against 1 × 10<sup>5</sup> CFU/ml of test pathogens analyzed by one-way analysis of variance (ANOVA). <bold>A</bold> Antibacterial activity against the CFU/ml of <italic>S. aureus</italic>, <bold>B</bold> Antibacterial activity against the CFU/ml of <italic>E. coli</italic>. A 1 × PBS buffer (pH 7.2) and suspension of bacteria inoculum were used as negative control. The bars represent the mean remaining CFU/ml of the test pathogens from three replica plates and the error bars represent the standard deviation (SD) of the means. Asterisks indicate the statistical significance difference (all <italic>P</italic>-values < 0.05) between the peptide, the negative control, and kanamycin (positive control)</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>MICs of LL-37_Renalexin expressed in BL21(DE3) and SHuffle T7(DE3) <italic>E. coli</italic> strains</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\">Pathogens</th><th colspan=\"2\">MICs: LL-37_Renalexin (μM)</th></tr><tr><th><italic>E. coli</italic> BL21(DE3)</th><th><italic>E. coli</italic> SHuffle T7(DE3)</th></tr></thead><tbody><tr><td><italic>Staphylococcus aureus</italic></td><td>20.1</td><td>18.2</td></tr><tr><td><italic>Escherichia coli</italic></td><td>21.4</td><td>19.7</td></tr><tr><td>MRSA</td><td>N/A</td><td>17.5</td></tr><tr><td><italic>Klebsiella 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affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p><italic>N/A</italic> Not Analyzed (peptide expressed in <italic>E. coli</italic> SHuffle T7(DE3) was used for subsequent bioactivity)</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher’s note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"253_2023_12887_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"253_2023_12887_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"253_2023_12887_Fig3_HTML\" id=\"MO3\"/>","<graphic xlink:href=\"253_2023_12887_Fig4_HTML\" id=\"MO4\"/>","<graphic xlink:href=\"253_2023_12887_Fig5_HTML\" id=\"MO5\"/>","<graphic xlink:href=\"253_2023_12887_Fig6_HTML\" id=\"MO6\"/>","<graphic xlink:href=\"253_2023_12887_Fig7_HTML\" id=\"MO7\"/>","<graphic xlink:href=\"253_2023_12887_Fig8_HTML\" 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In the United States (US), it is estimated that by 2026, the number of cancer survivors will surpass 20 million, which can be attributed to the ongoing innovation of treatment and early disease detection [##REF##27253694##2##, ##REF##23678936##3##]. While this increase in survival is optimistic, this population requires additional healthcare services to prevent or manage chronic health conditions, sequelae of cancer treatment, and monitoring for cancer reoccurrence [##REF##23678936##3##]. Moreover, given that at least 50% of cancer survivors will experience physical and mental health consequences due to their disease or treatment [##REF##33033685##4##], is important to mitigate healthcare delays in this population.</p>","<p id=\"Par6\">Overall, healthcare delays (due to the domains of accessibility, financial burden, and social support) can significantly impact cancer survivors, most notably individuals who are ethnic minorities, lower SES, or uninsured [##REF##1899012##5##]. Cancer survivors who experience healthcare delays can significantly suffer the consequences as their care is typically time sensitive [##REF##33315115##6##]. Because timely cancer care is associated with a favorable prognosis, barriers or delays to treatment can result in a more advanced stage of cancer at the time of eventual care, thus, resulting in poorer outcomes [##REF##20564623##7##]. However, physical health is not the only aspect affected; mental health may also suffer at the hands of delayed care [##REF##35393637##8##]. For instance, COVID-19 led to numerous appointment cancelations for cancer survivor patients, including self-cancelations that were caused by depression and anxiety symptoms surrounding the pandemic and safe access to care [##REF##36209390##9##]. These missed appointments, in turn, exacerbated patients’ fears of cancer recurrence as their follow-up care were halted (e.g., laboratory testing, imaging, and appointments), impacting their overall well-being and physical and mental health [##REF##33624572##10##, ##REF##32772225##11##].</p>","<p id=\"Par7\">One factor associated with increases in healthcare delays among cancer survivors is lower SES status, which is associated with all-cause mortality risk, poorer mental (e.g., depression), and physical health outcomes [##REF##35448185##12##, ##REF##28408935##13##]. Similarly, SES has been linked to a range of cancer outcomes and higher SES (suggesting more financial resources and the ability to afford medical care) is associated with decreased in the length of healthcare delays [##REF##25591711##14##]. Because of the long-term and specialized care needed for cancer survivors, they are at risk of experiencing higher financial burden, which in turn, impacts receipt of survivorship care and increases the risk of mortality, and worsens quality of life [##REF##29596618##15##].</p>","<p id=\"Par8\">A second factor associated with healthcare delays among cancer survivors is a lack of health literacy, which presents a barrier in properly understanding, communicating, and obtaining information required to navigate the complexity of the healthcare systems efficiently to obtain the care needed and to make educated health decisions [##REF##33494122##16##, ##REF##33938484##17##]. Among the general population, low health literacy has been positively associated with various care delays, including seeking treatment, forgoing care, and struggling with accessibility to needed care and providers [##REF##27043757##18##]. As cancer survivors have complex healthcare needs, mastering the skills required for their continual care is important.</p>","<p id=\"Par9\">Being foreign-born (immigrant) brings an additional barrier to accessing healthcare and consequently promotes healthcare delays [##REF##33390160##19##]. These barriers among foreign-born may be partially attributed to a higher likelihood of lack of insurance, healthcare cultural perception, and English skills proficiency [##REF##21841297##20##]. Evidence suggests that cancer survivors who are immigrants have a lower quality of life and higher depression symptomology compared to native-born [##REF##23465493##21##]. Moreover, foreign-born individuals may experience unique barriers (e.g., language, discrimination, laws and regulations to qualify for services) that impact the quality of care they can receive. For instance, language barriers and insurance difficulties caused by laws and policies may make it much more difficult for an immigrant patient than a US-born patient to receive adequate needed care [##REF##26586971##22##].</p>","<p id=\"Par10\">While it is well established that SES barriers and a lack of health literacy are associated with healthcare delays, there is limited and inconsistent knowledge of how these associations differ by nativity. To guide the selection of our variables for our model, we used the theoretical framework from Wafula and Snipes for barriers to healthcare among Black immigrants in the US [##REF##24006174##23##]. Additionally, we adapted their framework to focus on assessing the association between the barrier factors (i.e., SES barriers, health literacy) and general healthcare delays among cancer survivors and tested the moderating effects of nativity between SES barriers and health literacy with general healthcare delays (Fig. ##FIG##0##1##). Thus, this study aims to contribute to the current literature by examining whether nativity status modifies the relationship between a combination of SES and health literacy barriers, and healthcare delays in a large national cohort.</p>"],"string":"[\n \"<title>Background</title>\",\n \"<p id=\\\"Par5\\\">According to the National Cancer Institute (NCI), an individual is considered a cancer survivor from diagnosis to the end of life [##UREF##0##1##]. In the United States (US), it is estimated that by 2026, the number of cancer survivors will surpass 20 million, which can be attributed to the ongoing innovation of treatment and early disease detection [##REF##27253694##2##, ##REF##23678936##3##]. While this increase in survival is optimistic, this population requires additional healthcare services to prevent or manage chronic health conditions, sequelae of cancer treatment, and monitoring for cancer reoccurrence [##REF##23678936##3##]. Moreover, given that at least 50% of cancer survivors will experience physical and mental health consequences due to their disease or treatment [##REF##33033685##4##], is important to mitigate healthcare delays in this population.</p>\",\n \"<p id=\\\"Par6\\\">Overall, healthcare delays (due to the domains of accessibility, financial burden, and social support) can significantly impact cancer survivors, most notably individuals who are ethnic minorities, lower SES, or uninsured [##REF##1899012##5##]. Cancer survivors who experience healthcare delays can significantly suffer the consequences as their care is typically time sensitive [##REF##33315115##6##]. Because timely cancer care is associated with a favorable prognosis, barriers or delays to treatment can result in a more advanced stage of cancer at the time of eventual care, thus, resulting in poorer outcomes [##REF##20564623##7##]. However, physical health is not the only aspect affected; mental health may also suffer at the hands of delayed care [##REF##35393637##8##]. For instance, COVID-19 led to numerous appointment cancelations for cancer survivor patients, including self-cancelations that were caused by depression and anxiety symptoms surrounding the pandemic and safe access to care [##REF##36209390##9##]. These missed appointments, in turn, exacerbated patients’ fears of cancer recurrence as their follow-up care were halted (e.g., laboratory testing, imaging, and appointments), impacting their overall well-being and physical and mental health [##REF##33624572##10##, ##REF##32772225##11##].</p>\",\n \"<p id=\\\"Par7\\\">One factor associated with increases in healthcare delays among cancer survivors is lower SES status, which is associated with all-cause mortality risk, poorer mental (e.g., depression), and physical health outcomes [##REF##35448185##12##, ##REF##28408935##13##]. Similarly, SES has been linked to a range of cancer outcomes and higher SES (suggesting more financial resources and the ability to afford medical care) is associated with decreased in the length of healthcare delays [##REF##25591711##14##]. Because of the long-term and specialized care needed for cancer survivors, they are at risk of experiencing higher financial burden, which in turn, impacts receipt of survivorship care and increases the risk of mortality, and worsens quality of life [##REF##29596618##15##].</p>\",\n \"<p id=\\\"Par8\\\">A second factor associated with healthcare delays among cancer survivors is a lack of health literacy, which presents a barrier in properly understanding, communicating, and obtaining information required to navigate the complexity of the healthcare systems efficiently to obtain the care needed and to make educated health decisions [##REF##33494122##16##, ##REF##33938484##17##]. Among the general population, low health literacy has been positively associated with various care delays, including seeking treatment, forgoing care, and struggling with accessibility to needed care and providers [##REF##27043757##18##]. As cancer survivors have complex healthcare needs, mastering the skills required for their continual care is important.</p>\",\n \"<p id=\\\"Par9\\\">Being foreign-born (immigrant) brings an additional barrier to accessing healthcare and consequently promotes healthcare delays [##REF##33390160##19##]. These barriers among foreign-born may be partially attributed to a higher likelihood of lack of insurance, healthcare cultural perception, and English skills proficiency [##REF##21841297##20##]. Evidence suggests that cancer survivors who are immigrants have a lower quality of life and higher depression symptomology compared to native-born [##REF##23465493##21##]. Moreover, foreign-born individuals may experience unique barriers (e.g., language, discrimination, laws and regulations to qualify for services) that impact the quality of care they can receive. For instance, language barriers and insurance difficulties caused by laws and policies may make it much more difficult for an immigrant patient than a US-born patient to receive adequate needed care [##REF##26586971##22##].</p>\",\n \"<p id=\\\"Par10\\\">While it is well established that SES barriers and a lack of health literacy are associated with healthcare delays, there is limited and inconsistent knowledge of how these associations differ by nativity. To guide the selection of our variables for our model, we used the theoretical framework from Wafula and Snipes for barriers to healthcare among Black immigrants in the US [##REF##24006174##23##]. Additionally, we adapted their framework to focus on assessing the association between the barrier factors (i.e., SES barriers, health literacy) and general healthcare delays among cancer survivors and tested the moderating effects of nativity between SES barriers and health literacy with general healthcare delays (Fig. ##FIG##0##1##). Thus, this study aims to contribute to the current literature by examining whether nativity status modifies the relationship between a combination of SES and health literacy barriers, and healthcare delays in a large national cohort.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<title>Data collection and sample</title>","<p id=\"Par11\">Cross-sectional data for this study were obtained from the “All of Us” research program collected by online survey between May 2018 and April 2021. Briefly, this program is open to individuals who are 18 and over and are living in the US. Participants signed a consent form following the Declaration of Helsinki for data collection. The participants’ data used are de-identified and available to approved researchers. The All of Us program was approved by the National Institutes of Health (NIH) Institutional Review Board (IRB).</p>","<p id=\"Par12\">In this study, cancer survivors were defined as those participants who indicated that they had ever been diagnosed with cancer. Inclusion criteria for our cohort included participants who were ever told by their healthcare provider that had/have cancer. Skin cancer is one of the most prevalent cancers in the US with most cases being reasonably benign basal cells, not often tracked on most cancer registries, and having over 90% 5-year survival rate [##REF##28885696##24##]. In addition, previous studies have excluded these cancer survivors as their follow-up care is often reasonably minor [##REF##33942535##25##]. Thus, we excluded those with skin cancer and participants with missing data on the healthcare delay survey questions.</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<title>Data collection and sample</title>\",\n \"<p id=\\\"Par11\\\">Cross-sectional data for this study were obtained from the “All of Us” research program collected by online survey between May 2018 and April 2021. Briefly, this program is open to individuals who are 18 and over and are living in the US. Participants signed a consent form following the Declaration of Helsinki for data collection. The participants’ data used are de-identified and available to approved researchers. The All of Us program was approved by the National Institutes of Health (NIH) Institutional Review Board (IRB).</p>\",\n \"<p id=\\\"Par12\\\">In this study, cancer survivors were defined as those participants who indicated that they had ever been diagnosed with cancer. Inclusion criteria for our cohort included participants who were ever told by their healthcare provider that had/have cancer. Skin cancer is one of the most prevalent cancers in the US with most cases being reasonably benign basal cells, not often tracked on most cancer registries, and having over 90% 5-year survival rate [##REF##28885696##24##]. In addition, previous studies have excluded these cancer survivors as their follow-up care is often reasonably minor [##REF##33942535##25##]. Thus, we excluded those with skin cancer and participants with missing data on the healthcare delay survey questions.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par18\">The median age of the study population (<italic>n</italic> = 10,020) was approximately 64 (interquartile range [IQR Q1, Q3] 55.5, 71.8) years. The majority of participants were female (66.1%), US-born (92%), and self-identified as White (82.3%). There was a higher distribution of females vs males and other sex, foreign-born vs US-born, and Black cancer survivors vs all other race/ethnicity categories that had three or more SES barriers (Table ##TAB##0##1##). While a higher proportion of females vs males and other sex, foreign-born vs US-born, and Hispanic cancer survivors vs all other race/ethnicity cancer survivors had one or more healthcare delays (Table ##TAB##1##2##).</p>","<p id=\"Par19\">Results from the multivariable-adjusted model showed that neither nativity (OR 1.04, 95% CI [0.87, 1.25]) nor health literacy (OR 1.20, 95% CI [0.89, 1.59]) were statistically significantly associated with healthcare delays (see Table ##TAB##2##3##). However, when assessing for a p-trend for health literacy, for every one-unit increase in health literacy there was an 8% (OR 0.92, 95% CI 0.89, 0.95) decrease in the likelihood to experience healthcare delays. Furthermore, compared to those who did not have any SES barriers, those who reported two or three or more were 65% (OR 1.65, 95% CI [1.43, 1.90]) and 118% (OR 2.18, 95% CI [1.84, 2.58]) more likely to experience delays in healthcare, respectively. In addition, using SES barriers as a continuous measure to test for a p-trend, we found that for every one additional barrier increase, there was a 29% increase (OR 1.29, 95% CI [1.23, 1.36]) in the likelihood of experiencing healthcare delays. The association between SES barriers and healthcare delays differs by nativity status (<italic>p</italic><sub>interaction</sub> = 0.02). The stratified model by nativity showed that among those who were foreign-born, those who experienced two or three or more SES barriers were almost three times (OR 4.35, 95% CI [2.61, 7.34] vs OR 1.53, 95% CI [1.32, 1.78]) and two times (OR 3.83, 95% CI [2.14, 6.98] vs OR 2.10, 95% CI [1.76, 2.50]) as likely to experience healthcare delays as their US-born counterparts who experienced the same levels of SES barriers, respectively (see Table ##TAB##2##3##).</p>","<p id=\"Par20\">Assessing for p-trend in the stratified model by nativity, we found that for every additional SES barrier experienced among foreign-born individuals, they were 72% (OR 1.72, 95% CI [1.43, 2.08] vs OR 1.27, 95% CI [1.21, 1.34]) more likely to experience healthcare delays compared to their US counterparts. Finally, in the stratified model, low health literacy was associated with a 41% (OR 1.41, 95% CI [1.02, 1.97]) increase in the likelihood of healthcare delays, and each one-point increase in health literacy score was associated with a 9% (OR 0.91, 95% CI [0.88, 0.94]) decrease in the odds of healthcare delays among US-born cancer survivors. While for foreign-born cancer survivors, low health literacy was not statistically significantly associated with experiencing healthcare delays (OR 0.66, 95% CI [0.34, 1.25]), and for every one-unit increase in health literacy score there was a 2% (OR 0.98, 95% CI [0.90, 1.07]) decrease in the odds of healthcare delays. Although this association did not reach statistical significance (see Table ##TAB##2##3##).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par18\\\">The median age of the study population (<italic>n</italic> = 10,020) was approximately 64 (interquartile range [IQR Q1, Q3] 55.5, 71.8) years. The majority of participants were female (66.1%), US-born (92%), and self-identified as White (82.3%). There was a higher distribution of females vs males and other sex, foreign-born vs US-born, and Black cancer survivors vs all other race/ethnicity categories that had three or more SES barriers (Table ##TAB##0##1##). While a higher proportion of females vs males and other sex, foreign-born vs US-born, and Hispanic cancer survivors vs all other race/ethnicity cancer survivors had one or more healthcare delays (Table ##TAB##1##2##).</p>\",\n \"<p id=\\\"Par19\\\">Results from the multivariable-adjusted model showed that neither nativity (OR 1.04, 95% CI [0.87, 1.25]) nor health literacy (OR 1.20, 95% CI [0.89, 1.59]) were statistically significantly associated with healthcare delays (see Table ##TAB##2##3##). However, when assessing for a p-trend for health literacy, for every one-unit increase in health literacy there was an 8% (OR 0.92, 95% CI 0.89, 0.95) decrease in the likelihood to experience healthcare delays. Furthermore, compared to those who did not have any SES barriers, those who reported two or three or more were 65% (OR 1.65, 95% CI [1.43, 1.90]) and 118% (OR 2.18, 95% CI [1.84, 2.58]) more likely to experience delays in healthcare, respectively. In addition, using SES barriers as a continuous measure to test for a p-trend, we found that for every one additional barrier increase, there was a 29% increase (OR 1.29, 95% CI [1.23, 1.36]) in the likelihood of experiencing healthcare delays. The association between SES barriers and healthcare delays differs by nativity status (<italic>p</italic><sub>interaction</sub> = 0.02). The stratified model by nativity showed that among those who were foreign-born, those who experienced two or three or more SES barriers were almost three times (OR 4.35, 95% CI [2.61, 7.34] vs OR 1.53, 95% CI [1.32, 1.78]) and two times (OR 3.83, 95% CI [2.14, 6.98] vs OR 2.10, 95% CI [1.76, 2.50]) as likely to experience healthcare delays as their US-born counterparts who experienced the same levels of SES barriers, respectively (see Table ##TAB##2##3##).</p>\",\n \"<p id=\\\"Par20\\\">Assessing for p-trend in the stratified model by nativity, we found that for every additional SES barrier experienced among foreign-born individuals, they were 72% (OR 1.72, 95% CI [1.43, 2.08] vs OR 1.27, 95% CI [1.21, 1.34]) more likely to experience healthcare delays compared to their US counterparts. Finally, in the stratified model, low health literacy was associated with a 41% (OR 1.41, 95% CI [1.02, 1.97]) increase in the likelihood of healthcare delays, and each one-point increase in health literacy score was associated with a 9% (OR 0.91, 95% CI [0.88, 0.94]) decrease in the odds of healthcare delays among US-born cancer survivors. While for foreign-born cancer survivors, low health literacy was not statistically significantly associated with experiencing healthcare delays (OR 0.66, 95% CI [0.34, 1.25]), and for every one-unit increase in health literacy score there was a 2% (OR 0.98, 95% CI [0.90, 1.07]) decrease in the odds of healthcare delays. Although this association did not reach statistical significance (see Table ##TAB##2##3##).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par21\">Using data from the All of Us research cohort, we aimed to investigate the associations between SES barriers and health literacy with healthcare delays. We also explored whether there were any differences in these associations by nativity. We found that among all cancer survivors, health literacy (binary) and nativity were not statistically significantly associated with healthcare delays. We also found that experiencing 2 or 3+ SES barriers was significantly associated with an increased likelihood of healthcare delays. Further, at equal levels of SES barriers, foreign-born individuals had significantly higher odds of healthcare delays when compared to US-born individuals. Lastly, in our separate models by nativity status assessing for a trend, we found that health literacy was inversely associated with healthcare delays among US-born cancer survivors only.</p>","<title>Socioeconomic barriers</title>","<p id=\"Par22\">Our multivariable model suggested that cancer survivors who experienced more than one SES barrier experienced an increase in the likelihood of healthcare delays compared to cancer survivors who experienced no SES barriers. These findings are consistent with previous research that found that low SES cancer survivors are more likely to not receive appropriate follow-up care [##REF##27006193##30##] and that those who experience financial, housing, and employment barriers have a greater likelihood of delaying needed care [##REF##35559872##31##–##REF##22412136##33##]. Similarly, in another study, cancer survivors from low SES who reported lower income and education levels were less likely to have follow-up care discussions with their medical providers [##REF##27006193##30##]. This lack of follow-up discussions can potentially contribute to prolonged delays in preventative care. Regarding education, in a previous study by Gonzalez and colleagues, cancer survivors who had a college degree or higher were more likely to have higher access to care but experienced more delays than those with less than or equal to a high school diploma [##REF##35576025##34##]. Perhaps belonging to a higher educational level improves health literacy, enabling cancer survivors to adequately make an informed decision when seeking the appropriate care needed.</p>","<p id=\"Par23\">Among cancer survivors, those with low SES barriers are more likely to delay medical care, preventive care (dental and vision care), and not fill prescription medications due to cost-related concerns [##REF##23907958##32##, ##REF##33043464##35##, ##REF##20549763##36##]. Furthermore, as cancer survivors are met with the unexpected financial costs of cancer treatment, they may worry about struggling to meet their housing and household bills payments, food insecurities, and retirement [##UREF##2##37##], thus, potentially forgoing or delaying the crucial care they are required to enhance their survival. Similarly, housing insecurities are linked with negative health outcomes and poor access and quality of healthcare [##REF##35559872##31##, ##REF##29299816##38##]. Lastly, the impact of modifiable factors such as education and SES are inversely associated with experiencing more unmet healthcare needs [##REF##15836548##39##] and a lack of health insurance [##REF##15836548##39##, ##REF##28766209##40##]. Hence, uninsured cancer survivors have a higher risk for comorbidities, bearing a greater mortality risk than uninsured non-cancer survivors [##REF##30461610##41##].</p>","<title>Nativity differences</title>","<p id=\"Par24\">Previous research among cancer survivors has shown nativity to be a factor in cost-related barriers among US-born Hispanic cancer survivors [##REF##24904178##42##]. In our study, we found that foreign-born individuals who experienced the same level of SES barriers had higher likelihood of experiencing healthcare delays than their native-born counterparts when compared to those who experienced no SES barriers. Previous research that explored nativity differences among cancer survivors is limited and inconsistent. For example, although the results did not reach statistical significance, Diamant and colleagues reported the opposite in a sample of non-cancer survivors, showing that non-native-born were less likely to report healthcare delays compared to native-born individuals [##REF##15117701##43##]. Whereas, in a study of female cancer survivors that assessed disparities in healthcare access and utilization, they found that non-US-born females were less likely to report having a routine place to go to meet their healthcare compared to US-born cancer survivors [##REF##34661881##44##]. This is important, as not having a primary healthcare office to seek care or have routine services can promote delaying accessing the extended care cancer survivors need. In addition, a higher prevalence of sociodemographic and SES barriers (e.g., income, education) was found among foreign-born individuals than among native-born in two North American countries (US and Canada), and disparities in healthcare access were higher among foreign- compared to native-born [##REF##21841297##20##]. Thus, supporting the role of SES barriers among foreign-born individuals.</p>","<title>Health literacy</title>","<p id=\"Par25\">Our study found that, after adjusting for confounding sociodemographic, nativity, and SES barriers, health literacy was not statistically significantly associated with healthcare delays in our entire study cohort. However, we found that solely among US-born participants, limited health literacy was associated with an increased likelihood of healthcare delays compared to adequate health literacy. We also saw a statistically significant monotonic relationship between increased health literacy scores and decreased odds of healthcare delays among US-born. While our findings only reached statistical significance among US-born cancer survivors, the magnitude of our findings was in the same direction among foreign-born cancer survivors. This indicates that regardless of nativity, increasing health literacy could mitigate the impact of healthcare delays. While we controlled for nativity, sociodemographic, and SES barriers, our findings suggest that health literacy could also be associated with cultural differences and language that we were not able to control in our study. For example, cancer survivors with low health literacy may suffer difficulties when trying to decode their symptoms or understand their diagnoses through communication with providers, which could result in healthcare delays and a later stage of disease at diagnosis [##REF##12018928##45##]. In the case of cancer survivors, there is a great need for complex health services after they have completed their primary treatment for their illness and subsequent management of their health [##REF##30561774##46##]. These deficiencies in communicating efficiently with their providers could increase the risk of healthcare delays.</p>","<title>Strengths and limitations</title>","<p id=\"Par26\">An important strength of this study is using data from the All of Us research program, as it has a high proportion of enrolled underrepresented minority populations, which increases our sample size and access to geographically and ethnically diverse populations. Similarly, we were able to analyze a relatively large sample with complete socioeconomic and sociodemographic data of foreign-born cancer survivors, which helps to expand the current cancer survivor, cancer health disparities, and health disparities research. Lastly, our results can be generalized to cancer survivors and individuals with similar characteristics and settings that experienced similar SES barriers in the US.</p>","<p id=\"Par27\">This study is not without limitations. The cross-sectional nature of the design does not allow us to establish a temporal or causal relationship. There is potential for misclassification for some of the factors included in our main independent variables as this data is from self-reported questionnaires. Our sample was also comprised of a higher distribution of individuals with a college degree or higher and a higher income. We were unable able to assess for acculturation; thus, future studies should account for it as it can be a potential confounder in these associations. Using a complete case (CC) analysis method may have introduced bias to our results. We addressed this concern by conducting a sensitivity analysis using multiple imputation (MI) method for our outcome variable. This analysis revealed that there was a slight overestimation in the relationship between SES barriers and healthcare delays among foreign-born cancer survivors, compared to their US-born counterparts with the same level of SES barriers. Despite the small differences observed in the effect estimates, the overall trend and interpretation of the results remained consistent across both the MI and CC analyses (Supplemental Table 3).</p>","<p id=\"Par28\">Although the All of Us collects data nationwide, these results cannot be generalizable to all cancer survivors in the United States. A clear example of this limitation can be seen in that our sample reported roughly 90% some college or higher degree, whereas in 2021, those who had completed some college, or more were approximately 63% in the general US population [##UREF##3##47##]. Lastly, an additional limitation is that during our study period, the COVID-19 pandemic may have worsened SES barriers and healthcare delays that may have contributed to our findings and may need further exploring.</p>","<title>Implications and future direction</title>","<p id=\"Par29\">Our results can help guide policymakers to promote the development of policies that aim at eliminating SES barriers. For example, many of the variables that are part of our SES index are system-modifiable factors. Implementation of laws that make education equitable, job creation and training, housing affordability, and universal healthcare are ways in which policies can aid in mitigating these SES barriers. At the healthcare system level, practitioners and systems should recognize that these SES barriers exist and promote solutions. For example, systems can offer transportation services to those who are experiencing SES barriers to lessen healthcare delays [##REF##34723394##48##]. Similarly, providing adult- and childcare services can help avoid delays in seeking care [##REF##36261213##49##].</p>","<p id=\"Par30\">As the All of Us continues to enroll participants and participants complete all surveys, future studies should reassess this association to determine if our findings remain true. Moreover, in future analyses, it is important to consider adjusting for acculturation, as well as other types of stressors such as discrimination, as these experiences can contribute to healthcare delays. Additionally, future studies should aim to explore racial differences between US-born and foreign-born cancer survivors. It is crucial to recognize that races and ethnicities such as Black, Hispanic, and Asian are heterogeneous, varying across cultural and socioeconomic aspects. Thus, understanding the SES barriers associated with healthcare delays among US-born ethnic minorities from different racial and ethnic backgrounds (e.g., Mexican-US-born, Guatemalan-US-born, Chinese-US-born, Nigerian-US-born) compared to foreign-born counterparts (e.g., Mexican-foreign-born, Guatemalan-foreign-born, Chinese-foreign-born, Nigerian-foreign-born) is of extreme importance.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par21\\\">Using data from the All of Us research cohort, we aimed to investigate the associations between SES barriers and health literacy with healthcare delays. We also explored whether there were any differences in these associations by nativity. We found that among all cancer survivors, health literacy (binary) and nativity were not statistically significantly associated with healthcare delays. We also found that experiencing 2 or 3+ SES barriers was significantly associated with an increased likelihood of healthcare delays. Further, at equal levels of SES barriers, foreign-born individuals had significantly higher odds of healthcare delays when compared to US-born individuals. Lastly, in our separate models by nativity status assessing for a trend, we found that health literacy was inversely associated with healthcare delays among US-born cancer survivors only.</p>\",\n \"<title>Socioeconomic barriers</title>\",\n \"<p id=\\\"Par22\\\">Our multivariable model suggested that cancer survivors who experienced more than one SES barrier experienced an increase in the likelihood of healthcare delays compared to cancer survivors who experienced no SES barriers. These findings are consistent with previous research that found that low SES cancer survivors are more likely to not receive appropriate follow-up care [##REF##27006193##30##] and that those who experience financial, housing, and employment barriers have a greater likelihood of delaying needed care [##REF##35559872##31##–##REF##22412136##33##]. Similarly, in another study, cancer survivors from low SES who reported lower income and education levels were less likely to have follow-up care discussions with their medical providers [##REF##27006193##30##]. This lack of follow-up discussions can potentially contribute to prolonged delays in preventative care. Regarding education, in a previous study by Gonzalez and colleagues, cancer survivors who had a college degree or higher were more likely to have higher access to care but experienced more delays than those with less than or equal to a high school diploma [##REF##35576025##34##]. Perhaps belonging to a higher educational level improves health literacy, enabling cancer survivors to adequately make an informed decision when seeking the appropriate care needed.</p>\",\n \"<p id=\\\"Par23\\\">Among cancer survivors, those with low SES barriers are more likely to delay medical care, preventive care (dental and vision care), and not fill prescription medications due to cost-related concerns [##REF##23907958##32##, ##REF##33043464##35##, ##REF##20549763##36##]. Furthermore, as cancer survivors are met with the unexpected financial costs of cancer treatment, they may worry about struggling to meet their housing and household bills payments, food insecurities, and retirement [##UREF##2##37##], thus, potentially forgoing or delaying the crucial care they are required to enhance their survival. Similarly, housing insecurities are linked with negative health outcomes and poor access and quality of healthcare [##REF##35559872##31##, ##REF##29299816##38##]. Lastly, the impact of modifiable factors such as education and SES are inversely associated with experiencing more unmet healthcare needs [##REF##15836548##39##] and a lack of health insurance [##REF##15836548##39##, ##REF##28766209##40##]. Hence, uninsured cancer survivors have a higher risk for comorbidities, bearing a greater mortality risk than uninsured non-cancer survivors [##REF##30461610##41##].</p>\",\n \"<title>Nativity differences</title>\",\n \"<p id=\\\"Par24\\\">Previous research among cancer survivors has shown nativity to be a factor in cost-related barriers among US-born Hispanic cancer survivors [##REF##24904178##42##]. In our study, we found that foreign-born individuals who experienced the same level of SES barriers had higher likelihood of experiencing healthcare delays than their native-born counterparts when compared to those who experienced no SES barriers. Previous research that explored nativity differences among cancer survivors is limited and inconsistent. For example, although the results did not reach statistical significance, Diamant and colleagues reported the opposite in a sample of non-cancer survivors, showing that non-native-born were less likely to report healthcare delays compared to native-born individuals [##REF##15117701##43##]. Whereas, in a study of female cancer survivors that assessed disparities in healthcare access and utilization, they found that non-US-born females were less likely to report having a routine place to go to meet their healthcare compared to US-born cancer survivors [##REF##34661881##44##]. This is important, as not having a primary healthcare office to seek care or have routine services can promote delaying accessing the extended care cancer survivors need. In addition, a higher prevalence of sociodemographic and SES barriers (e.g., income, education) was found among foreign-born individuals than among native-born in two North American countries (US and Canada), and disparities in healthcare access were higher among foreign- compared to native-born [##REF##21841297##20##]. Thus, supporting the role of SES barriers among foreign-born individuals.</p>\",\n \"<title>Health literacy</title>\",\n \"<p id=\\\"Par25\\\">Our study found that, after adjusting for confounding sociodemographic, nativity, and SES barriers, health literacy was not statistically significantly associated with healthcare delays in our entire study cohort. However, we found that solely among US-born participants, limited health literacy was associated with an increased likelihood of healthcare delays compared to adequate health literacy. We also saw a statistically significant monotonic relationship between increased health literacy scores and decreased odds of healthcare delays among US-born. While our findings only reached statistical significance among US-born cancer survivors, the magnitude of our findings was in the same direction among foreign-born cancer survivors. This indicates that regardless of nativity, increasing health literacy could mitigate the impact of healthcare delays. While we controlled for nativity, sociodemographic, and SES barriers, our findings suggest that health literacy could also be associated with cultural differences and language that we were not able to control in our study. For example, cancer survivors with low health literacy may suffer difficulties when trying to decode their symptoms or understand their diagnoses through communication with providers, which could result in healthcare delays and a later stage of disease at diagnosis [##REF##12018928##45##]. In the case of cancer survivors, there is a great need for complex health services after they have completed their primary treatment for their illness and subsequent management of their health [##REF##30561774##46##]. These deficiencies in communicating efficiently with their providers could increase the risk of healthcare delays.</p>\",\n \"<title>Strengths and limitations</title>\",\n \"<p id=\\\"Par26\\\">An important strength of this study is using data from the All of Us research program, as it has a high proportion of enrolled underrepresented minority populations, which increases our sample size and access to geographically and ethnically diverse populations. Similarly, we were able to analyze a relatively large sample with complete socioeconomic and sociodemographic data of foreign-born cancer survivors, which helps to expand the current cancer survivor, cancer health disparities, and health disparities research. Lastly, our results can be generalized to cancer survivors and individuals with similar characteristics and settings that experienced similar SES barriers in the US.</p>\",\n \"<p id=\\\"Par27\\\">This study is not without limitations. The cross-sectional nature of the design does not allow us to establish a temporal or causal relationship. There is potential for misclassification for some of the factors included in our main independent variables as this data is from self-reported questionnaires. Our sample was also comprised of a higher distribution of individuals with a college degree or higher and a higher income. We were unable able to assess for acculturation; thus, future studies should account for it as it can be a potential confounder in these associations. Using a complete case (CC) analysis method may have introduced bias to our results. We addressed this concern by conducting a sensitivity analysis using multiple imputation (MI) method for our outcome variable. This analysis revealed that there was a slight overestimation in the relationship between SES barriers and healthcare delays among foreign-born cancer survivors, compared to their US-born counterparts with the same level of SES barriers. Despite the small differences observed in the effect estimates, the overall trend and interpretation of the results remained consistent across both the MI and CC analyses (Supplemental Table 3).</p>\",\n \"<p id=\\\"Par28\\\">Although the All of Us collects data nationwide, these results cannot be generalizable to all cancer survivors in the United States. A clear example of this limitation can be seen in that our sample reported roughly 90% some college or higher degree, whereas in 2021, those who had completed some college, or more were approximately 63% in the general US population [##UREF##3##47##]. Lastly, an additional limitation is that during our study period, the COVID-19 pandemic may have worsened SES barriers and healthcare delays that may have contributed to our findings and may need further exploring.</p>\",\n \"<title>Implications and future direction</title>\",\n \"<p id=\\\"Par29\\\">Our results can help guide policymakers to promote the development of policies that aim at eliminating SES barriers. For example, many of the variables that are part of our SES index are system-modifiable factors. Implementation of laws that make education equitable, job creation and training, housing affordability, and universal healthcare are ways in which policies can aid in mitigating these SES barriers. At the healthcare system level, practitioners and systems should recognize that these SES barriers exist and promote solutions. For example, systems can offer transportation services to those who are experiencing SES barriers to lessen healthcare delays [##REF##34723394##48##]. Similarly, providing adult- and childcare services can help avoid delays in seeking care [##REF##36261213##49##].</p>\",\n \"<p id=\\\"Par30\\\">As the All of Us continues to enroll participants and participants complete all surveys, future studies should reassess this association to determine if our findings remain true. Moreover, in future analyses, it is important to consider adjusting for acculturation, as well as other types of stressors such as discrimination, as these experiences can contribute to healthcare delays. Additionally, future studies should aim to explore racial differences between US-born and foreign-born cancer survivors. It is crucial to recognize that races and ethnicities such as Black, Hispanic, and Asian are heterogeneous, varying across cultural and socioeconomic aspects. Thus, understanding the SES barriers associated with healthcare delays among US-born ethnic minorities from different racial and ethnic backgrounds (e.g., Mexican-US-born, Guatemalan-US-born, Chinese-US-born, Nigerian-US-born) compared to foreign-born counterparts (e.g., Mexican-foreign-born, Guatemalan-foreign-born, Chinese-foreign-born, Nigerian-foreign-born) is of extreme importance.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par31\">Using data from the All of Us research program, we found that SES-related barriers are significantly associated with healthcare delays in cancer survivors in our study. However, a greater impact was observed among those who were foreign-born. Similarly, we observed a possible protective effect of health literacy on healthcare delays among US-born only. Our study highlights that to mitigate the impact of delayed healthcare, both policymakers and healthcare providers must prioritize addressing the social determinants of health and promoting health literacy in these populations.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par31\\\">Using data from the All of Us research program, we found that SES-related barriers are significantly associated with healthcare delays in cancer survivors in our study. However, a greater impact was observed among those who were foreign-born. Similarly, we observed a possible protective effect of health literacy on healthcare delays among US-born only. Our study highlights that to mitigate the impact of delayed healthcare, both policymakers and healthcare providers must prioritize addressing the social determinants of health and promoting health literacy in these populations.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose</title>","<p id=\"Par1\">We aimed to assess whether nativity differences in socioeconomic (SES) barriers and health literacy were associated with healthcare delays among US cancer survivors.</p>","<title>Methods</title>","<p id=\"Par2\">“All of Us” survey data were analyzed among adult participants ever diagnosed with cancer. A binary measure of healthcare delay (1+ delays versus no delays) was created. Health literacy was assessed using the Brief Health Literacy Screen. A composite measure of SES barriers (education, employment, housing, income, and insurance statuses) was created as 0, 1, 2, or 3+. Multivariable logistic regression model tested the associations of (1) SES barriers and health literacy with healthcare delays, and (2) whether nativity modified this relationship.</p>","<title>Results</title>","<p id=\"Par3\">Median participant age was 64 years (<italic>n</italic> = 10,020), with 8% foreign-born and 18% ethnic minorities. Compared to survivors with no SES barriers, those with 3+ had higher likelihood of experiencing healthcare delays (OR 2.18, 95% CI 1.84, 2.58). For every additional barrier, the odds of healthcare delays were greater among foreign-born (1.72, 1.43, 2.08) than US-born (1.27, 1.21, 1.34). For every 1-unit increase in health literacy among US-born, the odds of healthcare delay decreased by 9% (0.91, 0.89, 0.94).</p>","<title>Conclusion</title>","<p id=\"Par4\">We found that SES barriers to healthcare delays have a greater impact among foreign-born than US-born cancer survivors. Higher health literacy may mitigate healthcare delays among US cancer survivors. Healthcare providers, systems and policymakers should assess and address social determinants of health and promote health literacy as a way to minimize healthcare delays among both foreign- and US-born cancer survivors.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s10552-023-01782-z.</p>","<title>Keywords</title>","<p>Open access funding provided by SCELC, Statewide California Electronic Library Consortium</p>"],"string":"[\n \"<title>Purpose</title>\",\n \"<p id=\\\"Par1\\\">We aimed to assess whether nativity differences in socioeconomic (SES) barriers and health literacy were associated with healthcare delays among US cancer survivors.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">“All of Us” survey data were analyzed among adult participants ever diagnosed with cancer. A binary measure of healthcare delay (1+ delays versus no delays) was created. Health literacy was assessed using the Brief Health Literacy Screen. A composite measure of SES barriers (education, employment, housing, income, and insurance statuses) was created as 0, 1, 2, or 3+. Multivariable logistic regression model tested the associations of (1) SES barriers and health literacy with healthcare delays, and (2) whether nativity modified this relationship.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">Median participant age was 64 years (<italic>n</italic> = 10,020), with 8% foreign-born and 18% ethnic minorities. Compared to survivors with no SES barriers, those with 3+ had higher likelihood of experiencing healthcare delays (OR 2.18, 95% CI 1.84, 2.58). For every additional barrier, the odds of healthcare delays were greater among foreign-born (1.72, 1.43, 2.08) than US-born (1.27, 1.21, 1.34). For every 1-unit increase in health literacy among US-born, the odds of healthcare delay decreased by 9% (0.91, 0.89, 0.94).</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par4\\\">We found that SES barriers to healthcare delays have a greater impact among foreign-born than US-born cancer survivors. Higher health literacy may mitigate healthcare delays among US cancer survivors. Healthcare providers, systems and policymakers should assess and address social determinants of health and promote health literacy as a way to minimize healthcare delays among both foreign- and US-born cancer survivors.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s10552-023-01782-z.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by SCELC, Statewide California Electronic Library Consortium</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Measures</title>","<title>Demographics</title>","<p id=\"Par13\">Personal level characteristics accounted for in this study were age (at survey completion), sex (male vs female), race (Asian, Black, Hispanic, White, multiracial/biracial, other [includes those who selected: none of this, another population, and prefer not to answer]), marital status (married [includes those who selected: married and living with a partner] vs single [includes those who selected: Single, divorced, widowed, and separated]), nativity (US- vs foreign-born), annual income (using quintiles, the lowest quintile were those who reported income of < 35 K vs quintile 2–5 ≥ 35 K), education (college or more vs ≤ high school or equivalent), insured (yes vs no), housing status (own vs rent/other arrangements), employed (yes vs no), current treatment (yes vs no), and cancer type (range of multiple cancer sites).</p>","<title>Health literacy</title>","<p id=\"Par14\">Health Literacy was assessed using the three-item Brief Health Literacy Screen (BHLS) [##REF##23918160##26##, ##UREF##1##27##], which measures individual needs for help with filling out forms (“How confident are you filling out medical forms by yourself?”), reading health-related documents (“How often do you have someone help you read health-related materials?”), and difficulty learning due to a lack of understanding of written medical documents (“How often do you have problems learning about your medical condition because of difficulty understanding written information?”). Response options were on a five-point Likert scale, with options for reading and understanding health documents including “Always,” “Often,” “Sometimes,” Occasionally,” and “Never” and for the need for help to fill forms were, “Extremely,” “Quite a bit,” “Somewhat,” “A little bit,” “Not at all.” The survey question measuring if the participant required help with forms was reversed coded. All items were then summed to create a composite score with higher scores (max = 15), indicating fewer health literacy problems. Following a previous study that assessed health literacy using the BHLS scale by Willens and colleagues, we dichotomized this score, with those who scored ≤ 9 as having limited health literacy and scores > 9 as having adequate health literacy [##REF##24093351##28##] (Supplemental Table 1).</p>","<title>Socioeconomic barriers</title>","<p id=\"Par15\">Five SES factors (education, income, insurance, housing, and employment status) were dichotomized to create a composite measure following a previous study [##REF##31472134##29##]. If individuals selected an income “ ≥ 35 K,” an education level of “college or more,” being insured, owning a home, and being employed, they were coded as having no SES barriers (0). Those who selected either one of the following: an income between “ < 35 k,” an educational level of “ ≤ high school or equivalent,” not being insured, not being employed, and/or having a housing status as rent/another arrangement, they were given an additive score ranging from 1 to 5 [##REF##31472134##29##]. Due to sparse counts in categories of four and five SES barriers, scores were truncated to range from 0 to 3 or more SES barriers (Supplemental Table 2).</p>","<title>Healthcare delays</title>","<p id=\"Par16\">Nine questions were used to assess healthcare delays. These questions were obtained from the National Health Interview Survey asking participants if they experienced delays in any healthcare received due to various reasons in the past 12 months [##REF##33942535##25##]. These reasons include transportation, living in a rural area where healthcare providers are too far, nervousness about seeing a healthcare provider, could not get time off work, could not get childcare, cannot leave adult unattended due to being a caretaker, could not afford copays, deductible was too high, and could not afford it or had to pay out of pocket for some or all procedures. Response options were “no,” “yes,” or “don’t know.” A dichotomized measure was created with those who responded “yes” to one or more reasons as having experienced healthcare delays, and those who responded “no” to all reasons as having experienced no delays, those who reported “don’t know” were not counted in this measure.</p>","<title>Statistical methods</title>","<p id=\"Par17\">To characterize the study population, descriptive statistics were calculated for all demographic variables and variables of interest (nativity, SES barriers, and health literacy). Listwise deletion method was used to address missing data (15.9%). We conducted a post-hoc sensitivity analysis to determine the direction of the potential bias introduced from our listwise deletion method for a complete case analysis. We used a multiple imputation analysis using chained equations (MICE). This approach allowed us to impute missing values based on observed data and estimate relationships between variables. To satisfy the safe data sharing policy of “All of Us,” groups with less than 20 participants are reported in tables as ≤ 20 or < x% with another category in the same column/row also showing ≥ (%) to ensure that another count value cannot be used to derive the exact count that is less than <italic>n</italic> = 20 in the suppressed group. First, a multivariable logistic regression model tested the hypothesized independent relationships between SES barriers, and health literacy with healthcare delays adjusted for covariates (age, sex, ethnicity/race, marital status, treatment status, and cancer type). Secondly, to assess nativity differences in the association between SES barriers and healthcare delays, a product interaction term for nativity and SES barriers (SES barriers*nativity) was included in the model. Furthermore, we assessed for a p-trend in the adjusted and stratified model by introducing SES barriers and health literacy as continuous measures in the model. All statistical analyses were performed using R Jupyter Notebooks embedded in the “All of Us” workbench, with a significance level at alpha 0.05. Odds ratios (ORs) with 95% confidence intervals (CI) and <italic>p</italic> value are reported.</p>","<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Measures</title>\",\n \"<title>Demographics</title>\",\n \"<p id=\\\"Par13\\\">Personal level characteristics accounted for in this study were age (at survey completion), sex (male vs female), race (Asian, Black, Hispanic, White, multiracial/biracial, other [includes those who selected: none of this, another population, and prefer not to answer]), marital status (married [includes those who selected: married and living with a partner] vs single [includes those who selected: Single, divorced, widowed, and separated]), nativity (US- vs foreign-born), annual income (using quintiles, the lowest quintile were those who reported income of < 35 K vs quintile 2–5 ≥ 35 K), education (college or more vs ≤ high school or equivalent), insured (yes vs no), housing status (own vs rent/other arrangements), employed (yes vs no), current treatment (yes vs no), and cancer type (range of multiple cancer sites).</p>\",\n \"<title>Health literacy</title>\",\n \"<p id=\\\"Par14\\\">Health Literacy was assessed using the three-item Brief Health Literacy Screen (BHLS) [##REF##23918160##26##, ##UREF##1##27##], which measures individual needs for help with filling out forms (“How confident are you filling out medical forms by yourself?”), reading health-related documents (“How often do you have someone help you read health-related materials?”), and difficulty learning due to a lack of understanding of written medical documents (“How often do you have problems learning about your medical condition because of difficulty understanding written information?”). Response options were on a five-point Likert scale, with options for reading and understanding health documents including “Always,” “Often,” “Sometimes,” Occasionally,” and “Never” and for the need for help to fill forms were, “Extremely,” “Quite a bit,” “Somewhat,” “A little bit,” “Not at all.” The survey question measuring if the participant required help with forms was reversed coded. All items were then summed to create a composite score with higher scores (max = 15), indicating fewer health literacy problems. Following a previous study that assessed health literacy using the BHLS scale by Willens and colleagues, we dichotomized this score, with those who scored ≤ 9 as having limited health literacy and scores > 9 as having adequate health literacy [##REF##24093351##28##] (Supplemental Table 1).</p>\",\n \"<title>Socioeconomic barriers</title>\",\n \"<p id=\\\"Par15\\\">Five SES factors (education, income, insurance, housing, and employment status) were dichotomized to create a composite measure following a previous study [##REF##31472134##29##]. If individuals selected an income “ ≥ 35 K,” an education level of “college or more,” being insured, owning a home, and being employed, they were coded as having no SES barriers (0). Those who selected either one of the following: an income between “ < 35 k,” an educational level of “ ≤ high school or equivalent,” not being insured, not being employed, and/or having a housing status as rent/another arrangement, they were given an additive score ranging from 1 to 5 [##REF##31472134##29##]. Due to sparse counts in categories of four and five SES barriers, scores were truncated to range from 0 to 3 or more SES barriers (Supplemental Table 2).</p>\",\n \"<title>Healthcare delays</title>\",\n \"<p id=\\\"Par16\\\">Nine questions were used to assess healthcare delays. These questions were obtained from the National Health Interview Survey asking participants if they experienced delays in any healthcare received due to various reasons in the past 12 months [##REF##33942535##25##]. These reasons include transportation, living in a rural area where healthcare providers are too far, nervousness about seeing a healthcare provider, could not get time off work, could not get childcare, cannot leave adult unattended due to being a caretaker, could not afford copays, deductible was too high, and could not afford it or had to pay out of pocket for some or all procedures. Response options were “no,” “yes,” or “don’t know.” A dichotomized measure was created with those who responded “yes” to one or more reasons as having experienced healthcare delays, and those who responded “no” to all reasons as having experienced no delays, those who reported “don’t know” were not counted in this measure.</p>\",\n \"<title>Statistical methods</title>\",\n \"<p id=\\\"Par17\\\">To characterize the study population, descriptive statistics were calculated for all demographic variables and variables of interest (nativity, SES barriers, and health literacy). Listwise deletion method was used to address missing data (15.9%). We conducted a post-hoc sensitivity analysis to determine the direction of the potential bias introduced from our listwise deletion method for a complete case analysis. We used a multiple imputation analysis using chained equations (MICE). This approach allowed us to impute missing values based on observed data and estimate relationships between variables. To satisfy the safe data sharing policy of “All of Us,” groups with less than 20 participants are reported in tables as ≤ 20 or < x% with another category in the same column/row also showing ≥ (%) to ensure that another count value cannot be used to derive the exact count that is less than <italic>n</italic> = 20 in the suppressed group. First, a multivariable logistic regression model tested the hypothesized independent relationships between SES barriers, and health literacy with healthcare delays adjusted for covariates (age, sex, ethnicity/race, marital status, treatment status, and cancer type). Secondly, to assess nativity differences in the association between SES barriers and healthcare delays, a product interaction term for nativity and SES barriers (SES barriers*nativity) was included in the model. Furthermore, we assessed for a p-trend in the adjusted and stratified model by introducing SES barriers and health literacy as continuous measures in the model. All statistical analyses were performed using R Jupyter Notebooks embedded in the “All of Us” workbench, with a significance level at alpha 0.05. Odds ratios (ORs) with 95% confidence intervals (CI) and <italic>p</italic> value are reported.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<p>The authors would like to thank all of the participants of the All of Us Research Program, Drs. Cecilia Patino-Sutton, and Elizabeth Burner. The All of Us Research Program is supported by the National Institutes of Health, Office of the Director: Regional Medical Centers: 1 OT2 OD026549; 1 OT2 OD026554; 1 OT2 OD026557; 1 OT2 OD026556; 1 OT2 OD026550; 1 OT2 OD 026552; 1 OT2 OD026553; 1 OT2 OD026548; 1 OT2 OD026551; 1 OT2 OD026555; IAA #: AOD 16037; Federally Qualified Health Centers: HHSN 263201600085U; Data and Research Center: 5 U2C OD023196; Biobank: 1 U24 OD023121; The Participant Center: U24 OD023176; Participant Technology Systems Center: 1 U24 OD023163; Communications and Engagement: 3 OT2 OD023205; 3 OT2 OD023206; and Community Partners: 1 OT2 OD025277; 3 OT2 OD025315; 1 OT2 OD025337; 1 OT2 OD025276. In addition, the All of Us Research Program would not be possible without the partnership of its participants.</p>","<title>Author contributions</title>","<p>AA: Conception/design, assembly of data, data analysis, writing, and approval of the final version. SN: Conception/design, writing and editing of the article, and approval of the final version. CYO: Conception/design, writing and editing the article, and approval of the final version. CR: Writing the article and approval of the final version. SEK: Writing and editing the article and approval of the final version. AJF: Conception/design, assembly of data, writing and editing, and approval of the final version.</p>","<title>Funding</title>","<p>Open access funding provided by SCELC, Statewide California Electronic Library Consortium. Carol Ochoa was supported by the National Cancer Institute to conduct this study (K00CA264294-02).</p>","<title>Data availability</title>","<p>Data from the All of Us Research Program can only be accessed through the Researcher Workbench (<ext-link ext-link-type=\"uri\" xlink:href=\"https://workbench.researchallofus.org/login\">https://workbench.researchallofus.org/login</ext-link>) as per the informed consent of program participants. The investigators are prohibited to share raw-level data of participants in accordance with the user agreement established by this program. Therefore, it is not possible to provide a de-identified dataset for this manuscript.</p>","<title>Declarations</title>","<title>Competing interests</title>","<p id=\"Par32\">The authors declare no competing interests.</p>","<title>Ethical approval</title>","<p id=\"Par34\">The All of Us Research Program Institutional Review Board (IRB) established that registered tier data available on the All of Us’ Workbench meets criteria for non-Human Subject Research. This project used registered tier data and required no IRB review. <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.researchallofus.org/faq/do-i-need-institutional-review-board-irb-approval-from-my-own-institution-in-order-to-access-this-data-through-the-researcher-workbench/\">https://www.researchallofus.org/faq/do-i-need-institutional-review-board-irb-approval-from-my-own-institution-in-order-to-access-this-data-through-the-researcher-workbench/</ext-link>.</p>"],"string":"[\n \"<p>The authors would like to thank all of the participants of the All of Us Research Program, Drs. Cecilia Patino-Sutton, and Elizabeth Burner. The All of Us Research Program is supported by the National Institutes of Health, Office of the Director: Regional Medical Centers: 1 OT2 OD026549; 1 OT2 OD026554; 1 OT2 OD026557; 1 OT2 OD026556; 1 OT2 OD026550; 1 OT2 OD 026552; 1 OT2 OD026553; 1 OT2 OD026548; 1 OT2 OD026551; 1 OT2 OD026555; IAA #: AOD 16037; Federally Qualified Health Centers: HHSN 263201600085U; Data and Research Center: 5 U2C OD023196; Biobank: 1 U24 OD023121; The Participant Center: U24 OD023176; Participant Technology Systems Center: 1 U24 OD023163; Communications and Engagement: 3 OT2 OD023205; 3 OT2 OD023206; and Community Partners: 1 OT2 OD025277; 3 OT2 OD025315; 1 OT2 OD025337; 1 OT2 OD025276. In addition, the All of Us Research Program would not be possible without the partnership of its participants.</p>\",\n \"<title>Author contributions</title>\",\n \"<p>AA: Conception/design, assembly of data, data analysis, writing, and approval of the final version. SN: Conception/design, writing and editing of the article, and approval of the final version. CYO: Conception/design, writing and editing the article, and approval of the final version. CR: Writing the article and approval of the final version. SEK: Writing and editing the article and approval of the final version. AJF: Conception/design, assembly of data, writing and editing, and approval of the final version.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by SCELC, Statewide California Electronic Library Consortium. Carol Ochoa was supported by the National Cancer Institute to conduct this study (K00CA264294-02).</p>\",\n \"<title>Data availability</title>\",\n \"<p>Data from the All of Us Research Program can only be accessed through the Researcher Workbench (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://workbench.researchallofus.org/login\\\">https://workbench.researchallofus.org/login</ext-link>) as per the informed consent of program participants. The investigators are prohibited to share raw-level data of participants in accordance with the user agreement established by this program. Therefore, it is not possible to provide a de-identified dataset for this manuscript.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par32\\\">The authors declare no competing interests.</p>\",\n \"<title>Ethical approval</title>\",\n \"<p id=\\\"Par34\\\">The All of Us Research Program Institutional Review Board (IRB) established that registered tier data available on the All of Us’ Workbench meets criteria for non-Human Subject Research. This project used registered tier data and required no IRB review. <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://www.researchallofus.org/faq/do-i-need-institutional-review-board-irb-approval-from-my-own-institution-in-order-to-access-this-data-through-the-researcher-workbench/\\\">https://www.researchallofus.org/faq/do-i-need-institutional-review-board-irb-approval-from-my-own-institution-in-order-to-access-this-data-through-the-researcher-workbench/</ext-link>.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Theoretical framework of the impact nativity has on healthcare delays</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Theoretical framework of the impact nativity has on healthcare delays</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Descriptive characteristics of the sample and their association with SES barriers (<italic>n</italic> = 10,020)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Variables</th><th align=\"left\" colspan=\"4\">Number of socioeconomic barriers<break/><italic>n</italic> (%)</th><th align=\"left\" rowspan=\"2\"><italic>p</italic> value</th></tr><tr><th align=\"left\">No barriers<break/>(<italic>n</italic> = 3,112)</th><th align=\"left\">1<break/>(<italic>n</italic> = 4,314)</th><th align=\"left\">2<break/>(<italic>n</italic> = 1,514)</th><th align=\"left\">3 or more<break/>(<italic>n</italic> = 1,080)</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"6\">Sex</td></tr><tr><td align=\"left\"> Female</td><td align=\"left\"> > 2100 (> 30%)</td><td align=\"left\">2688 (40.6%)</td><td align=\"left\"> > 1000 (> 15%)</td><td align=\"left\"> > 500 (> 10%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Male</td><td align=\"left\">991 (29.7%)</td><td align=\"left\">1596 (47.9%)</td><td align=\"left\">450 (13.5%)</td><td align=\"left\">295 (8.9%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>a</sup></td><td align=\"left\"> ≤ 20 (< 25%)</td><td align=\"left\">31 (47.7%)</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Age</td></tr><tr><td align=\"left\"> Median [Q1, Q3]</td><td align=\"left\">59.3 [51.3, 65.8]</td><td align=\"left\">68.1 [60.6, 73.2]</td><td align=\"left\">64.7 [52.9, 72.0]</td><td align=\"left\">58.9 [48.0, 67.3]</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\" colspan=\"6\">Race/ethnicity</td></tr><tr><td align=\"left\"> Asian</td><td align=\"left\">70 (35.7%)</td><td align=\"left\"> > 50 (> 40%)</td><td align=\"left\"> > 20 (> 10%)</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Black</td><td align=\"left\">113 (17.6%)</td><td align=\"left\">178 (27.7%)</td><td align=\"left\">150 (23.4%)</td><td align=\"left\">201 (31.3%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Hispanic</td><td align=\"left\">143 (23.4%)</td><td align=\"left\">164 (26.7%)</td><td align=\"left\">137 (22.3%)</td><td align=\"left\">171 (27.8%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> White</td><td align=\"left\">2687 (32.6%)</td><td align=\"left\">3759 (45.6%)</td><td align=\"left\">1154 (14.0%)</td><td align=\"left\">643 (7.8%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> More than one pop</td><td align=\"left\">52 (34.7%)</td><td align=\"left\"> > 50 (> 40%)</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Other<sup>b</sup></td><td align=\"left\">47 (26.9%)</td><td align=\"left\">62 (35.4%)</td><td align=\"left\">32 (18.3%)</td><td align=\"left\">34 (19.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Annual income<sup>e</sup></td></tr><tr><td align=\"left\"> < 35 K</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\">140 (7.7%)</td><td align=\"left\">703 (38.4%)</td><td align=\"left\"> > 900 (> 40%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> ≥ 35 K</td><td align=\"left\">2864 (39.6%)</td><td align=\"left\">3687 (51.0%)</td><td align=\"left\">641 (8.9%)</td><td align=\"left\">39 (0.5%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>c</sup></td><td align=\"left\"> > 200 (> 20%)</td><td align=\"left\">488 (50.8%)</td><td align=\"left\">170 (17.7%)</td><td align=\"left\"> ≤ 60 (< 10%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Marital status</td></tr><tr><td align=\"left\"> Single</td><td align=\"left\"> > 600 (> 15%)</td><td align=\"left\">1215 (35.1%)</td><td align=\"left\">778 (22.5%)</td><td align=\"left\"> > 700 (> 20%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Married/living with a partner</td><td align=\"left\">2405 (37.2%)</td><td align=\"left\">3071 (47.5%)</td><td align=\"left\">709 (11.0%)</td><td align=\"left\">282 (4.4%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>c</sup></td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\">29 (30.9%)</td><td align=\"left\">27 (28.7%)</td><td align=\"left\"> ≤ 20 (< 10.0%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Employed</td></tr><tr><td align=\"left\"> Yes</td><td align=\"left\"> > 2700 (> 50%)</td><td align=\"left\"> > 900 (> 20%)</td><td align=\"left\"> > 250 (> 5%)</td><td align=\"left\"> < 100 (< 5%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> No</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\">3273 (60.4%)</td><td align=\"left\">1161 (21.4%)</td><td align=\"left\">981 (18.1%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>c</sup></td><td align=\"left\">48 (55.2%)</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Educational level</td></tr><tr><td align=\"left\"> College or more</td><td align=\"left\">3097 (34.5%)</td><td align=\"left\">4133 (46.0%)</td><td align=\"left\"> > 1000 (> 10%)</td><td align=\"left\"> > 500 (> 5%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> ≤ High school or equivalent</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\">160 (16.2%)</td><td align=\"left\"> > 300 (> 30%)</td><td align=\"left\">484 (49.1%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>c</sup></td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\">22 (40.0%)</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\"> ≤ 20 (< 20%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Insured</td></tr><tr><td align=\"left\"> No</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\">29 (22.1%)</td><td align=\"left\"> > 50 (> 50%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Yes</td><td align=\"left\"> > 2500 (> 30%)</td><td align=\"left\"> > 4000 (> 40%)</td><td align=\"left\">1463 (14.9%)</td><td align=\"left\">975 (9.9%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>d</sup></td><td align=\"left\">20 (25.3%)</td><td align=\"left\"> > 20 (30%)</td><td align=\"left\">22 (27.8%)</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Treatment</td></tr><tr><td align=\"left\"> No</td><td align=\"left\"> > 2300 (> 25%)</td><td align=\"left\"> > 3300 (> 40%)</td><td align=\"left\"> > 1100 (> 10%)</td><td align=\"left\"> > 800 (> 10%)</td><td char=\".\" align=\"char\"> < 0.01</td></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">796 (34.1%)</td><td align=\"left\">953 (40.8%)</td><td align=\"left\">356 (15.2%)</td><td align=\"left\">233 (10.0%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Missing</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\"> ≤ 20 (< 50%)</td><td align=\"left\"> ≤ 20 (< 25%)</td><td align=\"left\"> ≤ 20 (< 10%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Nativity</td></tr><tr><td align=\"left\"> US-Born</td><td align=\"left\">2869 (31.2%)</td><td align=\"left\">4003 (43.5%)</td><td align=\"left\">1379 (15.0%)</td><td align=\"left\">955 (10.4%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Foreign-Born</td><td align=\"left\">243 (29.8%)</td><td align=\"left\">312 (38.3%)</td><td align=\"left\">135 (16.6%)</td><td align=\"left\">125 (15.3%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Housing status</td></tr><tr><td align=\"left\"> Own</td><td align=\"left\"> > 3000 (> 40%)</td><td align=\"left\">3534 (47.6%)</td><td align=\"left\"> < 800 (< 10%)</td><td align=\"left\">125 (1.7%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Rent/other arrangements</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\">734 (29.6%)</td><td align=\"left\">793 (32.0%)</td><td align=\"left\"> > 800 (> 35%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>c</sup></td><td align=\"left\">50 (40.0%)</td><td align=\"left\">47 (37.6%)</td><td align=\"left\"> > 20 (> 17%)</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Cancer type</td></tr><tr><td align=\"left\"> Bladder</td><td align=\"left\">65 (26.5%)</td><td align=\"left\">105 (42.9%)</td><td align=\"left\">53 (21.6%)</td><td align=\"left\">22 (9.0%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Blood</td><td align=\"left\">238 (31.3%)</td><td align=\"left\">350 (46.1%)</td><td align=\"left\">111 (14.6%)</td><td align=\"left\">61 (8.0%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Bone</td><td align=\"left\"> > 25 (> 25%)</td><td align=\"left\">39 (36.5%)</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Brain</td><td align=\"left\">35 (27.8%)</td><td align=\"left\">49 (38.9%)</td><td align=\"left\">22 (17.5%)</td><td align=\"left\">20 (15.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Breast</td><td align=\"left\">1031 (34.8%)</td><td align=\"left\">1283 (43.3%)</td><td align=\"left\">408 (13.8%)</td><td align=\"left\">240 (8.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Cervical</td><td align=\"left\">188 (27.3%)</td><td align=\"left\">202 (29.4%)</td><td align=\"left\">126 (18.3%)</td><td align=\"left\">172 (25.0%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Colon rectal</td><td align=\"left\">131 (30.6%)</td><td align=\"left\">168 (39.3%)</td><td align=\"left\">73 (17.1%)</td><td align=\"left\">56 (13.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Endocrine</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\">31 (46.3%)</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\"> ≤ 20 (< 30%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Endometrial</td><td align=\"left\">70 (25.6%)</td><td align=\"left\">128 (46.7%)</td><td align=\"left\">50 (18.3%)</td><td align=\"left\">26 (9.5%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Head neck</td><td align=\"left\"> > 50 (> 30%)</td><td align=\"left\">79 (42.9%)</td><td align=\"left\">25 (13.6%)</td><td align=\"left\"> ≤ 20 (< 10%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Kidney</td><td align=\"left\">89 (30.4%)</td><td align=\"left\">122 (41.6%)</td><td align=\"left\">48 (16.4%)</td><td align=\"left\">34 (11.6%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Lung</td><td align=\"left\">39 (16.1%)</td><td align=\"left\">109 (45.0%)</td><td align=\"left\">55 (21.7%)</td><td align=\"left\">39 (16.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Other</td><td align=\"left\">427 (29.8%)</td><td align=\"left\">611 (42.6%)</td><td align=\"left\">211 (14.7%)</td><td align=\"left\">186 (13.0%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Ovarian</td><td align=\"left\">60 (27.3%)</td><td align=\"left\">89 (41.5%)</td><td align=\"left\">29 (13.2%)</td><td align=\"left\">42 (19.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Prostate</td><td align=\"left\">382 (28.3%)</td><td align=\"left\">727 (53.9%)</td><td align=\"left\">165 (12.2%)</td><td align=\"left\">74 (5.5%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Thyroid</td><td align=\"left\">245 (38.2%)</td><td align=\"left\">223 (34.7%)</td><td align=\"left\">104 (16.2%)</td><td align=\"left\">70 (10.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Healthcare delay</td></tr><tr><td align=\"left\"> No</td><td align=\"left\">2178 (31.6%)</td><td align=\"left\">3286 (47.7%)</td><td align=\"left\">914 (13.3%)</td><td align=\"left\">514 (7.5%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">934 (29.9%)</td><td align=\"left\">1028 (32.9%)</td><td align=\"left\">600 (19.2%)</td><td align=\"left\">566 (18.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\">Health literacy</td></tr><tr><td align=\"left\"> ≤ 9</td><td align=\"left\"> ≤ 20 (< 10%)</td><td align=\"left\">52 (25.1%)</td><td align=\"left\"> > 20 (10%)</td><td align=\"left\">126 (44.9%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> > 9</td><td align=\"left\">3048 (31.8%)</td><td align=\"left\">4188 (43.7%)</td><td align=\"left\">1435 (14.9%)</td><td align=\"left\">922 (9.6%)</td><td align=\"left\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Descriptive characteristics of the sample and their association with healthcare delays (<italic>n</italic> = 10,020)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Variables</th><th align=\"left\">No healthcare delay<break/>(<italic>n</italic> = 6,892)</th><th align=\"left\">Any healthcare delay (<italic>n</italic> = 3,128)</th><th align=\"left\"><italic>p</italic> value</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"4\">Sex</td></tr><tr><td align=\"left\"> Female</td><td align=\"left\">4239 (64.0%)</td><td align=\"left\">2384 (36.0%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Male</td><td align=\"left\">2608 (78.3%)</td><td align=\"left\">724 (21.7%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>a</sup></td><td align=\"left\">45 (69.2%)</td><td align=\"left\">20 (30.8%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">Age</td></tr><tr><td align=\"left\"> Median [Q1, Q3]</td><td align=\"left\">66.4 [579, 72.3]</td><td align=\"left\">57.8 [46.7, 65.7]</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\" colspan=\"4\">Race/ethnicity</td></tr><tr><td align=\"left\"> Asian</td><td align=\"left\">132 (67.4%)</td><td align=\"left\">64 (32.7%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Black</td><td align=\"left\">374 (58.3%)</td><td align=\"left\">267 (41.7%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Hispanic</td><td align=\"left\">345 (56.1%)</td><td align=\"left\">270 (43.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> White</td><td align=\"left\">5856 (71.0%)</td><td align=\"left\">2387 (29.0%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> More than one pop</td><td align=\"left\">89 (59.3%)</td><td align=\"left\">61 (40.7%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Other<sup>b</sup></td><td align=\"left\">96 (54.9%)</td><td align=\"left\">79 (45.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">Annual income<sup>e</sup></td></tr><tr><td align=\"left\"> < 35 K</td><td align=\"left\">914 (50.0%)</td><td align=\"left\">916 (50.0%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> ≥ 35 K</td><td align=\"left\">5300 (73.3%)</td><td align=\"left\">1930 (26.7%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>c</sup></td><td align=\"left\">678 (70.6%)</td><td align=\"left\">282 (29.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">Marital status</td></tr><tr><td align=\"left\"> Single</td><td align=\"left\">2103 (60.8%)</td><td align=\"left\">1357 (39.2%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Married/living with a partner</td><td align=\"left\">4734 (73.2%)</td><td align=\"left\">1732 (26.8%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>c</sup></td><td align=\"left\">55 (58.5%)</td><td align=\"left\">39 (41.5%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">Employed</td></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">2911 (64.4%)</td><td align=\"left\">1608 (35.6%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> No</td><td align=\"left\">3923 (72.5%)</td><td align=\"left\">1491 (27.6%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>c</sup></td><td align=\"left\">58 (66.7%)</td><td align=\"left\">29 (33.3%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">Educational level</td></tr><tr><td align=\"left\"> College or more</td><td align=\"left\">6273 (69.9%)</td><td align=\"left\">2707 (30.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> ≤ High school or equivalent</td><td align=\"left\">585 (59.3%)</td><td align=\"left\">401 (40.7%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>c</sup></td><td align=\"left\">34 (61.8%)</td><td align=\"left\">21 (38.2%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">Insured</td></tr><tr><td align=\"left\"> No</td><td align=\"left\">50 (38.2%)</td><td align=\"left\">81 (61.8%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">6798 (69.3%)</td><td align=\"left\">3012 (30.7%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>d</sup></td><td align=\"left\">44 (55.7%)</td><td align=\"left\">35 (44.3%)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\" colspan=\"4\">Nativity</td></tr><tr><td align=\"left\"> US-Born</td><td align=\"left\">6364 (69.1%)</td><td align=\"left\">2841 (30.9%)</td><td char=\".\" align=\"char\">0.01</td></tr><tr><td align=\"left\"> Foreign-Born</td><td align=\"left\">528 (64.8%)</td><td align=\"left\">287 (35.2%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">Housing status</td></tr><tr><td align=\"left\"> Own</td><td align=\"left\">5521 (74.4%)</td><td align=\"left\">1898 (25.6%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Rent/other arrangement</td><td align=\"left\">1296 (52.3%)</td><td align=\"left\">1180 (47.7%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Other<sup>c</sup></td><td align=\"left\">75 (60.0%)</td><td align=\"left\">50 (40.0%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">Treatment</td></tr><tr><td align=\"left\"> No</td><td align=\"left\"> > 5200 (> 65%)</td><td align=\"left\"> > 2300 (> 30%)</td><td char=\".\" align=\"char\">0.96</td></tr><tr><td align=\"left\"> Yes</td><td align=\"left\">1610 (68.9%)</td><td align=\"left\">728 (31.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Missing</td><td align=\"left\"> ≤ 13 (< 65%)</td><td align=\"left\"> ≤ 20 (< 40%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">Cancer type</td></tr><tr><td align=\"left\"> Bladder</td><td align=\"left\">193 (78.8%)</td><td align=\"left\">52 (21.2%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> Blood</td><td align=\"left\">549 (72.2%)</td><td align=\"left\">210 (27.8%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Bone</td><td align=\"left\">67 (62.6%)</td><td align=\"left\">40 (37.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Brain</td><td align=\"left\">72 (57.1%)</td><td align=\"left\">54 (42.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Breast</td><td align=\"left\">2076 (70.1%)</td><td align=\"left\">886 (42.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Cervical</td><td align=\"left\">316 (45.9%)</td><td align=\"left\">372 (54.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Colon rectal</td><td align=\"left\">295 (68.9%)</td><td align=\"left\">133 (31.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Endocrine</td><td align=\"left\">46 (68.7%)</td><td align=\"left\">21 (31.3%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Endometrial</td><td align=\"left\">172 (62.8%)</td><td align=\"left\">102 (37.2%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Head neck</td><td align=\"left\">129 (70.1%)</td><td align=\"left\">55 (29.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Kidney</td><td align=\"left\">198 (67.6%)</td><td align=\"left\">95 (32.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Lung</td><td align=\"left\">176 (72.7%)</td><td align=\"left\">66 (27.3%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Other</td><td align=\"left\">979 (68.2%)</td><td align=\"left\">456 (31.8%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Ovarian</td><td align=\"left\">134 (60.9%)</td><td align=\"left\">86 (39.1%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Prostate</td><td align=\"left\">1120 (83.1%)</td><td align=\"left\">228 (16.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> Thyroid</td><td align=\"left\">370 (57.6%)</td><td align=\"left\">272 (42.4%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">Health literacy</td></tr><tr><td align=\"left\"> ≤ 9</td><td align=\"left\">178 (55.1%)</td><td align=\"left\">145 (44.9%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> > 9</td><td align=\"left\">6714 (69.2%)</td><td align=\"left\">2983 (30.8%)</td><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"4\">SES barrier</td></tr><tr><td align=\"left\"> 0</td><td align=\"left\">2178 (70.0%)</td><td align=\"left\">934 (30.0%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> 1</td><td align=\"left\">3286 (76.2%)</td><td align=\"left\">1028 (23.9%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> 2</td><td align=\"left\">914 (60.4%)</td><td align=\"left\">600 (39.6%)</td><td align=\"left\"/></tr><tr><td align=\"left\"> 3+</td><td align=\"left\">514 (47.6%)</td><td align=\"left\">566 (52.4%)</td><td align=\"left\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Results from the multivariable regression analysis of risk factors for health care delay and by nativity status among cancer survivors from the All of Us Research Program</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Variables</th><th align=\"left\">Adjusted odds ratios<break/><italic>n</italic> = 9,820<break/>OR (95%CI)</th><th align=\"left\">US-Born<break/><italic>n</italic> = 9,022<break/>OR (95%CI)</th><th align=\"left\">Foreign-Born<break/><italic>n</italic> = 798<break/>OR (95%CI)</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"4\">Nativity</td></tr><tr><td align=\"left\"> US-Born</td><td align=\"left\">Ref</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\"> Foreign-Born</td><td align=\"left\">1.04 (0.87–1.25)</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\" colspan=\"4\">Health literacy</td></tr><tr><td align=\"left\"> p-trend</td><td align=\"left\">0.92 (0.89–0.95)<italic>*</italic>*<italic>*</italic></td><td align=\"left\">0.91(0.88–0.94)***</td><td align=\"left\">0.98(0.90–1.07)</td></tr><tr><td align=\"left\"> ≤ 9</td><td align=\"left\">1.20 (0.89–1.59)</td><td align=\"left\">1.41 (1.02–1.97)*</td><td align=\"left\">0.66 (0.34–1.25)</td></tr><tr><td align=\"left\"> > 9</td><td align=\"left\">Ref</td><td align=\"left\">Ref</td><td align=\"left\">Ref</td></tr><tr><td align=\"left\" colspan=\"4\">SES barrier factors index</td></tr><tr><td align=\"left\"> p-trend</td><td align=\"left\">1.29 (1.23–1.36)***</td><td align=\"left\">1.27 (1.21–1.34)***</td><td align=\"left\">1.72 (1.43–2.08)***</td></tr><tr><td align=\"left\"> 0</td><td align=\"left\">Ref</td><td align=\"left\">Ref</td><td align=\"left\">Ref</td></tr><tr><td align=\"left\"> 1</td><td align=\"left\">0.98 (0.88–1.10)</td><td align=\"left\">0.97 (0.86–1.09)</td><td align=\"left\">1.15 (0.76–1.74)</td></tr><tr><td align=\"left\"> 2</td><td align=\"left\">1.65 (1.43–1.90)***</td><td align=\"left\">1.53 (1.32–1.78)***</td><td align=\"left\">4.35 (2.61–7.34)***</td></tr><tr><td align=\"left\"> 3+</td><td align=\"left\">2.18 (1.84–2.58)***</td><td align=\"left\">2.10 (1.76–2.50)***</td><td align=\"left\">3.83 (2.14–6.98)***</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Descriptive characteristics of the sample and their association with SES barriers (<italic>n</italic> = 10,020)</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" rowspan=\\\"2\\\">Variables</th><th align=\\\"left\\\" colspan=\\\"4\\\">Number of socioeconomic barriers<break/><italic>n</italic> (%)</th><th align=\\\"left\\\" rowspan=\\\"2\\\"><italic>p</italic> value</th></tr><tr><th align=\\\"left\\\">No barriers<break/>(<italic>n</italic> = 3,112)</th><th align=\\\"left\\\">1<break/>(<italic>n</italic> = 4,314)</th><th align=\\\"left\\\">2<break/>(<italic>n</italic> = 1,514)</th><th align=\\\"left\\\">3 or more<break/>(<italic>n</italic> = 1,080)</th></tr></thead><tbody><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Sex</td></tr><tr><td align=\\\"left\\\"> Female</td><td align=\\\"left\\\"> > 2100 (> 30%)</td><td align=\\\"left\\\">2688 (40.6%)</td><td align=\\\"left\\\"> > 1000 (> 15%)</td><td align=\\\"left\\\"> > 500 (> 10%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Male</td><td align=\\\"left\\\">991 (29.7%)</td><td align=\\\"left\\\">1596 (47.9%)</td><td align=\\\"left\\\">450 (13.5%)</td><td align=\\\"left\\\">295 (8.9%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>a</sup></td><td align=\\\"left\\\"> ≤ 20 (< 25%)</td><td align=\\\"left\\\">31 (47.7%)</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Age</td></tr><tr><td align=\\\"left\\\"> Median [Q1, Q3]</td><td align=\\\"left\\\">59.3 [51.3, 65.8]</td><td align=\\\"left\\\">68.1 [60.6, 73.2]</td><td align=\\\"left\\\">64.7 [52.9, 72.0]</td><td align=\\\"left\\\">58.9 [48.0, 67.3]</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Race/ethnicity</td></tr><tr><td align=\\\"left\\\"> Asian</td><td align=\\\"left\\\">70 (35.7%)</td><td align=\\\"left\\\"> > 50 (> 40%)</td><td align=\\\"left\\\"> > 20 (> 10%)</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Black</td><td align=\\\"left\\\">113 (17.6%)</td><td align=\\\"left\\\">178 (27.7%)</td><td align=\\\"left\\\">150 (23.4%)</td><td align=\\\"left\\\">201 (31.3%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Hispanic</td><td align=\\\"left\\\">143 (23.4%)</td><td align=\\\"left\\\">164 (26.7%)</td><td align=\\\"left\\\">137 (22.3%)</td><td align=\\\"left\\\">171 (27.8%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> White</td><td align=\\\"left\\\">2687 (32.6%)</td><td align=\\\"left\\\">3759 (45.6%)</td><td align=\\\"left\\\">1154 (14.0%)</td><td align=\\\"left\\\">643 (7.8%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> More than one pop</td><td align=\\\"left\\\">52 (34.7%)</td><td align=\\\"left\\\"> > 50 (> 40%)</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Other<sup>b</sup></td><td align=\\\"left\\\">47 (26.9%)</td><td align=\\\"left\\\">62 (35.4%)</td><td align=\\\"left\\\">32 (18.3%)</td><td align=\\\"left\\\">34 (19.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Annual income<sup>e</sup></td></tr><tr><td align=\\\"left\\\"> < 35 K</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\">140 (7.7%)</td><td align=\\\"left\\\">703 (38.4%)</td><td align=\\\"left\\\"> > 900 (> 40%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ≥ 35 K</td><td align=\\\"left\\\">2864 (39.6%)</td><td align=\\\"left\\\">3687 (51.0%)</td><td align=\\\"left\\\">641 (8.9%)</td><td align=\\\"left\\\">39 (0.5%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>c</sup></td><td align=\\\"left\\\"> > 200 (> 20%)</td><td align=\\\"left\\\">488 (50.8%)</td><td align=\\\"left\\\">170 (17.7%)</td><td align=\\\"left\\\"> ≤ 60 (< 10%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Marital status</td></tr><tr><td align=\\\"left\\\"> Single</td><td align=\\\"left\\\"> > 600 (> 15%)</td><td align=\\\"left\\\">1215 (35.1%)</td><td align=\\\"left\\\">778 (22.5%)</td><td align=\\\"left\\\"> > 700 (> 20%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Married/living with a partner</td><td align=\\\"left\\\">2405 (37.2%)</td><td align=\\\"left\\\">3071 (47.5%)</td><td align=\\\"left\\\">709 (11.0%)</td><td align=\\\"left\\\">282 (4.4%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>c</sup></td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\">29 (30.9%)</td><td align=\\\"left\\\">27 (28.7%)</td><td align=\\\"left\\\"> ≤ 20 (< 10.0%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Employed</td></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\"> > 2700 (> 50%)</td><td align=\\\"left\\\"> > 900 (> 20%)</td><td align=\\\"left\\\"> > 250 (> 5%)</td><td align=\\\"left\\\"> < 100 (< 5%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\">3273 (60.4%)</td><td align=\\\"left\\\">1161 (21.4%)</td><td align=\\\"left\\\">981 (18.1%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>c</sup></td><td align=\\\"left\\\">48 (55.2%)</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Educational level</td></tr><tr><td align=\\\"left\\\"> College or more</td><td align=\\\"left\\\">3097 (34.5%)</td><td align=\\\"left\\\">4133 (46.0%)</td><td align=\\\"left\\\"> > 1000 (> 10%)</td><td align=\\\"left\\\"> > 500 (> 5%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ≤ High school or equivalent</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\">160 (16.2%)</td><td align=\\\"left\\\"> > 300 (> 30%)</td><td align=\\\"left\\\">484 (49.1%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>c</sup></td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\">22 (40.0%)</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\"> ≤ 20 (< 20%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Insured</td></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\">29 (22.1%)</td><td align=\\\"left\\\"> > 50 (> 50%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\"> > 2500 (> 30%)</td><td align=\\\"left\\\"> > 4000 (> 40%)</td><td align=\\\"left\\\">1463 (14.9%)</td><td align=\\\"left\\\">975 (9.9%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>d</sup></td><td align=\\\"left\\\">20 (25.3%)</td><td align=\\\"left\\\"> > 20 (30%)</td><td align=\\\"left\\\">22 (27.8%)</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Treatment</td></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\"> > 2300 (> 25%)</td><td align=\\\"left\\\"> > 3300 (> 40%)</td><td align=\\\"left\\\"> > 1100 (> 10%)</td><td align=\\\"left\\\"> > 800 (> 10%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.01</td></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">796 (34.1%)</td><td align=\\\"left\\\">953 (40.8%)</td><td align=\\\"left\\\">356 (15.2%)</td><td align=\\\"left\\\">233 (10.0%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Missing</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\"> ≤ 20 (< 50%)</td><td align=\\\"left\\\"> ≤ 20 (< 25%)</td><td align=\\\"left\\\"> ≤ 20 (< 10%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Nativity</td></tr><tr><td align=\\\"left\\\"> US-Born</td><td align=\\\"left\\\">2869 (31.2%)</td><td align=\\\"left\\\">4003 (43.5%)</td><td align=\\\"left\\\">1379 (15.0%)</td><td align=\\\"left\\\">955 (10.4%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Foreign-Born</td><td align=\\\"left\\\">243 (29.8%)</td><td align=\\\"left\\\">312 (38.3%)</td><td align=\\\"left\\\">135 (16.6%)</td><td align=\\\"left\\\">125 (15.3%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Housing status</td></tr><tr><td align=\\\"left\\\"> Own</td><td align=\\\"left\\\"> > 3000 (> 40%)</td><td align=\\\"left\\\">3534 (47.6%)</td><td align=\\\"left\\\"> < 800 (< 10%)</td><td align=\\\"left\\\">125 (1.7%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Rent/other arrangements</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\">734 (29.6%)</td><td align=\\\"left\\\">793 (32.0%)</td><td align=\\\"left\\\"> > 800 (> 35%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>c</sup></td><td align=\\\"left\\\">50 (40.0%)</td><td align=\\\"left\\\">47 (37.6%)</td><td align=\\\"left\\\"> > 20 (> 17%)</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Cancer type</td></tr><tr><td align=\\\"left\\\"> Bladder</td><td align=\\\"left\\\">65 (26.5%)</td><td align=\\\"left\\\">105 (42.9%)</td><td align=\\\"left\\\">53 (21.6%)</td><td align=\\\"left\\\">22 (9.0%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Blood</td><td align=\\\"left\\\">238 (31.3%)</td><td align=\\\"left\\\">350 (46.1%)</td><td align=\\\"left\\\">111 (14.6%)</td><td align=\\\"left\\\">61 (8.0%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Bone</td><td align=\\\"left\\\"> > 25 (> 25%)</td><td align=\\\"left\\\">39 (36.5%)</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Brain</td><td align=\\\"left\\\">35 (27.8%)</td><td align=\\\"left\\\">49 (38.9%)</td><td align=\\\"left\\\">22 (17.5%)</td><td align=\\\"left\\\">20 (15.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Breast</td><td align=\\\"left\\\">1031 (34.8%)</td><td align=\\\"left\\\">1283 (43.3%)</td><td align=\\\"left\\\">408 (13.8%)</td><td align=\\\"left\\\">240 (8.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Cervical</td><td align=\\\"left\\\">188 (27.3%)</td><td align=\\\"left\\\">202 (29.4%)</td><td align=\\\"left\\\">126 (18.3%)</td><td align=\\\"left\\\">172 (25.0%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Colon rectal</td><td align=\\\"left\\\">131 (30.6%)</td><td align=\\\"left\\\">168 (39.3%)</td><td align=\\\"left\\\">73 (17.1%)</td><td align=\\\"left\\\">56 (13.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Endocrine</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\">31 (46.3%)</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\"> ≤ 20 (< 30%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Endometrial</td><td align=\\\"left\\\">70 (25.6%)</td><td align=\\\"left\\\">128 (46.7%)</td><td align=\\\"left\\\">50 (18.3%)</td><td align=\\\"left\\\">26 (9.5%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Head neck</td><td align=\\\"left\\\"> > 50 (> 30%)</td><td align=\\\"left\\\">79 (42.9%)</td><td align=\\\"left\\\">25 (13.6%)</td><td align=\\\"left\\\"> ≤ 20 (< 10%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Kidney</td><td align=\\\"left\\\">89 (30.4%)</td><td align=\\\"left\\\">122 (41.6%)</td><td align=\\\"left\\\">48 (16.4%)</td><td align=\\\"left\\\">34 (11.6%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Lung</td><td align=\\\"left\\\">39 (16.1%)</td><td align=\\\"left\\\">109 (45.0%)</td><td align=\\\"left\\\">55 (21.7%)</td><td align=\\\"left\\\">39 (16.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Other</td><td align=\\\"left\\\">427 (29.8%)</td><td align=\\\"left\\\">611 (42.6%)</td><td align=\\\"left\\\">211 (14.7%)</td><td align=\\\"left\\\">186 (13.0%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Ovarian</td><td align=\\\"left\\\">60 (27.3%)</td><td align=\\\"left\\\">89 (41.5%)</td><td align=\\\"left\\\">29 (13.2%)</td><td align=\\\"left\\\">42 (19.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Prostate</td><td align=\\\"left\\\">382 (28.3%)</td><td align=\\\"left\\\">727 (53.9%)</td><td align=\\\"left\\\">165 (12.2%)</td><td align=\\\"left\\\">74 (5.5%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Thyroid</td><td align=\\\"left\\\">245 (38.2%)</td><td align=\\\"left\\\">223 (34.7%)</td><td align=\\\"left\\\">104 (16.2%)</td><td align=\\\"left\\\">70 (10.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Healthcare delay</td></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\">2178 (31.6%)</td><td align=\\\"left\\\">3286 (47.7%)</td><td align=\\\"left\\\">914 (13.3%)</td><td align=\\\"left\\\">514 (7.5%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">934 (29.9%)</td><td align=\\\"left\\\">1028 (32.9%)</td><td align=\\\"left\\\">600 (19.2%)</td><td align=\\\"left\\\">566 (18.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"6\\\">Health literacy</td></tr><tr><td align=\\\"left\\\"> ≤ 9</td><td align=\\\"left\\\"> ≤ 20 (< 10%)</td><td align=\\\"left\\\">52 (25.1%)</td><td align=\\\"left\\\"> > 20 (10%)</td><td align=\\\"left\\\">126 (44.9%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> > 9</td><td align=\\\"left\\\">3048 (31.8%)</td><td align=\\\"left\\\">4188 (43.7%)</td><td align=\\\"left\\\">1435 (14.9%)</td><td align=\\\"left\\\">922 (9.6%)</td><td align=\\\"left\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Descriptive characteristics of the sample and their association with healthcare delays (<italic>n</italic> = 10,020)</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Variables</th><th align=\\\"left\\\">No healthcare delay<break/>(<italic>n</italic> = 6,892)</th><th align=\\\"left\\\">Any healthcare delay (<italic>n</italic> = 3,128)</th><th align=\\\"left\\\"><italic>p</italic> value</th></tr></thead><tbody><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Sex</td></tr><tr><td align=\\\"left\\\"> Female</td><td align=\\\"left\\\">4239 (64.0%)</td><td align=\\\"left\\\">2384 (36.0%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Male</td><td align=\\\"left\\\">2608 (78.3%)</td><td align=\\\"left\\\">724 (21.7%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>a</sup></td><td align=\\\"left\\\">45 (69.2%)</td><td align=\\\"left\\\">20 (30.8%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Age</td></tr><tr><td align=\\\"left\\\"> Median [Q1, Q3]</td><td align=\\\"left\\\">66.4 [579, 72.3]</td><td align=\\\"left\\\">57.8 [46.7, 65.7]</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Race/ethnicity</td></tr><tr><td align=\\\"left\\\"> Asian</td><td align=\\\"left\\\">132 (67.4%)</td><td align=\\\"left\\\">64 (32.7%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Black</td><td align=\\\"left\\\">374 (58.3%)</td><td align=\\\"left\\\">267 (41.7%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Hispanic</td><td align=\\\"left\\\">345 (56.1%)</td><td align=\\\"left\\\">270 (43.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> White</td><td align=\\\"left\\\">5856 (71.0%)</td><td align=\\\"left\\\">2387 (29.0%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> More than one pop</td><td align=\\\"left\\\">89 (59.3%)</td><td align=\\\"left\\\">61 (40.7%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Other<sup>b</sup></td><td align=\\\"left\\\">96 (54.9%)</td><td align=\\\"left\\\">79 (45.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Annual income<sup>e</sup></td></tr><tr><td align=\\\"left\\\"> < 35 K</td><td align=\\\"left\\\">914 (50.0%)</td><td align=\\\"left\\\">916 (50.0%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ≥ 35 K</td><td align=\\\"left\\\">5300 (73.3%)</td><td align=\\\"left\\\">1930 (26.7%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>c</sup></td><td align=\\\"left\\\">678 (70.6%)</td><td align=\\\"left\\\">282 (29.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Marital status</td></tr><tr><td align=\\\"left\\\"> Single</td><td align=\\\"left\\\">2103 (60.8%)</td><td align=\\\"left\\\">1357 (39.2%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Married/living with a partner</td><td align=\\\"left\\\">4734 (73.2%)</td><td align=\\\"left\\\">1732 (26.8%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>c</sup></td><td align=\\\"left\\\">55 (58.5%)</td><td align=\\\"left\\\">39 (41.5%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Employed</td></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">2911 (64.4%)</td><td align=\\\"left\\\">1608 (35.6%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\">3923 (72.5%)</td><td align=\\\"left\\\">1491 (27.6%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>c</sup></td><td align=\\\"left\\\">58 (66.7%)</td><td align=\\\"left\\\">29 (33.3%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Educational level</td></tr><tr><td align=\\\"left\\\"> College or more</td><td align=\\\"left\\\">6273 (69.9%)</td><td align=\\\"left\\\">2707 (30.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> ≤ High school or equivalent</td><td align=\\\"left\\\">585 (59.3%)</td><td align=\\\"left\\\">401 (40.7%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>c</sup></td><td align=\\\"left\\\">34 (61.8%)</td><td align=\\\"left\\\">21 (38.2%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Insured</td></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\">50 (38.2%)</td><td align=\\\"left\\\">81 (61.8%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">6798 (69.3%)</td><td align=\\\"left\\\">3012 (30.7%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>d</sup></td><td align=\\\"left\\\">44 (55.7%)</td><td align=\\\"left\\\">35 (44.3%)</td><td char=\\\".\\\" align=\\\"char\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Nativity</td></tr><tr><td align=\\\"left\\\"> US-Born</td><td align=\\\"left\\\">6364 (69.1%)</td><td align=\\\"left\\\">2841 (30.9%)</td><td char=\\\".\\\" align=\\\"char\\\">0.01</td></tr><tr><td align=\\\"left\\\"> Foreign-Born</td><td align=\\\"left\\\">528 (64.8%)</td><td align=\\\"left\\\">287 (35.2%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Housing status</td></tr><tr><td align=\\\"left\\\"> Own</td><td align=\\\"left\\\">5521 (74.4%)</td><td align=\\\"left\\\">1898 (25.6%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Rent/other arrangement</td><td align=\\\"left\\\">1296 (52.3%)</td><td align=\\\"left\\\">1180 (47.7%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Other<sup>c</sup></td><td align=\\\"left\\\">75 (60.0%)</td><td align=\\\"left\\\">50 (40.0%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Treatment</td></tr><tr><td align=\\\"left\\\"> No</td><td align=\\\"left\\\"> > 5200 (> 65%)</td><td align=\\\"left\\\"> > 2300 (> 30%)</td><td char=\\\".\\\" align=\\\"char\\\">0.96</td></tr><tr><td align=\\\"left\\\"> Yes</td><td align=\\\"left\\\">1610 (68.9%)</td><td align=\\\"left\\\">728 (31.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Missing</td><td align=\\\"left\\\"> ≤ 13 (< 65%)</td><td align=\\\"left\\\"> ≤ 20 (< 40%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Cancer type</td></tr><tr><td align=\\\"left\\\"> Bladder</td><td align=\\\"left\\\">193 (78.8%)</td><td align=\\\"left\\\">52 (21.2%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> Blood</td><td align=\\\"left\\\">549 (72.2%)</td><td align=\\\"left\\\">210 (27.8%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Bone</td><td align=\\\"left\\\">67 (62.6%)</td><td align=\\\"left\\\">40 (37.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Brain</td><td align=\\\"left\\\">72 (57.1%)</td><td align=\\\"left\\\">54 (42.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Breast</td><td align=\\\"left\\\">2076 (70.1%)</td><td align=\\\"left\\\">886 (42.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Cervical</td><td align=\\\"left\\\">316 (45.9%)</td><td align=\\\"left\\\">372 (54.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Colon rectal</td><td align=\\\"left\\\">295 (68.9%)</td><td align=\\\"left\\\">133 (31.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Endocrine</td><td align=\\\"left\\\">46 (68.7%)</td><td align=\\\"left\\\">21 (31.3%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Endometrial</td><td align=\\\"left\\\">172 (62.8%)</td><td align=\\\"left\\\">102 (37.2%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Head neck</td><td align=\\\"left\\\">129 (70.1%)</td><td align=\\\"left\\\">55 (29.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Kidney</td><td align=\\\"left\\\">198 (67.6%)</td><td align=\\\"left\\\">95 (32.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Lung</td><td align=\\\"left\\\">176 (72.7%)</td><td align=\\\"left\\\">66 (27.3%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Other</td><td align=\\\"left\\\">979 (68.2%)</td><td align=\\\"left\\\">456 (31.8%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Ovarian</td><td align=\\\"left\\\">134 (60.9%)</td><td align=\\\"left\\\">86 (39.1%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Prostate</td><td align=\\\"left\\\">1120 (83.1%)</td><td align=\\\"left\\\">228 (16.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> Thyroid</td><td align=\\\"left\\\">370 (57.6%)</td><td align=\\\"left\\\">272 (42.4%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Health literacy</td></tr><tr><td align=\\\"left\\\"> ≤ 9</td><td align=\\\"left\\\">178 (55.1%)</td><td align=\\\"left\\\">145 (44.9%)</td><td char=\\\".\\\" align=\\\"char\\\"> < 0.001</td></tr><tr><td align=\\\"left\\\"> > 9</td><td align=\\\"left\\\">6714 (69.2%)</td><td align=\\\"left\\\">2983 (30.8%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">SES barrier</td></tr><tr><td align=\\\"left\\\"> 0</td><td align=\\\"left\\\">2178 (70.0%)</td><td align=\\\"left\\\">934 (30.0%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> 1</td><td align=\\\"left\\\">3286 (76.2%)</td><td align=\\\"left\\\">1028 (23.9%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> 2</td><td align=\\\"left\\\">914 (60.4%)</td><td align=\\\"left\\\">600 (39.6%)</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\"> 3+</td><td align=\\\"left\\\">514 (47.6%)</td><td align=\\\"left\\\">566 (52.4%)</td><td align=\\\"left\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Results from the multivariable regression analysis of risk factors for health care delay and by nativity status among cancer survivors from the All of Us Research Program</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Variables</th><th align=\\\"left\\\">Adjusted odds ratios<break/><italic>n</italic> = 9,820<break/>OR (95%CI)</th><th align=\\\"left\\\">US-Born<break/><italic>n</italic> = 9,022<break/>OR (95%CI)</th><th align=\\\"left\\\">Foreign-Born<break/><italic>n</italic> = 798<break/>OR (95%CI)</th></tr></thead><tbody><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Nativity</td></tr><tr><td align=\\\"left\\\"> US-Born</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\"> Foreign-Born</td><td align=\\\"left\\\">1.04 (0.87–1.25)</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">Health literacy</td></tr><tr><td align=\\\"left\\\"> p-trend</td><td align=\\\"left\\\">0.92 (0.89–0.95)<italic>*</italic>*<italic>*</italic></td><td align=\\\"left\\\">0.91(0.88–0.94)***</td><td align=\\\"left\\\">0.98(0.90–1.07)</td></tr><tr><td align=\\\"left\\\"> ≤ 9</td><td align=\\\"left\\\">1.20 (0.89–1.59)</td><td align=\\\"left\\\">1.41 (1.02–1.97)*</td><td align=\\\"left\\\">0.66 (0.34–1.25)</td></tr><tr><td align=\\\"left\\\"> > 9</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">Ref</td></tr><tr><td align=\\\"left\\\" colspan=\\\"4\\\">SES barrier factors index</td></tr><tr><td align=\\\"left\\\"> p-trend</td><td align=\\\"left\\\">1.29 (1.23–1.36)***</td><td align=\\\"left\\\">1.27 (1.21–1.34)***</td><td align=\\\"left\\\">1.72 (1.43–2.08)***</td></tr><tr><td align=\\\"left\\\"> 0</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">Ref</td><td align=\\\"left\\\">Ref</td></tr><tr><td align=\\\"left\\\"> 1</td><td align=\\\"left\\\">0.98 (0.88–1.10)</td><td align=\\\"left\\\">0.97 (0.86–1.09)</td><td align=\\\"left\\\">1.15 (0.76–1.74)</td></tr><tr><td align=\\\"left\\\"> 2</td><td align=\\\"left\\\">1.65 (1.43–1.90)***</td><td align=\\\"left\\\">1.53 (1.32–1.78)***</td><td align=\\\"left\\\">4.35 (2.61–7.34)***</td></tr><tr><td align=\\\"left\\\"> 3+</td><td align=\\\"left\\\">2.18 (1.84–2.58)***</td><td align=\\\"left\\\">2.10 (1.76–2.50)***</td><td align=\\\"left\\\">3.83 (2.14–6.98)***</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p>Single = includes those who reported: divorced, widowed, separated, and never married. p-values were obtained from chi-square and Kruskal–Wallis tests. Per “All of Us” data use agreement policy, groups < 20 participants are shown as ≤ 20 (%) with a corresponding > (%) category to prevent deriving counts < 20 from other values. No all percentages equal to 100</p><p>Q1 = Quartile 1(25%), Q3 = Quartile 3(75%)</p><p><italic>SES</italic> socioeconomic include (education, income, insurance, housing, and employment status)</p><p>Data values included in these categories: <sup>a</sup>Other and missing</p><p><sup>b</sup>None of this, another population, and prefer not to answer</p><p><sup>c</sup>Prefer not to answer and missing</p><p><sup>d</sup>Missing, do not know, and prefer not to answer, NA = Missing</p><p><sup>e</sup>Income reported in US dollar, Cancer type “other” also includes esophageal, eye, pancreatic, and stomach cancers</p></table-wrap-foot>","<table-wrap-foot><p>Single = includes those who reported: divorced, widowed, separated, and never married. <italic>p</italic> values were obtained from chi-square and Mann–Whitney tests. Any healthcare delay includes delays due to transportation, living in a rural area where healthcare providers are too far, nervousness about seeing a healthcare provider, could not get time off work, could not get childcare, cannot leave adult unattended due to being a caretaker, could not afford copays, deductible was too high or could not afford it or had to pay out of pocket for some or all procedures. Per “All of Us” data use agreement policy, groups < 20 participants are shown as ≤ 20 (%) with a corresponding > (%) category to prevent deriving counts < 20 from other values. No all percentages equal to 100</p><p>Q1 = Quartile 1(25%), Q3 = Quartile 3(75%)</p><p><italic>SES</italic> socioeconomic</p><p>Data values included in these categories:<sup>a</sup>Other and missing</p><p><sup>b</sup>None of this, another population, and prefer not to answer</p><p><sup>c</sup>Prefer not to answer and missing</p><p><sup>d</sup>Missing, do not know, and prefer not to answer, NA = Missing</p><p><sup>e</sup>Income reported in US dollar, Cancer type “other”: includes esophageal, eye, pancreatic, and stomach cancers</p></table-wrap-foot>","<table-wrap-foot><p>p-trends were obtained by assessing SES barriers and Health Literacy as continuous measures</p><p>Adjusted odds ratios (OR) for: sex, race/ethnicity, age, marital status, active treatment, and cancer type</p><p><italic>SES</italic> socioeconomic, <italic>Ref</italic> reference group, <italic>CI</italic> confidence interval</p><p>Significant <italic>p</italic> values ***< 0.001, **< 0.01, *< 0.05</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>Single = includes those who reported: divorced, widowed, separated, and never married. p-values were obtained from chi-square and Kruskal–Wallis tests. Per “All of Us” data use agreement policy, groups < 20 participants are shown as ≤ 20 (%) with a corresponding > (%) category to prevent deriving counts < 20 from other values. No all percentages equal to 100</p><p>Q1 = Quartile 1(25%), Q3 = Quartile 3(75%)</p><p><italic>SES</italic> socioeconomic include (education, income, insurance, housing, and employment status)</p><p>Data values included in these categories: <sup>a</sup>Other and missing</p><p><sup>b</sup>None of this, another population, and prefer not to answer</p><p><sup>c</sup>Prefer not to answer and missing</p><p><sup>d</sup>Missing, do not know, and prefer not to answer, NA = Missing</p><p><sup>e</sup>Income reported in US dollar, Cancer type “other” also includes esophageal, eye, pancreatic, and stomach cancers</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>Single = includes those who reported: divorced, widowed, separated, and never married. <italic>p</italic> values were obtained from chi-square and Mann–Whitney tests. Any healthcare delay includes delays due to transportation, living in a rural area where healthcare providers are too far, nervousness about seeing a healthcare provider, could not get time off work, could not get childcare, cannot leave adult unattended due to being a caretaker, could not afford copays, deductible was too high or could not afford it or had to pay out of pocket for some or all procedures. Per “All of Us” data use agreement policy, groups < 20 participants are shown as ≤ 20 (%) with a corresponding > (%) category to prevent deriving counts < 20 from other values. No all percentages equal to 100</p><p>Q1 = Quartile 1(25%), Q3 = Quartile 3(75%)</p><p><italic>SES</italic> socioeconomic</p><p>Data values included in these categories:<sup>a</sup>Other and missing</p><p><sup>b</sup>None of this, another population, and prefer not to answer</p><p><sup>c</sup>Prefer not to answer and missing</p><p><sup>d</sup>Missing, do not know, and prefer not to answer, NA = Missing</p><p><sup>e</sup>Income reported in US dollar, Cancer type “other”: includes esophageal, eye, pancreatic, and stomach cancers</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>p-trends were obtained by assessing SES barriers and Health Literacy as continuous measures</p><p>Adjusted odds ratios (OR) for: sex, race/ethnicity, age, marital status, active treatment, and cancer type</p><p><italic>SES</italic> socioeconomic, <italic>Ref</italic> reference group, <italic>CI</italic> confidence interval</p><p>Significant <italic>p</italic> values ***< 0.001, **< 0.01, *< 0.05</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"10552_2023_1782_Fig1_HTML\" id=\"MO1\"/>"],"string":"[\n \"<graphic xlink:href=\\\"10552_2023_1782_Fig1_HTML\\\" id=\\\"MO1\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"10552_2023_1782_MOESM1_ESM.docx\"><caption><p>Supplementary file1 (DOCX 19 KB)</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"10552_2023_1782_MOESM1_ESM.docx\\\"><caption><p>Supplementary file1 (DOCX 19 KB)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"1.\"], \"mixed-citation\": [\"National Cancer Institute (2023) NCI dictionary of cancer terms [definition of survivor]. \"], \"ext-link\": [\"https://www.cancer.gov/publications/dictionaries/cancer-terms/def/survivor\"]}, {\"label\": [\"27.\"], \"ext-link\": [\"https://www.researchallofus.org/wp-content/themes/research-hub-wordpress-theme/media/2019/02/Overall_Health.pdf\"]}, {\"label\": [\"37.\"], \"surname\": [\"Zheng\"], \"given-names\": [\"Z\"], \"article-title\": [\"Worry about daily financial needs and food insecurity among cancer survivors in the United States\"], \"source\": [\"J Natl Compr Cancer Netw\"], \"year\": [\"2020\"], \"volume\": [\"18\"], \"fpage\": [\"315\"], \"lpage\": [\"327\"], \"pub-id\": [\"10.6004/jnccn.2019.7359\"]}, {\"label\": [\"47.\"], \"ext-link\": [\"https://www.census.gov/newsroom/press-releases/2022/educational-attainment.html\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":49,"string":"49"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Cancer Causes Control. 2024 Sep 7; 35(2):203-214"},"package_file":{"kind":"string","value":"oa_package/b4/1d/PMC10787892.tar.gz"}}},{"rowIdx":176,"cells":{"accession_id":{"kind":"string","value":"PMC10787893"},"pmid":{"kind":"string","value":"0"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par5\">As a major discovery in the twentieth century, antibiotics play an irreplaceable role in human health, animal epidemic prevention, and disease treatment (Coleman et al. ##REF##26196923##2015##). However, due to the overuse of antibiotics, a variety of bacteria have developed drug resistance, including the intensively studied <italic>Staphylococcus aureus</italic> (<italic>S. aureus</italic>) through a variety of escape routes to avoid drug access, which can cause various animal diseases such as mastitis, endometritis, sepsis, and other systemic symptoms, seriously endangering the production of livestock and poultry and the development of animal husbandry (Coleman et al. ##REF##26196923##2015##; Kim et al. ##REF##31358625##2019##). Drug resistance was transmitted through the environment, humans, and livestock (Davies and Davies ##REF##20805405##2010##), causing huge economic losses (El-Sayed Ahmed et al. ##UREF##1##2020##). Vancomycin is considered the last line of defense against infection by drug-resistant Gram-positive bacteria (G<sup>+</sup>), but resistance has quickly developed with the increase in drug use in clinical practice (Rao ##REF##7772805##1995##). Therefore, there is an urgent need for an antibiotic substitution to address this issue.</p>","<p id=\"Par6\">Antimicrobial peptides (AMPs), as an important part of the natural immune barrier, exhibit antibacterial characteristics, immune regulation, and multiple other functions (Shinohara et al. ##REF##7638204##1995##; Huttner and Bevins ##REF##10367766##1999##; Peters et al. ##REF##21060861##2010##; Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##). The DBAASP database contains 20,523 antibacterial peptides and derivatives (Pirtskhalava et al. ##REF##33151284##2021##), being considered a rich resource for AMPs screening (Pinto et al. ##REF##31127270##2019##). AMPs generally have the characteristics of cationic, hydrophobic, short sequence length (< 50aa), and unique three-dimensional (3D) structure including α-helix, β-sheet, and β-turn or coil; these play a vital role in the functional activity of AMPs for better understanding of the structure-activity relationship (Cao et al. ##REF##25261129##2015##; Wang et al. ##REF##29523806##2018##; Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##; Wu et al. ##REF##35725522##2022##). The AMPs, consisting of only amino acid in term of narrow definition, have multiple antibacterial mechanisms including destroying the cell membrane or wall or causing the contents to leak (Miao et al. ##UREF##8##2016##; Glukhov et al. ##REF##18186149##2008##; Jhong et al. ##REF##30380085##2019##; Lee et al. ##REF##20553419##2011##; Ma et al. ##REF##28885829##2017##); this is different from traditional heterocyclic peptide antibiotics, for which it is not easy to produce drug resistance. Therefore, they are becoming a research hotspot for the substitution of antibiotics (Gao et al. ##REF##34673132##2021##; Pinto et al. ##REF##31127270##2019##; Wu et al. ##UREF##13##2021##). Plectasin is a fungal defensin, isolated from <italic>Pseudoplectania nigrella</italic>, which can inhibit the synthesis of the bacterial cell wall by binding to the cell wall precursor Lipid II. It has a strong killing effect on G<sup>+</sup> (Mygind et al. ##REF##16222292##2005##; Schneider et al. ##REF##20508130##2010##) and the ability to treat peritonitis and pneumonia caused by <italic>Streptococcus pneumoniae</italic> <italic>and</italic>\n<italic>S.aureus</italic> (Mygind et al. ##REF##16222292##2005##). However, its high cost and low druggability limit its clinical applications. In order to solve the above problems, 12 kinds of plectasin-derived peptides were screened, analyzed, and verified in the DBAASP database (Zhang et al. ##REF##23624708##2014##, ##REF##33534018##2021##; Othman et al. ##UREF##9##2018##; Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##; Huang et al. ##REF##35524777##2022##), AP138 (DBAASPS_12115) created by Lociuro was predicted to be the best one among plectasin-derived peptides with high antimicrobial activity and a long postantibiotic effect (PAE) against G<sup>+</sup> bacteria including <italic>S. aureus</italic> and methicillin-resistant <italic>Staphylococcus aureus</italic> (MRSA) (Lociuro et al. ##UREF##6##2015##; Groo et al. ##REF##30532539##2018##). Compared with plectasin, there are three main differences in AP138: (i) Five amino acids, 9D, 13 M, 14Q, 17N, and 26 K, are mutated to 9S, 13L, 14R, 17R, and 26R, respectively; (ii) the positive charge increased from + 1 to + 4.5; (iii) the second structure α-helix (30.75 to 20.8%) and β-sheet (20.8 to 12.5%), β-turn (95.8 to 120.8%) changed obviously (Table ##TAB##0##1## and ##SUPPL##0##S1##). These studies and bioinformatics analysis all indicated that AP138 has research and development value in clinic therapeutics. However, AP138 is chemically synthesized due to D-type Arg at 26th site in sequence, so suffer from the heterologous expression difficulty and high cost (Koo and Seo ##UREF##5##2019##). Heterologous expressions may solve the problem of high cost, but the amino acid composition will be natural L-type amino acids (Lee et al. ##REF##20553419##2011##; Lin et al. ##REF##29675221##2018##). Therefore, the potential new different properties and mechanism of L-type AP138 (defined as AP138L-arg26 (DBAASPS_12115)) should be characterized as one of the major goals of this work (Groo et al. ##REF##30532539##2018##; Pinto et al. ##REF##31127270##2019##; Patel et al. ##REF##19805558##2009##; Umerska et al. ##REF##27113868##2016##; McEwen and Collignon ##UREF##7##2018##).\n</p>","<p id=\"Par7\">In this study, plectasin-derived peptide AP138L-arg26 was expressed in <italic>Pichia pastoris</italic> (<italic>P. pastoris</italic>) cells X-33 with a 5-L fermenter, and in vitro analysis of structure, antibacterial activity, stability, drug resistance, toxicity, and safety was performed. Finally, its bactericidal mechanism against <italic>S. aureus</italic> was revealed.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">As a major discovery in the twentieth century, antibiotics play an irreplaceable role in human health, animal epidemic prevention, and disease treatment (Coleman et al. ##REF##26196923##2015##). However, due to the overuse of antibiotics, a variety of bacteria have developed drug resistance, including the intensively studied <italic>Staphylococcus aureus</italic> (<italic>S. aureus</italic>) through a variety of escape routes to avoid drug access, which can cause various animal diseases such as mastitis, endometritis, sepsis, and other systemic symptoms, seriously endangering the production of livestock and poultry and the development of animal husbandry (Coleman et al. ##REF##26196923##2015##; Kim et al. ##REF##31358625##2019##). Drug resistance was transmitted through the environment, humans, and livestock (Davies and Davies ##REF##20805405##2010##), causing huge economic losses (El-Sayed Ahmed et al. ##UREF##1##2020##). Vancomycin is considered the last line of defense against infection by drug-resistant Gram-positive bacteria (G<sup>+</sup>), but resistance has quickly developed with the increase in drug use in clinical practice (Rao ##REF##7772805##1995##). Therefore, there is an urgent need for an antibiotic substitution to address this issue.</p>\",\n \"<p id=\\\"Par6\\\">Antimicrobial peptides (AMPs), as an important part of the natural immune barrier, exhibit antibacterial characteristics, immune regulation, and multiple other functions (Shinohara et al. ##REF##7638204##1995##; Huttner and Bevins ##REF##10367766##1999##; Peters et al. ##REF##21060861##2010##; Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##). The DBAASP database contains 20,523 antibacterial peptides and derivatives (Pirtskhalava et al. ##REF##33151284##2021##), being considered a rich resource for AMPs screening (Pinto et al. ##REF##31127270##2019##). AMPs generally have the characteristics of cationic, hydrophobic, short sequence length (< 50aa), and unique three-dimensional (3D) structure including α-helix, β-sheet, and β-turn or coil; these play a vital role in the functional activity of AMPs for better understanding of the structure-activity relationship (Cao et al. ##REF##25261129##2015##; Wang et al. ##REF##29523806##2018##; Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##; Wu et al. ##REF##35725522##2022##). The AMPs, consisting of only amino acid in term of narrow definition, have multiple antibacterial mechanisms including destroying the cell membrane or wall or causing the contents to leak (Miao et al. ##UREF##8##2016##; Glukhov et al. ##REF##18186149##2008##; Jhong et al. ##REF##30380085##2019##; Lee et al. ##REF##20553419##2011##; Ma et al. ##REF##28885829##2017##); this is different from traditional heterocyclic peptide antibiotics, for which it is not easy to produce drug resistance. Therefore, they are becoming a research hotspot for the substitution of antibiotics (Gao et al. ##REF##34673132##2021##; Pinto et al. ##REF##31127270##2019##; Wu et al. ##UREF##13##2021##). Plectasin is a fungal defensin, isolated from <italic>Pseudoplectania nigrella</italic>, which can inhibit the synthesis of the bacterial cell wall by binding to the cell wall precursor Lipid II. It has a strong killing effect on G<sup>+</sup> (Mygind et al. ##REF##16222292##2005##; Schneider et al. ##REF##20508130##2010##) and the ability to treat peritonitis and pneumonia caused by <italic>Streptococcus pneumoniae</italic> <italic>and</italic>\\n<italic>S.aureus</italic> (Mygind et al. ##REF##16222292##2005##). However, its high cost and low druggability limit its clinical applications. In order to solve the above problems, 12 kinds of plectasin-derived peptides were screened, analyzed, and verified in the DBAASP database (Zhang et al. ##REF##23624708##2014##, ##REF##33534018##2021##; Othman et al. ##UREF##9##2018##; Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##; Huang et al. ##REF##35524777##2022##), AP138 (DBAASPS_12115) created by Lociuro was predicted to be the best one among plectasin-derived peptides with high antimicrobial activity and a long postantibiotic effect (PAE) against G<sup>+</sup> bacteria including <italic>S. aureus</italic> and methicillin-resistant <italic>Staphylococcus aureus</italic> (MRSA) (Lociuro et al. ##UREF##6##2015##; Groo et al. ##REF##30532539##2018##). Compared with plectasin, there are three main differences in AP138: (i) Five amino acids, 9D, 13 M, 14Q, 17N, and 26 K, are mutated to 9S, 13L, 14R, 17R, and 26R, respectively; (ii) the positive charge increased from + 1 to + 4.5; (iii) the second structure α-helix (30.75 to 20.8%) and β-sheet (20.8 to 12.5%), β-turn (95.8 to 120.8%) changed obviously (Table ##TAB##0##1## and ##SUPPL##0##S1##). These studies and bioinformatics analysis all indicated that AP138 has research and development value in clinic therapeutics. However, AP138 is chemically synthesized due to D-type Arg at 26th site in sequence, so suffer from the heterologous expression difficulty and high cost (Koo and Seo ##UREF##5##2019##). Heterologous expressions may solve the problem of high cost, but the amino acid composition will be natural L-type amino acids (Lee et al. ##REF##20553419##2011##; Lin et al. ##REF##29675221##2018##). Therefore, the potential new different properties and mechanism of L-type AP138 (defined as AP138L-arg26 (DBAASPS_12115)) should be characterized as one of the major goals of this work (Groo et al. ##REF##30532539##2018##; Pinto et al. ##REF##31127270##2019##; Patel et al. ##REF##19805558##2009##; Umerska et al. ##REF##27113868##2016##; McEwen and Collignon ##UREF##7##2018##).\\n</p>\",\n \"<p id=\\\"Par7\\\">In this study, plectasin-derived peptide AP138L-arg26 was expressed in <italic>Pichia pastoris</italic> (<italic>P. pastoris</italic>) cells X-33 with a 5-L fermenter, and in vitro analysis of structure, antibacterial activity, stability, drug resistance, toxicity, and safety was performed. Finally, its bactericidal mechanism against <italic>S. aureus</italic> was revealed.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<title>Strains and cell lines</title>","<p id=\"Par8\">Gram-positive bacteria: <italic>S. aureus</italic> (ATCC 25923, 43300), <italic>Staphylococcus epidermidis</italic> (<italic>S. epidermidis</italic>) (ATCC 12228, 35984), and <italic>Streptococcus agalactiae</italic> (<italic>S. agalactiae</italic>) ATCC 13813 were purchased from American Type Culture Collection (ATCC). <italic>S. aureus</italic> CVCC 546 and <italic>Streptococcus dysgalactiae</italic> (<italic>S. dysgalactiae</italic>) CVCC 3938 were purchased from the China Veterinary Culture Collection Center (CVCC). <italic>S. aureus</italic> E48 was donated by Northwest Agriculture and Forestry University<italic>. S. agalactiae</italic> CAU-FRI-2022-01 and <italic>S. agalactiae</italic> CAU-FRI-2022-02 were donated by China Agricultural University. Gram-negative bacteria: <italic>Escherichia coli</italic> (<italic>E. coli</italic>) ATCC 25922 were purchased from ATCC. <italic>Salmonella enteritidis</italic> (<italic>S. enteritidis</italic>) CVCC 3377 and <italic>Salmonella pullorum</italic> (<italic>S. pullorum</italic>) CVCC1789 were purchased from CVCC. <italic>Shigella flexneri</italic> (<italic>S. flexneri</italic>) (CMCC 3926, 51571) were purchased from National Center for Medical Culture Collections (CMCC). <italic>E. coli</italic> O157 (CICC 21530), <italic>Pseudomonas aeruginosa</italic> (<italic>P. aeruginosa</italic>) (CICC 21625, CICC21630) were purchased from China Center of Industrial Culture Collection (CICC). <italic>S. aureus</italic> CAAS-FRI-2023-01 <italic>and</italic> CAAS-FRI-2023-02 were separated from Tianjin Aoxin Animal Husbandry Sheep Farm and Huanxian Sheep Farm, respectively. <italic>E. coli</italic> DH5α, <italic>P. pastoris</italic> X-33, and pPICZαA were purchased from Invitrogen (Beijing, China). RAW 264.7 mice macrophages were obtained from Peking Union Medical College. Bovine endometrial epithelial cell line BNCC35923 was purchased from by BeNa Culture Collection (Beijing, China).</p>","<title>Reagents</title>","<p id=\"Par9\">The recombinant plasmid pPICZαA-AP138L-arg26 was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Plasmid extraction kits, antibiotics (vancomycin and ceftiofur sodium), Dulbecco’s modified Eagle medium (DMEM), and fetal bovine serum (FBS) were purchased from Tiangen Co., Ltd, China Meilungel and Gibco (China), respectively. Other reagents were analytical grade.</p>","<title>Model animal</title>","<p id=\"Par10\">The female ICR mice (SPF, 6–8 weeks, 20–25 g/mouse) were purchased from the Vital River Laboratories (VRL, Beijing, China). Mice acclimated to the environment for 1 week before the experiment. Animal experiments strictly complied with the requirements for animal handling and welfare of the Laboratory Animal Ethical Committee and its Inspection of the Feed Research Institute of Chinese Academy of Agricultural Sciences (CAAS) (AEC-CAAS-20090609).</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<title>Strains and cell lines</title>\",\n \"<p id=\\\"Par8\\\">Gram-positive bacteria: <italic>S. aureus</italic> (ATCC 25923, 43300), <italic>Staphylococcus epidermidis</italic> (<italic>S. epidermidis</italic>) (ATCC 12228, 35984), and <italic>Streptococcus agalactiae</italic> (<italic>S. agalactiae</italic>) ATCC 13813 were purchased from American Type Culture Collection (ATCC). <italic>S. aureus</italic> CVCC 546 and <italic>Streptococcus dysgalactiae</italic> (<italic>S. dysgalactiae</italic>) CVCC 3938 were purchased from the China Veterinary Culture Collection Center (CVCC). <italic>S. aureus</italic> E48 was donated by Northwest Agriculture and Forestry University<italic>. S. agalactiae</italic> CAU-FRI-2022-01 and <italic>S. agalactiae</italic> CAU-FRI-2022-02 were donated by China Agricultural University. Gram-negative bacteria: <italic>Escherichia coli</italic> (<italic>E. coli</italic>) ATCC 25922 were purchased from ATCC. <italic>Salmonella enteritidis</italic> (<italic>S. enteritidis</italic>) CVCC 3377 and <italic>Salmonella pullorum</italic> (<italic>S. pullorum</italic>) CVCC1789 were purchased from CVCC. <italic>Shigella flexneri</italic> (<italic>S. flexneri</italic>) (CMCC 3926, 51571) were purchased from National Center for Medical Culture Collections (CMCC). <italic>E. coli</italic> O157 (CICC 21530), <italic>Pseudomonas aeruginosa</italic> (<italic>P. aeruginosa</italic>) (CICC 21625, CICC21630) were purchased from China Center of Industrial Culture Collection (CICC). <italic>S. aureus</italic> CAAS-FRI-2023-01 <italic>and</italic> CAAS-FRI-2023-02 were separated from Tianjin Aoxin Animal Husbandry Sheep Farm and Huanxian Sheep Farm, respectively. <italic>E. coli</italic> DH5α, <italic>P. pastoris</italic> X-33, and pPICZαA were purchased from Invitrogen (Beijing, China). RAW 264.7 mice macrophages were obtained from Peking Union Medical College. Bovine endometrial epithelial cell line BNCC35923 was purchased from by BeNa Culture Collection (Beijing, China).</p>\",\n \"<title>Reagents</title>\",\n \"<p id=\\\"Par9\\\">The recombinant plasmid pPICZαA-AP138L-arg26 was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Plasmid extraction kits, antibiotics (vancomycin and ceftiofur sodium), Dulbecco’s modified Eagle medium (DMEM), and fetal bovine serum (FBS) were purchased from Tiangen Co., Ltd, China Meilungel and Gibco (China), respectively. Other reagents were analytical grade.</p>\",\n \"<title>Model animal</title>\",\n \"<p id=\\\"Par10\\\">The female ICR mice (SPF, 6–8 weeks, 20–25 g/mouse) were purchased from the Vital River Laboratories (VRL, Beijing, China). Mice acclimated to the environment for 1 week before the experiment. Animal experiments strictly complied with the requirements for animal handling and welfare of the Laboratory Animal Ethical Committee and its Inspection of the Feed Research Institute of Chinese Academy of Agricultural Sciences (CAAS) (AEC-CAAS-20090609).</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Analysis of AP138 and designation of AP138L-arg26</title>","<p id=\"Par34\">Plectasin was searched for as a keyword in the DBAASP database, and 12 sequences were retrieved; these derived peptides, AP138, NZ2114, NZX, MP1102, and MP1106, and other reported peptides DLP4, ID3, and P2 were also collected. Their physicochemical properties are shown in Table ##TAB##0##1##. AP138L-arg26 (GFGCNGPWSEDDLRCHRHCKSIKGYR<sub>L26</sub>G GYCAKGGFVCKCY) was predicted to have the highest AMP possibility (0.992) and charge (+ 4.5) (Table ##TAB##0##1##) and so was considered the plectasin-derived peptide. Through secondary and 3D structure analysis, we found that AP138L-arg26 conformation changed greatly with reduced α-helix/β-sheet and increased β-turn compared with the parent peptide plectasin (Table ##TAB##0##1##). Meanwhile, the electron cloud of amino acids at the 9th, 13th, 14th, 17th, and 26th positions changed significantly with the mutant amino acids; especially at the 13th, 14th, and 17th positions, more than 2 electron clouds changed at α-Helix, which may affect the function of peptides (Table ##TAB##0##1## and Fig. ##FIG##0##1##).</p>","<title>Design, expression, and purification of AP138L-arg26</title>","<p id=\"Par35\">The recombinant plasmid pPICZαA-AP138L-arg26 was linearized and transferred into <italic>P. pastoris</italic> X-33 competent cells. The recombinant plasmid sequence length was 3000–5000 bp (Fig. ##FIG##1##2##a). The positive transformants were screened using the inhibition zone test, and transformants AP138L-arg26-8, AP138L-arg26-28, AP138L-arg26-68, and AP138L-arg26-94 had a good antibacterial effect with a large and clear inhibition zone (Fig. ##FIG##1##2##b). Tricine-SDS-PAGE analysis of AP138L-arg26 expression in shaking flask level showed that the protein band was around 4.6 kDa, which was consistent with the predicted value of 4.46 kDa (Fig. ##FIG##1##2##c). The positive transformants AP138L-arg26 with the highest effect (AP138L-arg26-8) was chosen and expressed with a 5-L fermenter, which displayed high production compared with the shaking flask (Fig. ##FIG##1##2##d, e). The total protein of fermentation supernatant was 3.1 mg/mL and the biomass was 0.36 g/mL after induction in a 5-L fermenter at 120 h (Fig. ##FIG##1##2##f). The peptide was purified using the cation-exchange column (AKTAxpress system), and only a target peptide was detected at around 4.6 kDa through Tricine-SDS-PAGE band and a single peak was detected in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) of 4464.21 Da (Fig. ##FIG##1##2##g, h).</p>","<title>MICs and MBCs</title>","<p id=\"Par36\">The results of MICs and MBCs showed that AP138L-arg26 had potent antimicrobial activity against G<sup>+</sup> bacteria such as <italic>S. aureus</italic> (2–16 µg/mL), <italic>Streptococcus</italic> (4 µg/mL), and <italic>S. epidermidi</italic><italic>s</italic> (4 or 8 µg/mL) with MIC values of 4–16 µg/mL, and the MBCs were around fourfold higher than MICs (Table ##TAB##1##2##). Antimicrobial activity was slightly lower than AP138 (Groo et al ##REF##30532539##2018##) and vancomycin, but AP138L-arg26 retained good activity. These results show that AP138L-arg26 has potential for drug development against G<sup>+</sup>.\n</p>","<title>Time killing curves and PAE</title>","<p id=\"Par37\">As shown in Fig. ##FIG##2##3##a, all selected concentrations of AP138L-arg26 (1 × , 2 × , 4 × MIC) could kill 99.99% <italic>S. aureus</italic> ATCC 43300 within 1.5 h, while the antibiotic vancomycin needed at least 6 h. Meanwhile, AP138L-arg26 had longer PAE with 0.9 h, 1.25 h, and 1.91 h at 1 × , 2 × , 4 × MIC than vancomycin (0.27, 0.45, 1.18) (Fig. ##FIG##2##3##b). These results suggest that AP138L-arg26 had a faster and longer bactericidal effect than vancomycin, even within a low concentration (1 × MIC), which may be related to the different bactericidal mechanisms of AP138L-arg26 and vancomycin.</p>","<title>Intracellular activity of AP138L-arg26 against <italic>S. aureus</italic></title>","<p id=\"Par38\">As shown in Fig. ##FIG##2##3##c, d, AP138L-arg26 could kill intracellular <italic>S. aureus</italic> CVCC 546 in mouse macrophages RAW264.7 and bovine endometrial epithelial cells BNCC359233. The peptide of AP138L-arg26 could kill more than 70% of bacteria at a concentration of 5 × MIC, and the bactericidal rate was up to 85% at 50 × MIC. Therefore, AP138L-arg26 exhibited potent intracellular bactericidal ability, laying the foundation for in vivo applications.\n</p>","<title>The synergism assay of AP138L-arg26 with antibiotic</title>","<p id=\"Par39\">As shown in Table ##TAB##2##3##, the FICI values of peptides AP138L-arg26 and vancomycin, ceftiofur sodium, ampicillin, or streptomycin sulfate ranged from 0.375 to 0.75, showing a synergistic effect. AP138L-arg26 had the lowest FICI value (0.375) with ceftiofur sodium. The bacterial growth curve results showed that AP138L-arg26 (8µg/mL, 2 × MIC) and sublethal levels of ceftiofur sodium alone (0.25 µg/mL, 1/8 MIC) had little effect on the growth of <italic>S. aureus</italic> CVCC 546 (Fig. ##FIG##2##3##e), but their combination sharply inhibited the growth of <italic>S. aureus</italic> CVCC 546. Therefore, the combination of AP138L-arg26 and ceftiofur sodium had the potential to be used to cure infection by <italic>S. aureus</italic>.\n</p>","<title>Stability in vitro assay</title>","<p id=\"Par40\">These results show that AP138L-arg26 maintained its original MIC value (8 µg/mL) after incubation at different temperatures (20 °C, 40 °C, 60 °C, 80 °C), but the antibacterial activity was lost (> 64 µg/mL) under 100 °C. The antimicrobial activity of AP138L-arg26 was unaffected by gastric juice and different pH values (2–10). AP138L-arg26 was sensitive to trypsin, losting activity within 30 min after incubation (> 64 µg/mL) (Table ##TAB##3##4##), this would be the key limitation factor at least special for its oral administration.\n</p>","<title>Structure analysis</title>","<p id=\"Par41\">The secondary structures of AP138L-arg26 were measured in a different environment of 20 mM SDS and 50% TFE solution, which was used to simulate the hydrophobic environment of eukaryotic cells and the bacterial cell membrane environment, respectively. The results showed a positive peak at 196 nm and two negative peaks at 208 nm and 228 in 50% TFE solution, indicating an α/β spatial structure in these environments (Figure ##SUPPL##0##S2##). However, the second structure of AP138L-arg26 had obviously changed (α-Helix, 4.72 to 6.06%; β-sheet, 47.78 to 42.82%; β-turn, 15.6 to 17.4%) in 20 mM SDS, especially for the increased α-Helix (1.34% increase), which could enhance membrane interactions between bacteria and AP138L-arg26 (Table ##SUPPL##0##S1##).</p>","<title>High safety of AP138L-arg26 in vitro and vivo</title>","<p id=\"Par42\">As shown in Fig. ##FIG##3##4##a, b, there was only 3% hemolysis and over 75% cell viability at a high concentration of AP138L-arg26 (256 µg/mL) in vitro, which indicated that AP138L-arg26 had low hemolysis and cytotoxicity. In vivo, mice (ICR, 6-8 weeks) were intraperitoneally injected with AP138L-arg26 at a concentration of 10 mg/kg (Fig. ##FIG##3##4##c–g). Compared with the control group, there were no significant differences in the whole blood detection indexes and biochemical indexes of the AP138L-arg26 treatment group. Moreover, the tissues of the AP138L-arg26 treatment group such as the heart, kidney, spleen, lung, and liver had no hyperemia, bleeding, hyperplasia, or other lesions in the full field, being almost same as those of the control group (Fig. ##FIG##3##4##d). These data indicate that the AP138L-arg26 peptide has excellent safety and good potential as clinical therapeutic drugs.</p>","<title>The mechanism of AP138L-arg26 against <italic>S. aureus</italic> CVCC 546</title>","<title>SEM</title>","<p id=\"Par43\">The morphology changes of bacteria after treatment with AP138L-arg26 were observed using SEM. As shown in Fig. ##FIG##4##5##a, the surface of <italic>S. aureus</italic> CVCC 546 (nearly 100%) was smooth and spherical without peptide treatment. However, 60–80% of cell structures changed significantly with surface roughness, granular secretion, perforation, and deformation after treatment with 2 × MIC peptide AP138 at 0.5, 1, and 2 h. More than 80% of <italic>S. aureus</italic> CVCC 546 had significantly changed structure at 1 h. The results showed that AP138L-arg26 could destroy the structure of <italic>S. aureus</italic>.</p>","<title>The effect of AP138L-arg26 on the cell membrane</title>","<p id=\"Par44\">According to the SEM results demonstrated that AP138L-arg26 caused disruption to the cell membrane or wall. Thus, a PI/SYTO9 assay was conducted to prove the destructive effect of AP138L-arg26 on <italic>S. aureus</italic> CVCC 546. The untreated group was almost stained green by SYTO9 (over 99%), demonstrating that they were predominantly living cells. The AP138L-arg26 treatment group could disrupt the cell membrane integrity with more than 50% cells stained red by PI (Fig. ##FIG##4##5##b). K<sup>+</sup> leakage further illustrates the disruptive effect of AP138L-arg26 on bacterial membrane, the results showed that the AP138L-arg26 treated group significantly increased the extracellular K<sup>+</sup> leakage level (0.05 mg/L) compared to the untreated bacteria (0.02 mg/L), similar to the positive control (0.1% TritonX-100) (Fig. ##FIG##4##5##d). It was also found that AP138L-arg26 could affect the membrane potential, as measured using DiSC<sub>3</sub>(5) fluorescence staining, the relative fluorescence units (RFU) values of the untreated and 1 × MIC AP138L-arg26-treated groups were around 2000, and the 4 × MIC AP138L-arg26 and nisin (positive control) groups were above 3000 (a 1000 increase) (Fig. ##FIG##4##5##c), which was mainly due to the depolarization caused by the destructive effect of the AP138L-arg26 on the bacterial membrane when the special compounds insert into lipid bilayers, they can change the membrane fluidity and disrupt the normal plasma membrane fluidity homeostasis, which further leads to leakage of cellular components and bacterial death. Therefore, a dye, Laurdan was used to measure the <italic>S. aureus</italic> membrane fluidity and quantified it using the Laurdan GP index. The results showed that the Laurdan GP of <italic>S. aureus</italic> decreased from 0.22 to 0.18 after treatment with 2 × MIC AP138L-arg26, which indicated an increase in the fluidity of the bacterial membrane due to the interaction of the AP138L-arg26 with the membrane (Fig. ##FIG##4##5##e). These results suggested that AP138L-arg26 could destroy the cell membrane of <italic>S. aureus</italic>.</p>","<title>The effect of AP138L-arg26 on bacterial metabolism</title>","<p id=\"Par45\">In order to further study whether AP138L-arg26 could affect the metabolism to kill bacteria, bacterial respiration was detected using an ATP kit. The results showed that AP138L-arg26 could increase intracellular ATP: the RLU values of 1 × , 2 × , 4 × MIC AP138L-arg26-treated groups (10,700, 11,500, and 11,900, respectively) were 7.6, 8.2, and 8.5 times higher than that of the untreated group (1400), which indicated a concentration-dependent pattern (Fig. ##FIG##5##6##a). The increase in ROS causes a series of cascading reactions such as oxidation of lipids, proteins, and damage to DNA, inducing bacteria death. After treatment with 1 × , 2 × , and 4 × MIC AP138L-arg26, the fluorescence values increased from 600 (blank control, CK) to 930, 1000, and 1400 with concentration-dependent (Fig. ##FIG##5##6##b). LDH levels decreased after antimicrobial peptide incubation. The LDH activity % of CK, 1 × , 2 × , and 4 × MIC AP138L-arg26 were 100%, 24%, 16%, and 16% respectively, which indicated a concentration-dependent pattern (Fig. ##FIG##5##6##c). The results showed that AP138 could induce the ROS generation of <italic>S. aureus</italic> CVCC 546, ultimately promoting cell death and reducing the probability of resistance generation.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Analysis of AP138 and designation of AP138L-arg26</title>\",\n \"<p id=\\\"Par34\\\">Plectasin was searched for as a keyword in the DBAASP database, and 12 sequences were retrieved; these derived peptides, AP138, NZ2114, NZX, MP1102, and MP1106, and other reported peptides DLP4, ID3, and P2 were also collected. Their physicochemical properties are shown in Table ##TAB##0##1##. AP138L-arg26 (GFGCNGPWSEDDLRCHRHCKSIKGYR<sub>L26</sub>G GYCAKGGFVCKCY) was predicted to have the highest AMP possibility (0.992) and charge (+ 4.5) (Table ##TAB##0##1##) and so was considered the plectasin-derived peptide. Through secondary and 3D structure analysis, we found that AP138L-arg26 conformation changed greatly with reduced α-helix/β-sheet and increased β-turn compared with the parent peptide plectasin (Table ##TAB##0##1##). Meanwhile, the electron cloud of amino acids at the 9th, 13th, 14th, 17th, and 26th positions changed significantly with the mutant amino acids; especially at the 13th, 14th, and 17th positions, more than 2 electron clouds changed at α-Helix, which may affect the function of peptides (Table ##TAB##0##1## and Fig. ##FIG##0##1##).</p>\",\n \"<title>Design, expression, and purification of AP138L-arg26</title>\",\n \"<p id=\\\"Par35\\\">The recombinant plasmid pPICZαA-AP138L-arg26 was linearized and transferred into <italic>P. pastoris</italic> X-33 competent cells. The recombinant plasmid sequence length was 3000–5000 bp (Fig. ##FIG##1##2##a). The positive transformants were screened using the inhibition zone test, and transformants AP138L-arg26-8, AP138L-arg26-28, AP138L-arg26-68, and AP138L-arg26-94 had a good antibacterial effect with a large and clear inhibition zone (Fig. ##FIG##1##2##b). Tricine-SDS-PAGE analysis of AP138L-arg26 expression in shaking flask level showed that the protein band was around 4.6 kDa, which was consistent with the predicted value of 4.46 kDa (Fig. ##FIG##1##2##c). The positive transformants AP138L-arg26 with the highest effect (AP138L-arg26-8) was chosen and expressed with a 5-L fermenter, which displayed high production compared with the shaking flask (Fig. ##FIG##1##2##d, e). The total protein of fermentation supernatant was 3.1 mg/mL and the biomass was 0.36 g/mL after induction in a 5-L fermenter at 120 h (Fig. ##FIG##1##2##f). The peptide was purified using the cation-exchange column (AKTAxpress system), and only a target peptide was detected at around 4.6 kDa through Tricine-SDS-PAGE band and a single peak was detected in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) of 4464.21 Da (Fig. ##FIG##1##2##g, h).</p>\",\n \"<title>MICs and MBCs</title>\",\n \"<p id=\\\"Par36\\\">The results of MICs and MBCs showed that AP138L-arg26 had potent antimicrobial activity against G<sup>+</sup> bacteria such as <italic>S. aureus</italic> (2–16 µg/mL), <italic>Streptococcus</italic> (4 µg/mL), and <italic>S. epidermidi</italic><italic>s</italic> (4 or 8 µg/mL) with MIC values of 4–16 µg/mL, and the MBCs were around fourfold higher than MICs (Table ##TAB##1##2##). Antimicrobial activity was slightly lower than AP138 (Groo et al ##REF##30532539##2018##) and vancomycin, but AP138L-arg26 retained good activity. These results show that AP138L-arg26 has potential for drug development against G<sup>+</sup>.\\n</p>\",\n \"<title>Time killing curves and PAE</title>\",\n \"<p id=\\\"Par37\\\">As shown in Fig. ##FIG##2##3##a, all selected concentrations of AP138L-arg26 (1 × , 2 × , 4 × MIC) could kill 99.99% <italic>S. aureus</italic> ATCC 43300 within 1.5 h, while the antibiotic vancomycin needed at least 6 h. Meanwhile, AP138L-arg26 had longer PAE with 0.9 h, 1.25 h, and 1.91 h at 1 × , 2 × , 4 × MIC than vancomycin (0.27, 0.45, 1.18) (Fig. ##FIG##2##3##b). These results suggest that AP138L-arg26 had a faster and longer bactericidal effect than vancomycin, even within a low concentration (1 × MIC), which may be related to the different bactericidal mechanisms of AP138L-arg26 and vancomycin.</p>\",\n \"<title>Intracellular activity of AP138L-arg26 against <italic>S. aureus</italic></title>\",\n \"<p id=\\\"Par38\\\">As shown in Fig. ##FIG##2##3##c, d, AP138L-arg26 could kill intracellular <italic>S. aureus</italic> CVCC 546 in mouse macrophages RAW264.7 and bovine endometrial epithelial cells BNCC359233. The peptide of AP138L-arg26 could kill more than 70% of bacteria at a concentration of 5 × MIC, and the bactericidal rate was up to 85% at 50 × MIC. Therefore, AP138L-arg26 exhibited potent intracellular bactericidal ability, laying the foundation for in vivo applications.\\n</p>\",\n \"<title>The synergism assay of AP138L-arg26 with antibiotic</title>\",\n \"<p id=\\\"Par39\\\">As shown in Table ##TAB##2##3##, the FICI values of peptides AP138L-arg26 and vancomycin, ceftiofur sodium, ampicillin, or streptomycin sulfate ranged from 0.375 to 0.75, showing a synergistic effect. AP138L-arg26 had the lowest FICI value (0.375) with ceftiofur sodium. The bacterial growth curve results showed that AP138L-arg26 (8µg/mL, 2 × MIC) and sublethal levels of ceftiofur sodium alone (0.25 µg/mL, 1/8 MIC) had little effect on the growth of <italic>S. aureus</italic> CVCC 546 (Fig. ##FIG##2##3##e), but their combination sharply inhibited the growth of <italic>S. aureus</italic> CVCC 546. Therefore, the combination of AP138L-arg26 and ceftiofur sodium had the potential to be used to cure infection by <italic>S. aureus</italic>.\\n</p>\",\n \"<title>Stability in vitro assay</title>\",\n \"<p id=\\\"Par40\\\">These results show that AP138L-arg26 maintained its original MIC value (8 µg/mL) after incubation at different temperatures (20 °C, 40 °C, 60 °C, 80 °C), but the antibacterial activity was lost (> 64 µg/mL) under 100 °C. The antimicrobial activity of AP138L-arg26 was unaffected by gastric juice and different pH values (2–10). AP138L-arg26 was sensitive to trypsin, losting activity within 30 min after incubation (> 64 µg/mL) (Table ##TAB##3##4##), this would be the key limitation factor at least special for its oral administration.\\n</p>\",\n \"<title>Structure analysis</title>\",\n \"<p id=\\\"Par41\\\">The secondary structures of AP138L-arg26 were measured in a different environment of 20 mM SDS and 50% TFE solution, which was used to simulate the hydrophobic environment of eukaryotic cells and the bacterial cell membrane environment, respectively. The results showed a positive peak at 196 nm and two negative peaks at 208 nm and 228 in 50% TFE solution, indicating an α/β spatial structure in these environments (Figure ##SUPPL##0##S2##). However, the second structure of AP138L-arg26 had obviously changed (α-Helix, 4.72 to 6.06%; β-sheet, 47.78 to 42.82%; β-turn, 15.6 to 17.4%) in 20 mM SDS, especially for the increased α-Helix (1.34% increase), which could enhance membrane interactions between bacteria and AP138L-arg26 (Table ##SUPPL##0##S1##).</p>\",\n \"<title>High safety of AP138L-arg26 in vitro and vivo</title>\",\n \"<p id=\\\"Par42\\\">As shown in Fig. ##FIG##3##4##a, b, there was only 3% hemolysis and over 75% cell viability at a high concentration of AP138L-arg26 (256 µg/mL) in vitro, which indicated that AP138L-arg26 had low hemolysis and cytotoxicity. In vivo, mice (ICR, 6-8 weeks) were intraperitoneally injected with AP138L-arg26 at a concentration of 10 mg/kg (Fig. ##FIG##3##4##c–g). Compared with the control group, there were no significant differences in the whole blood detection indexes and biochemical indexes of the AP138L-arg26 treatment group. Moreover, the tissues of the AP138L-arg26 treatment group such as the heart, kidney, spleen, lung, and liver had no hyperemia, bleeding, hyperplasia, or other lesions in the full field, being almost same as those of the control group (Fig. ##FIG##3##4##d). These data indicate that the AP138L-arg26 peptide has excellent safety and good potential as clinical therapeutic drugs.</p>\",\n \"<title>The mechanism of AP138L-arg26 against <italic>S. aureus</italic> CVCC 546</title>\",\n \"<title>SEM</title>\",\n \"<p id=\\\"Par43\\\">The morphology changes of bacteria after treatment with AP138L-arg26 were observed using SEM. As shown in Fig. ##FIG##4##5##a, the surface of <italic>S. aureus</italic> CVCC 546 (nearly 100%) was smooth and spherical without peptide treatment. However, 60–80% of cell structures changed significantly with surface roughness, granular secretion, perforation, and deformation after treatment with 2 × MIC peptide AP138 at 0.5, 1, and 2 h. More than 80% of <italic>S. aureus</italic> CVCC 546 had significantly changed structure at 1 h. The results showed that AP138L-arg26 could destroy the structure of <italic>S. aureus</italic>.</p>\",\n \"<title>The effect of AP138L-arg26 on the cell membrane</title>\",\n \"<p id=\\\"Par44\\\">According to the SEM results demonstrated that AP138L-arg26 caused disruption to the cell membrane or wall. Thus, a PI/SYTO9 assay was conducted to prove the destructive effect of AP138L-arg26 on <italic>S. aureus</italic> CVCC 546. The untreated group was almost stained green by SYTO9 (over 99%), demonstrating that they were predominantly living cells. The AP138L-arg26 treatment group could disrupt the cell membrane integrity with more than 50% cells stained red by PI (Fig. ##FIG##4##5##b). K<sup>+</sup> leakage further illustrates the disruptive effect of AP138L-arg26 on bacterial membrane, the results showed that the AP138L-arg26 treated group significantly increased the extracellular K<sup>+</sup> leakage level (0.05 mg/L) compared to the untreated bacteria (0.02 mg/L), similar to the positive control (0.1% TritonX-100) (Fig. ##FIG##4##5##d). It was also found that AP138L-arg26 could affect the membrane potential, as measured using DiSC<sub>3</sub>(5) fluorescence staining, the relative fluorescence units (RFU) values of the untreated and 1 × MIC AP138L-arg26-treated groups were around 2000, and the 4 × MIC AP138L-arg26 and nisin (positive control) groups were above 3000 (a 1000 increase) (Fig. ##FIG##4##5##c), which was mainly due to the depolarization caused by the destructive effect of the AP138L-arg26 on the bacterial membrane when the special compounds insert into lipid bilayers, they can change the membrane fluidity and disrupt the normal plasma membrane fluidity homeostasis, which further leads to leakage of cellular components and bacterial death. Therefore, a dye, Laurdan was used to measure the <italic>S. aureus</italic> membrane fluidity and quantified it using the Laurdan GP index. The results showed that the Laurdan GP of <italic>S. aureus</italic> decreased from 0.22 to 0.18 after treatment with 2 × MIC AP138L-arg26, which indicated an increase in the fluidity of the bacterial membrane due to the interaction of the AP138L-arg26 with the membrane (Fig. ##FIG##4##5##e). These results suggested that AP138L-arg26 could destroy the cell membrane of <italic>S. aureus</italic>.</p>\",\n \"<title>The effect of AP138L-arg26 on bacterial metabolism</title>\",\n \"<p id=\\\"Par45\\\">In order to further study whether AP138L-arg26 could affect the metabolism to kill bacteria, bacterial respiration was detected using an ATP kit. The results showed that AP138L-arg26 could increase intracellular ATP: the RLU values of 1 × , 2 × , 4 × MIC AP138L-arg26-treated groups (10,700, 11,500, and 11,900, respectively) were 7.6, 8.2, and 8.5 times higher than that of the untreated group (1400), which indicated a concentration-dependent pattern (Fig. ##FIG##5##6##a). The increase in ROS causes a series of cascading reactions such as oxidation of lipids, proteins, and damage to DNA, inducing bacteria death. After treatment with 1 × , 2 × , and 4 × MIC AP138L-arg26, the fluorescence values increased from 600 (blank control, CK) to 930, 1000, and 1400 with concentration-dependent (Fig. ##FIG##5##6##b). LDH levels decreased after antimicrobial peptide incubation. The LDH activity % of CK, 1 × , 2 × , and 4 × MIC AP138L-arg26 were 100%, 24%, 16%, and 16% respectively, which indicated a concentration-dependent pattern (Fig. ##FIG##5##6##c). The results showed that AP138 could induce the ROS generation of <italic>S. aureus</italic> CVCC 546, ultimately promoting cell death and reducing the probability of resistance generation.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par46\"><italic>S</italic>. <italic>aureus</italic> is a pathogenic bacterium that greatly threatens human health and the production efficiency of livestock and poultry. Many bacteria have developed drug resistance, particularly MRSA (Pinto et al. ##REF##31127270##2019##). The rise of drug resistance has triggered a public health crisis; therefore, there is an urgent need to develop new antibacterial agents to alleviate bacterial resistance (Peters et al. ##REF##21060861##2010##; Li et al. ##REF##32582062##2020##). The unique bactericidal mechanism and multiple functions of AMPs have been extensively studied as antibiotic substitutes (Rao ##REF##7772805##1995##; Peters et al. ##REF##21060861##2010##). Among them, plectasin is the first fungal defensin extracted from <italic>Pseudoplectania nigrella (Saprophytic ascomycetes)</italic>; it kills G<sup>+</sup> bacteria but has low activity and some cytotoxicity. Therefore, researchers have conducted in-depth studies on the peptides derived from plectasin (Mygind et al. ##REF##16222292##2005##; Schneider et al. ##REF##20508130##2010##). AP138 is a derived peptide with potential for clinical development, obtained by chemical synthesis due to the D type Arg at the 26th position of the sequence (Stecher et al. ##REF##24413669##2014##; Groo et al. ##REF##30532539##2018##). In this work, natural amino acid sequence (L-type amino acid) was obtained using heterologous expression to reduce the high cost of chemical synthesis, and the activity and bactericidal mechanism of the L-type AP138 were analyzed.</p>","<p id=\"Par47\">As can be seen in Table ##TAB##0##1##, it was predicted that AP138L-arg26 would have a high charge (+4.5) and antimicrobial peptide potential. Compared with plectasin, the substitution of five amino acids (D9S, M13L, Q14R, N17R, and K26R) significantly changed the physicochemical properties of AP138L-arg26 including an increased positive charge (+1 to +4.5) and hydrophobicity (32 to 33%) (Table ##TAB##0##1##), making it a good AMP possibility. Although D-type modification of the Arg in the sequence (D-type AP138) could effectively improve the tolerance of the antimicrobial peptide to trypsin which this property cannot be maintained in our AP138L-arg26, its chemical synthesis is difficult on a large scale and at low cost, increasing the challenges and difficulties of clinical development (Stecher et al. ##REF##24413669##2014##; Umerska et al. ##REF##27113868##2016##; Groo et al. ##REF##30532539##2018##; Koo and Seo ##UREF##5##2019##. Heterologous expression can achieve high yields, thus reducing the cost of developing the target protein. The exogenous expression technology of proteins in a y<italic>east</italic> expression system or <italic>E. coli</italic> expression system is relatively mature, but the expression of small molecule peptides (< 50 aa) is relatively difficult execpt few successful cases from fungal defensins and others (Zhang et al. ##REF##21558006##2011##; Zhang et al. ##REF##23624708##2014##; Cao et al. ##REF##25261129##2015##; Yang et al. ##REF##31025073##2019##; Shen et al. ##REF##33815324##2021##; Gries ##REF##36232802##2022##; Zeng et al. ##REF##34990807##2022##). In addition, the AMPs Retrocyclin-101, and Protegrin-1, and Abaecin was also expressed in a prokaryotic expression system with a low yield (Lee et al. ##REF##20553419##2011##). More details in our previous works, many antimicrobial peptides were successfully expressed in <italic>P. pastoris</italic> with high yield at level of 1.0-3.0 g/L (supernatant) and even higher level via high-density fermentation such as NZ2114, NZX, ID3 and NZL, with continous optimisation including enhancing the microbial biomass, controlling the fermentation and induction conditions, the increasing production capacity of equipment per unit volume and modificating expression system (Zhang et al. ##REF##23624708##2014##. Liu et al. ##REF##31900561##2020##; Feng et al. ##REF##22189867##2012##; Li et al ##REF##32582062##2020##; Shen et al. ##REF##33815324##2021##; Hao et al. ##UREF##3##2023##; Jin et al. ##UREF##4##2023##). In this study, the recombinant vector AP138L-arg26 was successfully constructed and expressed for the first time at high levels in <italic>P. pastoris</italic>. The total protein concentrations of the fermentation supernatant and microbial biomass were up to 3.1 mg/mL (95% purity) and 0.36 g/mL, respectively, after 120 h high density fermentation in a 5-L fermenter (Fig. ##FIG##1##2##), higher than those of NZ2114 (2.390 mg/mL, 94.8% purity) (Zhang et al. ##REF##23624708##2014##), and the production cost of AP138L-arg26 was significantly lower than that of chemical synthesis (solid phase).</p>","<p id=\"Par48\">The MIC is one of the key indicators for the early screening of active AMPs, for which values of MICs under 16 µg/mL may be clinically relevant (Rao ##REF##7772805##1995##; Patel et al. ##REF##19805558##2009##; Oh et al. ##REF##30936103##2019##). The MICs of AP138L-arg26 were 2–16 µg/mL (0.45–3.6 µM) against selected standard and clinical <italic>S. aureus</italic>, <italic>S. epidermidis</italic>, <italic>S. dysgalactiae</italic>, and <italic>S. agalactiae</italic> (Table ##TAB##1##2##). Although the MIC values were lower than those of D-type AP138 (0.125–4 µg/mL), it is still superior to lincomycin, which is mainly used clinically against G<sup>+</sup> bacteria. The structure and function of AMPs are closely related, and in-depth investigation of the structure-activity relationship is an essential step in studying AMPs (Rost and Sander ##REF##7892171##1994##; Rao ##REF##7772805##1995##). It was analyzed that AP138L-arg26 has a special CSαβ structure with +4.5 net charge and 33% hydrophobic ratio in a normal environment with ddH<sub>2</sub>O. In the simulated bacterial membrane structure (20 mM SDS), AP138L-arg26 showed significant secondary structure changes, increased α-helix structure (4.72 to 6.06%), which may be related to the bactericidal function of the peptide, while it does not change significantly in the simulated eukaryotic cell membrane structure (50% TFE buffer), suggesting no damage to cells (Figure ##SUPPL##0##S2##). The killing curves showed that 1 × , 2 × , 4 × MIC of AP138L-arg26 could kill all <italic>S. aureus</italic> ATCC 43300 within 1.5 h, which was shorter than that of vancomycin (6 h) (Fig. ##FIG##2##3##a). These results showed that the AMP AP138L-arg26 could rapidly killed bacteria. The PAE is an important guide for the rational use of clinical drugs, and the re-evaluation of adverse effects of antibiotics and combination drugs. In this study, the PAE of AP138L-arg26 was longer than that of antibiotics (Fig. ##FIG##2##3##b), indicating that low doses and long dosing intervals are feasible, which could reduce the amount of drugs used and alleviate the problem of drug resistance caused by the misuse of antibiotics (Li et al. ##REF##32582062##2020##). <italic>Staphylococcus</italic> pathgens can evade antibiotics by entering cells (Wang et al. ##REF##29523806##2018##). AP138L-arg26 had the ability to enter mammalian cells and can kill infected bacteria within cells such as <italic>S. aureus</italic> CVCC 546 in mouse macrophages RAW264.7 and bovine uterine epithelial cells in this study (Fig. ##FIG##3##4##c, d). Stability and safety are the factors that must be controlled when drugs enter the clinic, as high toxicity and low stability will hinder AMPs’ application (Koo and Seo ##UREF##5##2019##). AP138L-arg26 retained its antimicrobial activity in different concentrations of salt ions, pH, temperature, and pepsin, but it was sensitive to trypsin and lost antimicrobial activity for 30 min. These results showed that it remained the antimicrobial activity (MIC against <italic>S. aureus</italic> ATCC 43300, 8 µg/mL) and has the potential for topical drug development (Table ##TAB##1##2##), whereas it may need to be encapsulated for oral administration (Umerska et al. ##REF##28848347##2017##; Groo et al. ##REF##30532539##2018##). AP138L-arg26 had a better safety rating in vitro and in vivo, as reflected in the high cell survival (over 75% for mouse macrophages RAW264.7) and the low hemolysis (less than 3%) at a high concentration of 256 µg/mL of AP138L-arg26 in vitro (Fig. ##FIG##3##4##a, b). After intraperitoneal injection of 10 mg/kg AP138L-arg26 for 1 week, it was found that there were no obvious differences in whole blood detection indexes (< 4.5%), biochemical index (< 4%). The histological sections of the heart, liver, spleen, lung, and kidney were not damaged and retained a normal structure (no bleeding, hyperemia, or hyperplasia) indicating the safety of AP138L-arg26 in vivo (Fig. ##FIG##3##4##c–g). These results indicate that AP138L-arg26 has a high safety profile as a drug in <italic>vivo</italic> and in <italic>vitro</italic>.</p>","<p id=\"Par49\">In general, the researchers suggest that the unique positive charge and hydrophobic properties of most AMPs could interact with bacterial cell membranes and exert bactericidal functions (Gao et al. ##REF##34673132##2021##; Pinto et al. ##REF##31127270##2019##; Zheng et al. ##REF##35722282##2022##). Changes in bacterial morphology were first observed using SEM after treatment with 2 × MIC AP138L-arg26 for 1 h, and most bacteria had rough surfaces, depressions, and granular secretions (Fig. ##FIG##4##5##a). It was further shown that AP138L-arg26 had a directly destructive effect on the cell membrane through PI staining, K<sup>+</sup> leakage, and membrane fluidity assays (Fig. ##FIG##4##5##a–e). AP138L-arg26 might mainly be considered to interact with the negatively charged components on the surface of the bacterial membrane through itself charges, and then insert into the cell membrane, interfering with its orderly arrangement, causing further destruction (She et al. ##UREF##10##2022##; Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##). This was similar to the case with the antimicrobial peptide 5j and L007-0069 (She et al. ##UREF##10##2022##). AP138L-arg26 also affect the bacterial metabolism: (i) After the incubation of AP138L-arg26 with bacteria, intracellular ATP levels were elevated (maximum: 8.5-fold), which may cause bacteria to switch from a dormant state to an active state that is more conducive to them being killed (Shi et al. ##REF##36071151##2022##). (ii) Intracellular ROS were increased twofold with 2 × MIC of AP138L-arg26, which suggests that bacteria could be damaged through bacterial auto-oxidation (Shi et al. ##REF##36071151##2022##), this is an important way in which drugs could damage bacteria. (iii) At the same time, the amount of LDH decreased (lowest, 16%), which may limit the level of respiratory metabolism due to LDH being an essential enzyme in the respiratory chain (Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##) (Fig. ##FIG##5##6##). All in all, AP138L-arg26 may kill bacteria by damaging cell membranes and influencing bacterial metabolism (ATP, ROS, LDH).</p>","<p id=\"Par50\">Finally, we changed the D-type AP138D to L-type AP138L-arg26, and it was successfully expressed in <italic>P. pastoris</italic> with high production. It was demonstrated that AP138L-arg26 had a faster and longer bactericidal effect compared with conventional antibiotics, high stability, and excellent safety in vivo and in vitro. It was revealed that AP138L-arg26 has multiple bactericidal mechanisms including membrane rupture and metabolism imbalance, which is also an important reason that antimicrobial peptides do not tend to develop resistance. The better derived AMPs of plectasin will be worth of developing in the future so that the excellent property of high resistance to trypsin hydrolysis in AP138 construct could be merged and maintained in new AMP derivative with a high expression level.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par46\\\"><italic>S</italic>. <italic>aureus</italic> is a pathogenic bacterium that greatly threatens human health and the production efficiency of livestock and poultry. Many bacteria have developed drug resistance, particularly MRSA (Pinto et al. ##REF##31127270##2019##). The rise of drug resistance has triggered a public health crisis; therefore, there is an urgent need to develop new antibacterial agents to alleviate bacterial resistance (Peters et al. ##REF##21060861##2010##; Li et al. ##REF##32582062##2020##). The unique bactericidal mechanism and multiple functions of AMPs have been extensively studied as antibiotic substitutes (Rao ##REF##7772805##1995##; Peters et al. ##REF##21060861##2010##). Among them, plectasin is the first fungal defensin extracted from <italic>Pseudoplectania nigrella (Saprophytic ascomycetes)</italic>; it kills G<sup>+</sup> bacteria but has low activity and some cytotoxicity. Therefore, researchers have conducted in-depth studies on the peptides derived from plectasin (Mygind et al. ##REF##16222292##2005##; Schneider et al. ##REF##20508130##2010##). AP138 is a derived peptide with potential for clinical development, obtained by chemical synthesis due to the D type Arg at the 26th position of the sequence (Stecher et al. ##REF##24413669##2014##; Groo et al. ##REF##30532539##2018##). In this work, natural amino acid sequence (L-type amino acid) was obtained using heterologous expression to reduce the high cost of chemical synthesis, and the activity and bactericidal mechanism of the L-type AP138 were analyzed.</p>\",\n \"<p id=\\\"Par47\\\">As can be seen in Table ##TAB##0##1##, it was predicted that AP138L-arg26 would have a high charge (+4.5) and antimicrobial peptide potential. Compared with plectasin, the substitution of five amino acids (D9S, M13L, Q14R, N17R, and K26R) significantly changed the physicochemical properties of AP138L-arg26 including an increased positive charge (+1 to +4.5) and hydrophobicity (32 to 33%) (Table ##TAB##0##1##), making it a good AMP possibility. Although D-type modification of the Arg in the sequence (D-type AP138) could effectively improve the tolerance of the antimicrobial peptide to trypsin which this property cannot be maintained in our AP138L-arg26, its chemical synthesis is difficult on a large scale and at low cost, increasing the challenges and difficulties of clinical development (Stecher et al. ##REF##24413669##2014##; Umerska et al. ##REF##27113868##2016##; Groo et al. ##REF##30532539##2018##; Koo and Seo ##UREF##5##2019##. Heterologous expression can achieve high yields, thus reducing the cost of developing the target protein. The exogenous expression technology of proteins in a y<italic>east</italic> expression system or <italic>E. coli</italic> expression system is relatively mature, but the expression of small molecule peptides (< 50 aa) is relatively difficult execpt few successful cases from fungal defensins and others (Zhang et al. ##REF##21558006##2011##; Zhang et al. ##REF##23624708##2014##; Cao et al. ##REF##25261129##2015##; Yang et al. ##REF##31025073##2019##; Shen et al. ##REF##33815324##2021##; Gries ##REF##36232802##2022##; Zeng et al. ##REF##34990807##2022##). In addition, the AMPs Retrocyclin-101, and Protegrin-1, and Abaecin was also expressed in a prokaryotic expression system with a low yield (Lee et al. ##REF##20553419##2011##). More details in our previous works, many antimicrobial peptides were successfully expressed in <italic>P. pastoris</italic> with high yield at level of 1.0-3.0 g/L (supernatant) and even higher level via high-density fermentation such as NZ2114, NZX, ID3 and NZL, with continous optimisation including enhancing the microbial biomass, controlling the fermentation and induction conditions, the increasing production capacity of equipment per unit volume and modificating expression system (Zhang et al. ##REF##23624708##2014##. Liu et al. ##REF##31900561##2020##; Feng et al. ##REF##22189867##2012##; Li et al ##REF##32582062##2020##; Shen et al. ##REF##33815324##2021##; Hao et al. ##UREF##3##2023##; Jin et al. ##UREF##4##2023##). In this study, the recombinant vector AP138L-arg26 was successfully constructed and expressed for the first time at high levels in <italic>P. pastoris</italic>. The total protein concentrations of the fermentation supernatant and microbial biomass were up to 3.1 mg/mL (95% purity) and 0.36 g/mL, respectively, after 120 h high density fermentation in a 5-L fermenter (Fig. ##FIG##1##2##), higher than those of NZ2114 (2.390 mg/mL, 94.8% purity) (Zhang et al. ##REF##23624708##2014##), and the production cost of AP138L-arg26 was significantly lower than that of chemical synthesis (solid phase).</p>\",\n \"<p id=\\\"Par48\\\">The MIC is one of the key indicators for the early screening of active AMPs, for which values of MICs under 16 µg/mL may be clinically relevant (Rao ##REF##7772805##1995##; Patel et al. ##REF##19805558##2009##; Oh et al. ##REF##30936103##2019##). The MICs of AP138L-arg26 were 2–16 µg/mL (0.45–3.6 µM) against selected standard and clinical <italic>S. aureus</italic>, <italic>S. epidermidis</italic>, <italic>S. dysgalactiae</italic>, and <italic>S. agalactiae</italic> (Table ##TAB##1##2##). Although the MIC values were lower than those of D-type AP138 (0.125–4 µg/mL), it is still superior to lincomycin, which is mainly used clinically against G<sup>+</sup> bacteria. The structure and function of AMPs are closely related, and in-depth investigation of the structure-activity relationship is an essential step in studying AMPs (Rost and Sander ##REF##7892171##1994##; Rao ##REF##7772805##1995##). It was analyzed that AP138L-arg26 has a special CSαβ structure with +4.5 net charge and 33% hydrophobic ratio in a normal environment with ddH<sub>2</sub>O. In the simulated bacterial membrane structure (20 mM SDS), AP138L-arg26 showed significant secondary structure changes, increased α-helix structure (4.72 to 6.06%), which may be related to the bactericidal function of the peptide, while it does not change significantly in the simulated eukaryotic cell membrane structure (50% TFE buffer), suggesting no damage to cells (Figure ##SUPPL##0##S2##). The killing curves showed that 1 × , 2 × , 4 × MIC of AP138L-arg26 could kill all <italic>S. aureus</italic> ATCC 43300 within 1.5 h, which was shorter than that of vancomycin (6 h) (Fig. ##FIG##2##3##a). These results showed that the AMP AP138L-arg26 could rapidly killed bacteria. The PAE is an important guide for the rational use of clinical drugs, and the re-evaluation of adverse effects of antibiotics and combination drugs. In this study, the PAE of AP138L-arg26 was longer than that of antibiotics (Fig. ##FIG##2##3##b), indicating that low doses and long dosing intervals are feasible, which could reduce the amount of drugs used and alleviate the problem of drug resistance caused by the misuse of antibiotics (Li et al. ##REF##32582062##2020##). <italic>Staphylococcus</italic> pathgens can evade antibiotics by entering cells (Wang et al. ##REF##29523806##2018##). AP138L-arg26 had the ability to enter mammalian cells and can kill infected bacteria within cells such as <italic>S. aureus</italic> CVCC 546 in mouse macrophages RAW264.7 and bovine uterine epithelial cells in this study (Fig. ##FIG##3##4##c, d). Stability and safety are the factors that must be controlled when drugs enter the clinic, as high toxicity and low stability will hinder AMPs’ application (Koo and Seo ##UREF##5##2019##). AP138L-arg26 retained its antimicrobial activity in different concentrations of salt ions, pH, temperature, and pepsin, but it was sensitive to trypsin and lost antimicrobial activity for 30 min. These results showed that it remained the antimicrobial activity (MIC against <italic>S. aureus</italic> ATCC 43300, 8 µg/mL) and has the potential for topical drug development (Table ##TAB##1##2##), whereas it may need to be encapsulated for oral administration (Umerska et al. ##REF##28848347##2017##; Groo et al. ##REF##30532539##2018##). AP138L-arg26 had a better safety rating in vitro and in vivo, as reflected in the high cell survival (over 75% for mouse macrophages RAW264.7) and the low hemolysis (less than 3%) at a high concentration of 256 µg/mL of AP138L-arg26 in vitro (Fig. ##FIG##3##4##a, b). After intraperitoneal injection of 10 mg/kg AP138L-arg26 for 1 week, it was found that there were no obvious differences in whole blood detection indexes (< 4.5%), biochemical index (< 4%). The histological sections of the heart, liver, spleen, lung, and kidney were not damaged and retained a normal structure (no bleeding, hyperemia, or hyperplasia) indicating the safety of AP138L-arg26 in vivo (Fig. ##FIG##3##4##c–g). These results indicate that AP138L-arg26 has a high safety profile as a drug in <italic>vivo</italic> and in <italic>vitro</italic>.</p>\",\n \"<p id=\\\"Par49\\\">In general, the researchers suggest that the unique positive charge and hydrophobic properties of most AMPs could interact with bacterial cell membranes and exert bactericidal functions (Gao et al. ##REF##34673132##2021##; Pinto et al. ##REF##31127270##2019##; Zheng et al. ##REF##35722282##2022##). Changes in bacterial morphology were first observed using SEM after treatment with 2 × MIC AP138L-arg26 for 1 h, and most bacteria had rough surfaces, depressions, and granular secretions (Fig. ##FIG##4##5##a). It was further shown that AP138L-arg26 had a directly destructive effect on the cell membrane through PI staining, K<sup>+</sup> leakage, and membrane fluidity assays (Fig. ##FIG##4##5##a–e). AP138L-arg26 might mainly be considered to interact with the negatively charged components on the surface of the bacterial membrane through itself charges, and then insert into the cell membrane, interfering with its orderly arrangement, causing further destruction (She et al. ##UREF##10##2022##; Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##). This was similar to the case with the antimicrobial peptide 5j and L007-0069 (She et al. ##UREF##10##2022##). AP138L-arg26 also affect the bacterial metabolism: (i) After the incubation of AP138L-arg26 with bacteria, intracellular ATP levels were elevated (maximum: 8.5-fold), which may cause bacteria to switch from a dormant state to an active state that is more conducive to them being killed (Shi et al. ##REF##36071151##2022##). (ii) Intracellular ROS were increased twofold with 2 × MIC of AP138L-arg26, which suggests that bacteria could be damaged through bacterial auto-oxidation (Shi et al. ##REF##36071151##2022##), this is an important way in which drugs could damage bacteria. (iii) At the same time, the amount of LDH decreased (lowest, 16%), which may limit the level of respiratory metabolism due to LDH being an essential enzyme in the respiratory chain (Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##) (Fig. ##FIG##5##6##). All in all, AP138L-arg26 may kill bacteria by damaging cell membranes and influencing bacterial metabolism (ATP, ROS, LDH).</p>\",\n \"<p id=\\\"Par50\\\">Finally, we changed the D-type AP138D to L-type AP138L-arg26, and it was successfully expressed in <italic>P. pastoris</italic> with high production. It was demonstrated that AP138L-arg26 had a faster and longer bactericidal effect compared with conventional antibiotics, high stability, and excellent safety in vivo and in vitro. It was revealed that AP138L-arg26 has multiple bactericidal mechanisms including membrane rupture and metabolism imbalance, which is also an important reason that antimicrobial peptides do not tend to develop resistance. The better derived AMPs of plectasin will be worth of developing in the future so that the excellent property of high resistance to trypsin hydrolysis in AP138 construct could be merged and maintained in new AMP derivative with a high expression level.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<title>Abstract</title>","<p id=\"Par1\">The low activity and yield of antimicrobial peptides (AMPs) are pressing problems. The improvement of activity and yield through modification and heterologous expression, a potential way to solve the problem, is a research hot-pot. In this work, a new plectasin-derived variant L-type AP138 (AP138L-arg26) was constructed for the study of recombination expression and druggablity. As a result, the total protein concentration of AP138L-arg26 was 3.1 mg/mL in <italic>Pichia pastoris</italic> X-33 supernatant after 5 days of induction expression in a 5-L fermenter. The recombinant peptide AP138L-arg26 has potential antibacterial activity against selected standard and clinical Gram-positive bacteria (G<sup>+</sup>, minimum inhibitory concentration (MIC) 2–16 µg/mL) and high stability under different conditions (temperature, pH, ion concentration) and 2 × MIC of AP138L-arg26 could rapidly kill <italic>Staphylococcus aureus</italic> (<italic>S. aureus</italic>) (> 99.99%) within 1.5 h. It showed a high safety in vivo and in vivo and a long post-antibiotic effect (PAE, 1.91 h) compared with vancomycin (1.2 h). Furthermore, the bactericidal mechanism was revealed from two dimensions related to its disruption of the cell membrane resulting in intracellular potassium leakage (2.5-fold higher than control), and an increase in intracellular adenosine triphosphate (ATP), and reactive oxygen species (ROS), the decrease of lactate dehydrogenase (LDH) and further intervening metabolism in <italic>S. aureus</italic>. These results indicate that AP138L-arg26 as a new peptide candidate could be used for more in-depth development in the future.</p>","<title>Key points</title>","<p id=\"Par2\">\n<italic>• The AP138L-arg26 was expressed in the P. pastoris expression system with high yield</italic>\n</p>","<p id=\"Par3\">\n<italic>• The AP138 L-arg26 showed high stability and safety in vitro and in vivo</italic>\n</p>","<p id=\"Par4\">\n<italic>• The AP138L-arg26 killed S. aureus by affecting cell membranes and metabolism</italic>\n</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00253-023-12947-w.</p>","<title>Keywords</title>"],"string":"[\n \"<title>Abstract</title>\",\n \"<p id=\\\"Par1\\\">The low activity and yield of antimicrobial peptides (AMPs) are pressing problems. The improvement of activity and yield through modification and heterologous expression, a potential way to solve the problem, is a research hot-pot. In this work, a new plectasin-derived variant L-type AP138 (AP138L-arg26) was constructed for the study of recombination expression and druggablity. As a result, the total protein concentration of AP138L-arg26 was 3.1 mg/mL in <italic>Pichia pastoris</italic> X-33 supernatant after 5 days of induction expression in a 5-L fermenter. The recombinant peptide AP138L-arg26 has potential antibacterial activity against selected standard and clinical Gram-positive bacteria (G<sup>+</sup>, minimum inhibitory concentration (MIC) 2–16 µg/mL) and high stability under different conditions (temperature, pH, ion concentration) and 2 × MIC of AP138L-arg26 could rapidly kill <italic>Staphylococcus aureus</italic> (<italic>S. aureus</italic>) (> 99.99%) within 1.5 h. It showed a high safety in vivo and in vivo and a long post-antibiotic effect (PAE, 1.91 h) compared with vancomycin (1.2 h). Furthermore, the bactericidal mechanism was revealed from two dimensions related to its disruption of the cell membrane resulting in intracellular potassium leakage (2.5-fold higher than control), and an increase in intracellular adenosine triphosphate (ATP), and reactive oxygen species (ROS), the decrease of lactate dehydrogenase (LDH) and further intervening metabolism in <italic>S. aureus</italic>. These results indicate that AP138L-arg26 as a new peptide candidate could be used for more in-depth development in the future.</p>\",\n \"<title>Key points</title>\",\n \"<p id=\\\"Par2\\\">\\n<italic>• The AP138L-arg26 was expressed in the P. pastoris expression system with high yield</italic>\\n</p>\",\n \"<p id=\\\"Par3\\\">\\n<italic>• The AP138 L-arg26 showed high stability and safety in vitro and in vivo</italic>\\n</p>\",\n \"<p id=\\\"Par4\\\">\\n<italic>• The AP138L-arg26 killed S. aureus by affecting cell membranes and metabolism</italic>\\n</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00253-023-12947-w.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Biological information analysis of AP138L-arg26</title>","<p id=\"Par11\">AP138L-arg26 and plectasin derived AMP sequences were obtained from DBAASP database (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.dbaasp.org/home\">https://www.dbaasp.org/home</ext-link>) (Pirtskhalava et al. ##REF##33151284##2021##), their physical and chemical properties, 3D structure, and antimicrobial peptide possibility were predicted by APD database (<ext-link ext-link-type=\"uri\" xlink:href=\"https://aps.unmc.edu/AP/\">https://aps.unmc.edu/AP/</ext-link>) (Wang et al. ##UREF##12##2016##), I-TASSER (<ext-link ext-link-type=\"uri\" xlink:href=\"https://zhanggroup.org/I-TASSER/\">https://zhanggroup.org/I-TASSER/</ext-link>) (Yang and Zhang ##REF##25883148##2015##), and CAMP database (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.camp.bicnirrh.res.in/\">http://www.camp.bicnirrh.res.in/</ext-link>), respectively (Waghu et al. ##UREF##11##2014##).</p>","<title>Expression, purification, and identification of AP138L-arg26</title>","<p id=\"Par12\">The nucleic acid sequence of AP138L-arg26 (GenBank ID: 2736833) was subjected to codon preference using the Reverse Translate Tool (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.bioinformati\">http://www.bioinformati</ext-link> cs.org/sms2/rev_trans.html), then a Kex2 signaling peptide cleavage site was added at its N-terminus (Figure ##SUPPL##0##S1##) and inserted into the eukaryotic expression vector pPICZαA between the double enzyme (<italic>Xho</italic>I and <italic>Xba</italic>I) cleavage site, to construct the plasmid pPICZαA-AP138L-arg26<italic>.</italic> Additionally, the pPICZαA-AP138L-arg26 was digested using <italic>Pme</italic>I and transformed into competent <italic>P. pastoris</italic> X-33 cells via electroporation. Peptide purification was carried out with the AKTAxpress system. Expression of AMP AP138L-arg26 was identified using Tricine-SDS-PAGE firstly, and the purified AP138L-arg26 was identified using MALDI-TOF/TOF MS (Ultraflextreme, Bruker, Germany), finally, the concentration was detected using Bradford assay kits.</p>","<title>Antimicrobial activity and pharmacodynamic analysis in vitro</title>","<title>Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)</title>","<p id=\"Par13\">To determine the MIC values of AP138L-arg26, we used the broth microdilution technique as previously described (Andrews. ##REF##11420333##2001##; Wiegand et al. ##REF##18274517##2008##). In brief, AP138L-arg26 peptide were diluted from 1280 to 2.5 µg/mL and added to a 96-cells plate with 10 µL per well. The bacteria in the logarithmic stage were diluted to 10<sup>5</sup> CFU/mL with 90 µL per well. All plates were cultured in a 37 °C constant-temperature incubator; no visible growth of bacteria was the MIC after 18 h incubation. The MBC method was also based on the CLSI 2021 guidelines. In brief, after the bacteria were incubated at 1 × , 2 × , 4 × , and 8 × MIC of AP138L-arg26, the cultures were coated with Mueller-Hinton Agar (MHA) plates, the concentrations of AP138L-arg26 with 99.99% the bacteria killed was defined as MBC.</p>","<title>Time-killing curve assay</title>","<p id=\"Par14\">The time-killing curve of AP138L-arg26 against MRSA ATCC 43300 was used to analyze the pharmacodynamics. The method was based on previous laboratory experiments (Flamm et al. ##UREF##2##2019##; Yang et al. ##REF##31025073##2019##). Briefly, exponential-phase bacteria were diluted to 1 × 10<sup>5</sup> CFU/mL, and peptides underwent a twofold gradient dilution setting of 4 × , 2 × , and 1 × MIC. There was incubation in a 37 °C thermostatic shaker at 200 rpm for 0–24 h to collect samples, which were plated on MHA plates. Colony numbers were recorded at all collected time points.</p>","<title>PAE assay</title>","<p id=\"Par15\">The method of PAE of AP138L-arg26 against MRSA ATCC 43300 or <italic>S. aureus</italic> CVCC 546 was described previously (Wang et al. ##REF##29523806##2018##). The formula is PAE = <italic>T</italic> − <italic>C</italic> (<italic>T</italic> is the time required for the number of colonies in the sample treatment group to increase by tenfold, and <italic>C</italic> is the time required for the untreated group).</p>","<title>Intracellular antibacterial activity</title>","<p id=\"Par16\">The method of intracellular bactericidal effect was described previously (Wang et al. ##REF##29523806##2018##). Only the number of cells and bacteria changed from 2.5 × 10<sup>5</sup> to 2 × 10<sup>5</sup> cells/mL.</p>","<title>Synergism with antibiotics</title>","<title>Fractional inhibitory concentration index (FICI)</title>","<p id=\"Par17\">The synergistic effects of AP138L-arg26 with different antibiotics were evaluated using a checkerboard assay. The synergistic effect was calculated using the FICI as follows: FICI = FIC of AP138L-arg26 + FIC of antibiotic; FIC = MIC<sub>c</sub>/MIC<sub>a</sub>, where MIC<sub>c</sub> is the MIC of the peptide and antibiotic in combination, and MIC<sub>a</sub> is the MIC of the peptide/antibiotic alone (Blier et al. ##REF##20008946##2010##). Three parallel experiments were performed for each group. The efficacy of combination therapy was defined as FICI ≤ 0.5, 0.5 < FICI ≤ 1, 1 < FICI ≤ 4, and FICI > 4 indicating synergy, addition, no difference, and antagonism, respectively.</p>","<title>Growth curve of <italic>S. aureus</italic></title>","<p id=\"Par18\">Samples were taken during the logarithmic growth of <italic>S. aureus</italic> and diluted to 1 × 10<sup>5</sup> CFU/mL (Delpech et al. ##UREF##0##2019##). Different concentrations of AP138L-arg26, ceftiofur sodium, or a combination of them with an equal volume of bacteria were added to a 96-well microplate. The growth curves were recorded using a fully automatic growth curve recording instrument.</p>","<title>Stability analysis</title>","<title>Artificial gastric and intestinal juice stability</title>","<p id=\"Par19\">AP138L-arg26 was incubated in artificial gastric and intestinal juice (Beijing Coolaber Technology Co., Ltd., Beijing, China) for 0.083, 0.167, 0.25, 0.75, and 1 h. The antimicrobial activity of AP138L-arg26 against MRSA ATCC 43300 was tested through the MIC assay.</p>","<title>Thermal, pH, and salt stability</title>","<p id=\"Par20\">The thermal, pH, and salt stability of AMPs were studied previously (Zhang et al. ##REF##21558006##2011##). Briefly, the AP138L-arg26 was incubated at different temperatures (4/20/40/60/80/100 °C), pH values (2/4/6/8/10), and kinds of salt ions (50/100/150/200/300 mM NaCl, 1.25/2.5/5 mM KCl, MgCl<sub>2</sub>, CaCl<sub>2</sub>) for 1 h at 37 °C, respectively. The antimicrobial activity of AP138L-arg26 against MRSA ATCC 43300 was tested using MIC assay.</p>","<title>Circular dichroism (CD) spectrum assay</title>","<p id=\"Par21\">The secondary structures of AP138L-arg26 were detected using CD (Bio-Logic MOS450 spectropolarimeter, France) in a simulated eukaryotic cell environment (50% trifluoroethanol, TFE) and bacterial membrane environment (20 mM sodium dodecyl sulfate, SDS) (Yao et al. ##REF##28870607##2018##).</p>","<title>Safety evaluation in vitro and in vivo</title>","<title>Hemolysis activity</title>","<p id=\"Par22\">The hemolysis of AP138L-arg26 to fresh ICR mouse erythrocytes was described previously (Zheng et al. ##REF##34491399##2021##). Briefly, 8% of mouse erythrocytes were incubated with different concentrations of antimicrobial peptide AP138L-arg26 (256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 µg/mL) in equal volumes. The blank and positive control was phosphate buffered saline (PBS) and 0.1% Triton X-100, respectively.</p>","<title>Cytotoxicity</title>","<p id=\"Par23\">The cytotoxicity of AP138L-arg26 to mouse macrophages RAW264.7 was detected by the thiazolyl blue tetrazolium bromide (MTT) method as described previously (Shen et al. ##REF##33815324##2021##).</p>","<title>Acute toxicity in mice</title>","<p id=\"Par24\">AP138L-arg26 was administrated by intraperitoneal injection (<italic>n</italic> = 4 per group, 10 mg/kg, body weight 25 g) every day for 1 week (Shi et al. ##REF##36071151##2022##). After 7 days, the mice were euthanized to collect anticoagulant whole-blood and tissues (liver, spleen, kidney, and lung), which were used for whole blood and biochemical detection, or tissue sections (hematoxylin-eosin (HE) staining), respectively.</p>","<title>Antimicrobial mechanism of peptides</title>","<title>Scanning electron microscopy (SEM) assay</title>","<p id=\"Par25\">The effect of peptides on changes in bacterial morphology was observed by scanning electron microscopy. Briefly the exponential phase <italic>S. aureus</italic> CVCC 546 cells (1 × 10<sup>9</sup> CFU/mL) were incubated with 2 × MIC peptides at 37 °C for 0.5 h, 1 h, and 2 h. The untreated bacteria were the negative control. The processing methods and steps of the samples were described in detail in our previous study (Li et al. ##REF##32582062##2020##). Briefly, there were two important steps: (1) Sample preparation: incubation, cleaning, fixation, drying, gold spraying. (2) Observation using microscope. Only one concentration was used to deal with bacteria at different times (0.5, 1, and 2 h).</p>","<title>SYTO9/propidium iodide (PI) assay</title>","<p id=\"Par26\">To explore the disruption of bacterial cell membranes by the AP138L-arg26, a SYTO9/PI kit was used to detect the integrity of cell membranes (L7007 LIVE/DEAD<sup>R</sup> BacLight™ Bacerial Viability Kits). The unique feature of this kit is that SYTO9 alone can pass through integral cell membranes, while PI can only penetrate damaged cell membranes. Briefly, the <italic>S. aureus</italic> CVCC 546 cells (1 × 10<sup>9</sup> CFU/mL) were incubated with AP138L-arg26 (2 × MIC, 37 °C for 1 h), and a volume of 3 µL SYTO9/PI (1.5 µL: 1.5 µL) was added to 1 mL of bacterial suspension. Finally, the results were observed using fluorescence microscopy.</p>","<title>Membrane fluidity assay</title>","<p id=\"Par27\">The final concentration of 10 µM Laurdan was used to detect the effect of AP138L-arg26 on bacterial cell membrane fluidity (Shi et al. ##REF##36071151##2022##). Briefly, (1) co-incubation of bacteria with Laurdan; (2) the stained bacteria are co-incubated with the peptide; (3) spectrophotometer (Tecan, Männedorf, Switzerland) detection. The calculation formula: generalized polarization (GP) = (I435 − I490)/(I435 + I490).</p>","<title>Membrane depolarization assay</title>","<p id=\"Par28\">To further explore the effect of AP138L-arg26 on the bacterial membrane, the membrane probe DiSC<sub>3</sub>(5) was selected to detect the change in membrane potential (Wang et al. ##REF##31974490##2020##). Briefly, <italic>S. aureus</italic> was washed and resuspended in PBS to 1 × 10<sup>8</sup> CFU/mL and incubated with 0.5 mM membrane dyes DiSC<sub>3</sub>(5) at 37 °C for 1 h in the dark. Then, 90 µL of stained bacterial suspension and 10 µL of AP138 L-arg26 (1 × , 2 × , 4 × MIC) were mixed and added to black and clean 96-cell plates and the fluorescence intensity was determined using a spectrophotometer (excitation wavelength 622/emission wavelength 670 nm, Infinite M200).</p>","<title>Potassium ion (K<sup>+</sup>) leakage</title>","<p id=\"Par29\">The integrity effect of antimicrobial peptide AP138L-arg26 on bacterial cell membranes was further verified, and the K<sup>+</sup> leakage assay was previously described (Li et al. ##REF##32582062##2020##). Briefly, the main procedures were as follows: Firstly, prepare the <italic>S. aureus</italic> CVCC 546 bacteria suspension at a concentration of 1 × 10<sup>8</sup> CFU/mL. Secondly, incubate the bacterial suspension with 2 × MIC AP138L-arg26 at different times (15/30/60/90/120 min) at 37 °C, with untreated cells and nisin used as negative and positive controls. Finally, the supernatants were detected using inductively coupled plasma mass spectrometry (ICP-MS) (SantaClara, CA, USA).</p>","<title>Intracellular adenosine triphosphate (ATP) determination</title>","<p id=\"Par30\">As ATP is the most direct source of energy for living organisms, the effect of antimicrobial peptides on ATP was explored with an ATP assay kit (Beyotime, Shanghai, China). Briefly, exponential-stage <italic>S. aureus</italic> CVCC 546 (1 × 10<sup>8</sup> CFU/mL) were incubated with different concentrations of AP138L-arg26 (1 × , 2 × , 4 × MIC) for 1 h, and the pellets were collected, lysed, and centrifuged to harvest the intracellular supernatant. The luminescence was detected using an Infinite M200 Microplate reader (Tecan, Luminescence signals).</p>","<title>Lactic dehydrogenase (LDH) activity</title>","<p id=\"Par31\">As disruption of the cell membrane structure leads to the release of LDH from the cytoplasm into the culture medium, the detection of LDH in bacteria can further reveal the antibacterial mechanism (Shi et al. ##REF##36071151##2022##). Briefly, <italic>S. aureus</italic> cells were co-incubated with AP138L-arg26 for 6 h. Later, the pellets were collected and sonicated (3 s/10 s, 30 times). The intracellular LDH activity was detected using an Infinite M200 Microplate reader (Tecan, Luminescence signals). The calculated results of LDH% = treated cells/the control.</p>","<title>Reactive oxygen species (ROS) measurements</title>","<p id=\"Par32\">The probe 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure the level of ROS in bacteria after incubated with AP138L-arg26 (Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##; Shi et al. ##REF##36071151##2022##). Briefly, exponential stage bacteria were incubated with DCFH-DA (10 µM, 37 °C, 0.5 h), and the stained bacteria were treated with AP138L-arg26 (1 × , 2 × , 4 × MIC) for 1 h. The ROS were detected at excitation wavelength (488 nm) and emission wavelength (525 nm) using a microplate reader (Tecan, Männedorf, Switzerland).</p>","<title>Statistical analysis</title>","<p id=\"Par33\">The software GraphPad Prism (version 8, USA) was used to analyze all data, and ANOVA was the method to determine the statistical significance. The results are presented as means ± standard deviation (SD). A <italic>P</italic> value of < 0.05 was considered statistically significant.</p>","<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Biological information analysis of AP138L-arg26</title>\",\n \"<p id=\\\"Par11\\\">AP138L-arg26 and plectasin derived AMP sequences were obtained from DBAASP database (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://www.dbaasp.org/home\\\">https://www.dbaasp.org/home</ext-link>) (Pirtskhalava et al. ##REF##33151284##2021##), their physical and chemical properties, 3D structure, and antimicrobial peptide possibility were predicted by APD database (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://aps.unmc.edu/AP/\\\">https://aps.unmc.edu/AP/</ext-link>) (Wang et al. ##UREF##12##2016##), I-TASSER (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://zhanggroup.org/I-TASSER/\\\">https://zhanggroup.org/I-TASSER/</ext-link>) (Yang and Zhang ##REF##25883148##2015##), and CAMP database (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"http://www.camp.bicnirrh.res.in/\\\">http://www.camp.bicnirrh.res.in/</ext-link>), respectively (Waghu et al. ##UREF##11##2014##).</p>\",\n \"<title>Expression, purification, and identification of AP138L-arg26</title>\",\n \"<p id=\\\"Par12\\\">The nucleic acid sequence of AP138L-arg26 (GenBank ID: 2736833) was subjected to codon preference using the Reverse Translate Tool (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"http://www.bioinformati\\\">http://www.bioinformati</ext-link> cs.org/sms2/rev_trans.html), then a Kex2 signaling peptide cleavage site was added at its N-terminus (Figure ##SUPPL##0##S1##) and inserted into the eukaryotic expression vector pPICZαA between the double enzyme (<italic>Xho</italic>I and <italic>Xba</italic>I) cleavage site, to construct the plasmid pPICZαA-AP138L-arg26<italic>.</italic> Additionally, the pPICZαA-AP138L-arg26 was digested using <italic>Pme</italic>I and transformed into competent <italic>P. pastoris</italic> X-33 cells via electroporation. Peptide purification was carried out with the AKTAxpress system. Expression of AMP AP138L-arg26 was identified using Tricine-SDS-PAGE firstly, and the purified AP138L-arg26 was identified using MALDI-TOF/TOF MS (Ultraflextreme, Bruker, Germany), finally, the concentration was detected using Bradford assay kits.</p>\",\n \"<title>Antimicrobial activity and pharmacodynamic analysis in vitro</title>\",\n \"<title>Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)</title>\",\n \"<p id=\\\"Par13\\\">To determine the MIC values of AP138L-arg26, we used the broth microdilution technique as previously described (Andrews. ##REF##11420333##2001##; Wiegand et al. ##REF##18274517##2008##). In brief, AP138L-arg26 peptide were diluted from 1280 to 2.5 µg/mL and added to a 96-cells plate with 10 µL per well. The bacteria in the logarithmic stage were diluted to 10<sup>5</sup> CFU/mL with 90 µL per well. All plates were cultured in a 37 °C constant-temperature incubator; no visible growth of bacteria was the MIC after 18 h incubation. The MBC method was also based on the CLSI 2021 guidelines. In brief, after the bacteria were incubated at 1 × , 2 × , 4 × , and 8 × MIC of AP138L-arg26, the cultures were coated with Mueller-Hinton Agar (MHA) plates, the concentrations of AP138L-arg26 with 99.99% the bacteria killed was defined as MBC.</p>\",\n \"<title>Time-killing curve assay</title>\",\n \"<p id=\\\"Par14\\\">The time-killing curve of AP138L-arg26 against MRSA ATCC 43300 was used to analyze the pharmacodynamics. The method was based on previous laboratory experiments (Flamm et al. ##UREF##2##2019##; Yang et al. ##REF##31025073##2019##). Briefly, exponential-phase bacteria were diluted to 1 × 10<sup>5</sup> CFU/mL, and peptides underwent a twofold gradient dilution setting of 4 × , 2 × , and 1 × MIC. There was incubation in a 37 °C thermostatic shaker at 200 rpm for 0–24 h to collect samples, which were plated on MHA plates. Colony numbers were recorded at all collected time points.</p>\",\n \"<title>PAE assay</title>\",\n \"<p id=\\\"Par15\\\">The method of PAE of AP138L-arg26 against MRSA ATCC 43300 or <italic>S. aureus</italic> CVCC 546 was described previously (Wang et al. ##REF##29523806##2018##). The formula is PAE = <italic>T</italic> − <italic>C</italic> (<italic>T</italic> is the time required for the number of colonies in the sample treatment group to increase by tenfold, and <italic>C</italic> is the time required for the untreated group).</p>\",\n \"<title>Intracellular antibacterial activity</title>\",\n \"<p id=\\\"Par16\\\">The method of intracellular bactericidal effect was described previously (Wang et al. ##REF##29523806##2018##). Only the number of cells and bacteria changed from 2.5 × 10<sup>5</sup> to 2 × 10<sup>5</sup> cells/mL.</p>\",\n \"<title>Synergism with antibiotics</title>\",\n \"<title>Fractional inhibitory concentration index (FICI)</title>\",\n \"<p id=\\\"Par17\\\">The synergistic effects of AP138L-arg26 with different antibiotics were evaluated using a checkerboard assay. The synergistic effect was calculated using the FICI as follows: FICI = FIC of AP138L-arg26 + FIC of antibiotic; FIC = MIC<sub>c</sub>/MIC<sub>a</sub>, where MIC<sub>c</sub> is the MIC of the peptide and antibiotic in combination, and MIC<sub>a</sub> is the MIC of the peptide/antibiotic alone (Blier et al. ##REF##20008946##2010##). Three parallel experiments were performed for each group. The efficacy of combination therapy was defined as FICI ≤ 0.5, 0.5 < FICI ≤ 1, 1 < FICI ≤ 4, and FICI > 4 indicating synergy, addition, no difference, and antagonism, respectively.</p>\",\n \"<title>Growth curve of <italic>S. aureus</italic></title>\",\n \"<p id=\\\"Par18\\\">Samples were taken during the logarithmic growth of <italic>S. aureus</italic> and diluted to 1 × 10<sup>5</sup> CFU/mL (Delpech et al. ##UREF##0##2019##). Different concentrations of AP138L-arg26, ceftiofur sodium, or a combination of them with an equal volume of bacteria were added to a 96-well microplate. The growth curves were recorded using a fully automatic growth curve recording instrument.</p>\",\n \"<title>Stability analysis</title>\",\n \"<title>Artificial gastric and intestinal juice stability</title>\",\n \"<p id=\\\"Par19\\\">AP138L-arg26 was incubated in artificial gastric and intestinal juice (Beijing Coolaber Technology Co., Ltd., Beijing, China) for 0.083, 0.167, 0.25, 0.75, and 1 h. The antimicrobial activity of AP138L-arg26 against MRSA ATCC 43300 was tested through the MIC assay.</p>\",\n \"<title>Thermal, pH, and salt stability</title>\",\n \"<p id=\\\"Par20\\\">The thermal, pH, and salt stability of AMPs were studied previously (Zhang et al. ##REF##21558006##2011##). Briefly, the AP138L-arg26 was incubated at different temperatures (4/20/40/60/80/100 °C), pH values (2/4/6/8/10), and kinds of salt ions (50/100/150/200/300 mM NaCl, 1.25/2.5/5 mM KCl, MgCl<sub>2</sub>, CaCl<sub>2</sub>) for 1 h at 37 °C, respectively. The antimicrobial activity of AP138L-arg26 against MRSA ATCC 43300 was tested using MIC assay.</p>\",\n \"<title>Circular dichroism (CD) spectrum assay</title>\",\n \"<p id=\\\"Par21\\\">The secondary structures of AP138L-arg26 were detected using CD (Bio-Logic MOS450 spectropolarimeter, France) in a simulated eukaryotic cell environment (50% trifluoroethanol, TFE) and bacterial membrane environment (20 mM sodium dodecyl sulfate, SDS) (Yao et al. ##REF##28870607##2018##).</p>\",\n \"<title>Safety evaluation in vitro and in vivo</title>\",\n \"<title>Hemolysis activity</title>\",\n \"<p id=\\\"Par22\\\">The hemolysis of AP138L-arg26 to fresh ICR mouse erythrocytes was described previously (Zheng et al. ##REF##34491399##2021##). Briefly, 8% of mouse erythrocytes were incubated with different concentrations of antimicrobial peptide AP138L-arg26 (256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 µg/mL) in equal volumes. The blank and positive control was phosphate buffered saline (PBS) and 0.1% Triton X-100, respectively.</p>\",\n \"<title>Cytotoxicity</title>\",\n \"<p id=\\\"Par23\\\">The cytotoxicity of AP138L-arg26 to mouse macrophages RAW264.7 was detected by the thiazolyl blue tetrazolium bromide (MTT) method as described previously (Shen et al. ##REF##33815324##2021##).</p>\",\n \"<title>Acute toxicity in mice</title>\",\n \"<p id=\\\"Par24\\\">AP138L-arg26 was administrated by intraperitoneal injection (<italic>n</italic> = 4 per group, 10 mg/kg, body weight 25 g) every day for 1 week (Shi et al. ##REF##36071151##2022##). After 7 days, the mice were euthanized to collect anticoagulant whole-blood and tissues (liver, spleen, kidney, and lung), which were used for whole blood and biochemical detection, or tissue sections (hematoxylin-eosin (HE) staining), respectively.</p>\",\n \"<title>Antimicrobial mechanism of peptides</title>\",\n \"<title>Scanning electron microscopy (SEM) assay</title>\",\n \"<p id=\\\"Par25\\\">The effect of peptides on changes in bacterial morphology was observed by scanning electron microscopy. Briefly the exponential phase <italic>S. aureus</italic> CVCC 546 cells (1 × 10<sup>9</sup> CFU/mL) were incubated with 2 × MIC peptides at 37 °C for 0.5 h, 1 h, and 2 h. The untreated bacteria were the negative control. The processing methods and steps of the samples were described in detail in our previous study (Li et al. ##REF##32582062##2020##). Briefly, there were two important steps: (1) Sample preparation: incubation, cleaning, fixation, drying, gold spraying. (2) Observation using microscope. Only one concentration was used to deal with bacteria at different times (0.5, 1, and 2 h).</p>\",\n \"<title>SYTO9/propidium iodide (PI) assay</title>\",\n \"<p id=\\\"Par26\\\">To explore the disruption of bacterial cell membranes by the AP138L-arg26, a SYTO9/PI kit was used to detect the integrity of cell membranes (L7007 LIVE/DEAD<sup>R</sup> BacLight™ Bacerial Viability Kits). The unique feature of this kit is that SYTO9 alone can pass through integral cell membranes, while PI can only penetrate damaged cell membranes. Briefly, the <italic>S. aureus</italic> CVCC 546 cells (1 × 10<sup>9</sup> CFU/mL) were incubated with AP138L-arg26 (2 × MIC, 37 °C for 1 h), and a volume of 3 µL SYTO9/PI (1.5 µL: 1.5 µL) was added to 1 mL of bacterial suspension. Finally, the results were observed using fluorescence microscopy.</p>\",\n \"<title>Membrane fluidity assay</title>\",\n \"<p id=\\\"Par27\\\">The final concentration of 10 µM Laurdan was used to detect the effect of AP138L-arg26 on bacterial cell membrane fluidity (Shi et al. ##REF##36071151##2022##). Briefly, (1) co-incubation of bacteria with Laurdan; (2) the stained bacteria are co-incubated with the peptide; (3) spectrophotometer (Tecan, Männedorf, Switzerland) detection. The calculation formula: generalized polarization (GP) = (I435 − I490)/(I435 + I490).</p>\",\n \"<title>Membrane depolarization assay</title>\",\n \"<p id=\\\"Par28\\\">To further explore the effect of AP138L-arg26 on the bacterial membrane, the membrane probe DiSC<sub>3</sub>(5) was selected to detect the change in membrane potential (Wang et al. ##REF##31974490##2020##). Briefly, <italic>S. aureus</italic> was washed and resuspended in PBS to 1 × 10<sup>8</sup> CFU/mL and incubated with 0.5 mM membrane dyes DiSC<sub>3</sub>(5) at 37 °C for 1 h in the dark. Then, 90 µL of stained bacterial suspension and 10 µL of AP138 L-arg26 (1 × , 2 × , 4 × MIC) were mixed and added to black and clean 96-cell plates and the fluorescence intensity was determined using a spectrophotometer (excitation wavelength 622/emission wavelength 670 nm, Infinite M200).</p>\",\n \"<title>Potassium ion (K<sup>+</sup>) leakage</title>\",\n \"<p id=\\\"Par29\\\">The integrity effect of antimicrobial peptide AP138L-arg26 on bacterial cell membranes was further verified, and the K<sup>+</sup> leakage assay was previously described (Li et al. ##REF##32582062##2020##). Briefly, the main procedures were as follows: Firstly, prepare the <italic>S. aureus</italic> CVCC 546 bacteria suspension at a concentration of 1 × 10<sup>8</sup> CFU/mL. Secondly, incubate the bacterial suspension with 2 × MIC AP138L-arg26 at different times (15/30/60/90/120 min) at 37 °C, with untreated cells and nisin used as negative and positive controls. Finally, the supernatants were detected using inductively coupled plasma mass spectrometry (ICP-MS) (SantaClara, CA, USA).</p>\",\n \"<title>Intracellular adenosine triphosphate (ATP) determination</title>\",\n \"<p id=\\\"Par30\\\">As ATP is the most direct source of energy for living organisms, the effect of antimicrobial peptides on ATP was explored with an ATP assay kit (Beyotime, Shanghai, China). Briefly, exponential-stage <italic>S. aureus</italic> CVCC 546 (1 × 10<sup>8</sup> CFU/mL) were incubated with different concentrations of AP138L-arg26 (1 × , 2 × , 4 × MIC) for 1 h, and the pellets were collected, lysed, and centrifuged to harvest the intracellular supernatant. The luminescence was detected using an Infinite M200 Microplate reader (Tecan, Luminescence signals).</p>\",\n \"<title>Lactic dehydrogenase (LDH) activity</title>\",\n \"<p id=\\\"Par31\\\">As disruption of the cell membrane structure leads to the release of LDH from the cytoplasm into the culture medium, the detection of LDH in bacteria can further reveal the antibacterial mechanism (Shi et al. ##REF##36071151##2022##). Briefly, <italic>S. aureus</italic> cells were co-incubated with AP138L-arg26 for 6 h. Later, the pellets were collected and sonicated (3 s/10 s, 30 times). The intracellular LDH activity was detected using an Infinite M200 Microplate reader (Tecan, Luminescence signals). The calculated results of LDH% = treated cells/the control.</p>\",\n \"<title>Reactive oxygen species (ROS) measurements</title>\",\n \"<p id=\\\"Par32\\\">The probe 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure the level of ROS in bacteria after incubated with AP138L-arg26 (Wang et al. ##REF##34090965##2021a##, ##REF##34009979##b##; Shi et al. ##REF##36071151##2022##). Briefly, exponential stage bacteria were incubated with DCFH-DA (10 µM, 37 °C, 0.5 h), and the stained bacteria were treated with AP138L-arg26 (1 × , 2 × , 4 × MIC) for 1 h. The ROS were detected at excitation wavelength (488 nm) and emission wavelength (525 nm) using a microplate reader (Tecan, Männedorf, Switzerland).</p>\",\n \"<title>Statistical analysis</title>\",\n \"<p id=\\\"Par33\\\">The software GraphPad Prism (version 8, USA) was used to analyze all data, and ANOVA was the method to determine the statistical significance. The results are presented as means ± standard deviation (SD). A <italic>P</italic> value of < 0.05 was considered statistically significant.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>We acknowledge Chunli Li from the Core Facility at the Institute of Microbiology at the Chinese Academy of Sciences (CAS) for the technical support with SEM and Tong Zhao for her technical support with FACS analysis.</p>","<title>Author contribution</title>","<p>KZ and JW conceived and designed the research. DT, RM, NY, and YH conducted experiments. KZ, NY, and JW evaluated data. KZ, NY, and JW wrote and revised the manuscript. All authors read and approved the manuscript.</p>","<title>Funding</title>","<p>This work was supported by the National Natural Science Foundation of China (Grant No. 31872393), National Key Research and Development Plan—High Expression of Thiopeptides and their Analogs (Grant No. 2022YFC2105000-03, 2022–2026), and the Innovation Program of Agricultural Science and Technology (ASTIP) in CAAS (Grant No. CAAS-ASTIP-2017-FRI-02) and its key projects (Grant No. CAAS-ZDRW202111 and Grant No. CAAS-ZDXT 201808).</p>","<title>Data availability</title>","<p>All data generated or analyzed during this study are included in this published article (and its supplementary information files).</p>","<title>Declarations</title>","<title>Ethical approval</title>","<p id=\"Par51\">The mouse experiment was performed according to the Animal Care and Use Committee of the Feed Research Institute of the Chinese Academy of Agricultural Sciences (CAAS) and approved by the Laboratory Animal Ethical Committee and its Inspection of the Feed Research Institute of CAAS (AEC-CAAS-20090609).</p>","<title>Competing interests</title>","<p id=\"Par52\">The authors declare no competing interests.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>We acknowledge Chunli Li from the Core Facility at the Institute of Microbiology at the Chinese Academy of Sciences (CAS) for the technical support with SEM and Tong Zhao for her technical support with FACS analysis.</p>\",\n \"<title>Author contribution</title>\",\n \"<p>KZ and JW conceived and designed the research. DT, RM, NY, and YH conducted experiments. KZ, NY, and JW evaluated data. KZ, NY, and JW wrote and revised the manuscript. All authors read and approved the manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>This work was supported by the National Natural Science Foundation of China (Grant No. 31872393), National Key Research and Development Plan—High Expression of Thiopeptides and their Analogs (Grant No. 2022YFC2105000-03, 2022–2026), and the Innovation Program of Agricultural Science and Technology (ASTIP) in CAAS (Grant No. CAAS-ASTIP-2017-FRI-02) and its key projects (Grant No. CAAS-ZDRW202111 and Grant No. CAAS-ZDXT 201808).</p>\",\n \"<title>Data availability</title>\",\n \"<p>All data generated or analyzed during this study are included in this published article (and its supplementary information files).</p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethical approval</title>\",\n \"<p id=\\\"Par51\\\">The mouse experiment was performed according to the Animal Care and Use Committee of the Feed Research Institute of the Chinese Academy of Agricultural Sciences (CAAS) and approved by the Laboratory Animal Ethical Committee and its Inspection of the Feed Research Institute of CAAS (AEC-CAAS-20090609).</p>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par52\\\">The authors declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>The 3D structure analysis of plectasin (left) and AP138L-arg26 (right). <bold>a</bold> Molecular modeling of plectasin and AP138L-arg26. <bold>b</bold> Electrostatic surface of plectasin (left) and AP138L-arg26 (right). Blue and red represent positive and negative charge, respectively</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>The construction of the pPICZαA-AP138L-arg26 plasmid and the expression of AP138L-arg26 in <italic>P. pastoris</italic> X-33 at the shaking flask and fermenter level. <bold>a</bold> Recombinant plasmids pPICZαA-AP138L-arg26 and linearized gel electrophoresis, lanes 1–2 were pPICZαA-AP138L-arg26 (left) and linearized plasmid. <bold>b</bold> Screening better AP138L-arg26 positive transformants AP138L-arg26-8, AP138L-arg26-28, AP138L-arg26-68, and AP138L-arg26-94 by inhibition zone against <italic>S. aureus</italic> ATCC 43300. <bold>c</bold> Tricine-SDS-PAGE analysis of AP138L-arg26 expression in shaking flask level, lanes 1–4 were fermentation supernatants of induction at 0 h, 24 h, 48 h, and 96 h, respectively. <bold>d</bold> Antimicrobial activity against <italic>S. aureus</italic> ATCC 43300 of AP138L-arg26 in fermenter level was analyzed by inhibition zone assay at 0 h, 24 h, 48 h, 96 h, and 120 h, respectively. <bold>e</bold> Tricine-SDS-PAGE analysis of AP138L-arg26 expression in fermenter level, lanes 1–6 were fermentation supernatants of induction at 0 h, 24 h, 48 h, 96 h, and 120 h, respectively. <bold>f</bold> Time curves of the total protein levels and cell wet weights at 0 h, 24 h, 48 h, 96 h, and 120 h, respectively. <bold>g</bold> Tricine-SDS-PAGE analysis of AP138L-arg26 purification. <bold>h</bold> MALDI-TOF MS analysis of the purified AP138L-arg26</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>The pharmacodynamic evaluation of antimicrobial peptide AP138L-arg26 in extracellular and intracellular activity. <bold>a</bold> Time killing curve of AP138L-arg26 against <italic>S. aureus</italic> ATCC 43300, vancomycin (Van) as positive control, untreated group (CK) as blank control. <bold>b</bold> Post-antibiotic effect of AP138L-arg26 against standard <italic>S. aureus</italic> ATCC 43300, Van as control. Intracellular activity of AP138L-arg26 against <italic>S. aureus</italic> CVCC 546 in <bold>c</bold> mouse macrophage RAW 264.7 and <bold>d</bold> Bovine uterine epithelial cells (BEND). Asterisk (*) indicates the significance between control and treatment groups. ***<italic>P</italic> < 0.001. Results are expressed as the means from three biological replicates ± SD (<italic>n</italic> = 3). <bold>e</bold> Growth curve of <italic>S. aureus</italic> CVCC 546 after treatment with different combination of AP138L-arg26 and Ceftiofur Sodium (Cef)</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>The safety of AP138L-arg26 in vitro and in vivo. <bold>a</bold> Hemolytic activity of AP138L-arg26 against fresh mouse red blood cells. <bold>b</bold> Cytotoxicity of the AP138L-arg26 against RAW264.7 cells. ICR female mice (<italic>n</italic> = 4) were intraperitoneally administered with AP138L-arg26 (10 mg/kg) daily for a week. <bold>c</bold>–<bold>g</bold> Histology images (H&E) stained (<bold>d</bold>), whole-blood cell profiles (<bold>e</bold>, <bold>f</bold>), serum biochemical 7 index (g) of mice after treatment with AP138L-arg26 for a week. CK, untreated group; scale bar, 100 µm</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Study on the effect of AP138L-arg26 on cell membranes. <bold>a</bold> The morphological changes of <italic>S. aureus</italic> CVCC 546 were observed by SEM after AP138L-arg26 treatment. <bold>b</bold> The destruction of cell membranes by antimicrobial peptide AP138L-arg26 was observed by SYTO9/PI. <bold>c</bold> Detection of cell membrane potential after incubation of AP138L-arg26 with <italic>S. aureus</italic> CVCC 546. <bold>d</bold> The K<sup>+</sup> leakage after incubation of AP138L-arg26 with <italic>S. aureus</italic> CVCC 546. <bold>e</bold> Membrane fluidity. Asterisk (*) indicates the significance between control and treatment groups, <italic>P</italic> < 0.05; (ns) indicates the no significance between control and treatment groups. CK, untreated group</p></caption></fig>","<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Effects of AP138L-arg26 on cell metabolism. <bold>a</bold> the level of Intracellular ATP, <bold>b</bold> the level of ROS, <bold>c</bold> the level of LDH. CK, untreated group. Asterisk (*) indicates the significance between control and treatment groups. ***<italic>P</italic> < 0.001. Results are expressed as the means from three biological replicates ± SD (<italic>n</italic> = 3)</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>The 3D structure analysis of plectasin (left) and AP138L-arg26 (right). <bold>a</bold> Molecular modeling of plectasin and AP138L-arg26. <bold>b</bold> Electrostatic surface of plectasin (left) and AP138L-arg26 (right). Blue and red represent positive and negative charge, respectively</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>The construction of the pPICZαA-AP138L-arg26 plasmid and the expression of AP138L-arg26 in <italic>P. pastoris</italic> X-33 at the shaking flask and fermenter level. <bold>a</bold> Recombinant plasmids pPICZαA-AP138L-arg26 and linearized gel electrophoresis, lanes 1–2 were pPICZαA-AP138L-arg26 (left) and linearized plasmid. <bold>b</bold> Screening better AP138L-arg26 positive transformants AP138L-arg26-8, AP138L-arg26-28, AP138L-arg26-68, and AP138L-arg26-94 by inhibition zone against <italic>S. aureus</italic> ATCC 43300. <bold>c</bold> Tricine-SDS-PAGE analysis of AP138L-arg26 expression in shaking flask level, lanes 1–4 were fermentation supernatants of induction at 0 h, 24 h, 48 h, and 96 h, respectively. <bold>d</bold> Antimicrobial activity against <italic>S. aureus</italic> ATCC 43300 of AP138L-arg26 in fermenter level was analyzed by inhibition zone assay at 0 h, 24 h, 48 h, 96 h, and 120 h, respectively. <bold>e</bold> Tricine-SDS-PAGE analysis of AP138L-arg26 expression in fermenter level, lanes 1–6 were fermentation supernatants of induction at 0 h, 24 h, 48 h, 96 h, and 120 h, respectively. <bold>f</bold> Time curves of the total protein levels and cell wet weights at 0 h, 24 h, 48 h, 96 h, and 120 h, respectively. <bold>g</bold> Tricine-SDS-PAGE analysis of AP138L-arg26 purification. <bold>h</bold> MALDI-TOF MS analysis of the purified AP138L-arg26</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>The pharmacodynamic evaluation of antimicrobial peptide AP138L-arg26 in extracellular and intracellular activity. <bold>a</bold> Time killing curve of AP138L-arg26 against <italic>S. aureus</italic> ATCC 43300, vancomycin (Van) as positive control, untreated group (CK) as blank control. <bold>b</bold> Post-antibiotic effect of AP138L-arg26 against standard <italic>S. aureus</italic> ATCC 43300, Van as control. Intracellular activity of AP138L-arg26 against <italic>S. aureus</italic> CVCC 546 in <bold>c</bold> mouse macrophage RAW 264.7 and <bold>d</bold> Bovine uterine epithelial cells (BEND). Asterisk (*) indicates the significance between control and treatment groups. ***<italic>P</italic> < 0.001. Results are expressed as the means from three biological replicates ± SD (<italic>n</italic> = 3). <bold>e</bold> Growth curve of <italic>S. aureus</italic> CVCC 546 after treatment with different combination of AP138L-arg26 and Ceftiofur Sodium (Cef)</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>The safety of AP138L-arg26 in vitro and in vivo. <bold>a</bold> Hemolytic activity of AP138L-arg26 against fresh mouse red blood cells. <bold>b</bold> Cytotoxicity of the AP138L-arg26 against RAW264.7 cells. ICR female mice (<italic>n</italic> = 4) were intraperitoneally administered with AP138L-arg26 (10 mg/kg) daily for a week. <bold>c</bold>–<bold>g</bold> Histology images (H&E) stained (<bold>d</bold>), whole-blood cell profiles (<bold>e</bold>, <bold>f</bold>), serum biochemical 7 index (g) of mice after treatment with AP138L-arg26 for a week. CK, untreated group; scale bar, 100 µm</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><p>Study on the effect of AP138L-arg26 on cell membranes. <bold>a</bold> The morphological changes of <italic>S. aureus</italic> CVCC 546 were observed by SEM after AP138L-arg26 treatment. <bold>b</bold> The destruction of cell membranes by antimicrobial peptide AP138L-arg26 was observed by SYTO9/PI. <bold>c</bold> Detection of cell membrane potential after incubation of AP138L-arg26 with <italic>S. aureus</italic> CVCC 546. <bold>d</bold> The K<sup>+</sup> leakage after incubation of AP138L-arg26 with <italic>S. aureus</italic> CVCC 546. <bold>e</bold> Membrane fluidity. Asterisk (*) indicates the significance between control and treatment groups, <italic>P</italic> < 0.05; (ns) indicates the no significance between control and treatment groups. CK, untreated group</p></caption></fig>\",\n \"<fig id=\\\"Fig6\\\"><label>Fig. 6</label><caption><p>Effects of AP138L-arg26 on cell metabolism. <bold>a</bold> the level of Intracellular ATP, <bold>b</bold> the level of ROS, <bold>c</bold> the level of LDH. CK, untreated group. Asterisk (*) indicates the significance between control and treatment groups. ***<italic>P</italic> < 0.001. Results are expressed as the means from three biological replicates ± SD (<italic>n</italic> = 3)</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Physicochemical properties and likelihood prediction of antimicrobial peptide</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Peptide</th><th align=\"left\">Sequence</th><th align=\"left\">AMP probability</th><th align=\"left\">Hydrophobic ratio</th><th align=\"left\">Net charge + </th><th align=\"left\">GRAVY − </th><th align=\"left\">Hydrophobicity</th><th align=\"left\">Amphiphilicity index</th><th align=\"left\">Protein-binding potential</th><th align=\"left\">Molecular weight</th></tr></thead><tbody><tr><td align=\"left\">Plectasin</td><td align=\"left\">GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY</td><td char=\".\" align=\"char\">0.9495</td><td align=\"left\">32%</td><td align=\"left\">1</td><td char=\".\" align=\"char\">0.695</td><td char=\".\" align=\"char\">—</td><td char=\".\" align=\"char\">1.15</td><td char=\".\" align=\"char\">1.4</td><td char=\".\" align=\"char\">4407.99</td></tr><tr><td align=\"left\">AP138L-arg26</td><td align=\"left\">GFGCNGPWSEDDLRCHRHCKSIKGYRGGYCAKGGFVCKCY</td><td char=\".\" align=\"char\">0.992</td><td align=\"left\">33%</td><td align=\"left\">4.5</td><td char=\".\" align=\"char\">0.674</td><td char=\".\" align=\"char\">3.42</td><td char=\".\" align=\"char\">1.25</td><td char=\".\" align=\"char\">1.88</td><td char=\".\" align=\"char\">4460.125</td></tr><tr><td align=\"left\">NZ2114</td><td align=\"left\">GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCY</td><td char=\".\" align=\"char\">0.9895</td><td align=\"left\">33%</td><td align=\"left\">3.5</td><td char=\".\" align=\"char\">0.672</td><td char=\".\" align=\"char\">3.5</td><td char=\".\" align=\"char\">1.18</td><td char=\".\" align=\"char\">1.52</td><td char=\".\" align=\"char\">4417.049</td></tr><tr><td align=\"left\">NZX</td><td align=\"left\">GFGCNGPWSEDDIQCHNHCKSIKGYKGGYCARGGFVCKCY</td><td char=\".\" align=\"char\">0.9355</td><td align=\"left\">33%</td><td align=\"left\">2.5</td><td char=\".\" align=\"char\">0.5775</td><td char=\".\" align=\"char\">3.05</td><td char=\".\" align=\"char\">1.12</td><td char=\".\" align=\"char\">1.43</td><td char=\".\" align=\"char\">4389.981</td></tr><tr><td align=\"left\">MP1102</td><td align=\"left\">GFGCNGPWQEDDVKCHNHCKSIKGYKGGYCAKGGFVCKCY</td><td char=\".\" align=\"char\">0.88</td><td align=\"left\">33%</td><td align=\"left\">3.5</td><td char=\".\" align=\"char\">0.6475</td><td char=\".\" align=\"char\">4.47</td><td char=\".\" align=\"char\">1.24</td><td char=\".\" align=\"char\">1.28</td><td char=\".\" align=\"char\">4460.125</td></tr><tr><td align=\"left\">DLP4</td><td align=\"left\">ATCDLLSPFKVGHAACAAHCIARGKRGGWCDKRAVCNCRK</td><td char=\".\" align=\"char\">0.9455</td><td align=\"left\">50%</td><td align=\"left\">6.0</td><td char=\".\" align=\"char\">0.13</td><td char=\".\" align=\"char\">—</td><td char=\".\" align=\"char\">0.86</td><td char=\".\" align=\"char\">1.72</td><td char=\".\" align=\"char\">4275.26</td></tr><tr><td align=\"left\">ID13</td><td align=\"left\">ATCDLLSPFKVGHAACAAHCIARGKRGGWCDGRAVCNCRK</td><td char=\".\" align=\"char\">0.96</td><td align=\"left\">50%</td><td align=\"left\">5.5</td><td char=\".\" align=\"char\">0.0425</td><td char=\".\" align=\"char\">5.68</td><td char=\".\" align=\"char\">0.77</td><td char=\".\" align=\"char\">1.56</td><td char=\".\" align=\"char\">4203.96</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>The MIC and MBC of AP138L-arg26 and vancomycin</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Strain</th><th align=\"left\" colspan=\"2\">MIC (µg/mL)</th><th align=\"left\" colspan=\"2\">MBC (µg/mL)</th></tr><tr><th align=\"left\">AP138L-arg26</th><th align=\"left\">Van</th><th align=\"left\">AP138L-arg26</th><th align=\"left\">Van</th></tr></thead><tbody><tr><td align=\"left\"><italic>Staphylococcus aureus</italic> ATCC 25923</td><td align=\"left\">16</td><td align=\"left\">1</td><td align=\"left\">32</td><td align=\"left\">2</td></tr><tr><td align=\"left\"><italic>Staphylococcus aureus</italic> ATCC 43300</td><td align=\"left\">8</td><td align=\"left\">1</td><td align=\"left\">8</td><td align=\"left\">1</td></tr><tr><td align=\"left\"><italic>Staphylococcus aureus</italic> E48</td><td align=\"left\">2</td><td align=\"left\">1</td><td align=\"left\">8</td><td align=\"left\">8</td></tr><tr><td align=\"left\"><italic>Staphylococcus aureus</italic> CVCC 546</td><td align=\"left\">4</td><td align=\"left\">1</td><td align=\"left\">32</td><td align=\"left\">1</td></tr><tr><td align=\"left\"><italic>Staphylococcus aureus</italic> CAAS-FRI-2023-01</td><td align=\"left\">4</td><td align=\"left\">1</td><td align=\"left\">16</td><td align=\"left\">2</td></tr><tr><td align=\"left\"><italic>Staphylococcus aureus</italic> CAAS-FRI-2023-02</td><td align=\"left\">8</td><td align=\"left\">2</td><td align=\"left\">32</td><td align=\"left\">8</td></tr><tr><td align=\"left\"><italic>Staphylococcus epidermidis</italic> ATCC 12228</td><td align=\"left\">4</td><td align=\"left\">2</td><td align=\"left\">16</td><td align=\"left\">4</td></tr><tr><td align=\"left\"><italic>Staphylococcus epidermidis</italic> ATCC 35984</td><td align=\"left\">8</td><td align=\"left\">2</td><td align=\"left\">16</td><td align=\"left\">4</td></tr><tr><td align=\"left\"><italic>Streptococcus dysgalactiae</italic> CVCC 3938</td><td align=\"left\">4</td><td align=\"left\">2</td><td align=\"left\">32</td><td align=\"left\">2</td></tr><tr><td align=\"left\"><italic>Streptococcus agalactiae</italic> ATCC 13813</td><td align=\"left\">4</td><td align=\"left\">2</td><td align=\"left\">8</td><td align=\"left\">4</td></tr><tr><td align=\"left\"><italic>Streptococcus agalactiae</italic> CAU-FRI-2022-01</td><td align=\"left\">4</td><td align=\"left\">2</td><td align=\"left\">32</td><td align=\"left\">8</td></tr><tr><td align=\"left\"><italic>Streptococcus agalactiae</italic> CAU-FRI-2022-02</td><td align=\"left\">4</td><td align=\"left\">2</td><td align=\"left\">32</td><td align=\"left\">4</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Synergism of peptide AP138L-arg26 with antibiotics</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Combination</th><th align=\"left\" rowspan=\"2\">Variety</th><th align=\"left\" colspan=\"4\"><italic>S. aureus</italic> CVCC 546</th></tr><tr><th align=\"left\">MIC<sub>a</sub> (µg/mL)</th><th align=\"left\">MIC<sub>c</sub> (µg/mL)</th><th align=\"left\">FIC</th><th align=\"left\">FICI index</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"2\">AP138L-arg26-vancomycin</td><td align=\"left\">AP138L-arg26</td><td align=\"left\">4</td><td align=\"left\">1</td><td align=\"left\">0.25</td><td align=\"left\">0.5</td></tr><tr><td align=\"left\">Vancomycin</td><td align=\"left\">1</td><td align=\"left\">0.25</td><td align=\"left\">0.25</td><td align=\"left\"/></tr><tr><td align=\"left\" rowspan=\"2\">AP138L-arg26-Streptomycin sulfate</td><td align=\"left\">AP138L-arg26</td><td align=\"left\">4</td><td align=\"left\">2</td><td align=\"left\">0.5</td><td align=\"left\">1</td></tr><tr><td align=\"left\">Streptomycin sulfate</td><td align=\"left\">1</td><td align=\"left\">0.5</td><td align=\"left\">0.5</td><td align=\"left\"/></tr><tr><td align=\"left\" rowspan=\"2\">AP138L-arg26-ceftiofur sodium</td><td align=\"left\">AP138L-arg26</td><td align=\"left\">4</td><td align=\"left\">0.25</td><td align=\"left\">0.0625</td><td align=\"left\">0.3125</td></tr><tr><td align=\"left\">Ceftiofur sodium</td><td align=\"left\">1</td><td align=\"left\">0.25</td><td align=\"left\">0.25</td><td align=\"left\"/></tr><tr><td align=\"left\" rowspan=\"2\">AP138L-arg26-ampicillin</td><td align=\"left\">AP138L-arg26</td><td align=\"left\">4</td><td align=\"left\">4</td><td align=\"left\">1</td><td align=\"left\">1.0625</td></tr><tr><td align=\"left\">Ampicillin</td><td align=\"left\">0.125</td><td align=\"left\">0.0078</td><td align=\"left\">0.0625</td><td align=\"left\"/></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>The stability of AMPs in different environments</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"6\">AP138L-arg26 against <italic>S. aureus</italic> CVCC 546</th></tr><tr><th align=\"left\">Condition</th><th align=\"left\">MIC</th><th align=\"left\">Condition</th><th align=\"left\">MIC</th><th align=\"left\">Condition</th><th align=\"left\">MIC</th></tr></thead><tbody><tr><td align=\"left\">Heat (20 °C)</td><td align=\"left\">8</td><td align=\"left\">KCl (1.25 mM)</td><td align=\"left\">8</td><td align=\"left\">50% serum (0.5 h)</td><td align=\"left\">8</td></tr><tr><td align=\"left\">Heat (40 °C)</td><td align=\"left\">8</td><td align=\"left\">KCl (2.5 mM)</td><td align=\"left\">8</td><td align=\"left\">50% serum (1 h)</td><td align=\"left\">8</td></tr><tr><td align=\"left\">Heat (60 °C)</td><td align=\"left\">8</td><td align=\"left\">KCl (5 mM)</td><td align=\"left\">8</td><td align=\"left\">50% serum (2 h)</td><td align=\"left\">8</td></tr><tr><td align=\"left\">Heat (80 °C)</td><td align=\"left\">8</td><td align=\"left\">CaCl<sub>2</sub> (1.25 mM)</td><td align=\"left\">8</td><td align=\"left\">50% serum (4 h)</td><td align=\"left\">8</td></tr><tr><td align=\"left\">Heat (100 °C)</td><td align=\"left\"> > 64</td><td align=\"left\">CaCl<sub>2</sub> (2.5 mM)</td><td align=\"left\">8</td><td align=\"left\">50% serum (6 h)</td><td align=\"left\">8</td></tr><tr><td align=\"left\">pH 2</td><td align=\"left\">8</td><td align=\"left\">CaCl<sub>2</sub> (5 mM)</td><td align=\"left\">16</td><td align=\"left\">50% serum (8 h)</td><td align=\"left\">8</td></tr><tr><td align=\"left\">pH 4</td><td align=\"left\">16</td><td align=\"left\">MgCl<sub>2</sub> (1.25 mM)</td><td align=\"left\">8</td><td align=\"left\">Gastric juice (0.25 h)</td><td align=\"left\">4</td></tr><tr><td align=\"left\">pH 6</td><td align=\"left\">16</td><td align=\"left\">MgCl<sub>2</sub> (2.5 mM)</td><td align=\"left\">16</td><td align=\"left\">Gastric juice (0.5 h)</td><td align=\"left\">4</td></tr><tr><td align=\"left\">pH 8</td><td align=\"left\">8</td><td align=\"left\">MgCl<sub>2</sub> (5 mM)</td><td align=\"left\">16</td><td align=\"left\">Gastric juice (1 h)</td><td align=\"left\">4</td></tr><tr><td align=\"left\">pH 10</td><td align=\"left\">16</td><td align=\"left\">25% serum (0.5 h)</td><td align=\"left\">8</td><td align=\"left\">Gastric juice (2 h)</td><td align=\"left\">8</td></tr><tr><td align=\"left\">NaCl (50 mM)</td><td align=\"left\">8</td><td align=\"left\">25% serum (1 h)</td><td align=\"left\">16</td><td align=\"left\">Intestinal juice (0.25 h)</td><td align=\"left\" rowspan=\"4\"> > 64</td></tr><tr><td align=\"left\">NaCl (100 mM)</td><td align=\"left\">8</td><td align=\"left\">25% serum (2 h)</td><td align=\"left\">16</td><td align=\"left\">Intestinal juice (0.5 h)</td></tr><tr><td align=\"left\">NaCl (150 mM)</td><td align=\"left\">8</td><td align=\"left\">25% serum (4 h)</td><td align=\"left\">8</td><td align=\"left\">Intestinal juice (1 h)</td></tr><tr><td align=\"left\">NaCl (200 mM)</td><td align=\"left\">16</td><td align=\"left\">25% serum (6 h)</td><td align=\"left\">16</td><td align=\"left\">Intestinal juice (2 h)</td></tr><tr><td align=\"left\">NaCl (300 mM)</td><td align=\"left\">16</td><td align=\"left\">25% serum (8 h)</td><td align=\"left\">8</td><td align=\"left\">Control (without treatment)</td><td align=\"left\">8</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Physicochemical properties and likelihood prediction of antimicrobial peptide</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Peptide</th><th align=\\\"left\\\">Sequence</th><th align=\\\"left\\\">AMP probability</th><th align=\\\"left\\\">Hydrophobic ratio</th><th align=\\\"left\\\">Net charge + </th><th align=\\\"left\\\">GRAVY − </th><th align=\\\"left\\\">Hydrophobicity</th><th align=\\\"left\\\">Amphiphilicity index</th><th align=\\\"left\\\">Protein-binding potential</th><th align=\\\"left\\\">Molecular weight</th></tr></thead><tbody><tr><td align=\\\"left\\\">Plectasin</td><td align=\\\"left\\\">GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY</td><td char=\\\".\\\" align=\\\"char\\\">0.9495</td><td align=\\\"left\\\">32%</td><td align=\\\"left\\\">1</td><td char=\\\".\\\" align=\\\"char\\\">0.695</td><td char=\\\".\\\" align=\\\"char\\\">—</td><td char=\\\".\\\" align=\\\"char\\\">1.15</td><td char=\\\".\\\" align=\\\"char\\\">1.4</td><td char=\\\".\\\" align=\\\"char\\\">4407.99</td></tr><tr><td align=\\\"left\\\">AP138L-arg26</td><td align=\\\"left\\\">GFGCNGPWSEDDLRCHRHCKSIKGYRGGYCAKGGFVCKCY</td><td char=\\\".\\\" align=\\\"char\\\">0.992</td><td align=\\\"left\\\">33%</td><td align=\\\"left\\\">4.5</td><td char=\\\".\\\" align=\\\"char\\\">0.674</td><td char=\\\".\\\" align=\\\"char\\\">3.42</td><td char=\\\".\\\" align=\\\"char\\\">1.25</td><td char=\\\".\\\" align=\\\"char\\\">1.88</td><td char=\\\".\\\" align=\\\"char\\\">4460.125</td></tr><tr><td align=\\\"left\\\">NZ2114</td><td align=\\\"left\\\">GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCY</td><td char=\\\".\\\" align=\\\"char\\\">0.9895</td><td align=\\\"left\\\">33%</td><td align=\\\"left\\\">3.5</td><td char=\\\".\\\" align=\\\"char\\\">0.672</td><td char=\\\".\\\" align=\\\"char\\\">3.5</td><td char=\\\".\\\" align=\\\"char\\\">1.18</td><td char=\\\".\\\" align=\\\"char\\\">1.52</td><td char=\\\".\\\" align=\\\"char\\\">4417.049</td></tr><tr><td align=\\\"left\\\">NZX</td><td align=\\\"left\\\">GFGCNGPWSEDDIQCHNHCKSIKGYKGGYCARGGFVCKCY</td><td char=\\\".\\\" align=\\\"char\\\">0.9355</td><td align=\\\"left\\\">33%</td><td align=\\\"left\\\">2.5</td><td char=\\\".\\\" align=\\\"char\\\">0.5775</td><td char=\\\".\\\" align=\\\"char\\\">3.05</td><td char=\\\".\\\" align=\\\"char\\\">1.12</td><td char=\\\".\\\" align=\\\"char\\\">1.43</td><td char=\\\".\\\" align=\\\"char\\\">4389.981</td></tr><tr><td align=\\\"left\\\">MP1102</td><td align=\\\"left\\\">GFGCNGPWQEDDVKCHNHCKSIKGYKGGYCAKGGFVCKCY</td><td char=\\\".\\\" align=\\\"char\\\">0.88</td><td align=\\\"left\\\">33%</td><td align=\\\"left\\\">3.5</td><td char=\\\".\\\" align=\\\"char\\\">0.6475</td><td char=\\\".\\\" align=\\\"char\\\">4.47</td><td char=\\\".\\\" align=\\\"char\\\">1.24</td><td char=\\\".\\\" align=\\\"char\\\">1.28</td><td char=\\\".\\\" align=\\\"char\\\">4460.125</td></tr><tr><td align=\\\"left\\\">DLP4</td><td align=\\\"left\\\">ATCDLLSPFKVGHAACAAHCIARGKRGGWCDKRAVCNCRK</td><td char=\\\".\\\" align=\\\"char\\\">0.9455</td><td align=\\\"left\\\">50%</td><td align=\\\"left\\\">6.0</td><td char=\\\".\\\" align=\\\"char\\\">0.13</td><td char=\\\".\\\" align=\\\"char\\\">—</td><td char=\\\".\\\" align=\\\"char\\\">0.86</td><td char=\\\".\\\" align=\\\"char\\\">1.72</td><td char=\\\".\\\" align=\\\"char\\\">4275.26</td></tr><tr><td align=\\\"left\\\">ID13</td><td align=\\\"left\\\">ATCDLLSPFKVGHAACAAHCIARGKRGGWCDGRAVCNCRK</td><td char=\\\".\\\" align=\\\"char\\\">0.96</td><td align=\\\"left\\\">50%</td><td align=\\\"left\\\">5.5</td><td char=\\\".\\\" align=\\\"char\\\">0.0425</td><td char=\\\".\\\" align=\\\"char\\\">5.68</td><td char=\\\".\\\" align=\\\"char\\\">0.77</td><td char=\\\".\\\" align=\\\"char\\\">1.56</td><td char=\\\".\\\" align=\\\"char\\\">4203.96</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>The MIC and MBC of AP138L-arg26 and vancomycin</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" rowspan=\\\"2\\\">Strain</th><th align=\\\"left\\\" colspan=\\\"2\\\">MIC (µg/mL)</th><th align=\\\"left\\\" colspan=\\\"2\\\">MBC (µg/mL)</th></tr><tr><th align=\\\"left\\\">AP138L-arg26</th><th align=\\\"left\\\">Van</th><th align=\\\"left\\\">AP138L-arg26</th><th align=\\\"left\\\">Van</th></tr></thead><tbody><tr><td align=\\\"left\\\"><italic>Staphylococcus aureus</italic> ATCC 25923</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">32</td><td align=\\\"left\\\">2</td></tr><tr><td align=\\\"left\\\"><italic>Staphylococcus aureus</italic> ATCC 43300</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">1</td></tr><tr><td align=\\\"left\\\"><italic>Staphylococcus aureus</italic> E48</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">8</td></tr><tr><td align=\\\"left\\\"><italic>Staphylococcus aureus</italic> CVCC 546</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">32</td><td align=\\\"left\\\">1</td></tr><tr><td align=\\\"left\\\"><italic>Staphylococcus aureus</italic> CAAS-FRI-2023-01</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">2</td></tr><tr><td align=\\\"left\\\"><italic>Staphylococcus aureus</italic> CAAS-FRI-2023-02</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">32</td><td align=\\\"left\\\">8</td></tr><tr><td align=\\\"left\\\"><italic>Staphylococcus epidermidis</italic> ATCC 12228</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">4</td></tr><tr><td align=\\\"left\\\"><italic>Staphylococcus epidermidis</italic> ATCC 35984</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">4</td></tr><tr><td align=\\\"left\\\"><italic>Streptococcus dysgalactiae</italic> CVCC 3938</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">32</td><td align=\\\"left\\\">2</td></tr><tr><td align=\\\"left\\\"><italic>Streptococcus agalactiae</italic> ATCC 13813</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">4</td></tr><tr><td align=\\\"left\\\"><italic>Streptococcus agalactiae</italic> CAU-FRI-2022-01</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">32</td><td align=\\\"left\\\">8</td></tr><tr><td align=\\\"left\\\"><italic>Streptococcus agalactiae</italic> CAU-FRI-2022-02</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">32</td><td align=\\\"left\\\">4</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Synergism of peptide AP138L-arg26 with antibiotics</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" rowspan=\\\"2\\\">Combination</th><th align=\\\"left\\\" rowspan=\\\"2\\\">Variety</th><th align=\\\"left\\\" colspan=\\\"4\\\"><italic>S. aureus</italic> CVCC 546</th></tr><tr><th align=\\\"left\\\">MIC<sub>a</sub> (µg/mL)</th><th align=\\\"left\\\">MIC<sub>c</sub> (µg/mL)</th><th align=\\\"left\\\">FIC</th><th align=\\\"left\\\">FICI index</th></tr></thead><tbody><tr><td align=\\\"left\\\" rowspan=\\\"2\\\">AP138L-arg26-vancomycin</td><td align=\\\"left\\\">AP138L-arg26</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">0.25</td><td align=\\\"left\\\">0.5</td></tr><tr><td align=\\\"left\\\">Vancomycin</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">0.25</td><td align=\\\"left\\\">0.25</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" rowspan=\\\"2\\\">AP138L-arg26-Streptomycin sulfate</td><td align=\\\"left\\\">AP138L-arg26</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">2</td><td align=\\\"left\\\">0.5</td><td align=\\\"left\\\">1</td></tr><tr><td align=\\\"left\\\">Streptomycin sulfate</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">0.5</td><td align=\\\"left\\\">0.5</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" rowspan=\\\"2\\\">AP138L-arg26-ceftiofur sodium</td><td align=\\\"left\\\">AP138L-arg26</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">0.25</td><td align=\\\"left\\\">0.0625</td><td align=\\\"left\\\">0.3125</td></tr><tr><td align=\\\"left\\\">Ceftiofur sodium</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">0.25</td><td align=\\\"left\\\">0.25</td><td align=\\\"left\\\"/></tr><tr><td align=\\\"left\\\" rowspan=\\\"2\\\">AP138L-arg26-ampicillin</td><td align=\\\"left\\\">AP138L-arg26</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">4</td><td align=\\\"left\\\">1</td><td align=\\\"left\\\">1.0625</td></tr><tr><td align=\\\"left\\\">Ampicillin</td><td align=\\\"left\\\">0.125</td><td align=\\\"left\\\">0.0078</td><td align=\\\"left\\\">0.0625</td><td align=\\\"left\\\"/></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab4\\\"><label>Table 4</label><caption><p>The stability of AMPs in different environments</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" colspan=\\\"6\\\">AP138L-arg26 against <italic>S. aureus</italic> CVCC 546</th></tr><tr><th align=\\\"left\\\">Condition</th><th align=\\\"left\\\">MIC</th><th align=\\\"left\\\">Condition</th><th align=\\\"left\\\">MIC</th><th align=\\\"left\\\">Condition</th><th align=\\\"left\\\">MIC</th></tr></thead><tbody><tr><td align=\\\"left\\\">Heat (20 °C)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">KCl (1.25 mM)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">50% serum (0.5 h)</td><td align=\\\"left\\\">8</td></tr><tr><td align=\\\"left\\\">Heat (40 °C)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">KCl (2.5 mM)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">50% serum (1 h)</td><td align=\\\"left\\\">8</td></tr><tr><td align=\\\"left\\\">Heat (60 °C)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">KCl (5 mM)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">50% serum (2 h)</td><td align=\\\"left\\\">8</td></tr><tr><td align=\\\"left\\\">Heat (80 °C)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">CaCl<sub>2</sub> (1.25 mM)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">50% serum (4 h)</td><td align=\\\"left\\\">8</td></tr><tr><td align=\\\"left\\\">Heat (100 °C)</td><td align=\\\"left\\\"> > 64</td><td align=\\\"left\\\">CaCl<sub>2</sub> (2.5 mM)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">50% serum (6 h)</td><td align=\\\"left\\\">8</td></tr><tr><td align=\\\"left\\\">pH 2</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">CaCl<sub>2</sub> (5 mM)</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">50% serum (8 h)</td><td align=\\\"left\\\">8</td></tr><tr><td align=\\\"left\\\">pH 4</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">MgCl<sub>2</sub> (1.25 mM)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">Gastric juice (0.25 h)</td><td align=\\\"left\\\">4</td></tr><tr><td align=\\\"left\\\">pH 6</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">MgCl<sub>2</sub> (2.5 mM)</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">Gastric juice (0.5 h)</td><td align=\\\"left\\\">4</td></tr><tr><td align=\\\"left\\\">pH 8</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">MgCl<sub>2</sub> (5 mM)</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">Gastric juice (1 h)</td><td align=\\\"left\\\">4</td></tr><tr><td align=\\\"left\\\">pH 10</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">25% serum (0.5 h)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">Gastric juice (2 h)</td><td align=\\\"left\\\">8</td></tr><tr><td align=\\\"left\\\">NaCl (50 mM)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">25% serum (1 h)</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">Intestinal juice (0.25 h)</td><td align=\\\"left\\\" rowspan=\\\"4\\\"> > 64</td></tr><tr><td align=\\\"left\\\">NaCl (100 mM)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">25% serum (2 h)</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">Intestinal juice (0.5 h)</td></tr><tr><td align=\\\"left\\\">NaCl (150 mM)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">25% serum (4 h)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">Intestinal juice (1 h)</td></tr><tr><td align=\\\"left\\\">NaCl (200 mM)</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">25% serum (6 h)</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">Intestinal juice (2 h)</td></tr><tr><td align=\\\"left\\\">NaCl (300 mM)</td><td align=\\\"left\\\">16</td><td align=\\\"left\\\">25% serum (8 h)</td><td align=\\\"left\\\">8</td><td align=\\\"left\\\">Control (without treatment)</td><td align=\\\"left\\\">8</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p>The line means no results; all data in this table were predicted by online software (APD, DBAASP, and CAMP)</p></table-wrap-foot>","<table-wrap-foot><p>MICa, the MIC of peptide/antibiotic alone; MICc, the MIC of the peptide and antibiotic in combination</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>The line means no results; all data in this table were predicted by online software (APD, DBAASP, and CAMP)</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>MICa, the MIC of peptide/antibiotic alone; MICc, the MIC of the peptide and antibiotic in combination</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"253_2023_12947_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"253_2023_12947_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"253_2023_12947_Fig3_HTML\" id=\"MO3\"/>","<graphic xlink:href=\"253_2023_12947_Fig4_HTML\" id=\"MO4\"/>","<graphic xlink:href=\"253_2023_12947_Fig5_HTML\" id=\"MO5\"/>","<graphic xlink:href=\"253_2023_12947_Fig6_HTML\" id=\"MO6\"/>"],"string":"[\n \"<graphic xlink:href=\\\"253_2023_12947_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12947_Fig2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12947_Fig3_HTML\\\" id=\\\"MO3\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12947_Fig4_HTML\\\" id=\\\"MO4\\\"/>\",\n \"<graphic xlink:href=\\\"253_2023_12947_Fig5_HTML\\\" id=\\\"MO5\\\"/>\",\n \"<graphic 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(2020) Colistin and its role in the era of antibiotic resistance: an extended review (2000\\u20132019).\\u00a0Emerg Microbes Infect\\u00a09(1):868\\u2013885\"]}, {\"surname\": [\"Flamm\", \"Rhomberg\", \"Lindley\", \"Sweeney\", \"Ellis-Grosse\", \"Shortridge\"], \"given-names\": [\"RK\", \"PR\", \"JM\", \"K\", \"EJ\", \"D\"], \"article-title\": [\"Evaluation of the bactericidal activity of fosfomycin in combination with selected antimicrobial comparison agents tested against Gram-negative bacterial strains by using time-kill curves\"], \"source\": [\"Antimicrob Agents Chemother\"], \"year\": [\"2019\"], \"volume\": [\"5\"], \"fpage\": [\"63\"]}, {\"mixed-citation\": [\"Hao Y, Teng D, Mao R, Yang N, Wang J (2023) Site mutation improves the expression and antimicrobial properties of fungal defense. Antibiotics (Basel) 12(8):1283\"]}, {\"mixed-citation\": [\"Jin Y, Yang N, Teng D, Hao Y, Mao R, Wang J (2023) Molecular modification of kex2 P1' site enhances expression and druggability of fungal defensin. Antibiotics (Basel) 12(4):786\"]}, {\"surname\": [\"Koo\", \"Seo\"], \"given-names\": [\"HB\", \"J\"], \"article-title\": [\"Antimicrobial peptides under clinical investigation\"], \"source\": [\"Peptide Sci\"], \"year\": [\"2019\"], \"volume\": [\"111\"], \"issue\": [\"5\"], \"fpage\": [\"e24122\"], \"pub-id\": [\"10.1002/pep2.24122\"]}, {\"mixed-citation\": [\"Lociuro S, Neve S, Zuegg J, Edwards IA, Cain AK, Boinett CJ, Barquist L, Lundberg CV, Steen J, Butler MS, Mobli M, Porter KM, Blaskovich MAT (2015) AP138, a second generation plectasin, shows good bactericidal properties and long post-antibiotic effect.\\u00a0Final Programme. ECCMID\"]}, {\"surname\": [\"McEwen\", \"Collignon\"], \"given-names\": [\"SA\", \"PJ\"], \"article-title\": [\"Antimicrobial resistance: a One Health Perspective\"], \"source\": [\"Microbiol Spectr\"], \"year\": [\"2018\"], \"volume\": [\"111\"], \"issue\": [\"6\"], \"fpage\": [\"255\"], \"lpage\": [\"260\"]}, {\"surname\": [\"Miao\", \"Zhou\", \"Liu\", \"Chen\", \"Chen\", \"Gao\", \"Dxion\", \"Song\", \"Xiao\", \"Cao\"], \"given-names\": [\"JY\", \"JL\", \"G\", \"FL\", \"YJ\", \"XY\", \"XY\", \"MY\", \"H\", \"Y\"], \"article-title\": [\"Membrane disruption and DNA binding of \"], \"italic\": [\"Staphylococcus aureus\", \"Lactobacillus paracasei\"], \"source\": [\"Food Control\"], \"year\": [\"2016\"], \"volume\": [\"59\"], \"fpage\": [\"609\"], \"lpage\": [\"613\"], \"pub-id\": [\"10.1016/j.foodcont.2015.06.044\"]}, {\"mixed-citation\": [\"Othman M, S Ratna, Tewari A, Kang A, Du K, Vaisman I (2018) Machine learning classification of antimicrobial peptides using reduced alphabets. The 2018 ACM International Conference 18: 548\"]}, {\"surname\": [\"She\", \"LiuYQ\", \"Li\", \"Li\", \"Liu\", \"Li\", \"Yang\", \"Zhou\", \"Wu\"], \"given-names\": [\"PF\", \"XuLL\", \"ZH\", \"YM\", \"SS\", \"LH\", \"YF\", \"LY\", \"Y\"], \"article-title\": [\"L007\\u20130069 kills \"], \"italic\": [\"Staphylococcus aureus\"], \"source\": [\"Cell Mol Life Sci\"], \"year\": [\"2022\"], \"volume\": [\"79\"], \"issue\": [\"11\"], \"fpage\": [\"1\"], \"lpage\": [\"16\"]}, {\"surname\": [\"Waghu\", \"Gopi\", \"Barai\", \"Ramteke\", \"Nizami\", \"Idicula-Thomas\"], \"given-names\": [\"FH\", \"L\", \"RS\", \"P\", \"B\", \"S\"], \"article-title\": [\"CAMP: collection of sequences and structures of antimicrobial peptides\"], \"source\": [\"Nucleic Acids Res\"], \"year\": [\"2014\"], \"volume\": [\"D1\"], \"fpage\": [\"D1154\"], \"lpage\": [\"D1158\"], \"pub-id\": [\"10.1093/nar/gkt1157\"]}, {\"surname\": [\"Wang\", \"Li\", \"Wang\"], \"given-names\": [\"G\", \"X\", \"Z\"], \"article-title\": [\"APD3: the antimicrobial peptide database as a tool for research and education\"], \"source\": [\"Nucleic Acids Res\"], \"year\": [\"2016\"], \"volume\": [\"D1\"], \"fpage\": [\"D1087\"], \"lpage\": [\"D1093\"], \"pub-id\": [\"10.1093/nar/gkv1278\"]}, {\"surname\": [\"Wu\", \"Khodaparast\", \"Khodaparast\", \"De Vleeschouwer\", \"Housmans\", \"Houben\", \"Schymkowitz\", \"Rousseau\"], \"given-names\": [\"G\", \"L\", \"L\", \"M\", \"J\", \"B\", \"J\", \"F\"], \"article-title\": [\"Investigating the mechanism of action of aggregation-inducing antimicrobial Pept-ins\"], \"source\": [\"Cell Chem Biol\"], \"year\": [\"2021\"], \"volume\": [\"3\"], \"issue\": [\"2\"], \"fpage\": [\"163\"], \"lpage\": [\"175\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":63,"string":"63"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Appl Microbiol Biotechnol. 2024 Jan 13; 108(1):1-18"},"package_file":{"kind":"string","value":"oa_package/45/d3/PMC10787893.tar.gz"}}},{"rowIdx":177,"cells":{"accession_id":{"kind":"string","value":"PMC10787894"},"pmid":{"kind":"string","value":"38217709"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par26\">Neprilysin (NEP) is a ubiquitous membrane-bound zinc-dependent metalloprotease that hydrolyzes numerous regulatory peptides including vasoactive peptides such as natriuretic peptides, adrenomedullin, angiotensins I and II, bradykinin, and endothelin-1 as well as non-vasoactive peptides such as beta amyloid, glucagon, enkephalins, and substance P [reviewed in ##UREF##0##1##–##REF##32987038##3##]. NEP’s broad tissue distribution and wide range of substrates portend its regulatory functions and involvement in the pathology of cardiovascular, renal, respiratory, brain, and nervous systems. NEP has a short cytoplasmic N-terminal domain of 27 amino acids, a single transmembrane region of 23 residues and a large C-terminal ectodomain harboring the catalytic site that extends into the extracellular space. As such, a catalytically active and soluble form of NEP (sNEP) can be released into the circulation by proteolytic ectodomain shedding [##REF##24495806##4##]. NEP-bound exosomes may also be released from endothelial cells and human adipose-derived mesenchymal stem cells [##REF##24495806##4##, ##REF##23378928##5##]. Recent mass spectrometry analysis of NEP antibody pull-down material from human plasma detected peptides that map to the N-terminal cytoplasmic domain, further corroborating possible NEP entry into circulation via exosomes [##REF##35331754##6##].</p>","<p id=\"Par27\">NEP is an emerging biomarker for various diseases including heart failure (HF), cardiovascular diseases, diabetic kidney disease, and metabolic syndrome [##REF##32423555##7##]. Most studies use commercially available research-use-only (RUO) ELISA kits for quantifying sNEP in circulation and data comparisons show strikingly poor correlations between assays [##UREF##0##1##]. It is not surprising, therefore, that published findings on the concentration of plasma sNEP and its clinical associations in health and disease lack agreement [##UREF##0##1##, ##REF##35331754##6##, ##REF##32944293##8##]. Although discrepant findings may in part be attributed to different cohort populations or disease subtypes studied in these reports, two important issues need to be addressed to help resolve discordant observations. First, significant areas of concern exist with NEP quantification, and in fact with many other protein biomarkers, where RUO ELISAs are used for measurement. These assays are often employed with little or no manufacturer’s information on the immunogen sequence, antibody type (polyclonal versus monoclonal and animal of origin), antibody specificity and binding site on the target, and nature of the calibrator (protein sequence and source). Second, little is known about the circulating forms of NEP in humans. A previous study suggested the presence of both catalytically active and inactive forms of NEP in circulation [##REF##35331754##6##]. The quaternary structure of NEP in humans is also unclear although it appears to exist as a monomer in rabbits [##REF##4214106##9##], while non-covalently associated homodimers have been reported in pigs [##REF##6349615##10##]. NEP is also heavily glycosylated with experimentally published N-linked glycosylation sites at N145, N285, N294, N325, and N628 [##REF##10669592##11##–##REF##24190977##14##]. Differences in glycosylation contribute to the range of NEP molecular weights (85–110 kDa) observed in different tissue sources [##UREF##3##15##]. Clearly, correct interpretation of immunoassay data requires the availability of highly specific antibodies with well-defined binding footprints and at least some information on the circulating NEP moieties detected by the antibody pairs.</p>","<p id=\"Par28\">Previously, we described an epitope-directed monoclonal antibody (mAb) production method that facilitated antibody characterization and validation [##REF##33824395##16##]. Using a similar approach, we generated and characterized mAbs directed against multiple non-overlapping preselected peptide regions on NEP. To facilitate the generation of glycosylation-sensitive mAbs, an epitope harboring experimentally verified glycosites was deliberately selected for immunogen preparation. Such mAbs may constitute useful tools for probing deficiencies in glycosylation site occupancy and elucidating how this property relates to health and in disease. The best mAb pair was validated for applications in western blotting and ELISA. The profile of circulating NEP moieties was elucidated using these two mAbs and compared against that obtained with a previously validated commercial polyclonal antibody (PE pAb) [##REF##35331754##6##]. These newly developed binding agents provide fresh insights into circulating forms of NEP and are useful tools for future research into mechanistic pathways of NEP regulation and its relationship in disease states.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par26\\\">Neprilysin (NEP) is a ubiquitous membrane-bound zinc-dependent metalloprotease that hydrolyzes numerous regulatory peptides including vasoactive peptides such as natriuretic peptides, adrenomedullin, angiotensins I and II, bradykinin, and endothelin-1 as well as non-vasoactive peptides such as beta amyloid, glucagon, enkephalins, and substance P [reviewed in ##UREF##0##1##–##REF##32987038##3##]. NEP’s broad tissue distribution and wide range of substrates portend its regulatory functions and involvement in the pathology of cardiovascular, renal, respiratory, brain, and nervous systems. NEP has a short cytoplasmic N-terminal domain of 27 amino acids, a single transmembrane region of 23 residues and a large C-terminal ectodomain harboring the catalytic site that extends into the extracellular space. As such, a catalytically active and soluble form of NEP (sNEP) can be released into the circulation by proteolytic ectodomain shedding [##REF##24495806##4##]. NEP-bound exosomes may also be released from endothelial cells and human adipose-derived mesenchymal stem cells [##REF##24495806##4##, ##REF##23378928##5##]. Recent mass spectrometry analysis of NEP antibody pull-down material from human plasma detected peptides that map to the N-terminal cytoplasmic domain, further corroborating possible NEP entry into circulation via exosomes [##REF##35331754##6##].</p>\",\n \"<p id=\\\"Par27\\\">NEP is an emerging biomarker for various diseases including heart failure (HF), cardiovascular diseases, diabetic kidney disease, and metabolic syndrome [##REF##32423555##7##]. Most studies use commercially available research-use-only (RUO) ELISA kits for quantifying sNEP in circulation and data comparisons show strikingly poor correlations between assays [##UREF##0##1##]. It is not surprising, therefore, that published findings on the concentration of plasma sNEP and its clinical associations in health and disease lack agreement [##UREF##0##1##, ##REF##35331754##6##, ##REF##32944293##8##]. Although discrepant findings may in part be attributed to different cohort populations or disease subtypes studied in these reports, two important issues need to be addressed to help resolve discordant observations. First, significant areas of concern exist with NEP quantification, and in fact with many other protein biomarkers, where RUO ELISAs are used for measurement. These assays are often employed with little or no manufacturer’s information on the immunogen sequence, antibody type (polyclonal versus monoclonal and animal of origin), antibody specificity and binding site on the target, and nature of the calibrator (protein sequence and source). Second, little is known about the circulating forms of NEP in humans. A previous study suggested the presence of both catalytically active and inactive forms of NEP in circulation [##REF##35331754##6##]. The quaternary structure of NEP in humans is also unclear although it appears to exist as a monomer in rabbits [##REF##4214106##9##], while non-covalently associated homodimers have been reported in pigs [##REF##6349615##10##]. NEP is also heavily glycosylated with experimentally published N-linked glycosylation sites at N145, N285, N294, N325, and N628 [##REF##10669592##11##–##REF##24190977##14##]. Differences in glycosylation contribute to the range of NEP molecular weights (85–110 kDa) observed in different tissue sources [##UREF##3##15##]. Clearly, correct interpretation of immunoassay data requires the availability of highly specific antibodies with well-defined binding footprints and at least some information on the circulating NEP moieties detected by the antibody pairs.</p>\",\n \"<p id=\\\"Par28\\\">Previously, we described an epitope-directed monoclonal antibody (mAb) production method that facilitated antibody characterization and validation [##REF##33824395##16##]. Using a similar approach, we generated and characterized mAbs directed against multiple non-overlapping preselected peptide regions on NEP. To facilitate the generation of glycosylation-sensitive mAbs, an epitope harboring experimentally verified glycosites was deliberately selected for immunogen preparation. Such mAbs may constitute useful tools for probing deficiencies in glycosylation site occupancy and elucidating how this property relates to health and in disease. The best mAb pair was validated for applications in western blotting and ELISA. The profile of circulating NEP moieties was elucidated using these two mAbs and compared against that obtained with a previously validated commercial polyclonal antibody (PE pAb) [##REF##35331754##6##]. These newly developed binding agents provide fresh insights into circulating forms of NEP and are useful tools for future research into mechanistic pathways of NEP regulation and its relationship in disease states.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<title>Peptide design and purification</title>","<p id=\"Par29\">The protein sequence of human NEP (GenBank Accession no. NP000893) was analyzed using the B Cell Linear Epitope Prediction tool on the Immune Epitope Database Analysis Resource server (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.iedb.org\">http://www.iedb.org</ext-link>). Four putative antigenic sequences, VATENWEQKYGASW (residues 169–182; designated AgNEP-1), YIKKNGEEKLL (residues 665–675; designated AgNEP-2), EIANATAKPEDRNDP (residues 282–296; designated AgNEP-3) and EKVDKDEWIS (residues 528–537; designated AgNEP-4) were chosen. Each sequence was fused as independent three-copy inserts separated by short glycine-serine linkers into the surface-exposed active loop of thioredoxin (Trx) harboring a C-terminal polyhistidine tag as described elsewhere [##REF##33824395##16##]. These constructs were used for preparation of the immunogens. A further composite construct for antibody screening, designated Trx-AgNEP(1–4), containing single copies of all four antigenic sequences arranged in tandem within the active loop of thioredoxin was also generated. The coding sequences of all constructs were verified by DNA sequencing. All thioredoxin fusion proteins were expressed in <italic>Escherichia coli</italic> BL21 (DE3) trxB and purified by immobilized metal affinity chromatography (IMAC) under native or denaturing conditions as previously described [##REF##33824395##16##, ##REF##24291344##17##].</p>","<title>Antibody generation and characterization</title>","<p id=\"Par30\">A cocktail comprising equimolar concentrations of Trx-AgNEP-1, -2, -3 and -4 (final total concentration 1 mg/ml) was added to an equal volume of Sigma Adjuvant System (Sigma Aldrich) and mixed to homogeneity for immunization into Balb/c mice. The immunization scheme and method for generating, screening, and selecting the hybridoma clonal cell lines are as previously described [##REF##33824395##16##]. All approved animal experiments were performed in compliance with A*STAR Institutional Animal Care and Use Committee (IACUC) regulations.</p>","<p id=\"Par31\">Antibody purification, isotyping, characterization by surface plasmon resonance (SPR), and epitope mapping by alanine scan analysis were performed as previously described [##REF##33824395##16##]. The association (k<sub>a</sub>), dissociation (k<sub>d</sub>), and equilibrium (KD) constants of mAbs were determined using the ProteOn XPR36 (Bio-Rad, Hercules, USA) and tested against recombinant human sNEP (HEK293 human cell line-derived NEP ectodomain spanning Y52-W750; Aviscera Bioscience, USA; product code 00724-06-10) and the cognate thioredoxin-fused AgNEP antigen. Critical residues for mAb binding were determined using the Multipin system (Mimotopes, Australia) comprising a library of peptides corresponding to the wild-type sequences of the NEP antigens and their analogs containing one alanine substitution in the peptide sequence.</p>","<p id=\"Par32\">Immunoprecipitation from 40 ml of pooled HF human plasma (NEP concentration at ~ 600 pg/ml as determined by sandwich ELISA using the mAb pair of 17E11 and 31E1 described below) was performed using biotinylated mAb 17E11 (raised against Trx-AgNEP-4)/streptavidin magnetic beads as described previously [##REF##33824395##16##]. Proteomic processing (reduction, alkylation and trypsin digestion) was performed on the pull-down eluate using the S-TRAP Micro column (Protifi) following manufacturer’s instructions. Peptides were then acidified to a final concentration of 0.1% formic acid. For online liquid chromatography–mass spectrometry (LCMS) analysis, 1 µg of peptides were injected into an eksigent 425 nano-LC system fitted with a ProteoCol C18P trap column (3 μm 120 Å, 300 μm × 10 mm; Trajan) and an Acclaim PepMap100 C18 analytical column (3 μm 100 Å, 75 μm × 250 mm; Thermo Scientific). The peptides were separated at 300 nL/min, using 0.1% formic acid in water and 0.1% formic acid in acetonitrile as mobile phase A and B, respectively, using a gradient of 5–15% B over 60 min and 15–30% B over the next 60 min. MS data were acquired on a TripleTOF 6600 mass spectrometer (SCIEX) in high-resolution multiple reaction monitoring (MRM<sup>HR</sup>) mode. The MRM<sup>HR</sup> analysis consisted of a TOF–MS scan across 400–1600 m<italic>/z</italic> with 50 ms accumulation time, followed by 15 product ion scans across 100–1800 m<italic>/z</italic> with 85 ms accumulation time each. The target NEP peptides were chosen based on predicted tryptic peptides that are close to the epitope of mAb 17E11. The product ion scan parameters are detailed in Table ##TAB##0##1##. MRM<sup>HR</sup> data were processed using the PeakView 2.2 software with the Bio Tool Kit 2.2 plugin (SCIEX). Extracted ion chromatograms (XIC) of all predicted b and y fragment ions from each targeted NEP peptide were generated with 0.05 Da width. Spectrum matching was performed using a mass tolerance of 0.05 Da, and against all charge states of b and y fragment ions.</p>","<title>Sandwich ELISA</title>","<p id=\"Par33\">Antibody pairs that gave the strongest positive signal readout against Trx-AgNEP(1–4) at 8000 pg/ml were identified by ELISA using a checkerboard screening method as described previously [##REF##33824395##16##]. With capture and detection antibody concentrations kept constant, Trx-AgNEP(1–4) was titrated to generate a seven-point standard curve ranging between 125 and 8000 pg/ml. Colorimetric signal was generated with streptavidin–HRP in conjunction with the chromogenic substrate, tetramethylbenzidine (TMB). To find the antibody pair yielding the maximum signal-to-noise ratio, absorbance values measured at 450 nm on the Enspire Multi-mode microplate reader (Perkin Elmer) with background subtraction at 570 nm were plotted against Trx-AgNEP(1–4) concentrations and fitted against a five-parameter logistic (5PL) model using the Enspire® software.</p>","<p id=\"Par34\">The optimal antibody pair comprising mAb 17E11 as capture antibody and biotinylated mAb 31E1 for detection was used to develop a two-site sandwich ELISA for human NEP. Calibration curves were generated with eight calibrators at 8000, 3200, 1280, 512, 205, 81.9, 32.8, and 13.1 pg/ml of Trx-AgNEP(1–4) prepared in 2.7% bovine serum albumin (BSA)/phosphate buffered saline (PBS). Assessment of assay cross-reactivity to structurally similar M13 family endopeptidases is as described previously [##REF##35331754##6##]: human endothelin converting enzyme 1 (ECE-1), human endothelin converting enzyme 2 (ECE-2), human phosphate-regulating neutral endopeptidase (PHEX), and human endothelin converting enzyme like 1 (ECEL-1). Spike and recovery testing was performed using recombinant Trx-AgNEP(1–4) stock solution and a plasma sample with very high endogenous NEP (23,000 pg/ml) spiked at two different concentrations into three plasma samples. Parallelism experiments to ascertain that the binding characteristics of the antibody pair to endogenous NEP and Trx-AgNEP(1–4) are comparable were performed using non-HF and HF plasma EDTA samples. Six plasma samples with endogenous NEP levels within the assay range when diluted with 2.7% BSA/PBS in the range of 2–24-folds were tested. Human test samples used were as previously described and in accord with relevant ethics approval and written informed consent [##REF##35331754##6##].</p>","<title>Western blotting</title>","<p id=\"Par35\">The reactivity of the mAbs to various protein sample types was determined by simple western analysis using the Jess fully automated system (ProteinSimple; Bio-Techne, USA) following the manufacturer’s instructions. Recombinant human sNEP (2 ng/well; Aviscera Bioscience, USA), purified recombinant Trx-AgNEP(1–4) (2 ng/well), whole cell lysates (1.2 µg/well) from human prostate adenocarcinoma LNCaP (Santa Cruz Biotechnology, USA; product number sc-2231) and human prostate cancer PC-3 (Santa Cruz Biotechnology, USA; product number sc-2220) cell lines, and human plasma samples (both individual and pooled (<italic>n</italic> > 100) samples; 1.2 µg/well) were used for western analysis. The previously validated polyclonal antibody (PE pAb) from the Neprilysin AlphaLISA® kit (Perkin Elmer, MA, USA; Cat# AL337HV) was used as the positive control for comparing immunoreactivity to the same samples [##REF##35331754##6##]. The chemiluminescence assay was used following the manufacturer’s instructions. Samples were processed as follows: 5 × master mix was prepared using reagents provided by Bio-Techne (EZ Standard Pack 1, cat. no.: PS-ST01EZ-8). Protein samples were mixed 1:4 with 5 × master mix, heated at 95 °C for 5 min and stored in ice. Samples were loaded onto the 12–230 kDa Jess/Wes Separation Module where protein separation and immobilization, immunoprobing, washing, and detection take place in capillary tubes. The incubation time of the primary and the secondary antibodies was 30 min. Concentration of all mAbs used was 5 µg/ml (when probing against recombinant sNEP and Trx-AgNEP(1–4)) and 10 µg/ml (when probing against whole cell lysates and human plasma samples). Concentration of PE pAb used was 5 µg/ml for all protein sample types except human plasma samples where antibody concentration at 10 µg/ml was used. The amount of total plasma proteins loaded in each capillary was 4 µg. All other samples were loaded at total protein of 1.2 µg per capillary. For the secondary antibody, ready-to-use HRP-conjugated anti-mouse or anti-goat antibody was used, depending on the primary antibody. Peptide competition assays were performed to validate the immunodetected bands in human plasma samples and Trx-AgNEP(1–4) was included as a positive control. Western blotting was performed exactly as described above except that the primary antibodies (mAbs 31E1 and 17E11) were pre-blocked overnight with matched peptides (AgNEP3 and AgNEP4, respectively) or an unmatched peptide (DLNAKDREGDTPLH) at tenfold excess concentration relative to that of the primary antibody. Peptide competition assays by two-site ELISA (mAb 17E11/31E1 as capture and detection antibodies and the reverse pairing) were also performed to corroborate the effect of matched/unmatched peptide blocking of mAb 31E1 and 17E11 on the detection of Trx-AgNEP(1–4) using 10X excess concentration of peptide relative to the detection antibody.</p>","<title>Glycosylation sensitivity of mAb 31E1</title>","<p id=\"Par36\">To provide direct evidence that mAb 31E1 binds only to glycan-deficient recombinant sNEP, PNGase F (New England Biolabs, Cat# P0704S) was used to fully remove N-glycan moieties from HEK293 cell line-derived recombinant sNEP (Aviscera Bioscience, USA) under denaturing conditions according to manufacturer’s instructions. PNGase F-treated recombinant sNEP and a paired sample with no glycosidase as control was separated on 12% SDS-PAGE and transblotted onto a PVDF membrane. Detection of recombinant sNEP was performed using purified mAb 31E1 (1.5 µg/ml) and a goat anti-mouse polyclonal antibody conjugated to horse-radish peroxidase (diluted 1:20,000; Abcam cat# AB205719). Protein bands were visualized using Supersignal™ West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific, USA).</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<title>Peptide design and purification</title>\",\n \"<p id=\\\"Par29\\\">The protein sequence of human NEP (GenBank Accession no. NP000893) was analyzed using the B Cell Linear Epitope Prediction tool on the Immune Epitope Database Analysis Resource server (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"http://www.iedb.org\\\">http://www.iedb.org</ext-link>). Four putative antigenic sequences, VATENWEQKYGASW (residues 169–182; designated AgNEP-1), YIKKNGEEKLL (residues 665–675; designated AgNEP-2), EIANATAKPEDRNDP (residues 282–296; designated AgNEP-3) and EKVDKDEWIS (residues 528–537; designated AgNEP-4) were chosen. Each sequence was fused as independent three-copy inserts separated by short glycine-serine linkers into the surface-exposed active loop of thioredoxin (Trx) harboring a C-terminal polyhistidine tag as described elsewhere [##REF##33824395##16##]. These constructs were used for preparation of the immunogens. A further composite construct for antibody screening, designated Trx-AgNEP(1–4), containing single copies of all four antigenic sequences arranged in tandem within the active loop of thioredoxin was also generated. The coding sequences of all constructs were verified by DNA sequencing. All thioredoxin fusion proteins were expressed in <italic>Escherichia coli</italic> BL21 (DE3) trxB and purified by immobilized metal affinity chromatography (IMAC) under native or denaturing conditions as previously described [##REF##33824395##16##, ##REF##24291344##17##].</p>\",\n \"<title>Antibody generation and characterization</title>\",\n \"<p id=\\\"Par30\\\">A cocktail comprising equimolar concentrations of Trx-AgNEP-1, -2, -3 and -4 (final total concentration 1 mg/ml) was added to an equal volume of Sigma Adjuvant System (Sigma Aldrich) and mixed to homogeneity for immunization into Balb/c mice. The immunization scheme and method for generating, screening, and selecting the hybridoma clonal cell lines are as previously described [##REF##33824395##16##]. All approved animal experiments were performed in compliance with A*STAR Institutional Animal Care and Use Committee (IACUC) regulations.</p>\",\n \"<p id=\\\"Par31\\\">Antibody purification, isotyping, characterization by surface plasmon resonance (SPR), and epitope mapping by alanine scan analysis were performed as previously described [##REF##33824395##16##]. The association (k<sub>a</sub>), dissociation (k<sub>d</sub>), and equilibrium (KD) constants of mAbs were determined using the ProteOn XPR36 (Bio-Rad, Hercules, USA) and tested against recombinant human sNEP (HEK293 human cell line-derived NEP ectodomain spanning Y52-W750; Aviscera Bioscience, USA; product code 00724-06-10) and the cognate thioredoxin-fused AgNEP antigen. Critical residues for mAb binding were determined using the Multipin system (Mimotopes, Australia) comprising a library of peptides corresponding to the wild-type sequences of the NEP antigens and their analogs containing one alanine substitution in the peptide sequence.</p>\",\n \"<p id=\\\"Par32\\\">Immunoprecipitation from 40 ml of pooled HF human plasma (NEP concentration at ~ 600 pg/ml as determined by sandwich ELISA using the mAb pair of 17E11 and 31E1 described below) was performed using biotinylated mAb 17E11 (raised against Trx-AgNEP-4)/streptavidin magnetic beads as described previously [##REF##33824395##16##]. Proteomic processing (reduction, alkylation and trypsin digestion) was performed on the pull-down eluate using the S-TRAP Micro column (Protifi) following manufacturer’s instructions. Peptides were then acidified to a final concentration of 0.1% formic acid. For online liquid chromatography–mass spectrometry (LCMS) analysis, 1 µg of peptides were injected into an eksigent 425 nano-LC system fitted with a ProteoCol C18P trap column (3 μm 120 Å, 300 μm × 10 mm; Trajan) and an Acclaim PepMap100 C18 analytical column (3 μm 100 Å, 75 μm × 250 mm; Thermo Scientific). The peptides were separated at 300 nL/min, using 0.1% formic acid in water and 0.1% formic acid in acetonitrile as mobile phase A and B, respectively, using a gradient of 5–15% B over 60 min and 15–30% B over the next 60 min. MS data were acquired on a TripleTOF 6600 mass spectrometer (SCIEX) in high-resolution multiple reaction monitoring (MRM<sup>HR</sup>) mode. The MRM<sup>HR</sup> analysis consisted of a TOF–MS scan across 400–1600 m<italic>/z</italic> with 50 ms accumulation time, followed by 15 product ion scans across 100–1800 m<italic>/z</italic> with 85 ms accumulation time each. The target NEP peptides were chosen based on predicted tryptic peptides that are close to the epitope of mAb 17E11. The product ion scan parameters are detailed in Table ##TAB##0##1##. MRM<sup>HR</sup> data were processed using the PeakView 2.2 software with the Bio Tool Kit 2.2 plugin (SCIEX). Extracted ion chromatograms (XIC) of all predicted b and y fragment ions from each targeted NEP peptide were generated with 0.05 Da width. Spectrum matching was performed using a mass tolerance of 0.05 Da, and against all charge states of b and y fragment ions.</p>\",\n \"<title>Sandwich ELISA</title>\",\n \"<p id=\\\"Par33\\\">Antibody pairs that gave the strongest positive signal readout against Trx-AgNEP(1–4) at 8000 pg/ml were identified by ELISA using a checkerboard screening method as described previously [##REF##33824395##16##]. With capture and detection antibody concentrations kept constant, Trx-AgNEP(1–4) was titrated to generate a seven-point standard curve ranging between 125 and 8000 pg/ml. Colorimetric signal was generated with streptavidin–HRP in conjunction with the chromogenic substrate, tetramethylbenzidine (TMB). To find the antibody pair yielding the maximum signal-to-noise ratio, absorbance values measured at 450 nm on the Enspire Multi-mode microplate reader (Perkin Elmer) with background subtraction at 570 nm were plotted against Trx-AgNEP(1–4) concentrations and fitted against a five-parameter logistic (5PL) model using the Enspire® software.</p>\",\n \"<p id=\\\"Par34\\\">The optimal antibody pair comprising mAb 17E11 as capture antibody and biotinylated mAb 31E1 for detection was used to develop a two-site sandwich ELISA for human NEP. Calibration curves were generated with eight calibrators at 8000, 3200, 1280, 512, 205, 81.9, 32.8, and 13.1 pg/ml of Trx-AgNEP(1–4) prepared in 2.7% bovine serum albumin (BSA)/phosphate buffered saline (PBS). Assessment of assay cross-reactivity to structurally similar M13 family endopeptidases is as described previously [##REF##35331754##6##]: human endothelin converting enzyme 1 (ECE-1), human endothelin converting enzyme 2 (ECE-2), human phosphate-regulating neutral endopeptidase (PHEX), and human endothelin converting enzyme like 1 (ECEL-1). Spike and recovery testing was performed using recombinant Trx-AgNEP(1–4) stock solution and a plasma sample with very high endogenous NEP (23,000 pg/ml) spiked at two different concentrations into three plasma samples. Parallelism experiments to ascertain that the binding characteristics of the antibody pair to endogenous NEP and Trx-AgNEP(1–4) are comparable were performed using non-HF and HF plasma EDTA samples. Six plasma samples with endogenous NEP levels within the assay range when diluted with 2.7% BSA/PBS in the range of 2–24-folds were tested. Human test samples used were as previously described and in accord with relevant ethics approval and written informed consent [##REF##35331754##6##].</p>\",\n \"<title>Western blotting</title>\",\n \"<p id=\\\"Par35\\\">The reactivity of the mAbs to various protein sample types was determined by simple western analysis using the Jess fully automated system (ProteinSimple; Bio-Techne, USA) following the manufacturer’s instructions. Recombinant human sNEP (2 ng/well; Aviscera Bioscience, USA), purified recombinant Trx-AgNEP(1–4) (2 ng/well), whole cell lysates (1.2 µg/well) from human prostate adenocarcinoma LNCaP (Santa Cruz Biotechnology, USA; product number sc-2231) and human prostate cancer PC-3 (Santa Cruz Biotechnology, USA; product number sc-2220) cell lines, and human plasma samples (both individual and pooled (<italic>n</italic> > 100) samples; 1.2 µg/well) were used for western analysis. The previously validated polyclonal antibody (PE pAb) from the Neprilysin AlphaLISA® kit (Perkin Elmer, MA, USA; Cat# AL337HV) was used as the positive control for comparing immunoreactivity to the same samples [##REF##35331754##6##]. The chemiluminescence assay was used following the manufacturer’s instructions. Samples were processed as follows: 5 × master mix was prepared using reagents provided by Bio-Techne (EZ Standard Pack 1, cat. no.: PS-ST01EZ-8). Protein samples were mixed 1:4 with 5 × master mix, heated at 95 °C for 5 min and stored in ice. Samples were loaded onto the 12–230 kDa Jess/Wes Separation Module where protein separation and immobilization, immunoprobing, washing, and detection take place in capillary tubes. The incubation time of the primary and the secondary antibodies was 30 min. Concentration of all mAbs used was 5 µg/ml (when probing against recombinant sNEP and Trx-AgNEP(1–4)) and 10 µg/ml (when probing against whole cell lysates and human plasma samples). Concentration of PE pAb used was 5 µg/ml for all protein sample types except human plasma samples where antibody concentration at 10 µg/ml was used. The amount of total plasma proteins loaded in each capillary was 4 µg. All other samples were loaded at total protein of 1.2 µg per capillary. For the secondary antibody, ready-to-use HRP-conjugated anti-mouse or anti-goat antibody was used, depending on the primary antibody. Peptide competition assays were performed to validate the immunodetected bands in human plasma samples and Trx-AgNEP(1–4) was included as a positive control. Western blotting was performed exactly as described above except that the primary antibodies (mAbs 31E1 and 17E11) were pre-blocked overnight with matched peptides (AgNEP3 and AgNEP4, respectively) or an unmatched peptide (DLNAKDREGDTPLH) at tenfold excess concentration relative to that of the primary antibody. Peptide competition assays by two-site ELISA (mAb 17E11/31E1 as capture and detection antibodies and the reverse pairing) were also performed to corroborate the effect of matched/unmatched peptide blocking of mAb 31E1 and 17E11 on the detection of Trx-AgNEP(1–4) using 10X excess concentration of peptide relative to the detection antibody.</p>\",\n \"<title>Glycosylation sensitivity of mAb 31E1</title>\",\n \"<p id=\\\"Par36\\\">To provide direct evidence that mAb 31E1 binds only to glycan-deficient recombinant sNEP, PNGase F (New England Biolabs, Cat# P0704S) was used to fully remove N-glycan moieties from HEK293 cell line-derived recombinant sNEP (Aviscera Bioscience, USA) under denaturing conditions according to manufacturer’s instructions. PNGase F-treated recombinant sNEP and a paired sample with no glycosidase as control was separated on 12% SDS-PAGE and transblotted onto a PVDF membrane. Detection of recombinant sNEP was performed using purified mAb 31E1 (1.5 µg/ml) and a goat anti-mouse polyclonal antibody conjugated to horse-radish peroxidase (diluted 1:20,000; Abcam cat# AB205719). Protein bands were visualized using Supersignal™ West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific, USA).</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Recombinant protein design and preparation</title>","<p id=\"Par37\">Sixteen antigenic sites (8–24 amino acids long) on human NEP were identified using the BepiPred B-cell linear epitope prediction tool [##REF##16635264##18##]. Four of these (designated AgNEP-1, -2, -3, and -4) were selected on the basis of their non-overlapping surface locations with respect to the 3D structure of NEP modeled against template 6suk.1.A using SWISS-MODEL (Fig. ##FIG##0##1##a). AgNEP-1, -2, and -4 do not contain any experimentally verified or predicted post-translational modifications. AgNEP-3 was deliberately selected as it contained two experimentally-verified N-glycosylation sites at positions N285 and N294 and the mAbs generated could be useful in revealing subtle effects of NEP glycosylation on its biological actions, clinical associations, and circulating concentrations. Figure ##FIG##0##1##b shows the secondary structure elements (helix, strand, and others) and solvent accessibility (buried versus exposed residues) of these selected epitopes using the PredictProtein analysis tool [##REF##33999203##19##].</p>","<p id=\"Par38\">All Trx-tripeptide proteins were highly expressed in <italic>E. coli</italic> and accounted for 21–29% of total bacterial protein (Fig. ##SUPPL##0##S1##). Trx-AgNEP-1 was produced in the insoluble form while Trx-AgNEP-2 accumulated equally in both soluble and insoluble forms. Trx-AgNEP-3 and -4 were mainly expressed in the soluble form. Trx-AgNEP-1 was purified by denaturing IMAC and refolded into phosphate buffer while the other three antigens were purified by native IMAC to recover only the soluble form. Protein yields ranged from 15 to 60 mg/l bacterial culture (Table ##SUPPL##0##S1##). These four antigens were then mixed in equimolar concentrations and used as an immunogen cocktail for animal immunization. The composite construct, Trx-AgNEP(1–4), was produced primarily as a soluble protein with yield at 42 mg/l of bacterial culture.</p>","<title>Hybridoma screening and selection</title>","<p id=\"Par39\">A single fusion experiment of splenocytes from two mouse spleens with SP2/0-Ag14 myeloma cells produced a total of 3840 hybridoma clones. Initial screening was performed using validated 96-well DEXT microplates as described previously [##REF##33824395##16##]. Hybridoma clones producing mAbs that reacted strongly to only one of the four coated antigens were selected for further expansion. A total of 7, 41, 77, and 67 clones were found to react strongly to Trx-AgNEP-1, -2, -3 or -4, respectively. Of these, ~ 90% were unstable and exhibited significant loss of reactivity after a second round of sub-culturing. Finally, a total of 16 stable primary parent clones of high specificity to their cognate antigen (Trx-AgNEP-3 or -4) were obtained. No stable clones with specificity against Trx-AgNEP-1 and -2 were obtained. Ten stable high-yielding hybridoma cell lines were chosen and sub-cloned for further analysis. Antibodies were purified by Protein A affinity chromatography with yields ranging between 32 and 92 μg/ml of hybridoma culture supernatant.</p>","<title>Monoclonal antibody characterization</title>","<p id=\"Par40\">Functional characteristics of the selected mAbs are summarized in Table ##TAB##1##2##. All ten mAbs were of the IgG-kappa class and could be classified into three isotypes, IgG<sub>1</sub>, IgG<sub>2a</sub>, and IgG<sub>2b</sub>. Binding characteristics of the mAbs to both recombinant sNEP as well as their cognate Trx-fused peptide (AgNEP-3 or AgNEP-4) were evaluated by surface plasmon resonance (SPR) analysis (Fig. S2). Two out of five mAbs (33G5 and 31E1) against AgNEP-3 were able to bind Trx-AgNEP-3 while all five mAbs against AgNEP-4 were able to bind Trx-AgNEP-4. Of these, three mAbs (33G5, 17E11, and 3E12) were able to bind recombinant human sNEP with monovalent binding affinities (KD) measuring 4.8 nM or lower. The importance of individual amino acid side chains on the epitopes was assessed by performing an alanine scan, allowing for direct identification of residues that are critical for antigen–antibody binding (Fig. S3). It was observed that the functional epitopes of the mAbs involved 2–6 key interacting residues.</p>","<p id=\"Par41\">Immunoprecipitation from human plasma was performed using biotinylated mAb 17E11 and streptavidin magnetic beads. Targeted MRM<sup>HR</sup> mass spectrometry analysis of the antibody pull-down material detected 2 putative hits out of 15 NEP peptides targeted as determined by co-eluting peaks in the extracted ion chromatogram: DEWISGAAVVNAFYSSGR, which partially coincides with the epitope for mAb 17E11, and an upstream sequence, IGYPDDIVSNDNK. However, upon manual curation, these peptide matches were considered of low confidence due to the poor peak matching assignments (Fig. S4).</p>","<title>ELISA development</title>","<title>Antibody pairing</title>","<p id=\"Par42\">Twelve positive antibody pairs (signal-to-noise ratio > 10) were found from 10 × 10 capture/detector mAb checkboard combinations by ELISA using Trx-AgNEP(1–4) as test analyte (Table S2). The antibody pair that gave the highest signal comprised AgNEP-4/mAb 17E11 as capture and AgNEP-3/mAb 31E1 as detector while the pair that gave the highest signal-to-noise ratio comprised AgNEP-3/mAb 31E1 as capture and AgNEP-4/mAb 12A10 as detector. AgNEP-3/mAb 31E1, AgNEP-4/mAb 12A10, and AgNEP-4/mAb 17E11 displayed dual utilities as both capture and detector while AgNEP-3/mAb 25A5 was more useful as a capture antibody only. Among the 12 positive antibody pairs, 5 pairs displayed raw absorbance (450 nm) values greater than 1.0 (for the highest concentration of 8000 pg/ml) and were further analyzed. All five antibody pairs demonstrated a linear dose–response relationship over a concentration range of 125–8000 pg/ml with R-squared values exceeding 0.99 (Fig. ##FIG##1##2##). No antibody pairs were found when using cell-line-derived recombinant human sNEP as test analyte. Hence, Trx-AgNEP(1–4) was used as calibrator in the ELISA developed and represents a surrogate for relative quantification of circulating NEP.</p>","<title>Assay performance and validation</title>","<p id=\"Par43\">The antibody pair with the highest signal comprising AgNEP-4/mAb 17E11 as capture and AgNEP-3/mAb 31E1 as detection antibody was chosen for the development of an immunoassay to measure plasma NEP. The assay does not cross-react with all tested closely related endopeptidases spiked at concentrations between 5 and 100 ng/ml (Fig. ##FIG##2##3##a). The assay working range of 13.1–8000 pg/ml was established based on the precision profile whereby the intra-assay coefficient of variation (CV) for each Trx-AgNEP(1–4) calibrator point did not exceed 20% in 18 independent assays performed. The limit of detection (LOD), defined as the concentration derived from the mean absorbance values of 18 zero standard replicates, was 2.15 pg/ml. The lower limit of quantification (LLOQ), defined as the lowest analyte concentration at which intra-assay CV was below 20% over 18 independent assays, was 13.1 pg/ml. Intra-assay CVs ranged from 1.7 to 3.2% for three different human plasma samples between the range of 151 and 1678 pg/ml of NEP. Inter-assay CVs (<italic>n</italic> = 18) for NEP concentrations between 142 and 2076 pg/ml were 10.8–15.4%. Spike and recovery analyses showed poor recovery of between 51 and 64% when plasma samples were spiked with Trx-AgNEP(1–4). However, acceptable recovery of 108 to 118% was obtained when spiking with endogenous NEP from a high concentration plasma sample (Table ##TAB##2##3##). One possible explanation for poor recovery of Trx-AgNEP(1–4) is that the analyte from an exogenous source was mostly bound by unknown matrix component(s) rendering it unavailable for antibody binding. The dilution curves for six human plasma EDTA samples were parallel to the Trx-AgNEP(1–4) calibrator response curve, indicating that the recombinant calibrator was suitable for measuring endogenous human NEP (Fig. ##FIG##2##3##b). NEP ELISA characteristics and assay performance are summarized in Table ##TAB##3##4##.</p>","<title>Western blot</title>","<title>Reactivity toward recombinant sNEP, Trx-AgNEP(1–4), and endogenous cellular NEP</title>","<p id=\"Par44\">All ten mAbs were tested for their reactivity toward recombinant human sNEP, recombinant Trx-AgNEP(1–4) (Fig. ##FIG##3##4##a), and endogenous human NEP in LNCaP (human prostate adenocarcinoma) cell lysate (Fig. ##FIG##3##4##b and c) using capillary western blot. Two of the mAbs (12A10 and 17E11) were reactive toward recombinant sNEP (~ 109 kDa) as well as endogenous human NEP (~ 130 kDa) while three of the mAbs (12A10, 17E11, and 31E1) were reactive toward Trx-AgNEP(1–4) detected at the ~ 28 kDa molecular weight position. The oxidizing environment of the mutant <italic>trxB</italic> bacteria host used for Trx-AgNEP(1–4) expression allowed dimerization via intermolecular disulphide bond formation and a second minor band at ~ 51 kDa was also detected. The apparent deviation in estimated molecular weight of immunodetected Trx-AgNEP(1–4) by capillary electrophoresis (CE) on the Jess/Wes Separation Module compared with SDS-PAGE (Fig. ##SUPPL##0##S1##i; theoretical molecular weight ~ 20.4 kDa) may be attributed to the different CE separation matrix and running conditions used in the former. Although molecular weight determination by CE and SDS-PAGE is generally congruent, deviation by as much as 30–40% of calculated molecular weight based on amino acid sequence can occur for some proteins [##REF##33185281##20##]. PE pAb, previously validated by immunoprecipitation-mass spectrometry analysis to pull down NEP from human plasma [##REF##35331754##6##], was used as the positive control antibody. All mAbs and PE pAb showed no immunoreactivity to the NEP-negative PC3-cell lysate. Results are summarized in Table ##TAB##4##5##.</p>","<title>Reactivity toward circulating NEP</title>","<p id=\"Par45\">The circulating profile of NEP in human plasma was probed with mAb 17E11 and 31E1. PE pAb was also included for comparison. Plasma samples tested were obtained from pooled as well as individual HF patients with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF) and non-HF controls (CTRL). All three antibodies immunodetected four common bands at 57–60, 101, 143, and 177 kDa, albeit at different band intensities with weakest detection for the high-molecular-weight moieties by mAb 31E1 (Fig. ##FIG##4##5##a). mAb 17E11 and mAb 31E1 share a common band at 33 kDa that was not apparent with PE pAb. A unique band at the 84 kDa position was recognized by mAb 17E11 alone. Fluctuating band thickness was observed for the immunodetected 57–60 kDa moiety which could be suggestive of variable post-translational modification and/or antibody reactivity against this fragment. Blocking of mAb 17E11 with the matched peptide completely abolished detection of Trx-AgNEP(1–4) as well as the 101, 143, and 177 kDa bands of the plasma samples while weak traces of the 33 and 57 kDa bands remained (Fig. ##FIG##4##5##b). In the case of mAb 31E1, the matched peptide significantly blocked detection of only the 57–60 kDa band, albeit not completely to leave a weak band at 57 kDa. All other immunoreactive plasma sample bands were still weakly detected. Surprisingly, blocking of mAb 31E1 with the matched peptide marginally reduced detection of Trx-AgNEP(1–4). Increasing the concentration of the blocking peptide did not change the results for the plasma samples (Fig. S5). However, a reduction in the intensity of Trx-AgNEP(1–4) band was observed when mAb 31E1 was blocked at higher matched peptide concentration. ELISA peptide competition experiments corroborate the ability of the matched peptide to completely block Trx-AgNEP(1–4) detection by mAb 17E11 and only partially by mAb 31E1 (Fig. ##FIG##5##6##).</p>","<title>Reactivity of mAb 31E1 to glycan-deficient recombinant sNEP</title>","<p id=\"Par46\">HEK293 cell-line-derived recombinant sNEP is expected to be fully glycosylated and this is corroborated by its migration at a higher molecular weight position than its theoretical molecular mass of ~ 79.8 kDa on SDS-PAGE (Fig. ##FIG##6##7##a, untreated). The band appears smeared between 90 and 110 kDa due to heterogeneity of the glycosites and glycan structures at each site. Following digestion with PNGase F, recombinant sNEP migrates as a sharp band at a lower molecular weight position (~ 78.2 kDa) compared with the untreated sample, confirming complete N-glycan removal. Western blot analysis confirms that mAb 31E1 does not bind to recombinant sNEP without PNGase F treatment but is able to detect deglycosylated recombinant sNEP in a concentration-dependent manner (Fig. ##FIG##6##7##b).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Recombinant protein design and preparation</title>\",\n \"<p id=\\\"Par37\\\">Sixteen antigenic sites (8–24 amino acids long) on human NEP were identified using the BepiPred B-cell linear epitope prediction tool [##REF##16635264##18##]. Four of these (designated AgNEP-1, -2, -3, and -4) were selected on the basis of their non-overlapping surface locations with respect to the 3D structure of NEP modeled against template 6suk.1.A using SWISS-MODEL (Fig. ##FIG##0##1##a). AgNEP-1, -2, and -4 do not contain any experimentally verified or predicted post-translational modifications. AgNEP-3 was deliberately selected as it contained two experimentally-verified N-glycosylation sites at positions N285 and N294 and the mAbs generated could be useful in revealing subtle effects of NEP glycosylation on its biological actions, clinical associations, and circulating concentrations. Figure ##FIG##0##1##b shows the secondary structure elements (helix, strand, and others) and solvent accessibility (buried versus exposed residues) of these selected epitopes using the PredictProtein analysis tool [##REF##33999203##19##].</p>\",\n \"<p id=\\\"Par38\\\">All Trx-tripeptide proteins were highly expressed in <italic>E. coli</italic> and accounted for 21–29% of total bacterial protein (Fig. ##SUPPL##0##S1##). Trx-AgNEP-1 was produced in the insoluble form while Trx-AgNEP-2 accumulated equally in both soluble and insoluble forms. Trx-AgNEP-3 and -4 were mainly expressed in the soluble form. Trx-AgNEP-1 was purified by denaturing IMAC and refolded into phosphate buffer while the other three antigens were purified by native IMAC to recover only the soluble form. Protein yields ranged from 15 to 60 mg/l bacterial culture (Table ##SUPPL##0##S1##). These four antigens were then mixed in equimolar concentrations and used as an immunogen cocktail for animal immunization. The composite construct, Trx-AgNEP(1–4), was produced primarily as a soluble protein with yield at 42 mg/l of bacterial culture.</p>\",\n \"<title>Hybridoma screening and selection</title>\",\n \"<p id=\\\"Par39\\\">A single fusion experiment of splenocytes from two mouse spleens with SP2/0-Ag14 myeloma cells produced a total of 3840 hybridoma clones. Initial screening was performed using validated 96-well DEXT microplates as described previously [##REF##33824395##16##]. Hybridoma clones producing mAbs that reacted strongly to only one of the four coated antigens were selected for further expansion. A total of 7, 41, 77, and 67 clones were found to react strongly to Trx-AgNEP-1, -2, -3 or -4, respectively. Of these, ~ 90% were unstable and exhibited significant loss of reactivity after a second round of sub-culturing. Finally, a total of 16 stable primary parent clones of high specificity to their cognate antigen (Trx-AgNEP-3 or -4) were obtained. No stable clones with specificity against Trx-AgNEP-1 and -2 were obtained. Ten stable high-yielding hybridoma cell lines were chosen and sub-cloned for further analysis. Antibodies were purified by Protein A affinity chromatography with yields ranging between 32 and 92 μg/ml of hybridoma culture supernatant.</p>\",\n \"<title>Monoclonal antibody characterization</title>\",\n \"<p id=\\\"Par40\\\">Functional characteristics of the selected mAbs are summarized in Table ##TAB##1##2##. All ten mAbs were of the IgG-kappa class and could be classified into three isotypes, IgG<sub>1</sub>, IgG<sub>2a</sub>, and IgG<sub>2b</sub>. Binding characteristics of the mAbs to both recombinant sNEP as well as their cognate Trx-fused peptide (AgNEP-3 or AgNEP-4) were evaluated by surface plasmon resonance (SPR) analysis (Fig. S2). Two out of five mAbs (33G5 and 31E1) against AgNEP-3 were able to bind Trx-AgNEP-3 while all five mAbs against AgNEP-4 were able to bind Trx-AgNEP-4. Of these, three mAbs (33G5, 17E11, and 3E12) were able to bind recombinant human sNEP with monovalent binding affinities (KD) measuring 4.8 nM or lower. The importance of individual amino acid side chains on the epitopes was assessed by performing an alanine scan, allowing for direct identification of residues that are critical for antigen–antibody binding (Fig. S3). It was observed that the functional epitopes of the mAbs involved 2–6 key interacting residues.</p>\",\n \"<p id=\\\"Par41\\\">Immunoprecipitation from human plasma was performed using biotinylated mAb 17E11 and streptavidin magnetic beads. Targeted MRM<sup>HR</sup> mass spectrometry analysis of the antibody pull-down material detected 2 putative hits out of 15 NEP peptides targeted as determined by co-eluting peaks in the extracted ion chromatogram: DEWISGAAVVNAFYSSGR, which partially coincides with the epitope for mAb 17E11, and an upstream sequence, IGYPDDIVSNDNK. However, upon manual curation, these peptide matches were considered of low confidence due to the poor peak matching assignments (Fig. S4).</p>\",\n \"<title>ELISA development</title>\",\n \"<title>Antibody pairing</title>\",\n \"<p id=\\\"Par42\\\">Twelve positive antibody pairs (signal-to-noise ratio > 10) were found from 10 × 10 capture/detector mAb checkboard combinations by ELISA using Trx-AgNEP(1–4) as test analyte (Table S2). The antibody pair that gave the highest signal comprised AgNEP-4/mAb 17E11 as capture and AgNEP-3/mAb 31E1 as detector while the pair that gave the highest signal-to-noise ratio comprised AgNEP-3/mAb 31E1 as capture and AgNEP-4/mAb 12A10 as detector. AgNEP-3/mAb 31E1, AgNEP-4/mAb 12A10, and AgNEP-4/mAb 17E11 displayed dual utilities as both capture and detector while AgNEP-3/mAb 25A5 was more useful as a capture antibody only. Among the 12 positive antibody pairs, 5 pairs displayed raw absorbance (450 nm) values greater than 1.0 (for the highest concentration of 8000 pg/ml) and were further analyzed. All five antibody pairs demonstrated a linear dose–response relationship over a concentration range of 125–8000 pg/ml with R-squared values exceeding 0.99 (Fig. ##FIG##1##2##). No antibody pairs were found when using cell-line-derived recombinant human sNEP as test analyte. Hence, Trx-AgNEP(1–4) was used as calibrator in the ELISA developed and represents a surrogate for relative quantification of circulating NEP.</p>\",\n \"<title>Assay performance and validation</title>\",\n \"<p id=\\\"Par43\\\">The antibody pair with the highest signal comprising AgNEP-4/mAb 17E11 as capture and AgNEP-3/mAb 31E1 as detection antibody was chosen for the development of an immunoassay to measure plasma NEP. The assay does not cross-react with all tested closely related endopeptidases spiked at concentrations between 5 and 100 ng/ml (Fig. ##FIG##2##3##a). The assay working range of 13.1–8000 pg/ml was established based on the precision profile whereby the intra-assay coefficient of variation (CV) for each Trx-AgNEP(1–4) calibrator point did not exceed 20% in 18 independent assays performed. The limit of detection (LOD), defined as the concentration derived from the mean absorbance values of 18 zero standard replicates, was 2.15 pg/ml. The lower limit of quantification (LLOQ), defined as the lowest analyte concentration at which intra-assay CV was below 20% over 18 independent assays, was 13.1 pg/ml. Intra-assay CVs ranged from 1.7 to 3.2% for three different human plasma samples between the range of 151 and 1678 pg/ml of NEP. Inter-assay CVs (<italic>n</italic> = 18) for NEP concentrations between 142 and 2076 pg/ml were 10.8–15.4%. Spike and recovery analyses showed poor recovery of between 51 and 64% when plasma samples were spiked with Trx-AgNEP(1–4). However, acceptable recovery of 108 to 118% was obtained when spiking with endogenous NEP from a high concentration plasma sample (Table ##TAB##2##3##). One possible explanation for poor recovery of Trx-AgNEP(1–4) is that the analyte from an exogenous source was mostly bound by unknown matrix component(s) rendering it unavailable for antibody binding. The dilution curves for six human plasma EDTA samples were parallel to the Trx-AgNEP(1–4) calibrator response curve, indicating that the recombinant calibrator was suitable for measuring endogenous human NEP (Fig. ##FIG##2##3##b). NEP ELISA characteristics and assay performance are summarized in Table ##TAB##3##4##.</p>\",\n \"<title>Western blot</title>\",\n \"<title>Reactivity toward recombinant sNEP, Trx-AgNEP(1–4), and endogenous cellular NEP</title>\",\n \"<p id=\\\"Par44\\\">All ten mAbs were tested for their reactivity toward recombinant human sNEP, recombinant Trx-AgNEP(1–4) (Fig. ##FIG##3##4##a), and endogenous human NEP in LNCaP (human prostate adenocarcinoma) cell lysate (Fig. ##FIG##3##4##b and c) using capillary western blot. Two of the mAbs (12A10 and 17E11) were reactive toward recombinant sNEP (~ 109 kDa) as well as endogenous human NEP (~ 130 kDa) while three of the mAbs (12A10, 17E11, and 31E1) were reactive toward Trx-AgNEP(1–4) detected at the ~ 28 kDa molecular weight position. The oxidizing environment of the mutant <italic>trxB</italic> bacteria host used for Trx-AgNEP(1–4) expression allowed dimerization via intermolecular disulphide bond formation and a second minor band at ~ 51 kDa was also detected. The apparent deviation in estimated molecular weight of immunodetected Trx-AgNEP(1–4) by capillary electrophoresis (CE) on the Jess/Wes Separation Module compared with SDS-PAGE (Fig. ##SUPPL##0##S1##i; theoretical molecular weight ~ 20.4 kDa) may be attributed to the different CE separation matrix and running conditions used in the former. Although molecular weight determination by CE and SDS-PAGE is generally congruent, deviation by as much as 30–40% of calculated molecular weight based on amino acid sequence can occur for some proteins [##REF##33185281##20##]. PE pAb, previously validated by immunoprecipitation-mass spectrometry analysis to pull down NEP from human plasma [##REF##35331754##6##], was used as the positive control antibody. All mAbs and PE pAb showed no immunoreactivity to the NEP-negative PC3-cell lysate. Results are summarized in Table ##TAB##4##5##.</p>\",\n \"<title>Reactivity toward circulating NEP</title>\",\n \"<p id=\\\"Par45\\\">The circulating profile of NEP in human plasma was probed with mAb 17E11 and 31E1. PE pAb was also included for comparison. Plasma samples tested were obtained from pooled as well as individual HF patients with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF) and non-HF controls (CTRL). All three antibodies immunodetected four common bands at 57–60, 101, 143, and 177 kDa, albeit at different band intensities with weakest detection for the high-molecular-weight moieties by mAb 31E1 (Fig. ##FIG##4##5##a). mAb 17E11 and mAb 31E1 share a common band at 33 kDa that was not apparent with PE pAb. A unique band at the 84 kDa position was recognized by mAb 17E11 alone. Fluctuating band thickness was observed for the immunodetected 57–60 kDa moiety which could be suggestive of variable post-translational modification and/or antibody reactivity against this fragment. Blocking of mAb 17E11 with the matched peptide completely abolished detection of Trx-AgNEP(1–4) as well as the 101, 143, and 177 kDa bands of the plasma samples while weak traces of the 33 and 57 kDa bands remained (Fig. ##FIG##4##5##b). In the case of mAb 31E1, the matched peptide significantly blocked detection of only the 57–60 kDa band, albeit not completely to leave a weak band at 57 kDa. All other immunoreactive plasma sample bands were still weakly detected. Surprisingly, blocking of mAb 31E1 with the matched peptide marginally reduced detection of Trx-AgNEP(1–4). Increasing the concentration of the blocking peptide did not change the results for the plasma samples (Fig. S5). However, a reduction in the intensity of Trx-AgNEP(1–4) band was observed when mAb 31E1 was blocked at higher matched peptide concentration. ELISA peptide competition experiments corroborate the ability of the matched peptide to completely block Trx-AgNEP(1–4) detection by mAb 17E11 and only partially by mAb 31E1 (Fig. ##FIG##5##6##).</p>\",\n \"<title>Reactivity of mAb 31E1 to glycan-deficient recombinant sNEP</title>\",\n \"<p id=\\\"Par46\\\">HEK293 cell-line-derived recombinant sNEP is expected to be fully glycosylated and this is corroborated by its migration at a higher molecular weight position than its theoretical molecular mass of ~ 79.8 kDa on SDS-PAGE (Fig. ##FIG##6##7##a, untreated). The band appears smeared between 90 and 110 kDa due to heterogeneity of the glycosites and glycan structures at each site. Following digestion with PNGase F, recombinant sNEP migrates as a sharp band at a lower molecular weight position (~ 78.2 kDa) compared with the untreated sample, confirming complete N-glycan removal. Western blot analysis confirms that mAb 31E1 does not bind to recombinant sNEP without PNGase F treatment but is able to detect deglycosylated recombinant sNEP in a concentration-dependent manner (Fig. ##FIG##6##7##b).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par47\">Careful selection of peptide immunogens is an important first step in the entire antibody production workflow from antigen preparation to antibody characterization and validation. The B-cell linear epitope prediction tool used in this study is trained to locate antigenic peptides that tend to be surface accessible, hydrophilic, and flexible. These physicochemical properties coincide with parameters for soluble expression in <italic>E. coli</italic>, an important advantage for easy protein production and purification, and is corroborated in our previous work where epitope-predicted peptides on human ankyrin repeat domain 1 (hANKRD1) were all produced primarily as soluble Trx fusion proteins in the bacteria host [##REF##33824395##16##]. Surprisingly, Trx-AgNEP-1 was produced primarily in the insoluble form while Trx-AgNEP-2 and -3 were partitioned into both soluble and insoluble fractions, with the former showing greater insolubility. We used the NetSolP analysis tool [##REF##35088833##21##], a protein language model for in silico prediction of the solubility of expressed proteins in <italic>E. coli</italic>, to cross-check the predicted solubility of the Trx-AgNEP antigens. Indeed, Trx-AgNEP-1, -2, -3, and -4 achieved comparable high solubility scores of 0.8634, 0.8812, 0.8740, and 0.8817, respectively. We posit that the presence of a helical secondary element, composed of at least four amino acids sufficient to form one helical turn and located in a central position on the antigenic peptide as in the case of AgNEP-1 (see Fig. ##FIG##0##1##b), contributes to the propensity for insoluble protein expression in <italic>E. coli</italic>. Although AgNEP-2 and -3 contained 3–4 amino acids that overlap from a predicted alpha-helix structure in the native protein, these are located to one end of the antigenic peptides and presumably exerts less influence in promoting protein insolubility. Concordant with the supposition that the presence of a helical secondary element contributes to insoluble protein expression, Trx-AgNEP-4 contained no predicted alpha-helix and was produced almost entirely in the soluble form. In further support of this postulation, re-examination of our three previously reported hANKRD1 antigenic peptides using the PredictProtein analysis tool showed only one peptide containing three amino acids that overlap from an alpha-helix region in the native hANKRD1 protein and hence is not expected to have a strong effect on Trx-peptide solubility. Indeed, all three Trx-peptide fusions were highly soluble in <italic>E. coli</italic> expression [##REF##33824395##16##]. In view of these observations, we suggest that secondary structure predictions could also be incorporated prior to final selection of in silico predicted antigenic peptides to increase the likelihood of soluble recombinant immunogen production, making the protein expression and purification workflow more straightforward and efficient.</p>","<p id=\"Par48\">We show that our previously reported Trx-tripeptide fusion strategy offers an effective antigen presentation scheme that consistently leads to successful generation of protein-reactive anti-peptide mAbs that exhibit high specificity and affinity under native and denaturing conditions. In this study, we applied a similar approach to generating protein-reactive mAbs targeting multiple non-overlapping sites on human NEP. The selected in silico predicted epitopes corresponding to short (10–15 residues) surface-exposed peptides initially yielded a total of 192 hybridoma clones (7, 41, 77, and 67 for AgNEP-1, -2, -3, and -4, respectively) that reacted strongly only with the peptide that they were generated against. However, after a second round of sub-culturing, only 16 hybridoma clones remained stable. All 16 clones were reactive toward either Trx-AgNEP-3 or -4. Of these, 10 mAbs were selected for detailed evaluation and application-specific validation. A few mAbs demonstrated protein reactivity toward recombinant sNEP by SPR (mAb 33G5, mAb 3E12 and mAb 17E11) and/or western blot (mAb 12A10 and mAb 17E11). Interestingly, although mAb 33G5 demonstrates strong affinity toward recombinant sNEP and Trx-AgNEP-3 by SPR, it was unable to detect these targets under denaturing conditions on western blot conditions. In contrast, mAb 12A10 detected only Trx-AgNEP-4 and showed no binding to sNEP by SPR but was able to detect both targets by Western blotting. These observations highlight differences in antibody reactivity to the same target in its native and denatured forms, and emphasizes the importance of careful selection and application-specific validation of antibodies for downstream deployment. In addition, 12 antibody pairs were found to be positive for two-site ELISA with Trx-AgNEP(1–4) but not recombinant sNEP as target analyte. Since mAb 33G5 and mAb 17E11 bind to recombinant sNEP in SPR, it is logical to expect some signal response in ELISA with this antibody pair. However, mAb 33G5 does not work well whether as a capture or detection antibody in ELISA as indicated by the checkerboard antibody screening results. When used as a capture antibody, poor performance may relate to partial denaturation of mAb 33G5 as it spreads over the microplate surface during passive adsorption via hydrophobic and hydrophilic interactions. Poor analyte binding by mAb 33G5 as a detection antibody may be attributed to changes in analyte conformation after its interaction with the capture antibody as exemplified in a precedent example [##REF##24291344##17##].</p>","<p id=\"Par49\">We examined our NEP mAbs for suitability in Western immunoassay applications. Although all ten selected mAbs were able to bind to their cognate peptide in ELISA testing with the Multipin system, only three mAbs (31E1, 12A10, and 17E11) were able to detect denatured Trx-AgNEP(1–4) by western analysis. The high number of mAbs failing to immunodetect Trx-AgNEP(1–4) may be attributed to the preferred conformational fold adopted by the immobilized cognate peptide, whether influenced by other flanking peptides alone or in combination with the Trx carrier, lacking steric complementarity with the antibody combining site [##REF##2446325##22##, ##REF##11851317##23##]. This result reiterates earlier observations that mAbs differ in their ability to bind to native and denatured forms of their antigen peptide sequence and this property governs their suitability for downstream applications. More importantly, the practical usefulness of anti-peptide antibodies resides in their ability to bind to the native protein from which its peptide sequence was derived. Two mAbs raised against AgNEP-4 (12A10 and 17E11) were able to detect Trx-AgNEP(1–4), recombinant sNEP and endogenous LNCaP NEP. However, mAb 31E1 derived from the AgNEP-3 peptide antigen detected Trx-AgNEP(1–4) but not recombinant sNEP or endogenous NEP. It is noteworthy that two reported glycosylation sites at N285 (in LNCaP [##REF##12754519##12##] and B cell lymphoma cell lines [##REF##24190977##14##]) and N294 (in B cell lymphoma cell lines [##REF##24190977##14##]) are present on the epitope of mAb 31E1. Glycosylation at N294 would likely have a greater influence on antibody binding since it is flanked on both sides by the critical binding residues of the antibody. However, published mass spectrometry evidence for the N294 glycosite is weak as the fragment ion matches are quite sparse and the fragment ion for deglycosylated N294 was not detected [Supplemental data in reference 14]. Furthermore, the sequence context of N294 does not fall within the canonical glycosylation consensus motif (N-X-T/S where X is not a proline) nor to known atypical sequons [##REF##27246700##24##, ##REF##26593774##25##]. Despite the uncertainty of glycosylation at N294, modification at the N285 glycosite might still be sufficient to abolish mAb 31E1 binding to NEP derived from eukaryotic sources since glycosylation at nearby sites outside of an epitope have been shown to be able to completely hinder antibody binding [##REF##25093517##26##]. In addition, western blot analysis provided direct evidence that mAb 31E1 can bind to HEK293 cell-line-derived sNEP only after N-glycan removal by PNGase F digestion. Proteins that are glycosylated at multiple sites can assume a variety of glycoforms depending on site occupancy and glycan structure at each glycosite [##REF##26555091##27##]. Proteins from different tissues of origin and disease states have been shown to exhibit variable glycosylation site occupancy [##REF##17823199##28##, ##UREF##4##29##]. Hence, the sensitivity of mAb 31E1 to glycosylation is of special interest in probing the significance of N285 and/or N294 glycan-deficient NEP in health and disease.</p>","<p id=\"Par50\">At this juncture, it is worth highlighting a seeming conundrum in the differences in peptide and protein reactivity of mAb 33G5 and 31E1. Both mAbs were raised against the same AgNEP-3 peptide antigen and share exactly the same critical binding residues on the epitope. Yet, mAb 33G5 was able to detect cell-line-derived recombinant sNEP and its cognate Trx-peptide by SPR but not under western blot conditions. This antibody also showed poor utility, whether as a capture or detection antibody, in two-site ELISA with low signal response to Trx-AgNEP(1–4) as analyte. In contrast, mAb 31E1 exhibited good binding only to Trx-AgNEP(1–4), but not recombinant sNEP, by SPR, western blotting, and ELISA. How could the inhibitory effect of glycosylation on mAb 31E1 protein reactivity be reconciled with the immunity of mAb 33G5 to do so by SPR? First, glycosylation does not necessarily always impose steric hindrance to antibody binding but can even enhance antibody affinity to its antigen. It has been reported that glycosylation at one specific location on the MUC-16 epitope increased antibody recognition while modifications at other positions within the epitope were inhibitory to antibody binding [##REF##25093517##26##]. Second, N-glycosylation has been observed to occur on the antibody variable light-chain domain. Such modifications can change the conformation of the antigen-binding region and increase or decrease subsequent antibody–antigen interactions [##REF##10024532##30##, ##REF##8925140##31##]. Thus, it is possible that mAb 33G5 and 31E1 may differ in terms of the glycosylation profile of their variable light chains which in turn alters their peptide/protein-reactive properties and glycosylation sensitivity.</p>","<p id=\"Par51\">We next investigated whether the mAbs 17E11 and 31E1 (the antibody pair used to develop the NEP ELISA) were able to detect circulating NEP in plasma by western blot analysis. Interestingly, both mAbs directed against different regions on NEP share four common immunodetected bands with the validated PE pAb, adding to confidence in antibody specificity for the target. Since NEP is highly glycosylated and can conceptually exist as monomers or dimers of the full-length protein or its ectodomain, it is difficult to interpret what the high-molecular-weight bands (> 100 kDa) represent. Since recombinant sNEP and LNCaP-derived endogenous NEP was found to migrate at the 109 and 130 kDa position, respectively, it is reasonable to postulate that the 101, 143, and 177 kDa bands detected in plasma might be glycosylated forms of monomeric NEP ectodomain, monomeric full-length NEP, and dimeric NEP ectodomain, respectively. PE pAb, mAb 31E1, and 17E11 consistently detected one major broad band at the 57–60 kDa position in all samples tested. This band exhibited slight differences in mobility and band thickness between samples, possibly reflecting a plethora of subtle nuances in post-translational modifications and/or antibody reactivity. Clear differences in antibody reactivity are exemplified in the case of mAb 17E11 and 31E1 binding to Trx-AgNEP(1–4) whereby more intense and broader immunodetected bands were obtained with mAb 31E1 under the same image exposure setting, especially for the higher molecular weight dimeric form of the recombinant protein. Peptide competition with the matched peptide for mAb 31E1 and 17E11 resulted in significant blocking of the 57–60 kDa band, though a weak residual band at the 57 kDa position could still be observed even at 25-fold excess of blocking peptide used. The poor ability of the free matched peptide to block the reaction between mAb 31E1 and the bound Trx-AgNEP(1–4), whether fixed within capillary tubes in western experiments or bound by the capture antibody in ELISA, suggests that the immobilized epitope sequence takes on a conformation that displayed much stronger affinity for mAb 31E1 [##REF##16737525##32##]. Identifying the biological processes that generate moieties in the 57–60 kDa region may reveal important information on the regulatory cascades modulating NEP activity and implications in health and disease. Thus, efforts to elucidate the identity of this fragment constitute a worthwhile pursuit in further work.</p>","<p id=\"Par52\">Finally, mAb 17E11 and 31E1 shared a band immunodetected at the ~ 33 kDa position that is apparently not recognized by PE pAb. The validated sandwich ELISA developed from this mAb pair will likely be predominantly measuring the 33 and 57–60 kDa NEP fragments in circulation. Using the SitePrediction tool [##REF##19546006##33##], we searched for candidate protease cut sites predicted from cleavage site entries in the MEROPS database [##REF##19892822##34##, ##REF##24157837##35##] with species specificity restricted to Homo sapiens. The analysis suggests that NEP can potentially be cleaved by numerous endopeptidases such as matrix metalloproteinases, cathepsins, and caspases at multiple sites. The SitePrediction output predicted matrix metalloproteinase 2 (MMP 2) to cleave at S<sub>65</sub>AAR↓LI<sub>70</sub>, A<sub>187</sub>IAQ↓LN<sub>192</sub>, S<sub>251</sub>VAR↓LI<sub>256</sub> (all three sites with > 99% specificity) and F<sub>555</sub>PAG↓IL<sub>560</sub> (> 99.9% specificity) (Fig. ##FIG##7##8##). Cleavage at S<sub>65</sub>AAR↓LI<sub>70</sub> and F<sub>555</sub>PAG↓IL<sub>560</sub> would result in a predicted 56.2 kDa (calculated from the amino acid sequence alone with no N-linked glycosylation at N145, N285, N294 or N325) fragment that could account for the 57 kDa immunodetected band. On the other hand, cleavage at A<sub>187</sub>IAQ↓LN<sub>192</sub> or S<sub>251</sub>VAR↓LI<sub>256</sub> and F<sub>555</sub>PAG↓IL<sub>560</sub> is expected to produce fragments of calculated molecular weights of 42.5 and 35.2 kDa, respectively, with the balance mass to make up the final mass of 57–60 kDa accounted for by the presence glycan chains at any of the abovementioned N-linked glycosylation sites. Cathepsin K (CatK), a cysteine protease with important functional roles in bone resorption and far-reaching action on other organs including the cardiovascular system [##REF##32582709##36##], was also predicted to cleave NEP at ten different sites with a specificity ≥ 99%. Two of these cleavage sites at L<sub>262</sub>PID↓EN<sub>267</sub> and V<sub>554</sub>FPA↓GI<sub>559</sub> would generate a ~ 33.7 kDa (assuming no N-linked glycosylation at N285 or N294) fragment, representing the minimal sequence that contains both the epitopes of mAbs 17E11 and 31E1 (Fig. ##FIG##7##8##). This also harmonizes with the direct evidence from PNGase F experiments that mAb 31E1 is glycosylation sensitive and would bind only to glycan-deficient NEP fragments. Hence, the ~ 33 and 57–60 kDa NEP bands recognized by both mAbs could, respectively, be generated by CatK or MMP2 cleavage alone or in combination with each other. It can be further deduced that NEP concentration measured by our two-site ELISA will not correlate with NEP activity since the predominantly detected fragments do not harbor residues that make up the active site.</p>","<p id=\"Par53\">A schematic representation of NEP and its fragments in circulation is depicted in Fig. ##FIG##8##9##. The complexity of circulating forms of NEP may help explain why previous NEP studies gave conflicting results from immunoassay data. For example, it was reported that circulating NEP concentrations were elevated in HF patients compared with non-HF controls [##REF##35331754##6##] and was positively associated with adverse outcomes [##REF##26251092##37##, ##REF##25677426##38##]. Yet, in other studies, soluble NEP levels were found to be significantly lower in HF patients compared with non-HF controls [##REF##31392960##39##] and not a prognosticator of adverse outcomes [##REF##26725876##40##, ##REF##32427034##41##]. One of the contributing factors for these contradictory results could be the use of different ELISA kits in the various studies. Hence, depending on the antibody pair used in the immunoassay, different results may be obtained. Clearly, our data showed that different NEP antibodies recognized different subsets of circulating NEP forms. Disease severity may be correlated with a reduction or increase in N-linked glycosylation site occupancy of serum proteins [##UREF##4##29##]. In addition, N-linked glycosylation is known to modulate protein function by altering the interaction of a cell-surface protein with its ligand [##REF##33664400##42##]. The new mAb pair generated in this work will add to the toolbox of well-defined binders to probe dynamic changes in NEP concentration and glycosylation site occupancy in health and disease. It remains to be determined which specific fragment(s) of NEP is useful as a biomarker and this would certainly be an important area requiring further research.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par47\\\">Careful selection of peptide immunogens is an important first step in the entire antibody production workflow from antigen preparation to antibody characterization and validation. The B-cell linear epitope prediction tool used in this study is trained to locate antigenic peptides that tend to be surface accessible, hydrophilic, and flexible. These physicochemical properties coincide with parameters for soluble expression in <italic>E. coli</italic>, an important advantage for easy protein production and purification, and is corroborated in our previous work where epitope-predicted peptides on human ankyrin repeat domain 1 (hANKRD1) were all produced primarily as soluble Trx fusion proteins in the bacteria host [##REF##33824395##16##]. Surprisingly, Trx-AgNEP-1 was produced primarily in the insoluble form while Trx-AgNEP-2 and -3 were partitioned into both soluble and insoluble fractions, with the former showing greater insolubility. We used the NetSolP analysis tool [##REF##35088833##21##], a protein language model for in silico prediction of the solubility of expressed proteins in <italic>E. coli</italic>, to cross-check the predicted solubility of the Trx-AgNEP antigens. Indeed, Trx-AgNEP-1, -2, -3, and -4 achieved comparable high solubility scores of 0.8634, 0.8812, 0.8740, and 0.8817, respectively. We posit that the presence of a helical secondary element, composed of at least four amino acids sufficient to form one helical turn and located in a central position on the antigenic peptide as in the case of AgNEP-1 (see Fig. ##FIG##0##1##b), contributes to the propensity for insoluble protein expression in <italic>E. coli</italic>. Although AgNEP-2 and -3 contained 3–4 amino acids that overlap from a predicted alpha-helix structure in the native protein, these are located to one end of the antigenic peptides and presumably exerts less influence in promoting protein insolubility. Concordant with the supposition that the presence of a helical secondary element contributes to insoluble protein expression, Trx-AgNEP-4 contained no predicted alpha-helix and was produced almost entirely in the soluble form. In further support of this postulation, re-examination of our three previously reported hANKRD1 antigenic peptides using the PredictProtein analysis tool showed only one peptide containing three amino acids that overlap from an alpha-helix region in the native hANKRD1 protein and hence is not expected to have a strong effect on Trx-peptide solubility. Indeed, all three Trx-peptide fusions were highly soluble in <italic>E. coli</italic> expression [##REF##33824395##16##]. In view of these observations, we suggest that secondary structure predictions could also be incorporated prior to final selection of in silico predicted antigenic peptides to increase the likelihood of soluble recombinant immunogen production, making the protein expression and purification workflow more straightforward and efficient.</p>\",\n \"<p id=\\\"Par48\\\">We show that our previously reported Trx-tripeptide fusion strategy offers an effective antigen presentation scheme that consistently leads to successful generation of protein-reactive anti-peptide mAbs that exhibit high specificity and affinity under native and denaturing conditions. In this study, we applied a similar approach to generating protein-reactive mAbs targeting multiple non-overlapping sites on human NEP. The selected in silico predicted epitopes corresponding to short (10–15 residues) surface-exposed peptides initially yielded a total of 192 hybridoma clones (7, 41, 77, and 67 for AgNEP-1, -2, -3, and -4, respectively) that reacted strongly only with the peptide that they were generated against. However, after a second round of sub-culturing, only 16 hybridoma clones remained stable. All 16 clones were reactive toward either Trx-AgNEP-3 or -4. Of these, 10 mAbs were selected for detailed evaluation and application-specific validation. A few mAbs demonstrated protein reactivity toward recombinant sNEP by SPR (mAb 33G5, mAb 3E12 and mAb 17E11) and/or western blot (mAb 12A10 and mAb 17E11). Interestingly, although mAb 33G5 demonstrates strong affinity toward recombinant sNEP and Trx-AgNEP-3 by SPR, it was unable to detect these targets under denaturing conditions on western blot conditions. In contrast, mAb 12A10 detected only Trx-AgNEP-4 and showed no binding to sNEP by SPR but was able to detect both targets by Western blotting. These observations highlight differences in antibody reactivity to the same target in its native and denatured forms, and emphasizes the importance of careful selection and application-specific validation of antibodies for downstream deployment. In addition, 12 antibody pairs were found to be positive for two-site ELISA with Trx-AgNEP(1–4) but not recombinant sNEP as target analyte. Since mAb 33G5 and mAb 17E11 bind to recombinant sNEP in SPR, it is logical to expect some signal response in ELISA with this antibody pair. However, mAb 33G5 does not work well whether as a capture or detection antibody in ELISA as indicated by the checkerboard antibody screening results. When used as a capture antibody, poor performance may relate to partial denaturation of mAb 33G5 as it spreads over the microplate surface during passive adsorption via hydrophobic and hydrophilic interactions. Poor analyte binding by mAb 33G5 as a detection antibody may be attributed to changes in analyte conformation after its interaction with the capture antibody as exemplified in a precedent example [##REF##24291344##17##].</p>\",\n \"<p id=\\\"Par49\\\">We examined our NEP mAbs for suitability in Western immunoassay applications. Although all ten selected mAbs were able to bind to their cognate peptide in ELISA testing with the Multipin system, only three mAbs (31E1, 12A10, and 17E11) were able to detect denatured Trx-AgNEP(1–4) by western analysis. The high number of mAbs failing to immunodetect Trx-AgNEP(1–4) may be attributed to the preferred conformational fold adopted by the immobilized cognate peptide, whether influenced by other flanking peptides alone or in combination with the Trx carrier, lacking steric complementarity with the antibody combining site [##REF##2446325##22##, ##REF##11851317##23##]. This result reiterates earlier observations that mAbs differ in their ability to bind to native and denatured forms of their antigen peptide sequence and this property governs their suitability for downstream applications. More importantly, the practical usefulness of anti-peptide antibodies resides in their ability to bind to the native protein from which its peptide sequence was derived. Two mAbs raised against AgNEP-4 (12A10 and 17E11) were able to detect Trx-AgNEP(1–4), recombinant sNEP and endogenous LNCaP NEP. However, mAb 31E1 derived from the AgNEP-3 peptide antigen detected Trx-AgNEP(1–4) but not recombinant sNEP or endogenous NEP. It is noteworthy that two reported glycosylation sites at N285 (in LNCaP [##REF##12754519##12##] and B cell lymphoma cell lines [##REF##24190977##14##]) and N294 (in B cell lymphoma cell lines [##REF##24190977##14##]) are present on the epitope of mAb 31E1. Glycosylation at N294 would likely have a greater influence on antibody binding since it is flanked on both sides by the critical binding residues of the antibody. However, published mass spectrometry evidence for the N294 glycosite is weak as the fragment ion matches are quite sparse and the fragment ion for deglycosylated N294 was not detected [Supplemental data in reference 14]. Furthermore, the sequence context of N294 does not fall within the canonical glycosylation consensus motif (N-X-T/S where X is not a proline) nor to known atypical sequons [##REF##27246700##24##, ##REF##26593774##25##]. Despite the uncertainty of glycosylation at N294, modification at the N285 glycosite might still be sufficient to abolish mAb 31E1 binding to NEP derived from eukaryotic sources since glycosylation at nearby sites outside of an epitope have been shown to be able to completely hinder antibody binding [##REF##25093517##26##]. In addition, western blot analysis provided direct evidence that mAb 31E1 can bind to HEK293 cell-line-derived sNEP only after N-glycan removal by PNGase F digestion. Proteins that are glycosylated at multiple sites can assume a variety of glycoforms depending on site occupancy and glycan structure at each glycosite [##REF##26555091##27##]. Proteins from different tissues of origin and disease states have been shown to exhibit variable glycosylation site occupancy [##REF##17823199##28##, ##UREF##4##29##]. Hence, the sensitivity of mAb 31E1 to glycosylation is of special interest in probing the significance of N285 and/or N294 glycan-deficient NEP in health and disease.</p>\",\n \"<p id=\\\"Par50\\\">At this juncture, it is worth highlighting a seeming conundrum in the differences in peptide and protein reactivity of mAb 33G5 and 31E1. Both mAbs were raised against the same AgNEP-3 peptide antigen and share exactly the same critical binding residues on the epitope. Yet, mAb 33G5 was able to detect cell-line-derived recombinant sNEP and its cognate Trx-peptide by SPR but not under western blot conditions. This antibody also showed poor utility, whether as a capture or detection antibody, in two-site ELISA with low signal response to Trx-AgNEP(1–4) as analyte. In contrast, mAb 31E1 exhibited good binding only to Trx-AgNEP(1–4), but not recombinant sNEP, by SPR, western blotting, and ELISA. How could the inhibitory effect of glycosylation on mAb 31E1 protein reactivity be reconciled with the immunity of mAb 33G5 to do so by SPR? First, glycosylation does not necessarily always impose steric hindrance to antibody binding but can even enhance antibody affinity to its antigen. It has been reported that glycosylation at one specific location on the MUC-16 epitope increased antibody recognition while modifications at other positions within the epitope were inhibitory to antibody binding [##REF##25093517##26##]. Second, N-glycosylation has been observed to occur on the antibody variable light-chain domain. Such modifications can change the conformation of the antigen-binding region and increase or decrease subsequent antibody–antigen interactions [##REF##10024532##30##, ##REF##8925140##31##]. Thus, it is possible that mAb 33G5 and 31E1 may differ in terms of the glycosylation profile of their variable light chains which in turn alters their peptide/protein-reactive properties and glycosylation sensitivity.</p>\",\n \"<p id=\\\"Par51\\\">We next investigated whether the mAbs 17E11 and 31E1 (the antibody pair used to develop the NEP ELISA) were able to detect circulating NEP in plasma by western blot analysis. Interestingly, both mAbs directed against different regions on NEP share four common immunodetected bands with the validated PE pAb, adding to confidence in antibody specificity for the target. Since NEP is highly glycosylated and can conceptually exist as monomers or dimers of the full-length protein or its ectodomain, it is difficult to interpret what the high-molecular-weight bands (> 100 kDa) represent. Since recombinant sNEP and LNCaP-derived endogenous NEP was found to migrate at the 109 and 130 kDa position, respectively, it is reasonable to postulate that the 101, 143, and 177 kDa bands detected in plasma might be glycosylated forms of monomeric NEP ectodomain, monomeric full-length NEP, and dimeric NEP ectodomain, respectively. PE pAb, mAb 31E1, and 17E11 consistently detected one major broad band at the 57–60 kDa position in all samples tested. This band exhibited slight differences in mobility and band thickness between samples, possibly reflecting a plethora of subtle nuances in post-translational modifications and/or antibody reactivity. Clear differences in antibody reactivity are exemplified in the case of mAb 17E11 and 31E1 binding to Trx-AgNEP(1–4) whereby more intense and broader immunodetected bands were obtained with mAb 31E1 under the same image exposure setting, especially for the higher molecular weight dimeric form of the recombinant protein. Peptide competition with the matched peptide for mAb 31E1 and 17E11 resulted in significant blocking of the 57–60 kDa band, though a weak residual band at the 57 kDa position could still be observed even at 25-fold excess of blocking peptide used. The poor ability of the free matched peptide to block the reaction between mAb 31E1 and the bound Trx-AgNEP(1–4), whether fixed within capillary tubes in western experiments or bound by the capture antibody in ELISA, suggests that the immobilized epitope sequence takes on a conformation that displayed much stronger affinity for mAb 31E1 [##REF##16737525##32##]. Identifying the biological processes that generate moieties in the 57–60 kDa region may reveal important information on the regulatory cascades modulating NEP activity and implications in health and disease. Thus, efforts to elucidate the identity of this fragment constitute a worthwhile pursuit in further work.</p>\",\n \"<p id=\\\"Par52\\\">Finally, mAb 17E11 and 31E1 shared a band immunodetected at the ~ 33 kDa position that is apparently not recognized by PE pAb. The validated sandwich ELISA developed from this mAb pair will likely be predominantly measuring the 33 and 57–60 kDa NEP fragments in circulation. Using the SitePrediction tool [##REF##19546006##33##], we searched for candidate protease cut sites predicted from cleavage site entries in the MEROPS database [##REF##19892822##34##, ##REF##24157837##35##] with species specificity restricted to Homo sapiens. The analysis suggests that NEP can potentially be cleaved by numerous endopeptidases such as matrix metalloproteinases, cathepsins, and caspases at multiple sites. The SitePrediction output predicted matrix metalloproteinase 2 (MMP 2) to cleave at S<sub>65</sub>AAR↓LI<sub>70</sub>, A<sub>187</sub>IAQ↓LN<sub>192</sub>, S<sub>251</sub>VAR↓LI<sub>256</sub> (all three sites with > 99% specificity) and F<sub>555</sub>PAG↓IL<sub>560</sub> (> 99.9% specificity) (Fig. ##FIG##7##8##). Cleavage at S<sub>65</sub>AAR↓LI<sub>70</sub> and F<sub>555</sub>PAG↓IL<sub>560</sub> would result in a predicted 56.2 kDa (calculated from the amino acid sequence alone with no N-linked glycosylation at N145, N285, N294 or N325) fragment that could account for the 57 kDa immunodetected band. On the other hand, cleavage at A<sub>187</sub>IAQ↓LN<sub>192</sub> or S<sub>251</sub>VAR↓LI<sub>256</sub> and F<sub>555</sub>PAG↓IL<sub>560</sub> is expected to produce fragments of calculated molecular weights of 42.5 and 35.2 kDa, respectively, with the balance mass to make up the final mass of 57–60 kDa accounted for by the presence glycan chains at any of the abovementioned N-linked glycosylation sites. Cathepsin K (CatK), a cysteine protease with important functional roles in bone resorption and far-reaching action on other organs including the cardiovascular system [##REF##32582709##36##], was also predicted to cleave NEP at ten different sites with a specificity ≥ 99%. Two of these cleavage sites at L<sub>262</sub>PID↓EN<sub>267</sub> and V<sub>554</sub>FPA↓GI<sub>559</sub> would generate a ~ 33.7 kDa (assuming no N-linked glycosylation at N285 or N294) fragment, representing the minimal sequence that contains both the epitopes of mAbs 17E11 and 31E1 (Fig. ##FIG##7##8##). This also harmonizes with the direct evidence from PNGase F experiments that mAb 31E1 is glycosylation sensitive and would bind only to glycan-deficient NEP fragments. Hence, the ~ 33 and 57–60 kDa NEP bands recognized by both mAbs could, respectively, be generated by CatK or MMP2 cleavage alone or in combination with each other. It can be further deduced that NEP concentration measured by our two-site ELISA will not correlate with NEP activity since the predominantly detected fragments do not harbor residues that make up the active site.</p>\",\n \"<p id=\\\"Par53\\\">A schematic representation of NEP and its fragments in circulation is depicted in Fig. ##FIG##8##9##. The complexity of circulating forms of NEP may help explain why previous NEP studies gave conflicting results from immunoassay data. For example, it was reported that circulating NEP concentrations were elevated in HF patients compared with non-HF controls [##REF##35331754##6##] and was positively associated with adverse outcomes [##REF##26251092##37##, ##REF##25677426##38##]. Yet, in other studies, soluble NEP levels were found to be significantly lower in HF patients compared with non-HF controls [##REF##31392960##39##] and not a prognosticator of adverse outcomes [##REF##26725876##40##, ##REF##32427034##41##]. One of the contributing factors for these contradictory results could be the use of different ELISA kits in the various studies. Hence, depending on the antibody pair used in the immunoassay, different results may be obtained. Clearly, our data showed that different NEP antibodies recognized different subsets of circulating NEP forms. Disease severity may be correlated with a reduction or increase in N-linked glycosylation site occupancy of serum proteins [##UREF##4##29##]. In addition, N-linked glycosylation is known to modulate protein function by altering the interaction of a cell-surface protein with its ligand [##REF##33664400##42##]. The new mAb pair generated in this work will add to the toolbox of well-defined binders to probe dynamic changes in NEP concentration and glycosylation site occupancy in health and disease. It remains to be determined which specific fragment(s) of NEP is useful as a biomarker and this would certainly be an important area requiring further research.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par54\">We show that the thioredoxin scaffold constitutes an effective strategy for surface presentation of <italic>in silic</italic>o predicted peptides as immunogens to generate high-affinity mAbs with well-defined epitope binding footprint. The validated antibody pair comprising mAb 17E11 and mAb 31E1 can be applied in Western blot and ELISA applications, adding to the arsenal of much needed reliable binders to unravel NEP biology, regulation, and function in the context of health and disease. Successful generation of glycosylation-sensitive mAb 31E1 demonstrates the power of epitope-directed antibody production by allowing peptides harboring post-translational modifications of interest to be preselected to meet experimental objectives. New information on the complexity of circulating forms of NEP and the consequent implications for correctly interpreting immunoassay data have been highlighted. We are now poised for applying our NEP ELISA on clinical HF samples to elucidate dynamic changes in NEP concentration in disease and how it might associate with adverse outcomes.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par54\\\">We show that the thioredoxin scaffold constitutes an effective strategy for surface presentation of <italic>in silic</italic>o predicted peptides as immunogens to generate high-affinity mAbs with well-defined epitope binding footprint. The validated antibody pair comprising mAb 17E11 and mAb 31E1 can be applied in Western blot and ELISA applications, adding to the arsenal of much needed reliable binders to unravel NEP biology, regulation, and function in the context of health and disease. Successful generation of glycosylation-sensitive mAb 31E1 demonstrates the power of epitope-directed antibody production by allowing peptides harboring post-translational modifications of interest to be preselected to meet experimental objectives. New information on the complexity of circulating forms of NEP and the consequent implications for correctly interpreting immunoassay data have been highlighted. We are now poised for applying our NEP ELISA on clinical HF samples to elucidate dynamic changes in NEP concentration in disease and how it might associate with adverse outcomes.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p id=\"Par1\">Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1–8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57–60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00018-023-05083-1.</p>","<title>Keywords</title>"],"string":"[\n \"<p id=\\\"Par1\\\">Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1–8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57–60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00018-023-05083-1.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>Funding support for this work was provided under the Centre Grant (NMRC/CG21APR1008) awarded to A. Mark Richards by the National Medical Research Council, Singapore.</p>","<title>Author contribution</title>","<p>OWL and SSML designed and developed the antibody production methodologies, performed most of the experiments, analyzed the data, and wrote the paper; LS, JYX, JPCC performed hybridoma screening and analysis of results; QFL, XEY, TKL, and QSL performed the tryptic digest and mass spectrometry analysis of immunoprecipitated eluates for antibody verification; AMR contributed key intellectual inputs to the manuscript and is the principal investigator of the grant that supported the projected. All authors have contributed, discussed, and approved the final version of the manuscript.</p>","<title>Funding</title>","<p>Funding support for this work was provided under the Centre Grant (NMRC/CG21APR1008) awarded to A. Mark Richards by the National Medical Research Council, Singapore.</p>","<title>Data availability</title>","<p>All data are available from the corresponding author upon reasonable request.</p>","<title>Declarations</title>","<title>Conflict of interests</title>","<p id=\"Par55\">All authors declare no competing interests.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>Funding support for this work was provided under the Centre Grant (NMRC/CG21APR1008) awarded to A. Mark Richards by the National Medical Research Council, Singapore.</p>\",\n \"<title>Author contribution</title>\",\n \"<p>OWL and SSML designed and developed the antibody production methodologies, performed most of the experiments, analyzed the data, and wrote the paper; LS, JYX, JPCC performed hybridoma screening and analysis of results; QFL, XEY, TKL, and QSL performed the tryptic digest and mass spectrometry analysis of immunoprecipitated eluates for antibody verification; AMR contributed key intellectual inputs to the manuscript and is the principal investigator of the grant that supported the projected. All authors have contributed, discussed, and approved the final version of the manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Funding support for this work was provided under the Centre Grant (NMRC/CG21APR1008) awarded to A. Mark Richards by the National Medical Research Council, Singapore.</p>\",\n \"<title>Data availability</title>\",\n \"<p>All data are available from the corresponding author upon reasonable request.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interests</title>\",\n \"<p id=\\\"Par55\\\">All authors declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Properties of selected antigen peptides. <bold>a</bold> 3D-ribbon structure of the NEP extracellular domain using PDB entry 6suk.1.A as template. The locations of the antigen sequences with their start residues in bold, AgNEP-1 (V<sub>169</sub>ATENWEQKYGASW)<sub>,</sub> AgNEP-2 (Y<sub>665</sub>IKKNGEEKKL), AGNEP-3 (E<sub>282</sub>IANATAKPEDRNDP), and AgNEP-4 (E<sub>528</sub>KVDKDEWIS), are shown in red. <bold>b</bold> Predicted secondary structural elements (pink: helix; orange: strand; green: others) and solvent accessibility (buried: blue; yellow: exposed) of each antigen peptide</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Screening of antibody pairs by ELISA. Antibody-pair dose–response is plotted against Trx-AgNEP(1–4) concentration. The legend lists each capture/detection antibody matched pair used. A linear curve fit over analyte concentration ranging from 125 to 8000 pg/ml is applied to the antibody pairs</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Assessment of assay specificity and selectivity by cross-reactivity and parallelism testing. <bold>a</bold> Cross-reactivity assessment of M13 endopeptidases on NEP ELISA. <bold>b</bold> Serial dilution response curves of calibrator Trx-AgNEP(1–4), and human plasma samples plotted in the log10 scale for both axes. Data points representing 2-, 3-, 4-, 6-, 8-, 12-, and 24-fold dilutions were fitted with a power-law regression where the exponent of function x represents the slope of the curve. The NEP concentration of each sample at twofold dilution is indicated in brackets</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Antibody validation by western blot analysis. Reactivity of mAbs (indicated above each panel) to <bold>a</bold> recombinant human sNEP (Aviscera Bioscience) and Trx-AgNEP(1–4) was determined using western blot performed with automated Jess system (ProteinSimple). The ability of AgNEP-3 <bold>(b)</bold> and AgNEP-4 <bold>(c)</bold> mAbs to detect endogenous human NEP was determined by probing them against NEP-positive LNCaP cell lysate (L) and NEP-negative PC-3 cell lysate (P). PE pAb was used as the positive control. Ladder: 12–230 kDa biotinylated molecular weight ladder (ProteinSimple). The primary target band of each protein sample type is indicated with a black arrow. Image exposure setting: High Dynamic Range 4.0 except for the Trx-AgNEP(1–4) panel where Exposure 5: 16 secs was used</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Circulating forms of plasma NEP as revealed by western blot analysis. <bold>a</bold> Individual (#1, #2) and pooled (<italic>n</italic> > 100) HFrEF, HFpEF, and CTRL (non-HF) plasma samples were probed separately with mAb 17E11 and mAb 31E1 and compared against the profile obtained with PE pAb. Ladder: 12–230 kDa biotinylated molecular weight ladder (ProteinSimple). Image exposure setting: High Dynamic Range 4.0. <bold>b</bold> Effect of peptide competition on immunodetected bands where interaction of the detection mAb with NEP targets is blocked in the absence (None) or presence of matched or unmatched peptides at tenfold excess concentration. The 57 kDa band position is indicated by black arrows. Image exposure setting: Exposure 8: 128 s. All images are representative of three independent experiments</p></caption></fig>","<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Effect of peptide competition on ELISA. The interaction of detection mAb (indicated at the top of each panel) with different concentrations of Trx-AgNEP(1–4) is blocked in the absence (None) or presence of matched or unmatched peptides</p></caption></fig>","<fig id=\"Fig7\"><label>Fig. 7</label><caption><p>HEK293 cell-line-derived recombinant sNEP, untreated (‒) or treated with PNGase F ( +). <bold>a</bold> SDS-PAGE of the untreated lane shows a smear representing glycosylated recombinant sNEP. Treatment with PNGase F removes N-glycans and results in a sharp band of smaller molecular weight representing deglycosylated recombinant sNEP (treated lane). <bold>b</bold> Western blot of untreated and PNGase F-treated recombinant sNEP probed with mAb 31E1 shows antibody binding only after N-glycan removal (indicated by arrow head). M, All-Blue Precision Plus Protein Standards (BioRad). The amount of recombinant sNEP loaded per well is indicated above each lane. Images are representative of three independent experiments</p></caption></fig>","<fig id=\"Fig8\"><label>Fig. 8</label><caption><p>Overlay of relevant protease labile and glycosylation sites with respect to mAb epitopes on the amino acid sequence of human neprilysin. The binding sites of mAb 31E1 and mAb 17E11 are indicated by dash boxes with critical residues for antibody binding bolded in red. Experimentally reported glycosylation sites are indicated by green arrows. Predicted cleavage sites of CatK and MMP2 are indicated by red and blue arrows, respectively. Cleavage by CatK alone or in combination with MMP2 at the F<sub>555</sub>PAG↓IL<sub>560</sub> site would generate a 33.7 kDa NEP fragment (underlined in black). Amino acid residues involved in zinc binding within the NEP catalytic pocket are highlighted in blue</p></caption></fig>","<fig id=\"Fig9\"><label>Fig. 9</label><caption><p>Schematic depiction of NEP and its fragments in circulation. Full-length NEP may circulate as a membrane-associated protein present on the surface of exosomes and neutrophils. Ectodomain shedding releases a non-membrane soluble form of NEP (sNEP) that is biologically active. Further proteolytic cleavage results in smaller fragments that may lack the catalytic site. mAb 17E11 will bind to NEP and its fragments harboring the AG4 epitope. mAb 31E1 is glycosylation sensitive and will only bind to NEP and fragments that contain N284/295 glycan-deficient AG3 epitopes. Two-site ELISA will only measure NEP moieties that contain the N284/295 glycan-deficient AG3 and AG4 epitopes. Figure schematics were generated from template elements available on Servier Medical Art licensed under a Creative Commons Attribution 3.0 Unported License (<ext-link ext-link-type=\"uri\" xlink:href=\"https://smart.servier.com\">https://smart.servier.com</ext-link>)</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Properties of selected antigen peptides. <bold>a</bold> 3D-ribbon structure of the NEP extracellular domain using PDB entry 6suk.1.A as template. The locations of the antigen sequences with their start residues in bold, AgNEP-1 (V<sub>169</sub>ATENWEQKYGASW)<sub>,</sub> AgNEP-2 (Y<sub>665</sub>IKKNGEEKKL), AGNEP-3 (E<sub>282</sub>IANATAKPEDRNDP), and AgNEP-4 (E<sub>528</sub>KVDKDEWIS), are shown in red. <bold>b</bold> Predicted secondary structural elements (pink: helix; orange: strand; green: others) and solvent accessibility (buried: blue; yellow: exposed) of each antigen peptide</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Screening of antibody pairs by ELISA. Antibody-pair dose–response is plotted against Trx-AgNEP(1–4) concentration. The legend lists each capture/detection antibody matched pair used. A linear curve fit over analyte concentration ranging from 125 to 8000 pg/ml is applied to the antibody pairs</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Assessment of assay specificity and selectivity by cross-reactivity and parallelism testing. <bold>a</bold> Cross-reactivity assessment of M13 endopeptidases on NEP ELISA. <bold>b</bold> Serial dilution response curves of calibrator Trx-AgNEP(1–4), and human plasma samples plotted in the log10 scale for both axes. Data points representing 2-, 3-, 4-, 6-, 8-, 12-, and 24-fold dilutions were fitted with a power-law regression where the exponent of function x represents the slope of the curve. The NEP concentration of each sample at twofold dilution is indicated in brackets</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Antibody validation by western blot analysis. Reactivity of mAbs (indicated above each panel) to <bold>a</bold> recombinant human sNEP (Aviscera Bioscience) and Trx-AgNEP(1–4) was determined using western blot performed with automated Jess system (ProteinSimple). The ability of AgNEP-3 <bold>(b)</bold> and AgNEP-4 <bold>(c)</bold> mAbs to detect endogenous human NEP was determined by probing them against NEP-positive LNCaP cell lysate (L) and NEP-negative PC-3 cell lysate (P). PE pAb was used as the positive control. Ladder: 12–230 kDa biotinylated molecular weight ladder (ProteinSimple). The primary target band of each protein sample type is indicated with a black arrow. Image exposure setting: High Dynamic Range 4.0 except for the Trx-AgNEP(1–4) panel where Exposure 5: 16 secs was used</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><p>Circulating forms of plasma NEP as revealed by western blot analysis. <bold>a</bold> Individual (#1, #2) and pooled (<italic>n</italic> > 100) HFrEF, HFpEF, and CTRL (non-HF) plasma samples were probed separately with mAb 17E11 and mAb 31E1 and compared against the profile obtained with PE pAb. Ladder: 12–230 kDa biotinylated molecular weight ladder (ProteinSimple). Image exposure setting: High Dynamic Range 4.0. <bold>b</bold> Effect of peptide competition on immunodetected bands where interaction of the detection mAb with NEP targets is blocked in the absence (None) or presence of matched or unmatched peptides at tenfold excess concentration. The 57 kDa band position is indicated by black arrows. Image exposure setting: Exposure 8: 128 s. All images are representative of three independent experiments</p></caption></fig>\",\n \"<fig id=\\\"Fig6\\\"><label>Fig. 6</label><caption><p>Effect of peptide competition on ELISA. The interaction of detection mAb (indicated at the top of each panel) with different concentrations of Trx-AgNEP(1–4) is blocked in the absence (None) or presence of matched or unmatched peptides</p></caption></fig>\",\n \"<fig id=\\\"Fig7\\\"><label>Fig. 7</label><caption><p>HEK293 cell-line-derived recombinant sNEP, untreated (‒) or treated with PNGase F ( +). <bold>a</bold> SDS-PAGE of the untreated lane shows a smear representing glycosylated recombinant sNEP. Treatment with PNGase F removes N-glycans and results in a sharp band of smaller molecular weight representing deglycosylated recombinant sNEP (treated lane). <bold>b</bold> Western blot of untreated and PNGase F-treated recombinant sNEP probed with mAb 31E1 shows antibody binding only after N-glycan removal (indicated by arrow head). M, All-Blue Precision Plus Protein Standards (BioRad). The amount of recombinant sNEP loaded per well is indicated above each lane. Images are representative of three independent experiments</p></caption></fig>\",\n \"<fig id=\\\"Fig8\\\"><label>Fig. 8</label><caption><p>Overlay of relevant protease labile and glycosylation sites with respect to mAb epitopes on the amino acid sequence of human neprilysin. The binding sites of mAb 31E1 and mAb 17E11 are indicated by dash boxes with critical residues for antibody binding bolded in red. Experimentally reported glycosylation sites are indicated by green arrows. Predicted cleavage sites of CatK and MMP2 are indicated by red and blue arrows, respectively. Cleavage by CatK alone or in combination with MMP2 at the F<sub>555</sub>PAG↓IL<sub>560</sub> site would generate a 33.7 kDa NEP fragment (underlined in black). Amino acid residues involved in zinc binding within the NEP catalytic pocket are highlighted in blue</p></caption></fig>\",\n \"<fig id=\\\"Fig9\\\"><label>Fig. 9</label><caption><p>Schematic depiction of NEP and its fragments in circulation. Full-length NEP may circulate as a membrane-associated protein present on the surface of exosomes and neutrophils. Ectodomain shedding releases a non-membrane soluble form of NEP (sNEP) that is biologically active. Further proteolytic cleavage results in smaller fragments that may lack the catalytic site. mAb 17E11 will bind to NEP and its fragments harboring the AG4 epitope. mAb 31E1 is glycosylation sensitive and will only bind to NEP and fragments that contain N284/295 glycan-deficient AG3 epitopes. Two-site ELISA will only measure NEP moieties that contain the N284/295 glycan-deficient AG3 and AG4 epitopes. Figure schematics were generated from template elements available on Servier Medical Art licensed under a Creative Commons Attribution 3.0 Unported License (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://smart.servier.com\\\">https://smart.servier.com</ext-link>)</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>MRM<sup>HR</sup> product ion scan parameters of neprilysin peptides</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Neprilysin peptide sequences</th><th align=\"left\">Precursor charge</th><th align=\"left\">Precursor theoretical <italic>m/z</italic></th><th align=\"left\">CE</th></tr></thead><tbody><tr><td align=\"left\">VDKDEWISGAAVVNAFYSSGR</td><td align=\"left\">3</td><td char=\".\" align=\"char\">757.7061768</td><td char=\".\" align=\"char\">34.4</td></tr><tr><td align=\"left\">DEWISGAAVVNAFYSSGR</td><td align=\"left\">2</td><td char=\".\" align=\"char\">964.9605</td><td char=\".\" align=\"char\">46.3</td></tr><tr><td align=\"left\">DEWISGAAVVNAFYSSGR</td><td align=\"left\">3</td><td char=\".\" align=\"char\">643.6428</td><td char=\".\" align=\"char\">28.9</td></tr><tr><td align=\"left\">DEWISGAAVVNAFYSSGR</td><td align=\"left\">4</td><td char=\".\" align=\"char\">482.9839</td><td char=\".\" align=\"char\">22.1</td></tr><tr><td align=\"left\">LNNEYLELNYK</td><td align=\"left\">2</td><td char=\".\" align=\"char\">706.8564</td><td char=\".\" align=\"char\">33.6</td></tr><tr><td align=\"left\">LNNEYLELNYK</td><td align=\"left\">3</td><td char=\".\" align=\"char\">471.5734</td><td char=\".\" align=\"char\">20.6</td></tr><tr><td align=\"left\">EDEYFENIIQNLK</td><td align=\"left\">2</td><td char=\".\" align=\"char\">827.9016</td><td char=\".\" align=\"char\">39.6</td></tr><tr><td align=\"left\">EDEYFENIIQNLK</td><td align=\"left\">3</td><td char=\".\" align=\"char\">552.2701</td><td char=\".\" align=\"char\">24.5</td></tr><tr><td align=\"left\">NQIVFPAGILQPPFFSAQQSNSLNYGGIGMVIGHEITHGFDDNGR</td><td align=\"left\">4</td><td char=\".\" align=\"char\">1211.5976</td><td char=\".\" align=\"char\">58.6</td></tr><tr><td align=\"left\">NQIVFPAGILQPPFFSAQQSNSLNYGGIGMVIGHEITHGFDDNGR</td><td align=\"left\">5</td><td char=\".\" align=\"char\">969.4795</td><td char=\".\" align=\"char\">46.5</td></tr><tr><td align=\"left\">NSVNHVIHIDQPR</td><td align=\"left\">4</td><td char=\".\" align=\"char\">382.9549255</td><td char=\".\" align=\"char\">17.1</td></tr><tr><td align=\"left\">IGYPDDIVSNDNK</td><td align=\"left\">2</td><td char=\".\" align=\"char\">725.3464355</td><td char=\".\" align=\"char\">34.5</td></tr><tr><td align=\"left\">DLQNLMSWR</td><td align=\"left\">2</td><td char=\".\" align=\"char\">581.7872925</td><td char=\".\" align=\"char\">27.5</td></tr><tr><td align=\"left\">FIMDLVSSLSR</td><td align=\"left\">2</td><td char=\".\" align=\"char\">634.3393555</td><td char=\".\" align=\"char\">30.1</td></tr><tr><td align=\"left\">LLPGLDLNHK</td><td align=\"left\">3</td><td char=\".\" align=\"char\">373.8888855</td><td char=\".\" align=\"char\">15.9</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Characterization of mAbs by isotyping, SPR, and alanine scan analysis</p></caption></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Mean recovery of NEP (n = 4) spiked with recombinant Trx-AgNEP(1–4) and endogenous NEP at two different concentrations into three human plasma samples</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Sample*</th><th align=\"left\" colspan=\"2\">Trx-AgNEP(1–4)</th><th align=\"left\" colspan=\"2\">Endogenous NEP</th></tr><tr><th align=\"left\">500 pg/ml</th><th align=\"left\">2100 pg/ml</th><th align=\"left\">366 pg/ml</th><th align=\"left\">1440 pg/ml</th></tr></thead><tbody><tr><td align=\"left\">A (1779 pg/ml)</td><td char=\".\" align=\"char\">57.8%</td><td char=\".\" align=\"char\">52.4%</td><td char=\".\" align=\"char\">113%</td><td char=\".\" align=\"char\">110%</td></tr><tr><td align=\"left\">B (640 pg/ml)</td><td char=\".\" align=\"char\">50.8%</td><td char=\".\" align=\"char\">61.3%</td><td char=\".\" align=\"char\">111%</td><td char=\".\" align=\"char\">118%</td></tr><tr><td align=\"left\">C (177 pg/ml)</td><td char=\".\" align=\"char\">52.6%</td><td char=\".\" align=\"char\">64.4%</td><td char=\".\" align=\"char\">108%</td><td char=\".\" align=\"char\">112%</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Summary of NEP assay characteristics and performance parameters</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Assay parameter</th><th align=\"left\">Assay characteristics/performance</th></tr></thead><tbody><tr><td align=\"left\">Format</td><td align=\"left\">Sandwich ELISA/colorimetric</td></tr><tr><td align=\"left\">Target</td><td align=\"left\">Human sNEP</td></tr><tr><td align=\"left\">Assay time</td><td align=\"left\">6 h</td></tr><tr><td align=\"left\">Calibrator</td><td align=\"left\">Trx-AgNEP(1–4)</td></tr><tr><td align=\"left\">Assay range</td><td align=\"left\">13.1 – 8000 pg/ml</td></tr><tr><td align=\"left\">Sample type</td><td align=\"left\">Plasma (EDTA)</td></tr><tr><td align=\"left\">Sample dilution</td><td align=\"left\">3 – 12 fold dilution</td></tr><tr><td align=\"left\">Total sample volume</td><td align=\"left\">100 µL per well</td></tr><tr><td align=\"left\"><p>LOD (18 replicates)</p><p>LLOQ (2 replicates, 18 independent assays)</p></td><td align=\"left\"><p>2.15 pg/ml</p><p>13.1 pg/ml</p></td></tr><tr><td align=\"left\">Calibration curve <italic>R</italic><sup><italic>2</italic></sup></td><td align=\"left\"> > 0.99</td></tr><tr><td align=\"left\" colspan=\"2\">Intra-assay variation</td></tr><tr><td align=\"left\"> Sample 1 (<italic>n</italic> = 8)</td><td align=\"left\">Mean = 1678 pg/ml; %CV = 1.7</td></tr><tr><td align=\"left\"> Sample 2 (<italic>n</italic> = 8)</td><td align=\"left\">Mean = 592 pg/ml; %CV = 2.3</td></tr><tr><td align=\"left\"> Sample 3 (<italic>n</italic> = 8)</td><td align=\"left\">Mean = 151 pg/ml; %CV = 3.2</td></tr><tr><td align=\"left\" colspan=\"2\">Inter-assay variation* (18 independent assays)</td></tr><tr><td align=\"left\"> Sample 1 (<italic>n</italic> = 2)</td><td align=\"left\">Mean = 2076 pg/ml; %CV = 11.3</td></tr><tr><td align=\"left\"> Sample 2 (<italic>n</italic> = 2)</td><td align=\"left\">Mean = 632 pg/ml; %CV = 10.8</td></tr><tr><td align=\"left\"> Sample 3 (<italic>n</italic> = 2)</td><td align=\"left\">Mean = 142 pg/ml; %CV = 15.4</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab5\"><label>Table 5</label><caption><p>Antibody reactivity to recombinant and endogenous NEP by western analysis</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Target</th><th align=\"left\">Antibody tested</th><th align=\"left\">Recombinant human sNEP</th><th align=\"left\">Recombinant Trx-AgNEP(1–4)</th><th align=\"left\">LNCaP<break/>NEP-positive cell lysate</th><th align=\"left\">PC-3<break/>NEP-negative cell lysate</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"5\">AgNEP-3</td><td align=\"left\">mAb 25A5</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">mAb 4G4</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">mAb 33G5</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">mAb 11C5</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">mAb 31E1</td><td align=\"left\">–</td><td align=\"left\">+</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\" rowspan=\"5\">AgNEP-4</td><td align=\"left\">mAb 13H8</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">mAb 12A10</td><td align=\"left\"> + </td><td align=\"left\">+ </td><td align=\"left\"> + </td><td align=\"left\">–</td></tr><tr><td align=\"left\">mAb 20H5</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">mAb 17E11</td><td align=\"left\"> + </td><td align=\"left\">+ </td><td align=\"left\"> + </td><td align=\"left\">–</td></tr><tr><td align=\"left\">mAb 3E12</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Human NEP</td><td align=\"left\">PE pAb</td><td align=\"left\"> + </td><td align=\"left\">+ </td><td align=\"left\"> + </td><td align=\"left\">–</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>MRM<sup>HR</sup> product ion scan parameters of neprilysin peptides</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Neprilysin peptide sequences</th><th align=\\\"left\\\">Precursor charge</th><th align=\\\"left\\\">Precursor theoretical <italic>m/z</italic></th><th align=\\\"left\\\">CE</th></tr></thead><tbody><tr><td align=\\\"left\\\">VDKDEWISGAAVVNAFYSSGR</td><td align=\\\"left\\\">3</td><td char=\\\".\\\" align=\\\"char\\\">757.7061768</td><td char=\\\".\\\" align=\\\"char\\\">34.4</td></tr><tr><td align=\\\"left\\\">DEWISGAAVVNAFYSSGR</td><td align=\\\"left\\\">2</td><td char=\\\".\\\" align=\\\"char\\\">964.9605</td><td char=\\\".\\\" align=\\\"char\\\">46.3</td></tr><tr><td align=\\\"left\\\">DEWISGAAVVNAFYSSGR</td><td align=\\\"left\\\">3</td><td char=\\\".\\\" align=\\\"char\\\">643.6428</td><td char=\\\".\\\" align=\\\"char\\\">28.9</td></tr><tr><td align=\\\"left\\\">DEWISGAAVVNAFYSSGR</td><td align=\\\"left\\\">4</td><td char=\\\".\\\" align=\\\"char\\\">482.9839</td><td char=\\\".\\\" align=\\\"char\\\">22.1</td></tr><tr><td align=\\\"left\\\">LNNEYLELNYK</td><td align=\\\"left\\\">2</td><td char=\\\".\\\" align=\\\"char\\\">706.8564</td><td char=\\\".\\\" align=\\\"char\\\">33.6</td></tr><tr><td align=\\\"left\\\">LNNEYLELNYK</td><td align=\\\"left\\\">3</td><td char=\\\".\\\" align=\\\"char\\\">471.5734</td><td char=\\\".\\\" align=\\\"char\\\">20.6</td></tr><tr><td align=\\\"left\\\">EDEYFENIIQNLK</td><td align=\\\"left\\\">2</td><td char=\\\".\\\" align=\\\"char\\\">827.9016</td><td char=\\\".\\\" align=\\\"char\\\">39.6</td></tr><tr><td align=\\\"left\\\">EDEYFENIIQNLK</td><td align=\\\"left\\\">3</td><td char=\\\".\\\" align=\\\"char\\\">552.2701</td><td char=\\\".\\\" align=\\\"char\\\">24.5</td></tr><tr><td align=\\\"left\\\">NQIVFPAGILQPPFFSAQQSNSLNYGGIGMVIGHEITHGFDDNGR</td><td align=\\\"left\\\">4</td><td char=\\\".\\\" align=\\\"char\\\">1211.5976</td><td char=\\\".\\\" align=\\\"char\\\">58.6</td></tr><tr><td align=\\\"left\\\">NQIVFPAGILQPPFFSAQQSNSLNYGGIGMVIGHEITHGFDDNGR</td><td align=\\\"left\\\">5</td><td char=\\\".\\\" align=\\\"char\\\">969.4795</td><td char=\\\".\\\" align=\\\"char\\\">46.5</td></tr><tr><td align=\\\"left\\\">NSVNHVIHIDQPR</td><td align=\\\"left\\\">4</td><td char=\\\".\\\" align=\\\"char\\\">382.9549255</td><td char=\\\".\\\" align=\\\"char\\\">17.1</td></tr><tr><td align=\\\"left\\\">IGYPDDIVSNDNK</td><td align=\\\"left\\\">2</td><td char=\\\".\\\" align=\\\"char\\\">725.3464355</td><td char=\\\".\\\" align=\\\"char\\\">34.5</td></tr><tr><td align=\\\"left\\\">DLQNLMSWR</td><td align=\\\"left\\\">2</td><td char=\\\".\\\" align=\\\"char\\\">581.7872925</td><td char=\\\".\\\" align=\\\"char\\\">27.5</td></tr><tr><td align=\\\"left\\\">FIMDLVSSLSR</td><td align=\\\"left\\\">2</td><td char=\\\".\\\" align=\\\"char\\\">634.3393555</td><td char=\\\".\\\" align=\\\"char\\\">30.1</td></tr><tr><td align=\\\"left\\\">LLPGLDLNHK</td><td align=\\\"left\\\">3</td><td char=\\\".\\\" align=\\\"char\\\">373.8888855</td><td char=\\\".\\\" align=\\\"char\\\">15.9</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Characterization of mAbs by isotyping, SPR, and alanine scan analysis</p></caption></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Mean recovery of NEP (n = 4) spiked with recombinant Trx-AgNEP(1–4) and endogenous NEP at two different concentrations into three human plasma samples</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" rowspan=\\\"2\\\">Sample*</th><th align=\\\"left\\\" colspan=\\\"2\\\">Trx-AgNEP(1–4)</th><th align=\\\"left\\\" colspan=\\\"2\\\">Endogenous NEP</th></tr><tr><th align=\\\"left\\\">500 pg/ml</th><th align=\\\"left\\\">2100 pg/ml</th><th align=\\\"left\\\">366 pg/ml</th><th align=\\\"left\\\">1440 pg/ml</th></tr></thead><tbody><tr><td align=\\\"left\\\">A (1779 pg/ml)</td><td char=\\\".\\\" align=\\\"char\\\">57.8%</td><td char=\\\".\\\" align=\\\"char\\\">52.4%</td><td char=\\\".\\\" align=\\\"char\\\">113%</td><td char=\\\".\\\" align=\\\"char\\\">110%</td></tr><tr><td align=\\\"left\\\">B (640 pg/ml)</td><td char=\\\".\\\" align=\\\"char\\\">50.8%</td><td char=\\\".\\\" align=\\\"char\\\">61.3%</td><td char=\\\".\\\" align=\\\"char\\\">111%</td><td char=\\\".\\\" align=\\\"char\\\">118%</td></tr><tr><td align=\\\"left\\\">C (177 pg/ml)</td><td char=\\\".\\\" align=\\\"char\\\">52.6%</td><td char=\\\".\\\" align=\\\"char\\\">64.4%</td><td char=\\\".\\\" align=\\\"char\\\">108%</td><td char=\\\".\\\" align=\\\"char\\\">112%</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab4\\\"><label>Table 4</label><caption><p>Summary of NEP assay characteristics and performance parameters</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Assay parameter</th><th align=\\\"left\\\">Assay characteristics/performance</th></tr></thead><tbody><tr><td align=\\\"left\\\">Format</td><td align=\\\"left\\\">Sandwich ELISA/colorimetric</td></tr><tr><td align=\\\"left\\\">Target</td><td align=\\\"left\\\">Human sNEP</td></tr><tr><td align=\\\"left\\\">Assay time</td><td align=\\\"left\\\">6 h</td></tr><tr><td align=\\\"left\\\">Calibrator</td><td align=\\\"left\\\">Trx-AgNEP(1–4)</td></tr><tr><td align=\\\"left\\\">Assay range</td><td align=\\\"left\\\">13.1 – 8000 pg/ml</td></tr><tr><td align=\\\"left\\\">Sample type</td><td align=\\\"left\\\">Plasma (EDTA)</td></tr><tr><td align=\\\"left\\\">Sample dilution</td><td align=\\\"left\\\">3 – 12 fold dilution</td></tr><tr><td align=\\\"left\\\">Total sample volume</td><td align=\\\"left\\\">100 µL per well</td></tr><tr><td align=\\\"left\\\"><p>LOD (18 replicates)</p><p>LLOQ (2 replicates, 18 independent assays)</p></td><td align=\\\"left\\\"><p>2.15 pg/ml</p><p>13.1 pg/ml</p></td></tr><tr><td align=\\\"left\\\">Calibration curve <italic>R</italic><sup><italic>2</italic></sup></td><td align=\\\"left\\\"> > 0.99</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Intra-assay variation</td></tr><tr><td align=\\\"left\\\"> Sample 1 (<italic>n</italic> = 8)</td><td align=\\\"left\\\">Mean = 1678 pg/ml; %CV = 1.7</td></tr><tr><td align=\\\"left\\\"> Sample 2 (<italic>n</italic> = 8)</td><td align=\\\"left\\\">Mean = 592 pg/ml; %CV = 2.3</td></tr><tr><td align=\\\"left\\\"> Sample 3 (<italic>n</italic> = 8)</td><td align=\\\"left\\\">Mean = 151 pg/ml; %CV = 3.2</td></tr><tr><td align=\\\"left\\\" colspan=\\\"2\\\">Inter-assay variation* (18 independent assays)</td></tr><tr><td align=\\\"left\\\"> Sample 1 (<italic>n</italic> = 2)</td><td align=\\\"left\\\">Mean = 2076 pg/ml; %CV = 11.3</td></tr><tr><td align=\\\"left\\\"> Sample 2 (<italic>n</italic> = 2)</td><td align=\\\"left\\\">Mean = 632 pg/ml; %CV = 10.8</td></tr><tr><td align=\\\"left\\\"> Sample 3 (<italic>n</italic> = 2)</td><td align=\\\"left\\\">Mean = 142 pg/ml; %CV = 15.4</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab5\\\"><label>Table 5</label><caption><p>Antibody reactivity to recombinant and endogenous NEP by western analysis</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Target</th><th align=\\\"left\\\">Antibody tested</th><th align=\\\"left\\\">Recombinant human sNEP</th><th align=\\\"left\\\">Recombinant Trx-AgNEP(1–4)</th><th align=\\\"left\\\">LNCaP<break/>NEP-positive cell lysate</th><th align=\\\"left\\\">PC-3<break/>NEP-negative cell lysate</th></tr></thead><tbody><tr><td align=\\\"left\\\" rowspan=\\\"5\\\">AgNEP-3</td><td align=\\\"left\\\">mAb 25A5</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">mAb 4G4</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">mAb 33G5</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">mAb 11C5</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">mAb 31E1</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">+</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\" rowspan=\\\"5\\\">AgNEP-4</td><td align=\\\"left\\\">mAb 13H8</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">mAb 12A10</td><td align=\\\"left\\\"> + </td><td align=\\\"left\\\">+ </td><td align=\\\"left\\\"> + </td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">mAb 20H5</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">mAb 17E11</td><td align=\\\"left\\\"> + </td><td align=\\\"left\\\">+ </td><td align=\\\"left\\\"> + </td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">mAb 3E12</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Human NEP</td><td align=\\\"left\\\">PE pAb</td><td align=\\\"left\\\"> + </td><td align=\\\"left\\\">+ </td><td align=\\\"left\\\"> + </td><td align=\\\"left\\\">–</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p>Kinetic rate constants were obtained from the interaction between recombinant sNEP and the respective mAb. Critical residues (highlighted in enlarged bold typeset in red) are defined as those that reduce the ELISA colorimetric response to < 50% of the wild-type sequence (Fig. S3)</p><p><italic>k</italic><sub><italic>a</italic></sub> association constant, <italic>k</italic><sub><italic>d</italic></sub> dissociation constant, <italic>KD</italic> equilibrium constant, <italic>NB</italic> no binding</p></table-wrap-foot>","<table-wrap-foot><p>*Concentration of endogenous NEP in the unspiked sample is shown in brackets</p></table-wrap-foot>","<table-wrap-foot><p>*Samples were measured in duplicates (<italic>n</italic> = 2) in 18 separate assays performed on different days. A mean concentration was obtained from each duplicate reading and the inter-assay %CV for each sample was derived from the 18 mean concentrations</p></table-wrap-foot>","<table-wrap-foot><p>Positive reactivity against the respective proteins is indicated by a “+” sign. Negative reactivity is indicated by a “–” sign</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>Kinetic rate constants were obtained from the interaction between recombinant sNEP and the respective mAb. Critical residues (highlighted in enlarged bold typeset in red) are defined as those that reduce the ELISA colorimetric response to < 50% of the wild-type sequence (Fig. S3)</p><p><italic>k</italic><sub><italic>a</italic></sub> association constant, <italic>k</italic><sub><italic>d</italic></sub> dissociation constant, <italic>KD</italic> equilibrium constant, <italic>NB</italic> no binding</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>*Concentration of endogenous NEP in the unspiked sample is shown in brackets</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>*Samples were measured in duplicates (<italic>n</italic> = 2) in 18 separate assays performed on different days. A mean concentration was obtained from each duplicate reading and the inter-assay %CV for each sample was derived from the 18 mean concentrations</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>Positive reactivity against the respective proteins is indicated by a “+” sign. Negative reactivity is indicated by a “–” sign</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"18_2023_5083_Fig1_HTML\" id=\"MO1\"/>","<graphic position=\"anchor\" xlink:href=\"18_2023_5083_Tab2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"18_2023_5083_Fig2_HTML\" id=\"MO3\"/>","<graphic xlink:href=\"18_2023_5083_Fig3_HTML\" id=\"MO4\"/>","<graphic xlink:href=\"18_2023_5083_Fig4_HTML\" id=\"MO5\"/>","<graphic xlink:href=\"18_2023_5083_Fig5_HTML\" id=\"MO6\"/>","<graphic xlink:href=\"18_2023_5083_Fig6_HTML\" id=\"MO7\"/>","<graphic xlink:href=\"18_2023_5083_Fig7_HTML\" id=\"MO8\"/>","<graphic xlink:href=\"18_2023_5083_Fig8_HTML\" id=\"MO9\"/>","<graphic xlink:href=\"18_2023_5083_Fig9_HTML\" id=\"MO10\"/>"],"string":"[\n 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xlink:href=\\\"18_2023_5083_MOESM1_ESM.docx\\\"><caption><p>Supplementary file1 (DOCX 7385 KB)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"1.\"], \"surname\": [\"Bayes-Genis\", \"Barallat\", \"Richards\"], \"given-names\": [\"A\", \"J\", \"AM\"], \"article-title\": [\"A test in context: neprilysin: function, inhibition, and biomarker\"], \"source\": [\"J Am Coll Cardiol\"], \"year\": [\"2006\"], \"volume\": [\"68\"], \"issue\": [\"6\"], \"fpage\": [\"639\"], \"lpage\": [\"653\"], \"pub-id\": [\"10.1016/j.jacc.2016.04.060\"]}, {\"label\": [\"2.\"], \"surname\": [\"Feygina\", \"Katrukha\", \"Semenov\"], \"given-names\": [\"EE\", \"AG\", \"AG\"], \"article-title\": [\"Neutral endopeptidase (neprilysin) in therapy and diagnostics: yin and yang\"], \"source\": [\"Biochem Mosc\"], \"year\": [\"2019\"], \"volume\": [\"84\"], \"issue\": [\"11\"], \"fpage\": [\"1346\"], \"lpage\": [\"1358\"], \"pub-id\": [\"10.1134/S0006297919110105\"]}, {\"label\": [\"13.\"], \"surname\": [\"Sato\", \"Katagiri\", \"Iijima\", \"Yamada\", \"Ito\", \"Kawasaki\"], \"given-names\": [\"B\", \"YU\", \"K\", \"H\", \"S\", \"N\"], \"article-title\": [\"The human CD10 lacking an \"], \"italic\": [\"N\"], \"source\": [\"Biochim Biophys Acta, Gen Subj\"], \"year\": [\"2012\"], \"volume\": [\"1820\"], \"fpage\": [\"1715\"], \"lpage\": [\"1723\"], \"pub-id\": [\"10.1016/j.bbagen.2012.06.017\"]}, {\"label\": [\"15.\"], \"surname\": [\"Nalivaeva\", \"Turner\", \"Rawlings\", \"Salvesen\"], \"given-names\": [\"NN\", \"AJ\", \"ND\", \"G\"], \"article-title\": [\"Chapter 127\\u2014neprilysin\"], \"source\": [\"Handbook of proteolytic enzymes\"], \"year\": [\"2013\"], \"edition\": [\"3\"], \"publisher-name\": [\"Academic Press\"], \"fpage\": [\"612\"], \"lpage\": [\"619\"]}, {\"label\": [\"29.\"], \"surname\": [\"H\\u00fclsmeier\", \"Tobler\", \"Burda\", \"Hennet\"], \"given-names\": [\"AJ\", \"M\", \"P\", \"T\"], \"article-title\": [\"Glycosylation site occupancy in health, congenital disorder of glycosylation and fatty liver disease\"], \"source\": [\"Sci Rep\"], \"year\": [\"2016\"], \"volume\": [\"11\"], \"issue\": [\"6\"], \"fpage\": [\"33927\"], \"pub-id\": [\"10.1038/srep33927\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [\n \"NEP\",\n \"HF\",\n \"RUO\",\n \"ELISA\",\n \"MAb\",\n \"pAb\",\n \"Trx\",\n \"IMAC\",\n \"SPR\",\n \"MRMHR\",\n \"BSA\",\n \"PBS\",\n \"ECE-1\",\n \"ECE-2\",\n \"PHEX\",\n \"ECEL-1\",\n \"LOD\",\n \"LLOQ\",\n \"CV\",\n \"rEF\",\n \"pEF\",\n \"CTRL\",\n \"CE\"\n ],\n \"definition\": [\n \"Neprilysin\",\n \"Heart failure\",\n \"Research-use-only\",\n \"Enzyme-linked immunosorbent assay\",\n \"Monoclonal antibody\",\n \"Polyclonal antibody\",\n \"Thioredoxin\",\n \"Immobilized metal affinity chromatography\",\n \"Surface plasmon resonance\",\n \"High-resolution multiple reaction monitoring\",\n \"Bovine serum albumin\",\n \"Phosphate buffered saline\",\n \"Human endothelin converting enzyme 1\",\n \"Human endothelin converting enzyme 2\",\n \"Human phosphate-regulating neutral endopeptidase\",\n \"Human endothelin converting enzyme like 1\",\n \"Limit of detection\",\n \"Lower limit of quantitation\",\n \"Coefficient of variation\",\n \"Reduced ejection fraction\",\n \"Preserved ejection fraction\",\n \"Non-HF control\",\n \"Capillary electrophoresis\"\n ]\n}"},"n_references":{"kind":"number","value":42,"string":"42"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Cell Mol Life Sci. 2024 Jan 13; 81(1):42"},"package_file":{"kind":"string","value":"oa_package/2d/2b/PMC10787894.tar.gz"}}},{"rowIdx":178,"cells":{"accession_id":{"kind":"string","value":"PMC10787895"},"pmid":{"kind":"string","value":"38051494"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par4\">Interferons (IFN) are a widely expressed family of cytokines. They are categorised, based on their receptor signalling, into types I, II, and III [<xref ref-type=\"bibr\" rid=\"CR1\">1</xref>]. IFN-I signal via a heterodimeric receptor composed of two distinct multi-chain structures, IFN-α receptor 1 and 2 (IFNAR-1 and IFNAR-2). The former is constitutively associated with tyrosine kinase 2 (TYK2) and the latter associated with Janus Kinase 1 (JAK1) [<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>]. IFN-I are produced as part of the innate immune response to infection and possess potent antiviral effects [<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>]. Triggers of IFN-I production and subsequent downstream signalling have been recently reviewed in [<xref ref-type=\"bibr\" rid=\"CR3\">3</xref>] and is summarised in Fig. ##FIG##0##1##. Similarly, IFN-II and IFN-III signal via their own unique heterodimeric receptors composed of IFN-γ receptors 1 and 2 (IFNGR-1 and IFNGR-2), IFNLR1 (IFN lambda receptor-1), and IL-10R2 (interleukin-10 receptor 2) subunits, respectively [<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>••]. Both of which subsequently lead to downstream signalling and potential induction of interferon inducible genes. In this review, we explore what role IFN-I, particularly IFN-α, may play in rheumatoid arthritis (RA) pathophysiology.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par4\\\">Interferons (IFN) are a widely expressed family of cytokines. They are categorised, based on their receptor signalling, into types I, II, and III [<xref ref-type=\\\"bibr\\\" rid=\\\"CR1\\\">1</xref>]. IFN-I signal via a heterodimeric receptor composed of two distinct multi-chain structures, IFN-α receptor 1 and 2 (IFNAR-1 and IFNAR-2). The former is constitutively associated with tyrosine kinase 2 (TYK2) and the latter associated with Janus Kinase 1 (JAK1) [<xref ref-type=\\\"bibr\\\" rid=\\\"CR2\\\">2</xref>]. IFN-I are produced as part of the innate immune response to infection and possess potent antiviral effects [<xref ref-type=\\\"bibr\\\" rid=\\\"CR2\\\">2</xref>]. Triggers of IFN-I production and subsequent downstream signalling have been recently reviewed in [<xref ref-type=\\\"bibr\\\" rid=\\\"CR3\\\">3</xref>] and is summarised in Fig. ##FIG##0##1##. Similarly, IFN-II and IFN-III signal via their own unique heterodimeric receptors composed of IFN-γ receptors 1 and 2 (IFNGR-1 and IFNGR-2), IFNLR1 (IFN lambda receptor-1), and IL-10R2 (interleukin-10 receptor 2) subunits, respectively [<xref ref-type=\\\"bibr\\\" rid=\\\"CR4\\\">4</xref>••]. Both of which subsequently lead to downstream signalling and potential induction of interferon inducible genes. In this review, we explore what role IFN-I, particularly IFN-α, may play in rheumatoid arthritis (RA) pathophysiology.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p id=\"Par34\">There is growing evidence that IFN-α plays an important role in early RA pathophysiology and Fig. ##FIG##4##5## summarises a working paradigm on IFN-α influencing RA progression. However, the triggers of IFN-α production and its timing in relation to early immune dysregulation or symptom onset remain unclear. Further work focusing on early disease or at-risk populations with a focus on genetic and epigenetic factors is likely to be informative. Despite mechanistic uncertainties, there is clear rationale to further test IFN-α targeting therapies in early RA, potentially using the IGS as a theragnostic biomarker, or to use the IGS as a biomarker for more intensive initial therapy. The heterogeneity and variety of IGSs remain challenging with regard to clinical utility, but recent progress in the international community on IGS stratification and uniform application of standardised measures of IFN-I signalling is encouraging [<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>••, <xref ref-type=\"bibr\" rid=\"CR5\">5</xref>••, <xref ref-type=\"bibr\" rid=\"CR124\">124</xref>, <xref ref-type=\"bibr\" rid=\"CR125\">125</xref>], and its use in this capacity may be on the horizon.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p id=\\\"Par34\\\">There is growing evidence that IFN-α plays an important role in early RA pathophysiology and Fig. ##FIG##4##5## summarises a working paradigm on IFN-α influencing RA progression. However, the triggers of IFN-α production and its timing in relation to early immune dysregulation or symptom onset remain unclear. Further work focusing on early disease or at-risk populations with a focus on genetic and epigenetic factors is likely to be informative. Despite mechanistic uncertainties, there is clear rationale to further test IFN-α targeting therapies in early RA, potentially using the IGS as a theragnostic biomarker, or to use the IGS as a biomarker for more intensive initial therapy. The heterogeneity and variety of IGSs remain challenging with regard to clinical utility, but recent progress in the international community on IGS stratification and uniform application of standardised measures of IFN-I signalling is encouraging [<xref ref-type=\\\"bibr\\\" rid=\\\"CR4\\\">4</xref>••, <xref ref-type=\\\"bibr\\\" rid=\\\"CR5\\\">5</xref>••, <xref ref-type=\\\"bibr\\\" rid=\\\"CR124\\\">124</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR125\\\">125</xref>], and its use in this capacity may be on the horizon.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose of Review</title>","<p id=\"Par1\">Type 1 interferons (IFN-I) are of increasing interest across a wide range of autoimmune rheumatic diseases. Historically, research into their role in rheumatoid arthritis (RA) has been relatively neglected, but recent work continues to highlight a potential contribution to RA pathophysiology.</p>","<title>Recent Findings</title>","<p id=\"Par2\">We emphasise the importance of disease stage when examining IFN-I in RA and provide an overview on how IFN-I may have a direct role on a variety of relevant cellular functions. We explore how clinical trajectory may be influenced by increased IFN-I signalling, and also, the limitations of scores composed of interferon response genes. Relevant environmental triggers and inheritable RA genetic risk relating to IFN-I signalling are explored with emphasis on intriguing data potentially linking IFN-I exposure, epigenetic changes, and disease relevant processes.</p>","<title>Summary</title>","<p id=\"Par3\">Whilst these data cumulatively illustrate a likely role for IFN-I in RA, they also highlight the knowledge gaps, particularly in populations at risk for RA, and suggest directions for future research to both better understand IFN-I biology and inform targeted therapeutic strategies.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s11926-023-01125-6.</p>","<title>Keywords</title>"],"string":"[\n \"<title>Purpose of Review</title>\",\n \"<p id=\\\"Par1\\\">Type 1 interferons (IFN-I) are of increasing interest across a wide range of autoimmune rheumatic diseases. Historically, research into their role in rheumatoid arthritis (RA) has been relatively neglected, but recent work continues to highlight a potential contribution to RA pathophysiology.</p>\",\n \"<title>Recent Findings</title>\",\n \"<p id=\\\"Par2\\\">We emphasise the importance of disease stage when examining IFN-I in RA and provide an overview on how IFN-I may have a direct role on a variety of relevant cellular functions. We explore how clinical trajectory may be influenced by increased IFN-I signalling, and also, the limitations of scores composed of interferon response genes. Relevant environmental triggers and inheritable RA genetic risk relating to IFN-I signalling are explored with emphasis on intriguing data potentially linking IFN-I exposure, epigenetic changes, and disease relevant processes.</p>\",\n \"<title>Summary</title>\",\n \"<p id=\\\"Par3\\\">Whilst these data cumulatively illustrate a likely role for IFN-I in RA, they also highlight the knowledge gaps, particularly in populations at risk for RA, and suggest directions for future research to both better understand IFN-I biology and inform targeted therapeutic strategies.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s11926-023-01125-6.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>The Interferon Gene Signature (IGS)</title>","<p id=\"Par5\">Measuring IFN-α protein <italic>in vivo</italic> has been historically challenging due to low circulating levels being frequently below the detection thresholds of standard assays. A solution was to infer IFN-I exposure, and hence levels, by measuring transcripts that reflect interferon stimulated or response genes (IRGs), and their cumulative expression was termed the interferon gene signature (IGS) (see Fig. ##FIG##1##2##). However, there are over 2000 IRGs and which IRGs are chosen to generate an IGS is lacking consensus across studies [<xref ref-type=\"bibr\" rid=\"CR5\">5</xref>••]. Despite this, an IGS is widely reported in autoimmune rheumatic diseases, and there are mutual IRGs increased in RA and other rheumatic diseases [<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>]. Nevertheless, some propose an exclusive and highly diverse IRG transcriptional profile in RA peripheral whole blood [<xref ref-type=\"bibr\" rid=\"CR7\">7</xref>] as well as in synovial biopsy samples [<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>], distinct from that found in SLE. However, IRG expression, and subsequently the calculated IGS, may vary between different cell types, suggesting that differences seen amongst related autoimmune diseases could be secondary to different immune cell proportions and signalling pathway activation [<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>]. Indeed, variation is seen in flow cytometry detected STAT class phosphorylation in CD4+ T cells, CD8+ T cells, B cells, and monocytes following IFN-I stimulation [<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>].</p>","<p id=\"Par6\">As IFN-I, IFN-II, or even IFN-III can induce IRGs (see Fig. ##FIG##0##1##), there has been a historical lack of clarity as to which IFN class was responsible for the IGS in RA. Indeed, it remains a controversial topic as IRG expression may be modulated by additional stimuli, such as TNF-α, with variable effects reported in monocytes vs T cells [<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>]. Nevertheless, in established RA, there was reportedly equal contribution of IFN-α and IFN-β to the whole blood IGS vs IFN-α exposure being dominant in SLE [<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>]. However, in a cohort of nearly 200 early drug naïve RA patients, circulating IFN-α protein and not IFN-β, IFN-II, or IFN-III nor any other circulating inflammatory cytokine uniquely correlated with the whole blood IGS [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>••]. This work remains to be validated, and reported differences may reflect disease stages, but does implicate predominantly IFN-α with the IGS in early RA.</p>","<p id=\"Par7\">Despite these caveats regarding its calculation, the IGS remains a useful tool in dissecting the role of IFN-I in RA, as explored below.</p>","<title>The IGS by Disease Phase</title>","<p id=\"Par8\">It is increasingly appreciated that disease processes in early RA are likely to be distinct from established RA. In early RA, a raised IGS (<italic>MxA</italic>, <italic>OAS1</italic>, <italic>ISG15</italic>, <italic>IFI44L</italic>, <italic>IFI6</italic>) was more prevalent compared with established RA, approximately 50% vs 20% of patients, respectively [<xref ref-type=\"bibr\" rid=\"CR13\">13</xref>], and fell with the initiation of therapy [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>••, <xref ref-type=\"bibr\" rid=\"CR13\">13</xref>]. Therapeutics may contribute to a reduced incidence in established RA as glucocorticoids, as well as disease modifying anti-rheumatoid drugs (DMARDs), can modify the IGS [<xref ref-type=\"bibr\" rid=\"CR14\">14</xref>]. Notably, this increase in early RA persists even after accounting for potential confounders such as disease stage dependant variation in cell subset proportions [<xref ref-type=\"bibr\" rid=\"CR15\">15</xref>].</p>","<p id=\"Par9\">Corroborating the raised IGS noted at disease onset, there is emerging data that IFNs may contribute to the transition from preclinical to sustained clinical disease. In ontology studies and network pathway analyses, the IGS distinguished DMARD-naïve early arthritis patients that developed a persistent inflammatory arthritis from those that had a self-limiting course [<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>]. In ACPA+ arthralgia populations, i.e. those who are at risk for developing RA, an IGS increases the chance of progression to synovitis, and its inclusion in outcome models improved its predictive capacity [<xref ref-type=\"bibr\" rid=\"CR17\">17</xref>, <xref ref-type=\"bibr\" rid=\"CR18\">18</xref>]. Even in healthy asymptomatic CCP+ individuals, there was evidence of increased IFN-α signalling which mirrored what was seen in early RA cohorts, and this, with other parameters, was able to differentiate progressors with a median of 4.1 years before symptom onset, from controls [<xref ref-type=\"bibr\" rid=\"CR19\">19</xref>, <xref ref-type=\"bibr\" rid=\"CR20\">20</xref>•]. In seropositive and seronegative RA, as well as in high-risk seropositive arthralgia patients, there was an overlap in circulating cytokine profiles with IFN-α, as well as IL-5, and TNF-α upregulated up to 50% in seropositive arthralgia and seropositive RA patients but not in seronegative RA [<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>] with an odds ratio (OR) of 21 for RA development in seropositive arthralgia patients [<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>, <xref ref-type=\"bibr\" rid=\"CR21\">21</xref>].</p>","<title>Clinical Characteristics and the IGS</title>","<p id=\"Par10\">There has been conflicting evidence around the impact of an IGS/IFN-I signalling on autoantibody production in RA. In established RA, there is a significant correlation between the IGS and ACPA titres and anti-carbamylated protein (anti-CarP) antibodies as well as with genes linked to B cell differentiation and antibody production [<xref ref-type=\"bibr\" rid=\"CR22\">22</xref>]. Conversely, others found no relation between the IGS and the presence and/or titres of ACPA and RF in established disease [<xref ref-type=\"bibr\" rid=\"CR23\">23</xref>]. Similarly, a 2016 systematic analysis, involving patients with established RA, found that there was no difference seen in the IGS between ACPA negative and ACPA positive patients [<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>]. Conversely, rheumatoid factor (RF) demonstrated a positive association between either the IGS or circulating IFN-α levels in both established and early RA as well as across several autoimmune rheumatic diseases [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>••, <xref ref-type=\"bibr\" rid=\"CR13\">13</xref>, <xref ref-type=\"bibr\" rid=\"CR25\">25</xref>]. These differences may reflect disease stage but may also reflect variability in the IRGs chosen to represent the IGS, with some using a combination of 19 IRGs [<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>] and others using only 6 (IFI27, IFI44L, IFIT1, ISG15, RSAD2 and SIGLEC1) [<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>] for example.</p>","<p id=\"Par11\">Multiple observational studies in established RA have found no association between the IGS and disease activity [<xref ref-type=\"bibr\" rid=\"CR13\">13</xref>, <xref ref-type=\"bibr\" rid=\"CR24\">24</xref>]. This contrasts with early drug naïve RA where in a number of prospective observational studies, a higher IGS in early drug naïve RA, were associated with increased baseline disease activity as well as a poorer response to initial therapies [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>, <xref ref-type=\"bibr\" rid=\"CR13\">13</xref>, <xref ref-type=\"bibr\" rid=\"CR15\">15</xref>] which was validated in additional cohorts for specific IGSs [<xref ref-type=\"bibr\" rid=\"CR26\">26</xref>].</p>","<p id=\"Par12\">RA, including early disease, is a risk factor for cardiovascular disease (CVD). [<xref ref-type=\"bibr\" rid=\"CR27\">27</xref>, <xref ref-type=\"bibr\" rid=\"CR28\">28</xref>]. In mouse and human <italic>in vitro</italic> models, IFN-α stimulation of macrophages resulted in significant metabolic rewiring with over 500 metabolic genes, including those related to key processes in the pathophysiology of CVD such as glycolysis, oxidative phosphorylation, fatty acid synthesis, and lipid metabolism [<xref ref-type=\"bibr\" rid=\"CR29\">29</xref>]. In addition, endothelial progenitor cells (EPCs), involved in vasculogenesis and repair, have impaired function in RA, with IFN-α implicated in both <italic>in vitro</italic> and <italic>in vivo</italic> studies [<xref ref-type=\"bibr\" rid=\"CR30\">30</xref>–<xref ref-type=\"bibr\" rid=\"CR32\">32</xref>]. In murine lupus models, prolonged and enhanced IFN-I exposure significantly reduced EPC numbers, with acute exposure affecting only EPC differentiation but not the cellular number [<xref ref-type=\"bibr\" rid=\"CR30\">30</xref>]. Finally, IFN-α may also influence CVD by promoting insulin resistance given, as early as the 1980s, IFN-α was shown to impair glucose tolerance and insulin sensitivity [<xref ref-type=\"bibr\" rid=\"CR33\">33</xref>] with reversal of this effect in IFNAR-/- mouse models [<xref ref-type=\"bibr\" rid=\"CR34\">34</xref>].</p>","<title>IFN-I and Its Effects on Cellular Function</title>","<title>B and T Cells</title>","<p id=\"Par13\">IFN-I can widely influence B cell activity which may contribute to RA pathophysiology, for example, by supporting B cell survival via increased monocyte B-lymphocyte stimulator (BLyS) production, by direct stimulation of B cells, and indirectly through T cell and Dendritic Cells (DCs) stimulation [<xref ref-type=\"bibr\" rid=\"CR35\">35</xref>]. Prolonged B cell survival can lead to increased differentiation into memory and plasma cells, immunoglobulin isotype switching, and autoantibody formation [<xref ref-type=\"bibr\" rid=\"CR36\">36</xref>, <xref ref-type=\"bibr\" rid=\"CR37\">37</xref>]. Furthermore, IFN-α modifies the plasma cell transcriptome towards a proinflammatory phenotype [<xref ref-type=\"bibr\" rid=\"CR38\">38</xref>]. IFN-I regulates BCR signalling, specifically via IFN-αR, which in turn may promote pathways involved in antibody formation and germinal centre development in murine models [<xref ref-type=\"bibr\" rid=\"CR39\">39</xref>]. IFN-I can also influence the differentiation of CD4+ T cells towards a Th1 response [<xref ref-type=\"bibr\" rid=\"CR40\">40</xref>], fostering B cell activation and subsequent activity [<xref ref-type=\"bibr\" rid=\"CR41\">41</xref>]. IFN-I also promotes CD8+ T cell survival and CD8+ cytotoxic T cell activity as well as prolonging the proliferation and expansion of CD8+ antigen specific T cells via inhibition of apoptosis [<xref ref-type=\"bibr\" rid=\"CR42\">42</xref>].</p>","<title>Dendritic Cells</title>","<p id=\"Par14\">Dendritic cells (DCs) upregulate HLA-DR, CD40, CD80, and CD86 expression upon IFN-I exposure [<xref ref-type=\"bibr\" rid=\"CR43\">43</xref>]. DC maturation and enhanced antigen presentation, in the context of increased co-stimulatory molecules, can result in the induction of autoimmunity in predisposed individuals via self-antigen presentation to low affinity autoreactive T cells [<xref ref-type=\"bibr\" rid=\"CR44\">44</xref>]. In SLE susceptible mice, IFN-I-treated DCs showed relative apoptosis resistance, this activated DC longevity potentially contributing to the development of autoimmunity [<xref ref-type=\"bibr\" rid=\"CR45\">45</xref>]. Conversely, in early drug naive RA, there was no association between the IGS and circulating CD1c or pDC frequency, but there was an inverse association with CD141+ DC frequency [<xref ref-type=\"bibr\" rid=\"CR46\">46</xref>]. This highlights the DC subset dependant complexity of IFN-I signalling <italic>in vivo</italic>.</p>","<title>Monocytes</title>","<p id=\"Par15\">Classical and non-classical monocytes have been implicated in RA pathogenesis [<xref ref-type=\"bibr\" rid=\"CR47\">47</xref>]. How the IGS affects monocyte function <italic>in vivo</italic> in RA remains to be fully examined but, when exposed to IFN-Is <italic>in vitro</italic>, monocytes upregulate TLR7 and IRF expression, resulting in increased responsiveness to subsequent immunostimulatory ligands [<xref ref-type=\"bibr\" rid=\"CR48\">48</xref>]. IFN-I exposure also increases expression of CD40, CD80, and CD86 and HLA-DR, ultimately promoting differentiation into a monocyte-derived dendritic cell, or mo-DC, with high capacity for antigen presentation [<xref ref-type=\"bibr\" rid=\"CR43\">43</xref>, <xref ref-type=\"bibr\" rid=\"CR49\">49</xref>]. Mo-DCs are also known to be increased in the RA synovial compartment and promote Th17 differentiation [<xref ref-type=\"bibr\" rid=\"CR50\">50</xref>]. However, as with DCs, what happens <italic>in vivo</italic> may be subset dependant as highlighted by enhanced responsiveness to IFN-α in murine proinflammatory monocytes secondary to increased IFNAR expression when compared with anti-inflammatory monocytes [<xref ref-type=\"bibr\" rid=\"CR51\">51</xref>].</p>","<title>Neutrophils</title>","<p id=\"Par16\">Neutrophils are one of the first cell types to enter the RA joint and may play an important role in the development and progression of RA [<xref ref-type=\"bibr\" rid=\"CR52\">52</xref>]. They are a major contributor to the whole blood IGS in RA, attributed to their uniquely upregulated IFNAR expression, a phenomenon not seen in either healthy controls or RA PBMCs [<xref ref-type=\"bibr\" rid=\"CR53\">53</xref>•]. Indeed, next generation sequencing of isolated blood neutrophils has found significantly upregulated IRGs in RA neutrophils compared to healthy controls [<xref ref-type=\"bibr\" rid=\"CR54\">54</xref>]. How this increased sensitivity to IFN-I influences neutrophil function is being explored, but, intriguingly, the pathogenic phenotype proposed for RA consists of delayed neutrophil apoptosis, increased ROS production and chemokine expression which, in part, can be recapitulated by IFN-I exposure <italic>in vitro</italic> [<xref ref-type=\"bibr\" rid=\"CR55\">55</xref>•].</p>","<title>Fibroblasts</title>","<p id=\"Par17\">Synovial fibroblasts are resident cells in the stroma of joints [<xref ref-type=\"bibr\" rid=\"CR56\">56</xref>], and we recently demonstrated comparable IFN-α levels in serum and early RA synovial fluid [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>••]. In RA, these fibroblasts have an activated phenotype, characterised by resistance to apoptosis, and increased proliferation and production of inflammatory mediators that promote immune cell differentiation and survival [<xref ref-type=\"bibr\" rid=\"CR57\">57</xref>]. Histology and RNA sequencing of early RA synovial tissue demonstrated three distinct pathotypes: fibroblastic pauci-immune, macrophage-rich diffuse myeloid, and a lympho-myeloid pathotype [<xref ref-type=\"bibr\" rid=\"CR58\">58</xref>••]. In the lympho-myeloid pathotype, seven out of the eight differentially expressed blood transcripts in synovial versus whole blood were IFN-I responses genes (IFI27, ISG15, IFI44L, OASL, USP18, RSAD2, LY6E) [<xref ref-type=\"bibr\" rid=\"CR58\">58</xref>•]. In addition, a pathogenic subset of sub-lining fibroblasts (<italic>THY1</italic><sup><italic>+</italic></sup><italic>HLA</italic><sup><italic>−</italic></sup><italic>DR</italic><sup><italic>high</italic></sup>) have increased IRG expression [<xref ref-type=\"bibr\" rid=\"CR59\">59</xref>]. However, this may not directly be secondary to IFN-I as TNF-α induced signalling co-opts the mTOR pathway to shift fibroblast like synoviocytes towards an IFN response [<xref ref-type=\"bibr\" rid=\"CR60\">60</xref>] which has been shown to be via secondary autocrine production of IFNβ and subsequent activation of the IRF1-IFNβ-IFNAR-JAK-STAT1 axis [<xref ref-type=\"bibr\" rid=\"CR61\">61</xref>]. Nevertheless, the role of IFN-α on fibroblast function in RA remains an important research question.</p>","<p id=\"Par18\">Cumulatively, these effects are likely to contribute to a highly activated and potentially autoimmune prone phenotype as summarised in Fig. ##FIG##2##3##.</p>","<title>Source of IFN-I in RA</title>","<p id=\"Par19\">pDCs, particularly in their immature state, are the main IFN-I producing cell, however, whether they are the primary source of IFN-α in RA remains unclear. In SLE, there is an element of so-called pDC fatigue, where the ability of the pDC to produce IFN-I reduces and other cells take over production [<xref ref-type=\"bibr\" rid=\"CR62\">62</xref>]. In early RA, circulating pDCs were not the primary source of <italic>IFNA</italic> transcript, with comparable expression in circulating lymphocytes. However, circulating pDC numbers were reduced with increased CCR7 expression inferring increased migration to the synovial compartment and target tissue [<xref ref-type=\"bibr\" rid=\"CR46\">46</xref>]. Indeed, in established RA patients, the synovial compartment has increased numbers of pDCs with reduced numbers seen in peripheral blood. However, those that remained in the circulation were immature with inferred increased IFN-I producing capacity [<xref ref-type=\"bibr\" rid=\"CR63\">63</xref>]. Nevertheless, RA synovial pDCs are potent producers of IFN-α [<xref ref-type=\"bibr\" rid=\"CR64\">64</xref>] and, in mice, intraarticular transfer of IFN-I producing dendritic cells was sufficient to propagate a persistent inflammatory arthritis [<xref ref-type=\"bibr\" rid=\"CR65\">65</xref>].</p>","<p id=\"Par20\">Conversely, after arthritogenic serum transfer in K/BxN serum-induced arthritis, collagen-induced arthritis, and human TNF transgene insertion, only pDC deficient mice showed exacerbations of symptoms and signs of inflammatory arthritis [<xref ref-type=\"bibr\" rid=\"CR66\">66</xref>] and topical imiquimod, a TLR7 agonist, increased pDC recruitment and activity which subsequently improved arthritis [<xref ref-type=\"bibr\" rid=\"CR66\">66</xref>]. Furthermore, transcriptomic analysis of circulating pDCs in early RA suggested enhanced tolerogenic function [<xref ref-type=\"bibr\" rid=\"CR46\">46</xref>]. These discrepancies may arise due to the complexities of DC development [<xref ref-type=\"bibr\" rid=\"CR67\">67</xref>] and cellular differences across species. Given their relative paucity <italic>in vivo</italic>, pDCs have been relatively neglected in RA research; however, better understanding of their complexity, particularly in relation to any location specific function, will help inform their role in RA and role in IFN-I production.</p>","<title>Potential Triggers of IFN Production</title>","<p id=\"Par21\">What drives the observed increased IGS/IFN-α in RA remains unclear; however, triggers may include viral infections or microbial DNA or antigen fragments, with these elements repeatedly reported in the joints of RA patients [<xref ref-type=\"bibr\" rid=\"CR68\">68</xref>–<xref ref-type=\"bibr\" rid=\"CR70\">70</xref>]. Retroelements are non-protein encoding portions of DNA derived from ancient transposable elements, such as retroviruses, that have been historically incorporated into the genome. Their activity can trigger intracellular viral sensors and thus promote local IFN-I production [<xref ref-type=\"bibr\" rid=\"CR71\">71</xref>]. In SLE and primary Sjogren’s syndrome, increased retrotransposon activity in disease relevant tissue associated with increased local IFN-α production [<xref ref-type=\"bibr\" rid=\"CR72\">72</xref>•], and, in established RA synovium, there is also increased retroelement expression [<xref ref-type=\"bibr\" rid=\"CR73\">73</xref>, <xref ref-type=\"bibr\" rid=\"CR74\">74</xref>]. Furthermore, in a subgroup of RA patients, a transcriptional profile was documented, reminiscent of a viral infection, which associated with both IFN-I signalling as well as increased ACPA titres [<xref ref-type=\"bibr\" rid=\"CR75\">75</xref>, <xref ref-type=\"bibr\" rid=\"CR76\">76</xref>]. How these retroelements may influence IFN-I production in RA remains to be seen.</p>","<p id=\"Par22\">Cell-free nucleic acids have been extensively implicated in IFN-I generation in SLE [<xref ref-type=\"bibr\" rid=\"CR77\">77</xref>] and monogenic interferonopathies [<xref ref-type=\"bibr\" rid=\"CR78\">78</xref>]. Mouse models with DNA clearance defects develop autoantibody-mediated chronic polyarthritis, resembling human RA [<xref ref-type=\"bibr\" rid=\"CR79\">79</xref>]. This corroborates RA human observational data, where evidence of raised levels of circulating cell -free DNA have been found in both peripheral blood [<xref ref-type=\"bibr\" rid=\"CR80\">80</xref>, <xref ref-type=\"bibr\" rid=\"CR81\">81</xref>] and synovial fluid [<xref ref-type=\"bibr\" rid=\"CR82\">82</xref>]. Although direct links with IFN-I were not made in these human RA studies, a similar mechanism to that described in SLE may be present<italic>.</italic></p>","<p id=\"Par23\">Neutrophils, found in high numbers in RA synovium, can undergo NETosis, a unique form of cell death which has been proposed as a potential trigger for IFN-I production [<xref ref-type=\"bibr\" rid=\"CR83\">83</xref>]. DNA from these NETs form complexes with antimicrobial peptides including LL37, secretory leukocyte protease inhibitor (SLPI), or with immunoglobulins to form immune complexes which facilitate pDC TLR7/9 signalling ultimately culminating in IFN-α production [<xref ref-type=\"bibr\" rid=\"CR84\">84</xref>–<xref ref-type=\"bibr\" rid=\"CR86\">86</xref>]. In RA, links between NETs and ACPA have been reported [<xref ref-type=\"bibr\" rid=\"CR87\">87</xref>, <xref ref-type=\"bibr\" rid=\"CR88\">88</xref>] and known pathogenic cytokines in RA, such as TNF-α and IL-17A, as well as IFN-a itself [<xref ref-type=\"bibr\" rid=\"CR89\">89</xref>], can also induce NETosis, potentially creating a vicious cycle of inflammation and disease activity [<xref ref-type=\"bibr\" rid=\"CR88\">88</xref>].</p>","<p id=\"Par24\">Other potential triggers include lifestyle and environmental factors. An inverse correlation between physical activity and IFN-I signalling has been reported [<xref ref-type=\"bibr\" rid=\"CR90\">90</xref>]. In addition, physical activity was associated with downregulation of TLR and IL-17R signalling and reduced inflammatory cytokines production, including IFN-I [<xref ref-type=\"bibr\" rid=\"CR90\">90</xref>].</p>","<p id=\"Par25\">Potential triggers are summarised in Fig. ##FIG##3##4##; however, much of the above involves extrapolation from other diseases, such as SLE, and caveats exist including differences in IRG expression and genetic risk between these diseases [<xref ref-type=\"bibr\" rid=\"CR91\">91</xref>]. Further work is needed to explore these pathways in RA specifically.</p>","<title>Heritable Genetic Risk and IFN-I Signalling</title>","<p id=\"Par26\">As genome-wide association studies (GWAS), and relevant data sets, become more available, numerous single nucleotide polymorphisms (SNPs) have been identified as contributing to the genetic risk of RA. Interestingly, a number of these SNPs are in genes related to the IFN-I response pathway including DNA-sensing proteins, toll-like receptors, and JAK-STAT protein mediators. These are summarised in Table ##TAB##0##1##. However, the functional consequences of these polymorphisms in RA with regards to IFN-I production or signalling are yet to be elucidated. Nevertheless, an overlap of certain at-risk genes associated with increased IFN-I signalling in SLE has also been linked to RA, for example SNPs in IRF5, STAT4, and PTPN22 [<xref ref-type=\"bibr\" rid=\"CR92\">92</xref>]. Some of these RA risk SNPs, such as IRF5 polymorphisms, associate with more severe or erosive disease [<xref ref-type=\"bibr\" rid=\"CR93\">93</xref>, <xref ref-type=\"bibr\" rid=\"CR94\">94</xref>], which may corroborate the clinical refractory disease phenotype observed in IGS high early RA patients [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>••]. Further work is needed to elucidate both the role of IFN-I on susceptible genetic backgrounds as well as the contribution of these SNPs to IFN-I production.</p>","<title>IFN-I and Epigenetics</title>","<p id=\"Par27\">Epigenetic changes are modifications that regulate genome activity, independent of DNA sequence. This occurs via molecular factors and processes, such as DNA methylation of CPG sites or chromatin conformational changes, which subsequently modulate transcription. They are frequently triggered by environmental factors or exposure to inflammatory stimuli, such as cytokines. Methylation changes are noted early in RA progression and vary by cell subset [<xref ref-type=\"bibr\" rid=\"CR95\">95</xref>••]. Furthermore, differential methylation has been implicated in initial response to methotrexate in early drug naïve RA patients as well as to certain biologics in established disease [<xref ref-type=\"bibr\" rid=\"CR95\">95</xref>••, <xref ref-type=\"bibr\" rid=\"CR96\">96</xref>–<xref ref-type=\"bibr\" rid=\"CR98\">98</xref>], and these processes are emerging as important modifiers of RA clinical progression and phenotype [<xref ref-type=\"bibr\" rid=\"CR99\">99</xref>].</p>","<p id=\"Par28\">Analysis of B and CD4 T cells from early drug naïve RA patients demonstrated differentially methylated CPG sites at disease relevant genes, such as PARP9, STAT1, and EPSTI between IGS high and low patients. It also implicated altered transcription factor binding, cumulatively promoting increased lymphocyte activation, and a proliferative phenotype in the IGS high cohort [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>••]. These data suggest that these changes may be IFN-α induced, and negatively influence clinical trajectory. In undifferentiated arthritis (UA) monocytes, methylation changes, which associated with disease progression and a poor prognosis, were partially recapitulated by monocyte exposure to IFN-α [<xref ref-type=\"bibr\" rid=\"CR100\">100</xref>•]. Furthermore, IFN-α treatment causes methylation changes in monocytes similar to those seen in established RA, which <italic>in vivo</italic> were themselves associated with increased disease activity [<xref ref-type=\"bibr\" rid=\"CR101\">101</xref>]. Intriguingly, in models of type 1 diabetes, where IFN-I plays a key part in disease initiation, exposure to IFN-α triggered increased TET2 expression. This prompted hypomethylation changes in genes controlling inflammatory and immune pathways, ultimately resulting in their increased expression and disease acceleration [<xref ref-type=\"bibr\" rid=\"CR102\">102</xref>]. TET proteins are key players in demethylation and are also increased in early drug naïve RA circulating lymphocytes [<xref ref-type=\"bibr\" rid=\"CR103\">103</xref>], however whether this is secondary to IFN-α is unknown.</p>","<p id=\"Par29\">It is important to acknowledge that CPG sites in IRGs themselves are frequently hypomethylated in autoimmune conditions, including RA [<xref ref-type=\"bibr\" rid=\"CR104\">104</xref>, <xref ref-type=\"bibr\" rid=\"CR105\">105</xref>]. In twin studies of CD4 T cells, hypomethylation of IRGs <italic>IFIT1</italic>, <italic>IRF7</italic>, <italic>MX1</italic>, <italic>OAS1</italic>, <italic>USP18</italic>, <italic>RSAD2,</italic> and <italic>IFI44L</italic> has even been proposed as biomarkers of progression to RA [<xref ref-type=\"bibr\" rid=\"CR104\">104</xref>]. This questions whether the IGS could be an artefact of altered gene expression secondary to hypomethylation caused by other circulating inflammatory cytokines, such as IL6, or due to increased IFN-α signalling itself. IFN-α protein is increased in early RA and uniquely correlates with the IGS [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>••], so the reality is likely to involve both mechanisms.</p>","<p id=\"Par30\">Although less extensively investigated, IFN-I-associated chromatin conformational changes may also be relevant to RA pathophysiology. There is variation in chromatin accessibility in RA synovial fibroblasts which is likely influenced by the synovial environment [<xref ref-type=\"bibr\" rid=\"CR106\">106</xref>], where IFN-α is known to be present [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>••]. In early RA, chromatin conformation changes in <italic>IFNAR2</italic> were associated with poorer outcomes [<xref ref-type=\"bibr\" rid=\"CR107\">107</xref>]. Furthermore, monocytes stimulated with IFN-α have increased trimethylated histone H3 Lys 4 (H3K4me3) which enhances transcription at promotors of genes that encode inflammatory mediators. In a more representative <italic>in vivo</italic> environment, incubation of monocytes with both IFN-α and TNF-α was associated with increased H3K4me3 that reduced monocyte tolerization to LPS and promoted an enhanced response to subsequent environmental challenges [<xref ref-type=\"bibr\" rid=\"CR108\">108</xref>]. This intriguingly implicates IFN-I, and chromatin-mediated modifications, with the induction of inflammatory genes beyond canonical IRGs. Indeed, instances where prior exposure to IFN-α can influence cellular response to additional stimuli are increasingly being reported [<xref ref-type=\"bibr\" rid=\"CR102\">102</xref>, <xref ref-type=\"bibr\" rid=\"CR109\">109</xref>–<xref ref-type=\"bibr\" rid=\"CR111\">111</xref>] and remain a potential mechanism whereby IFN-I can influence disease development in RA.</p>","<title>The IGS/IFN as a Therapeutic Target</title>","<p id=\"Par31\">Anifrolumab targets IFNAR1 and therefore blocks IFN-α and IFN-β signalling [<xref ref-type=\"bibr\" rid=\"CR112\">112</xref>]. In a pilot trial, seven established RA patients, all with a high IGS (<italic>IFI27, IFI44, IFI44L</italic> and <italic>RSAD2</italic>), and active diseases were randomised to anifrolumab or placebo [<xref ref-type=\"bibr\" rid=\"CR113\">113</xref>•]. The primary endpoint of an American College of Rheumatology (ACR) response of ≥ 20% after 24 weeks was achieved in patients receiving anifrolumab although only one patient in each arm completed the study despite a safety profile similar to that reported in SLE [<xref ref-type=\"bibr\" rid=\"CR114\">114</xref>]. Reasons for early discontinuation in the treatment group included lack of efficacy, hypersensitivity reaction, and infection whilst the control group participants stopped due to insufficient therapeutic response [<xref ref-type=\"bibr\" rid=\"CR113\">113</xref>•]. Larger trials are needed to assess the efficacy of this drug in RA.</p>","<p id=\"Par32\">Alternatively, JAK inhibitors (JAKi) suppress phosphorylation of STAT and thus affect downstream IFN signalling and reduce IRG expression [<xref ref-type=\"bibr\" rid=\"CR92\">92</xref>]. Indeed, <italic>in vitro</italic> JAKi reduce IFN-I driven plasmablast differentiation [<xref ref-type=\"bibr\" rid=\"CR115\">115</xref>], synovial BAFF expression, monocyte-derived DCs costimulatory molecule CD80/CD86 expression, and T cell differentiation into Th1 and Th17 cells [<xref ref-type=\"bibr\" rid=\"CR115\">115</xref>, <xref ref-type=\"bibr\" rid=\"CR116\">116</xref>] [<xref ref-type=\"bibr\" rid=\"CR61\">61</xref>]. The efficacy of JAKi in the treatment of established RA has been widely reported [<xref ref-type=\"bibr\" rid=\"CR117\">117</xref>], although how the IGS impacts on its effect has not been comprehensively examined. However, analysis of baricitinib SLE trial data demonstrated that clinical effect was independent of IGS reduction [<xref ref-type=\"bibr\" rid=\"CR118\">118</xref>].</p>","<p id=\"Par33\">Other inhibitors of downstream IFN-I signalling include a novel small molecule selective for JAK3/JAK1/TBK1 (tank-1 binding kinase), which, in mouse models, suppressed IFN-I production and osteoclast formation via TBK1 inhibition [<xref ref-type=\"bibr\" rid=\"CR119\">119</xref>]. Autoantibody dependent collagen-induced arthritis mice models confirmed the clinical benefit of TBK1 inhibition [<xref ref-type=\"bibr\" rid=\"CR120\">120</xref>, <xref ref-type=\"bibr\" rid=\"CR121\">121</xref>] and TBK1 deficient mice have reduced IRG and protein expression [<xref ref-type=\"bibr\" rid=\"CR122\">122</xref>]. These findings are yet to be reproduced in human studies, but given the interest in cancer regarding TBK1 inhibition [<xref ref-type=\"bibr\" rid=\"CR123\">123</xref>], this may provide a novel therapeutic approach.</p>","<title>Supplementary Information</title>","<p>\n</p>"],"string":"[\n \"<title>The Interferon Gene Signature (IGS)</title>\",\n \"<p id=\\\"Par5\\\">Measuring IFN-α protein <italic>in vivo</italic> has been historically challenging due to low circulating levels being frequently below the detection thresholds of standard assays. A solution was to infer IFN-I exposure, and hence levels, by measuring transcripts that reflect interferon stimulated or response genes (IRGs), and their cumulative expression was termed the interferon gene signature (IGS) (see Fig. ##FIG##1##2##). However, there are over 2000 IRGs and which IRGs are chosen to generate an IGS is lacking consensus across studies [<xref ref-type=\\\"bibr\\\" rid=\\\"CR5\\\">5</xref>••]. Despite this, an IGS is widely reported in autoimmune rheumatic diseases, and there are mutual IRGs increased in RA and other rheumatic diseases [<xref ref-type=\\\"bibr\\\" rid=\\\"CR6\\\">6</xref>]. Nevertheless, some propose an exclusive and highly diverse IRG transcriptional profile in RA peripheral whole blood [<xref ref-type=\\\"bibr\\\" rid=\\\"CR7\\\">7</xref>] as well as in synovial biopsy samples [<xref ref-type=\\\"bibr\\\" rid=\\\"CR8\\\">8</xref>], distinct from that found in SLE. However, IRG expression, and subsequently the calculated IGS, may vary between different cell types, suggesting that differences seen amongst related autoimmune diseases could be secondary to different immune cell proportions and signalling pathway activation [<xref ref-type=\\\"bibr\\\" rid=\\\"CR9\\\">9</xref>]. Indeed, variation is seen in flow cytometry detected STAT class phosphorylation in CD4+ T cells, CD8+ T cells, B cells, and monocytes following IFN-I stimulation [<xref ref-type=\\\"bibr\\\" rid=\\\"CR10\\\">10</xref>].</p>\",\n \"<p id=\\\"Par6\\\">As IFN-I, IFN-II, or even IFN-III can induce IRGs (see Fig. ##FIG##0##1##), there has been a historical lack of clarity as to which IFN class was responsible for the IGS in RA. Indeed, it remains a controversial topic as IRG expression may be modulated by additional stimuli, such as TNF-α, with variable effects reported in monocytes vs T cells [<xref ref-type=\\\"bibr\\\" rid=\\\"CR11\\\">11</xref>]. Nevertheless, in established RA, there was reportedly equal contribution of IFN-α and IFN-β to the whole blood IGS vs IFN-α exposure being dominant in SLE [<xref ref-type=\\\"bibr\\\" rid=\\\"CR9\\\">9</xref>]. However, in a cohort of nearly 200 early drug naïve RA patients, circulating IFN-α protein and not IFN-β, IFN-II, or IFN-III nor any other circulating inflammatory cytokine uniquely correlated with the whole blood IGS [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>••]. This work remains to be validated, and reported differences may reflect disease stages, but does implicate predominantly IFN-α with the IGS in early RA.</p>\",\n \"<p id=\\\"Par7\\\">Despite these caveats regarding its calculation, the IGS remains a useful tool in dissecting the role of IFN-I in RA, as explored below.</p>\",\n \"<title>The IGS by Disease Phase</title>\",\n \"<p id=\\\"Par8\\\">It is increasingly appreciated that disease processes in early RA are likely to be distinct from established RA. In early RA, a raised IGS (<italic>MxA</italic>, <italic>OAS1</italic>, <italic>ISG15</italic>, <italic>IFI44L</italic>, <italic>IFI6</italic>) was more prevalent compared with established RA, approximately 50% vs 20% of patients, respectively [<xref ref-type=\\\"bibr\\\" rid=\\\"CR13\\\">13</xref>], and fell with the initiation of therapy [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>••, <xref ref-type=\\\"bibr\\\" rid=\\\"CR13\\\">13</xref>]. Therapeutics may contribute to a reduced incidence in established RA as glucocorticoids, as well as disease modifying anti-rheumatoid drugs (DMARDs), can modify the IGS [<xref ref-type=\\\"bibr\\\" rid=\\\"CR14\\\">14</xref>]. Notably, this increase in early RA persists even after accounting for potential confounders such as disease stage dependant variation in cell subset proportions [<xref ref-type=\\\"bibr\\\" rid=\\\"CR15\\\">15</xref>].</p>\",\n \"<p id=\\\"Par9\\\">Corroborating the raised IGS noted at disease onset, there is emerging data that IFNs may contribute to the transition from preclinical to sustained clinical disease. In ontology studies and network pathway analyses, the IGS distinguished DMARD-naïve early arthritis patients that developed a persistent inflammatory arthritis from those that had a self-limiting course [<xref ref-type=\\\"bibr\\\" rid=\\\"CR16\\\">16</xref>]. In ACPA+ arthralgia populations, i.e. those who are at risk for developing RA, an IGS increases the chance of progression to synovitis, and its inclusion in outcome models improved its predictive capacity [<xref ref-type=\\\"bibr\\\" rid=\\\"CR17\\\">17</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR18\\\">18</xref>]. Even in healthy asymptomatic CCP+ individuals, there was evidence of increased IFN-α signalling which mirrored what was seen in early RA cohorts, and this, with other parameters, was able to differentiate progressors with a median of 4.1 years before symptom onset, from controls [<xref ref-type=\\\"bibr\\\" rid=\\\"CR19\\\">19</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR20\\\">20</xref>•]. In seropositive and seronegative RA, as well as in high-risk seropositive arthralgia patients, there was an overlap in circulating cytokine profiles with IFN-α, as well as IL-5, and TNF-α upregulated up to 50% in seropositive arthralgia and seropositive RA patients but not in seronegative RA [<xref ref-type=\\\"bibr\\\" rid=\\\"CR21\\\">21</xref>] with an odds ratio (OR) of 21 for RA development in seropositive arthralgia patients [<xref ref-type=\\\"bibr\\\" rid=\\\"CR18\\\">18</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR21\\\">21</xref>].</p>\",\n \"<title>Clinical Characteristics and the IGS</title>\",\n \"<p id=\\\"Par10\\\">There has been conflicting evidence around the impact of an IGS/IFN-I signalling on autoantibody production in RA. In established RA, there is a significant correlation between the IGS and ACPA titres and anti-carbamylated protein (anti-CarP) antibodies as well as with genes linked to B cell differentiation and antibody production [<xref ref-type=\\\"bibr\\\" rid=\\\"CR22\\\">22</xref>]. Conversely, others found no relation between the IGS and the presence and/or titres of ACPA and RF in established disease [<xref ref-type=\\\"bibr\\\" rid=\\\"CR23\\\">23</xref>]. Similarly, a 2016 systematic analysis, involving patients with established RA, found that there was no difference seen in the IGS between ACPA negative and ACPA positive patients [<xref ref-type=\\\"bibr\\\" rid=\\\"CR24\\\">24</xref>]. Conversely, rheumatoid factor (RF) demonstrated a positive association between either the IGS or circulating IFN-α levels in both established and early RA as well as across several autoimmune rheumatic diseases [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>••, <xref ref-type=\\\"bibr\\\" rid=\\\"CR13\\\">13</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR25\\\">25</xref>]. These differences may reflect disease stage but may also reflect variability in the IRGs chosen to represent the IGS, with some using a combination of 19 IRGs [<xref ref-type=\\\"bibr\\\" rid=\\\"CR24\\\">24</xref>] and others using only 6 (IFI27, IFI44L, IFIT1, ISG15, RSAD2 and SIGLEC1) [<xref ref-type=\\\"bibr\\\" rid=\\\"CR25\\\">25</xref>] for example.</p>\",\n \"<p id=\\\"Par11\\\">Multiple observational studies in established RA have found no association between the IGS and disease activity [<xref ref-type=\\\"bibr\\\" rid=\\\"CR13\\\">13</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR24\\\">24</xref>]. This contrasts with early drug naïve RA where in a number of prospective observational studies, a higher IGS in early drug naïve RA, were associated with increased baseline disease activity as well as a poorer response to initial therapies [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR13\\\">13</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR15\\\">15</xref>] which was validated in additional cohorts for specific IGSs [<xref ref-type=\\\"bibr\\\" rid=\\\"CR26\\\">26</xref>].</p>\",\n \"<p id=\\\"Par12\\\">RA, including early disease, is a risk factor for cardiovascular disease (CVD). [<xref ref-type=\\\"bibr\\\" rid=\\\"CR27\\\">27</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR28\\\">28</xref>]. In mouse and human <italic>in vitro</italic> models, IFN-α stimulation of macrophages resulted in significant metabolic rewiring with over 500 metabolic genes, including those related to key processes in the pathophysiology of CVD such as glycolysis, oxidative phosphorylation, fatty acid synthesis, and lipid metabolism [<xref ref-type=\\\"bibr\\\" rid=\\\"CR29\\\">29</xref>]. In addition, endothelial progenitor cells (EPCs), involved in vasculogenesis and repair, have impaired function in RA, with IFN-α implicated in both <italic>in vitro</italic> and <italic>in vivo</italic> studies [<xref ref-type=\\\"bibr\\\" rid=\\\"CR30\\\">30</xref>–<xref ref-type=\\\"bibr\\\" rid=\\\"CR32\\\">32</xref>]. In murine lupus models, prolonged and enhanced IFN-I exposure significantly reduced EPC numbers, with acute exposure affecting only EPC differentiation but not the cellular number [<xref ref-type=\\\"bibr\\\" rid=\\\"CR30\\\">30</xref>]. Finally, IFN-α may also influence CVD by promoting insulin resistance given, as early as the 1980s, IFN-α was shown to impair glucose tolerance and insulin sensitivity [<xref ref-type=\\\"bibr\\\" rid=\\\"CR33\\\">33</xref>] with reversal of this effect in IFNAR-/- mouse models [<xref ref-type=\\\"bibr\\\" rid=\\\"CR34\\\">34</xref>].</p>\",\n \"<title>IFN-I and Its Effects on Cellular Function</title>\",\n \"<title>B and T Cells</title>\",\n \"<p id=\\\"Par13\\\">IFN-I can widely influence B cell activity which may contribute to RA pathophysiology, for example, by supporting B cell survival via increased monocyte B-lymphocyte stimulator (BLyS) production, by direct stimulation of B cells, and indirectly through T cell and Dendritic Cells (DCs) stimulation [<xref ref-type=\\\"bibr\\\" rid=\\\"CR35\\\">35</xref>]. Prolonged B cell survival can lead to increased differentiation into memory and plasma cells, immunoglobulin isotype switching, and autoantibody formation [<xref ref-type=\\\"bibr\\\" rid=\\\"CR36\\\">36</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR37\\\">37</xref>]. Furthermore, IFN-α modifies the plasma cell transcriptome towards a proinflammatory phenotype [<xref ref-type=\\\"bibr\\\" rid=\\\"CR38\\\">38</xref>]. IFN-I regulates BCR signalling, specifically via IFN-αR, which in turn may promote pathways involved in antibody formation and germinal centre development in murine models [<xref ref-type=\\\"bibr\\\" rid=\\\"CR39\\\">39</xref>]. IFN-I can also influence the differentiation of CD4+ T cells towards a Th1 response [<xref ref-type=\\\"bibr\\\" rid=\\\"CR40\\\">40</xref>], fostering B cell activation and subsequent activity [<xref ref-type=\\\"bibr\\\" rid=\\\"CR41\\\">41</xref>]. IFN-I also promotes CD8+ T cell survival and CD8+ cytotoxic T cell activity as well as prolonging the proliferation and expansion of CD8+ antigen specific T cells via inhibition of apoptosis [<xref ref-type=\\\"bibr\\\" rid=\\\"CR42\\\">42</xref>].</p>\",\n \"<title>Dendritic Cells</title>\",\n \"<p id=\\\"Par14\\\">Dendritic cells (DCs) upregulate HLA-DR, CD40, CD80, and CD86 expression upon IFN-I exposure [<xref ref-type=\\\"bibr\\\" rid=\\\"CR43\\\">43</xref>]. DC maturation and enhanced antigen presentation, in the context of increased co-stimulatory molecules, can result in the induction of autoimmunity in predisposed individuals via self-antigen presentation to low affinity autoreactive T cells [<xref ref-type=\\\"bibr\\\" rid=\\\"CR44\\\">44</xref>]. In SLE susceptible mice, IFN-I-treated DCs showed relative apoptosis resistance, this activated DC longevity potentially contributing to the development of autoimmunity [<xref ref-type=\\\"bibr\\\" rid=\\\"CR45\\\">45</xref>]. Conversely, in early drug naive RA, there was no association between the IGS and circulating CD1c or pDC frequency, but there was an inverse association with CD141+ DC frequency [<xref ref-type=\\\"bibr\\\" rid=\\\"CR46\\\">46</xref>]. This highlights the DC subset dependant complexity of IFN-I signalling <italic>in vivo</italic>.</p>\",\n \"<title>Monocytes</title>\",\n \"<p id=\\\"Par15\\\">Classical and non-classical monocytes have been implicated in RA pathogenesis [<xref ref-type=\\\"bibr\\\" rid=\\\"CR47\\\">47</xref>]. How the IGS affects monocyte function <italic>in vivo</italic> in RA remains to be fully examined but, when exposed to IFN-Is <italic>in vitro</italic>, monocytes upregulate TLR7 and IRF expression, resulting in increased responsiveness to subsequent immunostimulatory ligands [<xref ref-type=\\\"bibr\\\" rid=\\\"CR48\\\">48</xref>]. IFN-I exposure also increases expression of CD40, CD80, and CD86 and HLA-DR, ultimately promoting differentiation into a monocyte-derived dendritic cell, or mo-DC, with high capacity for antigen presentation [<xref ref-type=\\\"bibr\\\" rid=\\\"CR43\\\">43</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR49\\\">49</xref>]. Mo-DCs are also known to be increased in the RA synovial compartment and promote Th17 differentiation [<xref ref-type=\\\"bibr\\\" rid=\\\"CR50\\\">50</xref>]. However, as with DCs, what happens <italic>in vivo</italic> may be subset dependant as highlighted by enhanced responsiveness to IFN-α in murine proinflammatory monocytes secondary to increased IFNAR expression when compared with anti-inflammatory monocytes [<xref ref-type=\\\"bibr\\\" rid=\\\"CR51\\\">51</xref>].</p>\",\n \"<title>Neutrophils</title>\",\n \"<p id=\\\"Par16\\\">Neutrophils are one of the first cell types to enter the RA joint and may play an important role in the development and progression of RA [<xref ref-type=\\\"bibr\\\" rid=\\\"CR52\\\">52</xref>]. They are a major contributor to the whole blood IGS in RA, attributed to their uniquely upregulated IFNAR expression, a phenomenon not seen in either healthy controls or RA PBMCs [<xref ref-type=\\\"bibr\\\" rid=\\\"CR53\\\">53</xref>•]. Indeed, next generation sequencing of isolated blood neutrophils has found significantly upregulated IRGs in RA neutrophils compared to healthy controls [<xref ref-type=\\\"bibr\\\" rid=\\\"CR54\\\">54</xref>]. How this increased sensitivity to IFN-I influences neutrophil function is being explored, but, intriguingly, the pathogenic phenotype proposed for RA consists of delayed neutrophil apoptosis, increased ROS production and chemokine expression which, in part, can be recapitulated by IFN-I exposure <italic>in vitro</italic> [<xref ref-type=\\\"bibr\\\" rid=\\\"CR55\\\">55</xref>•].</p>\",\n \"<title>Fibroblasts</title>\",\n \"<p id=\\\"Par17\\\">Synovial fibroblasts are resident cells in the stroma of joints [<xref ref-type=\\\"bibr\\\" rid=\\\"CR56\\\">56</xref>], and we recently demonstrated comparable IFN-α levels in serum and early RA synovial fluid [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>••]. In RA, these fibroblasts have an activated phenotype, characterised by resistance to apoptosis, and increased proliferation and production of inflammatory mediators that promote immune cell differentiation and survival [<xref ref-type=\\\"bibr\\\" rid=\\\"CR57\\\">57</xref>]. Histology and RNA sequencing of early RA synovial tissue demonstrated three distinct pathotypes: fibroblastic pauci-immune, macrophage-rich diffuse myeloid, and a lympho-myeloid pathotype [<xref ref-type=\\\"bibr\\\" rid=\\\"CR58\\\">58</xref>••]. In the lympho-myeloid pathotype, seven out of the eight differentially expressed blood transcripts in synovial versus whole blood were IFN-I responses genes (IFI27, ISG15, IFI44L, OASL, USP18, RSAD2, LY6E) [<xref ref-type=\\\"bibr\\\" rid=\\\"CR58\\\">58</xref>•]. In addition, a pathogenic subset of sub-lining fibroblasts (<italic>THY1</italic><sup><italic>+</italic></sup><italic>HLA</italic><sup><italic>−</italic></sup><italic>DR</italic><sup><italic>high</italic></sup>) have increased IRG expression [<xref ref-type=\\\"bibr\\\" rid=\\\"CR59\\\">59</xref>]. However, this may not directly be secondary to IFN-I as TNF-α induced signalling co-opts the mTOR pathway to shift fibroblast like synoviocytes towards an IFN response [<xref ref-type=\\\"bibr\\\" rid=\\\"CR60\\\">60</xref>] which has been shown to be via secondary autocrine production of IFNβ and subsequent activation of the IRF1-IFNβ-IFNAR-JAK-STAT1 axis [<xref ref-type=\\\"bibr\\\" rid=\\\"CR61\\\">61</xref>]. Nevertheless, the role of IFN-α on fibroblast function in RA remains an important research question.</p>\",\n \"<p id=\\\"Par18\\\">Cumulatively, these effects are likely to contribute to a highly activated and potentially autoimmune prone phenotype as summarised in Fig. ##FIG##2##3##.</p>\",\n \"<title>Source of IFN-I in RA</title>\",\n \"<p id=\\\"Par19\\\">pDCs, particularly in their immature state, are the main IFN-I producing cell, however, whether they are the primary source of IFN-α in RA remains unclear. In SLE, there is an element of so-called pDC fatigue, where the ability of the pDC to produce IFN-I reduces and other cells take over production [<xref ref-type=\\\"bibr\\\" rid=\\\"CR62\\\">62</xref>]. In early RA, circulating pDCs were not the primary source of <italic>IFNA</italic> transcript, with comparable expression in circulating lymphocytes. However, circulating pDC numbers were reduced with increased CCR7 expression inferring increased migration to the synovial compartment and target tissue [<xref ref-type=\\\"bibr\\\" rid=\\\"CR46\\\">46</xref>]. Indeed, in established RA patients, the synovial compartment has increased numbers of pDCs with reduced numbers seen in peripheral blood. However, those that remained in the circulation were immature with inferred increased IFN-I producing capacity [<xref ref-type=\\\"bibr\\\" rid=\\\"CR63\\\">63</xref>]. Nevertheless, RA synovial pDCs are potent producers of IFN-α [<xref ref-type=\\\"bibr\\\" rid=\\\"CR64\\\">64</xref>] and, in mice, intraarticular transfer of IFN-I producing dendritic cells was sufficient to propagate a persistent inflammatory arthritis [<xref ref-type=\\\"bibr\\\" rid=\\\"CR65\\\">65</xref>].</p>\",\n \"<p id=\\\"Par20\\\">Conversely, after arthritogenic serum transfer in K/BxN serum-induced arthritis, collagen-induced arthritis, and human TNF transgene insertion, only pDC deficient mice showed exacerbations of symptoms and signs of inflammatory arthritis [<xref ref-type=\\\"bibr\\\" rid=\\\"CR66\\\">66</xref>] and topical imiquimod, a TLR7 agonist, increased pDC recruitment and activity which subsequently improved arthritis [<xref ref-type=\\\"bibr\\\" rid=\\\"CR66\\\">66</xref>]. Furthermore, transcriptomic analysis of circulating pDCs in early RA suggested enhanced tolerogenic function [<xref ref-type=\\\"bibr\\\" rid=\\\"CR46\\\">46</xref>]. These discrepancies may arise due to the complexities of DC development [<xref ref-type=\\\"bibr\\\" rid=\\\"CR67\\\">67</xref>] and cellular differences across species. Given their relative paucity <italic>in vivo</italic>, pDCs have been relatively neglected in RA research; however, better understanding of their complexity, particularly in relation to any location specific function, will help inform their role in RA and role in IFN-I production.</p>\",\n \"<title>Potential Triggers of IFN Production</title>\",\n \"<p id=\\\"Par21\\\">What drives the observed increased IGS/IFN-α in RA remains unclear; however, triggers may include viral infections or microbial DNA or antigen fragments, with these elements repeatedly reported in the joints of RA patients [<xref ref-type=\\\"bibr\\\" rid=\\\"CR68\\\">68</xref>–<xref ref-type=\\\"bibr\\\" rid=\\\"CR70\\\">70</xref>]. Retroelements are non-protein encoding portions of DNA derived from ancient transposable elements, such as retroviruses, that have been historically incorporated into the genome. Their activity can trigger intracellular viral sensors and thus promote local IFN-I production [<xref ref-type=\\\"bibr\\\" rid=\\\"CR71\\\">71</xref>]. In SLE and primary Sjogren’s syndrome, increased retrotransposon activity in disease relevant tissue associated with increased local IFN-α production [<xref ref-type=\\\"bibr\\\" rid=\\\"CR72\\\">72</xref>•], and, in established RA synovium, there is also increased retroelement expression [<xref ref-type=\\\"bibr\\\" rid=\\\"CR73\\\">73</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR74\\\">74</xref>]. Furthermore, in a subgroup of RA patients, a transcriptional profile was documented, reminiscent of a viral infection, which associated with both IFN-I signalling as well as increased ACPA titres [<xref ref-type=\\\"bibr\\\" rid=\\\"CR75\\\">75</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR76\\\">76</xref>]. How these retroelements may influence IFN-I production in RA remains to be seen.</p>\",\n \"<p id=\\\"Par22\\\">Cell-free nucleic acids have been extensively implicated in IFN-I generation in SLE [<xref ref-type=\\\"bibr\\\" rid=\\\"CR77\\\">77</xref>] and monogenic interferonopathies [<xref ref-type=\\\"bibr\\\" rid=\\\"CR78\\\">78</xref>]. Mouse models with DNA clearance defects develop autoantibody-mediated chronic polyarthritis, resembling human RA [<xref ref-type=\\\"bibr\\\" rid=\\\"CR79\\\">79</xref>]. This corroborates RA human observational data, where evidence of raised levels of circulating cell -free DNA have been found in both peripheral blood [<xref ref-type=\\\"bibr\\\" rid=\\\"CR80\\\">80</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR81\\\">81</xref>] and synovial fluid [<xref ref-type=\\\"bibr\\\" rid=\\\"CR82\\\">82</xref>]. Although direct links with IFN-I were not made in these human RA studies, a similar mechanism to that described in SLE may be present<italic>.</italic></p>\",\n \"<p id=\\\"Par23\\\">Neutrophils, found in high numbers in RA synovium, can undergo NETosis, a unique form of cell death which has been proposed as a potential trigger for IFN-I production [<xref ref-type=\\\"bibr\\\" rid=\\\"CR83\\\">83</xref>]. DNA from these NETs form complexes with antimicrobial peptides including LL37, secretory leukocyte protease inhibitor (SLPI), or with immunoglobulins to form immune complexes which facilitate pDC TLR7/9 signalling ultimately culminating in IFN-α production [<xref ref-type=\\\"bibr\\\" rid=\\\"CR84\\\">84</xref>–<xref ref-type=\\\"bibr\\\" rid=\\\"CR86\\\">86</xref>]. In RA, links between NETs and ACPA have been reported [<xref ref-type=\\\"bibr\\\" rid=\\\"CR87\\\">87</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR88\\\">88</xref>] and known pathogenic cytokines in RA, such as TNF-α and IL-17A, as well as IFN-a itself [<xref ref-type=\\\"bibr\\\" rid=\\\"CR89\\\">89</xref>], can also induce NETosis, potentially creating a vicious cycle of inflammation and disease activity [<xref ref-type=\\\"bibr\\\" rid=\\\"CR88\\\">88</xref>].</p>\",\n \"<p id=\\\"Par24\\\">Other potential triggers include lifestyle and environmental factors. An inverse correlation between physical activity and IFN-I signalling has been reported [<xref ref-type=\\\"bibr\\\" rid=\\\"CR90\\\">90</xref>]. In addition, physical activity was associated with downregulation of TLR and IL-17R signalling and reduced inflammatory cytokines production, including IFN-I [<xref ref-type=\\\"bibr\\\" rid=\\\"CR90\\\">90</xref>].</p>\",\n \"<p id=\\\"Par25\\\">Potential triggers are summarised in Fig. ##FIG##3##4##; however, much of the above involves extrapolation from other diseases, such as SLE, and caveats exist including differences in IRG expression and genetic risk between these diseases [<xref ref-type=\\\"bibr\\\" rid=\\\"CR91\\\">91</xref>]. Further work is needed to explore these pathways in RA specifically.</p>\",\n \"<title>Heritable Genetic Risk and IFN-I Signalling</title>\",\n \"<p id=\\\"Par26\\\">As genome-wide association studies (GWAS), and relevant data sets, become more available, numerous single nucleotide polymorphisms (SNPs) have been identified as contributing to the genetic risk of RA. Interestingly, a number of these SNPs are in genes related to the IFN-I response pathway including DNA-sensing proteins, toll-like receptors, and JAK-STAT protein mediators. These are summarised in Table ##TAB##0##1##. However, the functional consequences of these polymorphisms in RA with regards to IFN-I production or signalling are yet to be elucidated. Nevertheless, an overlap of certain at-risk genes associated with increased IFN-I signalling in SLE has also been linked to RA, for example SNPs in IRF5, STAT4, and PTPN22 [<xref ref-type=\\\"bibr\\\" rid=\\\"CR92\\\">92</xref>]. Some of these RA risk SNPs, such as IRF5 polymorphisms, associate with more severe or erosive disease [<xref ref-type=\\\"bibr\\\" rid=\\\"CR93\\\">93</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR94\\\">94</xref>], which may corroborate the clinical refractory disease phenotype observed in IGS high early RA patients [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>••]. Further work is needed to elucidate both the role of IFN-I on susceptible genetic backgrounds as well as the contribution of these SNPs to IFN-I production.</p>\",\n \"<title>IFN-I and Epigenetics</title>\",\n \"<p id=\\\"Par27\\\">Epigenetic changes are modifications that regulate genome activity, independent of DNA sequence. This occurs via molecular factors and processes, such as DNA methylation of CPG sites or chromatin conformational changes, which subsequently modulate transcription. They are frequently triggered by environmental factors or exposure to inflammatory stimuli, such as cytokines. Methylation changes are noted early in RA progression and vary by cell subset [<xref ref-type=\\\"bibr\\\" rid=\\\"CR95\\\">95</xref>••]. Furthermore, differential methylation has been implicated in initial response to methotrexate in early drug naïve RA patients as well as to certain biologics in established disease [<xref ref-type=\\\"bibr\\\" rid=\\\"CR95\\\">95</xref>••, <xref ref-type=\\\"bibr\\\" rid=\\\"CR96\\\">96</xref>–<xref ref-type=\\\"bibr\\\" rid=\\\"CR98\\\">98</xref>], and these processes are emerging as important modifiers of RA clinical progression and phenotype [<xref ref-type=\\\"bibr\\\" rid=\\\"CR99\\\">99</xref>].</p>\",\n \"<p id=\\\"Par28\\\">Analysis of B and CD4 T cells from early drug naïve RA patients demonstrated differentially methylated CPG sites at disease relevant genes, such as PARP9, STAT1, and EPSTI between IGS high and low patients. It also implicated altered transcription factor binding, cumulatively promoting increased lymphocyte activation, and a proliferative phenotype in the IGS high cohort [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>••]. These data suggest that these changes may be IFN-α induced, and negatively influence clinical trajectory. In undifferentiated arthritis (UA) monocytes, methylation changes, which associated with disease progression and a poor prognosis, were partially recapitulated by monocyte exposure to IFN-α [<xref ref-type=\\\"bibr\\\" rid=\\\"CR100\\\">100</xref>•]. Furthermore, IFN-α treatment causes methylation changes in monocytes similar to those seen in established RA, which <italic>in vivo</italic> were themselves associated with increased disease activity [<xref ref-type=\\\"bibr\\\" rid=\\\"CR101\\\">101</xref>]. Intriguingly, in models of type 1 diabetes, where IFN-I plays a key part in disease initiation, exposure to IFN-α triggered increased TET2 expression. This prompted hypomethylation changes in genes controlling inflammatory and immune pathways, ultimately resulting in their increased expression and disease acceleration [<xref ref-type=\\\"bibr\\\" rid=\\\"CR102\\\">102</xref>]. TET proteins are key players in demethylation and are also increased in early drug naïve RA circulating lymphocytes [<xref ref-type=\\\"bibr\\\" rid=\\\"CR103\\\">103</xref>], however whether this is secondary to IFN-α is unknown.</p>\",\n \"<p id=\\\"Par29\\\">It is important to acknowledge that CPG sites in IRGs themselves are frequently hypomethylated in autoimmune conditions, including RA [<xref ref-type=\\\"bibr\\\" rid=\\\"CR104\\\">104</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR105\\\">105</xref>]. In twin studies of CD4 T cells, hypomethylation of IRGs <italic>IFIT1</italic>, <italic>IRF7</italic>, <italic>MX1</italic>, <italic>OAS1</italic>, <italic>USP18</italic>, <italic>RSAD2,</italic> and <italic>IFI44L</italic> has even been proposed as biomarkers of progression to RA [<xref ref-type=\\\"bibr\\\" rid=\\\"CR104\\\">104</xref>]. This questions whether the IGS could be an artefact of altered gene expression secondary to hypomethylation caused by other circulating inflammatory cytokines, such as IL6, or due to increased IFN-α signalling itself. IFN-α protein is increased in early RA and uniquely correlates with the IGS [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>••], so the reality is likely to involve both mechanisms.</p>\",\n \"<p id=\\\"Par30\\\">Although less extensively investigated, IFN-I-associated chromatin conformational changes may also be relevant to RA pathophysiology. There is variation in chromatin accessibility in RA synovial fibroblasts which is likely influenced by the synovial environment [<xref ref-type=\\\"bibr\\\" rid=\\\"CR106\\\">106</xref>], where IFN-α is known to be present [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>••]. In early RA, chromatin conformation changes in <italic>IFNAR2</italic> were associated with poorer outcomes [<xref ref-type=\\\"bibr\\\" rid=\\\"CR107\\\">107</xref>]. Furthermore, monocytes stimulated with IFN-α have increased trimethylated histone H3 Lys 4 (H3K4me3) which enhances transcription at promotors of genes that encode inflammatory mediators. In a more representative <italic>in vivo</italic> environment, incubation of monocytes with both IFN-α and TNF-α was associated with increased H3K4me3 that reduced monocyte tolerization to LPS and promoted an enhanced response to subsequent environmental challenges [<xref ref-type=\\\"bibr\\\" rid=\\\"CR108\\\">108</xref>]. This intriguingly implicates IFN-I, and chromatin-mediated modifications, with the induction of inflammatory genes beyond canonical IRGs. Indeed, instances where prior exposure to IFN-α can influence cellular response to additional stimuli are increasingly being reported [<xref ref-type=\\\"bibr\\\" rid=\\\"CR102\\\">102</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR109\\\">109</xref>–<xref ref-type=\\\"bibr\\\" rid=\\\"CR111\\\">111</xref>] and remain a potential mechanism whereby IFN-I can influence disease development in RA.</p>\",\n \"<title>The IGS/IFN as a Therapeutic Target</title>\",\n \"<p id=\\\"Par31\\\">Anifrolumab targets IFNAR1 and therefore blocks IFN-α and IFN-β signalling [<xref ref-type=\\\"bibr\\\" rid=\\\"CR112\\\">112</xref>]. In a pilot trial, seven established RA patients, all with a high IGS (<italic>IFI27, IFI44, IFI44L</italic> and <italic>RSAD2</italic>), and active diseases were randomised to anifrolumab or placebo [<xref ref-type=\\\"bibr\\\" rid=\\\"CR113\\\">113</xref>•]. The primary endpoint of an American College of Rheumatology (ACR) response of ≥ 20% after 24 weeks was achieved in patients receiving anifrolumab although only one patient in each arm completed the study despite a safety profile similar to that reported in SLE [<xref ref-type=\\\"bibr\\\" rid=\\\"CR114\\\">114</xref>]. Reasons for early discontinuation in the treatment group included lack of efficacy, hypersensitivity reaction, and infection whilst the control group participants stopped due to insufficient therapeutic response [<xref ref-type=\\\"bibr\\\" rid=\\\"CR113\\\">113</xref>•]. Larger trials are needed to assess the efficacy of this drug in RA.</p>\",\n \"<p id=\\\"Par32\\\">Alternatively, JAK inhibitors (JAKi) suppress phosphorylation of STAT and thus affect downstream IFN signalling and reduce IRG expression [<xref ref-type=\\\"bibr\\\" rid=\\\"CR92\\\">92</xref>]. Indeed, <italic>in vitro</italic> JAKi reduce IFN-I driven plasmablast differentiation [<xref ref-type=\\\"bibr\\\" rid=\\\"CR115\\\">115</xref>], synovial BAFF expression, monocyte-derived DCs costimulatory molecule CD80/CD86 expression, and T cell differentiation into Th1 and Th17 cells [<xref ref-type=\\\"bibr\\\" rid=\\\"CR115\\\">115</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR116\\\">116</xref>] [<xref ref-type=\\\"bibr\\\" rid=\\\"CR61\\\">61</xref>]. The efficacy of JAKi in the treatment of established RA has been widely reported [<xref ref-type=\\\"bibr\\\" rid=\\\"CR117\\\">117</xref>], although how the IGS impacts on its effect has not been comprehensively examined. However, analysis of baricitinib SLE trial data demonstrated that clinical effect was independent of IGS reduction [<xref ref-type=\\\"bibr\\\" rid=\\\"CR118\\\">118</xref>].</p>\",\n \"<p id=\\\"Par33\\\">Other inhibitors of downstream IFN-I signalling include a novel small molecule selective for JAK3/JAK1/TBK1 (tank-1 binding kinase), which, in mouse models, suppressed IFN-I production and osteoclast formation via TBK1 inhibition [<xref ref-type=\\\"bibr\\\" rid=\\\"CR119\\\">119</xref>]. Autoantibody dependent collagen-induced arthritis mice models confirmed the clinical benefit of TBK1 inhibition [<xref ref-type=\\\"bibr\\\" rid=\\\"CR120\\\">120</xref>, <xref ref-type=\\\"bibr\\\" rid=\\\"CR121\\\">121</xref>] and TBK1 deficient mice have reduced IRG and protein expression [<xref ref-type=\\\"bibr\\\" rid=\\\"CR122\\\">122</xref>]. These findings are yet to be reproduced in human studies, but given the interest in cancer regarding TBK1 inhibition [<xref ref-type=\\\"bibr\\\" rid=\\\"CR123\\\">123</xref>], this may provide a novel therapeutic approach.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>\\n</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Funding</title>","<p>This work was supported by the Research into Inflammatory Arthritis Centre Versus Arthritis (RACE) (grant number 22072) and the National Institute for Health and Care Research (NIHR) Newcastle Biomedical Research Centre for Ageing and Long-Term Conditions; views expressed are the authors’ and not necessarily those of the National Health Service, the National Institute of Health and Care Research, or the Department of Health.</p>","<title>Compliance with Ethical Standards</title>","<title>Conflict of Interest</title>","<p id=\"Par35\">JDI discloses research grants from Pfizer, Janssen, and GSK; conference support from Eli Lilly and Gilead; speaker/consulting fees from AbbVie, BMS, Gilead, Roche, and UCB. FAHC discloses speaker fees from AstraZeneca. The remaining authors have no competing interests.</p>","<title>Human and Animal Rights and Informed Consent</title>","<p id=\"Par36\"> This article does not contain any studies with human or animal subjects performed by any of the authors.</p>"],"string":"[\n \"<title>Funding</title>\",\n \"<p>This work was supported by the Research into Inflammatory Arthritis Centre Versus Arthritis (RACE) (grant number 22072) and the National Institute for Health and Care Research (NIHR) Newcastle Biomedical Research Centre for Ageing and Long-Term Conditions; views expressed are the authors’ and not necessarily those of the National Health Service, the National Institute of Health and Care Research, or the Department of Health.</p>\",\n \"<title>Compliance with Ethical Standards</title>\",\n \"<title>Conflict of Interest</title>\",\n \"<p id=\\\"Par35\\\">JDI discloses research grants from Pfizer, Janssen, and GSK; conference support from Eli Lilly and Gilead; speaker/consulting fees from AbbVie, BMS, Gilead, Roche, and UCB. FAHC discloses speaker fees from AstraZeneca. The remaining authors have no competing interests.</p>\",\n \"<title>Human and Animal Rights and Informed Consent</title>\",\n \"<p id=\\\"Par36\\\"> This article does not contain any studies with human or animal subjects performed by any of the authors.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Schematic of interferon (IFN) triggers and downstream signalling pathways. <bold>A</bold> The production of IFN-I can occur following recognition of pathogen-associated molecular patterns (PAMPs), often associated with foreign bacteria or viruses, such as cytosolic DNA and double stranded RNA. These are detected by pattern recognition receptors (PRRs) which comprise of a large repertoire of germline-encoded receptors. These PRRs can be divided into subclasses including cell surface toll-like receptors (TLRs), cytosolic nod-like receptors (NLRs), retinoic acid inducible gene I receptors (RLRs), AIM2 like receptors (ALRs), and cGAS-STING pathway. Recognition of damage-associated molecular patterns (DAMPs) or PAMPS by PRRs results in transcription factor activation, such as TRAF (tumour necrosis factor receptor-associated factor), NF-kB nuclear factor kappa B, activating protein-1 (AP-1), and interferon regulatory factors (IRFs), STING (stimulator of interferon genes), and TBK1 (tank binding kinase 1), all involved in the transcription of IFN-I genes. <bold>B</bold> IFNs are categorised based on their receptor signalling, into IFN-I, IFN-II, and IFN-III. IFN-I signal via a heterodimeric receptor composed of two distinct multi-chain structures, IFN-α receptor 1 and 2 (IFNAR-1 and IFNAR-2) subunits. IFNAR associates with Janus Kinases (JAKs), with the former constitutively associated with JAK1 and the latter associated with tyrosine kinase 2 (TYK2). In response to ligand binding, these JAKs undergo activation and phosphorylate two latent transcription factors, signal transducers, and activators of transcription 1 and 2 (STAT1 and STAT2), resulting in their activation and subsequent heterodimer formation. This binds with IRF9 (IFN regulatory factor 9) or p48 to form a multi-component transcription complex called interferon-stimulated gene factor 3 (ISGF3). This complex translocates to the nucleus and binds to specific sites called IFN-stimulated response elements (ISREs), leading to the transcriptional induction of several IRGs ultimately responsible for IFN-I’s antiviral and immunomodulatory properties. The phosphorylated STAT proteins can alternatively form STAT1-STAT1 homodimers which bind gamma-activated sequences (GASs) to induce pro-inflammatory genes. As IFN-II can also signal via this alternative route (via their own heterodimeric receptor, composed of IFNGR1 and IFNGR2 subunits and associated with JAK1 and JAK 2 signalling), there can be a crossover between IFN-I and IFN-II signalling. Finally, IFN-III signals via its own heterodimeric receptor composed of IL-10R2 and IFNLR1 subunits, associated with the activation of TYK2 and JAK1, respectively. This can result in the formation and activation of STAT1-STAT2 heterodimers which associate with IRF9 to form ISGF3 complexes, with subsequent signalling as per IFN-I. AP-1, activating protein-1; DNA, deoxyribonucleic acid; ER, endoplasmic reticulum; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLR, nod-like receptor; P, phosphate; RLR, rig-I-like receptor; RNA, ribonucleic acid; TRAF, tumour necrosis factor receptor-associated factor.</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Figure highlighting factors that may influence interferon response gene (IRG) expression, as well as additional aspects that can influence the subsequent calculation of the interferon gene signature (IGS). Primarily, class of IFN will dictate IRG expression and thus the resulting IGS calculated, however, additional contributory factors, for example genetic background or IFNAR expression, are highlighted. DAMP, damage associated molecular patterns; IFNAR, IFN alpha receptor; IFNGR, IFN gamma receptor; IFNLR, IFN lambda receptor; IGS, interferon gene signature; IRG, interferon response gene; PAMP, pattern associated molecular pattern; STAT, signal transducer and activators of transcription</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Schematic depicting interaction of cellular subsets in the presence of IFN-I. IFN-α influences the activity of surrounding innate and adaptive immune cells. It remains unknown what initially triggers the cascade of IFN production; however, it has been suggested that the generation of DNA/RNA via cell death pathways including apoptosis, necrosis, and NETosis (with subsequent ROS generation) plays a role. Exposure to these self-antigens increases the risk of developing autoantibodies, which form immune complexes that have potential to interact with IFN-producing cells to enhance further IFN-I production. Monocytes develop an inflammatory phenotype and activated cDCs promote activation of CD4+ and CD8+ T cell subsets. These T cells themselves upon exposure to IFN-I can further enhance B cell activation and mediation of cell death, respectively. cDCs, conventional dendritic cells; IFN-𝛂, interferon-𝛂; NET, neutrophil extracellular traps; ROS, reactive oxygen species</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Figure depicting some potential triggers of IFN-α production. Here, potential triggers are split into three subtypes: (1) cellular comprising of neutrophils, (2) environmental including infections increasing IFN-α production via cellular death and debris and a reduction in physical activity reportedly linked to increased IFN-α levels, and (3) proposed non-cellular host triggers including endogenous retroelement activity and the development of autoantibodies or immune complexes resulting in increased IFN-α production</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Schematic depicting associations between RA disease progression and IFN-𝛂 levels over time. There is increasing evidence that IFN-I is increased at RA disease onset and in at-risk cohorts. Proposed triggers include environmental influences including infection on the background of genetic risk; however, when these events may occur in relation to disease onset or initial immune dysfunction, with regards to autoantibody generation, is unclear. There is emerging evidence that this IFN-α exposure in early RA populations may cause potentially pathogenic epigenetic changes in key cellular subsets which could persist into established disease. IFN-𝛂, interferon-𝛂; RA, rheumatoid arthritis</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Schematic of interferon (IFN) triggers and downstream signalling pathways. <bold>A</bold> The production of IFN-I can occur following recognition of pathogen-associated molecular patterns (PAMPs), often associated with foreign bacteria or viruses, such as cytosolic DNA and double stranded RNA. These are detected by pattern recognition receptors (PRRs) which comprise of a large repertoire of germline-encoded receptors. These PRRs can be divided into subclasses including cell surface toll-like receptors (TLRs), cytosolic nod-like receptors (NLRs), retinoic acid inducible gene I receptors (RLRs), AIM2 like receptors (ALRs), and cGAS-STING pathway. Recognition of damage-associated molecular patterns (DAMPs) or PAMPS by PRRs results in transcription factor activation, such as TRAF (tumour necrosis factor receptor-associated factor), NF-kB nuclear factor kappa B, activating protein-1 (AP-1), and interferon regulatory factors (IRFs), STING (stimulator of interferon genes), and TBK1 (tank binding kinase 1), all involved in the transcription of IFN-I genes. <bold>B</bold> IFNs are categorised based on their receptor signalling, into IFN-I, IFN-II, and IFN-III. IFN-I signal via a heterodimeric receptor composed of two distinct multi-chain structures, IFN-α receptor 1 and 2 (IFNAR-1 and IFNAR-2) subunits. IFNAR associates with Janus Kinases (JAKs), with the former constitutively associated with JAK1 and the latter associated with tyrosine kinase 2 (TYK2). In response to ligand binding, these JAKs undergo activation and phosphorylate two latent transcription factors, signal transducers, and activators of transcription 1 and 2 (STAT1 and STAT2), resulting in their activation and subsequent heterodimer formation. This binds with IRF9 (IFN regulatory factor 9) or p48 to form a multi-component transcription complex called interferon-stimulated gene factor 3 (ISGF3). This complex translocates to the nucleus and binds to specific sites called IFN-stimulated response elements (ISREs), leading to the transcriptional induction of several IRGs ultimately responsible for IFN-I’s antiviral and immunomodulatory properties. The phosphorylated STAT proteins can alternatively form STAT1-STAT1 homodimers which bind gamma-activated sequences (GASs) to induce pro-inflammatory genes. As IFN-II can also signal via this alternative route (via their own heterodimeric receptor, composed of IFNGR1 and IFNGR2 subunits and associated with JAK1 and JAK 2 signalling), there can be a crossover between IFN-I and IFN-II signalling. Finally, IFN-III signals via its own heterodimeric receptor composed of IL-10R2 and IFNLR1 subunits, associated with the activation of TYK2 and JAK1, respectively. This can result in the formation and activation of STAT1-STAT2 heterodimers which associate with IRF9 to form ISGF3 complexes, with subsequent signalling as per IFN-I. AP-1, activating protein-1; DNA, deoxyribonucleic acid; ER, endoplasmic reticulum; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLR, nod-like receptor; P, phosphate; RLR, rig-I-like receptor; RNA, ribonucleic acid; TRAF, tumour necrosis factor receptor-associated factor.</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Figure highlighting factors that may influence interferon response gene (IRG) expression, as well as additional aspects that can influence the subsequent calculation of the interferon gene signature (IGS). Primarily, class of IFN will dictate IRG expression and thus the resulting IGS calculated, however, additional contributory factors, for example genetic background or IFNAR expression, are highlighted. DAMP, damage associated molecular patterns; IFNAR, IFN alpha receptor; IFNGR, IFN gamma receptor; IFNLR, IFN lambda receptor; IGS, interferon gene signature; IRG, interferon response gene; PAMP, pattern associated molecular pattern; STAT, signal transducer and activators of transcription</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Schematic depicting interaction of cellular subsets in the presence of IFN-I. IFN-α influences the activity of surrounding innate and adaptive immune cells. It remains unknown what initially triggers the cascade of IFN production; however, it has been suggested that the generation of DNA/RNA via cell death pathways including apoptosis, necrosis, and NETosis (with subsequent ROS generation) plays a role. Exposure to these self-antigens increases the risk of developing autoantibodies, which form immune complexes that have potential to interact with IFN-producing cells to enhance further IFN-I production. Monocytes develop an inflammatory phenotype and activated cDCs promote activation of CD4+ and CD8+ T cell subsets. These T cells themselves upon exposure to IFN-I can further enhance B cell activation and mediation of cell death, respectively. cDCs, conventional dendritic cells; IFN-𝛂, interferon-𝛂; NET, neutrophil extracellular traps; ROS, reactive oxygen species</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Figure depicting some potential triggers of IFN-α production. Here, potential triggers are split into three subtypes: (1) cellular comprising of neutrophils, (2) environmental including infections increasing IFN-α production via cellular death and debris and a reduction in physical activity reportedly linked to increased IFN-α levels, and (3) proposed non-cellular host triggers including endogenous retroelement activity and the development of autoantibodies or immune complexes resulting in increased IFN-α production</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><p>Schematic depicting associations between RA disease progression and IFN-𝛂 levels over time. There is increasing evidence that IFN-I is increased at RA disease onset and in at-risk cohorts. Proposed triggers include environmental influences including infection on the background of genetic risk; however, when these events may occur in relation to disease onset or initial immune dysfunction, with regards to autoantibody generation, is unclear. There is emerging evidence that this IFN-α exposure in early RA populations may cause potentially pathogenic epigenetic changes in key cellular subsets which could persist into established disease. IFN-𝛂, interferon-𝛂; RA, rheumatoid arthritis</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Known single nucleotide polymorphisms (SNPs) associated with RA genetic risk and how their function may affect IFN-I biology</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Gene with known RA risk variant</th><th>Role in IFN biology</th><th>Reference*</th></tr></thead><tbody><tr><td>TNFAIP3 (A20)</td><td>• NF-κB and other A20-regulated signalling molecules can induce IFN-I</td><td>1</td></tr><tr><td>PADI4</td><td>• PADI4 knockouts resulted in reduced IFN-I responses</td><td>2</td></tr><tr><td>STAT4</td><td>• STAT4 promotes RIG-I signalling independent of its classical activation pathway and promotes IFN-β production in myeloid innate cells</td><td>3</td></tr><tr><td>CD40</td><td>• CD40 can enhance STING-mediated IFN-I responses</td><td>4</td></tr><tr><td>UBE2L3</td><td>• UBE2L3 shown to negatively regulate IFN-I expression</td><td>5</td></tr><tr><td>IFNAR1/IFNGR2</td><td>• Encodes signalling receptors for IFN-I and IFN-II</td><td>6</td></tr><tr><td>ETS1</td><td>• ETS-1 suggested in SLE patient studies to be associated with IFN-I and to negatively regulate ISG3 and ISRE binding sites</td><td>7,8</td></tr><tr><td>PVT1</td><td><p>• PVT1 negative feedback mediator for IFN-I signalling via STAT1 interaction and subsequent reduction of its phosphorylation.</p><p>• IFN-α stimulation shown to upregulate PVT1 RNA expression</p></td><td>9,10</td></tr><tr><td>CDK6</td><td>• CKD6 regulates IFN-I signalling negative feedback loops</td><td>11</td></tr><tr><td>ETV7</td><td>• ETV7 negatively regulates IFN-I signalling</td><td>12</td></tr><tr><td>EOMES</td><td>• EOMES expression is driven by IFN-I signalling in CD8+ T cells which leads to regulation of memory-like CD8+ T cell homeostasis and function</td><td>13</td></tr><tr><td>TYK2</td><td>• TYK2 required for IFN-I induced activation of transcription factors STAT1-4 and downstream signalling</td><td>14</td></tr><tr><td>IRF8</td><td>• Regulates IFN-I production</td><td>15</td></tr><tr><td>Runx1</td><td>• RUNX1 upregulates IFNs and IRGs via IFN-I signalling</td><td>16</td></tr><tr><td>RCAN1</td><td>• RCAN1 protein stability negatively affected by IFN-α treatment via STAT2 activation</td><td>17</td></tr><tr><td>GATA3</td><td><p>• GATA3 overexpression promotes IFN-I expression</p><p>• IFN- α/β treatment suppresses GATA3 expression</p></td><td>18,19</td></tr><tr><td>DDX6</td><td><p>• DDX6 regulates RIG-I mediated IFN-I signalling</p><p>• DDX6 depletion leads to increased IRG expression</p></td><td>20,21</td></tr><tr><td>PRDM1</td><td><p>• Deletion results in impaired IFN-I production</p><p>• Control IKKα and IRF7 activation via direct suppression of Irak3, a negative regulator of TLR signalling</p></td><td>23</td></tr><tr><td>IRF5</td><td><p>• IRF5 shown to a positive regulator of IFN-I signalling</p><p>• Risk haplotype of IRF5 associated with SLE and with increased IFN-I production</p></td><td>24</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Known single nucleotide polymorphisms (SNPs) associated with RA genetic risk and how their function may affect IFN-I biology</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th>Gene with known RA risk variant</th><th>Role in IFN biology</th><th>Reference*</th></tr></thead><tbody><tr><td>TNFAIP3 (A20)</td><td>• NF-κB and other A20-regulated signalling molecules can induce IFN-I</td><td>1</td></tr><tr><td>PADI4</td><td>• PADI4 knockouts resulted in reduced IFN-I responses</td><td>2</td></tr><tr><td>STAT4</td><td>• STAT4 promotes RIG-I signalling independent of its classical activation pathway and promotes IFN-β production in myeloid innate cells</td><td>3</td></tr><tr><td>CD40</td><td>• CD40 can enhance STING-mediated IFN-I responses</td><td>4</td></tr><tr><td>UBE2L3</td><td>• UBE2L3 shown to negatively regulate IFN-I expression</td><td>5</td></tr><tr><td>IFNAR1/IFNGR2</td><td>• Encodes signalling receptors for IFN-I and IFN-II</td><td>6</td></tr><tr><td>ETS1</td><td>• ETS-1 suggested in SLE patient studies to be associated with IFN-I and to negatively regulate ISG3 and ISRE binding sites</td><td>7,8</td></tr><tr><td>PVT1</td><td><p>• PVT1 negative feedback mediator for IFN-I signalling via STAT1 interaction and subsequent reduction of its phosphorylation.</p><p>• IFN-α stimulation shown to upregulate PVT1 RNA expression</p></td><td>9,10</td></tr><tr><td>CDK6</td><td>• CKD6 regulates IFN-I signalling negative feedback loops</td><td>11</td></tr><tr><td>ETV7</td><td>• ETV7 negatively regulates IFN-I signalling</td><td>12</td></tr><tr><td>EOMES</td><td>• EOMES expression is driven by IFN-I signalling in CD8+ T cells which leads to regulation of memory-like CD8+ T cell homeostasis and function</td><td>13</td></tr><tr><td>TYK2</td><td>• TYK2 required for IFN-I induced activation of transcription factors STAT1-4 and downstream signalling</td><td>14</td></tr><tr><td>IRF8</td><td>• Regulates IFN-I production</td><td>15</td></tr><tr><td>Runx1</td><td>• RUNX1 upregulates IFNs and IRGs via IFN-I signalling</td><td>16</td></tr><tr><td>RCAN1</td><td>• RCAN1 protein stability negatively affected by IFN-α treatment via STAT2 activation</td><td>17</td></tr><tr><td>GATA3</td><td><p>• GATA3 overexpression promotes IFN-I expression</p><p>• IFN- α/β treatment suppresses GATA3 expression</p></td><td>18,19</td></tr><tr><td>DDX6</td><td><p>• DDX6 regulates RIG-I mediated IFN-I signalling</p><p>• DDX6 depletion leads to increased IRG expression</p></td><td>20,21</td></tr><tr><td>PRDM1</td><td><p>• Deletion results in impaired IFN-I production</p><p>• Control IKKα and IRF7 activation via direct suppression of Irak3, a negative regulator of TLR signalling</p></td><td>23</td></tr><tr><td>IRF5</td><td><p>• IRF5 shown to a positive regulator of IFN-I signalling</p><p>• Risk haplotype of IRF5 associated with SLE and with increased IFN-I production</p></td><td>24</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list 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BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Curr Rheumatol Rep. 2024 Dec 5; 26(2):37-52"},"package_file":{"kind":"string","value":"oa_package/5b/23/PMC10787895.tar.gz"}}},{"rowIdx":179,"cells":{"accession_id":{"kind":"string","value":"PMC10787896"},"pmid":{"kind":"string","value":"38217570"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par2\">Various technical and procedural modifications have been implemented intraoperatively to reduce fluoroscopy exposure to both urologists and patients, including proper shielding, low power fluoroscopy settings, and conservative use of fluoroscopy [##REF##26025493##1##]. Although these measures significantly reduce radiation exposure to the operating room staff and patient, the surgeon’s hand remains susceptible to radiation exposure using these methods. This is concerning as ionizing radiation is a risk factor for malignancy and many other adverse effects [##REF##18046031##2##, ##REF##20008690##3##]. Surgeons who frequently work within the direct radiation beam may experience skin and nail pigment abnormalities, joint pain, and osteoarthritic changes [##REF##33349545##4##–##REF##18406966##6##]. Furthermore, the full understanding of the health effects of radiation to the hand are currently limited in existing literature [##REF##20332170##7##, ##REF##18480143##8##].</p>","<p id=\"Par3\">Compared to shock wave lithotripsy (SWL) and ureteroscopy, percutaneous nephrolithotomy (PCNL) confers the highest radiation to both the surgeon and patient [##REF##22003848##9##–##REF##20952034##11##]. Radiation exposure to the surgeon includes both scatter and direct radiation with direct exposure being exponentially greater. The most common scenario when surgeons experience direct radiation exposure is while holding the needle during percutaneous access [##REF##22003848##9##, ##REF##26987619##12##]. This becomes even more important as the indications for PCNL expand and as a greater number of urologists obtain their own access [##REF##33430693##13##].</p>","<p id=\"Par4\">To mitigate this exposure, radiation-attenuating gloves and needle holders have been proposed as potential protective measures by reducing penetrating radiation and enabling removal of the hand from the direct radiation beam. However, their effectiveness during percutaneous renal access for PCNL has not been investigated. The aim of this study was to evaluate the effectiveness of a radiation-attenuating glove compared to a novel needle holder in reducing radiation exposure to the surgeon's hand.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par2\\\">Various technical and procedural modifications have been implemented intraoperatively to reduce fluoroscopy exposure to both urologists and patients, including proper shielding, low power fluoroscopy settings, and conservative use of fluoroscopy [##REF##26025493##1##]. Although these measures significantly reduce radiation exposure to the operating room staff and patient, the surgeon’s hand remains susceptible to radiation exposure using these methods. This is concerning as ionizing radiation is a risk factor for malignancy and many other adverse effects [##REF##18046031##2##, ##REF##20008690##3##]. Surgeons who frequently work within the direct radiation beam may experience skin and nail pigment abnormalities, joint pain, and osteoarthritic changes [##REF##33349545##4##–##REF##18406966##6##]. Furthermore, the full understanding of the health effects of radiation to the hand are currently limited in existing literature [##REF##20332170##7##, ##REF##18480143##8##].</p>\",\n \"<p id=\\\"Par3\\\">Compared to shock wave lithotripsy (SWL) and ureteroscopy, percutaneous nephrolithotomy (PCNL) confers the highest radiation to both the surgeon and patient [##REF##22003848##9##–##REF##20952034##11##]. Radiation exposure to the surgeon includes both scatter and direct radiation with direct exposure being exponentially greater. The most common scenario when surgeons experience direct radiation exposure is while holding the needle during percutaneous access [##REF##22003848##9##, ##REF##26987619##12##]. This becomes even more important as the indications for PCNL expand and as a greater number of urologists obtain their own access [##REF##33430693##13##].</p>\",\n \"<p id=\\\"Par4\\\">To mitigate this exposure, radiation-attenuating gloves and needle holders have been proposed as potential protective measures by reducing penetrating radiation and enabling removal of the hand from the direct radiation beam. However, their effectiveness during percutaneous renal access for PCNL has not been investigated. The aim of this study was to evaluate the effectiveness of a radiation-attenuating glove compared to a novel needle holder in reducing radiation exposure to the surgeon's hand.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<title>Study design and set up</title>","<p id=\"Par5\">After approval from the Loma Linda University’s Department of Pathology and Human Anatomy, and in compliance with institutional policies for use of anatomical specimens in research, a simulated percutaneous renal access for PCNL was performed. A male cadaver (body mass index 36.1) was positioned prone and draped in a manner typical for PCNL. A separate cadaveric right upper extremity, representing the surgeon's hand, was positioned to simulate percutaneous right renal access with an access needle (Fig. ##FIG##0##1##).\n</p>","<title>Radiation dose measurements</title>","<p id=\"Par6\">Landauer nanoDot optically stimulated luminescence dosimeter (OSLD) chips (Glenwood, Illinois) were affixed to four locations on the surgeon's hand: the thumb, middle finger, hypothenar eminence, and forearm (Fig. ##FIG##1##2##). Additionally, two OSLD chips were placed on the patient: one on the ventral surface and one on the dorsal surface of the skin directly in line with the right kidney. The index finger was positioned 7.6 cm above the skin overlying the right kidney.</p>","<p id=\"Par7\">Fluoroscopy was performed using a GE OEC 9900 portable C-arm system (GE Medical system, Inc., Salt Lake City, UT) using the default automatic exposure control (AEC) for all trials. The AEC adjusts the milliampere-seconds (mAs) and peak kilovoltage (kVp) based on the target density to provide optimal image quality [##REF##31088307##14##]. The C-arm was positioned over the right kidney with the X-ray source below the table and the image intensifier above the patient at a skin-to-source distance of 20 cm.</p>","<p id=\"Par8\">The study consisted of three groups. The first group served as the control with radiation exposure tested on the surgeon’s hand wearing conventional polyisoprene surgical gloves (Mölnlycke, Gothenburg, Sweden) directly holding the access needle. In the first experimental group, the renal access needle was held directly by the cadaveric arm using radiation-attenuating gloves (AliMed, Dedham, Massachusetts). The second experimental group employed a novel low radiodensity needle holder (Fig. ##FIG##0##1##), designed to facilitate PCNL access while ensuring that the surgeon’s hand is not directly in the line of the radiation beam.</p>","<p id=\"Par9\">Each treatment arm underwent five trials, with each trial having a fluoroscopy time of 300 s. This duration was chosen based on previous studies that reported the average fluoroscopy time during renal access for PCNL [##REF##31037404##15##–##REF##3877433##18##]. The OSLD chips used in the study were read with a microSTARii Dosimetry System (LANDAUER, Glenwood, Illinois). The absorbed dose measurements by OSLD chips were converted to equivalent doses in millisieverts (mSv) using the radiation weighting factor for fluoroscopy specified by the International Commission on Radiological Protection (ICRP) (<italic>w</italic><sub>R</sub> = 1) [##UREF##0##19##].</p>","<p id=\"Par10\">The radiation attenuating surgical gloves are constructed from a radiopaque proprietary material, which does not contain lead or latex. The interior is coated to allow easy donning. They are specifically designed to minimize the ionizing radiation penetrating the surgeon’s hand [##UREF##1##20##]. The needle holder employed in this study is 3D-printed with a 9-inch-long handle and a very low profile on fluoroscopy. The holder was specifically designed for use with a novel needle which includes a hub that securely interfaces with the needle holder for easy maneuverability and the potential for insertion using the handle. For consistency, this same needle was used in each of the three arms to eliminate it as a potential confounding variable.</p>","<p id=\"Par11\">The primary objective was to quantitatively measure and compare the radiation dose to the surgeon’s hand using the three different techniques for obtaining percutaneous renal access. The secondary outcomes were dose received by the patient, as well as the C-arm recorded cumulative radiation dose in mGy, current in mA, and voltage in kVp for each of the three arms.</p>","<title>Statistical analysis</title>","<p id=\"Par12\">The data was statistically analyzed using one-way analysis of variance (ANOVA) and Tukey’s <italic>B</italic> post hoc test using SPSS version 24 (IBM, Armonk, NY). The significance threshold was set at <italic>p</italic> < 0.05.</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<title>Study design and set up</title>\",\n \"<p id=\\\"Par5\\\">After approval from the Loma Linda University’s Department of Pathology and Human Anatomy, and in compliance with institutional policies for use of anatomical specimens in research, a simulated percutaneous renal access for PCNL was performed. A male cadaver (body mass index 36.1) was positioned prone and draped in a manner typical for PCNL. A separate cadaveric right upper extremity, representing the surgeon's hand, was positioned to simulate percutaneous right renal access with an access needle (Fig. ##FIG##0##1##).\\n</p>\",\n \"<title>Radiation dose measurements</title>\",\n \"<p id=\\\"Par6\\\">Landauer nanoDot optically stimulated luminescence dosimeter (OSLD) chips (Glenwood, Illinois) were affixed to four locations on the surgeon's hand: the thumb, middle finger, hypothenar eminence, and forearm (Fig. ##FIG##1##2##). Additionally, two OSLD chips were placed on the patient: one on the ventral surface and one on the dorsal surface of the skin directly in line with the right kidney. The index finger was positioned 7.6 cm above the skin overlying the right kidney.</p>\",\n \"<p id=\\\"Par7\\\">Fluoroscopy was performed using a GE OEC 9900 portable C-arm system (GE Medical system, Inc., Salt Lake City, UT) using the default automatic exposure control (AEC) for all trials. The AEC adjusts the milliampere-seconds (mAs) and peak kilovoltage (kVp) based on the target density to provide optimal image quality [##REF##31088307##14##]. The C-arm was positioned over the right kidney with the X-ray source below the table and the image intensifier above the patient at a skin-to-source distance of 20 cm.</p>\",\n \"<p id=\\\"Par8\\\">The study consisted of three groups. The first group served as the control with radiation exposure tested on the surgeon’s hand wearing conventional polyisoprene surgical gloves (Mölnlycke, Gothenburg, Sweden) directly holding the access needle. In the first experimental group, the renal access needle was held directly by the cadaveric arm using radiation-attenuating gloves (AliMed, Dedham, Massachusetts). The second experimental group employed a novel low radiodensity needle holder (Fig. ##FIG##0##1##), designed to facilitate PCNL access while ensuring that the surgeon’s hand is not directly in the line of the radiation beam.</p>\",\n \"<p id=\\\"Par9\\\">Each treatment arm underwent five trials, with each trial having a fluoroscopy time of 300 s. This duration was chosen based on previous studies that reported the average fluoroscopy time during renal access for PCNL [##REF##31037404##15##–##REF##3877433##18##]. The OSLD chips used in the study were read with a microSTARii Dosimetry System (LANDAUER, Glenwood, Illinois). The absorbed dose measurements by OSLD chips were converted to equivalent doses in millisieverts (mSv) using the radiation weighting factor for fluoroscopy specified by the International Commission on Radiological Protection (ICRP) (<italic>w</italic><sub>R</sub> = 1) [##UREF##0##19##].</p>\",\n \"<p id=\\\"Par10\\\">The radiation attenuating surgical gloves are constructed from a radiopaque proprietary material, which does not contain lead or latex. The interior is coated to allow easy donning. They are specifically designed to minimize the ionizing radiation penetrating the surgeon’s hand [##UREF##1##20##]. The needle holder employed in this study is 3D-printed with a 9-inch-long handle and a very low profile on fluoroscopy. The holder was specifically designed for use with a novel needle which includes a hub that securely interfaces with the needle holder for easy maneuverability and the potential for insertion using the handle. For consistency, this same needle was used in each of the three arms to eliminate it as a potential confounding variable.</p>\",\n \"<p id=\\\"Par11\\\">The primary objective was to quantitatively measure and compare the radiation dose to the surgeon’s hand using the three different techniques for obtaining percutaneous renal access. The secondary outcomes were dose received by the patient, as well as the C-arm recorded cumulative radiation dose in mGy, current in mA, and voltage in kVp for each of the three arms.</p>\",\n \"<title>Statistical analysis</title>\",\n \"<p id=\\\"Par12\\\">The data was statistically analyzed using one-way analysis of variance (ANOVA) and Tukey’s <italic>B</italic> post hoc test using SPSS version 24 (IBM, Armonk, NY). The significance threshold was set at <italic>p</italic> < 0.05.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par13\">During the 300 s of fluoroscopy with AEC settings, the surgeon’s hand in the control group received an average equivalent dose of 3.92 mSv (Table ##TAB##0##1##). Compared to the control, both the radiation-attenuating glove (2.48 mSv) and needle holder (1.37 mSv) reduced the average equivalent dose to the surgeon’s hand (<italic>p</italic> < 0.001; Table ##TAB##1##2## and Fig. ##FIG##2##3##). The needle holder resulted in a significantly lower dose to the surgeon's hand than the radiation-attenuating glove (<italic>p</italic> < 0.001). At all locations on the surgeon's hand, both the radiation-attenuating glove and needle holder had lower doses compared to the control (<italic>p</italic> < 0.05 for all). The needle holder demonstrated a reduced dose that was significant at all locations except the middle finger (<italic>p</italic> = 0.082).</p>","<p id=\"Par14\">Within the control group, the hypothenar eminence received the highest dose (5.64 mSv), while the forearm received the lowest (2.10 mSv; <italic>p</italic> < 0.001; Fig. ##FIG##2##3##). The doses for the middle finger (4.19 mSv) and the thumb (3.74 mSv) were similar (<italic>p</italic> = 0.207). In the radiation-attenuating glove condition, only the hypothenar eminence received a significantly higher dose compared to the other locations (Table ##TAB##0##1##). In the needle holder condition, the middle finger received the highest dose compared to the first digit and forearm, but it was not significantly greater than the hypothenar eminence (Table ##TAB##0##1##).</p>","<p id=\"Par15\">Compared to the control (8.17 mSv), the mean equivalent dose to the dorsal surface of the patient was greater when using a radiation-attenuating glove (8.43 mSv) and less when using the needle holder (7.03 mSv), though these differences were not significant (<italic>p</italic> > 0.05 for all; Table ##TAB##0##1## and Fig. ##FIG##3##4##). However, the radiation-attenuating glove resulted in a significant increase in equivalent dose to the dorsal surface of the patient compared to the needle holder (8.43 vs 7.03 mSv; <italic>p</italic> = 0.027). No significant differences were found in the equivalent dose to the ventral surface of the patient between surgeon hand conditions (<italic>p</italic> > 0.05 for all). Overall, the ventral surface had a significantly higher mean equivalent dose than the dorsal surface of the patient for the control (874.48 vs 8.17 mSv), radiation-attenuating glove (676.24 vs 8.43 mSv), and needle holder (632.75 vs 7.03 mSv) conditions (<italic>p</italic> < 0.001 for all).</p>","<p id=\"Par16\">Using a radiation-attenuating glove resulted in a significant increase in the radiation generated by the fluoroscopy machine compared to using a needle holder (83.49 vs 69.22 mGy; <italic>p</italic> = 0.019; Fig. ##FIG##4##5##). Compared to the control (79.00 mGy), the radiation produced was higher with a radiation-attenuating glove and lower with a needle holder (<italic>p</italic> < 0.05 for all; Table ##TAB##2##3##). Although the kVp and mA were higher when using the radiation-attenuating glove and lower when using the needle holder compared to the control, there was no statistically significant difference (Table ##TAB##2##3##).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par13\\\">During the 300 s of fluoroscopy with AEC settings, the surgeon’s hand in the control group received an average equivalent dose of 3.92 mSv (Table ##TAB##0##1##). Compared to the control, both the radiation-attenuating glove (2.48 mSv) and needle holder (1.37 mSv) reduced the average equivalent dose to the surgeon’s hand (<italic>p</italic> < 0.001; Table ##TAB##1##2## and Fig. ##FIG##2##3##). The needle holder resulted in a significantly lower dose to the surgeon's hand than the radiation-attenuating glove (<italic>p</italic> < 0.001). At all locations on the surgeon's hand, both the radiation-attenuating glove and needle holder had lower doses compared to the control (<italic>p</italic> < 0.05 for all). The needle holder demonstrated a reduced dose that was significant at all locations except the middle finger (<italic>p</italic> = 0.082).</p>\",\n \"<p id=\\\"Par14\\\">Within the control group, the hypothenar eminence received the highest dose (5.64 mSv), while the forearm received the lowest (2.10 mSv; <italic>p</italic> < 0.001; Fig. ##FIG##2##3##). The doses for the middle finger (4.19 mSv) and the thumb (3.74 mSv) were similar (<italic>p</italic> = 0.207). In the radiation-attenuating glove condition, only the hypothenar eminence received a significantly higher dose compared to the other locations (Table ##TAB##0##1##). In the needle holder condition, the middle finger received the highest dose compared to the first digit and forearm, but it was not significantly greater than the hypothenar eminence (Table ##TAB##0##1##).</p>\",\n \"<p id=\\\"Par15\\\">Compared to the control (8.17 mSv), the mean equivalent dose to the dorsal surface of the patient was greater when using a radiation-attenuating glove (8.43 mSv) and less when using the needle holder (7.03 mSv), though these differences were not significant (<italic>p</italic> > 0.05 for all; Table ##TAB##0##1## and Fig. ##FIG##3##4##). However, the radiation-attenuating glove resulted in a significant increase in equivalent dose to the dorsal surface of the patient compared to the needle holder (8.43 vs 7.03 mSv; <italic>p</italic> = 0.027). No significant differences were found in the equivalent dose to the ventral surface of the patient between surgeon hand conditions (<italic>p</italic> > 0.05 for all). Overall, the ventral surface had a significantly higher mean equivalent dose than the dorsal surface of the patient for the control (874.48 vs 8.17 mSv), radiation-attenuating glove (676.24 vs 8.43 mSv), and needle holder (632.75 vs 7.03 mSv) conditions (<italic>p</italic> < 0.001 for all).</p>\",\n \"<p id=\\\"Par16\\\">Using a radiation-attenuating glove resulted in a significant increase in the radiation generated by the fluoroscopy machine compared to using a needle holder (83.49 vs 69.22 mGy; <italic>p</italic> = 0.019; Fig. ##FIG##4##5##). Compared to the control (79.00 mGy), the radiation produced was higher with a radiation-attenuating glove and lower with a needle holder (<italic>p</italic> < 0.05 for all; Table ##TAB##2##3##). Although the kVp and mA were higher when using the radiation-attenuating glove and lower when using the needle holder compared to the control, there was no statistically significant difference (Table ##TAB##2##3##).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par17\">The existing literature extensively covers the various impacts of ionizing radiation such as DNA damage, malignancy, cataracts, dermatologic changes, and delayed wound healing [##REF##18046031##2##–##REF##18406966##6##, ##REF##31402748##21##]. Despite this research evidence, there remains a significant gap in our understanding of the full health effects of chronic low-level radiation exposure [##REF##20332170##7##, ##REF##18480143##8##]. One area of specific concern is the potential damage to the hands of surgeons due to prolonged exposure to significant doses of ionizing radiation during procedures involving fluoroscopic-guided imaging. Urologists, who frequently use fluoroscopy to perform essential procedures, are particularly at risk for repeated low-level radiation exposure. Consequently, it becomes imperative to acknowledge the potential harm associated with all levels of ionizing radiation and take every possible protective measure to reduce exposure to as low as reasonably achievable (ALARA) [##UREF##2##22##]. In addition to posing a significant risk of joint, vascular, and skin pathologies, any adverse effects on a surgeon’s hand can significantly impact their ability to provide effective care and perform surgical procedures safely. To address these concerns and minimize potential harm, the ICRP guidelines suggest not exceeding 20 mSv of average radiation exposure per year over a 5-year period and an annual limit of 50 mSv. Additionally, the ICRP advises maintaining an annual radiation dose limit of 500 mSv to the skin and extremities [##UREF##0##19##].</p>","<p id=\"Par18\">Our study aimed to investigate the efficacy of radiation-attenuating gloves and a novel needle-holder in reducing ionizing radiation exposure to the surgeon’s hands during urological procedures. Using a cadaver model, we evaluated different techniques to reduce radiation exposure during simulated percutaneous renal access and observed significant exposure reductions to the surgeon’s hand. The use of a radiation-attenuating glove resulted in a 37% reduction, while employing a needle holder led to a 65% reduction in the mean equivalent dose to the surgeon’s hand (Table ##TAB##0##1##). The 37% radiation reduction for the radiation-attenuating glove at 95.2 kVp is comparable to the manufacturer specifications of 33% reduction at 100 kVp [##UREF##1##20##]. The decreased radiation dose associated with the radiation-attenuating glove may be attributed to the material’s high attenuation coefficient which reduces penetrating radiation. Similar reductions in exposure have been reported in orthopedics, where radiation-attenuating gloves were found to decrease exposure by 61% in an anthropomorphic model [##REF##31402748##21##]. However, it is worth noting that their study used a mini-C arm with lower kVp and mA settings, unlike the GE OEC 9900 system at AEC settings used in our research. On the other hand, the needle holder further reduced exposure, likely by enabling the surgeon to remove their hand from the direct radiation beam. Notably, there is currently a lack of studies investigating the radiation-reducing effects of a needle holder during percutaneous renal access.</p>","<p id=\"Par19\">Simulated PCNL access demonstrated a mean radiation dose of 3.92 mSv to the hand without protection. Recent studies have reported similar hand radiation exposure during PCNL of 0.36–4.36 mSv [##REF##22003848##9##, ##REF##16868684##23##]. This wide range of hand exposure could be attributed to variations in fluoroscopy use, settings, and surgeon experience. Using the ICRP guidelines, approximately 127 PCNLs may be performed annually using surgical gloves without exceeding the dose limit for extremities. This number increases to 201 with radiation-attenuating gloves and 364 with the needle holder.</p>","<p id=\"Par20\">Custom-made needle holders have been specifically designed for the conventional “bullseye” technique in percutaneous renal access [##REF##31619863##24##, ##REF##17574053##25##]. As demonstrated in our study, these needle holders can potentially reduce radiation exposure to both the surgeon's hand and patient when constructed from a low radiation density material. However, literature on specialized needle holders for PCNL access is sparse. In contrast, the practice of utilizing needle holders has been extensively studied and more commonly employed in fields that frequently rely on fluoroscopic-guided imaging, such as interventional radiology. Studies investigating the use of improvised metal and custom-made plastic needle holders during fluoroscopic-guided interventions demonstrated significantly reduced radiation exposure to the user’s hand [##REF##29193644##26##, ##UREF##3##27##]. In the field of endourology, the utilization of specialized needle holders is not yet widespread, requiring further research to determine their efficacy and safety.</p>","<p id=\"Par21\">Considerable research has been dedicated to reducing radiation exposure during fluoroscopic procedures in the operating room [##REF##26025493##1##]. Implementation of simple yet effective measures, such as appropriate shielding, can lead to a significant reduction of up to 70-fold in radiation exposure [##REF##26025493##1##]. Similarly, operating the fluoroscopy machine at lower power settings is another effective strategy [##REF##26025493##1##]. However, adoption of these practices is not universal. A survey among endourologists revealed that lead aprons were worn in 99.3% of cases, thyroid shields in 98.7%, and radiation-attenuating gloves in only 9.7% [##REF##25917725##28##]. The underuse of radiation-attenuating gloves is most likely multifactorial in nature, potentially due to cost, unacquaintance, inconvenience, or believing further protection is unnecessary due to current occupational dose limit guidelines. For fluoroscopy settings, the AEC setting remains the most commonly used mode due to its ability to obtain optimal quality images [##REF##11158657##29##]. However, low dose modes and pulsed fluoroscopy are sufficient for many procedures [##REF##26025493##1##].</p>","<p id=\"Par22\">The radiation reduction techniques explored in this study had implications not only for the surgeon but also for the patient. While the use of a radiation-attenuating glove reduced radiation exposure to the surgeon's hand by 37%, it resulted in a 3% increase in dose to the patient's dorsal surface (Table ##TAB##0##1##). In contrast, utilizing the needle holder reduced exposure for both the surgeon's hand by 65% and the patient's dorsal surface by 14%. The use of radiation-attenuating gloves may offer protection for the surgeon, but at the cost of increasing the dose to the patient. This is likely due to the effect of introducing hyperdense objects, such as radiation-attenuating gloves, into the path of the fluoroscopy beam. This has been shown to increase the radiation produced by the machine when it is operating in the AEC setting [##REF##31088307##14##]. On the other hand, the needle holder is made from a low-density material and has a slim contour, which may have allowed the fluoroscopy machine to generate a lower radiation dose. This is supported by the findings regarding radiation dose, kVp, and mAs generated by the fluoroscopy machine in our study, which were found to be higher for the radiation-attenuating glove compared to the needle holder (Table ##TAB##2##3##).</p>","<p id=\"Par23\">Limitations of our study include the use of a cadaver model for simulated percutaneous renal access which cannot entirely replicate all aspects of the working environment encountered in a live PCNL. Nonetheless, this approach provided a controlled testing environment that allowed for accurate comparisons of radiation reduction techniques without resultant undue radiation exposure to human subjects. An additional limitation of our study is that it was designed to compare three different methods of holding the needle during fluoroscopic-guided access and did not include a comparison of ultrasound-guided access. We also used the AEC setting and a preset fluoroscopy time for simulated renal access. While the AEC setting is the most commonly used fluoroscopy setting, low-dose settings and pulsed fluoroscopy are also used in practice [##REF##26025493##1##, ##REF##11158657##29##]. The preset fluoroscopy time of 5 min in our study will not be representative of all practices and institutions. Finally, it is important to note that this needle holder and radiation-attenuating glove were only tested in a prone PCNL model, where the surgeon’s hand receives direct radiation exposure. In triangulation and supine PCNL the surgeon’s hand is less likely to encounter direct radiation exposure, and subsequently, these were not tested in our model. Despite these limitations, to our knowledge, this is the first study to assess and compare hand radiation reduction techniques during percutaneous renal access for PCNL in a controlled cadaver model.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par17\\\">The existing literature extensively covers the various impacts of ionizing radiation such as DNA damage, malignancy, cataracts, dermatologic changes, and delayed wound healing [##REF##18046031##2##–##REF##18406966##6##, ##REF##31402748##21##]. Despite this research evidence, there remains a significant gap in our understanding of the full health effects of chronic low-level radiation exposure [##REF##20332170##7##, ##REF##18480143##8##]. One area of specific concern is the potential damage to the hands of surgeons due to prolonged exposure to significant doses of ionizing radiation during procedures involving fluoroscopic-guided imaging. Urologists, who frequently use fluoroscopy to perform essential procedures, are particularly at risk for repeated low-level radiation exposure. Consequently, it becomes imperative to acknowledge the potential harm associated with all levels of ionizing radiation and take every possible protective measure to reduce exposure to as low as reasonably achievable (ALARA) [##UREF##2##22##]. In addition to posing a significant risk of joint, vascular, and skin pathologies, any adverse effects on a surgeon’s hand can significantly impact their ability to provide effective care and perform surgical procedures safely. To address these concerns and minimize potential harm, the ICRP guidelines suggest not exceeding 20 mSv of average radiation exposure per year over a 5-year period and an annual limit of 50 mSv. Additionally, the ICRP advises maintaining an annual radiation dose limit of 500 mSv to the skin and extremities [##UREF##0##19##].</p>\",\n \"<p id=\\\"Par18\\\">Our study aimed to investigate the efficacy of radiation-attenuating gloves and a novel needle-holder in reducing ionizing radiation exposure to the surgeon’s hands during urological procedures. Using a cadaver model, we evaluated different techniques to reduce radiation exposure during simulated percutaneous renal access and observed significant exposure reductions to the surgeon’s hand. The use of a radiation-attenuating glove resulted in a 37% reduction, while employing a needle holder led to a 65% reduction in the mean equivalent dose to the surgeon’s hand (Table ##TAB##0##1##). The 37% radiation reduction for the radiation-attenuating glove at 95.2 kVp is comparable to the manufacturer specifications of 33% reduction at 100 kVp [##UREF##1##20##]. The decreased radiation dose associated with the radiation-attenuating glove may be attributed to the material’s high attenuation coefficient which reduces penetrating radiation. Similar reductions in exposure have been reported in orthopedics, where radiation-attenuating gloves were found to decrease exposure by 61% in an anthropomorphic model [##REF##31402748##21##]. However, it is worth noting that their study used a mini-C arm with lower kVp and mA settings, unlike the GE OEC 9900 system at AEC settings used in our research. On the other hand, the needle holder further reduced exposure, likely by enabling the surgeon to remove their hand from the direct radiation beam. Notably, there is currently a lack of studies investigating the radiation-reducing effects of a needle holder during percutaneous renal access.</p>\",\n \"<p id=\\\"Par19\\\">Simulated PCNL access demonstrated a mean radiation dose of 3.92 mSv to the hand without protection. Recent studies have reported similar hand radiation exposure during PCNL of 0.36–4.36 mSv [##REF##22003848##9##, ##REF##16868684##23##]. This wide range of hand exposure could be attributed to variations in fluoroscopy use, settings, and surgeon experience. Using the ICRP guidelines, approximately 127 PCNLs may be performed annually using surgical gloves without exceeding the dose limit for extremities. This number increases to 201 with radiation-attenuating gloves and 364 with the needle holder.</p>\",\n \"<p id=\\\"Par20\\\">Custom-made needle holders have been specifically designed for the conventional “bullseye” technique in percutaneous renal access [##REF##31619863##24##, ##REF##17574053##25##]. As demonstrated in our study, these needle holders can potentially reduce radiation exposure to both the surgeon's hand and patient when constructed from a low radiation density material. However, literature on specialized needle holders for PCNL access is sparse. In contrast, the practice of utilizing needle holders has been extensively studied and more commonly employed in fields that frequently rely on fluoroscopic-guided imaging, such as interventional radiology. Studies investigating the use of improvised metal and custom-made plastic needle holders during fluoroscopic-guided interventions demonstrated significantly reduced radiation exposure to the user’s hand [##REF##29193644##26##, ##UREF##3##27##]. In the field of endourology, the utilization of specialized needle holders is not yet widespread, requiring further research to determine their efficacy and safety.</p>\",\n \"<p id=\\\"Par21\\\">Considerable research has been dedicated to reducing radiation exposure during fluoroscopic procedures in the operating room [##REF##26025493##1##]. Implementation of simple yet effective measures, such as appropriate shielding, can lead to a significant reduction of up to 70-fold in radiation exposure [##REF##26025493##1##]. Similarly, operating the fluoroscopy machine at lower power settings is another effective strategy [##REF##26025493##1##]. However, adoption of these practices is not universal. A survey among endourologists revealed that lead aprons were worn in 99.3% of cases, thyroid shields in 98.7%, and radiation-attenuating gloves in only 9.7% [##REF##25917725##28##]. The underuse of radiation-attenuating gloves is most likely multifactorial in nature, potentially due to cost, unacquaintance, inconvenience, or believing further protection is unnecessary due to current occupational dose limit guidelines. For fluoroscopy settings, the AEC setting remains the most commonly used mode due to its ability to obtain optimal quality images [##REF##11158657##29##]. However, low dose modes and pulsed fluoroscopy are sufficient for many procedures [##REF##26025493##1##].</p>\",\n \"<p id=\\\"Par22\\\">The radiation reduction techniques explored in this study had implications not only for the surgeon but also for the patient. While the use of a radiation-attenuating glove reduced radiation exposure to the surgeon's hand by 37%, it resulted in a 3% increase in dose to the patient's dorsal surface (Table ##TAB##0##1##). In contrast, utilizing the needle holder reduced exposure for both the surgeon's hand by 65% and the patient's dorsal surface by 14%. The use of radiation-attenuating gloves may offer protection for the surgeon, but at the cost of increasing the dose to the patient. This is likely due to the effect of introducing hyperdense objects, such as radiation-attenuating gloves, into the path of the fluoroscopy beam. This has been shown to increase the radiation produced by the machine when it is operating in the AEC setting [##REF##31088307##14##]. On the other hand, the needle holder is made from a low-density material and has a slim contour, which may have allowed the fluoroscopy machine to generate a lower radiation dose. This is supported by the findings regarding radiation dose, kVp, and mAs generated by the fluoroscopy machine in our study, which were found to be higher for the radiation-attenuating glove compared to the needle holder (Table ##TAB##2##3##).</p>\",\n \"<p id=\\\"Par23\\\">Limitations of our study include the use of a cadaver model for simulated percutaneous renal access which cannot entirely replicate all aspects of the working environment encountered in a live PCNL. Nonetheless, this approach provided a controlled testing environment that allowed for accurate comparisons of radiation reduction techniques without resultant undue radiation exposure to human subjects. An additional limitation of our study is that it was designed to compare three different methods of holding the needle during fluoroscopic-guided access and did not include a comparison of ultrasound-guided access. We also used the AEC setting and a preset fluoroscopy time for simulated renal access. While the AEC setting is the most commonly used fluoroscopy setting, low-dose settings and pulsed fluoroscopy are also used in practice [##REF##26025493##1##, ##REF##11158657##29##]. The preset fluoroscopy time of 5 min in our study will not be representative of all practices and institutions. Finally, it is important to note that this needle holder and radiation-attenuating glove were only tested in a prone PCNL model, where the surgeon’s hand receives direct radiation exposure. In triangulation and supine PCNL the surgeon’s hand is less likely to encounter direct radiation exposure, and subsequently, these were not tested in our model. Despite these limitations, to our knowledge, this is the first study to assess and compare hand radiation reduction techniques during percutaneous renal access for PCNL in a controlled cadaver model.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par24\">Protective measures during percutaneous renal access for PCNL can effectively reduce radiation exposure to a surgeon’s hand. Radiation-attenuating gloves showed a significant reduction in hand exposure but increased patient dose, while needle holders resulted in decreased exposure for both the surgeon and the patient. However, further research is needed to determine the efficacy and safety of radiation-attenuating gloves and needle holders in clinical urologic practice. These findings emphasize the importance of implementing radiation reduction techniques to enhance occupational safety during PCNL access.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par24\\\">Protective measures during percutaneous renal access for PCNL can effectively reduce radiation exposure to a surgeon’s hand. Radiation-attenuating gloves showed a significant reduction in hand exposure but increased patient dose, while needle holders resulted in decreased exposure for both the surgeon and the patient. However, further research is needed to determine the efficacy and safety of radiation-attenuating gloves and needle holders in clinical urologic practice. These findings emphasize the importance of implementing radiation reduction techniques to enhance occupational safety during PCNL access.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p id=\"Par1\">Percutaneous nephrolithotomy confers the highest radiation to the urologist’s hands compared to other urologic procedures. This study compares radiation exposure to the surgeon’s hand and patient’s body when utilizing three different techniques for needle insertion during renal access. Simulated percutaneous renal access was performed using a cadaveric patient and separate cadaveric forearm representing the surgeon’s hand. Three different needle-holding techniques were compared: conventional glove (control), a radiation-attenuating glove, and a novel needle holder. Five 300-s fluoroscopy trials were performed per treatment arm. The primary outcome was radiation dose (mSv) to the surgeon’s hand. The secondary outcome was radiation dose to the patient. One-way ANOVA and Tukey’s B post-hoc tests were performed with <italic>p</italic> < 0.05 considered significant. Compared to the control (3.92 mSv), both the radiation-attenuating glove (2.48 mSv) and the needle holder (1.37 mSv) reduced hand radiation exposure (<italic>p</italic> < 0.001). The needle holder reduced hand radiation compared to the radiation-attenuating glove (<italic>p</italic> < 0.001). The radiation-attenuating glove resulted in greater radiation produced by the C-arm compared to the needle holder (83.49 vs 69.22 mGy; <italic>p</italic> = 0.019). Patient radiation exposure was significantly higher with the radiation-attenuating glove compared to the needle holder (8.43 vs 7.03 mSv; <italic>p</italic> = 0.027). Though radiation-attenuating gloves decreased hand radiation dose by 37%, this came at the price of a 3% increase in patient exposure. In contrast, the needle holder reduced exposure to both the surgeon’s hand by 65% and the patient by 14%. Thus, a well-designed low-density needle holder could optimize radiation safety for both surgeon and patient.</p>","<title>Keywords</title>","<p>Open access funding provided by SCELC, Statewide California Electronic Library Consortium</p>"],"string":"[\n \"<p id=\\\"Par1\\\">Percutaneous nephrolithotomy confers the highest radiation to the urologist’s hands compared to other urologic procedures. This study compares radiation exposure to the surgeon’s hand and patient’s body when utilizing three different techniques for needle insertion during renal access. Simulated percutaneous renal access was performed using a cadaveric patient and separate cadaveric forearm representing the surgeon’s hand. Three different needle-holding techniques were compared: conventional glove (control), a radiation-attenuating glove, and a novel needle holder. Five 300-s fluoroscopy trials were performed per treatment arm. The primary outcome was radiation dose (mSv) to the surgeon’s hand. The secondary outcome was radiation dose to the patient. One-way ANOVA and Tukey’s B post-hoc tests were performed with <italic>p</italic> < 0.05 considered significant. Compared to the control (3.92 mSv), both the radiation-attenuating glove (2.48 mSv) and the needle holder (1.37 mSv) reduced hand radiation exposure (<italic>p</italic> < 0.001). The needle holder reduced hand radiation compared to the radiation-attenuating glove (<italic>p</italic> < 0.001). The radiation-attenuating glove resulted in greater radiation produced by the C-arm compared to the needle holder (83.49 vs 69.22 mGy; <italic>p</italic> = 0.019). Patient radiation exposure was significantly higher with the radiation-attenuating glove compared to the needle holder (8.43 vs 7.03 mSv; <italic>p</italic> = 0.027). Though radiation-attenuating gloves decreased hand radiation dose by 37%, this came at the price of a 3% increase in patient exposure. In contrast, the needle holder reduced exposure to both the surgeon’s hand by 65% and the patient by 14%. Thus, a well-designed low-density needle holder could optimize radiation safety for both surgeon and patient.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by SCELC, Statewide California Electronic Library Consortium</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":["<title>Author contributions</title>","<p>RC: Methodology, Formal Analysis, Investigation, Writing—Original Draft, Writing—Review and Editing. EJ: Methodology, Formal Analysis, Investigation, Writing—Original Draft, Project Administration. CB: Methodology, Investigation. JH: Investigation. ASA: Conceptualization, Methodology, Formal Analysis, Resources, Investigation, Writing—Review and Editing. KS: Writing—Review and Editing. JDB: Conceptualization, Methodology, Resources, Investigation. CR: Resources, Investigation. EAB: Investigation. ZO: Writing—Review and Editing. AF: Writing—Review and Editing. DDB: Conceptualization, Methodology, Validation, Resources, Investigation, Writing—Review and Editing, Supervision.</p>","<title>Funding</title>","<p>Open access funding provided by SCELC, Statewide California Electronic Library Consortium. No funding was received for this study.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par26\">D. D. B. is currently developing the needle and needler holder utilized in this study. No other authors have anything to disclose.</p>"],"string":"[\n \"<title>Author contributions</title>\",\n \"<p>RC: Methodology, Formal Analysis, Investigation, Writing—Original Draft, Writing—Review and Editing. EJ: Methodology, Formal Analysis, Investigation, Writing—Original Draft, Project Administration. CB: Methodology, Investigation. JH: Investigation. ASA: Conceptualization, Methodology, Formal Analysis, Resources, Investigation, Writing—Review and Editing. KS: Writing—Review and Editing. JDB: Conceptualization, Methodology, Resources, Investigation. CR: Resources, Investigation. EAB: Investigation. ZO: Writing—Review and Editing. AF: Writing—Review and Editing. DDB: Conceptualization, Methodology, Validation, Resources, Investigation, Writing—Review and Editing, Supervision.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by SCELC, Statewide California Electronic Library Consortium. No funding was received for this study.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par26\\\">D. D. B. is currently developing the needle and needler holder utilized in this study. No other authors have anything to disclose.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Cadaver hand representing a surgeon’s hand positioning and respective fluoroscopic images obtained during simulated percutaneous renal access on a cadaver patient model using <bold>A</bold> a surgical glove, <bold>B</bold> radiation-attenuating glove, and <bold>C</bold> needle holder</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Optically stimulated luminescence dosimeter (OSLD) chips (red boxes) were fixed on <bold>A</bold> four locations of the surgeon hand model: the lateral distal phalanx of the first digit, ventral distal phalanx of the third digit, hypothenar eminence, and forearm 5 cm proximal to the anterior surface of the radiocarpal joint; <bold>B</bold> two OSLD chips were fixed on the patient model: the ventral and dorsal surface of the skin directly in line with the right kidney</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Mean equivalent dose of the first digit, third digit, hypothenar eminence, forearm, and overall average dose to the surgeon’s hand during simulated percutaneous renal access. Error bars represent one standard deviation. *<italic>p</italic> < 0.05 for all pairwise comparisons between arms except for the 3rd digit between the radiation-attenuating glove versus needle holder arms</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Mean equivalent dose of <bold>A</bold> the dorsal and <bold>B</bold> ventral surface of the patient with either the control, radiation-attenuating glove, or needle holder experimental arms during renal access for percutaneous nephrolithotomy. *<italic>p</italic> < 0.05</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Mean radiation dose produced by fluoroscopy machine during the control, radiation-attenuating glove, and needle holder experimental arms for the surgeon hand model during simulated renal access for percutaneous nephrolithotomy</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Cadaver hand representing a surgeon’s hand positioning and respective fluoroscopic images obtained during simulated percutaneous renal access on a cadaver patient model using <bold>A</bold> a surgical glove, <bold>B</bold> radiation-attenuating glove, and <bold>C</bold> needle holder</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Optically stimulated luminescence dosimeter (OSLD) chips (red boxes) were fixed on <bold>A</bold> four locations of the surgeon hand model: the lateral distal phalanx of the first digit, ventral distal phalanx of the third digit, hypothenar eminence, and forearm 5 cm proximal to the anterior surface of the radiocarpal joint; <bold>B</bold> two OSLD chips were fixed on the patient model: the ventral and dorsal surface of the skin directly in line with the right kidney</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Mean equivalent dose of the first digit, third digit, hypothenar eminence, forearm, and overall average dose to the surgeon’s hand during simulated percutaneous renal access. Error bars represent one standard deviation. *<italic>p</italic> < 0.05 for all pairwise comparisons between arms except for the 3rd digit between the radiation-attenuating glove versus needle holder arms</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Mean equivalent dose of <bold>A</bold> the dorsal and <bold>B</bold> ventral surface of the patient with either the control, radiation-attenuating glove, or needle holder experimental arms during renal access for percutaneous nephrolithotomy. *<italic>p</italic> < 0.05</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><p>Mean radiation dose produced by fluoroscopy machine during the control, radiation-attenuating glove, and needle holder experimental arms for the surgeon hand model during simulated renal access for percutaneous nephrolithotomy</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Mean equivalent radiation dose and percent change versus control for the surgeon hand and patient between the control and radiation reduction techniques</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\">Control (<italic>n</italic> = 5)</th><th align=\"left\" colspan=\"2\">Radiation-attenuating glove (<italic>n</italic> = 5)</th><th align=\"left\" colspan=\"2\">Needle holder (<italic>n</italic> = 5)</th><th align=\"left\" rowspan=\"2\"><italic>p</italic> value</th></tr><tr><th align=\"left\">Mean mSv (SD)</th><th align=\"left\">% Change vs control</th><th align=\"left\">Mean mSv (SD)</th><th align=\"left\">% Change vs control</th><th align=\"left\">Mean mSv (SD)</th><th align=\"left\">% Change vs control</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"8\">Surgeon hand</td></tr><tr><td align=\"left\"> Average</td><td char=\"(\" align=\"char\">3.92 (0.13)</td><td align=\"left\">–</td><td char=\"(\" align=\"char\">2.48 (0.23)</td><td char=\".\" align=\"char\">− 37</td><td char=\"(\" align=\"char\">1.37 (0.11)</td><td char=\".\" align=\"char\">− 65</td><td char=\".\" align=\"char\"><bold> < 0.001*</bold></td></tr><tr><td align=\"left\"> Thumb</td><td char=\"(\" align=\"char\">3.74 (0.23)</td><td align=\"left\">–</td><td char=\"(\" align=\"char\">2.39 (0.41)</td><td char=\".\" align=\"char\">− 36</td><td char=\"(\" align=\"char\">1.04 (0.27)</td><td char=\".\" align=\"char\">− 72</td><td char=\".\" align=\"char\"><bold> < 0.001*</bold></td></tr><tr><td align=\"left\"> Middle finger</td><td char=\"(\" align=\"char\">4.19 (0.34)</td><td align=\"left\">–</td><td char=\"(\" align=\"char\">2.29 (0.37)</td><td char=\".\" align=\"char\">− 45</td><td char=\"(\" align=\"char\">1.83 (0.14)</td><td char=\".\" align=\"char\">− 56</td><td char=\".\" align=\"char\"><bold>< 0.001*</bold></td></tr><tr><td align=\"left\"> Hypothenar eminence</td><td char=\"(\" align=\"char\">5.64 (0.54)</td><td align=\"left\">–</td><td char=\"(\" align=\"char\">3.85 (0.38)</td><td char=\".\" align=\"char\">− 32</td><td char=\"(\" align=\"char\">1.63 (0.10)</td><td char=\".\" align=\"char\">− 71</td><td char=\".\" align=\"char\"><bold>< 0.001*</bold></td></tr><tr><td align=\"left\"> Forearm</td><td char=\"(\" align=\"char\">2.10 (0.10)</td><td align=\"left\">–</td><td char=\"(\" align=\"char\">1.42 (0.24)</td><td char=\".\" align=\"char\">− 33</td><td char=\"(\" align=\"char\">1.00 (0.17)</td><td char=\".\" align=\"char\">− 53</td><td char=\".\" align=\"char\"><bold>< 0.001*</bold></td></tr><tr><td align=\"left\" colspan=\"8\">Patient</td></tr><tr><td align=\"left\"> Dorsal surface</td><td char=\"(\" align=\"char\">8.17 (0.83)</td><td align=\"left\">–</td><td char=\"(\" align=\"char\">8.43 (0.90)</td><td char=\".\" align=\"char\">3</td><td char=\"(\" align=\"char\">7.03 (0.31)</td><td char=\".\" align=\"char\">− 14</td><td char=\".\" align=\"char\"><bold>0.020*</bold></td></tr><tr><td align=\"left\"> Ventral surface</td><td char=\"(\" align=\"char\">874.48 (44.92)</td><td align=\"left\">–</td><td char=\"(\" align=\"char\">676.24 (303.801)</td><td char=\".\" align=\"char\">− 23</td><td char=\"(\" align=\"char\">632.75 (269.67)</td><td char=\".\" align=\"char\">− 28</td><td char=\".\" align=\"char\">0.158</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Tukey post-hoc testing comparison of mean equivalent dose to the surgeon hand and patient model between the control and radiation reduction techniques</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Control</th><th align=\"left\">Radiation-attenuating glove</th><th align=\"left\">Needle holder</th></tr></thead><tbody><tr><td align=\"left\">Surgeon hand average dose (mSv)*</td><td align=\"left\">3.92</td><td align=\"left\">2.48</td><td align=\"left\">1.37</td></tr><tr><td align=\"left\"> Control</td><td align=\"left\">–</td><td align=\"left\"><bold><italic>p</italic></bold><bold> < 0.001</bold><bold>**</bold></td><td align=\"left\"><bold><italic>p</italic></bold><bold> < 0.001</bold><bold>**</bold></td></tr><tr><td align=\"left\"> Radiation-attenuating glove</td><td align=\"left\"/><td align=\"left\">–</td><td align=\"left\"><bold><italic>p</italic></bold><bold> < 0.001</bold><bold>**</bold></td></tr><tr><td align=\"left\"> Needle Holder</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">–</td></tr><tr><td align=\"left\">Patient dorsal surface dose (mSv)</td><td align=\"left\">8.17</td><td align=\"left\">8.43</td><td align=\"left\">7.03</td></tr><tr><td align=\"left\"> Control</td><td align=\"left\">–</td><td align=\"left\"><italic>p</italic> = 0.840</td><td align=\"left\"><italic>p</italic> = 0.073</td></tr><tr><td align=\"left\"> Radiation-attenuating glove</td><td align=\"left\"/><td align=\"left\">–</td><td align=\"left\"><bold><italic>p</italic></bold><bold> = 0.027</bold><bold>**</bold></td></tr><tr><td align=\"left\"> Needle holder</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">–</td></tr><tr><td align=\"left\">Patient ventral surface dose (mSv)</td><td align=\"left\">874.48</td><td align=\"left\">676.24</td><td align=\"left\">632.75</td></tr><tr><td align=\"left\"> Control</td><td align=\"left\">–</td><td align=\"left\"><italic>p</italic> = 0.407</td><td align=\"left\"><italic>p</italic> = 0.275</td></tr><tr><td align=\"left\"> Radiation-attenuating glove</td><td align=\"left\"/><td align=\"left\">–</td><td align=\"left\"><italic>p</italic> = 0.954</td></tr><tr><td align=\"left\"> Needle holder</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">–</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Mean radiation dose, kVp, and mAs produced by the fluoroscopy machine between the control and radiation reduction techniques</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\">Control (<italic>n</italic> = 5)</th><th align=\"left\" colspan=\"2\">Radiation-attenuating glove (<italic>n</italic> = 5)</th><th align=\"left\" colspan=\"2\">Needle holder (<italic>n</italic> = 5)</th><th align=\"left\" rowspan=\"2\"><italic>p</italic> value</th></tr><tr><th align=\"left\">Mean mGy (SD)</th><th align=\"left\">% Change vs control</th><th align=\"left\">Mean mGy (SD)</th><th align=\"left\">% Change vs control</th><th align=\"left\">Mean mGy (SD)</th><th align=\"left\">% Change vs control</th></tr></thead><tbody><tr><td align=\"left\">Radiation produced (mGy)</td><td char=\"(\" align=\"char\">79.00 (6.57)</td><td align=\"left\">–</td><td char=\"(\" align=\"char\">83.49 (9.0)</td><td char=\".\" align=\"char\">5.68</td><td char=\"(\" align=\"char\">69.24 (4.75)</td><td char=\".\" align=\"char\">− 12.37</td><td char=\".\" align=\"char\"><bold>0.026*</bold></td></tr><tr><td align=\"left\">kVp</td><td char=\"(\" align=\"char\">93.60 (2.88)</td><td align=\"left\">–</td><td char=\"(\" align=\"char\">95.2 (2.9)</td><td char=\".\" align=\"char\">1.71</td><td char=\"(\" align=\"char\">90.4 (2.2)</td><td char=\".\" align=\"char\">− 3.42</td><td char=\".\" align=\"char\">0.083</td></tr><tr><td align=\"left\">mAs</td><td char=\"(\" align=\"char\">3.09 (0.11)</td><td align=\"left\">–</td><td char=\"(\" align=\"char\">3.12 (0.12)</td><td char=\".\" align=\"char\">1.29</td><td char=\"(\" align=\"char\">2.95 (0.12)</td><td char=\".\" align=\"char\">− 4.53</td><td char=\".\" align=\"char\">0.116</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Mean equivalent radiation dose and percent change versus control for the surgeon hand and patient between the control and radiation reduction techniques</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" rowspan=\\\"2\\\"/><th align=\\\"left\\\" colspan=\\\"2\\\">Control (<italic>n</italic> = 5)</th><th align=\\\"left\\\" colspan=\\\"2\\\">Radiation-attenuating glove (<italic>n</italic> = 5)</th><th align=\\\"left\\\" colspan=\\\"2\\\">Needle holder (<italic>n</italic> = 5)</th><th align=\\\"left\\\" rowspan=\\\"2\\\"><italic>p</italic> value</th></tr><tr><th align=\\\"left\\\">Mean mSv (SD)</th><th align=\\\"left\\\">% Change vs control</th><th align=\\\"left\\\">Mean mSv (SD)</th><th align=\\\"left\\\">% Change vs control</th><th align=\\\"left\\\">Mean mSv (SD)</th><th align=\\\"left\\\">% Change vs control</th></tr></thead><tbody><tr><td align=\\\"left\\\" colspan=\\\"8\\\">Surgeon hand</td></tr><tr><td align=\\\"left\\\"> Average</td><td char=\\\"(\\\" align=\\\"char\\\">3.92 (0.13)</td><td align=\\\"left\\\">–</td><td char=\\\"(\\\" align=\\\"char\\\">2.48 (0.23)</td><td char=\\\".\\\" align=\\\"char\\\">− 37</td><td char=\\\"(\\\" align=\\\"char\\\">1.37 (0.11)</td><td char=\\\".\\\" align=\\\"char\\\">− 65</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001*</bold></td></tr><tr><td align=\\\"left\\\"> Thumb</td><td char=\\\"(\\\" align=\\\"char\\\">3.74 (0.23)</td><td align=\\\"left\\\">–</td><td char=\\\"(\\\" align=\\\"char\\\">2.39 (0.41)</td><td char=\\\".\\\" align=\\\"char\\\">− 36</td><td char=\\\"(\\\" align=\\\"char\\\">1.04 (0.27)</td><td char=\\\".\\\" align=\\\"char\\\">− 72</td><td char=\\\".\\\" align=\\\"char\\\"><bold> < 0.001*</bold></td></tr><tr><td align=\\\"left\\\"> Middle finger</td><td char=\\\"(\\\" align=\\\"char\\\">4.19 (0.34)</td><td align=\\\"left\\\">–</td><td char=\\\"(\\\" align=\\\"char\\\">2.29 (0.37)</td><td char=\\\".\\\" align=\\\"char\\\">− 45</td><td char=\\\"(\\\" align=\\\"char\\\">1.83 (0.14)</td><td char=\\\".\\\" align=\\\"char\\\">− 56</td><td char=\\\".\\\" align=\\\"char\\\"><bold>< 0.001*</bold></td></tr><tr><td align=\\\"left\\\"> Hypothenar eminence</td><td char=\\\"(\\\" align=\\\"char\\\">5.64 (0.54)</td><td align=\\\"left\\\">–</td><td char=\\\"(\\\" align=\\\"char\\\">3.85 (0.38)</td><td char=\\\".\\\" align=\\\"char\\\">− 32</td><td char=\\\"(\\\" align=\\\"char\\\">1.63 (0.10)</td><td char=\\\".\\\" align=\\\"char\\\">− 71</td><td char=\\\".\\\" align=\\\"char\\\"><bold>< 0.001*</bold></td></tr><tr><td align=\\\"left\\\"> Forearm</td><td char=\\\"(\\\" align=\\\"char\\\">2.10 (0.10)</td><td align=\\\"left\\\">–</td><td char=\\\"(\\\" align=\\\"char\\\">1.42 (0.24)</td><td char=\\\".\\\" align=\\\"char\\\">− 33</td><td char=\\\"(\\\" align=\\\"char\\\">1.00 (0.17)</td><td char=\\\".\\\" align=\\\"char\\\">− 53</td><td char=\\\".\\\" align=\\\"char\\\"><bold>< 0.001*</bold></td></tr><tr><td align=\\\"left\\\" colspan=\\\"8\\\">Patient</td></tr><tr><td align=\\\"left\\\"> Dorsal surface</td><td char=\\\"(\\\" align=\\\"char\\\">8.17 (0.83)</td><td align=\\\"left\\\">–</td><td char=\\\"(\\\" align=\\\"char\\\">8.43 (0.90)</td><td char=\\\".\\\" align=\\\"char\\\">3</td><td char=\\\"(\\\" align=\\\"char\\\">7.03 (0.31)</td><td char=\\\".\\\" align=\\\"char\\\">− 14</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.020*</bold></td></tr><tr><td align=\\\"left\\\"> Ventral surface</td><td char=\\\"(\\\" align=\\\"char\\\">874.48 (44.92)</td><td align=\\\"left\\\">–</td><td char=\\\"(\\\" align=\\\"char\\\">676.24 (303.801)</td><td char=\\\".\\\" align=\\\"char\\\">− 23</td><td char=\\\"(\\\" align=\\\"char\\\">632.75 (269.67)</td><td char=\\\".\\\" align=\\\"char\\\">− 28</td><td char=\\\".\\\" align=\\\"char\\\">0.158</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Tukey post-hoc testing comparison of mean equivalent dose to the surgeon hand and patient model between the control and radiation reduction techniques</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\"/><th align=\\\"left\\\">Control</th><th align=\\\"left\\\">Radiation-attenuating glove</th><th align=\\\"left\\\">Needle holder</th></tr></thead><tbody><tr><td align=\\\"left\\\">Surgeon hand average dose (mSv)*</td><td align=\\\"left\\\">3.92</td><td align=\\\"left\\\">2.48</td><td align=\\\"left\\\">1.37</td></tr><tr><td align=\\\"left\\\"> Control</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\"><bold><italic>p</italic></bold><bold> < 0.001</bold><bold>**</bold></td><td align=\\\"left\\\"><bold><italic>p</italic></bold><bold> < 0.001</bold><bold>**</bold></td></tr><tr><td align=\\\"left\\\"> Radiation-attenuating glove</td><td align=\\\"left\\\"/><td align=\\\"left\\\">–</td><td align=\\\"left\\\"><bold><italic>p</italic></bold><bold> < 0.001</bold><bold>**</bold></td></tr><tr><td align=\\\"left\\\"> Needle Holder</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Patient dorsal surface dose (mSv)</td><td align=\\\"left\\\">8.17</td><td align=\\\"left\\\">8.43</td><td align=\\\"left\\\">7.03</td></tr><tr><td align=\\\"left\\\"> Control</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\"><italic>p</italic> = 0.840</td><td align=\\\"left\\\"><italic>p</italic> = 0.073</td></tr><tr><td align=\\\"left\\\"> Radiation-attenuating glove</td><td align=\\\"left\\\"/><td align=\\\"left\\\">–</td><td align=\\\"left\\\"><bold><italic>p</italic></bold><bold> = 0.027</bold><bold>**</bold></td></tr><tr><td align=\\\"left\\\"> Needle holder</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Patient ventral surface dose (mSv)</td><td align=\\\"left\\\">874.48</td><td align=\\\"left\\\">676.24</td><td align=\\\"left\\\">632.75</td></tr><tr><td align=\\\"left\\\"> Control</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\"><italic>p</italic> = 0.407</td><td align=\\\"left\\\"><italic>p</italic> = 0.275</td></tr><tr><td align=\\\"left\\\"> Radiation-attenuating glove</td><td align=\\\"left\\\"/><td align=\\\"left\\\">–</td><td align=\\\"left\\\"><italic>p</italic> = 0.954</td></tr><tr><td align=\\\"left\\\"> Needle holder</td><td align=\\\"left\\\"/><td align=\\\"left\\\"/><td align=\\\"left\\\">–</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Mean radiation dose, kVp, and mAs produced by the fluoroscopy machine between the control and radiation reduction techniques</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" rowspan=\\\"2\\\"/><th align=\\\"left\\\" colspan=\\\"2\\\">Control (<italic>n</italic> = 5)</th><th align=\\\"left\\\" colspan=\\\"2\\\">Radiation-attenuating glove (<italic>n</italic> = 5)</th><th align=\\\"left\\\" colspan=\\\"2\\\">Needle holder (<italic>n</italic> = 5)</th><th align=\\\"left\\\" rowspan=\\\"2\\\"><italic>p</italic> value</th></tr><tr><th align=\\\"left\\\">Mean mGy (SD)</th><th align=\\\"left\\\">% Change vs control</th><th align=\\\"left\\\">Mean mGy (SD)</th><th align=\\\"left\\\">% Change vs control</th><th align=\\\"left\\\">Mean mGy (SD)</th><th align=\\\"left\\\">% Change vs control</th></tr></thead><tbody><tr><td align=\\\"left\\\">Radiation produced (mGy)</td><td char=\\\"(\\\" align=\\\"char\\\">79.00 (6.57)</td><td align=\\\"left\\\">–</td><td char=\\\"(\\\" align=\\\"char\\\">83.49 (9.0)</td><td char=\\\".\\\" align=\\\"char\\\">5.68</td><td char=\\\"(\\\" align=\\\"char\\\">69.24 (4.75)</td><td char=\\\".\\\" align=\\\"char\\\">− 12.37</td><td char=\\\".\\\" align=\\\"char\\\"><bold>0.026*</bold></td></tr><tr><td align=\\\"left\\\">kVp</td><td char=\\\"(\\\" align=\\\"char\\\">93.60 (2.88)</td><td align=\\\"left\\\">–</td><td char=\\\"(\\\" align=\\\"char\\\">95.2 (2.9)</td><td char=\\\".\\\" align=\\\"char\\\">1.71</td><td char=\\\"(\\\" align=\\\"char\\\">90.4 (2.2)</td><td char=\\\".\\\" align=\\\"char\\\">− 3.42</td><td char=\\\".\\\" align=\\\"char\\\">0.083</td></tr><tr><td align=\\\"left\\\">mAs</td><td char=\\\"(\\\" align=\\\"char\\\">3.09 (0.11)</td><td align=\\\"left\\\">–</td><td char=\\\"(\\\" align=\\\"char\\\">3.12 (0.12)</td><td char=\\\".\\\" align=\\\"char\\\">1.29</td><td char=\\\"(\\\" align=\\\"char\\\">2.95 (0.12)</td><td char=\\\".\\\" align=\\\"char\\\">− 4.53</td><td char=\\\".\\\" align=\\\"char\\\">0.116</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p>Bold indicates statsitically significant <italic>p</italic>-value</p><p><italic>n</italic> number of patients, <italic>mSv</italic> millisievert, <italic>SD</italic> standard deviation</p><p>*<italic>p</italic> < 0.05</p></table-wrap-foot>","<table-wrap-foot><p>Bold indicates statsitically significant <italic>p</italic>-value</p><p><italic>mSv</italic> millisievert</p><p>*All other pairwise comparisons for surgeon hand dose were statistically significant except for third digit for radiation-attenuating glove vs needle holder</p><p>**<italic>p</italic> < 0.05</p></table-wrap-foot>","<table-wrap-foot><p>Bold indicates statsitically significant <italic>p</italic>-value</p><p><italic>kVp</italic> peak kilovoltage, <italic>mAs</italic> milliampere-seconds, <italic>n</italic> number of patients, <italic>mGy</italic> milligray, <italic>SD</italic> standard deviation</p><p>*<italic>p</italic> < 0.05</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>Bold indicates statsitically significant <italic>p</italic>-value</p><p><italic>n</italic> number of patients, <italic>mSv</italic> millisievert, <italic>SD</italic> standard deviation</p><p>*<italic>p</italic> < 0.05</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>Bold indicates statsitically significant <italic>p</italic>-value</p><p><italic>mSv</italic> millisievert</p><p>*All other pairwise comparisons for surgeon hand dose were statistically significant except for third digit for radiation-attenuating glove vs needle holder</p><p>**<italic>p</italic> < 0.05</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>Bold indicates statsitically significant <italic>p</italic>-value</p><p><italic>kVp</italic> peak kilovoltage, <italic>mAs</italic> milliampere-seconds, <italic>n</italic> number of patients, <italic>mGy</italic> milligray, <italic>SD</italic> standard deviation</p><p>*<italic>p</italic> < 0.05</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"240_2023_1510_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"240_2023_1510_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"240_2023_1510_Fig3_HTML\" id=\"MO3\"/>","<graphic xlink:href=\"240_2023_1510_Fig4_HTML\" id=\"MO4\"/>","<graphic xlink:href=\"240_2023_1510_Fig5_HTML\" id=\"MO5\"/>"],"string":"[\n \"<graphic xlink:href=\\\"240_2023_1510_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"240_2023_1510_Fig2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic xlink:href=\\\"240_2023_1510_Fig3_HTML\\\" id=\\\"MO3\\\"/>\",\n \"<graphic 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Ann ICRP. International Commission on Radiological Protection, Sutton (UK), United Kingdom, Pergamon Press 14(4)\"]}, {\"label\": [\"27.\"], \"surname\": [\"Stoeckelhuber\", \"Leibecke\", \"Schulz\"], \"given-names\": [\"BM\", \"T\", \"E\"], \"article-title\": [\"Radiation dose to the radiologist\\u2019s hand during continuous CT fluoroscopy-guided interventions\"], \"source\": [\"CardioVasc Interv Radiol\"], \"year\": [\"2005\"], \"volume\": [\"28\"], \"issue\": [\"5\"], \"fpage\": [\"589\"], \"lpage\": [\"594\"], \"pub-id\": [\"10.1007/s00270-005-0104-2\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":29,"string":"29"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Urolithiasis. 2024 Jan 13; 52(1):27"},"package_file":{"kind":"string","value":"oa_package/3b/6b/PMC10787896.tar.gz"}}},{"rowIdx":180,"cells":{"accession_id":{"kind":"string","value":"PMC10787897"},"pmid":{"kind":"string","value":"38217735"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par3\">The genome integrity of all living organisms is constantly threatened by endogenous and exogenous sources of DNA damage (Lindahl ##REF##8469282##1993##). Endogenous sources include oxidation by reactive oxygen species (ROS), alkylation, mismatch of DNA bases, topoisomerase-DNA complexes, spontaneous base deamination and abasic (apurinic/apyrimidinic, AP) sites. Important exogenous sources are ionizing radiation (IR), ultraviolet (UV) radiation and exposure to chemical agents with genotoxic capacity (Chatterjee and Walker ##REF##28485537##2017##). To cope with such variety of DNA insults, cells have developed a robust DNA damage response (DDR) that activates several DNA repair pathways and DNA damage checkpoints. In addition, certain types of DNA lesions are substrates for DNA damage tolerance pathways (Chatterjee and Walker ##REF##28485537##2017##). The major DNA repair pathways comprise direct reversal of DNA damage, base excision repair (BER), single-strand break repair (SSBR), nucleotide excision repair (NER), mismatch repair (MMR) and double-strand break repair (DSBR) by homologous recombination (HR) or non-homologous end joining (NHEJ). Amongst such mechanisms, BER is required for the repair of a broad range of modified DNA bases (alkylated, oxidized or deaminated) and AP sites (Krokan and Bjoras ##REF##23545420##2013##).</p>","<p id=\"Par4\">BER is initiated by DNA glycosylases that remove the damaged or modified bases generating AP sites, that are repaired either by AP endonucleases or by the AP lyase activity associated with bifunctional DNA glycosylases (Demple and Harrison ##REF##7979257##1994##; Krokan and Bjoras ##REF##23545420##2013##; Seeberg et al. ##UREF##1##2000##). AP endonucleases hydrolyze DNA at the 5′-side of the AP site, leaving 3′-hydroxyl (3′-OH) and 5′-deoxyribose phosphate (5′-dRP) termini (Levin and Demple ##REF##1698278##1990##). AP lyases cleave 3′ to the AP site by β-elimination, generating 3′-phospho-α, β-unsaturated aldehyde (3′-PUA) and 5′-phosphate (5′-P) termini. A subset of AP lyases catalyze β-δ-elimination and generate 3′-phosphate (3′-P) termini (Levin and Demple ##REF##1698278##1990##). Therefore, AP endonucleases and AP lyases generate single-nucleotide gaps with 5′- and 3′-blocked ends, respectively. Once the blocked termini have been processed to canonical 5′-P and 3′-OH ends, gap filling may proceed by insertion of either one nucleotide (short-patch BER, SP-BER) or several nucleotides (long-patch BER, LP-BER) (Cordoba-Cañero et al. ##REF##19682284##2009##; Fortini and Dogliotti ##REF##17129767##2007##). In mammals, DNA polymerase β is involved in gap filling during SP-BER (Srivastava et al. ##REF##9694877##1998##), whereas LP-BER requires replicative DNA polymerases Pol δ and Pol ε (Levin et al. ##REF##9371766##1997##). The last BER step is DNA ligation that, in mammals, is carried out by the complex of XRCC1 and LigIIIα during SP-BER (Nash et al. ##REF##9136882##1997##). In LP-BER Pol δ/ε displace the strand containing the 5′-dRP terminus generating a flap structure that is processed by the flap endonuclease (FEN1), creating a nick that is sealed by LIG1 (Levin et al. ##REF##9371766##1997##).</p>","<p id=\"Par5\">Since AP endonucleases generate a 5′-dRP blocked end, SP-BER requires a 5′-dRP lyase activity to produce 5′-P ends amenable to DNA ligation. In mammals, the mayor dRP lyase activity is intrinsic to Pol β (Srivastava et al. ##REF##9694877##1998##), through an N-terminal 8-kDa domain characteristic of X family of DNA polymerases (Beard and Wilson ##REF##10946231##2000##). The members of the mammalian X family of DNA polymerases are Pol β, Pol λ, Pol μ and terminal deoxyribonucleotidyl transferase (TdT), and they are mainly involved in gap filling during DNA repair (Beard and Wilson ##REF##10946231##2000##). The X family proteins are distributive polymerases involved in synthesis of short segments of DNA. All these enzymes are composed of a DNA polymerization domain at the C-terminus, a DNA binding domain in the central region of the protein and, except for Pol β, a BRCT (BRCA1 C-terminal) domain at the N-terminus (Uchiyama et al. ##REF##18706967##2009##). Both mammalian Pol β and Pol λ display DNA polymerase and 5′-dRP lyase activity (Garcia-Diaz et al. ##REF##11457865##2001##; Matsumoto and Kim ##REF##7624801##1995##), which is needed for SP-BER (Braithwaite et al. ##REF##20805875##2010##). Pol λ, Pol μ and TdT, through their BRCT domain, are involved in NHEJ to repair double-strand DNA breaks generated by DNA damage, and/or V(D)J recombination to create diversity in the immunoglobulins (Fan and Wu ##REF##15451442##2004##; Lee et al. ##REF##14561766##2004##; Mahajan et al. ##REF##12077346##2002##; Nick McElhinny et al. ##REF##16061182##2005##).</p>","<p id=\"Par6\">Plants possess homologs of most BER genes found in other organisms, but there are exceptions. For instance, Pol β and Lig III, involved in SP-BER in mammals, are absent in plants. On the other hand, some BER proteins are only found in plants, indicating that there are some plant-specific BER features that have appeared during evolution (Roldan-Arjona et al. ##REF##31543887##2019##). Since there are no plant homologs of Pol β and LigIII, it was initially believed that plants, unlike mammals, do not use SP-BER to repair damaged bases (Uchiyama et al. ##REF##18247046##2008##). However, it has been reported that <italic>Arabidopsis</italic> cell extracts catalyze DNA repair of uracil and AP sites by both LP- and SP-BER (Cordoba-Cañero et al. ##REF##19682284##2009##). However, the identity of the DNA polymerase(s) involved in plant BER is still unknown.</p>","<p id=\"Par7\">The only member of the X family of DNA polymerases present in higher plants is Pol λ (Uchiyama et al. ##REF##15206945##2004##). By contrast, in the unicellular alga <italic>Chlamydomonas reinhardtii</italic>, in addition to Pol λ, sequences with similarity to X family members Pol μ and TdT have been identified (Morales-Ruiz et al. ##REF##29547780##2018##)<italic>.</italic> The possible role of plant Pol λ in DNA repair has been studied both in rice and <italic>Arabidopsis</italic> (Amoroso et al. ##REF##21325140##2011##; Furukawa et al. ##REF##26074930##2015##; Garcia-Diaz et al. ##REF##10966791##2000##; Roy et al. ##REF##21227935##2011##; Uchiyama et al. ##REF##15206945##2004##). In <italic>Arabidopsis</italic> it has been reported that, as its mammalian homolog, Pol λ performs error-free translesion synthesis past 8-oxoguanine (8-oxoG) (Amoroso et al. ##REF##21325140##2011##; Maga et al. ##REF##19104052##2008##). Both <italic>Arabidopsis</italic> and rice Pol λ have been described to have a highly conserved PCNA-interacting protein-box (PIP box) motif. In <italic>Arabidopsis</italic> the PIP box is involved in mediating the interaction of Pol λ with PCNA2 (proliferating cell nuclear antigen 2) to enhance the efficiency and fidelity of translesion synthesis (Amoroso et al. ##REF##21325140##2011##). Moreover, it has been shown that <italic>Arabidopsis</italic> Pol λ plays a role in NER of UV-B induced DNA damage (Roy et al. ##REF##21227935##2011##). It has been suggested that <italic>Arabidopsis</italic> Pol λ also participates in double-strand break repair. Specifically, an <italic>Arabidopsis</italic> Pol λ T-DNA insertion mutant showed sensitivity to both gamma-irradiation and treatment with radiomimetic agents, such as bleomycin, but not to others DNA damaging treatments including methyl methanesulfonate (MMS) and mitomycin-c (MMC) (Furukawa et al. ##REF##26074930##2015##). All these reports suggest that Pol λ is an important component of the plant DNA damage response. However, our knowledge about the role of plant Pol λ in specific DNA repair processes, such as BER, is still very limited. The biochemical characterization of rice Pol λ indicates that it displays dRP lyase activity (Uchiyama et al. ##REF##15206945##2004##). Nonetheless, although some biochemical properties of <italic>Arabidopsis</italic> Pol λ have been described, there is no evidence reported of its dRP lyase activity (Amoroso et al. ##REF##21325140##2011##; Roy et al. ##REF##21227935##2011##). In this work, we have biochemically characterized the enzymatic activity of <italic>Arabidopsis</italic> Pol λ and, importantly, we have found two residues, K248 and K255, within the 8-kDa domain characteristic of the X family of DNA polymerases, that are needed to repair 5′-dRP blocked ends. Our results show that <italic>Arabidopsis</italic> Pol λ displays both DNA polymerization and dRP lyase activities on DNA substrates mimicking DNA repair intermediates and suggest it could play an important role in plant BER.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par3\\\">The genome integrity of all living organisms is constantly threatened by endogenous and exogenous sources of DNA damage (Lindahl ##REF##8469282##1993##). Endogenous sources include oxidation by reactive oxygen species (ROS), alkylation, mismatch of DNA bases, topoisomerase-DNA complexes, spontaneous base deamination and abasic (apurinic/apyrimidinic, AP) sites. Important exogenous sources are ionizing radiation (IR), ultraviolet (UV) radiation and exposure to chemical agents with genotoxic capacity (Chatterjee and Walker ##REF##28485537##2017##). To cope with such variety of DNA insults, cells have developed a robust DNA damage response (DDR) that activates several DNA repair pathways and DNA damage checkpoints. In addition, certain types of DNA lesions are substrates for DNA damage tolerance pathways (Chatterjee and Walker ##REF##28485537##2017##). The major DNA repair pathways comprise direct reversal of DNA damage, base excision repair (BER), single-strand break repair (SSBR), nucleotide excision repair (NER), mismatch repair (MMR) and double-strand break repair (DSBR) by homologous recombination (HR) or non-homologous end joining (NHEJ). Amongst such mechanisms, BER is required for the repair of a broad range of modified DNA bases (alkylated, oxidized or deaminated) and AP sites (Krokan and Bjoras ##REF##23545420##2013##).</p>\",\n \"<p id=\\\"Par4\\\">BER is initiated by DNA glycosylases that remove the damaged or modified bases generating AP sites, that are repaired either by AP endonucleases or by the AP lyase activity associated with bifunctional DNA glycosylases (Demple and Harrison ##REF##7979257##1994##; Krokan and Bjoras ##REF##23545420##2013##; Seeberg et al. ##UREF##1##2000##). AP endonucleases hydrolyze DNA at the 5′-side of the AP site, leaving 3′-hydroxyl (3′-OH) and 5′-deoxyribose phosphate (5′-dRP) termini (Levin and Demple ##REF##1698278##1990##). AP lyases cleave 3′ to the AP site by β-elimination, generating 3′-phospho-α, β-unsaturated aldehyde (3′-PUA) and 5′-phosphate (5′-P) termini. A subset of AP lyases catalyze β-δ-elimination and generate 3′-phosphate (3′-P) termini (Levin and Demple ##REF##1698278##1990##). Therefore, AP endonucleases and AP lyases generate single-nucleotide gaps with 5′- and 3′-blocked ends, respectively. Once the blocked termini have been processed to canonical 5′-P and 3′-OH ends, gap filling may proceed by insertion of either one nucleotide (short-patch BER, SP-BER) or several nucleotides (long-patch BER, LP-BER) (Cordoba-Cañero et al. ##REF##19682284##2009##; Fortini and Dogliotti ##REF##17129767##2007##). In mammals, DNA polymerase β is involved in gap filling during SP-BER (Srivastava et al. ##REF##9694877##1998##), whereas LP-BER requires replicative DNA polymerases Pol δ and Pol ε (Levin et al. ##REF##9371766##1997##). The last BER step is DNA ligation that, in mammals, is carried out by the complex of XRCC1 and LigIIIα during SP-BER (Nash et al. ##REF##9136882##1997##). In LP-BER Pol δ/ε displace the strand containing the 5′-dRP terminus generating a flap structure that is processed by the flap endonuclease (FEN1), creating a nick that is sealed by LIG1 (Levin et al. ##REF##9371766##1997##).</p>\",\n \"<p id=\\\"Par5\\\">Since AP endonucleases generate a 5′-dRP blocked end, SP-BER requires a 5′-dRP lyase activity to produce 5′-P ends amenable to DNA ligation. In mammals, the mayor dRP lyase activity is intrinsic to Pol β (Srivastava et al. ##REF##9694877##1998##), through an N-terminal 8-kDa domain characteristic of X family of DNA polymerases (Beard and Wilson ##REF##10946231##2000##). The members of the mammalian X family of DNA polymerases are Pol β, Pol λ, Pol μ and terminal deoxyribonucleotidyl transferase (TdT), and they are mainly involved in gap filling during DNA repair (Beard and Wilson ##REF##10946231##2000##). The X family proteins are distributive polymerases involved in synthesis of short segments of DNA. All these enzymes are composed of a DNA polymerization domain at the C-terminus, a DNA binding domain in the central region of the protein and, except for Pol β, a BRCT (BRCA1 C-terminal) domain at the N-terminus (Uchiyama et al. ##REF##18706967##2009##). Both mammalian Pol β and Pol λ display DNA polymerase and 5′-dRP lyase activity (Garcia-Diaz et al. ##REF##11457865##2001##; Matsumoto and Kim ##REF##7624801##1995##), which is needed for SP-BER (Braithwaite et al. ##REF##20805875##2010##). Pol λ, Pol μ and TdT, through their BRCT domain, are involved in NHEJ to repair double-strand DNA breaks generated by DNA damage, and/or V(D)J recombination to create diversity in the immunoglobulins (Fan and Wu ##REF##15451442##2004##; Lee et al. ##REF##14561766##2004##; Mahajan et al. ##REF##12077346##2002##; Nick McElhinny et al. ##REF##16061182##2005##).</p>\",\n \"<p id=\\\"Par6\\\">Plants possess homologs of most BER genes found in other organisms, but there are exceptions. For instance, Pol β and Lig III, involved in SP-BER in mammals, are absent in plants. On the other hand, some BER proteins are only found in plants, indicating that there are some plant-specific BER features that have appeared during evolution (Roldan-Arjona et al. ##REF##31543887##2019##). Since there are no plant homologs of Pol β and LigIII, it was initially believed that plants, unlike mammals, do not use SP-BER to repair damaged bases (Uchiyama et al. ##REF##18247046##2008##). However, it has been reported that <italic>Arabidopsis</italic> cell extracts catalyze DNA repair of uracil and AP sites by both LP- and SP-BER (Cordoba-Cañero et al. ##REF##19682284##2009##). However, the identity of the DNA polymerase(s) involved in plant BER is still unknown.</p>\",\n \"<p id=\\\"Par7\\\">The only member of the X family of DNA polymerases present in higher plants is Pol λ (Uchiyama et al. ##REF##15206945##2004##). By contrast, in the unicellular alga <italic>Chlamydomonas reinhardtii</italic>, in addition to Pol λ, sequences with similarity to X family members Pol μ and TdT have been identified (Morales-Ruiz et al. ##REF##29547780##2018##)<italic>.</italic> The possible role of plant Pol λ in DNA repair has been studied both in rice and <italic>Arabidopsis</italic> (Amoroso et al. ##REF##21325140##2011##; Furukawa et al. ##REF##26074930##2015##; Garcia-Diaz et al. ##REF##10966791##2000##; Roy et al. ##REF##21227935##2011##; Uchiyama et al. ##REF##15206945##2004##). In <italic>Arabidopsis</italic> it has been reported that, as its mammalian homolog, Pol λ performs error-free translesion synthesis past 8-oxoguanine (8-oxoG) (Amoroso et al. ##REF##21325140##2011##; Maga et al. ##REF##19104052##2008##). Both <italic>Arabidopsis</italic> and rice Pol λ have been described to have a highly conserved PCNA-interacting protein-box (PIP box) motif. In <italic>Arabidopsis</italic> the PIP box is involved in mediating the interaction of Pol λ with PCNA2 (proliferating cell nuclear antigen 2) to enhance the efficiency and fidelity of translesion synthesis (Amoroso et al. ##REF##21325140##2011##). Moreover, it has been shown that <italic>Arabidopsis</italic> Pol λ plays a role in NER of UV-B induced DNA damage (Roy et al. ##REF##21227935##2011##). It has been suggested that <italic>Arabidopsis</italic> Pol λ also participates in double-strand break repair. Specifically, an <italic>Arabidopsis</italic> Pol λ T-DNA insertion mutant showed sensitivity to both gamma-irradiation and treatment with radiomimetic agents, such as bleomycin, but not to others DNA damaging treatments including methyl methanesulfonate (MMS) and mitomycin-c (MMC) (Furukawa et al. ##REF##26074930##2015##). All these reports suggest that Pol λ is an important component of the plant DNA damage response. However, our knowledge about the role of plant Pol λ in specific DNA repair processes, such as BER, is still very limited. The biochemical characterization of rice Pol λ indicates that it displays dRP lyase activity (Uchiyama et al. ##REF##15206945##2004##). Nonetheless, although some biochemical properties of <italic>Arabidopsis</italic> Pol λ have been described, there is no evidence reported of its dRP lyase activity (Amoroso et al. ##REF##21325140##2011##; Roy et al. ##REF##21227935##2011##). In this work, we have biochemically characterized the enzymatic activity of <italic>Arabidopsis</italic> Pol λ and, importantly, we have found two residues, K248 and K255, within the 8-kDa domain characteristic of the X family of DNA polymerases, that are needed to repair 5′-dRP blocked ends. Our results show that <italic>Arabidopsis</italic> Pol λ displays both DNA polymerization and dRP lyase activities on DNA substrates mimicking DNA repair intermediates and suggest it could play an important role in plant BER.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Material and methods</title>","<title>DNA substrates</title>","<p id=\"Par8\">Oligonucleotides used (Supplementary Table ##SUPPL##0##S1##) were synthesized by Integrated DNA Technologies (IDT) and purified by PAGE or dual HPLC before use. Double-stranded DNA substrates were prepared by mixing a 5 µM solution of a fluorescein (Fl) or alexa (Al) labelled oligonucleotide with a 10 µM solution of an unlabelled complementary oligonucleotide. DNA substrates with one nucleotide gap with a 3′-OH end were prepared by mixing a 5 µM solution of a 5′-alexa (Al) labelled oligonucleotide (Al_28-OH) with a 10 µM solution of both unlabelled oligonucleotide, corresponding to the complementary strand (CGR_G, CGR_A, CGR_T or CGR_C) and to the oligonucleotide containing a 5′-P, 5′-OH or 5′-THF terminus (P_30-51, OH_30-51 or THF_30-51, respectively). Annealing was carried out by heating at 95 °C for 5 min followed by slowly cooling to room temperature. DNA substrates with a 5′-dRP end were generated by incubating an oligonucleotide duplex containing a U:G mispair with 0.5 U of <italic>Escherichia coli</italic> uracil DNA glycosylase (UDG) (New England Biolabs) and 10 U of human AP endonuclease 1 (APE-1) (New England Biolabs, NEB).</p>","<title>Protein expression and purification</title>","<p id=\"Par9\">The full-length <italic>Arabidopsis thaliana</italic> Pol λ (AtPol λ) cDNA, obtained from the <italic>Arabidopsis</italic> Biological Resource Center (pENTR_221-At1G10520), was subcloned into pET28a expression vector (Novagen) to add a polyhistidine (His<sub>6</sub>) tag at the N-terminus of AtPol λ protein. Expression was carried out in <italic>E. coli</italic> BL21 (DE3) <italic>dcm</italic><sup><italic>−</italic></sup> Codon Plus cells (Stratagene) by adding 1 mM isopropyl-1-thio-β-D-galactopyranoside. His-AtPol λ was purified by affinity chromatography on a Ni<sup>2+</sup>-Sepharose column (HisTrap HP; GE Healthcare). Protein was eluted with a 5 mM to 1 M gradient of imidazole and analyzed by SDS/PAGE (10%) using broad-range molecular weight standards (Bio-Rad).</p>","<title>Other enzymes and reagents</title>","<p id=\"Par10\"><italic>Escherichia coli</italic> UDG and human APE1 were obtained from NEB. Human Pol β was purchased from Trevigen, T4 DNA ligase was obtained from Promega and Taq DNA Polymerase from Bioline. Anti-AtPol λ antibodies were generated by injecting rabbits with His-AtPol λ.</p>","<title>Site-directed mutagenesis</title>","<p id=\"Par11\">Site-directed mutagenesis was performed using the Quick-Change II XL kit (Stratagene). The mutations were introduced into the expression vector pET28a (Novagen) containing the full-length wild-type (WT) <italic>AtPol</italic> λ cDNA using specific oligonucleotides (Supplementary Table S2). Mutational changes were confirmed by DNA sequencing and the constructs were used to transform <italic>Escherichia coli</italic> BL21 (DE3) <italic>dcm</italic><sup><italic>−</italic></sup> Codon Plus cells (Stratagene). Mutant proteins were expressed and purified as describe above.</p>","<title>Gap-filling assay</title>","<p id=\"Par12\">Reactions (10 μl) contained 50 mM Tris HCl pH 7.5, 1 mM DTT, 0.2 mg/ml BSA, 2% glycerol, 10 mM MgCl<sub>2</sub>, 100 nM DNA substrate, the indicated amount of AtPol λ and dCTP, dNTPs (deoxynucleotides) or ddNTPs (dideoxynucleotides). When indicated, AtPol λ was pre-incubated with pre-immune serum or anti-AtPol λ antibody for one hour at 4 °C. After incubation at 37 °C for the indicated times, reactions were stopped by adding 20 mM EDTA, 0.6% SDS and 0.5 mg/ml proteinase K, and mixtures were incubated at 37 °C for 30 min. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitated at -20 °C in the presence of 0.3 mM NaCl and 16 mg/ml glycogen. Samples were resuspended in 10 µl of 90% formamide and heated at 95 °C for 5 min. Reaction products were separated in a 12% denaturing polyacrylamide gel containing 7 M urea. Labelled DNA was visualized using FLA-5100 imager (Fujifilm) and analyzed using Multi Gauge software version 3.0 (Fujifilm).</p>","<title>dRP lyase assay</title>","<p id=\"Par13\">Reactions (50 µl) contained 45 mM HEPES–KOH pH 7.8, 70 mM KCl, 5 mM MgCl<sub>2,</sub> 1 mM DTT, 0.4 mM EDTA, 2 mM ATP, 36 µg BSA, 1 mM NAD, 0.2% glycerol, 22 mM phosphocreatine, 2.5 ng creatine kinase, 0.02 mM dCTP, and a DNA substrate with a 5′-dRP (100 nM) prepared as described above. When indicated, human Pol β (2.4 U) or AtPol λ (10 nM), were added. After incubation at 30 °C for 90 min, reaction products were stabilized by the addition of freshly prepared sodium borohydride (NaBH<sub>4</sub>; Sigma-Aldrich) to a final concentration of 300 mM and incubated on ice for 30 min. Subsequently, the samples were desalted using microspin G-25 columns (GE Healthcare). The reactions were stopped, DNA was extracted, and samples were processed as described above.</p>","<title>In vitro reconstitution of base excision repair</title>","<p id=\"Par14\">BER reactions (50 μl) were performed in 45 mM HEPES–KOH pH 7.8, 70 mM KCl, 5 mM MgCl<sub>2</sub>, 1 mM DTT, 0.4 mM EDTA, 2 mM ATP, 36 μg BSA, 1 mM NAD, 0.2% glycerol, 22 mM phosphocreatine, 2.5 ng creatine kinase, 0.02 mM dCTP, and a DNA duplex containing a U:G mispair (100 nM). All reactions contained UDG (0.5 U) and APE1 (10 U). When indicated, human Pol β (2.4 U), AtPol λ (10 nM), and/or T4 DNA ligase (1.5 U), were added. Mixtures were incubated at 30 °C for 90 min, and the reactions were stopped, DNA was extracted, and samples were processed as described above.</p>"],"string":"[\n \"<title>Material and methods</title>\",\n \"<title>DNA substrates</title>\",\n \"<p id=\\\"Par8\\\">Oligonucleotides used (Supplementary Table ##SUPPL##0##S1##) were synthesized by Integrated DNA Technologies (IDT) and purified by PAGE or dual HPLC before use. Double-stranded DNA substrates were prepared by mixing a 5 µM solution of a fluorescein (Fl) or alexa (Al) labelled oligonucleotide with a 10 µM solution of an unlabelled complementary oligonucleotide. DNA substrates with one nucleotide gap with a 3′-OH end were prepared by mixing a 5 µM solution of a 5′-alexa (Al) labelled oligonucleotide (Al_28-OH) with a 10 µM solution of both unlabelled oligonucleotide, corresponding to the complementary strand (CGR_G, CGR_A, CGR_T or CGR_C) and to the oligonucleotide containing a 5′-P, 5′-OH or 5′-THF terminus (P_30-51, OH_30-51 or THF_30-51, respectively). Annealing was carried out by heating at 95 °C for 5 min followed by slowly cooling to room temperature. DNA substrates with a 5′-dRP end were generated by incubating an oligonucleotide duplex containing a U:G mispair with 0.5 U of <italic>Escherichia coli</italic> uracil DNA glycosylase (UDG) (New England Biolabs) and 10 U of human AP endonuclease 1 (APE-1) (New England Biolabs, NEB).</p>\",\n \"<title>Protein expression and purification</title>\",\n \"<p id=\\\"Par9\\\">The full-length <italic>Arabidopsis thaliana</italic> Pol λ (AtPol λ) cDNA, obtained from the <italic>Arabidopsis</italic> Biological Resource Center (pENTR_221-At1G10520), was subcloned into pET28a expression vector (Novagen) to add a polyhistidine (His<sub>6</sub>) tag at the N-terminus of AtPol λ protein. Expression was carried out in <italic>E. coli</italic> BL21 (DE3) <italic>dcm</italic><sup><italic>−</italic></sup> Codon Plus cells (Stratagene) by adding 1 mM isopropyl-1-thio-β-D-galactopyranoside. His-AtPol λ was purified by affinity chromatography on a Ni<sup>2+</sup>-Sepharose column (HisTrap HP; GE Healthcare). Protein was eluted with a 5 mM to 1 M gradient of imidazole and analyzed by SDS/PAGE (10%) using broad-range molecular weight standards (Bio-Rad).</p>\",\n \"<title>Other enzymes and reagents</title>\",\n \"<p id=\\\"Par10\\\"><italic>Escherichia coli</italic> UDG and human APE1 were obtained from NEB. Human Pol β was purchased from Trevigen, T4 DNA ligase was obtained from Promega and Taq DNA Polymerase from Bioline. Anti-AtPol λ antibodies were generated by injecting rabbits with His-AtPol λ.</p>\",\n \"<title>Site-directed mutagenesis</title>\",\n \"<p id=\\\"Par11\\\">Site-directed mutagenesis was performed using the Quick-Change II XL kit (Stratagene). The mutations were introduced into the expression vector pET28a (Novagen) containing the full-length wild-type (WT) <italic>AtPol</italic> λ cDNA using specific oligonucleotides (Supplementary Table S2). Mutational changes were confirmed by DNA sequencing and the constructs were used to transform <italic>Escherichia coli</italic> BL21 (DE3) <italic>dcm</italic><sup><italic>−</italic></sup> Codon Plus cells (Stratagene). Mutant proteins were expressed and purified as describe above.</p>\",\n \"<title>Gap-filling assay</title>\",\n \"<p id=\\\"Par12\\\">Reactions (10 μl) contained 50 mM Tris HCl pH 7.5, 1 mM DTT, 0.2 mg/ml BSA, 2% glycerol, 10 mM MgCl<sub>2</sub>, 100 nM DNA substrate, the indicated amount of AtPol λ and dCTP, dNTPs (deoxynucleotides) or ddNTPs (dideoxynucleotides). When indicated, AtPol λ was pre-incubated with pre-immune serum or anti-AtPol λ antibody for one hour at 4 °C. After incubation at 37 °C for the indicated times, reactions were stopped by adding 20 mM EDTA, 0.6% SDS and 0.5 mg/ml proteinase K, and mixtures were incubated at 37 °C for 30 min. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitated at -20 °C in the presence of 0.3 mM NaCl and 16 mg/ml glycogen. Samples were resuspended in 10 µl of 90% formamide and heated at 95 °C for 5 min. Reaction products were separated in a 12% denaturing polyacrylamide gel containing 7 M urea. Labelled DNA was visualized using FLA-5100 imager (Fujifilm) and analyzed using Multi Gauge software version 3.0 (Fujifilm).</p>\",\n \"<title>dRP lyase assay</title>\",\n \"<p id=\\\"Par13\\\">Reactions (50 µl) contained 45 mM HEPES–KOH pH 7.8, 70 mM KCl, 5 mM MgCl<sub>2,</sub> 1 mM DTT, 0.4 mM EDTA, 2 mM ATP, 36 µg BSA, 1 mM NAD, 0.2% glycerol, 22 mM phosphocreatine, 2.5 ng creatine kinase, 0.02 mM dCTP, and a DNA substrate with a 5′-dRP (100 nM) prepared as described above. When indicated, human Pol β (2.4 U) or AtPol λ (10 nM), were added. After incubation at 30 °C for 90 min, reaction products were stabilized by the addition of freshly prepared sodium borohydride (NaBH<sub>4</sub>; Sigma-Aldrich) to a final concentration of 300 mM and incubated on ice for 30 min. Subsequently, the samples were desalted using microspin G-25 columns (GE Healthcare). The reactions were stopped, DNA was extracted, and samples were processed as described above.</p>\",\n \"<title>In vitro reconstitution of base excision repair</title>\",\n \"<p id=\\\"Par14\\\">BER reactions (50 μl) were performed in 45 mM HEPES–KOH pH 7.8, 70 mM KCl, 5 mM MgCl<sub>2</sub>, 1 mM DTT, 0.4 mM EDTA, 2 mM ATP, 36 μg BSA, 1 mM NAD, 0.2% glycerol, 22 mM phosphocreatine, 2.5 ng creatine kinase, 0.02 mM dCTP, and a DNA duplex containing a U:G mispair (100 nM). All reactions contained UDG (0.5 U) and APE1 (10 U). When indicated, human Pol β (2.4 U), AtPol λ (10 nM), and/or T4 DNA ligase (1.5 U), were added. Mixtures were incubated at 30 °C for 90 min, and the reactions were stopped, DNA was extracted, and samples were processed as described above.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Characterization of AtPol λ gap filling activity</title>","<p id=\"Par15\">In order to characterize the catalytic activity of <italic>Arabidopsis thaliana</italic> Pol λ, a recombinant protein (AtPol λ) was expressed in <italic>E. coli</italic> and purified. Subsequently, optimal conditions for detecting polymerase activity were determined by performing polymerization assays with varying enzyme concentrations. A DNA duplex with the upper strand labelled at the 5′-end and containing a single nucleotide gap flanked by 3′-OH and 5′-P ends was used as substrate (Fig. ##FIG##0##1##a). AtPol λ already showed DNA polymerase activity in a 10 min reaction at a 2 nM concentration of recombinant protein (Fig. ##FIG##0##1##b, lane 6). To confirm that the polymerase activity detected was intrinsic to AtPol λ and not due to contamination with bacterial polymerases, further polymerization assays were carried out in which AtPol λ was pre-incubated with serum generated against AtPol λ (anti-AtPol λ) (Fig. ##FIG##0##1##c). We found a complete inhibition of the polymerase gap-filling capacity in the presence of the anti-AtPol λ serum (Fig. ##FIG##0##1##c, lane 4), suggesting that the DNA polymerase activity is intrinsic to AtPol λ.</p>","<p id=\"Par16\">To determine whether AtPol λ is able to insert the correct nucleotide in a gapped DNA mimicking a BER intermediate, we performed polymerization assays using a DNA substrate containing a single-nucleotide gap opposite G (Fig. ##FIG##0##1##a) in the presence of dATP, dCTP, dGTP, dTTP or a mixture of all four dNTPs (Fig. ##FIG##0##1##d). AtPol λ only incorporated a nucleotide opposite G when dCTP or dNTPs were present in the reaction (Fig. ##FIG##0##1##d, lanes 3 and 6). We also examined how the structure of the deoxyribose affects nucleotide incorporation by AtPol λ by analyzing the effect of dideoxynucleotides (ddNTPs) on its DNA polymerase activity. DNA polymerization is blocked during replication when a ddNTP is incorporated due to the lack of a canonical 3′-OH. While replicative DNA polymerases are resistant to ddNTP inhibition because they fail to bind the nucleotide analogue, human Pol β is ddNTP-sensitive because its active site can accommodate ddNTPs and incorporate them as well as dNTPs (Cavanaugh et al. ##UREF##0##2010##). Previous studies have shown that human and <italic>O. sativa</italic> Pol λ are also sensitive to ddNTPs and therefore unable to discriminate between dNTPs and ddNTPs (Garcia-Diaz et al. ##REF##11821417##2002##; Uchiyama et al. ##REF##15206945##2004##). Additionally, we have previously shown that BER catalyzed by <italic>Arabidopsis</italic> whole cell extracts is partially sensitive to ddNTPs (Cordoba-Cañero et al. ##REF##19682284##2009##). To investigate whether AtPol λ is affected by ddNTPs, a polymerization assay was performed using a primer-template DNA substrate in which dGMP insertion requires the previous incorporation of dCMP (Fig. ##FIG##1##2##). In the absence of ddCTP, AtPol λ generated products of 29 and 30 nucleotides, corresponding to the insertion of dCMP and dGMP, respectively (Fig. ##FIG##1##2##, lane 3). However, in the presence of increasing ddCTP:dCTP ratios, a near complete inhibition of AtPol λ activity was observed (Fig. ##FIG##1##2##, lanes 4–8). A strong inhibition was already detected with a 1:1 ddCTP:dCTP ratio (Fig. ##FIG##1##2##, lane 4). Therefore, AtPol λ exhibits high sensitivity to ddNTPs, in agreement with the weak discrimination of the 3′-OH group of the nucleotide to be inserted that is displayed by the X family of DNA polymerases (Banos et al. ##REF##18938175##2008##; Garcia-Diaz et al. ##REF##11821417##2002##; Prasad et al. ##REF##8265341##1993##).</p>","<title>Effect of the template base on gap filling activity of AtPol λ</title>","<p id=\"Par17\">To further analyze the DNA polymerase activity of AtPol λ, we performed a time-course DNA polymerization assay using DNA duplexes with a gap flanked by 3′-OH and 5′-P ends containing different bases (A, T, C, or G) on the complementary strand (Fig. ##FIG##2##3##a). As expected, AtPol λ performed gap-filling in all four substrates, although the efficiency of DNA synthesis was somewhat different depending on the template base (Fig. ##FIG##2##3##a-b). We found that AtPol λ displays a significant preference for C as template in comparison with A (Fig. ##FIG##2##3##b; t-test p < 0,05 and < 0,01 at 20 and 40 min, respectively). In the DNA substrate containing C as template, the presence of another C downstream in the complementary strand allowed limited strand displacement accompanied of a second dGMP insertion (Fig. ##FIG##2##3##a, lanes 12–13).</p>","<p id=\"Par18\">Some DNA polymerases involved in BER are capable of bypassing lesions on the template strand, resulting in both error-prone and error-free DNA synthesis (Krokan and Bjoras ##REF##23545420##2013##). The ubiquitous oxidative DNA lesion 8-oxoG is highly mutagenic, as it can pair with both cytosine and adenine, leading to G:C → T:A transversions after replication (Krokan and Bjoras ##REF##23545420##2013##). To analyze the translesion DNA synthesis capacity of AtPol λ, we performed a gap-filling assay with a DNA substrate containing 8-oxoG opposite the gap in the presence of either dCTP or dATP (Fig. ##FIG##2##3##c). The results indicate that AtPol λ can effectively bypass 8-oxoG by incorporating dCMP or dAMP with similar efficiencies (Fig. ##FIG##2##3##c).</p>","<title>Effect of the 5′-end group on the gap-filling activity of AtPol λ</title>","<p id=\"Par19\">Human Pol λ and Pol β possess high affinity for DNA substrates containing gaps flanked by 3′-OH and 5′-P ends. It has been suggested that this preference is due to the interaction between the 8-kDa domain of the protein and the phosphate group at the 5′-end of the gap (Garcia-Diaz et al. ##REF##11821417##2002##; Singhal and Wilson ##REF##8340415##1993##). In mammalian SP-BER initiated by AP endonucleases, the processing of the 5′-dRP group to generate a 5′-P end is a limiting step (Srivastava et al. ##REF##9694877##1998##) and it has been reported that the presence of a 5′-dRP group moderately decreases the DNA polymerization activity of human Pol λ (Duym et al. ##REF##17005572##2006##). To analyze the effect of the 5′-end group on the gap-filling activity of AtPol λ, we carried out DNA polymerization assays on gaps with a 5′-phosphate (5′-P), a 5′-hydroxyl (5′-OH) or a tetrahydrofuran residue (5′-THF) mimicking a 5′-dRP, which is resistant to dRP lyase activity. Time-course reactions with each substrate were performed in the presence of different dCTP concentrations (Fig. ##FIG##3##4##a–c).</p>","<p id=\"Par20\">When a canonical 5′-P end was used (Fig. ##FIG##3##4##a), AtPol λ incorporated a nucleotide at the shortest tested time (5 min) and at the higher dCTP concentrations of 100 and 10 μM (Fig. ##FIG##3##4##a, lanes 2 and 6), while at a lower dCTP concentrations, longer times were needed for the gap filling to occur with the same efficiency (Fig. ##FIG##3##4##a, lanes 11 and 17). However, in the presence of DNA substrates containing a 5′-OH end (Fig. ##FIG##3##4##b), AtPol λ required a longer time (20 min) for gap filling at 100 μM of dCTP (Fig. ##FIG##3##4##b, lane 4). Moreover, at lower dCTP concentrations (10, 5 and 1 μM) the percentage of product was around 80%, 60% and 20%, respectively, at the longest tested time (Fig. ##FIG##3##4##b, lanes 9, 13 and 17). When AtPol λ was incubated with the substrate containing a 5′-THF end and the lowest dCTP concentration (1 μM) the polymerase was able to fill the gap as well as with the 5′-P end (Fig. ##FIG##3##4##c, lane 17), and the minor differences were not statistically significant. At higher dCTP concentrations (100, 10 and 5 μM), the polymerase performed gap filling generating a product of 29 nt in 5, 10 and 20 min, respectively (Fig. ##FIG##3##4##c, lanes 2, 7 and 12). Altogether, these results suggest that the gap-filling capacity of AtPol λ is highest on a gap with a 5′-P end, and strongly inhibited by a 5′-OH end (Fig. ##FIG##3##4##d). Importantly, the presence of a THF group at the 5′-side of the gap did not have any inhibitory effect on the enzyme efficiency, suggesting that AtPol λ can effectively fill DNA repair gaps with a 5′-dRP.</p>","<title>AtPol λ possesses an intrinsic dRP lyase activity</title>","<p id=\"Par21\">A limiting step of SP-BER is the removal of the 5′-dRP generated after the processing of abasic sites by AP endonucleases. In humans, the removal of this end is mainly carried out by the dRP lyase activity of polymerase β (Beard and Wilson ##REF##10946231##2000##). No homologs of Pol β have been described in plants and AtPol λ is the only member of the X family described so far (Uchiyama et al. ##REF##18706967##2009##), suggesting that this polymerase might play a role in SP-BER. The 8-kDa domain present in all members of this family is responsible for the dRP lyase activity, which is catalyzed through β-elimination via a Schiff base intermediate between a nucleophile lysine residue and DNA (Matsumoto and Kim ##REF##7624801##1995##). Specific lysine residues responsible for this activity have been identified in human Pol β (K72) (Deterding et al. ##REF##10744736##2000##) and Pol λ (K312) (Garcia-Diaz et al. ##REF##11457865##2001##).</p>","<p id=\"Par22\">In order to test whether AtPol λ has intrinsic dRP lyase activity, we first identified candidate nucleophile residues in its 8-kDa domain. By multiple sequence alignment we found that AtPol λ has a histidine (H260) at the orthologous position of human Pol β K72 and Pol λ K312 (Supplementary Fig. ##SUPPL##0##S1##). We therefore focused on two AtPol λ lysine residues located nearby. One of such residues (K255) is conserved in all Pol λ orthologs, but not in Pol β enzymes, whereas the other one (K248) is only conserved in plant Pol λ proteins (Supplementary Fig. ##SUPPL##0##S1##). To examine the role of K248 and K255, two mutant derivatives (AtPol λ K248A and AtPol λ K255A) were expressed and purified as recombinant His tagged proteins. Subsequently, dRP lyase activity assays were performed using AtPol λ and its mutant versions (Fig. ##FIG##4##5##a). The removal of the 5′-dRP group generates a fragment with a 5′-P end that exhibits greater electrophoretic mobility. Although the non-processed 5′dRP end was stabilized by treatment with borohydride, spontaneous appearance of 5′-P was observed in the absence of any enzyme (Fig. ##FIG##4##5##b, lane 1) and in the presence of a DNA polymerase without dRP lyase activity (Fig. ##FIG##4##5##b, lane 2), suggesting that some spontaneous 5′-dRP hydrolysis takes place during the incubation time. We found that AtPol λ exhibited a clearly detectable dRP lyase activity (Fig. ##FIG##4##5##b, lane 6) that was significantly reduced in the mutant versions K248A and, particularly, K255A (Fig. ##FIG##4##5##b-c). These results indicate that AtPol λ possesses an intrinsic dRP lyase activity and suggest that both K248 and K255 are involved in this enzymatic function.</p>","<p id=\"Par23\">Next, we asked whether mutations in K248 or K255 have any effect on the DNA polymerase activity of AtPol λ. For this purpose, a polymerization assay was carried out using a DNA substrate containing a gap flanked by 3′-OH and 5′-P ends (Fig. ##FIG##5##6##). As compared with Klenow DNA polymerase, which achieved full-length DNA synthesis by displacement of the top strand (Fig. ##FIG##5##6##, lane 9), AtPol λ exhibited a limited capacity for strand displacement inserting only between 8 and 10 nucleotides (Fig. ##FIG##5##6##, lane 5). AtPol λ did not exhibited 3′-5′ exonuclease activity when the incubations were carried out in the absence of dNTPs (Fig. ##FIG##5##6##, lane 4). In comparison, mutant AtPol λ K248 showed a DNA polymerization activity similar to that of the WT protein, but a significantly lower capacity for strand displacement (Fig. ##FIG##5##6##, lane 3). Also, it exhibited a detectable 3′-5′ exonuclease activity in the absence of dNTPs (Fig. ##FIG##5##6##, lane 2). The K255 mutant version of AtPol λ displayed a stronger reduction in strand displacement capacity (Fig. ##FIG##5##6##, lane 7), but did not show detectable 3′-5′ exonuclease activity (Fig. ##FIG##5##6##, lane 6). These results suggest that residues K248 and K255 are not required for DNA polymerase activity but modulate the capacity of AtPol λ to perform strand displacement during gap-filling.</p>","<title>AtPol λ 5′-dRP lyase activity is required for efficient completion of SP-BER in vitro</title>","<p id=\"Par24\">The dRP lyase activity of human Pol β, which removes the blocking dRP group at the 5′-end of the gap, is required to allow repair completion during SP-BER (Srivastava et al. ##REF##9694877##1998##). To determine whether AtPol λ also carries out this function, an in vitro SP-BER reconstitution assay was performed using recombinant proteins and a DNA substrate containing a U:G mispair (Fig. ##FIG##6##7##). In this assay, uracil is removed by UDG leaving an intact AP site that is cleaved by APE1 at the 5′-side generating a gap flanked by 3′-OH and 5′-dRP ends. After nucleotide insertion by a DNA polymerase, a dRP lyase activity is required before DNA ligation completes repair (Fig. ##FIG##6##7##a). The results show that reactions containing both DNA ligase and AtPol λ, but not those with DNA ligase alone, generated a fully repaired product detected as a 51-nucleotide fragment (Fig. ##FIG##6##7##b, lanes 1 and 4). Although lower, repair levels achieved with AtPol λ were close to those observed with Pol β (Fig. ##FIG##6##7##c). Importantly, we found that the levels of fully repaired products were significantly lower in reactions containing the AtPol λ K255A mutant protein (Fig. ##FIG##6##7##c). Taken together, our results suggest that AtPol λ can support SP-BER, and that its dRP lyase activity is required for efficient completion of repair.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Characterization of AtPol λ gap filling activity</title>\",\n \"<p id=\\\"Par15\\\">In order to characterize the catalytic activity of <italic>Arabidopsis thaliana</italic> Pol λ, a recombinant protein (AtPol λ) was expressed in <italic>E. coli</italic> and purified. Subsequently, optimal conditions for detecting polymerase activity were determined by performing polymerization assays with varying enzyme concentrations. A DNA duplex with the upper strand labelled at the 5′-end and containing a single nucleotide gap flanked by 3′-OH and 5′-P ends was used as substrate (Fig. ##FIG##0##1##a). AtPol λ already showed DNA polymerase activity in a 10 min reaction at a 2 nM concentration of recombinant protein (Fig. ##FIG##0##1##b, lane 6). To confirm that the polymerase activity detected was intrinsic to AtPol λ and not due to contamination with bacterial polymerases, further polymerization assays were carried out in which AtPol λ was pre-incubated with serum generated against AtPol λ (anti-AtPol λ) (Fig. ##FIG##0##1##c). We found a complete inhibition of the polymerase gap-filling capacity in the presence of the anti-AtPol λ serum (Fig. ##FIG##0##1##c, lane 4), suggesting that the DNA polymerase activity is intrinsic to AtPol λ.</p>\",\n \"<p id=\\\"Par16\\\">To determine whether AtPol λ is able to insert the correct nucleotide in a gapped DNA mimicking a BER intermediate, we performed polymerization assays using a DNA substrate containing a single-nucleotide gap opposite G (Fig. ##FIG##0##1##a) in the presence of dATP, dCTP, dGTP, dTTP or a mixture of all four dNTPs (Fig. ##FIG##0##1##d). AtPol λ only incorporated a nucleotide opposite G when dCTP or dNTPs were present in the reaction (Fig. ##FIG##0##1##d, lanes 3 and 6). We also examined how the structure of the deoxyribose affects nucleotide incorporation by AtPol λ by analyzing the effect of dideoxynucleotides (ddNTPs) on its DNA polymerase activity. DNA polymerization is blocked during replication when a ddNTP is incorporated due to the lack of a canonical 3′-OH. While replicative DNA polymerases are resistant to ddNTP inhibition because they fail to bind the nucleotide analogue, human Pol β is ddNTP-sensitive because its active site can accommodate ddNTPs and incorporate them as well as dNTPs (Cavanaugh et al. ##UREF##0##2010##). Previous studies have shown that human and <italic>O. sativa</italic> Pol λ are also sensitive to ddNTPs and therefore unable to discriminate between dNTPs and ddNTPs (Garcia-Diaz et al. ##REF##11821417##2002##; Uchiyama et al. ##REF##15206945##2004##). Additionally, we have previously shown that BER catalyzed by <italic>Arabidopsis</italic> whole cell extracts is partially sensitive to ddNTPs (Cordoba-Cañero et al. ##REF##19682284##2009##). To investigate whether AtPol λ is affected by ddNTPs, a polymerization assay was performed using a primer-template DNA substrate in which dGMP insertion requires the previous incorporation of dCMP (Fig. ##FIG##1##2##). In the absence of ddCTP, AtPol λ generated products of 29 and 30 nucleotides, corresponding to the insertion of dCMP and dGMP, respectively (Fig. ##FIG##1##2##, lane 3). However, in the presence of increasing ddCTP:dCTP ratios, a near complete inhibition of AtPol λ activity was observed (Fig. ##FIG##1##2##, lanes 4–8). A strong inhibition was already detected with a 1:1 ddCTP:dCTP ratio (Fig. ##FIG##1##2##, lane 4). Therefore, AtPol λ exhibits high sensitivity to ddNTPs, in agreement with the weak discrimination of the 3′-OH group of the nucleotide to be inserted that is displayed by the X family of DNA polymerases (Banos et al. ##REF##18938175##2008##; Garcia-Diaz et al. ##REF##11821417##2002##; Prasad et al. ##REF##8265341##1993##).</p>\",\n \"<title>Effect of the template base on gap filling activity of AtPol λ</title>\",\n \"<p id=\\\"Par17\\\">To further analyze the DNA polymerase activity of AtPol λ, we performed a time-course DNA polymerization assay using DNA duplexes with a gap flanked by 3′-OH and 5′-P ends containing different bases (A, T, C, or G) on the complementary strand (Fig. ##FIG##2##3##a). As expected, AtPol λ performed gap-filling in all four substrates, although the efficiency of DNA synthesis was somewhat different depending on the template base (Fig. ##FIG##2##3##a-b). We found that AtPol λ displays a significant preference for C as template in comparison with A (Fig. ##FIG##2##3##b; t-test p < 0,05 and < 0,01 at 20 and 40 min, respectively). In the DNA substrate containing C as template, the presence of another C downstream in the complementary strand allowed limited strand displacement accompanied of a second dGMP insertion (Fig. ##FIG##2##3##a, lanes 12–13).</p>\",\n \"<p id=\\\"Par18\\\">Some DNA polymerases involved in BER are capable of bypassing lesions on the template strand, resulting in both error-prone and error-free DNA synthesis (Krokan and Bjoras ##REF##23545420##2013##). The ubiquitous oxidative DNA lesion 8-oxoG is highly mutagenic, as it can pair with both cytosine and adenine, leading to G:C → T:A transversions after replication (Krokan and Bjoras ##REF##23545420##2013##). To analyze the translesion DNA synthesis capacity of AtPol λ, we performed a gap-filling assay with a DNA substrate containing 8-oxoG opposite the gap in the presence of either dCTP or dATP (Fig. ##FIG##2##3##c). The results indicate that AtPol λ can effectively bypass 8-oxoG by incorporating dCMP or dAMP with similar efficiencies (Fig. ##FIG##2##3##c).</p>\",\n \"<title>Effect of the 5′-end group on the gap-filling activity of AtPol λ</title>\",\n \"<p id=\\\"Par19\\\">Human Pol λ and Pol β possess high affinity for DNA substrates containing gaps flanked by 3′-OH and 5′-P ends. It has been suggested that this preference is due to the interaction between the 8-kDa domain of the protein and the phosphate group at the 5′-end of the gap (Garcia-Diaz et al. ##REF##11821417##2002##; Singhal and Wilson ##REF##8340415##1993##). In mammalian SP-BER initiated by AP endonucleases, the processing of the 5′-dRP group to generate a 5′-P end is a limiting step (Srivastava et al. ##REF##9694877##1998##) and it has been reported that the presence of a 5′-dRP group moderately decreases the DNA polymerization activity of human Pol λ (Duym et al. ##REF##17005572##2006##). To analyze the effect of the 5′-end group on the gap-filling activity of AtPol λ, we carried out DNA polymerization assays on gaps with a 5′-phosphate (5′-P), a 5′-hydroxyl (5′-OH) or a tetrahydrofuran residue (5′-THF) mimicking a 5′-dRP, which is resistant to dRP lyase activity. Time-course reactions with each substrate were performed in the presence of different dCTP concentrations (Fig. ##FIG##3##4##a–c).</p>\",\n \"<p id=\\\"Par20\\\">When a canonical 5′-P end was used (Fig. ##FIG##3##4##a), AtPol λ incorporated a nucleotide at the shortest tested time (5 min) and at the higher dCTP concentrations of 100 and 10 μM (Fig. ##FIG##3##4##a, lanes 2 and 6), while at a lower dCTP concentrations, longer times were needed for the gap filling to occur with the same efficiency (Fig. ##FIG##3##4##a, lanes 11 and 17). However, in the presence of DNA substrates containing a 5′-OH end (Fig. ##FIG##3##4##b), AtPol λ required a longer time (20 min) for gap filling at 100 μM of dCTP (Fig. ##FIG##3##4##b, lane 4). Moreover, at lower dCTP concentrations (10, 5 and 1 μM) the percentage of product was around 80%, 60% and 20%, respectively, at the longest tested time (Fig. ##FIG##3##4##b, lanes 9, 13 and 17). When AtPol λ was incubated with the substrate containing a 5′-THF end and the lowest dCTP concentration (1 μM) the polymerase was able to fill the gap as well as with the 5′-P end (Fig. ##FIG##3##4##c, lane 17), and the minor differences were not statistically significant. At higher dCTP concentrations (100, 10 and 5 μM), the polymerase performed gap filling generating a product of 29 nt in 5, 10 and 20 min, respectively (Fig. ##FIG##3##4##c, lanes 2, 7 and 12). Altogether, these results suggest that the gap-filling capacity of AtPol λ is highest on a gap with a 5′-P end, and strongly inhibited by a 5′-OH end (Fig. ##FIG##3##4##d). Importantly, the presence of a THF group at the 5′-side of the gap did not have any inhibitory effect on the enzyme efficiency, suggesting that AtPol λ can effectively fill DNA repair gaps with a 5′-dRP.</p>\",\n \"<title>AtPol λ possesses an intrinsic dRP lyase activity</title>\",\n \"<p id=\\\"Par21\\\">A limiting step of SP-BER is the removal of the 5′-dRP generated after the processing of abasic sites by AP endonucleases. In humans, the removal of this end is mainly carried out by the dRP lyase activity of polymerase β (Beard and Wilson ##REF##10946231##2000##). No homologs of Pol β have been described in plants and AtPol λ is the only member of the X family described so far (Uchiyama et al. ##REF##18706967##2009##), suggesting that this polymerase might play a role in SP-BER. The 8-kDa domain present in all members of this family is responsible for the dRP lyase activity, which is catalyzed through β-elimination via a Schiff base intermediate between a nucleophile lysine residue and DNA (Matsumoto and Kim ##REF##7624801##1995##). Specific lysine residues responsible for this activity have been identified in human Pol β (K72) (Deterding et al. ##REF##10744736##2000##) and Pol λ (K312) (Garcia-Diaz et al. ##REF##11457865##2001##).</p>\",\n \"<p id=\\\"Par22\\\">In order to test whether AtPol λ has intrinsic dRP lyase activity, we first identified candidate nucleophile residues in its 8-kDa domain. By multiple sequence alignment we found that AtPol λ has a histidine (H260) at the orthologous position of human Pol β K72 and Pol λ K312 (Supplementary Fig. ##SUPPL##0##S1##). We therefore focused on two AtPol λ lysine residues located nearby. One of such residues (K255) is conserved in all Pol λ orthologs, but not in Pol β enzymes, whereas the other one (K248) is only conserved in plant Pol λ proteins (Supplementary Fig. ##SUPPL##0##S1##). To examine the role of K248 and K255, two mutant derivatives (AtPol λ K248A and AtPol λ K255A) were expressed and purified as recombinant His tagged proteins. Subsequently, dRP lyase activity assays were performed using AtPol λ and its mutant versions (Fig. ##FIG##4##5##a). The removal of the 5′-dRP group generates a fragment with a 5′-P end that exhibits greater electrophoretic mobility. Although the non-processed 5′dRP end was stabilized by treatment with borohydride, spontaneous appearance of 5′-P was observed in the absence of any enzyme (Fig. ##FIG##4##5##b, lane 1) and in the presence of a DNA polymerase without dRP lyase activity (Fig. ##FIG##4##5##b, lane 2), suggesting that some spontaneous 5′-dRP hydrolysis takes place during the incubation time. We found that AtPol λ exhibited a clearly detectable dRP lyase activity (Fig. ##FIG##4##5##b, lane 6) that was significantly reduced in the mutant versions K248A and, particularly, K255A (Fig. ##FIG##4##5##b-c). These results indicate that AtPol λ possesses an intrinsic dRP lyase activity and suggest that both K248 and K255 are involved in this enzymatic function.</p>\",\n \"<p id=\\\"Par23\\\">Next, we asked whether mutations in K248 or K255 have any effect on the DNA polymerase activity of AtPol λ. For this purpose, a polymerization assay was carried out using a DNA substrate containing a gap flanked by 3′-OH and 5′-P ends (Fig. ##FIG##5##6##). As compared with Klenow DNA polymerase, which achieved full-length DNA synthesis by displacement of the top strand (Fig. ##FIG##5##6##, lane 9), AtPol λ exhibited a limited capacity for strand displacement inserting only between 8 and 10 nucleotides (Fig. ##FIG##5##6##, lane 5). AtPol λ did not exhibited 3′-5′ exonuclease activity when the incubations were carried out in the absence of dNTPs (Fig. ##FIG##5##6##, lane 4). In comparison, mutant AtPol λ K248 showed a DNA polymerization activity similar to that of the WT protein, but a significantly lower capacity for strand displacement (Fig. ##FIG##5##6##, lane 3). Also, it exhibited a detectable 3′-5′ exonuclease activity in the absence of dNTPs (Fig. ##FIG##5##6##, lane 2). The K255 mutant version of AtPol λ displayed a stronger reduction in strand displacement capacity (Fig. ##FIG##5##6##, lane 7), but did not show detectable 3′-5′ exonuclease activity (Fig. ##FIG##5##6##, lane 6). These results suggest that residues K248 and K255 are not required for DNA polymerase activity but modulate the capacity of AtPol λ to perform strand displacement during gap-filling.</p>\",\n \"<title>AtPol λ 5′-dRP lyase activity is required for efficient completion of SP-BER in vitro</title>\",\n \"<p id=\\\"Par24\\\">The dRP lyase activity of human Pol β, which removes the blocking dRP group at the 5′-end of the gap, is required to allow repair completion during SP-BER (Srivastava et al. ##REF##9694877##1998##). To determine whether AtPol λ also carries out this function, an in vitro SP-BER reconstitution assay was performed using recombinant proteins and a DNA substrate containing a U:G mispair (Fig. ##FIG##6##7##). In this assay, uracil is removed by UDG leaving an intact AP site that is cleaved by APE1 at the 5′-side generating a gap flanked by 3′-OH and 5′-dRP ends. After nucleotide insertion by a DNA polymerase, a dRP lyase activity is required before DNA ligation completes repair (Fig. ##FIG##6##7##a). The results show that reactions containing both DNA ligase and AtPol λ, but not those with DNA ligase alone, generated a fully repaired product detected as a 51-nucleotide fragment (Fig. ##FIG##6##7##b, lanes 1 and 4). Although lower, repair levels achieved with AtPol λ were close to those observed with Pol β (Fig. ##FIG##6##7##c). Importantly, we found that the levels of fully repaired products were significantly lower in reactions containing the AtPol λ K255A mutant protein (Fig. ##FIG##6##7##c). Taken together, our results suggest that AtPol λ can support SP-BER, and that its dRP lyase activity is required for efficient completion of repair.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par25\">In this work we have performed a biochemical characterization of <italic>A. thaliana</italic> DNA polymerase λ and explored its possible role in BER. Firstly, we have shown that, as the rice and human orthologs (Garcia-Diaz et al. ##REF##11821417##2002##; Uchiyama et al. ##REF##15206945##2004##), AtPol λ inserts the correct nucleotide in a single-nucleotide gap. We have also shown that AtPol λ is able to catalyze DNA synthesis at the lowest dNTP concentration tested (1 μM), which is consistent with the high affinity for dNTPs observed in human Pol λ (Garcia-Diaz et al. ##REF##11821417##2002##). We also showed that AtPol λ is inhibited by ddCTP, even at low ddCTP: dCTP ratios. This result suggests a low discrimination for the presence of the 3′-OH group in the sugar of the incoming deoxynucleotide, as it has been found in other X family DNA polymerases (Banos et al. ##REF##18938175##2008##; Garcia-Diaz et al. ##REF##11821417##2002##; Prasad et al. ##REF##8265341##1993##). We have found that, like human Pol β and Pol λ, AtPol λ does not possess an intrinsic 3′-5′ exonuclease activity that might remove a nucleotide with the wrong sugar. The ddNTP resistance of high-fidelity DNA polymerases is not due to their 3′-5′-exonuclease activity, but to an exquisite structural selectivity that prevents productive binding to deoxynucleotides lacking hydroxyl at C3′ (Wang et al. ##UREF##2##2012##). Such selectivity is absent in ddNTP-sensitive DNA polymerases. For Pol β and other members of the X family, it has been proposed that a conserved Arg residue (R183 in human Pol β and R420 in human Pol λ) is sufficient to stabilize the incoming nucleotide in the absence of O3′ (Cavanaugh et al. ##UREF##0##2010##). Interestingly, this residue is also conserved in AtPol λ (R370), which suggests that the structural basis for low deoxynucleotide discrimination is conserved across Pol β and Pol λ proteins.</p>","<p id=\"Par26\">Interestingly, we found an effect of the base opposite the gap on the polymerization efficiency of AtPol λ. The rate of nucleotide incorporation catalyzed by the enzyme was higher in substrates containing a C in the template strand, as compared to G, T or A. Such preference might be related to the type of initial DNA damage that leads the generation of the repair gap. A cytosine opposite the gap is found in DNA intermediates arising during repair of 8-oxoG. This suggests that AtPol λ could participate, preferably but not exclusively, in the repair of certain DNA lesions. In human cells, Pol β has been implicated in 8-oxoG BER initiated by OGG1 DNA glycosylase (Dianov et al. ##REF##9837971##1998##; Fortini et al. ##REF##10329732##1999##). Plants possess orthologs of OGG1 and FPG, both of which are involved in 8-oxoG repair (Cordoba-Cañero et al. ##REF##24934622##2014##). Therefore, it will be interesting to analyse whether AtPol λ functions in DNA repair of 8-oxoG initiated by OGG1 and/or FPG.</p>","<p id=\"Par27\">Besides DNA repair functions, Pol λ enzymes are thought to play a role in DNA damage tolerance through DNA translesion synthesis. Thus, mammalian and <italic>Arabidopsis</italic> Pol λ have been reported to perform both error-free and error-prone bypass of 8-oxoG lesions (Amoroso et al. ##REF##21325140##2011##; Picher and Blanco ##REF##17686665##2007##). Here, we have confirmed that AtPol λ can catalyze both the correct incorporation of C and misincorporation of A opposite 8-oxoG. In our analysis, we have used a one-nucleotide gap mimicking a DNA intermediate generated during BER of 8-oxoG:A mispairs. In human cells such mispairs are targets of MUTYH DNA glycosylase, which specifically excises the misincorporated A and leaves 8-oxoG in the repair-template strand (Slupska et al. ##REF##8682794##1996##). Since <italic>Arabidopsis</italic> possesses a MUTYH ortholog (Roldan-Arjona et al. ##REF##31543887##2019##), the capacity of AtPol λ to misincorporate A opposite 8-oxoG must be somehow reduced in vivo in order to avoid futile cycles of 8-oxoG:A repair. In fact, it has been reported that PCNA2 increases AtPol λ fidelity in translesion DNA synthesis (Amoroso et al. ##REF##21325140##2011##).</p>","<p id=\"Par28\">It has been previously shown that human Pol λ is a distributive DNA polymerase on a template-primer substrate but processive in short gaps containing a phosphate group at its 5′-end (Garcia-Diaz et al. ##REF##11821417##2002##). This is believed to be a consequence of the additional contacts that are made between the N-terminal 8-kDa domain of the protein and the 5′-end of the downstream strand in the gapped substrate (Garcia-Diaz et al. ##REF##14992725##2004##). Here we have shown that AtPol λ is able to perform gap-filling in the presence of different groups at the 5′-end of the gap but shows the highest polymerization efficiency when the 5′-end contains a phosphate group. This finding suggests that plant Pol λ is not specialized in DNA substrates with a canonical 5′-P end and might fulfils diverse functions by being able to process substrates with different 5′-termini. We found a much-reduced, but still significant, gap-filling activity in gapped substrates containing an OH group at the 5′-terminus of the downstream strand. Such 5′-OH blocked ends are found at DNA breaks induced by abortive activity of topoisomerase I (TOP1) which are generated by TOP1 inhibitors, reactive oxygen species, and other genotoxins (Caldecott ##REF##18626472##2008##; Pommier et al. ##REF##14643436##2003##). TOP1 cleavage complexes are repaired by tyrosyl-DNA phosphodiesterase 1 (TDP1), that removes topoisomerase I from the 3′-termini at the gap, generating a gap with 3′-P and 5′-OH ends that are converted to 3′-OH and 5′-P by the DNA phosphatase and kinase activities of PNKP, respectively (Caldecott ##REF##18626472##2008##; Jilani et al. ##REF##10446192##1999##; Pouliot et al. ##REF##11532027##2001##). In mammals, the repair is completed in most cases by short patch gap filling mediated by Pol β (Krokan and Bjoras ##REF##23545420##2013##; Kubota et al. ##REF##8978692##1996##; Srivastava et al. ##REF##9694877##1998##). The <italic>Arabidopsis</italic> ortholog of PNKP lacks kinase activity (Martinez-Macias et al. ##REF##22325353##2012##; Petrucco et al. ##REF##11948185##2002##), and the mechanism responsible for processing 5′-OH ends in gapped DNA repair intermediates in plants remains unidentified. It is also unknown if AtPol λ plays a role in the processing of TOP1 cleavage complexes. Interestingly, we found that the nucleotide insertion efficiency of AtPol λ when the gap contains a 5′-dRP-mimicking group (THF), is comparable to that observed with a 5′-P end. BER DNA intermediates frequently harbour 5′-dRP blocking termini that arise upon cleavage of abasic sites by APE1 (Krokan and Bjoras ##REF##23545420##2013##; Levin and Demple ##REF##1698278##1990##) and are processed by the dRP lyase activity of Pol β (Matsumoto and Kim ##REF##7624801##1995##; Srivastava et al. ##REF##9694877##1998##). However, it has been reported that the dRP lyase activity of Pol β is rate-limiting during BER, and that its DNA polymerase activity performs gap-filling prior to removal of the dRP group (Srivastava et al. ##REF##9694877##1998##). Our results suggest that AtPol λ may perform efficient DNA synthesis in gapped DNA repair intermediates before elimination of the 5′-dRP moiety.</p>","<p id=\"Par29\">Like Pol β, human Pol λ possesses an 8-kDa domain responsible for dRP lyase activity (Garcia-Diaz et al. ##REF##10966791##2000##). The human Pol λ K312 residue, which is structurally homologous to Pol β K72, is crucial for this enzymatic function, suggesting that this is the main nucleophile responsible for the reaction (Garcia-Diaz et al. ##REF##11457865##2001##). Interestingly, our multiple sequence analysis revealed that human Pol λ K312 and Pol β K72 are replaced by histidine in plant Pol λ proteins. In our search for alternative candidate residues, we have found that AtPol λ K248 and K255 are important for the dRP lyase reaction. Replacement of these lysine residues by alanine caused a significant reduction of the dRP lyase activity of AtPol λ, which was nearly abolished in the K255A mutant protein. Alanine substitution for K248 resulted in a lesser, but still important reduction of activity. These results suggest that K255 is the preferred but no required nucleophile responsible for the dRP lyase reaction catalyzed by AtPol λ. Interestingly, AtPol λ K255 is conserved in plant and animal Pol λ enzymes, but not in Pol β proteins, whereas AtPol λ K248 is conserved in plant Pol λ and in Pol β proteins, but not in metazoan Pol λ. The residue aligned with AtPol λ K255 in human Pol β is K60, and its replacement by alanine also results in a significant reduction in Pol β dRP lyase activity (Prasad et al. ##REF##9556598##1998##).</p>","<p id=\"Par30\">We found that the DNA polymerase activity of AtPol λ K248A and K255A mutant proteins was not affected, but their ability to extend DNA synthesis in a single-nucleotide gap, with the consequent displacement of the downstream strand, was impaired. This result agrees with the fact that residues in the 8-kDa domain of human Pol λ make specific contacts with the 5′-end of the downstream strand in gapped substrates (Garcia-Diaz et al. ##REF##14992725##2004##). The strand-displacement ability of AtPol λ might allow its participation in LP-BER. Although this has already been suggested for human Pol λ by some authors (Garcia-Diaz et al. ##REF##11457865##2001##), to date there is no evidence that AtPol λ is involved in plant LP-BER.</p>","<p id=\"Par31\">In this work we have also shown that the dRP lyase activity of AtPol λ is required to complete SP-BER of uracil in a reconstituted repair reaction. In the BER reconstitution assay, AtPol λ was able to perform both the gap-filling and dRP removal steps that allow the final DNA ligation of the processed strand. In contrast, the ability of AtPol λ K248A and K255A mutant proteins to promote the repair of the BER intermediate was significantly impaired. Importantly, the capacity of the mutant AtPol λ versions to support SP-BER was inversely correlated with their dRP lyase activity and was strongly reduced in the AtPol λ K255A protein. In mammalian cells, removal of the dRP group is a critical BER step in vivo, since only the dRP lyase activity of Pol β, and not its DNA polymerization capacity, is required to reverse the DNA damage sensitivity of Pol β-null cells (Sobol et al. ##REF##10866204##2000##). Since plants lack Pol β homologues, our results suggest that the dRP lyase activity of AtPol λ might play a role in <italic>Arabidopsis</italic> SP-BER in vivo.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par25\\\">In this work we have performed a biochemical characterization of <italic>A. thaliana</italic> DNA polymerase λ and explored its possible role in BER. Firstly, we have shown that, as the rice and human orthologs (Garcia-Diaz et al. ##REF##11821417##2002##; Uchiyama et al. ##REF##15206945##2004##), AtPol λ inserts the correct nucleotide in a single-nucleotide gap. We have also shown that AtPol λ is able to catalyze DNA synthesis at the lowest dNTP concentration tested (1 μM), which is consistent with the high affinity for dNTPs observed in human Pol λ (Garcia-Diaz et al. ##REF##11821417##2002##). We also showed that AtPol λ is inhibited by ddCTP, even at low ddCTP: dCTP ratios. This result suggests a low discrimination for the presence of the 3′-OH group in the sugar of the incoming deoxynucleotide, as it has been found in other X family DNA polymerases (Banos et al. ##REF##18938175##2008##; Garcia-Diaz et al. ##REF##11821417##2002##; Prasad et al. ##REF##8265341##1993##). We have found that, like human Pol β and Pol λ, AtPol λ does not possess an intrinsic 3′-5′ exonuclease activity that might remove a nucleotide with the wrong sugar. The ddNTP resistance of high-fidelity DNA polymerases is not due to their 3′-5′-exonuclease activity, but to an exquisite structural selectivity that prevents productive binding to deoxynucleotides lacking hydroxyl at C3′ (Wang et al. ##UREF##2##2012##). Such selectivity is absent in ddNTP-sensitive DNA polymerases. For Pol β and other members of the X family, it has been proposed that a conserved Arg residue (R183 in human Pol β and R420 in human Pol λ) is sufficient to stabilize the incoming nucleotide in the absence of O3′ (Cavanaugh et al. ##UREF##0##2010##). Interestingly, this residue is also conserved in AtPol λ (R370), which suggests that the structural basis for low deoxynucleotide discrimination is conserved across Pol β and Pol λ proteins.</p>\",\n \"<p id=\\\"Par26\\\">Interestingly, we found an effect of the base opposite the gap on the polymerization efficiency of AtPol λ. The rate of nucleotide incorporation catalyzed by the enzyme was higher in substrates containing a C in the template strand, as compared to G, T or A. Such preference might be related to the type of initial DNA damage that leads the generation of the repair gap. A cytosine opposite the gap is found in DNA intermediates arising during repair of 8-oxoG. This suggests that AtPol λ could participate, preferably but not exclusively, in the repair of certain DNA lesions. In human cells, Pol β has been implicated in 8-oxoG BER initiated by OGG1 DNA glycosylase (Dianov et al. ##REF##9837971##1998##; Fortini et al. ##REF##10329732##1999##). Plants possess orthologs of OGG1 and FPG, both of which are involved in 8-oxoG repair (Cordoba-Cañero et al. ##REF##24934622##2014##). Therefore, it will be interesting to analyse whether AtPol λ functions in DNA repair of 8-oxoG initiated by OGG1 and/or FPG.</p>\",\n \"<p id=\\\"Par27\\\">Besides DNA repair functions, Pol λ enzymes are thought to play a role in DNA damage tolerance through DNA translesion synthesis. Thus, mammalian and <italic>Arabidopsis</italic> Pol λ have been reported to perform both error-free and error-prone bypass of 8-oxoG lesions (Amoroso et al. ##REF##21325140##2011##; Picher and Blanco ##REF##17686665##2007##). Here, we have confirmed that AtPol λ can catalyze both the correct incorporation of C and misincorporation of A opposite 8-oxoG. In our analysis, we have used a one-nucleotide gap mimicking a DNA intermediate generated during BER of 8-oxoG:A mispairs. In human cells such mispairs are targets of MUTYH DNA glycosylase, which specifically excises the misincorporated A and leaves 8-oxoG in the repair-template strand (Slupska et al. ##REF##8682794##1996##). Since <italic>Arabidopsis</italic> possesses a MUTYH ortholog (Roldan-Arjona et al. ##REF##31543887##2019##), the capacity of AtPol λ to misincorporate A opposite 8-oxoG must be somehow reduced in vivo in order to avoid futile cycles of 8-oxoG:A repair. In fact, it has been reported that PCNA2 increases AtPol λ fidelity in translesion DNA synthesis (Amoroso et al. ##REF##21325140##2011##).</p>\",\n \"<p id=\\\"Par28\\\">It has been previously shown that human Pol λ is a distributive DNA polymerase on a template-primer substrate but processive in short gaps containing a phosphate group at its 5′-end (Garcia-Diaz et al. ##REF##11821417##2002##). This is believed to be a consequence of the additional contacts that are made between the N-terminal 8-kDa domain of the protein and the 5′-end of the downstream strand in the gapped substrate (Garcia-Diaz et al. ##REF##14992725##2004##). Here we have shown that AtPol λ is able to perform gap-filling in the presence of different groups at the 5′-end of the gap but shows the highest polymerization efficiency when the 5′-end contains a phosphate group. This finding suggests that plant Pol λ is not specialized in DNA substrates with a canonical 5′-P end and might fulfils diverse functions by being able to process substrates with different 5′-termini. We found a much-reduced, but still significant, gap-filling activity in gapped substrates containing an OH group at the 5′-terminus of the downstream strand. Such 5′-OH blocked ends are found at DNA breaks induced by abortive activity of topoisomerase I (TOP1) which are generated by TOP1 inhibitors, reactive oxygen species, and other genotoxins (Caldecott ##REF##18626472##2008##; Pommier et al. ##REF##14643436##2003##). TOP1 cleavage complexes are repaired by tyrosyl-DNA phosphodiesterase 1 (TDP1), that removes topoisomerase I from the 3′-termini at the gap, generating a gap with 3′-P and 5′-OH ends that are converted to 3′-OH and 5′-P by the DNA phosphatase and kinase activities of PNKP, respectively (Caldecott ##REF##18626472##2008##; Jilani et al. ##REF##10446192##1999##; Pouliot et al. ##REF##11532027##2001##). In mammals, the repair is completed in most cases by short patch gap filling mediated by Pol β (Krokan and Bjoras ##REF##23545420##2013##; Kubota et al. ##REF##8978692##1996##; Srivastava et al. ##REF##9694877##1998##). The <italic>Arabidopsis</italic> ortholog of PNKP lacks kinase activity (Martinez-Macias et al. ##REF##22325353##2012##; Petrucco et al. ##REF##11948185##2002##), and the mechanism responsible for processing 5′-OH ends in gapped DNA repair intermediates in plants remains unidentified. It is also unknown if AtPol λ plays a role in the processing of TOP1 cleavage complexes. Interestingly, we found that the nucleotide insertion efficiency of AtPol λ when the gap contains a 5′-dRP-mimicking group (THF), is comparable to that observed with a 5′-P end. BER DNA intermediates frequently harbour 5′-dRP blocking termini that arise upon cleavage of abasic sites by APE1 (Krokan and Bjoras ##REF##23545420##2013##; Levin and Demple ##REF##1698278##1990##) and are processed by the dRP lyase activity of Pol β (Matsumoto and Kim ##REF##7624801##1995##; Srivastava et al. ##REF##9694877##1998##). However, it has been reported that the dRP lyase activity of Pol β is rate-limiting during BER, and that its DNA polymerase activity performs gap-filling prior to removal of the dRP group (Srivastava et al. ##REF##9694877##1998##). Our results suggest that AtPol λ may perform efficient DNA synthesis in gapped DNA repair intermediates before elimination of the 5′-dRP moiety.</p>\",\n \"<p id=\\\"Par29\\\">Like Pol β, human Pol λ possesses an 8-kDa domain responsible for dRP lyase activity (Garcia-Diaz et al. ##REF##10966791##2000##). The human Pol λ K312 residue, which is structurally homologous to Pol β K72, is crucial for this enzymatic function, suggesting that this is the main nucleophile responsible for the reaction (Garcia-Diaz et al. ##REF##11457865##2001##). Interestingly, our multiple sequence analysis revealed that human Pol λ K312 and Pol β K72 are replaced by histidine in plant Pol λ proteins. In our search for alternative candidate residues, we have found that AtPol λ K248 and K255 are important for the dRP lyase reaction. Replacement of these lysine residues by alanine caused a significant reduction of the dRP lyase activity of AtPol λ, which was nearly abolished in the K255A mutant protein. Alanine substitution for K248 resulted in a lesser, but still important reduction of activity. These results suggest that K255 is the preferred but no required nucleophile responsible for the dRP lyase reaction catalyzed by AtPol λ. Interestingly, AtPol λ K255 is conserved in plant and animal Pol λ enzymes, but not in Pol β proteins, whereas AtPol λ K248 is conserved in plant Pol λ and in Pol β proteins, but not in metazoan Pol λ. The residue aligned with AtPol λ K255 in human Pol β is K60, and its replacement by alanine also results in a significant reduction in Pol β dRP lyase activity (Prasad et al. ##REF##9556598##1998##).</p>\",\n \"<p id=\\\"Par30\\\">We found that the DNA polymerase activity of AtPol λ K248A and K255A mutant proteins was not affected, but their ability to extend DNA synthesis in a single-nucleotide gap, with the consequent displacement of the downstream strand, was impaired. This result agrees with the fact that residues in the 8-kDa domain of human Pol λ make specific contacts with the 5′-end of the downstream strand in gapped substrates (Garcia-Diaz et al. ##REF##14992725##2004##). The strand-displacement ability of AtPol λ might allow its participation in LP-BER. Although this has already been suggested for human Pol λ by some authors (Garcia-Diaz et al. ##REF##11457865##2001##), to date there is no evidence that AtPol λ is involved in plant LP-BER.</p>\",\n \"<p id=\\\"Par31\\\">In this work we have also shown that the dRP lyase activity of AtPol λ is required to complete SP-BER of uracil in a reconstituted repair reaction. In the BER reconstitution assay, AtPol λ was able to perform both the gap-filling and dRP removal steps that allow the final DNA ligation of the processed strand. In contrast, the ability of AtPol λ K248A and K255A mutant proteins to promote the repair of the BER intermediate was significantly impaired. Importantly, the capacity of the mutant AtPol λ versions to support SP-BER was inversely correlated with their dRP lyase activity and was strongly reduced in the AtPol λ K255A protein. In mammalian cells, removal of the dRP group is a critical BER step in vivo, since only the dRP lyase activity of Pol β, and not its DNA polymerization capacity, is required to reverse the DNA damage sensitivity of Pol β-null cells (Sobol et al. ##REF##10866204##2000##). Since plants lack Pol β homologues, our results suggest that the dRP lyase activity of AtPol λ might play a role in <italic>Arabidopsis</italic> SP-BER in vivo.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<p id=\"Par1\">Base excision repair (BER) generates gapped DNA intermediates containing a 5′-terminal 2-deoxyribose-5-phosphate (5′-dRP) group. In mammalian cells, gap filling and dRP removal are catalyzed by Pol β, which belongs to the X family of DNA polymerases. In higher plants, the only member of the X family of DNA polymerases is Pol λ. Although it is generally believed that plant Pol λ participates in BER, there is limited experimental evidence for this hypothesis. Here we have characterized the biochemical properties of <italic>Arabidopsis thaliana</italic> Pol λ (AtPol λ) in a BER context, using a variety of DNA repair intermediates. We have found that AtPol λ performs gap filling inserting the correct nucleotide, and that the rate of nucleotide incorporation is higher in substrates containing a C in the template strand. Gap filling catalyzed by AtPol λ is most efficient with a phosphate at the 5′-end of the gap and is not inhibited by the presence of a 5′-dRP mimic. We also show that AtPol λ possesses an intrinsic dRP lyase activity that is reduced by mutations at two lysine residues in its 8-kDa domain, one of which is present in Pol λ exclusively and not in any Pol β homolog. Importantly, we also found that the dRP lyase activity of AtPol λ allows efficient completion of uracil repair in a reconstituted short-patch BER reaction. These results suggest that AtPol λ plays an important role in plant BER.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s11103-023-01407-8.</p>","<title>Key message</title>","<p id=\"Par2\">Pol λ, the only X family DNA polymerase present in plants, possesses a dRP lyase activity that is required for completion of base excision repair in <italic>Arabidopsis</italic>.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s11103-023-01407-8.</p>","<title>Keywords</title>","<p>Funding for open access publishing: Universidad de Córdoba/CBUA</p>"],"string":"[\n \"<p id=\\\"Par1\\\">Base excision repair (BER) generates gapped DNA intermediates containing a 5′-terminal 2-deoxyribose-5-phosphate (5′-dRP) group. In mammalian cells, gap filling and dRP removal are catalyzed by Pol β, which belongs to the X family of DNA polymerases. In higher plants, the only member of the X family of DNA polymerases is Pol λ. Although it is generally believed that plant Pol λ participates in BER, there is limited experimental evidence for this hypothesis. Here we have characterized the biochemical properties of <italic>Arabidopsis thaliana</italic> Pol λ (AtPol λ) in a BER context, using a variety of DNA repair intermediates. We have found that AtPol λ performs gap filling inserting the correct nucleotide, and that the rate of nucleotide incorporation is higher in substrates containing a C in the template strand. Gap filling catalyzed by AtPol λ is most efficient with a phosphate at the 5′-end of the gap and is not inhibited by the presence of a 5′-dRP mimic. We also show that AtPol λ possesses an intrinsic dRP lyase activity that is reduced by mutations at two lysine residues in its 8-kDa domain, one of which is present in Pol λ exclusively and not in any Pol β homolog. Importantly, we also found that the dRP lyase activity of AtPol λ allows efficient completion of uracil repair in a reconstituted short-patch BER reaction. These results suggest that AtPol λ plays an important role in plant BER.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s11103-023-01407-8.</p>\",\n \"<title>Key message</title>\",\n \"<p id=\\\"Par2\\\">Pol λ, the only X family DNA polymerase present in plants, possesses a dRP lyase activity that is required for completion of base excision repair in <italic>Arabidopsis</italic>.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s11103-023-01407-8.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Funding for open access publishing: Universidad de Córdoba/CBUA</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>We are grateful to members of our lab for helpful criticism and advice. We thank Dr. Luis Blanco (Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain) for his advice on amino acid conservation analysis in Pol λ proteins.</p>","<title>Author contributions</title>","<p>DCC, TMR, TRA and RRA designed research; CBM, DOP and JALM. performed experiments; DCC, TMR, MIMM, RRA. and TRA analyzed data; DCC, TMR, MIMM, TRA and RRA wrote the manuscript. All authors read and approved the final manuscript.</p>","<title>Funding</title>","<p>Funding for open access publishing: Universidad de Córdoba/CBUA. This work was supported by the Spanish Ministry of Science and Innovation (MICINN) under grant number PID2019-109967 GB-I00. Funding for open access charge: Universidad de Córdoba / CBUA.</p>","<title>Data availability</title>","<p>The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par32\">The authors have no financial interests to disclose.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>We are grateful to members of our lab for helpful criticism and advice. We thank Dr. Luis Blanco (Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain) for his advice on amino acid conservation analysis in Pol λ proteins.</p>\",\n \"<title>Author contributions</title>\",\n \"<p>DCC, TMR, TRA and RRA designed research; CBM, DOP and JALM. performed experiments; DCC, TMR, MIMM, RRA. and TRA analyzed data; DCC, TMR, MIMM, TRA and RRA wrote the manuscript. All authors read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Funding for open access publishing: Universidad de Córdoba/CBUA. This work was supported by the Spanish Ministry of Science and Innovation (MICINN) under grant number PID2019-109967 GB-I00. Funding for open access charge: Universidad de Córdoba / CBUA.</p>\",\n \"<title>Data availability</title>\",\n \"<p>The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par32\\\">The authors have no financial interests to disclose.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Arabidopsis Pol λ gap filling activity. <bold>a</bold>\n<italic>Schematic diagram of substrate and product</italic>. <bold>b</bold>\n<italic>AtPol λ</italic>\n<italic>gap filling assay</italic>. DNA substrates (100 nM) were incubated at 30 °C with AtPol <italic>λ</italic> at the concentration and time indicated in reaction mixtures containing dCTP (100 μM). <bold>c</bold>\n<italic>Inhibition of DNA synthesis by anti-AtPol λ antiserum</italic>. AtPol λ protein (10 nM) was pre-incubated with pre-immune serum or anti-AtPol λ antiserum for one hour at 4 °C and then incubated for 20 min at 30 °C with the DNA substrate (100 nM) in reaction mixtures containing dCTP (100 μM). <bold>d</bold>\n<italic>DNA synthesis catalyzed by AtPol λ is template-directed</italic>. AtPol λ protein (10 nM) was incubated with DNA substrates (100 nM) at 30 °C for 30 min in reaction mixtures containing 20 μM of dATP, dCTP, dGTP or dTTP, as indicated</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>AtPol λ sensitivity to dideoxynucleotides.<bold> a</bold>\n<italic>Schematic diagram of molecules used as DNA substrates.</italic>\n<bold>b</bold>\n<italic>DNA polymerase assay.</italic> DNA substrate (100 nM) was incubated with AtPol λ protein (10 nM) for 60 min at 30 °C in reaction mixtures containing dCTP (10 μM), dGTP (10 μM) and increasing levels of ddCTP (0, 10, 25, 50, 250 and 500 μM) as indicated</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Effect of the template base on AtPol λ gap-filling activity. <bold>a</bold><italic>Gap-filling assay.</italic> DNA substrates (100 nM) containing a single-nucleotide gap with A, T, C or G as template were incubated with AtPol λ (10 nM) for 5, 10, 20 and 40 min at 30 °C in reaction mixtures containing 1 μM of the appropriate complementary deoxynucleotide. <bold>b</bold>\n<italic>Percentage of nucleotide insertion in gaps with A, T, C or G as template</italic>. Values are the mean with standard error from three independent experiments. <bold>c</bold>\n<italic>Bypass of 8-oxoG by AtPol λ</italic>. DNA substrate with a single-nucleotide gap opposite 8-oxoG (100 nM) was incubated with AtPol λ (10 nM) for 30 min at 30 °C in reaction mixtures containing increasing levels of dCTP or dATP (0, 1, 5, 10 and 50 μM) as indicated</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Effect the 5′-end group on AtPol λ gap-filling activity.<bold> a–c</bold>\n<italic>Gap-filling assay.</italic> DNA substrates (100 nM) containing a single-nucleotide gap with 5′-P (<bold>a</bold>), 5′-OH (<bold>b</bold>), or 5′-THF end (<bold>c</bold>) were incubated with AtPol λ (10 nM) for the indicated times at 30 °C in reaction mixtures containing dCTP at the indicated concentrations. <bold>d</bold>\n<italic>Percentage of dCMP insertion for each DNA substrate</italic>. Values are means ± standard errors from two independent experiments</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>AtPol λ dRP lyase activity. <bold>a</bold>\n<italic>Schematic diagram of substrate and product</italic>. <bold>b</bold>\n<italic>dRP lyase assay.</italic> DNA (100 nM) was incubated at 30 °C for 90 min in reaction mixtures containing the indicated DNA polymerase: Taq (1 U); human Pol β (2.4 U); WT or mutant versions of AtPol λ (10 nM). <bold>c</bold>\n<italic>Percentage of 5′-dRP lyase activity</italic>. The percentage of 5′-dRP lyase activity was calculated as the percentage of 5′-P product generated by each sample subtracting the 5′-P product generated by spontaneous hydrolysis of the 5′-dRP end in the absence of enzyme. Values are means ± standard errors from three independent experiments. Asterisks indicate statistically significant differences (P < 0.05; Student’s t-test)</p></caption></fig>","<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>DNA polymerase activity of AtPol λ K248A and K255A mutant proteins. <bold>a</bold>\n<italic>Schematic diagram of the DNA substrate</italic>. <bold>b</bold>\n<italic>DNA polymerase assay.</italic> DNA (100 nM) was incubated for 30 min at 30 °C with AtPol λ WT or mutant versions (10 nM) in reaction mixtures with or without dNTPs (100 μM), as indicated. Klenow DNA polymerase (2.5 U) was used as a control</p></caption></fig>","<fig id=\"Fig7\"><label>Fig. 7</label><caption><p>AtPol λ dRP lyase activity is required for SP-BER in vitro. <bold>a</bold>\n<italic>Schematic diagram of the BER reconstitution assay</italic>. <bold>b</bold>\n<italic>BER assay.</italic> DNA (100 nM) was incubated at 30 °C for 90 min in reaction mixtures containing 20 μM dCTP, <italic>E. coli</italic> UDG (0.5 U), human APE1 (10 U) and, when indicated, T4 DNA Ligase (1.5 U), human Pol β (2.4 U), or AtPol λ (10 nM). <bold>c</bold>\n<italic>Percentage of fully repaired DNA products</italic>. Values are means ± standard errors from two independent experiments. Asterisks indicate statistically significant differences (P < 0.05; Student’s t-test)</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Arabidopsis Pol λ gap filling activity. <bold>a</bold>\\n<italic>Schematic diagram of substrate and product</italic>. <bold>b</bold>\\n<italic>AtPol λ</italic>\\n<italic>gap filling assay</italic>. DNA substrates (100 nM) were incubated at 30 °C with AtPol <italic>λ</italic> at the concentration and time indicated in reaction mixtures containing dCTP (100 μM). <bold>c</bold>\\n<italic>Inhibition of DNA synthesis by anti-AtPol λ antiserum</italic>. AtPol λ protein (10 nM) was pre-incubated with pre-immune serum or anti-AtPol λ antiserum for one hour at 4 °C and then incubated for 20 min at 30 °C with the DNA substrate (100 nM) in reaction mixtures containing dCTP (100 μM). <bold>d</bold>\\n<italic>DNA synthesis catalyzed by AtPol λ is template-directed</italic>. AtPol λ protein (10 nM) was incubated with DNA substrates (100 nM) at 30 °C for 30 min in reaction mixtures containing 20 μM of dATP, dCTP, dGTP or dTTP, as indicated</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>AtPol λ sensitivity to dideoxynucleotides.<bold> a</bold>\\n<italic>Schematic diagram of molecules used as DNA substrates.</italic>\\n<bold>b</bold>\\n<italic>DNA polymerase assay.</italic> DNA substrate (100 nM) was incubated with AtPol λ protein (10 nM) for 60 min at 30 °C in reaction mixtures containing dCTP (10 μM), dGTP (10 μM) and increasing levels of ddCTP (0, 10, 25, 50, 250 and 500 μM) as indicated</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Effect of the template base on AtPol λ gap-filling activity. <bold>a</bold><italic>Gap-filling assay.</italic> DNA substrates (100 nM) containing a single-nucleotide gap with A, T, C or G as template were incubated with AtPol λ (10 nM) for 5, 10, 20 and 40 min at 30 °C in reaction mixtures containing 1 μM of the appropriate complementary deoxynucleotide. <bold>b</bold>\\n<italic>Percentage of nucleotide insertion in gaps with A, T, C or G as template</italic>. Values are the mean with standard error from three independent experiments. <bold>c</bold>\\n<italic>Bypass of 8-oxoG by AtPol λ</italic>. DNA substrate with a single-nucleotide gap opposite 8-oxoG (100 nM) was incubated with AtPol λ (10 nM) for 30 min at 30 °C in reaction mixtures containing increasing levels of dCTP or dATP (0, 1, 5, 10 and 50 μM) as indicated</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Effect the 5′-end group on AtPol λ gap-filling activity.<bold> a–c</bold>\\n<italic>Gap-filling assay.</italic> DNA substrates (100 nM) containing a single-nucleotide gap with 5′-P (<bold>a</bold>), 5′-OH (<bold>b</bold>), or 5′-THF end (<bold>c</bold>) were incubated with AtPol λ (10 nM) for the indicated times at 30 °C in reaction mixtures containing dCTP at the indicated concentrations. <bold>d</bold>\\n<italic>Percentage of dCMP insertion for each DNA substrate</italic>. Values are means ± standard errors from two independent experiments</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><p>AtPol λ dRP lyase activity. <bold>a</bold>\\n<italic>Schematic diagram of substrate and product</italic>. <bold>b</bold>\\n<italic>dRP lyase assay.</italic> DNA (100 nM) was incubated at 30 °C for 90 min in reaction mixtures containing the indicated DNA polymerase: Taq (1 U); human Pol β (2.4 U); WT or mutant versions of AtPol λ (10 nM). <bold>c</bold>\\n<italic>Percentage of 5′-dRP lyase activity</italic>. The percentage of 5′-dRP lyase activity was calculated as the percentage of 5′-P product generated by each sample subtracting the 5′-P product generated by spontaneous hydrolysis of the 5′-dRP end in the absence of enzyme. Values are means ± standard errors from three independent experiments. Asterisks indicate statistically significant differences (P < 0.05; Student’s t-test)</p></caption></fig>\",\n \"<fig id=\\\"Fig6\\\"><label>Fig. 6</label><caption><p>DNA polymerase activity of AtPol λ K248A and K255A mutant proteins. <bold>a</bold>\\n<italic>Schematic diagram of the DNA substrate</italic>. <bold>b</bold>\\n<italic>DNA polymerase assay.</italic> DNA (100 nM) was incubated for 30 min at 30 °C with AtPol λ WT or mutant versions (10 nM) in reaction mixtures with or without dNTPs (100 μM), as indicated. Klenow DNA polymerase (2.5 U) was used as a control</p></caption></fig>\",\n \"<fig id=\\\"Fig7\\\"><label>Fig. 7</label><caption><p>AtPol λ dRP lyase activity is required for SP-BER in vitro. <bold>a</bold>\\n<italic>Schematic diagram of the BER reconstitution assay</italic>. <bold>b</bold>\\n<italic>BER assay.</italic> DNA (100 nM) was incubated at 30 °C for 90 min in reaction mixtures containing 20 μM dCTP, <italic>E. coli</italic> UDG (0.5 U), human APE1 (10 U) and, when indicated, T4 DNA Ligase (1.5 U), human Pol β (2.4 U), or AtPol λ (10 nM). <bold>c</bold>\\n<italic>Percentage of fully repaired DNA products</italic>. Values are means ± standard errors from two independent experiments. Asterisks indicate statistically significant differences (P < 0.05; Student’s t-test)</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":[],"string":"[]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"11103_2023_1407_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"11103_2023_1407_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"11103_2023_1407_Fig3_HTML\" id=\"MO3\"/>","<graphic xlink:href=\"11103_2023_1407_Fig4_HTML\" id=\"MO4\"/>","<graphic xlink:href=\"11103_2023_1407_Fig5_HTML\" id=\"MO5\"/>","<graphic xlink:href=\"11103_2023_1407_Fig6_HTML\" id=\"MO6\"/>","<graphic xlink:href=\"11103_2023_1407_Fig7_HTML\" id=\"MO7\"/>"],"string":"[\n \"<graphic xlink:href=\\\"11103_2023_1407_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"11103_2023_1407_Fig2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic xlink:href=\\\"11103_2023_1407_Fig3_HTML\\\" id=\\\"MO3\\\"/>\",\n \"<graphic xlink:href=\\\"11103_2023_1407_Fig4_HTML\\\" id=\\\"MO4\\\"/>\",\n \"<graphic xlink:href=\\\"11103_2023_1407_Fig5_HTML\\\" id=\\\"MO5\\\"/>\",\n \"<graphic xlink:href=\\\"11103_2023_1407_Fig6_HTML\\\" id=\\\"MO6\\\"/>\",\n \"<graphic xlink:href=\\\"11103_2023_1407_Fig7_HTML\\\" id=\\\"MO7\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"11103_2023_1407_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 1763 KB)</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"11103_2023_1407_MOESM1_ESM.pdf\\\"><caption><p>Supplementary file1 (PDF 1763 KB)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"surname\": [\"Cavanaugh\", \"Beard\", \"Wilson\"], \"given-names\": [\"NA\", \"WA\", \"SHJJOBC\"], \"article-title\": [\"DNA polymerase \\u03b2 ribonucleotide discrimination: insertion, misinsertion, extension, and coding\"], \"source\": [\"Nat Rev Genet\"], \"year\": [\"2010\"], \"volume\": [\"285\"], \"fpage\": [\"24457\"], \"lpage\": [\"24465\"], \"pub-id\": [\"10.1074/jbc.M110.132407\"]}, {\"mixed-citation\": [\"Seeberg E, Luna L, Morland I, Eide L, Johnsen B, Larsen E, Alseth I, Dantzer F, Baynton K, Aamodt R (2000) Base removers and strand scissors: Different strategies employed in base excision and strand incision at modified base residues in DNA. Paper presented at: Cold Spring Harbor Symposia on Quantitative Biology (Cold Spring Harbor Laboratory Press). 10.1101/sqb.2000.65.135\"]}, {\"surname\": [\"Wang\", \"Wu\", \"Hellinga\", \"Beese\"], \"given-names\": [\"W\", \"EY\", \"HW\", \"LSJJOBC\"], \"article-title\": [\"Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides\"], \"source\": [\"Biochimie\"], \"year\": [\"2012\"], \"volume\": [\"287\"], \"fpage\": [\"28215\"], \"lpage\": [\"28226\"], \"pub-id\": [\"10.1074/jbc.M112.366609\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":52,"string":"52"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Plant Mol Biol. 2024 Jan 13; 114(1):3"},"package_file":{"kind":"string","value":"oa_package/d6/08/PMC10787897.tar.gz"}}},{"rowIdx":181,"cells":{"accession_id":{"kind":"string","value":"PMC10787898"},"pmid":{"kind":"string","value":"38217571"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par2\">Sensing and regulating the tension of the plasma membrane is crucial for cells to function effectively, especially when challenged by changes in the local micromechanical environment. Whether during malignant transformation, cytokinesis or morphogenesis, altered cell–cell and cell–substrate adhesivity are coupled to the dynamic cell signalling state defining these processes [##REF##31937589##28##, ##REF##11285293##48##]. A remodelled micro-environment can influence the cell membrane's mechanical state, changing its flexibility or tension. Processes involved in membrane homeostasis would ensure the maintenance of membrane tension or conservation of the microscopic fluctuations, providing the plasma membrane’s effective tautness or excess area (difference between microscopic area and projected area per unit microscopic area) [##REF##11220366##46##, ##UREF##8##64##]. While enhancing tension can affect endocytosis [##REF##28923837##18##, ##REF##31925316##29##] and pit-formation [##REF##29317637##9##], as displayed for clathrin-mediated endocytosis (CME) [##REF##36213118##2##], conversely, cells can also alter their endocytosis/exocytosis rates to regulate tension. Studies have addressed the role of membrane trafficking in tension regulation, either in general [##REF##15157483##65##] or focussing on particular pathways such as the CLIC-GEEC (CG) pathway [##REF##29317637##74##], CME [##REF##33788963##17##, ##REF##31391241##39##] or those using caveolae [##UREF##4##45##]. Flattening of caveolae can regulate tension surge [##REF##21295700##69##] suggesting that pits that participate in endocytosis may directly regulate tension.</p>","<p id=\"Par3\">Since pit-formation and internalization are common to all pathways and sometimes also regulated similarly, it becomes essential to understand whether the formation of endocytic pits in general can initiate tension regulation or their subsequent internalization is also essential. Addressing such questions would require mechanical perturbation of cells and following membrane mechanics and endocytosis state over time. Additionally, perturbing molecules necessary for pit formation or internalization can further elucidate their individual roles.</p>","<p id=\"Par4\">To quantify endocytosis, specific cargoes (e.g. Transferrin for CME) are usually labelled to quantify its corresponding pathways. However, Rab5 labels early endosomes of most pathways and, thus, reports the general state of endocytic trafficking [##REF##1516130##8##]. A fraction of endosomes are recycled back to the plasma membrane using a fast-recycling arm, Rab4 [##REF##10811830##71##] which can accumulate if either excess endosomes are formed, or the fusion with the plasma membrane is hampered [##REF##19924646##30##]. Therefore, following labelled cargo (like Transferrin), as well as Rab5 and Rab4, together with well-established Transferrin uptake assays, can collectively describe the trafficking state of the basal plasma membrane.</p>","<p id=\"Par5\">For perturbing endocytosis, molecules utilized by multiple pathways can be targeted. For example, many constitutive pathways in HeLa cells (e.g. CME, caveolae) require dynamin for the scission of their endocytic pits, making it an important target. Dynamin's GTPase action can be inhibited by Dynasore, an agent that can prevent scission and, thus, internalization of pits in these pathways without affecting pit formation [##REF##16740485##40##]. However, certain constitutive pathways, like CG, could still be operational even on Dynasore treatment—although its presence in HeLa cells is debated [##REF##31283376##75##], while other dynamin-independent constitutive pathways are not well classified. Knocking down adaptor protein AP2 would directly impact the pit-formation step of CME [##UREF##2##38##, ##REF##21883765##50##, ##REF##34774130##77##]. Cholesterol is required very early for the primary clustering of lipids/proteins in several pathways [##REF##7577971##32##]. The formation of endocytic carriers critically requires cholesterol for pathways such as CG pathway and caveolae, while assembly of clathrin and membrane curving in CME mostly requires adapters like AP2 [##REF##22863004##11##, ##REF##14990728##27##, ##REF##34774130##77##]. Depletion of Cholesterol (by MβCD) thus, can arrest the formation of new pits of the CG pathway and caveolae. However, it can also enhance tension [##REF##30979551##7##], and has been reported to cause a substantial reduction in the formation and budding of deep pits of CME without completely blocking CME [##REF##36213118##2##, ##REF##34919799##4##, ##REF##10198050##58##, ##REF##10359788##73##]. The use of ATP-depletion can reduce both the pinching-off of a majority of pathways as well as the formation of pits in pathways like CME [##REF##2126013##62##] and caveolae [##REF##21295700##69##]. Thus, ATP depletion could suppress the completion of a multitude of constitutive endocytic pathways and, therefore, could be used to study the role of scission in general. The alterations of these endocytic events need to be understood in response to perturbations to the mechanical microenvironment.</p>","<p id=\"Par6\">Mounting evidence [##REF##31843255##19##, ##REF##34346220##36##, ##REF##35532004##79##] shows significant crosstalk between endocytic machinery and cell adhesion molecules and its role in regulating cellular homeostasis [##UREF##11##80##]. While cell–substrate adhesion profiles of single cells have been reported to determine the distribution of endocytosis sites [##REF##24366944##22##], studies have also addressed if de-adhesion can trigger mechano-regulation via endocytosis [##REF##29317637##74##] or blebbing [##REF##20858416##49##]. Although measurements of apparent tension have been reported during cell spreading [##REF##21808040##20##, ##REF##21295700##69##] similar studies involving tension measurements during de-adhesion have not been performed, to the best of our knowledge. Measurement of membrane mechanics can be either done at specific points by optical tweezer-based tether pulling or by studying the spontaneous fluctuations of the membrane measured using interference reflection microscopy (IRM). Several studies [##REF##30979551##7##, ##REF##30392960##66##] have proposed spatial heterogeneities in tension. Multi-point measurements would enable accessing the local distribution of excess membrane undulations or tension in cells during different phases of de-adhesion.</p>","<p id=\"Par7\">In this study, we address the contribution of endocytosis to mechano-regulation during de-adhesion by employing the IRM-based measurement of effective fluctuation–tension of de-adhering HeLa cells. IRM [##REF##1169157##1##, ##REF##22174897##10##, ##REF##14126869##12##, ##REF##2768185##21##, ##REF##19816893##37##, ##UREF##6##54##] primarily provides information about the distance of the basal membrane from the substrate—also called the membrane ‘height’. Spatio-temporal measurements of height provide fluctuation amplitude (SD<sub>time</sub>) excess area and enable deriving mechanical parameters like fluctuation-tension [##REF##30979551##7##, ##REF##26796575##67##]. Spatial maps of fluctuation-tension (also termed tension in the rest of the manuscript) and their temporal evolution help us quantify the changes.</p>","<p id=\"Par8\">We use total internal reflection fluorescence (TIRF) microscopy to image Rab5 and Rab4 labelled structures, labelling the early endosomes and rapidly recycling endosomes, respectively. This measures how de-adhesion alters internalization during endocytosis. Fluorescent cargo—transferrin—is used to further validate the involvement of endocytosis while Dynasore, dynamin-mutants, knockdown of AP2, Cholesterol and ATP depletion are used to perturb steps/pathways of endocytosis to assess their contribution to the mechano-regulation.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par2\\\">Sensing and regulating the tension of the plasma membrane is crucial for cells to function effectively, especially when challenged by changes in the local micromechanical environment. Whether during malignant transformation, cytokinesis or morphogenesis, altered cell–cell and cell–substrate adhesivity are coupled to the dynamic cell signalling state defining these processes [##REF##31937589##28##, ##REF##11285293##48##]. A remodelled micro-environment can influence the cell membrane's mechanical state, changing its flexibility or tension. Processes involved in membrane homeostasis would ensure the maintenance of membrane tension or conservation of the microscopic fluctuations, providing the plasma membrane’s effective tautness or excess area (difference between microscopic area and projected area per unit microscopic area) [##REF##11220366##46##, ##UREF##8##64##]. While enhancing tension can affect endocytosis [##REF##28923837##18##, ##REF##31925316##29##] and pit-formation [##REF##29317637##9##], as displayed for clathrin-mediated endocytosis (CME) [##REF##36213118##2##], conversely, cells can also alter their endocytosis/exocytosis rates to regulate tension. Studies have addressed the role of membrane trafficking in tension regulation, either in general [##REF##15157483##65##] or focussing on particular pathways such as the CLIC-GEEC (CG) pathway [##REF##29317637##74##], CME [##REF##33788963##17##, ##REF##31391241##39##] or those using caveolae [##UREF##4##45##]. Flattening of caveolae can regulate tension surge [##REF##21295700##69##] suggesting that pits that participate in endocytosis may directly regulate tension.</p>\",\n \"<p id=\\\"Par3\\\">Since pit-formation and internalization are common to all pathways and sometimes also regulated similarly, it becomes essential to understand whether the formation of endocytic pits in general can initiate tension regulation or their subsequent internalization is also essential. Addressing such questions would require mechanical perturbation of cells and following membrane mechanics and endocytosis state over time. Additionally, perturbing molecules necessary for pit formation or internalization can further elucidate their individual roles.</p>\",\n \"<p id=\\\"Par4\\\">To quantify endocytosis, specific cargoes (e.g. Transferrin for CME) are usually labelled to quantify its corresponding pathways. However, Rab5 labels early endosomes of most pathways and, thus, reports the general state of endocytic trafficking [##REF##1516130##8##]. A fraction of endosomes are recycled back to the plasma membrane using a fast-recycling arm, Rab4 [##REF##10811830##71##] which can accumulate if either excess endosomes are formed, or the fusion with the plasma membrane is hampered [##REF##19924646##30##]. Therefore, following labelled cargo (like Transferrin), as well as Rab5 and Rab4, together with well-established Transferrin uptake assays, can collectively describe the trafficking state of the basal plasma membrane.</p>\",\n \"<p id=\\\"Par5\\\">For perturbing endocytosis, molecules utilized by multiple pathways can be targeted. For example, many constitutive pathways in HeLa cells (e.g. CME, caveolae) require dynamin for the scission of their endocytic pits, making it an important target. Dynamin's GTPase action can be inhibited by Dynasore, an agent that can prevent scission and, thus, internalization of pits in these pathways without affecting pit formation [##REF##16740485##40##]. However, certain constitutive pathways, like CG, could still be operational even on Dynasore treatment—although its presence in HeLa cells is debated [##REF##31283376##75##], while other dynamin-independent constitutive pathways are not well classified. Knocking down adaptor protein AP2 would directly impact the pit-formation step of CME [##UREF##2##38##, ##REF##21883765##50##, ##REF##34774130##77##]. Cholesterol is required very early for the primary clustering of lipids/proteins in several pathways [##REF##7577971##32##]. The formation of endocytic carriers critically requires cholesterol for pathways such as CG pathway and caveolae, while assembly of clathrin and membrane curving in CME mostly requires adapters like AP2 [##REF##22863004##11##, ##REF##14990728##27##, ##REF##34774130##77##]. Depletion of Cholesterol (by MβCD) thus, can arrest the formation of new pits of the CG pathway and caveolae. However, it can also enhance tension [##REF##30979551##7##], and has been reported to cause a substantial reduction in the formation and budding of deep pits of CME without completely blocking CME [##REF##36213118##2##, ##REF##34919799##4##, ##REF##10198050##58##, ##REF##10359788##73##]. The use of ATP-depletion can reduce both the pinching-off of a majority of pathways as well as the formation of pits in pathways like CME [##REF##2126013##62##] and caveolae [##REF##21295700##69##]. Thus, ATP depletion could suppress the completion of a multitude of constitutive endocytic pathways and, therefore, could be used to study the role of scission in general. The alterations of these endocytic events need to be understood in response to perturbations to the mechanical microenvironment.</p>\",\n \"<p id=\\\"Par6\\\">Mounting evidence [##REF##31843255##19##, ##REF##34346220##36##, ##REF##35532004##79##] shows significant crosstalk between endocytic machinery and cell adhesion molecules and its role in regulating cellular homeostasis [##UREF##11##80##]. While cell–substrate adhesion profiles of single cells have been reported to determine the distribution of endocytosis sites [##REF##24366944##22##], studies have also addressed if de-adhesion can trigger mechano-regulation via endocytosis [##REF##29317637##74##] or blebbing [##REF##20858416##49##]. Although measurements of apparent tension have been reported during cell spreading [##REF##21808040##20##, ##REF##21295700##69##] similar studies involving tension measurements during de-adhesion have not been performed, to the best of our knowledge. Measurement of membrane mechanics can be either done at specific points by optical tweezer-based tether pulling or by studying the spontaneous fluctuations of the membrane measured using interference reflection microscopy (IRM). Several studies [##REF##30979551##7##, ##REF##30392960##66##] have proposed spatial heterogeneities in tension. Multi-point measurements would enable accessing the local distribution of excess membrane undulations or tension in cells during different phases of de-adhesion.</p>\",\n \"<p id=\\\"Par7\\\">In this study, we address the contribution of endocytosis to mechano-regulation during de-adhesion by employing the IRM-based measurement of effective fluctuation–tension of de-adhering HeLa cells. IRM [##REF##1169157##1##, ##REF##22174897##10##, ##REF##14126869##12##, ##REF##2768185##21##, ##REF##19816893##37##, ##UREF##6##54##] primarily provides information about the distance of the basal membrane from the substrate—also called the membrane ‘height’. Spatio-temporal measurements of height provide fluctuation amplitude (SD<sub>time</sub>) excess area and enable deriving mechanical parameters like fluctuation-tension [##REF##30979551##7##, ##REF##26796575##67##]. Spatial maps of fluctuation-tension (also termed tension in the rest of the manuscript) and their temporal evolution help us quantify the changes.</p>\",\n \"<p id=\\\"Par8\\\">We use total internal reflection fluorescence (TIRF) microscopy to image Rab5 and Rab4 labelled structures, labelling the early endosomes and rapidly recycling endosomes, respectively. This measures how de-adhesion alters internalization during endocytosis. Fluorescent cargo—transferrin—is used to further validate the involvement of endocytosis while Dynasore, dynamin-mutants, knockdown of AP2, Cholesterol and ATP depletion are used to perturb steps/pathways of endocytosis to assess their contribution to the mechano-regulation.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<title>Cell line</title>","<p id=\"Par52\">HeLa cell line (CCL-2, ATCC) was used to perform all the experimental studies.</p>","<title>Cell culture</title>","<p id=\"Par53\">HeLa cells were grown in Dulbecco’s Modified Essential Medium (DMEM, Gibco, Life Technologies, USA) with 10% foetal bovine serum (FBS, Gibco) and 1% Anti-Anti (Gibco) at 95% humidity, 5% CO<sub>2</sub> and 37 °C. Experiments were always performed after 16–18 h of cell seeding.</p>","<title>Pharmacological treatments</title>","<p id=\"Par54\">To de-adhere cells from the substrate, HeLa cells were incubated with 0.05% or 0.25% Trypsin–EDTA solution (Gibco) at 37 °C on the onstage microscope incubator. To inhibit all dynamin-dependent endocytic pathways, we incubated cells with Dynasore hydrate (80 μM; Sigma) in serum-free media for 20 min [##REF##20098746##5##, ##REF##18413242##31##, ##REF##16740485##40##]. HeLa cells are incubated with ML-141 (10 μM; Sigma) for 30 min in serum-free media to inhibit CLIC-GEEC endocytic pathways [##REF##29317637##74##]. For ATP depletion, cells are incubated for 1 h with 10 mM Sodium Azide (Sigma-Aldrich) and 10 mM 2- deoxy-D- glucose (Sigma-Aldrich) dissolved in M1 media composed of 150 mM NaCl (Sigma-Aldrich), 1 mM MgCl<sub>2</sub> (Merck)<sub>,</sub> 20 mM HEPES (Sigma) [##REF##29045871##6##, ##REF##9425152##78##]. 10 mM Methyl-ß-cyclodextrin (Sigma-Aldrich) was used in serum-free media for 50 min to deplete Cholesterol [##REF##30979551##7##]. For inhibiting Actin filament polymerization, cells were kept in 5 μM Cytochalasin D (Sigma-Aldrich) for 1 h in serum-free media [##REF##29045871##6##]. Cells were de-adhered by TrypLE Express (Gibco). This was used as an alternative to Trypsin–EDTA for de-adhesion experiments [##REF##29317637##74##]. 1 mM EDTA was used for de-adhering cells [##REF##31348954##34##]. All the treatments were incubated at 37 °C inside the incubator. During imaging and de-adhesion, all treatments were maintained at the same concentration. For Calcein AM staining, 2 μM Calcein AM was added in serum free media and incubated for 30. After incubation media was discarded and fresh media was added for the imaging. For endocytosis uptake assay, 10 μg/ml Transferrin Alexa fluor 568 was incubated for 5 min at 37 °C. For stopping endocytosis, HEPES based buffer was used at ice cold temperature. To remove external Tf fluorescence, ascorbate buffer was used at 4 °C [##REF##29317637##74##]. Cells were fixed using ice cold 4% Paraformaldehyde. To follow Transferrin uptake during de-adhesion or in non-de-adhered cells (in normal or dynamin-inhibited conditions), cells were first incubated with 25 nM of Transferrin for 5 min. Subsequently, Transferrin was washed off and de-adhesion was followed. For dynamin inhibition cells were pre-treated with 80 μM Dynasore hydrate and then Transferrin was added.</p>","<title>Immunostaining</title>","<p id=\"Par55\">For immunostaining, cells were first fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min and subsequently washed twice with phosphate-buffered saline (PBS, Sigma-Aldrich). Next, cells were incubated in 0.1 M glycine (Sigma-Aldrich) for 5 min and then washed again with PBS. Triton-X was used for 2 min and then washed with PBS. For blocking, cells were incubated with 3 ml of 0.2% Gelatin (Sigma-Aldrich) solution for 3 h at room temperature. Primary antibody treatment was done with Recombinant Anti-Rab4 antibody (Abcam) at 1:200 dilution in Gelatin and kept overnight at 4 °C to mark recycling endosomes. Goat Anti -Rabbit IgG H&L, Alexa Fluor 488 secondary antibody (Abcam) was used at 1:500 dilution at Gelatin for 2 h after washing with PBS. Subsequently, cells were imaged in 2 ml of PBS.</p>","<title>Transfection</title>","<p id=\"Par56\">EGFP-Rab5 (Addgene) was a gift from Arnab Gupta. Cells were transfected with 1 μg of Rab5 plasmid DNA to label early endosomes, respectively, by using Lipofectamine 3000 (Invitrogen). EGFP-CAAX [##REF##27633000##41##] was a gift from Lei Lu. Cells were transfected with 1 μg EGFP-CAAX (Addgene) plasmid to mark the membrane. Cells were transfected with 1 μg of mCherry- Clathrin LC-15 (Addgene) to label clathrin-coated vesicles. To mark fast recycling endosomes, cells were transfected with 1 μg of mCherry- Rab 4a plasmid. Other treatments, if required, were performed 16 h after transfection.</p>","<p id=\"Par57\">Fixation of cells was performed by using 4% paraformaldehyde (Sigma-Aldrich) for 15 min at 37°C temperature.</p>","<title>siRNA-based knockdown</title>","<p id=\"Par58\">HeLa cells are transfected with 10 nM of AP2 siRNA total for 72 h. At 0 h first siRNA transfection was done and at 48 h fresh media was added with 10 nM of AP2 siRNA booster dose. To validate the knockdown of AP2, cells are immunostained with Anti AP2 antibody to check intensity at the basal membrane by TIRF microscopy. Western blotting was also performed using established protocols to validate the knockdown.</p>","<title>IRM imaging</title>","<p id=\"Par59\">Cells were imaged in Nikon Eclipse Ti-E motorized inverted microscope (Nikon, Japan) equipped with adjustable field and aperture diaphragms, 60X Plan Apo (NA 1.22, water immersion) and a 1.5X external magnification on an onstage 37 °C incubator (Tokai Hit, Japan). Either an EMCCD (Evolve 512 Delta, Photometrics, USA) or an s-CMOS camera (ORCA Flash 4.0, Hamamatsu, Japan) was used for imaging. A 100 W mercury arc lamp, an interference filter (546 ± 12 nm) and a 50–50 beam splitter were used [##REF##29045871##6##]. For IRM, movies consisted of 2048 frames (19.91 frames/s, EMCCD and 20 frames/s for s-CMOS) recorded at EM 30 and an exposure time of 50 ms.</p>","<title>Optical trap experiment</title>","<p id=\"Par60\">For optical trap-based tether-pulling experiments, a 2 µm polystyrene bead was trapped by focusing a 1064 nm Laser (Coherent, Sweden) of 1000 W power at source using a 100 × objective. The back-aperture of the objective was over filled after beam expansion and using mirrors and a 50/50 beam-splitter for beam manipulation. The trap stiffness (<italic>k</italic>) was calibrated from trajectories of the trapped bead using the equipartition approach where <italic>x</italic> is the displacement of the bead from the trap centre, <italic>k</italic><sub><italic>B</italic></sub> is the Boltzmann constant and T the temperature in the Kelvin scale. For analysis, the bead was detected as an object (MATLAB, Fiji), and its centre was tracked with time. The bead’s displacement from the centre of the trap and the spring constant of the trap was used to get the force (). For every cell, a tether was pulled at a constant velocity of 0.5 µm/s up to a distance of 40 µm (LabVIEW, National Instruments, USA). Tether force was calculated from the average bead position during the period when it was parked with the tether pulled (Fig. S3c) for ~ 50 s. Imaging was done at 200 frames per sec. The apparent membrane tension of the apical section of the cell was derived from the force using the Canham-Helfrich equation , where <italic>κ</italic> is the bending rigidity and taken to be 15 k<sub>B</sub>T [##REF##15695629##13##], and σ<sub>A</sub> denotes the apparent membrane tension.</p>","<title>TIRF imaging</title>","<p id=\"Par61\">For TIRF Microscopy, an inverted microscope (Olympus IX-83, Olympus, Japan) was used with a 100X 1.49 NA oil immersion TIRF objective (PlanApo, Olympus). An s-CMOS camera (ORCA Flash 4.0, Hamamatsu, Japan) and 488 nm, as well as 561 nm laser sources, were used. Images were acquired using an exposure time of 300 ms with ~ 70 nm penetration depth.</p>","<title>Confocal imaging</title>","<p id=\"Par62\">For confocal imaging, a Leica confocal microscope (Leica SP8) was used with a 63X oil objective lens (NA 1.4). A step size (in z) of 250 nm is used for imaging with a pixel size of 45 nm and deconvoluted (Leica Lightning Software).</p>","<title>STED imaging</title>","<p id=\"Par63\">An Abberior Facility Line system with an Olympus IX83 microscope (Abberior Instruments) was used for STED imaging. Abberior autoalignment sample was utilized for alignment of the STED and confocal channels. 15 nm pixel size was maintained during imaging. A pulsed STED line at 775 nm was used for depletion and STAR Red and Alexa 568 conjugated secondary antibodies were used for imaging Clathrin and AP2, respectively.</p>","<title>Analysis of IRM images</title>","<p id=\"Par64\">The intensity of IRM images was converted to height (wherever applicable) as reported [##REF##29045871##6##]. The amplitude of spatial undulations spatial (SD<sub>space</sub>) was obtained from the standard deviation (SD) of relative heights across all pixels in an FBR after averaging it over 20 frames. For obtaining the amplitude of temporal fluctuations, SD<sub>time</sub>, the SD of the relative heights over 2048 frames in each pixel was calculated and averaged across all pixels in an FBR. The power spectral density (PSD) of individual pixels was obtained from the temporal relative height time series using either the FFT method or the covariance method (MATLAB). PSDs of all pixels in an FBR were averaged to obtain the PSD for that FBR. For obtaining mechanical parameters, the PSDs were fitted to [##REF##25902428##3##, ##REF##29045871##6##, ##UREF##1##23##] where active temperature (A), effective cytoplasmic viscosity (<italic>η</italic><sub>eff</sub>), confinement (<italic>γ</italic>) and membrane tension (<italic>σ</italic>) were used as fitting parameters. The bending rigidity (<italic>κ</italic>) was fixed at 15 k<sub>B</sub>T [##UREF##9##68##]. For obtaining excess area fraction () over an FBR, the flat area of the FBR is taken as A<sub>P</sub> (= <italic>L</italic><sup>2</sup> when patch/FBR is a square of side <italic>L</italic>) and <italic>A</italic> is the sum of all <italic>dA</italic> calculated for each pixel by comparing the height at that pixel with its neighbours () [##UREF##0##16##]. The excess area is represented in figures as the percentage excess area ().</p>","<p id=\"Par65\">The activity per FBR is calculated as the lower bound of the entropy generation rate at each FBR. For obtaining the entropy generation rate, data pooled from all pixels of the FBR were taken through dimensional reduction using principal component analysis. The time series (2048 frames) of one of the principal components were built and analyzed as described recently [##UREF##3##43##] using the short-time inference scheme reported earlier [##REF##32281844##44##].</p>","<title>Tension mapping</title>","<p id=\"Par66\">For tension mapping (Fig. ##FIG##1##2##c), PSD was calculated for either for each pixel, and either directly used (pixel-wise tension mapping) or averaged over all pixels in each FBR (FBR-wise tension mapping). PSDs, thus, obtained are fitted and every parameter extracted from fits—including tension and <italic>R</italic><sup>2</sup> were mapped on to the same location as the pixel/FBR.</p>","<title>Fluorescence image analysis</title>","<p id=\"Par67\">For endosomal or puncta counting (MATLAB), first, a Gaussian blur operation was performed to spatially average out the image. The Gaussian-blur is next subtracted from the original image, thus enhancing local contrast. The subtracted image is normalized between 0 and 1, and thresholding was performed using the appropriate threshold, resulting in a binary image. Next, a mask was applied over the image to select the cell. Single pixels were removed using serial erosion and dilation, and subsequently, the binary image was used to detect objects. The area fraction is calculated by dividing the total area of detected objects () by the total area of the ROI or cell (). Similar approach was used for calculating colocalization. Objects were detected for each channel separately. Total area of the pixels overlapping in the two binary images was considered to be colocalizing area (). Colocalizing area divided by the total area of the ROI was used as colocalizing area fraction (). Colocalizing area divided by object-covered area of any particular was used as the percentage colocalization of that channel (). Mander’s coefficient was obtained using ImageJ.</p>","<title>Counting tubules from confocal z-slices</title>","<p id=\"Par68\">For counting tubules, Z-stack images of cells were captured. Line scans were drawn at the cortex regions of cells (ImageJ/Fiji), and the intensities of these line scans were taken from the intensity plot profile of each line scan to plot the internal intensity. Peak analysis (MATLAB) involved evaluating the intensity line scans for peaks of minimum prominence of ~ 6 and width of ~ 5. Peaks were counted as tubules. Length of tubules were obtained by drawing line ROIs along tubules and measuring their length.</p>","<title>Statistical analysis</title>","<p id=\"Par69\">Every IRM experiment was preceded by imaging beads. Every experiment was repeated at least thrice involving multiple cells and FBRs (Table ##SUPPL##0##S1##). A Mann–Whitney <italic>U</italic> test was performed for statistical significance testing (ns denotes <italic>p</italic> > 0.05, *denotes <italic>p</italic> < 0.05, **denoted <italic>p</italic> < 0.001). When indicated, a linear mixed effect model (LMM, MATLAB) was also used (using the fixed effect of time and random effects grouped under replicate set number of the experiments and cell number) to quantify the significance of the observed changes in logarithm of tension values (Table S2). This helped avoid the effect of the high sample size of FBRs that could influence hypothesis testing. LMM has been used for comparisons in other high-sampling mechanical measurements [##REF##29937952##24##, ##UREF##7##56##].</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<title>Cell line</title>\",\n \"<p id=\\\"Par52\\\">HeLa cell line (CCL-2, ATCC) was used to perform all the experimental studies.</p>\",\n \"<title>Cell culture</title>\",\n \"<p id=\\\"Par53\\\">HeLa cells were grown in Dulbecco’s Modified Essential Medium (DMEM, Gibco, Life Technologies, USA) with 10% foetal bovine serum (FBS, Gibco) and 1% Anti-Anti (Gibco) at 95% humidity, 5% CO<sub>2</sub> and 37 °C. Experiments were always performed after 16–18 h of cell seeding.</p>\",\n \"<title>Pharmacological treatments</title>\",\n \"<p id=\\\"Par54\\\">To de-adhere cells from the substrate, HeLa cells were incubated with 0.05% or 0.25% Trypsin–EDTA solution (Gibco) at 37 °C on the onstage microscope incubator. To inhibit all dynamin-dependent endocytic pathways, we incubated cells with Dynasore hydrate (80 μM; Sigma) in serum-free media for 20 min [##REF##20098746##5##, ##REF##18413242##31##, ##REF##16740485##40##]. HeLa cells are incubated with ML-141 (10 μM; Sigma) for 30 min in serum-free media to inhibit CLIC-GEEC endocytic pathways [##REF##29317637##74##]. For ATP depletion, cells are incubated for 1 h with 10 mM Sodium Azide (Sigma-Aldrich) and 10 mM 2- deoxy-D- glucose (Sigma-Aldrich) dissolved in M1 media composed of 150 mM NaCl (Sigma-Aldrich), 1 mM MgCl<sub>2</sub> (Merck)<sub>,</sub> 20 mM HEPES (Sigma) [##REF##29045871##6##, ##REF##9425152##78##]. 10 mM Methyl-ß-cyclodextrin (Sigma-Aldrich) was used in serum-free media for 50 min to deplete Cholesterol [##REF##30979551##7##]. For inhibiting Actin filament polymerization, cells were kept in 5 μM Cytochalasin D (Sigma-Aldrich) for 1 h in serum-free media [##REF##29045871##6##]. Cells were de-adhered by TrypLE Express (Gibco). This was used as an alternative to Trypsin–EDTA for de-adhesion experiments [##REF##29317637##74##]. 1 mM EDTA was used for de-adhering cells [##REF##31348954##34##]. All the treatments were incubated at 37 °C inside the incubator. During imaging and de-adhesion, all treatments were maintained at the same concentration. For Calcein AM staining, 2 μM Calcein AM was added in serum free media and incubated for 30. After incubation media was discarded and fresh media was added for the imaging. For endocytosis uptake assay, 10 μg/ml Transferrin Alexa fluor 568 was incubated for 5 min at 37 °C. For stopping endocytosis, HEPES based buffer was used at ice cold temperature. To remove external Tf fluorescence, ascorbate buffer was used at 4 °C [##REF##29317637##74##]. Cells were fixed using ice cold 4% Paraformaldehyde. To follow Transferrin uptake during de-adhesion or in non-de-adhered cells (in normal or dynamin-inhibited conditions), cells were first incubated with 25 nM of Transferrin for 5 min. Subsequently, Transferrin was washed off and de-adhesion was followed. For dynamin inhibition cells were pre-treated with 80 μM Dynasore hydrate and then Transferrin was added.</p>\",\n \"<title>Immunostaining</title>\",\n \"<p id=\\\"Par55\\\">For immunostaining, cells were first fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min and subsequently washed twice with phosphate-buffered saline (PBS, Sigma-Aldrich). Next, cells were incubated in 0.1 M glycine (Sigma-Aldrich) for 5 min and then washed again with PBS. Triton-X was used for 2 min and then washed with PBS. For blocking, cells were incubated with 3 ml of 0.2% Gelatin (Sigma-Aldrich) solution for 3 h at room temperature. Primary antibody treatment was done with Recombinant Anti-Rab4 antibody (Abcam) at 1:200 dilution in Gelatin and kept overnight at 4 °C to mark recycling endosomes. Goat Anti -Rabbit IgG H&L, Alexa Fluor 488 secondary antibody (Abcam) was used at 1:500 dilution at Gelatin for 2 h after washing with PBS. Subsequently, cells were imaged in 2 ml of PBS.</p>\",\n \"<title>Transfection</title>\",\n \"<p id=\\\"Par56\\\">EGFP-Rab5 (Addgene) was a gift from Arnab Gupta. Cells were transfected with 1 μg of Rab5 plasmid DNA to label early endosomes, respectively, by using Lipofectamine 3000 (Invitrogen). EGFP-CAAX [##REF##27633000##41##] was a gift from Lei Lu. Cells were transfected with 1 μg EGFP-CAAX (Addgene) plasmid to mark the membrane. Cells were transfected with 1 μg of mCherry- Clathrin LC-15 (Addgene) to label clathrin-coated vesicles. To mark fast recycling endosomes, cells were transfected with 1 μg of mCherry- Rab 4a plasmid. Other treatments, if required, were performed 16 h after transfection.</p>\",\n \"<p id=\\\"Par57\\\">Fixation of cells was performed by using 4% paraformaldehyde (Sigma-Aldrich) for 15 min at 37°C temperature.</p>\",\n \"<title>siRNA-based knockdown</title>\",\n \"<p id=\\\"Par58\\\">HeLa cells are transfected with 10 nM of AP2 siRNA total for 72 h. At 0 h first siRNA transfection was done and at 48 h fresh media was added with 10 nM of AP2 siRNA booster dose. To validate the knockdown of AP2, cells are immunostained with Anti AP2 antibody to check intensity at the basal membrane by TIRF microscopy. Western blotting was also performed using established protocols to validate the knockdown.</p>\",\n \"<title>IRM imaging</title>\",\n \"<p id=\\\"Par59\\\">Cells were imaged in Nikon Eclipse Ti-E motorized inverted microscope (Nikon, Japan) equipped with adjustable field and aperture diaphragms, 60X Plan Apo (NA 1.22, water immersion) and a 1.5X external magnification on an onstage 37 °C incubator (Tokai Hit, Japan). Either an EMCCD (Evolve 512 Delta, Photometrics, USA) or an s-CMOS camera (ORCA Flash 4.0, Hamamatsu, Japan) was used for imaging. A 100 W mercury arc lamp, an interference filter (546 ± 12 nm) and a 50–50 beam splitter were used [##REF##29045871##6##]. For IRM, movies consisted of 2048 frames (19.91 frames/s, EMCCD and 20 frames/s for s-CMOS) recorded at EM 30 and an exposure time of 50 ms.</p>\",\n \"<title>Optical trap experiment</title>\",\n \"<p id=\\\"Par60\\\">For optical trap-based tether-pulling experiments, a 2 µm polystyrene bead was trapped by focusing a 1064 nm Laser (Coherent, Sweden) of 1000 W power at source using a 100 × objective. The back-aperture of the objective was over filled after beam expansion and using mirrors and a 50/50 beam-splitter for beam manipulation. The trap stiffness (<italic>k</italic>) was calibrated from trajectories of the trapped bead using the equipartition approach where <italic>x</italic> is the displacement of the bead from the trap centre, <italic>k</italic><sub><italic>B</italic></sub> is the Boltzmann constant and T the temperature in the Kelvin scale. For analysis, the bead was detected as an object (MATLAB, Fiji), and its centre was tracked with time. The bead’s displacement from the centre of the trap and the spring constant of the trap was used to get the force (). For every cell, a tether was pulled at a constant velocity of 0.5 µm/s up to a distance of 40 µm (LabVIEW, National Instruments, USA). Tether force was calculated from the average bead position during the period when it was parked with the tether pulled (Fig. S3c) for ~ 50 s. Imaging was done at 200 frames per sec. The apparent membrane tension of the apical section of the cell was derived from the force using the Canham-Helfrich equation , where <italic>κ</italic> is the bending rigidity and taken to be 15 k<sub>B</sub>T [##REF##15695629##13##], and σ<sub>A</sub> denotes the apparent membrane tension.</p>\",\n \"<title>TIRF imaging</title>\",\n \"<p id=\\\"Par61\\\">For TIRF Microscopy, an inverted microscope (Olympus IX-83, Olympus, Japan) was used with a 100X 1.49 NA oil immersion TIRF objective (PlanApo, Olympus). An s-CMOS camera (ORCA Flash 4.0, Hamamatsu, Japan) and 488 nm, as well as 561 nm laser sources, were used. Images were acquired using an exposure time of 300 ms with ~ 70 nm penetration depth.</p>\",\n \"<title>Confocal imaging</title>\",\n \"<p id=\\\"Par62\\\">For confocal imaging, a Leica confocal microscope (Leica SP8) was used with a 63X oil objective lens (NA 1.4). A step size (in z) of 250 nm is used for imaging with a pixel size of 45 nm and deconvoluted (Leica Lightning Software).</p>\",\n \"<title>STED imaging</title>\",\n \"<p id=\\\"Par63\\\">An Abberior Facility Line system with an Olympus IX83 microscope (Abberior Instruments) was used for STED imaging. Abberior autoalignment sample was utilized for alignment of the STED and confocal channels. 15 nm pixel size was maintained during imaging. A pulsed STED line at 775 nm was used for depletion and STAR Red and Alexa 568 conjugated secondary antibodies were used for imaging Clathrin and AP2, respectively.</p>\",\n \"<title>Analysis of IRM images</title>\",\n \"<p id=\\\"Par64\\\">The intensity of IRM images was converted to height (wherever applicable) as reported [##REF##29045871##6##]. The amplitude of spatial undulations spatial (SD<sub>space</sub>) was obtained from the standard deviation (SD) of relative heights across all pixels in an FBR after averaging it over 20 frames. For obtaining the amplitude of temporal fluctuations, SD<sub>time</sub>, the SD of the relative heights over 2048 frames in each pixel was calculated and averaged across all pixels in an FBR. The power spectral density (PSD) of individual pixels was obtained from the temporal relative height time series using either the FFT method or the covariance method (MATLAB). PSDs of all pixels in an FBR were averaged to obtain the PSD for that FBR. For obtaining mechanical parameters, the PSDs were fitted to [##REF##25902428##3##, ##REF##29045871##6##, ##UREF##1##23##] where active temperature (A), effective cytoplasmic viscosity (<italic>η</italic><sub>eff</sub>), confinement (<italic>γ</italic>) and membrane tension (<italic>σ</italic>) were used as fitting parameters. The bending rigidity (<italic>κ</italic>) was fixed at 15 k<sub>B</sub>T [##UREF##9##68##]. For obtaining excess area fraction () over an FBR, the flat area of the FBR is taken as A<sub>P</sub> (= <italic>L</italic><sup>2</sup> when patch/FBR is a square of side <italic>L</italic>) and <italic>A</italic> is the sum of all <italic>dA</italic> calculated for each pixel by comparing the height at that pixel with its neighbours () [##UREF##0##16##]. The excess area is represented in figures as the percentage excess area ().</p>\",\n \"<p id=\\\"Par65\\\">The activity per FBR is calculated as the lower bound of the entropy generation rate at each FBR. For obtaining the entropy generation rate, data pooled from all pixels of the FBR were taken through dimensional reduction using principal component analysis. The time series (2048 frames) of one of the principal components were built and analyzed as described recently [##UREF##3##43##] using the short-time inference scheme reported earlier [##REF##32281844##44##].</p>\",\n \"<title>Tension mapping</title>\",\n \"<p id=\\\"Par66\\\">For tension mapping (Fig. ##FIG##1##2##c), PSD was calculated for either for each pixel, and either directly used (pixel-wise tension mapping) or averaged over all pixels in each FBR (FBR-wise tension mapping). PSDs, thus, obtained are fitted and every parameter extracted from fits—including tension and <italic>R</italic><sup>2</sup> were mapped on to the same location as the pixel/FBR.</p>\",\n \"<title>Fluorescence image analysis</title>\",\n \"<p id=\\\"Par67\\\">For endosomal or puncta counting (MATLAB), first, a Gaussian blur operation was performed to spatially average out the image. The Gaussian-blur is next subtracted from the original image, thus enhancing local contrast. The subtracted image is normalized between 0 and 1, and thresholding was performed using the appropriate threshold, resulting in a binary image. Next, a mask was applied over the image to select the cell. Single pixels were removed using serial erosion and dilation, and subsequently, the binary image was used to detect objects. The area fraction is calculated by dividing the total area of detected objects () by the total area of the ROI or cell (). Similar approach was used for calculating colocalization. Objects were detected for each channel separately. Total area of the pixels overlapping in the two binary images was considered to be colocalizing area (). Colocalizing area divided by the total area of the ROI was used as colocalizing area fraction (). Colocalizing area divided by object-covered area of any particular was used as the percentage colocalization of that channel (). Mander’s coefficient was obtained using ImageJ.</p>\",\n \"<title>Counting tubules from confocal z-slices</title>\",\n \"<p id=\\\"Par68\\\">For counting tubules, Z-stack images of cells were captured. Line scans were drawn at the cortex regions of cells (ImageJ/Fiji), and the intensities of these line scans were taken from the intensity plot profile of each line scan to plot the internal intensity. Peak analysis (MATLAB) involved evaluating the intensity line scans for peaks of minimum prominence of ~ 6 and width of ~ 5. Peaks were counted as tubules. Length of tubules were obtained by drawing line ROIs along tubules and measuring their length.</p>\",\n \"<title>Statistical analysis</title>\",\n \"<p id=\\\"Par69\\\">Every IRM experiment was preceded by imaging beads. Every experiment was repeated at least thrice involving multiple cells and FBRs (Table ##SUPPL##0##S1##). A Mann–Whitney <italic>U</italic> test was performed for statistical significance testing (ns denotes <italic>p</italic> > 0.05, *denotes <italic>p</italic> < 0.05, **denoted <italic>p</italic> < 0.001). When indicated, a linear mixed effect model (LMM, MATLAB) was also used (using the fixed effect of time and random effects grouped under replicate set number of the experiments and cell number) to quantify the significance of the observed changes in logarithm of tension values (Table S2). This helped avoid the effect of the high sample size of FBRs that could influence hypothesis testing. LMM has been used for comparisons in other high-sampling mechanical measurements [##REF##29937952##24##, ##UREF##7##56##].</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>De-adhesion-mediated increase in membrane fluctuations is actin-dependent</title>","<p id=\"Par9\">IRM images reflect the distance of the cellular basal membrane from the glass coverslip and, thus, can be used to study the spatiotemporal basal PM height fluctuations in adherent cells [##REF##29045871##6##]. Qualitatively, darker regions were closer to the coverslip while intensity increases with height till ~ 100 nm and periodically oscillates. Calibration with standards (beads) was performed to quantify the relative heights, followed by selecting regions where the intensity-height conversion was possible—usually, regions that are ~ 100 nm from the coverslip and fall under the first branch of the intensity profile (that displays bands) [##REF##29045871##6##]. The selected regions (square membrane patches) were termed first-branch regions (FBRs). Multiple such regions were marked for every cell.</p>","<p id=\"Par10\">The time series of membrane height fluctuations (obtained from single pixels) were used to get mean height and standard deviation (SD) of height and termed SD<sub>time</sub>. Pixel-wise measurements were averaged over neighbouring pixels to get “FBR-wise” data. SD<sub>space</sub> was obtained from a snapshot—comparing height across NxN pixels of any FBR where N was usually 12 in this study (see \"<xref rid=\"Sec16\" ref-type=\"sec\">Methods</xref>\"). These parameters depicted the amplitude of fluctuations. Averaging over all FBRs in a cell and pooling such data from all cells provided the “cell-wise” data. We compared these parameters between control and treated cells to understand how a mechanical perturbation like de-adhesion altered membrane mechanics.</p>","<p id=\"Par11\">IRM provides the ability to map fluctuation-tension, get its distribution in the cell, and thus differentiate between large global and local changes. In this study, we have compared changes at the level of whole cells (termed cell-wise) and those that show up in the local distribution of tension in single cells. However, while comparing local data, to ensure that the repeated measurements per cell do not falsely strengthen the statistics, we have employed linear mixed models (LMM) as usually used [##REF##29937952##24##, ##UREF##7##56##] to account for the nested grouping of replicate measurements.</p>","<p id=\"Par12\">Finally, it is important to note certain limitations. Fluctuations reported by IRM are mainly thermal but can have contributions from active (ATP-dependent, non-thermal) processes in cells [##UREF##10##76##]. Using a model-free method, a recently developed algorithm [##UREF##3##43##, ##REF##32281844##44##], quantified the effect of active forces on membrane fluctuations (termed activity) and revealed local membrane fluctuations to be weakly active. However, fluctuation-tension solely should not be used for drawing inferences.</p>","<p id=\"Par13\">We deal with this primarily by using direct measurements of fluctuations amplitude. We also compare activity at different time points of de-adhesion to record the level of deviation of the measured fluctuations from equilibrium. Finally, we corroborate the main effect of de-adhesion on fluctuation-tension with apparent membrane tension measurement using the generally accepted optical-trap-based tether extraction method.</p>","<p id=\"Par14\">HeLa cells were treated with either a low (0.05% Trypsin–EDTA) or high concentration (0.25% Trypsin–EDTA) of de-adhering solution and imaged by IRM (Fig. ##FIG##0##1##a) to study the changes in membrane fluctuations and mechanics in the basal membrane during de-adhesion. We observed high variability in the time cells took to de-adhere (Fig. ##FIG##0##1##b). To quantify the rate of de-adhesion, the time taken by each cell to reduce their spread area to 67% of the initial was calculated (Fig. ##FIG##0##1##c). We found that, among the various treatments used, Cytochalasin D (Cyto D)—an agent reducing actin polymerization [##REF##7199055##61##] of the actin cortex resulted in a very fast de-adhesion. Therefore, a lower concentration of de-adhering solution of Trypsin (0.05%) was used for Cyto D experiments. This was in line with the understanding that during de-adhesion, the contractile cortex caused lateral retraction [##REF##31348954##34##, ##REF##21297858##63##]. At lower trypsin concentrations, the process could be slowed down such that the decay times for Control and Cyto D were similar (Fig. ##FIG##0##1##c).</p>","<p id=\"Par15\">In general, de-adhesion caused an increase in temporal fluctuations (Fig. ##FIG##0##1##a—7 min), followed by lateral retraction (Fig. ##FIG##0##1##a—9–18 min). The increase in fluctuation amplitude (SD<sub>time</sub>) could be visualized from the maps (Fig. ##FIG##0##1##a bottom, right). Following the lateral retraction of the edge using a kymograph) of the IRM intensity (along the white line, Fig. ##FIG##0##1##d), we found that as the edges retract inwards, intensity patterns lying inward also moved. Membrane height (IRM intensity) increased in a cluster close to the edge (white oval, Fig. ##FIG##0##1##d) as the retraction progressed. This accumulation faded away with time (section below the oval, Fig. ##FIG##0##1##d). While the membrane undulations were locally enhanced in control cells, such local increase was less prominent in Cyto D-treated cells (Fig. ##FIG##0##1##d). Thus, as de-adhesion progressed, cytoskeleton-dependent transient accumulation of membrane folds point to the ongoing membrane remodelling.</p>","<p id=\"Par16\">We proceeded to measure the fluctuations and effective membrane-mechanical parameters from the fluctuations during the different phases and over time to underpin the effect of de-adhesion on membrane mechanics quantitatively.</p>","<title>The initial rise in fluctuations is regulated back</title>","<p id=\"Par17\">Since the rates of de-adhesion in cells were different, we classified the data in this and the following sections based on the level of reduction in spread area. For every cell, we divided the process of de-adhesion into four phases. The “C” phase was defined as the period for which the cell had not been treated with the de-adhering agent (Trypsin). The “P1” phase was demarcated as the slow de-adhesion phase before the exponential fall of the spread area started. Typically, the spread area in this phase remained within 90% of the initial cell spread area. The “P2” phase was defined as the period when the spread area exponentially reduced, at least to ~ 67% of the original value. The “P3” phase marked the period when the low spread area had stabilized to ~ 40–20% of the initial (Fig. ##FIG##0##1##b).</p>","<p id=\"Par18\">Height fluctuations were measured before (phase: C) and then every few minutes after adding de-adhering medium from image stacks acquired at every time-point, where each movie lasted for ~ 102 s. Only membrane regions that remained adhered through the movies were analyzed. On following a representative cell in time (Fig. ##FIG##1##2##a; same cell as shown in Fig. ##FIG##0##1##a) or averaging over a population (Fig. ##FIG##1##2##b), we found that the amplitude of temporal height fluctuations (SD<sub>time</sub>) first increased, followed by a decrease/saturation on de-adhesion. The reverse was observed for fluctuation-tension (Fig. ##FIG##1##2##a). Maps of FBR-wise fluctuation-tension (Fig. ##FIG##1##2##c) revealed the lowered tension state of the cell followed by an increase in the regions that remained adhered. Strictly for visualization purposes, we also created a pixel-wise map of tension (Fig. ##FIG##1##2##c). Mapping tension showed a reduction in the initial heterogeneity when cells are at phase P2. The global trend (Fig. ##FIG##1##2##b<bold>)</bold> was corroborated by the distribution of local (FBR-wise) fluctuation amplitude and tension for single cells. Clearly, the changes were greater than the error calculated while averaging the distributions over multiple cells and repeats (Fig. ##FIG##1##2##d).</p>","<p id=\"Par19\">We confirmed that in the absence of de-adhesion media, the fluctuations or tension of these cells did not change over 20 min (Fig. ##SUPPL##0##S1##). Following the same regions in single cells through de-adhesion also showed the dip and recovery of tension (Fig. S2), confirming that regions that remain adhered also underwent these changes. Although the spatial height variation (SD<sub>space</sub>) and excess area showed a decreasing trend in contrast to SD<sub>time</sub> (Fig. S3a), using a gentler substrate detachment reagent—TrypLE (Fig. S3b) or at lower trypsin concentration, their trends matched [Fig. S3 c, d (0.05% Tryp)].</p>","<p id=\"Par20\">The corresponding entropy-generation rate obtained from fluctuations of de-adhering cells changed mildly (Fig S3a), implying that the measured fluctuations did not capture any major enhancement in non-equilibrium activity in the frequencies assayed. Since actin remodelling was expected during de-adhesion, its impact on tension reduction was next studied.</p>","<title>The initial rise in fluctuations is weaker on cortex disruption</title>","<p id=\"Par21\">We used Cyto D to weaken the cortical actin. At 0.05% Trypsin, the spread area reduction rate was similar for Control and Cyto D, but cells de-adhered properly. Tension reduction in P2 was not substantial in the presence of Cyto D or reduced filamentous cortical actin (Fig. ##FIG##1##2##e, f). Although fluctuations were enhanced weakly (than Control (Fig. ##FIG##1##2##f, S4) at P2, by P3, unlike in Control, Cyto D showed similar fluctuations as C. In line with Fig. ##FIG##0##1##d, these data also suggest that disruption of cortical actin reduced membrane accumulation or enhancement of fluctuations.</p>","<p id=\"Par22\">De-adhesion only occurs at the basal membrane. To study its effect on the apical membrane, we next extracted membrane tethers from cells and measured tether forces. These were measured on single cells—before and after initiating de-adhesion (Fig. ##FIG##1##2##f top, Fig. S5) within the first—3–7 min. There was a reduction in force for 8/10 cells, which showed a 2–52% reduction in force (or effectively a 4–77% reduction in apparent tension).</p>","<p id=\"Par23\">Next, we examined whether the tension reduction and subsequent regulation correlated with endocytic activity.</p>","<title>De-adhesion increases early endocytic and fast recycling endosomes near the basal plasma membrane</title>","<p id=\"Par24\">To quantify endocytosis, we utilized three strategies—labelling the cargo of an endocytic pathway, labelling early endosomes using Rab5 and labelling recycling endosomes with Rab4 (Fig. ##FIG##2##3##a). For analyzing fluorescent puncta imaged in TIRF, the puncta were detected as objects and analyzed (Fig S6a). Since nearby objects could be distinguished from connected ones, we scored for the area fraction or the area covered by the puncta per µm<sup>2</sup> of the analyzed area. At first, we followed a cargo of CME—Transferrin (Tf)—added in Control and de-adhering cells (Fig. ##FIG##2##3##b, c) and found a clear increase in Tf's internalization (Fig. ##FIG##2##3##b) as well incorporation (per μm<sup>2</sup>) in the plasma membrane as clusters in de-adhering cells (Fig. ##FIG##2##3##c, d, Fig S6 b–d). Analyzing their disappearance indicated enhanced short-time dynamics on de-adhesion (Fig. S6. E–i) validating increase in pits not plaques [##REF##19809571##60##].</p>","<p id=\"Par25\">The involvement of the endocytic machinery was next confirmed by labelling early endosomes by Rab5 [##REF##1516130##8##] and the short-loop (fast) recycling endosomes by Rab4 [##REF##10811830##71##]. Near the plasma membrane, endosomes mainly contain these two labels [##REF##10811830##71##]. The Rab4-containing endosomes emerge from the same endosomes as Rab5 [##REF##10811830##71##] and thus are studied to evaluate the state of endocytic and recycling activity. We imaged EGFP-Rab5 and Rab4 -mCherry using TIRF microscopy (Fig. ##FIG##2##3##e, f, Fig S6). The area fraction of early endosomes (Fig. S6), calculated for whole cells, showed an increase and a subsequent tapering off/decrease (Fig. ##FIG##2##3##f). The increase was anti-correlated initially with the reduction in spread area (Fig. ##FIG##2##3##f). However, it was difficult to discount the effect of heterogenous de-adhesion in the observed trend since some regions had denser features than others. Thus, we looked at regions that stayed on through the observed time scale (15 min). We followed the area fraction of Rab5 for the same sub-cellular region of interest (ROI) (Fig. ##FIG##2##3##g). The rise and saturation were found to be consistent (Fig. ##FIG##2##3##g).</p>","<p id=\"Par26\">To further validate if endocytosis was ramped up, we analyzed clathrin-coated pits (Fig. S6j). Imaging cells fixed before or after faster de-adhesion. Although an increase and saturation in the area fraction of these pits were observed, the changes were not appreciable. However, on evaluating with super-resolution using Stimulated Emission Depletion (STED) microscopy, we found that clathrin was less colocalized with its adaptor AP2 (Fig. ##FIG##2##3##h) on de-adhesion. As reported earlier, AP2 was observed either well-colocalized with smaller clathrin clusters or at the edges of larger structures (Fig. ##FIG##2##3##h, zoomed-in sections) and expected to be more curved [##REF##28346440##70##]. On increasing tension, previous studies report a higher fraction of AP2 with clathrin indicative of flat structures [##REF##29317637##9##]. Our observation of AP2 localized at edges (Fig. ##FIG##2##3##i, j) and showing lesser colocalization (Fig. ##FIG##2##3##k, l) might indicate that more fraction of clathrin structures are pits on de-adhesion. We also find an increase in the distance between clathrin and AP2 clusters after de-adhesion, comparing pairs already with 525 nm of each other (Fig. ##FIG##2##3##m) or higher. The significance holds true even while comparing pairs within 165 nm but does not show any difference when only those lying within 150 nm (close to the size of well-formed pits, Fig. ##FIG##2##3##i<bold>,</bold> yellow arrow) are compared, clearly indicating the population with AP2 at edges start making this difference significant.</p>","<p id=\"Par27\">Together, the data showed that triggering de-adhesion reduced tension, and cells ramped up the frequency of their endocytosis events. We also know from following the fluctuations that the lowering of tension was also stalled or recovered back at later stages of de-adhesion (Fig. ##FIG##1##2##b). To understand if endocytosis affected tension regulation and how we next used pharmacological agents to block endocytosis and followed treated cells on de-adhesion.</p>","<title>Blocking dynamin-dependent pathways reduces de-adhesion-triggered endocytosis</title>","<p id=\"Par28\">We studied the effect of blocking dynamin-dependent pathways (Fig. ##FIG##3##4##, Fig. S6, S7) by treating HeLa cells with Dynasore [##REF##18413242##31##]. Specifically, it does not stop the formation of pits (clathrin-coated or caveolae, among others) but prevents dynamin’s function in the scission of pits (Fig. ##FIG##3##4##a). Through Tf-uptake assay Dynasore’s effect on stalling of pit scission is clear (Fig. S6 e, h, i). Instead of the rise in Rab5’s area fraction on de-adhesion, an initial drop was observed with de-adhesion as expected since early endosomes were expected to contain Rab5 [##REF##24307937##53##]. (Fig. ##FIG##3##4##b, c, Fig. S7 a, b). Failure of fission caused tubes to form, which also contained Rab5 (arrows, Fig. ##FIG##3##4##b, Fig. S7a). The increase in Tf puncta in Dynasore-treated cells was enhanced in de-adhering cells (Fig. ##FIG##3##4##c, d).</p>","<p id=\"Par29\">Rab4 labelling (immunofluorescence) revealed an increase during the de-adhesion (Fig. ##FIG##3##4##b, Fig. S7b). This data suggested that the intermittent accumulation could be due to its inability to fuse normally with the plasma membrane (with reduced tension). This aligns with studies that have reported reduced recycling on inhibiting Dynamin [##REF##12372835##14##, ##REF##11809831##15##].</p>","<p id=\"Par30\">Thus, Dynasore drastically reduced the formation of new early endosomes while also inhibiting the fusion of recycling endosomes with the plasma membrane. Together, these indicate that Dynasore effectively brought down de-adhesion-triggered endocytosis in the cell. We next studied how such blocking of endocytosis would affect the tension regulation during de-adhesion.</p>","<title>Inactivating dynamin does not stop tension recovery, but knocking down AP2 does</title>","<p id=\"Par31\">The single cell distribution of fluctuation amplitude and tension clearly changes as de-adhesion progresses to P2, implying that the tension reduced on de-adhesion of Dynasore treated cells (Fig. ##FIG##3##4##e–f, Fig S7d–i). Interestingly, instead of preventing tension recovery, it was enhanced in Dynasore-treated cells (Fig. ##FIG##3##4##e) in comparison to untreated cells (Fig. ##FIG##1##2##d). Normalized cell averages (Fig. ##FIG##3##4##g, Fig S7 f) point to the augmented mechanical regulation operating from P2 to P3 in Dynasore-treated cells. Maps help us visualize this (Fig. ##FIG##3##4##f, Fig S7e). On checking the state of activity (entropy generation rate), we found a lowering of activity on Dynasore treatment (Fig. S7g).</p>","<p id=\"Par32\">To further validate, we performed experiments with a dominant-mutant of dynamin that was transiently transfected in cells. We found that even in these cells, the increase in tension or reduction in SD<sub>time</sub> was more substantial than in the control condition (Fig. S8). Clearly, the tension at P3 was higher than C, unlike in control sets.</p>","<p id=\"Par33\">The data imply that although endosome formation is reduced, membrane mechanics regulation during de-adhesion is not stopped. This clearly implies that the formation of pits has a direct role in tension recovery. To verify, we next compared the excess areas from IRM images to find the impact of Dynasore on membrane smoothness. There was a significant reduction in the excess area in Dynasore-treated cells, implying a smoother membrane (Fig. ##FIG##3##4##g), supporting the hypothesis that pit formation can reduce excess area and enhance the effective tension. As a control, we checked how excess area changed on inhibiting Cdc42 by ML141. Cdc42 is required for the formation and scission of endocytic pits of the CG pathway. Inhibiting it for 30 min enhanced the excess area (Fig. S7h), further supporting the hypothesis that pit-formation reduces excess area.</p>","<p id=\"Par34\">To validate further the role of pit-formation in tension recovery, we used siRNA-mediated knocked-down AP2. The knock-down was confirmed by immunofluorescence (Fig. S7j) and western blot (Fig. S7k) using control cells and cells treated with either scrambled siRNA (Scramble) or AP2-siRNA (AP2-siRNA). Depletion of AP2 by siRNA has been shown [##REF##12960147##25##] to reduce membrane clathrin as well drastically reduce coated pits on the plasma membrane. We observe that AP2-siRNA significantly reduced tension and enhanced fluctuations and excess area (Fig. ##FIG##3##4##i top) of cells. During de-adhesion (Fig. ##FIG##3##4##i bottom), the tension reduced in the P2 phase but failed to be regulated back in the P3 phase (Fig. ##FIG##3##4##h, i). SD<sub>time</sub> increased with de-adhesion (Table S2), although when compared using cell-wise statistics, no significant change was noted (Fig. ##FIG##3##4##i). However, it was clearly not regulated back. The response of AP2-siRNA-treated cells were marked different than control cells or cells treated with scramble siRNA. Together, the Dynasore, dynamin mutant and AP2 siRNA data clearly establish the pit formation step of clathrin-mediated endocytosis to contribute to tension regulation during de-adhesion.</p>","<p id=\"Par35\">Having provided evidence suggesting pit formation as the critical step in endocytosis to cause tension increase, we next aimed to understand the nature of processes used for the initial pit formation. Dynamin-dependent pathways are not limited to major pathways like CME/caveolae. To understand the dependency on pathways that use key energy-consuming molecules like dynamin or actin, we next check the regulation’s dependency on ATP. ATP-dependent-pit formation is common (CME and caveolae [##REF##21295700##69##], for example). However, multiple pathways, like once induced by Shiga toxins, are ATP-independent [##REF##25517096##57##].</p>","<title>ATP-depletion does not block tension regulation</title>","<p id=\"Par36\">We used ATP depletion prior to de-adhesion to investigate the role of active processes and actin polymerization in the mechanical changes observed during de-adhesion (Fig. ##FIG##4##5##a, b). ATP-depleted cells showed low initial fluctuations but a similar trend of increase in fluctuations followed by a decrease as observed for control cells (Fig. ##FIG##4##5##a, b, Fig. S9a, Table ##SUPPL##0##S1##). The effective tension reduced and then increased. The distribution of local values in single cells also captured the changes which were found to be significant. No appreciable change was observed in the area fraction of Rab5 in ATP-depleted cells as de-adhesion progressed (Fig. ##FIG##4##5##c, d, Fig S9b). The area fraction of RAb5 in ATP-depleted cells was lower than in control cells (Fig. S9b), expected from reduction in active endocytosis.</p>","<p id=\"Par37\">Therefore, the data here suggest the use of mechanisms that start recovering the tension drop without critically requiring ATP. As the first steps to identify the involved pathway, we next checked the dependence of tension regulation on cholesterol in the ATP-compromised condition. We do so because known pathways (like FEME used to internalize Shiga toxin) using ATP-independent pit formation are cholesterol-dependent and depend on the ability of cholesterol to cluster lipids and initiate the creation of invaginations [##REF##11739634##33##].</p>","<title>Tension regulation in ATP-depleted cells is cholesterol-dependent</title>","<p id=\"Par38\">We used ATP depletion and cholesterol depletion as controls (Fig. ##FIG##5##6##a, S9e) to assess the role of cholesterol in tension regulation in ATP-depleted cells during de-adhesion. Comparing single-cell distributions or cell-averaged values (Fig. ##FIG##5##6##b–d) showed that while the initial (C-P2) fluctuation (SD<sub>time</sub>) increase displayed the same trend as for the controls, fluctuations from P2 to P3 were not reduced efficiently on combined depletion of ATP and cholesterol. Depleting ATP or cholesterol enhanced the recovery of tension (from Control), but on dual depletion of ATP and cholesterol, there was a further reduction (Fig. ##FIG##5##6##a, c, Fig S9 c, d) [clear from distributions of local measurements, tension maps (Fig. S9e) and the LMM analysis of the local values (Fig. ##FIG##5##6##d)].</p>","<p id=\"Par39\">Together, we showed (Fig. ##FIG##5##6##e) that the tension drop was significant even on perturbing scission, ATP or cholesterol content and faster in the case of the latter two. The recovery was also significant and faster for these perturbations but could not be effective under reduced ATP and cholesterol-depleted conditions. Treating tension reached by the control cells at the P3 phase as a reference, Dynasore treatment effectively tilted the balance towards a larger tension recovery rate while dual depletion of ATP and cholesterol resulted in lower and, therefore, appreciably slower recovery.</p>","<p id=\"Par40\">Therefore, the observations strengthen the hypothesis that in the absence of ATP, cells also use cholesterol-dependent processes for mechano-regulation. Since in pathways involved in Shiga-toxin's endocytosis, toxin-rich tubules have been reported to be formed even in ATP-depleted cells/giant unilamellar vesicles [##REF##24703428##59##], we next checked if ATP-depleted cells formed tubules when tension was lowered by de-adhesion. For this, we imaged cells transfected with EGFP-CAAX and quantified the membrane cross-section at higher planes in ATP-depleted and ATP and cholesterol-depleted cells using confocal microscopy.</p>","<title>On de-adhesion, ATP-depleted cells make more cholesterol-dependent tubular invaginations</title>","<p id=\"Par41\">Cells were transfected with EGFP-CAAX [##REF##27633000##41##] to label the plasma membrane and taken through different treatments (Fig. ##FIG##6##7##, S10a). They were subsequently fixed (at 3, 6 min after treatment). Since fixation is not instantaneous, we measured the spread area of cells after fixation to determine the decrease in spread area (Fig. S10b). We observed a reduction to ~ 40% of initial when fixed at 3 min and ~ 17% of initial when fixed at 6 min. Therefore, they were classified as P2 and P3. Cells were subsequently imaged in confocal microscopy at a final resolution of ~ 120 nm in <italic>x</italic> and <italic>y</italic> directions (Fig. ##FIG##6##7##a, b, S10). From the intensities obtained from scans under the membrane (Fig. ##FIG##6##7##b, Fig. S10 c, d) along multiple line regions of interest (ROI), each of length ~ 4 µm, peaks were detected with larger widths and heights (from basal intensity) than set thresholds from the line scans (Fig. ##FIG##6##7##c). After de-adhesion, ATP-depleted cells displayed significantly more internal surface-connected structures than ATP + cholesterol-depleted cells (Fig. ##FIG##6##7##d) at the P2 and P3 phases. It should be noted that on cholesterol deletion (without ATP depletion, Fig. S10 e) or in control cells (no drug treatment, Fig. S10 f), there were much fewer tubules (Fig. S10 g), whose number also did not appreciably increase on de-adhesion (Fig. ##FIG##6##7##d, Fig. S10 e–g). The number of peaks transiently increased with de-adhesion in ATP-depleted cells but, in contrast, decreased for cells also depleted of cholesterol (Fig. ##FIG##6##7##e, Fig. S10). Tubule length also increased from before trypsinization to 3 min post-trypsinization. Hence, we concluded that the ATP-depleted cells might gain more cholesterol-dependent tubules on de-adhesion, thereby causing the tension surge.</p>","<p id=\"Par42\">In conclusion, we show that de-adhesion induces a tension drop in the whole cell due to an altered adhesion state. The regulation sets in soon and engages endocytic pathways that internalize the membrane but also populate a recycling pool. Tension reduction is stalled but must be balanced by the recycling back of the membrane, because perturbations blocking internalization enhance the tension recovery rate. ATP and cholesterol depletion inhibits tension recovery, and this inhibition cannot be achieved by either preventing internalization or depleting only ATP or cholesterol individually.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>De-adhesion-mediated increase in membrane fluctuations is actin-dependent</title>\",\n \"<p id=\\\"Par9\\\">IRM images reflect the distance of the cellular basal membrane from the glass coverslip and, thus, can be used to study the spatiotemporal basal PM height fluctuations in adherent cells [##REF##29045871##6##]. Qualitatively, darker regions were closer to the coverslip while intensity increases with height till ~ 100 nm and periodically oscillates. Calibration with standards (beads) was performed to quantify the relative heights, followed by selecting regions where the intensity-height conversion was possible—usually, regions that are ~ 100 nm from the coverslip and fall under the first branch of the intensity profile (that displays bands) [##REF##29045871##6##]. The selected regions (square membrane patches) were termed first-branch regions (FBRs). Multiple such regions were marked for every cell.</p>\",\n \"<p id=\\\"Par10\\\">The time series of membrane height fluctuations (obtained from single pixels) were used to get mean height and standard deviation (SD) of height and termed SD<sub>time</sub>. Pixel-wise measurements were averaged over neighbouring pixels to get “FBR-wise” data. SD<sub>space</sub> was obtained from a snapshot—comparing height across NxN pixels of any FBR where N was usually 12 in this study (see \\\"<xref rid=\\\"Sec16\\\" ref-type=\\\"sec\\\">Methods</xref>\\\"). These parameters depicted the amplitude of fluctuations. Averaging over all FBRs in a cell and pooling such data from all cells provided the “cell-wise” data. We compared these parameters between control and treated cells to understand how a mechanical perturbation like de-adhesion altered membrane mechanics.</p>\",\n \"<p id=\\\"Par11\\\">IRM provides the ability to map fluctuation-tension, get its distribution in the cell, and thus differentiate between large global and local changes. In this study, we have compared changes at the level of whole cells (termed cell-wise) and those that show up in the local distribution of tension in single cells. However, while comparing local data, to ensure that the repeated measurements per cell do not falsely strengthen the statistics, we have employed linear mixed models (LMM) as usually used [##REF##29937952##24##, ##UREF##7##56##] to account for the nested grouping of replicate measurements.</p>\",\n \"<p id=\\\"Par12\\\">Finally, it is important to note certain limitations. Fluctuations reported by IRM are mainly thermal but can have contributions from active (ATP-dependent, non-thermal) processes in cells [##UREF##10##76##]. Using a model-free method, a recently developed algorithm [##UREF##3##43##, ##REF##32281844##44##], quantified the effect of active forces on membrane fluctuations (termed activity) and revealed local membrane fluctuations to be weakly active. However, fluctuation-tension solely should not be used for drawing inferences.</p>\",\n \"<p id=\\\"Par13\\\">We deal with this primarily by using direct measurements of fluctuations amplitude. We also compare activity at different time points of de-adhesion to record the level of deviation of the measured fluctuations from equilibrium. Finally, we corroborate the main effect of de-adhesion on fluctuation-tension with apparent membrane tension measurement using the generally accepted optical-trap-based tether extraction method.</p>\",\n \"<p id=\\\"Par14\\\">HeLa cells were treated with either a low (0.05% Trypsin–EDTA) or high concentration (0.25% Trypsin–EDTA) of de-adhering solution and imaged by IRM (Fig. ##FIG##0##1##a) to study the changes in membrane fluctuations and mechanics in the basal membrane during de-adhesion. We observed high variability in the time cells took to de-adhere (Fig. ##FIG##0##1##b). To quantify the rate of de-adhesion, the time taken by each cell to reduce their spread area to 67% of the initial was calculated (Fig. ##FIG##0##1##c). We found that, among the various treatments used, Cytochalasin D (Cyto D)—an agent reducing actin polymerization [##REF##7199055##61##] of the actin cortex resulted in a very fast de-adhesion. Therefore, a lower concentration of de-adhering solution of Trypsin (0.05%) was used for Cyto D experiments. This was in line with the understanding that during de-adhesion, the contractile cortex caused lateral retraction [##REF##31348954##34##, ##REF##21297858##63##]. At lower trypsin concentrations, the process could be slowed down such that the decay times for Control and Cyto D were similar (Fig. ##FIG##0##1##c).</p>\",\n \"<p id=\\\"Par15\\\">In general, de-adhesion caused an increase in temporal fluctuations (Fig. ##FIG##0##1##a—7 min), followed by lateral retraction (Fig. ##FIG##0##1##a—9–18 min). The increase in fluctuation amplitude (SD<sub>time</sub>) could be visualized from the maps (Fig. ##FIG##0##1##a bottom, right). Following the lateral retraction of the edge using a kymograph) of the IRM intensity (along the white line, Fig. ##FIG##0##1##d), we found that as the edges retract inwards, intensity patterns lying inward also moved. Membrane height (IRM intensity) increased in a cluster close to the edge (white oval, Fig. ##FIG##0##1##d) as the retraction progressed. This accumulation faded away with time (section below the oval, Fig. ##FIG##0##1##d). While the membrane undulations were locally enhanced in control cells, such local increase was less prominent in Cyto D-treated cells (Fig. ##FIG##0##1##d). Thus, as de-adhesion progressed, cytoskeleton-dependent transient accumulation of membrane folds point to the ongoing membrane remodelling.</p>\",\n \"<p id=\\\"Par16\\\">We proceeded to measure the fluctuations and effective membrane-mechanical parameters from the fluctuations during the different phases and over time to underpin the effect of de-adhesion on membrane mechanics quantitatively.</p>\",\n \"<title>The initial rise in fluctuations is regulated back</title>\",\n \"<p id=\\\"Par17\\\">Since the rates of de-adhesion in cells were different, we classified the data in this and the following sections based on the level of reduction in spread area. For every cell, we divided the process of de-adhesion into four phases. The “C” phase was defined as the period for which the cell had not been treated with the de-adhering agent (Trypsin). The “P1” phase was demarcated as the slow de-adhesion phase before the exponential fall of the spread area started. Typically, the spread area in this phase remained within 90% of the initial cell spread area. The “P2” phase was defined as the period when the spread area exponentially reduced, at least to ~ 67% of the original value. The “P3” phase marked the period when the low spread area had stabilized to ~ 40–20% of the initial (Fig. ##FIG##0##1##b).</p>\",\n \"<p id=\\\"Par18\\\">Height fluctuations were measured before (phase: C) and then every few minutes after adding de-adhering medium from image stacks acquired at every time-point, where each movie lasted for ~ 102 s. Only membrane regions that remained adhered through the movies were analyzed. On following a representative cell in time (Fig. ##FIG##1##2##a; same cell as shown in Fig. ##FIG##0##1##a) or averaging over a population (Fig. ##FIG##1##2##b), we found that the amplitude of temporal height fluctuations (SD<sub>time</sub>) first increased, followed by a decrease/saturation on de-adhesion. The reverse was observed for fluctuation-tension (Fig. ##FIG##1##2##a). Maps of FBR-wise fluctuation-tension (Fig. ##FIG##1##2##c) revealed the lowered tension state of the cell followed by an increase in the regions that remained adhered. Strictly for visualization purposes, we also created a pixel-wise map of tension (Fig. ##FIG##1##2##c). Mapping tension showed a reduction in the initial heterogeneity when cells are at phase P2. The global trend (Fig. ##FIG##1##2##b<bold>)</bold> was corroborated by the distribution of local (FBR-wise) fluctuation amplitude and tension for single cells. Clearly, the changes were greater than the error calculated while averaging the distributions over multiple cells and repeats (Fig. ##FIG##1##2##d).</p>\",\n \"<p id=\\\"Par19\\\">We confirmed that in the absence of de-adhesion media, the fluctuations or tension of these cells did not change over 20 min (Fig. ##SUPPL##0##S1##). Following the same regions in single cells through de-adhesion also showed the dip and recovery of tension (Fig. S2), confirming that regions that remain adhered also underwent these changes. Although the spatial height variation (SD<sub>space</sub>) and excess area showed a decreasing trend in contrast to SD<sub>time</sub> (Fig. S3a), using a gentler substrate detachment reagent—TrypLE (Fig. S3b) or at lower trypsin concentration, their trends matched [Fig. S3 c, d (0.05% Tryp)].</p>\",\n \"<p id=\\\"Par20\\\">The corresponding entropy-generation rate obtained from fluctuations of de-adhering cells changed mildly (Fig S3a), implying that the measured fluctuations did not capture any major enhancement in non-equilibrium activity in the frequencies assayed. Since actin remodelling was expected during de-adhesion, its impact on tension reduction was next studied.</p>\",\n \"<title>The initial rise in fluctuations is weaker on cortex disruption</title>\",\n \"<p id=\\\"Par21\\\">We used Cyto D to weaken the cortical actin. At 0.05% Trypsin, the spread area reduction rate was similar for Control and Cyto D, but cells de-adhered properly. Tension reduction in P2 was not substantial in the presence of Cyto D or reduced filamentous cortical actin (Fig. ##FIG##1##2##e, f). Although fluctuations were enhanced weakly (than Control (Fig. ##FIG##1##2##f, S4) at P2, by P3, unlike in Control, Cyto D showed similar fluctuations as C. In line with Fig. ##FIG##0##1##d, these data also suggest that disruption of cortical actin reduced membrane accumulation or enhancement of fluctuations.</p>\",\n \"<p id=\\\"Par22\\\">De-adhesion only occurs at the basal membrane. To study its effect on the apical membrane, we next extracted membrane tethers from cells and measured tether forces. These were measured on single cells—before and after initiating de-adhesion (Fig. ##FIG##1##2##f top, Fig. S5) within the first—3–7 min. There was a reduction in force for 8/10 cells, which showed a 2–52% reduction in force (or effectively a 4–77% reduction in apparent tension).</p>\",\n \"<p id=\\\"Par23\\\">Next, we examined whether the tension reduction and subsequent regulation correlated with endocytic activity.</p>\",\n \"<title>De-adhesion increases early endocytic and fast recycling endosomes near the basal plasma membrane</title>\",\n \"<p id=\\\"Par24\\\">To quantify endocytosis, we utilized three strategies—labelling the cargo of an endocytic pathway, labelling early endosomes using Rab5 and labelling recycling endosomes with Rab4 (Fig. ##FIG##2##3##a). For analyzing fluorescent puncta imaged in TIRF, the puncta were detected as objects and analyzed (Fig S6a). Since nearby objects could be distinguished from connected ones, we scored for the area fraction or the area covered by the puncta per µm<sup>2</sup> of the analyzed area. At first, we followed a cargo of CME—Transferrin (Tf)—added in Control and de-adhering cells (Fig. ##FIG##2##3##b, c) and found a clear increase in Tf's internalization (Fig. ##FIG##2##3##b) as well incorporation (per μm<sup>2</sup>) in the plasma membrane as clusters in de-adhering cells (Fig. ##FIG##2##3##c, d, Fig S6 b–d). Analyzing their disappearance indicated enhanced short-time dynamics on de-adhesion (Fig. S6. E–i) validating increase in pits not plaques [##REF##19809571##60##].</p>\",\n \"<p id=\\\"Par25\\\">The involvement of the endocytic machinery was next confirmed by labelling early endosomes by Rab5 [##REF##1516130##8##] and the short-loop (fast) recycling endosomes by Rab4 [##REF##10811830##71##]. Near the plasma membrane, endosomes mainly contain these two labels [##REF##10811830##71##]. The Rab4-containing endosomes emerge from the same endosomes as Rab5 [##REF##10811830##71##] and thus are studied to evaluate the state of endocytic and recycling activity. We imaged EGFP-Rab5 and Rab4 -mCherry using TIRF microscopy (Fig. ##FIG##2##3##e, f, Fig S6). The area fraction of early endosomes (Fig. S6), calculated for whole cells, showed an increase and a subsequent tapering off/decrease (Fig. ##FIG##2##3##f). The increase was anti-correlated initially with the reduction in spread area (Fig. ##FIG##2##3##f). However, it was difficult to discount the effect of heterogenous de-adhesion in the observed trend since some regions had denser features than others. Thus, we looked at regions that stayed on through the observed time scale (15 min). We followed the area fraction of Rab5 for the same sub-cellular region of interest (ROI) (Fig. ##FIG##2##3##g). The rise and saturation were found to be consistent (Fig. ##FIG##2##3##g).</p>\",\n \"<p id=\\\"Par26\\\">To further validate if endocytosis was ramped up, we analyzed clathrin-coated pits (Fig. S6j). Imaging cells fixed before or after faster de-adhesion. Although an increase and saturation in the area fraction of these pits were observed, the changes were not appreciable. However, on evaluating with super-resolution using Stimulated Emission Depletion (STED) microscopy, we found that clathrin was less colocalized with its adaptor AP2 (Fig. ##FIG##2##3##h) on de-adhesion. As reported earlier, AP2 was observed either well-colocalized with smaller clathrin clusters or at the edges of larger structures (Fig. ##FIG##2##3##h, zoomed-in sections) and expected to be more curved [##REF##28346440##70##]. On increasing tension, previous studies report a higher fraction of AP2 with clathrin indicative of flat structures [##REF##29317637##9##]. Our observation of AP2 localized at edges (Fig. ##FIG##2##3##i, j) and showing lesser colocalization (Fig. ##FIG##2##3##k, l) might indicate that more fraction of clathrin structures are pits on de-adhesion. We also find an increase in the distance between clathrin and AP2 clusters after de-adhesion, comparing pairs already with 525 nm of each other (Fig. ##FIG##2##3##m) or higher. The significance holds true even while comparing pairs within 165 nm but does not show any difference when only those lying within 150 nm (close to the size of well-formed pits, Fig. ##FIG##2##3##i<bold>,</bold> yellow arrow) are compared, clearly indicating the population with AP2 at edges start making this difference significant.</p>\",\n \"<p id=\\\"Par27\\\">Together, the data showed that triggering de-adhesion reduced tension, and cells ramped up the frequency of their endocytosis events. We also know from following the fluctuations that the lowering of tension was also stalled or recovered back at later stages of de-adhesion (Fig. ##FIG##1##2##b). To understand if endocytosis affected tension regulation and how we next used pharmacological agents to block endocytosis and followed treated cells on de-adhesion.</p>\",\n \"<title>Blocking dynamin-dependent pathways reduces de-adhesion-triggered endocytosis</title>\",\n \"<p id=\\\"Par28\\\">We studied the effect of blocking dynamin-dependent pathways (Fig. ##FIG##3##4##, Fig. S6, S7) by treating HeLa cells with Dynasore [##REF##18413242##31##]. Specifically, it does not stop the formation of pits (clathrin-coated or caveolae, among others) but prevents dynamin’s function in the scission of pits (Fig. ##FIG##3##4##a). Through Tf-uptake assay Dynasore’s effect on stalling of pit scission is clear (Fig. S6 e, h, i). Instead of the rise in Rab5’s area fraction on de-adhesion, an initial drop was observed with de-adhesion as expected since early endosomes were expected to contain Rab5 [##REF##24307937##53##]. (Fig. ##FIG##3##4##b, c, Fig. S7 a, b). Failure of fission caused tubes to form, which also contained Rab5 (arrows, Fig. ##FIG##3##4##b, Fig. S7a). The increase in Tf puncta in Dynasore-treated cells was enhanced in de-adhering cells (Fig. ##FIG##3##4##c, d).</p>\",\n \"<p id=\\\"Par29\\\">Rab4 labelling (immunofluorescence) revealed an increase during the de-adhesion (Fig. ##FIG##3##4##b, Fig. S7b). This data suggested that the intermittent accumulation could be due to its inability to fuse normally with the plasma membrane (with reduced tension). This aligns with studies that have reported reduced recycling on inhibiting Dynamin [##REF##12372835##14##, ##REF##11809831##15##].</p>\",\n \"<p id=\\\"Par30\\\">Thus, Dynasore drastically reduced the formation of new early endosomes while also inhibiting the fusion of recycling endosomes with the plasma membrane. Together, these indicate that Dynasore effectively brought down de-adhesion-triggered endocytosis in the cell. We next studied how such blocking of endocytosis would affect the tension regulation during de-adhesion.</p>\",\n \"<title>Inactivating dynamin does not stop tension recovery, but knocking down AP2 does</title>\",\n \"<p id=\\\"Par31\\\">The single cell distribution of fluctuation amplitude and tension clearly changes as de-adhesion progresses to P2, implying that the tension reduced on de-adhesion of Dynasore treated cells (Fig. ##FIG##3##4##e–f, Fig S7d–i). Interestingly, instead of preventing tension recovery, it was enhanced in Dynasore-treated cells (Fig. ##FIG##3##4##e) in comparison to untreated cells (Fig. ##FIG##1##2##d). Normalized cell averages (Fig. ##FIG##3##4##g, Fig S7 f) point to the augmented mechanical regulation operating from P2 to P3 in Dynasore-treated cells. Maps help us visualize this (Fig. ##FIG##3##4##f, Fig S7e). On checking the state of activity (entropy generation rate), we found a lowering of activity on Dynasore treatment (Fig. S7g).</p>\",\n \"<p id=\\\"Par32\\\">To further validate, we performed experiments with a dominant-mutant of dynamin that was transiently transfected in cells. We found that even in these cells, the increase in tension or reduction in SD<sub>time</sub> was more substantial than in the control condition (Fig. S8). Clearly, the tension at P3 was higher than C, unlike in control sets.</p>\",\n \"<p id=\\\"Par33\\\">The data imply that although endosome formation is reduced, membrane mechanics regulation during de-adhesion is not stopped. This clearly implies that the formation of pits has a direct role in tension recovery. To verify, we next compared the excess areas from IRM images to find the impact of Dynasore on membrane smoothness. There was a significant reduction in the excess area in Dynasore-treated cells, implying a smoother membrane (Fig. ##FIG##3##4##g), supporting the hypothesis that pit formation can reduce excess area and enhance the effective tension. As a control, we checked how excess area changed on inhibiting Cdc42 by ML141. Cdc42 is required for the formation and scission of endocytic pits of the CG pathway. Inhibiting it for 30 min enhanced the excess area (Fig. S7h), further supporting the hypothesis that pit-formation reduces excess area.</p>\",\n \"<p id=\\\"Par34\\\">To validate further the role of pit-formation in tension recovery, we used siRNA-mediated knocked-down AP2. The knock-down was confirmed by immunofluorescence (Fig. S7j) and western blot (Fig. S7k) using control cells and cells treated with either scrambled siRNA (Scramble) or AP2-siRNA (AP2-siRNA). Depletion of AP2 by siRNA has been shown [##REF##12960147##25##] to reduce membrane clathrin as well drastically reduce coated pits on the plasma membrane. We observe that AP2-siRNA significantly reduced tension and enhanced fluctuations and excess area (Fig. ##FIG##3##4##i top) of cells. During de-adhesion (Fig. ##FIG##3##4##i bottom), the tension reduced in the P2 phase but failed to be regulated back in the P3 phase (Fig. ##FIG##3##4##h, i). SD<sub>time</sub> increased with de-adhesion (Table S2), although when compared using cell-wise statistics, no significant change was noted (Fig. ##FIG##3##4##i). However, it was clearly not regulated back. The response of AP2-siRNA-treated cells were marked different than control cells or cells treated with scramble siRNA. Together, the Dynasore, dynamin mutant and AP2 siRNA data clearly establish the pit formation step of clathrin-mediated endocytosis to contribute to tension regulation during de-adhesion.</p>\",\n \"<p id=\\\"Par35\\\">Having provided evidence suggesting pit formation as the critical step in endocytosis to cause tension increase, we next aimed to understand the nature of processes used for the initial pit formation. Dynamin-dependent pathways are not limited to major pathways like CME/caveolae. To understand the dependency on pathways that use key energy-consuming molecules like dynamin or actin, we next check the regulation’s dependency on ATP. ATP-dependent-pit formation is common (CME and caveolae [##REF##21295700##69##], for example). However, multiple pathways, like once induced by Shiga toxins, are ATP-independent [##REF##25517096##57##].</p>\",\n \"<title>ATP-depletion does not block tension regulation</title>\",\n \"<p id=\\\"Par36\\\">We used ATP depletion prior to de-adhesion to investigate the role of active processes and actin polymerization in the mechanical changes observed during de-adhesion (Fig. ##FIG##4##5##a, b). ATP-depleted cells showed low initial fluctuations but a similar trend of increase in fluctuations followed by a decrease as observed for control cells (Fig. ##FIG##4##5##a, b, Fig. S9a, Table ##SUPPL##0##S1##). The effective tension reduced and then increased. The distribution of local values in single cells also captured the changes which were found to be significant. No appreciable change was observed in the area fraction of Rab5 in ATP-depleted cells as de-adhesion progressed (Fig. ##FIG##4##5##c, d, Fig S9b). The area fraction of RAb5 in ATP-depleted cells was lower than in control cells (Fig. S9b), expected from reduction in active endocytosis.</p>\",\n \"<p id=\\\"Par37\\\">Therefore, the data here suggest the use of mechanisms that start recovering the tension drop without critically requiring ATP. As the first steps to identify the involved pathway, we next checked the dependence of tension regulation on cholesterol in the ATP-compromised condition. We do so because known pathways (like FEME used to internalize Shiga toxin) using ATP-independent pit formation are cholesterol-dependent and depend on the ability of cholesterol to cluster lipids and initiate the creation of invaginations [##REF##11739634##33##].</p>\",\n \"<title>Tension regulation in ATP-depleted cells is cholesterol-dependent</title>\",\n \"<p id=\\\"Par38\\\">We used ATP depletion and cholesterol depletion as controls (Fig. ##FIG##5##6##a, S9e) to assess the role of cholesterol in tension regulation in ATP-depleted cells during de-adhesion. Comparing single-cell distributions or cell-averaged values (Fig. ##FIG##5##6##b–d) showed that while the initial (C-P2) fluctuation (SD<sub>time</sub>) increase displayed the same trend as for the controls, fluctuations from P2 to P3 were not reduced efficiently on combined depletion of ATP and cholesterol. Depleting ATP or cholesterol enhanced the recovery of tension (from Control), but on dual depletion of ATP and cholesterol, there was a further reduction (Fig. ##FIG##5##6##a, c, Fig S9 c, d) [clear from distributions of local measurements, tension maps (Fig. S9e) and the LMM analysis of the local values (Fig. ##FIG##5##6##d)].</p>\",\n \"<p id=\\\"Par39\\\">Together, we showed (Fig. ##FIG##5##6##e) that the tension drop was significant even on perturbing scission, ATP or cholesterol content and faster in the case of the latter two. The recovery was also significant and faster for these perturbations but could not be effective under reduced ATP and cholesterol-depleted conditions. Treating tension reached by the control cells at the P3 phase as a reference, Dynasore treatment effectively tilted the balance towards a larger tension recovery rate while dual depletion of ATP and cholesterol resulted in lower and, therefore, appreciably slower recovery.</p>\",\n \"<p id=\\\"Par40\\\">Therefore, the observations strengthen the hypothesis that in the absence of ATP, cells also use cholesterol-dependent processes for mechano-regulation. Since in pathways involved in Shiga-toxin's endocytosis, toxin-rich tubules have been reported to be formed even in ATP-depleted cells/giant unilamellar vesicles [##REF##24703428##59##], we next checked if ATP-depleted cells formed tubules when tension was lowered by de-adhesion. For this, we imaged cells transfected with EGFP-CAAX and quantified the membrane cross-section at higher planes in ATP-depleted and ATP and cholesterol-depleted cells using confocal microscopy.</p>\",\n \"<title>On de-adhesion, ATP-depleted cells make more cholesterol-dependent tubular invaginations</title>\",\n \"<p id=\\\"Par41\\\">Cells were transfected with EGFP-CAAX [##REF##27633000##41##] to label the plasma membrane and taken through different treatments (Fig. ##FIG##6##7##, S10a). They were subsequently fixed (at 3, 6 min after treatment). Since fixation is not instantaneous, we measured the spread area of cells after fixation to determine the decrease in spread area (Fig. S10b). We observed a reduction to ~ 40% of initial when fixed at 3 min and ~ 17% of initial when fixed at 6 min. Therefore, they were classified as P2 and P3. Cells were subsequently imaged in confocal microscopy at a final resolution of ~ 120 nm in <italic>x</italic> and <italic>y</italic> directions (Fig. ##FIG##6##7##a, b, S10). From the intensities obtained from scans under the membrane (Fig. ##FIG##6##7##b, Fig. S10 c, d) along multiple line regions of interest (ROI), each of length ~ 4 µm, peaks were detected with larger widths and heights (from basal intensity) than set thresholds from the line scans (Fig. ##FIG##6##7##c). After de-adhesion, ATP-depleted cells displayed significantly more internal surface-connected structures than ATP + cholesterol-depleted cells (Fig. ##FIG##6##7##d) at the P2 and P3 phases. It should be noted that on cholesterol deletion (without ATP depletion, Fig. S10 e) or in control cells (no drug treatment, Fig. S10 f), there were much fewer tubules (Fig. S10 g), whose number also did not appreciably increase on de-adhesion (Fig. ##FIG##6##7##d, Fig. S10 e–g). The number of peaks transiently increased with de-adhesion in ATP-depleted cells but, in contrast, decreased for cells also depleted of cholesterol (Fig. ##FIG##6##7##e, Fig. S10). Tubule length also increased from before trypsinization to 3 min post-trypsinization. Hence, we concluded that the ATP-depleted cells might gain more cholesterol-dependent tubules on de-adhesion, thereby causing the tension surge.</p>\",\n \"<p id=\\\"Par42\\\">In conclusion, we show that de-adhesion induces a tension drop in the whole cell due to an altered adhesion state. The regulation sets in soon and engages endocytic pathways that internalize the membrane but also populate a recycling pool. Tension reduction is stalled but must be balanced by the recycling back of the membrane, because perturbations blocking internalization enhance the tension recovery rate. ATP and cholesterol depletion inhibits tension recovery, and this inhibition cannot be achieved by either preventing internalization or depleting only ATP or cholesterol individually.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par43\">Loss or remodelling of the adhesion machinery are known determinants of malignant transformation [##REF##19909018##42##, ##REF##26919980##47##], while regulated de-adhesion has been shown to aid tumour invasion [##REF##35838828##26##]. In this study, we aimed to understand the basic steps of endocytosis-mediated tension regulation during de-adhesion using IRM as the primary tool. It should be noted that tension was derived using a model that does not account for the frequency-dependent activity factor. We have evidenced that membrane fluctuations assayed at such small patches (0.75 μm<sup>2</sup>) has overall weak activity [##UREF##3##43##] as inferred from measurements of the lower bound of the entropy generation rate. Using the same algorithm [##UREF##3##43##], change in activity is clear when endocytic activity is blocked by Dynasore (Fig. S7g), indicating that the technique can resolve small changes. The excess area was observed to be reduced, and fluctuation-tension was enhanced (Fig. S7). While incorporating the frequency dependence of activity factor would be ideal while extracting fluctuation-tension, such formulations are currently not yet feasible for easy application to our data.</p>","<p id=\"Par44\">Hence, direct measurements of fluctuations (SD<sub>time</sub>) are first used to comment on the state of the membrane and the effective fluctuation-tension, excess area and functional state of the membrane (endocytic activity) used for further inferences. We also observed that during de-adhesion, the active nature of fluctuations did not significantly increase (Fig. S3c). SD<sub>time</sub> supported inferences about fluctuation-tension, while apparent tension—measured by optical trapping experiments—validated the initial tension drop on de-adhesion. Furthermore, we not only reported changes in these parameters but also presented functional evidence of the altered physical state. Lowered tension state correlated with enhanced endocytosis (accumulation of Rab5 near the plasma membrane, Fig. ##FIG##2##3##g) and reduced recycling (accumulation of Rab4 near the plasma membrane, Fig. ##FIG##2##3##g). Importantly, we present straightforward evidence of the functional connection between higher fluctuations (and lower effective fluctuation-tension) leading to enhanced endocytosis which in turn leads to reduction of fluctuations. Active models of cell membrane undergoing endocytosis and exocytosis had been used in theoretical work [##REF##11580560##55##] showing that active endo/exocytosis could give rise to an effective membrane tension. Their application to IRM data was not possible, but the pattern of changes of fluctuations (or effective fluctuation-tension) and Rab5/Tf punctas indicate a similar relationship. Future studies measuring the evolution of fluctuations and endocytosis in the same cells would further clarify their local dependence.</p>","<p id=\"Par45\">Our data first highlight that the local (at the basal membrane) build-up of membrane fluctuations is actin-dependent and not solely dependent on de-adhesion/retraction. The changes in fluctuations were quick to resolve, and tension was negligibly altered in Cyto D-treated cells. While this aligns with recent work implicating the cytoskeleton–membrane connections in delaying tension flow and, therefore, its equilibration, further studies are needed to prove this conclusively.</p>","<p id=\"Par46\">Identifying the stage when tension recovery was initiated was our primary target of investigations. We believe that the data strongly suggest that the formation of new invaginations starts the tension recovery. Even in the absence of de-adhesion, data in this manuscript (Fig. S7d) and reported earlier show that enhanced pit-formation (Dynasore treatment) could lead to a reduction of fluctuations and increase in tension (Fig. S7) while suppressing pit-formation by knocking down AP2 increases excess area, decreasing tension (Fig. ##FIG##3##4##i). We have also observed that Shiga toxin created tubules and increased tension of ATP-depleted HeLa cells that contained GB3 but did not either create tubules or enhance tension in HeLa cells lacking GB3 (data not shown). Such tubules have also been reported to passively regulate the area of lipid bilayers on being strained [##REF##21562210##72##]. The reverse, where flattening of invagination helps cells buffer a tension surge, has already been demonstrated. Hence, it is not far-fetched or physically impossible to use invaginations to perform this task. We believe it is possible that clustered lipids/proteins that initiate the pit formation can already start damping the fluctuations, although reports suggest so in simulations [##REF##27943675##52##]. Clustering of resources, as reported for hotspots of CME [##REF##27431447##35##, ##REF##21883765##50##] could also be ideal sites where pit-formation could remove the excess area. Pinching-off of endocytic buds or other ATP-dependent mechanisms, therefore, may not be critically essential for enhancing tension but required to prevent an excessive rise in tension, which could potentially inhibit many membrane processes or start unwanted processes. ATP-dependent machinery, we propose, acts as mechano-stats in the cell. They could be vital for keeping tension surges in check rather than being required only for enhancing tension. Our hypothesis is strengthened by the observation (Fig. ##FIG##5##6##e) that the rate of tension-lowering is enhanced in cholesterol-depleted cells, which incidentally, also have a higher fraction of membrane covered by actin-membrane linker—Ezrin (Fig. S11). Whether other lipid-raft-dependent mechanisms are also at play [##UREF##5##51##] or cholesterol-dependent mechanisms downstream of membrane and actin-remodelling cannot be ruled out.</p>","<p id=\"Par47\">Finally, some conclusions may be drawn about the relevance of the different pathways of endocytosis in tension regulation during de-adhesion. Assuming the three main pathways to be possibly the CG pathway, CME and caveolae-dependent pathway, cholesterol depletion is expected to block the CG and caveolae-dependent pathways even before pit formation. Cholesterol depletion, despite being reported to drastically (~ 80%) reduce internalization of CME, still is expected to allow ~ 50% of deep pits to form [##REF##10359788##73##]. We observe that cholesterol depletion does not abolish tension recovery or its maintenance close to the Control's response (Fig. ##FIG##5##6##e, C-P3). However, knocking down AP2, an adaptor protein required for the formation of CCPs during constitutive endocytosis, completely impairs the tension recovery. A lower colocalization with AP2 and more edge localization further confirm enhanced pit formation on de-adhesion. Together, this provides compelling evidence that pit-formation for CME significantly contributes to the mechano-regulation in de-adhering HeLa cells. The passive (ATP-independent mechanisms but cholesterol-dependent) pathways may also contribute by the induction of tubulated structures capturing excess membrane. We suggest that spontaneously pre-clustered platforms poised for other functions might tubulate on sudden tension reduction and contribute to the passive arm of the regulation.</p>","<p id=\"Par48\">In conclusion, in this paper, we have presented the effect of de-adhesion on spatiotemporal fluctuations and effective cell membrane mechanics. We have demonstrated that an initial membrane slack is drastically reduced when the actin cortical network is weak. Pit formation on the plasma membrane has been shown to be a determining factor in regulating tension/fluctuations during the cellular process of de-adhesion. The restoration of the low effective tension used endocytic pit formation for increasing the tension, while the complete regulatory cycle utilized active as well as cholesterol-dependent passive forms of mechano-regulation (Fig. ##FIG##7##8##).</p>","<title>Lead contact</title>","<p id=\"Par49\">More detailed information and requests for the resources should be directed to and will be fulfilled by the lead contact, Bidisha Sinha (<underline>bidisha.sinha@iiserkol.ac.in</underline>).</p>","<title>Material availability</title>","<p id=\"Par50\">New materials and methods used in these studies will be available upon request to Bidisha Sinha.</p>","<title>Data and code availability</title>","<p id=\"Par51\">Data and codes used in this study for analysis purposes will be available upon request to the lead contact, Bidisha Sinha (<underline>bidisha.sinha@iiserkol.ac.in</underline>).</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par43\\\">Loss or remodelling of the adhesion machinery are known determinants of malignant transformation [##REF##19909018##42##, ##REF##26919980##47##], while regulated de-adhesion has been shown to aid tumour invasion [##REF##35838828##26##]. In this study, we aimed to understand the basic steps of endocytosis-mediated tension regulation during de-adhesion using IRM as the primary tool. It should be noted that tension was derived using a model that does not account for the frequency-dependent activity factor. We have evidenced that membrane fluctuations assayed at such small patches (0.75 μm<sup>2</sup>) has overall weak activity [##UREF##3##43##] as inferred from measurements of the lower bound of the entropy generation rate. Using the same algorithm [##UREF##3##43##], change in activity is clear when endocytic activity is blocked by Dynasore (Fig. S7g), indicating that the technique can resolve small changes. The excess area was observed to be reduced, and fluctuation-tension was enhanced (Fig. S7). While incorporating the frequency dependence of activity factor would be ideal while extracting fluctuation-tension, such formulations are currently not yet feasible for easy application to our data.</p>\",\n \"<p id=\\\"Par44\\\">Hence, direct measurements of fluctuations (SD<sub>time</sub>) are first used to comment on the state of the membrane and the effective fluctuation-tension, excess area and functional state of the membrane (endocytic activity) used for further inferences. We also observed that during de-adhesion, the active nature of fluctuations did not significantly increase (Fig. S3c). SD<sub>time</sub> supported inferences about fluctuation-tension, while apparent tension—measured by optical trapping experiments—validated the initial tension drop on de-adhesion. Furthermore, we not only reported changes in these parameters but also presented functional evidence of the altered physical state. Lowered tension state correlated with enhanced endocytosis (accumulation of Rab5 near the plasma membrane, Fig. ##FIG##2##3##g) and reduced recycling (accumulation of Rab4 near the plasma membrane, Fig. ##FIG##2##3##g). Importantly, we present straightforward evidence of the functional connection between higher fluctuations (and lower effective fluctuation-tension) leading to enhanced endocytosis which in turn leads to reduction of fluctuations. Active models of cell membrane undergoing endocytosis and exocytosis had been used in theoretical work [##REF##11580560##55##] showing that active endo/exocytosis could give rise to an effective membrane tension. Their application to IRM data was not possible, but the pattern of changes of fluctuations (or effective fluctuation-tension) and Rab5/Tf punctas indicate a similar relationship. Future studies measuring the evolution of fluctuations and endocytosis in the same cells would further clarify their local dependence.</p>\",\n \"<p id=\\\"Par45\\\">Our data first highlight that the local (at the basal membrane) build-up of membrane fluctuations is actin-dependent and not solely dependent on de-adhesion/retraction. The changes in fluctuations were quick to resolve, and tension was negligibly altered in Cyto D-treated cells. While this aligns with recent work implicating the cytoskeleton–membrane connections in delaying tension flow and, therefore, its equilibration, further studies are needed to prove this conclusively.</p>\",\n \"<p id=\\\"Par46\\\">Identifying the stage when tension recovery was initiated was our primary target of investigations. We believe that the data strongly suggest that the formation of new invaginations starts the tension recovery. Even in the absence of de-adhesion, data in this manuscript (Fig. S7d) and reported earlier show that enhanced pit-formation (Dynasore treatment) could lead to a reduction of fluctuations and increase in tension (Fig. S7) while suppressing pit-formation by knocking down AP2 increases excess area, decreasing tension (Fig. ##FIG##3##4##i). We have also observed that Shiga toxin created tubules and increased tension of ATP-depleted HeLa cells that contained GB3 but did not either create tubules or enhance tension in HeLa cells lacking GB3 (data not shown). Such tubules have also been reported to passively regulate the area of lipid bilayers on being strained [##REF##21562210##72##]. The reverse, where flattening of invagination helps cells buffer a tension surge, has already been demonstrated. Hence, it is not far-fetched or physically impossible to use invaginations to perform this task. We believe it is possible that clustered lipids/proteins that initiate the pit formation can already start damping the fluctuations, although reports suggest so in simulations [##REF##27943675##52##]. Clustering of resources, as reported for hotspots of CME [##REF##27431447##35##, ##REF##21883765##50##] could also be ideal sites where pit-formation could remove the excess area. Pinching-off of endocytic buds or other ATP-dependent mechanisms, therefore, may not be critically essential for enhancing tension but required to prevent an excessive rise in tension, which could potentially inhibit many membrane processes or start unwanted processes. ATP-dependent machinery, we propose, acts as mechano-stats in the cell. They could be vital for keeping tension surges in check rather than being required only for enhancing tension. Our hypothesis is strengthened by the observation (Fig. ##FIG##5##6##e) that the rate of tension-lowering is enhanced in cholesterol-depleted cells, which incidentally, also have a higher fraction of membrane covered by actin-membrane linker—Ezrin (Fig. S11). Whether other lipid-raft-dependent mechanisms are also at play [##UREF##5##51##] or cholesterol-dependent mechanisms downstream of membrane and actin-remodelling cannot be ruled out.</p>\",\n \"<p id=\\\"Par47\\\">Finally, some conclusions may be drawn about the relevance of the different pathways of endocytosis in tension regulation during de-adhesion. Assuming the three main pathways to be possibly the CG pathway, CME and caveolae-dependent pathway, cholesterol depletion is expected to block the CG and caveolae-dependent pathways even before pit formation. Cholesterol depletion, despite being reported to drastically (~ 80%) reduce internalization of CME, still is expected to allow ~ 50% of deep pits to form [##REF##10359788##73##]. We observe that cholesterol depletion does not abolish tension recovery or its maintenance close to the Control's response (Fig. ##FIG##5##6##e, C-P3). However, knocking down AP2, an adaptor protein required for the formation of CCPs during constitutive endocytosis, completely impairs the tension recovery. A lower colocalization with AP2 and more edge localization further confirm enhanced pit formation on de-adhesion. Together, this provides compelling evidence that pit-formation for CME significantly contributes to the mechano-regulation in de-adhering HeLa cells. The passive (ATP-independent mechanisms but cholesterol-dependent) pathways may also contribute by the induction of tubulated structures capturing excess membrane. We suggest that spontaneously pre-clustered platforms poised for other functions might tubulate on sudden tension reduction and contribute to the passive arm of the regulation.</p>\",\n \"<p id=\\\"Par48\\\">In conclusion, in this paper, we have presented the effect of de-adhesion on spatiotemporal fluctuations and effective cell membrane mechanics. We have demonstrated that an initial membrane slack is drastically reduced when the actin cortical network is weak. Pit formation on the plasma membrane has been shown to be a determining factor in regulating tension/fluctuations during the cellular process of de-adhesion. The restoration of the low effective tension used endocytic pit formation for increasing the tension, while the complete regulatory cycle utilized active as well as cholesterol-dependent passive forms of mechano-regulation (Fig. ##FIG##7##8##).</p>\",\n \"<title>Lead contact</title>\",\n \"<p id=\\\"Par49\\\">More detailed information and requests for the resources should be directed to and will be fulfilled by the lead contact, Bidisha Sinha (<underline>bidisha.sinha@iiserkol.ac.in</underline>).</p>\",\n \"<title>Material availability</title>\",\n \"<p id=\\\"Par50\\\">New materials and methods used in these studies will be available upon request to Bidisha Sinha.</p>\",\n \"<title>Data and code availability</title>\",\n \"<p id=\\\"Par51\\\">Data and codes used in this study for analysis purposes will be available upon request to the lead contact, Bidisha Sinha (<underline>bidisha.sinha@iiserkol.ac.in</underline>).</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<p id=\"Par1\">Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms, including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis are poorly understood. To investigate this, we studied the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes, as well as transferrin-labelled pits close to the basal plasma membrane, also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked, but knocking down AP2-dependent pit formation stopped the tension recovery. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested by depleting both. The data strongly supports Clathrin and AP2-dependent pit-formation to be central to the reduction in fluctuations confirmed by super-resolution microscopy. Furthermore, we propose that cholesterol-dependent pits spontaneously regulate tension under ATP-depleted conditions.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00018-023-05072-4.</p>","<title>Keywords</title>"],"string":"[\n \"<p id=\\\"Par1\\\">Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms, including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis are poorly understood. To investigate this, we studied the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes, as well as transferrin-labelled pits close to the basal plasma membrane, also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked, but knocking down AP2-dependent pit formation stopped the tension recovery. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested by depleting both. The data strongly supports Clathrin and AP2-dependent pit-formation to be central to the reduction in fluctuations confirmed by super-resolution microscopy. Furthermore, we propose that cholesterol-dependent pits spontaneously regulate tension under ATP-depleted conditions.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00018-023-05072-4.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>BS acknowledges support from Wellcome Trust/DBT India Alliance fellowship (Grant number IA/I/13/1/500885), SERB (Grant number SERB_CRG_2458) and CEFIPRA (Grant number 6303-1). The authors are grateful to CSIR and IISER Kolkata for providing scholarships to TM and AB, respectively. TG thanks CEFIPRA for providing his fellowship. The authors are also thankful to the DBT Wellcome Imaging Facility (IA/I/16/1/502369) for confocal imaging, the Builder Imaging facility (BT/INF/22/SP45383/2022) for super-resolution STED imaging and the DIRAC supercomputing facility for tension mapping.</p>","<title>Author contributions</title>","<p>TM: investigation, formal analysis, data curation, writing (original draft), AB: investigation, methodology, software, formal analysis, writing (original draft), TG: methodology, software, investigation, formal analysis, SM: resources, AK: methodology, AB: methodology, resources, DM: resources, BS: conceptualization, methodology, writing (original draft), funding acquisition. All authors edited the manuscript.</p>","<title>Funding</title>","<p>Wellcome Trust/DBT India Alliance fellowship (grant number IA/I/13/1/500885), SERB (grant number SERB_CRG_2458) and CEFIPRA (grant number 6303-1). CSIR and IISER Kolkata for providing scholarships to TM and AB, respectively. TG fellowship is provided by CEFIPRA.</p>","<title>Data availability</title>","<p>The data that support the findings of this study are available from the corresponding author upon reasonable request. Codes used for this study are available at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/BidishaSinha/Mechano-regulation-by-pit-formation-during-De-adhesion\">https://github.com/BidishaSinha/Mechano-regulation-by-pit-formation-during-De-adhesion</ext-link>.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par70\">The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.</p>","<title>Ethical approval and consent to participate</title>","<p id=\"Par71\">Not applicable.</p>","<title>Consent for publication</title>","<p id=\"Par72\">All authors agree to submit the manuscript for publications.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>BS acknowledges support from Wellcome Trust/DBT India Alliance fellowship (Grant number IA/I/13/1/500885), SERB (Grant number SERB_CRG_2458) and CEFIPRA (Grant number 6303-1). The authors are grateful to CSIR and IISER Kolkata for providing scholarships to TM and AB, respectively. TG thanks CEFIPRA for providing his fellowship. The authors are also thankful to the DBT Wellcome Imaging Facility (IA/I/16/1/502369) for confocal imaging, the Builder Imaging facility (BT/INF/22/SP45383/2022) for super-resolution STED imaging and the DIRAC supercomputing facility for tension mapping.</p>\",\n \"<title>Author contributions</title>\",\n \"<p>TM: investigation, formal analysis, data curation, writing (original draft), AB: investigation, methodology, software, formal analysis, writing (original draft), TG: methodology, software, investigation, formal analysis, SM: resources, AK: methodology, AB: methodology, resources, DM: resources, BS: conceptualization, methodology, writing (original draft), funding acquisition. All authors edited the manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Wellcome Trust/DBT India Alliance fellowship (grant number IA/I/13/1/500885), SERB (grant number SERB_CRG_2458) and CEFIPRA (grant number 6303-1). CSIR and IISER Kolkata for providing scholarships to TM and AB, respectively. TG fellowship is provided by CEFIPRA.</p>\",\n \"<title>Data availability</title>\",\n \"<p>The data that support the findings of this study are available from the corresponding author upon reasonable request. Codes used for this study are available at: <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://github.com/BidishaSinha/Mechano-regulation-by-pit-formation-during-De-adhesion\\\">https://github.com/BidishaSinha/Mechano-regulation-by-pit-formation-during-De-adhesion</ext-link>.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par70\\\">The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.</p>\",\n \"<title>Ethical approval and consent to participate</title>\",\n \"<p id=\\\"Par71\\\">Not applicable.</p>\",\n \"<title>Consent for publication</title>\",\n \"<p id=\\\"Par72\\\">All authors agree to submit the manuscript for publications.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Phases and variability in de-adhesion. <bold>a</bold> IRM images of a representative cell before and after Trypsin–EDTA addition at 0.25% (faster) concentrations and corresponding temporal fluctuation maps. The time after de-adhering solution addition is mentioned. Scale: 10 μm. Zoomed-in views of SD<sub>time</sub> maps (right). <bold>b</bold> Profiles of spread area for 6 representative cells in each concentration with time after Trypsin–EDTA addition in the two different concentrations. <bold>c</bold> Time taken to de-adhere 67% of spread area of cell. N<sub>cell</sub>: Control = 15, Dyna = 8, Cyto D = 17, ATP Dep = 20, Chol Dep = 18, ATP Dep + Chol Dep = 11. Control = 11, Cyto D = 18. <bold>d</bold> Representative colour-coded kymographs of IRM intensity of Control and Cyto D-treated cell. ROIS drawn perpendicular to the de-adhering front of a cell. Scale: 10 μm</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Membrane slacks transiently.<bold> a</bold> Representative time series of indicated parameters for a single cell during fast de-adhesion. <bold>b</bold> Comparison of normalized amplitudes of fluctuations and tension following the same cells across the indicated phases of de-adhesion. Data represent average median values calculated for 22 cells using FBRs of sizes 0.75 μm<sup>2</sup>. Normalization is performed by dividing any particular cell’s measurement at P2 or P3 by the measurement at C. Table ##SUPPL##0##S1## provides the minimum number of FBRs used per cell in each mechanism used for calculating the average. <bold>c</bold> IRM images of a representative cell at different phases of de-adhesion, and the corresponding FBR-wise tension values mapped back on the cell outline. The dark blue background represents the cell, and coloured boxes denote tension values derived from averaged PSDs from the indicated regions. Lower panel indicates pixel wise tension map and corresponding <italic>R</italic><sup>2</sup> map. <bold>d</bold> Comparison of probability of logarithm of temporal fluctuation and tension across the de-adhesion phases using 0.25% Trypsin–EDTA. Distributions were obtained from FBR-wise values for each cell and averaged. Shaded regions denote the SEM. <bold>e</bold> Comparison of probability of logarithm of tension measures across the cells (FBR wise) across the de-adhesion phases of Control (left) N<sub>cell</sub> = 18, and Cytochalasin D (right) treated cells N<sub>cell</sub> = 25 using 0.05% Trypsin–EDTA. <bold>f</bold> Comparison of fold change in the mean amplitude of fluctuations and median tension for the same cells followed over time of Control and Cyto D-treated cells. Normalization is performed by dividing any cell's measurement at P2 or P3 by the measurement at C. <bold>g</bold> Left: Typical image of a cell used for the tether-pulling experiment. N<sub>cell</sub> = 10. The trapped bead is marked out with an arrow. Right: Fold reduction of force after Trypsin addition (higher concentration: 0.25%). Black *denotes Mann–Whitney <italic>U</italic> statistical significance test with Bonferroni correction is performed, * denotes <italic>p</italic> values < 0.016, and **denotes <italic>p</italic> value < 0.001. Scale bar = 10 μm</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>De-adhesion induces endocytosis. <bold>a</bold> Schematic of formation of early endosomes and maturation into recycling endosomes. <bold>b</bold> Transferrin uptake assay in Normal and 0.25% Trypsinised cells N<sub>cell:</sub> Control = 116, Trypsinised = 125. <bold>c</bold> Representative TIRF images of same HeLa cells followed through time, puncta marked with Transferrin- 568 with 0.25% Trypsin (upper panel), and without Trypsin (bottom panel). Scale bar = 10 µm. <bold>d</bold> Normalized area fraction of Transferrin-marked puncta followed through time with and without Trypsin. N<sub>cell:</sub> Trypsin = 53, without Trypsin = 16. Shaded regions denote SEM measure while average per distribution. <bold>e</bold> Representative TIRF images of the same HeLa cells transiently expressing EGFP -Rab5 (upper panel) or mCherry -Rab4 (lower panel) before (0 min) and after administration of de-adhesion media. Bottom panel represents zoomed-in view (Scale bar = 5 µm). <bold>f</bold> Change in area fraction of Rab5 (left) and Rab4 (right) as spread area reduces on de-adhesion for typical single cells. <bold>g</bold> Normalized area fraction of Rab5 and Rab4 through different time points before and after addition of 0.25% Trypsin. N<sub>cell:</sub> Rab5 = 15, Rab4 = 43. Shaded regions denote SEM. Table ##SUPPL##0##S1## provides the list of the number of cells and other statistical parameters. <bold>h</bold> Representative STED images of Clathrin (Clathrin heavy chain) and AP2 (α subunit) Scale bar = 3 µm. <bold>i</bold> Zoomed in sections (ROIs marked in h with colours as indicated) Scale bar = 100 nm. Yellow arrow-head points at edge-localized AP2 next to well-matured calthrin-coated pit with distinctly distributed clathrin. Pink arrow-head points out smaller clathrin puncta much better colocalized. <bold>j</bold> Other randomly selected ROIs from other cells. Scale bar = 1 µm <bold>k</bold> Quantification of colocalization using Mander’s coefficient from 10 cells and 27 ROIs for Control, and 20 cells and 32 rois for + Trypsin condition. <bold>l</bold> Colocalization from object detection from N<sub>cell</sub> = 15 and 28 for Control and trypsinized conditions with 70 and 75 ROIs, respectively. <bold>m</bold> Comparison of distance between clathrin punctas and the nearest AP2 puncta for only those pairs that lie within 525 nm of each other. > 40,000 clathrin puncta and > 14,000 AP2 puncta were used. Data (h-m) are representative of 3 independent sets with 45 and > 60 cells imaged in each condition</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Blocking endocytosis does not stop tension recovery. <bold>a</bold> Schematic diagram of pit formation, scission, recycling and inhibition of dynamin. <bold>b</bold> TIRF images of Dynasore-treated cells transfected with EGFP-Rab5 or immune-stained with Rab4 before and after de-adhesion. Scale bar = 10 μm. Bottom: Normalized area fraction of Rab5 marked early endosomes (left) and Rab4 marked recycling endosomes (right) of Control and treated with Dynamin inhibitor- Dynasore. N<sub>cells(Rab5):</sub> Control ~ 57, Dynasore ~ 43, N<sub>cells(Rab4):</sub> Control ~ 70, Dynasore ~ 103. <bold>c</bold> Representative TIRF images of Dynamin-inhibited HeLa cells followed through time, puncta marked with Transferrin- 568 without Trypsin or with 0.25% Trypsin. Scale bar = 10 µm. <bold>d</bold> Normalized area fraction of Transferrin-marked puncta followed through time with and without Trypsin. N<sub>cell</sub>: Trypsin = 12, without Trypsin = 8. Shaded regions denote SEM. <bold>e</bold> Probability distribution of FBR wise log temporal fluctuations and tension of Dynasore treated same HeLa cell followed through different phases of De-adhesion. N<sub>cell:</sub> Dynasore = 11. Shaded regions denote SEM. <bold>f</bold> Typical tension map of Dynasore-treated cells in the different phases of de-adhesion. Scale bar = 10 μm. (Upper). <bold>g</bold> Fold change in cell-averaged parameters comparing each cell with its own measurements at different phases. N<sub>cell:</sub> Control = 20, Dynasore = 11. For excess area, FBR-wise comparison is presented with red *denoting statistical significance obtained from LMM. <bold>h</bold> FBR-wise tension map of a representative AP2 knockdown cell in different phases. <bold>i</bold> Top: Cell-wise comparison of tension and SD<sub>time</sub> of Control, scramble (scrambled siRNA), AP2 siRNA-treated cells without de-adhesion. For excess area, FBR-wise comparison is presented. Bottom: Fold change in cell-averaged parameters comparing each cell with its own measurements at different phases of de-adhesion. N<sub>cell:</sub> Control = 9, Scramble = 5, AP2 siRNA = 14. <italic>n</italic> = 3 independent experiments. One-way ANOVA with Bonferroni correction is performed for SD<sub>time</sub> since the data are normal. For tension, the Mann–Whitney <italic>U</italic> test is performed and *denotes a <italic>p</italic> value < 0.016 (adjusted by group size of 3 per experiment). <italic>N</italic> = 3 independent experiments. Table ##SUPPL##0##S1## provides a list of the number of FBRs</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Tension recovery can start without ATP. <bold>a</bold> Time series boxplots and median (with median absolute deviation (MAD) as error bar, lower panel) for different parameters for Control and ATP-depleted cells on de-adhesion using an FBR size of 4.67 μm<sup>2</sup>. N<sub>cells(control)</sub>:6 N<sub>cells(ATP Depleted)</sub>: 9. <bold>b</bold> Probability distribution of FBR-wise log temporal fluctuations and tension of Control and ATP depleted same HeLa cell followed through different phases of De-adhesion. Shaded regions denote SEM. <bold>c</bold> Zoomed-in TIRF images of different cells (transfected with EGFP-Rab5) at different stages of de-adhesion in ATP-depleted condition. <bold>d</bold> Normalized area fraction of Rab5 in ATP-depleted condition. Shaded regions denote SEM. N<sub>cells</sub>: C = 26, P2 = 41, P3 = 39</p></caption></fig>","<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Passive regulation is cholesterol-dependent. <bold>a</bold> Representative tension maps of cholesterol-depleted (Upper Panel) and ATP-depleted as well as cholesterol-depleted cells (Lower Panel) in three phases of de-adhesion<bold>.</bold> Scale bar = 10 μm. <bold>b</bold> Probability distribution of FBR-wise values of log temporal fluctuations and tension of cholesterol-depleted HeLa cell followed through different phases of de-adhesion. Shaded regions denote SEM. <bold>c</bold> Fold change in cell-averaged parameters comparing each cell with its own measurements at different phases. N<sub>cells</sub>: Control = 11, ATP dep = 9, Chol dep = 16, ATP dep + Chol dep = 15. <bold>d</bold> Probability distribution of FBR-wise values of log temporal fluctuations and tension of ATP depleted as well cholesterol-depleted same HeLa cells followed through different phases of de-adhesion. Shaded regions denote SEM. <bold>e</bold> Plot of rate of fractional change of tension when cells transit between different phases (mentioned). One-way Anova with Bonferroni correction is performed for SD<sub>time</sub> since the data are normal. For tension, the Mann–Whitney <italic>U</italic> test is performed and *denotes <italic>p</italic> value < 0.016 (adjusted by group size of 3 per experiment). <italic>n</italic> = 3 independent experiments. N<sub>cells</sub> mentioned in Table ##SUPPL##0##S1##</p></caption></fig>","<fig id=\"Fig7\"><label>Fig. 7</label><caption><p>Membrane imaging reveals cholesterol-dependent tubules. <bold>a</bold> Representative zoomed in colour-coded confocal images of cells [ATP-depleted (left) and ATP-depleted as well as cholesterol-depleted (right)]. Scale bar = 2 μm. <bold>b</bold> Typical line scans are performed parallel to the membrane on the cytosolic side. Scale bar = 2 μm <bold>c</bold> Intensity profile of typical line scans of ATP-depleted and ATP Dep + Chol Dep condition with triangles pointing out detected peaks with minimal width and height. <bold>d</bold> Box plots (left) and line plot (centre) comparing number of peaks/μm in ATP-depleted and ATP + Cholesterol depleted through different time points of de-adhesion. Number of cells = 20, 18, 28 for 0, 3, 6 min, respectively (ATP-depleted), 21, 24, 21 for 0, 3, 6 min, respectively (ATP + cholesterol-depleted). Effect of de-adhesion on only cholesterol-depleted cells were evaluated using 97 and 113 ROIs from 8 and 9 cells. <bold>e</bold> Comparison of intensity detected per μm of various 4 μm lines drawn as explained in (<bold>b</bold>). <bold>f</bold> Evaluation of length of tubules using analysis of 68 and 58 ROIs from 18 and 15 cells of ATP-depleted and ATP + Cholesterol-depleted cells, respectively. **denote <italic>p</italic> value < 0.001 calculated using Mann–Whitney <italic>U</italic> test. Table ##SUPPL##0##S1## lists the number of peaks</p></caption></fig>","<fig id=\"Fig8\"><label>Fig. 8</label><caption><p>Schematic diagram. <bold>a</bold> Membrane remodelling by active and passive regulation. Schematic shows that membrane fluctuations enhance in the P1 and P2 phase. However, while in passive condition (low ATP), cholesterol-dependent tubules reduce the membrane fluctuations, in normal conditions, active regulation entails formation of pits and their internalization in the P2 phase which transiently accumulate in early and recycling endosomal structures till the tension enhances back and fusion of recycling membrane keeps the increasing tension in check. <bold>b</bold> The decrease in tension by de-adhesion favours curving of clathrin-coated pits—moving AP2 to the edges. Pit formation increases tension. Dynamin-dependent scission and Rab 4-based recycling helps maintain homeostatic tension. Lack of AP2 decreases abrogates regulation by pit formation while lack of functional dynamin affects maintenance of tension and results in much higher tension. <bold>c</bold> Regulation of plasma membrane excess area by formation of pits, early and recycling endosomes. Schematic depicts that higher membrane excess area at the PM favours formation of pits as observed in this study. Such pits can be static or actively internalized to add to the early endosomal pool as shown in this study. Part of the early endosomal pool get converted to the recycling pool which is depleted when some structures fuse back with the PM. At higher membrane excess area, this fusion is disfavoured which can cause accumulation of the recycling endosome as shown in a and observed in this study</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Phases and variability in de-adhesion. <bold>a</bold> IRM images of a representative cell before and after Trypsin–EDTA addition at 0.25% (faster) concentrations and corresponding temporal fluctuation maps. The time after de-adhering solution addition is mentioned. Scale: 10 μm. Zoomed-in views of SD<sub>time</sub> maps (right). <bold>b</bold> Profiles of spread area for 6 representative cells in each concentration with time after Trypsin–EDTA addition in the two different concentrations. <bold>c</bold> Time taken to de-adhere 67% of spread area of cell. N<sub>cell</sub>: Control = 15, Dyna = 8, Cyto D = 17, ATP Dep = 20, Chol Dep = 18, ATP Dep + Chol Dep = 11. Control = 11, Cyto D = 18. <bold>d</bold> Representative colour-coded kymographs of IRM intensity of Control and Cyto D-treated cell. ROIS drawn perpendicular to the de-adhering front of a cell. Scale: 10 μm</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Membrane slacks transiently.<bold> a</bold> Representative time series of indicated parameters for a single cell during fast de-adhesion. <bold>b</bold> Comparison of normalized amplitudes of fluctuations and tension following the same cells across the indicated phases of de-adhesion. Data represent average median values calculated for 22 cells using FBRs of sizes 0.75 μm<sup>2</sup>. Normalization is performed by dividing any particular cell’s measurement at P2 or P3 by the measurement at C. Table ##SUPPL##0##S1## provides the minimum number of FBRs used per cell in each mechanism used for calculating the average. <bold>c</bold> IRM images of a representative cell at different phases of de-adhesion, and the corresponding FBR-wise tension values mapped back on the cell outline. The dark blue background represents the cell, and coloured boxes denote tension values derived from averaged PSDs from the indicated regions. Lower panel indicates pixel wise tension map and corresponding <italic>R</italic><sup>2</sup> map. <bold>d</bold> Comparison of probability of logarithm of temporal fluctuation and tension across the de-adhesion phases using 0.25% Trypsin–EDTA. Distributions were obtained from FBR-wise values for each cell and averaged. Shaded regions denote the SEM. <bold>e</bold> Comparison of probability of logarithm of tension measures across the cells (FBR wise) across the de-adhesion phases of Control (left) N<sub>cell</sub> = 18, and Cytochalasin D (right) treated cells N<sub>cell</sub> = 25 using 0.05% Trypsin–EDTA. <bold>f</bold> Comparison of fold change in the mean amplitude of fluctuations and median tension for the same cells followed over time of Control and Cyto D-treated cells. Normalization is performed by dividing any cell's measurement at P2 or P3 by the measurement at C. <bold>g</bold> Left: Typical image of a cell used for the tether-pulling experiment. N<sub>cell</sub> = 10. The trapped bead is marked out with an arrow. Right: Fold reduction of force after Trypsin addition (higher concentration: 0.25%). Black *denotes Mann–Whitney <italic>U</italic> statistical significance test with Bonferroni correction is performed, * denotes <italic>p</italic> values < 0.016, and **denotes <italic>p</italic> value < 0.001. Scale bar = 10 μm</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>De-adhesion induces endocytosis. <bold>a</bold> Schematic of formation of early endosomes and maturation into recycling endosomes. <bold>b</bold> Transferrin uptake assay in Normal and 0.25% Trypsinised cells N<sub>cell:</sub> Control = 116, Trypsinised = 125. <bold>c</bold> Representative TIRF images of same HeLa cells followed through time, puncta marked with Transferrin- 568 with 0.25% Trypsin (upper panel), and without Trypsin (bottom panel). Scale bar = 10 µm. <bold>d</bold> Normalized area fraction of Transferrin-marked puncta followed through time with and without Trypsin. N<sub>cell:</sub> Trypsin = 53, without Trypsin = 16. Shaded regions denote SEM measure while average per distribution. <bold>e</bold> Representative TIRF images of the same HeLa cells transiently expressing EGFP -Rab5 (upper panel) or mCherry -Rab4 (lower panel) before (0 min) and after administration of de-adhesion media. Bottom panel represents zoomed-in view (Scale bar = 5 µm). <bold>f</bold> Change in area fraction of Rab5 (left) and Rab4 (right) as spread area reduces on de-adhesion for typical single cells. <bold>g</bold> Normalized area fraction of Rab5 and Rab4 through different time points before and after addition of 0.25% Trypsin. N<sub>cell:</sub> Rab5 = 15, Rab4 = 43. Shaded regions denote SEM. Table ##SUPPL##0##S1## provides the list of the number of cells and other statistical parameters. <bold>h</bold> Representative STED images of Clathrin (Clathrin heavy chain) and AP2 (α subunit) Scale bar = 3 µm. <bold>i</bold> Zoomed in sections (ROIs marked in h with colours as indicated) Scale bar = 100 nm. Yellow arrow-head points at edge-localized AP2 next to well-matured calthrin-coated pit with distinctly distributed clathrin. Pink arrow-head points out smaller clathrin puncta much better colocalized. <bold>j</bold> Other randomly selected ROIs from other cells. Scale bar = 1 µm <bold>k</bold> Quantification of colocalization using Mander’s coefficient from 10 cells and 27 ROIs for Control, and 20 cells and 32 rois for + Trypsin condition. <bold>l</bold> Colocalization from object detection from N<sub>cell</sub> = 15 and 28 for Control and trypsinized conditions with 70 and 75 ROIs, respectively. <bold>m</bold> Comparison of distance between clathrin punctas and the nearest AP2 puncta for only those pairs that lie within 525 nm of each other. > 40,000 clathrin puncta and > 14,000 AP2 puncta were used. Data (h-m) are representative of 3 independent sets with 45 and > 60 cells imaged in each condition</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Blocking endocytosis does not stop tension recovery. <bold>a</bold> Schematic diagram of pit formation, scission, recycling and inhibition of dynamin. <bold>b</bold> TIRF images of Dynasore-treated cells transfected with EGFP-Rab5 or immune-stained with Rab4 before and after de-adhesion. Scale bar = 10 μm. Bottom: Normalized area fraction of Rab5 marked early endosomes (left) and Rab4 marked recycling endosomes (right) of Control and treated with Dynamin inhibitor- Dynasore. N<sub>cells(Rab5):</sub> Control ~ 57, Dynasore ~ 43, N<sub>cells(Rab4):</sub> Control ~ 70, Dynasore ~ 103. <bold>c</bold> Representative TIRF images of Dynamin-inhibited HeLa cells followed through time, puncta marked with Transferrin- 568 without Trypsin or with 0.25% Trypsin. Scale bar = 10 µm. <bold>d</bold> Normalized area fraction of Transferrin-marked puncta followed through time with and without Trypsin. N<sub>cell</sub>: Trypsin = 12, without Trypsin = 8. Shaded regions denote SEM. <bold>e</bold> Probability distribution of FBR wise log temporal fluctuations and tension of Dynasore treated same HeLa cell followed through different phases of De-adhesion. N<sub>cell:</sub> Dynasore = 11. Shaded regions denote SEM. <bold>f</bold> Typical tension map of Dynasore-treated cells in the different phases of de-adhesion. Scale bar = 10 μm. (Upper). <bold>g</bold> Fold change in cell-averaged parameters comparing each cell with its own measurements at different phases. N<sub>cell:</sub> Control = 20, Dynasore = 11. For excess area, FBR-wise comparison is presented with red *denoting statistical significance obtained from LMM. <bold>h</bold> FBR-wise tension map of a representative AP2 knockdown cell in different phases. <bold>i</bold> Top: Cell-wise comparison of tension and SD<sub>time</sub> of Control, scramble (scrambled siRNA), AP2 siRNA-treated cells without de-adhesion. For excess area, FBR-wise comparison is presented. Bottom: Fold change in cell-averaged parameters comparing each cell with its own measurements at different phases of de-adhesion. N<sub>cell:</sub> Control = 9, Scramble = 5, AP2 siRNA = 14. <italic>n</italic> = 3 independent experiments. One-way ANOVA with Bonferroni correction is performed for SD<sub>time</sub> since the data are normal. For tension, the Mann–Whitney <italic>U</italic> test is performed and *denotes a <italic>p</italic> value < 0.016 (adjusted by group size of 3 per experiment). <italic>N</italic> = 3 independent experiments. Table ##SUPPL##0##S1## provides a list of the number of FBRs</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><p>Tension recovery can start without ATP. <bold>a</bold> Time series boxplots and median (with median absolute deviation (MAD) as error bar, lower panel) for different parameters for Control and ATP-depleted cells on de-adhesion using an FBR size of 4.67 μm<sup>2</sup>. N<sub>cells(control)</sub>:6 N<sub>cells(ATP Depleted)</sub>: 9. <bold>b</bold> Probability distribution of FBR-wise log temporal fluctuations and tension of Control and ATP depleted same HeLa cell followed through different phases of De-adhesion. Shaded regions denote SEM. <bold>c</bold> Zoomed-in TIRF images of different cells (transfected with EGFP-Rab5) at different stages of de-adhesion in ATP-depleted condition. <bold>d</bold> Normalized area fraction of Rab5 in ATP-depleted condition. Shaded regions denote SEM. N<sub>cells</sub>: C = 26, P2 = 41, P3 = 39</p></caption></fig>\",\n \"<fig id=\\\"Fig6\\\"><label>Fig. 6</label><caption><p>Passive regulation is cholesterol-dependent. <bold>a</bold> Representative tension maps of cholesterol-depleted (Upper Panel) and ATP-depleted as well as cholesterol-depleted cells (Lower Panel) in three phases of de-adhesion<bold>.</bold> Scale bar = 10 μm. <bold>b</bold> Probability distribution of FBR-wise values of log temporal fluctuations and tension of cholesterol-depleted HeLa cell followed through different phases of de-adhesion. Shaded regions denote SEM. <bold>c</bold> Fold change in cell-averaged parameters comparing each cell with its own measurements at different phases. N<sub>cells</sub>: Control = 11, ATP dep = 9, Chol dep = 16, ATP dep + Chol dep = 15. <bold>d</bold> Probability distribution of FBR-wise values of log temporal fluctuations and tension of ATP depleted as well cholesterol-depleted same HeLa cells followed through different phases of de-adhesion. Shaded regions denote SEM. <bold>e</bold> Plot of rate of fractional change of tension when cells transit between different phases (mentioned). One-way Anova with Bonferroni correction is performed for SD<sub>time</sub> since the data are normal. For tension, the Mann–Whitney <italic>U</italic> test is performed and *denotes <italic>p</italic> value < 0.016 (adjusted by group size of 3 per experiment). <italic>n</italic> = 3 independent experiments. N<sub>cells</sub> mentioned in Table ##SUPPL##0##S1##</p></caption></fig>\",\n \"<fig id=\\\"Fig7\\\"><label>Fig. 7</label><caption><p>Membrane imaging reveals cholesterol-dependent tubules. <bold>a</bold> Representative zoomed in colour-coded confocal images of cells [ATP-depleted (left) and ATP-depleted as well as cholesterol-depleted (right)]. Scale bar = 2 μm. <bold>b</bold> Typical line scans are performed parallel to the membrane on the cytosolic side. Scale bar = 2 μm <bold>c</bold> Intensity profile of typical line scans of ATP-depleted and ATP Dep + Chol Dep condition with triangles pointing out detected peaks with minimal width and height. <bold>d</bold> Box plots (left) and line plot (centre) comparing number of peaks/μm in ATP-depleted and ATP + Cholesterol depleted through different time points of de-adhesion. Number of cells = 20, 18, 28 for 0, 3, 6 min, respectively (ATP-depleted), 21, 24, 21 for 0, 3, 6 min, respectively (ATP + cholesterol-depleted). Effect of de-adhesion on only cholesterol-depleted cells were evaluated using 97 and 113 ROIs from 8 and 9 cells. <bold>e</bold> Comparison of intensity detected per μm of various 4 μm lines drawn as explained in (<bold>b</bold>). <bold>f</bold> Evaluation of length of tubules using analysis of 68 and 58 ROIs from 18 and 15 cells of ATP-depleted and ATP + Cholesterol-depleted cells, respectively. **denote <italic>p</italic> value < 0.001 calculated using Mann–Whitney <italic>U</italic> test. Table ##SUPPL##0##S1## lists the number of peaks</p></caption></fig>\",\n \"<fig id=\\\"Fig8\\\"><label>Fig. 8</label><caption><p>Schematic diagram. <bold>a</bold> Membrane remodelling by active and passive regulation. Schematic shows that membrane fluctuations enhance in the P1 and P2 phase. However, while in passive condition (low ATP), cholesterol-dependent tubules reduce the membrane fluctuations, in normal conditions, active regulation entails formation of pits and their internalization in the P2 phase which transiently accumulate in early and recycling endosomal structures till the tension enhances back and fusion of recycling membrane keeps the increasing tension in check. <bold>b</bold> The decrease in tension by de-adhesion favours curving of clathrin-coated pits—moving AP2 to the edges. Pit formation increases tension. Dynamin-dependent scission and Rab 4-based recycling helps maintain homeostatic tension. Lack of AP2 decreases abrogates regulation by pit formation while lack of functional dynamin affects maintenance of tension and results in much higher tension. <bold>c</bold> Regulation of plasma membrane excess area by formation of pits, early and recycling endosomes. Schematic depicts that higher membrane excess area at the PM favours formation of pits as observed in this study. Such pits can be static or actively internalized to add to the early endosomal pool as shown in this study. Part of the early endosomal pool get converted to the recycling pool which is depleted when some structures fuse back with the PM. At higher membrane excess area, this fusion is disfavoured which can cause accumulation of the recycling endosome as shown in a and observed in this study</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Taba\"><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Resource availability reagent or resource</th><th align=\"left\">Source</th><th align=\"left\">Identifier</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"3\">Pharmacological reagents</td></tr><tr><td align=\"left\"> 2-Deoxy-D-glucose</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat# D8375; LOT# WXBB7357V</td></tr><tr><td align=\"left\"> Sodium azide</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\"><p>Cat# 438456; LOT#</p><p>MKBL3422V</p></td></tr><tr><td align=\"left\"> Potassium chloride</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat# P9541</td></tr><tr><td align=\"left\"> EDTA</td><td align=\"left\">SRL</td><td align=\"left\">Cat# 054448</td></tr><tr><td align=\"left\"> L-ascorbic acid</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat#A92902; LOT# BCBT6894</td></tr><tr><td align=\"left\"> L-ascorbic acid sodium salt extrapure</td><td align=\"left\">SRL</td><td align=\"left\">Cat# 65265</td></tr><tr><td align=\"left\"> Calcium chloride</td><td align=\"left\">Sigma</td><td align=\"left\">Cat#C5670; LOT# SLBJ2662V</td></tr><tr><td align=\"left\"> Dextrose anhydrous purified</td><td align=\"left\">Merck</td><td align=\"left\">Cat#61780905001730</td></tr><tr><td align=\"left\"> Sodium chloride</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat# S7653; LOT# 081M0051V</td></tr><tr><td align=\"left\"> Methyl-ß-cyclodextrin</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat# 332615; LOT# STBH0439</td></tr><tr><td align=\"left\"> Dynasore hydrate</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\"><p>Cat# D7693; LOT#</p><p>036M4609V</p></td></tr><tr><td align=\"left\"> ML-141</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat# SML0407</td></tr><tr><td align=\"left\"> Transferrin from human serum, alexa fluor 568 conjugate</td><td align=\"left\">Invitrogen</td><td align=\"left\"><p>Cat# T2365; LOT#</p><p>2136786</p></td></tr><tr><td align=\"left\"> Calcein AM</td><td align=\"left\">Invitrogen</td><td align=\"left\"><p>Cat# C3099; LOT#</p><p>2335625</p></td></tr><tr><td align=\"left\"> Cytochalasin D</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\"><p>Cat# C2618; LOT#</p><p>0000084039</p></td></tr><tr><td align=\"left\"><p> Magnesium chloride hexahydrate</p><p>Cryst. Purified</p></td><td align=\"left\">Merck</td><td align=\"left\">Cat# 442615; CAS# 7791-18-6;</td></tr><tr><td align=\"left\"> Dimethyl sulfoxide</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat# 276855; LOT# SHBC3339V;</td></tr><tr><td align=\"left\"> HEPES</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\"><p>Cat# H3375; LOT#</p><p>SLBN0452V</p></td></tr><tr><td align=\"left\"> Gelatin</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat# G2500; LOT# SLBX2973</td></tr><tr><td align=\"left\"> Glycine</td><td align=\"left\">Merck</td><td align=\"left\">Cat# 56-40-6</td></tr><tr><td align=\"left\"> TritonX-100</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat# 101371902</td></tr><tr><td align=\"left\"><p> Recombinant Anti-Rab4 antibody</p><p>[EPR3043]</p></td><td align=\"left\">Abcam</td><td align=\"left\"><p>Cat#ab109009</p><p>RRID# AB_10887396</p></td></tr><tr><td align=\"left\"> AP2A1 Rabbit mAb</td><td align=\"left\">Abclonal</td><td align=\"left\">Cat#A4403</td></tr><tr><td align=\"left\"> Goat Anti -Rabbit IgG, Alexa Fluor 568</td><td align=\"left\">Abcam</td><td align=\"left\">Cat#ab175471</td></tr><tr><td align=\"left\"> Goat Anti -Rabbit IgG H&L, Alexa Fluor 488</td><td align=\"left\">Abcam</td><td align=\"left\"><p>Cat#ab150077</p><p>RRID# AB_2630356</p></td></tr><tr><td align=\"left\"> Anti-Clathrin heavy chain antibody</td><td align=\"left\">Abcam</td><td align=\"left\">Cat#ab2731</td></tr><tr><td align=\"left\" colspan=\"3\">Cell culture</td></tr><tr><td align=\"left\"> Cell line (HeLa)</td><td align=\"left\">ATCC</td><td align=\"left\"><p>Cat# CCL-2;</p><p>RRID# CVCL_0030</p></td></tr><tr><td align=\"left\"> Dulbecco’s Modified Eagle’s Medium (DMEM, High Glucose)</td><td align=\"left\">Gibco</td><td align=\"left\">Cat# 11965092</td></tr><tr><td align=\"left\"> Fetal Bovine Serum, certified, heat-inactivated, US</td><td align=\"left\">Gibco</td><td align=\"left\">Cat# 10082147</td></tr><tr><td align=\"left\"> Antibiotic–Antimycotic (100X)</td><td align=\"left\">Gibco</td><td align=\"left\">Cat# 15240062</td></tr><tr><td align=\"left\"> TrypLE Express</td><td align=\"left\">Gibco</td><td align=\"left\"><p>Cat# 12605-028;</p><p>LOT# 2323753</p></td></tr><tr><td align=\"left\"> Trypsin–EDTA (0.05%), phenol red</td><td align=\"left\">Gibco</td><td align=\"left\"><p>Cat# 25300062; LOT#</p><p>2193180</p></td></tr><tr><td align=\"left\"> Trypsin–EDTA (0.25%), phenol red</td><td align=\"left\">Gibco</td><td align=\"left\">Cat# 25200072</td></tr><tr><td align=\"left\"> Paraformaldehyde</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat# P6148</td></tr><tr><td align=\"left\"> Phosphate Buffered Saline</td><td align=\"left\">Sigma-Aldrich</td><td align=\"left\">Cat# P3813</td></tr><tr><td align=\"left\"> Dulbecco’s Modified Eagle's Medium (DMEM, Phenol Red free)</td><td align=\"left\">Gibco</td><td align=\"left\"><p>Cat# 21063-029; LOT#</p><p>2239707</p></td></tr><tr><td align=\"left\"> Opti-MEM (Reduced serum medium)</td><td align=\"left\">Gibco</td><td align=\"left\"><p>Cat# 31985-070; LOT#</p><p>2192861</p></td></tr><tr><td align=\"left\"><p> Lipofectamine 3000</p><p>Transfection kit</p></td><td align=\"left\">Invitrogen</td><td align=\"left\"><p>Cat# L3000-015; LOT#</p><p>2145954</p></td></tr><tr><td align=\"left\" colspan=\"3\">Plasmids</td></tr><tr><td align=\"left\"> mCherry- Clathrin LC-15</td><td align=\"left\">Addgene</td><td align=\"left\">Plasmid# 55019</td></tr><tr><td align=\"left\"> EGFP-Rab5</td><td align=\"left\">Addgene</td><td align=\"left\">Plasmid# 49888</td></tr><tr><td align=\"left\"> mCherry-Rab4a</td><td align=\"left\">Addgene</td><td align=\"left\">Plasmid# 55125</td></tr><tr><td align=\"left\"> EGFP-CAAX</td><td align=\"left\">Addgene</td><td align=\"left\">Plasmid# 86056</td></tr><tr><td align=\"left\"> AP2 siRNA</td><td align=\"left\">IDT</td><td align=\"left\">Design id# hs.Ri.AP2A1.13.1</td></tr><tr><td align=\"left\" colspan=\"3\">For calibration</td></tr><tr><td align=\"left\"> NIST traceable particle size standard, 60 µm</td><td align=\"left\">Bangs laboratories</td><td align=\"left\">Cat# L130806L; LOT# 11247</td></tr><tr><td align=\"left\" colspan=\"3\">Software and algorithms</td></tr><tr><td align=\"left\"> ImageJ (FIJI)</td><td align=\"left\">NIH</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\"> Origin</td><td align=\"left\">OriginLab Corporation</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\"> MATLAB</td><td align=\"left\">The Mathworks, Inc</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\"> Labview</td><td align=\"left\">National Instruments</td><td align=\"left\"/></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Taba\\\"><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Resource availability reagent or resource</th><th align=\\\"left\\\">Source</th><th align=\\\"left\\\">Identifier</th></tr></thead><tbody><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Pharmacological reagents</td></tr><tr><td align=\\\"left\\\"> 2-Deoxy-D-glucose</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat# D8375; LOT# WXBB7357V</td></tr><tr><td align=\\\"left\\\"> Sodium azide</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\"><p>Cat# 438456; LOT#</p><p>MKBL3422V</p></td></tr><tr><td align=\\\"left\\\"> Potassium chloride</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat# P9541</td></tr><tr><td align=\\\"left\\\"> EDTA</td><td align=\\\"left\\\">SRL</td><td align=\\\"left\\\">Cat# 054448</td></tr><tr><td align=\\\"left\\\"> L-ascorbic acid</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat#A92902; LOT# BCBT6894</td></tr><tr><td align=\\\"left\\\"> L-ascorbic acid sodium salt extrapure</td><td align=\\\"left\\\">SRL</td><td align=\\\"left\\\">Cat# 65265</td></tr><tr><td align=\\\"left\\\"> Calcium chloride</td><td align=\\\"left\\\">Sigma</td><td align=\\\"left\\\">Cat#C5670; LOT# SLBJ2662V</td></tr><tr><td align=\\\"left\\\"> Dextrose anhydrous purified</td><td align=\\\"left\\\">Merck</td><td align=\\\"left\\\">Cat#61780905001730</td></tr><tr><td align=\\\"left\\\"> Sodium chloride</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat# S7653; LOT# 081M0051V</td></tr><tr><td align=\\\"left\\\"> Methyl-ß-cyclodextrin</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat# 332615; LOT# STBH0439</td></tr><tr><td align=\\\"left\\\"> Dynasore hydrate</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\"><p>Cat# D7693; LOT#</p><p>036M4609V</p></td></tr><tr><td align=\\\"left\\\"> ML-141</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat# SML0407</td></tr><tr><td align=\\\"left\\\"> Transferrin from human serum, alexa fluor 568 conjugate</td><td align=\\\"left\\\">Invitrogen</td><td align=\\\"left\\\"><p>Cat# T2365; LOT#</p><p>2136786</p></td></tr><tr><td align=\\\"left\\\"> Calcein AM</td><td align=\\\"left\\\">Invitrogen</td><td align=\\\"left\\\"><p>Cat# C3099; LOT#</p><p>2335625</p></td></tr><tr><td align=\\\"left\\\"> Cytochalasin D</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\"><p>Cat# C2618; LOT#</p><p>0000084039</p></td></tr><tr><td align=\\\"left\\\"><p> Magnesium chloride hexahydrate</p><p>Cryst. Purified</p></td><td align=\\\"left\\\">Merck</td><td align=\\\"left\\\">Cat# 442615; CAS# 7791-18-6;</td></tr><tr><td align=\\\"left\\\"> Dimethyl sulfoxide</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat# 276855; LOT# SHBC3339V;</td></tr><tr><td align=\\\"left\\\"> HEPES</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\"><p>Cat# H3375; LOT#</p><p>SLBN0452V</p></td></tr><tr><td align=\\\"left\\\"> Gelatin</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat# G2500; LOT# SLBX2973</td></tr><tr><td align=\\\"left\\\"> Glycine</td><td align=\\\"left\\\">Merck</td><td align=\\\"left\\\">Cat# 56-40-6</td></tr><tr><td align=\\\"left\\\"> TritonX-100</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat# 101371902</td></tr><tr><td align=\\\"left\\\"><p> Recombinant Anti-Rab4 antibody</p><p>[EPR3043]</p></td><td align=\\\"left\\\">Abcam</td><td align=\\\"left\\\"><p>Cat#ab109009</p><p>RRID# AB_10887396</p></td></tr><tr><td align=\\\"left\\\"> AP2A1 Rabbit mAb</td><td align=\\\"left\\\">Abclonal</td><td align=\\\"left\\\">Cat#A4403</td></tr><tr><td align=\\\"left\\\"> Goat Anti -Rabbit IgG, Alexa Fluor 568</td><td align=\\\"left\\\">Abcam</td><td align=\\\"left\\\">Cat#ab175471</td></tr><tr><td align=\\\"left\\\"> Goat Anti -Rabbit IgG H&L, Alexa Fluor 488</td><td align=\\\"left\\\">Abcam</td><td align=\\\"left\\\"><p>Cat#ab150077</p><p>RRID# AB_2630356</p></td></tr><tr><td align=\\\"left\\\"> Anti-Clathrin heavy chain antibody</td><td align=\\\"left\\\">Abcam</td><td align=\\\"left\\\">Cat#ab2731</td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Cell culture</td></tr><tr><td align=\\\"left\\\"> Cell line (HeLa)</td><td align=\\\"left\\\">ATCC</td><td align=\\\"left\\\"><p>Cat# CCL-2;</p><p>RRID# CVCL_0030</p></td></tr><tr><td align=\\\"left\\\"> Dulbecco’s Modified Eagle’s Medium (DMEM, High Glucose)</td><td align=\\\"left\\\">Gibco</td><td align=\\\"left\\\">Cat# 11965092</td></tr><tr><td align=\\\"left\\\"> Fetal Bovine Serum, certified, heat-inactivated, US</td><td align=\\\"left\\\">Gibco</td><td align=\\\"left\\\">Cat# 10082147</td></tr><tr><td align=\\\"left\\\"> Antibiotic–Antimycotic (100X)</td><td align=\\\"left\\\">Gibco</td><td align=\\\"left\\\">Cat# 15240062</td></tr><tr><td align=\\\"left\\\"> TrypLE Express</td><td align=\\\"left\\\">Gibco</td><td align=\\\"left\\\"><p>Cat# 12605-028;</p><p>LOT# 2323753</p></td></tr><tr><td align=\\\"left\\\"> Trypsin–EDTA (0.05%), phenol red</td><td align=\\\"left\\\">Gibco</td><td align=\\\"left\\\"><p>Cat# 25300062; LOT#</p><p>2193180</p></td></tr><tr><td align=\\\"left\\\"> Trypsin–EDTA (0.25%), phenol red</td><td align=\\\"left\\\">Gibco</td><td align=\\\"left\\\">Cat# 25200072</td></tr><tr><td align=\\\"left\\\"> Paraformaldehyde</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat# P6148</td></tr><tr><td align=\\\"left\\\"> Phosphate Buffered Saline</td><td align=\\\"left\\\">Sigma-Aldrich</td><td align=\\\"left\\\">Cat# P3813</td></tr><tr><td align=\\\"left\\\"> Dulbecco’s Modified Eagle's Medium (DMEM, Phenol Red free)</td><td align=\\\"left\\\">Gibco</td><td align=\\\"left\\\"><p>Cat# 21063-029; LOT#</p><p>2239707</p></td></tr><tr><td align=\\\"left\\\"> Opti-MEM (Reduced serum medium)</td><td align=\\\"left\\\">Gibco</td><td align=\\\"left\\\"><p>Cat# 31985-070; LOT#</p><p>2192861</p></td></tr><tr><td align=\\\"left\\\"><p> Lipofectamine 3000</p><p>Transfection kit</p></td><td align=\\\"left\\\">Invitrogen</td><td align=\\\"left\\\"><p>Cat# L3000-015; LOT#</p><p>2145954</p></td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Plasmids</td></tr><tr><td align=\\\"left\\\"> mCherry- Clathrin LC-15</td><td align=\\\"left\\\">Addgene</td><td align=\\\"left\\\">Plasmid# 55019</td></tr><tr><td align=\\\"left\\\"> EGFP-Rab5</td><td align=\\\"left\\\">Addgene</td><td align=\\\"left\\\">Plasmid# 49888</td></tr><tr><td align=\\\"left\\\"> mCherry-Rab4a</td><td align=\\\"left\\\">Addgene</td><td align=\\\"left\\\">Plasmid# 55125</td></tr><tr><td align=\\\"left\\\"> EGFP-CAAX</td><td align=\\\"left\\\">Addgene</td><td align=\\\"left\\\">Plasmid# 86056</td></tr><tr><td align=\\\"left\\\"> AP2 siRNA</td><td align=\\\"left\\\">IDT</td><td align=\\\"left\\\">Design id# hs.Ri.AP2A1.13.1</td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">For calibration</td></tr><tr><td align=\\\"left\\\"> NIST traceable particle size standard, 60 µm</td><td align=\\\"left\\\">Bangs laboratories</td><td align=\\\"left\\\">Cat# L130806L; LOT# 11247</td></tr><tr><td align=\\\"left\\\" colspan=\\\"3\\\">Software and algorithms</td></tr><tr><td align=\\\"left\\\"> ImageJ (FIJI)</td><td align=\\\"left\\\">NIH</td><td align=\\\"left\\\">N/A</td></tr><tr><td align=\\\"left\\\"> Origin</td><td align=\\\"left\\\">OriginLab Corporation</td><td align=\\\"left\\\">N/A</td></tr><tr><td align=\\\"left\\\"> MATLAB</td><td align=\\\"left\\\">The Mathworks, Inc</td><td align=\\\"left\\\">N/A</td></tr><tr><td align=\\\"left\\\"> Labview</td><td align=\\\"left\\\">National Instruments</td><td align=\\\"left\\\"/></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":["<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$k=\\frac{{k}_{B}T}{\\langle {x}^{2}\\rangle }$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mrow><mml:mi>k</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi>k</mml:mi><mml:mi>B</mml:mi></mml:msub><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mo 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See \"], \"ext-link\": [\"https://www.cmu.edu/biolphys/deserno/pdf/membrane_theory.pdf\"]}, {\"label\": [\"23.\"], \"surname\": [\"Helfrich\"], \"given-names\": [\"W\"], \"article-title\": [\"Elastic properties of lipid bilayers elastic properties of lipid bilayers: theory and possible experiments\"], \"source\": [\"Z Naturforsch\"], \"year\": [\"1973\"], \"volume\": [\"28\"], \"fpage\": [\"3\"], \"lpage\": [\"7\"]}, {\"label\": [\"38.\"], \"surname\": [\"Loerke\", \"Mettlen\", \"Yarar\", \"Jaqaman\", \"Jaqaman\", \"Danuser\", \"Schmid\"], \"given-names\": [\"D\", \"M\", \"D\", \"K\", \"H\", \"G\", \"SL\"], \"article-title\": [\"Cargo and dynamin regulate clathrin-coated pit maturation\"], \"source\": [\"PLoS Biol\"], \"year\": [\"2009\"], \"volume\": [\"7\"], \"fpage\": [\"0628\"], \"lpage\": [\"0639\"], \"pub-id\": [\"10.1371/journal.pbio.1000057\"]}, {\"label\": [\"43.\"], \"mixed-citation\": [\"Manikandan SK, Ghosh T, Mandal T, Biswas A, Sinha B, Mitra D (2022) Estimate of entropy generation rate can spatiotemporally resolve the active nature of cell flickering. doi: 10.48550/arxiv.2205.12849\"]}, {\"label\": [\"45.\"], \"surname\": [\"Mayor\", \"Parton\", \"Donaldson\"], \"given-names\": [\"S\", \"RG\", \"JG\"], \"article-title\": [\"Clathrin-independent pathways of endocytosis\"], \"source\": [\"Cold Spring Harb Perspect Biol\"], \"year\": [\"2014\"], \"volume\": [\"6\"], \"fpage\": [\"1\"], \"lpage\": [\"20\"], \"pub-id\": [\"10.1101/cshperspect.a016758\"]}, {\"label\": [\"51.\"], \"surname\": [\"Petersen\", \"Chung\", \"Nayebosadri\", \"Hansen\"], \"given-names\": [\"EN\", \"HW\", \"A\", \"SB\"], \"article-title\": [\"Kinetic disruption of lipid rafts is a mechanosensor for phospholipase D\"], \"source\": [\"Nat Commun\"], \"year\": [\"2016\"], \"volume\": [\"7\"], \"fpage\": [\"1\"], \"lpage\": [\"8\"], \"pub-id\": [\"10.1038/ncomms13873\"]}, {\"label\": [\"54.\"], \"surname\": [\"R\\u00e4dler\", \"Sackmann\"], \"given-names\": [\"JO\", \"E\"], \"article-title\": [\"Imaging optical thicknesses and separation distances of phospholipid vesicles at solid surfaces\"], \"source\": [\"J Phys II\"], \"year\": [\"1993\"], \"volume\": [\"3\"], \"fpage\": [\"727\"], \"lpage\": [\"748\"], \"pub-id\": [\"10.1051/jp2:1993163\"]}, {\"label\": [\"56.\"], \"surname\": [\"Reichel\", \"Kr\\u00e4ter\", \"Peikert\", \"Gla\\u00df\", \"Rosendahl\", \"Herbig\", \"Rivera Prieto\", \"Kihm\", \"Bosman\", \"Kaestner\", \"Hermann\", \"Guck\"], \"given-names\": [\"F\", \"M\", \"K\", \"H\", \"P\", \"M\", \"A\", \"A\", \"G\", \"L\", \"A\", \"J\"], \"article-title\": [\"Changes in blood cell deformability in chorea-acanthocytosis and effects of treatment with dasatinib or lithium\"], \"source\": [\"Front Physiol\"], \"year\": [\"2022\"], \"volume\": [\"13\"], \"fpage\": [\"556\"], \"pub-id\": [\"10.3389/FPHYS.2022.852946/BIBTEX\"]}, {\"label\": [\"64.\"], \"mixed-citation\": [\"Shan-Shan, L., J. Jopjin, and A.P. Liu. 2022. Exocytosis: From Molecules to Cells. A. Anantharam and J. Knight, editors. IOP Publishing. 6\\u20131\\u20136\\u201325 pp.\"]}, {\"label\": [\"68.\"], \"surname\": [\"Simunovic\", \"Voth\"], \"given-names\": [\"M\", \"GA\"], \"article-title\": [\"Membrane tension controls the assembly of curvature-generating proteins\"], \"source\": [\"Nat Commun\"], \"year\": [\"2015\"], \"volume\": [\"6\"], \"fpage\": [\"1\"], \"lpage\": [\"8\"], \"pub-id\": [\"10.1038/ncomms8219\"]}, {\"label\": [\"76.\"], \"surname\": [\"Turlier\", \"Betz\"], \"given-names\": [\"H\", \"T\"], \"article-title\": [\"Unveiling the active nature of living-membrane fluctuations and mechanics\"], \"source\": [\"Annu Rev Condens Matter Phys\"], \"year\": [\"2019\"], \"pub-id\": [\"10.1146/annurev-conmatphys-031218-013757\"]}, {\"label\": [\"80.\"], \"surname\": [\"Zuidema\", \"Wang\", \"Sonnenberg\"], \"given-names\": [\"A\", \"W\", \"A\"], \"article-title\": [\"Crosstalk between cell adhesion complexes in regulation of mechanotransduction\"], \"source\": [\"BioEssays\"], \"year\": [\"2020\"], \"volume\": [\"42\"], \"fpage\": [\"2000119\"], \"pub-id\": [\"10.1002/BIES.202000119\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":80,"string":"80"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Cell Mol Life Sci. 2024 Jan 13; 81(1):43"},"package_file":{"kind":"string","value":"oa_package/7f/66/PMC10787898.tar.gz"}}},{"rowIdx":182,"cells":{"accession_id":{"kind":"string","value":"PMC10787899"},"pmid":{"kind":"string","value":"38222211"},"introduction":{"kind":"list like","value":[],"string":"[]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<p>Tuberculosis is one of the oldest known infective diseases. It continues to pose a major threat to global health. In low- and middle-income countries, drug-resistant strains of tuberculosis have made the disease increasingly dangerous. In high-burden countries like India, people with tuberculosis may not always have a comprehensive examination for severe illness (requiring hospitalization management) due to oversaturated outpatient departments, inadequate diagnostic capacity, and a lack of manpower. Oftentimes, these patients are initiated on treatment, and detailed assessments for severe illnesses are missed. This is particularly important in a country with the greatest tuberculosis burden, where two tuberculosis-related deaths occur every three minutes. The present article throws light on this grave issue, emphasizing the need for early triage at the time of diagnosis, which would ultimately impact overall mortality and treatment outcomes.</p>"],"string":"[\n \"<p>Tuberculosis is one of the oldest known infective diseases. It continues to pose a major threat to global health. In low- and middle-income countries, drug-resistant strains of tuberculosis have made the disease increasingly dangerous. In high-burden countries like India, people with tuberculosis may not always have a comprehensive examination for severe illness (requiring hospitalization management) due to oversaturated outpatient departments, inadequate diagnostic capacity, and a lack of manpower. Oftentimes, these patients are initiated on treatment, and detailed assessments for severe illnesses are missed. This is particularly important in a country with the greatest tuberculosis burden, where two tuberculosis-related deaths occur every three minutes. The present article throws light on this grave issue, emphasizing the need for early triage at the time of diagnosis, which would ultimately impact overall mortality and treatment outcomes.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Editorial</title>","<p>Tuberculosis (TB) is still an immense threat to health in endemic nations. Drug-resistant strains of mycobacteria have made matters worse, mandating creative approaches for efficient treatment [##UREF##0##1##]. At the national level, according to the National TB Prevalence Survey (NATBPS) 2019-2021, the estimated point prevalence of microbiologically confirmed pulmonary tuberculosis among individuals over 15 years of age was 316 per lakh population. It was predicted that there were 312 cases of all forms of tuberculosis per lakh people. Overall, in the year 2022, out of the total TB cases notified, 14,71,190 (61%) were male, 9,48,190 (39%) were female, and 1,023 (<1%) belonged to lesbian, gay, bisexual, transgender, queer, intersex, asexual, and other identities [##UREF##1##2##].</p>","<p>As per the World Health Organization's (WHO) recently released Global TB Report 2023, India contributes 27% of the total cases of tuberculosis, with 22% mortality worldwide. India seeks to eliminate tuberculosis by the year 2025. The goal of the National Strategic Plan 2017-2025 was to achieve 44 new cases of tuberculosis per lakh of citizens by the end of 2025. This figure, according to the 2023 report, is 199 cases per lakh. Hitting this goal will prove difficult because, by 2023, the plan called for an incidence of only 77 cases per lakh population. By 2025, the program also seeks to lower mortality to three deaths per lakh of people. The WHO has acknowledged the updated numbers for India; yet, this still comes out to 23 per lakh people [##UREF##2##3##].</p>","<p>With the current data on significant mortality in tuberculosis, it becomes important to assess the determinants for the same. One of the factors was an improper assessment of the severity of the disease at the time of diagnosis. In a study by Shewade et al., out of 3,010 cases of tuberculosis, 1,529 (50.8%) were screened at the time of diagnosis or notification, of whom 537 (35.1%) had a high risk of severe illness. Further, when comparing individuals without a high risk of severe illness (3.8%) to those with a high risk of severe illness (8.9%) at diagnosis, the incidence of early fatalities was considerably greater. Furthermore, early mortality was highest in the first two weeks of the disease and was highly correlated with a high risk of severe illness upon diagnosis or notification [##UREF##3##4##].</p>","<p>The burden of severe disease at notification or diagnosis and the viability of gathering these data in standard program settings are topics that have received little attention in the literature. The National Tuberculosis Elimination Program guidelines for 2021 in India, unequivocally advise conducting a severity evaluation as soon as feasible upon diagnosis and referring patients for inpatient care if they are very sick. However, this guideline needs clinical, laboratory, and radiographic examinations, which might be difficult in peripheral health institutions [##UREF##3##4##]. To address the issues in severity evaluation in the National Tuberculosis Elimination Program guidelines for 2021, a 'Differentiated TB Care Model' was introduced, which was basically a simplified approach with criteria for hospitalization [##UREF##4##5##].</p>","<p>As recommended from 16 districts of Karnataka in the findings of the study by Shewade et al., an easy and quick method for early assessment of the severity of illness based on vital signs, body mass index (BMI), and the inability to stand without assistance are simple ways to screen for the severity of the illness. These indicators are well-known risk factors for death that are straightforward to evaluate and understand. Patients who exhibit any of these signs may be directed to more advanced facilities, like a tertiary care hospital, for inpatient care and an extensive clinical evaluation [##UREF##3##4##].</p>","<p>A similar initiative is planned for Delhi, involving all 25 chest clinics or district tuberculosis centers. Beginning January 1, 2024, all adults (more than or equal to 15 years of age) who will be diagnosed with tuberculosis, both drug-sensitive and drug-resistant, notified by public health institutes will be triaged. This activity will be started as a pilot project on December 15, 2023. This triage will be done by assessing five indicators (Table ##TAB##0##1##).</p>","<p>This triaging will be done as soon as possible at the diagnosing facility without even waiting for a formal notification of the patient or at the next earliest opportunity (at the home visit or start of the treatment or at the time of baseline investigations for tuberculosis). Two categories for severity of illness are defined based on the triage: either a triage positive or a triage negative. All the cases marked as triage positive will be registered in the notification registers at the health facility diagnosing the case and referred immediately to the designated tertiary care hospital in the national capital of India. Two hospitals, the National Institute of Tuberculosis and Respiratory Diseases and the Rajan Babu Institute of Pulmonary Medicine and Tuberculosis, are part of this initiative where these referrals will be made and a specific number of beds with trained staff designated to take care of these severe cases are planned. The five indicators mentioned in Table ##TAB##0##1## would help in detecting three conditions at the time of diagnosis (Table ##TAB##1##2##).</p>","<p>In India and other high-burden nations, there is a paucity of data on screening for severe illness on diagnosis or notification and the correlation between severe illness and early tuberculosis death in programmatic settings [##UREF##3##4##]. In a large study from Andhra Pradesh by Jonnalagada et al. on 8,240 tuberculosis patients, 50% of early deaths were reported within the first four weeks of treatment [##REF##22166132##6##]. This data rose to 75% in the study from Karnataka [##UREF##3##4##]. Besides, another study from the Indian state of Tamil Nadu (during April-June 2022) implemented a differentiated care strategy called Tamil Nadu-Kasanoi Erappila Thittam (TN-KET) for all adults aged 15 years and older with drug-susceptible tuberculosis notified by public health facilities. </p>","<p>Following notification of 14,961 TB patients, 11,599 (78%) underwent triage. Out of the people who were triaged, 1,509 (13%) had a high risk of developing a severe illness; of these, 1,128 (75%) underwent a thorough clinical evaluation in a nodal inpatient care health facility. In this study, 909 (92%) out of 993 confirmed as severely ill, were admitted, with 8% unfavorable admission outcomes, including 4% deaths [##UREF##5##7##].</p>","<p>To conclude, initial screening for severe sickness as an alternative strategy to reduce tuberculosis fatalities has existed since 2021, but a quicker and more targeted approach considering the issues at the grassroots level is warranted. This initiative in Delhi would be a remarkable step toward reducing mortality due to tuberculosis and achieving the desired aims of the National Strategic Plan 2017-2025. Additionally, this is the first such initiative where both drug-sensitive and drug-resistant tuberculosis cases will be triaged. </p>"],"string":"[\n \"<title>Editorial</title>\",\n \"<p>Tuberculosis (TB) is still an immense threat to health in endemic nations. Drug-resistant strains of mycobacteria have made matters worse, mandating creative approaches for efficient treatment [##UREF##0##1##]. At the national level, according to the National TB Prevalence Survey (NATBPS) 2019-2021, the estimated point prevalence of microbiologically confirmed pulmonary tuberculosis among individuals over 15 years of age was 316 per lakh population. It was predicted that there were 312 cases of all forms of tuberculosis per lakh people. Overall, in the year 2022, out of the total TB cases notified, 14,71,190 (61%) were male, 9,48,190 (39%) were female, and 1,023 (<1%) belonged to lesbian, gay, bisexual, transgender, queer, intersex, asexual, and other identities [##UREF##1##2##].</p>\",\n \"<p>As per the World Health Organization's (WHO) recently released Global TB Report 2023, India contributes 27% of the total cases of tuberculosis, with 22% mortality worldwide. India seeks to eliminate tuberculosis by the year 2025. The goal of the National Strategic Plan 2017-2025 was to achieve 44 new cases of tuberculosis per lakh of citizens by the end of 2025. This figure, according to the 2023 report, is 199 cases per lakh. Hitting this goal will prove difficult because, by 2023, the plan called for an incidence of only 77 cases per lakh population. By 2025, the program also seeks to lower mortality to three deaths per lakh of people. The WHO has acknowledged the updated numbers for India; yet, this still comes out to 23 per lakh people [##UREF##2##3##].</p>\",\n \"<p>With the current data on significant mortality in tuberculosis, it becomes important to assess the determinants for the same. One of the factors was an improper assessment of the severity of the disease at the time of diagnosis. In a study by Shewade et al., out of 3,010 cases of tuberculosis, 1,529 (50.8%) were screened at the time of diagnosis or notification, of whom 537 (35.1%) had a high risk of severe illness. Further, when comparing individuals without a high risk of severe illness (3.8%) to those with a high risk of severe illness (8.9%) at diagnosis, the incidence of early fatalities was considerably greater. Furthermore, early mortality was highest in the first two weeks of the disease and was highly correlated with a high risk of severe illness upon diagnosis or notification [##UREF##3##4##].</p>\",\n \"<p>The burden of severe disease at notification or diagnosis and the viability of gathering these data in standard program settings are topics that have received little attention in the literature. The National Tuberculosis Elimination Program guidelines for 2021 in India, unequivocally advise conducting a severity evaluation as soon as feasible upon diagnosis and referring patients for inpatient care if they are very sick. However, this guideline needs clinical, laboratory, and radiographic examinations, which might be difficult in peripheral health institutions [##UREF##3##4##]. To address the issues in severity evaluation in the National Tuberculosis Elimination Program guidelines for 2021, a 'Differentiated TB Care Model' was introduced, which was basically a simplified approach with criteria for hospitalization [##UREF##4##5##].</p>\",\n \"<p>As recommended from 16 districts of Karnataka in the findings of the study by Shewade et al., an easy and quick method for early assessment of the severity of illness based on vital signs, body mass index (BMI), and the inability to stand without assistance are simple ways to screen for the severity of the illness. These indicators are well-known risk factors for death that are straightforward to evaluate and understand. Patients who exhibit any of these signs may be directed to more advanced facilities, like a tertiary care hospital, for inpatient care and an extensive clinical evaluation [##UREF##3##4##].</p>\",\n \"<p>A similar initiative is planned for Delhi, involving all 25 chest clinics or district tuberculosis centers. Beginning January 1, 2024, all adults (more than or equal to 15 years of age) who will be diagnosed with tuberculosis, both drug-sensitive and drug-resistant, notified by public health institutes will be triaged. This activity will be started as a pilot project on December 15, 2023. This triage will be done by assessing five indicators (Table ##TAB##0##1##).</p>\",\n \"<p>This triaging will be done as soon as possible at the diagnosing facility without even waiting for a formal notification of the patient or at the next earliest opportunity (at the home visit or start of the treatment or at the time of baseline investigations for tuberculosis). Two categories for severity of illness are defined based on the triage: either a triage positive or a triage negative. All the cases marked as triage positive will be registered in the notification registers at the health facility diagnosing the case and referred immediately to the designated tertiary care hospital in the national capital of India. Two hospitals, the National Institute of Tuberculosis and Respiratory Diseases and the Rajan Babu Institute of Pulmonary Medicine and Tuberculosis, are part of this initiative where these referrals will be made and a specific number of beds with trained staff designated to take care of these severe cases are planned. The five indicators mentioned in Table ##TAB##0##1## would help in detecting three conditions at the time of diagnosis (Table ##TAB##1##2##).</p>\",\n \"<p>In India and other high-burden nations, there is a paucity of data on screening for severe illness on diagnosis or notification and the correlation between severe illness and early tuberculosis death in programmatic settings [##UREF##3##4##]. In a large study from Andhra Pradesh by Jonnalagada et al. on 8,240 tuberculosis patients, 50% of early deaths were reported within the first four weeks of treatment [##REF##22166132##6##]. This data rose to 75% in the study from Karnataka [##UREF##3##4##]. Besides, another study from the Indian state of Tamil Nadu (during April-June 2022) implemented a differentiated care strategy called Tamil Nadu-Kasanoi Erappila Thittam (TN-KET) for all adults aged 15 years and older with drug-susceptible tuberculosis notified by public health facilities. </p>\",\n \"<p>Following notification of 14,961 TB patients, 11,599 (78%) underwent triage. Out of the people who were triaged, 1,509 (13%) had a high risk of developing a severe illness; of these, 1,128 (75%) underwent a thorough clinical evaluation in a nodal inpatient care health facility. In this study, 909 (92%) out of 993 confirmed as severely ill, were admitted, with 8% unfavorable admission outcomes, including 4% deaths [##UREF##5##7##].</p>\",\n \"<p>To conclude, initial screening for severe sickness as an alternative strategy to reduce tuberculosis fatalities has existed since 2021, but a quicker and more targeted approach considering the issues at the grassroots level is warranted. This initiative in Delhi would be a remarkable step toward reducing mortality due to tuberculosis and achieving the desired aims of the National Strategic Plan 2017-2025. Additionally, this is the first such initiative where both drug-sensitive and drug-resistant tuberculosis cases will be triaged. </p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<p>Acknowledge the Tamil Nadu Kasanoi Erappila Thittam (TN-KET) team.</p>"],"string":"[\n \"<p>Acknowledge the Tamil Nadu Kasanoi Erappila Thittam (TN-KET) team.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":[],"string":"[]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Five indicators for assessment of the severity of illness.</title><p>BMI: Body mass index.</p><p>Reference [##UREF##3##4##].</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Indicator</td><td rowspan=\"1\" colspan=\"1\">Assessment</td></tr><tr><td rowspan=\"1\" colspan=\"1\">BMI (1)</td><td rowspan=\"1\" colspan=\"1\">Less than or equal to 14.0 kg/m<sup>2</sup>\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">BMI (2)</td><td rowspan=\"1\" colspan=\"1\">14.1 to 16.0 kg/m<sup>2 </sup>with swelling in the legs</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Respiratory rate (3)</td><td rowspan=\"1\" colspan=\"1\">More than 24/minute</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Oxygen saturation (4)</td><td rowspan=\"1\" colspan=\"1\">Less than 94% on room air</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ability to stand (5)</td><td rowspan=\"1\" colspan=\"1\">Without support, i.e., standing with support/squatting/sitting/bedridden</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Conditions were detected based on the presence of indicators.</title><p>Reference [##UREF##3##4##].</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Presence of indicator</td><td rowspan=\"1\" colspan=\"1\">Condition detected</td></tr><tr><td rowspan=\"1\" colspan=\"1\">1 or 2</td><td rowspan=\"1\" colspan=\"1\">Very severe undernutrition</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">3 or 4</td><td rowspan=\"1\" colspan=\"1\">Respiratory insufficiency</td></tr><tr><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">Poor performance status</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB1\\\"><label>Table 1</label><caption><title>Five indicators for assessment of the severity of illness.</title><p>BMI: Body mass index.</p><p>Reference [##UREF##3##4##].</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Indicator</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Assessment</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">BMI (1)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Less than or equal to 14.0 kg/m<sup>2</sup>\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">BMI (2)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14.1 to 16.0 kg/m<sup>2 </sup>with swelling in the legs</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Respiratory rate (3)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">More than 24/minute</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Oxygen saturation (4)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Less than 94% on room air</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Ability to stand (5)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Without support, i.e., standing with support/squatting/sitting/bedridden</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB2\\\"><label>Table 2</label><caption><title>Conditions were detected based on the presence of indicators.</title><p>Reference [##UREF##3##4##].</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Presence of indicator</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Condition detected</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1 or 2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Very severe undernutrition</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3 or 4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Respiratory insufficiency</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Poor performance status</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Sankalp Yadav</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Sankalp Yadav</p><p><bold>Drafting of the manuscript:</bold> Sankalp Yadav</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Sankalp Yadav</p><p><bold>Supervision:</bold> Sankalp Yadav</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Concept and design:</bold> Sankalp Yadav</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Sankalp Yadav</p><p><bold>Drafting of the manuscript:</bold> Sankalp Yadav</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Sankalp Yadav</p><p><bold>Supervision:</bold> Sankalp Yadav</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":[],"string":"[]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"1\"], \"article-title\": [\"India\\u2019s decision to deny an extension of patent for bedaquiline: a public health imperative\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Yadav\", \"Rawal\", \"Jeyaraman\"], \"given-names\": [\"S\", \"G\", \"M\"], \"fpage\": [\"0\"], \"volume\": [\"15\"], \"year\": [\"2023\"]}, {\"label\": [\"2\"], \"article-title\": [\"Leading the way: India TB report 2023\"], \"source\": [\"india tb report\"], \"date-in-citation\": [\"\\n\"], \"month\": [\"12\"], \"year\": [\"2023\", \"2023\"], \"uri\": [\"https://tbcindia.gov.in/WriteReadData/l892s/5646719104TB%20AR-2023_23-%2003-2023_LRP.pdf\"]}, {\"label\": [\"3\"], \"article-title\": [\"What WHO\\u2019s report has said on reduction in deaths due to TB in India, its treatments\"], \"date-in-citation\": [\"\\n\"], \"month\": [\"12\"], \"year\": [\"2024\", \"2023\"], \"uri\": [\"https://indianexpress.com/article/explained/explained-health/indias-tb-mortality-report-2023-explained-9023266/\"]}, {\"label\": [\"4\"], \"article-title\": [\"Screening for severe illness at diagnosis has the potential to prevent early TB deaths: programmatic experience from Karnataka, India\"], \"source\": [\"Glob Health: Sci Pract\"], \"person-group\": [\"\\n\"], \"surname\": [\"Shewade\", \"Nagaraja\", \"Vanitha\"], \"given-names\": [\"HD\", \"SB\", \"B\"], \"fpage\": [\"0\"], \"volume\": [\"10\"], \"year\": [\"2022\"]}, {\"label\": [\"5\"], \"article-title\": [\"Differentiated TB care model\"], \"date-in-citation\": [\"\\n\"], \"month\": [\"12\"], \"year\": [\"2023\", \"2023\"], \"uri\": [\"https://vikaspedia.in/health/health-campaigns/community-engagement-in-ending-tb/differentiated-tb-care-model\"]}, {\"label\": [\"7\"], \"article-title\": [\"The first differentiated TB care model from India: delays and predictors of losses in the care cascade\"], \"source\": [\"Glob Health Sci Pract\"], \"person-group\": [\"\\n\"], \"surname\": [\"Shewade\", \"Frederick\", \"Kiruthika\"], \"given-names\": [\"HD\", \"A\", \"G\"], \"fpage\": [\"0\"], \"volume\": [\"11\"], \"year\": [\"2023\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":7,"string":"7"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50550"},"package_file":{"kind":"string","value":"oa_package/4e/0e/PMC10787899.tar.gz"}}},{"rowIdx":183,"cells":{"accession_id":{"kind":"string","value":"PMC10787900"},"pmid":{"kind":"string","value":"38222222"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>A ureterocele is a cystic dilatation of the distal submucosal ureter, frequently located within the bladder [##REF##29043421##1##]. The development of tumors within the ureterocele is very rare; there are few reports in the literature, and the majority are of urothelial carcinomas [##REF##26913072##2##]. Urothelial cancer is developed countries' sixth most common cancer [##REF##33433946##3##]. Bladder cancer (BC) accounts for 90-95%, upper tract urothelial cancer (UTUC) accounts for 5-10% [##REF##33433946##3##], and urethral urothelial carcinoma is rare, less than 1% of all genitourinary malignancies [##REF##22033323##4##]. We report a case of a man with urothelial carcinoma of a ureterocele and review the literature, especially concerning diagnosis and treatment.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>A ureterocele is a cystic dilatation of the distal submucosal ureter, frequently located within the bladder [##REF##29043421##1##]. The development of tumors within the ureterocele is very rare; there are few reports in the literature, and the majority are of urothelial carcinomas [##REF##26913072##2##]. Urothelial cancer is developed countries' sixth most common cancer [##REF##33433946##3##]. Bladder cancer (BC) accounts for 90-95%, upper tract urothelial cancer (UTUC) accounts for 5-10% [##REF##33433946##3##], and urethral urothelial carcinoma is rare, less than 1% of all genitourinary malignancies [##REF##22033323##4##]. We report a case of a man with urothelial carcinoma of a ureterocele and review the literature, especially concerning diagnosis and treatment.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>A ureterocele is a congenital abnormality with an incidence as high as 1 in 500 in autopsy and a female preponderance four to six times greater than in men [##REF##12406111##5##]. It can appear in single or duplex systems associated with the upper pole system, as in the Weigert-Meyer rule [##REF##29043421##1##]. The insertion can be orthotopic in the bladder or ectopic, more frequently on the bladder neck or urethra, among others in the ectopic pathway [##REF##29043421##1##]. In adults, it is usually asymptomatic. However, when symptomatic, it is associated with upper urinary tract obstruction. Other symptoms are bladder neck obstruction, calculus formation, and recurrent infections [##REF##2188012##6##]. Their walls are made of two layers of urothelium (bladder and ureter) with muscle and collagen in the middle [##REF##12406111##5##].</p>","<p>Perego et al. first described a tumor on a ureterocele in 1974 [##UREF##0##7##]. Few cases have been reported since, and Astigueta et a. in 2016 published 10 cases [##REF##26913072##2##], and since then, only three more cases have been reported [##REF##28792188##8##, ####REF##31440452##9##, ##REF##36205289##10####36205289##10##]. We did a table summarizing the cases reported in the literature, including ours (Table ##TAB##0##1##).</p>","<p>The main clinical presentation is hematuria, followed by lower urinary tract symptoms (LUTS) and dysuria [##REF##26913072##2##]. Other symptoms, such as lower back and supra-pubic pain, were sporadic [##REF##26913072##2##].</p>","<p>Many imaging studies are used: intravenous urography (IVU), ultrasound (US), computed tomography (CT) urography, and MRI. The IVU is a historical exam with the typical alteration of the cobra-head sign in simple ureteroceles, with a distal ureter dilated in the bladder, surrounded by a thin and regular lucent line [##REF##36967359##18##]. If there is a thickening or irregularity, it may suggest a pseudoureterocele [##REF##26913072##2##]. A pseudoureterocele may be due to a tumor or edema from a stone in the ureterocele [##REF##26913072##2##]. The ultrasound detects ureteroceles with the typical image of a \"cyst within a cyst\" located in the posterior lateral wall of the bladder. A bladder tumor is an echogenic, fixed mass in the bladder wall without acoustic shadow. A tumor inside should be excluded if these alterations appear in a ureterocele [##REF##2188012##6##]. The CT urography detects the same alterations of the IVU with greater detail and can also show enhancement with contrast and exclude extravesical disease [##REF##26913072##2##]. The MRI is usually not used, but it should detect the same findings as a CT urography [##REF##28792188##8##].</p>","<p>Cystoscopy is used to confirm the diagnosis [##REF##26913072##2##, ##REF##36205289##10##]. However, in some reports, the cystoscopic appearance is similar to a simple ureterocele if the tumor is completely inside the ureterocele [##REF##28792188##8##, ####REF##31440452##9####31440452##9##], as in our case.</p>","<p>The management varies, and there are no guidelines. Most reports used the same guidelines as in BC [##REF##36205289##10##].</p>","<p>A TUR is useful for unroofing the ureterocele and removing the tumor for histological evaluation [##REF##26913072##2##, ##REF##12406111##5##, ####REF##2188012##6##, ##UREF##0##7##, ##REF##28792188##8####28792188##8##]. A more definitive and radical treatment was conducted in some reports. The most commonly used was a ureterocele resection and distal urethrectomy with ureteral reimplantation [##REF##26913072##2##, ##UREF##0##7##]. Other surgical options were RNU and even radical cystectomy [##REF##26913072##2##]. Non-surgical options were adjuvant treatment with bladder instillations with bacillus Calmette-Guérin (BCG) [##REF##31440452##9##, ##REF##36205289##10##], while other patients remained on surveillance only [##REF##26913072##2##, ##REF##28792188##8##]. The histological evaluation was essential in most of these decisions, in which the tumor was non-invasive in the majority. </p>","<p>In our report, since the tumor evolved into the distal ureter, we followed the guidelines for UTUC management with a single post-operative bladder instillation of MMC [##REF##12461261##19##]. The follow-up scheme adopted was similar to UTUC with cystoscopy and URS [##REF##12461261##19##], which is suggested by some authors [##REF##36205289##10##]. Although the recurrence was a low-grade lesion on biopsy, the previous tumor was classified as a high-risk UTUC, and therefore, RNU was proposed. The RNU identified a single 15 mm low-grade pTa urothelial tumor with a clear surgical margin.</p>","<p>There is no long-term follow-up data from the reports, so there are no recommendations concerning the best treatment option.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>A ureterocele is a congenital abnormality with an incidence as high as 1 in 500 in autopsy and a female preponderance four to six times greater than in men [##REF##12406111##5##]. It can appear in single or duplex systems associated with the upper pole system, as in the Weigert-Meyer rule [##REF##29043421##1##]. The insertion can be orthotopic in the bladder or ectopic, more frequently on the bladder neck or urethra, among others in the ectopic pathway [##REF##29043421##1##]. In adults, it is usually asymptomatic. However, when symptomatic, it is associated with upper urinary tract obstruction. Other symptoms are bladder neck obstruction, calculus formation, and recurrent infections [##REF##2188012##6##]. Their walls are made of two layers of urothelium (bladder and ureter) with muscle and collagen in the middle [##REF##12406111##5##].</p>\",\n \"<p>Perego et al. first described a tumor on a ureterocele in 1974 [##UREF##0##7##]. Few cases have been reported since, and Astigueta et a. in 2016 published 10 cases [##REF##26913072##2##], and since then, only three more cases have been reported [##REF##28792188##8##, ####REF##31440452##9##, ##REF##36205289##10####36205289##10##]. We did a table summarizing the cases reported in the literature, including ours (Table ##TAB##0##1##).</p>\",\n \"<p>The main clinical presentation is hematuria, followed by lower urinary tract symptoms (LUTS) and dysuria [##REF##26913072##2##]. Other symptoms, such as lower back and supra-pubic pain, were sporadic [##REF##26913072##2##].</p>\",\n \"<p>Many imaging studies are used: intravenous urography (IVU), ultrasound (US), computed tomography (CT) urography, and MRI. The IVU is a historical exam with the typical alteration of the cobra-head sign in simple ureteroceles, with a distal ureter dilated in the bladder, surrounded by a thin and regular lucent line [##REF##36967359##18##]. If there is a thickening or irregularity, it may suggest a pseudoureterocele [##REF##26913072##2##]. A pseudoureterocele may be due to a tumor or edema from a stone in the ureterocele [##REF##26913072##2##]. The ultrasound detects ureteroceles with the typical image of a \\\"cyst within a cyst\\\" located in the posterior lateral wall of the bladder. A bladder tumor is an echogenic, fixed mass in the bladder wall without acoustic shadow. A tumor inside should be excluded if these alterations appear in a ureterocele [##REF##2188012##6##]. The CT urography detects the same alterations of the IVU with greater detail and can also show enhancement with contrast and exclude extravesical disease [##REF##26913072##2##]. The MRI is usually not used, but it should detect the same findings as a CT urography [##REF##28792188##8##].</p>\",\n \"<p>Cystoscopy is used to confirm the diagnosis [##REF##26913072##2##, ##REF##36205289##10##]. However, in some reports, the cystoscopic appearance is similar to a simple ureterocele if the tumor is completely inside the ureterocele [##REF##28792188##8##, ####REF##31440452##9####31440452##9##], as in our case.</p>\",\n \"<p>The management varies, and there are no guidelines. Most reports used the same guidelines as in BC [##REF##36205289##10##].</p>\",\n \"<p>A TUR is useful for unroofing the ureterocele and removing the tumor for histological evaluation [##REF##26913072##2##, ##REF##12406111##5##, ####REF##2188012##6##, ##UREF##0##7##, ##REF##28792188##8####28792188##8##]. A more definitive and radical treatment was conducted in some reports. The most commonly used was a ureterocele resection and distal urethrectomy with ureteral reimplantation [##REF##26913072##2##, ##UREF##0##7##]. Other surgical options were RNU and even radical cystectomy [##REF##26913072##2##]. Non-surgical options were adjuvant treatment with bladder instillations with bacillus Calmette-Guérin (BCG) [##REF##31440452##9##, ##REF##36205289##10##], while other patients remained on surveillance only [##REF##26913072##2##, ##REF##28792188##8##]. The histological evaluation was essential in most of these decisions, in which the tumor was non-invasive in the majority. </p>\",\n \"<p>In our report, since the tumor evolved into the distal ureter, we followed the guidelines for UTUC management with a single post-operative bladder instillation of MMC [##REF##12461261##19##]. The follow-up scheme adopted was similar to UTUC with cystoscopy and URS [##REF##12461261##19##], which is suggested by some authors [##REF##36205289##10##]. Although the recurrence was a low-grade lesion on biopsy, the previous tumor was classified as a high-risk UTUC, and therefore, RNU was proposed. The RNU identified a single 15 mm low-grade pTa urothelial tumor with a clear surgical margin.</p>\",\n \"<p>There is no long-term follow-up data from the reports, so there are no recommendations concerning the best treatment option.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>Tumors in ureteroceles are very rare, and there are no guidelines for diagnosis, management, and follow-up. The diagnosis is challenging with the help of imaging studies and cystoscopy, although a normal exam may not exclude this diagnosis. Management varies according to histological results and imaging studies, from TUR and ureterocele resection to RNU. The follow-up data is unavailable; therefore, no evidence of the long-term outcomes is available in the literature.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>Tumors in ureteroceles are very rare, and there are no guidelines for diagnosis, management, and follow-up. The diagnosis is challenging with the help of imaging studies and cystoscopy, although a normal exam may not exclude this diagnosis. Management varies according to histological results and imaging studies, from TUR and ureterocele resection to RNU. The follow-up data is unavailable; therefore, no evidence of the long-term outcomes is available in the literature.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Urothelial carcinoma on a ureterocele is extremely rare in the literature, and few case reports have been reported. There are no guidelines for diagnosis and management, and current practice is extrapolated from bladder and upper urothelial tract carcinoma. We present a case from a 61-year-old man with urothelial carcinoma on a ureterocele treated with ureterocele resection, distal urethrectomy, and reimplantation on the bladder. We also review the literature concerning diagnostic approaches and management.</p>"],"string":"[\n \"<p>Urothelial carcinoma on a ureterocele is extremely rare in the literature, and few case reports have been reported. There are no guidelines for diagnosis and management, and current practice is extrapolated from bladder and upper urothelial tract carcinoma. We present a case from a 61-year-old man with urothelial carcinoma on a ureterocele treated with ureterocele resection, distal urethrectomy, and reimplantation on the bladder. We also review the literature concerning diagnostic approaches and management.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Case presentation</title>","<p>A 61-year-old caucasian man presented in the outpatient clinic with episodes of macroscopic hematuria for one year. The patient had no relevant past medical history and had no other complaints, and the physical examination was innocent. On blood analyses, there were no relevant alterations. </p>","<p>On an excretory CT, the right collecting system had incomplete duplicity with hydronephrosis of the upper pole collecting system and merged in a single meatus with a ureterocele. The ureterocele presented a thickening of the wall protruding inside, extending to the distal ureter with 30/6 mm with contrast enhancement, compatible with a tumor inside the ureterocele (Figure ##FIG##0##1##). No other lesions were found. </p>","<p>On cystoscopy, a single right meatus was found on a ureterocele; no papillary lesions came from that orifice, and clear urine was extracted during the exam; the left meatus and the bladder had no other alterations.</p>","<p>The patient underwent resection of the ureterocele with distal urethrectomy of both duplicated systems and common-sheath reimplantation on the bladder dome with the Psoas-Hitch technique (Figure ##FIG##1##2##). The pathology report identified a high-grade urothelial carcinoma pT1 with clear surgical margins (Figure ##FIG##2##3##). An intravesical instillation of mitomycin C (MMC) was made post-operatively on the eighth day, extrapolating data from the UTUC management. </p>","<p>A ureterorenoscopy (URS) was made at the three months of follow-up without any evidence of recurrence. At six months of follow-up, the patient was asymptomatic but presented on cystoscopy with bladder and ureteral recurrence (visible from the reimplanted ureter). The transurethral resection (TUR) of the bladder revealed a low-grade pTa urothelial bladder tumor, and a URS identified a ureteral tumor on the upper duplex system near the anastomosis, which was biopsied and identified as a low-grade ureteral tumor. The patient was submitted to a right radical nephroureterectomy (RNU) with the identification of a 15 mm low-grade pTa urothelial tumor with a clear surgical margin. An intravesical instillation of MMC was made post-operatively on the fifth day. The patient is on follow-up without any complaint or evidence of recurrence or progression.</p>"],"string":"[\n \"<title>Case presentation</title>\",\n \"<p>A 61-year-old caucasian man presented in the outpatient clinic with episodes of macroscopic hematuria for one year. The patient had no relevant past medical history and had no other complaints, and the physical examination was innocent. On blood analyses, there were no relevant alterations. </p>\",\n \"<p>On an excretory CT, the right collecting system had incomplete duplicity with hydronephrosis of the upper pole collecting system and merged in a single meatus with a ureterocele. The ureterocele presented a thickening of the wall protruding inside, extending to the distal ureter with 30/6 mm with contrast enhancement, compatible with a tumor inside the ureterocele (Figure ##FIG##0##1##). No other lesions were found. </p>\",\n \"<p>On cystoscopy, a single right meatus was found on a ureterocele; no papillary lesions came from that orifice, and clear urine was extracted during the exam; the left meatus and the bladder had no other alterations.</p>\",\n \"<p>The patient underwent resection of the ureterocele with distal urethrectomy of both duplicated systems and common-sheath reimplantation on the bladder dome with the Psoas-Hitch technique (Figure ##FIG##1##2##). The pathology report identified a high-grade urothelial carcinoma pT1 with clear surgical margins (Figure ##FIG##2##3##). An intravesical instillation of mitomycin C (MMC) was made post-operatively on the eighth day, extrapolating data from the UTUC management. </p>\",\n \"<p>A ureterorenoscopy (URS) was made at the three months of follow-up without any evidence of recurrence. At six months of follow-up, the patient was asymptomatic but presented on cystoscopy with bladder and ureteral recurrence (visible from the reimplanted ureter). The transurethral resection (TUR) of the bladder revealed a low-grade pTa urothelial bladder tumor, and a URS identified a ureteral tumor on the upper duplex system near the anastomosis, which was biopsied and identified as a low-grade ureteral tumor. The patient was submitted to a right radical nephroureterectomy (RNU) with the identification of a 15 mm low-grade pTa urothelial tumor with a clear surgical margin. An intravesical instillation of MMC was made post-operatively on the fifth day. The patient is on follow-up without any complaint or evidence of recurrence or progression.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Axial (A) and sagittal (B) contrast-enhanced CT on the excretory phase with a right parietal thickening of a ureterocele (yellow circle) with hydroureter associated (yellow arrows)</title></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Surgical specimen of resection of the ureterocele with distal urethrectomy of both duplicated systems</title></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG3\"><label>Figure 3</label><caption><title>Invasive conventional urothelial carcinoma displaying high-grade papillary morphology, characterized by disordered architecture, loss of polarity, nuclear pleomorphism with nuclear hyperchromasia, and a high nuclear-to-cytoplasm ratio. Mitotic figures are present. Notably, observe the lamina propria invasion (yellow arrow)</title><p>Hematoxylin and Eosin stain, 100x magnification</p></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>Axial (A) and sagittal (B) contrast-enhanced CT on the excretory phase with a right parietal thickening of a ureterocele (yellow circle) with hydroureter associated (yellow arrows)</title></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG2\\\"><label>Figure 2</label><caption><title>Surgical specimen of resection of the ureterocele with distal urethrectomy of both duplicated systems</title></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG3\\\"><label>Figure 3</label><caption><title>Invasive conventional urothelial carcinoma displaying high-grade papillary morphology, characterized by disordered architecture, loss of polarity, nuclear pleomorphism with nuclear hyperchromasia, and a high nuclear-to-cytoplasm ratio. Mitotic figures are present. Notably, observe the lamina propria invasion (yellow arrow)</title><p>Hematoxylin and Eosin stain, 100x magnification</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>List of reports of urothelial carcinomas on ureteroceles</title><p>CT - computed tomography, DUR - distal ureter resection, F - female, IVU/P - intravenous urography/pyelography, L - left, M - male, NMI - non-muscle invasive, MI - muscle invasive, R - right, RC - radical cystectomy, RNU - radical nephroureterectomy, SCBC - small cell bladder cancer, TUR - transurethral resection, UC - ureterocele, URR - ureterocele resection and reimplantation, US - ultrasound, LUTS - lower urinary tract symptoms</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Author (year)</td><td rowspan=\"1\" colspan=\"1\">Age</td><td rowspan=\"1\" colspan=\"1\">Sex</td><td rowspan=\"1\" colspan=\"1\">Initial symptoms</td><td rowspan=\"1\" colspan=\"1\">IVU/P</td><td rowspan=\"1\" colspan=\"1\">Bladder US</td><td rowspan=\"1\" colspan=\"1\">CT</td><td rowspan=\"1\" colspan=\"1\">Cystoscopy</td><td rowspan=\"1\" colspan=\"1\">Side</td><td rowspan=\"1\" colspan=\"1\">Inner/Outer mucosa</td><td rowspan=\"1\" colspan=\"1\">TUR </td><td rowspan=\"1\" colspan=\"1\">Treatment</td><td rowspan=\"1\" colspan=\"1\">Pathology</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Perego et al. (1974) [##UREF##0##7##]</td><td rowspan=\"1\" colspan=\"1\">68</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">Hematuria, dysuria</td><td rowspan=\"1\" colspan=\"1\">Simple UC - non-suspicious</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">Inner</td><td rowspan=\"1\" colspan=\"1\">No</td><td rowspan=\"1\" colspan=\"1\">URR</td><td rowspan=\"1\" colspan=\"1\">NMI</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Heyman et al. (1984) [##REF##6464253##11##]</td><td rowspan=\"1\" colspan=\"1\">57</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">Hematuria </td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">Solid mass in the UC</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">Simple UC</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">Inner</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">RNU</td><td rowspan=\"1\" colspan=\"1\">NMI</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Andrew et al. (1985) [##REF##3880929##12##]</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">Solid mass in the UC</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">Inner</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">URR</td><td rowspan=\"1\" colspan=\"1\">–</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Nakajima et al. (1986) [##REF##3565187##13##]</td><td rowspan=\"1\" colspan=\"1\">35</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">LUTS - dysuria</td><td rowspan=\"1\" colspan=\"1\">Simple UC - non-suspicious</td><td rowspan=\"1\" colspan=\"1\">Calculi in the UC</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">Tumor inside UC</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">Inner</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">URR</td><td rowspan=\"1\" colspan=\"1\">NMI</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Forer et al. (1990)[##REF##2188012##6##]</td><td rowspan=\"1\" colspan=\"1\">62</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">Hematuria</td><td rowspan=\"1\" colspan=\"1\">UC - suspicious </td><td rowspan=\"1\" colspan=\"1\">Complex cystic mass</td><td rowspan=\"1\" colspan=\"1\">UC - suspicious wall thickening</td><td rowspan=\"1\" colspan=\"1\">UC with tumor outside</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">Outer</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">URR</td><td rowspan=\"1\" colspan=\"1\">NMI</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Fukunaga et al. (1993) [##UREF##1##14##]</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">F</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ishida et al. (2002) [##UREF##2##15##]</td><td rowspan=\"1\" colspan=\"1\">45</td><td rowspan=\"1\" colspan=\"1\">F</td><td rowspan=\"1\" colspan=\"1\">Hematuria</td><td rowspan=\"1\" colspan=\"1\">Simple UC - non-suspicious</td><td rowspan=\"1\" colspan=\"1\">Simple UC</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">Simple UC</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">Surveillance</td><td rowspan=\"1\" colspan=\"1\">–</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Garcia et al. (2002) [##REF##12094491##16##]</td><td rowspan=\"1\" colspan=\"1\">74</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">Hematuria</td><td rowspan=\"1\" colspan=\"1\">UC - suspicious </td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">UC with solid content </td><td rowspan=\"1\" colspan=\"1\">Simple UC</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">Inner</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">RC + RNU + DUR</td><td rowspan=\"1\" colspan=\"1\">MI</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kadono et al. (2004) [##REF##15198005##17##]</td><td rowspan=\"1\" colspan=\"1\">62</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">Hematuria</td><td rowspan=\"1\" colspan=\"1\">Simple UC - non-suspicious</td><td rowspan=\"1\" colspan=\"1\">Suspicious of BC</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">UC with tumor outside</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">Outer</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">Surveillance</td><td rowspan=\"1\" colspan=\"1\">NMI</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Astigueta et al. (2015) [##REF##26913072##2##]</td><td rowspan=\"1\" colspan=\"1\">71</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">Hematuria, lumbar pain</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">Simple UC</td><td rowspan=\"1\" colspan=\"1\">UC with solid content </td><td rowspan=\"1\" colspan=\"1\">Tumor inside UC</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">Inner</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">Surveillance</td><td rowspan=\"1\" colspan=\"1\">NMI</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Law et al. (2017) [##REF##28792188##8##]</td><td rowspan=\"1\" colspan=\"1\">67</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">Hematuria</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">UC with solid content </td><td rowspan=\"1\" colspan=\"1\">Simple UC</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">Inner</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">Surveillance</td><td rowspan=\"1\" colspan=\"1\">NMI</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Burity et al. (2019)[##REF##31440452##9##]</td><td rowspan=\"1\" colspan=\"1\">68</td><td rowspan=\"1\" colspan=\"1\">F</td><td rowspan=\"1\" colspan=\"1\">Hematuria </td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">UC with solid content </td><td rowspan=\"1\" colspan=\"1\">Simple UC</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">Inner</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">BCG</td><td rowspan=\"1\" colspan=\"1\">SCBC</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Karakose et al. (2022) [##REF##36205289##10##] </td><td rowspan=\"1\" colspan=\"1\">65</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">Hematuria, suprapubic pain</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">UC - suspicious wall thickening</td><td rowspan=\"1\" colspan=\"1\">UC with tumor outside</td><td rowspan=\"1\" colspan=\"1\">L</td><td rowspan=\"1\" colspan=\"1\">Outer</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">BCG</td><td rowspan=\"1\" colspan=\"1\">NMI</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Pinheiro et al. (2023)</td><td rowspan=\"1\" colspan=\"1\">61</td><td rowspan=\"1\" colspan=\"1\">M</td><td rowspan=\"1\" colspan=\"1\">Hematuria</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">UC with solid content </td><td rowspan=\"1\" colspan=\"1\">Simple UC</td><td rowspan=\"1\" colspan=\"1\">R</td><td rowspan=\"1\" colspan=\"1\">Inner</td><td rowspan=\"1\" colspan=\"1\">No</td><td rowspan=\"1\" colspan=\"1\">URR</td><td rowspan=\"1\" colspan=\"1\">NMI</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB1\\\"><label>Table 1</label><caption><title>List of reports of urothelial carcinomas on ureteroceles</title><p>CT - computed tomography, DUR - distal ureter resection, F - female, IVU/P - intravenous urography/pyelography, L - left, M - male, NMI - non-muscle invasive, MI - muscle invasive, R - right, RC - radical cystectomy, RNU - radical nephroureterectomy, SCBC - small cell bladder cancer, TUR - transurethral resection, UC - ureterocele, URR - ureterocele resection and reimplantation, US - ultrasound, LUTS - lower urinary tract symptoms</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Author (year)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Age</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Sex</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Initial symptoms</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">IVU/P</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Bladder US</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">CT</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cystoscopy</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Side</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inner/Outer mucosa</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">TUR </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Treatment</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Pathology</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Perego et al. (1974) [##UREF##0##7##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">68</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria, dysuria</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC - non-suspicious</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inner</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">No</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">URR</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NMI</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Heyman et al. (1984) [##REF##6464253##11##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">57</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Solid mass in the UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inner</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">RNU</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NMI</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Andrew et al. (1985) [##REF##3880929##12##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Solid mass in the UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inner</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">URR</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Nakajima et al. (1986) [##REF##3565187##13##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">35</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">LUTS - dysuria</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC - non-suspicious</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Calculi in the UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Tumor inside UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inner</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">URR</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NMI</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Forer et al. (1990)[##REF##2188012##6##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">62</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC - suspicious </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Complex cystic mass</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC - suspicious wall thickening</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC with tumor outside</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Outer</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">URR</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NMI</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Fukunaga et al. (1993) [##UREF##1##14##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">F</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Ishida et al. (2002) [##UREF##2##15##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">45</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">F</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC - non-suspicious</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Surveillance</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Garcia et al. (2002) [##REF##12094491##16##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">74</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC - suspicious </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC with solid content </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inner</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">RC + RNU + DUR</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">MI</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kadono et al. (2004) [##REF##15198005##17##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">62</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC - non-suspicious</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Suspicious of BC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC with tumor outside</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Outer</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Surveillance</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NMI</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Astigueta et al. (2015) [##REF##26913072##2##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">71</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria, lumbar pain</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC with solid content </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Tumor inside UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inner</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Surveillance</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NMI</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Law et al. (2017) [##REF##28792188##8##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">67</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC with solid content </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inner</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Surveillance</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NMI</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Burity et al. (2019)[##REF##31440452##9##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">68</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">F</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC with solid content </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inner</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">BCG</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">SCBC</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Karakose et al. (2022) [##REF##36205289##10##] </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">65</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria, suprapubic pain</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC - suspicious wall thickening</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC with tumor outside</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">L</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Outer</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">BCG</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NMI</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Pinheiro et al. (2023)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">61</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">M</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hematuria</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">–</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UC with solid content </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Simple UC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">R</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inner</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">No</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">URR</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NMI</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Antonio M. Pinheiro, Pedro Bargão, Fernando Ribeiro, Filipa Galante Pereira, Ana Germano</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Antonio M. Pinheiro, Pedro Bargão, Filipa Galante Pereira, Ana Germano</p><p><bold>Drafting of the manuscript:</bold> Antonio M. Pinheiro, Filipa Galante Pereira</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Antonio M. Pinheiro, Pedro Bargão, Fernando Ribeiro, Filipa Galante Pereira, Ana Germano</p><p><bold>Supervision:</bold> Pedro Bargão, Fernando Ribeiro</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Concept and design:</bold> Antonio M. Pinheiro, Pedro Bargão, Fernando Ribeiro, Filipa Galante Pereira, Ana Germano</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Antonio M. Pinheiro, Pedro Bargão, Filipa Galante Pereira, Ana Germano</p><p><bold>Drafting of the manuscript:</bold> Antonio M. Pinheiro, Filipa Galante Pereira</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Antonio M. Pinheiro, Pedro Bargão, Fernando Ribeiro, Filipa Galante Pereira, Ana Germano</p><p><bold>Supervision:</bold> Pedro Bargão, Fernando Ribeiro</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0015-00000050549-i01\" position=\"float\"/>","<graphic xlink:href=\"cureus-0015-00000050549-i02\" position=\"float\"/>","<graphic xlink:href=\"cureus-0015-00000050549-i03\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0015-00000050549-i01\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0015-00000050549-i02\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0015-00000050549-i03\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"7\"], \"article-title\": [\"Carcinoma in ureterocele: caso clinico\"], \"source\": [\"Urologia\"], \"person-group\": [\"\\n\"], \"surname\": [\"Perego\", \"Marini\"], \"given-names\": [\"S\", \"F\"], \"fpage\": [\"117\"], \"lpage\": [\"119\"], \"volume\": [\"41\"], \"year\": [\"1974\"]}, {\"label\": [\"14\"], \"article-title\": [\"Transitional cell carcinoma in ureterocele associated with double ureter: a case report\"], \"source\": [\"Nishinihon J Urol\"], \"person-group\": [\"\\n\"], \"surname\": [\"Fukunaga\", \"Takahashi\", \"Kawano\", \"Nomura\"], \"given-names\": [\"Y\", \"S\", \"S\", \"Y\"], \"fpage\": [\"1513\"], \"lpage\": [\"1517\"], \"volume\": [\"55\"], \"year\": [\"1993\"], \"uri\": [\"https://www.researchgate.net/publication/298255994_Transitional_cell_carcinoma_in_ureterocele_associated_with_double_ureter_A_case_report\"]}, {\"label\": [\"15\"], \"article-title\": [\"Transitional cell carcinoma arising from a simple ureterocele: a case report\"], \"source\": [\"Nishinihon J Urol\"], \"person-group\": [\"\\n\"], \"surname\": [\"Ishida\", \"Tawada\", \"Muranaka\", \"Tanase\"], \"given-names\": [\"H\", \"M\", \"K\", \"K\"], \"fpage\": [\"667\"], \"lpage\": [\"669\"], \"volume\": [\"64\"], \"year\": [\"2002\"], \"uri\": [\"https://mol.medicalonline.jp/en/archive/search?jo=er9niuro&ye=2002&vo=64&issue=11\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":19,"string":"19"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50549"},"package_file":{"kind":"string","value":"oa_package/d9/20/PMC10787900.tar.gz"}}},{"rowIdx":184,"cells":{"accession_id":{"kind":"string","value":"PMC10787901"},"pmid":{"kind":"string","value":"38222195"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Iron accumulation in the brain, especially in the basal ganglia, frequently leads to neurodegeneration [##REF##26136767##1##]. Mutations in C19orf12 have recently been identified in individuals with a diagnosis of neurodegeneration with brain iron accumulation (NBIA) [##REF##23269600##2##]. The C19orf12 gene, which is localized in mitochondria and the endoplasmic reticulum, encodes a mitochondrial membrane protein. Mutations in this gene have been hypothesized to lead to the disruption of lipid homeostasis within the mitochondria, resulting in the onset of mitochondrial membrane protein-related neurodegeneration (MPAN) [##REF##26136767##1##, ####REF##23269600##2##, ##REF##33688131##3####33688131##3##]. Patients diagnosed with MPAN typically experience gait disturbance as a result of lower limb spasticity and dystonia [##REF##25819119##4##]. Other symptoms include parkinsonism, spastic paresis, dysarthria, cerebellar ataxia, behavioral problems, dementia, psychomotor delay, peripheral neuropathy, visual changes such as optic atrophy, and bowel/bladder incontinence [##REF##25819119##4##, ####REF##23278385##5##, ##REF##35188090##6####35188090##6##]. MPAN cases exhibit more variability than other NBIA cases, however, in terms of age of onset (between three and 30 years) and rate of progression [##REF##27671242##7##].</p>","<p>There is no rehabilitation protocol in the literature regarding exercise practices for MPAN syndrome. This study aims to reveal the effect of an 18-week therapeutic intervention on balance, coordination, and postural control in MPAN syndrome.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Iron accumulation in the brain, especially in the basal ganglia, frequently leads to neurodegeneration [##REF##26136767##1##]. Mutations in C19orf12 have recently been identified in individuals with a diagnosis of neurodegeneration with brain iron accumulation (NBIA) [##REF##23269600##2##]. The C19orf12 gene, which is localized in mitochondria and the endoplasmic reticulum, encodes a mitochondrial membrane protein. Mutations in this gene have been hypothesized to lead to the disruption of lipid homeostasis within the mitochondria, resulting in the onset of mitochondrial membrane protein-related neurodegeneration (MPAN) [##REF##26136767##1##, ####REF##23269600##2##, ##REF##33688131##3####33688131##3##]. Patients diagnosed with MPAN typically experience gait disturbance as a result of lower limb spasticity and dystonia [##REF##25819119##4##]. Other symptoms include parkinsonism, spastic paresis, dysarthria, cerebellar ataxia, behavioral problems, dementia, psychomotor delay, peripheral neuropathy, visual changes such as optic atrophy, and bowel/bladder incontinence [##REF##25819119##4##, ####REF##23278385##5##, ##REF##35188090##6####35188090##6##]. MPAN cases exhibit more variability than other NBIA cases, however, in terms of age of onset (between three and 30 years) and rate of progression [##REF##27671242##7##].</p>\",\n \"<p>There is no rehabilitation protocol in the literature regarding exercise practices for MPAN syndrome. This study aims to reveal the effect of an 18-week therapeutic intervention on balance, coordination, and postural control in MPAN syndrome.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>This is the first clinical case report to investigate the effects of a physiotherapy and rehabilitation program on body structure, function, and levels of activity and participation in a child with MPAN. The syndrome presents a variety of symptoms, ranging from gait disturbances to vision problems. Implementing a multidisciplinary approach to rehabilitation is therefore crucial in managing the syndrome.</p>","<p>Prevalent physical disorders in cases of MPAN include spasticity, ataxia, muscle weakness, and associated gait issues. In our two cases, there was a crouch gait present due to the shortening of the hamstrings and gastro-soleus [##REF##25819119##4##, ####REF##23278385##5##, ##REF##35188090##6####35188090##6##]. Initially, the disease appeared to have a spastic diplegic appearance. It was generally thought by pediatricians at the time that the children may have had cerebral palsy prior to the gene screening. However, based on the electroencephalogram reports and the bilateral involvement of the substantia nigra, red nucleus, and dentate nucleus in both siblings, as well as putamen involvement in the older sibling and globus pallidus in the younger sibling, the presence of brainstem involvement in our patients signifies the differentiation between MPAN disease and classical spastic diplegic cerebral palsy.</p>","<p>One of the important features of the disease is that it can occur in a wide age range (from three to 30 years), as reported in the literature [##REF##27671242##7##]. In our cases, diseases such as Duchenne muscular dystrophy, cerebral palsy, and hereditary spastic paraplegia were suspected until the older sibling reached the age of seven. However, the presence of the condition was confirmed through genetic screening.</p>","<p>The rehabilitation program was developed to address the main issues of the cases. Both participants were attending school and aimed for academic success. Moreover, the families’ expectation was for their children to lead as independent a life as possible. Particularly in terms of joining friends at school, problems arose due to balance and coordination difficulties. In developing the rehabilitation program, we took into account other pediatric neurological studies and the typical symptoms experienced by our patients [##UREF##1##16##, ####REF##2658915##17##, ##REF##25523410##18####25523410##18##].</p>","<p>The significance of maintaining trunk control for balance is crucial for both adults and children [##REF##31803048##19##]. Trunk control is an important milestone that supports all rehabilitation processes, particularly in children affected by neuromuscular disorders such as cerebral palsy [##UREF##2##20##]. Postural control in children with cerebral palsy worsens in comparison to their typically developing peers, potentially due to delayed and impaired development of neural motor control mechanisms and common secondary musculoskeletal abnormalities. After 18 weeks, we have made progress in parameters relating to balance and independence, a result of implementing a program focused on balance and coordination development. Although not evaluated in the study, the family reported a decrease in falls and improved running speed for both siblings following the treatment. We believe that this is closely linked to the use of balance exercises that involve trunk control.</p>","<p>The study had several limitations. The rehabilitation facility was used by patients and physiotherapists concurrently. This made it challenging to hold the attention of patients during specific exercises. Additionally, varying motivations among the patients represented a significant limitation. For instance, the older brother was resistant to implementing the rehabilitation program, despite having more severe issues. Taking breaks during exercise is vital for this reason. This study is a case report, and further testing of the methods is necessary in evidence-based, randomized studies involving multiple participants.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>This is the first clinical case report to investigate the effects of a physiotherapy and rehabilitation program on body structure, function, and levels of activity and participation in a child with MPAN. The syndrome presents a variety of symptoms, ranging from gait disturbances to vision problems. Implementing a multidisciplinary approach to rehabilitation is therefore crucial in managing the syndrome.</p>\",\n \"<p>Prevalent physical disorders in cases of MPAN include spasticity, ataxia, muscle weakness, and associated gait issues. In our two cases, there was a crouch gait present due to the shortening of the hamstrings and gastro-soleus [##REF##25819119##4##, ####REF##23278385##5##, ##REF##35188090##6####35188090##6##]. Initially, the disease appeared to have a spastic diplegic appearance. It was generally thought by pediatricians at the time that the children may have had cerebral palsy prior to the gene screening. However, based on the electroencephalogram reports and the bilateral involvement of the substantia nigra, red nucleus, and dentate nucleus in both siblings, as well as putamen involvement in the older sibling and globus pallidus in the younger sibling, the presence of brainstem involvement in our patients signifies the differentiation between MPAN disease and classical spastic diplegic cerebral palsy.</p>\",\n \"<p>One of the important features of the disease is that it can occur in a wide age range (from three to 30 years), as reported in the literature [##REF##27671242##7##]. In our cases, diseases such as Duchenne muscular dystrophy, cerebral palsy, and hereditary spastic paraplegia were suspected until the older sibling reached the age of seven. However, the presence of the condition was confirmed through genetic screening.</p>\",\n \"<p>The rehabilitation program was developed to address the main issues of the cases. Both participants were attending school and aimed for academic success. Moreover, the families’ expectation was for their children to lead as independent a life as possible. Particularly in terms of joining friends at school, problems arose due to balance and coordination difficulties. In developing the rehabilitation program, we took into account other pediatric neurological studies and the typical symptoms experienced by our patients [##UREF##1##16##, ####REF##2658915##17##, ##REF##25523410##18####25523410##18##].</p>\",\n \"<p>The significance of maintaining trunk control for balance is crucial for both adults and children [##REF##31803048##19##]. Trunk control is an important milestone that supports all rehabilitation processes, particularly in children affected by neuromuscular disorders such as cerebral palsy [##UREF##2##20##]. Postural control in children with cerebral palsy worsens in comparison to their typically developing peers, potentially due to delayed and impaired development of neural motor control mechanisms and common secondary musculoskeletal abnormalities. After 18 weeks, we have made progress in parameters relating to balance and independence, a result of implementing a program focused on balance and coordination development. Although not evaluated in the study, the family reported a decrease in falls and improved running speed for both siblings following the treatment. We believe that this is closely linked to the use of balance exercises that involve trunk control.</p>\",\n \"<p>The study had several limitations. The rehabilitation facility was used by patients and physiotherapists concurrently. This made it challenging to hold the attention of patients during specific exercises. Additionally, varying motivations among the patients represented a significant limitation. For instance, the older brother was resistant to implementing the rehabilitation program, despite having more severe issues. Taking breaks during exercise is vital for this reason. This study is a case report, and further testing of the methods is necessary in evidence-based, randomized studies involving multiple participants.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>The purpose of the investigation was to evaluate the impact of an 18-week rehabilitation program, comprising balance and coordination exercises, on two siblings diagnosed with neurodegeneration caused by iron accumulation in the brain. Comprehensive understanding of these conditions is imperative to determine the most appropriate therapy. Hence, balance and coordination-based exercise regimes have become a vital element of clinical intervention for patients affected by MPAN syndrome. It is crucial that rehabilitation programs are tailored according to the fundamental requirements of the patients or their families. The 18-week rehabilitation program for MPAN syndrome in this study has beneficial effects on muscle strength, balance parameters, and independence.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>The purpose of the investigation was to evaluate the impact of an 18-week rehabilitation program, comprising balance and coordination exercises, on two siblings diagnosed with neurodegeneration caused by iron accumulation in the brain. Comprehensive understanding of these conditions is imperative to determine the most appropriate therapy. Hence, balance and coordination-based exercise regimes have become a vital element of clinical intervention for patients affected by MPAN syndrome. It is crucial that rehabilitation programs are tailored according to the fundamental requirements of the patients or their families. The 18-week rehabilitation program for MPAN syndrome in this study has beneficial effects on muscle strength, balance parameters, and independence.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>This case report reports the effects of an 18-week physiotherapy program in children with mitochondrial membrane protein-associated neurodegeneration (MPAN). The study involved two brothers, aged 11 and 12, who had been diagnosed with MPAN. The physiotherapy program was divided into three phases and consisted of 18 weeks of training with a pediatric physiotherapist, including balance, coordination, and strengthening exercises. Muscle strength was assessed using pediatric manual muscle testing, functional balance using the Pediatric Berg Balance Test (PBBT), static balance using the Single-Leg Stance Test, dynamic balance using the Functional Reach Test, postural control using the 5-Time Sit-to-Stand Test, and independence using the Functional Independence Measure for Children (WeeFIM). Positive changes were observed in muscle strength, balance, and independence. After Phase I, PBBT scores (younger sibling +4, 8.1%; older +3, 6.8%) were higher than the minimal clinically important difference (MCID=3.66-5.83). After Phase III, although the PBBT scores improved (younger +2, 4.05%; older +1, 2.3%), the older sibling’s score was not higher than the MCID. Thus, the two children showed visible improvements in both body structure and function, as well as activity and participation levels.</p>"],"string":"[\n \"<p>This case report reports the effects of an 18-week physiotherapy program in children with mitochondrial membrane protein-associated neurodegeneration (MPAN). The study involved two brothers, aged 11 and 12, who had been diagnosed with MPAN. The physiotherapy program was divided into three phases and consisted of 18 weeks of training with a pediatric physiotherapist, including balance, coordination, and strengthening exercises. Muscle strength was assessed using pediatric manual muscle testing, functional balance using the Pediatric Berg Balance Test (PBBT), static balance using the Single-Leg Stance Test, dynamic balance using the Functional Reach Test, postural control using the 5-Time Sit-to-Stand Test, and independence using the Functional Independence Measure for Children (WeeFIM). Positive changes were observed in muscle strength, balance, and independence. After Phase I, PBBT scores (younger sibling +4, 8.1%; older +3, 6.8%) were higher than the minimal clinically important difference (MCID=3.66-5.83). After Phase III, although the PBBT scores improved (younger +2, 4.05%; older +1, 2.3%), the older sibling’s score was not higher than the MCID. Thus, the two children showed visible improvements in both body structure and function, as well as activity and participation levels.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Case presentation</title>","<p>Two brothers with MPAN, aged 11 and 12, who were born at term with birth weights of 2,000 g (36 weeks) and 3,500 g (40 weeks), respectively, presented to the clinic with a complaint of falling. Muscle weakness, loss of coordination and balance, and gait disturbance were observed during the initial assessment (Table ##TAB##0##1##).</p>","<p>The mother experienced her first pregnancy at the age of 25 (150 cm, 55 kg). Jaundice or trauma was not encountered in either child post-delivery. Apart from the premature birth of the younger sibling, no substantial birth difficulties were observed. The younger sibling did not demonstrate any motor developmental delays and acquired head control at three months and sitting at five months. The older sibling began walking at 14 months, whereas the younger sibling started walking at just nine months, without crawling first. At the age of five years, the older brother began experiencing falls, prompting his mother to seek medical attention. The younger brother underwent a check-up due to concerns raised by his teacher regarding vision and reading difficulties during his second year of primary school. When the older brother was seven years old, a physical therapist was consulted due to frequent falls and pes planus. Genetic screening was performed on the entire family, resulting in the detection of related gene damage in both siblings. Vitamin supplements were administered, and rehabilitation was recommended.</p>","<p>Assessments</p>","<p>Body Structures and Functions</p>","<p>Assessments were based on the International Classification of Functioning, Disability and Health Child and Youth Version (ICF-CY). Hip extensor and abdominal muscle strength was evaluated by manual muscle testing (MMT) and scored from 0 to 5. Standard positions and procedures were used [##UREF##0##8##]. In pediatric research, instrumental maximum voluntary contraction should be preferred over MMT scoring [##REF##29400673##9##]. Quantitative methods, however, have the disadvantage of requiring a specific tool rather than a clinician’s hands [##REF##11360262##10##].</p>","<p>Functional balance was evaluated with the Pediatric Berg Balance Test (PBBT). Reliability testing for PBBT, performed with a sample of 20 children aged five to 15 years with mild to moderate motor impairments, showed good test-retest reliability (intraclass correlation coefficient [ICC] = 0.998) and good interrater reliability (ICC = 0.997) [##REF##17057441##11##]. Validity testing for PBBT, performed with a sample of 30 children aged four to 10 years with spastic cerebral palsy in Gross Motor Function Classification System (GMFCS) Levels I-III, showed a strong correlation between the Pediatric Balance Scale and the self-care (r = 0.73, p < 0.001) and mobility (r = 0.82, p < 0.001) dimensions of the Pediatric Disability Evaluation Inventory [##REF##25013281##12##]. After completing the 12-week program encompassing phases I and II, postural control and static and dynamic balance assessments were included. We assessed static balance using the single-leg stance test and found a standard error of measurement (SEM) of 8.7 s and a minimum detectable change (MDC) of 24.1 s at a 95% confidence level. We also assessed dynamic balance using the Functional Reach Test and postural control using five repetitions of the Sit-to-Stand Test.</p>","<p>Activity and Participation</p>","<p>Participation in activities of daily living (ADL) was evaluated with the Functional Independence Measure for Children (WeeFIM). WeeFIM is easy and efficient to administer and is a valuable predictor of function in children with disabilities. WeeFIM includes 18 items with a 20-minute administration time. As WeeFIM is shorter and quicker to administer, it lends itself to assessment of functional outcome in pediatric rehabilitation. Higher scores represent more independence in participation [##REF##19207295##13##]. Test-retest for the six domains range from r = 0.83 to r = 0.99. Internal consistency (Cronbach’s alpha, or ICC) of the WeeFIM motor and cognitive scales was high (>0.90) and consistent for individual use. Interrater reliability was excellent, with ICC values of 0.98 and 0.93 for the motor and cognitive scales, respectively.</p>","<p>Therapeutic management</p>","<p>The initial phase of the rehabilitation program was structured according to the patients’ main problems. The primary concern was the impairment of balance during regular daily activities, followed by loss of muscle strength in the abdominals and hip extensors. It was identified that implementing exercises aimed at improving balance and coordination while walking was an important step forward [##REF##23653605##14##]. Following these objectives, the Phase I scheme was developed in accordance with Table ##TAB##1##2##.</p>","<p>Results of Phase I</p>","<p>Following the initial phase, which involved muscle strengthening and balance training, the children demonstrated improvement in several measures, including the PBBT, WeeFIM scores, and the strength of their abdominals and hip muscles. Notably, the increase in the PBBT score was significantly greater than the MCID reported in the literature for children with cerebral palsy [##REF##23291508##15##]. In the older brother, there was an increase in the ability to stand on one leg (PBBT score from 1 to 2), complete a 360-degree turn (from 3 to 4), and reach forward while standing (from 3 to 4). The younger brother showed an improvement in unsupported standing with one foot forward position (from 0 to 4). The observed 2-point increase in WeeFIM scores for both siblings was attributed to an increase in stair-climbing activity. For detailed findings, please refer to Table ##TAB##2##3##.</p>","<p>Phase II (six to 12 weeks/at home) initiative was based on the caregiver's ability and the suitability of home conditions. Exercises were prescribed for home-based practice, as stiffness of the muscles around the feet and ankles in the morning and balance and coordination irregularities were observed. Following clinical conditions and legal regulations, a six-week hiatus was taken, and the objective was to preserve the previous progress through home-based exercises during this period. The home exercise regimen consists of straight leg lifts, side leg lifts, wrist flexor and gastro-soleus muscle stretches, standing up and sitting down repeatedly, and 20 minutes of cycling on an ergometer and light jogging every day.</p>","<p>The younger sibling increased functional independence by 9 points, while the older sibling experienced an increase of 1 point before Phase III. Both siblings experienced a 2-point increase in balance; however, this improvement was below the MCID. As muscle strength was already gained to the desired extent during Phase I and there was no observed loss in relevant muscle strength before Phase III, strengthening exercises were not added to the rehabilitation program. Phase III was designed according to Table ##TAB##3##4##.</p>","<p>Results of Phase III</p>","<p>The assessment conducted after Phase III showed that progress had been made in all measures except for the older sibling’s ability to stand on one leg and complete the 5-Time Sit-to-Stand Test. Among these achievements, only the dynamic balance metric for the younger sibling exceeded the minimum clinical significance threshold (Table ##TAB##4##5##).</p>"],"string":"[\n \"<title>Case presentation</title>\",\n \"<p>Two brothers with MPAN, aged 11 and 12, who were born at term with birth weights of 2,000 g (36 weeks) and 3,500 g (40 weeks), respectively, presented to the clinic with a complaint of falling. Muscle weakness, loss of coordination and balance, and gait disturbance were observed during the initial assessment (Table ##TAB##0##1##).</p>\",\n \"<p>The mother experienced her first pregnancy at the age of 25 (150 cm, 55 kg). Jaundice or trauma was not encountered in either child post-delivery. Apart from the premature birth of the younger sibling, no substantial birth difficulties were observed. The younger sibling did not demonstrate any motor developmental delays and acquired head control at three months and sitting at five months. The older sibling began walking at 14 months, whereas the younger sibling started walking at just nine months, without crawling first. At the age of five years, the older brother began experiencing falls, prompting his mother to seek medical attention. The younger brother underwent a check-up due to concerns raised by his teacher regarding vision and reading difficulties during his second year of primary school. When the older brother was seven years old, a physical therapist was consulted due to frequent falls and pes planus. Genetic screening was performed on the entire family, resulting in the detection of related gene damage in both siblings. Vitamin supplements were administered, and rehabilitation was recommended.</p>\",\n \"<p>Assessments</p>\",\n \"<p>Body Structures and Functions</p>\",\n \"<p>Assessments were based on the International Classification of Functioning, Disability and Health Child and Youth Version (ICF-CY). Hip extensor and abdominal muscle strength was evaluated by manual muscle testing (MMT) and scored from 0 to 5. Standard positions and procedures were used [##UREF##0##8##]. In pediatric research, instrumental maximum voluntary contraction should be preferred over MMT scoring [##REF##29400673##9##]. Quantitative methods, however, have the disadvantage of requiring a specific tool rather than a clinician’s hands [##REF##11360262##10##].</p>\",\n \"<p>Functional balance was evaluated with the Pediatric Berg Balance Test (PBBT). Reliability testing for PBBT, performed with a sample of 20 children aged five to 15 years with mild to moderate motor impairments, showed good test-retest reliability (intraclass correlation coefficient [ICC] = 0.998) and good interrater reliability (ICC = 0.997) [##REF##17057441##11##]. Validity testing for PBBT, performed with a sample of 30 children aged four to 10 years with spastic cerebral palsy in Gross Motor Function Classification System (GMFCS) Levels I-III, showed a strong correlation between the Pediatric Balance Scale and the self-care (r = 0.73, p < 0.001) and mobility (r = 0.82, p < 0.001) dimensions of the Pediatric Disability Evaluation Inventory [##REF##25013281##12##]. After completing the 12-week program encompassing phases I and II, postural control and static and dynamic balance assessments were included. We assessed static balance using the single-leg stance test and found a standard error of measurement (SEM) of 8.7 s and a minimum detectable change (MDC) of 24.1 s at a 95% confidence level. We also assessed dynamic balance using the Functional Reach Test and postural control using five repetitions of the Sit-to-Stand Test.</p>\",\n \"<p>Activity and Participation</p>\",\n \"<p>Participation in activities of daily living (ADL) was evaluated with the Functional Independence Measure for Children (WeeFIM). WeeFIM is easy and efficient to administer and is a valuable predictor of function in children with disabilities. WeeFIM includes 18 items with a 20-minute administration time. As WeeFIM is shorter and quicker to administer, it lends itself to assessment of functional outcome in pediatric rehabilitation. Higher scores represent more independence in participation [##REF##19207295##13##]. Test-retest for the six domains range from r = 0.83 to r = 0.99. Internal consistency (Cronbach’s alpha, or ICC) of the WeeFIM motor and cognitive scales was high (>0.90) and consistent for individual use. Interrater reliability was excellent, with ICC values of 0.98 and 0.93 for the motor and cognitive scales, respectively.</p>\",\n \"<p>Therapeutic management</p>\",\n \"<p>The initial phase of the rehabilitation program was structured according to the patients’ main problems. The primary concern was the impairment of balance during regular daily activities, followed by loss of muscle strength in the abdominals and hip extensors. It was identified that implementing exercises aimed at improving balance and coordination while walking was an important step forward [##REF##23653605##14##]. Following these objectives, the Phase I scheme was developed in accordance with Table ##TAB##1##2##.</p>\",\n \"<p>Results of Phase I</p>\",\n \"<p>Following the initial phase, which involved muscle strengthening and balance training, the children demonstrated improvement in several measures, including the PBBT, WeeFIM scores, and the strength of their abdominals and hip muscles. Notably, the increase in the PBBT score was significantly greater than the MCID reported in the literature for children with cerebral palsy [##REF##23291508##15##]. In the older brother, there was an increase in the ability to stand on one leg (PBBT score from 1 to 2), complete a 360-degree turn (from 3 to 4), and reach forward while standing (from 3 to 4). The younger brother showed an improvement in unsupported standing with one foot forward position (from 0 to 4). The observed 2-point increase in WeeFIM scores for both siblings was attributed to an increase in stair-climbing activity. For detailed findings, please refer to Table ##TAB##2##3##.</p>\",\n \"<p>Phase II (six to 12 weeks/at home) initiative was based on the caregiver's ability and the suitability of home conditions. Exercises were prescribed for home-based practice, as stiffness of the muscles around the feet and ankles in the morning and balance and coordination irregularities were observed. Following clinical conditions and legal regulations, a six-week hiatus was taken, and the objective was to preserve the previous progress through home-based exercises during this period. The home exercise regimen consists of straight leg lifts, side leg lifts, wrist flexor and gastro-soleus muscle stretches, standing up and sitting down repeatedly, and 20 minutes of cycling on an ergometer and light jogging every day.</p>\",\n \"<p>The younger sibling increased functional independence by 9 points, while the older sibling experienced an increase of 1 point before Phase III. Both siblings experienced a 2-point increase in balance; however, this improvement was below the MCID. As muscle strength was already gained to the desired extent during Phase I and there was no observed loss in relevant muscle strength before Phase III, strengthening exercises were not added to the rehabilitation program. Phase III was designed according to Table ##TAB##3##4##.</p>\",\n \"<p>Results of Phase III</p>\",\n \"<p>The assessment conducted after Phase III showed that progress had been made in all measures except for the older sibling’s ability to stand on one leg and complete the 5-Time Sit-to-Stand Test. Among these achievements, only the dynamic balance metric for the younger sibling exceeded the minimum clinical significance threshold (Table ##TAB##4##5##).</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":[],"string":"[]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Timeline of events related to patient treatment</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Events</td><td rowspan=\"1\" colspan=\"1\">Date</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Recognizing toe walking</td><td rowspan=\"1\" colspan=\"1\">2017</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Rehabilitation in special clinics</td><td rowspan=\"1\" colspan=\"1\">2017–2018</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Genetic screening for the family</td><td rowspan=\"1\" colspan=\"1\">2020</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Rehabilitation in special clinics and hospital</td><td rowspan=\"1\" colspan=\"1\">2020–2022</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Applying for the hospital</td><td rowspan=\"1\" colspan=\"1\">11.20.2022</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Phase I</td><td rowspan=\"1\" colspan=\"1\">01.06.2023–02.21.2023</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Inpatient treatment</td><td rowspan=\"1\" colspan=\"1\">07.05.2023–08.20.2023</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Phase II home exercises program</td><td rowspan=\"1\" colspan=\"1\">09.20.2023–10.20.2023</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Phase III</td><td rowspan=\"1\" colspan=\"1\">10.23.2023–12.01.2023</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Phase I (0 to six weeks/in clinic) rehabilitation program</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Problem identified</td><td rowspan=\"1\" colspan=\"1\">Goals</td><td rowspan=\"1\" colspan=\"1\">Physiotherapy intervention</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Muscle weakness in abdominals and hip extensors</td><td rowspan=\"1\" colspan=\"1\">To increase muscle strength and prevent anterior pelvic tilt</td><td rowspan=\"1\" colspan=\"1\">Abdominal muscle-strengthening exercises were prescribed to both siblings to perform 10 times per day, 5 days a week. In the supine position, the patients were asked to lie on their knees with the knees flexed. The hip extensors were trained in the prone position on the bed by resisting hip extension.</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Loss of balance and coordination</td><td rowspan=\"1\" colspan=\"1\">To improve balance and coordination and therefore independence in walking</td><td rowspan=\"1\" colspan=\"1\">The following exercises were applied 5 times a week, with 10 repetition once a day: Clapping hands while marching, standing on one leg in front of the mirror, walking in tandem, rhythmic dynamic stabilization, swiss ball exercises (eyes open and closed)</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB3\"><label>Table 3</label><caption><title>Results of Phase I rehabilitation program</title><p>Y: Younger brother, O: Older brother, MCID: Minimal Clinical Important Difference, WeeFIM: Functional Independence Measure for Children</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Component</td><td rowspan=\"1\" colspan=\"1\">Measurement method</td><td rowspan=\"1\" colspan=\"1\">Parameters</td><td rowspan=\"1\" colspan=\"1\">Subject</td><td rowspan=\"1\" colspan=\"1\">Pre- rehabilitation of Phase I</td><td rowspan=\"1\" colspan=\"1\">Post-rehabilitation of Phase I</td><td rowspan=\"1\" colspan=\"1\">Change (%)</td><td rowspan=\"1\" colspan=\"1\">MCID</td></tr><tr><td rowspan=\"4\" colspan=\"1\">Muscle strength</td><td rowspan=\"4\" colspan=\"1\">Pediatric manual muscle testing</td><td rowspan=\"2\" colspan=\"1\">Abdominals</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">+2 (%66)</td><td rowspan=\"4\" colspan=\"1\">-</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">O</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">+1 (%25)</td></tr><tr><td rowspan=\"2\" colspan=\"1\">Hip extensors</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">+1 (%33)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">O</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">+2 (%66)</td></tr><tr><td rowspan=\"2\" colspan=\"1\">Functional balance</td><td rowspan=\"2\" colspan=\"1\">Pediatric Berg Balance Test</td><td rowspan=\"2\" colspan=\"1\">Total score</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">49</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">+4 (%8.1)</td><td rowspan=\"2\" colspan=\"1\">3.66-5.83</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">O</td><td rowspan=\"1\" colspan=\"1\">44</td><td rowspan=\"1\" colspan=\"1\">47</td><td rowspan=\"1\" colspan=\"1\">+3 (%6.8)</td></tr><tr><td rowspan=\"2\" colspan=\"1\">Independence</td><td rowspan=\"2\" colspan=\"1\">WeeFIM</td><td rowspan=\"2\" colspan=\"1\">Total score</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">108</td><td rowspan=\"1\" colspan=\"1\">110</td><td rowspan=\"1\" colspan=\"1\">+2 (%1.6)</td><td rowspan=\"2\" colspan=\"1\">-</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">O</td><td rowspan=\"1\" colspan=\"1\">119</td><td rowspan=\"1\" colspan=\"1\">121</td><td rowspan=\"1\" colspan=\"1\">+2 (%1.6)</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB4\"><label>Table 4</label><caption><title>Phase III (12–18 weeks/in clinic) rehabilitation program</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Problem identified</td><td rowspan=\"1\" colspan=\"1\">Goals</td><td rowspan=\"1\" colspan=\"1\">Physiotherapy intervention</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Persistent disturbances in balance and coordination</td><td rowspan=\"1\" colspan=\"1\">To increase walking independence by enhancing balance and coordination</td><td rowspan=\"1\" colspan=\"1\">The following exercises were performed 5 times a week, once a day, with the specified number of repetitions: Heel to toe raise (20 times), stand up and turn each side (5 times), single-leg stance with chair support initially (10 times), tandem walking (5 m), marching with opposite arm lifts (soldier walking)(5 mm), side lunges (10 times), weight bearing for both sides (10 times), giant step backward (10 times), crossing one leg in front of or behind the other in a continuous manner (5 m)</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB5\"><label>Table 5</label><caption><title>Results of Phase III rehabilitation program</title><p>Y: Younger brother, O: Older brother, MCID: Minimal Clinical Important Difference, WeeFIM: Functional Independence Measure for Children</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Component</td><td rowspan=\"1\" colspan=\"1\">Measurement method</td><td rowspan=\"1\" colspan=\"1\">Parameters</td><td rowspan=\"1\" colspan=\"1\">Subject</td><td rowspan=\"1\" colspan=\"1\">Pre- rehabilitation of Phase III</td><td rowspan=\"1\" colspan=\"1\">Post-rehabilitation of Phase III</td><td rowspan=\"1\" colspan=\"1\">Change</td><td rowspan=\"1\" colspan=\"1\">MCID</td></tr><tr><td rowspan=\"4\" colspan=\"1\">Static balance</td><td rowspan=\"4\" colspan=\"1\">Single-Leg Stance Test</td><td rowspan=\"2\" colspan=\"1\">Right</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">3.9 s</td><td rowspan=\"1\" colspan=\"1\">10 s</td><td rowspan=\"1\" colspan=\"1\">+6.1</td><td rowspan=\"4\" colspan=\"1\">24.1 s</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">O</td><td rowspan=\"1\" colspan=\"1\">1.5 s</td><td rowspan=\"1\" colspan=\"1\">2.45 s</td><td rowspan=\"1\" colspan=\"1\">+0.95</td></tr><tr><td rowspan=\"2\" colspan=\"1\">Left</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">2.18 s</td><td rowspan=\"1\" colspan=\"1\">7.5 s</td><td rowspan=\"1\" colspan=\"1\">+5.32</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">O</td><td rowspan=\"1\" colspan=\"1\">3.45 s</td><td rowspan=\"1\" colspan=\"1\">2.32 s</td><td rowspan=\"1\" colspan=\"1\">−1.33</td></tr><tr><td rowspan=\"2\" colspan=\"1\">Independence</td><td rowspan=\"2\" colspan=\"1\">WeeFIM</td><td rowspan=\"2\" colspan=\"1\">Total score</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">119</td><td rowspan=\"1\" colspan=\"1\">123</td><td rowspan=\"1\" colspan=\"1\">+4</td><td rowspan=\"2\" colspan=\"1\">-</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">O</td><td rowspan=\"1\" colspan=\"1\">122</td><td rowspan=\"1\" colspan=\"1\">125</td><td rowspan=\"1\" colspan=\"1\">+3</td></tr><tr><td rowspan=\"2\" colspan=\"1\">Functional balance</td><td rowspan=\"2\" colspan=\"1\">Pediatric Berg Balance Test</td><td rowspan=\"2\" colspan=\"1\">Total score</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">52</td><td rowspan=\"1\" colspan=\"1\">54</td><td rowspan=\"1\" colspan=\"1\">+2</td><td rowspan=\"2\" colspan=\"1\">3.66-5.83</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">O</td><td rowspan=\"1\" colspan=\"1\">49</td><td rowspan=\"1\" colspan=\"1\">50</td><td rowspan=\"1\" colspan=\"1\">+1</td></tr><tr><td rowspan=\"2\" colspan=\"1\">Dynamic balance</td><td rowspan=\"2\" colspan=\"1\">Functional Reach Test</td><td rowspan=\"2\" colspan=\"1\">Total score</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">18 cm</td><td rowspan=\"1\" colspan=\"1\">24 cm</td><td rowspan=\"1\" colspan=\"1\">+6</td><td rowspan=\"2\" colspan=\"1\">4-11 cm</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">O</td><td rowspan=\"1\" colspan=\"1\">18 cm</td><td rowspan=\"1\" colspan=\"1\">18 cm</td><td rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td rowspan=\"2\" colspan=\"1\">Postural control</td><td rowspan=\"2\" colspan=\"1\">5 Times Sit-to-Stand Test</td><td rowspan=\"2\" colspan=\"1\">Total score</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">8.01 s</td><td rowspan=\"1\" colspan=\"1\">8.91 s</td><td rowspan=\"1\" colspan=\"1\">+0.9</td><td rowspan=\"2\" colspan=\"1\">2.5 s</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">O</td><td rowspan=\"1\" colspan=\"1\">10.29 s</td><td rowspan=\"1\" colspan=\"1\">9.58 s</td><td rowspan=\"1\" colspan=\"1\">−0.71</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB1\\\"><label>Table 1</label><caption><title>Timeline of events related to patient treatment</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Events</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Date</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Recognizing toe walking</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2017</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Rehabilitation in special clinics</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2017–2018</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Genetic screening for the family</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2020</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Rehabilitation in special clinics and hospital</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2020–2022</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Applying for the hospital</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">11.20.2022</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Phase I</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">01.06.2023–02.21.2023</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Inpatient treatment</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">07.05.2023–08.20.2023</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Phase II home exercises program</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">09.20.2023–10.20.2023</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Phase III</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10.23.2023–12.01.2023</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB2\\\"><label>Table 2</label><caption><title>Phase I (0 to six weeks/in clinic) rehabilitation program</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Problem identified</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Goals</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Physiotherapy intervention</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Muscle weakness in abdominals and hip extensors</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">To increase muscle strength and prevent anterior pelvic tilt</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Abdominal muscle-strengthening exercises were prescribed to both siblings to perform 10 times per day, 5 days a week. In the supine position, the patients were asked to lie on their knees with the knees flexed. The hip extensors were trained in the prone position on the bed by resisting hip extension.</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Loss of balance and coordination</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">To improve balance and coordination and therefore independence in walking</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The following exercises were applied 5 times a week, with 10 repetition once a day: Clapping hands while marching, standing on one leg in front of the mirror, walking in tandem, rhythmic dynamic stabilization, swiss ball exercises (eyes open and closed)</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB3\\\"><label>Table 3</label><caption><title>Results of Phase I rehabilitation program</title><p>Y: Younger brother, O: Older brother, MCID: Minimal Clinical Important Difference, WeeFIM: Functional Independence Measure for Children</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Component</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Measurement method</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Parameters</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Subject</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Pre- rehabilitation of Phase I</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Post-rehabilitation of Phase I</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Change (%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">MCID</td></tr><tr><td rowspan=\\\"4\\\" colspan=\\\"1\\\">Muscle strength</td><td rowspan=\\\"4\\\" colspan=\\\"1\\\">Pediatric manual muscle testing</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Abdominals</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Y</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+2 (%66)</td><td rowspan=\\\"4\\\" colspan=\\\"1\\\">-</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">O</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+1 (%25)</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Hip extensors</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Y</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+1 (%33)</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">O</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+2 (%66)</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Functional balance</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Pediatric Berg Balance Test</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Total score</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Y</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">49</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">53</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+4 (%8.1)</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">3.66-5.83</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">O</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">44</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">47</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+3 (%6.8)</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Independence</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">WeeFIM</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Total score</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Y</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">108</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">110</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+2 (%1.6)</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">-</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">O</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">119</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">121</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+2 (%1.6)</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB4\\\"><label>Table 4</label><caption><title>Phase III (12–18 weeks/in clinic) rehabilitation program</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Problem identified</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Goals</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Physiotherapy intervention</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Persistent disturbances in balance and coordination</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">To increase walking independence by enhancing balance and coordination</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The following exercises were performed 5 times a week, once a day, with the specified number of repetitions: Heel to toe raise (20 times), stand up and turn each side (5 times), single-leg stance with chair support initially (10 times), tandem walking (5 m), marching with opposite arm lifts (soldier walking)(5 mm), side lunges (10 times), weight bearing for both sides (10 times), giant step backward (10 times), crossing one leg in front of or behind the other in a continuous manner (5 m)</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB5\\\"><label>Table 5</label><caption><title>Results of Phase III rehabilitation program</title><p>Y: Younger brother, O: Older brother, MCID: Minimal Clinical Important Difference, WeeFIM: Functional Independence Measure for Children</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Component</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Measurement method</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Parameters</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Subject</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Pre- rehabilitation of Phase III</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Post-rehabilitation of Phase III</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Change</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">MCID</td></tr><tr><td rowspan=\\\"4\\\" colspan=\\\"1\\\">Static balance</td><td rowspan=\\\"4\\\" colspan=\\\"1\\\">Single-Leg Stance Test</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Right</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Y</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.9 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+6.1</td><td rowspan=\\\"4\\\" colspan=\\\"1\\\">24.1 s</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">O</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.5 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.45 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+0.95</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Left</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Y</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.18 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.5 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+5.32</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">O</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.45 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.32 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">−1.33</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Independence</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">WeeFIM</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Total score</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Y</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">119</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">123</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+4</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">-</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">O</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">122</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">125</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+3</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Functional balance</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Pediatric Berg Balance Test</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Total score</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Y</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">52</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">54</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+2</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">3.66-5.83</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">O</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">49</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">50</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+1</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Dynamic balance</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Functional Reach Test</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Total score</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Y</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">18 cm</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">24 cm</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+6</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">4-11 cm</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">O</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">18 cm</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">18 cm</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Postural control</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">5 Times Sit-to-Stand Test</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Total score</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Y</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.01 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.91 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">+0.9</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">2.5 s</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">O</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10.29 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">9.58 s</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">−0.71</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Ömer Faruk Özçelep, Atahan Turhan</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Ömer Faruk Özçelep, Sibel Fi̇dan, Safiye Kandemir</p><p><bold>Drafting of the manuscript:</bold> Ömer Faruk Özçelep, Atahan Turhan, Sibel Fi̇dan, Safiye Kandemir</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Ömer Faruk Özçelep, Atahan Turhan, Sibel Fi̇dan, Safiye Kandemir</p><p><bold>Supervision:</bold> Ömer Faruk Özçelep, Atahan Turhan</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study. Ethics Committee of the Non-invasive Clinical Trials of Haliç University issued approval 2023-03/62. This study was approved by the Ethics Committee of the Non-invasive Clinical Trials of Haliç University (decision number: 2023-03/62) and registered in Clinical Trials under the number NCT05678790.</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Concept and design:</bold> Ömer Faruk Özçelep, Atahan Turhan</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Ömer Faruk Özçelep, Sibel Fi̇dan, Safiye Kandemir</p><p><bold>Drafting of the manuscript:</bold> Ömer Faruk Özçelep, Atahan Turhan, Sibel Fi̇dan, Safiye Kandemir</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Ömer Faruk Özçelep, Atahan Turhan, Sibel Fi̇dan, Safiye Kandemir</p><p><bold>Supervision:</bold> Ömer Faruk Özçelep, Atahan Turhan</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study. Ethics Committee of the Non-invasive Clinical Trials of Haliç University issued approval 2023-03/62. This study was approved by the Ethics Committee of the Non-invasive Clinical Trials of Haliç University (decision number: 2023-03/62) and registered in Clinical Trials under the number NCT05678790.</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":[],"string":"[]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"8\"], \"article-title\": [\"Daniels and Worthingham\\u2019s Muscle Testing-Techniques of Manual Examination and Performance Testing\"], \"person-group\": [\"\\n\"], \"surname\": [\"Brown\", \"Avers\"], \"given-names\": [\"M\", \"D\"], \"publisher-loc\": [\"Saunders\"], \"year\": [\"2018\"], \"uri\": [\"https://shop.elsevier.com/books/daniels-and-worthinghams-muscle-testing/brown/978-0-323-56914-9\"]}, {\"label\": [\"16\"], \"article-title\": [\"The effect of neuro-physiotherapy on gross motor function in a male child with spastic diplegic cerebral palsy: a case report\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Harjpal\", \"Raipure\", \"Kovela\", \"Qureshi\"], \"given-names\": [\"P\", \"A\", \"RK\", \"MI\"], \"fpage\": [\"0\"], \"volume\": [\"14\"], \"year\": [\"2022\"]}, {\"label\": [\"20\"], \"article-title\": [\"Effects of individually structured trunk training on body function and structures in children with spastic cerebral palsy: a stratified randomized controlled trial\"], \"source\": [\"Turkish J Physiother Rehabil\"], \"person-group\": [\"\\n\"], \"surname\": [\"Akba\\u015f\", \"Kerem G\\u00fcnel\"], \"given-names\": [\"AN\", \"M\"], \"fpage\": [\"11\"], \"lpage\": [\"22\"], \"volume\": [\"30\"], \"year\": [\"2019\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":20,"string":"20"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50540"},"package_file":{"kind":"string","value":"oa_package/c5/4c/PMC10787901.tar.gz"}}},{"rowIdx":185,"cells":{"accession_id":{"kind":"string","value":"PMC10787902"},"pmid":{"kind":"string","value":"38222127"},"introduction":{"kind":"list like","value":["<title>Introduction and background</title>","<p>Helicobacter pylori peptic ulcers may be treated with one of less than ten medications, including metronidazole, amoxicillin, clarithromycin, furazolidone, levofloxacin, and tetracycline. Antimicrobial resistance may be avoided and effective eradication is achieved by combining these antibiotics with bismuth salts and proton pump inhibitors (PPIs) [##REF##36079146##1##,##REF##30736338##2##]. The World Health Organisation has designated the study and development of novel medicines for H. pylori species that are resistant to clarithromycin as a \"high priority'' [##UREF##0##3##]. Primary antibiotic resistance is the problem caused by poor adherence to medication, which may result in treatment failure and subsequent resistance. Clarithromycin and metronidazole resistance is a worldwide crisis that must be addressed because chronic H. pylori infection is associated with gastric adenocarcinoma and localized B-cell lymphoma of the stomach (formerly known as gastric MALT lymphomas) [##REF##16404738##4##,##REF##28071659##5##,##REF##29990487##6##,##REF##16847081##7##]. H. pylori infections have been linked to numerous extragastric symptoms, such as ischemic heart disease and refractory anemia, according to recent studies [##UREF##1##8##,##REF##19483158##9##]. Despite a global drop in the percentage of H. pylori infections successfully treated, antibiotic resistance, particularly to highly effective broad-spectrum antibiotics, has been rapidly increasing [##REF##29990487##6##,##REF##26694080##10##,##REF##22017749##11##]. Tetracycline and amoxicillin have lower rates of resistance [##REF##29990487##6##]. The World Health Organisation published a study in 2018 on the worldwide spread of antibiotic resistance, and they discovered that resistance to levofloxacin, metronidazole, and clarithromycin had reached 15% worldwide. However, metronidazole and clarithromycin secondary resistance rates in certain areas were from 30 to 70%. Most often owing to initial clarithromycin resistance, eradication efforts with the most frequently used first-line regimens of clarithromycin, amoxicillin, and/or metronidazole failed more than 20-30% of the time [##REF##27707777##12##]. Despite the bismuth quadruple regimen (PPI, bismuth salts, and two antibiotics) being successful as a first- or second-line therapy, adverse effects have been recorded in 16-33% of instances, resulting in treatment cessation and failure [##REF##23376004##13##]. With high levels of resistance in H. pylori infections, both first and second-line treatment options have shown minimal elimination of the organism, making it a particularly challenging situation with limited alternative options. Primarily, combinations of antibiotics are being used for this purpose. Rifabutin, a rifamycin derivative, has emerged as an alternative to rifampicin in treating Mycobacterium tuberculosis. Its effect in the prophylaxis for Mycobacterium avium complex in immunocompromised patients is well known [##REF##33379336##14##,##REF##32365359##15##]. In recent years, novel regimens incorporating Rifabutin have been extensively studied for their efficacy in eliminating H. pylori, showing promising results even after multiple treatment failures [##REF##30736338##2##,##REF##32365359##15##,##REF##22129228##16##]. Furthermore, rifabutin resistance in vivo is rare, and the risk can be significantly reduced when Rifabutin is administered in combination with other antibiotics [##REF##32365359##15##]. Previous trials have used Rifabutin in various combinations, doses, and treatment durations. However, comprehensive reviews of multiple regimens still need to be included. This review aims to determine the most effective rifabutin-based regimen by considering its benefits and potential drawbacks for treating this infection.</p>"],"string":"[\n \"<title>Introduction and background</title>\",\n \"<p>Helicobacter pylori peptic ulcers may be treated with one of less than ten medications, including metronidazole, amoxicillin, clarithromycin, furazolidone, levofloxacin, and tetracycline. Antimicrobial resistance may be avoided and effective eradication is achieved by combining these antibiotics with bismuth salts and proton pump inhibitors (PPIs) [##REF##36079146##1##,##REF##30736338##2##]. The World Health Organisation has designated the study and development of novel medicines for H. pylori species that are resistant to clarithromycin as a \\\"high priority'' [##UREF##0##3##]. Primary antibiotic resistance is the problem caused by poor adherence to medication, which may result in treatment failure and subsequent resistance. Clarithromycin and metronidazole resistance is a worldwide crisis that must be addressed because chronic H. pylori infection is associated with gastric adenocarcinoma and localized B-cell lymphoma of the stomach (formerly known as gastric MALT lymphomas) [##REF##16404738##4##,##REF##28071659##5##,##REF##29990487##6##,##REF##16847081##7##]. H. pylori infections have been linked to numerous extragastric symptoms, such as ischemic heart disease and refractory anemia, according to recent studies [##UREF##1##8##,##REF##19483158##9##]. Despite a global drop in the percentage of H. pylori infections successfully treated, antibiotic resistance, particularly to highly effective broad-spectrum antibiotics, has been rapidly increasing [##REF##29990487##6##,##REF##26694080##10##,##REF##22017749##11##]. Tetracycline and amoxicillin have lower rates of resistance [##REF##29990487##6##]. The World Health Organisation published a study in 2018 on the worldwide spread of antibiotic resistance, and they discovered that resistance to levofloxacin, metronidazole, and clarithromycin had reached 15% worldwide. However, metronidazole and clarithromycin secondary resistance rates in certain areas were from 30 to 70%. Most often owing to initial clarithromycin resistance, eradication efforts with the most frequently used first-line regimens of clarithromycin, amoxicillin, and/or metronidazole failed more than 20-30% of the time [##REF##27707777##12##]. Despite the bismuth quadruple regimen (PPI, bismuth salts, and two antibiotics) being successful as a first- or second-line therapy, adverse effects have been recorded in 16-33% of instances, resulting in treatment cessation and failure [##REF##23376004##13##]. With high levels of resistance in H. pylori infections, both first and second-line treatment options have shown minimal elimination of the organism, making it a particularly challenging situation with limited alternative options. Primarily, combinations of antibiotics are being used for this purpose. Rifabutin, a rifamycin derivative, has emerged as an alternative to rifampicin in treating Mycobacterium tuberculosis. Its effect in the prophylaxis for Mycobacterium avium complex in immunocompromised patients is well known [##REF##33379336##14##,##REF##32365359##15##]. In recent years, novel regimens incorporating Rifabutin have been extensively studied for their efficacy in eliminating H. pylori, showing promising results even after multiple treatment failures [##REF##30736338##2##,##REF##32365359##15##,##REF##22129228##16##]. Furthermore, rifabutin resistance in vivo is rare, and the risk can be significantly reduced when Rifabutin is administered in combination with other antibiotics [##REF##32365359##15##]. Previous trials have used Rifabutin in various combinations, doses, and treatment durations. However, comprehensive reviews of multiple regimens still need to be included. This review aims to determine the most effective rifabutin-based regimen by considering its benefits and potential drawbacks for treating this infection.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>In conclusion, therapies that can successfully eliminate this infection in the first attempt must be considered the gold standard when prescribing medications. However, the rising global resistance to antibiotics like amoxicillin, metronidazole, clarithromycin, tetracycline, and levofloxacin raises concerns about their efficacy. Due to this, there is an increased need for developing efficient rescue regimens that can successfully eradicate this organism and prevent treatment failure. Our systematic review provides evidence showing that rifabutin regimens can be used to eliminate this infection successfully. Nearly all studies have shown that rifabutin-based regimens exhibited the lowest resistance rates compared to other drugs included in the therapy. It is noteworthy that rifabutin therapy has shown efficacy even in individuals with initial resistance to levofloxacin, clarithromycin, and metronidazole. Without relying on bacterial culture, it may be prudent to consider the empirical use of rifabutin as ''rescue therapy\" in circumstances where these antibiotics have failed to work. Therefore, rifabutin-containing regimens should only be used as a fourth or subsequent therapy if early eradication regimens using other first-line antibiotics such as metronidazole, amoxicillin, clarithromycin, tetracycline, and levofloxacin fail. The microbial resistance, possible adverse effects, and the availability and efficacy of alternative drugs should all be carefully considered before choosing rifabutin as a first-line therapy choice for H. pylori infection.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>In conclusion, therapies that can successfully eliminate this infection in the first attempt must be considered the gold standard when prescribing medications. However, the rising global resistance to antibiotics like amoxicillin, metronidazole, clarithromycin, tetracycline, and levofloxacin raises concerns about their efficacy. Due to this, there is an increased need for developing efficient rescue regimens that can successfully eradicate this organism and prevent treatment failure. Our systematic review provides evidence showing that rifabutin regimens can be used to eliminate this infection successfully. Nearly all studies have shown that rifabutin-based regimens exhibited the lowest resistance rates compared to other drugs included in the therapy. It is noteworthy that rifabutin therapy has shown efficacy even in individuals with initial resistance to levofloxacin, clarithromycin, and metronidazole. Without relying on bacterial culture, it may be prudent to consider the empirical use of rifabutin as ''rescue therapy\\\" in circumstances where these antibiotics have failed to work. Therefore, rifabutin-containing regimens should only be used as a fourth or subsequent therapy if early eradication regimens using other first-line antibiotics such as metronidazole, amoxicillin, clarithromycin, tetracycline, and levofloxacin fail. The microbial resistance, possible adverse effects, and the availability and efficacy of alternative drugs should all be carefully considered before choosing rifabutin as a first-line therapy choice for H. pylori infection.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Helicobacter pylori has been reported as a health problem worldwide, affecting a sizable portion of people. Peptic ulcers, gastric cancer, and various extra gastric conditions are associated with this bacterium. The rampant overprescribing of antibiotics has led to the emergence of H. pylori strains resistant to multiple antibiotics, causing a decline in the effectiveness of current treatments. Recently, there has been growing interest in researching alternative treatment options for H. pylori infections that do not respond to initial therapy. Rifabutin, a rifamycin derivative initially designed for tuberculosis treatment and preventing Mycobacterium avium complex infection, has gained attention as a potential rescue medication. It has shown efficacy against H. pylori and the potential to eradicate the bacterium when combined with other antibiotics. This systematic review article focuses on using rifabutin-based regimens as a treatment option after initial treatments have failed. The authors screened literature published in the last five years, between 2017 and 2022, across various search engines and closely examined relevant studies following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria. The search covered a variety of electronic databases and focused on H. pylori gastritis, rifabutin-based treatment plans, and in vivo investigations in healthy individuals. The comprehensive review provides convincing evidence that rifabutin-based regimens are effective rescue treatments for H. pylori infections. Multiple studies in various areas consistently demonstrated high eradication rates, ranging from 70% to 90%, when rifabutin-containing regimens were used. The analysis found that only a tiny percentage of H. pylori strains (1%) were resistant to rifabutin therapy, further supporting the viability of Rifabutin as an alternative when other antibiotics failed to eradicate H. pylori. The cost of Rifabutin is a significant factor that may limit its accessibility, particularly in resource-constrained settings where H. pylori infection is common. Moreover, the potential side effects of Rifabutin, such as hematological problems, rashes, and digestive issues, need to be considered. However, these side effects are typically manageable and can be reduced by combining Rifabutin with other antibiotics. In conclusion, this systematic review provides evidence supporting the effectiveness of regimens derived from Rifabutin in eliminating H. pylori infections after initial therapy failure. Due to the observation that Rifabutin effectively eradicates resistant H. pylori infections, it can be considered a suitable choice for rescue therapy. Rifabutin-containing regimens should be reserved as fourth- or later-line therapy options, considering economic factors, the risk of microbial resistance, potential side effects, and the availability of alternative medications. Future research should focus on optimizing rifabutin-based regimens and investigating combination therapies that have better H. pylori eradication rates while also addressing the problem of resistant strains.</p>"],"string":"[\n \"<p>Helicobacter pylori has been reported as a health problem worldwide, affecting a sizable portion of people. Peptic ulcers, gastric cancer, and various extra gastric conditions are associated with this bacterium. The rampant overprescribing of antibiotics has led to the emergence of H. pylori strains resistant to multiple antibiotics, causing a decline in the effectiveness of current treatments. Recently, there has been growing interest in researching alternative treatment options for H. pylori infections that do not respond to initial therapy. Rifabutin, a rifamycin derivative initially designed for tuberculosis treatment and preventing Mycobacterium avium complex infection, has gained attention as a potential rescue medication. It has shown efficacy against H. pylori and the potential to eradicate the bacterium when combined with other antibiotics. This systematic review article focuses on using rifabutin-based regimens as a treatment option after initial treatments have failed. The authors screened literature published in the last five years, between 2017 and 2022, across various search engines and closely examined relevant studies following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria. The search covered a variety of electronic databases and focused on H. pylori gastritis, rifabutin-based treatment plans, and in vivo investigations in healthy individuals. The comprehensive review provides convincing evidence that rifabutin-based regimens are effective rescue treatments for H. pylori infections. Multiple studies in various areas consistently demonstrated high eradication rates, ranging from 70% to 90%, when rifabutin-containing regimens were used. The analysis found that only a tiny percentage of H. pylori strains (1%) were resistant to rifabutin therapy, further supporting the viability of Rifabutin as an alternative when other antibiotics failed to eradicate H. pylori. The cost of Rifabutin is a significant factor that may limit its accessibility, particularly in resource-constrained settings where H. pylori infection is common. Moreover, the potential side effects of Rifabutin, such as hematological problems, rashes, and digestive issues, need to be considered. However, these side effects are typically manageable and can be reduced by combining Rifabutin with other antibiotics. In conclusion, this systematic review provides evidence supporting the effectiveness of regimens derived from Rifabutin in eliminating H. pylori infections after initial therapy failure. Due to the observation that Rifabutin effectively eradicates resistant H. pylori infections, it can be considered a suitable choice for rescue therapy. Rifabutin-containing regimens should be reserved as fourth- or later-line therapy options, considering economic factors, the risk of microbial resistance, potential side effects, and the availability of alternative medications. Future research should focus on optimizing rifabutin-based regimens and investigating combination therapies that have better H. pylori eradication rates while also addressing the problem of resistant strains.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Review</title>","<p>Methods</p>","<p>The systematic review was done in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A rigorous search strategy was developed to test this hypothesis and identify relevant literature. A comprehensive search of the various search engines and databases was done to include relevant literature.</p>","<p>A PubMed search was conducted using the Mesh terminology ''Rifabutin or N'-acetyl rifabutin or Mycobutin or ansatipine or ansamycin or LM 427, H pylori or helicobacter pylori, Therapy or therapeutic* or regimen* or protocol*, ''Rifabutin/administration and dosage''[Mesh] OR ''Rifabutin/adverse effects''[Mesh] OR ''Rifabutin/metabolism''[Mesh] OR ''Rifabutin/pharmacokinetics''[Mesh] OR ''Rifabutin/therapeutic use''[Mesh] OR ''Rifabutin/toxicity''[Mesh]. The MeSH methods are listed in Table ##TAB##0##1##.</p>","<p>Data extracted via the PubMed search strategy is shown in Table ##TAB##1##2##.</p>","<p>The authors wrote inclusion criteria, and articles were then chosen. This criterion was laid down based on various factors, including those related to H. pylori gastritis, papers discussing the research subject, publications published in English, and other specific criteria. These criteria included selecting papers with full-text articles, studies involving in vivo investigations, papers focusing on patients without comorbidities, publications published within the last five years (2016-2022), papers specifically addressing the treatment of Helicobacter gastritis, and studies examining the use of rifabutin for treating this infection, among others. We thus included randomized control trials and observational studies in this systematic review. Articles published in languages other than English, those not related to the research topic, those without full-text availability, and those based solely on in vitro studies were not included in the analysis. Patients with other gastrointestinal (GIT) diseases and comorbidities, MALTomas, and gastric adenocarcinomas were also not included. PRISMA chart is given in Figure ##FIG##0##1##.</p>","<p>The authors independently reviewed the research design and the possibility of bias in selection or publication. Any disagreements in the reviews of the writers were settled by consensus. A comprehensive quality assessment of the retrieved publications was conducted using the appropriate methodologies for each type of study. The results of critical appraisal using the NewCastle-Ottawa Quality Assessment Tool evaluation for case-control and cohort studies and the Cochrane Risk of Bias Tool for RCT are given in Table ##TAB##2##3##.</p>","<p>Result</p>","<p>Fifteen studies were finalized following multiple readings of the full text of the papers and discussions among co-authors. Study characteristics are given in Table ##TAB##3##4##.</p>","<p>Erick A. Argueta did research in the USA to see how new antibiotic resistance would affect efforts to eradicate H. pylori infection [##REF##33577874##17##]. In a study including 101 patients, the bismuth quadruple (BQ) regimen was used. This treatment plan includes the use of bismuth, tetracycline, metronidazole, and PPIs. Eighty-six patients were given the triple regimen, which included PPIs, clarithromycin, and either amoxicillin, metronidazole, or rifabutin. The eradication rate for Cohort 2 (treated with the triple regimen) was 60.5%, whereas the eradication rate for Cohort 1 (treated with the BQ regimen) was 88.1%. The research indicated that of the antibiotics tested, metronidazole had the greatest resistance rate (33.3%), followed by clarithromycin (30%), levofloxacin (29.6%), amoxicillin (1.0%), rifabutin (0.5%), and tetracycline (0.5%). Not only that but multidrug resistance (MDR) was found in 65.6% of bacterial isolates.</p>","<p>Youn I Choi conducted a study in Korea to identify potential candidates for a rescue regimen in cases of antibiotic-resistant H. pylori infections [##REF##31360270##18##]. Nine patients showed no elimination when just one antibiotic was used, while 22 were resistant to several. Interestingly, none of the subjects showed resistance to rifabutin or furazolidone. However, this gastric infection was completely eradicated in every patient in both cohorts, showing a 100% success rate. The study also listed the rates of resistance for several antibiotics, with clarithromycin having the highest rate at 71.1%, followed by metronidazole at 67.7%, levofloxacin at 41.9%, and amoxicillin at 22.6%. Rifabutin and furazolidone showed no signs of resistance.</p>","<p>In a Spanish study, susceptibility testing was used to identify treatment plans for 68 individuals with dual antibiotic resistance [##REF##28566898##19##]. OAL (omeprazole, amoxicillin, levofloxacin) was administered to 43 patients, OAC (omeprazole, amoxicillin, clarithromycin) to 12 patients, and OAM (omeprazole, amoxicillin, metronidazole) to 13 patients for those with dual-resistant infections. The OAR (omeprazole, amoxicillin, rifabutin) regimen was used to treat 12 patients with triple antibiotic resistance. The study reported high eradication rates for the various regimens in both cohorts. Notably, dual antibiotic-resistant patients had a resistance rate of less than 10% following medication, indicating effective eradication. However, the resistance rate in the triple antibiotic-resistant group receiving the rifabutin-based regimen was 41.7%, indicating a lower level of effectiveness in this cohort.</p>","<p>David Y. Graham looked into the Rifabutin-Based Triple Therapy (RHB-105) to see if it was beneficial in treating H. pylori infection [##REF##32365359##15##]. Two hundred and twenty-eight patients were given the RHB-105 regimen (amoxicillin, omeprazole, and Rifabutin) while 227 patients were given an active comparator regimen (amoxicillin and omeprazole). The study used an intention-to-treat (ITT) methodology to evaluate the eradication rates. The RHB-105 regimen had an eradication percentage of 83.8%, while the active comparator had a rate of 57.7%. According to these results, RHB-105 significantly outperformed the active comparator in eliminating H. pylori. Rifabutin showed a lower resistance rate than the active comparator, highlighting the potential benefit of utilizing it to treat infections with H. pylori.</p>","<p>The effects of the EAR treatment regimen (esomeprazole, amoxicillin, Rifabutin) were investigated by Kazumi Inokuchi using a historical control group of 12 patients [##REF##30736338##2##]. Vonoprazan, amoxicillin, and rifabutin (VAR) were used to treat 57 different individuals. The research assessed the efficacy of two strategies for treating this infection, contrasting the ''intention-to-treat'' (ITT) approach with the ''per protocol'' (PP) approach. The case group had a greater rate of H. pylori eradication after receiving the triple therapy based on low-dose rifabutin. The 10-day regimen had a resistance rate of 16.7%, whereas the seven-day regimen had a resistance rate of 8.8%, according to the study's analysis of rifabutin regimen resistance rates.</p>","<p>Triple rifabutin (RHB-105) was studied by Ira N. Klafus in the USA to determine its efficacy in treating this illness [##REF##33050205##21##]. Seventy-seven people were given the RHB-105 regimen (which contained amoxicillin, omeprazole, and rifabutin), and 41 people were given a placebo. In both the ITT and PP analyses, the RHB-105 regimen showed statistically significant eradication. The resistance rate to the RHB-105 regimen was 10.6%, which could affect the treatment's efficacy.</p>","<p>Chia Jung Kuo sought to understand better the clinical difficulties associated with multidrug-resistant infections. The trend of resistance being faced by the population was evaluated, with clarithromycin (92.7%) and levofloxacin (85.4%) having the highest resistance rates [##REF##33840604##22##]. Rifabutin showed a resistance rate of 29.3%. Notably, the study disclosed neither the eradication rates attained nor the treatment regimens utilized. Instead, it focused on describing the MDR patterns and H. pylori's antibiotic susceptibility in the research population.</p>","<p>A study by Chang Ming Lee had 323 participants divided into two cohorts [##UREF##3##23##]. The moxifloxacin-rifabutin triple therapy (MRT) treatment regimen was administered to 71 individuals who were unsuccessful with the first-line clarithromycin regimen (Cohort 1), while 252 patients with the same condition received the BQ treatment regimen (Cohort 2). The study found that eradication rates could differ depending on the treatment plan. MRT and BQ regimens showed effectiveness as second-line treatments for peptic ulcer cases unresponsive to the first-line clarithromycin regimen.</p>","<p>Miftahussurur conducted research to evaluate different H. pylori infection treatment plans in Indonesian areas where metronidazole and levofloxacin resistance are common [##REF##30774400##24##]. The study evaluated the susceptibility and resistance of five different antibiotics, furazolidone, sitafloxacin, garenoxacin, rifaximin, and rififabutin, to different strains of H. pylori bacteria. Among the participants, 61 out of 105 (58.1%) were responsive to each of the five antibiotics tested, indicating that these people may benefit from different eradication regimens that include these antibiotics. Notably, none of the organisms resisted sitafloxacin, furazolidone, or rifabutin. However, the study found various degrees of resistance to rifaximin and garenoxacin. Notably, 40 out of 105 subjects (38.9%) and seven out of 105 people (6.7%) were resistant to rifaximin and garenoxacin, respectively. This shows that these drugs are highly resistant in the Indonesian regions under study. The results do, however, indicate that adding furazolidone, rifabutin, and sitafloxacin to regimens can be successful in regions with great resistance to some antibiotics like levofloxacin and metronidazole.</p>","<p>Another study took place in the Southeast Asian countries of Bangladesh and Nepal [##REF##30815255##25##]. The study assessed the regions of Nepal where there was high antibiotic resistance. Garenoxacin resistance was seen with the tested antibiotics in 12 out of 42 people (28.6%), whereas sitafloxacin resistance was seen in two out of 42 participants (4.8%). Furazolidone and Rifabutin, however, did not show any signs of resistance. Another antibiotic, rifaximin, was tested, and 22 out of 42 people (52.4%) showed resistance. A similar evaluation was carried out in Bangladesh, demonstrating the antibiotic resistance patterns in these strains. Not more than one of the 56 subjects (1.8%) demonstrated resistance to sitafloxacin, compared to 29 out of 56 patients (51.8%) who showed resistance to garenoxacin. No rifabutin or furazolidone resistance was found, similar to Nepal. Resistance to rifaximin was seen in 36 out of 56 subjects (64.3%). Importantly, none of the tested strains in either nation showed rifabutin or furazolidone resistance. This shows that despite the significant resistance seen for other regularly used antibiotics such as garenoxacin, sitafloxacin, and rifaximin, these two antibiotics could be possible candidates for effective therapy regimens against H. pylori infection in Bangladesh and Nepal.</p>","<p>Miftahussurur conducted a study in the Dominican Republic to find substitute antibiotics to deal with increased resistance to levofloxacin and metronidazole in treating this infection [##UREF##4##26##]. The distribution of strains of this organism in the Dominican Republic that were resistant to several antibiotics was evaluated. Rifaximin resistance was present in 52 out of 62 persons (82.5%) among the antibiotics tested, while garenoxacin resistance was seen in 22 out of 63 participants (34.9%). There was no resistance to sitafloxacin or furazolidone among the individuals. Rifabutin showed no resistance in any of the 63 participants. The study also found that 18.6% of the 63 patients exhibited double antibiotic resistance to rifaximin and garenoxacin. The study's conclusions are essential as they demonstrate how rare antibiotic resistance to rifabutin, sitafloxacin, and furazolidone is in the Dominican Republic. Therefore, these three medicines could be used to treat this infection in this area.</p>","<p>To assess whether triple therapy based on rifabutin was efficient as a third- and fourth-line treatments for this infection, H. Mori conducted a study in Japan [##UREF##5##27##]. The 10-day and 14-day cohorts were each given a group of subjects. Twelve patients were administered esomeprazole, amoxicillin, and rifabutin four times daily in the 10-day group. The same drugs were given to 17 individuals in the 14-day group; however, rifabutin was only given once daily. In this study, we used ITT and PP analyses to evaluate the success rate at curing H. pylori infection as our major end measure. In the ITT analysis, the eradication rate for the 10-day group was 83.3%, while in the PP analysis, it was just 81.8%. Eighty percent of patients saw improvement after 10 days of treatment. ITT analysis showed a 94.1% eradication rate in the 14-day group, and PP analysis showed a 91.7% eradication rate. The 14-day treatment plan was successful in curing 90% of the illness. No one in the 10-day group showed resistance to rifabutin, whereas 5.9% of those in the 14-day group did. Rifabutin-based triple therapy showed promise in the study as a therapeutic option when first- and second-line treatments failed to eradicate this infection.</p>","<p>In a sizable cohort of people suffering from this infection who had not responded to other treatment options, a trial by David et al. in Italy examined how effective this rifabutin-based rescue therapy was for treating this infection. For 14 days, all individuals received treatment with rifabutin, amoxicillin, and a PPI. ITT analysis was used to determine the primary objective: the percentage of these infections that had been eliminated completely. Male participants in the ITT study had an eradication rate of 70.6%, while female participants had an eradication rate of 72.1%. It is noted that 19 patients had adverse reactions, indicating side effects from the medication. The study stated that rifabutin resistance was minimal, but no information was given regarding its prevalence or extent. Overall, the study showed that rifabutin-based rescue therapy produced reasonably high eradication rates in patients with this infection who were challenging to treat.</p>","<p>The research conducted in Italy by Saracino looked into antibiotic resistance and treatment results in individuals who had failed to eliminate this infection [##REF##32183165##28##]. The participants then got categorized depending on the number of treatment regimen failures they had experienced. Among the participants with only one treatment regimen failure (n=415), the failed regimens included sequential therapy in 67 patients, levofloxacin-based therapy in 221 patients, rifabutin-based therapy in 109 patients, and pylera therapy in 18 patients. The second group consisted of patients with two treatment regimen failures (n=310), and the third group comprised patients who had experienced three or more treatment regimen failures (n=312). The specific failed regimens in these groups were similar to the first group, with varying frequencies. The trial gave the recruited subjects a 10- to 14-day therapy period. According to both resistance rates and minimum inhibitory concentration (MIC) values, the study found a considerable rise in secondary resistance to the tested antibiotics, including Rifabutin. This rise in resistance was closely related to how frequently the patients' treatments failed. The study brought attention to the fact that continued treatments that were eventually not able to eliminate the organism only led to an increase in building up resistance. Notably, the resistance to rifabutin grew with each succeeding treatment failure, reaching resistance rates of 26% after one unsuccessful treatment regimen, 41.6% after two unsuccessful regimens, and 69.2% after three unsuccessful regimens or more.</p>","<p>YI Choi's study looked at possible rescue treatments for these infection strains that were resistant to antibiotics in China and Korea [##UREF##6##29##]. The esomeprazole, amoxicillin, and clarithromycin (EAC) group had 12 patients, the esomeprazole, amoxicillin, and metronidazole (EAM) group had 13 patients, the esomeprazole, amoxicillin, and levofloxacin (EAL) group had 31 patients, and the esomeprazole, bismuth, amoxicillin, and metronidazole (EBAM) group had 144 patients. These groups were divided based on the rescue regimens given. For a total of 14 days, the subjects underwent their rescue regimens. As per the analysis, the study discovered high eradication rates in all groups. For the EAC group, the eradication rate was 91.7%; for the EAM group, it was 92.3%; for the EAL group, it was 93.5%; and for the EBAM group, it was 95.1%. These findings show that susceptibility-guided therapy with the rescue regimens was very successful in individuals with several past H. pylori treatment failures.</p>","<p>Among patients who had previously taken these antibiotics as part of their treatments, resistance levels for clarithromycin, metronidazole, and levofloxacin were 93.9%, 99.2%, and 98.3%, respectively. This emphasizes the significance of modifying treatment plans in accordance with profiles of antibiotic susceptibility to increase effectiveness.</p>","<p>Overall, the research points to rifabutin and furazolidone as potential treatments for this infection that are resistant to antibiotics when used in rescue regimens. Research like this highlights the need to tailor antibiotic treatment plans to each patient's unique resistance profile in order to effectively eliminate H. pylori.</p>","<p>Finally, the study and the resources we selected shed insight into the difficulties of antibiotic resistance in the treatment of H. pylori infections. Antibiotic resistance is a major problem that threatens the success of treatment plans. Rifabutin and furazolidone are two medicines that have shown promise as effective alternatives for treating H. pylori strains that are resistant to traditional antibiotics.</p>","<p>These findings demonstrate the critical importance that individualized treatment regimens and susceptibility-guided therapy play in obtaining effective eradication rates. Healthcare providers may increase the likelihood of effective treatment results by choosing the most suitable antibiotics by analyzing the resistance patterns of specific patients.</p>","<p>Further research and ongoing surveillance of antibiotic resistance patterns in this microorganism are essential to continuously refine treatment strategies and combat the growing problem of antibiotic resistance. Additionally, developing new antibiotics and innovative treatment approaches may be necessary to ensure effective management of these infections in the future.</p>","<p>Discussion</p>","<p>This 18-month study investigated the effect of treatment failure on 187 US patients diagnosed with H. pylori infection [##REF##33577874##17##]. Next-generation sequencing was applied in this study to determine H. pylori's susceptibility to different types of antibiotics. Researchers believed this procedure was quite effective because it worked in 95% of the cases and yielded results in 72 hours. Sequencing led to the administration of two separate 14-day treatment regimens to groups of 101 and 86 patients, respectively; these were the BQ regimen and the triple regimen. After this intervention, it was observed that H. pylori exhibited antibiotic resistance rates ranging from 29.6% to 33.3% to levofloxacin, clarithromycin, and metronidazole. The rifabutin-PPI-amoxicillin triple regimen was administered to two individuals. With the BQ regimen, the overall success in eliminating was 88.1%, and with the triple regimen, it was 60.5%. Rifabutin, tetracycline, and amoxicillin resistance rates varied from 0.5% to 1.1%. These rates were lower than the other antimicrobial medications used in this trial. As a result, researchers concluded that early guidance on regimens reliant on these medications led to the efficient eradication of H. pylori.</p>","<p>Youn I Choi et al. conducted a study on 31 patients in Korea to determine the optimal antibiotic treatment strategy for eradicating multidrug-resistant H. pylori [##REF##31360270##18##]. The agar dilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI), was used to determine the lowest dilution concentration (MIC) for determining antibiotic resistance. The MIC of an antimicrobial agent is the lowest concentration at which the growth of bacterial colonies is inhibited. The CLSI guidelines were used to create the criteria for determining resistance to individual antimicrobials. In this case, the MIC for clarithromycin is higher than 1 g/mL. The resistance criteria were set at larger than 0.5 g/mL for amoxicillin, 8 mg/mL for metronidazole, 4 g/mL for tetracycline, 1 g/mL for levofloxacin antibiotics, 0.25 g/mL for rifabutin, and 4 g/mL for furazolidone. MDR is the presence of resistance to two or more antimicrobials. This traditional approach of gathering data on resistance opposes that of the study by Argueta et al. [##REF##33577874##17##]. Of the 31 isolated strains, around 71.0% were resistant to more than two antibiotics. There were 13 that were resistant to two antibiotics, seven that were resistant to three, and two that were resistant to four. Only nine were immune to at least one antibiotic. The most prevalent combination of medication resistance was resistance to both clarithromycin and metronidazole (two strains, 0%). According to the in vitro results, the MIC for rifabutin ranged from 0.00098 g/mL to 0.0078 g/mL, while the MIC for furazolidone was higher. No rifabutin- or furazolidone-resistant isolates were detected, despite the fact that the concentration range on agar was lower than the CLSI-established threshold. Therefore, whether used together or individually, their regimen completely eliminates even the most resilient strains of multidrug-resistant H. pylori. To fully understand the risks associated with these drugs, however, further research is necessary.</p>","<p>The aim of this 12-month study by Cosmo et al. in Spain was to assess the use of giving only those antibiotics that had shown susceptibility to treat the infection [##REF##28566898##19##]. This approach involved the administration of a PPI and two other antibiotics for a specific period as a new treatment option in patients with significantly high resistance to more than one drug. In this study, all participants underwent stomach biopsy procedures to obtain microorganisms for bacterial culture and E-test (BioMerieux) susceptibility testing, which included 68 people with dual antibiotic resistance to clarithromycin, levofloxacin, and metronidazole who received triple regimens (OAL/OAC/OAM). A rifabutin regimen (OAR) was administered to 12 patients who had developed antibiotic resistance to all three medicines: clarithromycin, levofloxacin, and metronidazole. Following a 10-day course of medication with strict adherence to the prescribed regimens, it was discovered that 10% of patients with dual resistance and 41.7% of patients with triple resistance still had H. pylori, which had not been completely eradicated. The utilization of rifabutin in this trial specifically targeted cases with triple resistance that had been scientifically proven, resulting in different outcomes compared to studies conducted in the USA and Korea [##REF##33577874##17##,##REF##31360270##18##]. Consequently, rifabutin advice was not provided at an earlier stage than in previous studies. In this study, the success of eliminating the infection was 95.5% when a combination of PPI and two antibiotics was used in those who already had resistance to more than one drug.</p>","<p>Another study by David Y. Graham on 455 patients with this infection in the USA aimed to assess how good the RHB-105 regimen was in eliminating this infection [##REF##32365359##15##]. No treatment plans were recommended for the participant's symptoms. They were between the ages of 18 and 70 when they presented with dyspepsia that had lasted for at least two weeks. Upper endoscopy and 13C urea breath test (UBT) confirmed the diagnosis. Everyone with a favorable culture, histology, or rapid urease result was eligible for randomization. The RHB-105 treatment plan called for the use of omeprazole 120 mg, clavulanic acid 3 g, and rifabutin 150 mg. Two hundred and twenty-eight patients were given this regimen for 14 days, whereas another 227 patients were given an active comparator consisting of amoxicillin and omeprazole alone. In the ITT analysis, the RHB-105 regimen significantly outperformed the active comparator regarding the H. pylori eradication rate (83.8% vs. 57.7%). Susceptibility testing was performed for patients who experienced treatment failures; however, unlike in other studies from the USA, Korea, and Spain, the method employed in this study was not disclosed [##REF##33577874##17##,##REF##31360270##18##]. Based on the standard followed in this study, first-line treatment for any infection should have the highest probability of effectiveness. This is true for antimicrobial therapies. Unlike previous research, this study focused on the care of uninformed patients and concluded that the RHB-105 regimen could successfully eradicate H. pylori. This suggests that the problem of resistance to existing anti-H. pylori treatment may be handled by giving rifabutin and equivalent regimens early in the course of the illness.</p>","<p>A new low-dose regimen of rifabutin with vonoprazan and amoxicillin was given for seven days, and effects studies by Inokuchi et al. on 69 patients with resistant gastric infections in Japan [##UREF##2##20##]. In contrast to a study conducted in the USA, the patients in this trial had treatment failure up to the sixth therapeutic regimen [##REF##32365359##15##]. According to the MIC criteria established by the CSIL and utilized in the study by Youn I Choi et al., antimicrobial susceptibility was evaluated [##REF##31360270##18##]. Twelve patients served as the historical control group and were given the 10-day course of the EAR therapy regimen (esomeprazole, amoxicillin, rifabutin). However, 57 patients were treated with the VAR regimen (lansoprazole, amoxicillin, and rifabutin) for seven days. ITT analysis showed that the eradication rate for VAR therapy was 91.2%, while it was only 83.3% for the EAR treatment group.</p>","<p>The median rifabutin MIC for all strains was 0.00 0.01 g/mL. When comparing the two groups' safety profiles, those in the seven-day VAR group fared worst, with 31.6% (18/57) reporting side effects and two people dropping out of the study due to adverse reactions. Both patients stopped taking the medication on day four; one owing to stomach pain and the other due to headache and conjunctival hyperemia. There were no serious adverse events, and the symptoms of each patient diminished when the medicine was used up. Unlike previous studies conducted in the USA, South Korea, Japan, and Spain, this one really assessed potential risks. In this study, a seven-day low-dose rifabutin-based VAR treatment was shown to be a reliable and secure way to get rid of H. pylori. With the use of early advice for rifabutin regimens, this infection might be treated and symptoms reduced sooner rather than later.</p>","<p>Isolates of H. pylori from individuals with refractory infections showed extremely high levels of antibiotic resistance, which greatly hampered effective therapy. The study discovered that resistance rates were quite high for well-known drugs such as metronidazole, levofloxacin, amoxicillin, and clarithromycin. Many people had dual resistance to clarithromycin and levofloxacin. The results highlighted the urgent need for alternative H. pylori treatment methods [##REF##33050205##21##]. When clarithromycin failed to completely cure H. pylori infection, the efficacy of BQ treatment and MRT was examined. The results showed that BQ treatment, compared to MRT therapy, was more effective in curing the infection in patients with peptic ulcers. MRT should seldom be considered when BQ therapy is not viable [##REF##33840604##22##].</p>","<p>In conclusion, the rising likelihood of antibiotic resistance among patients with refractory H. pylori infection highlights the urgent need for novel treatment modalities. According to the trial's findings, BQ therapy is superior to MRT as a second-line therapy for this condition. Even yet, further investigation is necessary to identify complementary treatments for those with resistant strains of H. pylori [##REF##33050205##21##,##REF##33840604##22##].</p>","<p>This retrospective research was carried out by Chang Ming Lee et al. [##UREF##3##23##]. Due to limited patient compliance and a convoluted dose strategy, finding a suitable third-line treatment when BQ therapy fails is difficult. Several Korean regimens failed to completely eradicate the illness. Therefore, the researchers looked at how well BQ treatment for peptic ulcers compared to the second-line MRT combination for curing the infection. Between January 2013 and December 2019, 665 H. pylori patients who had failed to respond to first-line clarithromycin were continually recruited at Gyeongsang National University Hospital. A total of 665 participants were included in the trial, with 71 receiving BQ and 252 receiving MRT. In order to conduct the peptic ulcer subgroup analysis, 132 patients from the BQ group and 51 patients from the MRT group were randomly selected. The study included 323 patients who met the inclusion criteria. Using an age and gender matching mechanism, 45 patients were examined, 15 from each group. Sixty-nine point zero percent of the cases were eradicated in the MRT group. These rates were much lower than the eradication rate seen in the BQ group, indicating that the treatment was far more successful. Analysis of patients who suffered from peptic ulcers revealed that those in the BQ group fared much better than those in the MRT group. Thirteen of the 14 patients who did not improve with MRT were cured after switching to a BQ regimen in the third line of treatment. BQ was effectively eradicated at a 90% rate after MRT had failed. From these findings, the researchers drew the conclusion that MRT treatment is not as successful as BQ therapy and should only be explored in cases when BQ is not possible.</p>","<p>Miftahussurur et al. did research to determine effective therapy for this illness in areas of Indonesia where metronidazole and levofloxacin resistance are prevalent [##REF##30774400##24##]. Clarithromycin and metronidazole resistance are serious problems in Indonesia. Resistance to levofloxacin and bismuth therapy is rising, which has resulted in a rise in the use of other antibiotic treatments. The dilution test was performed to determine how resistant H. pylori was to five different antibiotics. Additionally, next-generation sequencing was employed to identify mutations associated with antibiotic resistance. After being acquired from 1039 adult dyspeptic sufferers, 106 strains were tested, none showing furazolidone resistance. Furthermore, all the tested strains showed sensitivity to both rifabutin and sitafloxacin. In contrast, rifaximin and garenoxacin exhibited high resistance rates. Notably, garenoxacin-sensitive organisms have been found in places with high levels of clarithromycin resistance. However, rifaximin might not be a good antibiotic choice due to the prevalence of clarithromycin resistance. Finally, it was seen that rifabutin-based therapies were effective in eliminating this infection compared to the conventional protocols. Sitafloxacin should be explicitly considered to eradicate levofloxacin-resistant strains, while garenoxacin should be avoided. In the study conducted by Miftahussurur and colleagues, the researchers looked at successful treatment techniques for H. pylori infections in Bangladesh and Nepal, two countries where there was a considerable amount of resistance to the primary medicines [##REF##30815255##25##]. Patients with adult dyspepsia who were undergoing endoscopies at the Tribhuvan University Teaching Hospital (TUTH) were included in this study. Patients came from both Nepal and Bangladesh. After homogenizing the antral biopsy tissues, 42 strains were successfully recovered from Nepal (146 patients), while 56 strains were successfully recovered from Bangladesh. These strains were then cultivated at 37°C for up to 10 days. Subcultures of H. pylori were grown in the same microaerophilic conditions on antibiotic-free Mueller-Hinton II agar medium with 7% horse blood. Brucella broth with 10% dimethyl sulfoxide and 10% horse serum was used to keep the H. pylori solution at an ideal temperature of 80°C. Two-fold agar dilution was employed to determine antibiotic susceptibility. The five antibiotics examined were rifaximin, sitafloxacin, furazolidone, garenoxacin, and rifabutin.</p>","<p>Rifabutin and furazolidone were not resistant; however, sitafloxacin susceptibility was very high. In contrast, rifaximin was highly resistant. Garenoxacin resistance is also more prevalent in Bangladesh than in Nepal because of its association with levofloxacin resistance. As a remedy for this infection, Rifabutin should be taken cautiously, according to the authors, because of the drug's interaction with Bangladesh's widespread tuberculosis. The high susceptibility to furazolidone and sitafloxacin increased the prospects for their future use in Bangladesh and Nepal.</p>","<p>In a study, Muhammad Miftahussurur et al. sought to identify alternative H. pylori antibiotics for the Dominican Republic to mitigate resistance, which is widespread throughout the entire nation [##UREF##4##26##]. The MICs of five different antibiotics were determined using a two-fold agar dilution method against a panel of 63 different strains from the Dominican Republic. We employed next-generation sequencing to analyze genomic changes associated with antibiotic resistance. Rifabutin, furazolidone, and sitafloxacin were active against all 63 strains. On the other hand, resistance to rifaximin and garenoxacin was widespread (82.5% and 34.9%, respectively). Garenoxacin resistance (8/9, 88.9%, OR=45.33, P=0.002) and dual resistance to antibiotics (7/9, 77.8%, OR=31.5, P=0.009) were more likely in individuals older than 60 years old (P=0.004, r=0.357). More than three changes in rpoB were present in most rifaximin-resistant strains, with the most common new variants being S352L, I2726L, and V2465A. A strong correlation between the vacA genotype and rifaximin resistance was found (P=0.042). The lowest inhibitory concentration was significantly higher among the 23 levofloxacin-resistant organisms (39.1%, 9/23) and was positively correlated with resistance (P=0.001, r=0.84). Additionally, the vast majority of these organisms were resistant to garenoxacin (82.6%, 19/23, P=0.001). In a study with 10 strains, 16 (or 84.2% of the total) exhibited gyrA mutations at D91 and N87, making them resistant to garenoxacin.</p>","<p>The study exhibited several limitations, outlined as follows: first, due to the limited sample size and the exclusive focus on samples collected solely from the capital of the Dominican Republic, the generalizability of the results to the rest of Latin America was compromised. Second, the study examined only a subset of the numerous H. pylori genes that could potentially be associated with antibiotic resistance, thus offering a limited understanding of the topic. Third, the resistance rates of the five different antibiotics were solely estimated through in vitro experimentation. Alternatives to levofloxacin and metronidazole that could be incorporated into the eradication strategy include sitafloxacin, rifabutin, and furazolidone. Such regions include the Dominican Republic, which has a high rate of resistance to these medications.</p>","<p>The purpose of the prospective randomized investigation by Hideki Mori et al. is to determine whether rifabutin, used as part of a triple therapy regimen, is effective as a third- or fourth-line rescue medication [##UREF##5##27##]. Participants in the trial were those who had not improved after receiving both primary and secondary eradication treatments. A stomach biopsy was carried out to ascertain whether the rpoB mutation was present. For 10 or 14 days, patients took eradication medication with rifabutin (300 mg once daily), amoxicillin (500 mg four times daily), and esomeprazole (20 mg four times daily). Eighty percent of the sample reported that they did not take their medication as prescribed. A 13C UBT or a stool antigen test was used to establish if H. pylori had been successfully eradicated from the body after therapy had concluded. There were a total of 22 people who took part in the experiment, 12 of whom were placed in the 10-day group and 17 in the 14-day group. In the 14-day group, the success rate of eradicating the infection was 94.1% while in the 10-day group it was 81.8% (depending on the technique of analysis). Additionally, between 8% and fewer than 30% of patients in the two cohorts stopped taking their prescriptions as a result of side effects including diarrhea.</p>","<p>However, more patients in the 10-day group finished the fourth eradication medication, suggesting that the 14-day group's success rate may have been overestimated. In addition, there was no reliable way to assess eradication rates, rpoB mutations, or rifabutin MICs. Finally, we can say that both the 10-day and 14-day rescue regimens worked, with the latter reaching an eradication rate of over 90%. However, when therapeutic tolerance is taken into account, it is possible that a 10-day course of treatment is all that is necessary for complete eradication.</p>","<p>A few issues with this study should be noted. The self-reported approach for assessing therapy compliance was the first. This approach may be limited by its dubious precision and subpar assessment accuracy. Second, there needs to be more resistance data due to the exclusive reliance on clinical assessment without including susceptibility testing. As a result, the reasons for earlier treatment failures in these patients (lack of drug adherence or a rise in antibiotic resistance) are unknown. Furthermore, the study population exhibited a lack of complete homogeneity due to various characteristics of the study design.</p>","<p>In summary, our study demonstrated that, when repeated attempts to eradicate H. pylori using standard antibiotics failed, rifabutin-based rescue therapy is a practical and secure option. A sizable group of people who were very difficult to treat participated in the trial. In addition, Saracino et al. carried out a retrospective analysis to examine how antibiotic resistance emerged, how therapy progressed, and how MIC values changed over time in patients who had been unable to eradicate H. pylori with at least one medication [##REF##32183165##28##]. The effectiveness of levofloxacin, metronidazole, and clarithromycin against H. pylori bacteria was examined in a study including individuals who had UGIE after prior medication. Pylera® or a susceptibility-guided treatment plan was provided to the patients.</p>","<p>Antibiotic susceptibility data was provided for 1037 of the 1223 patients who tested positive for H. pylori. Antibiotic resistance was found to be at substantially higher rates, MIC values were much lower than expected, and the number of failed treatment efforts was much higher than expected. Except for when sequential therapy is utilized as a third-line therapy, the eradication rates of antibiogram-tailored drugs remained unchanged. It was found that when Pylera® therapy was utilized as a last resort, it resulted in cure rates comparable to those of other culture-guided treatments. Unfortunately, the Pylera® regimen is difficult to administer and more frequently leads to adverse effects than alternative treatments. Up to 14 pills taken four times over the course may be necessary, and more than 30% of patients experience side effects, which increases the likelihood that therapy will be stopped early.</p>","<p>In conclusion, the number of therapeutic failures was inversely linked to the rate and MIC values of secondary resistance to the three tested antibiotics. The eradication rates from Pylera® therapy were comparable to those from treatments tailored to a particular culture. Rifabutin, furazolidone, and other antibacterial medications were tested for their antimicrobial efficacy in a second prospective trial by Youn I Choi et al. with 200 participants to investigate their potential efficacy as therapies for multidrug-resistant H. pylori along with H. pylori strains, which are resistant to other antibiotics [##UREF##6##29##]. After conducting a retrospective analysis of the information available at the Helicobacter Registry of two famous medical centers, along with four reference strains, a total of 31 single- or multidrug-resistant H. pylori bacteria were selected for testing. The strains were subjected to the broth microdilution technique to evaluate their resistance to a number of different antibiotics.</p>","<p>No rifabutin- or furazolidone-resistant H. pylori isolates were discovered among the 31 antibiotic-resistant strains. Only one of the strains tested positive for tetracycline resistance. Rifabutin and furazolidone are more effective at curing the infection than amoxicillin and levofloxacin, which have resistance rates of 22.6% and 1.9%, respectively. Only 3.2% of H. pylori bacteria were resistant to the antibiotic tetracycline. Since the eradication rate was not demonstrated by treating actual patients, it is possible that the in vitro results do not reflect what happens in vivo. It is also important to remember that there may be a disparity between the results of the study and the actual prevalence of antibiotic-resistant H. pylori in Korea due to selection bias. Clinicians should exercise caution before extrapolating the findings to the general public due to ethical considerations, and the research's lack of information on antibiotic resistance in H. pylori is another drawback.</p>","<p>In conclusion, rifabutin, furazolidone, and tetracycline can be used to eliminate this infection successfully. They can be used as solitary treatment options or together as a complete regimen in regions where species of this organism have developed very high rates of resistance. However, further research and careful consideration are warranted before implementing these findings on a broader scale. As H. pylori becomes increasingly resistant to medications, new approaches are required to treat this persistent infection.</p>","<p>The review of key data presented in this research sheds light on the challenges posed by antibiotic-resistant H. pylori and the need to develop new therapeutic techniques. In view of the proliferation of multidrug-resistant strains of H. pylori, the development of new drugs and treatment methods is essential for the successful elimination of this pathogen. Rifabutin, furazolidone, and sitafloxacin have shown promise as potential therapeutic options, especially in regions where resistance to conventional antibiotics is prevalent.</p>","<p>Healthcare providers must keep abreast of the most recent findings on H. pylori resistance patterns and treatment choices in order to give patients with H. pylori infection effective and individualized therapeutic interventions. Furthermore, larger-scale studies with a more representative sample of the general population are required to demonstrate the efficacy and safety of these supplementary drugs.</p>","<p>In conclusion, combating H. pylori infection and the ensuing rise in antibiotic resistance requires an all-encompassing strategy that includes continual monitoring, the cautious use of drugs, and the exploration of potential innovative treatments. The medical community has to make considerable efforts worldwide to improve H. pylori infection treatment and reduce the prevalence of related illnesses.</p>","<p>Limitations</p>","<p>There were no in vitro clinical trials included in this systematic review. We might have overlooked a few crucial publications because we could not obtain a few articles as the complete text was unavailable, and these were not included in our study.</p>"],"string":"[\n \"<title>Review</title>\",\n \"<p>Methods</p>\",\n \"<p>The systematic review was done in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A rigorous search strategy was developed to test this hypothesis and identify relevant literature. A comprehensive search of the various search engines and databases was done to include relevant literature.</p>\",\n \"<p>A PubMed search was conducted using the Mesh terminology ''Rifabutin or N'-acetyl rifabutin or Mycobutin or ansatipine or ansamycin or LM 427, H pylori or helicobacter pylori, Therapy or therapeutic* or regimen* or protocol*, ''Rifabutin/administration and dosage''[Mesh] OR ''Rifabutin/adverse effects''[Mesh] OR ''Rifabutin/metabolism''[Mesh] OR ''Rifabutin/pharmacokinetics''[Mesh] OR ''Rifabutin/therapeutic use''[Mesh] OR ''Rifabutin/toxicity''[Mesh]. The MeSH methods are listed in Table ##TAB##0##1##.</p>\",\n \"<p>Data extracted via the PubMed search strategy is shown in Table ##TAB##1##2##.</p>\",\n \"<p>The authors wrote inclusion criteria, and articles were then chosen. This criterion was laid down based on various factors, including those related to H. pylori gastritis, papers discussing the research subject, publications published in English, and other specific criteria. These criteria included selecting papers with full-text articles, studies involving in vivo investigations, papers focusing on patients without comorbidities, publications published within the last five years (2016-2022), papers specifically addressing the treatment of Helicobacter gastritis, and studies examining the use of rifabutin for treating this infection, among others. We thus included randomized control trials and observational studies in this systematic review. Articles published in languages other than English, those not related to the research topic, those without full-text availability, and those based solely on in vitro studies were not included in the analysis. Patients with other gastrointestinal (GIT) diseases and comorbidities, MALTomas, and gastric adenocarcinomas were also not included. PRISMA chart is given in Figure ##FIG##0##1##.</p>\",\n \"<p>The authors independently reviewed the research design and the possibility of bias in selection or publication. Any disagreements in the reviews of the writers were settled by consensus. A comprehensive quality assessment of the retrieved publications was conducted using the appropriate methodologies for each type of study. The results of critical appraisal using the NewCastle-Ottawa Quality Assessment Tool evaluation for case-control and cohort studies and the Cochrane Risk of Bias Tool for RCT are given in Table ##TAB##2##3##.</p>\",\n \"<p>Result</p>\",\n \"<p>Fifteen studies were finalized following multiple readings of the full text of the papers and discussions among co-authors. Study characteristics are given in Table ##TAB##3##4##.</p>\",\n \"<p>Erick A. Argueta did research in the USA to see how new antibiotic resistance would affect efforts to eradicate H. pylori infection [##REF##33577874##17##]. In a study including 101 patients, the bismuth quadruple (BQ) regimen was used. This treatment plan includes the use of bismuth, tetracycline, metronidazole, and PPIs. Eighty-six patients were given the triple regimen, which included PPIs, clarithromycin, and either amoxicillin, metronidazole, or rifabutin. The eradication rate for Cohort 2 (treated with the triple regimen) was 60.5%, whereas the eradication rate for Cohort 1 (treated with the BQ regimen) was 88.1%. The research indicated that of the antibiotics tested, metronidazole had the greatest resistance rate (33.3%), followed by clarithromycin (30%), levofloxacin (29.6%), amoxicillin (1.0%), rifabutin (0.5%), and tetracycline (0.5%). Not only that but multidrug resistance (MDR) was found in 65.6% of bacterial isolates.</p>\",\n \"<p>Youn I Choi conducted a study in Korea to identify potential candidates for a rescue regimen in cases of antibiotic-resistant H. pylori infections [##REF##31360270##18##]. Nine patients showed no elimination when just one antibiotic was used, while 22 were resistant to several. Interestingly, none of the subjects showed resistance to rifabutin or furazolidone. However, this gastric infection was completely eradicated in every patient in both cohorts, showing a 100% success rate. The study also listed the rates of resistance for several antibiotics, with clarithromycin having the highest rate at 71.1%, followed by metronidazole at 67.7%, levofloxacin at 41.9%, and amoxicillin at 22.6%. Rifabutin and furazolidone showed no signs of resistance.</p>\",\n \"<p>In a Spanish study, susceptibility testing was used to identify treatment plans for 68 individuals with dual antibiotic resistance [##REF##28566898##19##]. OAL (omeprazole, amoxicillin, levofloxacin) was administered to 43 patients, OAC (omeprazole, amoxicillin, clarithromycin) to 12 patients, and OAM (omeprazole, amoxicillin, metronidazole) to 13 patients for those with dual-resistant infections. The OAR (omeprazole, amoxicillin, rifabutin) regimen was used to treat 12 patients with triple antibiotic resistance. The study reported high eradication rates for the various regimens in both cohorts. Notably, dual antibiotic-resistant patients had a resistance rate of less than 10% following medication, indicating effective eradication. However, the resistance rate in the triple antibiotic-resistant group receiving the rifabutin-based regimen was 41.7%, indicating a lower level of effectiveness in this cohort.</p>\",\n \"<p>David Y. Graham looked into the Rifabutin-Based Triple Therapy (RHB-105) to see if it was beneficial in treating H. pylori infection [##REF##32365359##15##]. Two hundred and twenty-eight patients were given the RHB-105 regimen (amoxicillin, omeprazole, and Rifabutin) while 227 patients were given an active comparator regimen (amoxicillin and omeprazole). The study used an intention-to-treat (ITT) methodology to evaluate the eradication rates. The RHB-105 regimen had an eradication percentage of 83.8%, while the active comparator had a rate of 57.7%. According to these results, RHB-105 significantly outperformed the active comparator in eliminating H. pylori. Rifabutin showed a lower resistance rate than the active comparator, highlighting the potential benefit of utilizing it to treat infections with H. pylori.</p>\",\n \"<p>The effects of the EAR treatment regimen (esomeprazole, amoxicillin, Rifabutin) were investigated by Kazumi Inokuchi using a historical control group of 12 patients [##REF##30736338##2##]. Vonoprazan, amoxicillin, and rifabutin (VAR) were used to treat 57 different individuals. The research assessed the efficacy of two strategies for treating this infection, contrasting the ''intention-to-treat'' (ITT) approach with the ''per protocol'' (PP) approach. The case group had a greater rate of H. pylori eradication after receiving the triple therapy based on low-dose rifabutin. The 10-day regimen had a resistance rate of 16.7%, whereas the seven-day regimen had a resistance rate of 8.8%, according to the study's analysis of rifabutin regimen resistance rates.</p>\",\n \"<p>Triple rifabutin (RHB-105) was studied by Ira N. Klafus in the USA to determine its efficacy in treating this illness [##REF##33050205##21##]. Seventy-seven people were given the RHB-105 regimen (which contained amoxicillin, omeprazole, and rifabutin), and 41 people were given a placebo. In both the ITT and PP analyses, the RHB-105 regimen showed statistically significant eradication. The resistance rate to the RHB-105 regimen was 10.6%, which could affect the treatment's efficacy.</p>\",\n \"<p>Chia Jung Kuo sought to understand better the clinical difficulties associated with multidrug-resistant infections. The trend of resistance being faced by the population was evaluated, with clarithromycin (92.7%) and levofloxacin (85.4%) having the highest resistance rates [##REF##33840604##22##]. Rifabutin showed a resistance rate of 29.3%. Notably, the study disclosed neither the eradication rates attained nor the treatment regimens utilized. Instead, it focused on describing the MDR patterns and H. pylori's antibiotic susceptibility in the research population.</p>\",\n \"<p>A study by Chang Ming Lee had 323 participants divided into two cohorts [##UREF##3##23##]. The moxifloxacin-rifabutin triple therapy (MRT) treatment regimen was administered to 71 individuals who were unsuccessful with the first-line clarithromycin regimen (Cohort 1), while 252 patients with the same condition received the BQ treatment regimen (Cohort 2). The study found that eradication rates could differ depending on the treatment plan. MRT and BQ regimens showed effectiveness as second-line treatments for peptic ulcer cases unresponsive to the first-line clarithromycin regimen.</p>\",\n \"<p>Miftahussurur conducted research to evaluate different H. pylori infection treatment plans in Indonesian areas where metronidazole and levofloxacin resistance are common [##REF##30774400##24##]. The study evaluated the susceptibility and resistance of five different antibiotics, furazolidone, sitafloxacin, garenoxacin, rifaximin, and rififabutin, to different strains of H. pylori bacteria. Among the participants, 61 out of 105 (58.1%) were responsive to each of the five antibiotics tested, indicating that these people may benefit from different eradication regimens that include these antibiotics. Notably, none of the organisms resisted sitafloxacin, furazolidone, or rifabutin. However, the study found various degrees of resistance to rifaximin and garenoxacin. Notably, 40 out of 105 subjects (38.9%) and seven out of 105 people (6.7%) were resistant to rifaximin and garenoxacin, respectively. This shows that these drugs are highly resistant in the Indonesian regions under study. The results do, however, indicate that adding furazolidone, rifabutin, and sitafloxacin to regimens can be successful in regions with great resistance to some antibiotics like levofloxacin and metronidazole.</p>\",\n \"<p>Another study took place in the Southeast Asian countries of Bangladesh and Nepal [##REF##30815255##25##]. The study assessed the regions of Nepal where there was high antibiotic resistance. Garenoxacin resistance was seen with the tested antibiotics in 12 out of 42 people (28.6%), whereas sitafloxacin resistance was seen in two out of 42 participants (4.8%). Furazolidone and Rifabutin, however, did not show any signs of resistance. Another antibiotic, rifaximin, was tested, and 22 out of 42 people (52.4%) showed resistance. A similar evaluation was carried out in Bangladesh, demonstrating the antibiotic resistance patterns in these strains. Not more than one of the 56 subjects (1.8%) demonstrated resistance to sitafloxacin, compared to 29 out of 56 patients (51.8%) who showed resistance to garenoxacin. No rifabutin or furazolidone resistance was found, similar to Nepal. Resistance to rifaximin was seen in 36 out of 56 subjects (64.3%). Importantly, none of the tested strains in either nation showed rifabutin or furazolidone resistance. This shows that despite the significant resistance seen for other regularly used antibiotics such as garenoxacin, sitafloxacin, and rifaximin, these two antibiotics could be possible candidates for effective therapy regimens against H. pylori infection in Bangladesh and Nepal.</p>\",\n \"<p>Miftahussurur conducted a study in the Dominican Republic to find substitute antibiotics to deal with increased resistance to levofloxacin and metronidazole in treating this infection [##UREF##4##26##]. The distribution of strains of this organism in the Dominican Republic that were resistant to several antibiotics was evaluated. Rifaximin resistance was present in 52 out of 62 persons (82.5%) among the antibiotics tested, while garenoxacin resistance was seen in 22 out of 63 participants (34.9%). There was no resistance to sitafloxacin or furazolidone among the individuals. Rifabutin showed no resistance in any of the 63 participants. The study also found that 18.6% of the 63 patients exhibited double antibiotic resistance to rifaximin and garenoxacin. The study's conclusions are essential as they demonstrate how rare antibiotic resistance to rifabutin, sitafloxacin, and furazolidone is in the Dominican Republic. Therefore, these three medicines could be used to treat this infection in this area.</p>\",\n \"<p>To assess whether triple therapy based on rifabutin was efficient as a third- and fourth-line treatments for this infection, H. Mori conducted a study in Japan [##UREF##5##27##]. The 10-day and 14-day cohorts were each given a group of subjects. Twelve patients were administered esomeprazole, amoxicillin, and rifabutin four times daily in the 10-day group. The same drugs were given to 17 individuals in the 14-day group; however, rifabutin was only given once daily. In this study, we used ITT and PP analyses to evaluate the success rate at curing H. pylori infection as our major end measure. In the ITT analysis, the eradication rate for the 10-day group was 83.3%, while in the PP analysis, it was just 81.8%. Eighty percent of patients saw improvement after 10 days of treatment. ITT analysis showed a 94.1% eradication rate in the 14-day group, and PP analysis showed a 91.7% eradication rate. The 14-day treatment plan was successful in curing 90% of the illness. No one in the 10-day group showed resistance to rifabutin, whereas 5.9% of those in the 14-day group did. Rifabutin-based triple therapy showed promise in the study as a therapeutic option when first- and second-line treatments failed to eradicate this infection.</p>\",\n \"<p>In a sizable cohort of people suffering from this infection who had not responded to other treatment options, a trial by David et al. in Italy examined how effective this rifabutin-based rescue therapy was for treating this infection. For 14 days, all individuals received treatment with rifabutin, amoxicillin, and a PPI. ITT analysis was used to determine the primary objective: the percentage of these infections that had been eliminated completely. Male participants in the ITT study had an eradication rate of 70.6%, while female participants had an eradication rate of 72.1%. It is noted that 19 patients had adverse reactions, indicating side effects from the medication. The study stated that rifabutin resistance was minimal, but no information was given regarding its prevalence or extent. Overall, the study showed that rifabutin-based rescue therapy produced reasonably high eradication rates in patients with this infection who were challenging to treat.</p>\",\n \"<p>The research conducted in Italy by Saracino looked into antibiotic resistance and treatment results in individuals who had failed to eliminate this infection [##REF##32183165##28##]. The participants then got categorized depending on the number of treatment regimen failures they had experienced. Among the participants with only one treatment regimen failure (n=415), the failed regimens included sequential therapy in 67 patients, levofloxacin-based therapy in 221 patients, rifabutin-based therapy in 109 patients, and pylera therapy in 18 patients. The second group consisted of patients with two treatment regimen failures (n=310), and the third group comprised patients who had experienced three or more treatment regimen failures (n=312). The specific failed regimens in these groups were similar to the first group, with varying frequencies. The trial gave the recruited subjects a 10- to 14-day therapy period. According to both resistance rates and minimum inhibitory concentration (MIC) values, the study found a considerable rise in secondary resistance to the tested antibiotics, including Rifabutin. This rise in resistance was closely related to how frequently the patients' treatments failed. The study brought attention to the fact that continued treatments that were eventually not able to eliminate the organism only led to an increase in building up resistance. Notably, the resistance to rifabutin grew with each succeeding treatment failure, reaching resistance rates of 26% after one unsuccessful treatment regimen, 41.6% after two unsuccessful regimens, and 69.2% after three unsuccessful regimens or more.</p>\",\n \"<p>YI Choi's study looked at possible rescue treatments for these infection strains that were resistant to antibiotics in China and Korea [##UREF##6##29##]. The esomeprazole, amoxicillin, and clarithromycin (EAC) group had 12 patients, the esomeprazole, amoxicillin, and metronidazole (EAM) group had 13 patients, the esomeprazole, amoxicillin, and levofloxacin (EAL) group had 31 patients, and the esomeprazole, bismuth, amoxicillin, and metronidazole (EBAM) group had 144 patients. These groups were divided based on the rescue regimens given. For a total of 14 days, the subjects underwent their rescue regimens. As per the analysis, the study discovered high eradication rates in all groups. For the EAC group, the eradication rate was 91.7%; for the EAM group, it was 92.3%; for the EAL group, it was 93.5%; and for the EBAM group, it was 95.1%. These findings show that susceptibility-guided therapy with the rescue regimens was very successful in individuals with several past H. pylori treatment failures.</p>\",\n \"<p>Among patients who had previously taken these antibiotics as part of their treatments, resistance levels for clarithromycin, metronidazole, and levofloxacin were 93.9%, 99.2%, and 98.3%, respectively. This emphasizes the significance of modifying treatment plans in accordance with profiles of antibiotic susceptibility to increase effectiveness.</p>\",\n \"<p>Overall, the research points to rifabutin and furazolidone as potential treatments for this infection that are resistant to antibiotics when used in rescue regimens. Research like this highlights the need to tailor antibiotic treatment plans to each patient's unique resistance profile in order to effectively eliminate H. pylori.</p>\",\n \"<p>Finally, the study and the resources we selected shed insight into the difficulties of antibiotic resistance in the treatment of H. pylori infections. Antibiotic resistance is a major problem that threatens the success of treatment plans. Rifabutin and furazolidone are two medicines that have shown promise as effective alternatives for treating H. pylori strains that are resistant to traditional antibiotics.</p>\",\n \"<p>These findings demonstrate the critical importance that individualized treatment regimens and susceptibility-guided therapy play in obtaining effective eradication rates. Healthcare providers may increase the likelihood of effective treatment results by choosing the most suitable antibiotics by analyzing the resistance patterns of specific patients.</p>\",\n \"<p>Further research and ongoing surveillance of antibiotic resistance patterns in this microorganism are essential to continuously refine treatment strategies and combat the growing problem of antibiotic resistance. Additionally, developing new antibiotics and innovative treatment approaches may be necessary to ensure effective management of these infections in the future.</p>\",\n \"<p>Discussion</p>\",\n \"<p>This 18-month study investigated the effect of treatment failure on 187 US patients diagnosed with H. pylori infection [##REF##33577874##17##]. Next-generation sequencing was applied in this study to determine H. pylori's susceptibility to different types of antibiotics. Researchers believed this procedure was quite effective because it worked in 95% of the cases and yielded results in 72 hours. Sequencing led to the administration of two separate 14-day treatment regimens to groups of 101 and 86 patients, respectively; these were the BQ regimen and the triple regimen. After this intervention, it was observed that H. pylori exhibited antibiotic resistance rates ranging from 29.6% to 33.3% to levofloxacin, clarithromycin, and metronidazole. The rifabutin-PPI-amoxicillin triple regimen was administered to two individuals. With the BQ regimen, the overall success in eliminating was 88.1%, and with the triple regimen, it was 60.5%. Rifabutin, tetracycline, and amoxicillin resistance rates varied from 0.5% to 1.1%. These rates were lower than the other antimicrobial medications used in this trial. As a result, researchers concluded that early guidance on regimens reliant on these medications led to the efficient eradication of H. pylori.</p>\",\n \"<p>Youn I Choi et al. conducted a study on 31 patients in Korea to determine the optimal antibiotic treatment strategy for eradicating multidrug-resistant H. pylori [##REF##31360270##18##]. The agar dilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI), was used to determine the lowest dilution concentration (MIC) for determining antibiotic resistance. The MIC of an antimicrobial agent is the lowest concentration at which the growth of bacterial colonies is inhibited. The CLSI guidelines were used to create the criteria for determining resistance to individual antimicrobials. In this case, the MIC for clarithromycin is higher than 1 g/mL. The resistance criteria were set at larger than 0.5 g/mL for amoxicillin, 8 mg/mL for metronidazole, 4 g/mL for tetracycline, 1 g/mL for levofloxacin antibiotics, 0.25 g/mL for rifabutin, and 4 g/mL for furazolidone. MDR is the presence of resistance to two or more antimicrobials. This traditional approach of gathering data on resistance opposes that of the study by Argueta et al. [##REF##33577874##17##]. Of the 31 isolated strains, around 71.0% were resistant to more than two antibiotics. There were 13 that were resistant to two antibiotics, seven that were resistant to three, and two that were resistant to four. Only nine were immune to at least one antibiotic. The most prevalent combination of medication resistance was resistance to both clarithromycin and metronidazole (two strains, 0%). According to the in vitro results, the MIC for rifabutin ranged from 0.00098 g/mL to 0.0078 g/mL, while the MIC for furazolidone was higher. No rifabutin- or furazolidone-resistant isolates were detected, despite the fact that the concentration range on agar was lower than the CLSI-established threshold. Therefore, whether used together or individually, their regimen completely eliminates even the most resilient strains of multidrug-resistant H. pylori. To fully understand the risks associated with these drugs, however, further research is necessary.</p>\",\n \"<p>The aim of this 12-month study by Cosmo et al. in Spain was to assess the use of giving only those antibiotics that had shown susceptibility to treat the infection [##REF##28566898##19##]. This approach involved the administration of a PPI and two other antibiotics for a specific period as a new treatment option in patients with significantly high resistance to more than one drug. In this study, all participants underwent stomach biopsy procedures to obtain microorganisms for bacterial culture and E-test (BioMerieux) susceptibility testing, which included 68 people with dual antibiotic resistance to clarithromycin, levofloxacin, and metronidazole who received triple regimens (OAL/OAC/OAM). A rifabutin regimen (OAR) was administered to 12 patients who had developed antibiotic resistance to all three medicines: clarithromycin, levofloxacin, and metronidazole. Following a 10-day course of medication with strict adherence to the prescribed regimens, it was discovered that 10% of patients with dual resistance and 41.7% of patients with triple resistance still had H. pylori, which had not been completely eradicated. The utilization of rifabutin in this trial specifically targeted cases with triple resistance that had been scientifically proven, resulting in different outcomes compared to studies conducted in the USA and Korea [##REF##33577874##17##,##REF##31360270##18##]. Consequently, rifabutin advice was not provided at an earlier stage than in previous studies. In this study, the success of eliminating the infection was 95.5% when a combination of PPI and two antibiotics was used in those who already had resistance to more than one drug.</p>\",\n \"<p>Another study by David Y. Graham on 455 patients with this infection in the USA aimed to assess how good the RHB-105 regimen was in eliminating this infection [##REF##32365359##15##]. No treatment plans were recommended for the participant's symptoms. They were between the ages of 18 and 70 when they presented with dyspepsia that had lasted for at least two weeks. Upper endoscopy and 13C urea breath test (UBT) confirmed the diagnosis. Everyone with a favorable culture, histology, or rapid urease result was eligible for randomization. The RHB-105 treatment plan called for the use of omeprazole 120 mg, clavulanic acid 3 g, and rifabutin 150 mg. Two hundred and twenty-eight patients were given this regimen for 14 days, whereas another 227 patients were given an active comparator consisting of amoxicillin and omeprazole alone. In the ITT analysis, the RHB-105 regimen significantly outperformed the active comparator regarding the H. pylori eradication rate (83.8% vs. 57.7%). Susceptibility testing was performed for patients who experienced treatment failures; however, unlike in other studies from the USA, Korea, and Spain, the method employed in this study was not disclosed [##REF##33577874##17##,##REF##31360270##18##]. Based on the standard followed in this study, first-line treatment for any infection should have the highest probability of effectiveness. This is true for antimicrobial therapies. Unlike previous research, this study focused on the care of uninformed patients and concluded that the RHB-105 regimen could successfully eradicate H. pylori. This suggests that the problem of resistance to existing anti-H. pylori treatment may be handled by giving rifabutin and equivalent regimens early in the course of the illness.</p>\",\n \"<p>A new low-dose regimen of rifabutin with vonoprazan and amoxicillin was given for seven days, and effects studies by Inokuchi et al. on 69 patients with resistant gastric infections in Japan [##UREF##2##20##]. In contrast to a study conducted in the USA, the patients in this trial had treatment failure up to the sixth therapeutic regimen [##REF##32365359##15##]. According to the MIC criteria established by the CSIL and utilized in the study by Youn I Choi et al., antimicrobial susceptibility was evaluated [##REF##31360270##18##]. Twelve patients served as the historical control group and were given the 10-day course of the EAR therapy regimen (esomeprazole, amoxicillin, rifabutin). However, 57 patients were treated with the VAR regimen (lansoprazole, amoxicillin, and rifabutin) for seven days. ITT analysis showed that the eradication rate for VAR therapy was 91.2%, while it was only 83.3% for the EAR treatment group.</p>\",\n \"<p>The median rifabutin MIC for all strains was 0.00 0.01 g/mL. When comparing the two groups' safety profiles, those in the seven-day VAR group fared worst, with 31.6% (18/57) reporting side effects and two people dropping out of the study due to adverse reactions. Both patients stopped taking the medication on day four; one owing to stomach pain and the other due to headache and conjunctival hyperemia. There were no serious adverse events, and the symptoms of each patient diminished when the medicine was used up. Unlike previous studies conducted in the USA, South Korea, Japan, and Spain, this one really assessed potential risks. In this study, a seven-day low-dose rifabutin-based VAR treatment was shown to be a reliable and secure way to get rid of H. pylori. With the use of early advice for rifabutin regimens, this infection might be treated and symptoms reduced sooner rather than later.</p>\",\n \"<p>Isolates of H. pylori from individuals with refractory infections showed extremely high levels of antibiotic resistance, which greatly hampered effective therapy. The study discovered that resistance rates were quite high for well-known drugs such as metronidazole, levofloxacin, amoxicillin, and clarithromycin. Many people had dual resistance to clarithromycin and levofloxacin. The results highlighted the urgent need for alternative H. pylori treatment methods [##REF##33050205##21##]. When clarithromycin failed to completely cure H. pylori infection, the efficacy of BQ treatment and MRT was examined. The results showed that BQ treatment, compared to MRT therapy, was more effective in curing the infection in patients with peptic ulcers. MRT should seldom be considered when BQ therapy is not viable [##REF##33840604##22##].</p>\",\n \"<p>In conclusion, the rising likelihood of antibiotic resistance among patients with refractory H. pylori infection highlights the urgent need for novel treatment modalities. According to the trial's findings, BQ therapy is superior to MRT as a second-line therapy for this condition. Even yet, further investigation is necessary to identify complementary treatments for those with resistant strains of H. pylori [##REF##33050205##21##,##REF##33840604##22##].</p>\",\n \"<p>This retrospective research was carried out by Chang Ming Lee et al. [##UREF##3##23##]. Due to limited patient compliance and a convoluted dose strategy, finding a suitable third-line treatment when BQ therapy fails is difficult. Several Korean regimens failed to completely eradicate the illness. Therefore, the researchers looked at how well BQ treatment for peptic ulcers compared to the second-line MRT combination for curing the infection. Between January 2013 and December 2019, 665 H. pylori patients who had failed to respond to first-line clarithromycin were continually recruited at Gyeongsang National University Hospital. A total of 665 participants were included in the trial, with 71 receiving BQ and 252 receiving MRT. In order to conduct the peptic ulcer subgroup analysis, 132 patients from the BQ group and 51 patients from the MRT group were randomly selected. The study included 323 patients who met the inclusion criteria. Using an age and gender matching mechanism, 45 patients were examined, 15 from each group. Sixty-nine point zero percent of the cases were eradicated in the MRT group. These rates were much lower than the eradication rate seen in the BQ group, indicating that the treatment was far more successful. Analysis of patients who suffered from peptic ulcers revealed that those in the BQ group fared much better than those in the MRT group. Thirteen of the 14 patients who did not improve with MRT were cured after switching to a BQ regimen in the third line of treatment. BQ was effectively eradicated at a 90% rate after MRT had failed. From these findings, the researchers drew the conclusion that MRT treatment is not as successful as BQ therapy and should only be explored in cases when BQ is not possible.</p>\",\n \"<p>Miftahussurur et al. did research to determine effective therapy for this illness in areas of Indonesia where metronidazole and levofloxacin resistance are prevalent [##REF##30774400##24##]. Clarithromycin and metronidazole resistance are serious problems in Indonesia. Resistance to levofloxacin and bismuth therapy is rising, which has resulted in a rise in the use of other antibiotic treatments. The dilution test was performed to determine how resistant H. pylori was to five different antibiotics. Additionally, next-generation sequencing was employed to identify mutations associated with antibiotic resistance. After being acquired from 1039 adult dyspeptic sufferers, 106 strains were tested, none showing furazolidone resistance. Furthermore, all the tested strains showed sensitivity to both rifabutin and sitafloxacin. In contrast, rifaximin and garenoxacin exhibited high resistance rates. Notably, garenoxacin-sensitive organisms have been found in places with high levels of clarithromycin resistance. However, rifaximin might not be a good antibiotic choice due to the prevalence of clarithromycin resistance. Finally, it was seen that rifabutin-based therapies were effective in eliminating this infection compared to the conventional protocols. Sitafloxacin should be explicitly considered to eradicate levofloxacin-resistant strains, while garenoxacin should be avoided. In the study conducted by Miftahussurur and colleagues, the researchers looked at successful treatment techniques for H. pylori infections in Bangladesh and Nepal, two countries where there was a considerable amount of resistance to the primary medicines [##REF##30815255##25##]. Patients with adult dyspepsia who were undergoing endoscopies at the Tribhuvan University Teaching Hospital (TUTH) were included in this study. Patients came from both Nepal and Bangladesh. After homogenizing the antral biopsy tissues, 42 strains were successfully recovered from Nepal (146 patients), while 56 strains were successfully recovered from Bangladesh. These strains were then cultivated at 37°C for up to 10 days. Subcultures of H. pylori were grown in the same microaerophilic conditions on antibiotic-free Mueller-Hinton II agar medium with 7% horse blood. Brucella broth with 10% dimethyl sulfoxide and 10% horse serum was used to keep the H. pylori solution at an ideal temperature of 80°C. Two-fold agar dilution was employed to determine antibiotic susceptibility. The five antibiotics examined were rifaximin, sitafloxacin, furazolidone, garenoxacin, and rifabutin.</p>\",\n \"<p>Rifabutin and furazolidone were not resistant; however, sitafloxacin susceptibility was very high. In contrast, rifaximin was highly resistant. Garenoxacin resistance is also more prevalent in Bangladesh than in Nepal because of its association with levofloxacin resistance. As a remedy for this infection, Rifabutin should be taken cautiously, according to the authors, because of the drug's interaction with Bangladesh's widespread tuberculosis. The high susceptibility to furazolidone and sitafloxacin increased the prospects for their future use in Bangladesh and Nepal.</p>\",\n \"<p>In a study, Muhammad Miftahussurur et al. sought to identify alternative H. pylori antibiotics for the Dominican Republic to mitigate resistance, which is widespread throughout the entire nation [##UREF##4##26##]. The MICs of five different antibiotics were determined using a two-fold agar dilution method against a panel of 63 different strains from the Dominican Republic. We employed next-generation sequencing to analyze genomic changes associated with antibiotic resistance. Rifabutin, furazolidone, and sitafloxacin were active against all 63 strains. On the other hand, resistance to rifaximin and garenoxacin was widespread (82.5% and 34.9%, respectively). Garenoxacin resistance (8/9, 88.9%, OR=45.33, P=0.002) and dual resistance to antibiotics (7/9, 77.8%, OR=31.5, P=0.009) were more likely in individuals older than 60 years old (P=0.004, r=0.357). More than three changes in rpoB were present in most rifaximin-resistant strains, with the most common new variants being S352L, I2726L, and V2465A. A strong correlation between the vacA genotype and rifaximin resistance was found (P=0.042). The lowest inhibitory concentration was significantly higher among the 23 levofloxacin-resistant organisms (39.1%, 9/23) and was positively correlated with resistance (P=0.001, r=0.84). Additionally, the vast majority of these organisms were resistant to garenoxacin (82.6%, 19/23, P=0.001). In a study with 10 strains, 16 (or 84.2% of the total) exhibited gyrA mutations at D91 and N87, making them resistant to garenoxacin.</p>\",\n \"<p>The study exhibited several limitations, outlined as follows: first, due to the limited sample size and the exclusive focus on samples collected solely from the capital of the Dominican Republic, the generalizability of the results to the rest of Latin America was compromised. Second, the study examined only a subset of the numerous H. pylori genes that could potentially be associated with antibiotic resistance, thus offering a limited understanding of the topic. Third, the resistance rates of the five different antibiotics were solely estimated through in vitro experimentation. Alternatives to levofloxacin and metronidazole that could be incorporated into the eradication strategy include sitafloxacin, rifabutin, and furazolidone. Such regions include the Dominican Republic, which has a high rate of resistance to these medications.</p>\",\n \"<p>The purpose of the prospective randomized investigation by Hideki Mori et al. is to determine whether rifabutin, used as part of a triple therapy regimen, is effective as a third- or fourth-line rescue medication [##UREF##5##27##]. Participants in the trial were those who had not improved after receiving both primary and secondary eradication treatments. A stomach biopsy was carried out to ascertain whether the rpoB mutation was present. For 10 or 14 days, patients took eradication medication with rifabutin (300 mg once daily), amoxicillin (500 mg four times daily), and esomeprazole (20 mg four times daily). Eighty percent of the sample reported that they did not take their medication as prescribed. A 13C UBT or a stool antigen test was used to establish if H. pylori had been successfully eradicated from the body after therapy had concluded. There were a total of 22 people who took part in the experiment, 12 of whom were placed in the 10-day group and 17 in the 14-day group. In the 14-day group, the success rate of eradicating the infection was 94.1% while in the 10-day group it was 81.8% (depending on the technique of analysis). Additionally, between 8% and fewer than 30% of patients in the two cohorts stopped taking their prescriptions as a result of side effects including diarrhea.</p>\",\n \"<p>However, more patients in the 10-day group finished the fourth eradication medication, suggesting that the 14-day group's success rate may have been overestimated. In addition, there was no reliable way to assess eradication rates, rpoB mutations, or rifabutin MICs. Finally, we can say that both the 10-day and 14-day rescue regimens worked, with the latter reaching an eradication rate of over 90%. However, when therapeutic tolerance is taken into account, it is possible that a 10-day course of treatment is all that is necessary for complete eradication.</p>\",\n \"<p>A few issues with this study should be noted. The self-reported approach for assessing therapy compliance was the first. This approach may be limited by its dubious precision and subpar assessment accuracy. Second, there needs to be more resistance data due to the exclusive reliance on clinical assessment without including susceptibility testing. As a result, the reasons for earlier treatment failures in these patients (lack of drug adherence or a rise in antibiotic resistance) are unknown. Furthermore, the study population exhibited a lack of complete homogeneity due to various characteristics of the study design.</p>\",\n \"<p>In summary, our study demonstrated that, when repeated attempts to eradicate H. pylori using standard antibiotics failed, rifabutin-based rescue therapy is a practical and secure option. A sizable group of people who were very difficult to treat participated in the trial. In addition, Saracino et al. carried out a retrospective analysis to examine how antibiotic resistance emerged, how therapy progressed, and how MIC values changed over time in patients who had been unable to eradicate H. pylori with at least one medication [##REF##32183165##28##]. The effectiveness of levofloxacin, metronidazole, and clarithromycin against H. pylori bacteria was examined in a study including individuals who had UGIE after prior medication. Pylera® or a susceptibility-guided treatment plan was provided to the patients.</p>\",\n \"<p>Antibiotic susceptibility data was provided for 1037 of the 1223 patients who tested positive for H. pylori. Antibiotic resistance was found to be at substantially higher rates, MIC values were much lower than expected, and the number of failed treatment efforts was much higher than expected. Except for when sequential therapy is utilized as a third-line therapy, the eradication rates of antibiogram-tailored drugs remained unchanged. It was found that when Pylera® therapy was utilized as a last resort, it resulted in cure rates comparable to those of other culture-guided treatments. Unfortunately, the Pylera® regimen is difficult to administer and more frequently leads to adverse effects than alternative treatments. Up to 14 pills taken four times over the course may be necessary, and more than 30% of patients experience side effects, which increases the likelihood that therapy will be stopped early.</p>\",\n \"<p>In conclusion, the number of therapeutic failures was inversely linked to the rate and MIC values of secondary resistance to the three tested antibiotics. The eradication rates from Pylera® therapy were comparable to those from treatments tailored to a particular culture. Rifabutin, furazolidone, and other antibacterial medications were tested for their antimicrobial efficacy in a second prospective trial by Youn I Choi et al. with 200 participants to investigate their potential efficacy as therapies for multidrug-resistant H. pylori along with H. pylori strains, which are resistant to other antibiotics [##UREF##6##29##]. After conducting a retrospective analysis of the information available at the Helicobacter Registry of two famous medical centers, along with four reference strains, a total of 31 single- or multidrug-resistant H. pylori bacteria were selected for testing. The strains were subjected to the broth microdilution technique to evaluate their resistance to a number of different antibiotics.</p>\",\n \"<p>No rifabutin- or furazolidone-resistant H. pylori isolates were discovered among the 31 antibiotic-resistant strains. Only one of the strains tested positive for tetracycline resistance. Rifabutin and furazolidone are more effective at curing the infection than amoxicillin and levofloxacin, which have resistance rates of 22.6% and 1.9%, respectively. Only 3.2% of H. pylori bacteria were resistant to the antibiotic tetracycline. Since the eradication rate was not demonstrated by treating actual patients, it is possible that the in vitro results do not reflect what happens in vivo. It is also important to remember that there may be a disparity between the results of the study and the actual prevalence of antibiotic-resistant H. pylori in Korea due to selection bias. Clinicians should exercise caution before extrapolating the findings to the general public due to ethical considerations, and the research's lack of information on antibiotic resistance in H. pylori is another drawback.</p>\",\n \"<p>In conclusion, rifabutin, furazolidone, and tetracycline can be used to eliminate this infection successfully. They can be used as solitary treatment options or together as a complete regimen in regions where species of this organism have developed very high rates of resistance. However, further research and careful consideration are warranted before implementing these findings on a broader scale. As H. pylori becomes increasingly resistant to medications, new approaches are required to treat this persistent infection.</p>\",\n \"<p>The review of key data presented in this research sheds light on the challenges posed by antibiotic-resistant H. pylori and the need to develop new therapeutic techniques. In view of the proliferation of multidrug-resistant strains of H. pylori, the development of new drugs and treatment methods is essential for the successful elimination of this pathogen. Rifabutin, furazolidone, and sitafloxacin have shown promise as potential therapeutic options, especially in regions where resistance to conventional antibiotics is prevalent.</p>\",\n \"<p>Healthcare providers must keep abreast of the most recent findings on H. pylori resistance patterns and treatment choices in order to give patients with H. pylori infection effective and individualized therapeutic interventions. Furthermore, larger-scale studies with a more representative sample of the general population are required to demonstrate the efficacy and safety of these supplementary drugs.</p>\",\n \"<p>In conclusion, combating H. pylori infection and the ensuing rise in antibiotic resistance requires an all-encompassing strategy that includes continual monitoring, the cautious use of drugs, and the exploration of potential innovative treatments. The medical community has to make considerable efforts worldwide to improve H. pylori infection treatment and reduce the prevalence of related illnesses.</p>\",\n \"<p>Limitations</p>\",\n \"<p>There were no in vitro clinical trials included in this systematic review. We might have overlooked a few crucial publications because we could not obtain a few articles as the complete text was unavailable, and these were not included in our study.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<p>We would like to express our gratitude to all those who contributed to the successful completion of this systematic review article. It is through the collective efforts and dedication of each author that this work has come to fruition.</p>"],"string":"[\n \"<p>We would like to express our gratitude to all those who contributed to the successful completion of this systematic review article. It is through the collective efforts and dedication of each author that this work has come to fruition.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>PRISMA chart</title><p>PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses</p></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>PRISMA chart</title><p>PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Data extraction using the MeSH strategy</title><p>MeSH, Medical Subject Headings</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">MeSH Strategy Used</td><td rowspan=\"1\" colspan=\"1\">Results Obtained</td></tr><tr><td rowspan=\"1\" colspan=\"1\">''Helicobacter/genetics''[Mesh] OR ''Helicobacter/isolation and purification''[Mesh] OR ''Helicobacter/pathogenicity''[Mesh] OR ''Helicobacter/physiology''[Mesh] ) OR ( ''Helicobacter pylori/analysis''[Mesh] OR ''Helicobacter pylori/drug effects''[Mesh] OR ''Helicobacter pylori/etiology''[Mesh] OR ''Helicobacter pylori/genetics''[Mesh] OR ''Helicobacter pylori/isolation and purification''[Mesh] OR ''Helicobacter pylori/pathogenicity''[Mesh] OR ''Helicobacter pylori/physiology''[Mesh] )) AND ( ''Rifabutin/administration and dosage''[Mesh] OR ''Rifabutin/adverse effects''[Mesh] OR ''Rifabutin/analogs and derivatives''[Mesh] OR ''Rifabutin/analysis''[Mesh] OR ''Rifabutin/antagonists and inhibitors''[Mesh] OR ''Rifabutin/economics''[Mesh] OR ''Rifabutin/etiology''[Mesh] OR ''Rifabutin/immunology''[Mesh] OR ''Rifabutin/metabolism''[Mesh] OR ''Rifabutin/organization and administration''[Mesh] OR ''Rifabutin/pharmacokinetics''[Mesh] OR ''Rifabutin/pharmacology''[Mesh] OR ''Rifabutin/physiology''[Mesh] OR ''Rifabutin/statistics and numerical data''[Mesh] OR ''Rifabutin/supply and distribution''[Mesh] OR ''Rifabutin/therapeutic use''[Mesh] OR ''Rifabutin/toxicity''[Mesh] ) AND (treatment* OR regimen* OR therap* OR Clinical Protocols OR Treatment outcome OR Treatment Failure OR Disease Eradication OR Eradication OR Drug Therapy, Combination)</td><td rowspan=\"1\" colspan=\"1\">62</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Rifabutin/administration and dosage''[Mesh] OR ''Rifabutin/adverse effects''[Mesh] OR ''Rifabutin/metabolism''[Mesh] OR ''Rifabutin/pharmacokinetics''[Mesh] OR ''Rifabutin/therapeutic use''[Mesh] OR ''Rifabutin/toxicity''[Mesh]</td><td rowspan=\"1\" colspan=\"1\">47</td></tr><tr><td rowspan=\"1\" colspan=\"1\">The number of titles obtained.</td><td rowspan=\"1\" colspan=\"1\">109</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>PubMed search strategy</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">PubMed Search Strategy</td><td rowspan=\"1\" colspan=\"1\">Results Obtained</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Rifabutin AND peptic ulcer</td><td rowspan=\"1\" colspan=\"1\">7</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Vonoprazan AND rifabutin AND Helicobacter</td><td rowspan=\"1\" colspan=\"1\">10</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Helicobacter AND Rifabutin</td><td rowspan=\"1\" colspan=\"1\">48</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">The number of titles obtained.</td><td rowspan=\"1\" colspan=\"1\">65</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB3\"><label>Table 3</label><caption><title>Critical appraisal using NewCastle-Ottawa Quality Assessment Tool evaluation for case control and cohort studies and Cochrane Risk of Bias Tool for RCT</title><p>RCT, randomized control trial</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n<sup>Study</sup>\n</td><td rowspan=\"1\" colspan=\"1\">\n<sup>Non-Bias Percentage</sup>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">DG Ribaldone et al., 2019 [##REF##30736338##2##]</td><td rowspan=\"1\" colspan=\"1\">100%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">David Y Graham et al., 2020 [##REF##32365359##15##] \n</td><td rowspan=\"1\" colspan=\"1\">66.66%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Erick A Argueta et al., 2022 [##REF##33577874##17##]</td><td rowspan=\"1\" colspan=\"1\">77.77%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Youn I Choi et al., 2019 [##REF##31360270##18##]</td><td rowspan=\"1\" colspan=\"1\">66.66%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Angel Cosme et al., 2017 [##REF##28566898##19##]</td><td rowspan=\"1\" colspan=\"1\">66.66%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Kazumi Inokuchi et al., 2022 [##UREF##2##20##]</td><td rowspan=\"1\" colspan=\"1\">66.66%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ira N Kalfus et al., 2020 [##REF##33050205##21##]</td><td rowspan=\"1\" colspan=\"1\">83.33%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Chia-Jung Kuo et al., 2021 [##REF##33840604##22##]</td><td rowspan=\"1\" colspan=\"1\">88.88%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Lee CM et al., 2022 [##UREF##3##23##]</td><td rowspan=\"1\" colspan=\"1\">55.55%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Miftahussurur et al., 2019 [##REF##30774400##24##]</td><td rowspan=\"1\" colspan=\"1\">77.77%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Miftahussurur et al., 2019 [##REF##30815255##25##]</td><td rowspan=\"1\" colspan=\"1\">77.77%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Miftahussurur et al., 2019 [##UREF##4##26##]</td><td rowspan=\"1\" colspan=\"1\">77.77%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hideki Mori et al., 2016 [##UREF##5##27##]</td><td rowspan=\"1\" colspan=\"1\">66.66%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Saracino IM et al., 2020 [##REF##32183165##28##]</td><td rowspan=\"1\" colspan=\"1\">44.44%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Youn I Choi et al., 2019 [##UREF##6##29##]</td><td rowspan=\"1\" colspan=\"1\">66.66%</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB4\"><label>Table 4</label><caption><title>Result table with study characteristics</title><p>RCT, randomized control trial; OAL, omeprazole, amoxicillin, and levofloxacin; OAC, omeprazole, amoxicillin, and clarithromycin; OAM, omeprazole, amoxicillin, and metronidazole; EAR, esomeprazole, amoxicillin, and rifabutin; MRT, moxifloxacin-rifabutin triple therapy; EAC, esomeprazole, amoxicillin, and clarithromycin; EAM, esomeprazole, amoxicillin, and metronidazole; EAL, esomeprazole, amoxicillin, and levofloxacin; EBAM, esomeprazole, bismuth, amoxicillin, and metronidazole; VAR, vonoprazan, amoxicillin, rifabutin; ITT, intention to treat; PP, per protocol</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n<ext-link xlink:href=\"https://Sr.no\" ext-link-type=\"uri\">Sr.no</ext-link>.</td><td rowspan=\"1\" colspan=\"1\">Number of Studies As in the References</td><td rowspan=\"1\" colspan=\"1\">Study Setting</td><td rowspan=\"1\" colspan=\"1\">Total no. of Participants</td><td rowspan=\"1\" colspan=\"1\">Type of Study</td><td rowspan=\"1\" colspan=\"1\">Mean Age</td><td rowspan=\"1\" colspan=\"1\">Antibiotic Regimen Used Cohort 1/Control Group/Group 1</td><td rowspan=\"1\" colspan=\"1\">Cohort 2/Case Group/Group 2</td><td rowspan=\"1\" colspan=\"1\">Mean Dose</td><td rowspan=\"1\" colspan=\"1\">Mean Duration</td><td rowspan=\"1\" colspan=\"1\">Effectiveness of Eradication in Cohort 1/Control Group</td><td rowspan=\"1\" colspan=\"1\">Effectiveness of Eradication in Cohort 2/Case Group</td><td rowspan=\"1\" colspan=\"1\">Dropouts</td><td rowspan=\"1\" colspan=\"1\">Resistance to Antimicrobial Drugs (%)</td><td rowspan=\"1\" colspan=\"1\">Year</td></tr><tr><td rowspan=\"1\" colspan=\"1\">1.</td><td rowspan=\"1\" colspan=\"1\">[##REF##30736338##2##]</td><td rowspan=\"1\" colspan=\"1\">Italy</td><td rowspan=\"1\" colspan=\"1\">302</td><td rowspan=\"1\" colspan=\"1\">Cohort</td><td rowspan=\"1\" colspan=\"1\">57±13 years</td><td rowspan=\"1\" colspan=\"1\">Men (n=135). Patients received twice-daily treatments of rifabutin 150 mg, amoxicillin 1 g, and a PPI </td><td rowspan=\"1\" colspan=\"1\">Women (n=167). Rifabutin 150 mg, 1 g of amoxicillin, and a PPI were given to the patients twice daily </td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">14 days</td><td rowspan=\"1\" colspan=\"1\">In the ITT analysis, the eradication rate was 84/119 (70.6%). The remaining 19 patients had adverse reactions</td><td rowspan=\"1\" colspan=\"1\">In the ITT analysis, the eradication rate was 132/183 (72.1%)</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">Resistance to rifabutin was low</td><td rowspan=\"1\" colspan=\"1\">2019</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">2.</td><td rowspan=\"1\" colspan=\"1\">[##REF##32365359##15##]</td><td rowspan=\"1\" colspan=\"1\">USA</td><td rowspan=\"1\" colspan=\"1\">455</td><td rowspan=\"1\" colspan=\"1\">RCT</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">The regimen used was amoxicillin 3 g, omeprazole 120 mg, and rifabutin 150 mg, administered to 228 patients </td><td rowspan=\"1\" colspan=\"1\">Here amoxicillin 3 g and omeprazole 120 mg were administered to 227 individuals</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">14 days</td><td rowspan=\"1\" colspan=\"1\">As per ITT analysis, the eradication rate with RHB-105 was 83.8%</td><td rowspan=\"1\" colspan=\"1\">As per ITT analysis, the eradication rate with the active comparator was 57.7%</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Resistant to rifabutin regimen=16.2%. Resistant to active comparator=42.3%</td><td rowspan=\"1\" colspan=\"1\">2020</td></tr><tr><td rowspan=\"1\" colspan=\"1\">3.</td><td rowspan=\"1\" colspan=\"1\">[##REF##33577874##17##]</td><td rowspan=\"1\" colspan=\"1\">USA</td><td rowspan=\"1\" colspan=\"1\">187</td><td rowspan=\"1\" colspan=\"1\">Cohort study</td><td rowspan=\"1\" colspan=\"1\">45.2 years</td><td rowspan=\"1\" colspan=\"1\">101 patients on BQ regimen: PPI-tetracycline-metronidazole-bismuth (79), PPI-doxycycline- metronidazole-bismuth (18), PPI-clarithromycin-amoxicillin-metronidazole (4)</td><td rowspan=\"1\" colspan=\"1\">Eighty-six patients on triple regimen. PPI-clarithromycin-amoxicillin (62), PPI-clarithromycin-metronidazole (18), PPI-amoxicillin-metronidazole (4), and PPI-rifabutin-amoxicillin (2)</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">14 days</td><td rowspan=\"1\" colspan=\"1\">Cohort 1: 89 (88.1%)</td><td rowspan=\"1\" colspan=\"1\">Cohort 2: 52 (60.5%) PP-rifabutin-amoxicillin (50%)</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Resistance to ≥1 antimicrobials=65.6, metronidazole=33.3, clarithromycin=30.0, levofloxacin=29.6, amoxicillin=1.1, rifabutin=0.5, tetracycline=0.5</td><td rowspan=\"1\" colspan=\"1\">2022</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">4.</td><td rowspan=\"1\" colspan=\"1\">[##REF##31360270##18##]</td><td rowspan=\"1\" colspan=\"1\">Korea</td><td rowspan=\"1\" colspan=\"1\">31</td><td rowspan=\"1\" colspan=\"1\">Cohort study</td><td rowspan=\"1\" colspan=\"1\">58.2±10.3 years</td><td rowspan=\"1\" colspan=\"1\">Nine resistant to one antibiotic; however, no resistance developed to rifabutin and furazolidone</td><td rowspan=\"1\" colspan=\"1\">22 resistant to more than one antibiotic; however, no resistance developed to rifabutin and furazolidone</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Cohort 1: 9 (100%)</td><td rowspan=\"1\" colspan=\"1\">Cohort 2: 22 (100%)</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Clarithromycin=71.1, levofloxacin=41.9, metronidazole=67.7, amoxicillin=22.6, rifabutin=0, furazolidone=0</td><td rowspan=\"1\" colspan=\"1\">2019</td></tr><tr><td rowspan=\"1\" colspan=\"1\">5.</td><td rowspan=\"1\" colspan=\"1\">[##REF##28566898##19##]</td><td rowspan=\"1\" colspan=\"1\">Spain</td><td rowspan=\"1\" colspan=\"1\">80</td><td rowspan=\"1\" colspan=\"1\">Cohort study</td><td rowspan=\"1\" colspan=\"1\">51.92 years</td><td rowspan=\"1\" colspan=\"1\">Based on susceptibility, 68 patients with dual antibiotic resistance were given triple regimens: OAL (43), OAC (12), and OAM (13).</td><td rowspan=\"1\" colspan=\"1\">12 patients with triple antibiotic resistance were treated with OAR</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">10 days</td><td rowspan=\"1\" colspan=\"1\">Cohort 1: OAL (97.6%), OAC (92.3%), and OAM (91.6%)</td><td rowspan=\"1\" colspan=\"1\">Cohort 2: OAR (58.3%)</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Dual antibiotic-resistant patients after therapy=<10% resistant. Triple antibiotic-resistant patients after treatment with rifabutin regimen=41.7%</td><td rowspan=\"1\" colspan=\"1\">2017</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">6.</td><td rowspan=\"1\" colspan=\"1\">[##UREF##2##20##]</td><td rowspan=\"1\" colspan=\"1\">Japan</td><td rowspan=\"1\" colspan=\"1\">69</td><td rowspan=\"1\" colspan=\"1\">Case control</td><td rowspan=\"1\" colspan=\"1\">Control: 50.3±13.9. Case group: 52.5±9.5</td><td rowspan=\"1\" colspan=\"1\">12 patients in historical control were given EAR therapy regimen</td><td rowspan=\"1\" colspan=\"1\">57 patients were given VAR therapy regimen</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Control, 10 days. Case Group, 7 days</td><td rowspan=\"1\" colspan=\"1\">Control group: as per ITT analysis, the eradication rate was 83.3%, and as per PP analysis, the eradication rate was 81.8%</td><td rowspan=\"1\" colspan=\"1\">Case group: as per ITT analysis, the eradication rate was 91.2%, and as per PP analysis, eradication rate was 92.7%.</td><td rowspan=\"1\" colspan=\"1\">18 dropouts in the case group</td><td rowspan=\"1\" colspan=\"1\">Resistance to 10 say rifabutin regimen=16.7%. Resistance to 7-day rifabutin regimen=8.8%</td><td rowspan=\"1\" colspan=\"1\">2022</td></tr><tr><td rowspan=\"1\" colspan=\"1\">7.</td><td rowspan=\"1\" colspan=\"1\">[##REF##33050205##21##]</td><td rowspan=\"1\" colspan=\"1\">USA</td><td rowspan=\"1\" colspan=\"1\">118</td><td rowspan=\"1\" colspan=\"1\">RCT</td><td rowspan=\"1\" colspan=\"1\">46.0±10.18</td><td rowspan=\"1\" colspan=\"1\">RHB-105 (amoxicillin 3 g, omeprazole 120 mg, and rifabutin 150 mg) was administered to n=77 individuals </td><td rowspan=\"1\" colspan=\"1\">n=41. Patients were treated with placebo</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">14 days</td><td rowspan=\"1\" colspan=\"1\">As per ITT analysis, 59/66, the eradication rate was 89.4%, and as per PP analysis, 56/63, eradication rate was 88.9%</td><td rowspan=\"1\" colspan=\"1\">As per ITT analysis, 17/33, i.e., eradication rate was 51.5%.</td><td rowspan=\"1\" colspan=\"1\">In the RHB-105 group: 11 dropouts, and in the placebo group: 36 dropouts</td><td rowspan=\"1\" colspan=\"1\">Resistance to rifabutin regimen=10.6%</td><td rowspan=\"1\" colspan=\"1\">2020</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">8.</td><td rowspan=\"1\" colspan=\"1\"> [##REF##33840604##22##]</td><td rowspan=\"1\" colspan=\"1\">Taiwan</td><td rowspan=\"1\" colspan=\"1\">41</td><td rowspan=\"1\" colspan=\"1\">Cohort</td><td rowspan=\"1\" colspan=\"1\">53.8 years</td><td rowspan=\"1\" colspan=\"1\">Antimicrobial resistance: amoxicillin (34.1%), clarithromycin (92.7%), metronidazole (65.9%), tetracycline (2.4%), levofloxacin (85.4%), and rifabutin (29.3%) </td><td rowspan=\"1\" colspan=\"1\">Dual antibiotic resistance to clarithromycin+levofloxacin (73.2%)</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">10-14 days</td><td rowspan=\"1\" colspan=\"1\">Antimicrobial susceptibility: amoxicillin (65.9%), clarithromycin (7.3%), metronidazole (34.1%), tetracycline (97.6%), levofloxacin (14.6%), rifabutin (70.7%) </td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Resistance to rifabutin=29.3%, amoxicillin=34.1%, clarithromycin=92.7%, metronidazole=65.9%, tetracycline=2.4%, levofloxacin=85.4%</td><td rowspan=\"1\" colspan=\"1\">2021</td></tr><tr><td rowspan=\"1\" colspan=\"1\">9.</td><td rowspan=\"1\" colspan=\"1\">[##UREF##3##23##]</td><td rowspan=\"1\" colspan=\"1\">Korea</td><td rowspan=\"1\" colspan=\"1\">323</td><td rowspan=\"1\" colspan=\"1\">Cohort</td><td rowspan=\"1\" colspan=\"1\">Cohort 1: 61.7±12.6. Cohort 2: 58.0±12.1</td><td rowspan=\"1\" colspan=\"1\">MRT therapy regimen was administered to 71 patients following failure of the first-line clarithromycin regimen</td><td rowspan=\"1\" colspan=\"1\">BQ therapy regimen was administered to 252 patients following failure of the first-line clarithromycin regimen</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Cohort 1, 7 days. Cohort 2, 14 days</td><td rowspan=\"1\" colspan=\"1\">69% eradication rate in ITT analysis and 77.8% in PP analysis </td><td rowspan=\"1\" colspan=\"1\">82.5% eradication rate in the ITT analysis, and 89.3% in the PP analysis</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Resistance to MRT regimen=22.2%</td><td rowspan=\"1\" colspan=\"1\">2022</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">10.</td><td rowspan=\"1\" colspan=\"1\">[##REF##30774400##24##]</td><td rowspan=\"1\" colspan=\"1\">Java Island</td><td rowspan=\"1\" colspan=\"1\">105</td><td rowspan=\"1\" colspan=\"1\">Cohort</td><td rowspan=\"1\" colspan=\"1\">Males range in age from 17 to 88; the mean age is (46.14±13.63). Females ranged in age from 14 to 80; the mean was 47.79±14.4 years </td><td rowspan=\"1\" colspan=\"1\">61/105 ( 58.1%) were sensitive to all five antibiotics examined: furazolidone, sitafloxacin, garenoxacin, rifaximin, and rifabutin. 7/105 (6.7%) were resistant to garenoxacin, and 40/105 (38.9%) were resistant to rifaximin</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">10 days</td><td rowspan=\"1\" colspan=\"1\">No isolates were tested for resistance to furazolidone, rifabutin, or sitafloxacin </td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Resistance to rifabutin=0, furazolidone=0, sitafloxacin=0, garenoxacin=6.7%, rifaximin=38.9% .</td><td rowspan=\"1\" colspan=\"1\">2019</td></tr><tr><td rowspan=\"1\" colspan=\"1\">11.</td><td rowspan=\"1\" colspan=\"1\">[##REF##30815255##25##]</td><td rowspan=\"1\" colspan=\"1\">Bangladesh and Nepal</td><td rowspan=\"1\" colspan=\"1\">98</td><td rowspan=\"1\" colspan=\"1\">Cohort</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Nepal (n=42). Antibiotic resistance was as follows: garenoxacin 12/42 (28.6%), sitafloxacin 2/42 (4.8%), furazolidone 0/42 (0.0%), rifabutin 0/42 (0.0%), rifaximin 22/42 (52.4%)</td><td rowspan=\"1\" colspan=\"1\">Bangladesh (n=56). Antibiotic resistance was as follows: garenoxacin 29/56 (51.8%), sitafloxacin 1/56 (1.8%), furazolidone 0/56 (0.0%), rifabutin 0/56 (0.0%), and rifaximin 36/56 (64.3%)</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">10 days</td><td rowspan=\"1\" colspan=\"1\">None of the examined strains exhibited resistance to furazolidone or rifabutin</td><td rowspan=\"1\" colspan=\"1\">None of the examined strains exhibited resistance to furazolidone or rifabutin</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Resistance in Bangladesh and Nepal to rifabutin=0, furazolidone=0</td><td rowspan=\"1\" colspan=\"1\">2019</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">12.</td><td rowspan=\"1\" colspan=\"1\">[##UREF##4##26##]</td><td rowspan=\"1\" colspan=\"1\">Dominican Republic</td><td rowspan=\"1\" colspan=\"1\">63</td><td rowspan=\"1\" colspan=\"1\">Cohort</td><td rowspan=\"1\" colspan=\"1\">40-49 years</td><td rowspan=\"1\" colspan=\"1\">Antibiotic resistance was as follows: rifaximin 52/62 (82.5%), garenoxacin 22/63 (34.9%), sitafloxacin 0/63 (0.0%), furazolidone 0/63 (0.0%), rifabutin 0/63 (0.0%)</td><td rowspan=\"1\" colspan=\"1\">Antibiotic resistance to rifaximin and garenoxacin is doubled (18/63) or 28.6% </td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">10 days</td><td rowspan=\"1\" colspan=\"1\">Rifabutin, sitafloxacin, and furazolidone are not resistant to antibiotics in the Dominican Republic. All strains were found to be responsive to rifabutin in the study, confirming the recommendation of rifabutin as a possible antibiotic to treat H. pylori infection </td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Resistance to rifabutin=0, sitafloxacin=0</td><td rowspan=\"1\" colspan=\"1\">2019</td></tr><tr><td rowspan=\"1\" colspan=\"1\">13.</td><td rowspan=\"1\" colspan=\"1\">[##UREF##5##27##]</td><td rowspan=\"1\" colspan=\"1\">Japan</td><td rowspan=\"1\" colspan=\"1\">29</td><td rowspan=\"1\" colspan=\"1\">Cohort</td><td rowspan=\"1\" colspan=\"1\">48.0±11.1 years</td><td rowspan=\"1\" colspan=\"1\">In the 10-day group, 12 patients received esomeprazole 20 mg q.i.d., amoxicillin 500 mg q.i.d., and rifabutin 300mg q.i.d. </td><td rowspan=\"1\" colspan=\"1\">The 14-day group, which included 17 patients, received esomeprazole 20 mg q.i.d., amoxicillin 500 mg q.i.d., and rifabutin 300 mg q.i.d. </td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Cohort 1= 10 days. Cohort 2= 14 days</td><td rowspan=\"1\" colspan=\"1\">In the ITT analysis, the eradication rate was 10/12, i.e., 83.3%, and in the PP analysis, the eradication rate was 9/11, i.e., 81.8%. Overall eradication rate was 80% </td><td rowspan=\"1\" colspan=\"1\">In the ITT analysis, the eradication rate was 16/17, i.e., (94.1%), and in the PP analysis, the eradication rate was 11/12, i.e., (91.7%). Overall eradication rate was 90%</td><td rowspan=\"1\" colspan=\"1\">Cohort 1=1 Cohort 2=5</td><td rowspan=\"1\" colspan=\"1\">Resistance to rifabutin in 10 day regimen=0, in 14 day regimen=5.9%</td><td rowspan=\"1\" colspan=\"1\">2016</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">14.</td><td rowspan=\"1\" colspan=\"1\">[##REF##32183165##28##]</td><td rowspan=\"1\" colspan=\"1\">Italy</td><td rowspan=\"1\" colspan=\"1\">1037</td><td rowspan=\"1\" colspan=\"1\">Single-center, open-label, single-arm prospective interventional trial </td><td rowspan=\"1\" colspan=\"1\">52 years</td><td rowspan=\"1\" colspan=\"1\">n=415. These are the patients with only one treatment regimen failure. The regimens that failed were as follows: sequential, 67/415; levofloxacin-based, 221/415; rifabutin-based, 109/415; pylera, 18/415</td><td rowspan=\"1\" colspan=\"1\">n= 310. These are the patients with two treatment regimen failures. The regimens that failed were as follows: sequential, 29/310; levofloxacin-based, 132/310; rifabutin-based, 129/310; pylera 20/310. n=312. These are the patients with ≥3 treatment regimen failures. The regimens that failed were as follows: sequential, 18/312; levofloxacin-based, 63/312; rifabutin-based, 216/312; pylera, 15/312</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">10-14 days</td><td rowspan=\"1\" colspan=\"1\">A significant increase in the secondary resistance toward the three tested antibiotics was observed, both as rate and MIC values, in correlation with the number of therapy failures</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Resistance to rifabutin after one therapy regimen failure=26%; Resistance to rifabutin after two therapy regimen failur=41.6%. Resistance to rifabutin after ≥3 therapy regimens failur=69.2%</td><td rowspan=\"1\" colspan=\"1\">2020</td></tr><tr><td rowspan=\"1\" colspan=\"1\">15.</td><td rowspan=\"1\" colspan=\"1\">[##UREF##6##29##]</td><td rowspan=\"1\" colspan=\"1\">Korea China</td><td rowspan=\"1\" colspan=\"1\">200</td><td rowspan=\"1\" colspan=\"1\">Single-center, prospective, open-label, single-arm interventional study </td><td rowspan=\"1\" colspan=\"1\">47.9 years</td><td rowspan=\"1\" colspan=\"1\">n=12 for EAC. n=13 for EAM. n=31 for EAL. EBAM, n=144 </td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">14 days</td><td rowspan=\"1\" colspan=\"1\">According to ITT analysis, the following regimens had the highest rates of eradication: EAC Group 11/12 (91.7%), EAM Group 12/13 (92.3%), EAL Group 29/31 (93.5%), and EBAM Group 137/144 (95.1%). Thus, despite numerous earlier H. pylori treatment failures, susceptibility-guided therapy proved highly efficient</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">The resistance rates for clarithromycin, metronidazole, and levofloxacin were 98.3%, 99.2%, and 93.9%, respectively, among patients who had previously received these antibiotics as part of their treatments</td><td rowspan=\"1\" colspan=\"1\">2019</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB1\\\"><label>Table 1</label><caption><title>Data extraction using the MeSH strategy</title><p>MeSH, Medical Subject Headings</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">MeSH Strategy Used</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Results Obtained</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">''Helicobacter/genetics''[Mesh] OR ''Helicobacter/isolation and purification''[Mesh] OR ''Helicobacter/pathogenicity''[Mesh] OR ''Helicobacter/physiology''[Mesh] ) OR ( ''Helicobacter pylori/analysis''[Mesh] OR ''Helicobacter pylori/drug effects''[Mesh] OR ''Helicobacter pylori/etiology''[Mesh] OR ''Helicobacter pylori/genetics''[Mesh] OR ''Helicobacter pylori/isolation and purification''[Mesh] OR ''Helicobacter pylori/pathogenicity''[Mesh] OR ''Helicobacter pylori/physiology''[Mesh] )) AND ( ''Rifabutin/administration and dosage''[Mesh] OR ''Rifabutin/adverse effects''[Mesh] OR ''Rifabutin/analogs and derivatives''[Mesh] OR ''Rifabutin/analysis''[Mesh] OR ''Rifabutin/antagonists and inhibitors''[Mesh] OR ''Rifabutin/economics''[Mesh] OR ''Rifabutin/etiology''[Mesh] OR ''Rifabutin/immunology''[Mesh] OR ''Rifabutin/metabolism''[Mesh] OR ''Rifabutin/organization and administration''[Mesh] OR ''Rifabutin/pharmacokinetics''[Mesh] OR ''Rifabutin/pharmacology''[Mesh] OR ''Rifabutin/physiology''[Mesh] OR ''Rifabutin/statistics and numerical data''[Mesh] OR ''Rifabutin/supply and distribution''[Mesh] OR ''Rifabutin/therapeutic use''[Mesh] OR ''Rifabutin/toxicity''[Mesh] ) AND (treatment* OR regimen* OR therap* OR Clinical Protocols OR Treatment outcome OR Treatment Failure OR Disease Eradication OR Eradication OR Drug Therapy, Combination)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">62</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Rifabutin/administration and dosage''[Mesh] OR ''Rifabutin/adverse effects''[Mesh] OR ''Rifabutin/metabolism''[Mesh] OR ''Rifabutin/pharmacokinetics''[Mesh] OR ''Rifabutin/therapeutic use''[Mesh] OR ''Rifabutin/toxicity''[Mesh]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">47</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The number of titles obtained.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">109</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB2\\\"><label>Table 2</label><caption><title>PubMed search strategy</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">PubMed Search Strategy</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Results Obtained</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Rifabutin AND peptic ulcer</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Vonoprazan AND rifabutin AND Helicobacter</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Helicobacter AND Rifabutin</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">48</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The number of titles obtained.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">65</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB3\\\"><label>Table 3</label><caption><title>Critical appraisal using NewCastle-Ottawa Quality Assessment Tool evaluation for case control and cohort studies and Cochrane Risk of Bias Tool for RCT</title><p>RCT, randomized control trial</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<sup>Study</sup>\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<sup>Non-Bias Percentage</sup>\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DG Ribaldone et al., 2019 [##REF##30736338##2##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">100%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">David Y Graham et al., 2020 [##REF##32365359##15##] \\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">66.66%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Erick A Argueta et al., 2022 [##REF##33577874##17##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">77.77%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Youn I Choi et al., 2019 [##REF##31360270##18##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">66.66%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Angel Cosme et al., 2017 [##REF##28566898##19##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">66.66%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kazumi Inokuchi et al., 2022 [##UREF##2##20##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">66.66%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Ira N Kalfus et al., 2020 [##REF##33050205##21##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">83.33%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Chia-Jung Kuo et al., 2021 [##REF##33840604##22##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">88.88%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Lee CM et al., 2022 [##UREF##3##23##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">55.55%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Miftahussurur et al., 2019 [##REF##30774400##24##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">77.77%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Miftahussurur et al., 2019 [##REF##30815255##25##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">77.77%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Miftahussurur et al., 2019 [##UREF##4##26##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">77.77%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hideki Mori et al., 2016 [##UREF##5##27##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">66.66%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Saracino IM et al., 2020 [##REF##32183165##28##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">44.44%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Youn I Choi et al., 2019 [##UREF##6##29##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">66.66%</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB4\\\"><label>Table 4</label><caption><title>Result table with study characteristics</title><p>RCT, randomized control trial; OAL, omeprazole, amoxicillin, and levofloxacin; OAC, omeprazole, amoxicillin, and clarithromycin; OAM, omeprazole, amoxicillin, and metronidazole; EAR, esomeprazole, amoxicillin, and rifabutin; MRT, moxifloxacin-rifabutin triple therapy; EAC, esomeprazole, amoxicillin, and clarithromycin; EAM, esomeprazole, amoxicillin, and metronidazole; EAL, esomeprazole, amoxicillin, and levofloxacin; EBAM, esomeprazole, bismuth, amoxicillin, and metronidazole; VAR, vonoprazan, amoxicillin, rifabutin; ITT, intention to treat; PP, per protocol</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<ext-link xlink:href=\\\"https://Sr.no\\\" ext-link-type=\\\"uri\\\">Sr.no</ext-link>.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Number of Studies As in the References</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Study Setting</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Total no. of Participants</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Type of Study</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Mean Age</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Antibiotic Regimen Used Cohort 1/Control Group/Group 1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 2/Case Group/Group 2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Mean Dose</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Mean Duration</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Effectiveness of Eradication in Cohort 1/Control Group</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Effectiveness of Eradication in Cohort 2/Case Group</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Dropouts</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to Antimicrobial Drugs (%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Year</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##REF##30736338##2##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Italy</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">302</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">57±13 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Men (n=135). Patients received twice-daily treatments of rifabutin 150 mg, amoxicillin 1 g, and a PPI </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Women (n=167). Rifabutin 150 mg, 1 g of amoxicillin, and a PPI were given to the patients twice daily </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">In the ITT analysis, the eradication rate was 84/119 (70.6%). The remaining 19 patients had adverse reactions</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">In the ITT analysis, the eradication rate was 132/183 (72.1%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to rifabutin was low</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2019</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##REF##32365359##15##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">USA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">455</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">RCT</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The regimen used was amoxicillin 3 g, omeprazole 120 mg, and rifabutin 150 mg, administered to 228 patients </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Here amoxicillin 3 g and omeprazole 120 mg were administered to 227 individuals</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">As per ITT analysis, the eradication rate with RHB-105 was 83.8%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">As per ITT analysis, the eradication rate with the active comparator was 57.7%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistant to rifabutin regimen=16.2%. Resistant to active comparator=42.3%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2020</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##REF##33577874##17##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">USA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">187</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort study</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">45.2 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">101 patients on BQ regimen: PPI-tetracycline-metronidazole-bismuth (79), PPI-doxycycline- metronidazole-bismuth (18), PPI-clarithromycin-amoxicillin-metronidazole (4)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Eighty-six patients on triple regimen. PPI-clarithromycin-amoxicillin (62), PPI-clarithromycin-metronidazole (18), PPI-amoxicillin-metronidazole (4), and PPI-rifabutin-amoxicillin (2)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 1: 89 (88.1%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 2: 52 (60.5%) PP-rifabutin-amoxicillin (50%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to ≥1 antimicrobials=65.6, metronidazole=33.3, clarithromycin=30.0, levofloxacin=29.6, amoxicillin=1.1, rifabutin=0.5, tetracycline=0.5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2022</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##REF##31360270##18##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Korea</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">31</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort study</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">58.2±10.3 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Nine resistant to one antibiotic; however, no resistance developed to rifabutin and furazolidone</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">22 resistant to more than one antibiotic; however, no resistance developed to rifabutin and furazolidone</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 1: 9 (100%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 2: 22 (100%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Clarithromycin=71.1, levofloxacin=41.9, metronidazole=67.7, amoxicillin=22.6, rifabutin=0, furazolidone=0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2019</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##REF##28566898##19##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Spain</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">80</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort study</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">51.92 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Based on susceptibility, 68 patients with dual antibiotic resistance were given triple regimens: OAL (43), OAC (12), and OAM (13).</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12 patients with triple antibiotic resistance were treated with OAR</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 1: OAL (97.6%), OAC (92.3%), and OAM (91.6%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 2: OAR (58.3%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Dual antibiotic-resistant patients after therapy=<10% resistant. Triple antibiotic-resistant patients after treatment with rifabutin regimen=41.7%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2017</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##UREF##2##20##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Japan</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">69</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Case control</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Control: 50.3±13.9. Case group: 52.5±9.5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12 patients in historical control were given EAR therapy regimen</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">57 patients were given VAR therapy regimen</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Control, 10 days. Case Group, 7 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Control group: as per ITT analysis, the eradication rate was 83.3%, and as per PP analysis, the eradication rate was 81.8%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Case group: as per ITT analysis, the eradication rate was 91.2%, and as per PP analysis, eradication rate was 92.7%.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">18 dropouts in the case group</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to 10 say rifabutin regimen=16.7%. Resistance to 7-day rifabutin regimen=8.8%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2022</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##REF##33050205##21##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">USA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">118</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">RCT</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">46.0±10.18</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">RHB-105 (amoxicillin 3 g, omeprazole 120 mg, and rifabutin 150 mg) was administered to n=77 individuals </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">n=41. Patients were treated with placebo</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">As per ITT analysis, 59/66, the eradication rate was 89.4%, and as per PP analysis, 56/63, eradication rate was 88.9%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">As per ITT analysis, 17/33, i.e., eradication rate was 51.5%.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">In the RHB-105 group: 11 dropouts, and in the placebo group: 36 dropouts</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to rifabutin regimen=10.6%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2020</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\"> [##REF##33840604##22##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Taiwan</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">41</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">53.8 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Antimicrobial resistance: amoxicillin (34.1%), clarithromycin (92.7%), metronidazole (65.9%), tetracycline (2.4%), levofloxacin (85.4%), and rifabutin (29.3%) </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Dual antibiotic resistance to clarithromycin+levofloxacin (73.2%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10-14 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Antimicrobial susceptibility: amoxicillin (65.9%), clarithromycin (7.3%), metronidazole (34.1%), tetracycline (97.6%), levofloxacin (14.6%), rifabutin (70.7%) </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to rifabutin=29.3%, amoxicillin=34.1%, clarithromycin=92.7%, metronidazole=65.9%, tetracycline=2.4%, levofloxacin=85.4%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2021</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">9.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##UREF##3##23##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Korea</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">323</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 1: 61.7±12.6. Cohort 2: 58.0±12.1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">MRT therapy regimen was administered to 71 patients following failure of the first-line clarithromycin regimen</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">BQ therapy regimen was administered to 252 patients following failure of the first-line clarithromycin regimen</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 1, 7 days. Cohort 2, 14 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">69% eradication rate in ITT analysis and 77.8% in PP analysis </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">82.5% eradication rate in the ITT analysis, and 89.3% in the PP analysis</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to MRT regimen=22.2%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2022</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##REF##30774400##24##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Java Island</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">105</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Males range in age from 17 to 88; the mean age is (46.14±13.63). Females ranged in age from 14 to 80; the mean was 47.79±14.4 years </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">61/105 ( 58.1%) were sensitive to all five antibiotics examined: furazolidone, sitafloxacin, garenoxacin, rifaximin, and rifabutin. 7/105 (6.7%) were resistant to garenoxacin, and 40/105 (38.9%) were resistant to rifaximin</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">No isolates were tested for resistance to furazolidone, rifabutin, or sitafloxacin </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to rifabutin=0, furazolidone=0, sitafloxacin=0, garenoxacin=6.7%, rifaximin=38.9% .</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2019</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">11.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##REF##30815255##25##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Bangladesh and Nepal</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">98</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Nepal (n=42). Antibiotic resistance was as follows: garenoxacin 12/42 (28.6%), sitafloxacin 2/42 (4.8%), furazolidone 0/42 (0.0%), rifabutin 0/42 (0.0%), rifaximin 22/42 (52.4%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Bangladesh (n=56). Antibiotic resistance was as follows: garenoxacin 29/56 (51.8%), sitafloxacin 1/56 (1.8%), furazolidone 0/56 (0.0%), rifabutin 0/56 (0.0%), and rifaximin 36/56 (64.3%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">None of the examined strains exhibited resistance to furazolidone or rifabutin</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">None of the examined strains exhibited resistance to furazolidone or rifabutin</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance in Bangladesh and Nepal to rifabutin=0, furazolidone=0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2019</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##UREF##4##26##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Dominican Republic</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">63</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">40-49 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Antibiotic resistance was as follows: rifaximin 52/62 (82.5%), garenoxacin 22/63 (34.9%), sitafloxacin 0/63 (0.0%), furazolidone 0/63 (0.0%), rifabutin 0/63 (0.0%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Antibiotic resistance to rifaximin and garenoxacin is doubled (18/63) or 28.6% </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Rifabutin, sitafloxacin, and furazolidone are not resistant to antibiotics in the Dominican Republic. All strains were found to be responsive to rifabutin in the study, confirming the recommendation of rifabutin as a possible antibiotic to treat H. pylori infection </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to rifabutin=0, sitafloxacin=0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2019</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">13.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##UREF##5##27##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Japan</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">29</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">48.0±11.1 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">In the 10-day group, 12 patients received esomeprazole 20 mg q.i.d., amoxicillin 500 mg q.i.d., and rifabutin 300mg q.i.d. </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The 14-day group, which included 17 patients, received esomeprazole 20 mg q.i.d., amoxicillin 500 mg q.i.d., and rifabutin 300 mg q.i.d. </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 1= 10 days. Cohort 2= 14 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">In the ITT analysis, the eradication rate was 10/12, i.e., 83.3%, and in the PP analysis, the eradication rate was 9/11, i.e., 81.8%. Overall eradication rate was 80% </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">In the ITT analysis, the eradication rate was 16/17, i.e., (94.1%), and in the PP analysis, the eradication rate was 11/12, i.e., (91.7%). Overall eradication rate was 90%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Cohort 1=1 Cohort 2=5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to rifabutin in 10 day regimen=0, in 14 day regimen=5.9%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2016</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##REF##32183165##28##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Italy</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1037</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Single-center, open-label, single-arm prospective interventional trial </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">52 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">n=415. These are the patients with only one treatment regimen failure. The regimens that failed were as follows: sequential, 67/415; levofloxacin-based, 221/415; rifabutin-based, 109/415; pylera, 18/415</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">n= 310. These are the patients with two treatment regimen failures. The regimens that failed were as follows: sequential, 29/310; levofloxacin-based, 132/310; rifabutin-based, 129/310; pylera 20/310. n=312. These are the patients with ≥3 treatment regimen failures. The regimens that failed were as follows: sequential, 18/312; levofloxacin-based, 63/312; rifabutin-based, 216/312; pylera, 15/312</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10-14 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">A significant increase in the secondary resistance toward the three tested antibiotics was observed, both as rate and MIC values, in correlation with the number of therapy failures</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Resistance to rifabutin after one therapy regimen failure=26%; Resistance to rifabutin after two therapy regimen failur=41.6%. Resistance to rifabutin after ≥3 therapy regimens failur=69.2%</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2020</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">15.</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">[##UREF##6##29##]</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Korea China</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">200</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Single-center, prospective, open-label, single-arm interventional study </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">47.9 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">n=12 for EAC. n=13 for EAM. n=31 for EAL. EBAM, n=144 </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14 days</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">According to ITT analysis, the following regimens had the highest rates of eradication: EAC Group 11/12 (91.7%), EAM Group 12/13 (92.3%), EAL Group 29/31 (93.5%), and EBAM Group 137/144 (95.1%). Thus, despite numerous earlier H. pylori treatment failures, susceptibility-guided therapy proved highly efficient</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">NA</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The resistance rates for clarithromycin, metronidazole, and levofloxacin were 98.3%, 99.2%, and 93.9%, respectively, among patients who had previously received these antibiotics as part of their treatments</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2019</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0015-00000050541-i01\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0015-00000050541-i01\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"3\"], \"article-title\": [\"List of Bacteria for Which New Antibiotics Are Urgently Needed\"], \"source\": [\"WHO\"], \"date-in-citation\": [\"\\n\"], \"month\": [\"11\"], \"year\": [\"2022\", \"2017\"], \"person-group\": [\"\\n\"], \"surname\": [\"WHO\"], \"given-names\": [\"(2017)\"], \"fpage\": [\"4\"], \"volume\": [\"38\"], \"uri\": [\"https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed\"]}, {\"label\": [\"8\"], \"article-title\": [\"Extragastric manifestations of Helicobacter pylori infection\"], \"source\": [\"Helicobacter\"], \"person-group\": [\"\\n\"], \"surname\": [\"Bani\\u0107\", \"Franceschi\", \"Babi\\u0107\", \"Gasbarrini\"], \"given-names\": [\"M\", \"F\", \"Z\", \"A\"], \"fpage\": [\"49\"], \"lpage\": [\"55\"], \"volume\": [\"17\"], \"year\": [\"2012\"]}, {\"label\": [\"20\"], \"article-title\": [\"Efficacy and safety of low-dose rifabutin-based 7-day triple therapy as a third- or later-line Helicobacter pylori eradication regimen\"], \"source\": [\"Helicobacter\"], \"person-group\": [\"\\n\"], \"surname\": [\"Inokuchi\", \"Mori\", \"Matsuzaki\"], \"given-names\": [\"K\", \"H\", \"J\"], \"fpage\": [\"0\"], \"volume\": [\"27\"], \"year\": [\"2022\"]}, {\"label\": [\"23\"], \"article-title\": [\"Comparison of eradication rates of moxifloxacin-rifabutin triple therapy and bismuth quadruple therapy as second-line regimens in patients with peptic ulcers\"], \"source\": [\"Health Sci Rep\"], \"person-group\": [\"\\n\"], \"surname\": [\"Lee\", \"Kim\", \"Hah\"], \"given-names\": [\"CM\", \"SJ\", \"SI\"], \"fpage\": [\"0\"], \"volume\": [\"5\"], \"year\": [\"2022\"]}, {\"label\": [\"26\"], \"article-title\": [\"Five alternative Helicobacter pylori antibiotics to counter high levofloxacin and metronidazole resistance in the Dominican Republic\"], \"source\": [\"PLoS One\"], \"person-group\": [\"\\n\"], \"surname\": [\"Miftahussurur\", \"Cruz\", \"Doohan\"], \"given-names\": [\"M\", \"M\", \"D\"], \"fpage\": [\"0\"], \"volume\": [\"14\"], \"year\": [\"2019\"]}, {\"label\": [\"27\"], \"article-title\": [\"Rifabutin-based 10-day and 14-day triple therapy as a third-line and fourth-line regimen for Helicobacter pylori eradication: a pilot study\"], \"source\": [\"United European Gastroenterol J\"], \"person-group\": [\"\\n\"], \"surname\": [\"Mori\", \"Suzuki\", \"Matsuzaki\"], \"given-names\": [\"H\", \"H\", \"J\"], \"fpage\": [\"380\"], \"lpage\": [\"387\"], \"volume\": [\"4\"], \"year\": [\"2016\"]}, {\"label\": [\"29\"], \"article-title\": [\"Susceptibility-guided therapy for Helicobacter pylori infection treatment failures\"], \"source\": [\"Therap Adv Gastroenterol\"], \"person-group\": [\"\\n\"], \"surname\": [\"Yu\", \"Luo\", \"Long\"], \"given-names\": [\"L\", \"L\", \"X\"], \"volume\": [\"12\"], \"year\": [\"2019\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":29,"string":"29"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50541"},"package_file":{"kind":"string","value":"oa_package/28/bf/PMC10787902.tar.gz"}}},{"rowIdx":186,"cells":{"accession_id":{"kind":"string","value":"PMC10787903"},"pmid":{"kind":"string","value":"38217706"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par5\">Successful treatment of urethral stricture disease requires not only adequate surgical experience but also appropriate preoperative diagnosis. The basic tools widely used for the initial evaluation of patients with suspicion of urethral stricture (US) are uroflowmetry, supplemented with the IPSS (International Prostate Symptom Score) questionnaire. However, these non-invasive tests remain only supplements to the available imaging methods. Currently, the standard imaging of the urethra includes urethroscopy, cystourethrography (CUG) with voiding cystourethrography (VCUG), and increasingly used sonourethrography (SUG) and magnetic resonance urethrography (MRU) [##REF##12835971##1##–##REF##17874650##3##]. Comprehensive data collection is of utmost importance prior to an operation, because factors such as stricture length, location, and extent of periurethral pathology have a key impact on the choice of surgical approach, reconstruction technique, and the final outcome. The implementation of SUG has been already described more than 30 years ago, yet importantly, this method is still evolving. Compared to the first data provided by McAninch in 1988, who was the first to describe implementation of SUG in US diagnosis, the currently widely available high-quality ultrasound devices offer incomparable image quality and detail in the assessment of pathological tissue [##REF##3276926##4##]. The main limitation of the ultrasound technique includes operator dependence and lower sensitivity for evaluation of posterior urethra—a limitation that according to some authors can partly be overcome by the use of transrectal ultrasound [##UREF##1##5##]. Sonourethrography has shown significant value in several studies and in the light of the growing interest in the application of this method, this narrative review provides a summary of the available literature on the diagnostic role of SUG in the management of urethral strictures. The aim of this review is a thorough analysis of the SUG including technical aspects of the procedure, operator dependency, advantages, and limitations.</p>","<title>Pathophysiology of urethral stricture disease</title>","<p id=\"Par6\">The pathophysiology of urethral stenosis is linked to excessive fibrotic growth at the level of the corpus spongiosum. The result of this pathological process is known as “spongiofibrosis”. In contrast to the normal urethral wall, the epithelial layer at the site of stricture is much thicker. Dense packing of elastin fibers around the narrowed urethra causes the loss of natural elasticity of the urethra until they finally prevent proper urination [##UREF##2##6##]. Fluid’s irritative effect at the site of urethral damage may theoretically intensify the process, but this mechanism has not been practically explored in human studies [##UREF##3##7##]. It is yet noteworthy, that the first murine model for urinary extravasation revealed that mesenchymal spongiofibrosis can be induced by urethral injury with subsequent extravasation. Understanding of this cause-and-effect sequence explains the need to look for more accurate diagnostic methods that provide information on pathology beyond the urethral lumen [##UREF##4##8##].</p>","<title>Conventional urethral imaging techniques: urethrocystography and urethroscopy</title>","<p id=\"Par7\">Cystourethrography and voiding cystourethrography have been the oldest and most used imaging modalities for patients with US, still being the “gold standard”. The examination is widely accessible and the location and length of the stricture can be evaluated instantly and at a relatively low cost. A great advantage of this method is the ability to assess the entire length of the urethra including the posterior urethra. In the case of complete obliteration of the urethra, in patients who are already on a suprapubic catheter—the proximal segment can be visualized by performing the antegrade urethrogram. Furthermore, CUG/VCUG also detects presence of diverticula, stones, fistula or false path. The main limitation is lack of information about the tissue beyond the lumen of the urethra; thus, information on spongiofibrosis cannot be obtained. Some comparable studies suggest that CUG/VCUG underestimates stricture length [##REF##30534649##9##–##REF##28698753##14##]. Moreover, both the patient and physician may be exposed to ionizing radiation during the procedure, unless an infusion line is used to fill the urethra and bladder. The impact of radiation can be especially significant when repeated examinations are necessary. On the other hand, urethroscopy enables a real-time endoscopic visualization of the urethral lumen without exposure to harmful radiation. Noteworthy, urethrocystoscopy and CUG/VCUG have been considered the preferred tools in post-urethroplasty follow-up protocols to detect a recurrent stricture [##REF##30712091##15##, ##REF##24275285##16##]. Yet, urethroscopy rarely enables assessment of the stricture length as the caliber of symptomatic strictures is usually narrower than the standard cystoscopes used [##REF##30584423##17##]. Moreover, urethroscopy is limited in providing a clear diagnosis in complex cases such as multiple strictures, or complete urethral obliteration.</p>","<title>Novel urethral imaging technique: magnetic resonance urethrography</title>","<p id=\"Par8\">Magnetic resonance urethrography stands out among the methods used in the diagnosis of urethral stricture, because it provides three-dimensional images of urethral stricture disease, including data on the tissue surrounding the urethra. One of the major differences, which also determines the choice of one of these methods, is the range of urethra evaluation. Magnetic resonance urethrography was found to be accurate in assessment of both anterior and posterior urethra. The value of MRU is particularly emphasized for the evaluation of the posterior urethra, because preoperative assessment of these strictures correlated more closely with operative findings compared to RUG/VCUG [##UREF##9##18##–##REF##19608363##20##].</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par5\\\">Successful treatment of urethral stricture disease requires not only adequate surgical experience but also appropriate preoperative diagnosis. The basic tools widely used for the initial evaluation of patients with suspicion of urethral stricture (US) are uroflowmetry, supplemented with the IPSS (International Prostate Symptom Score) questionnaire. However, these non-invasive tests remain only supplements to the available imaging methods. Currently, the standard imaging of the urethra includes urethroscopy, cystourethrography (CUG) with voiding cystourethrography (VCUG), and increasingly used sonourethrography (SUG) and magnetic resonance urethrography (MRU) [##REF##12835971##1##–##REF##17874650##3##]. Comprehensive data collection is of utmost importance prior to an operation, because factors such as stricture length, location, and extent of periurethral pathology have a key impact on the choice of surgical approach, reconstruction technique, and the final outcome. The implementation of SUG has been already described more than 30 years ago, yet importantly, this method is still evolving. Compared to the first data provided by McAninch in 1988, who was the first to describe implementation of SUG in US diagnosis, the currently widely available high-quality ultrasound devices offer incomparable image quality and detail in the assessment of pathological tissue [##REF##3276926##4##]. The main limitation of the ultrasound technique includes operator dependence and lower sensitivity for evaluation of posterior urethra—a limitation that according to some authors can partly be overcome by the use of transrectal ultrasound [##UREF##1##5##]. Sonourethrography has shown significant value in several studies and in the light of the growing interest in the application of this method, this narrative review provides a summary of the available literature on the diagnostic role of SUG in the management of urethral strictures. The aim of this review is a thorough analysis of the SUG including technical aspects of the procedure, operator dependency, advantages, and limitations.</p>\",\n \"<title>Pathophysiology of urethral stricture disease</title>\",\n \"<p id=\\\"Par6\\\">The pathophysiology of urethral stenosis is linked to excessive fibrotic growth at the level of the corpus spongiosum. The result of this pathological process is known as “spongiofibrosis”. In contrast to the normal urethral wall, the epithelial layer at the site of stricture is much thicker. Dense packing of elastin fibers around the narrowed urethra causes the loss of natural elasticity of the urethra until they finally prevent proper urination [##UREF##2##6##]. Fluid’s irritative effect at the site of urethral damage may theoretically intensify the process, but this mechanism has not been practically explored in human studies [##UREF##3##7##]. It is yet noteworthy, that the first murine model for urinary extravasation revealed that mesenchymal spongiofibrosis can be induced by urethral injury with subsequent extravasation. Understanding of this cause-and-effect sequence explains the need to look for more accurate diagnostic methods that provide information on pathology beyond the urethral lumen [##UREF##4##8##].</p>\",\n \"<title>Conventional urethral imaging techniques: urethrocystography and urethroscopy</title>\",\n \"<p id=\\\"Par7\\\">Cystourethrography and voiding cystourethrography have been the oldest and most used imaging modalities for patients with US, still being the “gold standard”. The examination is widely accessible and the location and length of the stricture can be evaluated instantly and at a relatively low cost. A great advantage of this method is the ability to assess the entire length of the urethra including the posterior urethra. In the case of complete obliteration of the urethra, in patients who are already on a suprapubic catheter—the proximal segment can be visualized by performing the antegrade urethrogram. Furthermore, CUG/VCUG also detects presence of diverticula, stones, fistula or false path. The main limitation is lack of information about the tissue beyond the lumen of the urethra; thus, information on spongiofibrosis cannot be obtained. Some comparable studies suggest that CUG/VCUG underestimates stricture length [##REF##30534649##9##–##REF##28698753##14##]. Moreover, both the patient and physician may be exposed to ionizing radiation during the procedure, unless an infusion line is used to fill the urethra and bladder. The impact of radiation can be especially significant when repeated examinations are necessary. On the other hand, urethroscopy enables a real-time endoscopic visualization of the urethral lumen without exposure to harmful radiation. Noteworthy, urethrocystoscopy and CUG/VCUG have been considered the preferred tools in post-urethroplasty follow-up protocols to detect a recurrent stricture [##REF##30712091##15##, ##REF##24275285##16##]. Yet, urethroscopy rarely enables assessment of the stricture length as the caliber of symptomatic strictures is usually narrower than the standard cystoscopes used [##REF##30584423##17##]. Moreover, urethroscopy is limited in providing a clear diagnosis in complex cases such as multiple strictures, or complete urethral obliteration.</p>\",\n \"<title>Novel urethral imaging technique: magnetic resonance urethrography</title>\",\n \"<p id=\\\"Par8\\\">Magnetic resonance urethrography stands out among the methods used in the diagnosis of urethral stricture, because it provides three-dimensional images of urethral stricture disease, including data on the tissue surrounding the urethra. One of the major differences, which also determines the choice of one of these methods, is the range of urethra evaluation. Magnetic resonance urethrography was found to be accurate in assessment of both anterior and posterior urethra. The value of MRU is particularly emphasized for the evaluation of the posterior urethra, because preoperative assessment of these strictures correlated more closely with operative findings compared to RUG/VCUG [##UREF##9##18##–##REF##19608363##20##].</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<p id=\"Par9\">A comprehensive literature search was performed using the Medline and Cochrane databases in October 2022. Studies that evaluated the use of SUG in the diagnosis of urethral stricture disease were included in the analysis. Prospective studies were selected for this review to obtain the most informative data possible. This exclusion of case reports, editorials, and commentaries, while potentially limiting the scope of the review, was deemed necessary to ensure the highest quality and clinical relevance of the findings. Articles were screened by two reviewers (M.F. and K.M.) who followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement. The selection process is presented in the PRISMA flowchart (Fig. ##FIG##0##1##) [##UREF##10##21##, ##REF##25554246##22##]. The articles were screened using the keywords “sonourethrography”, “urethral ultrasound”, “urethral stricture”, and “SUG”. Only human studies and articles in English were included. Case reports, conference abstracts, editorials, and comments were excluded from detailed analysis.</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<p id=\\\"Par9\\\">A comprehensive literature search was performed using the Medline and Cochrane databases in October 2022. Studies that evaluated the use of SUG in the diagnosis of urethral stricture disease were included in the analysis. Prospective studies were selected for this review to obtain the most informative data possible. This exclusion of case reports, editorials, and commentaries, while potentially limiting the scope of the review, was deemed necessary to ensure the highest quality and clinical relevance of the findings. Articles were screened by two reviewers (M.F. and K.M.) who followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement. The selection process is presented in the PRISMA flowchart (Fig. ##FIG##0##1##) [##UREF##10##21##, ##REF##25554246##22##]. The articles were screened using the keywords “sonourethrography”, “urethral ultrasound”, “urethral stricture”, and “SUG”. Only human studies and articles in English were included. Case reports, conference abstracts, editorials, and comments were excluded from detailed analysis.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par10\">Seventeen papers were selected as a result of our literature review on the use of SUG in assessing urethral strictures. Final analysis is based on prospective studies, the majority of which are limited by a small patient population (number of patients varied from 28 to 113). Nine studies included patients with urethral stricture located in anterior urethra and eight studies included patients regardless of the stricture location. As most of the available literature assesses the value of ultrasound based on comparing this method to other modalities and/or surgical findings, the collected data are presented in the Table ##TAB##0##1##. As shown in the table, the diagnostic accuracy of SUG was compared to RUG/VCUG, MRU, and sonoelastography. The accuracy of SUG was generally high, with most studies reporting a sensitivity of over 80% and a specificity of over 90%. However, there was some variability between studies, with the accuracy of SUG being lower for strictures in the posterior urethra.</p>","<title>Sonourethrography technique</title>","<p id=\"Par11\">The technique of the procedure itself has not changed much since its introduction and most of the authors follow the same steps. The ultrasound transducer should be positioned on the perineal area, and high-frequency ultrasound waves are directed into the urethral tissue. The ultrasound frequencies should be adjusted in different parts of the urethra—15–18 MHz for the penile urethra (from meatus to the distal bulbar urethra) and 9–12 MHz for the bulbar urethra (up to the urethral external sphincter). Special attention should be paid to the impact of the examiner’s pressure created with the transducer against the skin, as too much pressure may generate an impression of a false stricture. Moreover, to avoid artefacts and evaluate the dynamic view of the intraurethral flow, the urethra should be filled during the examination.</p>","<p id=\"Par12\">The following is a general description of the steps involved in injecting contrast for sonourethrography:<list list-type=\"order\"><list-item><p id=\"Par13\">Patient preparation: Patient is positioned in a lithotomy position, with legs supported and separated. Perineal area is cleansed with an antiseptic solution.</p></list-item><list-item><p id=\"Par14\">Filling the urethra: A tip of a thin catheter is inserted into the urethral meatus, and the saline is prepared in a syringe. In case of a distal urethra stricture, a blunt plastic cannula can be used. Saline is slowly administered into the urethral lumen, typically in small portions. Any discomfort or adverse reactions should always be noted.</p></list-item><list-item><p id=\"Par15\">Ultrasound imaging: The ultrasound transducer is positioned and moved from the urethral meatus toward the perineal area, and the saline-filled urethra is imaged in real-time. The physician should carefully assess the images for any abnormalities or areas of narrowing. The direction of examination is not relevant; however, the entire length of the urethra available for examination should always be assessed.</p></list-item><list-item><p id=\"Par16\">Post-procedure: Once the imaging is completed, the catheter or cannula is removed, and the patient is instructed to void.</p></list-item></list></p>","<title>Anatomy of male urethra on sonourethrography</title>","<p id=\"Par17\">Normal urethra as seen on Fig. ##FIG##1##2## presents as an anechoic tubular area, with smooth outline, usually of 8–10 mm in diameter [##UREF##11##23##]. If saline is introduced, small hyperechoic echoes may be visible within the urethral lumen (Fig. ##FIG##2##3##). Alterations in course of spongiofibrosis present as hyperechogenic areas in comparison to the normal echogenicity of corpus spongiosum (Fig. ##FIG##3##4##). Calcifications may be encountered. Ultrasound also allows the evaluation of mucosa and its abnormalities, lumen abnormalities such as diverticula, Cowper glands, paraurethral soft tissues and/or perineal masses, posttraumatic changes, etc., as well as imaging of the bladder (which may show a thickened trabeculated bladder wall in case of high-pressure voiding due to the presence of stricture).</p>","<title>Additional ultrasound techniques</title>","<p id=\"Par18\">Sonoelastography also known as virtual or electronic palpation is a novel technique used for measurement of tissue stiffness. Talreja et al., in a study on 77 patients with clinical features of anterior urethral stricture disease concluded that sonoelastography estimates stricture site and length better in comparison with RUG/VCUG and SUG. It estimates the degree of spongiofibrosis which serves as an important prognostic factor for stricture recurrence more accurately than SUG. Despite several subsequent studies, it is not widely used [##REF##15618383##24##–##REF##34428090##29##]. Bosio described contrast-enhanced voiding urosonography (CE-VSUG) via the transperineal approach in a pediatric population after catheter filling of the bladder with ultrasound contrast diluted in serum, and its use for assessing posterior urethral anomalies and the degree of vesicoureteral reflux in children has become widespread [##REF##9545481##30##].</p>","<title>Sonourethrography vs. other imaging methods</title>","<title>Diagnostic accuracy of sonourethrography compared to other methods and surgical findings</title>","<p id=\"Par19\">Most of the studies compared SUG findings with that of RUG/VCUG in the diagnosis of urethral stricture. In two studies SUG was found to be more accurate at diagnosing stricture presence and estimating the stricture length compared to RUG [##REF##30534649##9##, ##UREF##5##10##]. Yet, the sensitivity in detecting the stricture and estimating its length using the SUG largely depends on the part of the urethra where the stricture is located. In six studies, SUG has been found to be superior to RUG for anterior urethral strictures [##REF##30534649##9##, ##UREF##5##10##, ##REF##27274893##26##, ##REF##26005985##31##–##REF##15262549##33##]</p>","<p id=\"Par20\">The highest correlation for stricture length at operation was for strictures located in the penile urethra [##UREF##2##6##]. Another early study comparing SUG to conventional RUG found that RUG tended to underestimate actual stricture length as compared to SUG [##REF##16831160##32##, ##REF##8478455##34##]. Tembhekar and colleagues evaluated the role of SUG in 70 male patients referred to the urology department for symptoms suggestive of urethral stricture disease. This study diagnosed 39 strictures in 33 patients. RUG/VCUG and SUG were equally efficacious in diagnosing anterior urethral strictures; however, only one of three (33.3%) posterior urethral strictures were adequately visualized on SUG. The group also concluded that SUG was superior in evaluating spongiofibrosis; however, this appeared to be subjective, based on authors’ opinion. Interestingly, 61 of the 70 (87%) of patients involved in this study preferred SUG over conventional RUG, as it was felt to be less invasive and caused less discomfort [##UREF##12##35##, ##REF##22674713##36##]. Only in one study, SUG was the least accurate method compared with RUG/VCUG and MRU with average overestimation of 2 mm as related to the operative measure [##UREF##9##18##]. Despite high accuracy of SUG in most patients, the authors of this study experienced some notable outliers in the SUG measurements. None of these problems occurred in the penile urethra; instead, they were all exclusive to the bulbar or membranous urethra. This accurately depicts the technical challenges of performing SUG in the posterior urethra, which is nearly impossible despite optimal patient placement and considerable operator expertise [##UREF##9##18##, ##REF##14504903##37##]. Also, it was discovered that in 44 out of 232 (19%) patients undergoing anterior urethral reconstruction included in the study, the results of the intraoperative SUG changed the planned reconstructive technique (based on the preoperative RUG). The authors of this study described criteria to perform an anastomotic urethroplasty based on the intraoperative urethral ultrasonogram findings demonstrating a bulbar urethral stricture length of < 25 mm on aggressive urethral distension [##REF##21615851##38##].</p>","<title>Sonourethrography for the assessment of spongiofibrosis</title>","<p id=\"Par21\">Most authors concluded that SUG enables the evaluation of spongiofibrosis in the anterior urethra and provides similar accuracy as compared to MRU. More anatomical detail is MRU’s principal benefit, which is offset by the cost of the modality and the difficulty of image interpretation. A qualitative and quantitative evaluation of spongiofibrosis may also be provided by SUG incorporating real-time elastography [##REF##27274893##26##, ##REF##9545481##30##, ##REF##14504903##37##]. It is yet unknown whether determining the exact extent of spongiofibrosis before the surgery has significant clinical value and is still to be investigated in further research. However, most authors agree that it has an influence on the choice of surgical technique as excision of the fibrotic fragment and end-to-end anastomosis is preferred in the case of extensive spongiofibrosis [##REF##21615851##38##]. In a study by Ravikumur et al. [##REF##26005985##31##], SUG appeared to more accurately depict stricture length, stricture diameter, and degree of spongiofibrosis when correlated with cystoscopic and intraoperative findings.</p>","<title>Sonourethrography as a sole imaging technique</title>","<p id=\"Par22\">Most of the articles that have been published demonstrate the value of SUG as an auxiliary modality in addition to the standard methods of diagnosing urethral strictures such as RUG or urethroscopy. However, in a recent study, Bryk and colleagues evaluated the viability of using SUG as the sole imaging technique for diagnosing urethral strictures prior to surgical treatment. This study demonstrates that, in a high-volume center with an experienced team, SUG may be the sole imaging modality needed to plan a definitive urethral reconstruction. It should be highlighted that this study only included patients with anterior urethral strictures. In comparison to RUG, which was 90% accurate in this study of 30 men who underwent both procedures, SUG was 100% correct for anterior urethral strictures, but only 60% accurate for posterior urethral strictures. Hence, as the authors concluded, it is not recommended to extend these findings to the posterior urethra. In the light of available data on SUG, because of its limited value in detecting posterior urethral strictures, the standard urethrography should remain the basic ‘road-map’ prior to surgery, particularly in patients with suspected urethral stricture undergoing initial diagnosis [##REF##26993351##39##].</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par10\\\">Seventeen papers were selected as a result of our literature review on the use of SUG in assessing urethral strictures. Final analysis is based on prospective studies, the majority of which are limited by a small patient population (number of patients varied from 28 to 113). Nine studies included patients with urethral stricture located in anterior urethra and eight studies included patients regardless of the stricture location. As most of the available literature assesses the value of ultrasound based on comparing this method to other modalities and/or surgical findings, the collected data are presented in the Table ##TAB##0##1##. As shown in the table, the diagnostic accuracy of SUG was compared to RUG/VCUG, MRU, and sonoelastography. The accuracy of SUG was generally high, with most studies reporting a sensitivity of over 80% and a specificity of over 90%. However, there was some variability between studies, with the accuracy of SUG being lower for strictures in the posterior urethra.</p>\",\n \"<title>Sonourethrography technique</title>\",\n \"<p id=\\\"Par11\\\">The technique of the procedure itself has not changed much since its introduction and most of the authors follow the same steps. The ultrasound transducer should be positioned on the perineal area, and high-frequency ultrasound waves are directed into the urethral tissue. The ultrasound frequencies should be adjusted in different parts of the urethra—15–18 MHz for the penile urethra (from meatus to the distal bulbar urethra) and 9–12 MHz for the bulbar urethra (up to the urethral external sphincter). Special attention should be paid to the impact of the examiner’s pressure created with the transducer against the skin, as too much pressure may generate an impression of a false stricture. Moreover, to avoid artefacts and evaluate the dynamic view of the intraurethral flow, the urethra should be filled during the examination.</p>\",\n \"<p id=\\\"Par12\\\">The following is a general description of the steps involved in injecting contrast for sonourethrography:<list list-type=\\\"order\\\"><list-item><p id=\\\"Par13\\\">Patient preparation: Patient is positioned in a lithotomy position, with legs supported and separated. Perineal area is cleansed with an antiseptic solution.</p></list-item><list-item><p id=\\\"Par14\\\">Filling the urethra: A tip of a thin catheter is inserted into the urethral meatus, and the saline is prepared in a syringe. In case of a distal urethra stricture, a blunt plastic cannula can be used. Saline is slowly administered into the urethral lumen, typically in small portions. Any discomfort or adverse reactions should always be noted.</p></list-item><list-item><p id=\\\"Par15\\\">Ultrasound imaging: The ultrasound transducer is positioned and moved from the urethral meatus toward the perineal area, and the saline-filled urethra is imaged in real-time. The physician should carefully assess the images for any abnormalities or areas of narrowing. The direction of examination is not relevant; however, the entire length of the urethra available for examination should always be assessed.</p></list-item><list-item><p id=\\\"Par16\\\">Post-procedure: Once the imaging is completed, the catheter or cannula is removed, and the patient is instructed to void.</p></list-item></list></p>\",\n \"<title>Anatomy of male urethra on sonourethrography</title>\",\n \"<p id=\\\"Par17\\\">Normal urethra as seen on Fig. ##FIG##1##2## presents as an anechoic tubular area, with smooth outline, usually of 8–10 mm in diameter [##UREF##11##23##]. If saline is introduced, small hyperechoic echoes may be visible within the urethral lumen (Fig. ##FIG##2##3##). Alterations in course of spongiofibrosis present as hyperechogenic areas in comparison to the normal echogenicity of corpus spongiosum (Fig. ##FIG##3##4##). Calcifications may be encountered. Ultrasound also allows the evaluation of mucosa and its abnormalities, lumen abnormalities such as diverticula, Cowper glands, paraurethral soft tissues and/or perineal masses, posttraumatic changes, etc., as well as imaging of the bladder (which may show a thickened trabeculated bladder wall in case of high-pressure voiding due to the presence of stricture).</p>\",\n \"<title>Additional ultrasound techniques</title>\",\n \"<p id=\\\"Par18\\\">Sonoelastography also known as virtual or electronic palpation is a novel technique used for measurement of tissue stiffness. Talreja et al., in a study on 77 patients with clinical features of anterior urethral stricture disease concluded that sonoelastography estimates stricture site and length better in comparison with RUG/VCUG and SUG. It estimates the degree of spongiofibrosis which serves as an important prognostic factor for stricture recurrence more accurately than SUG. Despite several subsequent studies, it is not widely used [##REF##15618383##24##–##REF##34428090##29##]. Bosio described contrast-enhanced voiding urosonography (CE-VSUG) via the transperineal approach in a pediatric population after catheter filling of the bladder with ultrasound contrast diluted in serum, and its use for assessing posterior urethral anomalies and the degree of vesicoureteral reflux in children has become widespread [##REF##9545481##30##].</p>\",\n \"<title>Sonourethrography vs. other imaging methods</title>\",\n \"<title>Diagnostic accuracy of sonourethrography compared to other methods and surgical findings</title>\",\n \"<p id=\\\"Par19\\\">Most of the studies compared SUG findings with that of RUG/VCUG in the diagnosis of urethral stricture. In two studies SUG was found to be more accurate at diagnosing stricture presence and estimating the stricture length compared to RUG [##REF##30534649##9##, ##UREF##5##10##]. Yet, the sensitivity in detecting the stricture and estimating its length using the SUG largely depends on the part of the urethra where the stricture is located. In six studies, SUG has been found to be superior to RUG for anterior urethral strictures [##REF##30534649##9##, ##UREF##5##10##, ##REF##27274893##26##, ##REF##26005985##31##–##REF##15262549##33##]</p>\",\n \"<p id=\\\"Par20\\\">The highest correlation for stricture length at operation was for strictures located in the penile urethra [##UREF##2##6##]. Another early study comparing SUG to conventional RUG found that RUG tended to underestimate actual stricture length as compared to SUG [##REF##16831160##32##, ##REF##8478455##34##]. Tembhekar and colleagues evaluated the role of SUG in 70 male patients referred to the urology department for symptoms suggestive of urethral stricture disease. This study diagnosed 39 strictures in 33 patients. RUG/VCUG and SUG were equally efficacious in diagnosing anterior urethral strictures; however, only one of three (33.3%) posterior urethral strictures were adequately visualized on SUG. The group also concluded that SUG was superior in evaluating spongiofibrosis; however, this appeared to be subjective, based on authors’ opinion. Interestingly, 61 of the 70 (87%) of patients involved in this study preferred SUG over conventional RUG, as it was felt to be less invasive and caused less discomfort [##UREF##12##35##, ##REF##22674713##36##]. Only in one study, SUG was the least accurate method compared with RUG/VCUG and MRU with average overestimation of 2 mm as related to the operative measure [##UREF##9##18##]. Despite high accuracy of SUG in most patients, the authors of this study experienced some notable outliers in the SUG measurements. None of these problems occurred in the penile urethra; instead, they were all exclusive to the bulbar or membranous urethra. This accurately depicts the technical challenges of performing SUG in the posterior urethra, which is nearly impossible despite optimal patient placement and considerable operator expertise [##UREF##9##18##, ##REF##14504903##37##]. Also, it was discovered that in 44 out of 232 (19%) patients undergoing anterior urethral reconstruction included in the study, the results of the intraoperative SUG changed the planned reconstructive technique (based on the preoperative RUG). The authors of this study described criteria to perform an anastomotic urethroplasty based on the intraoperative urethral ultrasonogram findings demonstrating a bulbar urethral stricture length of < 25 mm on aggressive urethral distension [##REF##21615851##38##].</p>\",\n \"<title>Sonourethrography for the assessment of spongiofibrosis</title>\",\n \"<p id=\\\"Par21\\\">Most authors concluded that SUG enables the evaluation of spongiofibrosis in the anterior urethra and provides similar accuracy as compared to MRU. More anatomical detail is MRU’s principal benefit, which is offset by the cost of the modality and the difficulty of image interpretation. A qualitative and quantitative evaluation of spongiofibrosis may also be provided by SUG incorporating real-time elastography [##REF##27274893##26##, ##REF##9545481##30##, ##REF##14504903##37##]. It is yet unknown whether determining the exact extent of spongiofibrosis before the surgery has significant clinical value and is still to be investigated in further research. However, most authors agree that it has an influence on the choice of surgical technique as excision of the fibrotic fragment and end-to-end anastomosis is preferred in the case of extensive spongiofibrosis [##REF##21615851##38##]. In a study by Ravikumur et al. [##REF##26005985##31##], SUG appeared to more accurately depict stricture length, stricture diameter, and degree of spongiofibrosis when correlated with cystoscopic and intraoperative findings.</p>\",\n \"<title>Sonourethrography as a sole imaging technique</title>\",\n \"<p id=\\\"Par22\\\">Most of the articles that have been published demonstrate the value of SUG as an auxiliary modality in addition to the standard methods of diagnosing urethral strictures such as RUG or urethroscopy. However, in a recent study, Bryk and colleagues evaluated the viability of using SUG as the sole imaging technique for diagnosing urethral strictures prior to surgical treatment. This study demonstrates that, in a high-volume center with an experienced team, SUG may be the sole imaging modality needed to plan a definitive urethral reconstruction. It should be highlighted that this study only included patients with anterior urethral strictures. In comparison to RUG, which was 90% accurate in this study of 30 men who underwent both procedures, SUG was 100% correct for anterior urethral strictures, but only 60% accurate for posterior urethral strictures. Hence, as the authors concluded, it is not recommended to extend these findings to the posterior urethra. In the light of available data on SUG, because of its limited value in detecting posterior urethral strictures, the standard urethrography should remain the basic ‘road-map’ prior to surgery, particularly in patients with suspected urethral stricture undergoing initial diagnosis [##REF##26993351##39##].</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par27\">Sonourethrography assessment of the male anterior urethra in patients with anterior urethral strictures is a safe, well-tolerated, minimally invasive and cost-effective diagnostic modality. For the posterior urethra, this technique cannot be recommended, based on the available published evidence. While more studies are needed to better characterize SUG, it could be proposed as an additional diagnostic modality, especially in severe and recurrent cases. More evidence on SUG and more data from studies with larger patients' groups need to be collected in the next future, as so far no randomized clinical trials have been published. Although in the future, SUG might replace CUG/VCUG as the investigation of choice in the diagnosis of anterior urethra strictures, at present, combining RUG/VCUG still remains the gold standard in evaluating urethral stricture disease.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par27\\\">Sonourethrography assessment of the male anterior urethra in patients with anterior urethral strictures is a safe, well-tolerated, minimally invasive and cost-effective diagnostic modality. For the posterior urethra, this technique cannot be recommended, based on the available published evidence. While more studies are needed to better characterize SUG, it could be proposed as an additional diagnostic modality, especially in severe and recurrent cases. More evidence on SUG and more data from studies with larger patients' groups need to be collected in the next future, as so far no randomized clinical trials have been published. Although in the future, SUG might replace CUG/VCUG as the investigation of choice in the diagnosis of anterior urethra strictures, at present, combining RUG/VCUG still remains the gold standard in evaluating urethral stricture disease.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose</title>","<p id=\"Par1\">To synthetize the current scientific knowledge on the use of ultrasound of the male urethra for evaluation of urethral stricture disease. This review aims to provide a detailed description of the technical aspects of ultrasonography, and provides some indications on clinical applications of it, based on the evidence available from the selected prospective studies. Advantages and limitations of the technique are also provided.</p>","<title>Methods</title>","<p id=\"Par2\">A comprehensive literature search was performed using the Medline and Cochrane databases on October 2022. The articles were searched using the keywords “sonourethrography”, “urethral ultrasound”, “urethral stricture” and “SUG”. Only human studies and articles in English were included. Articles were screened by two reviewers (M.F. and K.M.).</p>","<title>Results</title>","<p id=\"Par3\">Our literature search reporting on the role of sonourethrography in evaluating urethral strictures resulted in selection of 17 studies, all prospective, even if of limited quality due to the small patients’ number (varied from 28 to 113). Nine studies included patients with urethral stricture located in anterior urethra and eight studies included patients regardless of the stricture location. Final analysis was based on selected prospective studies, whose power was limited by the small patients’ groups.</p>","<title>Conclusion</title>","<p id=\"Par4\">Sonourethrography is a cost-effective and safe technique allowing for a dynamic and three-dimensional urethra assessment. Yet, because of its limited value in detecting posterior urethral strictures, the standard urethrography should remain the basic ‘road-map’ prior to surgery. It is an operator-dependent technique, which can provide detailed information on the length, location, and extent of spongiofibrosis without risks of exposure to ionizing radiation.</p>","<title>Keywords</title>","<p>Open Access funding enabled and organized by Projekt DEAL.</p>"],"string":"[\n \"<title>Purpose</title>\",\n \"<p id=\\\"Par1\\\">To synthetize the current scientific knowledge on the use of ultrasound of the male urethra for evaluation of urethral stricture disease. This review aims to provide a detailed description of the technical aspects of ultrasonography, and provides some indications on clinical applications of it, based on the evidence available from the selected prospective studies. Advantages and limitations of the technique are also provided.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">A comprehensive literature search was performed using the Medline and Cochrane databases on October 2022. The articles were searched using the keywords “sonourethrography”, “urethral ultrasound”, “urethral stricture” and “SUG”. Only human studies and articles in English were included. Articles were screened by two reviewers (M.F. and K.M.).</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">Our literature search reporting on the role of sonourethrography in evaluating urethral strictures resulted in selection of 17 studies, all prospective, even if of limited quality due to the small patients’ number (varied from 28 to 113). Nine studies included patients with urethral stricture located in anterior urethra and eight studies included patients regardless of the stricture location. Final analysis was based on selected prospective studies, whose power was limited by the small patients’ groups.</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par4\\\">Sonourethrography is a cost-effective and safe technique allowing for a dynamic and three-dimensional urethra assessment. Yet, because of its limited value in detecting posterior urethral strictures, the standard urethrography should remain the basic ‘road-map’ prior to surgery. It is an operator-dependent technique, which can provide detailed information on the length, location, and extent of spongiofibrosis without risks of exposure to ionizing radiation.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open Access funding enabled and organized by Projekt DEAL.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Highlights and clinical indications</title>","<p id=\"Par23\">Retrograde urethrography has historically been the gold standard for identifying urethral strictures; however, because of its certain drawbacks, novel imaging techniques have been investigated and evaluated. Before deciding on surgical intervention, it is crucial to thoroughly consider the length, location, number of the strictures and their morphology since each may affect the choice of the treatment method. Modern high-resolution ultrasound is widely available; thus, the quality of data provided by this diagnostic method has improved significantly since the first description several decades ago. Sonourethrography has nowadays become a viable supplement to the standard modalities and provides additional valuable information. Fibrous scarring of the corpus spongiosum leading to a decrease in the urethral lumen is the fundamental theory explaining the pathogenesis of urethral stricture disease. Sonourethrography provides data on spongiofibrosis with satisfactory accuracy making this method widely used mostly in specialized reconstructive urology centers. As a high-resolution, multi-planar, and cost-effective technique that can be performed in an outpatient setting, SUG has found its place in the new standards of diagnostics of anterior urethral strictures. It is safe for both the patient and the physician because neither are exposed to radiation. Moreover, the possibility of using saline instead of iodine contrast makes it applicable also for allergic patients.</p>","<p id=\"Par24\">However, knowing in which clinical situations SUG is of the greatest value is crucial. As proven in numerous publications, the satisfactory accuracy of the SUG refers primarily to the penile urethra. Some authors question the value of the radiological assessment of strictures of the distal urethra and its impact on the choice of surgical technique. These strictures are often extensive or multiple, rather than single as mostly observed in iatrogenic bulbar strictures. Thus, regardless of length and extent of spongiofibrosis, these strictures often require onlay urethroplasty with opening the urethral lumen when most accurate assessment of the pathology may be achieved during the surgery. On the other hand, in these cases, SUG seems to be the best method to show the periurethral pathology up to the urethral opening with high accuracy, allows discussion of the surgical plan with the patient before the surgery. Moreover, SUG can be of particular use to calculate the flap width in the pendulous urethra, where fasciocutaneous flaps are frequently used for reconstruction. For this purpose, Morey and McAninch proposed a straightforward formula 26–3 D (where D is the urethral diameter in mm) [##REF##10737469##40##]. The lumen diameter can be measured with satisfactory accuracy with ultrasonography. This prevents excessive flap width from causing urine pooling and enables the fasciocutaneous flap to be harvested before the urethra is opened.</p>","<p id=\"Par25\">Furthermore, SUG can be particularly valuable in cases when conventional ascending urethrography is challenging or impossible due to the anomalous anatomy of the distal urethra. This is particularly the case in patients with hypospadias, when both the native and reconstructed urethra are often extremely difficult to evaluate. While descending SUG avoids the need to inject a contrast agent, micturating SUG, although challenging, is feasible even in very complex cases and does not require catheterization of the urethra. The use of SUG in these patients should also be particularly considered as a follow-up tool—without exposing the patient to radiation. Thus, future research should investigate the accuracy of sonourethrography in the follow-up of patients after urethral stricture surgery. This could be a way to detect early recurrence of the stricture. In addition, research is ongoing to develop new ultrasound techniques that can improve the accuracy and clinical utility of sonourethrography. For example, researchers are exploring the use of three-dimensional ultrasound and contrast-enhanced ultrasound.</p>","<p id=\"Par26\">One of the significant limitations of SUG is operator dependency and although the statistical analysis on the issue is scarce or non-existent, nearly all papers stress that despite wide availability of ultrasound and inclusion of the technique in both urethral stricture diagnostic algorithms and guidelines, it has not yet been entirely incorporated into urological everyday practice [##UREF##13##41##–##REF##7776459##43##]. Also the issues of long learning curve, limitation in evaluating the posterior urethra, technical aspects of the examination, such as patient preparation and the length of the examination itself are being raised [##REF##7776459##43##, ##UREF##14##44##].</p>"],"string":"[\n \"<title>Highlights and clinical indications</title>\",\n \"<p id=\\\"Par23\\\">Retrograde urethrography has historically been the gold standard for identifying urethral strictures; however, because of its certain drawbacks, novel imaging techniques have been investigated and evaluated. Before deciding on surgical intervention, it is crucial to thoroughly consider the length, location, number of the strictures and their morphology since each may affect the choice of the treatment method. Modern high-resolution ultrasound is widely available; thus, the quality of data provided by this diagnostic method has improved significantly since the first description several decades ago. Sonourethrography has nowadays become a viable supplement to the standard modalities and provides additional valuable information. Fibrous scarring of the corpus spongiosum leading to a decrease in the urethral lumen is the fundamental theory explaining the pathogenesis of urethral stricture disease. Sonourethrography provides data on spongiofibrosis with satisfactory accuracy making this method widely used mostly in specialized reconstructive urology centers. As a high-resolution, multi-planar, and cost-effective technique that can be performed in an outpatient setting, SUG has found its place in the new standards of diagnostics of anterior urethral strictures. It is safe for both the patient and the physician because neither are exposed to radiation. Moreover, the possibility of using saline instead of iodine contrast makes it applicable also for allergic patients.</p>\",\n \"<p id=\\\"Par24\\\">However, knowing in which clinical situations SUG is of the greatest value is crucial. As proven in numerous publications, the satisfactory accuracy of the SUG refers primarily to the penile urethra. Some authors question the value of the radiological assessment of strictures of the distal urethra and its impact on the choice of surgical technique. These strictures are often extensive or multiple, rather than single as mostly observed in iatrogenic bulbar strictures. Thus, regardless of length and extent of spongiofibrosis, these strictures often require onlay urethroplasty with opening the urethral lumen when most accurate assessment of the pathology may be achieved during the surgery. On the other hand, in these cases, SUG seems to be the best method to show the periurethral pathology up to the urethral opening with high accuracy, allows discussion of the surgical plan with the patient before the surgery. Moreover, SUG can be of particular use to calculate the flap width in the pendulous urethra, where fasciocutaneous flaps are frequently used for reconstruction. For this purpose, Morey and McAninch proposed a straightforward formula 26–3 D (where D is the urethral diameter in mm) [##REF##10737469##40##]. The lumen diameter can be measured with satisfactory accuracy with ultrasonography. This prevents excessive flap width from causing urine pooling and enables the fasciocutaneous flap to be harvested before the urethra is opened.</p>\",\n \"<p id=\\\"Par25\\\">Furthermore, SUG can be particularly valuable in cases when conventional ascending urethrography is challenging or impossible due to the anomalous anatomy of the distal urethra. This is particularly the case in patients with hypospadias, when both the native and reconstructed urethra are often extremely difficult to evaluate. While descending SUG avoids the need to inject a contrast agent, micturating SUG, although challenging, is feasible even in very complex cases and does not require catheterization of the urethra. The use of SUG in these patients should also be particularly considered as a follow-up tool—without exposing the patient to radiation. Thus, future research should investigate the accuracy of sonourethrography in the follow-up of patients after urethral stricture surgery. This could be a way to detect early recurrence of the stricture. In addition, research is ongoing to develop new ultrasound techniques that can improve the accuracy and clinical utility of sonourethrography. For example, researchers are exploring the use of three-dimensional ultrasound and contrast-enhanced ultrasound.</p>\",\n \"<p id=\\\"Par26\\\">One of the significant limitations of SUG is operator dependency and although the statistical analysis on the issue is scarce or non-existent, nearly all papers stress that despite wide availability of ultrasound and inclusion of the technique in both urethral stricture diagnostic algorithms and guidelines, it has not yet been entirely incorporated into urological everyday practice [##UREF##13##41##–##REF##7776459##43##]. Also the issues of long learning curve, limitation in evaluating the posterior urethra, technical aspects of the examination, such as patient preparation and the length of the examination itself are being raised [##REF##7776459##43##, ##UREF##14##44##].</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Author’s contribution</title>","<p>MF: protocol/project development, data Collection, manuscript writing/editing. MV: protocol/project development, data Collection, manuscript writing/editing. KM: data Collection, manuscript writing/editing. JA: data Collection, manuscript writing/editing. FC-J: protocol/project development, data collection. AC: protocol/project development, data collection. CMR: data collection, manuscript writing/editing. WV: protocol/project development, data collection, manuscript writing/editing. MW: protocol/project development, data collection. GM: protocol/project development, data collection, manuscript writing/editing. MM: protocol/project development, data collection, manuscript writing/editing.</p>","<title>Funding</title>","<p>Open Access funding enabled and organized by Projekt DEAL.</p>","<title>Data availability</title>","<p>This article is a narrative review that synthesizes findings from existing literature. It does not contain any new data generated by the authors. Data supporting the findings of this review are available within the cited articles. Readers are referred to these original publications for access to the specific datasets analyzed.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par28\">There are no potential conflicts of interest.</p>","<title>Research involving human participants and/or animals</title>","<p id=\"Par29\">Not applicable.</p>","<title>Informed consent</title>","<p id=\"Par30\">Informed consent was not required for this study.</p>"],"string":"[\n \"<title>Author’s contribution</title>\",\n \"<p>MF: protocol/project development, data Collection, manuscript writing/editing. MV: protocol/project development, data Collection, manuscript writing/editing. KM: data Collection, manuscript writing/editing. JA: data Collection, manuscript writing/editing. FC-J: protocol/project development, data collection. AC: protocol/project development, data collection. CMR: data collection, manuscript writing/editing. WV: protocol/project development, data collection, manuscript writing/editing. MW: protocol/project development, data collection. GM: protocol/project development, data collection, manuscript writing/editing. MM: protocol/project development, data collection, manuscript writing/editing.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open Access funding enabled and organized by Projekt DEAL.</p>\",\n \"<title>Data availability</title>\",\n \"<p>This article is a narrative review that synthesizes findings from existing literature. It does not contain any new data generated by the authors. Data supporting the findings of this review are available within the cited articles. Readers are referred to these original publications for access to the specific datasets analyzed.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par28\\\">There are no potential conflicts of interest.</p>\",\n \"<title>Research involving human participants and/or animals</title>\",\n \"<p id=\\\"Par29\\\">Not applicable.</p>\",\n \"<title>Informed consent</title>\",\n \"<p id=\\\"Par30\\\">Informed consent was not required for this study.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>PRISMA flowchart</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Ultrasound image of bulbar urethra in longitudinal scan shown within the white box (Figure provided by the authors) <italic>U</italic> urethra. <italic>CS</italic> corpus spongiosum. <italic>BSM</italic> bulbospongiosus muscle. Thin white line—urethral epithelium, thick white line —Buck’s fascia, dotted line— <italic>DPF</italic> deep perineal fascia</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Echoes from saline bubbles are visible within urethral lumen in a patient with stricture. Figure provided by the authors</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Urethral stricture with spongiofibrosis. Figure provided by the authors</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>PRISMA flowchart</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Ultrasound image of bulbar urethra in longitudinal scan shown within the white box (Figure provided by the authors) <italic>U</italic> urethra. <italic>CS</italic> corpus spongiosum. <italic>BSM</italic> bulbospongiosus muscle. Thin white line—urethral epithelium, thick white line —Buck’s fascia, dotted line— <italic>DPF</italic> deep perineal fascia</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Echoes from saline bubbles are visible within urethral lumen in a patient with stricture. Figure provided by the authors</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Urethral stricture with spongiofibrosis. Figure provided by the authors</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Diagnostic accuracy of sonourethrography compared to other modalities and surgical findings if applicable</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Study</th><th align=\"left\" rowspan=\"2\">Study type</th><th align=\"left\" rowspan=\"2\">Number of patients</th><th align=\"left\" rowspan=\"2\">Evaluated segments of the urethra</th><th align=\"left\" rowspan=\"2\">Compared studies</th><th align=\"left\" rowspan=\"2\">Intraoperative stricture length measurement</th><th align=\"left\" colspan=\"5\">Accuracy of SUG</th></tr><tr><th align=\"left\">Diagnosis</th><th align=\"left\">Location</th><th align=\"left\">Length</th><th align=\"left\">Diameter</th><th align=\"left\">Recurrence</th></tr></thead><tbody><tr><td align=\"left\">Chung PH et al. 2022</td><td align=\"left\">Prospective</td><td align=\"left\">28</td><td align=\"left\">Bulbar urethra</td><td align=\"left\">CEUS SUG, SWE vs. SUG and RUG</td><td align=\"left\">Yes</td><td align=\"left\"><p>CEUS SUG best correlated with intraoperative stricture length</p><p>(<italic>R</italic><sup>2</sup> 0.709)</p></td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">CEUS SUG 80% sensitivity, 100% specificity, 93% accuracy</td></tr><tr><td align=\"left\">Orakzai ZJ et al. 2022</td><td align=\"left\">Prospective</td><td align=\"left\">77</td><td align=\"left\">Anterior and posterior</td><td align=\"left\">SUG vs. RUG</td><td align=\"left\">Yes</td><td align=\"left\">–</td><td align=\"left\">SUG showed better results than RUG</td><td align=\"left\"><p>SUG:</p><p>62–85% sensitivity,</p><p>92–100% specificity,</p><p>92–98% accuracy</p></td><td align=\"left\"><p>SUG:</p><p>69–82% sensitivity,</p><p>95–98% specificity,</p><p>83–95% accuracy</p></td><td align=\"left\">–</td></tr><tr><td align=\"left\">Frankiewicz et al. 2021</td><td align=\"left\">Prospective</td><td align=\"left\">55</td><td align=\"left\">Anterior and posterior</td><td align=\"left\">SUG vs. VCUG, MRU</td><td align=\"left\">Yes</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">SUG least accurate</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Oyelowo N et al. 2021</td><td align=\"left\">Prospective</td><td align=\"left\">84</td><td align=\"left\">Anterior and posterior</td><td align=\"left\">SUG</td><td align=\"left\">Yes</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\"><p>SUG</p><p>84.6% sensitivity, 82.7% specificity</p></td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Jesrani A et al. 2020</td><td align=\"left\">Prospective</td><td align=\"left\">50</td><td align=\"left\">Anterior urethra</td><td align=\"left\">SUG vs. RUG</td><td align=\"left\">Yes</td><td align=\"left\">77.5% sensitivity, 96.8% specificity, 90% accuracy</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Noori D et al. 2020</td><td align=\"left\">Prospective</td><td align=\"left\">30 with stricture 30 healthy control group</td><td align=\"left\">Anterior and posterior</td><td align=\"left\">SUG vs. RUG</td><td align=\"left\">No</td><td align=\"left\"><p>SUG</p><p>96.87% accuracy</p></td><td align=\"left\">–</td><td align=\"left\">SUG length significantly longer than in RUG (<italic>p</italic> = 0.045)</td><td align=\"left\">SUG stricture caliber significantly larger than in RUG (<italic>p</italic> = 0.05)</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Berna- Mestre et al. 2018</td><td align=\"left\">Prospective</td><td align=\"left\">113</td><td align=\"left\">Anterior and posterior</td><td align=\"left\">SUG vs. RUG, VCUG</td><td align=\"left\">No</td><td align=\"left\">SUG more accurate than RUG (<italic>p</italic> < 0.05)</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Kalabhavi S et al. 2018</td><td align=\"left\">Prospective</td><td align=\"left\">30</td><td align=\"left\">Anterior urethra</td><td align=\"left\">SUG vs. RUG</td><td align=\"left\">Yes</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">SUG 95% accuracy (<italic>p</italic> < 0.001), RUG 57% (<italic>p</italic> < 0.001)</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Krukowski et al.2018</td><td align=\"left\">Prospective</td><td align=\"left\">66</td><td align=\"left\">Anterior urethra</td><td align=\"left\">SUG vs. RUG</td><td align=\"left\">Yes</td><td align=\"left\"><p>SUG 100%</p><p>RUG 97%</p></td><td align=\"left\">SUG better correlation in penile urethra <italic>R</italic> = 0.86 (<italic>p</italic> < 0.001), RUG <italic>R</italic> = 0.66 (<italic>p</italic> < 0.001)</td><td align=\"left\"><p>SUG better correlation <italic>R</italic> = 0.73 (<italic>p</italic> < 0.001),</p><p>RUG R = 0.55 (<italic>p</italic> < 0.001)</p></td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Shahsavari R et al. 2017</td><td align=\"left\">Prospective</td><td align=\"left\">97</td><td align=\"left\">Anterior urethra</td><td align=\"left\">SUG vs. RUG</td><td align=\"left\">No</td><td align=\"left\"><p>SUG</p><p>86.6% sensitivity 94.6% specificity</p></td><td align=\"left\">–</td><td align=\"left\">The length of the stricture in RUG significantly longer than in SUG (<italic>p</italic> = 0.025)</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Bryk D et al. 2016</td><td align=\"left\">Prospective</td><td align=\"left\">35</td><td align=\"left\">Anterior urethra</td><td align=\"left\">SUG</td><td align=\"left\">Yes</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">No significant differences in SUG and intraoperatively (<italic>p</italic> = 0.10) with correlation coefficient of 0.84 (<italic>p</italic> < 0.001)</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Talreja S et al. 2016</td><td align=\"left\">Prospective</td><td align=\"left\">77</td><td align=\"left\">Anterior urethra</td><td align=\"left\">SUG vs. RUG and SE</td><td align=\"left\">Yes</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\"><p>SUG 82–92.7% accuracy,</p><p>RUG 69.5–89% accuracy, SE 87.84–100%</p><p>accuracy. SE higher accuracy in intermediate and long segment strictures</p></td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Ravikumar et al. 2015</td><td align=\"left\">Prospective</td><td align=\"left\">40</td><td align=\"left\">Anterior and posterior</td><td align=\"left\">SUG vs. RUG</td><td align=\"left\">Yes</td><td align=\"left\"><p>SUG</p><p>anterior urethra: 100% sensitivity, 100% specificity; posterior urethra 75% sensitivity,</p><p>50% specificity</p><p>RUG</p><p>100% sensitivity and specificity</p></td><td align=\"left\">–</td><td align=\"left\">More precise estimation with SUG than RUG</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">El-Ghar et al. 2011</td><td align=\"left\">Prospective</td><td align=\"left\">30</td><td align=\"left\">Anterior and posterior</td><td align=\"left\">SUG vs. RUG vs. MRU</td><td align=\"left\">Yes</td><td align=\"left\"><p>Anterior urethra:</p><p>SUG</p><p>100% accuracy,</p><p>RUG</p><p>91% sensitivity,</p><p>90% specificity,</p><p>90% accuracy</p><p>Posterior urethra:</p><p>SUG</p><p>60% accuracy</p><p>RUG</p><p>89% sensitivity, 91.7% specificity, 90% accuracy</p><p>MRU</p><p>100% sensitivity, 91.7% specificity, 95% accuracy</p></td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Gupta N et al. 2006</td><td align=\"left\">Prospective</td><td align=\"left\">52</td><td align=\"left\">Anterior urethra</td><td align=\"left\">SUG vs. RUG</td><td align=\"left\">Yes</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">SUG more precise than RUG</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Choudhary S et al. 2004</td><td align=\"left\">Prospective</td><td align=\"left\">70</td><td align=\"left\">Anterior urethra</td><td align=\"left\">SUG vs. RUG</td><td align=\"left\">Yes</td><td align=\"left\">SUG and RUG equally efficacious</td><td align=\"left\">–</td><td align=\"left\">RUG lower sensitivity (60–80%) for lengths 1–4 cm compared with SUG (73.3–100%)</td><td align=\"left\"><p>SUG</p><p>50–88% sensitivity, 92–97% specificity, 90–96% accuracy</p><p>RUG</p><p>50–78% sensitivity, 86–96% specificity, 81–94% accuracy</p></td><td align=\"left\">–</td></tr><tr><td align=\"left\">Peskar D et al. 2004</td><td align=\"left\">Prospective</td><td align=\"left\">51</td><td align=\"left\">Anterior and posterior</td><td align=\"left\">SUG vs. RUG</td><td align=\"left\">Yes</td><td align=\"left\">SUG identifies 98.4% of RUG strictures</td><td align=\"left\">–</td><td align=\"left\">No major differences</td><td align=\"left\">no major differences</td><td align=\"left\">–</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Diagnostic accuracy of sonourethrography compared to other modalities and surgical findings if applicable</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\" rowspan=\\\"2\\\">Study</th><th align=\\\"left\\\" rowspan=\\\"2\\\">Study type</th><th align=\\\"left\\\" rowspan=\\\"2\\\">Number of patients</th><th align=\\\"left\\\" rowspan=\\\"2\\\">Evaluated segments of the urethra</th><th align=\\\"left\\\" rowspan=\\\"2\\\">Compared studies</th><th align=\\\"left\\\" rowspan=\\\"2\\\">Intraoperative stricture length measurement</th><th align=\\\"left\\\" colspan=\\\"5\\\">Accuracy of SUG</th></tr><tr><th align=\\\"left\\\">Diagnosis</th><th align=\\\"left\\\">Location</th><th align=\\\"left\\\">Length</th><th align=\\\"left\\\">Diameter</th><th align=\\\"left\\\">Recurrence</th></tr></thead><tbody><tr><td align=\\\"left\\\">Chung PH et al. 2022</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">28</td><td align=\\\"left\\\">Bulbar urethra</td><td align=\\\"left\\\">CEUS SUG, SWE vs. SUG and RUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\"><p>CEUS SUG best correlated with intraoperative stricture length</p><p>(<italic>R</italic><sup>2</sup> 0.709)</p></td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">CEUS SUG 80% sensitivity, 100% specificity, 93% accuracy</td></tr><tr><td align=\\\"left\\\">Orakzai ZJ et al. 2022</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">77</td><td align=\\\"left\\\">Anterior and posterior</td><td align=\\\"left\\\">SUG vs. RUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">SUG showed better results than RUG</td><td align=\\\"left\\\"><p>SUG:</p><p>62–85% sensitivity,</p><p>92–100% specificity,</p><p>92–98% accuracy</p></td><td align=\\\"left\\\"><p>SUG:</p><p>69–82% sensitivity,</p><p>95–98% specificity,</p><p>83–95% accuracy</p></td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Frankiewicz et al. 2021</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">55</td><td align=\\\"left\\\">Anterior and posterior</td><td align=\\\"left\\\">SUG vs. VCUG, MRU</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">SUG least accurate</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Oyelowo N et al. 2021</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">84</td><td align=\\\"left\\\">Anterior and posterior</td><td align=\\\"left\\\">SUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\"><p>SUG</p><p>84.6% sensitivity, 82.7% specificity</p></td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Jesrani A et al. 2020</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">50</td><td align=\\\"left\\\">Anterior urethra</td><td align=\\\"left\\\">SUG vs. RUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\">77.5% sensitivity, 96.8% specificity, 90% accuracy</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Noori D et al. 2020</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">30 with stricture 30 healthy control group</td><td align=\\\"left\\\">Anterior and posterior</td><td align=\\\"left\\\">SUG vs. RUG</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\"><p>SUG</p><p>96.87% accuracy</p></td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">SUG length significantly longer than in RUG (<italic>p</italic> = 0.045)</td><td align=\\\"left\\\">SUG stricture caliber significantly larger than in RUG (<italic>p</italic> = 0.05)</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Berna- Mestre et al. 2018</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">113</td><td align=\\\"left\\\">Anterior and posterior</td><td align=\\\"left\\\">SUG vs. RUG, VCUG</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\">SUG more accurate than RUG (<italic>p</italic> < 0.05)</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Kalabhavi S et al. 2018</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">30</td><td align=\\\"left\\\">Anterior urethra</td><td align=\\\"left\\\">SUG vs. RUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">SUG 95% accuracy (<italic>p</italic> < 0.001), RUG 57% (<italic>p</italic> < 0.001)</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Krukowski et al.2018</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">66</td><td align=\\\"left\\\">Anterior urethra</td><td align=\\\"left\\\">SUG vs. RUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\"><p>SUG 100%</p><p>RUG 97%</p></td><td align=\\\"left\\\">SUG better correlation in penile urethra <italic>R</italic> = 0.86 (<italic>p</italic> < 0.001), RUG <italic>R</italic> = 0.66 (<italic>p</italic> < 0.001)</td><td align=\\\"left\\\"><p>SUG better correlation <italic>R</italic> = 0.73 (<italic>p</italic> < 0.001),</p><p>RUG R = 0.55 (<italic>p</italic> < 0.001)</p></td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Shahsavari R et al. 2017</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">97</td><td align=\\\"left\\\">Anterior urethra</td><td align=\\\"left\\\">SUG vs. RUG</td><td align=\\\"left\\\">No</td><td align=\\\"left\\\"><p>SUG</p><p>86.6% sensitivity 94.6% specificity</p></td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">The length of the stricture in RUG significantly longer than in SUG (<italic>p</italic> = 0.025)</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Bryk D et al. 2016</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">35</td><td align=\\\"left\\\">Anterior urethra</td><td align=\\\"left\\\">SUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">No significant differences in SUG and intraoperatively (<italic>p</italic> = 0.10) with correlation coefficient of 0.84 (<italic>p</italic> < 0.001)</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Talreja S et al. 2016</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">77</td><td align=\\\"left\\\">Anterior urethra</td><td align=\\\"left\\\">SUG vs. RUG and SE</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\"><p>SUG 82–92.7% accuracy,</p><p>RUG 69.5–89% accuracy, SE 87.84–100%</p><p>accuracy. SE higher accuracy in intermediate and long segment strictures</p></td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Ravikumar et al. 2015</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">40</td><td align=\\\"left\\\">Anterior and posterior</td><td align=\\\"left\\\">SUG vs. RUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\"><p>SUG</p><p>anterior urethra: 100% sensitivity, 100% specificity; posterior urethra 75% sensitivity,</p><p>50% specificity</p><p>RUG</p><p>100% sensitivity and specificity</p></td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">More precise estimation with SUG than RUG</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">El-Ghar et al. 2011</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">30</td><td align=\\\"left\\\">Anterior and posterior</td><td align=\\\"left\\\">SUG vs. RUG vs. MRU</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\"><p>Anterior urethra:</p><p>SUG</p><p>100% accuracy,</p><p>RUG</p><p>91% sensitivity,</p><p>90% specificity,</p><p>90% accuracy</p><p>Posterior urethra:</p><p>SUG</p><p>60% accuracy</p><p>RUG</p><p>89% sensitivity, 91.7% specificity, 90% accuracy</p><p>MRU</p><p>100% sensitivity, 91.7% specificity, 95% accuracy</p></td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Gupta N et al. 2006</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">52</td><td align=\\\"left\\\">Anterior urethra</td><td align=\\\"left\\\">SUG vs. RUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">SUG more precise than RUG</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Choudhary S et al. 2004</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">70</td><td align=\\\"left\\\">Anterior urethra</td><td align=\\\"left\\\">SUG vs. RUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\">SUG and RUG equally efficacious</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">RUG lower sensitivity (60–80%) for lengths 1–4 cm compared with SUG (73.3–100%)</td><td align=\\\"left\\\"><p>SUG</p><p>50–88% sensitivity, 92–97% specificity, 90–96% accuracy</p><p>RUG</p><p>50–78% sensitivity, 86–96% specificity, 81–94% accuracy</p></td><td align=\\\"left\\\">–</td></tr><tr><td align=\\\"left\\\">Peskar D et al. 2004</td><td align=\\\"left\\\">Prospective</td><td align=\\\"left\\\">51</td><td align=\\\"left\\\">Anterior and posterior</td><td align=\\\"left\\\">SUG vs. RUG</td><td align=\\\"left\\\">Yes</td><td align=\\\"left\\\">SUG identifies 98.4% of RUG strictures</td><td align=\\\"left\\\">–</td><td align=\\\"left\\\">No major differences</td><td align=\\\"left\\\">no major differences</td><td align=\\\"left\\\">–</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p>CEUS—contrast-enhanced ultrasonography, SUG—sonourethrography, RUG—retrograde urethrography, VCUG—voiding cystourethrography, MRU—magnetic resonance urethrography, SE—sonoelastography, SWE—shear wave elastography</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>CEUS—contrast-enhanced ultrasonography, SUG—sonourethrography, RUG—retrograde urethrography, VCUG—voiding cystourethrography, MRU—magnetic resonance urethrography, SE—sonoelastography, SWE—shear wave elastography</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"345_2023_4760_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"345_2023_4760_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"345_2023_4760_Fig3_HTML\" id=\"MO3\"/>","<graphic xlink:href=\"345_2023_4760_Fig4_HTML\" id=\"MO4\"/>"],"string":"[\n \"<graphic xlink:href=\\\"345_2023_4760_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic 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prostatectomy\"], \"source\": [\"Technol Heal Care\"], \"year\": [\"2016\"], \"volume\": [\"24\"], \"fpage\": [\"S487\"], \"lpage\": [\"S492\"], \"pub-id\": [\"10.3233/THC-161172\"]}, {\"label\": [\"6.\"], \"surname\": [\"Baskin\", \"Constantinescu\", \"Howard\"], \"given-names\": [\"LS\", \"SC\", \"PS\"], \"article-title\": [\"Biochemical characterization and quantitation of the collagenous components of urethral stricture tissue\"], \"source\": [\"J Urol\"], \"year\": [\"1993\"], \"volume\": [\"150\"], \"fpage\": [\"426\"], \"lpage\": [\"647\"], \"pub-id\": [\"10.1016/S0022-5347(17)35572-6\"]}, {\"label\": [\"7.\"], \"surname\": [\"Kaplan\", \"Brock\", \"Fisch\", \"Koraitim\", \"Snyder\"], \"given-names\": [\"GW\", \"JW\", \"M\", \"MM\", \"HM\"], \"article-title\": [\"SIU/ICUD consultation on urethral strictures: urethral strictures in children\"], \"source\": [\"Urology\"], \"year\": [\"2014\"], \"volume\": [\"83\"], \"fpage\": [\"71\"], \"lpage\": [\"73\"], \"pub-id\": [\"10.1016/j.urology.2013.09.010\"]}, {\"label\": [\"8.\"], \"surname\": [\"Hyuga\", \"Fujimoto\", \"Hashimoto\", \"Tanabe\", \"Kubo\", \"Nakamura\"], \"given-names\": [\"T\", \"K\", \"D\", \"K\", \"T\", \"S\"], \"article-title\": [\"Wound healing responses of urinary extravasation after urethral injury\"], \"source\": [\"Sci Rep\"], \"year\": [\"2013\"], \"volume\": [\"2023\"], \"fpage\": [\"1\"], \"lpage\": [\"12\"], \"pub-id\": [\"10.1038/s41598-023-37610-2\"]}, {\"label\": [\"10.\"], \"surname\": [\"Kalabhavi\", \"Jayaram\", \"Nagaraja\"], \"given-names\": [\"S\", \"S\", \"NH\"], \"article-title\": [\"Role of sonourethrogram in evaluation of anterior urethral stricture and its correlation with retrograde urethrogram and intraoperative findings\\u2014a prospective study\"], \"source\": [\"J Clin Diag Res\"], \"year\": [\"2018\"], \"volume\": [\"12\"], \"fpage\": [\"PC01\"]}, {\"label\": [\"11.\"], \"surname\": [\"Orakzai\", \"Nighat\", \"Sharif\", \"Zahra\"], \"given-names\": [\"JZ\", \"S\", \"S\", \"M\"], \"article-title\": [\"A comparison of sonourethrography and retrograde urethrography in evaluation of anterior urethral strictures: sonourethrography and retrograde urethrography in evaluation of anterior urethral strictures\"], \"source\": [\"Pak BioMed J\"], \"year\": [\"2022\"], \"volume\": [\"5\"], \"fpage\": [\"6\"], \"pub-id\": [\"10.54393/pbmj.v5i6.576\"]}, {\"label\": [\"12.\"], \"surname\": [\"Jesrani\", \"Muhammad\", \"Hussain\", \"Lalwani\", \"Farid\", \"Saad\"], \"given-names\": [\"A\", \"FS\", \"M\", \"A\", \"M\", \"M\"], \"article-title\": [\"Comparison of sonourethrography and retrograde urethrography in anterior uretheral strictures taking surgical findings as gold standard\"], \"source\": [\"Pak J Radiol\"], \"year\": [\"2020\"], \"volume\": [\"30\"], \"issue\": [\"3\"], \"fpage\": [\"175\"], \"lpage\": [\"179\"]}, {\"label\": [\"13.\"], \"surname\": [\"Noori\", \"Ali\", \"Abdulazeez\"], \"given-names\": [\"FA\", \"AAB\", \"RA\"], \"article-title\": [\"Value of the perineal ultrasound in the diagnosis of anterior urethral stricture in male patient\"], \"source\": [\"Medico Legal Update\"], \"year\": [\"2006\"], \"volume\": [\"20\"], \"fpage\": [\"4\"], \"pub-id\": [\"10.37506/mlu.v20i4.2138\"]}, {\"label\": [\"18.\"], \"surname\": [\"Frankiewicz\", \"Markiet\", \"Kozak\", \"Krukowski\", \"Ka\\u0142u\\u017cny\"], \"given-names\": [\"M\", \"K\", \"O\", \"J\", \"A\"], \"article-title\": [\"Retrograde urethrography, sonourethrography and magnetic resonance urethrography in evaluation of male urethral strictures. Should the novel methods become the new standard in radiological diagnosis of urethral stricture disease?\"], \"source\": [\"Int Urol Nephrol\"], \"year\": [\"2021\"], \"pub-id\": [\"10.1007/s11255-021-02994-5\"]}, {\"label\": [\"21.\"], \"surname\": [\"Moher\", \"Liberati\", \"Tetzlaff\", \"Altman\"], \"given-names\": [\"D\", \"A\", \"J\", \"DG\"], \"collab\": [\"The PRISMA Group\"], \"article-title\": [\"Preferred reporting items for systematic reviews and meta-analyses: The PRISMA statement\"], \"source\": [\"PLoS Med\"], \"year\": [\"2009\"], \"volume\": [\"2009\"], \"issue\": [\"6\"], \"fpage\": [\"e1000097\"], \"pub-id\": [\"10.1371/journal.pmed.1000097\"]}, {\"label\": [\"23.\"], \"surname\": [\"Galosi\", \"Atti\"], \"given-names\": [\"AB\", \"LD\"], \"article-title\": [\"Atlas of ultrasonography in urology, andrology, and nephrology atlas ultrason urol andrology\"], \"source\": [\"Nephrology\"], \"year\": [\"2017\"], \"pub-id\": [\"10.1007/978-3-319-40782-1\"]}, {\"label\": [\"35.\"], \"surname\": [\"Tembhekar\", \"Sonawane\", \"Mashirkar\", \"Titare\", \"Anand\", \"Rathod\"], \"given-names\": [\"NG\", \"B\", \"A\", \"PU\", \"A\", \"PB\"], \"article-title\": [\"Sonourethragraphic assessment of urethral obstruction\"], \"source\": [\"JEMDS\"], \"year\": [\"2014\"], \"volume\": [\"3\"], \"issue\": [\"64\"], \"fpage\": [\"13971\"], \"lpage\": [\"13978\"], \"pub-id\": [\"10.14260/jemds/2014/3870\"]}, {\"label\": [\"41.\"], \"mixed-citation\": [\"EAU Guidelines (2022) Edn presented at the EAU Annual Congress Amsterdam 2022. \"], \"ext-link\": [\"https://uroweb.org/guidelines\"]}, {\"label\": [\"44.\"], \"surname\": [\"Sheehan\", \"Naringrekar\", \"Misiura\"], \"given-names\": [\"JL\", \"HV\", \"AK\"], \"article-title\": [\"The pre-operative and post-operative imaging appearances of urethral strictures and surgical techniques\"], \"source\": [\"Abdom Radiol\"], \"year\": [\"2021\"], \"volume\": [\"46\"], \"issue\": [\"5\"], \"fpage\": [\"2115\"], \"lpage\": [\"2126\"], \"pub-id\": [\"10.1007/s00261-020-02879-8\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":44,"string":"44"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"World J Urol. 2024 Jan 13; 42(1):32"},"package_file":{"kind":"string","value":"oa_package/ec/0f/PMC10787903.tar.gz"}}},{"rowIdx":187,"cells":{"accession_id":{"kind":"string","value":"PMC10787904"},"pmid":{"kind":"string","value":"38112900"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par4\">Urinary tract infections (UTIs) are the most prevalent outpatient illnesses and affect about 50% of the population at some point in their lives [<xref ref-type=\"bibr\" rid=\"CR1\">1</xref>]. The incidence of UTIs is increasing with age, and close to 10% of postmenopausal women indicate that they had a UTI in the previous year [<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>]. Moreover, it is a frequent emergency department (ED) diagnosis with reportedly high diagnostic inaccuracy [<xref ref-type=\"bibr\" rid=\"CR3\">3</xref>]. According to clinical criteria alone, the diagnosis of UTI has a diagnostic error rate of approximately 33% [<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>]. Different classification systems for UTI exist. Despite this diversity, defining UTI is reduced to the presence of bacteria in the urinary tract accompanied by related symptoms and dividing UTI into noncomplicated and complicated groups, with the latter leading to severe consequences, such as urosepsis, if untreated [<xref ref-type=\"bibr\" rid=\"CR5\">5</xref>••]. The algorithm for diagnosing patients with suspected UTIs consists of several stages; each of the subsequent ones allows for more reasoned further diagnostics to make the correct diagnosis. Figure ##FIG##0##1## shows the artificial intelligence (AI)–based treatment and diagnosis of UTIs.</p>","<p id=\"Par5\">Liquid-based laboratories, such as urine analyses with microscopy and culturing, represent the standard for the initial diagnosis of UTI to suspect pathologies of the urinary system and to specify indications for an instrumental approach [<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>•, <xref ref-type=\"bibr\" rid=\"CR7\">7</xref>]. Currently, it is well known that UTI-associated urine changes could be present in non-infection urinary tract pathologies, leading to decreased urine microscopy accuracy [<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>]. Moreover, urine culture suffers from several shortcomings, such as being time-consuming and highly susceptible to contamination, leading to incorrect antibiotic prescription, overutilization, antibiotic resistance, and postponed treatment [<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>]. The use of AI in the medical industry has grown and expanded over time. Among them, the development of intelligent decision-making for clinical medicine is the fastest [<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>]. Currently, it has already been stated that AI-based models can significantly improve physicians’ workflow when examining patients with UTI [<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>••]. However, most contemporary reviews focus on examining AI usage with a restricted quantity of data, analyzing only a subset of AI algorithms, or performing narrative work without analyzing all dedicated studies. Given the preceding, the goal of this work was to conduct a mini-review to determine the current state of AI-based systems as a support in UTI diagnosis.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par4\\\">Urinary tract infections (UTIs) are the most prevalent outpatient illnesses and affect about 50% of the population at some point in their lives [<xref ref-type=\\\"bibr\\\" rid=\\\"CR1\\\">1</xref>]. The incidence of UTIs is increasing with age, and close to 10% of postmenopausal women indicate that they had a UTI in the previous year [<xref ref-type=\\\"bibr\\\" rid=\\\"CR2\\\">2</xref>]. Moreover, it is a frequent emergency department (ED) diagnosis with reportedly high diagnostic inaccuracy [<xref ref-type=\\\"bibr\\\" rid=\\\"CR3\\\">3</xref>]. According to clinical criteria alone, the diagnosis of UTI has a diagnostic error rate of approximately 33% [<xref ref-type=\\\"bibr\\\" rid=\\\"CR4\\\">4</xref>]. Different classification systems for UTI exist. Despite this diversity, defining UTI is reduced to the presence of bacteria in the urinary tract accompanied by related symptoms and dividing UTI into noncomplicated and complicated groups, with the latter leading to severe consequences, such as urosepsis, if untreated [<xref ref-type=\\\"bibr\\\" rid=\\\"CR5\\\">5</xref>••]. The algorithm for diagnosing patients with suspected UTIs consists of several stages; each of the subsequent ones allows for more reasoned further diagnostics to make the correct diagnosis. Figure ##FIG##0##1## shows the artificial intelligence (AI)–based treatment and diagnosis of UTIs.</p>\",\n \"<p id=\\\"Par5\\\">Liquid-based laboratories, such as urine analyses with microscopy and culturing, represent the standard for the initial diagnosis of UTI to suspect pathologies of the urinary system and to specify indications for an instrumental approach [<xref ref-type=\\\"bibr\\\" rid=\\\"CR6\\\">6</xref>•, <xref ref-type=\\\"bibr\\\" rid=\\\"CR7\\\">7</xref>]. Currently, it is well known that UTI-associated urine changes could be present in non-infection urinary tract pathologies, leading to decreased urine microscopy accuracy [<xref ref-type=\\\"bibr\\\" rid=\\\"CR8\\\">8</xref>]. Moreover, urine culture suffers from several shortcomings, such as being time-consuming and highly susceptible to contamination, leading to incorrect antibiotic prescription, overutilization, antibiotic resistance, and postponed treatment [<xref ref-type=\\\"bibr\\\" rid=\\\"CR9\\\">9</xref>]. The use of AI in the medical industry has grown and expanded over time. Among them, the development of intelligent decision-making for clinical medicine is the fastest [<xref ref-type=\\\"bibr\\\" rid=\\\"CR10\\\">10</xref>]. Currently, it has already been stated that AI-based models can significantly improve physicians’ workflow when examining patients with UTI [<xref ref-type=\\\"bibr\\\" rid=\\\"CR11\\\">11</xref>••]. However, most contemporary reviews focus on examining AI usage with a restricted quantity of data, analyzing only a subset of AI algorithms, or performing narrative work without analyzing all dedicated studies. Given the preceding, the goal of this work was to conduct a mini-review to determine the current state of AI-based systems as a support in UTI diagnosis.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Material and Methods</title>","<title>Search Strategy</title>","<p id=\"Par6\">In July 2023, the systematic publication search was done in several databases, including ACM Digital Library, CINAHL, IEEE Xplore, PubMed, and Google Scholar via Boolean operators with the use of the following terms: “AI,””artificial intelligence,” “UTI,” “urinary tract infection,” “cystitis,” “pyelonephritis,” “prostatitis,” “orchitis,” “epididymitis,” “urine,” “urinalysis,” and “urine culture.”</p>","<p id=\"Par7\"><italic>Inclusion criteria</italic>: description of the development and validation of AI-based approaches for UTI diagnosis based on clinical and/or laboratory and/or instrumental data, description of the AI model used, presence of performance metrics; publication date within 5 years from the search time; English-written papers; accessibility of full papers.</p>","<p id=\"Par8\"><italic>Exclusion criteria</italic>: papers not in the English language; papers published more than 5 years ago. Also, papers describing solely the technological aspects of the proposed method without its clinical implementation were excluded.</p>","<title>Studies Process</title>","<p id=\"Par9\">Two reviewers (A. T. and N. N.) independently identified all papers. All studies fitting the inclusion criteria were selected for full review. If there was disagreement or discrepancy, the senior author (B. K. S.) made the final decision.</p>","<title>Data Extraction and Analysis</title>","<p id=\"Par10\">We reviewed studies and extracted information related to the objective, dataset volume, data used for the training, AI approach with precise classification or networks used, performance metrics, outcomes, and validation type. In papers comparing several AI-based models, the most accurate was included in the table. After investigation of the included papers, we divided the described AI models into basic clinical scenarios where they are supposed to be used. This study was reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines.</p>"],"string":"[\n \"<title>Material and Methods</title>\",\n \"<title>Search Strategy</title>\",\n \"<p id=\\\"Par6\\\">In July 2023, the systematic publication search was done in several databases, including ACM Digital Library, CINAHL, IEEE Xplore, PubMed, and Google Scholar via Boolean operators with the use of the following terms: “AI,””artificial intelligence,” “UTI,” “urinary tract infection,” “cystitis,” “pyelonephritis,” “prostatitis,” “orchitis,” “epididymitis,” “urine,” “urinalysis,” and “urine culture.”</p>\",\n \"<p id=\\\"Par7\\\"><italic>Inclusion criteria</italic>: description of the development and validation of AI-based approaches for UTI diagnosis based on clinical and/or laboratory and/or instrumental data, description of the AI model used, presence of performance metrics; publication date within 5 years from the search time; English-written papers; accessibility of full papers.</p>\",\n \"<p id=\\\"Par8\\\"><italic>Exclusion criteria</italic>: papers not in the English language; papers published more than 5 years ago. Also, papers describing solely the technological aspects of the proposed method without its clinical implementation were excluded.</p>\",\n \"<title>Studies Process</title>\",\n \"<p id=\\\"Par9\\\">Two reviewers (A. T. and N. N.) independently identified all papers. All studies fitting the inclusion criteria were selected for full review. If there was disagreement or discrepancy, the senior author (B. K. S.) made the final decision.</p>\",\n \"<title>Data Extraction and Analysis</title>\",\n \"<p id=\\\"Par10\\\">We reviewed studies and extracted information related to the objective, dataset volume, data used for the training, AI approach with precise classification or networks used, performance metrics, outcomes, and validation type. In papers comparing several AI-based models, the most accurate was included in the table. After investigation of the included papers, we divided the described AI models into basic clinical scenarios where they are supposed to be used. This study was reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par11\">Out of the 782 papers that were considered, only 14 studies on AI models in the area of UTIs met the criteria for inclusion (Fig. ##FIG##0##1##). These can be grouped according to the scenarios the AI models were developed for, namely, (1) diagnosis of uncomplicated UTI and symptoms checkers, (2) diagnosis of complicated UTI, and (3) diagnosis of UTIs in specific population groups. Among models, 12 and two papers described machine and deep learning approaches, respectively. The most popular machine learning model was the artificial neural network (ANN) described in six studies, followed by extreme gradient boosting (XGBoost) (<italic>n</italic> = 3), support vector machine (SVM) (<italic>n</italic> = 1), CatBoost (<italic>n</italic> = 1), and ensemble learning model (ELM) (<italic>n</italic> = 1). Among predictive inputs, demographic parameters were used in 10/14 (71.4%) studies and mostly in the view of age (<italic>n</italic> = 9), gender (9), race, and weight (<italic>n</italic> = 1). Notably, the latter are included in papers with pediatric patients. Anamnesis was considered in 7/14 (50%) papers, namely, history of previous UTIs (<italic>n</italic> = 4), history of previous antibiotic treatment failure (<italic>n</italic> = 1), history of previous urine culture results (<italic>n</italic> = 1), and invasive urethral procedures (<italic>n</italic> = 1). Comorbidities were used in 3/14 (21.4%) studies: diabetes (<italic>n</italic> = 2), pneumonia (<italic>n</italic> = 2), classification of stroke (<italic>n</italic> = 1), and the presence of mixed cerebrovascular disease (<italic>n</italic> = 1). Logically, the last two were used in developing AI decision support for stroke patients. UTI-associated symptoms were included in 7/14 (50%) papers: dysuria (<italic>n</italic> = 4), fever (<italic>n</italic> = 3), suprapubic pain (<italic>n</italic> = 3), frequency and urgency (<italic>n</italic> = 1), pollakiuria (<italic>n</italic> = 1), and urine incontinence (<italic>n</italic> = 1). Urinalysis was used, and prognostic input was provided in 7/14 (50%) papers; two of them included dipstick tests only. When urine microscopy was used, red blood cells (RBC), white blood cells (WBC), bacteria’s presence, nitrites, epithelial cells, and glucose were analyzed in 2, 3, 2, 1, 1, and 1 studies, respectively. The study used urine cloudiness as one of its features. Imaging data were used in 3/14 (21.4%): one study analyzed the cystoscopic appearance of the lower urinary tract, and two papers described ultrasound imaging usage (for estimation of hydronephrosis and vesicoureteral reflux grades, respectively). Also, there were other inputs not related to the abovementioned groups: length of stay (LOS) (<italic>n</italic> = 3), length of urethral catheterization (<italic>n</italic> = 2), immunological urine markers (<italic>n</italic> = 1), ward (<italic>n</italic> = 1), serum creatinine and albumin (<italic>n</italic> = 1 and <italic>n</italic> = 1), glucocorticosteroid use (<italic>n</italic> = 1), and duration of immobility (<italic>n</italic> = 1). Performance metrics, validation type as well, and the abovementioned data arranged to include studies are discussed and presented in the review.</p>","<title>Uncomplicated UTI AI-Based Diagnosis and Symptom Checkers</title>","<p id=\"Par12\">Research on AI-based models for uncomplicated UTI diagnosis and symptom checking is listed in Table ##TAB##0##1##. The study by Ozkan et al. [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>••] sought to determine the accuracy of several artificial intelligence models in predicting the likelihood of cystitis and non-specific urethritis disorders, given similar symptoms from the urinary system. Anamnesis, urinalysis, and ultrasound results from 59 individuals were gathered as a training and validation dataset for the study. Four distinct artificial intelligence techniques were applied: decision trees (DT), random forests (RF), support vector machines (SVM), and artificial neural networks (ANN). When these models were compared, it became evident that ANN had the greatest accuracy for UTI detection, with a result of 98.3%. This ANN model only requires the variables pollakiuria, erythrocyturia, and suprapubic pain to acquire a diagnosis with comparable accuracy to a clinical-based diagnosis (Fig. ##FIG##1##2##).\n</p>","<p id=\"Par13\">It was demonstrated that the ANN-based model structure could categorize UTIs without the requirement for expensive laboratory testing, ultrasounds, or invasive methods. Hence, it results in a cheaper diagnostic cost and a quicker decision-making process.</p>","<p id=\"Par14\">The motivation behind Gadalla et al.’s [<xref ref-type=\"bibr\" rid=\"CR13\">13</xref>] paper is that women with uncomplicated UTI symptoms are frequently treated with empirical antibiotics, leading to antibiotic misuse and the development of antimicrobial resistance. The authors looked into 17 clinical and 42 immunological potential predictors for bacterial culture using a random forest or support vector machine (SVM) paired with recursive feature removal (RFE). The most effective clinical predictor to rule in and rule out UTI was urine cloudiness. Interestingly, adding the selected immunological biomarkers to the model with clinical features (including cloudiness or turbidity) did not improve the predictive properties. Dhanda et al. [<xref ref-type=\"bibr\" rid=\"CR14\">14</xref>] described the NoMicro model, which does not take into account urine microscopy. Instead, the results of the urine dipstick test are used. Moreover, the authors generated NoMicro models based on several machine learning classificators, namely XGBoost, RF, and ANN, and compared their efficiency. The primary outcome was a pathogenic urine culture growing ≥ 100,000 colony-forming units. Predictor variables included age; gender; dipstick urinalysis nitrites, leukocytes, clarity, glucose, protein, and blood; dysuria; abdominal pain; and history of UTI. According to the results, the AUC of the NoMicro approach reached 0.85 in external validation and did not statistically differ from the version considering urine microscopy results. Arches et al. [<xref ref-type=\"bibr\" rid=\"CR15\">15</xref>] described an application providing an analysis of the urine test strip using smartphones. According to the results, among the 65 participants, the confirmed UTI AI model achieved an overall accuracy rate of 96.03% and an overall reliability rate of ≥ 0.9, which is interpreted as excellent.</p>","<title>Complicated UTI AI-Based Diagnosis</title>","<p id=\"Par15\">Research describing AI-based models for complicated UTI diagnosis is listed in Table ##TAB##1##2##. Møller et al. [<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>] aimed to develop two predictive models, using data from the index admission as well as historic data on a patient, to predict the development of UTI at the time of entry to the hospital and after 48 h of admission (HA-UTI). The ultimate goal was to assess the individual patient’s risk. The methodology included developing five machine learning models using features such as demographic information, laboratory results, past medical history, and clinical data. The unstructured features, such as the narrative text in electronic medical records, were preprocessed and converted to structured form by natural language processing. The area under the curve ranged from 0.82 to 0.84 for the entry model (<italic>t</italic> = 0 h) and 0.71 to 0.77 for the model predicting HA-UTI.\n</p>","<p id=\"Par16\">Taylor et al. [<xref ref-type=\"bibr\" rid=\"CR17\">17</xref>] performed a single-center, multi-site, retrospective cohort analysis of adults who visited the emergency department based on urine culture results, clinical symptoms, and blood tests. Using both laboratory and clinical data, models for UTI prediction were created using six machine learning algorithms: RF, XGBoost, SVM, adaptive boosting, elastic net, and ANN. A full set of 211 variables and a reduced set of 10 variables (age, gender, history of UTI, dysuria, the presence of nitrites in urine, white blood cells (WBC), red blood cells (RBC), bacteria, and epithelial cells) were both used to develop the models. Comparisons between the UTI predictions and previously recorded UTI diagnoses were made. XGBoost, which has an area under the curve of 0.904, was found to be the best-performing method. It was also shown to have greater sensitivity when compared to the documentation of the UTI diagnosis. According to the results obtained, in practical application, approximately 1 in 4 patients will be re-classified from false positive to true negative, and 1 in 11 patients will be re-categorized from false negative to true positive on account of implementing the algorithm. Mancini et al. [<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>] created a machine learning model that can forecast a patient’s likelihood of developing a multidrug-resistant (MDR) UTI after being admitted to the hospital. The paper added a user-friendly cloud platform called DSaaS (Data Science as a Service), which is ideal for hospital organizations where healthcare operators might not have specialized programming language skills but need to analyze data, via machine learning techniques including CatBoost, SVM, and ANN. The paper employed DSaaS on a real antibiotic stewardship dataset. The development of an MDR UTI was predicted using data related to 1486 hospitalized patients, namely, sex, age, age class, ward, and time period. According to the results obtained, CatBoost exhibited the best predictive results, with the highest value in every metric used. Cai et al. [<xref ref-type=\"bibr\" rid=\"CR19\">19</xref>] described two models based on ANN for predicting fluoroquinolone-based therapy failure (model 1) and fosfomycin-based therapy failure (model 2) among patients with recurrent UTI. Input data mostly consisted of previous urine culture profiles as well as types of antibiotic therapy failures. After the completion of the ANN learning and prediction processes, our neural network showed a sensitivity of 87.8% and a specificity of 97.3% in predicting the clinical efficacy of empirical therapy. Interestingly, the previous use of a specific class of antibiotic was not a risk factor for developing bacterial resistance to the same class (except for the fluoroquinolones), but instead, the most important risk factor for predicting resistance is the use of other classes of antibiotics.</p>","<p id=\"Par17\">Chen et al. [<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>•] compared models based on LR and ANN in defining UTI risk after cystoscopy to reduce antibiotic overuse. As input data, previous UTI history as well as cystoscopic findings such as benign prostatic hyperplasia (BPH), diverticulum, trabeculation, blood clot, cystocele, stone, and tumor was selected. The neural network model had a high accuracy of 85%, sensitivity of 80%, and specificity of 88%. Hong et al. [<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>] constructed a prediction model for urosepsis risk for patients with upper urinary tract calculi with the use of a machine learning ANN model. Several clinical and laboratory features, as well as a hydronephrosis degree based in the USA, were taken as predictive inputs. The area under the receiver operating curve in the validation set was 0.95. According to the results, the proposed model could provide risk assessments for urosepsis in patients with upper urinary tract calculi.</p>","<title>UTI AI-Based Diagnosis in Susceptible Subgroups</title>","<p id=\"Par18\">Papers describing AI-based models for uncomplicated UTI diagnosis in susceptible subgroups are listed in Table ##TAB##2##3##. Pregnant women and children represent a separate subgroup of patients more susceptible to UTIs and requiring specific diagnostic flow and treatment. Pregnancy immunologic and urinary tract alterations predispose women to UTIs. Progesterone-induced smooth muscle relaxation and gravid uterine compression cause ureter and renal calyces dilatation. Also, vesicoureteral reflux may occur. These modifications exacerbate urinary tract infections [<xref ref-type=\"bibr\" rid=\"CR22\">22</xref>]. In turn, UTIs are among the most prevalent bacterial pediatric infections. They are equally prevalent in males and girls during the first year of life but become more prevalent in girls following the first year [<xref ref-type=\"bibr\" rid=\"CR23\">23</xref>]. This high susceptibility makes the development of decision support models based on AI even more relevant. Bertsimas et al. [<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>] developed a machine learning model to better stratify pediatric patients with vesicoureteral reflux complicated by UTI according to the effect of continuous antibiotic prophylaxis. The authors used the following data as input: vesicoureteral reflux grade, serum creatinine, race/gender, fever, dysuria, and weight, and achieved an AUC of 0.82. The described model allows better identification of patients for whom continuous antibiotic prophylaxis will be more effective, thereby providing a personalized approach, while minimizing use in those with the least need. A study by Burton et al. [<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>] aimed at introducing a way to increase the efficiency of urine culture results among pregnant women and children by reducing the number of query samples to be cultured and enabling diagnostic services to concentrate on those in which there are true microbial infections.\n</p>","<p id=\"Par19\">This research discussed two methods of classification to test: one is a heuristic approach using a combination of features such as urine WBC and bacterial counts, and the second is testing typical machine learning models such as random forest, neural network, and XGBoost using independent features such as demographics, previous urine culture results, and clinical details as well. The most optimal solution found was three separate XGBoost algorithms trained separately for pregnant patients, children, and the rest of the categories. Combining the three models yielded a workload reduction of 41% and a sensitivity of 95% for each patient group. The work shows the possibility of using supervised machine learning models to improve service efficiency in situations where demand exceeds the number of resources available to public healthcare providers.</p>","<p id=\"Par20\">Immobile stroke patients also represent a highly susceptible patient subgroup. The prevalence of urinary tract infections is approximately 19%. In addition, the occurrence of an infection can exacerbate the physical harm caused by a stroke, forming a vicious circle with the stroke [<xref ref-type=\"bibr\" rid=\"CR26\">26</xref>]. Zhu et al. [<xref ref-type=\"bibr\" rid=\"CR27\">27</xref>] aimed to develop a prognostic model to define the risk of UTI among immobile stroke patients. Six machine learning models and an ensemble learning model were derived and evaluated. The latter achieved the best performance metrics both in internal and external validation sets, with an AUC of up to 0.82. Xu et al. [<xref ref-type=\"bibr\" rid=\"CR28\">28</xref>] created an effective prediction model for identifying UTI risk in immobile stroke patients and compared its prediction performance to establish machine learning algorithms. They addressed this issue by developing a Siamese network that employed commonly used clinical criteria to identify patients at risk of UTIs. The model was developed and validated using a countrywide dataset of 3982 Chinese patients. A Siamese network is a deep neural network architecture with two or more identical subnetworks that are commonly employed in object detection. With an AUC of 0.83, the Siamese deep learning network did better than all the other machine learning–based models at predicting UTIs in stroke patients who were unable to move.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par11\\\">Out of the 782 papers that were considered, only 14 studies on AI models in the area of UTIs met the criteria for inclusion (Fig. ##FIG##0##1##). These can be grouped according to the scenarios the AI models were developed for, namely, (1) diagnosis of uncomplicated UTI and symptoms checkers, (2) diagnosis of complicated UTI, and (3) diagnosis of UTIs in specific population groups. Among models, 12 and two papers described machine and deep learning approaches, respectively. The most popular machine learning model was the artificial neural network (ANN) described in six studies, followed by extreme gradient boosting (XGBoost) (<italic>n</italic> = 3), support vector machine (SVM) (<italic>n</italic> = 1), CatBoost (<italic>n</italic> = 1), and ensemble learning model (ELM) (<italic>n</italic> = 1). Among predictive inputs, demographic parameters were used in 10/14 (71.4%) studies and mostly in the view of age (<italic>n</italic> = 9), gender (9), race, and weight (<italic>n</italic> = 1). Notably, the latter are included in papers with pediatric patients. Anamnesis was considered in 7/14 (50%) papers, namely, history of previous UTIs (<italic>n</italic> = 4), history of previous antibiotic treatment failure (<italic>n</italic> = 1), history of previous urine culture results (<italic>n</italic> = 1), and invasive urethral procedures (<italic>n</italic> = 1). Comorbidities were used in 3/14 (21.4%) studies: diabetes (<italic>n</italic> = 2), pneumonia (<italic>n</italic> = 2), classification of stroke (<italic>n</italic> = 1), and the presence of mixed cerebrovascular disease (<italic>n</italic> = 1). Logically, the last two were used in developing AI decision support for stroke patients. UTI-associated symptoms were included in 7/14 (50%) papers: dysuria (<italic>n</italic> = 4), fever (<italic>n</italic> = 3), suprapubic pain (<italic>n</italic> = 3), frequency and urgency (<italic>n</italic> = 1), pollakiuria (<italic>n</italic> = 1), and urine incontinence (<italic>n</italic> = 1). Urinalysis was used, and prognostic input was provided in 7/14 (50%) papers; two of them included dipstick tests only. When urine microscopy was used, red blood cells (RBC), white blood cells (WBC), bacteria’s presence, nitrites, epithelial cells, and glucose were analyzed in 2, 3, 2, 1, 1, and 1 studies, respectively. The study used urine cloudiness as one of its features. Imaging data were used in 3/14 (21.4%): one study analyzed the cystoscopic appearance of the lower urinary tract, and two papers described ultrasound imaging usage (for estimation of hydronephrosis and vesicoureteral reflux grades, respectively). Also, there were other inputs not related to the abovementioned groups: length of stay (LOS) (<italic>n</italic> = 3), length of urethral catheterization (<italic>n</italic> = 2), immunological urine markers (<italic>n</italic> = 1), ward (<italic>n</italic> = 1), serum creatinine and albumin (<italic>n</italic> = 1 and <italic>n</italic> = 1), glucocorticosteroid use (<italic>n</italic> = 1), and duration of immobility (<italic>n</italic> = 1). Performance metrics, validation type as well, and the abovementioned data arranged to include studies are discussed and presented in the review.</p>\",\n \"<title>Uncomplicated UTI AI-Based Diagnosis and Symptom Checkers</title>\",\n \"<p id=\\\"Par12\\\">Research on AI-based models for uncomplicated UTI diagnosis and symptom checking is listed in Table ##TAB##0##1##. The study by Ozkan et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>••] sought to determine the accuracy of several artificial intelligence models in predicting the likelihood of cystitis and non-specific urethritis disorders, given similar symptoms from the urinary system. Anamnesis, urinalysis, and ultrasound results from 59 individuals were gathered as a training and validation dataset for the study. Four distinct artificial intelligence techniques were applied: decision trees (DT), random forests (RF), support vector machines (SVM), and artificial neural networks (ANN). When these models were compared, it became evident that ANN had the greatest accuracy for UTI detection, with a result of 98.3%. This ANN model only requires the variables pollakiuria, erythrocyturia, and suprapubic pain to acquire a diagnosis with comparable accuracy to a clinical-based diagnosis (Fig. ##FIG##1##2##).\\n</p>\",\n \"<p id=\\\"Par13\\\">It was demonstrated that the ANN-based model structure could categorize UTIs without the requirement for expensive laboratory testing, ultrasounds, or invasive methods. Hence, it results in a cheaper diagnostic cost and a quicker decision-making process.</p>\",\n \"<p id=\\\"Par14\\\">The motivation behind Gadalla et al.’s [<xref ref-type=\\\"bibr\\\" rid=\\\"CR13\\\">13</xref>] paper is that women with uncomplicated UTI symptoms are frequently treated with empirical antibiotics, leading to antibiotic misuse and the development of antimicrobial resistance. The authors looked into 17 clinical and 42 immunological potential predictors for bacterial culture using a random forest or support vector machine (SVM) paired with recursive feature removal (RFE). The most effective clinical predictor to rule in and rule out UTI was urine cloudiness. Interestingly, adding the selected immunological biomarkers to the model with clinical features (including cloudiness or turbidity) did not improve the predictive properties. Dhanda et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR14\\\">14</xref>] described the NoMicro model, which does not take into account urine microscopy. Instead, the results of the urine dipstick test are used. Moreover, the authors generated NoMicro models based on several machine learning classificators, namely XGBoost, RF, and ANN, and compared their efficiency. The primary outcome was a pathogenic urine culture growing ≥ 100,000 colony-forming units. Predictor variables included age; gender; dipstick urinalysis nitrites, leukocytes, clarity, glucose, protein, and blood; dysuria; abdominal pain; and history of UTI. According to the results, the AUC of the NoMicro approach reached 0.85 in external validation and did not statistically differ from the version considering urine microscopy results. Arches et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR15\\\">15</xref>] described an application providing an analysis of the urine test strip using smartphones. According to the results, among the 65 participants, the confirmed UTI AI model achieved an overall accuracy rate of 96.03% and an overall reliability rate of ≥ 0.9, which is interpreted as excellent.</p>\",\n \"<title>Complicated UTI AI-Based Diagnosis</title>\",\n \"<p id=\\\"Par15\\\">Research describing AI-based models for complicated UTI diagnosis is listed in Table ##TAB##1##2##. Møller et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR16\\\">16</xref>] aimed to develop two predictive models, using data from the index admission as well as historic data on a patient, to predict the development of UTI at the time of entry to the hospital and after 48 h of admission (HA-UTI). The ultimate goal was to assess the individual patient’s risk. The methodology included developing five machine learning models using features such as demographic information, laboratory results, past medical history, and clinical data. The unstructured features, such as the narrative text in electronic medical records, were preprocessed and converted to structured form by natural language processing. The area under the curve ranged from 0.82 to 0.84 for the entry model (<italic>t</italic> = 0 h) and 0.71 to 0.77 for the model predicting HA-UTI.\\n</p>\",\n \"<p id=\\\"Par16\\\">Taylor et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR17\\\">17</xref>] performed a single-center, multi-site, retrospective cohort analysis of adults who visited the emergency department based on urine culture results, clinical symptoms, and blood tests. Using both laboratory and clinical data, models for UTI prediction were created using six machine learning algorithms: RF, XGBoost, SVM, adaptive boosting, elastic net, and ANN. A full set of 211 variables and a reduced set of 10 variables (age, gender, history of UTI, dysuria, the presence of nitrites in urine, white blood cells (WBC), red blood cells (RBC), bacteria, and epithelial cells) were both used to develop the models. Comparisons between the UTI predictions and previously recorded UTI diagnoses were made. XGBoost, which has an area under the curve of 0.904, was found to be the best-performing method. It was also shown to have greater sensitivity when compared to the documentation of the UTI diagnosis. According to the results obtained, in practical application, approximately 1 in 4 patients will be re-classified from false positive to true negative, and 1 in 11 patients will be re-categorized from false negative to true positive on account of implementing the algorithm. Mancini et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR18\\\">18</xref>] created a machine learning model that can forecast a patient’s likelihood of developing a multidrug-resistant (MDR) UTI after being admitted to the hospital. The paper added a user-friendly cloud platform called DSaaS (Data Science as a Service), which is ideal for hospital organizations where healthcare operators might not have specialized programming language skills but need to analyze data, via machine learning techniques including CatBoost, SVM, and ANN. The paper employed DSaaS on a real antibiotic stewardship dataset. The development of an MDR UTI was predicted using data related to 1486 hospitalized patients, namely, sex, age, age class, ward, and time period. According to the results obtained, CatBoost exhibited the best predictive results, with the highest value in every metric used. Cai et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR19\\\">19</xref>] described two models based on ANN for predicting fluoroquinolone-based therapy failure (model 1) and fosfomycin-based therapy failure (model 2) among patients with recurrent UTI. Input data mostly consisted of previous urine culture profiles as well as types of antibiotic therapy failures. After the completion of the ANN learning and prediction processes, our neural network showed a sensitivity of 87.8% and a specificity of 97.3% in predicting the clinical efficacy of empirical therapy. Interestingly, the previous use of a specific class of antibiotic was not a risk factor for developing bacterial resistance to the same class (except for the fluoroquinolones), but instead, the most important risk factor for predicting resistance is the use of other classes of antibiotics.</p>\",\n \"<p id=\\\"Par17\\\">Chen et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR20\\\">20</xref>•] compared models based on LR and ANN in defining UTI risk after cystoscopy to reduce antibiotic overuse. As input data, previous UTI history as well as cystoscopic findings such as benign prostatic hyperplasia (BPH), diverticulum, trabeculation, blood clot, cystocele, stone, and tumor was selected. The neural network model had a high accuracy of 85%, sensitivity of 80%, and specificity of 88%. Hong et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR21\\\">21</xref>] constructed a prediction model for urosepsis risk for patients with upper urinary tract calculi with the use of a machine learning ANN model. Several clinical and laboratory features, as well as a hydronephrosis degree based in the USA, were taken as predictive inputs. The area under the receiver operating curve in the validation set was 0.95. According to the results, the proposed model could provide risk assessments for urosepsis in patients with upper urinary tract calculi.</p>\",\n \"<title>UTI AI-Based Diagnosis in Susceptible Subgroups</title>\",\n \"<p id=\\\"Par18\\\">Papers describing AI-based models for uncomplicated UTI diagnosis in susceptible subgroups are listed in Table ##TAB##2##3##. Pregnant women and children represent a separate subgroup of patients more susceptible to UTIs and requiring specific diagnostic flow and treatment. Pregnancy immunologic and urinary tract alterations predispose women to UTIs. Progesterone-induced smooth muscle relaxation and gravid uterine compression cause ureter and renal calyces dilatation. Also, vesicoureteral reflux may occur. These modifications exacerbate urinary tract infections [<xref ref-type=\\\"bibr\\\" rid=\\\"CR22\\\">22</xref>]. In turn, UTIs are among the most prevalent bacterial pediatric infections. They are equally prevalent in males and girls during the first year of life but become more prevalent in girls following the first year [<xref ref-type=\\\"bibr\\\" rid=\\\"CR23\\\">23</xref>]. This high susceptibility makes the development of decision support models based on AI even more relevant. Bertsimas et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR24\\\">24</xref>] developed a machine learning model to better stratify pediatric patients with vesicoureteral reflux complicated by UTI according to the effect of continuous antibiotic prophylaxis. The authors used the following data as input: vesicoureteral reflux grade, serum creatinine, race/gender, fever, dysuria, and weight, and achieved an AUC of 0.82. The described model allows better identification of patients for whom continuous antibiotic prophylaxis will be more effective, thereby providing a personalized approach, while minimizing use in those with the least need. A study by Burton et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR25\\\">25</xref>] aimed at introducing a way to increase the efficiency of urine culture results among pregnant women and children by reducing the number of query samples to be cultured and enabling diagnostic services to concentrate on those in which there are true microbial infections.\\n</p>\",\n \"<p id=\\\"Par19\\\">This research discussed two methods of classification to test: one is a heuristic approach using a combination of features such as urine WBC and bacterial counts, and the second is testing typical machine learning models such as random forest, neural network, and XGBoost using independent features such as demographics, previous urine culture results, and clinical details as well. The most optimal solution found was three separate XGBoost algorithms trained separately for pregnant patients, children, and the rest of the categories. Combining the three models yielded a workload reduction of 41% and a sensitivity of 95% for each patient group. The work shows the possibility of using supervised machine learning models to improve service efficiency in situations where demand exceeds the number of resources available to public healthcare providers.</p>\",\n \"<p id=\\\"Par20\\\">Immobile stroke patients also represent a highly susceptible patient subgroup. The prevalence of urinary tract infections is approximately 19%. In addition, the occurrence of an infection can exacerbate the physical harm caused by a stroke, forming a vicious circle with the stroke [<xref ref-type=\\\"bibr\\\" rid=\\\"CR26\\\">26</xref>]. Zhu et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR27\\\">27</xref>] aimed to develop a prognostic model to define the risk of UTI among immobile stroke patients. Six machine learning models and an ensemble learning model were derived and evaluated. The latter achieved the best performance metrics both in internal and external validation sets, with an AUC of up to 0.82. Xu et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR28\\\">28</xref>] created an effective prediction model for identifying UTI risk in immobile stroke patients and compared its prediction performance to establish machine learning algorithms. They addressed this issue by developing a Siamese network that employed commonly used clinical criteria to identify patients at risk of UTIs. The model was developed and validated using a countrywide dataset of 3982 Chinese patients. A Siamese network is a deep neural network architecture with two or more identical subnetworks that are commonly employed in object detection. With an AUC of 0.83, the Siamese deep learning network did better than all the other machine learning–based models at predicting UTIs in stroke patients who were unable to move.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par25\">AI-driven UTI detection is a promising new area of healthcare research; however, it is still in the exploratory rather than implementation phase. To fully realize AI’s potential for enhancing UTI diagnosis, more study is needed, ideally guided by larger, more diversified datasets and rigorous validation techniques. By resolving these issues, we can bring AI to bear on this important aspect of healthcare, which will improve patient care, cut costs, and slow the spread of antibiotic resistance. Further studies utilizing large, heterogeneous, prospectively collected datasets, as well as external validations, are required to define the actual clinical workflow value of artificial intelligence.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par25\\\">AI-driven UTI detection is a promising new area of healthcare research; however, it is still in the exploratory rather than implementation phase. To fully realize AI’s potential for enhancing UTI diagnosis, more study is needed, ideally guided by larger, more diversified datasets and rigorous validation techniques. By resolving these issues, we can bring AI to bear on this important aspect of healthcare, which will improve patient care, cut costs, and slow the spread of antibiotic resistance. Further studies utilizing large, heterogeneous, prospectively collected datasets, as well as external validations, are required to define the actual clinical workflow value of artificial intelligence.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose of Review</title>","<p id=\"Par1\">Artificial intelligence (AI) can significantly improve physicians’ workflow when examining patients with UTI. However, most contemporary reviews are focused on examining the usage of AI with a restricted quantity of data, analyzing only a subset of AI algorithms, or performing narrative work without analyzing all dedicated studies. Given the preceding, the goal of this work was to conduct a mini-review to determine the current state of AI-based systems as a support in UTI diagnosis.</p>","<title>Recent Findings</title>","<p id=\"Par2\">There are sufficient publications to comprehend the potential applications of artificial intelligence in the diagnosis of UTIs. Existing research in this field, in general, publishes performance metrics that are exemplary. However, upon closer inspection, many of the available publications are burdened with flaws associated with the improper use of artificial intelligence, such as the use of a small number of samples, their lack of heterogeneity, and the absence of external validation. AI-based models cannot be classified as full-fledged physician assistants in diagnosing UTIs due to the fact that these limitations and flaws represent only a portion of all potential obstacles. Instead, such studies should be evaluated as exploratory, with a focus on the importance of future work that complies with all rules governing the use of AI.</p>","<title>Summary</title>","<p id=\"Par3\">AI algorithms have demonstrated their potential for UTI diagnosis. However, further studies utilizing large, heterogeneous, prospectively collected datasets, as well as external validations, are required to define the actual clinical workflow value of artificial intelligence.</p>","<title>Keywords</title>","<p>Open access funding provided by Manipal Academy of Higher Education, Manipal</p>"],"string":"[\n \"<title>Purpose of Review</title>\",\n \"<p id=\\\"Par1\\\">Artificial intelligence (AI) can significantly improve physicians’ workflow when examining patients with UTI. However, most contemporary reviews are focused on examining the usage of AI with a restricted quantity of data, analyzing only a subset of AI algorithms, or performing narrative work without analyzing all dedicated studies. Given the preceding, the goal of this work was to conduct a mini-review to determine the current state of AI-based systems as a support in UTI diagnosis.</p>\",\n \"<title>Recent Findings</title>\",\n \"<p id=\\\"Par2\\\">There are sufficient publications to comprehend the potential applications of artificial intelligence in the diagnosis of UTIs. Existing research in this field, in general, publishes performance metrics that are exemplary. However, upon closer inspection, many of the available publications are burdened with flaws associated with the improper use of artificial intelligence, such as the use of a small number of samples, their lack of heterogeneity, and the absence of external validation. AI-based models cannot be classified as full-fledged physician assistants in diagnosing UTIs due to the fact that these limitations and flaws represent only a portion of all potential obstacles. Instead, such studies should be evaluated as exploratory, with a focus on the importance of future work that complies with all rules governing the use of AI.</p>\",\n \"<title>Summary</title>\",\n \"<p id=\\\"Par3\\\">AI algorithms have demonstrated their potential for UTI diagnosis. However, further studies utilizing large, heterogeneous, prospectively collected datasets, as well as external validations, are required to define the actual clinical workflow value of artificial intelligence.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by Manipal Academy of Higher Education, Manipal</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Limitations and Future Directions</title>","<p id=\"Par21\">AI algorithms can identify unique correlations between symptoms, urinalysis results, and inflammatory processes in the urinary tract, as well as concise variable sets that are accurate in predicting urinary tract infections. Unquestionably, artificial intelligence is a highly precise and reliable instrument for predicting various events in healthcare [<xref ref-type=\"bibr\" rid=\"CR29\">29</xref>]. In contrast to conventional statistics, artificial intelligence forecasts events by identifying distinct patterns. Sadly, along with new opportunities, associated difficulties with their application have emerged, necessitating a reduction in general optimism in order to comprehend the actual state of this technology, especially in the UTI field.</p>","<p id=\"Par22\">A sufficient amount of data is required for training neural networks to attain optimal performance metrics. In addition, limited dataset sizes may lead to estimation instability and overfitting [<xref ref-type=\"bibr\" rid=\"CR13\">13</xref>]. According to our review, 10 of the 14 studies included more than 1000 cases, which, at first sight, may be an argument in favor of the utility of AI in the context of UTIs. In addition to quantity, however, the dataset must also be of sufficient quality. To be generalizable, the data should ideally be multicenter and prospectively collected, as well as span multiple geographic regions [<xref ref-type=\"bibr\" rid=\"CR14\">14</xref>]. Furthermore, validation is an essential aspect of the reliability of the results. To obtain as objective and unbiased performance metrics as feasible, validation should be performed externally with samples that AI has never seen before [<xref ref-type=\"bibr\" rid=\"CR30\">30</xref>]. Only two works provided external validation results in our review. On the other hand, the disparity in laboratory thresholds between medical centers and guidelines further complicates the collection of multicenter datasets and the routine application of the resulting AI-based models. For instance, there is currently no accepted level for a positive urine culture, with published values ranging from 10^2 to 10^5 cfu/mL. Conceivably different thresholds would result in different test performances [<xref ref-type=\"bibr\" rid=\"CR17\">17</xref>].</p>","<p id=\"Par23\">The limitations outlined above represent only a small portion of the issues associated with the application of artificial intelligence and the interpretation of the results obtained. Despite this, the results of the studies included in this review demonstrate the potential utility of AI-based models for diagnosing UTIs. Clarifying the issues associated with the use of such technologies is an integral part of comprehending how the urological community should advance their sophistication. To facilitate the training of models, it is essential that as many medical centers around the world as possible converge on a common terminology for UTI, threshold values for various indicators, and research quality. To ensure generalizability, future studies should be prospective and multicenter to transition AI-based models from a stage of experimental development to a stage where they can be utilized in the clinical practice of urologists. Lastly, the advancement of AI in the field of UTIs is directly related to the general enhancement of diagnostic techniques. As new markers, new modalities, and improved interpretation become available, studies should be conducted to ascertain their utility in predicting UTIs using AI, thereby enhancing our knowledge of the future development of this technology.</p>","<p id=\"Par24\">There is great potential in using AI algorithms for the detection of urinary tract infections (UTIs), but there are also an array of challenges that need to be addressed. The investigations discussed demonstrate that machine and deep learning models have the potential to significantly improve UTI diagnosis, leading to faster, more precise diagnoses. The limitations and unknowns of their clinical influence, however, must be noticed. Although AI models have shown remarkable precision in specific settings, a wider variety of data is necessary to guarantee their consistency and generalizability. Larger datasets that comprise patients from many different backgrounds, ages, and locations fall under this category. To minimize errors and verify the efficacy of AI algorithms in actual clinical situations, prospective data collecting is essential. In addition, it is essential to acknowledge the value of external validation in making AI models robust and useful in various healthcare settings. Moving forward, healthcare facilities should work together to develop standardized diagnostic criteria and terminology for UTIs. This will help alleviate problems caused by disparities in laboratory methods and terminology. This will allow AI algorithms to be more seamlessly integrated into the diagnostic workflow, minimizing the need for intrusive and expensive laboratory testing and imaging.</p>"],"string":"[\n \"<title>Limitations and Future Directions</title>\",\n \"<p id=\\\"Par21\\\">AI algorithms can identify unique correlations between symptoms, urinalysis results, and inflammatory processes in the urinary tract, as well as concise variable sets that are accurate in predicting urinary tract infections. Unquestionably, artificial intelligence is a highly precise and reliable instrument for predicting various events in healthcare [<xref ref-type=\\\"bibr\\\" rid=\\\"CR29\\\">29</xref>]. In contrast to conventional statistics, artificial intelligence forecasts events by identifying distinct patterns. Sadly, along with new opportunities, associated difficulties with their application have emerged, necessitating a reduction in general optimism in order to comprehend the actual state of this technology, especially in the UTI field.</p>\",\n \"<p id=\\\"Par22\\\">A sufficient amount of data is required for training neural networks to attain optimal performance metrics. In addition, limited dataset sizes may lead to estimation instability and overfitting [<xref ref-type=\\\"bibr\\\" rid=\\\"CR13\\\">13</xref>]. According to our review, 10 of the 14 studies included more than 1000 cases, which, at first sight, may be an argument in favor of the utility of AI in the context of UTIs. In addition to quantity, however, the dataset must also be of sufficient quality. To be generalizable, the data should ideally be multicenter and prospectively collected, as well as span multiple geographic regions [<xref ref-type=\\\"bibr\\\" rid=\\\"CR14\\\">14</xref>]. Furthermore, validation is an essential aspect of the reliability of the results. To obtain as objective and unbiased performance metrics as feasible, validation should be performed externally with samples that AI has never seen before [<xref ref-type=\\\"bibr\\\" rid=\\\"CR30\\\">30</xref>]. Only two works provided external validation results in our review. On the other hand, the disparity in laboratory thresholds between medical centers and guidelines further complicates the collection of multicenter datasets and the routine application of the resulting AI-based models. For instance, there is currently no accepted level for a positive urine culture, with published values ranging from 10^2 to 10^5 cfu/mL. Conceivably different thresholds would result in different test performances [<xref ref-type=\\\"bibr\\\" rid=\\\"CR17\\\">17</xref>].</p>\",\n \"<p id=\\\"Par23\\\">The limitations outlined above represent only a small portion of the issues associated with the application of artificial intelligence and the interpretation of the results obtained. Despite this, the results of the studies included in this review demonstrate the potential utility of AI-based models for diagnosing UTIs. Clarifying the issues associated with the use of such technologies is an integral part of comprehending how the urological community should advance their sophistication. To facilitate the training of models, it is essential that as many medical centers around the world as possible converge on a common terminology for UTI, threshold values for various indicators, and research quality. To ensure generalizability, future studies should be prospective and multicenter to transition AI-based models from a stage of experimental development to a stage where they can be utilized in the clinical practice of urologists. Lastly, the advancement of AI in the field of UTIs is directly related to the general enhancement of diagnostic techniques. As new markers, new modalities, and improved interpretation become available, studies should be conducted to ascertain their utility in predicting UTIs using AI, thereby enhancing our knowledge of the future development of this technology.</p>\",\n \"<p id=\\\"Par24\\\">There is great potential in using AI algorithms for the detection of urinary tract infections (UTIs), but there are also an array of challenges that need to be addressed. The investigations discussed demonstrate that machine and deep learning models have the potential to significantly improve UTI diagnosis, leading to faster, more precise diagnoses. The limitations and unknowns of their clinical influence, however, must be noticed. Although AI models have shown remarkable precision in specific settings, a wider variety of data is necessary to guarantee their consistency and generalizability. Larger datasets that comprise patients from many different backgrounds, ages, and locations fall under this category. To minimize errors and verify the efficacy of AI algorithms in actual clinical situations, prospective data collecting is essential. In addition, it is essential to acknowledge the value of external validation in making AI models robust and useful in various healthcare settings. Moving forward, healthcare facilities should work together to develop standardized diagnostic criteria and terminology for UTIs. This will help alleviate problems caused by disparities in laboratory methods and terminology. This will allow AI algorithms to be more seamlessly integrated into the diagnostic workflow, minimizing the need for intrusive and expensive laboratory testing and imaging.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Author Contribution</title>","<p>NN—conception, data collection, data analysis, writing the main manuscript; AT- conception, data collection, data analysis, writing the main manuscript; DRS—data analysis, writing, and editing; BZH—conception, data collection, data analysis, writing main manuscript; RZ—data analysis, writing, and editing; BKS—conception, data collection, and analysis, writing, and editing, supervision.</p>","<title>Funding</title>","<p>Open access funding provided by Manipal Academy of Higher Education, Manipal</p>","<title>Data Availability</title>","<p>Data is available on request.</p>","<title>Declarations</title>","<title>Ethical Approval</title>","<p id=\"Par26\">Not applicable.</p>","<title>Conflict of Interest</title>","<p id=\"Par27\">Nil.</p>","<title>Human and Animal Rights and Informed Consent</title>","<p id=\"Par28\">This article does not contain any studies with human or animal subjects performed by any of the authors.</p>"],"string":"[\n \"<title>Author Contribution</title>\",\n \"<p>NN—conception, data collection, data analysis, writing the main manuscript; AT- conception, data collection, data analysis, writing the main manuscript; DRS—data analysis, writing, and editing; BZH—conception, data collection, data analysis, writing main manuscript; RZ—data analysis, writing, and editing; BKS—conception, data collection, and analysis, writing, and editing, supervision.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by Manipal Academy of Higher Education, Manipal</p>\",\n \"<title>Data Availability</title>\",\n \"<p>Data is available on request.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethical Approval</title>\",\n \"<p id=\\\"Par26\\\">Not applicable.</p>\",\n \"<title>Conflict of Interest</title>\",\n \"<p id=\\\"Par27\\\">Nil.</p>\",\n \"<title>Human and Animal Rights and Informed Consent</title>\",\n \"<p id=\\\"Par28\\\">This article does not contain any studies with human or animal subjects performed by any of the authors.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Artificial intelligence (AI)–based treatment and diagnosis of UTIs</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>PRISMA flowchart: AI-based approaches for UTI diagnosis</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Artificial intelligence (AI)–based treatment and diagnosis of UTIs</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>PRISMA flowchart: AI-based approaches for UTI diagnosis</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Uncomplicated UTI AI-based diagnosis and symptom checkers</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Author</th><th align=\"left\">Objective</th><th align=\"left\">Dataset (<italic>n</italic>)</th><th align=\"left\">AI model</th><th align=\"left\">Demographics</th><th align=\"left\">Anamnesis</th><th align=\"left\">Comorbidity</th><th align=\"left\">Symptoms</th><th align=\"left\">Urine analysis</th><th align=\"left\">Imaging</th><th align=\"left\">Other</th><th align=\"left\">Performance</th><th align=\"left\">Validation</th></tr></thead><tbody><tr><td align=\"left\">Ozkan et al. [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>••]</td><td align=\"left\">Prediction of cystitis and non-specific urethritis</td><td align=\"left\">59</td><td align=\"left\">ANN</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">Pollakiuria, suprapubic pain</td><td align=\"left\">RBC</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\"><p>Accuracy, 98.3%</p><p>Sensitivity, 97.7%</p><p>Specificity, 100%</p></td><td align=\"left\">Internal</td></tr><tr><td align=\"left\">Gadalla et al. [<xref ref-type=\"bibr\" rid=\"CR13\">13</xref>]</td><td align=\"left\">Prediction of UTI</td><td align=\"left\">183</td><td align=\"left\">SVM + RF</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">Cloudiness</td><td align=\"left\">-</td><td align=\"left\">Immunological markers</td><td align=\"left\">AUC, 0.86</td><td align=\"left\">Internal</td></tr><tr><td align=\"left\">Dhanda et al. [<xref ref-type=\"bibr\" rid=\"CR14\">14</xref>]</td><td align=\"left\">Prediction of UTI in ED</td><td align=\"left\">80,859</td><td align=\"left\">XGBoost</td><td align=\"left\">Age, gender</td><td align=\"left\">History of UTI</td><td align=\"left\">-</td><td align=\"left\">Dysuria, suprapubic pain</td><td align=\"left\">Dipstick test results</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">AUC, 0.85</td><td align=\"left\">External</td></tr><tr><td align=\"left\">Arches et al. [<xref ref-type=\"bibr\" rid=\"CR15\">15</xref>]</td><td align=\"left\">Prediction of UTI</td><td align=\"left\">65</td><td align=\"left\">CNN</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">Dipstick test results</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">Accuracy, 96%</td><td align=\"left\">Internal</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Complicated UTI AI-based diagnosis</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Author</th><th align=\"left\">Objective</th><th align=\"left\">Dataset (<italic>n</italic>)</th><th align=\"left\">AI model</th><th align=\"left\">Demographics</th><th align=\"left\">Anamnesis</th><th align=\"left\">Comorbidity</th><th align=\"left\">symptoms</th><th align=\"left\">Urine analysis</th><th align=\"left\">Imaging</th><th align=\"left\">Other</th><th align=\"left\">Performance</th><th align=\"left\">Validation</th></tr></thead><tbody><tr><td align=\"left\">Møller et al. [<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>]</td><td align=\"left\">Prediction of UTI (model 1) and hospital-acquired UTI (model 2)</td><td align=\"left\">300,000</td><td align=\"left\">ANN</td><td align=\"left\">Age, gender</td><td align=\"left\">History of UTI</td><td align=\"left\">-</td><td align=\"left\">Fever, dysuria, frequency, urgency, suprapubic pain</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">AUC, 0.84</td><td align=\"left\">Internal</td></tr><tr><td align=\"left\">Taylor et al. [<xref ref-type=\"bibr\" rid=\"CR17\">17</xref>]</td><td align=\"left\">Prediction of UTI in ED</td><td align=\"left\">80,387</td><td align=\"left\">XGBoost</td><td align=\"left\">Age, gender</td><td align=\"left\">History of UTI</td><td align=\"left\">-</td><td align=\"left\">Dysuria</td><td align=\"left\">Nitrites, WBC, RBC, bacteria, epithelial cells</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\"><p>AUC, 0.904</p><p>Sensitivity</p><p>Specificity:</p></td><td align=\"left\">Internal</td></tr><tr><td align=\"left\">Mancini et al. [<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>]</td><td align=\"left\">Prediction of hospital-acquired multidrug-resistant UTI</td><td align=\"left\">1486</td><td align=\"left\">CatBoost</td><td align=\"left\">Age, gender</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">Ward, length of stay</td><td align=\"left\"><p>AUC, 0.853</p><p>Sensitivity, 0.904</p></td><td align=\"left\">Internal</td></tr><tr><td align=\"left\">Cai et al. [<xref ref-type=\"bibr\" rid=\"CR19\">19</xref>]</td><td align=\"left\">Prediction of clinical efficacy of antibiotics in women with recurrent UTIs</td><td align=\"left\">1043</td><td align=\"left\">ANN</td><td align=\"left\">-</td><td align=\"left\"><p>Model 1: history of fluoroquinolones and cephalosporins failure, previously mentioned <italic>E. coli</italic> resistant to cotrimoxazole</p><p>Model 2: previously mentioned <italic>E. coli</italic> resistant to cotrimoxazole and amoxicillin-clavulanic acid</p></td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\"><p>For both:</p><p>AUC, 0.87</p><p>Sensitivity, 87.8%</p><p>Specificity, 97.3%</p></td><td align=\"left\">Internal</td></tr><tr><td align=\"left\">Chen et al. [<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>•]</td><td align=\"left\">Prediction of UTI after cystoscopy</td><td align=\"left\">1647</td><td align=\"left\">ANN</td><td align=\"left\">Age</td><td align=\"left\">History of UTI</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">Cystoscopy findings</td><td align=\"left\">-</td><td align=\"left\"><p>Accuracy, 91%</p><p>Sensitivity, 80%</p><p>Specificity, 88%</p></td><td align=\"left\">Internal</td></tr><tr><td align=\"left\">Hong et al. [<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>]</td><td align=\"left\">Prediction of urosepsis among patients with upper urinary tract calculi</td><td align=\"left\">1716</td><td align=\"left\">ANN</td><td align=\"left\">Age, gender</td><td align=\"left\">-</td><td align=\"left\">Diabetes</td><td align=\"left\">Fever</td><td align=\"left\">WBC, nitrites, glucose</td><td align=\"left\">Ultrasound (hydronephrosis grade)</td><td align=\"left\">-</td><td align=\"left\"><p>AUC, 0.95</p><p>Sensitivity, 80.4%</p><p>Specificity, 98.2%</p></td><td align=\"left\">Internal</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>UTI AI-based diagnosis in susceptible subgroups</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Author</th><th align=\"left\">Objective</th><th align=\"left\">Dataset (<italic>n</italic>)</th><th align=\"left\">AI model</th><th align=\"left\">Demographics</th><th align=\"left\">Anamnesis</th><th align=\"left\">Comorbidity</th><th align=\"left\">Symptoms</th><th align=\"left\">Urine analysis</th><th align=\"left\">Imaging</th><th align=\"left\">Other</th><th align=\"left\">Performance</th><th align=\"left\">Validation</th></tr></thead><tbody><tr><td align=\"left\">Bertsimas et al. [<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>]</td><td align=\"left\">Prediction of continuous antibiotic prophylaxis benefits among children with VUR and UTI</td><td align=\"left\">607</td><td align=\"left\">ANN</td><td align=\"left\">Race, gender, weight</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">Fever, dysuria</td><td align=\"left\">-</td><td align=\"left\">Ultrasound (vesicoureteral reflux grade)</td><td align=\"left\">Serum creatinine</td><td align=\"left\">AUC, 0.82</td><td align=\"left\">Internal</td></tr><tr><td align=\"left\">Burton et al. [<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>]</td><td align=\"left\">Prediction of urine culture in pregnant and children</td><td align=\"left\">212,554</td><td align=\"left\">XGBoost</td><td align=\"left\">Age, gender</td><td align=\"left\">History of urine culture results</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">WBC, bacteria</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\"><p>AUC, 91%</p><p>Sensitivity, 95%</p></td><td align=\"left\">Internal</td></tr><tr><td align=\"left\">Zhu et al. [<xref ref-type=\"bibr\" rid=\"CR27\">27</xref>]</td><td align=\"left\">Prediction of UTI among immobile stroke patients</td><td align=\"left\">7819</td><td align=\"left\">Ensemble learning model (ELM)</td><td align=\"left\">Age, gender</td><td align=\"left\">-</td><td align=\"left\">Pneumonia, mixed cerebrovascular disease</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">Length of stay, length of urethral catheterization, glucocorticoid use</td><td align=\"left\"><p>AUC, 0.82</p><p>Sensitivity, 80.9%</p></td><td align=\"left\">External</td></tr><tr><td align=\"left\">Xu et al. [<xref ref-type=\"bibr\" rid=\"CR28\">28</xref>]</td><td align=\"left\">Prediction of UTI among immobile stroke patients</td><td align=\"left\">3982</td><td align=\"left\">Siamese network</td><td align=\"left\">Age, gender</td><td align=\"left\">History of urethral invasive procedures</td><td align=\"left\">Classification of stroke, pneumonia, diabetes</td><td align=\"left\">Urine incontinence</td><td align=\"left\">-</td><td align=\"left\">-</td><td align=\"left\">Length of stay, duration of immobility, length of urethral catheterization, serum albumin</td><td align=\"left\"><p>AUC: 0.83</p><p>Accuracy: 74.2%</p><p>Sensitivity: 81%</p><p>Specificity: 74%</p></td><td align=\"left\">Internal</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Uncomplicated UTI AI-based diagnosis and symptom checkers</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Author</th><th align=\\\"left\\\">Objective</th><th align=\\\"left\\\">Dataset (<italic>n</italic>)</th><th align=\\\"left\\\">AI model</th><th align=\\\"left\\\">Demographics</th><th align=\\\"left\\\">Anamnesis</th><th align=\\\"left\\\">Comorbidity</th><th align=\\\"left\\\">Symptoms</th><th align=\\\"left\\\">Urine analysis</th><th align=\\\"left\\\">Imaging</th><th align=\\\"left\\\">Other</th><th align=\\\"left\\\">Performance</th><th align=\\\"left\\\">Validation</th></tr></thead><tbody><tr><td align=\\\"left\\\">Ozkan et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR12\\\">12</xref>••]</td><td align=\\\"left\\\">Prediction of cystitis and non-specific urethritis</td><td align=\\\"left\\\">59</td><td align=\\\"left\\\">ANN</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Pollakiuria, suprapubic pain</td><td align=\\\"left\\\">RBC</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\"><p>Accuracy, 98.3%</p><p>Sensitivity, 97.7%</p><p>Specificity, 100%</p></td><td align=\\\"left\\\">Internal</td></tr><tr><td align=\\\"left\\\">Gadalla et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR13\\\">13</xref>]</td><td align=\\\"left\\\">Prediction of UTI</td><td align=\\\"left\\\">183</td><td align=\\\"left\\\">SVM + RF</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Cloudiness</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Immunological markers</td><td align=\\\"left\\\">AUC, 0.86</td><td align=\\\"left\\\">Internal</td></tr><tr><td align=\\\"left\\\">Dhanda et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR14\\\">14</xref>]</td><td align=\\\"left\\\">Prediction of UTI in ED</td><td align=\\\"left\\\">80,859</td><td align=\\\"left\\\">XGBoost</td><td align=\\\"left\\\">Age, gender</td><td align=\\\"left\\\">History of UTI</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Dysuria, suprapubic pain</td><td align=\\\"left\\\">Dipstick test results</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">AUC, 0.85</td><td align=\\\"left\\\">External</td></tr><tr><td align=\\\"left\\\">Arches et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR15\\\">15</xref>]</td><td align=\\\"left\\\">Prediction of UTI</td><td align=\\\"left\\\">65</td><td align=\\\"left\\\">CNN</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Dipstick test results</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Accuracy, 96%</td><td align=\\\"left\\\">Internal</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Complicated UTI AI-based diagnosis</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Author</th><th align=\\\"left\\\">Objective</th><th align=\\\"left\\\">Dataset (<italic>n</italic>)</th><th align=\\\"left\\\">AI model</th><th align=\\\"left\\\">Demographics</th><th align=\\\"left\\\">Anamnesis</th><th align=\\\"left\\\">Comorbidity</th><th align=\\\"left\\\">symptoms</th><th align=\\\"left\\\">Urine analysis</th><th align=\\\"left\\\">Imaging</th><th align=\\\"left\\\">Other</th><th align=\\\"left\\\">Performance</th><th align=\\\"left\\\">Validation</th></tr></thead><tbody><tr><td align=\\\"left\\\">Møller et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR16\\\">16</xref>]</td><td align=\\\"left\\\">Prediction of UTI (model 1) and hospital-acquired UTI (model 2)</td><td align=\\\"left\\\">300,000</td><td align=\\\"left\\\">ANN</td><td align=\\\"left\\\">Age, gender</td><td align=\\\"left\\\">History of UTI</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Fever, dysuria, frequency, urgency, suprapubic pain</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">AUC, 0.84</td><td align=\\\"left\\\">Internal</td></tr><tr><td align=\\\"left\\\">Taylor et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR17\\\">17</xref>]</td><td align=\\\"left\\\">Prediction of UTI in ED</td><td align=\\\"left\\\">80,387</td><td align=\\\"left\\\">XGBoost</td><td align=\\\"left\\\">Age, gender</td><td align=\\\"left\\\">History of UTI</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Dysuria</td><td align=\\\"left\\\">Nitrites, WBC, RBC, bacteria, epithelial cells</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\"><p>AUC, 0.904</p><p>Sensitivity</p><p>Specificity:</p></td><td align=\\\"left\\\">Internal</td></tr><tr><td align=\\\"left\\\">Mancini et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR18\\\">18</xref>]</td><td align=\\\"left\\\">Prediction of hospital-acquired multidrug-resistant UTI</td><td align=\\\"left\\\">1486</td><td align=\\\"left\\\">CatBoost</td><td align=\\\"left\\\">Age, gender</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Ward, length of stay</td><td align=\\\"left\\\"><p>AUC, 0.853</p><p>Sensitivity, 0.904</p></td><td align=\\\"left\\\">Internal</td></tr><tr><td align=\\\"left\\\">Cai et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR19\\\">19</xref>]</td><td align=\\\"left\\\">Prediction of clinical efficacy of antibiotics in women with recurrent UTIs</td><td align=\\\"left\\\">1043</td><td align=\\\"left\\\">ANN</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\"><p>Model 1: history of fluoroquinolones and cephalosporins failure, previously mentioned <italic>E. coli</italic> resistant to cotrimoxazole</p><p>Model 2: previously mentioned <italic>E. coli</italic> resistant to cotrimoxazole and amoxicillin-clavulanic acid</p></td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\"><p>For both:</p><p>AUC, 0.87</p><p>Sensitivity, 87.8%</p><p>Specificity, 97.3%</p></td><td align=\\\"left\\\">Internal</td></tr><tr><td align=\\\"left\\\">Chen et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR20\\\">20</xref>•]</td><td align=\\\"left\\\">Prediction of UTI after cystoscopy</td><td align=\\\"left\\\">1647</td><td align=\\\"left\\\">ANN</td><td align=\\\"left\\\">Age</td><td align=\\\"left\\\">History of UTI</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Cystoscopy findings</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\"><p>Accuracy, 91%</p><p>Sensitivity, 80%</p><p>Specificity, 88%</p></td><td align=\\\"left\\\">Internal</td></tr><tr><td align=\\\"left\\\">Hong et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR21\\\">21</xref>]</td><td align=\\\"left\\\">Prediction of urosepsis among patients with upper urinary tract calculi</td><td align=\\\"left\\\">1716</td><td align=\\\"left\\\">ANN</td><td align=\\\"left\\\">Age, gender</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Diabetes</td><td align=\\\"left\\\">Fever</td><td align=\\\"left\\\">WBC, nitrites, glucose</td><td align=\\\"left\\\">Ultrasound (hydronephrosis grade)</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\"><p>AUC, 0.95</p><p>Sensitivity, 80.4%</p><p>Specificity, 98.2%</p></td><td align=\\\"left\\\">Internal</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>UTI AI-based diagnosis in susceptible subgroups</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Author</th><th align=\\\"left\\\">Objective</th><th align=\\\"left\\\">Dataset (<italic>n</italic>)</th><th align=\\\"left\\\">AI model</th><th align=\\\"left\\\">Demographics</th><th align=\\\"left\\\">Anamnesis</th><th align=\\\"left\\\">Comorbidity</th><th align=\\\"left\\\">Symptoms</th><th align=\\\"left\\\">Urine analysis</th><th align=\\\"left\\\">Imaging</th><th align=\\\"left\\\">Other</th><th align=\\\"left\\\">Performance</th><th align=\\\"left\\\">Validation</th></tr></thead><tbody><tr><td align=\\\"left\\\">Bertsimas et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR24\\\">24</xref>]</td><td align=\\\"left\\\">Prediction of continuous antibiotic prophylaxis benefits among children with VUR and UTI</td><td align=\\\"left\\\">607</td><td align=\\\"left\\\">ANN</td><td align=\\\"left\\\">Race, gender, weight</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Fever, dysuria</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Ultrasound (vesicoureteral reflux grade)</td><td align=\\\"left\\\">Serum creatinine</td><td align=\\\"left\\\">AUC, 0.82</td><td align=\\\"left\\\">Internal</td></tr><tr><td align=\\\"left\\\">Burton et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR25\\\">25</xref>]</td><td align=\\\"left\\\">Prediction of urine culture in pregnant and children</td><td align=\\\"left\\\">212,554</td><td align=\\\"left\\\">XGBoost</td><td align=\\\"left\\\">Age, gender</td><td align=\\\"left\\\">History of urine culture results</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">WBC, bacteria</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\"><p>AUC, 91%</p><p>Sensitivity, 95%</p></td><td align=\\\"left\\\">Internal</td></tr><tr><td align=\\\"left\\\">Zhu et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR27\\\">27</xref>]</td><td align=\\\"left\\\">Prediction of UTI among immobile stroke patients</td><td align=\\\"left\\\">7819</td><td align=\\\"left\\\">Ensemble learning model (ELM)</td><td align=\\\"left\\\">Age, gender</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Pneumonia, mixed cerebrovascular disease</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Length of stay, length of urethral catheterization, glucocorticoid use</td><td align=\\\"left\\\"><p>AUC, 0.82</p><p>Sensitivity, 80.9%</p></td><td align=\\\"left\\\">External</td></tr><tr><td align=\\\"left\\\">Xu et al. [<xref ref-type=\\\"bibr\\\" rid=\\\"CR28\\\">28</xref>]</td><td align=\\\"left\\\">Prediction of UTI among immobile stroke patients</td><td align=\\\"left\\\">3982</td><td align=\\\"left\\\">Siamese network</td><td align=\\\"left\\\">Age, gender</td><td align=\\\"left\\\">History of urethral invasive procedures</td><td align=\\\"left\\\">Classification of stroke, pneumonia, diabetes</td><td align=\\\"left\\\">Urine incontinence</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">-</td><td align=\\\"left\\\">Length of stay, duration of immobility, length of urethral catheterization, serum albumin</td><td align=\\\"left\\\"><p>AUC: 0.83</p><p>Accuracy: 74.2%</p><p>Sensitivity: 81%</p><p>Specificity: 74%</p></td><td align=\\\"left\\\">Internal</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"11934_2023_1192_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"11934_2023_1192_Fig2_HTML\" id=\"MO2\"/>"],"string":"[\n \"<graphic xlink:href=\\\"11934_2023_1192_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"11934_2023_1192_Fig2_HTML\\\" id=\\\"MO2\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":0,"string":"0"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Curr Urol Rep. 2024 Dec 19; 25(1):37-47"},"package_file":{"kind":"string","value":"oa_package/c0/67/PMC10787904.tar.gz"}}},{"rowIdx":188,"cells":{"accession_id":{"kind":"string","value":"PMC10787905"},"pmid":{"kind":"string","value":"38217694"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par6\">Schwannomas are peripheral nerve sheath tumors (PNST) found in cranial and spinal nerves throughout the body. Schwannomas of the orbit are rare, and there are less than 500 cases reported in the literature [##REF##33397169##16##]. It is estimated that these tumors constitute only about 1% of orbital tumors [##UREF##1##9##]. They are typically benign, slow growing, and encapsulated tumors that occur without any predilection for sex or age [##REF##33397169##16##]. The growth of these tumors has been associated to functional and/or aesthetic morbidity and malignant transformation seems to be exceedingly rare [##UREF##0##8##]. Symptoms and clinical signs vary depending on the size and location of the tumor and may include a palpable mass, bulb dislocation, ptosis, optic neuropathy, and diplopia.</p>","<p id=\"Par7\">Management of these tumors is challenging and often requires a multidisciplinary collaboration that involves ophthalmologists, neurosurgeons, otolaryngologists, and maxillofacial surgeons. When possible, surgery with gross total resection (GTR) is the treatment of choice [##REF##33072479##5##]. The orbit is a confined space delimited by bone, containing delicate neuro-ophthalmic structures and with a limited capacity to expand [##REF##19539832##15##]. In the context of orbital schwannomas, curative approaches are often pursued to prevent tumor growth and subsequent compression and injury to intra-orbital structures [##UREF##3##19##]. The majority of cases in the literature have been offered surgery and most of the remaining have been treated with radiation therapy or stereotactic radiosurgery [##REF##18653273##12##, ##REF##33397169##16##]. Nevertheless, the surgical risks inherent to the complexity of the neurovascular structures of the orbit are not negligible [##REF##18645431##2##, ##REF##31341800##6##, ##REF##29551422##10##, ##REF##1520662##18##] and warrant further investigation of the outcomes of conservative management. Recently, three separate reports have questioned the validity of an aggressive treatment strategy and advocate watchful waiting in selected cases [##REF##34229331##3##, ##REF##30073272##7##, ##REF##33397169##16##]. In addition, increased use, availability, and sensitivity of diagnostic imaging makes incidental findings of orbital tumors more common, which also indicates a need for conservative approaches.</p>","<p id=\"Par8\">In brief, the natural course of OS has been poorly studied and support for conservative management is consequently lacking. The aim of this study was to evaluate the national Swedish experience of surgical and conservative management of OS.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par6\\\">Schwannomas are peripheral nerve sheath tumors (PNST) found in cranial and spinal nerves throughout the body. Schwannomas of the orbit are rare, and there are less than 500 cases reported in the literature [##REF##33397169##16##]. It is estimated that these tumors constitute only about 1% of orbital tumors [##UREF##1##9##]. They are typically benign, slow growing, and encapsulated tumors that occur without any predilection for sex or age [##REF##33397169##16##]. The growth of these tumors has been associated to functional and/or aesthetic morbidity and malignant transformation seems to be exceedingly rare [##UREF##0##8##]. Symptoms and clinical signs vary depending on the size and location of the tumor and may include a palpable mass, bulb dislocation, ptosis, optic neuropathy, and diplopia.</p>\",\n \"<p id=\\\"Par7\\\">Management of these tumors is challenging and often requires a multidisciplinary collaboration that involves ophthalmologists, neurosurgeons, otolaryngologists, and maxillofacial surgeons. When possible, surgery with gross total resection (GTR) is the treatment of choice [##REF##33072479##5##]. The orbit is a confined space delimited by bone, containing delicate neuro-ophthalmic structures and with a limited capacity to expand [##REF##19539832##15##]. In the context of orbital schwannomas, curative approaches are often pursued to prevent tumor growth and subsequent compression and injury to intra-orbital structures [##UREF##3##19##]. The majority of cases in the literature have been offered surgery and most of the remaining have been treated with radiation therapy or stereotactic radiosurgery [##REF##18653273##12##, ##REF##33397169##16##]. Nevertheless, the surgical risks inherent to the complexity of the neurovascular structures of the orbit are not negligible [##REF##18645431##2##, ##REF##31341800##6##, ##REF##29551422##10##, ##REF##1520662##18##] and warrant further investigation of the outcomes of conservative management. Recently, three separate reports have questioned the validity of an aggressive treatment strategy and advocate watchful waiting in selected cases [##REF##34229331##3##, ##REF##30073272##7##, ##REF##33397169##16##]. In addition, increased use, availability, and sensitivity of diagnostic imaging makes incidental findings of orbital tumors more common, which also indicates a need for conservative approaches.</p>\",\n \"<p id=\\\"Par8\\\">In brief, the natural course of OS has been poorly studied and support for conservative management is consequently lacking. The aim of this study was to evaluate the national Swedish experience of surgical and conservative management of OS.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<title>Admission routine</title>","<p id=\"Par9\">The study center is the primary Swedish referral center for multidisciplinary management of orbital tumors, including schwannomas. During the period of 2005 to 2021, 16 patients with a new diagnosis of OS were managed. Other neurosurgical and neuro-ophthalmological centers in Sweden were contacted to identify cases that may have been overlooked. One neurosurgical center reported having managed up to five cases during the study period. Unfortunately, no data on patients treated outside the authors’ institution could be retrieved.</p>","<p id=\"Par10\">Once a patient with a lesion suspicious of schwannoma is referred to the study center, a complete neuroophthalmological examination is performed. This includes additional imaging, automated perimetry, Optical Coherence Tomography (OCT) and fine-needle aspiration biopsy if deemed necessary. Management is then discussed at a multidisciplinary conference of ophthalmologists, neuro-ophthalmologists, radiologists, and in some cases neurosurgeons. Typically, a conservative management is considered for all patients with OS if the indications for early surgery are not met.</p>","<title>Surgery and postoperative follow-up</title>","<p id=\"Par11\">Indications for early surgery are deformation of the globe or compression of the optic nerve leading to optic neuropathy. Relative indications for surgery may include impaired motility with double vision and cosmetic implications including proptosis, hyper- or hypoglobus, or ptosis with a palpable mass.</p>","<p id=\"Par12\">The surgical approach is determined by the surgeon’s preference and the tumor location, size, and extension. All procedures are performed with microsurgical techniques and under general anesthesia. Tumors extending into the orbital apex, or through the orbital fissure, or those causing severe thinning of adjacent bony structures are often operated jointly with a neurosurgeon. OS located in the superior part of the orbit can be accessed by an anterior orbitotomy through an eyelid crease incision. OS located posteriorly in the orbital apex, sometimes with skull base extensions, are often accessed transcranially via craniotomy.</p>","<p id=\"Par13\">In GTR, the nerve harboring the tumor is cut to allow complete removal of the tumor. In subtotal resection (STR), the tumor capsule is incised along the longitudinal axis of the nerve, and the schwannoma is excised leaving the capsule to retain the integrity of the nerve. This approach offers surgical plane separated from delicate structures surrounding the nerve, allowing safe tumor removal deep in the apex of the orbit.</p>","<p id=\"Par14\">A neuro-ophthalmic examination and MRI are performed at one-week post-surgery. If radicality is confirmed on MRI and the pathology report, no further follow-ups are required. However, in the case of STR, MRI is recommended at 3 months post-surgery and annually afterwards. In these cases, the follow-up is generally carried out at the primary referring clinic.</p>","<p id=\"Par15\">At follow-up, tumor growth was defined as the radiological growth of a tumor remnant following SRT, while tumor recurrence was defined as the reappearance of a tumor following GTR.</p>","<title>Conservative management and follow-up</title>","<p id=\"Par16\">Conservative management is offered to patients with mild symptoms, no signs of compressive optic neuropathy and low-risk of neuro-ophthalmic impairment. This encompasses individuals incidentally discovered, and those reporting minimal subjective discomfort, proptosis, globe displacement, or ocular motility issues that do not interfere with everyday tasks, such as medical requirements for driving. Low-risk neuro-ophthalmic impairment is defined as cases where the tumor is not in the apex or direct contact with the optic nerve.</p>","<p id=\"Par17\">For these patients, radiological and ophthalmological examinations are performed at regular intervals. In patients where the tumor is in the orbital apex or located in the vicinity of the optic nerve automated perimetry and, in later years, OCT with measurements of the peripapillary retinal nerve fiber layer, macular ganglion cell layer thickness and macular ganglion cell inner plexiform layer is added. OCT with these measurements is a useful tool as they provide early signs of compressive optic neuropathy often preceding changes on automated perimetry [##REF##35053482##1##, ##REF##33239763##17##]. Additionally, patients are encouraged to seek care upon onset or worsening of symptoms. There is no consensus on the intervals or the optimal length of follow-up. In our practice, patients with stable tumors are examined twice a year during the first 2 years after the diagnosis and then once a year, until 5 years. However, longer follow-ups may be indicated in selected cases, including children. In patients with slowly growing tumors, MRI is performed twice a year until cessation of tumor growth or surgery. After the follow-up period patients are encouraged to seek care for any eye-related symptoms.</p>","<title>Study setting</title>","<p id=\"Par18\">Patients included in this study were divided into 3 groups: patients with surgery as the primary management (group 1), patients converted to surgery after an initially conservative management (group 2), and patients conservatively treated throughout the entire follow-up period (group 3).</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<title>Admission routine</title>\",\n \"<p id=\\\"Par9\\\">The study center is the primary Swedish referral center for multidisciplinary management of orbital tumors, including schwannomas. During the period of 2005 to 2021, 16 patients with a new diagnosis of OS were managed. Other neurosurgical and neuro-ophthalmological centers in Sweden were contacted to identify cases that may have been overlooked. One neurosurgical center reported having managed up to five cases during the study period. Unfortunately, no data on patients treated outside the authors’ institution could be retrieved.</p>\",\n \"<p id=\\\"Par10\\\">Once a patient with a lesion suspicious of schwannoma is referred to the study center, a complete neuroophthalmological examination is performed. This includes additional imaging, automated perimetry, Optical Coherence Tomography (OCT) and fine-needle aspiration biopsy if deemed necessary. Management is then discussed at a multidisciplinary conference of ophthalmologists, neuro-ophthalmologists, radiologists, and in some cases neurosurgeons. Typically, a conservative management is considered for all patients with OS if the indications for early surgery are not met.</p>\",\n \"<title>Surgery and postoperative follow-up</title>\",\n \"<p id=\\\"Par11\\\">Indications for early surgery are deformation of the globe or compression of the optic nerve leading to optic neuropathy. Relative indications for surgery may include impaired motility with double vision and cosmetic implications including proptosis, hyper- or hypoglobus, or ptosis with a palpable mass.</p>\",\n \"<p id=\\\"Par12\\\">The surgical approach is determined by the surgeon’s preference and the tumor location, size, and extension. All procedures are performed with microsurgical techniques and under general anesthesia. Tumors extending into the orbital apex, or through the orbital fissure, or those causing severe thinning of adjacent bony structures are often operated jointly with a neurosurgeon. OS located in the superior part of the orbit can be accessed by an anterior orbitotomy through an eyelid crease incision. OS located posteriorly in the orbital apex, sometimes with skull base extensions, are often accessed transcranially via craniotomy.</p>\",\n \"<p id=\\\"Par13\\\">In GTR, the nerve harboring the tumor is cut to allow complete removal of the tumor. In subtotal resection (STR), the tumor capsule is incised along the longitudinal axis of the nerve, and the schwannoma is excised leaving the capsule to retain the integrity of the nerve. This approach offers surgical plane separated from delicate structures surrounding the nerve, allowing safe tumor removal deep in the apex of the orbit.</p>\",\n \"<p id=\\\"Par14\\\">A neuro-ophthalmic examination and MRI are performed at one-week post-surgery. If radicality is confirmed on MRI and the pathology report, no further follow-ups are required. However, in the case of STR, MRI is recommended at 3 months post-surgery and annually afterwards. In these cases, the follow-up is generally carried out at the primary referring clinic.</p>\",\n \"<p id=\\\"Par15\\\">At follow-up, tumor growth was defined as the radiological growth of a tumor remnant following SRT, while tumor recurrence was defined as the reappearance of a tumor following GTR.</p>\",\n \"<title>Conservative management and follow-up</title>\",\n \"<p id=\\\"Par16\\\">Conservative management is offered to patients with mild symptoms, no signs of compressive optic neuropathy and low-risk of neuro-ophthalmic impairment. This encompasses individuals incidentally discovered, and those reporting minimal subjective discomfort, proptosis, globe displacement, or ocular motility issues that do not interfere with everyday tasks, such as medical requirements for driving. Low-risk neuro-ophthalmic impairment is defined as cases where the tumor is not in the apex or direct contact with the optic nerve.</p>\",\n \"<p id=\\\"Par17\\\">For these patients, radiological and ophthalmological examinations are performed at regular intervals. In patients where the tumor is in the orbital apex or located in the vicinity of the optic nerve automated perimetry and, in later years, OCT with measurements of the peripapillary retinal nerve fiber layer, macular ganglion cell layer thickness and macular ganglion cell inner plexiform layer is added. OCT with these measurements is a useful tool as they provide early signs of compressive optic neuropathy often preceding changes on automated perimetry [##REF##35053482##1##, ##REF##33239763##17##]. Additionally, patients are encouraged to seek care upon onset or worsening of symptoms. There is no consensus on the intervals or the optimal length of follow-up. In our practice, patients with stable tumors are examined twice a year during the first 2 years after the diagnosis and then once a year, until 5 years. However, longer follow-ups may be indicated in selected cases, including children. In patients with slowly growing tumors, MRI is performed twice a year until cessation of tumor growth or surgery. After the follow-up period patients are encouraged to seek care for any eye-related symptoms.</p>\",\n \"<title>Study setting</title>\",\n \"<p id=\\\"Par18\\\">Patients included in this study were divided into 3 groups: patients with surgery as the primary management (group 1), patients converted to surgery after an initially conservative management (group 2), and patients conservatively treated throughout the entire follow-up period (group 3).</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Incidence</title>","<p id=\"Par19\">Considering the 16 documented cases of OS between the years of 2005 and 2021, the apparent incidence of OS in Sweden was estimated at 0.1 per million and year. However, the true incidence may be higher.</p>","<title>Baseline characteristics</title>","<p id=\"Par20\">A total of 16 patients with OS, diagnosed in Sweden between 2005 and 2021, were included in this study (Table ##TAB##0##1##). Patients were aged between 8 and 74 years (median 52) at the time of diagnosis and 50% (<italic>n</italic> = 8) were female. Six patients (37%) were referred due to the incidental detection of an orbital mass during imaging for unrelated causes, such as dementia, stroke, or hydrocephalus. The remainder of the patients actively sought care for ophthalmic symptoms. Patients were referred to the study center under preliminary diagnoses, including OS, cavernous malformation, or dermoid cysts. A final diagnosis of OS was established in all cases, based on either histology and imaging (<italic>n</italic> = 11; 69%), or imaging alone (<italic>n</italic> = 5; 31%).</p>","<p id=\"Par21\">Initial imaging revealed well-demarcated tumors of varying sizes, often heterogeneous (<italic>n</italic> = 11; 69%) and cystic (<italic>n</italic> = 11; 69%) on MRI. Half of the tumors exhibited lobular growth patterns (<italic>n</italic> = 8). The location of the tumor was extraconal in eight cases (50%), intraconal in six cases (38%), and mixed intra- and extraconal in 2 cases (12%). Extension of the tumor into the skull base was found in five cases (31%), where four passed through the orbital fissure and one through the orbital roof. Thinning of adjacent bone in conjunction with the slow growth of the tumor was seen in 10 patients (63%), involving the superior orbital fissure, the orbital roof, the medial, and lateral walls of the orbit, or the frontal bone (Table ##TAB##0##1##).</p>","<p id=\"Par22\">Surgical management was chosen in 4 patients (25%) and conservative management in 12 patients (75%). Among the 12 conservatively managed, later follow-ups resulted in delayed surgery in 3 patients (25%, Table ##TAB##0##1##).</p>","<title>Presenting signs and symptoms</title>","<p id=\"Par23\">Ten patients had symptoms on presentation (63%), the rest of the cases were discovered incidentally. Symptomatic patients reported an average symptom duration of 18 months prior to presentation and diagnosis. Proptosis was the most common finding (<italic>n</italic> = 12; 75%), followed by vertical displacement of the eye globe (<italic>n</italic> = 7; 44%) with all but one exhibiting hypoglobus rather than hyperglobus. Six of the patients (37%) presented with diplopia, five (31%) with pain or discomfort localized to the orbital region, four (25%) with impaired ocular motility and visual impairment, and three (19%) with ptosis. Presenting signs and symptoms were more common among patients who underwent surgery. More than half of the patients (56%) managed conservatively had been incidentally diagnosed with OS, as opposed to only one patient (14%) among those surgically managed (Table ##TAB##1##2##).</p>","<title>Outcomes</title>","<p id=\"Par24\">Four patients underwent early surgery (group 1), three with GTR (75%) and one with STR (25%). Three patients, initially treated conservatively, were later operated on with STR, (group 2). One of these three was initially offered surgery due to advanced tumor size and extent of symptoms but had declined. After 15 years of observation, the patient deteriorated due to tumor growth and underwent emergency surgery (Patient 8 in Table ##TAB##0##1##; and Fig. ##FIG##0##1##). The two other patients did not experience clinical worsening but were both offered surgery after 1.5 years of observation due to rapid tumor growth, increasing pressure on the eye globe, or thinning of surrounding bony structures (Patients 6 and 13 in Table ##TAB##1##2##). For groups 1 and 2, the mean postoperative radiological follow-up was 96 and 24 months, while the mean clinical follow-up was 81 and 44 months, respectively. In both groups, no tumor growth or recurrence were detected (two patients lost to radiological follow-up) and all patients had favorable postoperative outcomes characterized by complete symptom resolution (Table ##TAB##2##3##). One patient operated for an incidentally discovered tumor remained asymptomatic postoperatively. Among the patients conservatively managed (group 3), none experienced any worsening of symptoms at an average of 30 months of clinical follow-up. Moreover, only one of these patients (Patient 12, 11%) experienced tumor growth after 17 months, while the rest had no evidence of further growth over a radiographic follow-up period of 24 months (Table ##TAB##2##3##, Fig. ##FIG##1##2##).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Incidence</title>\",\n \"<p id=\\\"Par19\\\">Considering the 16 documented cases of OS between the years of 2005 and 2021, the apparent incidence of OS in Sweden was estimated at 0.1 per million and year. However, the true incidence may be higher.</p>\",\n \"<title>Baseline characteristics</title>\",\n \"<p id=\\\"Par20\\\">A total of 16 patients with OS, diagnosed in Sweden between 2005 and 2021, were included in this study (Table ##TAB##0##1##). Patients were aged between 8 and 74 years (median 52) at the time of diagnosis and 50% (<italic>n</italic> = 8) were female. Six patients (37%) were referred due to the incidental detection of an orbital mass during imaging for unrelated causes, such as dementia, stroke, or hydrocephalus. The remainder of the patients actively sought care for ophthalmic symptoms. Patients were referred to the study center under preliminary diagnoses, including OS, cavernous malformation, or dermoid cysts. A final diagnosis of OS was established in all cases, based on either histology and imaging (<italic>n</italic> = 11; 69%), or imaging alone (<italic>n</italic> = 5; 31%).</p>\",\n \"<p id=\\\"Par21\\\">Initial imaging revealed well-demarcated tumors of varying sizes, often heterogeneous (<italic>n</italic> = 11; 69%) and cystic (<italic>n</italic> = 11; 69%) on MRI. Half of the tumors exhibited lobular growth patterns (<italic>n</italic> = 8). The location of the tumor was extraconal in eight cases (50%), intraconal in six cases (38%), and mixed intra- and extraconal in 2 cases (12%). Extension of the tumor into the skull base was found in five cases (31%), where four passed through the orbital fissure and one through the orbital roof. Thinning of adjacent bone in conjunction with the slow growth of the tumor was seen in 10 patients (63%), involving the superior orbital fissure, the orbital roof, the medial, and lateral walls of the orbit, or the frontal bone (Table ##TAB##0##1##).</p>\",\n \"<p id=\\\"Par22\\\">Surgical management was chosen in 4 patients (25%) and conservative management in 12 patients (75%). Among the 12 conservatively managed, later follow-ups resulted in delayed surgery in 3 patients (25%, Table ##TAB##0##1##).</p>\",\n \"<title>Presenting signs and symptoms</title>\",\n \"<p id=\\\"Par23\\\">Ten patients had symptoms on presentation (63%), the rest of the cases were discovered incidentally. Symptomatic patients reported an average symptom duration of 18 months prior to presentation and diagnosis. Proptosis was the most common finding (<italic>n</italic> = 12; 75%), followed by vertical displacement of the eye globe (<italic>n</italic> = 7; 44%) with all but one exhibiting hypoglobus rather than hyperglobus. Six of the patients (37%) presented with diplopia, five (31%) with pain or discomfort localized to the orbital region, four (25%) with impaired ocular motility and visual impairment, and three (19%) with ptosis. Presenting signs and symptoms were more common among patients who underwent surgery. More than half of the patients (56%) managed conservatively had been incidentally diagnosed with OS, as opposed to only one patient (14%) among those surgically managed (Table ##TAB##1##2##).</p>\",\n \"<title>Outcomes</title>\",\n \"<p id=\\\"Par24\\\">Four patients underwent early surgery (group 1), three with GTR (75%) and one with STR (25%). Three patients, initially treated conservatively, were later operated on with STR, (group 2). One of these three was initially offered surgery due to advanced tumor size and extent of symptoms but had declined. After 15 years of observation, the patient deteriorated due to tumor growth and underwent emergency surgery (Patient 8 in Table ##TAB##0##1##; and Fig. ##FIG##0##1##). The two other patients did not experience clinical worsening but were both offered surgery after 1.5 years of observation due to rapid tumor growth, increasing pressure on the eye globe, or thinning of surrounding bony structures (Patients 6 and 13 in Table ##TAB##1##2##). For groups 1 and 2, the mean postoperative radiological follow-up was 96 and 24 months, while the mean clinical follow-up was 81 and 44 months, respectively. In both groups, no tumor growth or recurrence were detected (two patients lost to radiological follow-up) and all patients had favorable postoperative outcomes characterized by complete symptom resolution (Table ##TAB##2##3##). One patient operated for an incidentally discovered tumor remained asymptomatic postoperatively. Among the patients conservatively managed (group 3), none experienced any worsening of symptoms at an average of 30 months of clinical follow-up. Moreover, only one of these patients (Patient 12, 11%) experienced tumor growth after 17 months, while the rest had no evidence of further growth over a radiographic follow-up period of 24 months (Table ##TAB##2##3##, Fig. ##FIG##1##2##).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par25\">This study reports on a population-based cohort of orbital schwannomas. Of the 16 patients included, four (25%) underwent early surgery at the time of diagnosis, while three were surgically treated later, after initial conservative management (19%), and nine (56%) did not require any surgical intervention during the study period. Although no intraoperative complications occurred in this series, orbital surgery carries the risk of nerve, vessel, and extraocular muscle damage, which may result in both functional and cosmetic sequelae [##REF##29551422##10##, ##REF##1520662##18##]. For patients with no or mild symptoms or who are poor candidates for surgery, the risks of surgical treatment may outweigh the benefits. Conservative management with regular clinical and radiological follow-ups was therefore adopted. Additionally, patients were encouraged to immediately seek care in the advent of any new symptoms or worsening of previous symptoms.</p>","<p id=\"Par26\">Currently, it is a matter of debate whether GTR should be the gold standard for all patients diagnosed with OS [##REF##34229331##3##]. To date, there are only three cases of conservative management reported in the literature. One case was observed over a 4-year period due to the risk for cosmetic disfigurement in case of surgery. There was no deterioration or increase in tumor size during the observation period [##REF##33397169##16##]. In the second case, surgery was not offered given lack of symptoms and high risk of injury to orbital neurovascular structures. The patient’s neuro-ophthalmic status remained stable at 6 months of follow-up [##REF##30073272##7##]. The third case was an 8-year-old with an orbital schwannoma involving the extraocular muscles. However, outcomes at follow-up were poorly disclosed [##REF##2240140##4##]</p>","<p id=\"Par27\">Most schwannomas are benign and slow growing WHO grade I tumors. However, intraorbital space is limited and the contained structures are sensitive. To minimize the risk of damage to these structures, surgery is often performed promptly. Surgical tumor removal may come at the cost of injury to the intraorbital structures. Consequently, a conservative management strategy may provide a safer course in selected cases. Of the 12 conservatively managed patients, only three required delayed surgery due to tumor growth (<italic>n</italic> = 3) or worsening of symptoms (<italic>n</italic> = 1). The patient who experienced both tumor growth and worsening of symptoms was initially offered surgery but declined. The rest (<italic>n</italic> = 9) were conservatively managed without requiring further intervention during the study period. Among these patients, only one experienced tumor growth. However, the growth was mild (from 2.4×1.7×1.8 cm to 2.6×1.6×2.1cm) and the patient had no associated symptoms.</p>","<p id=\"Par28\">Patients presenting with signs of neuro-ophthalmic compromise were offered early surgery. At the study center, STR was often considered when the tumor reached deep into the apex or was adjacent to major neurovascular structures. Of the seven surgically treated patients, four underwent STR (57%) with no reported intraoperative complications, contrary to previous reports [##REF##18645431##2##, ##REF##31341800##6##, ##UREF##4##21##]. Despite STR, none of these patients experienced tumor growth at 65 months of radiological follow-up. In fact, only two cases of tumor recurrence have been reported in the literature, lending further support to the benign behavior of these tumors.[##UREF##2##13##] Taken together, this indicates that STR may be sufficient to secure tumor control when GTR may not be safely achievable.</p>","<p id=\"Par29\">Delaying surgery may arguably do harm through the prolonged compression of intra-orbital structures. However, conservative management allows for surgical intervention when indicated by changes in symptoms or imaging. Nonetheless, this strategy is best suited for tumors that do not compromise the optic nerve or the globe. The group that underwent late surgery had similar outcomes as the early surgical group, demonstrating the success of this strategy.</p>","<p id=\"Par30\">Radiosurgery, predominantly Gamma Knife surgery (GKS), is increasingly used for benign orbital tumors including schwannomas [##REF##21121785##20##]. However, doses greater than 12 Gy are considered unsafe due to the risk of optic neuropathy and visual acuity impairments are seen with doses ranging from 6 to 16 Gy [##REF##8584833##14##]. Several studies report good tumor control after GKS, however, there are no guidelines for when to pursue GKS over surgical resection [##REF##25931308##11##, ##REF##21121785##20##]. None of the patients in this study were considered for radio surgery, reflecting our own institutional practices. However, radiosurgery may be considered as a viable alternative to surgery or conservative treatment and should incorporated in future treatment guidelines.</p>","<p id=\"Par31\">In summary, the results of this study suggest that surgery may be avoided or delayed in a selected number of patients presenting with mild or no symptoms. Conservative management, with clinical and radiological follow-ups with gradually increased intervals are therefore advocated in these cases. In cases requiring surgery, STR offers tumor control with less surgical risks compared to GTR. Postoperative yearly radiological controls, for a period of 5 years, are suggested to detect rare cases of tumor growth or recurrence (Fig. ##FIG##2##3##). Yearly follow-ups are motivated by the rarity of the disease and the risk of permanent visual impairment.</p>","<title>Strengths and limitations</title>","<p id=\"Par32\">This population-based study is one of the largest series of reported OS with more than 5 years of follow-up. The retrospective study design and the small sample size, however, hamper the level of evidence. Since patients were referred from different regions, radiological and clinical follow-ups were not always consistent. The diagnosis of schwannoma was not histologically confirmed in four of the patients.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par25\\\">This study reports on a population-based cohort of orbital schwannomas. Of the 16 patients included, four (25%) underwent early surgery at the time of diagnosis, while three were surgically treated later, after initial conservative management (19%), and nine (56%) did not require any surgical intervention during the study period. Although no intraoperative complications occurred in this series, orbital surgery carries the risk of nerve, vessel, and extraocular muscle damage, which may result in both functional and cosmetic sequelae [##REF##29551422##10##, ##REF##1520662##18##]. For patients with no or mild symptoms or who are poor candidates for surgery, the risks of surgical treatment may outweigh the benefits. Conservative management with regular clinical and radiological follow-ups was therefore adopted. Additionally, patients were encouraged to immediately seek care in the advent of any new symptoms or worsening of previous symptoms.</p>\",\n \"<p id=\\\"Par26\\\">Currently, it is a matter of debate whether GTR should be the gold standard for all patients diagnosed with OS [##REF##34229331##3##]. To date, there are only three cases of conservative management reported in the literature. One case was observed over a 4-year period due to the risk for cosmetic disfigurement in case of surgery. There was no deterioration or increase in tumor size during the observation period [##REF##33397169##16##]. In the second case, surgery was not offered given lack of symptoms and high risk of injury to orbital neurovascular structures. The patient’s neuro-ophthalmic status remained stable at 6 months of follow-up [##REF##30073272##7##]. The third case was an 8-year-old with an orbital schwannoma involving the extraocular muscles. However, outcomes at follow-up were poorly disclosed [##REF##2240140##4##]</p>\",\n \"<p id=\\\"Par27\\\">Most schwannomas are benign and slow growing WHO grade I tumors. However, intraorbital space is limited and the contained structures are sensitive. To minimize the risk of damage to these structures, surgery is often performed promptly. Surgical tumor removal may come at the cost of injury to the intraorbital structures. Consequently, a conservative management strategy may provide a safer course in selected cases. Of the 12 conservatively managed patients, only three required delayed surgery due to tumor growth (<italic>n</italic> = 3) or worsening of symptoms (<italic>n</italic> = 1). The patient who experienced both tumor growth and worsening of symptoms was initially offered surgery but declined. The rest (<italic>n</italic> = 9) were conservatively managed without requiring further intervention during the study period. Among these patients, only one experienced tumor growth. However, the growth was mild (from 2.4×1.7×1.8 cm to 2.6×1.6×2.1cm) and the patient had no associated symptoms.</p>\",\n \"<p id=\\\"Par28\\\">Patients presenting with signs of neuro-ophthalmic compromise were offered early surgery. At the study center, STR was often considered when the tumor reached deep into the apex or was adjacent to major neurovascular structures. Of the seven surgically treated patients, four underwent STR (57%) with no reported intraoperative complications, contrary to previous reports [##REF##18645431##2##, ##REF##31341800##6##, ##UREF##4##21##]. Despite STR, none of these patients experienced tumor growth at 65 months of radiological follow-up. In fact, only two cases of tumor recurrence have been reported in the literature, lending further support to the benign behavior of these tumors.[##UREF##2##13##] Taken together, this indicates that STR may be sufficient to secure tumor control when GTR may not be safely achievable.</p>\",\n \"<p id=\\\"Par29\\\">Delaying surgery may arguably do harm through the prolonged compression of intra-orbital structures. However, conservative management allows for surgical intervention when indicated by changes in symptoms or imaging. Nonetheless, this strategy is best suited for tumors that do not compromise the optic nerve or the globe. The group that underwent late surgery had similar outcomes as the early surgical group, demonstrating the success of this strategy.</p>\",\n \"<p id=\\\"Par30\\\">Radiosurgery, predominantly Gamma Knife surgery (GKS), is increasingly used for benign orbital tumors including schwannomas [##REF##21121785##20##]. However, doses greater than 12 Gy are considered unsafe due to the risk of optic neuropathy and visual acuity impairments are seen with doses ranging from 6 to 16 Gy [##REF##8584833##14##]. Several studies report good tumor control after GKS, however, there are no guidelines for when to pursue GKS over surgical resection [##REF##25931308##11##, ##REF##21121785##20##]. None of the patients in this study were considered for radio surgery, reflecting our own institutional practices. However, radiosurgery may be considered as a viable alternative to surgery or conservative treatment and should incorporated in future treatment guidelines.</p>\",\n \"<p id=\\\"Par31\\\">In summary, the results of this study suggest that surgery may be avoided or delayed in a selected number of patients presenting with mild or no symptoms. Conservative management, with clinical and radiological follow-ups with gradually increased intervals are therefore advocated in these cases. In cases requiring surgery, STR offers tumor control with less surgical risks compared to GTR. Postoperative yearly radiological controls, for a period of 5 years, are suggested to detect rare cases of tumor growth or recurrence (Fig. ##FIG##2##3##). Yearly follow-ups are motivated by the rarity of the disease and the risk of permanent visual impairment.</p>\",\n \"<title>Strengths and limitations</title>\",\n \"<p id=\\\"Par32\\\">This population-based study is one of the largest series of reported OS with more than 5 years of follow-up. The retrospective study design and the small sample size, however, hamper the level of evidence. Since patients were referred from different regions, radiological and clinical follow-ups were not always consistent. The diagnosis of schwannoma was not histologically confirmed in four of the patients.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par33\">There were no differences in long term outcome between patients who had been managed with early surgery and those operated later after an initially conservative management. Conservatively treated patients had minimal to no symptoms and remained clinically stable throughout the follow-up period. Based on these findings, conservative management may successfully be adopted in cases with mild symptoms and low risk of neuro-ophthalmic impairment. Conversion to surgical management is indicated upon clinical deterioration or tumor growth.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par33\\\">There were no differences in long term outcome between patients who had been managed with early surgery and those operated later after an initially conservative management. Conservatively treated patients had minimal to no symptoms and remained clinically stable throughout the follow-up period. Based on these findings, conservative management may successfully be adopted in cases with mild symptoms and low risk of neuro-ophthalmic impairment. Conversion to surgical management is indicated upon clinical deterioration or tumor growth.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Abstract</title>","<title>Introduction</title>","<p id=\"Par1\">Orbital schwannomas (OS) are rare occurrences with no more than 500 cases reported in the literature. The tumor’s potential to compromise the delicate neuro-ophthalmic structures within the orbit prompts surgical removal. Tumor removal is performed by ophthalmologists, often requiring a multidisciplinary surgical approach. The literature contains a very limited number of cases managed non-surgically. However, the inherent risks of orbital surgery warrant a comparison of the outcomes of conservative and surgical management strategies.</p>","<title>Aims</title>","<p id=\"Par2\">To review the national Swedish experience with the management of orbital schwannomas.</p>","<title>Methods</title>","<p id=\"Par3\">The study center is the primary Swedish referral center for the multidisciplinary management of orbital tumors, including schwannomas. During the period of 2005 to 2021, 16 patients with an OS diagnosis were managed at the center.</p>","<title>Results</title>","<p id=\"Par4\">Four patients initially underwent surgery where gross total resection (GTR) was achieved in three (75%) and subtotal resection (STR) in one (25%) case. The remaining 12 patients, who had a low risk of neuro-ophthalmic impairment, were managed conservatively with radiological and clinical examinations at regular intervals. After an average follow-up of 17 months, surgery was performed in three of these cases (25%). No recurrences or tumor growths were detected on radiological follow-ups (mean 50 months), and all patients experienced postoperative improvement at clinical follow-up (mean 65 months). The remainder of the conservatively treated patients (<italic>n</italic>=9) experienced no clinical progression (mean 30 months). A slight radiological tumor progression was detected in one patient after 17 months.</p>","<title>Conclusion</title>","<p id=\"Par5\">There were no differences in long-term outcome between patients who had been managed with early surgery and those operated later after an initially conservative management. Conservatively treated patients had minimal to no symptoms and remained clinically stable throughout the follow-up period. Based on these findings, conservative management may successfully be adopted in cases with mild symptoms, no signs of compressive optic neuropathy and low risk of neuro-ophthalmic impairment. Conversion to surgical management is indicated upon clinical deterioration or tumor growth. Based on the findings of this study a decision tree for the management of orbital schwannomas is suggested.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00701-024-05899-1.</p>","<title>Keywords</title>","<p>Open access funding provided by Karolinska Institute.</p>"],"string":"[\n \"<title>Abstract</title>\",\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par1\\\">Orbital schwannomas (OS) are rare occurrences with no more than 500 cases reported in the literature. The tumor’s potential to compromise the delicate neuro-ophthalmic structures within the orbit prompts surgical removal. Tumor removal is performed by ophthalmologists, often requiring a multidisciplinary surgical approach. The literature contains a very limited number of cases managed non-surgically. However, the inherent risks of orbital surgery warrant a comparison of the outcomes of conservative and surgical management strategies.</p>\",\n \"<title>Aims</title>\",\n \"<p id=\\\"Par2\\\">To review the national Swedish experience with the management of orbital schwannomas.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par3\\\">The study center is the primary Swedish referral center for the multidisciplinary management of orbital tumors, including schwannomas. During the period of 2005 to 2021, 16 patients with an OS diagnosis were managed at the center.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par4\\\">Four patients initially underwent surgery where gross total resection (GTR) was achieved in three (75%) and subtotal resection (STR) in one (25%) case. The remaining 12 patients, who had a low risk of neuro-ophthalmic impairment, were managed conservatively with radiological and clinical examinations at regular intervals. After an average follow-up of 17 months, surgery was performed in three of these cases (25%). No recurrences or tumor growths were detected on radiological follow-ups (mean 50 months), and all patients experienced postoperative improvement at clinical follow-up (mean 65 months). The remainder of the conservatively treated patients (<italic>n</italic>=9) experienced no clinical progression (mean 30 months). A slight radiological tumor progression was detected in one patient after 17 months.</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par5\\\">There were no differences in long-term outcome between patients who had been managed with early surgery and those operated later after an initially conservative management. Conservatively treated patients had minimal to no symptoms and remained clinically stable throughout the follow-up period. Based on these findings, conservative management may successfully be adopted in cases with mild symptoms, no signs of compressive optic neuropathy and low risk of neuro-ophthalmic impairment. Conversion to surgical management is indicated upon clinical deterioration or tumor growth. Based on the findings of this study a decision tree for the management of orbital schwannomas is suggested.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00701-024-05899-1.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by Karolinska Institute.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary information</title>","<p>\n</p>"],"string":"[\n \"<title>Supplementary information</title>\",\n \"<p>\\n</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Author contribution</title>","<p>All authors approved of the submitted manuscript. All authors qualify for authorship according to the ICJME guidelines, as all have participated in the conceptualization, design, data extraction, writing, and drafting, as well as reviewing the manuscript. AET, EE, and EB supervised the work.</p>","<title>Funding</title>","<p>Open access funding provided by Karolinska Institute.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par34\">The authors declare no competing interests.</p>"],"string":"[\n \"<title>Author contribution</title>\",\n \"<p>All authors approved of the submitted manuscript. All authors qualify for authorship according to the ICJME guidelines, as all have participated in the conceptualization, design, data extraction, writing, and drafting, as well as reviewing the manuscript. AET, EE, and EB supervised the work.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by Karolinska Institute.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par34\\\">The authors declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p><bold>a</bold>) MRI 5 years after initial diagnosis of a large orbital schwannoma in a patient who had refused surgery (Patient 8), <bold>b</bold>) MRI of the same patient 10 years later showing further growth during the observation period</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p><bold>a</bold>) MRI on presentation supporting the diagnosis of orbital schwannoma (Patient 5), b) CT after 5 years showing no growth of the mass during the observation period</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Suggested decision tree for the management of orbital schwannomas. Clinical ophthalmological examination includes visual acuity, pupillary reactions, ocular motility, examination of anterior and posterior segment of the eye, and assessment of globe displacement. For patients with tumor located in the orbital apex or in the vicinity of the optic nerve automated perimetry and OCT is added</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p><bold>a</bold>) MRI 5 years after initial diagnosis of a large orbital schwannoma in a patient who had refused surgery (Patient 8), <bold>b</bold>) MRI of the same patient 10 years later showing further growth during the observation period</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p><bold>a</bold>) MRI on presentation supporting the diagnosis of orbital schwannoma (Patient 5), b) CT after 5 years showing no growth of the mass during the observation period</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Suggested decision tree for the management of orbital schwannomas. Clinical ophthalmological examination includes visual acuity, pupillary reactions, ocular motility, examination of anterior and posterior segment of the eye, and assessment of globe displacement. For patients with tumor located in the orbital apex or in the vicinity of the optic nerve automated perimetry and OCT is added</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Baseline characteristics of patients included in the study</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>ID</th><th>Sex, age, and chief complaint</th><th>Referral differential diagnoses</th><th>Tumor size (cm)</th><th>Location and extensions</th><th>Findings on MRI/CT</th><th>Basis of the final diagnosis</th><th>Management approach</th><th>Indications supporting the approach of choice</th></tr></thead><tbody><tr><td>1</td><td>M, 15, hypoglobus</td><td>1- Schwannoma 2- Cavernous venous malformation</td><td>4.5x2.5x2.5</td><td>Intra- & extraconal, superior, medial, anterior, extends to the apex into the superior orbital fissure</td><td>Demarcated, heterogenous, lobulated, cystic, erosion of the roof and lateral wall of the orbit</td><td>Histology</td><td>Surgery</td><td>History of symptom worsening, and extent of the tumor</td></tr><tr><td>2</td><td>M, 57, incidental</td><td>1- Schwannoma 2- Dermoid cyst</td><td>2.0x1.5x1.0</td><td>Extraconal, superior, anterior</td><td>Demarcated, heterogenous, cystic, no erosion</td><td>Imaging</td><td>Observation</td><td>Incidental finding, with minimal associated symptoms</td></tr><tr><td>3</td><td>F, 44, pain and discomfort in the orbital region</td><td>Schwannoma</td><td>4.0x2.0x1.5</td><td>Extraconal, superior, lateral, anterior, extends to the apex and through the orbital roof</td><td>Demarcated, heterogenous, lobulated, cystic, erosion of the orbital roof</td><td>Histology</td><td>Biopsy and surgery</td><td>Tumor extension into the anterior fossa due to severe erosion of the orbital roof</td></tr><tr><td>4</td><td>F, 57, sudden vision loss</td><td>Schwannoma</td><td>1.4x0.9x0.8</td><td>Intraconal, posterior, with extension into the cavernous sinus</td><td>Demarcated, homogenous, cystic, erosion of the orbital fissure</td><td>Imaging</td><td>Observation</td><td>Rapid, spontaneous resolution of initial presenting symptoms</td></tr><tr><td>5</td><td>M, 26, incidental</td><td>1- Schwannoma 2- Dermoid cyst 3- Orbital venous varices</td><td>3.0x1.5x1.0</td><td>Extraconal, superior, medial, anterior, extends into the superior eyelid</td><td>Demarcated, homogenous, lobulated, cystic, erosion of the orbital roof</td><td>Imaging</td><td>Observation</td><td>Initial observation: incidental finding, with no associated symptoms</td></tr><tr><td>6</td><td>M, 8, palpable eyelid mass</td><td>1- Dermoid cyst 2- Cavernous venous malformation 3- Rhabdomyosarcoma</td><td>1.7x1.9x1.9</td><td>Extraconal, superior, medial, anterior, extends into the superior eyelid</td><td>Demarcated, homogenous, lobulated, cystic, erosion of the medial orbital wall</td><td>Histology</td><td>Biopsy and observation for 1.5 years before surgery</td><td>1) Initial observation: minimal symptoms, and intricate surgery 2) Subsequent surgery: rapid tumor growth</td></tr><tr><td>7</td><td>F, 60, pain and discomfort in the orbital region</td><td>1- Schwannoma 2- Cavernous venous malformation</td><td>2.8x2.0x2.0</td><td>Intraconal, posterior, extends to the apex</td><td>Demarcated, heterogenous, lobulated, cystic, no erosion</td><td>Histology</td><td>Biopsy and observation</td><td>Minimal associated symptoms, and surgery contraindicated due to proximity to the optic nerve</td></tr><tr><td>8</td><td>M, 21, palpable eyelid mass</td><td><p>1- Cavernous venous malformation</p><p>2- Schwannoma 3- Dermoid cyst</p></td><td>4.0x2.0x1.8</td><td>Extraconal, superior, medial, anterior, extends to both; apex posteriorly and superior eyelid anteriorly</td><td>Demarcated, homogenous, lobulated, erosion of the roof and lateral wall of the orbit</td><td>Histology</td><td>Observation for 15 years before surgery</td><td>1) Initial observation: refusal of surgery against medical advice 2) Subsequent surgery: extreme tumor growth, pressure on the globe, and severe bony erosion of the roof and lateral wall of the orbit</td></tr><tr><td>9</td><td>M, 53, hypoglobus</td><td>cavernous venous malformation</td><td>1.4x1.7x1.6</td><td>Extraconal, superior, medial, anterior</td><td>Demarcated, heterogenous, cystic, erosion of the frontal bone</td><td>Histology</td><td>Biopsy and surgery</td><td>Displacement of the eye, with significant symptoms</td></tr><tr><td>10</td><td>F, 85, incidental</td><td>1- Lymphoma 2- Cavernous venous malformation</td><td>2.0x2.0x2.0</td><td>Intraconal, posterior, extends to the apex</td><td>Demarcated, heterogenous, cystic, no erosion</td><td>Histology</td><td>Biopsy and observation</td><td>Incidental finding, with no associated symptoms</td></tr><tr><td>11</td><td>M, 47, reduced visual acuity and proptosis</td><td>1- Cavernous venous malformation 2- Schwannoma</td><td>4.0x2.0x1.6</td><td>Intraconal, posterior, with extension into the cavernous sinus</td><td>Demarcated, heterogenous, erosion of the orbital fissure</td><td>Histology</td><td>Biopsy and observation</td><td>Surgery contraindicated due to proximity to the optic nerve, infiltration of cavernous sinus, as well as patient's severe cardiovascular comorbidities</td></tr><tr><td>12</td><td>F, 74, diplopia and headache</td><td>1- Metastasis 2- Malignant peripheral nerve sheath tumor</td><td>2.4x1.7x1.8</td><td>Intraconal, posterior, with extension into the cavernous sinus</td><td>Demarcated, heterogenous, erosion of the orbital fissure</td><td>Histology</td><td>Biopsy and observation</td><td>Minimal associated symptoms, and surgery contraindicated due to proximity to the optic nerve, and extension into cavernous sinus</td></tr><tr><td>13</td><td>F, 44, incidental</td><td>Schwannoma</td><td>1.8x1.4x1.1</td><td>Extraconal, superior, anterior, extends to the apex</td><td>Demarcated, heterogenous, lobulated, cystic, erosion of the orbital roof</td><td>Histology</td><td>Observation for 1.5 years before surgery</td><td>1) Initial observation: Incidental finding, minimal symptoms, and intricate surgery 2) Subsequent surgery: rapid tumor growth, and pressure on the globe</td></tr><tr><td>14</td><td>M, 84, incidental</td><td>1- Schwannoma 2- Cavernous venous malformation</td><td>1.3x1.0x1.2</td><td>Intraconal, posterior</td><td>Demarcated, heterogenous, no erosion</td><td>Imaging</td><td>Observation</td><td>No associated symptoms, as well as advanced patient age</td></tr><tr><td>15</td><td>F, 62, incidental</td><td>Schwannoma</td><td>1.3x0.9x0.9</td><td>Extraconal, posterior</td><td>Demarcated, heterogenous, lobulated, cystic, no erosion</td><td>Imaging</td><td>Observation</td><td>Minimal associated symptoms</td></tr><tr><td>16</td><td>F, 51, pain and discomfort in the orbital region</td><td>Cavernous venous malformation</td><td>1.8x1.4x1.8</td><td>Intra- & extraconal, posterior</td><td>Demarcated, homogenous, no erosion</td><td>Histology</td><td>Surgery</td><td>Pressure on the globe</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Presenting signs and symptoms in patients diagnosed with orbital schwannomas</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Whole cohort<break/>(<italic>n</italic> = 16)</th><th>Conservative<break/>(<italic>n</italic> = 9)</th><th>Surgery<break/>(<italic>n</italic> = 7)*</th></tr></thead><tbody><tr><td colspan=\"4\">Signs and symptoms</td></tr><tr><td> Proptosis (%)</td><td>12 (75%)</td><td>6 (67%)</td><td>6 (86%)</td></tr><tr><td> Vertical displacement of the globe (%)</td><td>7 (44%)</td><td>2 (22%)</td><td>5 (71%)</td></tr><tr><td> Diplopia (%)</td><td>6 (37%)</td><td>3 (33%)</td><td>3 (43%)</td></tr><tr><td> Pain or discomfort in the orbital region (%)</td><td>5 (31%)</td><td>2 (22%)</td><td>3 (43%)</td></tr><tr><td> Impaired ocular motility (%)</td><td>4 (25%)</td><td>3 (33%)</td><td>1 (14%)</td></tr><tr><td> Visual impairment (%)</td><td>4 (25%)</td><td>2 (22%)</td><td>2 (29%)</td></tr><tr><td> Ptosis (%)</td><td>3 (19%)</td><td>1 (11%)</td><td>2 (29%)</td></tr><tr><td>Incidental finding (%)</td><td>6 (37%)</td><td>5 (56%)</td><td>1 (14%)</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Outcomes in patients with OS depending on the management strategy</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Outcome endpoints</th><th>Group 1: Initial surgery (n = 4)</th><th>Group 2: Delayed surgery (n = 3)</th><th>Group 3: Conservative (n = 9)</th></tr></thead><tbody><tr><td rowspan=\"2\">Observation period</td><td>Radiological outcomes (mean radiographic FU)</td><td>n/a</td><td><p>3 (100%) had tumor growth (FU: 67 mos)</p><p>• Patient 6: 2.2x2.4x2.3cm</p><p>• Patient 8: 6.3x2.8x1.8cm</p><p>• Patient 13: 2.0x1.9x1.7cm</p></td><td><p>8 (89%) had unchanged tumor sizes (FU: 24 mos).</p><p>1 (11%) had mild tumor growth (FU: 17 mos)</p><p>• Patient 12: 2.6x1.6x2.1cm</p></td></tr><tr><td>Clinical outcomes (mean clinical FU)</td><td>n/a</td><td><p>2 (67%) were stable (FU: 20 mos).</p><p>1 (33%) experienced worsening (FU: 171 mos):</p><p>• Patient 8</p></td><td>9 (100%) were stable (FU: 30 mos).</td></tr><tr><td rowspan=\"3\">Postoperative period</td><td>Extent of resection</td><td>3 (75%) GTR and 1 (25%) STR</td><td>3 (100%) STR</td><td>n/a</td></tr><tr><td>Radiologic outcomes (mean postoperative radiographic FU)</td><td><p>Two patients operated by GTR (67%) had no evidence of recurrence (FU: 135 mos).</p><p>1 patient (33%) was lost to FU (Patient 1).</p><p>1 patient with STR (100%) had a stable local status (FU: 18 mos).</p></td><td><p>2 (67%) patients had stable local status (FU: 23 mos).</p><p>1 (33%) was lost to FU</p><p>(Patient 8).</p></td><td>n/a</td></tr><tr><td>Clinical outcomes (mean postoperative clinical FU)</td><td>4 (100%) experienced complete resolution of symptoms (median FU: 61 mos)</td><td><p>3 (100%) had favorable outcomes (FU: 44 mos):</p><p>• 2 experienced complete symptom resolution.</p><p>• 1 with no initial symptoms (incidental finding) remained stable.</p></td><td>n/a</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Baseline characteristics of patients included in the study</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th>ID</th><th>Sex, age, and chief complaint</th><th>Referral differential diagnoses</th><th>Tumor size (cm)</th><th>Location and extensions</th><th>Findings on MRI/CT</th><th>Basis of the final diagnosis</th><th>Management approach</th><th>Indications supporting the approach of choice</th></tr></thead><tbody><tr><td>1</td><td>M, 15, hypoglobus</td><td>1- Schwannoma 2- Cavernous venous malformation</td><td>4.5x2.5x2.5</td><td>Intra- & extraconal, superior, medial, anterior, extends to the apex into the superior orbital fissure</td><td>Demarcated, heterogenous, lobulated, cystic, erosion of the roof and lateral wall of the orbit</td><td>Histology</td><td>Surgery</td><td>History of symptom worsening, and extent of the tumor</td></tr><tr><td>2</td><td>M, 57, incidental</td><td>1- Schwannoma 2- Dermoid cyst</td><td>2.0x1.5x1.0</td><td>Extraconal, superior, anterior</td><td>Demarcated, heterogenous, cystic, no erosion</td><td>Imaging</td><td>Observation</td><td>Incidental finding, with minimal associated symptoms</td></tr><tr><td>3</td><td>F, 44, pain and discomfort in the orbital region</td><td>Schwannoma</td><td>4.0x2.0x1.5</td><td>Extraconal, superior, lateral, anterior, extends to the apex and through the orbital roof</td><td>Demarcated, heterogenous, lobulated, cystic, erosion of the orbital roof</td><td>Histology</td><td>Biopsy and surgery</td><td>Tumor extension into the anterior fossa due to severe erosion of the orbital roof</td></tr><tr><td>4</td><td>F, 57, sudden vision loss</td><td>Schwannoma</td><td>1.4x0.9x0.8</td><td>Intraconal, posterior, with extension into the cavernous sinus</td><td>Demarcated, homogenous, cystic, erosion of the orbital fissure</td><td>Imaging</td><td>Observation</td><td>Rapid, spontaneous resolution of initial presenting symptoms</td></tr><tr><td>5</td><td>M, 26, incidental</td><td>1- Schwannoma 2- Dermoid cyst 3- Orbital venous varices</td><td>3.0x1.5x1.0</td><td>Extraconal, superior, medial, anterior, extends into the superior eyelid</td><td>Demarcated, homogenous, lobulated, cystic, erosion of the orbital roof</td><td>Imaging</td><td>Observation</td><td>Initial observation: incidental finding, with no associated symptoms</td></tr><tr><td>6</td><td>M, 8, palpable eyelid mass</td><td>1- Dermoid cyst 2- Cavernous venous malformation 3- Rhabdomyosarcoma</td><td>1.7x1.9x1.9</td><td>Extraconal, superior, medial, anterior, extends into the superior eyelid</td><td>Demarcated, homogenous, lobulated, cystic, erosion of the medial orbital wall</td><td>Histology</td><td>Biopsy and observation for 1.5 years before surgery</td><td>1) Initial observation: minimal symptoms, and intricate surgery 2) Subsequent surgery: rapid tumor growth</td></tr><tr><td>7</td><td>F, 60, pain and discomfort in the orbital region</td><td>1- Schwannoma 2- Cavernous venous malformation</td><td>2.8x2.0x2.0</td><td>Intraconal, posterior, extends to the apex</td><td>Demarcated, heterogenous, lobulated, cystic, no erosion</td><td>Histology</td><td>Biopsy and observation</td><td>Minimal associated symptoms, and surgery contraindicated due to proximity to the optic nerve</td></tr><tr><td>8</td><td>M, 21, palpable eyelid mass</td><td><p>1- Cavernous venous malformation</p><p>2- Schwannoma 3- Dermoid cyst</p></td><td>4.0x2.0x1.8</td><td>Extraconal, superior, medial, anterior, extends to both; apex posteriorly and superior eyelid anteriorly</td><td>Demarcated, homogenous, lobulated, erosion of the roof and lateral wall of the orbit</td><td>Histology</td><td>Observation for 15 years before surgery</td><td>1) Initial observation: refusal of surgery against medical advice 2) Subsequent surgery: extreme tumor growth, pressure on the globe, and severe bony erosion of the roof and lateral wall of the orbit</td></tr><tr><td>9</td><td>M, 53, hypoglobus</td><td>cavernous venous malformation</td><td>1.4x1.7x1.6</td><td>Extraconal, superior, medial, anterior</td><td>Demarcated, heterogenous, cystic, erosion of the frontal bone</td><td>Histology</td><td>Biopsy and surgery</td><td>Displacement of the eye, with significant symptoms</td></tr><tr><td>10</td><td>F, 85, incidental</td><td>1- Lymphoma 2- Cavernous venous malformation</td><td>2.0x2.0x2.0</td><td>Intraconal, posterior, extends to the apex</td><td>Demarcated, heterogenous, cystic, no erosion</td><td>Histology</td><td>Biopsy and observation</td><td>Incidental finding, with no associated symptoms</td></tr><tr><td>11</td><td>M, 47, reduced visual acuity and proptosis</td><td>1- Cavernous venous malformation 2- Schwannoma</td><td>4.0x2.0x1.6</td><td>Intraconal, posterior, with extension into the cavernous sinus</td><td>Demarcated, heterogenous, erosion of the orbital fissure</td><td>Histology</td><td>Biopsy and observation</td><td>Surgery contraindicated due to proximity to the optic nerve, infiltration of cavernous sinus, as well as patient's severe cardiovascular comorbidities</td></tr><tr><td>12</td><td>F, 74, diplopia and headache</td><td>1- Metastasis 2- Malignant peripheral nerve sheath tumor</td><td>2.4x1.7x1.8</td><td>Intraconal, posterior, with extension into the cavernous sinus</td><td>Demarcated, heterogenous, erosion of the orbital fissure</td><td>Histology</td><td>Biopsy and observation</td><td>Minimal associated symptoms, and surgery contraindicated due to proximity to the optic nerve, and extension into cavernous sinus</td></tr><tr><td>13</td><td>F, 44, incidental</td><td>Schwannoma</td><td>1.8x1.4x1.1</td><td>Extraconal, superior, anterior, extends to the apex</td><td>Demarcated, heterogenous, lobulated, cystic, erosion of the orbital roof</td><td>Histology</td><td>Observation for 1.5 years before surgery</td><td>1) Initial observation: Incidental finding, minimal symptoms, and intricate surgery 2) Subsequent surgery: rapid tumor growth, and pressure on the globe</td></tr><tr><td>14</td><td>M, 84, incidental</td><td>1- Schwannoma 2- Cavernous venous malformation</td><td>1.3x1.0x1.2</td><td>Intraconal, posterior</td><td>Demarcated, heterogenous, no erosion</td><td>Imaging</td><td>Observation</td><td>No associated symptoms, as well as advanced patient age</td></tr><tr><td>15</td><td>F, 62, incidental</td><td>Schwannoma</td><td>1.3x0.9x0.9</td><td>Extraconal, posterior</td><td>Demarcated, heterogenous, lobulated, cystic, no erosion</td><td>Imaging</td><td>Observation</td><td>Minimal associated symptoms</td></tr><tr><td>16</td><td>F, 51, pain and discomfort in the orbital region</td><td>Cavernous venous malformation</td><td>1.8x1.4x1.8</td><td>Intra- & extraconal, posterior</td><td>Demarcated, homogenous, no erosion</td><td>Histology</td><td>Surgery</td><td>Pressure on the globe</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Presenting signs and symptoms in patients diagnosed with orbital schwannomas</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th/><th>Whole cohort<break/>(<italic>n</italic> = 16)</th><th>Conservative<break/>(<italic>n</italic> = 9)</th><th>Surgery<break/>(<italic>n</italic> = 7)*</th></tr></thead><tbody><tr><td colspan=\\\"4\\\">Signs and symptoms</td></tr><tr><td> Proptosis (%)</td><td>12 (75%)</td><td>6 (67%)</td><td>6 (86%)</td></tr><tr><td> Vertical displacement of the globe (%)</td><td>7 (44%)</td><td>2 (22%)</td><td>5 (71%)</td></tr><tr><td> Diplopia (%)</td><td>6 (37%)</td><td>3 (33%)</td><td>3 (43%)</td></tr><tr><td> Pain or discomfort in the orbital region (%)</td><td>5 (31%)</td><td>2 (22%)</td><td>3 (43%)</td></tr><tr><td> Impaired ocular motility (%)</td><td>4 (25%)</td><td>3 (33%)</td><td>1 (14%)</td></tr><tr><td> Visual impairment (%)</td><td>4 (25%)</td><td>2 (22%)</td><td>2 (29%)</td></tr><tr><td> Ptosis (%)</td><td>3 (19%)</td><td>1 (11%)</td><td>2 (29%)</td></tr><tr><td>Incidental finding (%)</td><td>6 (37%)</td><td>5 (56%)</td><td>1 (14%)</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Outcomes in patients with OS depending on the management strategy</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th/><th>Outcome endpoints</th><th>Group 1: Initial surgery (n = 4)</th><th>Group 2: Delayed surgery (n = 3)</th><th>Group 3: Conservative (n = 9)</th></tr></thead><tbody><tr><td rowspan=\\\"2\\\">Observation period</td><td>Radiological outcomes (mean radiographic FU)</td><td>n/a</td><td><p>3 (100%) had tumor growth (FU: 67 mos)</p><p>• Patient 6: 2.2x2.4x2.3cm</p><p>• Patient 8: 6.3x2.8x1.8cm</p><p>• Patient 13: 2.0x1.9x1.7cm</p></td><td><p>8 (89%) had unchanged tumor sizes (FU: 24 mos).</p><p>1 (11%) had mild tumor growth (FU: 17 mos)</p><p>• Patient 12: 2.6x1.6x2.1cm</p></td></tr><tr><td>Clinical outcomes (mean clinical FU)</td><td>n/a</td><td><p>2 (67%) were stable (FU: 20 mos).</p><p>1 (33%) experienced worsening (FU: 171 mos):</p><p>• Patient 8</p></td><td>9 (100%) were stable (FU: 30 mos).</td></tr><tr><td rowspan=\\\"3\\\">Postoperative period</td><td>Extent of resection</td><td>3 (75%) GTR and 1 (25%) STR</td><td>3 (100%) STR</td><td>n/a</td></tr><tr><td>Radiologic outcomes (mean postoperative radiographic FU)</td><td><p>Two patients operated by GTR (67%) had no evidence of recurrence (FU: 135 mos).</p><p>1 patient (33%) was lost to FU (Patient 1).</p><p>1 patient with STR (100%) had a stable local status (FU: 18 mos).</p></td><td><p>2 (67%) patients had stable local status (FU: 23 mos).</p><p>1 (33%) was lost to FU</p><p>(Patient 8).</p></td><td>n/a</td></tr><tr><td>Clinical outcomes (mean postoperative clinical FU)</td><td>4 (100%) experienced complete resolution of symptoms (median FU: 61 mos)</td><td><p>3 (100%) had favorable outcomes (FU: 44 mos):</p><p>• 2 experienced complete symptom resolution.</p><p>• 1 with no initial symptoms (incidental finding) remained stable.</p></td><td>n/a</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p><sup>*</sup>Including patients surgically treated after initially being managed conservatively</p></table-wrap-foot>","<table-wrap-foot><p>FU = Follow-up, n/a = Not Applicable</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p><sup>*</sup>Including patients surgically treated after initially being managed conservatively</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>FU = Follow-up, n/a = Not Applicable</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"701_2024_5899_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"701_2024_5899_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"701_2024_5899_Fig3_HTML\" id=\"MO3\"/>"],"string":"[\n \"<graphic xlink:href=\\\"701_2024_5899_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"701_2024_5899_Fig2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic xlink:href=\\\"701_2024_5899_Fig3_HTML\\\" id=\\\"MO3\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"701_2024_5899_MOESM1_ESM.docx\"><label>ESM 1</label><caption><p>Supplementary file 1: STROBE guidelines checklist. (DOCX 32 kb)</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"701_2024_5899_MOESM1_ESM.docx\\\"><label>ESM 1</label><caption><p>Supplementary file 1: STROBE guidelines checklist. (DOCX 32 kb)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"8.\"], \"mixed-citation\": [\"Ghaith AK, Johnson SE, El-Hajj VG et al (2023) Surgical management of malignant melanotic nerve sheath tumors: an institutional experience and systematic review of the literature. J Neurosurg Spine:1\\u201310\"]}, {\"label\": [\"9.\"], \"surname\": [\"Grover\", \"Rastogi\", \"Chaturvedi\", \"Gupta\"], \"given-names\": [\"AK\", \"A\", \"KU\", \"AK\"], \"article-title\": [\"Schwannoma of the Orbit\"], \"source\": [\"Arch Craniofac Surg\"], \"year\": [\"2015\"], \"volume\": [\"16\"], \"issue\": [\"2\"], \"fpage\": [\"128\"], \"lpage\": [\"129\"]}, {\"label\": [\"13.\"], \"mixed-citation\": [\"Kron M, Bohnsack BL, Archer SM, McHugh JB, Kahana A (2012) Recurrent orbital schwannomas: clinical course and histopathologic correlation. BMC Ophthalmol. 10.1186/1471-2415-12-44\"]}, {\"label\": [\"19.\"], \"surname\": [\"Shields\", \"Shields\"], \"given-names\": [\"JA\", \"CL\"], \"source\": [\"Eyelid, conjunctival, and orbital tumors: an atlas and textbook\"], \"year\": [\"2015\"], \"edition\": [\"3\"], \"publisher-loc\": [\"Philadelphia\"], \"publisher-name\": [\"Lippincott, Williams, & Wilkins\"]}, {\"label\": [\"21.\"], \"mixed-citation\": [\"Yong KL, Beckman TJ, Cranstoun M, Sullivan TJ (2020) Orbital Schwannoma - Management and Clinical Outcomes. Ophthalmic Plast Reconstr Surg:590\\u2013595\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":21,"string":"21"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Acta Neurochir (Wien). 2024 Jan 13; 166(1):9"},"package_file":{"kind":"string","value":"oa_package/a7/d3/PMC10787905.tar.gz"}}},{"rowIdx":189,"cells":{"accession_id":{"kind":"string","value":"PMC10787907"},"pmid":{"kind":"string","value":"38217801"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par6\">Periodontal disease is a chronic, multifactorial, and infectious disease caused by bacteria. It is characterized by the formation of an inflammatory response in the supporting bone and connective tissue against microbial dental plaque, and the nature of the resulting inflammatory response determines the course of periodontal disease [##REF##11585783##1##, ##REF##19388950##2##]. Although periodontitis is a disease that develops in response to bacteria and their products, the course of the disease is regulated by the host tissue response. Periodontal tissue may act as a source of endocrine-like inflammatory mediators (such as IL-1β, TNF-α, and IL-6) that are important for treating periodontal inflammation and can affect glucose and lipid metabolism [##REF##9722690##3##]. According to current study, Body Mass Index (BMI) was correlated with clinical attachment loss (CAL), pocket depth (PD), plaque index (PI), stage and grade of periodontitis, and number of remaining teeth [##REF##34709456##4##].</p>","<p id=\"Par7\">Adipose tissue, particularly white adipose tissue, is an endocrine organ in which a number of pro- or anti-inflammatory cytokines known as adipokines are produced, including adiponectin, visfatin, leptin, and resistin [##REF##20050744##5##, ##UREF##0##6##]. Additionally, periodontal diseases may have an effect on adipokines or on the periodontal response [##REF##21804283##7##]. Asprosin, a recently discovered glucogenic adipokine, is produced by the C-terminal cleavage product of profibrillin coded by profibrillin gene (FBN1), is mainly synthesized by white adipose tissue and released during fasting [##REF##27087445##8##]. Asprosin circulates in the blood at nanomolar levels and is taken to the liver, where it triggers the G protein-cAMP-PKA pathway, causing rapid glucose delivery into circulation [##REF##29106398##9##]. Asprosin is associated with diseases such as diabetes mellitus, obesity, polycystic ovary syndrome, and cardiovascular diseases [##REF##32153505##10##]. Recent research has revealed that asprosin is implicated in many cell types’ inflammatory responses. According to the TLR4-JNK pathway, asprosin causes pancreatic islet cells to become inflamed and dysfunctional [##REF##30853600##11##]. Moreover, asprosin stimulates endoplasmic reticulum stress-induced skeletal muscle inflammation in a PKC-dependent way [##UREF##1##12##]. We thought that periodontitis might have an impact on the level of circulating asprosin because of its systemic effects (similarly to other chronic inflammatory diseases). As far as the study’s authors are aware, no study could be found that investigated the relationship between periodontitis and asprosin, a hormone that may be helpful in the approach to periodontology as an additional biochemical marker to monitor the periodontal status and BMI of patients.</p>","<p id=\"Par8\">This study aimed to (I) investigate levels of serum and saliva asprosin in patients with periodontitis (II) determine whether asprosin levels were related to clinical periodontal parameters, and (III) investigate the relationship between serum and saliva asprosin levels and periodontitis by grouping it according to BMI.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par6\\\">Periodontal disease is a chronic, multifactorial, and infectious disease caused by bacteria. It is characterized by the formation of an inflammatory response in the supporting bone and connective tissue against microbial dental plaque, and the nature of the resulting inflammatory response determines the course of periodontal disease [##REF##11585783##1##, ##REF##19388950##2##]. Although periodontitis is a disease that develops in response to bacteria and their products, the course of the disease is regulated by the host tissue response. Periodontal tissue may act as a source of endocrine-like inflammatory mediators (such as IL-1β, TNF-α, and IL-6) that are important for treating periodontal inflammation and can affect glucose and lipid metabolism [##REF##9722690##3##]. According to current study, Body Mass Index (BMI) was correlated with clinical attachment loss (CAL), pocket depth (PD), plaque index (PI), stage and grade of periodontitis, and number of remaining teeth [##REF##34709456##4##].</p>\",\n \"<p id=\\\"Par7\\\">Adipose tissue, particularly white adipose tissue, is an endocrine organ in which a number of pro- or anti-inflammatory cytokines known as adipokines are produced, including adiponectin, visfatin, leptin, and resistin [##REF##20050744##5##, ##UREF##0##6##]. Additionally, periodontal diseases may have an effect on adipokines or on the periodontal response [##REF##21804283##7##]. Asprosin, a recently discovered glucogenic adipokine, is produced by the C-terminal cleavage product of profibrillin coded by profibrillin gene (FBN1), is mainly synthesized by white adipose tissue and released during fasting [##REF##27087445##8##]. Asprosin circulates in the blood at nanomolar levels and is taken to the liver, where it triggers the G protein-cAMP-PKA pathway, causing rapid glucose delivery into circulation [##REF##29106398##9##]. Asprosin is associated with diseases such as diabetes mellitus, obesity, polycystic ovary syndrome, and cardiovascular diseases [##REF##32153505##10##]. Recent research has revealed that asprosin is implicated in many cell types’ inflammatory responses. According to the TLR4-JNK pathway, asprosin causes pancreatic islet cells to become inflamed and dysfunctional [##REF##30853600##11##]. Moreover, asprosin stimulates endoplasmic reticulum stress-induced skeletal muscle inflammation in a PKC-dependent way [##UREF##1##12##]. We thought that periodontitis might have an impact on the level of circulating asprosin because of its systemic effects (similarly to other chronic inflammatory diseases). As far as the study’s authors are aware, no study could be found that investigated the relationship between periodontitis and asprosin, a hormone that may be helpful in the approach to periodontology as an additional biochemical marker to monitor the periodontal status and BMI of patients.</p>\",\n \"<p id=\\\"Par8\\\">This study aimed to (I) investigate levels of serum and saliva asprosin in patients with periodontitis (II) determine whether asprosin levels were related to clinical periodontal parameters, and (III) investigate the relationship between serum and saliva asprosin levels and periodontitis by grouping it according to BMI.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<p id=\"Par9\">Sixty-five systemically healthy individuals were recruited from the Clinic of Periodontology, Faculty of Dentistry, Atatürk University, between October 2021 and July 2022. The study population was designated into two groups: 1. Thirty-five patients with periodontitis (periodontitis group), 2. Thirty periodontally healthy patients (control group). This study was approved by the Institutional Internal Review and Ethics Board (AU-IIREB reference code: 511) and conducted according to the 2008 Declaration of Helsinki and its later amendments. Registration number (NCT05449821) was taken from <ext-link ext-link-type=\"uri\" xlink:href=\"http://clinicaltrials.gov\">ClinicalTrials.gov</ext-link> website. A signed consent form was obtained from all participants prior to the commencement of the study.</p>","<p id=\"Par10\">The inclusion criteria of the study were as follows: Participants were aged between 18 and 60 years old, were generally healthy, non-smoking, and none had undergone periodontal therapy and/or antibiotic therapy in the past 6 months [##REF##33184718##13##]. Patients with systemic diseases, such as diabetes mellitus, rheumatoid arthritis, and cancer, pregnant or breastfeeding women were excluded [##REF##31038568##14##].</p>","<title>Clinical examination</title>","<p id=\"Par11\">Complete clinical periodontal examination was performed by a single-blinded examiner (DÖE) using Williams probes (Williams, Hu-Friedy, Chicago, IL, USA). The periodontal parameters probing depth (PD), clinical attachment level (CAL), plaque index (PI)[##REF##14158464##15##], gingival index (GI) [##REF##14121956##16##] and bleeding on probing (BOP) were all measured during a full-mouth clinical examination of all patients. The measurements were recorded from 6 sites (disto-buccal, mesio-buccal, mid-buccal, and disto-palatal, mesio-palatal, mid-palatal). A sample group of 10 people with periodontitis who rejected to take part in the study served as the intra-examiner calibration sample prior to the study’s start. Within 24 h, each patient’s CAL and PD were measured twice at six different sites on each tooth. After the assessments achieved the established success criteria of CAL and PD (the percentage of agreement within 1 mm between repeated measurements should be at least 90%), they were considered to be reproducible. The BMI values of the participants were used using an electronic scale (Charder MS-3400-1, Taiwan) that can give height, weight and BMI values, and the method of dividing the weight by the square of the height was used [##REF##29926952##17##].</p>","<title>Study groups</title>","<p id=\"Par12\">Using the following criteria, the cases were allocated to the study groups:<list list-type=\"bullet\"><list-item><p id=\"Par13\">Control group: The sites presented BOP < 10% and PD ≤ 3 mm, no sites had attachment loss. There was no evidence of alveolar bone loss on radiographs or a history of periodontitis.</p></list-item><list-item><p id=\"Par14\">Periodontitis group: At least two non-adjacent teeth showed signs of interdental CAL or buccal or oral CAL ≥ 3 mm and PD >3 mm. In line with the periodontal parameters obtained, it was divided into stages according to the classification conducted by the 2018 EFP and AAP [##REF##29926952##17##].</p></list-item><list-item><p id=\"Par15\">Sample Collection</p></list-item></list></p>","<p id=\"Par16\">During the collection of the serum samples, blood samples were taken from the antecubital fossa while the patients remained in a sitting position for the purpose of standardization. Blood samples taken for biochemistry tests were kept for 20–30 min for coagulation. The tube was centrifuged at +4°C and 4000 rpm for 15 min while remaining upright [##REF##35844162##18##]. The collected serum samples were aliquoted, stored until analysis day at -80° in a deep freezer.</p>","<p id=\"Par17\">Between the hours of 9:00 AM and 10:00 AM, whole saliva samples were taken from the patients and controls and placed into weighted 5-ml sterile polypropylene tubes for 10 min. Ambient conditions were provided for all participants to sit comfortably in a resting position. For two hours prior to collection, no oral stimulation was allowed in order to rule out the influence of mastication or eating. During this time, the sitting patients collected their saliva, gathered it at the back of the mouth, and occasionally emptied it into a collection tube. For 5 minutes, the samples were centrifuged at 10,000 rpm [##REF##25164155##19##]. The final supernatant was kept at -80°C until it was time to use it.</p>","<title>Biochemical analysis</title>","<p id=\"Par18\">As directed by the manufacturer, the serum samples were examined using a “Human Asprosin ELISA Kit.” (BT LAB, Cat. No. E4095 Hu, China). This kit has a detection range of 0.5–100 ng/mL. This test has a sensitivity of 0.23 ng/mL. The kit manufacturer specifies that the asprosin measurement’s interassay and intraassay coefficients of variation are < 10% and <8%, respectively. In a summary, each sample and standard (128, 64, 32, 16, 8, and 4 ng/mL) were introduced to the appropriate well that had already been coated with human asprosin antibodies. The antibodies that were coated on the wells bound the asprosin that was present in the sample. After that, human asprosin antibodies that had been biotinylated were added and bound to the asprosin in the sample. The biotinylated asprosin antibodies were then combined with streptavidin-HRP and bound to them. Unbound streptavidin-HRP was removed following incubation. The substrate solution was then added, and the color of the mixture changed in direct proportion to the level of human asprosin present. By adding an acidic stop solution, the process was stopped, and absorbance at 450 nm was measured. The optical density (OD) of the asprosin samples was compared to the standard curve to estimate their levels [##UREF##2##20##].</p>","<title>Statistical analyses</title>","<p id=\"Par19\">The results were described as the mean ± standard deviation (SD). The normal distribution suitability of the parameters was determined using Kolmogorov-Smirnov tests. Since the asprosin values of the healthy and test groups were normally distributed, Student’s <italic>t-</italic>test was used to compare the asprosin levels of the two groups. BMI comparisons of the control and periodontitis groups were performed on an independent sample using Student’s <italic>t</italic>-test. The stage of the periodontitis group was analyzed statistically using One-way ANOVA. Also, analysis of covariance (ANCOVA) was used to investigate the effect of the body mass index as covariates. Pearson’s correlation test was also performed. ROC curve analysis was used to determine the discriminating power of asprosin in the diagnosis of periodontitis. The value of <italic>p < 0.05</italic> was statistically significant. Statistical analyses were performed by using the SPSS 20.0 statistical software program (SPSS Inc., Chicago, IL, USA).</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<p id=\\\"Par9\\\">Sixty-five systemically healthy individuals were recruited from the Clinic of Periodontology, Faculty of Dentistry, Atatürk University, between October 2021 and July 2022. The study population was designated into two groups: 1. Thirty-five patients with periodontitis (periodontitis group), 2. Thirty periodontally healthy patients (control group). This study was approved by the Institutional Internal Review and Ethics Board (AU-IIREB reference code: 511) and conducted according to the 2008 Declaration of Helsinki and its later amendments. Registration number (NCT05449821) was taken from <ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"http://clinicaltrials.gov\\\">ClinicalTrials.gov</ext-link> website. A signed consent form was obtained from all participants prior to the commencement of the study.</p>\",\n \"<p id=\\\"Par10\\\">The inclusion criteria of the study were as follows: Participants were aged between 18 and 60 years old, were generally healthy, non-smoking, and none had undergone periodontal therapy and/or antibiotic therapy in the past 6 months [##REF##33184718##13##]. Patients with systemic diseases, such as diabetes mellitus, rheumatoid arthritis, and cancer, pregnant or breastfeeding women were excluded [##REF##31038568##14##].</p>\",\n \"<title>Clinical examination</title>\",\n \"<p id=\\\"Par11\\\">Complete clinical periodontal examination was performed by a single-blinded examiner (DÖE) using Williams probes (Williams, Hu-Friedy, Chicago, IL, USA). The periodontal parameters probing depth (PD), clinical attachment level (CAL), plaque index (PI)[##REF##14158464##15##], gingival index (GI) [##REF##14121956##16##] and bleeding on probing (BOP) were all measured during a full-mouth clinical examination of all patients. The measurements were recorded from 6 sites (disto-buccal, mesio-buccal, mid-buccal, and disto-palatal, mesio-palatal, mid-palatal). A sample group of 10 people with periodontitis who rejected to take part in the study served as the intra-examiner calibration sample prior to the study’s start. Within 24 h, each patient’s CAL and PD were measured twice at six different sites on each tooth. After the assessments achieved the established success criteria of CAL and PD (the percentage of agreement within 1 mm between repeated measurements should be at least 90%), they were considered to be reproducible. The BMI values of the participants were used using an electronic scale (Charder MS-3400-1, Taiwan) that can give height, weight and BMI values, and the method of dividing the weight by the square of the height was used [##REF##29926952##17##].</p>\",\n \"<title>Study groups</title>\",\n \"<p id=\\\"Par12\\\">Using the following criteria, the cases were allocated to the study groups:<list list-type=\\\"bullet\\\"><list-item><p id=\\\"Par13\\\">Control group: The sites presented BOP < 10% and PD ≤ 3 mm, no sites had attachment loss. There was no evidence of alveolar bone loss on radiographs or a history of periodontitis.</p></list-item><list-item><p id=\\\"Par14\\\">Periodontitis group: At least two non-adjacent teeth showed signs of interdental CAL or buccal or oral CAL ≥ 3 mm and PD >3 mm. In line with the periodontal parameters obtained, it was divided into stages according to the classification conducted by the 2018 EFP and AAP [##REF##29926952##17##].</p></list-item><list-item><p id=\\\"Par15\\\">Sample Collection</p></list-item></list></p>\",\n \"<p id=\\\"Par16\\\">During the collection of the serum samples, blood samples were taken from the antecubital fossa while the patients remained in a sitting position for the purpose of standardization. Blood samples taken for biochemistry tests were kept for 20–30 min for coagulation. The tube was centrifuged at +4°C and 4000 rpm for 15 min while remaining upright [##REF##35844162##18##]. The collected serum samples were aliquoted, stored until analysis day at -80° in a deep freezer.</p>\",\n \"<p id=\\\"Par17\\\">Between the hours of 9:00 AM and 10:00 AM, whole saliva samples were taken from the patients and controls and placed into weighted 5-ml sterile polypropylene tubes for 10 min. Ambient conditions were provided for all participants to sit comfortably in a resting position. For two hours prior to collection, no oral stimulation was allowed in order to rule out the influence of mastication or eating. During this time, the sitting patients collected their saliva, gathered it at the back of the mouth, and occasionally emptied it into a collection tube. For 5 minutes, the samples were centrifuged at 10,000 rpm [##REF##25164155##19##]. The final supernatant was kept at -80°C until it was time to use it.</p>\",\n \"<title>Biochemical analysis</title>\",\n \"<p id=\\\"Par18\\\">As directed by the manufacturer, the serum samples were examined using a “Human Asprosin ELISA Kit.” (BT LAB, Cat. No. E4095 Hu, China). This kit has a detection range of 0.5–100 ng/mL. This test has a sensitivity of 0.23 ng/mL. The kit manufacturer specifies that the asprosin measurement’s interassay and intraassay coefficients of variation are < 10% and <8%, respectively. In a summary, each sample and standard (128, 64, 32, 16, 8, and 4 ng/mL) were introduced to the appropriate well that had already been coated with human asprosin antibodies. The antibodies that were coated on the wells bound the asprosin that was present in the sample. After that, human asprosin antibodies that had been biotinylated were added and bound to the asprosin in the sample. The biotinylated asprosin antibodies were then combined with streptavidin-HRP and bound to them. Unbound streptavidin-HRP was removed following incubation. The substrate solution was then added, and the color of the mixture changed in direct proportion to the level of human asprosin present. By adding an acidic stop solution, the process was stopped, and absorbance at 450 nm was measured. The optical density (OD) of the asprosin samples was compared to the standard curve to estimate their levels [##UREF##2##20##].</p>\",\n \"<title>Statistical analyses</title>\",\n \"<p id=\\\"Par19\\\">The results were described as the mean ± standard deviation (SD). The normal distribution suitability of the parameters was determined using Kolmogorov-Smirnov tests. Since the asprosin values of the healthy and test groups were normally distributed, Student’s <italic>t-</italic>test was used to compare the asprosin levels of the two groups. BMI comparisons of the control and periodontitis groups were performed on an independent sample using Student’s <italic>t</italic>-test. The stage of the periodontitis group was analyzed statistically using One-way ANOVA. Also, analysis of covariance (ANCOVA) was used to investigate the effect of the body mass index as covariates. Pearson’s correlation test was also performed. ROC curve analysis was used to determine the discriminating power of asprosin in the diagnosis of periodontitis. The value of <italic>p < 0.05</italic> was statistically significant. Statistical analyses were performed by using the SPSS 20.0 statistical software program (SPSS Inc., Chicago, IL, USA).</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par20\">Sixty-five patients were included in this study, and there was no statistically significant difference between the groups in terms of gender (<italic>p</italic>=0.97). Asprosin levels and other factors were compared between participants with periodontitis and those with control (Table ##TAB##0##1##). Both the serum and saliva asprosin levels were statistically significantly higher in the periodontitis group than in the control group (<italic>p</italic><0.001). Saliva asprosin and serum asprosin levels were statistically significantly different in the periodontitis group (<italic>p</italic><0.001). Saliva asprosin and serum asprosin levels were statistically significantly different in the control group (<italic>p</italic>=0.04).\n</p>","<p id=\"Par21\">In the periodontitis group, when classified as stage I, II, III and IV according to the 2018 EFP/ AAP classification, serum asprosin levels are respectively; 47.48 ± 2.19, 57.27 ± 8.21, 82.30 ± 24.67, 124.18 ± 38.43 and saliva asprosin levels are respectively; 34.91±5.23, 46.01±3.23, 54.50±2.07, 64.41±14.27. A statistically significant difference was found between both serum and saliva asprosin levels in the Stage IV periodontitis group compared to the Stage I and II periodontitis groups (<italic>p</italic><0.001) (Table ##TAB##1##2##).\n</p>","<p id=\"Par22\">The distribution of asprosin levels according to BMI in the periodontitis group are shown in Table ##TAB##2##3##. BMI was significantly higher in the periodontitis group than in the control group (<italic>p</italic>=0.017). A strong positive and significant correlation was found between the asprosin levels and BMI index (<italic>r</italic>=0.77, <italic>p</italic><0.001). Also, in an ANCOVA taking BMI as covariance the difference between periodontitis and control group level remained significant for saliva and serum asprosin level, (F: 161;61, respectively, <italic>p</italic><0.001).\n</p>","<p id=\"Par23\">The correlation of saliva and serum asprosin levels with other variables are shown in Table ##TAB##3##4##. There was a significant and positive correlation between the serum and saliva asprosin levels and PI, GI, BOP, CAL, and PD (<italic>p</italic><0.001). The correlation between the asprosin levels with CAL and PD was the strongest. There was a significant relationship between the serum and saliva asprosin levels <italic>r: 0.663</italic>, <italic>p</italic><0.001.\n</p>","<p id=\"Par24\">When the serum cut-off value was 25.36, the sensitivity was 94% and the specificity was 83% (AUC = 0.94, <italic>p</italic> < 0.001) (95% confidence interval, 0.88–1.0). When the saliva cut-off value was 19.81, the sensitivity was 89% and the specificity was 80% (AUC = 0.92, <italic>p</italic> < 0.001) (95% confidence interval, 0.85–0.99). The ROC curve is shown in Fig. ##FIG##0##1##.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par20\\\">Sixty-five patients were included in this study, and there was no statistically significant difference between the groups in terms of gender (<italic>p</italic>=0.97). Asprosin levels and other factors were compared between participants with periodontitis and those with control (Table ##TAB##0##1##). Both the serum and saliva asprosin levels were statistically significantly higher in the periodontitis group than in the control group (<italic>p</italic><0.001). Saliva asprosin and serum asprosin levels were statistically significantly different in the periodontitis group (<italic>p</italic><0.001). Saliva asprosin and serum asprosin levels were statistically significantly different in the control group (<italic>p</italic>=0.04).\\n</p>\",\n \"<p id=\\\"Par21\\\">In the periodontitis group, when classified as stage I, II, III and IV according to the 2018 EFP/ AAP classification, serum asprosin levels are respectively; 47.48 ± 2.19, 57.27 ± 8.21, 82.30 ± 24.67, 124.18 ± 38.43 and saliva asprosin levels are respectively; 34.91±5.23, 46.01±3.23, 54.50±2.07, 64.41±14.27. A statistically significant difference was found between both serum and saliva asprosin levels in the Stage IV periodontitis group compared to the Stage I and II periodontitis groups (<italic>p</italic><0.001) (Table ##TAB##1##2##).\\n</p>\",\n \"<p id=\\\"Par22\\\">The distribution of asprosin levels according to BMI in the periodontitis group are shown in Table ##TAB##2##3##. BMI was significantly higher in the periodontitis group than in the control group (<italic>p</italic>=0.017). A strong positive and significant correlation was found between the asprosin levels and BMI index (<italic>r</italic>=0.77, <italic>p</italic><0.001). Also, in an ANCOVA taking BMI as covariance the difference between periodontitis and control group level remained significant for saliva and serum asprosin level, (F: 161;61, respectively, <italic>p</italic><0.001).\\n</p>\",\n \"<p id=\\\"Par23\\\">The correlation of saliva and serum asprosin levels with other variables are shown in Table ##TAB##3##4##. There was a significant and positive correlation between the serum and saliva asprosin levels and PI, GI, BOP, CAL, and PD (<italic>p</italic><0.001). The correlation between the asprosin levels with CAL and PD was the strongest. There was a significant relationship between the serum and saliva asprosin levels <italic>r: 0.663</italic>, <italic>p</italic><0.001.\\n</p>\",\n \"<p id=\\\"Par24\\\">When the serum cut-off value was 25.36, the sensitivity was 94% and the specificity was 83% (AUC = 0.94, <italic>p</italic> < 0.001) (95% confidence interval, 0.88–1.0). When the saliva cut-off value was 19.81, the sensitivity was 89% and the specificity was 80% (AUC = 0.92, <italic>p</italic> < 0.001) (95% confidence interval, 0.85–0.99). The ROC curve is shown in Fig. ##FIG##0##1##.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par25\">Asprosin was first identified by Romere et al., who also verified that patients with neonatal progeroid syndrome have mutations at the 3' terminus of the fibrillin-1 (FBN-1) gene that cause asprosin to be absent near the C-terminal cleavage site [##REF##27087445##8##]. As a result of this study, it was found that both serum and saliva asprosin levels were higher in the periodontitis group compared to the control group. Based on the results of a literature search, it was concluded that this study is one of the first of its kind to investigate the serum and saliva asprosin levels of periodontitis patients.</p>","<p id=\"Par26\">Asprosin has been demonstrated to bind to hepatocyte membranes and stimulate glucose synthesis through the cyclic AMP (cAMP)-protein kinase A (PKA) pathway [##REF##27087445##8##]. According to a different study, asprosin affects mice through the olfactory receptor OLFR734 that is a G-protein coupled receptor [##REF##31230984##21##]. Li et al. demonstrated that asprosin therapy significantly decreased circulating glucose levels in OLFR734-knockdown mice compared to their wild-type counterparts. Additionally, asprosin enhanced circulation in response to fasting raised hepatic cAMP levels and glucose synthesis, however these effects were reportedly less pronounced in the OLRF734-knockdown mice [##REF##31230984##22##]. The role of asprosin in the inflammatory response in different cell types has been detailed in a number of recent research. According to research, asprosin activates the TLR4-JNK pathway, which results in inflammation and malfunction of pancreatic islet cells [##REF##30853600##11##]. Additionally, in vivo studies showed that asprosin increased endoplasmic reticulum stress, glucose intolerance, insulin resistance, and the flow of pro-inflammatory cytokines (monocyte chemoattractant protein-1, IL-6, and TNF) [##REF##30997682##23##]. Huang et al. reported that asprosin has a proinflammatory effect on the vascular endothelium, and also alleviates vascular endothelial inflammation caused by high-fat diet by neutralizing it with asprosin antibody. Asprosin has been reported to induce and exacerbate vascular endothelial inflammation through the IKKβ-NF-κBp65 pathway [##UREF##3##24##].Asprosin induced a pro-inflammatory response in THP-1 macrophages, as shown by Shabir et al, by greatly increasing the production and secretion of important pro-inflammatory mediators such TNF, IL-1, IL-8, and IL-12 [##UREF##1##12##].</p>","<p id=\"Par27\">Periodontitis is a chronic inflammatory condition that produces an inflammatory and immune response through the interaction of bacterial products and multiple cell populations [##UREF##4##25##]. Since periodontitis is a chronic inflammatory disease, asprosin levels in both serum and saliva were higher in the periodontitis group compared to the control group. We considered that periodontitis might have an impact on the level of circulating asprosin because of its systemic effects (similarly to other chronic inflammatory diseases). Accordingly, asprosin may be a biomarker in detecting periodontitis and linking periodontitis with BMI status. In this study, periodontitis was staged, and it was investigated whether asprosin levels were affected according to the severity of the disease. When the serum asprosin levels were analyzed, a statistically significant difference was found between stages II and IV and between stages I and IV (<italic>p</italic><0.001). Accordingly, as the severity of periodontitis increased, the serum asprosin level increased. The increase in serum asprosin level according to the severity of periodontitis revealed the relationship between the severity of inflammation and asprosin levels. Additionally, asprosin levels had a significant and positive correlation with PI, GI, BOP, CAL, and PD. The correlation between asprosin levels with CAL and PD was the strongest. Periodontal disease severity and degree of damage are related to CAL and PD. The positive correlation of asprosin with clinical indicators suggests that this marker can be used to determine the severity of periodontal disease.</p>","<p id=\"Par28\">Asprosin and BMI index showed a substantial positive and significant association in this investigation. Uğur and Aydın reported that asprosin levels in serum and saliva increased with BMI, which is consistent with our study [##UREF##5##26##]. However, in addition to this study, it was determined by the help of ANCOVA analysis that there was no confounding factor of BMI with high asprosin levels in the periodontitis group in our study and that this disease was a result of periodontitis. Additionally, a number of clinical trials revealed that people who were overweight or obese had significantly higher serum asprosin levels [##UREF##6##27##–##UREF##8##29##]. Circulating asprosin was found to cross the blood-brain barrier and directly activate orexigenic AgRP+ neurons through a cAMP-dependent pathway in a study. It was also reported to trigger appetite and boost body weight by inhibiting anorexigenic proopiomelanocortin (POMC)-positive neurons in a downstream manner in a GABA-dependent manner [##REF##29106398##9##]. In humans, a genetic deficiency in asprosin causes NPS, which is characterized by anorexia and extreme weakness [##REF##27087445##8##]. However, there is conflicting information on the levels of circulating asprosin in obese kids. Children with obesity had considerably lower serum levels of asprosin than children of normal weight, according to studies by Long et al. and Corica et al. [##REF##31212299##30##, ##REF##33662891##31##]. Additionally, Silistre and Hatipoglu discovered no gender-related changes in the asprosin levels in the blood of children with obesity compared to the normal weight group [##REF##32003085##32##].</p>","<p id=\"Par29\">Many studies have reported that obesity and overweight are associated with the risk of periodontitis [##REF##19207883##33##, ##REF##26059115##34##]. The reason for this relationship may be the involvement of proinflammatory cytokines secreted from adipose tissue in the formation of excessive inflammatory response in periodontitis [##REF##17474931##35##]. It has been shown that the relationship between obesity and periodontitis is bidirectional, and the presence of inflammation in periodontal tissues may be a predisposing factor for obesity [##REF##19207875##36##]. As with obesity, adipokines and systemic low-grade inflammation may explain another mechanism by which periodontitis is associated with obesity. It has been suggested that obesity may contribute to periodontitis through altered adipokine levels [##UREF##9##37##]. Since there is no study of asprosin, a recently discovered adipokine, with periodontitis, there is no study in which we can compare the relationship of serum and saliva with BMI in individuals with periodontitis.</p>","<p id=\"Par30\">One of the limitations of this study was that the gender and age factors were not limited. The levels of adipokine hormones may differ between different genders and age groups. Age and gender standardized-controlled trials need to obtain precise data. Another limitation of the study was examining the serum and saliva asprosin levels using a limited sample size. For the objective of evaluating activity, it may be helpful to compare the levels of asprosin in the serum and saliva with those in GCF and gingival tissue (as well as research at how periodontal treatment affects asprosin levels) in patients with various types and degrees of periodontitis.\n</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par25\\\">Asprosin was first identified by Romere et al., who also verified that patients with neonatal progeroid syndrome have mutations at the 3' terminus of the fibrillin-1 (FBN-1) gene that cause asprosin to be absent near the C-terminal cleavage site [##REF##27087445##8##]. As a result of this study, it was found that both serum and saliva asprosin levels were higher in the periodontitis group compared to the control group. Based on the results of a literature search, it was concluded that this study is one of the first of its kind to investigate the serum and saliva asprosin levels of periodontitis patients.</p>\",\n \"<p id=\\\"Par26\\\">Asprosin has been demonstrated to bind to hepatocyte membranes and stimulate glucose synthesis through the cyclic AMP (cAMP)-protein kinase A (PKA) pathway [##REF##27087445##8##]. According to a different study, asprosin affects mice through the olfactory receptor OLFR734 that is a G-protein coupled receptor [##REF##31230984##21##]. Li et al. demonstrated that asprosin therapy significantly decreased circulating glucose levels in OLFR734-knockdown mice compared to their wild-type counterparts. Additionally, asprosin enhanced circulation in response to fasting raised hepatic cAMP levels and glucose synthesis, however these effects were reportedly less pronounced in the OLRF734-knockdown mice [##REF##31230984##22##]. The role of asprosin in the inflammatory response in different cell types has been detailed in a number of recent research. According to research, asprosin activates the TLR4-JNK pathway, which results in inflammation and malfunction of pancreatic islet cells [##REF##30853600##11##]. Additionally, in vivo studies showed that asprosin increased endoplasmic reticulum stress, glucose intolerance, insulin resistance, and the flow of pro-inflammatory cytokines (monocyte chemoattractant protein-1, IL-6, and TNF) [##REF##30997682##23##]. Huang et al. reported that asprosin has a proinflammatory effect on the vascular endothelium, and also alleviates vascular endothelial inflammation caused by high-fat diet by neutralizing it with asprosin antibody. Asprosin has been reported to induce and exacerbate vascular endothelial inflammation through the IKKβ-NF-κBp65 pathway [##UREF##3##24##].Asprosin induced a pro-inflammatory response in THP-1 macrophages, as shown by Shabir et al, by greatly increasing the production and secretion of important pro-inflammatory mediators such TNF, IL-1, IL-8, and IL-12 [##UREF##1##12##].</p>\",\n \"<p id=\\\"Par27\\\">Periodontitis is a chronic inflammatory condition that produces an inflammatory and immune response through the interaction of bacterial products and multiple cell populations [##UREF##4##25##]. Since periodontitis is a chronic inflammatory disease, asprosin levels in both serum and saliva were higher in the periodontitis group compared to the control group. We considered that periodontitis might have an impact on the level of circulating asprosin because of its systemic effects (similarly to other chronic inflammatory diseases). Accordingly, asprosin may be a biomarker in detecting periodontitis and linking periodontitis with BMI status. In this study, periodontitis was staged, and it was investigated whether asprosin levels were affected according to the severity of the disease. When the serum asprosin levels were analyzed, a statistically significant difference was found between stages II and IV and between stages I and IV (<italic>p</italic><0.001). Accordingly, as the severity of periodontitis increased, the serum asprosin level increased. The increase in serum asprosin level according to the severity of periodontitis revealed the relationship between the severity of inflammation and asprosin levels. Additionally, asprosin levels had a significant and positive correlation with PI, GI, BOP, CAL, and PD. The correlation between asprosin levels with CAL and PD was the strongest. Periodontal disease severity and degree of damage are related to CAL and PD. The positive correlation of asprosin with clinical indicators suggests that this marker can be used to determine the severity of periodontal disease.</p>\",\n \"<p id=\\\"Par28\\\">Asprosin and BMI index showed a substantial positive and significant association in this investigation. Uğur and Aydın reported that asprosin levels in serum and saliva increased with BMI, which is consistent with our study [##UREF##5##26##]. However, in addition to this study, it was determined by the help of ANCOVA analysis that there was no confounding factor of BMI with high asprosin levels in the periodontitis group in our study and that this disease was a result of periodontitis. Additionally, a number of clinical trials revealed that people who were overweight or obese had significantly higher serum asprosin levels [##UREF##6##27##–##UREF##8##29##]. Circulating asprosin was found to cross the blood-brain barrier and directly activate orexigenic AgRP+ neurons through a cAMP-dependent pathway in a study. It was also reported to trigger appetite and boost body weight by inhibiting anorexigenic proopiomelanocortin (POMC)-positive neurons in a downstream manner in a GABA-dependent manner [##REF##29106398##9##]. In humans, a genetic deficiency in asprosin causes NPS, which is characterized by anorexia and extreme weakness [##REF##27087445##8##]. However, there is conflicting information on the levels of circulating asprosin in obese kids. Children with obesity had considerably lower serum levels of asprosin than children of normal weight, according to studies by Long et al. and Corica et al. [##REF##31212299##30##, ##REF##33662891##31##]. Additionally, Silistre and Hatipoglu discovered no gender-related changes in the asprosin levels in the blood of children with obesity compared to the normal weight group [##REF##32003085##32##].</p>\",\n \"<p id=\\\"Par29\\\">Many studies have reported that obesity and overweight are associated with the risk of periodontitis [##REF##19207883##33##, ##REF##26059115##34##]. The reason for this relationship may be the involvement of proinflammatory cytokines secreted from adipose tissue in the formation of excessive inflammatory response in periodontitis [##REF##17474931##35##]. It has been shown that the relationship between obesity and periodontitis is bidirectional, and the presence of inflammation in periodontal tissues may be a predisposing factor for obesity [##REF##19207875##36##]. As with obesity, adipokines and systemic low-grade inflammation may explain another mechanism by which periodontitis is associated with obesity. It has been suggested that obesity may contribute to periodontitis through altered adipokine levels [##UREF##9##37##]. Since there is no study of asprosin, a recently discovered adipokine, with periodontitis, there is no study in which we can compare the relationship of serum and saliva with BMI in individuals with periodontitis.</p>\",\n \"<p id=\\\"Par30\\\">One of the limitations of this study was that the gender and age factors were not limited. The levels of adipokine hormones may differ between different genders and age groups. Age and gender standardized-controlled trials need to obtain precise data. Another limitation of the study was examining the serum and saliva asprosin levels using a limited sample size. For the objective of evaluating activity, it may be helpful to compare the levels of asprosin in the serum and saliva with those in GCF and gingival tissue (as well as research at how periodontal treatment affects asprosin levels) in patients with various types and degrees of periodontitis.\\n</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par31\">As a result of our study, asprosin may be a useful parameter as a biomarker in the course of periodontal diseases. However; BMI status should be considered when evaluating asprosin levels in patients with periodontitis. It is necessary to conduct molecular research to determine the function of asprosin in the inflammatory pathway in light of the pathophysiology of periodontal tissues.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par31\\\">As a result of our study, asprosin may be a useful parameter as a biomarker in the course of periodontal diseases. However; BMI status should be considered when evaluating asprosin levels in patients with periodontitis. It is necessary to conduct molecular research to determine the function of asprosin in the inflammatory pathway in light of the pathophysiology of periodontal tissues.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Objectives</title>","<p id=\"Par1\">A newly discovered adipokine known asprosin in serum and saliva in patients with periodontitis has not been explored. The aim of this study was to determine the relationship between serum and saliva asprosin levels and periodontitis by grouping it according to body mass index (BMI).</p>","<title>Materials and methods</title>","<p id=\"Par2\">The study was conducted on 65 systemically healthy patients (35 patients with periodontitis (periodontitis group), 30 periodontally healthy patients (control group)). In each patient, age, BMI, and clinical periodontal parameters (plaque index (PI), gingival index (GI), probing depth (PD), and clinical attachment level (CAL)) were evaluated. Statistical analyses were conducted utilizing the Student <italic>t</italic>-test, ANOVA, and Pearson correlation analysis. For the significance level of the tests, <italic>p</italic><0.05 were accepted.</p>","<title>Results</title>","<p id=\"Par3\">The serum and saliva were collected to assess asprosin levels. Both the serum and saliva asprosin levels were statistically significantly higher in the periodontitis group than in the control group (<italic>p</italic><0.001). Saliva and serum asprosin levels were directly proportional to the severity of the periodontal disease (<italic>p<0.05</italic>). Asprosin levels were higher in patients with a higher BMI (<italic>p<0.05</italic>).</p>","<title>Conclusion</title>","<p id=\"Par4\">Asprosin levels were increased in periodontitis, and even a high BMI status apparently affected the levels of this hormone. It is thought that asprosin may be a useful biomarker in evaluating the relationship between periodontal status and BMI.</p>","<title>Clinical relevance</title>","<p id=\"Par5\">\nAsprosin may be a useful parameter as a biomarker of periodontal disease progression. However, BMI status should be considered when evaluating asprosin levels in patients with periodontitis.</p>","<title>Keywords</title>","<p>Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK).</p>"],"string":"[\n \"<title>Objectives</title>\",\n \"<p id=\\\"Par1\\\">A newly discovered adipokine known asprosin in serum and saliva in patients with periodontitis has not been explored. The aim of this study was to determine the relationship between serum and saliva asprosin levels and periodontitis by grouping it according to body mass index (BMI).</p>\",\n \"<title>Materials and methods</title>\",\n \"<p id=\\\"Par2\\\">The study was conducted on 65 systemically healthy patients (35 patients with periodontitis (periodontitis group), 30 periodontally healthy patients (control group)). In each patient, age, BMI, and clinical periodontal parameters (plaque index (PI), gingival index (GI), probing depth (PD), and clinical attachment level (CAL)) were evaluated. Statistical analyses were conducted utilizing the Student <italic>t</italic>-test, ANOVA, and Pearson correlation analysis. For the significance level of the tests, <italic>p</italic><0.05 were accepted.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">The serum and saliva were collected to assess asprosin levels. Both the serum and saliva asprosin levels were statistically significantly higher in the periodontitis group than in the control group (<italic>p</italic><0.001). Saliva and serum asprosin levels were directly proportional to the severity of the periodontal disease (<italic>p<0.05</italic>). Asprosin levels were higher in patients with a higher BMI (<italic>p<0.05</italic>).</p>\",\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par4\\\">Asprosin levels were increased in periodontitis, and even a high BMI status apparently affected the levels of this hormone. It is thought that asprosin may be a useful biomarker in evaluating the relationship between periodontal status and BMI.</p>\",\n \"<title>Clinical relevance</title>\",\n \"<p id=\\\"Par5\\\">\\nAsprosin may be a useful parameter as a biomarker of periodontal disease progression. However, BMI status should be considered when evaluating asprosin levels in patients with periodontitis.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK).</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":["<title>Author contributions</title>","<p>SNSG worked on the conception and design of the study, interpretation of data, and drafted the manuscript. AD worked on the conception and design of the study and critically revised the manuscript. EL worked on the execution of biochemical analysis, interpretation of data, and critically revised manuscript. DÖE worked on sampling and interpretation of data and critically revised the manuscript. TA worked on the conception and design of the study and critically revised the manuscript. All authors contributed significantly to the drafting and revision of the manuscript and gave final approval of the version to be published. </p>","<title>Funding</title>","<p>Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK).</p>","<title>Declarations</title>","<title>Ethics approval</title>","<p id=\"Par32\">This study was approved by the Institutional Internal Review and Ethics Board (AU-IIREB reference code: 511).</p>","<title>Informed consent</title>","<p id=\"Par33\">Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.</p>","<title>Conflict of interest</title>","<p id=\"Par34\">The authors declare no competing interests.</p>"],"string":"[\n \"<title>Author contributions</title>\",\n \"<p>SNSG worked on the conception and design of the study, interpretation of data, and drafted the manuscript. AD worked on the conception and design of the study and critically revised the manuscript. EL worked on the execution of biochemical analysis, interpretation of data, and critically revised manuscript. DÖE worked on sampling and interpretation of data and critically revised the manuscript. TA worked on the conception and design of the study and critically revised the manuscript. All authors contributed significantly to the drafting and revision of the manuscript and gave final approval of the version to be published. </p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK).</p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethics approval</title>\",\n \"<p id=\\\"Par32\\\">This study was approved by the Institutional Internal Review and Ethics Board (AU-IIREB reference code: 511).</p>\",\n \"<title>Informed consent</title>\",\n \"<p id=\\\"Par33\\\">Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.</p>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par34\\\">The authors declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Determination of the diagnostic sensitivity and specificity of serum and saliva asprosin levels in periodontitis and control groups by ROC curve analysis. ROC: receiver-operating characteristic curve</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Determination of the diagnostic sensitivity and specificity of serum and saliva asprosin levels in periodontitis and control groups by ROC curve analysis. ROC: receiver-operating characteristic curve</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparison between control and periodontitis groups</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Control Group<break/>(<italic>n</italic>=30)<break/>Mean ± SD</th><th>Periodontitis Group<break/>(<italic>n</italic>=35)<break/>Mean ± SD</th><th><italic>p</italic></th></tr></thead><tbody><tr><td><italic>Salivary Asprosin (ng/mL)</italic></td><td>11.59±10.03</td><td>48.18±12.30</td><td><0.001</td></tr><tr><td><italic>Serum Asprosin(ng/mL)</italic></td><td>17.66±11.84</td><td>72.38±33.59</td><td><0.001</td></tr><tr><td><italic>BMI (kg/m</italic><sup><italic>2</italic></sup><italic>)</italic></td><td>21.06±3.45</td><td>23.55±4.54</td><td>0.017</td></tr><tr><td><italic>Age (years)</italic></td><td>33.63±11.38</td><td>38.23±6.95</td><td>0.05</td></tr><tr><td><italic>PI</italic></td><td>0.27±0.45</td><td>1.86±0.65</td><td><0.001</td></tr><tr><td><italic>GI</italic></td><td>0±0</td><td>1.71±0.62</td><td><0.001</td></tr><tr><td><italic>BOP (%)</italic></td><td>0±0</td><td>76.34±12.69</td><td><0.001</td></tr><tr><td><italic>CAL (mm)</italic></td><td>0±0</td><td>5.29±2.92</td><td><0.001</td></tr><tr><td><italic>PD (mm)</italic></td><td>2.63±0.49</td><td>5.83±2.22</td><td><0.001</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>The distribution of asprosin levels according to the stage of periodontitis group</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Salivary Asprosin Levels</th><th>Serum Asprosin Levels</th></tr></thead><tbody><tr><td>Stage I (n:10)</td><td>34.91±5.23</td><td>47.48 ± 2.19</td></tr><tr><td>Stage II (n:10)</td><td>46.01±3.23</td><td>57.27 ± 8.21</td></tr><tr><td>Stage III (n:9)</td><td>54.50±2.07</td><td>82.30 ± 24.67</td></tr><tr><td>Stage IV (n:6)</td><td>64.41±14.27</td><td>124.18 ± 38.43</td></tr><tr><td>I vs II</td><td><italic>p</italic><0.01</td><td><italic>p</italic><0.01</td></tr><tr><td>I vs III</td><td><italic>p</italic><0.001</td><td><italic>p</italic><0.01</td></tr><tr><td>I vs IV</td><td><italic>p</italic><0.001</td><td><italic>p</italic><0.001</td></tr><tr><td>II vs III</td><td><italic>p</italic><0.01</td><td><italic>p</italic><0.01</td></tr><tr><td>II vs IV</td><td><italic>p</italic><0.001</td><td><italic>p</italic><0.001</td></tr><tr><td>III vs IV</td><td><italic>p</italic><0.01</td><td><italic>p</italic><0.01</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>The distribution of asprosin levels according to BMI in the periodontitis group</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Salivary Asprosin Levels</th><th>Serum Asprosin Levels</th></tr></thead><tbody><tr><td>Normal (Group 1, n:15)</td><td>37.75±6.05</td><td>49.31±3.45</td></tr><tr><td>Overweight (Group 2, n:12)</td><td>51.92±3.38</td><td>65.74±13.28</td></tr><tr><td>Obese (Group 3, n: 8)</td><td>62.13±12.80</td><td>125.59±27.01</td></tr><tr><td>Statics</td><td><italic>p</italic></td><td><italic>p</italic></td></tr><tr><td>1 vs 2</td><td><italic>p</italic><0.001</td><td><italic>p</italic><0.05</td></tr><tr><td>1 vs 3</td><td><italic>p</italic><0.001</td><td><italic>p</italic><0.001</td></tr><tr><td>2 vs 3</td><td><italic>p</italic><0.05</td><td><italic>p</italic><0.001</td></tr></tbody></table></table-wrap>","<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Pearson correlation of serum and saliva asprosin levels with other variables</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th/><th>Serum Aspirosin</th><th>Saliva Asprosin</th></tr></thead><tbody><tr><td>PI</td><td><bold><italic>r</italic></bold></td><td>0.676<sup>**</sup></td><td>0.661<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td><0.001</td><td><0.001</td></tr><tr><td>GI</td><td><bold><italic>r</italic></bold></td><td>0.726<sup>**</sup></td><td>0.760<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td><0.001</td><td><0.001</td></tr><tr><td>BOP</td><td><bold><italic>r</italic></bold></td><td>0.715<sup>**</sup></td><td>0.804<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td><0.001</td><td><0.001</td></tr><tr><td>CAL</td><td><bold><italic>r</italic></bold></td><td>0.885<sup>**</sup></td><td>0.793<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td><0.001</td><td><0.001</td></tr><tr><td>PD</td><td><bold><italic>r</italic></bold></td><td>0.882<sup>**</sup></td><td>0.760<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td><0.001</td><td><0.001</td></tr><tr><td>Serum Asprosin</td><td><bold><italic>r</italic></bold></td><td/><td>0.663<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td/><td><0.001</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Comparison between control and periodontitis groups</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th/><th>Control Group<break/>(<italic>n</italic>=30)<break/>Mean ± SD</th><th>Periodontitis Group<break/>(<italic>n</italic>=35)<break/>Mean ± SD</th><th><italic>p</italic></th></tr></thead><tbody><tr><td><italic>Salivary Asprosin (ng/mL)</italic></td><td>11.59±10.03</td><td>48.18±12.30</td><td><0.001</td></tr><tr><td><italic>Serum Asprosin(ng/mL)</italic></td><td>17.66±11.84</td><td>72.38±33.59</td><td><0.001</td></tr><tr><td><italic>BMI (kg/m</italic><sup><italic>2</italic></sup><italic>)</italic></td><td>21.06±3.45</td><td>23.55±4.54</td><td>0.017</td></tr><tr><td><italic>Age (years)</italic></td><td>33.63±11.38</td><td>38.23±6.95</td><td>0.05</td></tr><tr><td><italic>PI</italic></td><td>0.27±0.45</td><td>1.86±0.65</td><td><0.001</td></tr><tr><td><italic>GI</italic></td><td>0±0</td><td>1.71±0.62</td><td><0.001</td></tr><tr><td><italic>BOP (%)</italic></td><td>0±0</td><td>76.34±12.69</td><td><0.001</td></tr><tr><td><italic>CAL (mm)</italic></td><td>0±0</td><td>5.29±2.92</td><td><0.001</td></tr><tr><td><italic>PD (mm)</italic></td><td>2.63±0.49</td><td>5.83±2.22</td><td><0.001</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>The distribution of asprosin levels according to the stage of periodontitis group</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th/><th>Salivary Asprosin Levels</th><th>Serum Asprosin Levels</th></tr></thead><tbody><tr><td>Stage I (n:10)</td><td>34.91±5.23</td><td>47.48 ± 2.19</td></tr><tr><td>Stage II (n:10)</td><td>46.01±3.23</td><td>57.27 ± 8.21</td></tr><tr><td>Stage III (n:9)</td><td>54.50±2.07</td><td>82.30 ± 24.67</td></tr><tr><td>Stage IV (n:6)</td><td>64.41±14.27</td><td>124.18 ± 38.43</td></tr><tr><td>I vs II</td><td><italic>p</italic><0.01</td><td><italic>p</italic><0.01</td></tr><tr><td>I vs III</td><td><italic>p</italic><0.001</td><td><italic>p</italic><0.01</td></tr><tr><td>I vs IV</td><td><italic>p</italic><0.001</td><td><italic>p</italic><0.001</td></tr><tr><td>II vs III</td><td><italic>p</italic><0.01</td><td><italic>p</italic><0.01</td></tr><tr><td>II vs IV</td><td><italic>p</italic><0.001</td><td><italic>p</italic><0.001</td></tr><tr><td>III vs IV</td><td><italic>p</italic><0.01</td><td><italic>p</italic><0.01</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>The distribution of asprosin levels according to BMI in the periodontitis group</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th/><th>Salivary Asprosin Levels</th><th>Serum Asprosin Levels</th></tr></thead><tbody><tr><td>Normal (Group 1, n:15)</td><td>37.75±6.05</td><td>49.31±3.45</td></tr><tr><td>Overweight (Group 2, n:12)</td><td>51.92±3.38</td><td>65.74±13.28</td></tr><tr><td>Obese (Group 3, n: 8)</td><td>62.13±12.80</td><td>125.59±27.01</td></tr><tr><td>Statics</td><td><italic>p</italic></td><td><italic>p</italic></td></tr><tr><td>1 vs 2</td><td><italic>p</italic><0.001</td><td><italic>p</italic><0.05</td></tr><tr><td>1 vs 3</td><td><italic>p</italic><0.001</td><td><italic>p</italic><0.001</td></tr><tr><td>2 vs 3</td><td><italic>p</italic><0.05</td><td><italic>p</italic><0.001</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap id=\\\"Tab4\\\"><label>Table 4</label><caption><p>Pearson correlation of serum and saliva asprosin levels with other variables</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th/><th/><th>Serum Aspirosin</th><th>Saliva Asprosin</th></tr></thead><tbody><tr><td>PI</td><td><bold><italic>r</italic></bold></td><td>0.676<sup>**</sup></td><td>0.661<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td><0.001</td><td><0.001</td></tr><tr><td>GI</td><td><bold><italic>r</italic></bold></td><td>0.726<sup>**</sup></td><td>0.760<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td><0.001</td><td><0.001</td></tr><tr><td>BOP</td><td><bold><italic>r</italic></bold></td><td>0.715<sup>**</sup></td><td>0.804<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td><0.001</td><td><0.001</td></tr><tr><td>CAL</td><td><bold><italic>r</italic></bold></td><td>0.885<sup>**</sup></td><td>0.793<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td><0.001</td><td><0.001</td></tr><tr><td>PD</td><td><bold><italic>r</italic></bold></td><td>0.882<sup>**</sup></td><td>0.760<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td><0.001</td><td><0.001</td></tr><tr><td>Serum Asprosin</td><td><bold><italic>r</italic></bold></td><td/><td>0.663<sup>**</sup></td></tr><tr><td/><td><bold><italic>p</italic></bold></td><td/><td><0.001</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<table-wrap-foot><p>Abbreviations: <italic>BMI</italic> body mass index, <italic>PI</italic> plaque index, <italic>GI</italic> gingival index, <italic>BOP</italic> bleeding on probing, <italic>CAL</italic> clinical attachment level, <italic>PD</italic> pocket depth, <italic>SD</italic> standart deviation</p></table-wrap-foot>","<table-wrap-foot><p>Abbreviations: <italic>PI</italic> plaque index, <italic>GI</italic> gingival index, <italic>BOP</italic> bleeding on probing, <italic>CAL</italic> clinical attachment level, <italic>PD</italic> pocket depth</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p>Abbreviations: <italic>BMI</italic> body mass index, <italic>PI</italic> plaque index, <italic>GI</italic> gingival index, <italic>BOP</italic> bleeding on probing, <italic>CAL</italic> clinical attachment level, <italic>PD</italic> pocket depth, <italic>SD</italic> standart deviation</p></table-wrap-foot>\",\n \"<table-wrap-foot><p>Abbreviations: <italic>PI</italic> plaque index, <italic>GI</italic> gingival index, <italic>BOP</italic> bleeding on probing, <italic>CAL</italic> clinical attachment level, <italic>PD</italic> pocket depth</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"784_2024_5494_Fig1_HTML\" id=\"MO1\"/>"],"string":"[\n \"<graphic xlink:href=\\\"784_2024_5494_Fig1_HTML\\\" id=\\\"MO1\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"6.\"], \"surname\": [\"Zhu\"], \"given-names\": [\"J\"], \"article-title\": [\"Association of circulating leptin and adiponectin with periodontitis: A systematic review and meta-analysis\"], \"source\": [\"BMC Oral Health\"], \"year\": [\"2017\"], \"volume\": [\"17\"], \"issue\": [\"1\"], \"fpage\": [\"1\"], \"lpage\": [\"14\"], \"pub-id\": [\"10.1186/S12903-017-0395-0/FIGURES/3\"]}, {\"label\": [\"12.\"], \"surname\": [\"Shabir\"], \"given-names\": [\"K\"], \"article-title\": [\"Asprosin Exerts Pro-Inflammatory Effects in THP-1 Macrophages Mediated via the Toll-like Receptor 4 (TLR4) Pathway\"], \"source\": [\"Int J Mol Sci\"], \"year\": [\"2023\"], \"volume\": [\"24\"], \"issue\": [\"1\"], \"fpage\": [\"227\"], \"pub-id\": [\"10.3390/IJMS24010227/S1\"]}, {\"label\": [\"20.\"], \"surname\": [\"Laboratory\"], \"given-names\": [\"BT\"], \"source\": [\"Human Asprosin ELISA kit\"], \"year\": [\"2021\"]}, {\"label\": [\"24.\"], \"mixed-citation\": [\"Huang Q et al (2022) Asprosin Exacerbates Endothelium Inflammation Induced by Hyperlipidemia Through Activating IKK\\u03b2-NF-\\u03baBp65 Pathway. Inflammation:1\\u201316. 10.1007/S10753-022-01761-7/FIGURES/8\"]}, {\"label\": [\"25.\"], \"mixed-citation\": [\"Yucel-Lindberg T, B\\u00e5ge T (2013) Inflammatory mediators in the pathogenesis of periodontitis. Expert Rev Mol Med 15. 10.1017/ERM.2013.8\"]}, {\"label\": [\"26.\"], \"mixed-citation\": [\"Ugur K, Aydin S (2019) Saliva and blood asprosin hormone concentration associated with obesity. Int J Endocrinol 2019. 10.1155/2019/2521096\"]}, {\"label\": [\"27.\"], \"mixed-citation\": [\"Ju X et al (2014) IL-6 regulates extracellular matrix remodeling associated with aortic dilation in a fibrillin-1 hypomorphic mgR/mgR mouse model of severe Marfan syndrome. J. Am. Heart Assoc. 3(1). 10.1161/JAHA.113.000476\"]}, {\"label\": [\"28.\"], \"surname\": [\"Wang\"], \"given-names\": [\"CY\"], \"article-title\": [\"Serum asprosin levels and bariatric surgery outcomes in obese adults\"], \"source\": [\"Int. J. Obes. 2018 435\"], \"year\": [\"2018\"], \"volume\": [\"43\"], \"issue\": [\"5\"], \"fpage\": [\"1019\"], \"lpage\": [\"1025\"], \"pub-id\": [\"10.1038/s41366-018-0248-1\"]}, {\"label\": [\"29.\"], \"mixed-citation\": [\"Ceylan H\\u0130, Sayg\\u0131n \\u00d6, \\u00d6zel T\\u00fcrkc\\u00fc \\u00dc (2020) Assessment of acute aerobic exercise in the morning versus evening on asprosin, spexin, lipocalin-2, and insulin level in overweight/obese versus normal weight adult men. 37(8):1252\\u20131268. 10.1080/07420528.2020.1792482\"]}, {\"label\": [\"37.\"], \"mixed-citation\": [\"Zhang L et al (2014) Adiponectin ameliorates experimental periodontitis in diet-induced obesity mice. PLoS One 9(5). 10.1371/JOURNAL.PONE.0097824\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":37,"string":"37"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Clin Oral Investig. 2024 Jan 13; 28(1):91"},"package_file":{"kind":"string","value":"oa_package/dc/a1/PMC10787907.tar.gz"}}},{"rowIdx":190,"cells":{"accession_id":{"kind":"string","value":"PMC10787908"},"pmid":{"kind":"string","value":"38217553"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p id=\"Par6\">Eating disorders (ED) are defined, according to the current edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5-TR) [##UREF##0##1##], by specific disturbances in eating behaviors, and by a persistent and undue influence of body weight or shape on the self-evaluation of the individual [##UREF##0##1##]. Rather than completely demarcated clinical entities, Anorexia Nervosa (AN), Bulimia Nervosa (BN), and Binge Eating Disorder (BED) may share a common psychopathological core [##UREF##1##2##–##REF##12711261##4##]. In fact, potential transitions between diagnoses have been shown to occur in patient during their lifetime [##REF##15800146##5##–##REF##35476589##7##].</p>","<p id=\"Par7\">Psychological treatments currently adopted for EDs—such as Cognitive Behavioral Therapy—posit the existence of a specific core of pathological beliefs shared across AN, BN and BED. This psychopathological core is represented by a primary low self-esteem, an over-evaluation of achievements, and a clinically relevant intolerance for adverse mood states, all driving the persistent and undue influence of body weight or shape on self-evaluation [##UREF##1##2##, ##REF##26275760##3##]. Phenomenological research has offered a novel perspective on this point, focusing on the role played by embodiment in shaping eating psychopathology [##UREF##2##8##–##REF##25720444##10##].</p>","<p id=\"Par8\">Traditionally, phenomenology has developed a distinction between “lived body” (<italic>Leib</italic>)<xref ref-type=\"fn\" rid=\"Fn1\">1</xref> and “physical body” (<italic>Koerper</italic>)<xref ref-type=\"fn\" rid=\"Fn2\">2</xref>. In this perspective, the “lived body” has been defined as the subjective preconscious, <italic>coenesthetic</italic>\n<xref ref-type=\"fn\" rid=\"Fn3\">3</xref>experience of one’s own body, while the “physical body” represents its material dimension [##REF##19293962##11##, ##UREF##4##12##]. Recently, Stanghellini and colleagues [##UREF##3##9##] attempted to conceptualize EDs psychopathology in terms of the “lived-body-for-others”—a concept which was first introduced by Sartre [##UREF##5##13##]. In addition to the previously described dimensions of corporeality, Sartre described that one can apprehend one’s own body from another point of view, as one’s own body when it is looked at by another person [##UREF##5##13##]. When we are looked by another person, the “lived body” is no longer a direct, first-personal experiential evidence, but it is an entity that exists as viewed from an external perspective. This third-person perspective of oneself is defined as the “gaze of the Other” (Fig. ##FIG##0##1##).</p>","<p id=\"Par9\">According to several observations [##REF##23744445##19##–##UREF##7##21##], individuals with EDs report a particular difficulty in experiencing their own body from within, or, in other words, from the coenesthetic perspective. The dialectic integration between the inputs arising from the “lived body” and those coming from the “physical body” is impaired. Stanghellini and colleagues [##UREF##3##9##] have hypothesized that people with ED tend to experience their own body first and foremost as an object looked by another person (the above-mentioned “lived-body-for-others”), rather than from a first-person perspective [##REF##31496104##14##]. This core psychopathological feature would explain the main characteristics of EDs, which are represented by the adoption of external, objective measures to define one’s self and for the clinically relevant preoccupation with controlling one’s own body shape and/or weight [##UREF##3##9##, ##REF##31496104##14##, ##UREF##6##15##].</p>","<p id=\"Par10\">In this perspective, symptoms such as severe dieting or obsessive weight-control might represent a dysfunctional coping strategy to manage the feelings of alienation and extraneousness towards one’s own corporeality. Individuals may report a divergence in the degree to which dieting attempts and eating restriction can be applied or followed [##REF##12711261##4##]. Nonetheless, even when impulsiveness and loss of control over eating represent the main features of the clinical presentation—such as in the case of BED—individuals frequently report a reduced possibility to feel their body from within [##UREF##3##9##], as well as a perceived similarity between their lived experiences and the lived experience of patients with AN or BN [##REF##32911574##16##]. According to Stanghellini et al. [##REF##30836317##17##], people with EDs define themselves reaching external measures. Thus, they perceive their body as an objectified entity to which aesthetic and moral judgments can be applied.</p>","<p id=\"Par11\">Further discussion should also be reserved for the process of perception itself. Contemporary experimental research, and previous theoretical studies, have questioned the hierarchy of psychological processes shaping individual perception. Sensory stimuli, rather than constituting the object of perception, can represent external constraints for internal representations [##REF##20068583##18##–##REF##31067416##20##]. In fact, according to recent theoretical frameworks (e.g., active inference), sensory stimuli are first integrated at a preconscious level, and only later cognitively appraised [##REF##20068583##18##]. Therefore, a perceptual and preconscious representation of one’s own body relies at the basis of a conscious experience of it [##UREF##7##21##]. This novel perspective on perception and consciousness has since triggered a wave of innovations in psychological sciences [##REF##31496104##14##, ##REF##31541638##22##, ##REF##33666885##23##]—while open questions remain in the field of EDs [##REF##33666885##23##].</p>","<p id=\"Par12\">For instance, as previously mentioned, an altered optical, visible and aesthetic self-appraisal of one’s own body has been postulated for ED<xref ref-type=\"fn\" rid=\"Fn4\">4</xref> [##REF##30836317##17##, ##UREF##8##24##]. To individuals with ED, their body would principally be given as an object “to be seen”. The “gaze of the Other” would serve as an optical prosthesis to cope with an altered coenesthesia <sup>3</sup> and as a device through which persons with ED can define themselves. This hypothesis has been empirically supported [##REF##31496104##14##, ##REF##33666885##23##, ##REF##31619011##25##–##REF##35834362##28##], and embodiment disorders have been shown to play a role as maintain factors in longitudinal studies focusing on ED symptomatology [##REF##33534077##29##]. Nonetheless, this hypothesis does not yet address the significant gender gap observed within EDs and within clinical practice [##UREF##0##1##]. In addition, the role of socio-cultural factors in influencing both embodiment and psychopathological symptoms has not been fully previously embedded within this conceptual structure.</p>","<p id=\"Par13\">The present study thus aims at offering a novel perspective on the strong female preponderance for EDs [##UREF##0##1##], in light of the role played by the “gaze of Other” in eating psychopathology. For these reasons, a brief review is first offered on the role of socio-cultural factors as informing the lived experience of individuals. Subsequently, the role of gender identity in embodiment is discussed. Finally, the complex interaction between embodiment, gender identity, and psychopathology is presented.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p id=\\\"Par6\\\">Eating disorders (ED) are defined, according to the current edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5-TR) [##UREF##0##1##], by specific disturbances in eating behaviors, and by a persistent and undue influence of body weight or shape on the self-evaluation of the individual [##UREF##0##1##]. Rather than completely demarcated clinical entities, Anorexia Nervosa (AN), Bulimia Nervosa (BN), and Binge Eating Disorder (BED) may share a common psychopathological core [##UREF##1##2##–##REF##12711261##4##]. In fact, potential transitions between diagnoses have been shown to occur in patient during their lifetime [##REF##15800146##5##–##REF##35476589##7##].</p>\",\n \"<p id=\\\"Par7\\\">Psychological treatments currently adopted for EDs—such as Cognitive Behavioral Therapy—posit the existence of a specific core of pathological beliefs shared across AN, BN and BED. This psychopathological core is represented by a primary low self-esteem, an over-evaluation of achievements, and a clinically relevant intolerance for adverse mood states, all driving the persistent and undue influence of body weight or shape on self-evaluation [##UREF##1##2##, ##REF##26275760##3##]. Phenomenological research has offered a novel perspective on this point, focusing on the role played by embodiment in shaping eating psychopathology [##UREF##2##8##–##REF##25720444##10##].</p>\",\n \"<p id=\\\"Par8\\\">Traditionally, phenomenology has developed a distinction between “lived body” (<italic>Leib</italic>)<xref ref-type=\\\"fn\\\" rid=\\\"Fn1\\\">1</xref> and “physical body” (<italic>Koerper</italic>)<xref ref-type=\\\"fn\\\" rid=\\\"Fn2\\\">2</xref>. In this perspective, the “lived body” has been defined as the subjective preconscious, <italic>coenesthetic</italic>\\n<xref ref-type=\\\"fn\\\" rid=\\\"Fn3\\\">3</xref>experience of one’s own body, while the “physical body” represents its material dimension [##REF##19293962##11##, ##UREF##4##12##]. Recently, Stanghellini and colleagues [##UREF##3##9##] attempted to conceptualize EDs psychopathology in terms of the “lived-body-for-others”—a concept which was first introduced by Sartre [##UREF##5##13##]. In addition to the previously described dimensions of corporeality, Sartre described that one can apprehend one’s own body from another point of view, as one’s own body when it is looked at by another person [##UREF##5##13##]. When we are looked by another person, the “lived body” is no longer a direct, first-personal experiential evidence, but it is an entity that exists as viewed from an external perspective. This third-person perspective of oneself is defined as the “gaze of the Other” (Fig. ##FIG##0##1##).</p>\",\n \"<p id=\\\"Par9\\\">According to several observations [##REF##23744445##19##–##UREF##7##21##], individuals with EDs report a particular difficulty in experiencing their own body from within, or, in other words, from the coenesthetic perspective. The dialectic integration between the inputs arising from the “lived body” and those coming from the “physical body” is impaired. Stanghellini and colleagues [##UREF##3##9##] have hypothesized that people with ED tend to experience their own body first and foremost as an object looked by another person (the above-mentioned “lived-body-for-others”), rather than from a first-person perspective [##REF##31496104##14##]. This core psychopathological feature would explain the main characteristics of EDs, which are represented by the adoption of external, objective measures to define one’s self and for the clinically relevant preoccupation with controlling one’s own body shape and/or weight [##UREF##3##9##, ##REF##31496104##14##, ##UREF##6##15##].</p>\",\n \"<p id=\\\"Par10\\\">In this perspective, symptoms such as severe dieting or obsessive weight-control might represent a dysfunctional coping strategy to manage the feelings of alienation and extraneousness towards one’s own corporeality. Individuals may report a divergence in the degree to which dieting attempts and eating restriction can be applied or followed [##REF##12711261##4##]. Nonetheless, even when impulsiveness and loss of control over eating represent the main features of the clinical presentation—such as in the case of BED—individuals frequently report a reduced possibility to feel their body from within [##UREF##3##9##], as well as a perceived similarity between their lived experiences and the lived experience of patients with AN or BN [##REF##32911574##16##]. According to Stanghellini et al. [##REF##30836317##17##], people with EDs define themselves reaching external measures. Thus, they perceive their body as an objectified entity to which aesthetic and moral judgments can be applied.</p>\",\n \"<p id=\\\"Par11\\\">Further discussion should also be reserved for the process of perception itself. Contemporary experimental research, and previous theoretical studies, have questioned the hierarchy of psychological processes shaping individual perception. Sensory stimuli, rather than constituting the object of perception, can represent external constraints for internal representations [##REF##20068583##18##–##REF##31067416##20##]. In fact, according to recent theoretical frameworks (e.g., active inference), sensory stimuli are first integrated at a preconscious level, and only later cognitively appraised [##REF##20068583##18##]. Therefore, a perceptual and preconscious representation of one’s own body relies at the basis of a conscious experience of it [##UREF##7##21##]. This novel perspective on perception and consciousness has since triggered a wave of innovations in psychological sciences [##REF##31496104##14##, ##REF##31541638##22##, ##REF##33666885##23##]—while open questions remain in the field of EDs [##REF##33666885##23##].</p>\",\n \"<p id=\\\"Par12\\\">For instance, as previously mentioned, an altered optical, visible and aesthetic self-appraisal of one’s own body has been postulated for ED<xref ref-type=\\\"fn\\\" rid=\\\"Fn4\\\">4</xref> [##REF##30836317##17##, ##UREF##8##24##]. To individuals with ED, their body would principally be given as an object “to be seen”. The “gaze of the Other” would serve as an optical prosthesis to cope with an altered coenesthesia <sup>3</sup> and as a device through which persons with ED can define themselves. This hypothesis has been empirically supported [##REF##31496104##14##, ##REF##33666885##23##, ##REF##31619011##25##–##REF##35834362##28##], and embodiment disorders have been shown to play a role as maintain factors in longitudinal studies focusing on ED symptomatology [##REF##33534077##29##]. Nonetheless, this hypothesis does not yet address the significant gender gap observed within EDs and within clinical practice [##UREF##0##1##]. In addition, the role of socio-cultural factors in influencing both embodiment and psychopathological symptoms has not been fully previously embedded within this conceptual structure.</p>\",\n \"<p id=\\\"Par13\\\">The present study thus aims at offering a novel perspective on the strong female preponderance for EDs [##UREF##0##1##], in light of the role played by the “gaze of Other” in eating psychopathology. For these reasons, a brief review is first offered on the role of socio-cultural factors as informing the lived experience of individuals. Subsequently, the role of gender identity in embodiment is discussed. Finally, the complex interaction between embodiment, gender identity, and psychopathology is presented.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p id=\"Par36\">As long as the “feminine” is lived and conceptualized as the “Other”, the feminine gender will be associated with a higher risk to adopt a dysfunctional identification of the self through the “gaze of the Other”, especially in an ocular-centric society. Current theoretical models positing a role for embodiment in shaping eating psychopathology can be updated, appreciating the interplay between the feminine gender and the risk to engage in maladaptive self-objectification. This maladaptive self-objectification may be attempted through the subjugation to the visual representation of oneself through the “gaze of the Other”. The interplay between gender and EDs needs to be considered as embedded within a world of values—both at the individual and sociocultural level.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p id=\\\"Par36\\\">As long as the “feminine” is lived and conceptualized as the “Other”, the feminine gender will be associated with a higher risk to adopt a dysfunctional identification of the self through the “gaze of the Other”, especially in an ocular-centric society. Current theoretical models positing a role for embodiment in shaping eating psychopathology can be updated, appreciating the interplay between the feminine gender and the risk to engage in maladaptive self-objectification. This maladaptive self-objectification may be attempted through the subjugation to the visual representation of oneself through the “gaze of the Other”. The interplay between gender and EDs needs to be considered as embedded within a world of values—both at the individual and sociocultural level.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Background</title>","<p id=\"Par1\">Phenomenological research has enriched the scientific and clinical understanding of Eating Disorders (ED), describing the significant role played by disorders of embodiment in shaping the lived experience of patients with ED. According to the phenomenological perspective, disorders of embodiment in ED are associated with feelings of alienation from one’s own body, determining an excessive concern for external appearance as a form of dysfunctional coping. The purpose of the present narrative review is to address the role of gender identity as a risk factor for EDs in the light of phenomenological approaches.</p>","<title>Methods</title>","<p id=\"Par2\">Narrative review.</p>","<title>Results</title>","<p id=\"Par3\">The current study discusses the interplay between perception, gender identity, and embodiment, all posited to influence eating psychopathology. Internalized concerns for body appearance are described as potentially associated with self-objectification. Furthermore, concerns on body appearance are discussed in relation to gendered social expectations. The current review also explores how societal norms and gender stereotypes can contribute to dysfunctional self-identification with external appearances, particularly through an excessive focus on the optical dimension. The socio-cultural perspective on gender identity was considered as a further explanation of the lived experience of individuals with ED.</p>","<title>Conclusions</title>","<p id=\"Par4\">By acknowledging the interplay between these factors, clinicians and researchers can gain a deeper understanding of these disorders and develop more effective interventions for affected individuals.</p>","<title>Level of evidence</title>","<p id=\"Par5\">Level V narrative review.</p>","<title>Keywords</title>","<p>Open access funding provided by Università degli Studi di Firenze within the CRUI-CARE Agreement.</p>"],"string":"[\n \"<title>Background</title>\",\n \"<p id=\\\"Par1\\\">Phenomenological research has enriched the scientific and clinical understanding of Eating Disorders (ED), describing the significant role played by disorders of embodiment in shaping the lived experience of patients with ED. According to the phenomenological perspective, disorders of embodiment in ED are associated with feelings of alienation from one’s own body, determining an excessive concern for external appearance as a form of dysfunctional coping. The purpose of the present narrative review is to address the role of gender identity as a risk factor for EDs in the light of phenomenological approaches.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">Narrative review.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">The current study discusses the interplay between perception, gender identity, and embodiment, all posited to influence eating psychopathology. Internalized concerns for body appearance are described as potentially associated with self-objectification. Furthermore, concerns on body appearance are discussed in relation to gendered social expectations. The current review also explores how societal norms and gender stereotypes can contribute to dysfunctional self-identification with external appearances, particularly through an excessive focus on the optical dimension. The socio-cultural perspective on gender identity was considered as a further explanation of the lived experience of individuals with ED.</p>\",\n \"<title>Conclusions</title>\",\n \"<p id=\\\"Par4\\\">By acknowledging the interplay between these factors, clinicians and researchers can gain a deeper understanding of these disorders and develop more effective interventions for affected individuals.</p>\",\n \"<title>Level of evidence</title>\",\n \"<p id=\\\"Par5\\\">Level V narrative review.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by Università degli Studi di Firenze within the CRUI-CARE Agreement.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Lived experience and sociocultural factors</title>","<p id=\"Par14\">The present narrative review suggests a possible integration of the phenomenological approach with some elements of other available theoretical perspectives (e.g., cognitive–behavioral, psycho-dynamic, psychophysiological findings). Phenomenological explorations typically target the lived experience of individuals. The result is a rich and detailed collection of qualitative self-descriptions from patients. In an attempt to better grasp the pathogenesis of ED, it is crucial to shift the attention from abnormal eating behaviors to more complex and subtler psycho(patho)logical features, especially experiential ones.</p>","<p id=\"Par15\">For instance, caloric restriction and rapid weight loss are both frequently observed among athletes competing in a specific weight-class [##UREF##9##30##–##REF##33790193##32##]. These behaviors may even reach clinical significance—possibly inducing side-effects, such as amenorrhea—while still not been conceptualized as constituting a psychopathological disorder [##UREF##9##30##–##REF##33790193##32##], although potentially satisfying all criteria to be diagnosed with AN or BN. By contrast, a core difference in EDs is that eating behaviors transcend their original significance of nutrition, entertainment, pleasure, and social function. Thus, thinness becomes crucial for self-worth, dietary restraint for the need for control, and binge-eating to manage emotions [##REF##25720444##10##]. The personal meanings behind these behaviors might be analyzed in terms of individual history, and in terms of a dialectical interaction with sociocultural factors, which may vary across cultures and history.</p>","<title>Sociocultural factors and EDs</title>","<p id=\"Par16\">Some of the pathological connotations attributed to weight/food control by persons with ED are a product of the fashion industry or media [##REF##15847053##33##–##REF##18444705##37##]. Almost two centuries ago (after the industrial revolution) a sober and controlled alimentation became a spread and largely shared value, and a thin body was considered a symbol of efficiency [##UREF##11##38##, ##UREF##12##39##]. Across time the image of the ideal body changed, but it constantly mirrored social position and individual worth, with a number of studies documenting the trend of increasing thinness between the 1950s and the 1990s [##REF##7454902##40##, ##UREF##13##41##]. Contemporary estimates report a prevalence of 0.7% for EDs in Europe, and a rise of around 15% from 1990 to 2019 for this group of diagnoses in the same region [##REF##35392452##42##].</p>","<p id=\"Par17\">Common risk factors have been identified for AN, BN and BED [##REF##14717649##43##, ##UREF##14##44##]. One of the strongest factors were observed in relation to gender [##REF##14717649##43##] and cultural acculturation [##REF##14717649##43##, ##REF##35627600##45##]. Sociocultural influences for EDs have also been noted to interest certain professional sectors more specifically. In particular, those exposed to ideals of beauty and self-control, such as ballet dancers [##REF##7208724##46##–##REF##34021904##48##] and fashion models [##REF##32798930##49##]. Moreover, the importance given to thinness, as an expression of power and control over one’s self [##REF##26998306##50##], as well as a means to reach higher social desirability [##REF##7208724##46##, ##UREF##15##51##], has been observed as influencing the risk to develop EDs during the lifetime. Interestingly, preliminary evidence has also shown that this risk is influenced by a negative assessment of the position reserved to women in family or society [##REF##26998306##50##]. An effective appraisal of lived experiences along and not in contrast to physical and social determinants is, therefore, of primary importance to the advancement of psychiatry, and to our understanding of EDs in particular.</p>","<title>Gender identity and eating disorders</title>","<p id=\"Par18\">More than 70 years ago, Simone de Beauvoir published “Le Deuxième Sexe” (The Second Sex; [##UREF##16##52##]). Its second volume (“L'Expérience Vécue”, The Lived Experience) is a seminal book, which is arguably at the basis of contemporary thought on gender, gender identity and what a feminine gender entails in general [##UREF##17##53##–##UREF##19##55##]. The main focus of this second volume is to ponder “What is woman?” [##UREF##16##52##]. Beauvoir argues that a woman is, by definition, the “Other”:</p>","<p id=\"Par20\">Then, how can the “Other” define itself if not through a third-person view? Positing the feminine as the essential “Other”, Beauvoir implies that a woman is objectified, or, in other words, that a woman becomes connoted by passivity and thus alienated from her true self. Agency, and the active possibility for women to autonomously obtain self-representation and self-definition, is undermined [##UREF##20##56##]. The essential quality of “Other”-ness can also be internalized [##UREF##21##57##], with distinct expectations for what concerns gender roles within society and with an explicit focus on the visual representations of the self [##UREF##22##58##].</p>","<p id=\"Par21\">The higher prevalence of EDs in Western countries [##REF##35392452##42##], where gender equality is higher [##REF##33229558##59##], can then be interpreted in light of the influence exerted by gender stereotypes on social roles [##REF##33229558##59##]. In fact, occupational segregation between genders may be more readily appraised in more egalitarian and developed countries [##UREF##23##60##–##UREF##24##62##]. This cultural and social representation of gender stereotypes, in the occupational or educational sector, can partly explain the equality paradox<xref ref-type=\"fn\" rid=\"Fn5\">5</xref> [##REF##33229558##59##]. A common theoretical framework to explain this paradox is to posit that virtue-signaling or group-affiliation may shape personal identities, driving social roles to become increasingly more divergent between genders [##UREF##25##63##–##UREF##32##71##]. A commonly employed example would be relegating professions involving care to the feminine, and technical oriented careers to the masculine [##REF##29442575##61##].</p>","<p id=\"Par22\">According to the bio-psycho-social model for EDs [##REF##14717649##43##, ##UREF##14##44##], biological sex has been recognized as a risk factor for development of these disorders, as demonstrated in both clinical and non-clinical samples. A strong female to male ratio for EDs is estimated from population-based studies [##REF##35392452##42##]. In parallel, a higher risk for ED has consistently been observed at the epidemiological level for transgender women in comparison with transgender men [##REF##35524487##72##], irrespective of gender-affirming hormonal therapy or surgical interventions [##REF##31617014##73##]. Transgender women appear to be at a higher risk of being diagnosed with an ED also when accounting eating restraints aimed at modulating hormonal effects on body weight and shape [##REF##31617014##73##], that is when eating restraints are not secondary solely to gender-distress. Therefore, gender, and not solely sex, should also be recognized as influencing eating behaviors. For this reason, a perspective on lived experiences for women should move beyond mainly characterizing biological characteristics in relation to sex as informing the risk to develop an ED during the lifetime.</p>","<p id=\"Par23\">While genetic and hormonal factors have a role in eating psychopathology [##UREF##33##74##], the particular onset of most EDs around puberty [##UREF##0##1##] may also be appreciated beyond mechanistic or reductionist claims of hormonal influences on mental disorders [##REF##31025301##75##]. In fact, a child going through puberty may feel that their body is escaping them, that their body is no longer the clear expression of their individuality [##REF##35906856##76##, ##REF##35292195##77##]. This experience of alienation from one’s own body is also a function of social expectations for what concerns gender identity [##REF##32583040##78##–##UREF##34##81##]. In other words, female and male adolescents who do not fully conform to gendered expectations for what concerns primary or secondary sexual characteristics, or who do not fully conform to social expectations for what concerns gender roles or gender expression, may be at a higher risk of experiencing feelings of alienation from their body [##REF##28290118##82##–##UREF##35##85##].</p>","<p id=\"Par24\">In this instance, the body becomes foreign, and, at the same moment, it is grasped by others as an “object”. If the child is a woman, she may also more frequently be objectified or sexualized [##REF##31020943##86##]. The visual, or ocular characteristics, are those more readily grasped by the “gaze of the Other”, and may thus become a primary target for body modification goals, or concealment [##REF##31617014##73##, ##REF##32717626##87##].</p>","<title>Gender identity and embodiment</title>","<p id=\"Par25\">Since ancient times, the optical dimension has been specific to the feminine, and the mirror is the feminine utensil par excellence—at least in the stereotypical and common-sense meaning [##UREF##36##88##, ##UREF##37##89##]. It evokes the radiance of beauty, the charm of the gaze, seduction. To reflect oneself in the mirror is to project one's image before oneself, to split oneself into a self that is looked at and one that is looked at. The mirror is used to see, know, modify, and disguise oneself. The face in Greek is called prosopon—the figure that offers itself to the eyes of the “gaze of Other” as a seal of its own identity [##UREF##38##90##]. Female identity has thus always been linked to the optical dimension—both to one's own appearance reflected in the mirror and to one’s own appearance offered to the “gaze of the Other”.</p>","<p id=\"Par26\">This dependence of identity on gaze (not female only), and especially on the “gaze of the Other”, has not diminished in the course of history, but on the contrary has been further strengthened in the “society of the spectacle” [##UREF##39##91##] whose distinctive trait is, precisely, “ocular-centrism” [##UREF##40##92##]. This mode of access to oneself mediated by visual representations can turn out to be alienating, since images convey individual ghosts and cultural aspects, social prejudices, gender stereotypes. At the same time, the attempt to experience and define one’s own self through the “gaze of the Other” may be captivating or socially rewarding [##REF##33637702##93##]. However, defining one’s self in this manner exposes to the risk of being enthralled into an alienated representation of the self, fully enmeshed and intertwined within social expectations [##REF##38109874##94##], in complete opposition to an authentic definition of the self.</p>","<p id=\"Par27\">Theoretically, any individual can ultimately succeed in grasping itself only by alienating itself, positing oneself both as a subject and, vis-à-vis oneself, as an object [##UREF##5##13##]. In fact, the act of defining oneself is not solipsistic in nature, but requires another being to compare oneself, and to which to be compared [##REF##14643368##95##]. Moreover, the “Other”, as an existent being itself, can form a representation of us in its mind, and we, in turn, can define ourselves as a function of this representation [##UREF##41##96##].</p>","<p id=\"Par28\">Women have not been equally supported in the maturation of an autonomous definition of their identity by the presence of widespread, culturally and socially relevant gender models, by which to define themselves [##UREF##42##97##]. Similarly, gender minorities and non-stereotypical males may experience psychological distress secondary to social expectations in relation to their body weight or shape [##UREF##43##98##–##UREF##45##102##], as well as their gender roles or their gender expression [##UREF##46##103##]. Social expectations for what concerns “feminine” can strongly influence the lived experience of any individual [##UREF##16##52##, ##UREF##47##104##], and the essential quality of the “Other” internalized in relation to gender identity (Fig. ##FIG##1##2##).</p>","<title>Gender identity, embodiment, and psychopathology</title>","<p id=\"Par29\">The interplay between gender identity, embodiment and psychopathology has not yet been fully elucidated. Here, the authors posit that the “gaze of the Other” can exert a dual effect on the individual. On one hand, the “gaze of the Other” offers an external reality, capable to offer us self-recognition and validation [##UREF##48##105##]. On the other, it may be a source of distress, defining us, possibly in contrasts with our personal values [##UREF##5##13##].</p>","<p id=\"Par30\">The individual may react to the challenge posited by the “gaze of the Other” symmetrically and in a dual manner: either rejecting this external “definition” of itself, or, vice-versa, seeking it, to possibly grasp itself through a well-defined, stable and “objectified” representation [##UREF##49##106##–##UREF##51##108##]. A healthy well-being may result from the balance of these two processes. On the contrary, a maladaptive self-identification can be attempted in persons with ED [##REF##33666885##23##], fueled by a disproportion in the preconscious optical–coenesthetic experience of oneself. Nonetheless, the attempt to nullify oneself to resist an external definition may be observed within EDs as well. As the visible body is the channel by which oneself is subjugated by the “Other”, coercing it can diminish the possibility of being grasped. This process is more readily apparent in victims of abuse or sexual violence [##REF##30152723##27##, ##REF##35834362##28##, ##UREF##52##109##]. Nullifying one’s own body can represent a form of self-injury and self-preservation at the same time [##UREF##53##110##].</p>","<p id=\"Par31\">Repetition of trauma and hypersexuality may also be observed, even in individuals with a history of adverse experiences in the sexual domain [##REF##33229025##111##–##UREF##55##114##]. In fact, while previous theoretical contributions focused on the role of trauma in predisposing individuals for emotional dysregulation, and thus, potentially, hypersexuality, sexual activity can also represent an attempt to alienate and subjugate oneself in a perceptual manner [##REF##33229025##111##]. In addition, it may reflect an effort to experientially, affectively, seek an alternative encounter with one’s own body, thus escaping a dysfunctional optico-coenaesthesis<xref ref-type=\"fn\" rid=\"Fn6\">6</xref> [##REF##32858598##115##].</p>","<title>Strengths and limits</title>","<p id=\"Par32\">The strength of the current study lies in its phenomenological approach. The subjective experience of individuals with ED was considered, which allowed for a deeper and nuanced understanding of their lived corporeality. Previous theoretical contributions have been integrated with a sociocultural perspective, in light of self-objectification theory. A novel perspective on the gender gap observed within AN, BN and BED was reached. In summary, the current study proposes the interplay between perception, gender identity and embodiment as a potential target for future empirical research and clinical interventions.</p>","<p id=\"Par33\">The limits of the study, on the other hand, are represented by its narrative nature, relying on the existing literature. Furthermore, while the study discusses important theoretical implications of gender identity and perception on embodiment, it may lack full empirical evidence to support these claims, and its exploratory intent should be taken into consideration.</p>","<title>What is already known on this subject?</title>","<p id=\"Par34\">Some key features of embodiment disturbances in ED have been previously described, such as the experience of a distorted body image, or the lack of effective integration between interoceptive or visual stimuli within an appropriate cognitive appraisal of body weight and shape. In addition, individuals with ED report feelings of alienation from their own body, feeling their material self to be extraneous from themselves. For this reason, a subjective experience of ‘estrangement’ from their physical self is known to fuel distress in these individuals, and thus contribute to disordered eating patterns. As a response, individuals with ED frequently report relying on external measures to reach an effective definition of their corporeality.</p>","<title>What this study adds?</title>","<p id=\"Par35\">The present narrative review attempts to integrate phenomenological accounts with the psychosocial model for EDs, remarking the role of the broader social context in which these disorders develop and are experienced. Accordingly, the role of the feminine as the “Other” was discussed as shaping self-objectification among women, and as a partial explanation of gender discrepancies in the epidemiology of EDs. The current review also emphasizes the need to move beyond solely describing biological sex as contributing to the female/male disparity in EDs, advocating for a socio-cultural perspective on gender identity.</p>"],"string":"[\n \"<title>Lived experience and sociocultural factors</title>\",\n \"<p id=\\\"Par14\\\">The present narrative review suggests a possible integration of the phenomenological approach with some elements of other available theoretical perspectives (e.g., cognitive–behavioral, psycho-dynamic, psychophysiological findings). Phenomenological explorations typically target the lived experience of individuals. The result is a rich and detailed collection of qualitative self-descriptions from patients. In an attempt to better grasp the pathogenesis of ED, it is crucial to shift the attention from abnormal eating behaviors to more complex and subtler psycho(patho)logical features, especially experiential ones.</p>\",\n \"<p id=\\\"Par15\\\">For instance, caloric restriction and rapid weight loss are both frequently observed among athletes competing in a specific weight-class [##UREF##9##30##–##REF##33790193##32##]. These behaviors may even reach clinical significance—possibly inducing side-effects, such as amenorrhea—while still not been conceptualized as constituting a psychopathological disorder [##UREF##9##30##–##REF##33790193##32##], although potentially satisfying all criteria to be diagnosed with AN or BN. By contrast, a core difference in EDs is that eating behaviors transcend their original significance of nutrition, entertainment, pleasure, and social function. Thus, thinness becomes crucial for self-worth, dietary restraint for the need for control, and binge-eating to manage emotions [##REF##25720444##10##]. The personal meanings behind these behaviors might be analyzed in terms of individual history, and in terms of a dialectical interaction with sociocultural factors, which may vary across cultures and history.</p>\",\n \"<title>Sociocultural factors and EDs</title>\",\n \"<p id=\\\"Par16\\\">Some of the pathological connotations attributed to weight/food control by persons with ED are a product of the fashion industry or media [##REF##15847053##33##–##REF##18444705##37##]. Almost two centuries ago (after the industrial revolution) a sober and controlled alimentation became a spread and largely shared value, and a thin body was considered a symbol of efficiency [##UREF##11##38##, ##UREF##12##39##]. Across time the image of the ideal body changed, but it constantly mirrored social position and individual worth, with a number of studies documenting the trend of increasing thinness between the 1950s and the 1990s [##REF##7454902##40##, ##UREF##13##41##]. Contemporary estimates report a prevalence of 0.7% for EDs in Europe, and a rise of around 15% from 1990 to 2019 for this group of diagnoses in the same region [##REF##35392452##42##].</p>\",\n \"<p id=\\\"Par17\\\">Common risk factors have been identified for AN, BN and BED [##REF##14717649##43##, ##UREF##14##44##]. One of the strongest factors were observed in relation to gender [##REF##14717649##43##] and cultural acculturation [##REF##14717649##43##, ##REF##35627600##45##]. Sociocultural influences for EDs have also been noted to interest certain professional sectors more specifically. In particular, those exposed to ideals of beauty and self-control, such as ballet dancers [##REF##7208724##46##–##REF##34021904##48##] and fashion models [##REF##32798930##49##]. Moreover, the importance given to thinness, as an expression of power and control over one’s self [##REF##26998306##50##], as well as a means to reach higher social desirability [##REF##7208724##46##, ##UREF##15##51##], has been observed as influencing the risk to develop EDs during the lifetime. Interestingly, preliminary evidence has also shown that this risk is influenced by a negative assessment of the position reserved to women in family or society [##REF##26998306##50##]. An effective appraisal of lived experiences along and not in contrast to physical and social determinants is, therefore, of primary importance to the advancement of psychiatry, and to our understanding of EDs in particular.</p>\",\n \"<title>Gender identity and eating disorders</title>\",\n \"<p id=\\\"Par18\\\">More than 70 years ago, Simone de Beauvoir published “Le Deuxième Sexe” (The Second Sex; [##UREF##16##52##]). Its second volume (“L'Expérience Vécue”, The Lived Experience) is a seminal book, which is arguably at the basis of contemporary thought on gender, gender identity and what a feminine gender entails in general [##UREF##17##53##–##UREF##19##55##]. The main focus of this second volume is to ponder “What is woman?” [##UREF##16##52##]. Beauvoir argues that a woman is, by definition, the “Other”:</p>\",\n \"<p id=\\\"Par20\\\">Then, how can the “Other” define itself if not through a third-person view? Positing the feminine as the essential “Other”, Beauvoir implies that a woman is objectified, or, in other words, that a woman becomes connoted by passivity and thus alienated from her true self. Agency, and the active possibility for women to autonomously obtain self-representation and self-definition, is undermined [##UREF##20##56##]. The essential quality of “Other”-ness can also be internalized [##UREF##21##57##], with distinct expectations for what concerns gender roles within society and with an explicit focus on the visual representations of the self [##UREF##22##58##].</p>\",\n \"<p id=\\\"Par21\\\">The higher prevalence of EDs in Western countries [##REF##35392452##42##], where gender equality is higher [##REF##33229558##59##], can then be interpreted in light of the influence exerted by gender stereotypes on social roles [##REF##33229558##59##]. In fact, occupational segregation between genders may be more readily appraised in more egalitarian and developed countries [##UREF##23##60##–##UREF##24##62##]. This cultural and social representation of gender stereotypes, in the occupational or educational sector, can partly explain the equality paradox<xref ref-type=\\\"fn\\\" rid=\\\"Fn5\\\">5</xref> [##REF##33229558##59##]. A common theoretical framework to explain this paradox is to posit that virtue-signaling or group-affiliation may shape personal identities, driving social roles to become increasingly more divergent between genders [##UREF##25##63##–##UREF##32##71##]. A commonly employed example would be relegating professions involving care to the feminine, and technical oriented careers to the masculine [##REF##29442575##61##].</p>\",\n \"<p id=\\\"Par22\\\">According to the bio-psycho-social model for EDs [##REF##14717649##43##, ##UREF##14##44##], biological sex has been recognized as a risk factor for development of these disorders, as demonstrated in both clinical and non-clinical samples. A strong female to male ratio for EDs is estimated from population-based studies [##REF##35392452##42##]. In parallel, a higher risk for ED has consistently been observed at the epidemiological level for transgender women in comparison with transgender men [##REF##35524487##72##], irrespective of gender-affirming hormonal therapy or surgical interventions [##REF##31617014##73##]. Transgender women appear to be at a higher risk of being diagnosed with an ED also when accounting eating restraints aimed at modulating hormonal effects on body weight and shape [##REF##31617014##73##], that is when eating restraints are not secondary solely to gender-distress. Therefore, gender, and not solely sex, should also be recognized as influencing eating behaviors. For this reason, a perspective on lived experiences for women should move beyond mainly characterizing biological characteristics in relation to sex as informing the risk to develop an ED during the lifetime.</p>\",\n \"<p id=\\\"Par23\\\">While genetic and hormonal factors have a role in eating psychopathology [##UREF##33##74##], the particular onset of most EDs around puberty [##UREF##0##1##] may also be appreciated beyond mechanistic or reductionist claims of hormonal influences on mental disorders [##REF##31025301##75##]. In fact, a child going through puberty may feel that their body is escaping them, that their body is no longer the clear expression of their individuality [##REF##35906856##76##, ##REF##35292195##77##]. This experience of alienation from one’s own body is also a function of social expectations for what concerns gender identity [##REF##32583040##78##–##UREF##34##81##]. In other words, female and male adolescents who do not fully conform to gendered expectations for what concerns primary or secondary sexual characteristics, or who do not fully conform to social expectations for what concerns gender roles or gender expression, may be at a higher risk of experiencing feelings of alienation from their body [##REF##28290118##82##–##UREF##35##85##].</p>\",\n \"<p id=\\\"Par24\\\">In this instance, the body becomes foreign, and, at the same moment, it is grasped by others as an “object”. If the child is a woman, she may also more frequently be objectified or sexualized [##REF##31020943##86##]. The visual, or ocular characteristics, are those more readily grasped by the “gaze of the Other”, and may thus become a primary target for body modification goals, or concealment [##REF##31617014##73##, ##REF##32717626##87##].</p>\",\n \"<title>Gender identity and embodiment</title>\",\n \"<p id=\\\"Par25\\\">Since ancient times, the optical dimension has been specific to the feminine, and the mirror is the feminine utensil par excellence—at least in the stereotypical and common-sense meaning [##UREF##36##88##, ##UREF##37##89##]. It evokes the radiance of beauty, the charm of the gaze, seduction. To reflect oneself in the mirror is to project one's image before oneself, to split oneself into a self that is looked at and one that is looked at. The mirror is used to see, know, modify, and disguise oneself. The face in Greek is called prosopon—the figure that offers itself to the eyes of the “gaze of Other” as a seal of its own identity [##UREF##38##90##]. Female identity has thus always been linked to the optical dimension—both to one's own appearance reflected in the mirror and to one’s own appearance offered to the “gaze of the Other”.</p>\",\n \"<p id=\\\"Par26\\\">This dependence of identity on gaze (not female only), and especially on the “gaze of the Other”, has not diminished in the course of history, but on the contrary has been further strengthened in the “society of the spectacle” [##UREF##39##91##] whose distinctive trait is, precisely, “ocular-centrism” [##UREF##40##92##]. This mode of access to oneself mediated by visual representations can turn out to be alienating, since images convey individual ghosts and cultural aspects, social prejudices, gender stereotypes. At the same time, the attempt to experience and define one’s own self through the “gaze of the Other” may be captivating or socially rewarding [##REF##33637702##93##]. However, defining one’s self in this manner exposes to the risk of being enthralled into an alienated representation of the self, fully enmeshed and intertwined within social expectations [##REF##38109874##94##], in complete opposition to an authentic definition of the self.</p>\",\n \"<p id=\\\"Par27\\\">Theoretically, any individual can ultimately succeed in grasping itself only by alienating itself, positing oneself both as a subject and, vis-à-vis oneself, as an object [##UREF##5##13##]. In fact, the act of defining oneself is not solipsistic in nature, but requires another being to compare oneself, and to which to be compared [##REF##14643368##95##]. Moreover, the “Other”, as an existent being itself, can form a representation of us in its mind, and we, in turn, can define ourselves as a function of this representation [##UREF##41##96##].</p>\",\n \"<p id=\\\"Par28\\\">Women have not been equally supported in the maturation of an autonomous definition of their identity by the presence of widespread, culturally and socially relevant gender models, by which to define themselves [##UREF##42##97##]. Similarly, gender minorities and non-stereotypical males may experience psychological distress secondary to social expectations in relation to their body weight or shape [##UREF##43##98##–##UREF##45##102##], as well as their gender roles or their gender expression [##UREF##46##103##]. Social expectations for what concerns “feminine” can strongly influence the lived experience of any individual [##UREF##16##52##, ##UREF##47##104##], and the essential quality of the “Other” internalized in relation to gender identity (Fig. ##FIG##1##2##).</p>\",\n \"<title>Gender identity, embodiment, and psychopathology</title>\",\n \"<p id=\\\"Par29\\\">The interplay between gender identity, embodiment and psychopathology has not yet been fully elucidated. Here, the authors posit that the “gaze of the Other” can exert a dual effect on the individual. On one hand, the “gaze of the Other” offers an external reality, capable to offer us self-recognition and validation [##UREF##48##105##]. On the other, it may be a source of distress, defining us, possibly in contrasts with our personal values [##UREF##5##13##].</p>\",\n \"<p id=\\\"Par30\\\">The individual may react to the challenge posited by the “gaze of the Other” symmetrically and in a dual manner: either rejecting this external “definition” of itself, or, vice-versa, seeking it, to possibly grasp itself through a well-defined, stable and “objectified” representation [##UREF##49##106##–##UREF##51##108##]. A healthy well-being may result from the balance of these two processes. On the contrary, a maladaptive self-identification can be attempted in persons with ED [##REF##33666885##23##], fueled by a disproportion in the preconscious optical–coenesthetic experience of oneself. Nonetheless, the attempt to nullify oneself to resist an external definition may be observed within EDs as well. As the visible body is the channel by which oneself is subjugated by the “Other”, coercing it can diminish the possibility of being grasped. This process is more readily apparent in victims of abuse or sexual violence [##REF##30152723##27##, ##REF##35834362##28##, ##UREF##52##109##]. Nullifying one’s own body can represent a form of self-injury and self-preservation at the same time [##UREF##53##110##].</p>\",\n \"<p id=\\\"Par31\\\">Repetition of trauma and hypersexuality may also be observed, even in individuals with a history of adverse experiences in the sexual domain [##REF##33229025##111##–##UREF##55##114##]. In fact, while previous theoretical contributions focused on the role of trauma in predisposing individuals for emotional dysregulation, and thus, potentially, hypersexuality, sexual activity can also represent an attempt to alienate and subjugate oneself in a perceptual manner [##REF##33229025##111##]. In addition, it may reflect an effort to experientially, affectively, seek an alternative encounter with one’s own body, thus escaping a dysfunctional optico-coenaesthesis<xref ref-type=\\\"fn\\\" rid=\\\"Fn6\\\">6</xref> [##REF##32858598##115##].</p>\",\n \"<title>Strengths and limits</title>\",\n \"<p id=\\\"Par32\\\">The strength of the current study lies in its phenomenological approach. The subjective experience of individuals with ED was considered, which allowed for a deeper and nuanced understanding of their lived corporeality. Previous theoretical contributions have been integrated with a sociocultural perspective, in light of self-objectification theory. A novel perspective on the gender gap observed within AN, BN and BED was reached. In summary, the current study proposes the interplay between perception, gender identity and embodiment as a potential target for future empirical research and clinical interventions.</p>\",\n \"<p id=\\\"Par33\\\">The limits of the study, on the other hand, are represented by its narrative nature, relying on the existing literature. Furthermore, while the study discusses important theoretical implications of gender identity and perception on embodiment, it may lack full empirical evidence to support these claims, and its exploratory intent should be taken into consideration.</p>\",\n \"<title>What is already known on this subject?</title>\",\n \"<p id=\\\"Par34\\\">Some key features of embodiment disturbances in ED have been previously described, such as the experience of a distorted body image, or the lack of effective integration between interoceptive or visual stimuli within an appropriate cognitive appraisal of body weight and shape. In addition, individuals with ED report feelings of alienation from their own body, feeling their material self to be extraneous from themselves. For this reason, a subjective experience of ‘estrangement’ from their physical self is known to fuel distress in these individuals, and thus contribute to disordered eating patterns. As a response, individuals with ED frequently report relying on external measures to reach an effective definition of their corporeality.</p>\",\n \"<title>What this study adds?</title>\",\n \"<p id=\\\"Par35\\\">The present narrative review attempts to integrate phenomenological accounts with the psychosocial model for EDs, remarking the role of the broader social context in which these disorders develop and are experienced. Accordingly, the role of the feminine as the “Other” was discussed as shaping self-objectification among women, and as a partial explanation of gender discrepancies in the epidemiology of EDs. The current review also emphasizes the need to move beyond solely describing biological sex as contributing to the female/male disparity in EDs, advocating for a socio-cultural perspective on gender identity.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Author contributions</title>","<p>All authors equally contributed. L.T. wrote the first draft of the manuscript, under the supervision of G.S., V.R. and G.C.; all authors reviewed the manuscript and agree with its content.</p>","<title>Funding</title>","<p>Open access funding provided by Università degli Studi di Firenze within the CRUI-CARE Agreement. Open access funding provided by Università degli Studi di Firenze within the CRUI–CARE Agreement. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.</p>","<title>Data availability</title>","<p>Not applicable.</p>","<title>Declarations</title>","<title>Ethics approval and consent to participate</title>","<p id=\"Par37\">Not applicable.</p>","<title>Consent for publication</title>","<p id=\"Par38\">Not applicable.</p>","<title>Competing interests</title>","<p id=\"Par39\">The authors declare no potential conflict of interest.</p>"],"string":"[\n \"<title>Author contributions</title>\",\n \"<p>All authors equally contributed. L.T. wrote the first draft of the manuscript, under the supervision of G.S., V.R. and G.C.; all authors reviewed the manuscript and agree with its content.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by Università degli Studi di Firenze within the CRUI-CARE Agreement. Open access funding provided by Università degli Studi di Firenze within the CRUI–CARE Agreement. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.</p>\",\n \"<title>Data availability</title>\",\n \"<p>Not applicable.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethics approval and consent to participate</title>\",\n \"<p id=\\\"Par37\\\">Not applicable.</p>\",\n \"<title>Consent for publication</title>\",\n \"<p id=\\\"Par38\\\">Not applicable.</p>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par39\\\">The authors declare no potential conflict of interest.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Conceptual summary of key phenomenological concepts. The “physical body” (<italic>Koerper</italic>) is the material, third-person view of one’s own body. The “lived body” (<italic>Leib</italic>) represents the lived experience of it. A third dimension can also be described, which is the first-person view of one’s own body when it is looked by the other (“lived-body-for-others”). Coenesthesia is the integration of perceptual stimuli originating from the body, the foundation of one’s conscious appraisal of their own body. The “gaze of the Other” is an external point of view, subjectively experienced. It acts as a “filter” or “mirror”, through which one’s own body is experienced</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Proposed novel framework of interactions between embodiment and gender identity. The “gaze of the Other” can play a dual role. On one hand, it may exert a violent action, subjugating one’s own visual dimension as the forefront component of the self. On the other, reassuring, offering a cohesive representation of the self. The “lived-body-for-others” can thus become an external validation of one’s own gender identity, or impose socially sanctioned gender expectations, which may be experienced as distressing at the individual level</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Conceptual summary of key phenomenological concepts. The “physical body” (<italic>Koerper</italic>) is the material, third-person view of one’s own body. The “lived body” (<italic>Leib</italic>) represents the lived experience of it. A third dimension can also be described, which is the first-person view of one’s own body when it is looked by the other (“lived-body-for-others”). Coenesthesia is the integration of perceptual stimuli originating from the body, the foundation of one’s conscious appraisal of their own body. The “gaze of the Other” is an external point of view, subjectively experienced. It acts as a “filter” or “mirror”, through which one’s own body is experienced</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Proposed novel framework of interactions between embodiment and gender identity. The “gaze of the Other” can play a dual role. On one hand, it may exert a violent action, subjugating one’s own visual dimension as the forefront component of the self. On the other, reassuring, offering a cohesive representation of the self. The “lived-body-for-others” can thus become an external validation of one’s own gender identity, or impose socially sanctioned gender expectations, which may be experienced as distressing at the individual level</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":[],"string":"[]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":["<disp-quote><p id=\"Par19\">“(...) humanity is male, and man defines woman not herself, but as relative to him.\"</p></disp-quote>"],"string":"[\n \"<disp-quote><p id=\\\"Par19\\\">“(...) humanity is male, and man defines woman not herself, but as relative to him.\\\"</p></disp-quote>\"\n]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group><fn id=\"Fn1\"><label>1</label><p id=\"Par40\">One’s body experienced <italic>from within</italic>, i.e. from a first-person perspective. Also called the <italic>body-I-am</italic> or body-subject.</p></fn><fn id=\"Fn2\"><label>2</label><p id=\"Par41\">One’s body apprehended <italic>from without</italic>, i.e. from a third-person perspective. Also called the <italic>body-I-have</italic> or body-object.</p></fn><fn id=\"Fn3\"><label>3</label><p id=\"Par42\">The overall collection of perceptual stimuli originating from the body, which creates the foundation for one's consciousness of their own body or their own physical condition, such as the sense of well-being, energy, or alertness.</p></fn><fn id=\"Fn4\"><label>4</label><p id=\"Par43\"><italic>The Optical-Coenesthetic Disproportion Hypothesis</italic>. Under normal conditions, the apprehension of one’s body through coenesthesia and through the other’s look are in a dynamic balance. The <italic>optical-coenaesthetic proportion</italic> is a prerequisite for constructing a dependable sense of bodily self and personal identity. In persons with ED, this dialectic breaks down. Particularly relevant to understanding a person with ED is to envision in the “gaze of the Other” a kind of visual prosthesis that helps him/her feel his/her own body.</p></fn><fn id=\"Fn5\"><label>5</label><p id=\"Par44\">That is, the paradox that specific gender biases, such as the under-representation of women in math-related fields, seems more pronounced in more developed countries.</p></fn><fn id=\"Fn6\"><label>6</label><p id=\"Par45\">As in, the adoption of an optically-driven definition of one’s self, given the altered experience of one’s own body from within—the coenaesthetic perspective.</p></fn><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<fn-group><fn id=\\\"Fn1\\\"><label>1</label><p id=\\\"Par40\\\">One’s body experienced <italic>from within</italic>, i.e. from a first-person perspective. Also called the <italic>body-I-am</italic> or body-subject.</p></fn><fn id=\\\"Fn2\\\"><label>2</label><p id=\\\"Par41\\\">One’s body apprehended <italic>from without</italic>, i.e. from a third-person perspective. Also called the <italic>body-I-have</italic> or body-object.</p></fn><fn id=\\\"Fn3\\\"><label>3</label><p id=\\\"Par42\\\">The overall collection of perceptual stimuli originating from the body, which creates the foundation for one's consciousness of their own body or their own physical condition, such as the sense of well-being, energy, or alertness.</p></fn><fn id=\\\"Fn4\\\"><label>4</label><p id=\\\"Par43\\\"><italic>The Optical-Coenesthetic Disproportion Hypothesis</italic>. Under normal conditions, the apprehension of one’s body through coenesthesia and through the other’s look are in a dynamic balance. The <italic>optical-coenaesthetic proportion</italic> is a prerequisite for constructing a dependable sense of bodily self and personal identity. In persons with ED, this dialectic breaks down. Particularly relevant to understanding a person with ED is to envision in the “gaze of the Other” a kind of visual prosthesis that helps him/her feel his/her own body.</p></fn><fn id=\\\"Fn5\\\"><label>5</label><p id=\\\"Par44\\\">That is, the paradox that specific gender biases, such as the under-representation of women in math-related fields, seems more pronounced in more developed countries.</p></fn><fn id=\\\"Fn6\\\"><label>6</label><p id=\\\"Par45\\\">As in, the adoption of an optically-driven definition of one’s self, given the altered experience of one’s own body from within—the coenaesthetic perspective.</p></fn><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"40519_2023_1632_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"40519_2023_1632_Fig2_HTML\" id=\"MO2\"/>"],"string":"[\n \"<graphic xlink:href=\\\"40519_2023_1632_Fig1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic xlink:href=\\\"40519_2023_1632_Fig2_HTML\\\" id=\\\"MO2\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"1.\"], \"mixed-citation\": [\"American Psychiatric Association (2022) Diagnostic and Statistical Manual of Mental Disorders\\u202f: Fifth Edition Text Revision DSM-5-TR\"], \"sup\": [\"TM\"]}, {\"label\": [\"2.\"], \"surname\": [\"Fairburn\", \"Fairburn\"], \"given-names\": [\"CG\", \"CG\"], \"article-title\": [\"Eating disorders: the transdiagnostic view and the cognitive behavioral theory\"], \"source\": [\"Cognitive behavior therapy and eating disorders\"], \"year\": [\"2008\"], \"publisher-name\": [\"Guilford Press\"], \"fpage\": [\"7\"], \"lpage\": [\"22\"]}, {\"label\": [\"8.\"], \"mixed-citation\": [\"Fuchs T, Koch SC (2014) Embodied affectivity: on moving and being moved. Front Psychol 5:508. 10.3389/fpsyg.2014.00508\"]}, {\"label\": [\"9.\"], \"surname\": [\"Stanghellini\", \"Castellini\", \"Brogna\"], \"given-names\": [\"G\", \"G\", \"P\"], \"article-title\": [\"Identity and eating disorders (IDEA): a questionnaire evaluating identity and embodiment in eating disorder patients\"], \"source\": [\"PSP\"], \"year\": [\"2012\"], \"volume\": [\"45\"], \"fpage\": [\"147\"], \"lpage\": [\"158\"], \"pub-id\": [\"10.1159/000330258\"]}, {\"label\": [\"12.\"], \"surname\": [\"Karoblis\", \"Geniusas\"], \"given-names\": [\"G\", \"S\"], \"article-title\": [\"Kinesthesis and self-awareness\"], \"source\": [\"Varieties of self-awareness: new perspectives from phenomenology, hermeneutics, and comparative philosophy\"], \"year\": [\"2023\"], \"publisher-loc\": [\"Cham\"], \"publisher-name\": [\"Springer International Publishing\"], \"fpage\": [\"85\"], \"lpage\": [\"99\"]}, {\"label\": [\"13.\"], \"surname\": [\"Sartre\"], \"given-names\": [\"J-P\"], \"source\": [\"Being and nothingness: an essay on phenomenological ontology\"], \"year\": [\"1969\"], \"edition\": [\"1\"], \"publisher-loc\": [\"London, UK\"], \"publisher-name\": [\"Routledge\"]}, {\"label\": [\"15.\"], \"surname\": [\"Stanghellini\", \"Trisolini\", \"Castellini\"], \"given-names\": [\"G\", \"F\", \"G\"], \"article-title\": [\"Is feeling extraneous from one\\u2019s own body a core vulnerability feature in eating disorders?\"], \"source\": [\"PSP\"], \"year\": [\"2015\"], \"volume\": [\"48\"], \"fpage\": [\"18\"], \"lpage\": [\"24\"], \"pub-id\": [\"10.1159/000364882\"]}, {\"label\": [\"21.\"], \"surname\": [\"Merleau-Ponty\"], \"given-names\": [\"M\"], \"source\": [\"The primacy of perception: and other essays on phenomenological psychology, the philosophy of art, history and politics\"], \"year\": [\"1964\"], \"edition\": [\"1\"], \"publisher-loc\": [\"Evanston, Ill\"], \"publisher-name\": [\"Northwestern University Press\"]}, {\"label\": [\"24.\"], \"mixed-citation\": [\"Stanghellini G, Ballerini M, Mancini M (2019) The optical-coenaesthetic disproportion hypothesis of feeding and eating disorders in the light of neuroscience. Front Psychiatry 10:630. 10.3389/fpsyt.2019.00630\"]}, {\"label\": [\"30.\"], \"mixed-citation\": [\"Sundgot-Borgen J, Garthe I (2011) Elite athletes in aesthetic and Olympic weight-class sports and the challenge of body weight and body compositions. J Sports Sci 2011;29 Suppl 1:S101-14. 10.1080/02640414.2011.565783\"]}, {\"label\": [\"31.\"], \"mixed-citation\": [\"Ranisavljev M, Kuzmanovic J, Todorovic N, et al (2022) Rapid weight loss practices in grapplers competing in combat sports. Front Physiol 13:842992. 10.3389/fphys.2022.842992\"]}, {\"label\": [\"38.\"], \"surname\": [\"Vigarello\"], \"given-names\": [\"G\"], \"source\": [\"The metamorphoses of fat: a history of obesity\"], \"year\": [\"2016\"], \"publisher-loc\": [\"New York\"], \"publisher-name\": [\"Columbia University Press\"]}, {\"label\": [\"39.\"], \"surname\": [\"Eknoyan\"], \"given-names\": [\"G\"], \"article-title\": [\"A history of obesity, or how what was good became ugly and then bad\"], \"source\": [\"Adv Chron Kidney Dis\"], \"year\": [\"2006\"], \"volume\": [\"13\"], \"fpage\": [\"421\"], \"lpage\": [\"427\"], \"pub-id\": [\"10.1053/j.ackd.2006.07.002\"]}, {\"label\": [\"41.\"], \"surname\": [\"Wiseman\", \"Gray\", \"Mosimann\", \"Ahrens\"], \"given-names\": [\"CV\", \"JJ\", \"JE\", \"AH\"], \"article-title\": [\"Cultural expectations of thinness in women: an update\"], \"source\": [\"Int J Eat Disord\"], \"year\": [\"1992\"], \"volume\": [\"11\"], \"fpage\": [\"85\"], \"lpage\": [\"89\"], \"pub-id\": [\"10.1002/1098-108X(199201)11:1<85::AID-EAT2260110112>3.0.CO;2-T\"]}, {\"label\": [\"44.\"], \"surname\": [\"Weissman\"], \"given-names\": [\"RS\"], \"article-title\": [\"The role of sociocultural factors in the etiology of eating disorders\"], \"source\": [\"Psychiatr Clin\"], \"year\": [\"2019\"], \"volume\": [\"42\"], \"fpage\": [\"121\"], \"lpage\": [\"144\"], \"pub-id\": [\"10.1016/j.psc.2018.10.009\"]}, {\"label\": [\"51.\"], \"surname\": [\"Ferguson\", \"Winegard\", \"Winegard\"], \"given-names\": [\"CJ\", \"B\", \"BM\"], \"article-title\": [\"Who is the fairest one of all? 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Editions L\\u2019Harmattan\"]}, {\"label\": [\"112.\"], \"surname\": [\"Joseph\", \"Dalgleish\", \"Thrasher\", \"Yule\"], \"given-names\": [\"S\", \"T\", \"S\", \"W\"], \"article-title\": [\"Impulsivity and post-traumatic stress\"], \"source\": [\"Personal Individ Differ\"], \"year\": [\"1997\"], \"volume\": [\"22\"], \"fpage\": [\"279\"], \"lpage\": [\"281\"], \"pub-id\": [\"10.1016/S0191-8869(96)00213-9\"]}, {\"label\": [\"114.\"], \"surname\": [\"van der Kolk\"], \"given-names\": [\"BA\"], \"article-title\": [\"The compulsion to repeat the trauma. Re-enactment, revictimization, and masochism\"], \"source\": [\"Psychiatr Clin N Am\"], \"year\": [\"1989\"], \"volume\": [\"12\"], \"fpage\": [\"389\"], \"lpage\": [\"411\"], \"pub-id\": [\"10.1016/S0193-953X(18)30439-8\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":115,"string":"115"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Eat Weight Disord. 2024 Jan 13; 29(1):8"},"package_file":{"kind":"string","value":"oa_package/b1/93/PMC10787908.tar.gz"}}},{"rowIdx":191,"cells":{"accession_id":{"kind":"string","value":"PMC10787909"},"pmid":{"kind":"string","value":"38222998"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Constipation can notably affect children's health and their parents' quality of life. Functional constipation (FC) in children is defined as irregular or complex bowel movements without underlying systemic or anatomical causes [##UREF##0##1##]. Although the prevalence of constipation varies among societies based on many factors, including sociocultural and dietary habits, constipation is a common pediatric health problem, affecting between 0.7% and 29.6% of children [##REF##21382575##2##]. One study reported inadequate nutrition as a primary (58%) risk factor for constipation, with mental instability (21.2%) and genetic factors (3.5%) also identified [##REF##17032205##3##].</p>","<p>Worldwide, many diagnostic criteria have been approved for diagnosing functional constipation. However, the Rome III criteria and the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition (NASPGHAN) guidelines are the clinical criteria most often utilized in Saudi Arabia [##REF##26115431##4##]. Recently, the Rome IV criteria have been widely used to diagnose FC. According to Rome IV, the diagnosis of FC in children under the age of four requires the existence of at least two of the following symptoms for at least one month in the absence of an organic cause and with insufficient criteria to diagnose irritable bowel syndrome: maximum of two attempts to defecate per week, history of constipation, history of uncomfortable, painful bowel movements, history of hard or large stools, having a large fecal mass in the rectum. Rome IV also notes the following criteria for diagnosing FC in toilet-trained children over four years of age: one or more incontinence incidences per week and a history of having large, thick stools that could block the toilet [##REF##23535762##5##].</p>","<p>Fecal incontinence, frequent abdominal pain, bleeding of the rectum, enuresis, and urinary retention and infection are all well-recognized complications of FC in children [##REF##35611377##6##]. Fecal occlusion, intestinal/ileus blockage, toxic megacolon, and bowel perforation are the more catastrophic potential effects of FC [##REF##16606352##7##]. The North American and European Societies for Pediatric Gastroenterology, Hepatology, and Nutrition (NASPEGHAN/ESPGHAN) released updated clinical recommendations for diagnosing and managing FC in children. These include \"a normal intake of fibers and liquids, normal physical activity, and an extra pharmacological treatment for rectal fecal impaction followed by a pharmacological maintenance therapy.\" The societies also recommend polyethylene glycol (PEG) with or without electrolytes (0.2-0.8 g/kg) for maintenance therapy [##UREF##1##8##].</p>","<p>Providentially, FC is estimated to account for 90% of cases seen in healthcare centers, with the remaining 5-10% having organic causes [##UREF##2##9##]. Because FC is a relatively benign disorder, it often goes undiagnosed and untreated, which can lead to a variety of medical and psychosocial issues for children and their parents, cause concern for healthcare budgets, and, presumably, place a significant socioeconomic burden [##UREF##0##1##,##UREF##3##10##,##REF##31460507##11##]. A 2022 study in Saudi Arabia found that parents' knowledge of childhood constipation was linked to their practices in dealing with it. The study involved 568 participants and concluded that poor practices were due to inadequate knowledge [##UREF##4##12##]. A 2019 study in kindergartens in Jatinangor found that in 111 parents there was a correlation between parental knowledge and their children's toilet training practice and behavior. The study highlights the importance of educating parents about fecal continence in children [##UREF##5##13##]. In addition, a 2020 systematic review of 13 studies attempted to locate, organize, and synthesize the current data on the experiences of parents caring for children with FC and the information needed by such parents to facilitate successful treatment. The study revealed that most parents have inadequate knowledge of childhood constipation, with a relatively small percentage having sufficient and correct information about the condition [##REF##33026251##14##].</p>","<p>In this study, pediatricians were also found to have inadequate knowledge of FC in children, with notable differences in the pediatricians' knowledge and patterns of practice regarding childhood constipation [##REF##17032205##3##,##REF##26115431##4##,##REF##33026251##14##]. To the best of our knowledge, no studies in the Western region of Saudi Arabia assess the level of parents' knowledge regarding childhood FC. Thus, the study aimed to evaluate parents' knowledge, attitudes, and practices regarding childhood FC in Makkah, Saudi Arabia to identify knowledge gaps that can be filled to reduce morbidity through early detection, appropriate access to medical care, and increased public awareness.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Constipation can notably affect children's health and their parents' quality of life. Functional constipation (FC) in children is defined as irregular or complex bowel movements without underlying systemic or anatomical causes [##UREF##0##1##]. Although the prevalence of constipation varies among societies based on many factors, including sociocultural and dietary habits, constipation is a common pediatric health problem, affecting between 0.7% and 29.6% of children [##REF##21382575##2##]. One study reported inadequate nutrition as a primary (58%) risk factor for constipation, with mental instability (21.2%) and genetic factors (3.5%) also identified [##REF##17032205##3##].</p>\",\n \"<p>Worldwide, many diagnostic criteria have been approved for diagnosing functional constipation. However, the Rome III criteria and the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition (NASPGHAN) guidelines are the clinical criteria most often utilized in Saudi Arabia [##REF##26115431##4##]. Recently, the Rome IV criteria have been widely used to diagnose FC. According to Rome IV, the diagnosis of FC in children under the age of four requires the existence of at least two of the following symptoms for at least one month in the absence of an organic cause and with insufficient criteria to diagnose irritable bowel syndrome: maximum of two attempts to defecate per week, history of constipation, history of uncomfortable, painful bowel movements, history of hard or large stools, having a large fecal mass in the rectum. Rome IV also notes the following criteria for diagnosing FC in toilet-trained children over four years of age: one or more incontinence incidences per week and a history of having large, thick stools that could block the toilet [##REF##23535762##5##].</p>\",\n \"<p>Fecal incontinence, frequent abdominal pain, bleeding of the rectum, enuresis, and urinary retention and infection are all well-recognized complications of FC in children [##REF##35611377##6##]. Fecal occlusion, intestinal/ileus blockage, toxic megacolon, and bowel perforation are the more catastrophic potential effects of FC [##REF##16606352##7##]. The North American and European Societies for Pediatric Gastroenterology, Hepatology, and Nutrition (NASPEGHAN/ESPGHAN) released updated clinical recommendations for diagnosing and managing FC in children. These include \\\"a normal intake of fibers and liquids, normal physical activity, and an extra pharmacological treatment for rectal fecal impaction followed by a pharmacological maintenance therapy.\\\" The societies also recommend polyethylene glycol (PEG) with or without electrolytes (0.2-0.8 g/kg) for maintenance therapy [##UREF##1##8##].</p>\",\n \"<p>Providentially, FC is estimated to account for 90% of cases seen in healthcare centers, with the remaining 5-10% having organic causes [##UREF##2##9##]. Because FC is a relatively benign disorder, it often goes undiagnosed and untreated, which can lead to a variety of medical and psychosocial issues for children and their parents, cause concern for healthcare budgets, and, presumably, place a significant socioeconomic burden [##UREF##0##1##,##UREF##3##10##,##REF##31460507##11##]. A 2022 study in Saudi Arabia found that parents' knowledge of childhood constipation was linked to their practices in dealing with it. The study involved 568 participants and concluded that poor practices were due to inadequate knowledge [##UREF##4##12##]. A 2019 study in kindergartens in Jatinangor found that in 111 parents there was a correlation between parental knowledge and their children's toilet training practice and behavior. The study highlights the importance of educating parents about fecal continence in children [##UREF##5##13##]. In addition, a 2020 systematic review of 13 studies attempted to locate, organize, and synthesize the current data on the experiences of parents caring for children with FC and the information needed by such parents to facilitate successful treatment. The study revealed that most parents have inadequate knowledge of childhood constipation, with a relatively small percentage having sufficient and correct information about the condition [##REF##33026251##14##].</p>\",\n \"<p>In this study, pediatricians were also found to have inadequate knowledge of FC in children, with notable differences in the pediatricians' knowledge and patterns of practice regarding childhood constipation [##REF##17032205##3##,##REF##26115431##4##,##REF##33026251##14##]. To the best of our knowledge, no studies in the Western region of Saudi Arabia assess the level of parents' knowledge regarding childhood FC. Thus, the study aimed to evaluate parents' knowledge, attitudes, and practices regarding childhood FC in Makkah, Saudi Arabia to identify knowledge gaps that can be filled to reduce morbidity through early detection, appropriate access to medical care, and increased public awareness.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<p>Study design and participants</p>","<p>This web-based descriptive cross-sectional study evaluated parents' knowledge, attitude, and practice toward childhood constipation. The data were obtained through an online questionnaire directed to parents in Makkah, Saudi Arabia. Parents working in the medical field were excluded.</p>","<p>Ethical considerations and sampling technique</p>","<p>The researchers were committed to all ethical considerations required to conduct the research. The Biomedical Ethics Committee at Umm Al-Qura University provided ethical approval no. HAPO-02-K-012-2023-04-1539 to conduct this study. Participants’ privacy was maintained, and the responses remained confidential. Using the Raosoft sample size calculator (Seattle, WA: Raosoft, Inc.), the sample size was determined with a 95% confidence level, a 5% margin of error, and a 50% response distribution, and 385 participants were considered the minimal sample size [##REF##21382575##2##]. The overall sample size increased to a maximum of 824 participants in case of potential data loss and to generalize the study results more efficiently.</p>","<p>Study tool and data collection</p>","<p>A previously validated questionnaire has been utilized [##UREF##4##12##]. An online questionnaire was sent electronically through Google Forms to 824 parents; two opted out of the study and 26 were related to the health sector. After the exclusion of these 28 parents, a total of 796 parents satisfying the inclusion criteria were included in the analysis. The questionnaire is composed of three sections. The first section comprised parents' sociodemographic characteristics, such as age, gender, employment status, residence, and educational status. The second part included seven questions to evaluate parents' knowledge, causes, and symptoms. The third section assessed the response, treatment, and source of knowledge. The questionnaire has been closed-ended with predefined choices. The questionnaire was already prepared and translated into Arabic, the local language. Google Forms have been used for the design.</p>","<p>Statistical analysis</p>","<p>Descriptive and inferential statistical analyses of the data were carried out. Simple descriptive statistics of the sociodemographic characteristics and other categorical variables in the form of frequencies and percentages were calculated and tabulated. Continuous variables medians and interquartile ranges (IQRs) were reported as measures of central tendency and dispersion, respectively, owing to the relatively non-normal distribution of variables as determined by the Kolmogorov-Smirnov test (p<0.001). Seven questions assessed the parents' knowledge and awareness of childhood constipation, and one point was given for each correct response. These were summed up to obtain the total knowledge score of each participant. Because some questions involved multiple responses, the total possible knowledge score of a participant ranged from 0 to 18. Similarly, the practice scores of the participants ranged from 0 to 4. The scores of participants with different sociodemographic characteristics were compared using the non-parametric Mann-Whitney U test and the Kruskal-Wallis test. Additionally, non-parametric Spearman's rank correlation was applied to assess the correlation between knowledge and practice scores. Significance was established at a p-value of 0.05, indicating a 95% confidence interval. All statistical calculations were performed using SPSS version 27.0.1 (Armonk, NY: IBM Corp.).</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<p>Study design and participants</p>\",\n \"<p>This web-based descriptive cross-sectional study evaluated parents' knowledge, attitude, and practice toward childhood constipation. The data were obtained through an online questionnaire directed to parents in Makkah, Saudi Arabia. Parents working in the medical field were excluded.</p>\",\n \"<p>Ethical considerations and sampling technique</p>\",\n \"<p>The researchers were committed to all ethical considerations required to conduct the research. The Biomedical Ethics Committee at Umm Al-Qura University provided ethical approval no. HAPO-02-K-012-2023-04-1539 to conduct this study. Participants’ privacy was maintained, and the responses remained confidential. Using the Raosoft sample size calculator (Seattle, WA: Raosoft, Inc.), the sample size was determined with a 95% confidence level, a 5% margin of error, and a 50% response distribution, and 385 participants were considered the minimal sample size [##REF##21382575##2##]. The overall sample size increased to a maximum of 824 participants in case of potential data loss and to generalize the study results more efficiently.</p>\",\n \"<p>Study tool and data collection</p>\",\n \"<p>A previously validated questionnaire has been utilized [##UREF##4##12##]. An online questionnaire was sent electronically through Google Forms to 824 parents; two opted out of the study and 26 were related to the health sector. After the exclusion of these 28 parents, a total of 796 parents satisfying the inclusion criteria were included in the analysis. The questionnaire is composed of three sections. The first section comprised parents' sociodemographic characteristics, such as age, gender, employment status, residence, and educational status. The second part included seven questions to evaluate parents' knowledge, causes, and symptoms. The third section assessed the response, treatment, and source of knowledge. The questionnaire has been closed-ended with predefined choices. The questionnaire was already prepared and translated into Arabic, the local language. Google Forms have been used for the design.</p>\",\n \"<p>Statistical analysis</p>\",\n \"<p>Descriptive and inferential statistical analyses of the data were carried out. Simple descriptive statistics of the sociodemographic characteristics and other categorical variables in the form of frequencies and percentages were calculated and tabulated. Continuous variables medians and interquartile ranges (IQRs) were reported as measures of central tendency and dispersion, respectively, owing to the relatively non-normal distribution of variables as determined by the Kolmogorov-Smirnov test (p<0.001). Seven questions assessed the parents' knowledge and awareness of childhood constipation, and one point was given for each correct response. These were summed up to obtain the total knowledge score of each participant. Because some questions involved multiple responses, the total possible knowledge score of a participant ranged from 0 to 18. Similarly, the practice scores of the participants ranged from 0 to 4. The scores of participants with different sociodemographic characteristics were compared using the non-parametric Mann-Whitney U test and the Kruskal-Wallis test. Additionally, non-parametric Spearman's rank correlation was applied to assess the correlation between knowledge and practice scores. Significance was established at a p-value of 0.05, indicating a 95% confidence interval. All statistical calculations were performed using SPSS version 27.0.1 (Armonk, NY: IBM Corp.).</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p>Among 796 participants, a notable majority were female, accounting for 74.2% of the sample, while the remaining 25.8% were male. Each participant willingly agreed to partake in the survey. The age distribution varied, with the largest group falling in the 35-60 years category at 62.8%, followed by 25 to 34-year-olds at 22.0%; a smaller representation of individuals aged 15-24 years (6.7%) and those above 60 years (8.5%). The educational backgrounds of the participants were diverse, with a significant majority having a university education (67.5%), while 12.1% possessing postgraduate qualifications. The participants also came from various occupational backgrounds, with government employees making up the largest group (34.3%) and unemployed individuals accounting for 31.0%. Most participants were married (92.5%), while 7.5% were divorced. Additionally, 80.7% of the participants reported that their children had previously experienced constipation (Table ##TAB##0##1##).</p>","<p>Table ##TAB##1##2## presents the participants’ responses to knowledge questions regarding childhood constipation, highlighting the percentages of correct answers. Only 11.1% of participants correctly identified the definition of constipation as “less than three bowel movements per week,” while 63.6% correctly recognized it as a “symptom” rather than an illness. In terms of common causes, a significant 88.4% recognized “organic constipation” and 81.3% identified “functional constipation” as common causes. Regarding the causes of functional constipation in children, 84.3% correctly attributed it to a “low-fiber diet.” In terms of symptoms, substantial percentages correctly identified “hard, dry stool” (71.0%) and “painful defecation” (63.0%) as common symptoms. However, fewer participants recognized the symptom of “large diameter stool that may obstruct the toilet” (19.3%). Concerning the necessity of complete tests for constipated children, 38.7% correctly answered “no,” while 30.2% believed “yes” and 31.2% responded with “I don't know.” Finally, for complications, the majority correctly identified “painful anal fissures” (75.1%) and “fecal impaction” (60.6%) as potential complications, while fewer recognized “intestinal perforation” (7.3%) and “fecal incontinence” (8.3%). The participants’ total knowledge score had a median of 8.0, indicating a moderate overall understanding of childhood constipation. This summary emphasizes the varying levels of correct answers among the participants and underscores areas where educational efforts may be beneficial (Table ##TAB##1##2##).</p>","<p>Table ##TAB##2##3## provides insights into the participants’ responses regarding their practices related to childhood constipation, with a focus on the percentages of correct practices. When asked about the initial home treatment for their child’s constipation, 27.9% correctly suggested “giving him high-fiber food,” while 23.5% recognized the importance of “giving him plenty of fluids.” However, a notable 23.7% mentioned “giving him laxatives” as an initial treatment, which is not recommended without medical guidance. Furthermore, only a small percentage (10.6%) suggested “give him bananas or honey.” Regarding the treatment for fecal impaction and intestinal blockage, 42.8% correctly identified “do an enema to expel stool” as the most successful treatment, indicating a good understanding of this critical aspect. In terms of recommending fiber-rich foods for children with constipation, 71.7% rightly suggested “fruits like watermelon, apple, and banana” and 68.8% recognized “vegetables” as a suitable option. This indicates a relatively strong awareness of dietary recommendations for managing constipation. The participants’ total practice score had a median of 2.0 (IQR: 1.0-3.0), suggesting a moderate level of adherence to correct practices related to childhood constipation (Table ##TAB##2##3##).</p>","<p>When asked about their biggest concern regarding chronic childhood constipation, the majority (65.1%) expressed the fear of it “continuing into adulthood.” A significant portion also expressed concerns related to severe medical conditions, with 30.9% fearing “internal abdominal tumors” and 17.3% fearing “congenital abnormalities of the colon (stricture).” These responses highlight the substantial apprehension parents may have about the long-term implications of childhood constipation on their children’s health (Table ##TAB##3##4##).</p>","<p>Regarding sources of information about constipation, the internet emerged as the most commonly cited source, with 25.5% of the participants relying on online resources. Friends and relatives also played a significant role, with 23.6% turning to them for information. Doctors and medical staff were mentioned by 14.6% of the participants, while frequent medical practice was cited by 13.8%. Magazines, books, TV, and other sources made up the remainder of the information channels. This indicates a diverse range of sources from which parents seek information about childhood constipation, with the internet being a prominent choice. This underscores the importance of ensuring accurate and reliable online resources for parents seeking guidance on this health issue (Figure ##FIG##0##1##).</p>","<p>The findings indicated a significant difference in knowledge scores based on gender (p<0.001). Female participants had a higher median knowledge score of 8.0 (IQR: 7.0-10.0) compared to males, with a median score of 8.0 (IQR: 5.0-10.0). Regarding educational level, there was a significant difference in knowledge scores (p=0.001). Postgraduate participants exhibited the highest median knowledge score of 9.0 (IQR: 7.0-11.0), while those with a middle-level education had the lowest median score of 7.0 (IQR: 5.0-9.0). However, no significant associations were observed between knowledge scores and age, occupation, marital status, or whether their children had experienced constipation before.</p>","<p>In summary, parents and educational level were associated with variations in knowledge about childhood constipation among the participants. Female participants and those with postgraduate education tended to have higher knowledge scores (Table ##TAB##4##5##).</p>","<p>The analysis revealed some significant findings. First, there was a significant difference in practice scores based on age (p=0.007). The participants in the 25-34 years age group had a median practice score of 2.0 (IQR: 1.0-3.0; mean=2.0), and those in the 35-60 years group (mean=2.2) and more than 60 years age group (mean=2.4) also had a median score of 2.0 (IQR: 2.0-3.0). However, participants in the 15-24 years age group had a comparatively lower score of 2.0 (IQR: 1.0-3.0) (mean=1.9). Other sociodemographic characteristics, including gender, agreement to participate in the survey, educational level, occupation, marital status, and whether their children had experienced constipation before, did not show significant associations with practice scores (Table ##TAB##5##6##).</p>","<p>The analysis, performed using Spearman’s rank correlation coefficient, revealed a statistically significant positive correlation (ρ=0.328, p<0.001) between knowledge and practice scores among the 796 participants (Table ##TAB##6##7##). This finding indicates that the participants who demonstrated higher levels of knowledge about childhood constipation tended to exhibit better practices related to its management. In other words, as the participants’ knowledge scores increased, their practice scores also showed a positive trend. This suggests that improving knowledge about childhood constipation can lead to more effective and appropriate practices in its management and prevention (Figure ##FIG##1##2##).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p>Among 796 participants, a notable majority were female, accounting for 74.2% of the sample, while the remaining 25.8% were male. Each participant willingly agreed to partake in the survey. The age distribution varied, with the largest group falling in the 35-60 years category at 62.8%, followed by 25 to 34-year-olds at 22.0%; a smaller representation of individuals aged 15-24 years (6.7%) and those above 60 years (8.5%). The educational backgrounds of the participants were diverse, with a significant majority having a university education (67.5%), while 12.1% possessing postgraduate qualifications. The participants also came from various occupational backgrounds, with government employees making up the largest group (34.3%) and unemployed individuals accounting for 31.0%. Most participants were married (92.5%), while 7.5% were divorced. Additionally, 80.7% of the participants reported that their children had previously experienced constipation (Table ##TAB##0##1##).</p>\",\n \"<p>Table ##TAB##1##2## presents the participants’ responses to knowledge questions regarding childhood constipation, highlighting the percentages of correct answers. Only 11.1% of participants correctly identified the definition of constipation as “less than three bowel movements per week,” while 63.6% correctly recognized it as a “symptom” rather than an illness. In terms of common causes, a significant 88.4% recognized “organic constipation” and 81.3% identified “functional constipation” as common causes. Regarding the causes of functional constipation in children, 84.3% correctly attributed it to a “low-fiber diet.” In terms of symptoms, substantial percentages correctly identified “hard, dry stool” (71.0%) and “painful defecation” (63.0%) as common symptoms. However, fewer participants recognized the symptom of “large diameter stool that may obstruct the toilet” (19.3%). Concerning the necessity of complete tests for constipated children, 38.7% correctly answered “no,” while 30.2% believed “yes” and 31.2% responded with “I don't know.” Finally, for complications, the majority correctly identified “painful anal fissures” (75.1%) and “fecal impaction” (60.6%) as potential complications, while fewer recognized “intestinal perforation” (7.3%) and “fecal incontinence” (8.3%). The participants’ total knowledge score had a median of 8.0, indicating a moderate overall understanding of childhood constipation. This summary emphasizes the varying levels of correct answers among the participants and underscores areas where educational efforts may be beneficial (Table ##TAB##1##2##).</p>\",\n \"<p>Table ##TAB##2##3## provides insights into the participants’ responses regarding their practices related to childhood constipation, with a focus on the percentages of correct practices. When asked about the initial home treatment for their child’s constipation, 27.9% correctly suggested “giving him high-fiber food,” while 23.5% recognized the importance of “giving him plenty of fluids.” However, a notable 23.7% mentioned “giving him laxatives” as an initial treatment, which is not recommended without medical guidance. Furthermore, only a small percentage (10.6%) suggested “give him bananas or honey.” Regarding the treatment for fecal impaction and intestinal blockage, 42.8% correctly identified “do an enema to expel stool” as the most successful treatment, indicating a good understanding of this critical aspect. In terms of recommending fiber-rich foods for children with constipation, 71.7% rightly suggested “fruits like watermelon, apple, and banana” and 68.8% recognized “vegetables” as a suitable option. This indicates a relatively strong awareness of dietary recommendations for managing constipation. The participants’ total practice score had a median of 2.0 (IQR: 1.0-3.0), suggesting a moderate level of adherence to correct practices related to childhood constipation (Table ##TAB##2##3##).</p>\",\n \"<p>When asked about their biggest concern regarding chronic childhood constipation, the majority (65.1%) expressed the fear of it “continuing into adulthood.” A significant portion also expressed concerns related to severe medical conditions, with 30.9% fearing “internal abdominal tumors” and 17.3% fearing “congenital abnormalities of the colon (stricture).” These responses highlight the substantial apprehension parents may have about the long-term implications of childhood constipation on their children’s health (Table ##TAB##3##4##).</p>\",\n \"<p>Regarding sources of information about constipation, the internet emerged as the most commonly cited source, with 25.5% of the participants relying on online resources. Friends and relatives also played a significant role, with 23.6% turning to them for information. Doctors and medical staff were mentioned by 14.6% of the participants, while frequent medical practice was cited by 13.8%. Magazines, books, TV, and other sources made up the remainder of the information channels. This indicates a diverse range of sources from which parents seek information about childhood constipation, with the internet being a prominent choice. This underscores the importance of ensuring accurate and reliable online resources for parents seeking guidance on this health issue (Figure ##FIG##0##1##).</p>\",\n \"<p>The findings indicated a significant difference in knowledge scores based on gender (p<0.001). Female participants had a higher median knowledge score of 8.0 (IQR: 7.0-10.0) compared to males, with a median score of 8.0 (IQR: 5.0-10.0). Regarding educational level, there was a significant difference in knowledge scores (p=0.001). Postgraduate participants exhibited the highest median knowledge score of 9.0 (IQR: 7.0-11.0), while those with a middle-level education had the lowest median score of 7.0 (IQR: 5.0-9.0). However, no significant associations were observed between knowledge scores and age, occupation, marital status, or whether their children had experienced constipation before.</p>\",\n \"<p>In summary, parents and educational level were associated with variations in knowledge about childhood constipation among the participants. Female participants and those with postgraduate education tended to have higher knowledge scores (Table ##TAB##4##5##).</p>\",\n \"<p>The analysis revealed some significant findings. First, there was a significant difference in practice scores based on age (p=0.007). The participants in the 25-34 years age group had a median practice score of 2.0 (IQR: 1.0-3.0; mean=2.0), and those in the 35-60 years group (mean=2.2) and more than 60 years age group (mean=2.4) also had a median score of 2.0 (IQR: 2.0-3.0). However, participants in the 15-24 years age group had a comparatively lower score of 2.0 (IQR: 1.0-3.0) (mean=1.9). Other sociodemographic characteristics, including gender, agreement to participate in the survey, educational level, occupation, marital status, and whether their children had experienced constipation before, did not show significant associations with practice scores (Table ##TAB##5##6##).</p>\",\n \"<p>The analysis, performed using Spearman’s rank correlation coefficient, revealed a statistically significant positive correlation (ρ=0.328, p<0.001) between knowledge and practice scores among the 796 participants (Table ##TAB##6##7##). This finding indicates that the participants who demonstrated higher levels of knowledge about childhood constipation tended to exhibit better practices related to its management. In other words, as the participants’ knowledge scores increased, their practice scores also showed a positive trend. This suggests that improving knowledge about childhood constipation can lead to more effective and appropriate practices in its management and prevention (Figure ##FIG##1##2##).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>Our study aimed to determine parents’ knowledge, attitudes, and practices toward childhood constipation in a major city in Saudi Arabia. The dominance of female participants, constituting 74.2% of the sample, was a noteworthy observation.</p>","<p>Moreover, the high percentage of participants with a university education (67.5%) and postgraduate qualifications (12.1%) reflects a relatively well-educated sample. Educated parents are more likely to actively seek information and engage in health-related discussions [##UREF##6##15##]. However, the educational diversity within the sample may lead to varying levels of health literacy. This diversity highlights the importance of tailoring health education programs to different educational backgrounds to ensure effective communication and understanding.</p>","<p>This study provides valuable insights into the participants’ understanding of childhood constipation. While many respondents correctly identified common causes of constipation, such as “organic constipation” and “functional constipation,” there were noticeable gaps in their knowledge [##UREF##0##1##]. This indicates that constipation is perceived differently by individuals in the Makkah community. For instance, only 11.1% of persons correctly identified the definition of constipation as “less than three bowel movements per week,” consistent with the findings of Gray [##UREF##7##16##]. This lack of awareness regarding a fundamental aspect of constipation could have led to misconceptions and delayed recognition of the condition, which may have implications for children’s health. Thus, there is a lack of standardized definitions and a shared understanding of constipation within the Makkah community [##UREF##8##17##]. This lack of consensus could result in challenges in healthcare settings, as healthcare providers may need to clarify and educate patients and their families about what constipation entails.</p>","<p>Additionally, the relatively low recognition of specific symptoms, such as “large diameter stool that may obstruct the toilet,” and severe complications, such as “intestinal perforation,” underscores areas where parents may not be fully aware of the potential seriousness of childhood constipation, echoing concerns raised by the study conducted in Nigeria [##REF##20589146##18##]. This highlights the need for comprehensive education that not only addresses common aspects of the condition but also delves into less prevalent but critical facets to ensure a holistic understanding among parents in Makkah.</p>","<p>The practice assessment reflects the actions that parents take when their child experiences constipation. A significant percentage suggested giving high-fiber foods and plenty of fluids as initial home treatments, which aligns with evidence-based recommendations for managing childhood constipation [##REF##23326148##19##,##UREF##9##20##]. However, the finding that 23.7% of the participants mentioned “give him laxatives” as an initial treatment is concerning. This finding aligns with concerns about laxative misuse in similar populations, as identified by Roerig et al. [##REF##20687617##21##]. The inappropriate use of laxatives can lead to dependence, electrolyte imbalances, and other adverse effects, highlighting the need for caution in their administration. It is crucial to highlight the importance of clear and accessible guidance on appropriate home remedies to avoid potential harm to children. Education campaigns can play a pivotal role in promoting safe and effective home treatments.</p>","<p>The expression of concern by parents regarding the long-term consequences of childhood constipation is a significant finding. This reflects parental anxieties about their children’s health, which has been documented by Rajindrajith et al. [##REF##27570423##22##]. The fear of constipation continuing into adulthood and concerns about severe medical conditions indicate that parents in Makkah are deeply invested in their children’s well-being. This reflects a forward-looking approach to healthcare decisions. Healthcare providers should recognize and address these fears during consultations, offering reassurance and guidance to alleviate parental anxiety [##UREF##10##23##]. This personalized approach to addressing parental concerns can improve overall healthcare experiences for families in Makkah.</p>","<p>The analysis of associations among knowledge, practice, and sociodemographic factors revealed intriguing patterns. There was a significant difference in the knowledge scores based on gender and educational level. The female participants and those with postgraduate education tended to have higher knowledge scores. This suggests that tailored educational interventions can be particularly effective for these groups. Additionally, it highlights the importance of gender-inclusive health education efforts to bridge the knowledge gap among parents.</p>","<p>The positive correlation between knowledge and practice scores is a crucial finding. It suggests that when parents are better informed and have a deeper understanding of constipation, they are more likely to implement effective strategies for caring for their children with constipation. It emphasizes that improving parents’ knowledge about childhood constipation can lead directly to better practices in managing the condition, as indicated by Thompson et al. [##REF##33026251##14##]. This underlines the potential impact of educational interventions in enhancing the care and well-being of children with constipation in Makkah.</p>","<p>The limitations of this study should be acknowledged to provide a comprehensive assessment of its findings and implications. The study’s reliance on a self-report survey may introduce response bias, as the participants may provide socially desirable answers or unintentionally misrepresent their knowledge, attitudes, and practices related to childhood constipation. The research was conducted in Makkah, Saudi Arabia, and may not be fully representative of the broader Saudi population. The predominantly female sample may also introduce gender bias and limit the generalizability of the results. Third, the study’s cross-sectional design restricts the ability to establish causal relationships between sociodemographic factors and knowledge or practice scores. Additionally, the survey’s reliance on closed-ended questions may not capture the depth of the participants’ attitudes and practices, and qualitative research methods could complement future investigations in this regard.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>Our study aimed to determine parents’ knowledge, attitudes, and practices toward childhood constipation in a major city in Saudi Arabia. The dominance of female participants, constituting 74.2% of the sample, was a noteworthy observation.</p>\",\n \"<p>Moreover, the high percentage of participants with a university education (67.5%) and postgraduate qualifications (12.1%) reflects a relatively well-educated sample. Educated parents are more likely to actively seek information and engage in health-related discussions [##UREF##6##15##]. However, the educational diversity within the sample may lead to varying levels of health literacy. This diversity highlights the importance of tailoring health education programs to different educational backgrounds to ensure effective communication and understanding.</p>\",\n \"<p>This study provides valuable insights into the participants’ understanding of childhood constipation. While many respondents correctly identified common causes of constipation, such as “organic constipation” and “functional constipation,” there were noticeable gaps in their knowledge [##UREF##0##1##]. This indicates that constipation is perceived differently by individuals in the Makkah community. For instance, only 11.1% of persons correctly identified the definition of constipation as “less than three bowel movements per week,” consistent with the findings of Gray [##UREF##7##16##]. This lack of awareness regarding a fundamental aspect of constipation could have led to misconceptions and delayed recognition of the condition, which may have implications for children’s health. Thus, there is a lack of standardized definitions and a shared understanding of constipation within the Makkah community [##UREF##8##17##]. This lack of consensus could result in challenges in healthcare settings, as healthcare providers may need to clarify and educate patients and their families about what constipation entails.</p>\",\n \"<p>Additionally, the relatively low recognition of specific symptoms, such as “large diameter stool that may obstruct the toilet,” and severe complications, such as “intestinal perforation,” underscores areas where parents may not be fully aware of the potential seriousness of childhood constipation, echoing concerns raised by the study conducted in Nigeria [##REF##20589146##18##]. This highlights the need for comprehensive education that not only addresses common aspects of the condition but also delves into less prevalent but critical facets to ensure a holistic understanding among parents in Makkah.</p>\",\n \"<p>The practice assessment reflects the actions that parents take when their child experiences constipation. A significant percentage suggested giving high-fiber foods and plenty of fluids as initial home treatments, which aligns with evidence-based recommendations for managing childhood constipation [##REF##23326148##19##,##UREF##9##20##]. However, the finding that 23.7% of the participants mentioned “give him laxatives” as an initial treatment is concerning. This finding aligns with concerns about laxative misuse in similar populations, as identified by Roerig et al. [##REF##20687617##21##]. The inappropriate use of laxatives can lead to dependence, electrolyte imbalances, and other adverse effects, highlighting the need for caution in their administration. It is crucial to highlight the importance of clear and accessible guidance on appropriate home remedies to avoid potential harm to children. Education campaigns can play a pivotal role in promoting safe and effective home treatments.</p>\",\n \"<p>The expression of concern by parents regarding the long-term consequences of childhood constipation is a significant finding. This reflects parental anxieties about their children’s health, which has been documented by Rajindrajith et al. [##REF##27570423##22##]. The fear of constipation continuing into adulthood and concerns about severe medical conditions indicate that parents in Makkah are deeply invested in their children’s well-being. This reflects a forward-looking approach to healthcare decisions. Healthcare providers should recognize and address these fears during consultations, offering reassurance and guidance to alleviate parental anxiety [##UREF##10##23##]. This personalized approach to addressing parental concerns can improve overall healthcare experiences for families in Makkah.</p>\",\n \"<p>The analysis of associations among knowledge, practice, and sociodemographic factors revealed intriguing patterns. There was a significant difference in the knowledge scores based on gender and educational level. The female participants and those with postgraduate education tended to have higher knowledge scores. This suggests that tailored educational interventions can be particularly effective for these groups. Additionally, it highlights the importance of gender-inclusive health education efforts to bridge the knowledge gap among parents.</p>\",\n \"<p>The positive correlation between knowledge and practice scores is a crucial finding. It suggests that when parents are better informed and have a deeper understanding of constipation, they are more likely to implement effective strategies for caring for their children with constipation. It emphasizes that improving parents’ knowledge about childhood constipation can lead directly to better practices in managing the condition, as indicated by Thompson et al. [##REF##33026251##14##]. This underlines the potential impact of educational interventions in enhancing the care and well-being of children with constipation in Makkah.</p>\",\n \"<p>The limitations of this study should be acknowledged to provide a comprehensive assessment of its findings and implications. The study’s reliance on a self-report survey may introduce response bias, as the participants may provide socially desirable answers or unintentionally misrepresent their knowledge, attitudes, and practices related to childhood constipation. The research was conducted in Makkah, Saudi Arabia, and may not be fully representative of the broader Saudi population. The predominantly female sample may also introduce gender bias and limit the generalizability of the results. Third, the study’s cross-sectional design restricts the ability to establish causal relationships between sociodemographic factors and knowledge or practice scores. Additionally, the survey’s reliance on closed-ended questions may not capture the depth of the participants’ attitudes and practices, and qualitative research methods could complement future investigations in this regard.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>This study highlights the variability in knowledge levels among parents, with a moderate overall understanding of childhood constipation. It emphasizes a moderate level of adherence to recommended practices related to childhood constipation, with some room for improvement in certain areas. These findings underscore the importance of targeted educational efforts to improve parents’ understanding and behavior concerning childhood constipation in Makkah, Saudi Arabia. Additionally, the study emphasizes the role of the internet and interpersonal sources in disseminating information, highlighting the need for reliable online resources and healthcare professionals to provide accurate guidance to concerned parents.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>This study highlights the variability in knowledge levels among parents, with a moderate overall understanding of childhood constipation. It emphasizes a moderate level of adherence to recommended practices related to childhood constipation, with some room for improvement in certain areas. These findings underscore the importance of targeted educational efforts to improve parents’ understanding and behavior concerning childhood constipation in Makkah, Saudi Arabia. Additionally, the study emphasizes the role of the internet and interpersonal sources in disseminating information, highlighting the need for reliable online resources and healthcare professionals to provide accurate guidance to concerned parents.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Introduction</p>","<p>Functional constipation in children is described as irregular or difficult bowel motions without underlying systemic or anatomical causes. Although constipation can have a serious negative impact on a child's health and the lives of their parents. This study aimed to assess the knowledge of parents about childhood constipation, intending to reduce morbidity and mortality through increased public health education in Makkah, Saudi Arabia.</p>","<p>Methods</p>","<p>The current study was a web-based, descriptive cross-sectional study. The data were obtained from May 2023 to November 2023 through an online questionnaire directed to parents in Makkah, Saudi Arabia, and analyzed using SPSS version 27.0.1 (Armonk, NY: IBM Corp.).</p>","<p>Results</p>","<p>A total of 796 participants were included in the present study, of which 205 (25.8%) were males and 591 (74.2%) were females. The knowledge levels among them varied, with 11.1% correctly defining constipation and 63.6% recognizing it as a symptom. Common causes like organic and functional constipation were acknowledged by 88.4% and 81.3% of participants, respectively. Regarding practices, 27.9% recommended high-fiber foods for initial home treatment, and 42.8% acknowledged that an enema is effective for fecal impaction. In the dietary recommendations, 71.7% suggested fruits and 68.8% mentioned vegetables. Concerning attitudes, 65.1% expressed fear of childhood constipation continuing into adulthood, while 30.9% feared severe medical conditions. The internet (25.5%) and friends/relatives (23.6%) were the primary sources of information. Knowledge was significantly higher among females and those with postgraduate education.</p>","<p>Conclusion</p>","<p>This study highlights the variability in knowledge levels among parents, with an overall moderate understanding of childhood constipation. It emphasizes a moderate level of adherence to recommended practices related to childhood constipation, with some room for improvement in Makkah, Saudi Arabia.</p>"],"string":"[\n \"<p>Introduction</p>\",\n \"<p>Functional constipation in children is described as irregular or difficult bowel motions without underlying systemic or anatomical causes. Although constipation can have a serious negative impact on a child's health and the lives of their parents. This study aimed to assess the knowledge of parents about childhood constipation, intending to reduce morbidity and mortality through increased public health education in Makkah, Saudi Arabia.</p>\",\n \"<p>Methods</p>\",\n \"<p>The current study was a web-based, descriptive cross-sectional study. The data were obtained from May 2023 to November 2023 through an online questionnaire directed to parents in Makkah, Saudi Arabia, and analyzed using SPSS version 27.0.1 (Armonk, NY: IBM Corp.).</p>\",\n \"<p>Results</p>\",\n \"<p>A total of 796 participants were included in the present study, of which 205 (25.8%) were males and 591 (74.2%) were females. The knowledge levels among them varied, with 11.1% correctly defining constipation and 63.6% recognizing it as a symptom. Common causes like organic and functional constipation were acknowledged by 88.4% and 81.3% of participants, respectively. Regarding practices, 27.9% recommended high-fiber foods for initial home treatment, and 42.8% acknowledged that an enema is effective for fecal impaction. In the dietary recommendations, 71.7% suggested fruits and 68.8% mentioned vegetables. Concerning attitudes, 65.1% expressed fear of childhood constipation continuing into adulthood, while 30.9% feared severe medical conditions. The internet (25.5%) and friends/relatives (23.6%) were the primary sources of information. Knowledge was significantly higher among females and those with postgraduate education.</p>\",\n \"<p>Conclusion</p>\",\n \"<p>This study highlights the variability in knowledge levels among parents, with an overall moderate understanding of childhood constipation. It emphasizes a moderate level of adherence to recommended practices related to childhood constipation, with some room for improvement in Makkah, Saudi Arabia.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Source of information of the participants.</title></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Correlation of knowledge and practice scores of the participants.</title></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>Source of information of the participants.</title></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG2\\\"><label>Figure 2</label><caption><title>Correlation of knowledge and practice scores of the participants.</title></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Sociodemographic characteristics of the participants.</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"1\">Characteristics</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">%</td></tr><tr><td rowspan=\"2\" colspan=\"1\">Gender</td><td rowspan=\"1\" colspan=\"1\">Female</td><td rowspan=\"1\" colspan=\"1\">591</td><td rowspan=\"1\" colspan=\"1\">74.2%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Male</td><td rowspan=\"1\" colspan=\"1\">205</td><td rowspan=\"1\" colspan=\"1\">25.8%</td></tr><tr><td rowspan=\"4\" colspan=\"1\">Age</td><td rowspan=\"1\" colspan=\"1\">15-24 years</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">6.7%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">25-34 years</td><td rowspan=\"1\" colspan=\"1\">175</td><td rowspan=\"1\" colspan=\"1\">22.0%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">35-60 years</td><td rowspan=\"1\" colspan=\"1\">500</td><td rowspan=\"1\" colspan=\"1\">62.8%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">More than 60 years</td><td rowspan=\"1\" colspan=\"1\">68</td><td rowspan=\"1\" colspan=\"1\">8.5%</td></tr><tr><td rowspan=\"6\" colspan=\"1\">Educational level</td><td rowspan=\"1\" colspan=\"1\">Middle</td><td rowspan=\"1\" colspan=\"1\">21</td><td rowspan=\"1\" colspan=\"1\">2.6%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Nothing</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">0.3%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Postgraduate</td><td rowspan=\"1\" colspan=\"1\">96</td><td rowspan=\"1\" colspan=\"1\">12.1%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Primary</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">1.1%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Secondary</td><td rowspan=\"1\" colspan=\"1\">131</td><td rowspan=\"1\" colspan=\"1\">16.5%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">University education</td><td rowspan=\"1\" colspan=\"1\">537</td><td rowspan=\"1\" colspan=\"1\">67.5%</td></tr><tr><td rowspan=\"5\" colspan=\"1\">Occupation</td><td rowspan=\"1\" colspan=\"1\">An employee in the private sector</td><td rowspan=\"1\" colspan=\"1\">85</td><td rowspan=\"1\" colspan=\"1\">10.7%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Self-employed</td><td rowspan=\"1\" colspan=\"1\">45</td><td rowspan=\"1\" colspan=\"1\">5.7%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Government employee</td><td rowspan=\"1\" colspan=\"1\">273</td><td rowspan=\"1\" colspan=\"1\">34.3%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Retired</td><td rowspan=\"1\" colspan=\"1\">146</td><td rowspan=\"1\" colspan=\"1\">18.3%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Unemployed</td><td rowspan=\"1\" colspan=\"1\">247</td><td rowspan=\"1\" colspan=\"1\">31.0%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Marital status</td><td rowspan=\"1\" colspan=\"1\">Divorced</td><td rowspan=\"1\" colspan=\"1\">60</td><td rowspan=\"1\" colspan=\"1\">7.5%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Married</td><td rowspan=\"1\" colspan=\"1\">736</td><td rowspan=\"1\" colspan=\"1\">92.5%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Have any of your children suffered from constipation before?</td><td rowspan=\"1\" colspan=\"1\">No</td><td rowspan=\"1\" colspan=\"1\">154</td><td rowspan=\"1\" colspan=\"1\">19.3%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">642</td><td rowspan=\"1\" colspan=\"1\">80.7%</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Responses of the participants to knowledge questions.</title><p>*Correct answer.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"1\">Characteristics</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">%</td></tr><tr><td rowspan=\"4\" colspan=\"1\">Among the following, which is closest to the definition of constipation?</td><td rowspan=\"1\" colspan=\"1\">Difficulty in excretion</td><td rowspan=\"1\" colspan=\"1\">398</td><td rowspan=\"1\" colspan=\"1\">50.0%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Lack of daily output</td><td rowspan=\"1\" colspan=\"1\">173</td><td rowspan=\"1\" colspan=\"1\">21.7%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Less than three bowel movements per week*</td><td rowspan=\"1\" colspan=\"1\">88</td><td rowspan=\"1\" colspan=\"1\">11.1%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Metaphyseal ossification (stool)</td><td rowspan=\"1\" colspan=\"1\">137</td><td rowspan=\"1\" colspan=\"1\">17.2%</td></tr><tr><td rowspan=\"3\" colspan=\"1\">In your opinion, is constipation a disease or a symptom?</td><td rowspan=\"1\" colspan=\"1\">A symptom*</td><td rowspan=\"1\" colspan=\"1\">506</td><td rowspan=\"1\" colspan=\"1\">63.6%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">I don't know</td><td rowspan=\"1\" colspan=\"1\">102</td><td rowspan=\"1\" colspan=\"1\">12.8%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Illness</td><td rowspan=\"1\" colspan=\"1\">188</td><td rowspan=\"1\" colspan=\"1\">23.6%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"3\" colspan=\"1\">What is the common cause of constipation in children (multi-select question)?</td><td rowspan=\"1\" colspan=\"1\">Organic constipation*</td><td rowspan=\"1\" colspan=\"1\">704</td><td rowspan=\"1\" colspan=\"1\">88.4%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Functional constipation*</td><td rowspan=\"1\" colspan=\"1\">647</td><td rowspan=\"1\" colspan=\"1\">81.3%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">I don't know</td><td rowspan=\"1\" colspan=\"1\">101</td><td rowspan=\"1\" colspan=\"1\">12.7%</td></tr><tr><td rowspan=\"5\" colspan=\"1\">What is the cause of functional constipation in children (multi-select question)?</td><td rowspan=\"1\" colspan=\"1\">Stool withholding*</td><td rowspan=\"1\" colspan=\"1\">267</td><td rowspan=\"1\" colspan=\"1\">36.1%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Toilet training resistance*</td><td rowspan=\"1\" colspan=\"1\">147</td><td rowspan=\"1\" colspan=\"1\">19.9%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Low-fibers diet*</td><td rowspan=\"1\" colspan=\"1\">623</td><td rowspan=\"1\" colspan=\"1\">84.3%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Low physical activity*</td><td rowspan=\"1\" colspan=\"1\">252</td><td rowspan=\"1\" colspan=\"1\">34.1%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">I don't know</td><td rowspan=\"1\" colspan=\"1\">78</td><td rowspan=\"1\" colspan=\"1\">10.6%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"4\" colspan=\"1\">What are the most common symptoms of constipation (multi-select question)?</td><td rowspan=\"1\" colspan=\"1\">Abdominal pain*</td><td rowspan=\"1\" colspan=\"1\">390</td><td rowspan=\"1\" colspan=\"1\">50.1%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Painful defecation*</td><td rowspan=\"1\" colspan=\"1\">491</td><td rowspan=\"1\" colspan=\"1\">63.0%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Hard, dry stool*</td><td rowspan=\"1\" colspan=\"1\">553</td><td rowspan=\"1\" colspan=\"1\">71.0%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Large diameter stool that may obstruct the toilet*</td><td rowspan=\"1\" colspan=\"1\">150</td><td rowspan=\"1\" colspan=\"1\">19.3%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"3\" colspan=\"1\">Does a constipated child need complete tests at all times?</td><td rowspan=\"1\" colspan=\"1\">I don't know</td><td rowspan=\"1\" colspan=\"1\">248</td><td rowspan=\"1\" colspan=\"1\">31.2%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">No*</td><td rowspan=\"1\" colspan=\"1\">308</td><td rowspan=\"1\" colspan=\"1\">38.7%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">240</td><td rowspan=\"1\" colspan=\"1\">30.2%</td></tr><tr><td rowspan=\"5\" colspan=\"1\">What are the complications of constipation in children (multi-select question)?</td><td rowspan=\"1\" colspan=\"1\">Painful anal fissures (torn skin around the anus)*</td><td rowspan=\"1\" colspan=\"1\">598</td><td rowspan=\"1\" colspan=\"1\">75.1%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Rectal prolapse (intestine that protrudes from the anus)*</td><td rowspan=\"1\" colspan=\"1\">216</td><td rowspan=\"1\" colspan=\"1\">27.1%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Fecal impaction (stool that can't be expelled)*</td><td rowspan=\"1\" colspan=\"1\">482</td><td rowspan=\"1\" colspan=\"1\">60.6%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Fecal incontinence (leaking of watery stool from the bottom)*</td><td rowspan=\"1\" colspan=\"1\">66</td><td rowspan=\"1\" colspan=\"1\">8.3%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Intestinal perforation*</td><td rowspan=\"1\" colspan=\"1\">58</td><td rowspan=\"1\" colspan=\"1\">7.3%</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"1\">Total knowledge score - median (IQR)</td><td colspan=\"2\" rowspan=\"1\">8.0 (6.0-10.0)</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB3\"><label>Table 3</label><caption><title>Responses of the participants to practice questions.</title><p>*Correct practice.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"1\">Variables</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">%</td></tr><tr><td rowspan=\"6\" colspan=\"1\">What is the initial treatment that you do at home to treat your child’s constipation before taking him to the doctor?</td><td rowspan=\"1\" colspan=\"1\">Give him bananas or honey</td><td rowspan=\"1\" colspan=\"1\">84</td><td rowspan=\"1\" colspan=\"1\">10.6%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Give him laxatives</td><td rowspan=\"1\" colspan=\"1\">189</td><td rowspan=\"1\" colspan=\"1\">23.7%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Giving him high-fiber food*</td><td rowspan=\"1\" colspan=\"1\">222</td><td rowspan=\"1\" colspan=\"1\">27.9%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Giving him plenty of fluids</td><td rowspan=\"1\" colspan=\"1\">187</td><td rowspan=\"1\" colspan=\"1\">23.5%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Massage</td><td rowspan=\"1\" colspan=\"1\">72</td><td rowspan=\"1\" colspan=\"1\">9.0%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Warm bath</td><td rowspan=\"1\" colspan=\"1\">42</td><td rowspan=\"1\" colspan=\"1\">5.3%</td></tr><tr><td rowspan=\"4\" colspan=\"1\">In the event of fecal impaction and blockage of the intestine, in your opinion, what is the most successful treatment?</td><td rowspan=\"1\" colspan=\"1\">Do an enema to expel stool*</td><td rowspan=\"1\" colspan=\"1\">341</td><td rowspan=\"1\" colspan=\"1\">42.8%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Give him laxatives</td><td rowspan=\"1\" colspan=\"1\">292</td><td rowspan=\"1\" colspan=\"1\">36.7%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">High-fiber foods</td><td rowspan=\"1\" colspan=\"1\">117</td><td rowspan=\"1\" colspan=\"1\">14.7%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Manual emptying of rectal contents by a doctor</td><td rowspan=\"1\" colspan=\"1\">46</td><td rowspan=\"1\" colspan=\"1\">5.8%</td></tr><tr><td rowspan=\"5\" colspan=\"1\">From the list below, what is the fiber-rich food that you recommend giving to a child with constipation (multi-select question)?</td><td rowspan=\"1\" colspan=\"1\">Rice</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">6.7%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Fruit like watermelon, apple, and banana*</td><td rowspan=\"1\" colspan=\"1\">571</td><td rowspan=\"1\" colspan=\"1\">71.7%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Bread</td><td rowspan=\"1\" colspan=\"1\">45</td><td rowspan=\"1\" colspan=\"1\">5.7%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Vegetable*</td><td rowspan=\"1\" colspan=\"1\">548</td><td rowspan=\"1\" colspan=\"1\">68.8%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Potatoes</td><td rowspan=\"1\" colspan=\"1\">55</td><td rowspan=\"1\" colspan=\"1\">6.9%</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"1\">Practice score - median (IQR)</td><td colspan=\"2\" rowspan=\"1\">2.0 (1.0-3.0)</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB4\"><label>Table 4</label><caption><title>Responses of the participants to attitude questions.</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"1\">Variables</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">%</td></tr><tr><td rowspan=\"3\" colspan=\"1\">What is your biggest fear from chronic childhood constipation (multi-select question)?</td><td rowspan=\"1\" colspan=\"1\">The fear of being from congenital abnormalities of the colon (stricture)</td><td rowspan=\"1\" colspan=\"1\">138</td><td rowspan=\"1\" colspan=\"1\">17.3%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">The fear of being from internal abdominal tumors</td><td rowspan=\"1\" colspan=\"1\">246</td><td rowspan=\"1\" colspan=\"1\">30.9%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">The fear of continuing to adulthood</td><td rowspan=\"1\" colspan=\"1\">518</td><td rowspan=\"1\" colspan=\"1\">65.1%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"7\" colspan=\"1\">What is your source of information about constipation?</td><td rowspan=\"1\" colspan=\"1\">Doctors and medical staff</td><td rowspan=\"1\" colspan=\"1\">116</td><td rowspan=\"1\" colspan=\"1\">14.6%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Frequent medical practice</td><td rowspan=\"1\" colspan=\"1\">110</td><td rowspan=\"1\" colspan=\"1\">13.8%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Friends and relatives</td><td rowspan=\"1\" colspan=\"1\">188</td><td rowspan=\"1\" colspan=\"1\">23.6%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Internet</td><td rowspan=\"1\" colspan=\"1\">203</td><td rowspan=\"1\" colspan=\"1\">25.5%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Magazines and books</td><td rowspan=\"1\" colspan=\"1\">40</td><td rowspan=\"1\" colspan=\"1\">5.0%</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Other</td><td rowspan=\"1\" colspan=\"1\">108</td><td rowspan=\"1\" colspan=\"1\">13.6%</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">TV</td><td rowspan=\"1\" colspan=\"1\">31</td><td rowspan=\"1\" colspan=\"1\">3.9%</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB5\"><label>Table 5</label><caption><title>Association of knowledge score with sociodemographic characteristics.</title><p><sup>*</sup>Independent samples Mann-Whitney U test.</p><p><sup>**</sup>Independent samples Kruskal-Wallis test.</p><p><sup>***</sup>P-value <0.05 was considered significant.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"2\">Variables</td><td colspan=\"2\" rowspan=\"1\">Knowledge score</td><td rowspan=\"2\" colspan=\"1\">p-Value<sup>*,**</sup>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Median</td><td rowspan=\"1\" colspan=\"1\">IQR</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Gender</td><td rowspan=\"1\" colspan=\"1\">Female</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">7.0-10.0</td><td rowspan=\"2\" colspan=\"1\"><0.001***</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Male</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">5.0-10.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Do you agree to participate in the survey?</td><td rowspan=\"1\" colspan=\"1\">Yes, I agree</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-10.0</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"4\" colspan=\"1\">Age</td><td rowspan=\"1\" colspan=\"1\">0-24 years</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">5.0-10.0</td><td rowspan=\"4\" colspan=\"1\">0.764</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">25-34 years</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-10.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">35-60 years</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-10.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">More than 60 years</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-10.0</td></tr><tr><td rowspan=\"6\" colspan=\"1\">Educational level</td><td rowspan=\"1\" colspan=\"1\">Middle</td><td rowspan=\"1\" colspan=\"1\">7.0</td><td rowspan=\"1\" colspan=\"1\">5.0-9.0</td><td rowspan=\"6\" colspan=\"1\">0.001***</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Nothing</td><td rowspan=\"1\" colspan=\"1\">7.5</td><td rowspan=\"1\" colspan=\"1\">5.0-10.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Postgraduate</td><td rowspan=\"1\" colspan=\"1\">9.0</td><td rowspan=\"1\" colspan=\"1\">7.0-11.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Primary</td><td rowspan=\"1\" colspan=\"1\">7.0</td><td rowspan=\"1\" colspan=\"1\">6.0-7.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Secondary</td><td rowspan=\"1\" colspan=\"1\">7.0</td><td rowspan=\"1\" colspan=\"1\">5.0-10.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">University education</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">7.0-10.0</td></tr><tr><td rowspan=\"5\" colspan=\"1\">Occupation</td><td rowspan=\"1\" colspan=\"1\">An employee in the private sector</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-10.0</td><td rowspan=\"5\" colspan=\"1\">0.562</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Self-employed</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">5.0-10.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Government employee</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-11.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Retired</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-10.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Unemployed</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-10.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Marital status</td><td rowspan=\"1\" colspan=\"1\">Divorced</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">5.5-10.0</td><td rowspan=\"2\" colspan=\"1\">0.470</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Married</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-10.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Have any of your children suffered from constipation before?</td><td rowspan=\"1\" colspan=\"1\">No</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-10.0</td><td rowspan=\"2\" colspan=\"1\">0.162</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">8.0</td><td rowspan=\"1\" colspan=\"1\">6.0-10.0</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB6\"><label>Table 6</label><caption><title>Association of practice score with sociodemographic characteristics.</title><p><sup>*</sup>Independent samples Mann-Whitney U test.</p><p><sup>**</sup>Independent samples Kruskal-Wallis test.</p><p><sup>***</sup>P-value <0.05 was considered significant.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"2\">Variables</td><td colspan=\"2\" rowspan=\"1\">Practice score</td><td rowspan=\"2\" colspan=\"1\">p-Value<sup>*,**</sup>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Median</td><td rowspan=\"1\" colspan=\"1\">IQR</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Gender</td><td rowspan=\"1\" colspan=\"1\">Female</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">2.0-3.0</td><td rowspan=\"2\" colspan=\"1\">0.320</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Male</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Do you agree to participate in the survey?</td><td rowspan=\"1\" colspan=\"1\">Yes, I agree</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"4\" colspan=\"1\">Age</td><td rowspan=\"1\" colspan=\"1\">15-24 years</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td><td rowspan=\"4\" colspan=\"1\">0.007***</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">25-34 years</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">35-60 years</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">2.0-3.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">More than 60 years</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">2.0-3.0</td></tr><tr><td rowspan=\"6\" colspan=\"1\">Educational level</td><td rowspan=\"1\" colspan=\"1\">Middle</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">2.0-3.0</td><td rowspan=\"6\" colspan=\"1\">0.191</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Nothing</td><td rowspan=\"1\" colspan=\"1\">1.0</td><td rowspan=\"1\" colspan=\"1\">0.0-2.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Postgraduate</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">2.0-3.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Primary</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">0.0-2.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Secondary</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">University education</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td></tr><tr><td rowspan=\"5\" colspan=\"1\">Occupation</td><td rowspan=\"1\" colspan=\"1\">An employee in the private sector</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td><td rowspan=\"5\" colspan=\"1\">0.261</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Self-employed</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Government employee</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">2.0-3.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Retired</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">2.0-3.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Unemployed</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Marital status</td><td rowspan=\"1\" colspan=\"1\">Divorced</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td><td rowspan=\"2\" colspan=\"1\">0.853</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Married</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Have any of your children suffered from constipation before?</td><td rowspan=\"1\" colspan=\"1\">No</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">1.0-3.0</td><td rowspan=\"2\" colspan=\"1\">0.288</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">2.0-3.0</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB7\"><label>Table 7</label><caption><title>Correlation of knowledge and practice scores of the participants.</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"3\" rowspan=\"1\">Variables</td><td rowspan=\"1\" colspan=\"1\">Practice score</td></tr><tr><td rowspan=\"3\" colspan=\"1\">Spearman's rho</td><td rowspan=\"3\" colspan=\"1\">Knowledge score</td><td rowspan=\"1\" colspan=\"1\">Correlation coefficient</td><td rowspan=\"1\" colspan=\"1\">0.328</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Sig. (two-tailed)</td><td rowspan=\"1\" colspan=\"1\">0.000</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Number of participants (n)</td><td rowspan=\"1\" colspan=\"1\">796</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB1\\\"><label>Table 1</label><caption><title>Sociodemographic characteristics of the participants.</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Characteristics</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">N</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">%</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Gender</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Female</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">591</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">74.2%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Male</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">205</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">25.8%</td></tr><tr><td rowspan=\\\"4\\\" colspan=\\\"1\\\">Age</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">15-24 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">53</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.7%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">25-34 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">175</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">22.0%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">35-60 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">500</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">62.8%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">More than 60 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">68</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.5%</td></tr><tr><td rowspan=\\\"6\\\" colspan=\\\"1\\\">Educational level</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Middle</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">21</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.6%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Nothing</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.3%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Postgraduate</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">96</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12.1%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Primary</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">9</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.1%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Secondary</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">131</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">16.5%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">University education</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">537</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">67.5%</td></tr><tr><td rowspan=\\\"5\\\" colspan=\\\"1\\\">Occupation</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">An employee in the private sector</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">85</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10.7%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Self-employed</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">45</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.7%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Government employee</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">273</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">34.3%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Retired</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">146</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">18.3%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Unemployed</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">247</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">31.0%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Marital status</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Divorced</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">60</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.5%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Married</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">736</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">92.5%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Have any of your children suffered from constipation before?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">No</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">154</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">19.3%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">642</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">80.7%</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB2\\\"><label>Table 2</label><caption><title>Responses of the participants to knowledge questions.</title><p>*Correct answer.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Characteristics</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">N</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">%</td></tr><tr><td rowspan=\\\"4\\\" colspan=\\\"1\\\">Among the following, which is closest to the definition of constipation?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Difficulty in excretion</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">398</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">50.0%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Lack of daily output</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">173</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">21.7%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Less than three bowel movements per week*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">88</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">11.1%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Metaphyseal ossification (stool)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">137</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">17.2%</td></tr><tr><td rowspan=\\\"3\\\" colspan=\\\"1\\\">In your opinion, is constipation a disease or a symptom?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">A symptom*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">506</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">63.6%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">I don't know</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">102</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12.8%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Illness</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">188</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">23.6%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"3\\\" colspan=\\\"1\\\">What is the common cause of constipation in children (multi-select question)?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Organic constipation*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">704</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">88.4%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Functional constipation*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">647</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">81.3%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">I don't know</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">101</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12.7%</td></tr><tr><td rowspan=\\\"5\\\" colspan=\\\"1\\\">What is the cause of functional constipation in children (multi-select question)?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Stool withholding*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">267</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">36.1%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Toilet training resistance*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">147</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">19.9%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Low-fibers diet*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">623</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">84.3%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Low physical activity*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">252</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">34.1%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">I don't know</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">78</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10.6%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"4\\\" colspan=\\\"1\\\">What are the most common symptoms of constipation (multi-select question)?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Abdominal pain*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">390</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">50.1%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Painful defecation*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">491</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">63.0%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Hard, dry stool*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">553</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">71.0%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Large diameter stool that may obstruct the toilet*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">150</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">19.3%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"3\\\" colspan=\\\"1\\\">Does a constipated child need complete tests at all times?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">I don't know</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">248</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">31.2%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">No*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">308</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">38.7%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">240</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">30.2%</td></tr><tr><td rowspan=\\\"5\\\" colspan=\\\"1\\\">What are the complications of constipation in children (multi-select question)?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Painful anal fissures (torn skin around the anus)*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">598</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">75.1%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Rectal prolapse (intestine that protrudes from the anus)*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">216</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">27.1%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Fecal impaction (stool that can't be expelled)*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">482</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">60.6%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Fecal incontinence (leaking of watery stool from the bottom)*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">66</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.3%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Intestinal perforation*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">58</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.3%</td></tr><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Total knowledge score - median (IQR)</td><td colspan=\\\"2\\\" rowspan=\\\"1\\\">8.0 (6.0-10.0)</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB3\\\"><label>Table 3</label><caption><title>Responses of the participants to practice questions.</title><p>*Correct practice.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Variables</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">N</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">%</td></tr><tr><td rowspan=\\\"6\\\" colspan=\\\"1\\\">What is the initial treatment that you do at home to treat your child’s constipation before taking him to the doctor?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Give him bananas or honey</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">84</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10.6%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Give him laxatives</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">189</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">23.7%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Giving him high-fiber food*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">222</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">27.9%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Giving him plenty of fluids</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">187</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">23.5%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Massage</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">72</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">9.0%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Warm bath</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">42</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.3%</td></tr><tr><td rowspan=\\\"4\\\" colspan=\\\"1\\\">In the event of fecal impaction and blockage of the intestine, in your opinion, what is the most successful treatment?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Do an enema to expel stool*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">341</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">42.8%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Give him laxatives</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">292</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">36.7%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">High-fiber foods</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">117</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14.7%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Manual emptying of rectal contents by a doctor</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">46</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.8%</td></tr><tr><td rowspan=\\\"5\\\" colspan=\\\"1\\\">From the list below, what is the fiber-rich food that you recommend giving to a child with constipation (multi-select question)?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Rice</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">53</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.7%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Fruit like watermelon, apple, and banana*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">571</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">71.7%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Bread</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">45</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.7%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Vegetable*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">548</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">68.8%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Potatoes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">55</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.9%</td></tr><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Practice score - median (IQR)</td><td colspan=\\\"2\\\" rowspan=\\\"1\\\">2.0 (1.0-3.0)</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB4\\\"><label>Table 4</label><caption><title>Responses of the participants to attitude questions.</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Variables</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">N</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">%</td></tr><tr><td rowspan=\\\"3\\\" colspan=\\\"1\\\">What is your biggest fear from chronic childhood constipation (multi-select question)?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The fear of being from congenital abnormalities of the colon (stricture)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">138</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">17.3%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The fear of being from internal abdominal tumors</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">246</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">30.9%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The fear of continuing to adulthood</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">518</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">65.1%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"7\\\" colspan=\\\"1\\\">What is your source of information about constipation?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Doctors and medical staff</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">116</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14.6%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Frequent medical practice</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">110</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">13.8%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Friends and relatives</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">188</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">23.6%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Internet</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">203</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">25.5%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Magazines and books</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">40</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.0%</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Other</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">108</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">13.6%</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">TV</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">31</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.9%</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB5\\\"><label>Table 5</label><caption><title>Association of knowledge score with sociodemographic characteristics.</title><p><sup>*</sup>Independent samples Mann-Whitney U test.</p><p><sup>**</sup>Independent samples Kruskal-Wallis test.</p><p><sup>***</sup>P-value <0.05 was considered significant.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"2\\\">Variables</td><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Knowledge score</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">p-Value<sup>*,**</sup>\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Median</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">IQR</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Gender</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Female</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.0-10.0</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\"><0.001***</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Male</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.0-10.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Do you agree to participate in the survey?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes, I agree</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-10.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\"> </td></tr><tr><td rowspan=\\\"4\\\" colspan=\\\"1\\\">Age</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0-24 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.0-10.0</td><td rowspan=\\\"4\\\" colspan=\\\"1\\\">0.764</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">25-34 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-10.0</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">35-60 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-10.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">More than 60 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-10.0</td></tr><tr><td rowspan=\\\"6\\\" colspan=\\\"1\\\">Educational level</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Middle</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.0-9.0</td><td rowspan=\\\"6\\\" colspan=\\\"1\\\">0.001***</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Nothing</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.0-10.0</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Postgraduate</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">9.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.0-11.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Primary</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-7.0</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Secondary</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.0-10.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">University education</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.0-10.0</td></tr><tr><td rowspan=\\\"5\\\" colspan=\\\"1\\\">Occupation</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">An employee in the private sector</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-10.0</td><td rowspan=\\\"5\\\" colspan=\\\"1\\\">0.562</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Self-employed</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.0-10.0</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Government employee</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-11.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Retired</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-10.0</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Unemployed</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-10.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Marital status</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Divorced</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.5-10.0</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.470</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Married</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-10.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Have any of your children suffered from constipation before?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">No</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-10.0</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.162</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.0-10.0</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB6\\\"><label>Table 6</label><caption><title>Association of practice score with sociodemographic characteristics.</title><p><sup>*</sup>Independent samples Mann-Whitney U test.</p><p><sup>**</sup>Independent samples Kruskal-Wallis test.</p><p><sup>***</sup>P-value <0.05 was considered significant.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"2\\\">Variables</td><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Practice score</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">p-Value<sup>*,**</sup>\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Median</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">IQR</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Gender</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Female</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0-3.0</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.320</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Male</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Do you agree to participate in the survey?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes, I agree</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\"> </td></tr><tr><td rowspan=\\\"4\\\" colspan=\\\"1\\\">Age</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">15-24 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td><td rowspan=\\\"4\\\" colspan=\\\"1\\\">0.007***</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">25-34 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">35-60 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0-3.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">More than 60 years</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0-3.0</td></tr><tr><td rowspan=\\\"6\\\" colspan=\\\"1\\\">Educational level</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Middle</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0-3.0</td><td rowspan=\\\"6\\\" colspan=\\\"1\\\">0.191</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Nothing</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.0-2.0</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Postgraduate</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0-3.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Primary</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.0-2.0</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Secondary</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">University education</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td></tr><tr><td rowspan=\\\"5\\\" colspan=\\\"1\\\">Occupation</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">An employee in the private sector</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td><td rowspan=\\\"5\\\" colspan=\\\"1\\\">0.261</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Self-employed</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Government employee</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0-3.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Retired</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0-3.0</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Unemployed</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Marital status</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Divorced</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.853</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Married</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Have any of your children suffered from constipation before?</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">No</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1.0-3.0</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.288</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Yes</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.0-3.0</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB7\\\"><label>Table 7</label><caption><title>Correlation of knowledge and practice scores of the participants.</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"3\\\" rowspan=\\\"1\\\">Variables</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Practice score</td></tr><tr><td rowspan=\\\"3\\\" colspan=\\\"1\\\">Spearman's rho</td><td rowspan=\\\"3\\\" colspan=\\\"1\\\">Knowledge score</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Correlation coefficient</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.328</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Sig. (two-tailed)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.000</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Number of participants (n)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">796</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Albraa J. Khayyat, Moath A. Khayat, Refal T. Abumansour, Nada O. Almalayo, Raghad E. Saleh, Doaa S. Baashar, Musaad M. Almhmadi , Rayan O. Almalki, Mohammed Ageel</p><p><bold>Drafting of the manuscript:</bold> Albraa J. Khayyat, Moath A. Khayat, Refal T. Abumansour, Nada O. Almalayo, Raghad E. Saleh, Doaa S. Baashar, Musaad M. Almhmadi , Rayan O. Almalki</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Moath A. Khayat, Refal T. Abumansour, Nada O. Almalayo, Mohammed Ageel</p><p><bold>Supervision:</bold> Mohammed Ageel</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study. Umm Al-Qura University's Biomedical Ethics Committee issued approval #HAPO-02-K-012-2023-04-1539</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Concept and design:</bold> Albraa J. Khayyat, Moath A. Khayat, Refal T. Abumansour, Nada O. Almalayo, Raghad E. Saleh, Doaa S. Baashar, Musaad M. Almhmadi , Rayan O. Almalki, Mohammed Ageel</p><p><bold>Drafting of the manuscript:</bold> Albraa J. Khayyat, Moath A. Khayat, Refal T. Abumansour, Nada O. Almalayo, Raghad E. Saleh, Doaa S. Baashar, Musaad M. Almhmadi , Rayan O. Almalki</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Moath A. Khayat, Refal T. Abumansour, Nada O. Almalayo, Mohammed Ageel</p><p><bold>Supervision:</bold> Mohammed Ageel</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study. Umm Al-Qura University's Biomedical Ethics Committee issued approval #HAPO-02-K-012-2023-04-1539</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Animal Ethics</title><fn fn-type=\\\"other\\\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0016-00000052236-i01\" position=\"float\"/>","<graphic xlink:href=\"cureus-0016-00000052236-i02\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0016-00000052236-i01\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0016-00000052236-i02\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"1\"], \"article-title\": [\"Functional constipation in children: a cross-sectional study\"], \"source\": [\"Int J Sci Res\"], \"person-group\": [\"\\n\"], \"surname\": [\"Singh\", \"Singh\"], \"given-names\": [\"RD\", \"SK\"], \"volume\": [\"7\"], \"year\": [\"2018\"], \"uri\": [\"https://www.worldwidejournals.com/international-journal-of-scientific-research-(IJSR)/article/functional-constipation-in-children-a-crossandndash-sectional-study/MTQ0NTE=/?is=1&b1=1005&k=252\"]}, {\"label\": [\"8\"], \"article-title\": [\"Functional constipation in children\"], \"source\": [\"J Pediatr (Rio J)\"], \"person-group\": [\"\\n\"], \"surname\": [\"Vandenplas\", \"Devreker\"], \"given-names\": [\"Y\", \"T\"], \"fpage\": [\"1\"], \"lpage\": [\"3\"], \"volume\": [\"95\"], \"year\": [\"2019\"]}, {\"label\": [\"9\"], \"article-title\": [\"Functional constipation in children\"], \"source\": [\"SA Pharm J\"], \"person-group\": [\"\\n\"], \"surname\": [\"Meyer\", \"Mashaba\", \"Makhele\", \"Sibanda\"], \"given-names\": [\"JC\", \"T\", \"L\", \"M\"], \"fpage\": [\"51\"], \"lpage\": [\"55\"], \"volume\": [\"84\"], \"year\": [\"2017\"], \"uri\": [\"https://hdl.handle.net/10520/EJC-b02de813f\"]}, {\"label\": [\"10\"], \"article-title\": [\"Parental characteristics and functional constipation in children: a cross-sectional cohort study\"], \"source\": [\"BMJ Paediatr Open\"], \"person-group\": [\"\\n\"], \"surname\": [\"Peeters\", \"Vriesman\", \"Koppen\", \"van Dijk\", \"Grootenhuis\", \"Di Lorenzo\", \"Benninga\"], \"given-names\": [\"B\", \"MH\", \"IJ\", \"M\", \"MA\", \"C\", \"MA\"], \"volume\": [\"1\"], \"year\": [\"2017\"]}, {\"label\": [\"12\"], \"article-title\": [\"Parents\\u2019 knowledge, attitude, and practice towards childhood constipation in Al Baha, Saudi Arabia\"], \"source\": [\"Med Sci\"], \"person-group\": [\"\\n\"], \"surname\": [\"Salih\", \"Alghamdi\", \"Muaibid\", \"Alghmadi\"], \"given-names\": [\"EM\", \"JM\", \"AF\", \"AA\"], \"volume\": [\"26\"], \"year\": [\"2022\"]}, {\"label\": [\"13\"], \"article-title\": [\"Parents\\u2019 knowledge and children\\u2019s toilet training practices: study in kindergartens in Jatinangor\"], \"source\": [\"Althea Med J\"], \"person-group\": [\"\\n\"], \"surname\": [\"Shafira\", \"Aziz\", \"Ermaya\", \"Sari\"], \"given-names\": [\"A\", \"PC\", \"YS\", \"NM\"], \"fpage\": [\"86\"], \"lpage\": [\"90\"], \"volume\": [\"6\"], \"year\": [\"2019\"]}, {\"label\": [\"15\"], \"article-title\": [\"Online health information seeking by parents for their children: systematic review and agenda for further research\"], \"source\": [\"J Med Internet Res\"], \"person-group\": [\"\\n\"], \"surname\": [\"Kubb\", \"Foran\"], \"given-names\": [\"C\", \"HM\"], \"volume\": [\"22\"], \"year\": [\"2020\"]}, {\"label\": [\"16\"], \"article-title\": [\"What is chronic constipation? Definition and diagnosis\"], \"source\": [\"Can J Gastroenterol\"], \"person-group\": [\"\\n\"], \"surname\": [\"Gray\"], \"given-names\": [\"JR\"], \"fpage\": [\"7\"], \"lpage\": [\"10\"], \"volume\": [\"25\"], \"year\": [\"2011\"], \"uri\": [\"https://pubmed.ncbi.nlm.nih.gov/22114751/\"]}, {\"label\": [\"17\"], \"article-title\": [\"Awareness of the general population toward constipation and its complications in the western region, Saudi Arabia\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Hemdi\", \"Alkarmo\", \"Alahmadi\", \"Almajnoni\", \"Alharbi\", \"Alfahmi\", \"Almaghrabi\"], \"given-names\": [\"M\", \"MY\", \"RA\", \"RS\", \"JK\", \"AM\", \"HA\"], \"volume\": [\"15\"], \"year\": [\"2023\"]}, {\"label\": [\"20\"], \"article-title\": [\"Therapeutic benefits and dietary restrictions of fiber intake: a state of the art review\"], \"source\": [\"Nutrients\"], \"person-group\": [\"\\n\"], \"surname\": [\"Ioni\\u021b\\u0103-M\\u00eendrican\", \"Ziani\", \"Mititelu\"], \"given-names\": [\"CB\", \"K\", \"M\"], \"volume\": [\"14\"], \"year\": [\"2022\"]}, {\"label\": [\"23\"], \"article-title\": [\"Health-related quality of life in young adults with symptoms of constipation continuing from childhood into adulthood\"], \"source\": [\"Health Qual Life Outcomes\"], \"person-group\": [\"\\n\"], \"surname\": [\"Bongers\", \"Benninga\", \"Maurice-Stam\", \"Grootenhuis\"], \"given-names\": [\"ME\", \"MA\", \"H\", \"MA\"], \"volume\": [\"7\"], \"year\": [\"2009\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":23,"string":"23"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Cureus.; 16(1):e52236"},"package_file":{"kind":"string","value":"oa_package/56/1d/PMC10787909.tar.gz"}}},{"rowIdx":192,"cells":{"accession_id":{"kind":"string","value":"PMC10787910"},"pmid":{"kind":"string","value":"38217744"},"introduction":{"kind":"list like","value":["<title>Background</title>","<p id=\"Par6\">Colorectal cancer (CRC) is more common among men than women, and as of 2020, CRC had a 5-year prevalence rate of 51.9 per 100,000 people in Asia and an incidence rate of 68 per 100,000 in Hong Kong [##REF##31085968##1##, ##UREF##0##2##]. Surgical methods are commonly applied to remove tumors from the colon or rectum, including low anterior resection (LAR), laparoscopy, abdominoperineal resection, and transanal microsurgery. However, fecal incontinence (FI), defined as the “involuntary loss of feces when feces are solid and/or liquid,” commonly develops following surgical management of CRC and chemoradiation [##REF##30681183##3##]. Following surgery, FI can develop due to damage to muscular, fascial, or neural tissues during surgery [##REF##33111180##4##]. Seventy to 90% of the patients who undergo sphincter preserving surgery experience FI in addition to other symptoms such as incontinence for flatus, increased intestinal gas, and rectal urgency (commonly referred to known as LAR syndrome) [##REF##32530135##5##]. The reported incidence of FI in patients treated with pelvic chemoradiation ranges between 3 and 53% [##UREF##1##6##, ##REF##23891095##7##]. FI has a significant negative impact on the quality of life (QoL) among CRC survivors [##REF##19410574##8##]. The inability to control the involuntary leakage of stool can cause embarrassment and fear of such episodes may hinder social participation or physical activity, adversely affecting mental health [##REF##10813117##9##]. Therefore, the post-surgical management of FI is necessary.</p>","<p id=\"Par7\">Treatments for FI include both pharmaceutical and non-pharmaceutical interventions. Non-pharmaceutical treatments may include diet adjustments, anal plugs, or physiotherapy [##REF##12790954##10##, ##UREF##2##11##]. Physiotherapy interventions for the treatment of FI include pelvic floor muscle training (PFMT) with or without biofeedback, neuromuscular electrical stimulation (NMES), and acupuncture [##UREF##3##12##]. A recent review by Kim and Oh [##REF##36867038##13##] evaluated the effectiveness of PFMT on bowel function and health-related QoL among patients who have undergone LAR and included studies published until 2019. Since then, further studies have been published on physiotherapy interventions in the management of FI, and therefore, the review requires updating. The objective of this review is to investigate the effectiveness of physiotherapy interventions on FI and the QoL following colorectal surgery.</p>"],"string":"[\n \"<title>Background</title>\",\n \"<p id=\\\"Par6\\\">Colorectal cancer (CRC) is more common among men than women, and as of 2020, CRC had a 5-year prevalence rate of 51.9 per 100,000 people in Asia and an incidence rate of 68 per 100,000 in Hong Kong [##REF##31085968##1##, ##UREF##0##2##]. Surgical methods are commonly applied to remove tumors from the colon or rectum, including low anterior resection (LAR), laparoscopy, abdominoperineal resection, and transanal microsurgery. However, fecal incontinence (FI), defined as the “involuntary loss of feces when feces are solid and/or liquid,” commonly develops following surgical management of CRC and chemoradiation [##REF##30681183##3##]. Following surgery, FI can develop due to damage to muscular, fascial, or neural tissues during surgery [##REF##33111180##4##]. Seventy to 90% of the patients who undergo sphincter preserving surgery experience FI in addition to other symptoms such as incontinence for flatus, increased intestinal gas, and rectal urgency (commonly referred to known as LAR syndrome) [##REF##32530135##5##]. The reported incidence of FI in patients treated with pelvic chemoradiation ranges between 3 and 53% [##UREF##1##6##, ##REF##23891095##7##]. FI has a significant negative impact on the quality of life (QoL) among CRC survivors [##REF##19410574##8##]. The inability to control the involuntary leakage of stool can cause embarrassment and fear of such episodes may hinder social participation or physical activity, adversely affecting mental health [##REF##10813117##9##]. Therefore, the post-surgical management of FI is necessary.</p>\",\n \"<p id=\\\"Par7\\\">Treatments for FI include both pharmaceutical and non-pharmaceutical interventions. Non-pharmaceutical treatments may include diet adjustments, anal plugs, or physiotherapy [##REF##12790954##10##, ##UREF##2##11##]. Physiotherapy interventions for the treatment of FI include pelvic floor muscle training (PFMT) with or without biofeedback, neuromuscular electrical stimulation (NMES), and acupuncture [##UREF##3##12##]. A recent review by Kim and Oh [##REF##36867038##13##] evaluated the effectiveness of PFMT on bowel function and health-related QoL among patients who have undergone LAR and included studies published until 2019. Since then, further studies have been published on physiotherapy interventions in the management of FI, and therefore, the review requires updating. The objective of this review is to investigate the effectiveness of physiotherapy interventions on FI and the QoL following colorectal surgery.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<p id=\"Par8\">This systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [##REF##19621070##14##] and was registered in the International Prospective Register of Systematic Reviews (PROSPERO, CRD42022337084).</p>","<title>Search strategy</title>","<p id=\"Par9\">Electronic databases of English-language articles, including the Allied and Complementary Medicine Database (AMED), The Cochrane Central Register of Controlled Trials (CENTRAL), the Cumulative Index of Nursing and Allied Health (CINAHL), Embase, MEDLINE, Ovid, the Physiotherapy Evidence Database (PEDro), Scopus, and Web of Science, and electronic databases of Chinese-language articles, including Chinese National Knowledge Infrastructure (CNKI) and Wanfang Data, were searched to identify potentially relevant articles. Searches were conducted from database inception to December 2021 and updated in November 2022. Searches were conducted by two independent review authors (P.C. and C.T.C). The specific search strategy applied to the Medline database is presented in Supplementary Appendix ##SUPPL##0##1##. Four themes, “physiotherapy intervention,” “colorectal cancer and surgery,” “bowel incontinence,” and “randomized controlled trials,” were utilized to identify potentially relevant articles. Related terms associated with each theme were combined using the Boolean operator “OR.” All four themes were combined using the Boolean operator “AND.” EndNote 20 citation management software was used to archive and organize the search results and remove duplicates. Manual searches were also performed by examining the reference lists for each included trial and relevant systematic reviews to identify additional candidate trials.</p>","<title>Study eligibility criteria</title>","<p id=\"Par10\">Trials were included if they (1) were randomized controlled trials (RCTs, including pilot, cluster, or crossover trials) comparing physiotherapy interventions (acupuncture, biofeedback, electrical stimulation, aerobic exercises, resistance exercises, stretching exercises, manual therapy, PFMT, or yoga) with a control condition (no treatment, usual care [UC], placebo, or active control) on FI and QoL outcomes following colorectal surgery; (2) measured FI using the Cleveland Clinic Incontinence Score (CCIS, also known as the Wexner score), the low anterior resection syndrome (LARS) score, or anorectal manometry (ARM) or measured QoL using the Fecal Incontinence Quality of Life (FIQL) scale, the EuroQoL 5-Dimension (EQ-5D) scale, the European Organization for Research and Treatment of Cancer (EORTC), Quality of Life Questionnaire (QLQ) module for colorectal cancer (QLQ-CR29), the EORTC QLQ module for cancer (QLQ-C30), or the 36-Item Short Form Survey (SF-36); and (3) were available in full-text format in either English or Chinese (traditional or simplified). Unpublished theses were also included in the review if they met the review criteria described above. Studies were excluded if they were systematic reviews, RCT/systematic review protocols, or case reports. Trials that included subjects with FI associated with causes other than colorectal surgery or radiotherapy for CRC (such as labor, prostate cancer, malabsorption, spinal surgery, or congenital disorders) were excluded. Trials reporting data as median and interquartile range, trials that included subjects with FI secondary to other medical conditions, such as neurological diseases, and trials that evaluated the effectiveness of non-physiotherapy treatments (such as anal plugs, Chinese medication, herb medication, injection, acupressure, moxibustion) were also excluded.</p>","<title>Article screening</title>","<p id=\"Par11\">Articles identified through the electronic searches underwent a three-stage screening process, including title, abstract, and full-text screening. Studies were screened for inclusion by two reviewers (P.M.Y. and L.P.H.). Disagreements between reviewers were resolved by discussion, and a third reviewer (P.K.) was consulted if the disagreement remained unresolved after discussion.</p>","<title>Data extraction</title>","<p id=\"Par12\">Data extraction was conducted independently by three authors (P.C., P.K., and C.K.H). The following data were extracted from each included study: last name of the first author, publication year, country of origin, sample size, mean age/age range of study subjects, intervention and control, outcome measures, and pre- and post-treatment data (mean and standard deviation) of ARM measured by anal resting pressure (ARP) and maximum squeeze pressure (MSP) for FI, and lifestyle, coping behavior, depression, and embarrassment components of QoL. For post-treatment data, only the data associated with the longest follow-up period was extracted.</p>","<title>Quality assessment</title>","<p id=\"Par13\">Five authors (P.C., P.K., C.K.H., P.M.Y., and L.P.H) independently assessed the methodological quality of the included trials using the Revised Cochrane Risk-of-Bias (RoB 2) tool for randomized trials [##REF##31462531##15##]. Disagreements between reviewers were resolved by discussion. An additional reviewer (PK) was consulted for any unresolved disagreements. The RoB 2 categorizes the overall RoB level as high, moderate, or low, based on the RoB identified in five domains: (1) randomization process, (2) deviation from intended interventions, (3) missing outcome data, (4) outcome measurements, and (5) the selection of the reported results [##REF##31462531##15##]. The overall RoB evaluation was guided by signaling questions in the five domains [##REF##31462531##15##].</p>","<title>Data synthesis and statistical analysis</title>","<p id=\"Par14\">All meta-analyses were performed using Comprehensive Meta-Analysis software (CMA version 3.3.070, Biostat Inc., Englewood, NJ, USA). Statistical heterogeneity was measured using the <italic>I</italic><sup>2</sup> test. A random-effect model was applied for high heterogeneity (<italic>I</italic><sup>2</sup> > 50%); otherwise, a fixed-effect model was utilized [##REF##26061376##16##]. Trials evaluating similar physiotherapy interventions, control conditions, and outcome measures were grouped together for meta-analysis. For continuous data, the weighted mean difference (WMD) and 95% confidence interval (CI) were calculated. WMD was chosen because similar units of measurement were used for outcome measures in the included trials [##UREF##4##17##]. In the meta-analyses and throughout the “<xref rid=\"Sec9\" ref-type=\"sec\">Results</xref>” section, all data for ARP and MSP reported by Chen [##UREF##5##18##] were converted from mmHg to kPa (by multiplying by 0.133) [##UREF##6##19##] to standardize the unit for the calculation of WMD. Significance was defined as a <italic>p</italic>-value ≤ 0.05.</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<p id=\\\"Par8\\\">This systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [##REF##19621070##14##] and was registered in the International Prospective Register of Systematic Reviews (PROSPERO, CRD42022337084).</p>\",\n \"<title>Search strategy</title>\",\n \"<p id=\\\"Par9\\\">Electronic databases of English-language articles, including the Allied and Complementary Medicine Database (AMED), The Cochrane Central Register of Controlled Trials (CENTRAL), the Cumulative Index of Nursing and Allied Health (CINAHL), Embase, MEDLINE, Ovid, the Physiotherapy Evidence Database (PEDro), Scopus, and Web of Science, and electronic databases of Chinese-language articles, including Chinese National Knowledge Infrastructure (CNKI) and Wanfang Data, were searched to identify potentially relevant articles. Searches were conducted from database inception to December 2021 and updated in November 2022. Searches were conducted by two independent review authors (P.C. and C.T.C). The specific search strategy applied to the Medline database is presented in Supplementary Appendix ##SUPPL##0##1##. Four themes, “physiotherapy intervention,” “colorectal cancer and surgery,” “bowel incontinence,” and “randomized controlled trials,” were utilized to identify potentially relevant articles. Related terms associated with each theme were combined using the Boolean operator “OR.” All four themes were combined using the Boolean operator “AND.” EndNote 20 citation management software was used to archive and organize the search results and remove duplicates. Manual searches were also performed by examining the reference lists for each included trial and relevant systematic reviews to identify additional candidate trials.</p>\",\n \"<title>Study eligibility criteria</title>\",\n \"<p id=\\\"Par10\\\">Trials were included if they (1) were randomized controlled trials (RCTs, including pilot, cluster, or crossover trials) comparing physiotherapy interventions (acupuncture, biofeedback, electrical stimulation, aerobic exercises, resistance exercises, stretching exercises, manual therapy, PFMT, or yoga) with a control condition (no treatment, usual care [UC], placebo, or active control) on FI and QoL outcomes following colorectal surgery; (2) measured FI using the Cleveland Clinic Incontinence Score (CCIS, also known as the Wexner score), the low anterior resection syndrome (LARS) score, or anorectal manometry (ARM) or measured QoL using the Fecal Incontinence Quality of Life (FIQL) scale, the EuroQoL 5-Dimension (EQ-5D) scale, the European Organization for Research and Treatment of Cancer (EORTC), Quality of Life Questionnaire (QLQ) module for colorectal cancer (QLQ-CR29), the EORTC QLQ module for cancer (QLQ-C30), or the 36-Item Short Form Survey (SF-36); and (3) were available in full-text format in either English or Chinese (traditional or simplified). Unpublished theses were also included in the review if they met the review criteria described above. Studies were excluded if they were systematic reviews, RCT/systematic review protocols, or case reports. Trials that included subjects with FI associated with causes other than colorectal surgery or radiotherapy for CRC (such as labor, prostate cancer, malabsorption, spinal surgery, or congenital disorders) were excluded. Trials reporting data as median and interquartile range, trials that included subjects with FI secondary to other medical conditions, such as neurological diseases, and trials that evaluated the effectiveness of non-physiotherapy treatments (such as anal plugs, Chinese medication, herb medication, injection, acupressure, moxibustion) were also excluded.</p>\",\n \"<title>Article screening</title>\",\n \"<p id=\\\"Par11\\\">Articles identified through the electronic searches underwent a three-stage screening process, including title, abstract, and full-text screening. Studies were screened for inclusion by two reviewers (P.M.Y. and L.P.H.). Disagreements between reviewers were resolved by discussion, and a third reviewer (P.K.) was consulted if the disagreement remained unresolved after discussion.</p>\",\n \"<title>Data extraction</title>\",\n \"<p id=\\\"Par12\\\">Data extraction was conducted independently by three authors (P.C., P.K., and C.K.H). The following data were extracted from each included study: last name of the first author, publication year, country of origin, sample size, mean age/age range of study subjects, intervention and control, outcome measures, and pre- and post-treatment data (mean and standard deviation) of ARM measured by anal resting pressure (ARP) and maximum squeeze pressure (MSP) for FI, and lifestyle, coping behavior, depression, and embarrassment components of QoL. For post-treatment data, only the data associated with the longest follow-up period was extracted.</p>\",\n \"<title>Quality assessment</title>\",\n \"<p id=\\\"Par13\\\">Five authors (P.C., P.K., C.K.H., P.M.Y., and L.P.H) independently assessed the methodological quality of the included trials using the Revised Cochrane Risk-of-Bias (RoB 2) tool for randomized trials [##REF##31462531##15##]. Disagreements between reviewers were resolved by discussion. An additional reviewer (PK) was consulted for any unresolved disagreements. The RoB 2 categorizes the overall RoB level as high, moderate, or low, based on the RoB identified in five domains: (1) randomization process, (2) deviation from intended interventions, (3) missing outcome data, (4) outcome measurements, and (5) the selection of the reported results [##REF##31462531##15##]. The overall RoB evaluation was guided by signaling questions in the five domains [##REF##31462531##15##].</p>\",\n \"<title>Data synthesis and statistical analysis</title>\",\n \"<p id=\\\"Par14\\\">All meta-analyses were performed using Comprehensive Meta-Analysis software (CMA version 3.3.070, Biostat Inc., Englewood, NJ, USA). Statistical heterogeneity was measured using the <italic>I</italic><sup>2</sup> test. A random-effect model was applied for high heterogeneity (<italic>I</italic><sup>2</sup> > 50%); otherwise, a fixed-effect model was utilized [##REF##26061376##16##]. Trials evaluating similar physiotherapy interventions, control conditions, and outcome measures were grouped together for meta-analysis. For continuous data, the weighted mean difference (WMD) and 95% confidence interval (CI) were calculated. WMD was chosen because similar units of measurement were used for outcome measures in the included trials [##UREF##4##17##]. In the meta-analyses and throughout the “<xref rid=\\\"Sec9\\\" ref-type=\\\"sec\\\">Results</xref>” section, all data for ARP and MSP reported by Chen [##UREF##5##18##] were converted from mmHg to kPa (by multiplying by 0.133) [##UREF##6##19##] to standardize the unit for the calculation of WMD. Significance was defined as a <italic>p</italic>-value ≤ 0.05.</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p id=\"Par15\">The study selection process, which followed the PRISMA approach, is summarized in Fig. ##FIG##0##1##. The studies that were excluded at the full-text screening stage and the reasons for exclusion are listed in Supplementary Appendix ##SUPPL##0##2##. A total of 4413 articles were identified via electronic and manual searches. After all screening stages were applied, only 10 trials met the inclusion criteria for the meta-analytic review.</p>","<title>Characteristics of the included trials</title>","<p id=\"Par16\">Table ##TAB##0##1## summarizes the characteristics of the included trials. Across all 10 trials, data were extracted for 608 subjects. The sample sizes of the included trials ranged from 12 to 100. The mean age of participants in the included trials ranged from 45.6 to 66.8 years. All subjects in the included studies had fecal incontinence following surgery for CRC. The interventions assessed in the included trials were PFMT (<italic>n</italic> = 2), biofeedback (<italic>n</italic> = 4), and biofeedback combined with PFMT (<italic>n</italic> = 4). Among the 10 included trials, three trials reported QoL outcomes, and eight trials reported FI outcomes.\n</p>","<title>Risk of bias in the included trials</title>","<p id=\"Par17\">Figure ##FIG##1##2##A shows the distributions of RoB levels across each domain, and Fig. ##FIG##1##2##B shows the overall RoB levels assessed for all included trials. Of the 10 included trials, three (30%) reported adequate randomization processes, one (10%) had few to no deviations from the intended intervention, six (60%) were free of missing outcome data, seven (70%) blinded the outcome assessors, and eight (80%) had reported all planned outcomes. Of the 10 included trials, four had moderate RoB, and six had high RoB.</p>","<title>Effects of interventions on QoL among individuals with FI after CRC surgery</title>","<title>PFMT versus UC</title>","<p id=\"Par18\">Two trials compared the effects of PFMT with those of UC on QoL [##REF##27461451##20##, ##UREF##7##21##]. The RoB for these two trials was high. Both trials [##REF##27461451##20##, ##UREF##7##21##] measured QoL using the FIQL and reported the lifestyle, coping behavior, depression, and embarrassment components. PFMT was performed daily in both trials, but the protocols varied. In Hung, Lin (20), participants performed four PMFT sessions per day, with each session consisting of 20 contractions. In Xia et al. [##UREF##7##21##], participants performed three PMFT sessions per day, with each session consisting of 20 contractions. The duration of PFMT ranged from 3 to 9 months. Meta-analysis of data from the two trials [##REF##27461451##20##, ##UREF##7##21##] (<italic>n</italic> = 112) revealed significant effects of the intervention compared with UC for the lifestyle (WMD 0.54; 95% CI 0.03, 1.05; <italic>p</italic> = 0.04; Fig. ##FIG##2##3##A), coping behavior (WMD 1.14; 95% CI 0.24, 2.04; <italic>p</italic> = 0.01; Fig. ##FIG##2##3##B), and embarrassment (WMD 0.417; 95% CI 0.14, 0.70; <italic>p</italic> = 0.00; Fig. ##FIG##2##3##C) components of the FIQL; however, no significant effect was observed for PFMT compared with UC on the depression component (WMD 0.424; 95% CI − 0.24, 1.09; <italic>p</italic> = 0.21; Fig. ##FIG##2##3##D).</p>","<title>Effect of interventions on FI</title>","<title>Biofeedback versus UC</title>","<p id=\"Par19\">Three trials compared the effects of biofeedback with those of UC on FI, measured by ARM [##UREF##8##22##–##UREF##10##24##]. The RoB for these three trials ranged from moderate to high. ARP and MSP were reported in all three trials, but only two trials reported RRP [##UREF##8##22##, ##UREF##10##24##]. In all three trials [##UREF##8##22##–##UREF##10##24##], electromyography (EMG) biofeedback therapy was implemented after colorectal surgery. One [##UREF##8##22##] of the three trials used an anal electrode inserted into the lower rectum, with adhesive electrodes placed on the external oblique muscles, forming a circuit to enable the detection of muscle activities during bowel movements. In this trial, daily biofeedback therapy, provided for 45–60 min per session, was performed for 15 days. Two trials [##UREF##9##23##, ##UREF##10##24##] provided insufficient descriptions of the method used to detect electrical activity during bowel movements or the treatment parameters. The meta-analysis of all three trials [##UREF##9##23##, ##UREF##10##24##] (<italic>n</italic> = 226) demonstrated a significant effect for biofeedback compared with UC for improving ARP (WMD 9.55; 95% CI 2.59, 16.51; <italic>p</italic> = 0.01; Fig. ##FIG##3##4##A) and MSP (WMD 25.29; 95% CI 4.08, 48.50; <italic>p</italic> = 0.02; Fig. ##FIG##3##4##B). The meta-analysis of the two trials reporting RRP [##UREF##8##22##, ##UREF##10##24##] (<italic>n</italic> = 152) found a significant effect for biofeedback compared with UC on RRP improvement (WMD 0.51; 95% CI 0.10, 0.92; <italic>p</italic> = 0.02; Fig. ##FIG##3##4##C).</p>","<title>PFMT plus biofeedback versus PFMT</title>","<p id=\"Par20\">The effects of PFMT alone compared with the effects of PFMT combined with biofeedback on anorectal dynamics were examined in three trials [##UREF##5##18##, ##UREF##11##25##, ##UREF##12##26##]. The RoB of these three trials varied from moderate to high. All three trials [##UREF##11##25##, ##UREF##12##26##] reported ARP and MSP as outcomes, but only two trials [##UREF##11##25##, ##UREF##12##26##] reported RRP. Chen (18) and Zheng, Wu (26) included EMG biofeedback therapy in addition to PFMT. The electrical activities of the pelvic floor muscles were measured by anal electrodes in all three trials. Zheng, Wu (26) also used adhesive electrodes to detect the electrical activity of external oblique muscles. Biofeedback therapy was performed two to three times per week for 20–30 min each time, with the total training period ranging from 3 to 13 months. In the trials by Chen (18) and Zheng, Wu (26), PFMT was performed daily, consisting of five sets of 10 repetitions consisting of contractions lasting 5–10 s per repetition, with 10 s of rest between repetitions. Subjects in the study by Yang, Wang (25) performed PFMT by contracting pelvic floor muscles for 10 s. The total PMFT training period varied from 16 months to the time subjects needed to be discharged [##UREF##5##18##, ##UREF##11##25##, ##UREF##12##26##]. The meta-analysis of the three trials [##UREF##5##18##, ##UREF##11##25##, ##UREF##12##26##] (<italic>n</italic> = 211) showed a significant effect for biofeedback combined with PFMT compared with PMFT alone on ARP (WMD 3.00; 95% CI 0.40, 5.59; <italic>p</italic> = 0.02, Fig. ##FIG##4##5##A) and MSP (WMD 9.35; 95% CI 0.17, 18.53; <italic>p</italic> = 0.05, Fig. ##FIG##4##5##B). The meta-analysis of the two trials reporting RRP [##UREF##11##25##, ##UREF##12##26##] (<italic>n</italic> = 135) revealed a significant effect for biofeedback combined with PMFT compared with PMFT alone on RRP (WMD 1.54; 95% CI 0.60, 2.48; <italic>p</italic> = 0.00, Fig. ##FIG##4##5##C).</p>","<title>Biofeedback versus PFMT</title>","<p id=\"Par21\">The effects of biofeedback therapy alone were compared with the effects of PFMT alone on FI using the CCIS in two trials [##REF##26361615##27##, ##REF##34768692##28##]. The RoB for the two trials varied from moderate to high. Both trials provided biofeedback therapy for the experimental group, whereas the control group performed Kegel exercises. Cho, Kim (28) provided the experimental group with biofeedback training designed to strengthen their external anal sphincter. Subjects were educated to slowly contract and relax the pelvic floor muscles and were presented with visual or audible signals proportional to their anal squeezing pressure. The training was provided one to two times per week for 6 months. Kim, Jeon (27) provided the experimental group with biofeedback training in which an anorectal probe was used to train subjects to achieve adequate squeeze pressure using a visual feedback display. Each training session lasted 10 to 30 min, and subjects were encouraged to repeat the exercises five times each day. The PFMT dosage used for the control groups was not specified in either trial [##REF##26361615##27##, ##REF##34768692##28##]. The meta-analysis of data from both trials [##REF##26361615##27##, ##REF##34768692##28##] (<italic>n</italic> = 168) showed a non-significant effect for biofeedback alone compared with PFMT alone on the CCIS (WMD 0.49; 95% CI − 1.68, 2.66; <italic>p</italic> = 0.66; Fig. ##FIG##5##6##).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p id=\\\"Par15\\\">The study selection process, which followed the PRISMA approach, is summarized in Fig. ##FIG##0##1##. The studies that were excluded at the full-text screening stage and the reasons for exclusion are listed in Supplementary Appendix ##SUPPL##0##2##. A total of 4413 articles were identified via electronic and manual searches. After all screening stages were applied, only 10 trials met the inclusion criteria for the meta-analytic review.</p>\",\n \"<title>Characteristics of the included trials</title>\",\n \"<p id=\\\"Par16\\\">Table ##TAB##0##1## summarizes the characteristics of the included trials. Across all 10 trials, data were extracted for 608 subjects. The sample sizes of the included trials ranged from 12 to 100. The mean age of participants in the included trials ranged from 45.6 to 66.8 years. All subjects in the included studies had fecal incontinence following surgery for CRC. The interventions assessed in the included trials were PFMT (<italic>n</italic> = 2), biofeedback (<italic>n</italic> = 4), and biofeedback combined with PFMT (<italic>n</italic> = 4). Among the 10 included trials, three trials reported QoL outcomes, and eight trials reported FI outcomes.\\n</p>\",\n \"<title>Risk of bias in the included trials</title>\",\n \"<p id=\\\"Par17\\\">Figure ##FIG##1##2##A shows the distributions of RoB levels across each domain, and Fig. ##FIG##1##2##B shows the overall RoB levels assessed for all included trials. Of the 10 included trials, three (30%) reported adequate randomization processes, one (10%) had few to no deviations from the intended intervention, six (60%) were free of missing outcome data, seven (70%) blinded the outcome assessors, and eight (80%) had reported all planned outcomes. Of the 10 included trials, four had moderate RoB, and six had high RoB.</p>\",\n \"<title>Effects of interventions on QoL among individuals with FI after CRC surgery</title>\",\n \"<title>PFMT versus UC</title>\",\n \"<p id=\\\"Par18\\\">Two trials compared the effects of PFMT with those of UC on QoL [##REF##27461451##20##, ##UREF##7##21##]. The RoB for these two trials was high. Both trials [##REF##27461451##20##, ##UREF##7##21##] measured QoL using the FIQL and reported the lifestyle, coping behavior, depression, and embarrassment components. PFMT was performed daily in both trials, but the protocols varied. In Hung, Lin (20), participants performed four PMFT sessions per day, with each session consisting of 20 contractions. In Xia et al. [##UREF##7##21##], participants performed three PMFT sessions per day, with each session consisting of 20 contractions. The duration of PFMT ranged from 3 to 9 months. Meta-analysis of data from the two trials [##REF##27461451##20##, ##UREF##7##21##] (<italic>n</italic> = 112) revealed significant effects of the intervention compared with UC for the lifestyle (WMD 0.54; 95% CI 0.03, 1.05; <italic>p</italic> = 0.04; Fig. ##FIG##2##3##A), coping behavior (WMD 1.14; 95% CI 0.24, 2.04; <italic>p</italic> = 0.01; Fig. ##FIG##2##3##B), and embarrassment (WMD 0.417; 95% CI 0.14, 0.70; <italic>p</italic> = 0.00; Fig. ##FIG##2##3##C) components of the FIQL; however, no significant effect was observed for PFMT compared with UC on the depression component (WMD 0.424; 95% CI − 0.24, 1.09; <italic>p</italic> = 0.21; Fig. ##FIG##2##3##D).</p>\",\n \"<title>Effect of interventions on FI</title>\",\n \"<title>Biofeedback versus UC</title>\",\n \"<p id=\\\"Par19\\\">Three trials compared the effects of biofeedback with those of UC on FI, measured by ARM [##UREF##8##22##–##UREF##10##24##]. The RoB for these three trials ranged from moderate to high. ARP and MSP were reported in all three trials, but only two trials reported RRP [##UREF##8##22##, ##UREF##10##24##]. In all three trials [##UREF##8##22##–##UREF##10##24##], electromyography (EMG) biofeedback therapy was implemented after colorectal surgery. One [##UREF##8##22##] of the three trials used an anal electrode inserted into the lower rectum, with adhesive electrodes placed on the external oblique muscles, forming a circuit to enable the detection of muscle activities during bowel movements. In this trial, daily biofeedback therapy, provided for 45–60 min per session, was performed for 15 days. Two trials [##UREF##9##23##, ##UREF##10##24##] provided insufficient descriptions of the method used to detect electrical activity during bowel movements or the treatment parameters. The meta-analysis of all three trials [##UREF##9##23##, ##UREF##10##24##] (<italic>n</italic> = 226) demonstrated a significant effect for biofeedback compared with UC for improving ARP (WMD 9.55; 95% CI 2.59, 16.51; <italic>p</italic> = 0.01; Fig. ##FIG##3##4##A) and MSP (WMD 25.29; 95% CI 4.08, 48.50; <italic>p</italic> = 0.02; Fig. ##FIG##3##4##B). The meta-analysis of the two trials reporting RRP [##UREF##8##22##, ##UREF##10##24##] (<italic>n</italic> = 152) found a significant effect for biofeedback compared with UC on RRP improvement (WMD 0.51; 95% CI 0.10, 0.92; <italic>p</italic> = 0.02; Fig. ##FIG##3##4##C).</p>\",\n \"<title>PFMT plus biofeedback versus PFMT</title>\",\n \"<p id=\\\"Par20\\\">The effects of PFMT alone compared with the effects of PFMT combined with biofeedback on anorectal dynamics were examined in three trials [##UREF##5##18##, ##UREF##11##25##, ##UREF##12##26##]. The RoB of these three trials varied from moderate to high. All three trials [##UREF##11##25##, ##UREF##12##26##] reported ARP and MSP as outcomes, but only two trials [##UREF##11##25##, ##UREF##12##26##] reported RRP. Chen (18) and Zheng, Wu (26) included EMG biofeedback therapy in addition to PFMT. The electrical activities of the pelvic floor muscles were measured by anal electrodes in all three trials. Zheng, Wu (26) also used adhesive electrodes to detect the electrical activity of external oblique muscles. Biofeedback therapy was performed two to three times per week for 20–30 min each time, with the total training period ranging from 3 to 13 months. In the trials by Chen (18) and Zheng, Wu (26), PFMT was performed daily, consisting of five sets of 10 repetitions consisting of contractions lasting 5–10 s per repetition, with 10 s of rest between repetitions. Subjects in the study by Yang, Wang (25) performed PFMT by contracting pelvic floor muscles for 10 s. The total PMFT training period varied from 16 months to the time subjects needed to be discharged [##UREF##5##18##, ##UREF##11##25##, ##UREF##12##26##]. The meta-analysis of the three trials [##UREF##5##18##, ##UREF##11##25##, ##UREF##12##26##] (<italic>n</italic> = 211) showed a significant effect for biofeedback combined with PFMT compared with PMFT alone on ARP (WMD 3.00; 95% CI 0.40, 5.59; <italic>p</italic> = 0.02, Fig. ##FIG##4##5##A) and MSP (WMD 9.35; 95% CI 0.17, 18.53; <italic>p</italic> = 0.05, Fig. ##FIG##4##5##B). The meta-analysis of the two trials reporting RRP [##UREF##11##25##, ##UREF##12##26##] (<italic>n</italic> = 135) revealed a significant effect for biofeedback combined with PMFT compared with PMFT alone on RRP (WMD 1.54; 95% CI 0.60, 2.48; <italic>p</italic> = 0.00, Fig. ##FIG##4##5##C).</p>\",\n \"<title>Biofeedback versus PFMT</title>\",\n \"<p id=\\\"Par21\\\">The effects of biofeedback therapy alone were compared with the effects of PFMT alone on FI using the CCIS in two trials [##REF##26361615##27##, ##REF##34768692##28##]. The RoB for the two trials varied from moderate to high. Both trials provided biofeedback therapy for the experimental group, whereas the control group performed Kegel exercises. Cho, Kim (28) provided the experimental group with biofeedback training designed to strengthen their external anal sphincter. Subjects were educated to slowly contract and relax the pelvic floor muscles and were presented with visual or audible signals proportional to their anal squeezing pressure. The training was provided one to two times per week for 6 months. Kim, Jeon (27) provided the experimental group with biofeedback training in which an anorectal probe was used to train subjects to achieve adequate squeeze pressure using a visual feedback display. Each training session lasted 10 to 30 min, and subjects were encouraged to repeat the exercises five times each day. The PFMT dosage used for the control groups was not specified in either trial [##REF##26361615##27##, ##REF##34768692##28##]. The meta-analysis of data from both trials [##REF##26361615##27##, ##REF##34768692##28##] (<italic>n</italic> = 168) showed a non-significant effect for biofeedback alone compared with PFMT alone on the CCIS (WMD 0.49; 95% CI − 1.68, 2.66; <italic>p</italic> = 0.66; Fig. ##FIG##5##6##).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par22\">This systematic review and meta-analysis investigated the effectiveness of physiotherapy interventions on FI and QoL following colorectal surgery. The literature searches identified 4413 potentially relevant articles indexed in both English- and Chinese-language databases; however, only 10 trials met the pre-defined inclusion criteria for the meta-analysis. The interventions examined in the included trials were PFMT alone, biofeedback therapy alone, and the combination of PFMT and biofeedback therapy. The RoB of the included trials ranged from moderate to high.</p>","<p id=\"Par23\">Meta-analysis of data from two trials [##REF##27461451##20##, ##UREF##7##21##] comparing PFMT alone with UC revealed a significant effect of the intervention on QoL components, including lifestyle, coping behavior, and embarrassment, as measured using the FIQL. The minimal clinically important difference (MCID) values reported for lifestyle, coping behavior, and embarrassment are 0.2, 0.3, and 0.2, respectively, among the noncancerous population [##REF##31021166##29##]. The mean estimated effects obtained in the studies included in the current review surpassed the MCID for lifestyle (0.54), coping behavior (1.14), and embarrassment (0.43), indicating that these effects might be clinically meaningful. Despite the significant results obtained for QoL in the current review, the findings are limited by the high RoB, the limited number of pooled trials (<italic>n</italic> = 2), and the varying PFMT protocols used by each included study. Nevertheless, considering the effect size and safety of PFMT [##REF##23076935##30##], this intervention should be considered a potential treatment option for improving QoL among individuals who experience FI following CRC surgery.</p>","<p id=\"Par24\">Meta-analysis of data from trials with moderate to high RoB comparing biofeedback alone with UC revealed significant effects of the intervention on the ARP, MSP [##UREF##8##22##–##UREF##10##24##], and RRP [##UREF##8##22##, ##UREF##10##24##] measures of ARM. Meta-analysis of data from trials [##UREF##5##18##, ##UREF##11##25##, ##UREF##12##26##] with moderate to high RoB identified similar significant effects on FI parameters when PFMT combined with biofeedback was compared with PFMT alone. ARM is a non-invasive procedure used to objectively quantify anorectal function and has been found to be clinically relevant for assessing the severity of FI in both children and adults [##REF##17445040##31##–##REF##31913322##33##]. No MCID has been established for ARM, preventing interpretation of the estimated effect size. However, the 95% CI values for both interventions (PFMT plus biofeedback and biofeedback alone) were below the MCID, indicating the potential for clinically trivial effects. Additional data examining the effects of these interventions would narrow the 95% CI and provide more precise estimates of the average effects of PFMT plus biofeedback and biofeedback alone for the treatment of FI following CRC surgery.</p>","<p id=\"Par25\">The results obtained in the current study for PFMT plus biofeedback agree with results reported in previous systematic reviews [##REF##24999460##34##, ##REF##23017030##35##] examining the effects of pelvic floor rehabilitation, including PFMT plus biofeedback, on improving anorectal function following rectal resection surgery. However, these prior systematic reviews [##REF##24999460##34##, ##REF##23017030##35##] did not include quantitative analyses, and both reviews included non-RCTs. By contrast, the current review quantitatively evaluated the efficacy of PFMT plus biofeedback and only included RCTs, offering a higher level of evidence [##REF##21701348##36##].</p>","<p id=\"Par26\">Although the current review found significant effects for biofeedback alone and PFMT plus biofeedback on FI as measured by ARM, these findings are limited by a considerably high RoB, large variations in the PFMT and biofeedback protocols described in the included trials, and the small number of trials included in the meta-analysis. However, considering the non-invasive nature of PFMT and the minimally invasive nature of biofeedback, both PFMT combined with biofeedback and biofeedback alone should be considered potential interventions for improving FI following CRC surgery. Future studies should investigate additional ARM other than ARP, MSP, and RRP, such as urge volume and volume of first sensation [##REF##26717931##37##], to obtain a more holistic understanding of the effects of these interventions on FI.</p>","<p id=\"Par27\">Meta-analysis of data from two trials [##REF##26361615##27##, ##REF##34768692##28##] comparing biofeedback alone with a PMFT alone revealed a non-significant effect of the intervention on FI following colorectal surgery. Based on these results, no recommendations can be made regarding the effectiveness of biofeedback alone compared with PFMT alone. Future studies of high methodological rigor are required to confirm the results obtained in this review for biofeedback alone compared with PFMT alone.</p>","<title>Strengths and limitations of the review</title>","<p id=\"Par28\">The current review has several strengths, including being the first review to include meta-analyses evaluating the effectiveness of various physiotherapy interventions on FI and QoL following CRC surgery. A comprehensive search strategy was applied to identify RCTs evaluating the effectiveness of various physiotherapy interventions for the treatment of FI following CRC surgery. Meta-analyses revealed significant effects for PFMT alone, biofeedback alone, and the combination of PFMT with biofeedback for improving QoL and FI following CRC surgery.</p>","<p id=\"Par29\">Our review also has some limitations. More than half of the included studies (6 of 10) were identified as having a high RoB. Other limitations are the inclusion of unpublished theses, which might hinder the study quality because unpublished studies may be of lower methodological quality than published studies [##UREF##13##38##], heterogeneity in terms of PFMT and biofeedback protocols utilized in the included trials, which minimizes the applicability of these findings to clinical settings; a high degree of statistical heterogeneity was evident across the pooled estimates as indicated by large <italic>I</italic><sup>2</sup> values, small sample sizes in some of the included trial;, and the small number of trials included in meta-analyses.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par22\\\">This systematic review and meta-analysis investigated the effectiveness of physiotherapy interventions on FI and QoL following colorectal surgery. The literature searches identified 4413 potentially relevant articles indexed in both English- and Chinese-language databases; however, only 10 trials met the pre-defined inclusion criteria for the meta-analysis. The interventions examined in the included trials were PFMT alone, biofeedback therapy alone, and the combination of PFMT and biofeedback therapy. The RoB of the included trials ranged from moderate to high.</p>\",\n \"<p id=\\\"Par23\\\">Meta-analysis of data from two trials [##REF##27461451##20##, ##UREF##7##21##] comparing PFMT alone with UC revealed a significant effect of the intervention on QoL components, including lifestyle, coping behavior, and embarrassment, as measured using the FIQL. The minimal clinically important difference (MCID) values reported for lifestyle, coping behavior, and embarrassment are 0.2, 0.3, and 0.2, respectively, among the noncancerous population [##REF##31021166##29##]. The mean estimated effects obtained in the studies included in the current review surpassed the MCID for lifestyle (0.54), coping behavior (1.14), and embarrassment (0.43), indicating that these effects might be clinically meaningful. Despite the significant results obtained for QoL in the current review, the findings are limited by the high RoB, the limited number of pooled trials (<italic>n</italic> = 2), and the varying PFMT protocols used by each included study. Nevertheless, considering the effect size and safety of PFMT [##REF##23076935##30##], this intervention should be considered a potential treatment option for improving QoL among individuals who experience FI following CRC surgery.</p>\",\n \"<p id=\\\"Par24\\\">Meta-analysis of data from trials with moderate to high RoB comparing biofeedback alone with UC revealed significant effects of the intervention on the ARP, MSP [##UREF##8##22##–##UREF##10##24##], and RRP [##UREF##8##22##, ##UREF##10##24##] measures of ARM. Meta-analysis of data from trials [##UREF##5##18##, ##UREF##11##25##, ##UREF##12##26##] with moderate to high RoB identified similar significant effects on FI parameters when PFMT combined with biofeedback was compared with PFMT alone. ARM is a non-invasive procedure used to objectively quantify anorectal function and has been found to be clinically relevant for assessing the severity of FI in both children and adults [##REF##17445040##31##–##REF##31913322##33##]. No MCID has been established for ARM, preventing interpretation of the estimated effect size. However, the 95% CI values for both interventions (PFMT plus biofeedback and biofeedback alone) were below the MCID, indicating the potential for clinically trivial effects. Additional data examining the effects of these interventions would narrow the 95% CI and provide more precise estimates of the average effects of PFMT plus biofeedback and biofeedback alone for the treatment of FI following CRC surgery.</p>\",\n \"<p id=\\\"Par25\\\">The results obtained in the current study for PFMT plus biofeedback agree with results reported in previous systematic reviews [##REF##24999460##34##, ##REF##23017030##35##] examining the effects of pelvic floor rehabilitation, including PFMT plus biofeedback, on improving anorectal function following rectal resection surgery. However, these prior systematic reviews [##REF##24999460##34##, ##REF##23017030##35##] did not include quantitative analyses, and both reviews included non-RCTs. By contrast, the current review quantitatively evaluated the efficacy of PFMT plus biofeedback and only included RCTs, offering a higher level of evidence [##REF##21701348##36##].</p>\",\n \"<p id=\\\"Par26\\\">Although the current review found significant effects for biofeedback alone and PFMT plus biofeedback on FI as measured by ARM, these findings are limited by a considerably high RoB, large variations in the PFMT and biofeedback protocols described in the included trials, and the small number of trials included in the meta-analysis. However, considering the non-invasive nature of PFMT and the minimally invasive nature of biofeedback, both PFMT combined with biofeedback and biofeedback alone should be considered potential interventions for improving FI following CRC surgery. Future studies should investigate additional ARM other than ARP, MSP, and RRP, such as urge volume and volume of first sensation [##REF##26717931##37##], to obtain a more holistic understanding of the effects of these interventions on FI.</p>\",\n \"<p id=\\\"Par27\\\">Meta-analysis of data from two trials [##REF##26361615##27##, ##REF##34768692##28##] comparing biofeedback alone with a PMFT alone revealed a non-significant effect of the intervention on FI following colorectal surgery. Based on these results, no recommendations can be made regarding the effectiveness of biofeedback alone compared with PFMT alone. Future studies of high methodological rigor are required to confirm the results obtained in this review for biofeedback alone compared with PFMT alone.</p>\",\n \"<title>Strengths and limitations of the review</title>\",\n \"<p id=\\\"Par28\\\">The current review has several strengths, including being the first review to include meta-analyses evaluating the effectiveness of various physiotherapy interventions on FI and QoL following CRC surgery. A comprehensive search strategy was applied to identify RCTs evaluating the effectiveness of various physiotherapy interventions for the treatment of FI following CRC surgery. Meta-analyses revealed significant effects for PFMT alone, biofeedback alone, and the combination of PFMT with biofeedback for improving QoL and FI following CRC surgery.</p>\",\n \"<p id=\\\"Par29\\\">Our review also has some limitations. More than half of the included studies (6 of 10) were identified as having a high RoB. Other limitations are the inclusion of unpublished theses, which might hinder the study quality because unpublished studies may be of lower methodological quality than published studies [##UREF##13##38##], heterogeneity in terms of PFMT and biofeedback protocols utilized in the included trials, which minimizes the applicability of these findings to clinical settings; a high degree of statistical heterogeneity was evident across the pooled estimates as indicated by large <italic>I</italic><sup>2</sup> values, small sample sizes in some of the included trial;, and the small number of trials included in meta-analyses.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusion</title>","<p id=\"Par30\">The current systematic review and meta-analysis identified significant improvements in the lifestyle, coping behavior, and embarrassment components of the FIQL among patients who received PFMT compared with those who received UC. Considering the non-invasive nature of PFMT and the sizes of the effects obtained for this intervention on different QoL components, PFMT should be considered an intervention that may improve QoL among individuals who experience FI following CRC surgery. Meta-analysis revealed that biofeedback alone was superior to UC and that PFMT plus biofeedback was superior to PFMT alone, with both superior interventions resulting in significant improvements in ARP, MSP, and RRP when assessed using ARM. Biofeedback is a minimally invasive intervention that can be applied alone or in combination with PFMT to treat FI following CRC surgery. However, the efficacy of biofeedback alone compared with PFMT alone remains inconclusive. Future high-quality RCTs remain necessary to standardize and optimize PFMT and biofeedback parameters for FI rehabilitation following CRC surgery and to confirm the results obtained in this review.</p>"],"string":"[\n \"<title>Conclusion</title>\",\n \"<p id=\\\"Par30\\\">The current systematic review and meta-analysis identified significant improvements in the lifestyle, coping behavior, and embarrassment components of the FIQL among patients who received PFMT compared with those who received UC. Considering the non-invasive nature of PFMT and the sizes of the effects obtained for this intervention on different QoL components, PFMT should be considered an intervention that may improve QoL among individuals who experience FI following CRC surgery. Meta-analysis revealed that biofeedback alone was superior to UC and that PFMT plus biofeedback was superior to PFMT alone, with both superior interventions resulting in significant improvements in ARP, MSP, and RRP when assessed using ARM. Biofeedback is a minimally invasive intervention that can be applied alone or in combination with PFMT to treat FI following CRC surgery. However, the efficacy of biofeedback alone compared with PFMT alone remains inconclusive. Future high-quality RCTs remain necessary to standardize and optimize PFMT and biofeedback parameters for FI rehabilitation following CRC surgery and to confirm the results obtained in this review.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose</title>","<p id=\"Par1\">To investigate the effectiveness of physiotherapy interventions compared to control conditions on fecal incontinence (FI) and quality of life (QoL) following colorectal surgery.</p>","<title>Methods</title>","<p id=\"Par2\">Electronic searches in English-language (Scopus, Web of Science, Embase, AMED, CENTRAL, CINAHL, MEDLINE, Ovid, and PEDro) and Chinese-language (CNKI, Wanfang Data) databases were conducted. Trials comparing physiotherapy interventions against control conditions and assessing FI and QoL outcomes were included in the review.</p>","<title>Results</title>","<p id=\"Par3\">Ten trials were included. Meta-analysis revealed statistically significant improvements in lifestyle (0.54; 95% CI 0.03, 1.05; <italic>p</italic> = 0.04), coping behavior (MD 1.136; 95% CI 0.24, 2.04; <italic>p</italic> = 0.01), and embarrassment (0.417; 95% CI 0.14, 0.70; <italic>p</italic> = 0.00) components of QoL among individuals receiving pelvic floor muscle training (PFMT) compared with those receiving usual care (UC). Meta-analysis showed biofeedback to be significantly more effective than UC in enhancing anal resting pressure (ARP; 9.551; 95% CI 2.60, 16.51; <italic>p</italic> = 0.007), maximum squeeze pressure (MSP; 25.29; 95% CI 4.08, 48.50; <italic>p</italic> = 0.02), and rectal resting pressure (RRP; 0.51; 95% CI 0.10, 0.9; <italic>p</italic> = 0.02). Meta-analysis also found PFMT combined with biofeedback to be significantly more effective than PFMT alone for ARP (3.00; 95% CI 0.40, 5.60; <italic>p</italic> = 0.02), MSP (9.35, 95% CI 0.17, 18.53; <italic>p</italic> = 0.05), and RRP (1.54; 95% CI 0.60, 2.47; <italic>p</italic> = 0.00).</p>","<title>Conclusions</title>","<p id=\"Par4\">PFMT combined with biofeedback was more effective than PFMT alone, but both interventions delivered alone were superior to UC. Future studies remain necessary to optimize and standardize the PFMT parameters for improving QoL among individuals who experience FI following CRC surgery.</p>","<title>Review registration</title>","<p id=\"Par5\">This systematic review is registered in the PROSPERO registry (Ref: CRD42022337084).</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s00520-023-08294-1.</p>","<title>Keywords</title>","<p>Open access funding provided by The Hong Kong Polytechnic University.</p>"],"string":"[\n \"<title>Purpose</title>\",\n \"<p id=\\\"Par1\\\">To investigate the effectiveness of physiotherapy interventions compared to control conditions on fecal incontinence (FI) and quality of life (QoL) following colorectal surgery.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">Electronic searches in English-language (Scopus, Web of Science, Embase, AMED, CENTRAL, CINAHL, MEDLINE, Ovid, and PEDro) and Chinese-language (CNKI, Wanfang Data) databases were conducted. Trials comparing physiotherapy interventions against control conditions and assessing FI and QoL outcomes were included in the review.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">Ten trials were included. Meta-analysis revealed statistically significant improvements in lifestyle (0.54; 95% CI 0.03, 1.05; <italic>p</italic> = 0.04), coping behavior (MD 1.136; 95% CI 0.24, 2.04; <italic>p</italic> = 0.01), and embarrassment (0.417; 95% CI 0.14, 0.70; <italic>p</italic> = 0.00) components of QoL among individuals receiving pelvic floor muscle training (PFMT) compared with those receiving usual care (UC). Meta-analysis showed biofeedback to be significantly more effective than UC in enhancing anal resting pressure (ARP; 9.551; 95% CI 2.60, 16.51; <italic>p</italic> = 0.007), maximum squeeze pressure (MSP; 25.29; 95% CI 4.08, 48.50; <italic>p</italic> = 0.02), and rectal resting pressure (RRP; 0.51; 95% CI 0.10, 0.9; <italic>p</italic> = 0.02). Meta-analysis also found PFMT combined with biofeedback to be significantly more effective than PFMT alone for ARP (3.00; 95% CI 0.40, 5.60; <italic>p</italic> = 0.02), MSP (9.35, 95% CI 0.17, 18.53; <italic>p</italic> = 0.05), and RRP (1.54; 95% CI 0.60, 2.47; <italic>p</italic> = 0.00).</p>\",\n \"<title>Conclusions</title>\",\n \"<p id=\\\"Par4\\\">PFMT combined with biofeedback was more effective than PFMT alone, but both interventions delivered alone were superior to UC. Future studies remain necessary to optimize and standardize the PFMT parameters for improving QoL among individuals who experience FI following CRC surgery.</p>\",\n \"<title>Review registration</title>\",\n \"<p id=\\\"Par5\\\">This systematic review is registered in the PROSPERO registry (Ref: CRD42022337084).</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s00520-023-08294-1.</p>\",\n \"<title>Keywords</title>\",\n \"<p>Open access funding provided by The Hong Kong Polytechnic University.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>The research team acknowledges Mr. Muhammad Usman Ali for his assistance and support.</p>","<title>Author contributions</title>","<p>P.K. contributed to the study’s conception and design.</p>","<p>P.C. and C.T.C performed electronic and hand searches.</p>","<p>P.M.Y. and L.P.H. completed the study screening.</p>","<p>P.C., P.K., and C.K.H performed data extraction. All authors conducted the risk of bias assessment and read and approved the final manuscript.</p>","<title>Funding</title>","<p>Open access funding provided by The Hong Kong Polytechnic University.</p>","<title>Declarations</title>","<title>Competing interests</title>","<p id=\"Par31\">The authors declare no competing interests.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>The research team acknowledges Mr. Muhammad Usman Ali for his assistance and support.</p>\",\n \"<title>Author contributions</title>\",\n \"<p>P.K. contributed to the study’s conception and design.</p>\",\n \"<p>P.C. and C.T.C performed electronic and hand searches.</p>\",\n \"<p>P.M.Y. and L.P.H. completed the study screening.</p>\",\n \"<p>P.C., P.K., and C.K.H performed data extraction. All authors conducted the risk of bias assessment and read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>Open access funding provided by The Hong Kong Polytechnic University.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par31\\\">The authors declare no competing interests.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Flow diagram of the study selection process applied for this review</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p><bold>A</bold> Assessments made by review authors for each risk-of-bias domain are presented as percentages across all included studies. <bold>B</bold> Risk-of-bias assessment for each included study (as assessed by the review authors)</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Effect of pelvic floor muscle training on quality-of-life measures assessed using Fecal Incontinence Quality of Life (FIQL) subscales</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Effect of biofeedback on fecal incontinence measures assessed by anorectal manometry. Abbreviations: ARP, anal resting pressure; MSP, maximum squeeze pressure; RRP, rectal resting pressure; CI, confidence interval</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Effect of pelvic floor muscle training plus biofeedback on fecal incontinence measures assessed by anorectal manometry. Abbreviations: ARP, anal resting pressure; MSP, maximal squeeze pressure; RRP, rectal resting pressure</p></caption></fig>","<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Effect of biofeedback on fecal incontinence measured by Cleveland Clinic Incontinence Score</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Flow diagram of the study selection process applied for this review</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p><bold>A</bold> Assessments made by review authors for each risk-of-bias domain are presented as percentages across all included studies. <bold>B</bold> Risk-of-bias assessment for each included study (as assessed by the review authors)</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Effect of pelvic floor muscle training on quality-of-life measures assessed using Fecal Incontinence Quality of Life (FIQL) subscales</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Effect of biofeedback on fecal incontinence measures assessed by anorectal manometry. Abbreviations: ARP, anal resting pressure; MSP, maximum squeeze pressure; RRP, rectal resting pressure; CI, confidence interval</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><p>Effect of pelvic floor muscle training plus biofeedback on fecal incontinence measures assessed by anorectal manometry. Abbreviations: ARP, anal resting pressure; MSP, maximal squeeze pressure; RRP, rectal resting pressure</p></caption></fig>\",\n \"<fig id=\\\"Fig6\\\"><label>Fig. 6</label><caption><p>Effect of biofeedback on fecal incontinence measured by Cleveland Clinic Incontinence Score</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Characteristics of included trials (<italic>n</italic> = 10)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">First author, year, country of study</th><th align=\"left\">Mean age of participants (SD); sample size of each group</th><th align=\"left\">Intervention</th><th align=\"left\">Control</th><th align=\"left\">Outcome measure(s)</th><th align=\"left\">Results (time points of assessment): mean (SD)</th></tr></thead><tbody><tr><td align=\"left\">Chai [##UREF##9##23##], 2018, China</td><td align=\"left\"><p>Exp: 62.9 (8.6)</p><p>Con: 62.7 (6.7)</p><p>Exp: <italic>n</italic> = 46</p><p>Con: <italic>n</italic> = 28</p></td><td align=\"left\"><p>BFT</p><p>- 3 months</p><p>Details NR</p></td><td align=\"left\">Usual care</td><td align=\"left\">FI: ARM (ARP, MSP)</td><td align=\"left\"><p><italic>ARM: ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 56.4 (8.7)</p><p>Con: 56.4 (8.7)</p><p><underline>Post (12 months)</underline></p><p>Exp: 47.6 (8.0)</p><p>Con: 28.7 (7.9)</p><p><italic>ARM: MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 234.2 (31.7)</p><p>Con: 244.2 (28.7)</p><p><underline>Post (12 months)</underline></p><p>Exp: 189.9 (36.4)</p><p>Con: 131.3 (36.1)</p></td></tr><tr><td align=\"left\">Chen [##UREF##5##18##], 2021, China</td><td align=\"left\"><p>Exp: 56.28 (8.07)</p><p>Con: 56.22 (8.14)</p><p>Exp: <italic>n</italic> = 38</p><p>Con: <italic>n</italic> = 38</p></td><td align=\"left\"><p>PFMT</p><p>- Contract pelvic floor muscle and hold for 5–10 s, rest for 10 s</p><p>- 10 reps/set × 5 sets/day</p><p>- Start on post-op 2nd week until discharge</p><p>EMG-BFT</p><p>- 20–30 min/session × 3 sessions/week</p><p>Start on post-op 2nd week until discharge</p></td><td align=\"left\"><p>PFMT</p><p>- Contract pelvic floor muscle and hold for 5–10 s, rest for 10 s</p><p>- 10 reps/set × 5 sets/day</p><p>Start on post-op 2nd week until discharge</p></td><td align=\"left\">FI: ARM (ARP, MSP)</td><td align=\"left\"><p><italic>ARM</italic><italic>: </italic><italic>ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 25.88 (15.83)</p><p>Con: 26.18 (15.75)</p><p><underline>Post (on discharge)</underline></p><p>Exp: 56.85 (18.45)</p><p>Con: 51.15 (17.78)</p><p><italic>ARM</italic><italic>: </italic><italic>MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 71.86 (29.40)</p><p>Con: 72.00 (29.33)</p><p><underline>Post (on discharge)</underline></p><p>Exp: 138.39 (33.53)</p><p>Con: 115.28 (34.65)</p></td></tr><tr><td align=\"left\">Cho [##REF##34768692##28##], 2021, Korea</td><td align=\"left\"><p>Exp: 61.7 (9.8)</p><p>Con: 64.5 (9.4)</p><p>Exp: <italic>n</italic> = 21</p><p>Con: <italic>n</italic> = 26</p></td><td align=\"left\"><p>BFT</p><p>- 1–2 times/week × 6 months</p><p>PFMT</p><p>Details NR</p></td><td align=\"left\"><p>PFMT</p><p>Details NR</p></td><td align=\"left\">FI: CCIS</td><td align=\"left\"><p><italic>CCIS</italic></p><p><underline>Pre</underline></p><p>NR</p><p><underline>Post (12 months)</underline></p><p>Exp: 10.05 (5.2)</p><p>Control: 10.17 (5.3)</p></td></tr><tr><td align=\"left\">Ng [##REF##27461451##20##], 2016, Taiwan</td><td align=\"left\"><p>All participants: 66.8 (12.5)*</p><p>Exp: <italic>n</italic> = 26</p><p>Con: <italic>n</italic> = 26</p></td><td align=\"left\"><p>PFMT</p><p>20 reps/session × 4 sessions/day × 9 months</p></td><td align=\"left\">Usual care</td><td align=\"left\">QoL: FIQL</td><td align=\"left\"><p><italic>FIQL: Lifestyle</italic></p><p><underline>Pre</underline></p><p>Exp: 3.29 (0.87)</p><p>Con: 2.99 (0.98)</p><p><underline>Post (6 months)</underline></p><p>Exp: 3.68 (0.55)</p><p>Con: 3.66 (0.68)</p><p><italic>FIQL: Coping behavior</italic></p><p><underline>Pre</underline></p><p>Exp: 3.41 (0.81)</p><p>Con: 2.85 (1.04)</p><p><underline>Post (6 months)</underline></p><p>Exp: 3.60 (0.68)</p><p>Con: 3.72 (0.64)</p><p><italic>FIQL: Depression</italic></p><p><underline>Pre</underline></p><p>Exp: 3.30 (0.50)</p><p>Con: 3.19 (0.61)</p><p><underline>Post (6 months)</underline></p><p>Exp: 3.43 (0.40)</p><p>Con: 3.44 (0.49)</p><p><italic>FIQL: Embarrassment</italic></p><p><underline>Pre</underline></p><p>Exp: 3.61 (0.71)</p><p>Con: 3.45 (0.71)</p><p><underline>Post (6 months)</underline></p><p>Exp: 3.73 (0.53)</p><p>Con: 3.85 (0.38)</p><p><italic>FIQL: Total</italic></p><p><underline>Pre</underline></p><p>Exp: 13.62 (2.50)</p><p>Con: 12.50 (2.93)</p><p><underline>Post (6 months)</underline></p><p>Exp: 14.44 (1.85)</p><p>Con: 12.50 (2.06)</p></td></tr><tr><td align=\"left\">Kim [##REF##26361615##27##], 2015, Korea</td><td align=\"left\"><p>Exp: 60.6 (6.0)</p><p>Con: 54.5 (10.1)</p><p>Exp: <italic>n</italic> = 6</p><p>Con: <italic>n</italic> = 6</p></td><td align=\"left\"><p>BFT</p><p>- Visual, auditory, and verbal biofeedback</p><p>- Subjects were instructed to breathe naturally without stopping during the pelvic muscle contraction exercise and to slowly contract and hold the muscle tightly, followed by a break</p><p>- Strength and the number of exercises gradually increased</p><p>- Twice per week before surgery</p><p>Lasted 4 weeks after surgery</p></td><td align=\"left\"><p>PFMT</p><p>Details NR</p></td><td align=\"left\"><p>FI: CCIS</p><p>QoL: FIQL</p></td><td align=\"left\"><p><italic>CCIS</italic></p><p><underline>Pre</underline></p><p>Exp: 9.0 (3.5)</p><p>Con: 10.7 (3.1)</p><p><underline>Post (12 months)</underline></p><p>Exp: 6.2 (3.1)</p><p>Con:7.0 (3.5)</p><p><italic>FIQL: Lifestyle</italic></p><p><underline>Pre</underline></p><p>Exp 22.2 (10.4)</p><p>Con: 20.5 (11.0)</p><p><underline>Post (12 months)</underline></p><p>Exp: 32.8 (4.7)</p><p>Con: 30.8 (8.1)</p><p><italic>FIQL: Coping behavior</italic></p><p><underline>Pre</underline></p><p>Exp 20.0 (7.3)</p><p>Con: 17.3 (8.0)</p><p><underline>Post (12 months)</underline></p><p>Exp: 26.5 (5.4)</p><p>Con: 26 (8)</p><p><italic>FIQL: depression</italic></p><p><underline>Pre</underline></p><p>Exp 17.8 (6.2)</p><p>Con: 14.3 (5.4)</p><p><underline>Post (12 months)</underline></p><p>Exp: 16.7 (4.2)</p><p>Con: 16.2 (3.2)</p><p><italic>FIQL: embarrassment</italic></p><p><underline>Pre</underline></p><p>Exp 8.5 (3.2)</p><p>Con: 8.3 (3.0)</p><p><underline>Post (12 months)</underline></p><p>Exp: 10.2 (1.7)</p><p>Con: 10.0 (2.1)</p></td></tr><tr><td align=\"left\">Xia [##UREF##7##21##], 2016, China</td><td align=\"left\"><p>Exp: 58.6 (11.4)</p><p>Con: 60.5 (12.1)</p><p>Exp: <italic>n</italic> = 30</p><p>Con: <italic>n</italic> = 30</p></td><td align=\"left\"><p>PFMT</p><p>- Hold > 10 s, rest > 10 s</p><p>20 reps/set × 3 sets/day × 12 weeks</p></td><td align=\"left\">Usual care</td><td align=\"left\">QoL: FIQL</td><td align=\"left\"><p><italic>FIQL: Lifestyle</italic></p><p><underline>Pre</underline></p><p>Exp: 2.4 (0.7)</p><p>Con: 2.6 (0.6)</p><p><underline>Post (3 months)</underline></p><p>Exp: 3.6 (1.1)</p><p>Con: 3.0 (0.9)</p><p><italic>FIQL: Coping behavior</italic></p><p><underline>Pre</underline></p><p>Exp: 2.8 (0.9)</p><p>Con: 2.9 (1.0)</p><p><underline>Post (3 months)</underline></p><p>Exp: 3.9 (1.0)</p><p>Con: 3.4 (0.9)</p><p><italic>FIQL: Depression</italic></p><p><underline>Pre</underline></p><p>Exp: 2.6 (1.0)</p><p>Con: 2.8 (0.8)</p><p><underline>Post (3 months)</underline></p><p>Exp: 3.9 (1.2)</p><p>Con: 3.3 (1.0)</p><p><italic>FIQL: Embarrassment</italic></p><p><underline>Pre</underline></p><p>Exp: 2.7 (0.8)</p><p>Con: 2.8 (0.9)</p><p><underline>Post (3 months)</underline></p><p>Exp: 3.9 (1.1)</p><p>Con: 3.3 (1.0)</p></td></tr><tr><td align=\"left\">Yang [##UREF##11##25##], 2020, China</td><td align=\"left\"><p>Exp: 61.31 (10.05)</p><p>Con: 61.26 (10.05)</p><p>Exp: <italic>n</italic> = 32</p><p>Con: <italic>n</italic> = 32</p></td><td align=\"left\"><p>EMG-BFT</p><p>- 20 min/day × 3 days/week × 3 months</p><p>PFMT</p><p>- Contract pelvic floor muscles in a comfortable position for 10 s repeatedly until post-op 16 weeks on rest days between sessions</p></td><td align=\"left\"><p>PFMT</p><p>- Contract pelvic floor muscles in a comfortable position for 10 s repeatedly until post-op 16 weeks on rest days between sessions</p></td><td align=\"left\">FI: ARM (ARP, MSP)</td><td align=\"left\"><p><italic>ARM</italic><italic>: </italic><italic>ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 45.32 (8.95)</p><p>Con: 45.26 (9.01)</p><p><underline>Post</underline></p><p>Exp: 44.21 (7.41)</p><p>Con: 40.26 (8.15)</p><p><italic>ARM</italic><italic>: </italic><italic>MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 131.89 (11.03)</p><p>Con: 132.56 (10.59)</p><p><underline>Post</underline></p><p>Exp: 129.64 (11.03)</p><p>Con: 120.69 (10.53)</p></td></tr><tr><td align=\"left\">You [##UREF##10##24##], 2018, China</td><td align=\"left\"><p>All subjects: 53.6 (3.7)*</p><p>Exp: <italic>n</italic> = 26</p><p>Con: <italic>n</italic> = 26</p></td><td align=\"left\"><p>BFT</p><p>Post-op 1st month until post-op 6th month</p></td><td align=\"left\">No treatment</td><td align=\"left\">FI: ARM (ARP, MSP, RRP)</td><td align=\"left\"><p><italic>ARM</italic><italic>: </italic><italic>ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 31.26 (6.15)</p><p>Con: 29.72 (5.61)</p><p><underline>Post (6 months)</underline></p><p>Exp: 42.07 (4.97)</p><p>Con: 35.49 (7.13)</p><p><italic>ARM</italic><italic>: </italic><italic>MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 116.34 (16.97)</p><p>Con: 117.42 (16.81)</p><p><underline>Post (6 months)</underline></p><p>Exp: 141.16 (5.28)</p><p>Con: 133.24 (4.89)</p><p><italic>ARM</italic><italic>: </italic><italic>RRP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 7.56 (1.39)</p><p>Con: 7.81 (1.93)</p><p><underline>Post (6 months)</underline></p><p>Exp: 6.07 (0.64)</p><p>Con: 7.21 (0.87)</p></td></tr><tr><td align=\"left\">Zhang [##UREF##8##22##], 2016, China</td><td align=\"left\"><p>All subjects: 45.6 (6.7)*</p><p>Exp: <italic>n</italic> = 50</p><p>Con: <italic>n</italic> = 50</p></td><td align=\"left\"><p>BFT</p><p>- Adhesive electrodes on the external oblique abdominis and anal electrode for the external anal sphincter</p><p>45–60 min/day × 1 month</p></td><td align=\"left\">Usual care</td><td align=\"left\">FI: ARM (ARP, MSP, RRP)</td><td align=\"left\"><p><italic>ARM: ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 33.52 (3.45)</p><p>Con: 32.67 (3.51)</p><p><underline>Post (9 months)</underline></p><p>Exp: 50.65 (5.61)</p><p>Con: 43.98 (4.36)</p><p><italic>ARM: MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 86.32 (8.84)</p><p>Con: 85.68 (4.31)</p><p><underline>Post (9 months)</underline></p><p>Exp: 110.8 (6.14)</p><p>Con: 102.14 (3.48)</p><p><italic>ARM: RRP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 2.66 (0.89)</p><p>Con: 2.61 (0.86)</p><p><underline>Post (9 months)</underline></p><p>Exp: 3.74 (1.52)</p><p>Con: 3.32 (1.26)</p></td></tr><tr><td align=\"left\">Zheng [##UREF##12##26##], 2019, China</td><td align=\"left\"><p>Exp: 54.34 (9.94)</p><p>Con: 52.50 (10.44)</p><p>Exp: <italic>n</italic> = 35</p><p>Con: <italic>n</italic> = 36</p></td><td align=\"left\"><p>PFMT</p><p>- Contract 5–10 s/rep × 10 reps/session × 5 sessions/day × 13 months</p><p>EMG-BFT</p><p>20 min/session × 3 sessions/week, × 16 weeks</p></td><td align=\"left\"><p>PFMT</p><p>- Contract 5–10 s/rep × 10 reps/session × 5 sessions/day × 13 months</p></td><td align=\"left\">FI: ARM (ARP, MSP, RRP)</td><td align=\"left\"><p><italic>ARM: ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 44.63 (8.71)</p><p>Con: 44.31 (6.69)</p><p><underline>Post (13 months)</underline></p><p>Exp: 44.83 (9.01)</p><p>Con: 42.92 (7.15)</p><p><italic>ARM: MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 131.66 (11.61)</p><p>Con: 131.08 (12.89)</p><p><underline>Post (13 months)</underline></p><p>Exp: 130.46 (10.00)</p><p>Con: 128.36 (9.91)</p><p><italic>ARM: RRP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 4.71 (2.24)</p><p>Con: 4.58 (2.26)</p><p><underline>Post (13 months)</underline></p><p>Exp: 4.31 (1.75)</p><p>Con: 5.72 (1.85)</p></td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Characteristics of included trials (<italic>n</italic> = 10)</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">First author, year, country of study</th><th align=\\\"left\\\">Mean age of participants (SD); sample size of each group</th><th align=\\\"left\\\">Intervention</th><th align=\\\"left\\\">Control</th><th align=\\\"left\\\">Outcome measure(s)</th><th align=\\\"left\\\">Results (time points of assessment): mean (SD)</th></tr></thead><tbody><tr><td align=\\\"left\\\">Chai [##UREF##9##23##], 2018, China</td><td align=\\\"left\\\"><p>Exp: 62.9 (8.6)</p><p>Con: 62.7 (6.7)</p><p>Exp: <italic>n</italic> = 46</p><p>Con: <italic>n</italic> = 28</p></td><td align=\\\"left\\\"><p>BFT</p><p>- 3 months</p><p>Details NR</p></td><td align=\\\"left\\\">Usual care</td><td align=\\\"left\\\">FI: ARM (ARP, MSP)</td><td align=\\\"left\\\"><p><italic>ARM: ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 56.4 (8.7)</p><p>Con: 56.4 (8.7)</p><p><underline>Post (12 months)</underline></p><p>Exp: 47.6 (8.0)</p><p>Con: 28.7 (7.9)</p><p><italic>ARM: MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 234.2 (31.7)</p><p>Con: 244.2 (28.7)</p><p><underline>Post (12 months)</underline></p><p>Exp: 189.9 (36.4)</p><p>Con: 131.3 (36.1)</p></td></tr><tr><td align=\\\"left\\\">Chen [##UREF##5##18##], 2021, China</td><td align=\\\"left\\\"><p>Exp: 56.28 (8.07)</p><p>Con: 56.22 (8.14)</p><p>Exp: <italic>n</italic> = 38</p><p>Con: <italic>n</italic> = 38</p></td><td align=\\\"left\\\"><p>PFMT</p><p>- Contract pelvic floor muscle and hold for 5–10 s, rest for 10 s</p><p>- 10 reps/set × 5 sets/day</p><p>- Start on post-op 2nd week until discharge</p><p>EMG-BFT</p><p>- 20–30 min/session × 3 sessions/week</p><p>Start on post-op 2nd week until discharge</p></td><td align=\\\"left\\\"><p>PFMT</p><p>- Contract pelvic floor muscle and hold for 5–10 s, rest for 10 s</p><p>- 10 reps/set × 5 sets/day</p><p>Start on post-op 2nd week until discharge</p></td><td align=\\\"left\\\">FI: ARM (ARP, MSP)</td><td align=\\\"left\\\"><p><italic>ARM</italic><italic>: </italic><italic>ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 25.88 (15.83)</p><p>Con: 26.18 (15.75)</p><p><underline>Post (on discharge)</underline></p><p>Exp: 56.85 (18.45)</p><p>Con: 51.15 (17.78)</p><p><italic>ARM</italic><italic>: </italic><italic>MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 71.86 (29.40)</p><p>Con: 72.00 (29.33)</p><p><underline>Post (on discharge)</underline></p><p>Exp: 138.39 (33.53)</p><p>Con: 115.28 (34.65)</p></td></tr><tr><td align=\\\"left\\\">Cho [##REF##34768692##28##], 2021, Korea</td><td align=\\\"left\\\"><p>Exp: 61.7 (9.8)</p><p>Con: 64.5 (9.4)</p><p>Exp: <italic>n</italic> = 21</p><p>Con: <italic>n</italic> = 26</p></td><td align=\\\"left\\\"><p>BFT</p><p>- 1–2 times/week × 6 months</p><p>PFMT</p><p>Details NR</p></td><td align=\\\"left\\\"><p>PFMT</p><p>Details NR</p></td><td align=\\\"left\\\">FI: CCIS</td><td align=\\\"left\\\"><p><italic>CCIS</italic></p><p><underline>Pre</underline></p><p>NR</p><p><underline>Post (12 months)</underline></p><p>Exp: 10.05 (5.2)</p><p>Control: 10.17 (5.3)</p></td></tr><tr><td align=\\\"left\\\">Ng [##REF##27461451##20##], 2016, Taiwan</td><td align=\\\"left\\\"><p>All participants: 66.8 (12.5)*</p><p>Exp: <italic>n</italic> = 26</p><p>Con: <italic>n</italic> = 26</p></td><td align=\\\"left\\\"><p>PFMT</p><p>20 reps/session × 4 sessions/day × 9 months</p></td><td align=\\\"left\\\">Usual care</td><td align=\\\"left\\\">QoL: FIQL</td><td align=\\\"left\\\"><p><italic>FIQL: Lifestyle</italic></p><p><underline>Pre</underline></p><p>Exp: 3.29 (0.87)</p><p>Con: 2.99 (0.98)</p><p><underline>Post (6 months)</underline></p><p>Exp: 3.68 (0.55)</p><p>Con: 3.66 (0.68)</p><p><italic>FIQL: Coping behavior</italic></p><p><underline>Pre</underline></p><p>Exp: 3.41 (0.81)</p><p>Con: 2.85 (1.04)</p><p><underline>Post (6 months)</underline></p><p>Exp: 3.60 (0.68)</p><p>Con: 3.72 (0.64)</p><p><italic>FIQL: Depression</italic></p><p><underline>Pre</underline></p><p>Exp: 3.30 (0.50)</p><p>Con: 3.19 (0.61)</p><p><underline>Post (6 months)</underline></p><p>Exp: 3.43 (0.40)</p><p>Con: 3.44 (0.49)</p><p><italic>FIQL: Embarrassment</italic></p><p><underline>Pre</underline></p><p>Exp: 3.61 (0.71)</p><p>Con: 3.45 (0.71)</p><p><underline>Post (6 months)</underline></p><p>Exp: 3.73 (0.53)</p><p>Con: 3.85 (0.38)</p><p><italic>FIQL: Total</italic></p><p><underline>Pre</underline></p><p>Exp: 13.62 (2.50)</p><p>Con: 12.50 (2.93)</p><p><underline>Post (6 months)</underline></p><p>Exp: 14.44 (1.85)</p><p>Con: 12.50 (2.06)</p></td></tr><tr><td align=\\\"left\\\">Kim [##REF##26361615##27##], 2015, Korea</td><td align=\\\"left\\\"><p>Exp: 60.6 (6.0)</p><p>Con: 54.5 (10.1)</p><p>Exp: <italic>n</italic> = 6</p><p>Con: <italic>n</italic> = 6</p></td><td align=\\\"left\\\"><p>BFT</p><p>- Visual, auditory, and verbal biofeedback</p><p>- Subjects were instructed to breathe naturally without stopping during the pelvic muscle contraction exercise and to slowly contract and hold the muscle tightly, followed by a break</p><p>- Strength and the number of exercises gradually increased</p><p>- Twice per week before surgery</p><p>Lasted 4 weeks after surgery</p></td><td align=\\\"left\\\"><p>PFMT</p><p>Details NR</p></td><td align=\\\"left\\\"><p>FI: CCIS</p><p>QoL: FIQL</p></td><td align=\\\"left\\\"><p><italic>CCIS</italic></p><p><underline>Pre</underline></p><p>Exp: 9.0 (3.5)</p><p>Con: 10.7 (3.1)</p><p><underline>Post (12 months)</underline></p><p>Exp: 6.2 (3.1)</p><p>Con:7.0 (3.5)</p><p><italic>FIQL: Lifestyle</italic></p><p><underline>Pre</underline></p><p>Exp 22.2 (10.4)</p><p>Con: 20.5 (11.0)</p><p><underline>Post (12 months)</underline></p><p>Exp: 32.8 (4.7)</p><p>Con: 30.8 (8.1)</p><p><italic>FIQL: Coping behavior</italic></p><p><underline>Pre</underline></p><p>Exp 20.0 (7.3)</p><p>Con: 17.3 (8.0)</p><p><underline>Post (12 months)</underline></p><p>Exp: 26.5 (5.4)</p><p>Con: 26 (8)</p><p><italic>FIQL: depression</italic></p><p><underline>Pre</underline></p><p>Exp 17.8 (6.2)</p><p>Con: 14.3 (5.4)</p><p><underline>Post (12 months)</underline></p><p>Exp: 16.7 (4.2)</p><p>Con: 16.2 (3.2)</p><p><italic>FIQL: embarrassment</italic></p><p><underline>Pre</underline></p><p>Exp 8.5 (3.2)</p><p>Con: 8.3 (3.0)</p><p><underline>Post (12 months)</underline></p><p>Exp: 10.2 (1.7)</p><p>Con: 10.0 (2.1)</p></td></tr><tr><td align=\\\"left\\\">Xia [##UREF##7##21##], 2016, China</td><td align=\\\"left\\\"><p>Exp: 58.6 (11.4)</p><p>Con: 60.5 (12.1)</p><p>Exp: <italic>n</italic> = 30</p><p>Con: <italic>n</italic> = 30</p></td><td align=\\\"left\\\"><p>PFMT</p><p>- Hold > 10 s, rest > 10 s</p><p>20 reps/set × 3 sets/day × 12 weeks</p></td><td align=\\\"left\\\">Usual care</td><td align=\\\"left\\\">QoL: FIQL</td><td align=\\\"left\\\"><p><italic>FIQL: Lifestyle</italic></p><p><underline>Pre</underline></p><p>Exp: 2.4 (0.7)</p><p>Con: 2.6 (0.6)</p><p><underline>Post (3 months)</underline></p><p>Exp: 3.6 (1.1)</p><p>Con: 3.0 (0.9)</p><p><italic>FIQL: Coping behavior</italic></p><p><underline>Pre</underline></p><p>Exp: 2.8 (0.9)</p><p>Con: 2.9 (1.0)</p><p><underline>Post (3 months)</underline></p><p>Exp: 3.9 (1.0)</p><p>Con: 3.4 (0.9)</p><p><italic>FIQL: Depression</italic></p><p><underline>Pre</underline></p><p>Exp: 2.6 (1.0)</p><p>Con: 2.8 (0.8)</p><p><underline>Post (3 months)</underline></p><p>Exp: 3.9 (1.2)</p><p>Con: 3.3 (1.0)</p><p><italic>FIQL: Embarrassment</italic></p><p><underline>Pre</underline></p><p>Exp: 2.7 (0.8)</p><p>Con: 2.8 (0.9)</p><p><underline>Post (3 months)</underline></p><p>Exp: 3.9 (1.1)</p><p>Con: 3.3 (1.0)</p></td></tr><tr><td align=\\\"left\\\">Yang [##UREF##11##25##], 2020, China</td><td align=\\\"left\\\"><p>Exp: 61.31 (10.05)</p><p>Con: 61.26 (10.05)</p><p>Exp: <italic>n</italic> = 32</p><p>Con: <italic>n</italic> = 32</p></td><td align=\\\"left\\\"><p>EMG-BFT</p><p>- 20 min/day × 3 days/week × 3 months</p><p>PFMT</p><p>- Contract pelvic floor muscles in a comfortable position for 10 s repeatedly until post-op 16 weeks on rest days between sessions</p></td><td align=\\\"left\\\"><p>PFMT</p><p>- Contract pelvic floor muscles in a comfortable position for 10 s repeatedly until post-op 16 weeks on rest days between sessions</p></td><td align=\\\"left\\\">FI: ARM (ARP, MSP)</td><td align=\\\"left\\\"><p><italic>ARM</italic><italic>: </italic><italic>ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 45.32 (8.95)</p><p>Con: 45.26 (9.01)</p><p><underline>Post</underline></p><p>Exp: 44.21 (7.41)</p><p>Con: 40.26 (8.15)</p><p><italic>ARM</italic><italic>: </italic><italic>MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 131.89 (11.03)</p><p>Con: 132.56 (10.59)</p><p><underline>Post</underline></p><p>Exp: 129.64 (11.03)</p><p>Con: 120.69 (10.53)</p></td></tr><tr><td align=\\\"left\\\">You [##UREF##10##24##], 2018, China</td><td align=\\\"left\\\"><p>All subjects: 53.6 (3.7)*</p><p>Exp: <italic>n</italic> = 26</p><p>Con: <italic>n</italic> = 26</p></td><td align=\\\"left\\\"><p>BFT</p><p>Post-op 1st month until post-op 6th month</p></td><td align=\\\"left\\\">No treatment</td><td align=\\\"left\\\">FI: ARM (ARP, MSP, RRP)</td><td align=\\\"left\\\"><p><italic>ARM</italic><italic>: </italic><italic>ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 31.26 (6.15)</p><p>Con: 29.72 (5.61)</p><p><underline>Post (6 months)</underline></p><p>Exp: 42.07 (4.97)</p><p>Con: 35.49 (7.13)</p><p><italic>ARM</italic><italic>: </italic><italic>MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 116.34 (16.97)</p><p>Con: 117.42 (16.81)</p><p><underline>Post (6 months)</underline></p><p>Exp: 141.16 (5.28)</p><p>Con: 133.24 (4.89)</p><p><italic>ARM</italic><italic>: </italic><italic>RRP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 7.56 (1.39)</p><p>Con: 7.81 (1.93)</p><p><underline>Post (6 months)</underline></p><p>Exp: 6.07 (0.64)</p><p>Con: 7.21 (0.87)</p></td></tr><tr><td align=\\\"left\\\">Zhang [##UREF##8##22##], 2016, China</td><td align=\\\"left\\\"><p>All subjects: 45.6 (6.7)*</p><p>Exp: <italic>n</italic> = 50</p><p>Con: <italic>n</italic> = 50</p></td><td align=\\\"left\\\"><p>BFT</p><p>- Adhesive electrodes on the external oblique abdominis and anal electrode for the external anal sphincter</p><p>45–60 min/day × 1 month</p></td><td align=\\\"left\\\">Usual care</td><td align=\\\"left\\\">FI: ARM (ARP, MSP, RRP)</td><td align=\\\"left\\\"><p><italic>ARM: ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 33.52 (3.45)</p><p>Con: 32.67 (3.51)</p><p><underline>Post (9 months)</underline></p><p>Exp: 50.65 (5.61)</p><p>Con: 43.98 (4.36)</p><p><italic>ARM: MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 86.32 (8.84)</p><p>Con: 85.68 (4.31)</p><p><underline>Post (9 months)</underline></p><p>Exp: 110.8 (6.14)</p><p>Con: 102.14 (3.48)</p><p><italic>ARM: RRP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 2.66 (0.89)</p><p>Con: 2.61 (0.86)</p><p><underline>Post (9 months)</underline></p><p>Exp: 3.74 (1.52)</p><p>Con: 3.32 (1.26)</p></td></tr><tr><td align=\\\"left\\\">Zheng [##UREF##12##26##], 2019, China</td><td align=\\\"left\\\"><p>Exp: 54.34 (9.94)</p><p>Con: 52.50 (10.44)</p><p>Exp: <italic>n</italic> = 35</p><p>Con: <italic>n</italic> = 36</p></td><td align=\\\"left\\\"><p>PFMT</p><p>- Contract 5–10 s/rep × 10 reps/session × 5 sessions/day × 13 months</p><p>EMG-BFT</p><p>20 min/session × 3 sessions/week, × 16 weeks</p></td><td align=\\\"left\\\"><p>PFMT</p><p>- Contract 5–10 s/rep × 10 reps/session × 5 sessions/day × 13 months</p></td><td align=\\\"left\\\">FI: ARM (ARP, MSP, RRP)</td><td align=\\\"left\\\"><p><italic>ARM: ARP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 44.63 (8.71)</p><p>Con: 44.31 (6.69)</p><p><underline>Post (13 months)</underline></p><p>Exp: 44.83 (9.01)</p><p>Con: 42.92 (7.15)</p><p><italic>ARM: MSP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 131.66 (11.61)</p><p>Con: 131.08 (12.89)</p><p><underline>Post (13 months)</underline></p><p>Exp: 130.46 (10.00)</p><p>Con: 128.36 (9.91)</p><p><italic>ARM: RRP (mmHg)</italic></p><p><underline>Pre</underline></p><p>Exp: 4.71 (2.24)</p><p>Con: 4.58 (2.26)</p><p><underline>Post (13 months)</underline></p><p>Exp: 4.31 (1.75)</p><p>Con: 5.72 (1.85)</p></td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material 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<italic>QoL</italic>, Quality of life; <italic>RRP</italic>, resting rectal pressure</p><p><sup>*</sup>Individual mean and standard deviation (SD) were not reported</p></table-wrap-foot>","<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<table-wrap-foot><p><italic>ARM</italic>, anorectal manometry; <italic>ARP</italic>, anal resting pressure; <italic>BFT</italic>, biofeedback therapy; <italic>CCIS</italic>, Cleveland Clinic Incontinence Score; <italic>Con</italic>, control group; <italic>EMG</italic>, electromyography; <italic>ES</italic>, electrical stimulation; <italic>Exp</italic>, experimental group; <italic>FI</italic>, fecal incontinence; <italic>FIQL</italic>, Fecal Incontinence Quality of Life scale; <italic>IQR</italic>, interquartile range; <italic>MSP</italic>, maximum squeeze pressure; <italic>NR</italic>, not reported; <italic>PFMT</italic>, pelvic floor muscle training; <italic>Pre</italic>, before therapy; <italic>Post</italic>, after therapy; <italic>QoL</italic>, Quality of life; <italic>RRP</italic>, resting rectal pressure</p><p><sup>*</sup>Individual mean and standard deviation (SD) were not reported</p></table-wrap-foot>\",\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"520_2023_8294_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"520_2023_8294_Fig2_HTML\" id=\"MO2\"/>","<graphic xlink:href=\"520_2023_8294_Fig3a_HTML\" id=\"d32e1281\"/>","<graphic xlink:href=\"520_2023_8294_Fig3b_HTML\" id=\"d32e1282\"/>","<graphic xlink:href=\"520_2023_8294_Fig4_HTML\" id=\"MO4\"/>","<graphic xlink:href=\"520_2023_8294_Fig5_HTML\" id=\"MO5\"/>","<graphic 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The study protocol was approved by University of Michigan Institutional Review Board for Health Sciences and Behavioral Sciences (IRB-HSBS). No data have been previously reported.</p>","<title>Participants</title>","<p id=\"Par19\">The final sample included 106 adolescents (57.5% girls, 38.7% boys, 3.8% other gender identity<xref ref-type=\"fn\" rid=\"Fn1\">1</xref>). Adolescents were between 9 and 18 years old (<italic>M</italic> = 13.34, <italic>SD</italic> = 1.92), and 75% were at least 12 years old. Most were White (75.5%) and not Latin(o/a) (91.5%), with others identifying as more than one race (17.9%), Black/African American (5.7%), or American Indian/Alaskan Native (0.9%). Families were recruited through social media, virtual flyers, and university-affiliated databases. Families were defined as one parent or legal guardian and two children (of any degree of genetic relatedness) between 8 and 21 years old, with at least one child between age 8 and 17. After enrollment, some adolescents did not complete the study or meet inclusion criteria for this paper (see below). Thus, the final sample reported here consists of 63 families with 43 sibling pairs and 20 singletons. The average daily response rate of included adolescents was 94%.</p>","<p id=\"Par21\">The full study sample consisted of 174 adolescents (54.9% girls, 42.9% boys, 2.2% other gender identity) aged 8 to 20 years old (<italic>M</italic> = 13.37, <italic>SD</italic> = 2.19). Of these adolescents, 6.3% withdrew from the study, 13.3% were dropped from the study because they failed to complete at least 50% of the daily diaries after the first 30 days, and 19.5% completed the study but were excluded from this paper because their response rate was less than 80%. This follows procedures and simulation results from past research on data fidelity and missingness (Rankin & Marsh, ##UREF##9##1985##; Wright et al., ##REF##30920277##2019##). Included participants did not differ from excluded participants demographically (i.e., gender, age, or race/ethnicity, all <italic>p</italic>s <underline>≥</underline> 0.05), however, they did report fewer impulsive behaviors as well as greater working memory and attentional control (all <italic>p</italic>s <underline>≤</underline> 0.01); they did not report differences in perceptual sensitivity (<italic>p</italic> > .05).</p>","<title>Procedure</title>","<p id=\"Par22\">The study was conducted virtually between March 2021 and August 2022. Families first completed a baseline session in which parents provided electronically signed informed consent for themselves and for their children under 18 years old, who also provided informed assent. Adolescents aged 18 years or older provided informed consent. Parents and adolescents then independently completed 90-minute online surveys (using any Internet-capable device) via Qualtrics. Surveys contained questions about their identities, feelings, and behaviors as well as cognitive tests. Then, every night for the next 100 days, adolescents completed a 20-minute online survey. Around 5:00PM, unique survey links were sent to the parent’s email address<xref ref-type=\"fn\" rid=\"Fn2\">2</xref>, who distributed them to their children. Adolescents were asked to take the survey at 8:00PM or after that day’s activities; survey links expired the next day at noon. Among other measures, the daily surveys included questions about externalizing behaviors and social experiences as well as the novel IC task. Each family member received $15 for completing the baseline survey. Adolescents received $1 for each completed daily survey, which doubled to $2 if they completed at least 80% of the surveys; they received a $35 bonus if they completed at least 90% of the daily surveys.</p>","<title>Measures</title>","<p id=\"Par24\">The focus of this paper is on the novel intensive longitudinal measure of IC. Baseline measures of cognition (i.e., task-based working memory and self-reported attentional control) and age were used to assess convergent validity; measures of perceptual sensitivity and gender were used to assess discriminant validity. To examine how IC’s daily average and fluctuations were linked to externalizing behaviors, baseline impulsive behaviors were used. Daily impulsive behaviors and social experiences were also used in illustrative person-specific analyses.</p>","<title>Baseline Measures</title>","<p id=\"Par25\"><bold>Task-Based Working Memory.</bold> The Symmetry Span task (Foster et al., ##REF##25217113##2015##) was used to assess working memory. Adolescents were shown a series of highlighted red squares in a 4 × 4 black and white matrix. Interspersed was a symmetry task in which adolescents had to judge whether a pattern composed of black and white squares was symmetrical along its vertical axis. Set lengths ranged from 2 to 5 symmetry-matrix combinations (12 trials total). At the end of each set, adolescents were asked to recall the location of each red square in the correct order. Adolescents received two practice trials and were required to maintain at least 85% accuracy on the symmetry trials. The absolute span score (i.e., sum of all perfectly recalled sets) was used in the subsequent analyses (Shipstead et al., ##REF##22409508##2012##).</p>","<p id=\"Par26\"><bold>Self-Reported Attentional Control.</bold> Attentional control was assessed using a subscale from the Early Adolescent Temperament Questionnaire-Revised (EATQ-R; Ellis & Rothbart ##REF##10474213##1999##). It contained six items on capacity to focus and shift attention (e.g., “It is easy for me to really concentrate on homework problems”; α = 0.75). Items were rated on a 5-point scale (1 = <italic>Almost always untrue</italic> to 5 = <italic>Almost always true</italic>) with higher scores reflecting greater control.</p>","<p id=\"Par27\"><bold>Self-Reported Perceptual Sensitivity.</bold> Perceptual sensitivity was assessed using another subscale from the EATQ-R (Ellis & Rothbart, ##REF##10474213##1999##). It contained four items on the detection or awareness of slight, low-intensity stimulation in the environment (e.g., “I am very aware of noises.”; α = 0.81). Higher scores reflect greater sensitivity.</p>","<p id=\"Par28\"><bold>Self-Reported Impulsive Behaviors.</bold> Impulsive behaviors were assessed with the 40-item UPPS-P Impulsive Behavior scale (Lynam et al., ##UREF##6##2006##). It has five subscales, measuring a lack of premeditation (e.g., “I am one of those people who blurt out things without thinking”; α = 0.76), lack of perseverance (e.g., “I tend to give up easily”; α = 0.76), sensation seeking (e.g., “I quite enjoy taking risks”; α = 0.87), positive urgency (e.g., “I tend to act without thinking when I am really excited”; α = 0.93), and negative urgency (e.g., “When I feel bad, I will often do things I later regret in order to make myself feel better now”; α = 0.89). Items were rated on a 4-point scale (1 = <italic>Not at all like me</italic> to 4 <italic>= Very much like me</italic>) with higher scores reflecting greater impulsivity.</p>","<title>Daily Measures</title>","<p id=\"Par29\"><bold>IC.</bold> The Stroop Color Word Test (Golden, ##REF##16367401##1975##) was adapted for the 100 daily assessments. In the classic measure, participants received 3 pages with 100 color words each (“red”, “green”, “blue”). On the first page, all color words were printed in black ink. On the second page, all color words were printed in congruent ink (e.g., the word “red” printed in red ink), and on the third page, all color words were printed in incongruent ink (e.g., “red” printed in green ink). Answer choices included each color word printed in black ink. Participants were given 45 seconds per page to circle as many answers corresponding to the ink color as possible. It was expected that more items would be correctly circled on congruent than incongruent pages; the number of correctly answered incongruent items was the IC score.</p>","<p id=\"Par30\">Since 1975, subtraction methods (e.g., comparing reaction time on incongruent and congruent trials) have gained popularity, but they have significant limitations for accurately detecting individual differences (Draheim et al., ##REF##30896187##2019##; Weigard et al., ##REF##34252724##2021##), and thus, intraindividual variation. Therefore, in this study, adolescents were presented with a randomized set of 100 color words (“red”, “green”, blue”, or “yellow”) in incongruent colors only (e.g., “red” in green font). Consistent with other work (Heitz & Engle, ##REF##17500647##2007##), there were no neutral or congruent conditions, and all combinations of words and font colors were presented the same number of times per day. As in the classic measure, adolescents indicated the font color of as many words as they could in 45 seconds by selecting the correct color word presented in black ink (see supplemental materials for task images)<xref ref-type=\"fn\" rid=\"Fn3\">3</xref>. The number of correct responses indexed each day’s IC score; this is consistent with the original measure and has been suggested as a viable alternative to reaction time-based indices (Khng & Lee, ##REF##24992683##2014##). On the first day, adolescents also completed five practice trials with feedback. The task is openly available (<ext-link ext-link-type=\"uri\" xlink:href=\"https://osf.io/9yabr/\">https://osf.io/9yabr/</ext-link>).</p>","<p id=\"Par32\">To ensure data fidelity, some trials were excluded for some adolescents. Days adolescents completed one or fewer trials correctly were excluded (<0.5%), as they likely reflected low effort or technical issues. Also, days adolescents completed nearly all 100 trials were winsorized to three standard deviations above that day’s average (<0.5%), as they likely reflected technical issues (e.g., screen freezing) and internal testing suggested it would be difficult to complete more than 75 trials.</p>","<p id=\"Par33\"><bold>Daily Impulsive and Social Experiences.</bold> Daily positive urgency and daily negative urgency (i.e., tendencies to act impulsively when experiencing positive and negative emotions, respectively) were assessed via the Short UPPS-P (Cyders et al., ##REF##24636739##2014##); each scale contained four items adapted to reflect adolescents’ impulsive behaviors that day (e.g., positive urgency: “<italic>Today</italic>, I tended to act without thinking when I was really excited”) and were rated on a 4-point scale (1 = <italic>Not at all like me</italic> to 4 = <italic>Very much like me</italic>). Similar measures have been adapted and used in intensive longitudinal studies (Sperry et al., ##UREF##12##2016##; Tomko et al., ##REF##24274047##2014##). Reliabilities were good, according to multilevel confirmatory factor analysis (Schuurman & Hamaker, ##REF##30188157##2019##). For daily positive urgency, between-person ω = 0.80 and within-person ω = 0.79. For daily negative urgency, between-person ω = 0.81 and within-person ω = 0.88.</p>","<p id=\"Par34\">Daily social experiences were assessed using a modified activity questionnaire (McHale et al., ##REF##10446731##1999##). Adolescents completed one item indicating how much time (in minutes) they spent visiting or hanging out that day on a sliding scale from 0 to 100. Responses were binned: 0 = <italic>Did not visit or hang out today</italic>; 2 (1–49 min) = <italic>Visited or hung out a little</italic>; 3 (50–99 min) = <italic>Visited or hung out a moderate amount</italic>; 4 (100 + minutes) = <italic>Visited or hung out a lot</italic>. Further details for the daily measures are provided in the supplemental materials.</p>","<title>Analytic Plan</title>","<p id=\"Par35\">Three sets of analyses were conducted. First, the reliability and validity of the novel IC measure were assessed. Second, average daily IC scores and fluctuations in those scores were associated with baseline impulsive behaviors. Finally, personalized network analyses (using GIMME; Gates & Molenaar ##REF##22732562##2012##) were conducted for a subset of individuals to illustrate the utility of intensive longitudinal data for person-specific inferences. Analyses were conducted in SPSS (version 26) and R (v4.1.2; R Core Team, ##UREF##13##2022##).</p>","<p id=\"Par36\"><bold>Reliability and Validity of Daily IC.</bold> Parallel forms reliability was assessed by comparing the interindividual IC means and standard deviations (<italic>SD</italic>s) across all 100 days. Daily means and <italic>SD</italic>s across participants for each day were expected to be approximately equal and normally distributed, suggesting that random differences in stimuli order did not systematically impact IC assessment. Intraclass correlation coefficients (ICCs) were also calculated, with values greater than 0.50 indicating moderate reliability, 0.75 good reliability, and 0.90 excellent reliability (Bartko, ##REF##5942109##1966##). Moderate to good reliability across days was expected (see Kelly & Beltz ##REF##33106024##2021##).</p>","<p id=\"Par37\">Convergent validity was assessed by correlating each day’s IC score with standard baseline measures of cognition (i.e., working memory and attentional control) across participants. Low to moderate correlations were expected because they assessed similar, but distinct domains of cognition, and in the case of attentional control, in a different modality (Toplak et al., ##REF##23057693##2013##). Convergent validity was also assessed by correlating each day’s IC score with age. Older youth were expected to have higher IC. Discriminant validity was assessed by correlating each day’s IC score with baseline perceptual sensitivity and via gender differences. No relations were expected given weak, null, and inconsistent findings in the extant literature (Weafer, ##REF##32462613##2020##). All reliability and validity analyses used listwise deletion for daily missing data, but there were at least 92 participants (87%) included in each day’s analysis. Effect sizes for independent analyses were evaluated using <italic>r</italic> and Cohen’s <italic>d</italic>, with small effect sizes corresponding to <italic>r</italic> = 0.1 and <italic>d</italic> = 0.2, medium to <italic>r</italic> = 0.3 and <italic>d</italic> = 0.5, and large to <italic>r</italic> = 0.5 and <italic>d</italic> = 0.8 (Cohen, ##UREF##2##1988##). Nested analyses (accounting for family dependencies) showed the same pattern of results as the independent analyses described in the main text; they are available in the supplemental materials.</p>","<p id=\"Par38\"><bold>Fluctuations in Daily IC and Links to Impulsive Behaviors.</bold> Each adolescent’s average or mean IC (i<italic>M</italic>) across the 100 days was calculated. Fluctuations were calculated using intraindividual standard deviations (i<italic>SD</italic>); smaller i<italic>SD</italic>s reflect fewer and/or smaller deviations from an adolescent’s own average, and larger i<italic>SD</italic>s reflect more and/or larger deviations from that average. For instance, an adolescent with i<italic>M</italic> = 34 and i<italic>SD</italic> = 2.7 demonstrates relatively consistent performance across days compared to an adolescent with i<italic>M</italic> = 34 and i<italic>SD</italic> = 7.5 who has the same level of performance but with larger variability, sometimes scoring well below or above their average. A one-sample <italic>t</italic>-test was used to determine whether the sample showed fluctuations (i.e., i<italic>SD</italic>s significantly different from zero). Gender and age effects were also explored. Multilevel models (nesting individuals within families) were then used to assess associations between baseline impulsive behaviors (i.e., lack of premeditation, lack of perseverance, sensation seeking, positive urgency, and negative urgency) and daily IC (i<italic>M</italic>, i<italic>SD</italic>). Each outcome was assessed in a separate model and all models included age and gender (0 = boys; 1 = girls)<xref ref-type=\"fn\" rid=\"Fn4\">4</xref>.</p>","<p id=\"Par40\"><bold>Illustrative Adolescent-Specific Network Analyses</bold>. Finally, illustrative adolescent-specific network analyses were conducted via GIMME to highlight the utility of intensive longitudinal methods for future work on externalizing behaviors. Specifically, 12 adolescents who reported any substance use (e.g., tobacco, marijuana, or alcohol) during the 100-day study were matched (see supplemental materials) with 12 adolescents who reported no substance use. This extreme groups comparison is ideal for highlighting heterogeneity among adolescents who use substances (and demographically similar youth who do not). Their daily IC, positive urgency, negative urgency, and social time were linearly detrended by day (as many time series approaches assume stationarity; Beltz & Gates ##REF##29161187##2017##), and then submitted to confirmatory subgrouping-GIMME (CS-GIMME; Henry et al., ##REF##30583065##2019##).</p>","<p id=\"Par0040\">CS-GIMME is a variant of GIMME which uses unified structural equation models (or uSEMs) in combination with a grouping algorithm to derive sparse, person-specific networks of directed relations among intensively measured variables (Gates & Molenaar, ##REF##22732562##2012##). It is unique among network approaches because it estimates contemporaneous (i.e., same day) and first order lagged (i.e., next-day) relations, providing some temporal indexing for relations, and because it provides both nomothetic and idiographic inferences via relations that can apply to the whole sample or to a single adolescent. As described in Fig. ##FIG##0##1##, GIMME derives person-specific networks through a multistep, data-driven process. The analysis begins with a null model. Then, a directed relation is added between two variables if it would significantly improve model fit for at least 75% of the sample (as determined by Lagrange Multiplier tests; Sörbom ##UREF##11##1989##); these group-level relations are iteratively added until none meets the 75% criterion. In this analysis, autoregressive relations (i.e., variables predicting themselves from one day to the next) are specified at the group-level to facilitate model fitting (a common procedure; Lane et al., ##REF##30124300##2019##). Individual-level relations are then added if an individual’s model does not fit well with only group-level relations. After each level is fit, models are pruned of relations that no longer meet criteria, and final models are evaluated using standard fit indices. In this study, CS-GIMME was used to permit <italic>a priori</italic> comparisons between adolescents who did and did not use substances over the 100 days. In CS-GIMME (Henry et al., 2019), subgroup-level relations are iteratively estimated after group-level relations and before individual-level relations (with a 51% criterion). This means that relations common among the majority of youth who used substances have the opportunity to be estimated separately from youth who did not use substances. Thus, each resulting network reflects a personalized set of relations with unique weights (some of which apply to everyone, some of which apply only to an adolescent’s subgroup, and some of which are unique to an adolescent). GIMME uses full information maximum likelihood, and only includes individuals with 80% or more of the daily dairies. GIMME, and its extensions, have been widely used and are well-supported by largescale simulations (see Gates & Molenaar ##REF##22732562##2012##; Henry et al., ##REF##30583065##2019##; Lane et al., ##REF##30124300##2019##).</p>","<p id=\"Par42\">After GIMME, network node centrality was calculated and compared across subgroups. Node centrality is the number of relations for each variable divided by the total number of network relations. It reflects the relative influence of each behavior in an adolescent’s person-specific model (Beltz & Gates, ##REF##29161187##2017##). Independent sample <italic>t</italic>-tests were conducted to determine whether node centrality differed across adolescents who used substances and those who did not.</p>","<p id=\"Par43\">\n\n</p>"],"string":"[\n \"<title>Method</title>\",\n \"<p id=\\\"Par18\\\">Data come from a 100-day intensive longitudinal study of adolescent daily experiences. The study protocol was approved by University of Michigan Institutional Review Board for Health Sciences and Behavioral Sciences (IRB-HSBS). No data have been previously reported.</p>\",\n \"<title>Participants</title>\",\n \"<p id=\\\"Par19\\\">The final sample included 106 adolescents (57.5% girls, 38.7% boys, 3.8% other gender identity<xref ref-type=\\\"fn\\\" rid=\\\"Fn1\\\">1</xref>). Adolescents were between 9 and 18 years old (<italic>M</italic> = 13.34, <italic>SD</italic> = 1.92), and 75% were at least 12 years old. Most were White (75.5%) and not Latin(o/a) (91.5%), with others identifying as more than one race (17.9%), Black/African American (5.7%), or American Indian/Alaskan Native (0.9%). Families were recruited through social media, virtual flyers, and university-affiliated databases. Families were defined as one parent or legal guardian and two children (of any degree of genetic relatedness) between 8 and 21 years old, with at least one child between age 8 and 17. After enrollment, some adolescents did not complete the study or meet inclusion criteria for this paper (see below). Thus, the final sample reported here consists of 63 families with 43 sibling pairs and 20 singletons. The average daily response rate of included adolescents was 94%.</p>\",\n \"<p id=\\\"Par21\\\">The full study sample consisted of 174 adolescents (54.9% girls, 42.9% boys, 2.2% other gender identity) aged 8 to 20 years old (<italic>M</italic> = 13.37, <italic>SD</italic> = 2.19). Of these adolescents, 6.3% withdrew from the study, 13.3% were dropped from the study because they failed to complete at least 50% of the daily diaries after the first 30 days, and 19.5% completed the study but were excluded from this paper because their response rate was less than 80%. This follows procedures and simulation results from past research on data fidelity and missingness (Rankin & Marsh, ##UREF##9##1985##; Wright et al., ##REF##30920277##2019##). Included participants did not differ from excluded participants demographically (i.e., gender, age, or race/ethnicity, all <italic>p</italic>s <underline>≥</underline> 0.05), however, they did report fewer impulsive behaviors as well as greater working memory and attentional control (all <italic>p</italic>s <underline>≤</underline> 0.01); they did not report differences in perceptual sensitivity (<italic>p</italic> > .05).</p>\",\n \"<title>Procedure</title>\",\n \"<p id=\\\"Par22\\\">The study was conducted virtually between March 2021 and August 2022. Families first completed a baseline session in which parents provided electronically signed informed consent for themselves and for their children under 18 years old, who also provided informed assent. Adolescents aged 18 years or older provided informed consent. Parents and adolescents then independently completed 90-minute online surveys (using any Internet-capable device) via Qualtrics. Surveys contained questions about their identities, feelings, and behaviors as well as cognitive tests. Then, every night for the next 100 days, adolescents completed a 20-minute online survey. Around 5:00PM, unique survey links were sent to the parent’s email address<xref ref-type=\\\"fn\\\" rid=\\\"Fn2\\\">2</xref>, who distributed them to their children. Adolescents were asked to take the survey at 8:00PM or after that day’s activities; survey links expired the next day at noon. Among other measures, the daily surveys included questions about externalizing behaviors and social experiences as well as the novel IC task. Each family member received $15 for completing the baseline survey. Adolescents received $1 for each completed daily survey, which doubled to $2 if they completed at least 80% of the surveys; they received a $35 bonus if they completed at least 90% of the daily surveys.</p>\",\n \"<title>Measures</title>\",\n \"<p id=\\\"Par24\\\">The focus of this paper is on the novel intensive longitudinal measure of IC. Baseline measures of cognition (i.e., task-based working memory and self-reported attentional control) and age were used to assess convergent validity; measures of perceptual sensitivity and gender were used to assess discriminant validity. To examine how IC’s daily average and fluctuations were linked to externalizing behaviors, baseline impulsive behaviors were used. Daily impulsive behaviors and social experiences were also used in illustrative person-specific analyses.</p>\",\n \"<title>Baseline Measures</title>\",\n \"<p id=\\\"Par25\\\"><bold>Task-Based Working Memory.</bold> The Symmetry Span task (Foster et al., ##REF##25217113##2015##) was used to assess working memory. Adolescents were shown a series of highlighted red squares in a 4 × 4 black and white matrix. Interspersed was a symmetry task in which adolescents had to judge whether a pattern composed of black and white squares was symmetrical along its vertical axis. Set lengths ranged from 2 to 5 symmetry-matrix combinations (12 trials total). At the end of each set, adolescents were asked to recall the location of each red square in the correct order. Adolescents received two practice trials and were required to maintain at least 85% accuracy on the symmetry trials. The absolute span score (i.e., sum of all perfectly recalled sets) was used in the subsequent analyses (Shipstead et al., ##REF##22409508##2012##).</p>\",\n \"<p id=\\\"Par26\\\"><bold>Self-Reported Attentional Control.</bold> Attentional control was assessed using a subscale from the Early Adolescent Temperament Questionnaire-Revised (EATQ-R; Ellis & Rothbart ##REF##10474213##1999##). It contained six items on capacity to focus and shift attention (e.g., “It is easy for me to really concentrate on homework problems”; α = 0.75). Items were rated on a 5-point scale (1 = <italic>Almost always untrue</italic> to 5 = <italic>Almost always true</italic>) with higher scores reflecting greater control.</p>\",\n \"<p id=\\\"Par27\\\"><bold>Self-Reported Perceptual Sensitivity.</bold> Perceptual sensitivity was assessed using another subscale from the EATQ-R (Ellis & Rothbart, ##REF##10474213##1999##). It contained four items on the detection or awareness of slight, low-intensity stimulation in the environment (e.g., “I am very aware of noises.”; α = 0.81). Higher scores reflect greater sensitivity.</p>\",\n \"<p id=\\\"Par28\\\"><bold>Self-Reported Impulsive Behaviors.</bold> Impulsive behaviors were assessed with the 40-item UPPS-P Impulsive Behavior scale (Lynam et al., ##UREF##6##2006##). It has five subscales, measuring a lack of premeditation (e.g., “I am one of those people who blurt out things without thinking”; α = 0.76), lack of perseverance (e.g., “I tend to give up easily”; α = 0.76), sensation seeking (e.g., “I quite enjoy taking risks”; α = 0.87), positive urgency (e.g., “I tend to act without thinking when I am really excited”; α = 0.93), and negative urgency (e.g., “When I feel bad, I will often do things I later regret in order to make myself feel better now”; α = 0.89). Items were rated on a 4-point scale (1 = <italic>Not at all like me</italic> to 4 <italic>= Very much like me</italic>) with higher scores reflecting greater impulsivity.</p>\",\n \"<title>Daily Measures</title>\",\n \"<p id=\\\"Par29\\\"><bold>IC.</bold> The Stroop Color Word Test (Golden, ##REF##16367401##1975##) was adapted for the 100 daily assessments. In the classic measure, participants received 3 pages with 100 color words each (“red”, “green”, “blue”). On the first page, all color words were printed in black ink. On the second page, all color words were printed in congruent ink (e.g., the word “red” printed in red ink), and on the third page, all color words were printed in incongruent ink (e.g., “red” printed in green ink). Answer choices included each color word printed in black ink. Participants were given 45 seconds per page to circle as many answers corresponding to the ink color as possible. It was expected that more items would be correctly circled on congruent than incongruent pages; the number of correctly answered incongruent items was the IC score.</p>\",\n \"<p id=\\\"Par30\\\">Since 1975, subtraction methods (e.g., comparing reaction time on incongruent and congruent trials) have gained popularity, but they have significant limitations for accurately detecting individual differences (Draheim et al., ##REF##30896187##2019##; Weigard et al., ##REF##34252724##2021##), and thus, intraindividual variation. Therefore, in this study, adolescents were presented with a randomized set of 100 color words (“red”, “green”, blue”, or “yellow”) in incongruent colors only (e.g., “red” in green font). Consistent with other work (Heitz & Engle, ##REF##17500647##2007##), there were no neutral or congruent conditions, and all combinations of words and font colors were presented the same number of times per day. As in the classic measure, adolescents indicated the font color of as many words as they could in 45 seconds by selecting the correct color word presented in black ink (see supplemental materials for task images)<xref ref-type=\\\"fn\\\" rid=\\\"Fn3\\\">3</xref>. The number of correct responses indexed each day’s IC score; this is consistent with the original measure and has been suggested as a viable alternative to reaction time-based indices (Khng & Lee, ##REF##24992683##2014##). On the first day, adolescents also completed five practice trials with feedback. The task is openly available (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://osf.io/9yabr/\\\">https://osf.io/9yabr/</ext-link>).</p>\",\n \"<p id=\\\"Par32\\\">To ensure data fidelity, some trials were excluded for some adolescents. Days adolescents completed one or fewer trials correctly were excluded (<0.5%), as they likely reflected low effort or technical issues. Also, days adolescents completed nearly all 100 trials were winsorized to three standard deviations above that day’s average (<0.5%), as they likely reflected technical issues (e.g., screen freezing) and internal testing suggested it would be difficult to complete more than 75 trials.</p>\",\n \"<p id=\\\"Par33\\\"><bold>Daily Impulsive and Social Experiences.</bold> Daily positive urgency and daily negative urgency (i.e., tendencies to act impulsively when experiencing positive and negative emotions, respectively) were assessed via the Short UPPS-P (Cyders et al., ##REF##24636739##2014##); each scale contained four items adapted to reflect adolescents’ impulsive behaviors that day (e.g., positive urgency: “<italic>Today</italic>, I tended to act without thinking when I was really excited”) and were rated on a 4-point scale (1 = <italic>Not at all like me</italic> to 4 = <italic>Very much like me</italic>). Similar measures have been adapted and used in intensive longitudinal studies (Sperry et al., ##UREF##12##2016##; Tomko et al., ##REF##24274047##2014##). Reliabilities were good, according to multilevel confirmatory factor analysis (Schuurman & Hamaker, ##REF##30188157##2019##). For daily positive urgency, between-person ω = 0.80 and within-person ω = 0.79. For daily negative urgency, between-person ω = 0.81 and within-person ω = 0.88.</p>\",\n \"<p id=\\\"Par34\\\">Daily social experiences were assessed using a modified activity questionnaire (McHale et al., ##REF##10446731##1999##). Adolescents completed one item indicating how much time (in minutes) they spent visiting or hanging out that day on a sliding scale from 0 to 100. Responses were binned: 0 = <italic>Did not visit or hang out today</italic>; 2 (1–49 min) = <italic>Visited or hung out a little</italic>; 3 (50–99 min) = <italic>Visited or hung out a moderate amount</italic>; 4 (100 + minutes) = <italic>Visited or hung out a lot</italic>. Further details for the daily measures are provided in the supplemental materials.</p>\",\n \"<title>Analytic Plan</title>\",\n \"<p id=\\\"Par35\\\">Three sets of analyses were conducted. First, the reliability and validity of the novel IC measure were assessed. Second, average daily IC scores and fluctuations in those scores were associated with baseline impulsive behaviors. Finally, personalized network analyses (using GIMME; Gates & Molenaar ##REF##22732562##2012##) were conducted for a subset of individuals to illustrate the utility of intensive longitudinal data for person-specific inferences. Analyses were conducted in SPSS (version 26) and R (v4.1.2; R Core Team, ##UREF##13##2022##).</p>\",\n \"<p id=\\\"Par36\\\"><bold>Reliability and Validity of Daily IC.</bold> Parallel forms reliability was assessed by comparing the interindividual IC means and standard deviations (<italic>SD</italic>s) across all 100 days. Daily means and <italic>SD</italic>s across participants for each day were expected to be approximately equal and normally distributed, suggesting that random differences in stimuli order did not systematically impact IC assessment. Intraclass correlation coefficients (ICCs) were also calculated, with values greater than 0.50 indicating moderate reliability, 0.75 good reliability, and 0.90 excellent reliability (Bartko, ##REF##5942109##1966##). Moderate to good reliability across days was expected (see Kelly & Beltz ##REF##33106024##2021##).</p>\",\n \"<p id=\\\"Par37\\\">Convergent validity was assessed by correlating each day’s IC score with standard baseline measures of cognition (i.e., working memory and attentional control) across participants. Low to moderate correlations were expected because they assessed similar, but distinct domains of cognition, and in the case of attentional control, in a different modality (Toplak et al., ##REF##23057693##2013##). Convergent validity was also assessed by correlating each day’s IC score with age. Older youth were expected to have higher IC. Discriminant validity was assessed by correlating each day’s IC score with baseline perceptual sensitivity and via gender differences. No relations were expected given weak, null, and inconsistent findings in the extant literature (Weafer, ##REF##32462613##2020##). All reliability and validity analyses used listwise deletion for daily missing data, but there were at least 92 participants (87%) included in each day’s analysis. Effect sizes for independent analyses were evaluated using <italic>r</italic> and Cohen’s <italic>d</italic>, with small effect sizes corresponding to <italic>r</italic> = 0.1 and <italic>d</italic> = 0.2, medium to <italic>r</italic> = 0.3 and <italic>d</italic> = 0.5, and large to <italic>r</italic> = 0.5 and <italic>d</italic> = 0.8 (Cohen, ##UREF##2##1988##). Nested analyses (accounting for family dependencies) showed the same pattern of results as the independent analyses described in the main text; they are available in the supplemental materials.</p>\",\n \"<p id=\\\"Par38\\\"><bold>Fluctuations in Daily IC and Links to Impulsive Behaviors.</bold> Each adolescent’s average or mean IC (i<italic>M</italic>) across the 100 days was calculated. Fluctuations were calculated using intraindividual standard deviations (i<italic>SD</italic>); smaller i<italic>SD</italic>s reflect fewer and/or smaller deviations from an adolescent’s own average, and larger i<italic>SD</italic>s reflect more and/or larger deviations from that average. For instance, an adolescent with i<italic>M</italic> = 34 and i<italic>SD</italic> = 2.7 demonstrates relatively consistent performance across days compared to an adolescent with i<italic>M</italic> = 34 and i<italic>SD</italic> = 7.5 who has the same level of performance but with larger variability, sometimes scoring well below or above their average. A one-sample <italic>t</italic>-test was used to determine whether the sample showed fluctuations (i.e., i<italic>SD</italic>s significantly different from zero). Gender and age effects were also explored. Multilevel models (nesting individuals within families) were then used to assess associations between baseline impulsive behaviors (i.e., lack of premeditation, lack of perseverance, sensation seeking, positive urgency, and negative urgency) and daily IC (i<italic>M</italic>, i<italic>SD</italic>). Each outcome was assessed in a separate model and all models included age and gender (0 = boys; 1 = girls)<xref ref-type=\\\"fn\\\" rid=\\\"Fn4\\\">4</xref>.</p>\",\n \"<p id=\\\"Par40\\\"><bold>Illustrative Adolescent-Specific Network Analyses</bold>. Finally, illustrative adolescent-specific network analyses were conducted via GIMME to highlight the utility of intensive longitudinal methods for future work on externalizing behaviors. Specifically, 12 adolescents who reported any substance use (e.g., tobacco, marijuana, or alcohol) during the 100-day study were matched (see supplemental materials) with 12 adolescents who reported no substance use. This extreme groups comparison is ideal for highlighting heterogeneity among adolescents who use substances (and demographically similar youth who do not). Their daily IC, positive urgency, negative urgency, and social time were linearly detrended by day (as many time series approaches assume stationarity; Beltz & Gates ##REF##29161187##2017##), and then submitted to confirmatory subgrouping-GIMME (CS-GIMME; Henry et al., ##REF##30583065##2019##).</p>\",\n \"<p id=\\\"Par0040\\\">CS-GIMME is a variant of GIMME which uses unified structural equation models (or uSEMs) in combination with a grouping algorithm to derive sparse, person-specific networks of directed relations among intensively measured variables (Gates & Molenaar, ##REF##22732562##2012##). It is unique among network approaches because it estimates contemporaneous (i.e., same day) and first order lagged (i.e., next-day) relations, providing some temporal indexing for relations, and because it provides both nomothetic and idiographic inferences via relations that can apply to the whole sample or to a single adolescent. As described in Fig. ##FIG##0##1##, GIMME derives person-specific networks through a multistep, data-driven process. The analysis begins with a null model. Then, a directed relation is added between two variables if it would significantly improve model fit for at least 75% of the sample (as determined by Lagrange Multiplier tests; Sörbom ##UREF##11##1989##); these group-level relations are iteratively added until none meets the 75% criterion. In this analysis, autoregressive relations (i.e., variables predicting themselves from one day to the next) are specified at the group-level to facilitate model fitting (a common procedure; Lane et al., ##REF##30124300##2019##). Individual-level relations are then added if an individual’s model does not fit well with only group-level relations. After each level is fit, models are pruned of relations that no longer meet criteria, and final models are evaluated using standard fit indices. In this study, CS-GIMME was used to permit <italic>a priori</italic> comparisons between adolescents who did and did not use substances over the 100 days. In CS-GIMME (Henry et al., 2019), subgroup-level relations are iteratively estimated after group-level relations and before individual-level relations (with a 51% criterion). This means that relations common among the majority of youth who used substances have the opportunity to be estimated separately from youth who did not use substances. Thus, each resulting network reflects a personalized set of relations with unique weights (some of which apply to everyone, some of which apply only to an adolescent’s subgroup, and some of which are unique to an adolescent). GIMME uses full information maximum likelihood, and only includes individuals with 80% or more of the daily dairies. GIMME, and its extensions, have been widely used and are well-supported by largescale simulations (see Gates & Molenaar ##REF##22732562##2012##; Henry et al., ##REF##30583065##2019##; Lane et al., ##REF##30124300##2019##).</p>\",\n \"<p id=\\\"Par42\\\">After GIMME, network node centrality was calculated and compared across subgroups. Node centrality is the number of relations for each variable divided by the total number of network relations. It reflects the relative influence of each behavior in an adolescent’s person-specific model (Beltz & Gates, ##REF##29161187##2017##). Independent sample <italic>t</italic>-tests were conducted to determine whether node centrality differed across adolescents who used substances and those who did not.</p>\",\n \"<p id=\\\"Par43\\\">\\n\\n</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Reliability and Validity of Daily IC</title>","<p id=\"Par44\">The interindividual means and <italic>SD</italic>s of IC across 100 days are shown in Fig. ##FIG##1##2##. Each bar represents <italic>that day’s</italic> IC averaged across all adolescents. The thick black line represents the overall IC score (<italic>M</italic> = 34.19), aggregated across adolescents and days, and the thin dashed line represents daily variation across adolescents’ scores (overall <italic>SD</italic> = 1.82).</p>","<p id=\"Par45\">\n\n</p>","<p id=\"Par46\">Overall skewness was 2.23 and kurtosis was 8.84, suggesting that the distribution was negatively skewed and heavy-tailed. This is easy to visualize in Fig. ##FIG##1##2##; IC notably increases over the first week (indicated by light gray bars). Excluding this week, the mean for the remaining 93 days was similar, and <italic>SD</italic> expectedly decreased (<italic>M</italic> = 34.53; <italic>SD</italic> = 1.14); the skewness and kurtosis also decreased to close to zero (skewness: 0.18, kurtosis: − 0.70), approximating a normal distribution. The ICC was 0.53 (95% CI = [0.47, 0.61]), suggesting relatively good reliability across days, and it was 0.56 (95% CI = [0.50, 0.63]) across the last 93 days.</p>","<p id=\"Par47\">Convergent and divergent validity were assessed via correlations of daily IC with standard baseline measures. These correlations are shown in Fig. ##FIG##2##3##. Regarding convergent validity, there were small-to-moderate, positive correlations. IC’s average correlation with working memory (Fig. ##FIG##2##3##, dark gray) was <italic>r</italic> = .16 (<italic>SD</italic> = 0.10; range: 0.03 - 0.37), and 37% of the correlations were significant at <italic>p</italic> < .05. IC’s average correlation with attentional control (Fig. ##FIG##2##3##, light gray) was <italic>r</italic> = .20 (<italic>SD</italic> = 0.07; range: 0.01 - 0.37), and 54% were significant. IC’s average correlation with age was <italic>r</italic> = .20 (<italic>SD</italic> = 0.07; range: 0.01 - 0.38), and 52% were significant; thus, older adolescents had higher IC. Regarding divergent validity, perceptual sensitivity (Fig. ##FIG##2##3##, black) was not significantly correlated with any day’s IC performance (average <italic>r </italic>= -.03, all <italic>p</italic>s > 0.05). On average, girls (<italic>M</italic> = 35.03; <italic>SD</italic> = 11.72) had better daily IC than boys (<italic>M</italic> = 33.43; <italic>SD</italic> = 11.30), but this difference was not significant (<italic>p</italic> > .05), and girls only significantly outperformed boys on a single day, which likely reflects Type I error. The average effect size (Cohen’s <italic>d</italic>) across all 100 days was small (<italic>d</italic> = 0.15; <italic>SD</italic> = 0.11; range: 0.002 - 0.56). Each daily correlation and multilevel models of the same relations adjusting for family dependencies are provided in supplemental materials.</p>","<p id=\"Par48\">\n\n</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Reliability and Validity of Daily IC</title>\",\n \"<p id=\\\"Par44\\\">The interindividual means and <italic>SD</italic>s of IC across 100 days are shown in Fig. ##FIG##1##2##. Each bar represents <italic>that day’s</italic> IC averaged across all adolescents. The thick black line represents the overall IC score (<italic>M</italic> = 34.19), aggregated across adolescents and days, and the thin dashed line represents daily variation across adolescents’ scores (overall <italic>SD</italic> = 1.82).</p>\",\n \"<p id=\\\"Par45\\\">\\n\\n</p>\",\n \"<p id=\\\"Par46\\\">Overall skewness was 2.23 and kurtosis was 8.84, suggesting that the distribution was negatively skewed and heavy-tailed. This is easy to visualize in Fig. ##FIG##1##2##; IC notably increases over the first week (indicated by light gray bars). Excluding this week, the mean for the remaining 93 days was similar, and <italic>SD</italic> expectedly decreased (<italic>M</italic> = 34.53; <italic>SD</italic> = 1.14); the skewness and kurtosis also decreased to close to zero (skewness: 0.18, kurtosis: − 0.70), approximating a normal distribution. The ICC was 0.53 (95% CI = [0.47, 0.61]), suggesting relatively good reliability across days, and it was 0.56 (95% CI = [0.50, 0.63]) across the last 93 days.</p>\",\n \"<p id=\\\"Par47\\\">Convergent and divergent validity were assessed via correlations of daily IC with standard baseline measures. These correlations are shown in Fig. ##FIG##2##3##. Regarding convergent validity, there were small-to-moderate, positive correlations. IC’s average correlation with working memory (Fig. ##FIG##2##3##, dark gray) was <italic>r</italic> = .16 (<italic>SD</italic> = 0.10; range: 0.03 - 0.37), and 37% of the correlations were significant at <italic>p</italic> < .05. IC’s average correlation with attentional control (Fig. ##FIG##2##3##, light gray) was <italic>r</italic> = .20 (<italic>SD</italic> = 0.07; range: 0.01 - 0.37), and 54% were significant. IC’s average correlation with age was <italic>r</italic> = .20 (<italic>SD</italic> = 0.07; range: 0.01 - 0.38), and 52% were significant; thus, older adolescents had higher IC. Regarding divergent validity, perceptual sensitivity (Fig. ##FIG##2##3##, black) was not significantly correlated with any day’s IC performance (average <italic>r </italic>= -.03, all <italic>p</italic>s > 0.05). On average, girls (<italic>M</italic> = 35.03; <italic>SD</italic> = 11.72) had better daily IC than boys (<italic>M</italic> = 33.43; <italic>SD</italic> = 11.30), but this difference was not significant (<italic>p</italic> > .05), and girls only significantly outperformed boys on a single day, which likely reflects Type I error. The average effect size (Cohen’s <italic>d</italic>) across all 100 days was small (<italic>d</italic> = 0.15; <italic>SD</italic> = 0.11; range: 0.002 - 0.56). Each daily correlation and multilevel models of the same relations adjusting for family dependencies are provided in supplemental materials.</p>\",\n \"<p id=\\\"Par48\\\">\\n\\n</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par54\">IC is often conceptualized as a relatively stable construct, but growing evidence suggests that cognition varies across contexts and time (Brose et al., ##REF##24364855##2014##; Sliwinski et al., ##REF##16953716##2006##). Although this has significant implications for adolescent externalizing behaviors, the relevant literature has been stagnated by a lack of suitable intensive longitudinal cognitive assessments. Thus, the present study considered the extent to which adolescent IC – captured by 100 novel daily measurements – fluctuates from day-to-day in ways that are meaningfully associated with impulsivity and differ by the daily experiences of adolescent who do and do not use substances.</p>","<title>IC Can Be Measured Daily</title>","<p id=\"Par55\">Findings suggest that the novel IC task is a reliable and valid method for assessing daily cognitive processes related to inhibition. Parallel forms reliability was determined by comparing <italic>M</italic>s and <italic>SD</italic>s across days and calculating ICCs. The 100-day distribution of scores was non-normal: Specifically, IC performance increased over the first week before stabilizing, which may be due to initial task-related learning. Indeed, when the first seven days were excluded, IC scores from the remaining 93 days approximated a normal distribution. Moreover, variation across participants increased over the course of the study until about day 70, indicating increasing then plateauing between-person differences in IC. This is consistent with the notion that contextual demands can reduce individual differences (Miller et al., ##REF##17225514##2006##), with the novelty of the study being an initially constraining context. ICCs also indicated that individuals’ scores displayed moderate reliability across days (Bartko, ##REF##5942109##1966##). This is aligned with expectations for daily studies of cognition - prior work has suggested moderate ICCs are likely well-suited to intensive longitudinal research, as they reflect both between-person stability of assessments and potentially true variation in those assessments within a person over time (Bolger & Laurenceau, ##UREF##0##2013##).</p>","<p id=\"Par56\">Notably, most other daily cognition validation studies have not shown similar first-week effects, although there was evidence for learning in a mental rotation task across a 75-day study (Kelly & Beltz, ##REF##33106024##2021##), and there was a short-term practice effect for executive function over the first 4 days of a recent 2-week study (Wang et al., ##UREF##15##2021##). Thus, the current findings may reflect something unique about executive function (or repeated assessments of it), as IC is typically thought to be a subdomain. Findings could also reflect something unique about adolescence. Younger participants may need more time to become accustomed to intensive assessments (in which case, the first week could reflect noise), or adolescents may be more flexible and malleable thinkers compared to young and older adults (Laube et al., ##REF##32072931##2020##), who have been the participants in most intensive longitudinal studies (in which case, the first week could reflect learning). This is an exciting area for future research.</p>","<p id=\"Par57\">Clearly, this finding has significant implications for the design and implementation of future intensive longitudinal research as well. Quite a few ‘intensive’ studies are 14 days long (McNeish et al., ##REF##34737528##2021##), which means that their first half could reflect task-related noise or learning effects rather than reflections of daily experiences. Extended training opportunities may be necessary in future studies, and data analytics that explicitly consider stationarity should be used. For instance, daily data detrending was used in this paper’s network analyses and other studies have excluded the first day (or several days) of data collection (e.g., Kelly & Beltz ##REF##33106024##2021##), but these approaches are debatable, and no consensus exists. If more than 14 days of data are available, then continuous time and other models of non-stationarity could be employed, which is an area ripe for future applications (Zhu et al., ##UREF##16##2021##).</p>","<p id=\"Par58\">Validity was assessed via correlations of daily IC with standard baseline measures (across participants, separately for each day). As expected, correlations with cognitive measures (i.e., working memory and attentional control) and age were low to moderate, but were always positive and were replicated in multilevel analyses adjusting for sibling dependencies; this provides some evidence of convergent validity. Also as expected, perceptual sensitivity and gender were not associated with IC, providing some evidence of divergent validity.</p>","<p id=\"Par59\">As higher correlations between daily IC and baseline cognition might have been expected, the reported moderate correlations could reflect some degree of unreliability or some influence of daily experiences. IC and working memory are distinct executive functioning skills that continue to differentiate during adolescence (Miyake & Friedman, ##REF##22773897##2012##), and low correlations (<italic>r</italic> = 0.20 - 0.30) are common in nomothetic and population-level studies (Chaku et al., ##REF##35238432##2022##; Ferguson et al., ##REF##33446798##2021##). Attentional control was also measured via self-report and low correlations are expected with cross-modality measures (Snyder et al., ##REF##33084353##2021##). In task-based measures, participants provide ‘objective’ data with narrowly defined goals, but in self-report measures, participants provide ‘subjective’ data about how they broadly utilize cognitive skills across situations and time (Dang et al., ##REF##32160564##2020##). One exciting direction for future intensive longitudinal research involves assessing multiple inhibitory control tasks, or even other executive functioning tasks. Indeed, a recent study assessing multiple daily executive function tasks over two weeks found that about 40% of daily variation in the latent construct could be attributed to within-person variability (Wang et al., ##UREF##15##2021##). Thus, future work could consider additional psychometric properties of daily IC or even <italic>individual differences in the structure of executive function</italic> via multilevel or person-specific factor analyses, respectively (Nesselroade & Ford, ##REF##3903891##1985##; Vogelsmeier et al., ##UREF##14##2019##).</p>","<p id=\"Par60\">Alternatively, moderate correlations between daily and baseline measures could reflect true variation. Indeed, unreliability and true daily fluctuation would both appear as occasion-to-occasion variation in daily data. The IC task was developed specifically to capture adolescents’ <italic>daily</italic> cognition. It was expected to fluctuate and change potentially in concert with adolescent experiences (Molenaar, ##UREF##7##2004##; Nesselroade, ##UREF##8##1991##), whereas existing, standard cognitive measures assume stability across contexts and that variation over short periods of time is merely noise (Bielak et al., ##REF##37771386##2017##). Given these conceptual differences, it is not surprising that daily IC exhibited low-to-moderate correlations with baseline cognition. They shared some degree of common variance, but daily IC ultimately provided distinct, complementary information. As this measure is openly available (<ext-link ext-link-type=\"uri\" xlink:href=\"https://osf.io/9yabr/\">https://osf.io/9yabr/</ext-link>), there is ample opportunity for future work to examine the extent to which IC fluctuations are <italic>truly</italic> explained by daily experiences.</p>","<title>Daily Fluctuations in IC Matter for Impulsive Behaviors</title>","<p id=\"Par61\">IC exhibited significant fluctuations across days. Gender was not associated with fluctuations, but older adolescents demonstrated fewer fluctuations than younger adolescents. This aligns with an extant literature showing that intraindividual variability decreases from childhood to adolescence, as cognitive skills become more efficient and stable (Dykiert et al., ##REF##23071524##2012##; Hultsch et al., ##UREF##4##2011##). Thus, intraindividual variability may contain valuable information about development (Lövden et al., ##UREF##5##2013##; Tamnes et al., ##REF##22262895##2012##) and IC fluctuations (i.e., i<italic>SD</italic>) may be a developmental marker – a construct independent of an individual’s level of IC (i.e., i<italic>M</italic>).</p>","<p id=\"Par62\">Links between daily IC and baseline impulsivity support this notion. Notably, fluctuations in IC were uniquely associated with greater lack of perseverance, suggesting that adolescents whose IC levels varied more across days were also more likely to give up during difficult tasks. This was true even when controlling for average IC, indicating that fluctuations in IC provide distinct information about constructs related to impulsivity. Adolescents who experience more fluctuations in IC may live in more chaotic environments or may be more susceptible to environmental changes (e.g., less sleep, more stress); thus, variability may be a developmental marker of stress reactivity or vulnerability (Nesselroade, ##UREF##8##1991##). Understanding how these fluctuations are linked to everyday experiences is just beginning to be realized and future time-indexed, multivariate studies would be well-poised to investigate these questions. Beyond fluctuations and perseverance, higher average IC was also associated with less positive and negative urgency, suggesting that average IC – assessed in the context of adolescents’ everyday lives – may be particularly relevant for impulsive behaviors that occur in emotional or motivational situations. This nomothetic inference is consistent with the larger literature suggesting that IC is particularly salient for externalizing behaviors (Heffer & Willoughby, ##REF##33401153##2021##).</p>","<title>Adolescents Are Unique</title>","<p id=\"Par63\">Research has largely focused on characterizing the ‘average’ adolescent, but averages cannot characterize the biopsychosocial experiences that are unique to a single adolescent (Molenaar, ##UREF##7##2004##), so a person-specific approach (i.e., GIMME) was used to accurately model developmental heterogeneity among the daily data of adolescents who used substances and those who did not (Gates & Molenaar, ##REF##22732562##2012##; Henry et al., ##REF##30583065##2019##). Even though GIMME prioritized commonalities across all adolescents and within subgroups during model building, none were found; in fact, no two individuals shared the exact same pattern of associations. This highlights the significant heterogeneity among adolescents who use substances and how links between IC and externalizing behaviors may unfold in unique ways for unique youth. As with all approaches, though, some caution is warranted because several features of the dataset (e.g., power or measurement intervals) could impact estimation of individual-level network features (see Weigard et al., ##REF##34252724##2021##).</p>","<p id=\"Par64\">Despite this, indices that quantified the importance of each variable in the adolescent-specific networks (i.e., centrality) suggested that IC played a more important role in the networks of adolescents who used substances than those who did not. Thus, IC may represent a hub that integrates and distributes information within and between externalizing and social behaviors for adolescents who use substances. Indeed, nomothetic studies have found that IC explains more variance (i.e., plays a central role) in externalizing behaviors than working memory or other cognitive skills (Young et al., ##REF##19222319##2009##). Future intensive longitudinal work could explore the specific influence of IC on externalizing behaviors, for example, by leveraging the direction of relations with IC to determine whether it is more likely to influence (i.e., out-degree) or be influenced by (i.e., in-degree) other network behaviors. Such directional inferences require specialized network analyses, such as the multiple solutions version of GIMME (Beltz & Molenaar, ##REF##27093380##2016##).</p>","<p id=\"Par65\">Although this GIMME analysis only focused on a subset of adolescents who used substances in order to emphasize insights into externalizing behaviors, it nonetheless illustrates one way in which intensive longitudinal data can be used to inform future personalized studies of externalizing behaviors. Clinical science already has a rich history of examining externalizing behaviors in context via EMA (reviewed in Votaw & Witkiewitz ##REF##34447615##2021##), and future research could benefit from intensively measuring individuals during interventions, paving the way forward for individualized, tailored, and even adaptive treatments (Ginexi et al., ##REF##24711629##2014##). For example, a randomized clinical trial used 2-week pre-treatment EMA data to develop individualized cognitive behavioral therapy plans for individuals struggling with substance use, and those strategies were utilized during temptation, according to post-treatment EMA data (Litt et al., ##REF##19712124##2009##). As EMA typically has fewer and shorter measures than the 100-day intensive longitudinal study described here, it is easy to envision the extensive opportunities for personalization in both basic and applied science.</p>","<p id=\"Par66\">There are also new and exciting questions that could be answered with personalized analyses of intensive longitudinal data. For instance, although relations between cognition and other behaviors (e.g., sleep, affect, and mood) are well-established at the between-person level, there is limited and somewhat contradictory, evidence at the within-person level (Hawks et al., ##REF##36922302##2022##; Neubauer et al., ##REF##30556707##2019##). As reliable and valid intensive cognitive assessments (like the IC task used here) become increasingly available, though, within-person insights may become clear. Another important direction concerns how intraindividual variation in IC (e.g., its stability or behavioral network centrality) over development is related to interindividual differences in sensation seeking and other externalizing behaviors (e.g., substance use, antisocial behavior, or conduct disorders) known to normatively shift across adolescence. IC’s daily fluctuation or centrality in a network may be a marker of development or maturation, which could be revealed in measurement burst designs that incorporate intensive measurement (e.g., over days or weeks) with traditional longitudinal measurement (e.g., annually; Sliwinski ##UREF##10##2008##).</p>","<title>Study Considerations</title>","<p id=\"Par67\">This study had several limitations that should be considered. First, data collection was completed during the novel coronavirus-19 (COVID-19) pandemic; inferences may have been impacted by this period of heightened instability. All testing was conducted during the pandemic, though, so effects were similarly distributed across adolescents, and intensive longitudinal studies emphasize intraindividual <italic>variation</italic> (not necessarily <italic>levels</italic>).</p>","<p id=\"Par0067\">Second, because all testing was conducted virtually, a small subset of days (representing < 0.5 of all data) may have been affected by technical difficulties (e.g., screen freezing or spotty connectivity). Further, it was difficult to examine potential impacts of the timing of daily survey completion (e.g., whether adolescents completed a daily survey the next morning) due to software limitations. This is somewhat common in intensive longitudinal studies (Keijsers et al., ##REF##34788708##2022##). Future research could use local application-based assessments (that do not require Internet connectivity) or ask participants to report technical difficulties, but these alternatives may be costly or burdensome for participants.</p>","<p id=\"Par68\">Third, the sample (<italic>N</italic> = 106) was smaller than in other validation studies (Collins et al., ##REF##26553135##2016##), including some that included intensive longitudinal cognitive measures (e.g., Kelly & Beltz ##REF##33106024##2021##), but the 100-day time series was longer than other intensive longitudinal studies, and retention was excellent. Of recruited adolescents, 61% completed over 80% of the daily diaries (with an average response rate of 94%). This completion rate is like other intensive longitudinal studies using adult samples (Teague et al., ##REF##30477443##2018##; Wright et al., ##REF##30920277##2019##) and better than others using adolescent samples (Keijsers et al., ##REF##34788708##2022##).</p>","<p id=\"Par69\">Fourth, although the sample was generally reflective of the surrounding area, there was limited race/ethnic diversity, with most adolescents endorsing White race/ethnicities (75.5%); thus, findings may not be generalizable to all adolescents and must be replicated in larger and more representative samples of uniquely diverse youth. Further, although excluded participants were similar in age, gender, and race/ethnicity to included participants, they did report more impulsive behaviors and lower cognitive skills, suggesting that study findings may also have limited generalizability to participants with high externalizing problems. This is unfortunately a common finding in traditional cross-sectional and longitudinal research even though other intensive longitudinal studies do not systematically report associations between psychological characteristics and participant retention (see Ewing et al., ##REF##29150307##2018##). Although not without trade-offs, ways to facilitate retention include using shorter daily surveys, collecting data for fewer days, implementing planned missingness or related designs (Yuan et al., ##REF##32367549##2020##), and leveraging passive sensing data (Ponnada et al., ##REF##35138253##2022##). The present study was designed to be 100 days because it provided enough within-person power to estimate person-specific models with 20% missing data (Rankin & Marsh, ##UREF##9##1985##); with a 30-day study, fewer data would likely be missing, but person-specific analyses could not be conducted.</p>","<p id=\"Par70\">Fifth, identical measures were not used at baseline and in the daily assessments, limiting direct comparisons, but this is true in almost all intensive longitudinal studies, as measures must be adapted for repeated use (Chaku & Beltz, ##UREF##1##2022##). Finally, only adolescents (e.g., not parents) reported on daily externalizing behaviors; there was low endorsement of externalizing behaviors generally, and clinically-significant externalizing disorders were not directly assessed. Only 12 adolescents endorsed using any type of substance during the study and rates of impulsive behaviors were generally low. This could be related to adolescent ability to access substances during the COVID-19 pandemic. Nonetheless, these are important avenues to explore in future research. Parent reports would be especially relevant for younger samples and replicating these analyses in clinical samples could shed light on patterns unique to those on the externalizing spectrum, as daily IC may represent different constructs in those with, for example, substance use disorders versus antisocial behavior.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par54\\\">IC is often conceptualized as a relatively stable construct, but growing evidence suggests that cognition varies across contexts and time (Brose et al., ##REF##24364855##2014##; Sliwinski et al., ##REF##16953716##2006##). Although this has significant implications for adolescent externalizing behaviors, the relevant literature has been stagnated by a lack of suitable intensive longitudinal cognitive assessments. Thus, the present study considered the extent to which adolescent IC – captured by 100 novel daily measurements – fluctuates from day-to-day in ways that are meaningfully associated with impulsivity and differ by the daily experiences of adolescent who do and do not use substances.</p>\",\n \"<title>IC Can Be Measured Daily</title>\",\n \"<p id=\\\"Par55\\\">Findings suggest that the novel IC task is a reliable and valid method for assessing daily cognitive processes related to inhibition. Parallel forms reliability was determined by comparing <italic>M</italic>s and <italic>SD</italic>s across days and calculating ICCs. The 100-day distribution of scores was non-normal: Specifically, IC performance increased over the first week before stabilizing, which may be due to initial task-related learning. Indeed, when the first seven days were excluded, IC scores from the remaining 93 days approximated a normal distribution. Moreover, variation across participants increased over the course of the study until about day 70, indicating increasing then plateauing between-person differences in IC. This is consistent with the notion that contextual demands can reduce individual differences (Miller et al., ##REF##17225514##2006##), with the novelty of the study being an initially constraining context. ICCs also indicated that individuals’ scores displayed moderate reliability across days (Bartko, ##REF##5942109##1966##). This is aligned with expectations for daily studies of cognition - prior work has suggested moderate ICCs are likely well-suited to intensive longitudinal research, as they reflect both between-person stability of assessments and potentially true variation in those assessments within a person over time (Bolger & Laurenceau, ##UREF##0##2013##).</p>\",\n \"<p id=\\\"Par56\\\">Notably, most other daily cognition validation studies have not shown similar first-week effects, although there was evidence for learning in a mental rotation task across a 75-day study (Kelly & Beltz, ##REF##33106024##2021##), and there was a short-term practice effect for executive function over the first 4 days of a recent 2-week study (Wang et al., ##UREF##15##2021##). Thus, the current findings may reflect something unique about executive function (or repeated assessments of it), as IC is typically thought to be a subdomain. Findings could also reflect something unique about adolescence. Younger participants may need more time to become accustomed to intensive assessments (in which case, the first week could reflect noise), or adolescents may be more flexible and malleable thinkers compared to young and older adults (Laube et al., ##REF##32072931##2020##), who have been the participants in most intensive longitudinal studies (in which case, the first week could reflect learning). This is an exciting area for future research.</p>\",\n \"<p id=\\\"Par57\\\">Clearly, this finding has significant implications for the design and implementation of future intensive longitudinal research as well. Quite a few ‘intensive’ studies are 14 days long (McNeish et al., ##REF##34737528##2021##), which means that their first half could reflect task-related noise or learning effects rather than reflections of daily experiences. Extended training opportunities may be necessary in future studies, and data analytics that explicitly consider stationarity should be used. For instance, daily data detrending was used in this paper’s network analyses and other studies have excluded the first day (or several days) of data collection (e.g., Kelly & Beltz ##REF##33106024##2021##), but these approaches are debatable, and no consensus exists. If more than 14 days of data are available, then continuous time and other models of non-stationarity could be employed, which is an area ripe for future applications (Zhu et al., ##UREF##16##2021##).</p>\",\n \"<p id=\\\"Par58\\\">Validity was assessed via correlations of daily IC with standard baseline measures (across participants, separately for each day). As expected, correlations with cognitive measures (i.e., working memory and attentional control) and age were low to moderate, but were always positive and were replicated in multilevel analyses adjusting for sibling dependencies; this provides some evidence of convergent validity. Also as expected, perceptual sensitivity and gender were not associated with IC, providing some evidence of divergent validity.</p>\",\n \"<p id=\\\"Par59\\\">As higher correlations between daily IC and baseline cognition might have been expected, the reported moderate correlations could reflect some degree of unreliability or some influence of daily experiences. IC and working memory are distinct executive functioning skills that continue to differentiate during adolescence (Miyake & Friedman, ##REF##22773897##2012##), and low correlations (<italic>r</italic> = 0.20 - 0.30) are common in nomothetic and population-level studies (Chaku et al., ##REF##35238432##2022##; Ferguson et al., ##REF##33446798##2021##). Attentional control was also measured via self-report and low correlations are expected with cross-modality measures (Snyder et al., ##REF##33084353##2021##). In task-based measures, participants provide ‘objective’ data with narrowly defined goals, but in self-report measures, participants provide ‘subjective’ data about how they broadly utilize cognitive skills across situations and time (Dang et al., ##REF##32160564##2020##). One exciting direction for future intensive longitudinal research involves assessing multiple inhibitory control tasks, or even other executive functioning tasks. Indeed, a recent study assessing multiple daily executive function tasks over two weeks found that about 40% of daily variation in the latent construct could be attributed to within-person variability (Wang et al., ##UREF##15##2021##). Thus, future work could consider additional psychometric properties of daily IC or even <italic>individual differences in the structure of executive function</italic> via multilevel or person-specific factor analyses, respectively (Nesselroade & Ford, ##REF##3903891##1985##; Vogelsmeier et al., ##UREF##14##2019##).</p>\",\n \"<p id=\\\"Par60\\\">Alternatively, moderate correlations between daily and baseline measures could reflect true variation. Indeed, unreliability and true daily fluctuation would both appear as occasion-to-occasion variation in daily data. The IC task was developed specifically to capture adolescents’ <italic>daily</italic> cognition. It was expected to fluctuate and change potentially in concert with adolescent experiences (Molenaar, ##UREF##7##2004##; Nesselroade, ##UREF##8##1991##), whereas existing, standard cognitive measures assume stability across contexts and that variation over short periods of time is merely noise (Bielak et al., ##REF##37771386##2017##). Given these conceptual differences, it is not surprising that daily IC exhibited low-to-moderate correlations with baseline cognition. They shared some degree of common variance, but daily IC ultimately provided distinct, complementary information. As this measure is openly available (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://osf.io/9yabr/\\\">https://osf.io/9yabr/</ext-link>), there is ample opportunity for future work to examine the extent to which IC fluctuations are <italic>truly</italic> explained by daily experiences.</p>\",\n \"<title>Daily Fluctuations in IC Matter for Impulsive Behaviors</title>\",\n \"<p id=\\\"Par61\\\">IC exhibited significant fluctuations across days. Gender was not associated with fluctuations, but older adolescents demonstrated fewer fluctuations than younger adolescents. This aligns with an extant literature showing that intraindividual variability decreases from childhood to adolescence, as cognitive skills become more efficient and stable (Dykiert et al., ##REF##23071524##2012##; Hultsch et al., ##UREF##4##2011##). Thus, intraindividual variability may contain valuable information about development (Lövden et al., ##UREF##5##2013##; Tamnes et al., ##REF##22262895##2012##) and IC fluctuations (i.e., i<italic>SD</italic>) may be a developmental marker – a construct independent of an individual’s level of IC (i.e., i<italic>M</italic>).</p>\",\n \"<p id=\\\"Par62\\\">Links between daily IC and baseline impulsivity support this notion. Notably, fluctuations in IC were uniquely associated with greater lack of perseverance, suggesting that adolescents whose IC levels varied more across days were also more likely to give up during difficult tasks. This was true even when controlling for average IC, indicating that fluctuations in IC provide distinct information about constructs related to impulsivity. Adolescents who experience more fluctuations in IC may live in more chaotic environments or may be more susceptible to environmental changes (e.g., less sleep, more stress); thus, variability may be a developmental marker of stress reactivity or vulnerability (Nesselroade, ##UREF##8##1991##). Understanding how these fluctuations are linked to everyday experiences is just beginning to be realized and future time-indexed, multivariate studies would be well-poised to investigate these questions. Beyond fluctuations and perseverance, higher average IC was also associated with less positive and negative urgency, suggesting that average IC – assessed in the context of adolescents’ everyday lives – may be particularly relevant for impulsive behaviors that occur in emotional or motivational situations. This nomothetic inference is consistent with the larger literature suggesting that IC is particularly salient for externalizing behaviors (Heffer & Willoughby, ##REF##33401153##2021##).</p>\",\n \"<title>Adolescents Are Unique</title>\",\n \"<p id=\\\"Par63\\\">Research has largely focused on characterizing the ‘average’ adolescent, but averages cannot characterize the biopsychosocial experiences that are unique to a single adolescent (Molenaar, ##UREF##7##2004##), so a person-specific approach (i.e., GIMME) was used to accurately model developmental heterogeneity among the daily data of adolescents who used substances and those who did not (Gates & Molenaar, ##REF##22732562##2012##; Henry et al., ##REF##30583065##2019##). Even though GIMME prioritized commonalities across all adolescents and within subgroups during model building, none were found; in fact, no two individuals shared the exact same pattern of associations. This highlights the significant heterogeneity among adolescents who use substances and how links between IC and externalizing behaviors may unfold in unique ways for unique youth. As with all approaches, though, some caution is warranted because several features of the dataset (e.g., power or measurement intervals) could impact estimation of individual-level network features (see Weigard et al., ##REF##34252724##2021##).</p>\",\n \"<p id=\\\"Par64\\\">Despite this, indices that quantified the importance of each variable in the adolescent-specific networks (i.e., centrality) suggested that IC played a more important role in the networks of adolescents who used substances than those who did not. Thus, IC may represent a hub that integrates and distributes information within and between externalizing and social behaviors for adolescents who use substances. Indeed, nomothetic studies have found that IC explains more variance (i.e., plays a central role) in externalizing behaviors than working memory or other cognitive skills (Young et al., ##REF##19222319##2009##). Future intensive longitudinal work could explore the specific influence of IC on externalizing behaviors, for example, by leveraging the direction of relations with IC to determine whether it is more likely to influence (i.e., out-degree) or be influenced by (i.e., in-degree) other network behaviors. Such directional inferences require specialized network analyses, such as the multiple solutions version of GIMME (Beltz & Molenaar, ##REF##27093380##2016##).</p>\",\n \"<p id=\\\"Par65\\\">Although this GIMME analysis only focused on a subset of adolescents who used substances in order to emphasize insights into externalizing behaviors, it nonetheless illustrates one way in which intensive longitudinal data can be used to inform future personalized studies of externalizing behaviors. Clinical science already has a rich history of examining externalizing behaviors in context via EMA (reviewed in Votaw & Witkiewitz ##REF##34447615##2021##), and future research could benefit from intensively measuring individuals during interventions, paving the way forward for individualized, tailored, and even adaptive treatments (Ginexi et al., ##REF##24711629##2014##). For example, a randomized clinical trial used 2-week pre-treatment EMA data to develop individualized cognitive behavioral therapy plans for individuals struggling with substance use, and those strategies were utilized during temptation, according to post-treatment EMA data (Litt et al., ##REF##19712124##2009##). As EMA typically has fewer and shorter measures than the 100-day intensive longitudinal study described here, it is easy to envision the extensive opportunities for personalization in both basic and applied science.</p>\",\n \"<p id=\\\"Par66\\\">There are also new and exciting questions that could be answered with personalized analyses of intensive longitudinal data. For instance, although relations between cognition and other behaviors (e.g., sleep, affect, and mood) are well-established at the between-person level, there is limited and somewhat contradictory, evidence at the within-person level (Hawks et al., ##REF##36922302##2022##; Neubauer et al., ##REF##30556707##2019##). As reliable and valid intensive cognitive assessments (like the IC task used here) become increasingly available, though, within-person insights may become clear. Another important direction concerns how intraindividual variation in IC (e.g., its stability or behavioral network centrality) over development is related to interindividual differences in sensation seeking and other externalizing behaviors (e.g., substance use, antisocial behavior, or conduct disorders) known to normatively shift across adolescence. IC’s daily fluctuation or centrality in a network may be a marker of development or maturation, which could be revealed in measurement burst designs that incorporate intensive measurement (e.g., over days or weeks) with traditional longitudinal measurement (e.g., annually; Sliwinski ##UREF##10##2008##).</p>\",\n \"<title>Study Considerations</title>\",\n \"<p id=\\\"Par67\\\">This study had several limitations that should be considered. First, data collection was completed during the novel coronavirus-19 (COVID-19) pandemic; inferences may have been impacted by this period of heightened instability. All testing was conducted during the pandemic, though, so effects were similarly distributed across adolescents, and intensive longitudinal studies emphasize intraindividual <italic>variation</italic> (not necessarily <italic>levels</italic>).</p>\",\n \"<p id=\\\"Par0067\\\">Second, because all testing was conducted virtually, a small subset of days (representing < 0.5 of all data) may have been affected by technical difficulties (e.g., screen freezing or spotty connectivity). Further, it was difficult to examine potential impacts of the timing of daily survey completion (e.g., whether adolescents completed a daily survey the next morning) due to software limitations. This is somewhat common in intensive longitudinal studies (Keijsers et al., ##REF##34788708##2022##). Future research could use local application-based assessments (that do not require Internet connectivity) or ask participants to report technical difficulties, but these alternatives may be costly or burdensome for participants.</p>\",\n \"<p id=\\\"Par68\\\">Third, the sample (<italic>N</italic> = 106) was smaller than in other validation studies (Collins et al., ##REF##26553135##2016##), including some that included intensive longitudinal cognitive measures (e.g., Kelly & Beltz ##REF##33106024##2021##), but the 100-day time series was longer than other intensive longitudinal studies, and retention was excellent. Of recruited adolescents, 61% completed over 80% of the daily diaries (with an average response rate of 94%). This completion rate is like other intensive longitudinal studies using adult samples (Teague et al., ##REF##30477443##2018##; Wright et al., ##REF##30920277##2019##) and better than others using adolescent samples (Keijsers et al., ##REF##34788708##2022##).</p>\",\n \"<p id=\\\"Par69\\\">Fourth, although the sample was generally reflective of the surrounding area, there was limited race/ethnic diversity, with most adolescents endorsing White race/ethnicities (75.5%); thus, findings may not be generalizable to all adolescents and must be replicated in larger and more representative samples of uniquely diverse youth. Further, although excluded participants were similar in age, gender, and race/ethnicity to included participants, they did report more impulsive behaviors and lower cognitive skills, suggesting that study findings may also have limited generalizability to participants with high externalizing problems. This is unfortunately a common finding in traditional cross-sectional and longitudinal research even though other intensive longitudinal studies do not systematically report associations between psychological characteristics and participant retention (see Ewing et al., ##REF##29150307##2018##). Although not without trade-offs, ways to facilitate retention include using shorter daily surveys, collecting data for fewer days, implementing planned missingness or related designs (Yuan et al., ##REF##32367549##2020##), and leveraging passive sensing data (Ponnada et al., ##REF##35138253##2022##). The present study was designed to be 100 days because it provided enough within-person power to estimate person-specific models with 20% missing data (Rankin & Marsh, ##UREF##9##1985##); with a 30-day study, fewer data would likely be missing, but person-specific analyses could not be conducted.</p>\",\n \"<p id=\\\"Par70\\\">Fifth, identical measures were not used at baseline and in the daily assessments, limiting direct comparisons, but this is true in almost all intensive longitudinal studies, as measures must be adapted for repeated use (Chaku & Beltz, ##UREF##1##2022##). Finally, only adolescents (e.g., not parents) reported on daily externalizing behaviors; there was low endorsement of externalizing behaviors generally, and clinically-significant externalizing disorders were not directly assessed. Only 12 adolescents endorsed using any type of substance during the study and rates of impulsive behaviors were generally low. This could be related to adolescent ability to access substances during the COVID-19 pandemic. Nonetheless, these are important avenues to explore in future research. Parent reports would be especially relevant for younger samples and replicating these analyses in clinical samples could shed light on patterns unique to those on the externalizing spectrum, as daily IC may represent different constructs in those with, for example, substance use disorders versus antisocial behavior.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p id=\"Par71\">Externalizing behaviors normatively increase during adolescence in ways that accentuate individual differences and implicate IC (Young et al., ##REF##19222319##2009##). Yet, IC – and its expression across different contexts and times – is thought to be stable even though growing evidence suggests it may vary within individuals (Brose et al., ##REF##24364855##2014##; Sliwinski et al., ##REF##16953716##2006##). Thus, single assessments that describe ‘average’ adolescents may not accurately capture the lived experiences of individuals, including their daily highs and lows. In an attempt to fill this knowledge gap, this intensive longitudinal study followed over 100 adolescents for 100 days while they completed a novel, openly available, IC task. The task was generally found to be reliable and valid, with daily levels of IC and fluctuations in IC associated with individual differences in impulsive behaviors. Moreover, an adolescent-specific network analysis comparing adolescents who used substances over the 100 days to those who did not revealed that IC was central to the daily interplay among impulsivity and adolescent social experiences, but only for substance users. Thus, the new IC measure – and the illustration of its use in adolescent intensive longitudinal research – encourages future innovation in the investigation of adolescent-specific externalizing behaviors.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p id=\\\"Par71\\\">Externalizing behaviors normatively increase during adolescence in ways that accentuate individual differences and implicate IC (Young et al., ##REF##19222319##2009##). Yet, IC – and its expression across different contexts and times – is thought to be stable even though growing evidence suggests it may vary within individuals (Brose et al., ##REF##24364855##2014##; Sliwinski et al., ##REF##16953716##2006##). Thus, single assessments that describe ‘average’ adolescents may not accurately capture the lived experiences of individuals, including their daily highs and lows. In an attempt to fill this knowledge gap, this intensive longitudinal study followed over 100 adolescents for 100 days while they completed a novel, openly available, IC task. The task was generally found to be reliable and valid, with daily levels of IC and fluctuations in IC associated with individual differences in impulsive behaviors. Moreover, an adolescent-specific network analysis comparing adolescents who used substances over the 100 days to those who did not revealed that IC was central to the daily interplay among impulsivity and adolescent social experiences, but only for substance users. Thus, the new IC measure – and the illustration of its use in adolescent intensive longitudinal research – encourages future innovation in the investigation of adolescent-specific externalizing behaviors.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p id=\"Par1\">Inhibitory control is a transdiagnostic risk factor for externalizing behaviors, particularly during adolescence. Despite advances in understanding links between inhibitory control and externalizing behaviors across youth <italic>on average</italic>, significant questions remain about how these links play out in the day-to-day lives of individual adolescents. The goals of the current study were to: (1) validate a novel 100-occasion measure of inhibitory control; (2) assess links between day-to-day fluctuations in inhibitory control and individual differences in externalizing behaviors; and (3) illustrate the potential of intensive longitudinal studies for person-specific analyses of adolescent externalizing behaviors. Participants were 106 youth (57.5% female, <italic>M</italic><sub><italic>age</italic> </sub>= 13.34 years; <italic>SD</italic><sub><italic>age </italic></sub>= 1.92) who completed a virtual baseline session followed by 100 daily surveys, including an adapted Stroop Color Word task designed to assess inhibitory control. Results suggested that the novel task was generally reliable and valid, and that inhibitory control fluctuated across days in ways that were meaningfully associated with individual differences in baseline impulsive behaviors. Results of illustrative personalized analyses suggested that inhibitory control had more influence in the daily networks of adolescents who used substances during the 100 days than in a matched set of adolescents who did not. This work marks a path forward in intensive longitudinal research by validating a novel inhibitory control measure, revealing that daily fluctuations in inhibitory control may be a unique construct broadly relevant to adolescent externalizing problems, and at the same time, highlighting that links between daily inhibitory control and impulsive behaviors are adolescent-specific.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s10802-023-01071-y.</p>","<title>Keywords</title>"],"string":"[\n \"<p id=\\\"Par1\\\">Inhibitory control is a transdiagnostic risk factor for externalizing behaviors, particularly during adolescence. Despite advances in understanding links between inhibitory control and externalizing behaviors across youth <italic>on average</italic>, significant questions remain about how these links play out in the day-to-day lives of individual adolescents. The goals of the current study were to: (1) validate a novel 100-occasion measure of inhibitory control; (2) assess links between day-to-day fluctuations in inhibitory control and individual differences in externalizing behaviors; and (3) illustrate the potential of intensive longitudinal studies for person-specific analyses of adolescent externalizing behaviors. Participants were 106 youth (57.5% female, <italic>M</italic><sub><italic>age</italic> </sub>= 13.34 years; <italic>SD</italic><sub><italic>age </italic></sub>= 1.92) who completed a virtual baseline session followed by 100 daily surveys, including an adapted Stroop Color Word task designed to assess inhibitory control. Results suggested that the novel task was generally reliable and valid, and that inhibitory control fluctuated across days in ways that were meaningfully associated with individual differences in baseline impulsive behaviors. Results of illustrative personalized analyses suggested that inhibitory control had more influence in the daily networks of adolescents who used substances during the 100 days than in a matched set of adolescents who did not. This work marks a path forward in intensive longitudinal research by validating a novel inhibitory control measure, revealing that daily fluctuations in inhibitory control may be a unique construct broadly relevant to adolescent externalizing problems, and at the same time, highlighting that links between daily inhibitory control and impulsive behaviors are adolescent-specific.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s10802-023-01071-y.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<p id=\"Par6\">Behaviors on the externalizing spectrum, such as impulsivity, substance use, and conduct problems, increase during adolescence. Although this increase is generally considered normative, individual differences in externalizing behaviors can result in social difficulties, school failure, and persistent deviance (Odgers et al., ##REF##18423100##2008##). Inhibitory control (IC; the ability to suppress prepotent responses) may play a key role in regulating externalizing behaviors (Young et al., ##REF##19222319##2009##). This field of research, however, has overwhelmingly relied on cross-sectional or a few longitudinal assessments, and typically represents IC as a trait or locally stable construct. Yet, growing evidence suggests that cognition fluctuates at granular timescales and is meaningfully linked to personal characteristics and wellbeing (Brose et al., ##REF##24364855##2014##; Sliwinski et al., ##REF##16953716##2006##). Thus, it is likely that IC is associated with externalizing behaviors in unique ways for individual adolescents, but that the research methods employed to-date could not empirically detect this.</p>","<p id=\"Par7\">Intensive longitudinal designs can be used to understand cognitive fluctuations and their psychological significance. These methods involve many repeated assessments of the same variables and participants over relatively short periods of time: from moment-to-moment (e.g., neural activity; Demidenko et al., ##REF##35043448##2022##), hour-to-hour (e.g., affect; Brose et al., ##REF##24364855##2014##), or day-to-day (e.g., personality; Kelly et al., ##REF##32736131##2020##), revealing how behavior, including cognition, unfolds within an individual. Intensive longitudinal designs can also support a range of inferences, including generalizable nomothetic inferences made from examinations of interindividual (i.e., between-person) variation, idiographic inferences about unique individuals made from examinations of intraindividual (i.e., within-person) variation, or a combination of the two (Molenaar, ##UREF##7##2004##).</p>","<p id=\"Par8\">Unfortunately, there is a lack of cognitive measures that are well-suited for intensive longitudinal assessment, potentially because highly controlled, lab-based measurement is at odds with mobile, ecologically-valid assessments (Roche et al., ##REF##27819470##2016##). Thus, the psychometric validation of intensive longitudinal cognitive assessments is a challenging, nascent area of research (but see Kelly & Beltz ##REF##33106024##2021##; Sliwinski et al., ##REF##27084835##2018##). The goals of the current study were to: (1) develop and validate a novel 100-occasion measure of IC; (2) assess how daily IC fluctuations were related to baseline impulsive behaviors; and (3) explicate the potential of idiographic analyses for revealing adolescent-specific links among daily IC and externalizing behavior in social contexts.</p>","<title>IC is a Risk Factor for Adolescent Externalizing Behaviors</title>","<p id=\"Par9\">IC is one of the most salient cognitive factors associated with externalizing behaviors (Young et al., ##REF##19222319##2009##). Although most work has focused on children (see Schoemaker et al., ##REF##23054130##2013##), IC plays an important role in adolescence, too. Deficits in IC reliably mark adolescent populations with clinically relevant problem behaviors. For instance, low IC predicts early onset of alcohol use-related problems (López-Caneda et al., ##REF##24243684##2014##), illicit drug use (Nigg et al., ##REF##16601652##2006##), and high likelihood of antisocial or oppositional defiant disorder diagnoses (Bonham et al., ##REF##33048265##2021##). Even in non-clinical samples, low IC is consistently linked with increased externalizing behaviors in both cross-sectional and longitudinal research (Kim-Spoon et al., ##REF##31073257##2019##).</p>","<p id=\"Par10\">IC may be particularly important for understanding adolescent behaviors because it continues to mature into early adulthood (Luna et al., ##REF##26154978##2015##) and occurs in the context of early adolescent increases in neural reactivity to socioemotional stimuli (Foulkes & Blakemore, ##REF##29403031##2018##), which may contribute to normative rises in externalizing behaviors (Lydon-Staley & Bassett, ##REF##30210404##2018##). Most studies on this topic to-date are cross-sectional and limited to nomothetic inferences even though they assume cognitive control and socioemotional reactivity change <italic>within individuals over time</italic> (see Beltz ##REF##29460359##2018##). Thus, there is theoretical acknowledgement of the importance of <italic>intra</italic>individual variation for adolescent externalizing behaviors, but there is little empirical evidence for it, as the extant literature primarily concerns <italic>inter</italic>individual variation (Chaku & Beltz, ##UREF##1##2022##). Intensive longitudinal research is needed to fill this critical knowledge gap.</p>","<title>From Nomothetic to Idiographic Assessments of IC</title>","<p id=\"Par11\">IC is often conceptualized as a relatively stable trait that can be assessed once and generalized across time. Converging evidence increasingly shows, however, that short-term fluctuations in cognition have significance for psychological wellbeing (Castellanos et al., ##REF##15950016##2005##; Hultsch et al., ##UREF##4##2011##). Numerous factors, such as interpersonal experiences, contextual demands, and emotional processes, can influence or be influenced by cognition at any given moment. For example, increased working memory fluctuations (measured across four weeks with four assessments a day) have been associated with poor sleep quality in early adolescents (Galeano-Keiner et al., ##UREF##3##2022##). Similarly, better working memory (measured daily over two weeks) has been associated with less negative mood and greater school belonging in late adolescence (Wang et al., ##UREF##15##2021##).</p>","<p id=\"Par12\">Unfortunately, work in this area is challenged by a lack of sufficient measurement tools. Current cognitive assessments (e.g., Stroop Color Word, Stop-Signal, and Go/No Go tasks) have primarily been used to capture normative IC-linked processes, but they have limitations (see Snyder et al., ##REF##25859234##2015##). Specifically, recent work suggests that the subtraction methods often used to analyze recorded task data (e.g., comparing reaction times on incongruent versus congruent trials) are unreliable and do not index individual differences well (Salthouse & Hedden, ##REF##12647765##2002##; Weigard et al., ##REF##34252724##2021##), which limits their suitability for studies of intraindividual variation.</p>","<p id=\"Par13\">Nonetheless, understanding short-term cognitive fluctuations is essential because patterns of intraindividual variability may reliably differ across individuals, potentially reflecting an enduring personal characteristic (Nesselroade, ##UREF##8##1991##). Although the extent to which fluctuations may reflect vulnerability versus resilience, and in turn adjustment, likely depends on the specific cognitive construct, context, or sample, the general importance of cognitive variation is being increasingly realized. For instance, greater response variability on a lab-based cognitive task was associated with lower perceptions of real-world risks (e.g., substance use) in 14-to-16-year-olds (Goldenberg et al., ##REF##27651539##2017##). Yet, most studies only capture trial-to-trial fluctuations during a single, highly controlled task, so little is known about how longer-term fluctuations in IC are associated with externalizing behaviors in everyday life. This is unfortunate because <italic>daily</italic> fluctuations are likely related to adolescents’ unique but recurring biopsychosocial experiences.</p>","<p id=\"Par14\">Beyond understanding fluctuations in cognition, daily data (if abundant) can also be used to make person-specific inferences. Adolescence is characterized by increased, variable opportunities for learning, adaptation, and exploration (Knoll et al., ##REF##27815519##2016##). These experiences do not just differ in degree, but also in nature and biopsychosocial context across individual adolescents (Chaku & Beltz, ##UREF##1##2022##). To capture this heterogeneity, averages (and even average fluctuations) must be eschewed in favor of person-specific models (Molenaar, ##UREF##7##2004##). For instance, group iterative multiple model estimation (GIMME; Gates & Molenaar ##REF##22732562##2012##) is a person-specific analytic technique that maps variation among pre-selected variables within a person over many relatively short intervals. Applied to daily adolescent behavior, GIMME considers each adolescent as if they are a sample of <italic>N</italic>=1, allowing for unique inferences about the timing of relations among variables (i.e., same-day or next-day) that potentially reflect adolescents’ real-world contexts. GIMME is unique among person-specific analytic approaches because it also affords some generalizable inferences. It does so without averaging by prioritizing relations that are common across a sample. Thus, when combined with daily data, GIMME and other person-specific techniques can be used to make potentially important insights into <italic>when</italic>, <italic>for whom</italic>, and <italic>in what situations</italic> links between IC and externalizing behaviors matter.</p>","<title>Intensive Longitudinal Measures of IC</title>","<p id=\"Par15\">The feasibility of intensive longitudinal studies is increasing due to wide accessibility of internet-capable devices and sophisticated analytic techniques for dependent data (Lydon-Staley & Bassett, ##REF##30210404##2018##). Still, there is a paucity of <italic>cognitive</italic> assessments available for inclusion in these studies. Beyond their traditionally restricted use in laboratories, this may be because cognitive measures cannot be intensively repeated due to trial counts, length, or practice effects, or because they require timing mechanisms that are difficult to apply in situ (Ladouce et al., ##REF##28127283##2016##).</p>","<p id=\"Par16\">Nonetheless, a few intensive cognitive assessments have been validated. For example, Sliwinski and colleagues (##REF##27084835##2018##) validated working memory (i.e., dot memory and n-Back) and perceptual speed (i.e., symbol search) tasks across 14-day ecological momentary assessment (EMA) bursts. Although links to other behaviors were not examined, the working memory task exhibited lower intraindividual reliability than the perceptual speed task. Recently, Kelly and Beltz (##REF##33106024##2021##) validated novel, openly available 75-occasion measures of delayed verbal recall (<ext-link ext-link-type=\"uri\" xlink:href=\"https://osf.io/vhr7u/\">https://osf.io/vhr7u/</ext-link>) and three-dimensional (3D) mental rotations (<ext-link ext-link-type=\"uri\" xlink:href=\"https://osf.io/m6ae8/\">https://osf.io/m6ae8/</ext-link>). This involved creating 75 unique sets of stimuli and establishing procedures for parallel forms reliability across sets. Overall, the 3D mental rotations measure had better psychometrics than the delayed verbal recall measure, however, there are significant challenges in distinguishing between unreliability and meaningful fluctuation in intensive longitudinal measurement (Keijsers et al., ##REF##34788708##2022##). Thus, the authors posed that verbal recall may be more influenced by daily experiences compared to mental rotations but did not empirically examine this possibility. This small literature suggests that intensive longitudinal measurement of cognition is feasible, but it has not yet been widely employed to study adolescent IC in everyday contexts.</p>","<title>The Current Study</title>","<p id=\"Par17\">IC likely varies from day-to-day in ways that have significance for externalizing behaviors in unique adolescents, but measures are not yet available to examine this. Thus, the overarching study goal was to reveal adolescents’ daily associations between IC and externalizing behaviors through three aims. First, a 100-occasion measure of IC was developed and psychometrically validated. Second, daily IC (both 100-day averages and fluctuations) were related to baseline impulsive behaviors; low averages and high fluctuations were expected to predict impulsivity. Third, the potential of person-specific analyses for revealing links between IC and externalizing behaviors was illustrated by comparing the daily networks of adolescents who used substances to the networks of a matched subsample who did not.</p>","<title>Fluctuations in Daily IC and Links to Impulsive Behaviors</title>","<p id=\"Par49\">Daily IC scores for 12 illustrative adolescents are shown in Fig. ##FIG##3##4## along with their i<italic>M</italic>s and i<italic>SD</italic>s. Adolescents demonstrated remarkably different trajectories. For example, ID 36 exhibited large day-to-day variation (i<italic>SD</italic> = 10.44), whereas the ID 73 exhibited far less variability (i<italic>SD</italic> = 3.16). Further, some adolescents were characterized by a relatively flat trajectory (e.g., ID 11) while others were better characterized by positive (e.g., ID 77), negative (e.g., ID 41), or non-linear (e.g., ID 29) trajectories. Across adolescents, the i<italic>M</italic> ranged from 12.79 to 55.58, and the i<italic>SD</italic> ranged from 3.16 to 15.44. A one-sample <italic>t</italic>-test indicated that the i<italic>SDs</italic> were significantly different from zero, <italic>t</italic>(105) = 31.53, <italic>p</italic> < .001, providing statistical evidence for IC fluctuations. The i<italic>M</italic> and i<italic>SD</italic> were not significantly correlated (<italic>r </italic>= -.01, <italic>p</italic> = .91). Although the i<italic>M</italic>s and i<italic>SD</italic>s did not vary by gender (<italic>p</italic>s > 0.05), they did covary with age, as older adolescents had higher scores (i<italic>M</italic>: <italic>r</italic> = .27, <italic>p</italic> = .01) and fewer fluctuations (i<italic>SD</italic>: <italic>r </italic>= -.19, <italic>p</italic> = .04).</p>","<p id=\"Par50\">\n\n</p>","<p id=\"Par51\">Multilevel models (nesting siblings within families) were then used to examine how i<italic>M</italic>s and i<italic>SD</italic>s were associated with impulsive behavior, accounting for gender and age. Higher i<italic>M</italic>s were associated with less positive urgency, <italic>b </italic>= -0.02(0.01), <italic>p</italic> = .03, and less negative urgency, <italic>b </italic>= -0.02(0.01), <italic>p</italic> = .04, suggesting that adolescents who had higher average daily IC tended to make less impulsive choices during extreme emotions. Higher i<italic>SD</italic>s were associated with lack of perseverance, <italic>b</italic> = 0.03(0.01), <italic>p</italic> = .03, even after controlling for i<italic>M</italic>s; this suggests that adolescents who had more variable IC were more likely to give up during difficult tasks. No significant links were found between daily IC and lack of premeditation or sensation seeking. Analyses were repeated excluding the first week of skewed assessments, and inferences were the same; see the supplemental materials.</p>","<title>Illustrative Adolescent-Specific Network Analyses</title>","<p id=\"Par531\">All adolescent-specific GIMME models fit the data well according to average indices: CFI = 0.99, NNFI = 1.00, RMSEA = 0.02, SRMR = 0.06. Surprisingly, there were no group- or subgroup-level relations (besides the specified autoregressions), indicating substantial heterogeneity among adolescents who used and did not use substances during the 100 days. Figure ##FIG##4##5##A shows the network of an adolescent (11.42-year-old male) who used alcohol. Circles represent variables and directed arrows represent relations between those variables; solid lines reflect same-day relations, dashed lines reflect next-day relations, red lines reflect positive relations, and blue lines reflect negative relations. Thus, this adolescent’s network contains eight relations, and only four were not specified <italic>a priori</italic> (i.e., the autoregressives). There is a positive, same-day relation between social time and positive urgency, indicating more impulsive behaviors due to positive emotions on days he was more social. There is also a pair of relations between positive and negative urgency, suggesting that negative urgency is related to same-day increases in positive urgency that propagate across time. Finally, there is an inverse, lagged relation between negative urgency and IC suggesting that increases in negative urgency are associated with IC declines the next day. This could reflect a downstream cognitive consequence of actions taken to alleviate unwanted emotions. Figure ##FIG##4##5##B shows the network of the matched adolescent (11.83-year-old male) who did not use substances. His network is less dense, with only two relations that are not autoregressives. Like the adolescent in Fig. ##FIG##4##5##A, there is a positive, same-day relation between negative and positive urgency. There is an additional positive, lagged relation between social time and negative urgency, though, potentially suggesting that his social interactions are related to next-day increases in negative urgency. Interestingly, however, his daily IC was not linked to externalizing or social experiences.</p>","<p id=\"Par53\">It is important to emphasize that the adolescents in Fig. ##FIG##4##5## do not characterize the all the adolescents in the sample nor do they characterize all the adolescents who used substances or those who did not in the sample; otherwise, the relations would have appeared at the group- or subgroup-level. Nonetheless, follow-up analyses suggested that adolescents who reported using substances tended to have more relations involving IC (i.e., greater IC centrality) compared to adolescents who did not use substances, <italic>t</italic>(22) = -2.24, <italic>p</italic> = .05, <italic>d</italic> = 0.74. Most (60%) relations with IC were negative (i.e., worse IC was associated with more negative urgency), suggesting that IC was more salient for the daily experiences of substance users.</p>","<p id=\"Par52\">\n\n</p>","<title>Electronic Supplementary Material</title>","<p>Below is the link to the electronic supplementary material.</p>","<p>\n\n</p>"],"string":"[\n \"<p id=\\\"Par6\\\">Behaviors on the externalizing spectrum, such as impulsivity, substance use, and conduct problems, increase during adolescence. Although this increase is generally considered normative, individual differences in externalizing behaviors can result in social difficulties, school failure, and persistent deviance (Odgers et al., ##REF##18423100##2008##). Inhibitory control (IC; the ability to suppress prepotent responses) may play a key role in regulating externalizing behaviors (Young et al., ##REF##19222319##2009##). This field of research, however, has overwhelmingly relied on cross-sectional or a few longitudinal assessments, and typically represents IC as a trait or locally stable construct. Yet, growing evidence suggests that cognition fluctuates at granular timescales and is meaningfully linked to personal characteristics and wellbeing (Brose et al., ##REF##24364855##2014##; Sliwinski et al., ##REF##16953716##2006##). Thus, it is likely that IC is associated with externalizing behaviors in unique ways for individual adolescents, but that the research methods employed to-date could not empirically detect this.</p>\",\n \"<p id=\\\"Par7\\\">Intensive longitudinal designs can be used to understand cognitive fluctuations and their psychological significance. These methods involve many repeated assessments of the same variables and participants over relatively short periods of time: from moment-to-moment (e.g., neural activity; Demidenko et al., ##REF##35043448##2022##), hour-to-hour (e.g., affect; Brose et al., ##REF##24364855##2014##), or day-to-day (e.g., personality; Kelly et al., ##REF##32736131##2020##), revealing how behavior, including cognition, unfolds within an individual. Intensive longitudinal designs can also support a range of inferences, including generalizable nomothetic inferences made from examinations of interindividual (i.e., between-person) variation, idiographic inferences about unique individuals made from examinations of intraindividual (i.e., within-person) variation, or a combination of the two (Molenaar, ##UREF##7##2004##).</p>\",\n \"<p id=\\\"Par8\\\">Unfortunately, there is a lack of cognitive measures that are well-suited for intensive longitudinal assessment, potentially because highly controlled, lab-based measurement is at odds with mobile, ecologically-valid assessments (Roche et al., ##REF##27819470##2016##). Thus, the psychometric validation of intensive longitudinal cognitive assessments is a challenging, nascent area of research (but see Kelly & Beltz ##REF##33106024##2021##; Sliwinski et al., ##REF##27084835##2018##). The goals of the current study were to: (1) develop and validate a novel 100-occasion measure of IC; (2) assess how daily IC fluctuations were related to baseline impulsive behaviors; and (3) explicate the potential of idiographic analyses for revealing adolescent-specific links among daily IC and externalizing behavior in social contexts.</p>\",\n \"<title>IC is a Risk Factor for Adolescent Externalizing Behaviors</title>\",\n \"<p id=\\\"Par9\\\">IC is one of the most salient cognitive factors associated with externalizing behaviors (Young et al., ##REF##19222319##2009##). Although most work has focused on children (see Schoemaker et al., ##REF##23054130##2013##), IC plays an important role in adolescence, too. Deficits in IC reliably mark adolescent populations with clinically relevant problem behaviors. For instance, low IC predicts early onset of alcohol use-related problems (López-Caneda et al., ##REF##24243684##2014##), illicit drug use (Nigg et al., ##REF##16601652##2006##), and high likelihood of antisocial or oppositional defiant disorder diagnoses (Bonham et al., ##REF##33048265##2021##). Even in non-clinical samples, low IC is consistently linked with increased externalizing behaviors in both cross-sectional and longitudinal research (Kim-Spoon et al., ##REF##31073257##2019##).</p>\",\n \"<p id=\\\"Par10\\\">IC may be particularly important for understanding adolescent behaviors because it continues to mature into early adulthood (Luna et al., ##REF##26154978##2015##) and occurs in the context of early adolescent increases in neural reactivity to socioemotional stimuli (Foulkes & Blakemore, ##REF##29403031##2018##), which may contribute to normative rises in externalizing behaviors (Lydon-Staley & Bassett, ##REF##30210404##2018##). Most studies on this topic to-date are cross-sectional and limited to nomothetic inferences even though they assume cognitive control and socioemotional reactivity change <italic>within individuals over time</italic> (see Beltz ##REF##29460359##2018##). Thus, there is theoretical acknowledgement of the importance of <italic>intra</italic>individual variation for adolescent externalizing behaviors, but there is little empirical evidence for it, as the extant literature primarily concerns <italic>inter</italic>individual variation (Chaku & Beltz, ##UREF##1##2022##). Intensive longitudinal research is needed to fill this critical knowledge gap.</p>\",\n \"<title>From Nomothetic to Idiographic Assessments of IC</title>\",\n \"<p id=\\\"Par11\\\">IC is often conceptualized as a relatively stable trait that can be assessed once and generalized across time. Converging evidence increasingly shows, however, that short-term fluctuations in cognition have significance for psychological wellbeing (Castellanos et al., ##REF##15950016##2005##; Hultsch et al., ##UREF##4##2011##). Numerous factors, such as interpersonal experiences, contextual demands, and emotional processes, can influence or be influenced by cognition at any given moment. For example, increased working memory fluctuations (measured across four weeks with four assessments a day) have been associated with poor sleep quality in early adolescents (Galeano-Keiner et al., ##UREF##3##2022##). Similarly, better working memory (measured daily over two weeks) has been associated with less negative mood and greater school belonging in late adolescence (Wang et al., ##UREF##15##2021##).</p>\",\n \"<p id=\\\"Par12\\\">Unfortunately, work in this area is challenged by a lack of sufficient measurement tools. Current cognitive assessments (e.g., Stroop Color Word, Stop-Signal, and Go/No Go tasks) have primarily been used to capture normative IC-linked processes, but they have limitations (see Snyder et al., ##REF##25859234##2015##). Specifically, recent work suggests that the subtraction methods often used to analyze recorded task data (e.g., comparing reaction times on incongruent versus congruent trials) are unreliable and do not index individual differences well (Salthouse & Hedden, ##REF##12647765##2002##; Weigard et al., ##REF##34252724##2021##), which limits their suitability for studies of intraindividual variation.</p>\",\n \"<p id=\\\"Par13\\\">Nonetheless, understanding short-term cognitive fluctuations is essential because patterns of intraindividual variability may reliably differ across individuals, potentially reflecting an enduring personal characteristic (Nesselroade, ##UREF##8##1991##). Although the extent to which fluctuations may reflect vulnerability versus resilience, and in turn adjustment, likely depends on the specific cognitive construct, context, or sample, the general importance of cognitive variation is being increasingly realized. For instance, greater response variability on a lab-based cognitive task was associated with lower perceptions of real-world risks (e.g., substance use) in 14-to-16-year-olds (Goldenberg et al., ##REF##27651539##2017##). Yet, most studies only capture trial-to-trial fluctuations during a single, highly controlled task, so little is known about how longer-term fluctuations in IC are associated with externalizing behaviors in everyday life. This is unfortunate because <italic>daily</italic> fluctuations are likely related to adolescents’ unique but recurring biopsychosocial experiences.</p>\",\n \"<p id=\\\"Par14\\\">Beyond understanding fluctuations in cognition, daily data (if abundant) can also be used to make person-specific inferences. Adolescence is characterized by increased, variable opportunities for learning, adaptation, and exploration (Knoll et al., ##REF##27815519##2016##). These experiences do not just differ in degree, but also in nature and biopsychosocial context across individual adolescents (Chaku & Beltz, ##UREF##1##2022##). To capture this heterogeneity, averages (and even average fluctuations) must be eschewed in favor of person-specific models (Molenaar, ##UREF##7##2004##). For instance, group iterative multiple model estimation (GIMME; Gates & Molenaar ##REF##22732562##2012##) is a person-specific analytic technique that maps variation among pre-selected variables within a person over many relatively short intervals. Applied to daily adolescent behavior, GIMME considers each adolescent as if they are a sample of <italic>N</italic>=1, allowing for unique inferences about the timing of relations among variables (i.e., same-day or next-day) that potentially reflect adolescents’ real-world contexts. GIMME is unique among person-specific analytic approaches because it also affords some generalizable inferences. It does so without averaging by prioritizing relations that are common across a sample. Thus, when combined with daily data, GIMME and other person-specific techniques can be used to make potentially important insights into <italic>when</italic>, <italic>for whom</italic>, and <italic>in what situations</italic> links between IC and externalizing behaviors matter.</p>\",\n \"<title>Intensive Longitudinal Measures of IC</title>\",\n \"<p id=\\\"Par15\\\">The feasibility of intensive longitudinal studies is increasing due to wide accessibility of internet-capable devices and sophisticated analytic techniques for dependent data (Lydon-Staley & Bassett, ##REF##30210404##2018##). Still, there is a paucity of <italic>cognitive</italic> assessments available for inclusion in these studies. Beyond their traditionally restricted use in laboratories, this may be because cognitive measures cannot be intensively repeated due to trial counts, length, or practice effects, or because they require timing mechanisms that are difficult to apply in situ (Ladouce et al., ##REF##28127283##2016##).</p>\",\n \"<p id=\\\"Par16\\\">Nonetheless, a few intensive cognitive assessments have been validated. For example, Sliwinski and colleagues (##REF##27084835##2018##) validated working memory (i.e., dot memory and n-Back) and perceptual speed (i.e., symbol search) tasks across 14-day ecological momentary assessment (EMA) bursts. Although links to other behaviors were not examined, the working memory task exhibited lower intraindividual reliability than the perceptual speed task. Recently, Kelly and Beltz (##REF##33106024##2021##) validated novel, openly available 75-occasion measures of delayed verbal recall (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://osf.io/vhr7u/\\\">https://osf.io/vhr7u/</ext-link>) and three-dimensional (3D) mental rotations (<ext-link ext-link-type=\\\"uri\\\" xlink:href=\\\"https://osf.io/m6ae8/\\\">https://osf.io/m6ae8/</ext-link>). This involved creating 75 unique sets of stimuli and establishing procedures for parallel forms reliability across sets. Overall, the 3D mental rotations measure had better psychometrics than the delayed verbal recall measure, however, there are significant challenges in distinguishing between unreliability and meaningful fluctuation in intensive longitudinal measurement (Keijsers et al., ##REF##34788708##2022##). Thus, the authors posed that verbal recall may be more influenced by daily experiences compared to mental rotations but did not empirically examine this possibility. This small literature suggests that intensive longitudinal measurement of cognition is feasible, but it has not yet been widely employed to study adolescent IC in everyday contexts.</p>\",\n \"<title>The Current Study</title>\",\n \"<p id=\\\"Par17\\\">IC likely varies from day-to-day in ways that have significance for externalizing behaviors in unique adolescents, but measures are not yet available to examine this. Thus, the overarching study goal was to reveal adolescents’ daily associations between IC and externalizing behaviors through three aims. First, a 100-occasion measure of IC was developed and psychometrically validated. Second, daily IC (both 100-day averages and fluctuations) were related to baseline impulsive behaviors; low averages and high fluctuations were expected to predict impulsivity. Third, the potential of person-specific analyses for revealing links between IC and externalizing behaviors was illustrated by comparing the daily networks of adolescents who used substances to the networks of a matched subsample who did not.</p>\",\n \"<title>Fluctuations in Daily IC and Links to Impulsive Behaviors</title>\",\n \"<p id=\\\"Par49\\\">Daily IC scores for 12 illustrative adolescents are shown in Fig. ##FIG##3##4## along with their i<italic>M</italic>s and i<italic>SD</italic>s. Adolescents demonstrated remarkably different trajectories. For example, ID 36 exhibited large day-to-day variation (i<italic>SD</italic> = 10.44), whereas the ID 73 exhibited far less variability (i<italic>SD</italic> = 3.16). Further, some adolescents were characterized by a relatively flat trajectory (e.g., ID 11) while others were better characterized by positive (e.g., ID 77), negative (e.g., ID 41), or non-linear (e.g., ID 29) trajectories. Across adolescents, the i<italic>M</italic> ranged from 12.79 to 55.58, and the i<italic>SD</italic> ranged from 3.16 to 15.44. A one-sample <italic>t</italic>-test indicated that the i<italic>SDs</italic> were significantly different from zero, <italic>t</italic>(105) = 31.53, <italic>p</italic> < .001, providing statistical evidence for IC fluctuations. The i<italic>M</italic> and i<italic>SD</italic> were not significantly correlated (<italic>r </italic>= -.01, <italic>p</italic> = .91). Although the i<italic>M</italic>s and i<italic>SD</italic>s did not vary by gender (<italic>p</italic>s > 0.05), they did covary with age, as older adolescents had higher scores (i<italic>M</italic>: <italic>r</italic> = .27, <italic>p</italic> = .01) and fewer fluctuations (i<italic>SD</italic>: <italic>r </italic>= -.19, <italic>p</italic> = .04).</p>\",\n \"<p id=\\\"Par50\\\">\\n\\n</p>\",\n \"<p id=\\\"Par51\\\">Multilevel models (nesting siblings within families) were then used to examine how i<italic>M</italic>s and i<italic>SD</italic>s were associated with impulsive behavior, accounting for gender and age. Higher i<italic>M</italic>s were associated with less positive urgency, <italic>b </italic>= -0.02(0.01), <italic>p</italic> = .03, and less negative urgency, <italic>b </italic>= -0.02(0.01), <italic>p</italic> = .04, suggesting that adolescents who had higher average daily IC tended to make less impulsive choices during extreme emotions. Higher i<italic>SD</italic>s were associated with lack of perseverance, <italic>b</italic> = 0.03(0.01), <italic>p</italic> = .03, even after controlling for i<italic>M</italic>s; this suggests that adolescents who had more variable IC were more likely to give up during difficult tasks. No significant links were found between daily IC and lack of premeditation or sensation seeking. Analyses were repeated excluding the first week of skewed assessments, and inferences were the same; see the supplemental materials.</p>\",\n \"<title>Illustrative Adolescent-Specific Network Analyses</title>\",\n \"<p id=\\\"Par531\\\">All adolescent-specific GIMME models fit the data well according to average indices: CFI = 0.99, NNFI = 1.00, RMSEA = 0.02, SRMR = 0.06. Surprisingly, there were no group- or subgroup-level relations (besides the specified autoregressions), indicating substantial heterogeneity among adolescents who used and did not use substances during the 100 days. Figure ##FIG##4##5##A shows the network of an adolescent (11.42-year-old male) who used alcohol. Circles represent variables and directed arrows represent relations between those variables; solid lines reflect same-day relations, dashed lines reflect next-day relations, red lines reflect positive relations, and blue lines reflect negative relations. Thus, this adolescent’s network contains eight relations, and only four were not specified <italic>a priori</italic> (i.e., the autoregressives). There is a positive, same-day relation between social time and positive urgency, indicating more impulsive behaviors due to positive emotions on days he was more social. There is also a pair of relations between positive and negative urgency, suggesting that negative urgency is related to same-day increases in positive urgency that propagate across time. Finally, there is an inverse, lagged relation between negative urgency and IC suggesting that increases in negative urgency are associated with IC declines the next day. This could reflect a downstream cognitive consequence of actions taken to alleviate unwanted emotions. Figure ##FIG##4##5##B shows the network of the matched adolescent (11.83-year-old male) who did not use substances. His network is less dense, with only two relations that are not autoregressives. Like the adolescent in Fig. ##FIG##4##5##A, there is a positive, same-day relation between negative and positive urgency. There is an additional positive, lagged relation between social time and negative urgency, though, potentially suggesting that his social interactions are related to next-day increases in negative urgency. Interestingly, however, his daily IC was not linked to externalizing or social experiences.</p>\",\n \"<p id=\\\"Par53\\\">It is important to emphasize that the adolescents in Fig. ##FIG##4##5## do not characterize the all the adolescents in the sample nor do they characterize all the adolescents who used substances or those who did not in the sample; otherwise, the relations would have appeared at the group- or subgroup-level. Nonetheless, follow-up analyses suggested that adolescents who reported using substances tended to have more relations involving IC (i.e., greater IC centrality) compared to adolescents who did not use substances, <italic>t</italic>(22) = -2.24, <italic>p</italic> = .05, <italic>d</italic> = 0.74. Most (60%) relations with IC were negative (i.e., worse IC was associated with more negative urgency), suggesting that IC was more salient for the daily experiences of substance users.</p>\",\n \"<p id=\\\"Par52\\\">\\n\\n</p>\",\n \"<title>Electronic Supplementary Material</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\",\n \"<p>\\n\\n</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>The authors thank members of the Methods, Sex differences, and Development – M(SD) – Lab at the University of Michigan, especially Amy Loviska who assisted with study implementation, Kaitlyn Zhao and Emma Donnelly-Sironen who assisted with participant testing, Jennifer Cleary and Heidi Westerman who assisted with clinical assessments, and Gwyn Reece who assisted with data management. The authors also thank Anthony Provenzola for providing programming expertise related to the novel inhibitory control task. Finally, the authors thank the adolescents and their families for graciously sharing their daily experiences.</p>","<title>Funding</title>","<p>This research and A.M.B. were supported by the Jacobs Foundation. This work was also supported in part by a James S. McDonnell Foundation Understanding Human Cognition Opportunity Award to A. Beltz (doi: 10.37717/2020-1145). N.C. was supported by NICHD T32 HD007109 (to Chris Monk, Vonnie McLoyd, and Susan Gelman). A.S.W was supported by K23 DA051561. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</p>","<title>Declarations</title>","<title>Conflict of interest</title>","<p id=\"Par3\">The authors have no competing interests to declare.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>The authors thank members of the Methods, Sex differences, and Development – M(SD) – Lab at the University of Michigan, especially Amy Loviska who assisted with study implementation, Kaitlyn Zhao and Emma Donnelly-Sironen who assisted with participant testing, Jennifer Cleary and Heidi Westerman who assisted with clinical assessments, and Gwyn Reece who assisted with data management. The authors also thank Anthony Provenzola for providing programming expertise related to the novel inhibitory control task. Finally, the authors thank the adolescents and their families for graciously sharing their daily experiences.</p>\",\n \"<title>Funding</title>\",\n \"<p>This research and A.M.B. were supported by the Jacobs Foundation. This work was also supported in part by a James S. McDonnell Foundation Understanding Human Cognition Opportunity Award to A. Beltz (doi: 10.37717/2020-1145). N.C. was supported by NICHD T32 HD007109 (to Chris Monk, Vonnie McLoyd, and Susan Gelman). A.S.W was supported by K23 DA051561. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Conflict of interest</title>\",\n \"<p id=\\\"Par3\\\">The authors have no competing interests to declare.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Mathematical equation, model fitting procedure, and graphical depiction of standard GIMME, which contains group- and individual-level relations; the extension of GIMME used in this paper also included confirmatory subgroup-level relations (see Henry et al., ##REF##30583065##2019##). Adapted with permission from Chaku, N., & Beltz, A. M. (2022). Using temporal network methods to reveal the idiographic nature of development. <italic>New Methods and Approaches for Studying Child Development</italic>, 159</p></caption></fig>","<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Daily means and standard deviations of the novel inhibitory control (IC) measure, across adolescent participants (<italic>N</italic> = 106). Each bar represents the average score for each of the 100 study days. The thick black line represents the average score across all participants and all study days. The dashed black line represents each day’s standard deviation, reflecting variability between participants. The thin black line represents the average standard deviation across participants and all study days. Means for the first seven days are depicted with light gray bars to emphasize performance differences early in the study</p></caption></fig>","<fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Average daily correlations between the novel inhibitory control (IC) measure and standard baseline measures: working memory (dark grey), attentional control (light grey), and perceptual sensitivity (black)</p></caption></fig>","<fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Plots of daily inhibitory control (IC) for 12 illustrative participants. Each plot shows the intensive longitudinal study data for one adolescent, with the IC score on the y-axis and study day on the x-axis. Grey lines show raw scores. Dashed black lines show i<italic>M</italic>s (i.e., each adolescent’s average IC score across all days), and dotted black lines show i<italic>SD</italic>s (i.e., each adolescent’s standard deviation across all days)</p></caption></fig>","<fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Illustrative person-specific GIMME networks of 100-day inhibitory control (IC), negative urgency (NU), positive urgency (PU), and social time (ST) for two adolescents matched on gender, age, and pubertal status. Circles represent variables. Solid lines represent contemporaneous (i.e., same-day) relations between variables, and dashed lines represent first order lagged (i.e., next-day) relations between variables. Blue lines represent inverse relations, and red lines represent positive relations. Line thickness corresponds to the magnitude (i.e., person-specific beta weight) of the relation. (<bold>A</bold>) Illustrative person-specific network for the adolescent (male, 11.42 years old, PDS = 2.00) who used substances during the 100-day study, <italic>χ</italic><sup><italic>2</italic></sup>(14) = 16.68, <italic>p</italic> = .27, CFI = 0.96, NNFI = 0.93, RMSEA = 0.04, SRMR = 0.08. Beside the autoregressive effect, IC centrality is 1. (<bold>B</bold>) Matched person-specific network for the adolescent (male, 11.83 years old, PDS = 2.40) who did not use substances during the study, <italic>χ</italic><sup><italic>2</italic></sup>(18) = 21.50, <italic>p</italic> = .26, CFI = 0.98, NNFI = 0.97, RMSEA = 0.04, SRMR = 0.05. Beside the autoregressive effect, IC centrality is 0. GIMME: Group Iterative Multiple Model Estimation; PDS: Pubertal Development Scale; CFI: Comparative Fit Index; NNFI: Non-Normed Fit Index; RMSEA: Root Mean Squared Error of Approximation; SRMR: Standardized Root Mean Squared Residual</p></caption></fig>"],"string":"[\n \"<fig id=\\\"Fig1\\\"><label>Fig. 1</label><caption><p>Mathematical equation, model fitting procedure, and graphical depiction of standard GIMME, which contains group- and individual-level relations; the extension of GIMME used in this paper also included confirmatory subgroup-level relations (see Henry et al., ##REF##30583065##2019##). Adapted with permission from Chaku, N., & Beltz, A. M. (2022). Using temporal network methods to reveal the idiographic nature of development. <italic>New Methods and Approaches for Studying Child Development</italic>, 159</p></caption></fig>\",\n \"<fig id=\\\"Fig2\\\"><label>Fig. 2</label><caption><p>Daily means and standard deviations of the novel inhibitory control (IC) measure, across adolescent participants (<italic>N</italic> = 106). Each bar represents the average score for each of the 100 study days. The thick black line represents the average score across all participants and all study days. The dashed black line represents each day’s standard deviation, reflecting variability between participants. The thin black line represents the average standard deviation across participants and all study days. Means for the first seven days are depicted with light gray bars to emphasize performance differences early in the study</p></caption></fig>\",\n \"<fig id=\\\"Fig3\\\"><label>Fig. 3</label><caption><p>Average daily correlations between the novel inhibitory control (IC) measure and standard baseline measures: working memory (dark grey), attentional control (light grey), and perceptual sensitivity (black)</p></caption></fig>\",\n \"<fig id=\\\"Fig4\\\"><label>Fig. 4</label><caption><p>Plots of daily inhibitory control (IC) for 12 illustrative participants. Each plot shows the intensive longitudinal study data for one adolescent, with the IC score on the y-axis and study day on the x-axis. Grey lines show raw scores. Dashed black lines show i<italic>M</italic>s (i.e., each adolescent’s average IC score across all days), and dotted black lines show i<italic>SD</italic>s (i.e., each adolescent’s standard deviation across all days)</p></caption></fig>\",\n \"<fig id=\\\"Fig5\\\"><label>Fig. 5</label><caption><p>Illustrative person-specific GIMME networks of 100-day inhibitory control (IC), negative urgency (NU), positive urgency (PU), and social time (ST) for two adolescents matched on gender, age, and pubertal status. Circles represent variables. Solid lines represent contemporaneous (i.e., same-day) relations between variables, and dashed lines represent first order lagged (i.e., next-day) relations between variables. Blue lines represent inverse relations, and red lines represent positive relations. Line thickness corresponds to the magnitude (i.e., person-specific beta weight) of the relation. (<bold>A</bold>) Illustrative person-specific network for the adolescent (male, 11.42 years old, PDS = 2.00) who used substances during the 100-day study, <italic>χ</italic><sup><italic>2</italic></sup>(14) = 16.68, <italic>p</italic> = .27, CFI = 0.96, NNFI = 0.93, RMSEA = 0.04, SRMR = 0.08. Beside the autoregressive effect, IC centrality is 1. (<bold>B</bold>) Matched person-specific network for the adolescent (male, 11.83 years old, PDS = 2.40) who did not use substances during the study, <italic>χ</italic><sup><italic>2</italic></sup>(18) = 21.50, <italic>p</italic> = .26, CFI = 0.98, NNFI = 0.97, RMSEA = 0.04, SRMR = 0.05. Beside the autoregressive effect, IC centrality is 0. GIMME: Group Iterative Multiple Model Estimation; PDS: Pubertal Development Scale; CFI: Comparative Fit Index; NNFI: Non-Normed Fit Index; RMSEA: Root Mean Squared Error of Approximation; SRMR: Standardized Root Mean Squared Residual</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":[],"string":"[]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group><fn id=\"Fn1\"><label>1</label><p id=\"Par20\"> This included adolescents with transgender, gender non-conforming, and genderfluid identities.</p></fn><fn id=\"Fn2\"><label>2</label><p id=\"Par23\"> Adolescents who were at least 18 years old could opt to receive their emails directly.</p></fn><fn id=\"Fn3\"><label>3</label><p id=\"Par31\"> Adolescents were allowed to unclick a response and select a new one.</p></fn><fn id=\"Fn4\"><label>4</label><p id=\"Par39\"> Adolescents who reported non-cisgender identities (<italic>n</italic> = 4) were excluded from these analyses.</p></fn><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<fn-group><fn id=\\\"Fn1\\\"><label>1</label><p id=\\\"Par20\\\"> This included adolescents with transgender, gender non-conforming, and genderfluid identities.</p></fn><fn id=\\\"Fn2\\\"><label>2</label><p id=\\\"Par23\\\"> Adolescents who were at least 18 years old could opt to receive their emails directly.</p></fn><fn id=\\\"Fn3\\\"><label>3</label><p id=\\\"Par31\\\"> Adolescents were allowed to unclick a response and select a new one.</p></fn><fn id=\\\"Fn4\\\"><label>4</label><p id=\\\"Par39\\\"> Adolescents who reported non-cisgender identities (<italic>n</italic> = 4) were excluded from these analyses.</p></fn><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"10802_2023_1071_Fig1_HTML\" id=\"d32e781\"/>","<graphic xlink:href=\"10802_2023_1071_Fig2_HTML\" id=\"d32e816\"/>","<graphic xlink:href=\"10802_2023_1071_Fig3_HTML\" id=\"d32e908\"/>","<graphic xlink:href=\"10802_2023_1071_Fig4_HTML\" id=\"d32e1005\"/>","<graphic xlink:href=\"10802_2023_1071_Fig5_HTML\" id=\"d32e1107\"/>"],"string":"[\n \"<graphic xlink:href=\\\"10802_2023_1071_Fig1_HTML\\\" id=\\\"d32e781\\\"/>\",\n \"<graphic xlink:href=\\\"10802_2023_1071_Fig2_HTML\\\" id=\\\"d32e816\\\"/>\",\n \"<graphic xlink:href=\\\"10802_2023_1071_Fig3_HTML\\\" id=\\\"d32e908\\\"/>\",\n \"<graphic xlink:href=\\\"10802_2023_1071_Fig4_HTML\\\" id=\\\"d32e1005\\\"/>\",\n \"<graphic xlink:href=\\\"10802_2023_1071_Fig5_HTML\\\" id=\\\"d32e1107\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"10802_2023_1071_MOESM1_ESM.docx\"><caption><p>Supplementary Material 1</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"10802_2023_1071_MOESM1_ESM.docx\\\"><caption><p>Supplementary Material 1</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"mixed-citation\": [\"Bolger, N., & Laurenceau, J. P. (2013). \"], \"italic\": [\"Intensive longitudinal methods: An introduction to diary and experience sampling research\"]}, {\"mixed-citation\": [\"Chaku, N., & Beltz, A. M. (2022). Using temporal network methods to reveal the idiographic nature of development. In J. J. Lockman & R. O. Gilmore (Eds.), \"], \"italic\": [\"New methods and approaches for studying child development\"]}, {\"mixed-citation\": [\"Cohen, J. (1988). Set correlation and contingency tables. \"], \"italic\": [\"Applied Psychological Measurement, 12\"]}, {\"surname\": [\"Galeano-Keiner\", \"Neubauer\", \"Schmidt\", \"Schmiedek\"], \"given-names\": [\"EM\", \"A\", \"A\", \"F\"], \"article-title\": [\"Daily fluctuations in children\\u2019s working memory accuracy and precision: Variability at multiple time scales and links to daily sleep behavior and fluid intelligence\"], \"source\": [\"Cognitive Development\"], \"year\": [\"2022\"], \"volume\": [\"64\"], \"fpage\": [\"101260\"], \"pub-id\": [\"10.1016/j.cogdev.2022.101260\"]}, {\"mixed-citation\": [\"Hultsch, D. F., Strauss, E., Hunter, M. A., & MacDonald, S. W. S. (2011). Intraindividual variability, cognition, and aging. In F. I. M. Craik & T. A. Salthouse (Eds.), \"], \"italic\": [\"The Handbook of Aging and Cognition\"]}, {\"mixed-citation\": [\"L\\u00f6vden, M., Wenger E, M\\u00e5rtensson J, Lindenberger U, & B\\u00e5ckman L. (2013). Structural brain plasticity in adult learning and development. \"], \"italic\": [\"Neuroscience & Biobehavioral Reviews, 37\"]}, {\"mixed-citation\": [\"Lynam, D. R., Smith, G. T., Whiteside, S. P., & Cyders, M. A. (2006). \"], \"italic\": [\"The UPPS-P: Assessing five personality pathways to impulsive behavior\"]}, {\"surname\": [\"Molenaar\"], \"given-names\": [\"PCM\"], \"article-title\": [\"A manifesto on psychology as idiographic science: Bringing the person back into scientific psychology, this time forever\"], \"source\": [\"Measurement: Interdisciplinary Research & Perspective\"], \"year\": [\"2004\"], \"volume\": [\"2\"], \"issue\": [\"4\"], \"fpage\": [\"201\"], \"lpage\": [\"218\"], \"pub-id\": [\"10.1207/s15366359mea0204_1\"]}, {\"mixed-citation\": [\"Nesselroade, J. R. (1991). Interindividual differences in intraindividual change. In L. M. Collins & J. L. Horn (Eds.), \"], \"italic\": [\"Best methods for the analysis of change: Recent advances, unanswered questions, future directions\"]}, {\"surname\": [\"Rankin\", \"Marsh\"], \"given-names\": [\"ED\", \"JC\"], \"article-title\": [\"Effects of missing data on the statistical analysis of clinical time series\"], \"source\": [\"Social Work Research and Abstracts\"], \"year\": [\"1985\"], \"volume\": [\"21\"], \"issue\": [\"2\"], \"fpage\": [\"13\"], \"lpage\": [\"16\"], \"pub-id\": [\"10.1093/swra/21.2.13\"]}, {\"surname\": [\"Sliwinski\"], \"given-names\": [\"MJ\"], \"article-title\": [\"Measurement-burst designs for social health research\"], \"source\": [\"Social and Personality Psychology Compass\"], \"year\": [\"2008\"], \"volume\": [\"2\"], \"issue\": [\"1\"], \"fpage\": [\"245\"], \"lpage\": [\"261\"], \"pub-id\": [\"10.1111/j.1751-9004.2007.00043.x\"]}, {\"surname\": [\"S\\u00f6rbom\"], \"given-names\": [\"D\"], \"article-title\": [\"Model modification\"], \"source\": [\"Psychometrika\"], \"year\": [\"1989\"], \"volume\": [\"54\"], \"issue\": [\"3\"], \"fpage\": [\"371\"], \"lpage\": [\"384\"], \"pub-id\": [\"10.1007/BF02294623\"]}, {\"surname\": [\"Sperry\", \"Lynam\", \"Walsh\", \"Horton\", \"Kwapil\"], \"given-names\": [\"SH\", \"DR\", \"MA\", \"LE\", \"TR\"], \"article-title\": [\"Examining the multidimensional structure of impulsivity in daily life\"], \"source\": [\"Personality and Individual Differences\"], \"year\": [\"2016\"], \"volume\": [\"94\"], \"fpage\": [\"153\"], \"lpage\": [\"158\"], \"pub-id\": [\"10.1016/j.paid.2016.01.018\"]}, {\"mixed-citation\": [\"Team, R. C. (2022). \"], \"italic\": [\"R: A language and environment for statistical computing\"], \"ext-link\": [\"https://www.R-project.org/\"]}, {\"surname\": [\"Vogelsmeier\", \"Vermunt\", \"van Roekel\", \"De Roover\"], \"given-names\": [\"LV\", \"JK\", \"E\", \"K\"], \"article-title\": [\"Latent Markov factor analysis for exploring measurement model changes in time-intensive longitudinal studies\"], \"source\": [\"Structural Equation Modeling: A Multidisciplinary Journal\"], \"year\": [\"2019\"], \"volume\": [\"26\"], \"issue\": [\"4\"], \"fpage\": [\"557\"], \"lpage\": [\"575\"], \"pub-id\": [\"10.1080/10705511.2018.1554445\"]}, {\"mixed-citation\": [\"Wang, Y., Zhang, Y., Chen, M., & Chen, Y. (2021). Adolescents\\u2019 daily executive function: Methodological considerations, daily variation, and associations with daily experiences. \"], \"italic\": [\"Psychological Assessment, 33\"]}, {\"surname\": [\"Zhu\", \"Zhang\", \"Li\", \"Leung\"], \"given-names\": [\"H\", \"G\", \"Y\", \"H\"], \"article-title\": [\"A novel robust Kalman filter with unknown non-stationary heavy-tailed noise\"], \"source\": [\"Automatica\"], \"year\": [\"2021\"], \"volume\": [\"127\"], \"fpage\": [\"109511\"], \"pub-id\": [\"10.1016/j.automatica.2021.109511\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":87,"string":"87"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:42:02"},"citation":{"kind":"string","value":"Res Child Adolesc Psychopathol. 2024 Jul 5; 52(1):93-110"},"package_file":{"kind":"string","value":"oa_package/6c/cb/PMC10787911.tar.gz"}}},{"rowIdx":194,"cells":{"accession_id":{"kind":"string","value":"PMC10787912"},"pmid":{"kind":"string","value":"37878150"},"introduction":{"kind":"list like","value":["<title>Background</title>","<p id=\"Par5\">Breast cancer affects almost 56,000 women every year in the United Kingdom (UK) [##UREF##0##1##] and despite improvements in treatment, approximately 40% of these will require mastectomy [##REF##11091232##2##]. Seroma formation following mastectomy and/or axillary clearance is common, with reported incidence in the literature varying between 10 and 85% [##REF##16286909##3##]. Although rarely a serious complication of breast surgery, seroma can cause delayed wound healing, require repeated aspiration with the risk of infection, and may delay the start of adjuvant treatments [##REF##15588301##4##, ##REF##21849243##5##].</p>","<p id=\"Par6\">Strategies to reduce the formation of seroma include the use of surgical drains and flap fixation methods such as quilting or tissue glue and external compression which all act by minimising the surgical dead-space and evidence to support the effectiveness of different approaches has been summarised in several systematic reviews [##REF##33051116##6##–##REF##29039118##10##]. Many of these reviews, however, have highlighted the lack of high-quality research to support practice and the need for future well-designed studies in this area.</p>","<p id=\"Par7\">For future research to be meaningful, it is vital that the study design should reflect current practice and address key uncertainties that are important to patients and the clinical community. We aimed to survey breast surgeons to determine current approaches to the management of seroma in the UK; particularly the use of drains after simple breast cancer surgery to inform the feasibility and design of a future randomised controlled trial (RCT).</p>"],"string":"[\n \"<title>Background</title>\",\n \"<p id=\\\"Par5\\\">Breast cancer affects almost 56,000 women every year in the United Kingdom (UK) [##UREF##0##1##] and despite improvements in treatment, approximately 40% of these will require mastectomy [##REF##11091232##2##]. Seroma formation following mastectomy and/or axillary clearance is common, with reported incidence in the literature varying between 10 and 85% [##REF##16286909##3##]. Although rarely a serious complication of breast surgery, seroma can cause delayed wound healing, require repeated aspiration with the risk of infection, and may delay the start of adjuvant treatments [##REF##15588301##4##, ##REF##21849243##5##].</p>\",\n \"<p id=\\\"Par6\\\">Strategies to reduce the formation of seroma include the use of surgical drains and flap fixation methods such as quilting or tissue glue and external compression which all act by minimising the surgical dead-space and evidence to support the effectiveness of different approaches has been summarised in several systematic reviews [##REF##33051116##6##–##REF##29039118##10##]. Many of these reviews, however, have highlighted the lack of high-quality research to support practice and the need for future well-designed studies in this area.</p>\",\n \"<p id=\\\"Par7\\\">For future research to be meaningful, it is vital that the study design should reflect current practice and address key uncertainties that are important to patients and the clinical community. We aimed to survey breast surgeons to determine current approaches to the management of seroma in the UK; particularly the use of drains after simple breast cancer surgery to inform the feasibility and design of a future randomised controlled trial (RCT).</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Methods</title>","<p id=\"Par8\">An online national practice survey was developed in REDCap<sup>®</sup> to capture current UK practice regarding the use of strategies to reduce seroma formation following mastectomy and axillary surgery including the use of drains and flap fixation techniques, and details of the patient pathway for the management of seroma post-operatively. Preliminary work suggested that drains were the most commonly used method of reducing seroma in the UK, so questions focussed on evaluating the feasibility of a future trial comparing the use of drains versus no drains and key elements of trial design including inclusion/exclusion criteria and selection of the primary outcome (Appendix 1).</p>","<p id=\"Par9\">All consultant breast surgeons and senior breast surgery trainees/fellows, defined as being within their final two years of training, were invited to complete the survey through the professional associations (Association of Breast Surgery (ABS) and the Mammary Fold (UK trainee breast surgery group) and via social media networks. The survey was open for a 4-month period (December 2021–March 2022) and regular reminders were sent to optimise participation. Simple descriptive summary statistics were calculated for each survey item and free text responses were analysed using content analysis [##REF##16204405##11##].</p>"],"string":"[\n \"<title>Methods</title>\",\n \"<p id=\\\"Par8\\\">An online national practice survey was developed in REDCap<sup>®</sup> to capture current UK practice regarding the use of strategies to reduce seroma formation following mastectomy and axillary surgery including the use of drains and flap fixation techniques, and details of the patient pathway for the management of seroma post-operatively. Preliminary work suggested that drains were the most commonly used method of reducing seroma in the UK, so questions focussed on evaluating the feasibility of a future trial comparing the use of drains versus no drains and key elements of trial design including inclusion/exclusion criteria and selection of the primary outcome (Appendix 1).</p>\",\n \"<p id=\\\"Par9\\\">All consultant breast surgeons and senior breast surgery trainees/fellows, defined as being within their final two years of training, were invited to complete the survey through the professional associations (Association of Breast Surgery (ABS) and the Mammary Fold (UK trainee breast surgery group) and via social media networks. The survey was open for a 4-month period (December 2021–March 2022) and regular reminders were sent to optimise participation. Simple descriptive summary statistics were calculated for each survey item and free text responses were analysed using content analysis [##REF##16204405##11##].</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<title>Respondent demographics</title>","<p id=\"Par10\">A total of 147 responses were received of which 97 were completed with data that could be included in the analysis. The partially complete responses were excluded from further analysis where they had very limited, or no data entered. Respondent demographics are summarised in Table ##TAB##0##1##. Most respondents were consultant breast surgeons (<italic>n</italic> = 75, 77%) with responses received from surgeons practicing across the UK (Table ##TAB##0##1##).</p>","<title>Use of peri-operative interventions to reduce seroma formation</title>","<p id=\"Par11\">Of the 97 surgeons who completed in the survey, the majority (<italic>n</italic> = 82, 85%) used drains either routinely (<italic>n</italic> = 38, 39%) or in certain circumstances (<italic>n</italic> = 44, 45%). This was most frequently a single drain, although when mastectomy was combined with an axillary node clearance (ANC), two drains were used by a proportion of surgeons (Table ##TAB##1##2##).</p>","<p id=\"Par12\">The indications for selective drain use included patient (e.g. age, high body mass index or large breasts) and treatment factors (e.g. extent of surgery or after neo-adjuvant chemotherapy). The most common reasons for not using a drain included non-compliance and patient preference (Table ##TAB##1##2##). Whilst further details on how patient preference influences drain use were not collected by this survey, drain placement is part of the informed consent process and some patients may therefore decline drain insertion despite the reasons for use being explained.</p>","<p id=\"Par13\">Few surgeons (<italic>n</italic> = 26/97, 27%) reported routinely using flap fixation methods to reduce seroma. The most commonly used methods were quilting (<italic>n</italic> = 11, 11.5%) and glue sealants (<italic>n</italic> = 8, 8.5%). These methods were more frequently used by surgeons who did not routinely use drains (<italic>n</italic> = 20, 34% vs <italic>n</italic> = 6, 16%) (Table ##TAB##1##2##).</p>","<title>Post-operative patient pathway</title>","<p id=\"Par14\">Significant variability in the post-operative management of drains and seromas was reported. Of the 79 respondents using drains, the majority (<italic>n</italic> = 59, 62%) removed them based on the volume of seroma drained per day, most commonly < 50 mls/24 h (Table ##TAB##2##3##). Drains were most frequently removed by nursing staff (<italic>n</italic> = 63/106, 60%) in breast clinic (<italic>n</italic> = 44, 56%).</p>","<p id=\"Par15\">Breast care nurses were most frequently responsible for assessing patients; deciding when seromas should be drained (77/199, 39%) and performing the procedure (72/250, 29%). Factors influencing decision-making regarding seroma drainage included patient symptoms (88/240, 37%) and assessment of actual (e.g. infection, 64/250, 26%) or impending (e.g. concerns re skin viability, 79/250, 33%) complications. Very few respondents reported draining all seromas (Table ##TAB##2##3##).</p>","<title>Feasibility and design of a future RCT</title>","<p id=\"Par16\">Of the 91 surgeons completing this section of the survey, just under half (<italic>n</italic> = 37, 41%) expressed uncertainty regarding the use of drains after routine breast cancer surgery. Of those indicating some uncertainty, this was mostly regarding the use of a drain following mastectomy and sentinel lymph node biopsy (SLNB) (<italic>n</italic> = 33, 70%) and isolated ANC (<italic>n</italic> = 29, 62%). Two-thirds of surgeons felt a trial comparing the use of drains vs no drains after simple breast cancer surgery was needed (n = 59, 65%).</p>","<p id=\"Par17\">Almost half of the surveyed surgeons (<italic>n</italic> = 45, 49%) indicated they would be willing to randomise all patients undergoing mastectomy ± axillary surgery in an RCT comparing the use of drains versus no drains. The remaining half expressed reluctance randomising specific groups of patients. These groups primarily included patients in whom the surgical dead-space was anticipated to be large, for example following mastectomy and ANC (29/83, 35%); in women with Body Mass Index (BMI) > 30 (16/83, 19%); in those with large breasts (12/83, 15%) or; in those perceived to be at higher risk of post-operative complications (13/83, 16% e.g. those with high risk of bleeding; post neo-adjuvant chemotherapy etc.) (Table ##TAB##3##4##).</p>","<p id=\"Par18\">There was a lack of consensus regarding the most suitable primary outcome for a future trial. Respondents were asked to select all outcomes of importance and/or interest in order to try and elucidate whether a single or co-primary endpoint might be necessary for a future trial. The most commonly selected outcome was the number of symptomatic seromas drained (67/302, 22%), followed by the number of hospital/healthcare provider visits (49/302, 16%) and patient reported outcomes such as patient satisfaction (<italic>n</italic> = 41/302, 14%) (Table ##TAB##3##4##). Almost three-quarters (<italic>n</italic> = 67, 73.5%) of respondents felt that a future trial was feasible and almost 80% would be definitely (<italic>n</italic> = 54) or possibly (<italic>n</italic> = 18) interested in recruiting patients to a future RCT.</p>","<p id=\"Par19\">Of 91 respondents, 52 (57%) provided free text comments relating to the feasibility and design of a future trial. Three key themes emerged (Table ##TAB##4##5##) from the content analysis. These were: 1. The need for high-quality evidence to inform, change and address variation in practice (specifically no longer using drains); 2. Clinical (personal and community) equipoise and; 3. Trial design, outcome selection and feasibility. Overall, respondents felt that as there was significant variability in UK practice and no clear evidence to support the use of drains, a trial was necessary.</p>"],"string":"[\n \"<title>Results</title>\",\n \"<title>Respondent demographics</title>\",\n \"<p id=\\\"Par10\\\">A total of 147 responses were received of which 97 were completed with data that could be included in the analysis. The partially complete responses were excluded from further analysis where they had very limited, or no data entered. Respondent demographics are summarised in Table ##TAB##0##1##. Most respondents were consultant breast surgeons (<italic>n</italic> = 75, 77%) with responses received from surgeons practicing across the UK (Table ##TAB##0##1##).</p>\",\n \"<title>Use of peri-operative interventions to reduce seroma formation</title>\",\n \"<p id=\\\"Par11\\\">Of the 97 surgeons who completed in the survey, the majority (<italic>n</italic> = 82, 85%) used drains either routinely (<italic>n</italic> = 38, 39%) or in certain circumstances (<italic>n</italic> = 44, 45%). This was most frequently a single drain, although when mastectomy was combined with an axillary node clearance (ANC), two drains were used by a proportion of surgeons (Table ##TAB##1##2##).</p>\",\n \"<p id=\\\"Par12\\\">The indications for selective drain use included patient (e.g. age, high body mass index or large breasts) and treatment factors (e.g. extent of surgery or after neo-adjuvant chemotherapy). The most common reasons for not using a drain included non-compliance and patient preference (Table ##TAB##1##2##). Whilst further details on how patient preference influences drain use were not collected by this survey, drain placement is part of the informed consent process and some patients may therefore decline drain insertion despite the reasons for use being explained.</p>\",\n \"<p id=\\\"Par13\\\">Few surgeons (<italic>n</italic> = 26/97, 27%) reported routinely using flap fixation methods to reduce seroma. The most commonly used methods were quilting (<italic>n</italic> = 11, 11.5%) and glue sealants (<italic>n</italic> = 8, 8.5%). These methods were more frequently used by surgeons who did not routinely use drains (<italic>n</italic> = 20, 34% vs <italic>n</italic> = 6, 16%) (Table ##TAB##1##2##).</p>\",\n \"<title>Post-operative patient pathway</title>\",\n \"<p id=\\\"Par14\\\">Significant variability in the post-operative management of drains and seromas was reported. Of the 79 respondents using drains, the majority (<italic>n</italic> = 59, 62%) removed them based on the volume of seroma drained per day, most commonly < 50 mls/24 h (Table ##TAB##2##3##). Drains were most frequently removed by nursing staff (<italic>n</italic> = 63/106, 60%) in breast clinic (<italic>n</italic> = 44, 56%).</p>\",\n \"<p id=\\\"Par15\\\">Breast care nurses were most frequently responsible for assessing patients; deciding when seromas should be drained (77/199, 39%) and performing the procedure (72/250, 29%). Factors influencing decision-making regarding seroma drainage included patient symptoms (88/240, 37%) and assessment of actual (e.g. infection, 64/250, 26%) or impending (e.g. concerns re skin viability, 79/250, 33%) complications. Very few respondents reported draining all seromas (Table ##TAB##2##3##).</p>\",\n \"<title>Feasibility and design of a future RCT</title>\",\n \"<p id=\\\"Par16\\\">Of the 91 surgeons completing this section of the survey, just under half (<italic>n</italic> = 37, 41%) expressed uncertainty regarding the use of drains after routine breast cancer surgery. Of those indicating some uncertainty, this was mostly regarding the use of a drain following mastectomy and sentinel lymph node biopsy (SLNB) (<italic>n</italic> = 33, 70%) and isolated ANC (<italic>n</italic> = 29, 62%). Two-thirds of surgeons felt a trial comparing the use of drains vs no drains after simple breast cancer surgery was needed (n = 59, 65%).</p>\",\n \"<p id=\\\"Par17\\\">Almost half of the surveyed surgeons (<italic>n</italic> = 45, 49%) indicated they would be willing to randomise all patients undergoing mastectomy ± axillary surgery in an RCT comparing the use of drains versus no drains. The remaining half expressed reluctance randomising specific groups of patients. These groups primarily included patients in whom the surgical dead-space was anticipated to be large, for example following mastectomy and ANC (29/83, 35%); in women with Body Mass Index (BMI) > 30 (16/83, 19%); in those with large breasts (12/83, 15%) or; in those perceived to be at higher risk of post-operative complications (13/83, 16% e.g. those with high risk of bleeding; post neo-adjuvant chemotherapy etc.) (Table ##TAB##3##4##).</p>\",\n \"<p id=\\\"Par18\\\">There was a lack of consensus regarding the most suitable primary outcome for a future trial. Respondents were asked to select all outcomes of importance and/or interest in order to try and elucidate whether a single or co-primary endpoint might be necessary for a future trial. The most commonly selected outcome was the number of symptomatic seromas drained (67/302, 22%), followed by the number of hospital/healthcare provider visits (49/302, 16%) and patient reported outcomes such as patient satisfaction (<italic>n</italic> = 41/302, 14%) (Table ##TAB##3##4##). Almost three-quarters (<italic>n</italic> = 67, 73.5%) of respondents felt that a future trial was feasible and almost 80% would be definitely (<italic>n</italic> = 54) or possibly (<italic>n</italic> = 18) interested in recruiting patients to a future RCT.</p>\",\n \"<p id=\\\"Par19\\\">Of 91 respondents, 52 (57%) provided free text comments relating to the feasibility and design of a future trial. Three key themes emerged (Table ##TAB##4##5##) from the content analysis. These were: 1. The need for high-quality evidence to inform, change and address variation in practice (specifically no longer using drains); 2. Clinical (personal and community) equipoise and; 3. Trial design, outcome selection and feasibility. Overall, respondents felt that as there was significant variability in UK practice and no clear evidence to support the use of drains, a trial was necessary.</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p id=\"Par20\">This survey has demonstrated considerable variation in the management of seromas following mastectomy and axillary surgery in the UK and the need for and potential feasibility of a large-scale pragmatic RCT to establish best practice. It is likely that a future trial would compare the use of drains vs no drains as this is currently the main strategy for reducing seroma development in the UK.</p>","<p id=\"Par21\">Surgeons’ attitudes to a potential trial in this area reflects the lack of high-quality evidence to support the use of drains following breast cancer surgery. A systematic review [##REF##33051116##6##] considered seroma formation in 1347 women following mastectomy (± axillary lymph node clearance) with and without suction drainage. The review included two RCTs [##REF##14716794##12##, ##REF##11872051##13##] and six non-randomised studies [##REF##26907221##14##–##REF##25486263##19##]. The data were found to be at a high risk of bias, heterogeneous with variable use of flap fixation methods and with an inconsistently defined primary outcome of seroma formation. The authors concluded that there was some evidence that drainage following mastectomy and axillary surgery could be safely omitted without increasing seroma formation or complications but highlighted the need for further high-quality research to determine the role of surgical drains following breast cancer surgery including outcomes of importance to patients. These findings were consistent with previous reviews [##UREF##1##9##, ##REF##20827578##20##, ##REF##19289285##21##] suggesting that drainage does appear to reduce seroma rates but may be associated with longer hospital stays. However, it should be noted that drain use increasingly may not affect hospital stay as significantly as it has done in the past. The 2021 Getting It Right First Time (GIRFT) report (Using Hospital Episode Statistics, HES Data April 2015-March 2018) [##UREF##2##22##], demonstrated that only 20% of mastectomies without reconstruction, were conducted as a day case and that rates vary widely across trusts from 0% to 78.28%. The report recommended that day case mastectomy rates should be increased to 75%. Increasing day case mastectomy has perhaps recently been driven by the COVID-19 pandemic, and the consequent need to avoid hospital stay and risk of infection. In this survey, 77% (58/75) of respondents reported that patients went home on the day of surgery more than 75% of the time (Table ##TAB##1##2##).</p>","<p id=\"Par22\">Several recent or ongoing European RCTs, as well as comprehensive literature reviews [##REF##24941989##23##] have considered techniques to reduce seroma formation and the need for drains after mastectomy. The Dutch SAM trial [##REF##33078318##24##, ##REF##30119663##25##] (NCT03305757), was a multicentre three arm RCT of flap fixation with sutures or tissue glue and conventional closure, with closed suction drains in all arms. This showed a significant reduction in clinically significant seroma in both flap fixation arms with the greatest reduction in the sutured flap fixation group [##REF##33078318##24##]. Ongoing RCTs include the single-centre Dutch SARA [##REF##32767988##26##] (NCT04035590) trial which will compare flap fixation with and without suction drainage; the multicentre Dutch QUILT (NCT05272904—not yet recruiting) trial comparing quilting without a drain and conventional closure; and the multicentre French QUISERMAS trial [##REF##27044574##27##] (NCT02263651) comparing conventional closure with a drain and flap fixation without a drain. This study completed in 2018 but has yet to report. None of these trials, however, reflect current UK practice or include outcomes of importance of patients. Quilting is not standard practice in the UK, perhaps due to the increased costs associated with the time quilting takes and the use of more expensive self-locking sutures to perform the procedure. In addition, there are perceived concerns regarding compromising skin flap viability, particularly where the skin flaps are thin or in those already deemed to be high risk for complications such as smokers.</p>","<p id=\"Par23\">Whilst the most common outcome for the trial suggested by surgeons in this survey was the number of symptomatic seromas drained (20% of respondents), this was closely followed by the number of hospital/healthcare provider visits (16% of respondents). Work with our patient and public involvement (PPI) group highlighted that hospital visits were perceived as a major burden to patients. This outcome would comprehensively evaluate drain-related issues, symptomatic seromas; wound complications and patient concerns which may require clinical evaluation while being objective and easy to measure. As such, hospital visits would pragmatically be the most appropriate primary outcome for a future trial.</p>","<p id=\"Par24\">This is a national practice survey with limitations that require consideration. Firstly, it only includes the views of a relatively small group of UK breast surgeons. From the Getting It Right First Time (GIRFT) report in 2021 [##UREF##2##22##], there were 130 breast surgery units in England, but this number varies year to year depending on service mergers and closures. The surgeons who responded, may be more engaged in research and thus may not be representative of the breast surgical community more broadly. Whilst this is possible, the survey has included surgeons from across the UK, in all major geographical areas, with various degrees of experience. Furthermore, this engaged group of surgeons is likely to include those who will open and recruit to any future study. It could therefore be argued that their views are the most relevant as they will determine whether a future study would be successful. It is, however, possible that willing to participate in a future RCT in principle, does not always translate into actual participation in practice.</p>","<p id=\"Par25\">Despite limitations, this work demonstrates there is a need for a high-quality RCT to determine if, when and in whom closed suction drains are necessary following mastectomy and axillary surgery in the UK. Perhaps more importantly, this is a question that is also meaningful to patients as in the recent James Lind Priority Setting Partnership (PSP) in breast cancer surgery [##REF##36319906##28##], one in three patient respondents submitted questions related to seroma and the benefits of drains after breast cancer surgery. Overall, this question was ranked as the 11<sup>th</sup> most important research priority to patients completing the survey and although it narrowly missed being considered for the top 10 research priorities [##REF##36319906##28##], it is clearly an area where more research is needed.</p>","<p id=\"Par26\">Work to design and gain funding for a future trial is now underway. Given the large volume of procedures performed, it is likely that that such a trial would recruit quickly and easily and utilisation of the breast trainee collaborative research network may represent a cost-effective means of delivering the study in a timely fashion [##REF##30132807##29##–##REF##30639093##31##]. If an RCT proves that drains are unnecessary in all or most patients undergoing mastectomy and/or axillary surgery, it will provide the necessary high-quality evidence to change practice. This will reduce NHS costs and the burden on scarce resources, but more importantly, improve patient experiences of breast cancer treatment.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p id=\\\"Par20\\\">This survey has demonstrated considerable variation in the management of seromas following mastectomy and axillary surgery in the UK and the need for and potential feasibility of a large-scale pragmatic RCT to establish best practice. It is likely that a future trial would compare the use of drains vs no drains as this is currently the main strategy for reducing seroma development in the UK.</p>\",\n \"<p id=\\\"Par21\\\">Surgeons’ attitudes to a potential trial in this area reflects the lack of high-quality evidence to support the use of drains following breast cancer surgery. A systematic review [##REF##33051116##6##] considered seroma formation in 1347 women following mastectomy (± axillary lymph node clearance) with and without suction drainage. The review included two RCTs [##REF##14716794##12##, ##REF##11872051##13##] and six non-randomised studies [##REF##26907221##14##–##REF##25486263##19##]. The data were found to be at a high risk of bias, heterogeneous with variable use of flap fixation methods and with an inconsistently defined primary outcome of seroma formation. The authors concluded that there was some evidence that drainage following mastectomy and axillary surgery could be safely omitted without increasing seroma formation or complications but highlighted the need for further high-quality research to determine the role of surgical drains following breast cancer surgery including outcomes of importance to patients. These findings were consistent with previous reviews [##UREF##1##9##, ##REF##20827578##20##, ##REF##19289285##21##] suggesting that drainage does appear to reduce seroma rates but may be associated with longer hospital stays. However, it should be noted that drain use increasingly may not affect hospital stay as significantly as it has done in the past. The 2021 Getting It Right First Time (GIRFT) report (Using Hospital Episode Statistics, HES Data April 2015-March 2018) [##UREF##2##22##], demonstrated that only 20% of mastectomies without reconstruction, were conducted as a day case and that rates vary widely across trusts from 0% to 78.28%. The report recommended that day case mastectomy rates should be increased to 75%. Increasing day case mastectomy has perhaps recently been driven by the COVID-19 pandemic, and the consequent need to avoid hospital stay and risk of infection. In this survey, 77% (58/75) of respondents reported that patients went home on the day of surgery more than 75% of the time (Table ##TAB##1##2##).</p>\",\n \"<p id=\\\"Par22\\\">Several recent or ongoing European RCTs, as well as comprehensive literature reviews [##REF##24941989##23##] have considered techniques to reduce seroma formation and the need for drains after mastectomy. The Dutch SAM trial [##REF##33078318##24##, ##REF##30119663##25##] (NCT03305757), was a multicentre three arm RCT of flap fixation with sutures or tissue glue and conventional closure, with closed suction drains in all arms. This showed a significant reduction in clinically significant seroma in both flap fixation arms with the greatest reduction in the sutured flap fixation group [##REF##33078318##24##]. Ongoing RCTs include the single-centre Dutch SARA [##REF##32767988##26##] (NCT04035590) trial which will compare flap fixation with and without suction drainage; the multicentre Dutch QUILT (NCT05272904—not yet recruiting) trial comparing quilting without a drain and conventional closure; and the multicentre French QUISERMAS trial [##REF##27044574##27##] (NCT02263651) comparing conventional closure with a drain and flap fixation without a drain. This study completed in 2018 but has yet to report. None of these trials, however, reflect current UK practice or include outcomes of importance of patients. Quilting is not standard practice in the UK, perhaps due to the increased costs associated with the time quilting takes and the use of more expensive self-locking sutures to perform the procedure. In addition, there are perceived concerns regarding compromising skin flap viability, particularly where the skin flaps are thin or in those already deemed to be high risk for complications such as smokers.</p>\",\n \"<p id=\\\"Par23\\\">Whilst the most common outcome for the trial suggested by surgeons in this survey was the number of symptomatic seromas drained (20% of respondents), this was closely followed by the number of hospital/healthcare provider visits (16% of respondents). Work with our patient and public involvement (PPI) group highlighted that hospital visits were perceived as a major burden to patients. This outcome would comprehensively evaluate drain-related issues, symptomatic seromas; wound complications and patient concerns which may require clinical evaluation while being objective and easy to measure. As such, hospital visits would pragmatically be the most appropriate primary outcome for a future trial.</p>\",\n \"<p id=\\\"Par24\\\">This is a national practice survey with limitations that require consideration. Firstly, it only includes the views of a relatively small group of UK breast surgeons. From the Getting It Right First Time (GIRFT) report in 2021 [##UREF##2##22##], there were 130 breast surgery units in England, but this number varies year to year depending on service mergers and closures. The surgeons who responded, may be more engaged in research and thus may not be representative of the breast surgical community more broadly. Whilst this is possible, the survey has included surgeons from across the UK, in all major geographical areas, with various degrees of experience. Furthermore, this engaged group of surgeons is likely to include those who will open and recruit to any future study. It could therefore be argued that their views are the most relevant as they will determine whether a future study would be successful. It is, however, possible that willing to participate in a future RCT in principle, does not always translate into actual participation in practice.</p>\",\n \"<p id=\\\"Par25\\\">Despite limitations, this work demonstrates there is a need for a high-quality RCT to determine if, when and in whom closed suction drains are necessary following mastectomy and axillary surgery in the UK. Perhaps more importantly, this is a question that is also meaningful to patients as in the recent James Lind Priority Setting Partnership (PSP) in breast cancer surgery [##REF##36319906##28##], one in three patient respondents submitted questions related to seroma and the benefits of drains after breast cancer surgery. Overall, this question was ranked as the 11<sup>th</sup> most important research priority to patients completing the survey and although it narrowly missed being considered for the top 10 research priorities [##REF##36319906##28##], it is clearly an area where more research is needed.</p>\",\n \"<p id=\\\"Par26\\\">Work to design and gain funding for a future trial is now underway. Given the large volume of procedures performed, it is likely that that such a trial would recruit quickly and easily and utilisation of the breast trainee collaborative research network may represent a cost-effective means of delivering the study in a timely fashion [##REF##30132807##29##–##REF##30639093##31##]. If an RCT proves that drains are unnecessary in all or most patients undergoing mastectomy and/or axillary surgery, it will provide the necessary high-quality evidence to change practice. This will reduce NHS costs and the burden on scarce resources, but more importantly, improve patient experiences of breast cancer treatment.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":[],"string":"[]","isConversational":false},"front":{"kind":"list like","value":["<title>Purpose</title>","<p id=\"Par1\">Up to 40% of the 56,000 women diagnosed with breast cancer each year in the UK undergo mastectomy. Seroma formation following surgery is common, may delay wound healing, and be uncomfortable or delay the start of adjuvant treatment. Multiple strategies to reduce seroma formation include surgical drains, flap fixation and external compression exist but evidence to support best practice is lacking. We aimed to survey UK breast surgeons to determine current practice to inform the feasibility of undertaking a future trial.</p>","<title>Methods</title>","<p id=\"Par2\">An online survey was developed and circulated to UK breast surgeons via professional and trainee associations and social media to explore current attitudes to drain use and management of post-operative seroma. Simple descriptive statistics were used to summarise the results.</p>","<title>Results</title>","<p id=\"Par3\">The majority of surgeons (82/97, 85%) reported using drains either routinely (38, 39%) or in certain circumstances (44, 45%). Other methods for reducing seroma such as flap fixation were less commonly used. Wide variation was reported in the assessment and management of post-operative seromas. Over half (47/91, 52%) of respondents felt there was some uncertainty about drain use after mastectomy and axillary surgery and two-thirds (59/91, 65%) felt that a trial evaluating the use of drains vs no drains after simple breast cancer surgery was needed.</p>","<title>Conclusions</title>","<p id=\"Par4\">There is a need for a large-scale UK-based RCT to determine if, when and in whom drains are necessary following mastectomy and axillary surgery. This work will inform the design and conduct of a future trial.</p>","<title>Supplementary Information</title>","<p>The online version contains supplementary material available at 10.1007/s10549-023-07042-7.</p>","<title>Keywords</title>"],"string":"[\n \"<title>Purpose</title>\",\n \"<p id=\\\"Par1\\\">Up to 40% of the 56,000 women diagnosed with breast cancer each year in the UK undergo mastectomy. Seroma formation following surgery is common, may delay wound healing, and be uncomfortable or delay the start of adjuvant treatment. Multiple strategies to reduce seroma formation include surgical drains, flap fixation and external compression exist but evidence to support best practice is lacking. We aimed to survey UK breast surgeons to determine current practice to inform the feasibility of undertaking a future trial.</p>\",\n \"<title>Methods</title>\",\n \"<p id=\\\"Par2\\\">An online survey was developed and circulated to UK breast surgeons via professional and trainee associations and social media to explore current attitudes to drain use and management of post-operative seroma. Simple descriptive statistics were used to summarise the results.</p>\",\n \"<title>Results</title>\",\n \"<p id=\\\"Par3\\\">The majority of surgeons (82/97, 85%) reported using drains either routinely (38, 39%) or in certain circumstances (44, 45%). Other methods for reducing seroma such as flap fixation were less commonly used. Wide variation was reported in the assessment and management of post-operative seromas. Over half (47/91, 52%) of respondents felt there was some uncertainty about drain use after mastectomy and axillary surgery and two-thirds (59/91, 65%) felt that a trial evaluating the use of drains vs no drains after simple breast cancer surgery was needed.</p>\",\n \"<title>Conclusions</title>\",\n \"<p id=\\\"Par4\\\">There is a need for a large-scale UK-based RCT to determine if, when and in whom drains are necessary following mastectomy and axillary surgery. This work will inform the design and conduct of a future trial.</p>\",\n \"<title>Supplementary Information</title>\",\n \"<p>The online version contains supplementary material available at 10.1007/s10549-023-07042-7.</p>\",\n \"<title>Keywords</title>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Supplementary Information</title>","<p>Below is the link to the electronic supplementary material.</p>"],"string":"[\n \"<title>Supplementary Information</title>\",\n \"<p>Below is the link to the electronic supplementary material.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":["<title>Acknowledgements</title>","<p>List of PUBMed citable collaborators (names as given in the online survey; some who completed did not leave a name). Nick Abbott, Raj Achuthan, Goran Ahmed, Rachel Ainsworth, Laura Arthur, Salena Bains, Zoe Barber, Jeremy Batt, Ashleigh Bell, Jane Carter, Alice Chambers, Anna Conway, Carol-Ann Courtney, Ian Daltrey, Raouf Daoud, Isabella Dash, Rajiv Dave, Julia Dicks, Urszula Donigiewicz, Hiba Fatayer, Daniel Glassman, Nikki Green, Eleanor Gutteridge, Ahmed Hamad, Anita Hargreaves, James Harvey, Shaziya Hassan Ali, Sophie Helme, Julia Henderson, Susan Hignett, Fiona Hoar, Jonathan Horsnell, Thomas Hubbard, Alex Humphreys, Javeria Iqbal, Omotayo Johnson, Meera Joshi, Charlotte Kallaway, Isabella Karat, Baek Kim, Eleftheria Kleidi, Manish Kothari, Chrissie Laban, Kelly Lambert, Siobhan Laws, Alexander Leeper, Serena Ledwidge, Valentina Lefemine, Jonathan Lund, E Jane Macaskill, Mariam Malik, James Mansell, Loaie Maraqa, Yazan Masannat, Julia Massey, Ross McLean, Jennifer McIlhenny, Colin McIlmunn, Louise Merker, Geraldine Mitchell, Jo Mondani, Elizabeth Morrow, Nabila Nasir, Olubunmi Odofin, Caroline Osborne, Polly Partlett, Anna Powell-Chandler, Sreekumar Sundara Rajan, Clare Rogers, Chandeena Roshanlall, Matthew Philip Rowland, Walid Abou Samra, Lucy Satherley, Brendan Skelly, Richard Sutton, Anne Tansley, Marios Konstantinos Tasoulis, Simon Timbrel, Nader Touqan, Alison Waterworth, Lisa Whisker, Kate Williams, Nihal Gonen Yildirim, Charles Zammit.</p>","<title>Previous communication</title>","<p id=\"Par27\">Abstract (Poster at Association of Breast Surgery Conference): Fairhurst, Katherine et al. <italic>Are drains necessary following mastectomy and axillary surgery? Feasibility work for the Diamond Study.</italic> European Journal of Surgical Oncology, Volume 48, Issue 5, e207.</p>","<title>Author contributions</title>","<p>All authors contributed to study conception and design. Material preparation and data collection were performed by Katherine Fairhurst. Data analysis was completed by Kirsty Roberts and Katherine Fairhurst. The first draft of the manuscript was written by Katherine Fairhurst and all authors read and approved the final manuscript.</p>","<title>Funding</title>","<p>This work was supported by an NIHR Academic Clinical Lectureship (CL-2020-25-002) for Katherine Fairhurst. Shelley Potter is an NIHR Clinician Scientist (CS-2016-16-019). The views expressed are those of the authors and not necessarily those of the UK National Health Service or National Institute for Health and Care Research.</p>","<title>Data availability</title>","<p>The datasets generated and analysed during this study are stored under the provisions of the National Data Protection Act and the University of Bristol requirements. Data may be made available to bona fida researchers only, on reasonable request to the corresponding author, after their host institution has signed a Data Access Agreement.</p>","<title>Declarations</title>","<title>Ethical approval</title>","<p id=\"Par28\">Not required.</p>","<title>Consent to participate/publish</title>","<p id=\"Par29\">All participants voluntarily participated and were made aware of potential publication. All data is presented anonymously, and no patient participants were involved.</p>","<title>Competing interests</title>","<p id=\"Par30\">The authors have no relevant financial or non-financial interests to disclose.</p>"],"string":"[\n \"<title>Acknowledgements</title>\",\n \"<p>List of PUBMed citable collaborators (names as given in the online survey; some who completed did not leave a name). Nick Abbott, Raj Achuthan, Goran Ahmed, Rachel Ainsworth, Laura Arthur, Salena Bains, Zoe Barber, Jeremy Batt, Ashleigh Bell, Jane Carter, Alice Chambers, Anna Conway, Carol-Ann Courtney, Ian Daltrey, Raouf Daoud, Isabella Dash, Rajiv Dave, Julia Dicks, Urszula Donigiewicz, Hiba Fatayer, Daniel Glassman, Nikki Green, Eleanor Gutteridge, Ahmed Hamad, Anita Hargreaves, James Harvey, Shaziya Hassan Ali, Sophie Helme, Julia Henderson, Susan Hignett, Fiona Hoar, Jonathan Horsnell, Thomas Hubbard, Alex Humphreys, Javeria Iqbal, Omotayo Johnson, Meera Joshi, Charlotte Kallaway, Isabella Karat, Baek Kim, Eleftheria Kleidi, Manish Kothari, Chrissie Laban, Kelly Lambert, Siobhan Laws, Alexander Leeper, Serena Ledwidge, Valentina Lefemine, Jonathan Lund, E Jane Macaskill, Mariam Malik, James Mansell, Loaie Maraqa, Yazan Masannat, Julia Massey, Ross McLean, Jennifer McIlhenny, Colin McIlmunn, Louise Merker, Geraldine Mitchell, Jo Mondani, Elizabeth Morrow, Nabila Nasir, Olubunmi Odofin, Caroline Osborne, Polly Partlett, Anna Powell-Chandler, Sreekumar Sundara Rajan, Clare Rogers, Chandeena Roshanlall, Matthew Philip Rowland, Walid Abou Samra, Lucy Satherley, Brendan Skelly, Richard Sutton, Anne Tansley, Marios Konstantinos Tasoulis, Simon Timbrel, Nader Touqan, Alison Waterworth, Lisa Whisker, Kate Williams, Nihal Gonen Yildirim, Charles Zammit.</p>\",\n \"<title>Previous communication</title>\",\n \"<p id=\\\"Par27\\\">Abstract (Poster at Association of Breast Surgery Conference): Fairhurst, Katherine et al. <italic>Are drains necessary following mastectomy and axillary surgery? Feasibility work for the Diamond Study.</italic> European Journal of Surgical Oncology, Volume 48, Issue 5, e207.</p>\",\n \"<title>Author contributions</title>\",\n \"<p>All authors contributed to study conception and design. Material preparation and data collection were performed by Katherine Fairhurst. Data analysis was completed by Kirsty Roberts and Katherine Fairhurst. The first draft of the manuscript was written by Katherine Fairhurst and all authors read and approved the final manuscript.</p>\",\n \"<title>Funding</title>\",\n \"<p>This work was supported by an NIHR Academic Clinical Lectureship (CL-2020-25-002) for Katherine Fairhurst. Shelley Potter is an NIHR Clinician Scientist (CS-2016-16-019). The views expressed are those of the authors and not necessarily those of the UK National Health Service or National Institute for Health and Care Research.</p>\",\n \"<title>Data availability</title>\",\n \"<p>The datasets generated and analysed during this study are stored under the provisions of the National Data Protection Act and the University of Bristol requirements. Data may be made available to bona fida researchers only, on reasonable request to the corresponding author, after their host institution has signed a Data Access Agreement.</p>\",\n \"<title>Declarations</title>\",\n \"<title>Ethical approval</title>\",\n \"<p id=\\\"Par28\\\">Not required.</p>\",\n \"<title>Consent to participate/publish</title>\",\n \"<p id=\\\"Par29\\\">All participants voluntarily participated and were made aware of potential publication. All data is presented anonymously, and no patient participants were involved.</p>\",\n \"<title>Competing interests</title>\",\n \"<p id=\\\"Par30\\\">The authors have no relevant financial or non-financial interests to disclose.</p>\"\n]","isConversational":false},"figure":{"kind":"list like","value":[],"string":"[]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Respondent demographics</p></caption></table-wrap>","<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Operative practice regarding drain use following mastectomy and axillary surgery</p></caption></table-wrap>","<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Post-operative patient pathway of care regarding drain removal following mastectomy and/or axillary surgery (<italic>n</italic> = 95 survey participant responses)</p></caption></table-wrap>","<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Feasibility of a trial examining comparing drains vs no drains following mastectomy and/or axillary surgery (<italic>n</italic> = 91 responses)</p></caption></table-wrap>","<table-wrap id=\"Tab5\"><label>Table 5</label><caption><p>Key themes emerging from content analysis of free text survey responses</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Theme</th><th align=\"left\" colspan=\"2\">Survey respondent quotes</th></tr></thead><tbody><tr><td align=\"left\">The need for high-quality evidence to inform best practice</td><td align=\"left\" colspan=\"2\"><p>Respondent 71: “To provide evidence regarding the indications and benefits of drain use where it is currently lacking. The avoidance of drains would offer an additional economic benefit.”</p><p>Respondent 69: “It would be very useful for our hospital bed capacity if we were confident that no drain use was safe and these 1–2 night stay patients could be considered for day case surgery.”</p><p>Respondent 66: “In my own practice I am satisfied that the seroma and wound complication rate without drains is lower than with drains, and has significantly lower patient discomfort. I am not sure the results of a randomised trial would be applicable to my own technique and would be unlikely to change practice, however a trial may be necessary for drains to be abandoned as a routine.”</p><p>Respondent 58: “…i think it is much less certain whether drains are required after an axillary node dissection and for my own practice a trial would be helpful to guide clinical practice”</p></td></tr><tr><td align=\"left\" rowspan=\"3\">Equipoise</td><td align=\"left\">Personal equipoise</td><td align=\"left\"><p>Respondent 19: “I really do not know if drains are beneficial but I am also unsure if not using them is the 'right' thing to do. I would very much support an RCT”</p><p>Respondent 83: “I would like confirmation of my assumption that drains are not helpful”</p></td></tr><tr><td align=\"left\">Lack of equipoise</td><td align=\"left\"><p>Respondent 34: “Not sure I'm willing to go back to using drains”</p><p>Respondent 37: “May be difficult to establish equipoise. A place I worked in trialled stopping using drains, but then felt they had more problematic haematomas/seromas so went back to using drains. They are unlikely to want to stop using drains.”</p></td></tr><tr><td align=\"left\">In equipoise in some settings</td><td align=\"left\">Respondent 139: “I am comfortable that in my practice drains are not needed for the vast majority of patients but there may be a subset where their use is indicated such as in obese patients.”</td></tr><tr><td align=\"left\">Trial design and feasibility</td><td align=\"left\" colspan=\"2\"><p>Respondent 133: “simple intervention, no high-quality evidence either way, high volume procedures”</p><p>Respondent 35: “Long overdue study. Would be easy to instigate but practice does seem to vary between consultants”</p><p>Respondent 42: “It's a good question that needs studying and there are enough surgeons wanting to answer the question.”</p><p>Respondent 103: “There are units who routinely use drains and others who doesn’t. So it should be possible to randomize and accrue sufficient number of people to achieve adequate power to detect a difference.”</p><p>Respondent 110: “Because there is currently no consensus and patients will generally be guided by surgeons on this so I would expect good compliance and no issues with recruitment numbers”</p><p>Respondent 83: “There is variation in practice so lack of consensus in surgical community. Patients would benefit and question is clear and so would not be difficult to recruit to”</p><p>Respondent 82: “Any trial would be valuable but I feel it is important for the data to capture how many times a patient WITHOUT any drain contacts and/or attends the breast clinic for seroma drainage or wound check, and if they perceive a less comfortable QOL existence in the first 1–2 weeks after surgery when seroma volume is maximal.”</p><p>Respondent 43: “I think no drain policy may increase load on BCN and surgeons for seroma aspiration</p></td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap id=\\\"Tab1\\\"><label>Table 1</label><caption><p>Respondent demographics</p></caption></table-wrap>\",\n \"<table-wrap id=\\\"Tab2\\\"><label>Table 2</label><caption><p>Operative practice regarding drain use following mastectomy and axillary surgery</p></caption></table-wrap>\",\n \"<table-wrap id=\\\"Tab3\\\"><label>Table 3</label><caption><p>Post-operative patient pathway of care regarding drain removal following mastectomy and/or axillary surgery (<italic>n</italic> = 95 survey participant responses)</p></caption></table-wrap>\",\n \"<table-wrap id=\\\"Tab4\\\"><label>Table 4</label><caption><p>Feasibility of a trial examining comparing drains vs no drains following mastectomy and/or axillary surgery (<italic>n</italic> = 91 responses)</p></caption></table-wrap>\",\n \"<table-wrap id=\\\"Tab5\\\"><label>Table 5</label><caption><p>Key themes emerging from content analysis of free text survey responses</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><thead><tr><th align=\\\"left\\\">Theme</th><th align=\\\"left\\\" colspan=\\\"2\\\">Survey respondent quotes</th></tr></thead><tbody><tr><td align=\\\"left\\\">The need for high-quality evidence to inform best practice</td><td align=\\\"left\\\" colspan=\\\"2\\\"><p>Respondent 71: “To provide evidence regarding the indications and benefits of drain use where it is currently lacking. The avoidance of drains would offer an additional economic benefit.”</p><p>Respondent 69: “It would be very useful for our hospital bed capacity if we were confident that no drain use was safe and these 1–2 night stay patients could be considered for day case surgery.”</p><p>Respondent 66: “In my own practice I am satisfied that the seroma and wound complication rate without drains is lower than with drains, and has significantly lower patient discomfort. I am not sure the results of a randomised trial would be applicable to my own technique and would be unlikely to change practice, however a trial may be necessary for drains to be abandoned as a routine.”</p><p>Respondent 58: “…i think it is much less certain whether drains are required after an axillary node dissection and for my own practice a trial would be helpful to guide clinical practice”</p></td></tr><tr><td align=\\\"left\\\" rowspan=\\\"3\\\">Equipoise</td><td align=\\\"left\\\">Personal equipoise</td><td align=\\\"left\\\"><p>Respondent 19: “I really do not know if drains are beneficial but I am also unsure if not using them is the 'right' thing to do. I would very much support an RCT”</p><p>Respondent 83: “I would like confirmation of my assumption that drains are not helpful”</p></td></tr><tr><td align=\\\"left\\\">Lack of equipoise</td><td align=\\\"left\\\"><p>Respondent 34: “Not sure I'm willing to go back to using drains”</p><p>Respondent 37: “May be difficult to establish equipoise. A place I worked in trialled stopping using drains, but then felt they had more problematic haematomas/seromas so went back to using drains. They are unlikely to want to stop using drains.”</p></td></tr><tr><td align=\\\"left\\\">In equipoise in some settings</td><td align=\\\"left\\\">Respondent 139: “I am comfortable that in my practice drains are not needed for the vast majority of patients but there may be a subset where their use is indicated such as in obese patients.”</td></tr><tr><td align=\\\"left\\\">Trial design and feasibility</td><td align=\\\"left\\\" colspan=\\\"2\\\"><p>Respondent 133: “simple intervention, no high-quality evidence either way, high volume procedures”</p><p>Respondent 35: “Long overdue study. Would be easy to instigate but practice does seem to vary between consultants”</p><p>Respondent 42: “It's a good question that needs studying and there are enough surgeons wanting to answer the question.”</p><p>Respondent 103: “There are units who routinely use drains and others who doesn’t. So it should be possible to randomize and accrue sufficient number of people to achieve adequate power to detect a difference.”</p><p>Respondent 110: “Because there is currently no consensus and patients will generally be guided by surgeons on this so I would expect good compliance and no issues with recruitment numbers”</p><p>Respondent 83: “There is variation in practice so lack of consensus in surgical community. Patients would benefit and question is clear and so would not be difficult to recruit to”</p><p>Respondent 82: “Any trial would be valuable but I feel it is important for the data to capture how many times a patient WITHOUT any drain contacts and/or attends the breast clinic for seroma drainage or wound check, and if they perceive a less comfortable QOL existence in the first 1–2 weeks after surgery when seroma volume is maximal.”</p><p>Respondent 43: “I think no drain policy may increase load on BCN and surgeons for seroma aspiration</p></td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":["<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"],"string":"[\n \"<supplementary-material content-type=\\\"local-data\\\" id=\\\"MOESM1\\\"></supplementary-material>\"\n]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"],"string":"[\n \"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic position=\"anchor\" xlink:href=\"10549_2023_7042_Tab1_HTML\" id=\"MO1\"/>","<graphic position=\"anchor\" xlink:href=\"10549_2023_7042_Tab2_HTML\" id=\"MO2\"/>","<graphic position=\"anchor\" xlink:href=\"10549_2023_7042_Tab3_HTML\" id=\"MO3\"/>","<graphic position=\"anchor\" xlink:href=\"10549_2023_7042_Tab4_HTML\" id=\"MO4\"/>"],"string":"[\n \"<graphic position=\\\"anchor\\\" xlink:href=\\\"10549_2023_7042_Tab1_HTML\\\" id=\\\"MO1\\\"/>\",\n \"<graphic position=\\\"anchor\\\" xlink:href=\\\"10549_2023_7042_Tab2_HTML\\\" id=\\\"MO2\\\"/>\",\n \"<graphic position=\\\"anchor\\\" xlink:href=\\\"10549_2023_7042_Tab3_HTML\\\" id=\\\"MO3\\\"/>\",\n \"<graphic position=\\\"anchor\\\" xlink:href=\\\"10549_2023_7042_Tab4_HTML\\\" id=\\\"MO4\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":["<media xlink:href=\"10549_2023_7042_MOESM1_ESM.docx\"><caption><p>Supplementary file1 (DOCX 215 KB)</p></caption></media>"],"string":"[\n \"<media xlink:href=\\\"10549_2023_7042_MOESM1_ESM.docx\\\"><caption><p>Supplementary file1 (DOCX 215 KB)</p></caption></media>\"\n]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"1.\"], \"mixed-citation\": [\"Cancer Research U.K. (2019). \"], \"ext-link\": [\"https://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/breast-cancer\"]}, {\"label\": [\"9.\"], \"surname\": [\"Thomson\", \"Sadideen\", \"Furniss\"], \"given-names\": [\"DR\", \"H\", \"D\"], \"article-title\": [\"Wound drainage after axillary dissection for carcinoma of the breast\"], \"source\": [\"Cochrane Database Syst Rev\"], \"year\": [\"2013\"], \"volume\": [\"10\"], \"fpage\": [\"CD006823\"]}, {\"label\": [\"22.\"], \"mixed-citation\": [\"MacNeill F, Irvine T (2021) Breast surgery, GIRFT Programme National Specialty Report. \"], \"ext-link\": [\"https://gettingitrightfirsttime.co.uk/wp-content/uploads/2021/09/BreastSurgeryReport-Jul21p.pdf\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":31,"string":"31"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:43:45"},"citation":{"kind":"string","value":"Breast Cancer Res Treat. 2024 Oct 25; 203(2):187-196"},"package_file":{"kind":"string","value":"oa_package/1d/dd/PMC10787912.tar.gz"}}},{"rowIdx":195,"cells":{"accession_id":{"kind":"string","value":"PMC10787939"},"pmid":{"kind":"string","value":"38222147"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Stress urinary incontinence (SUI) is a prevalent (4-35% prevalence rate) and often distressing condition that affects individuals, primarily women, across various age groups [##REF##12559262##1##]. It is characterized by the involuntary leakage of urine during activities that increase intra-abdominal pressure, such as sneezing, coughing, running, or lifting heavy objects [##UREF##0##2##]. The impact of SUI extends beyond the physical symptoms, as it can significantly disrupt an individual's social life and personal well-being, ultimately compromising their overall quality of life [##REF##18755458##3##].</p>","<p>Intriguingly, the prevalence of SUI appears to transcend age boundaries, affecting women not only as they age but also in their younger years [##REF##18339574##4##]. This widespread occurrence underscores the importance of addressing this condition comprehensively. However, in many societies, particularly in countries like India, women often endure urinary incontinence silently, with reluctance to discuss their health issues openly [##REF##18339574##4##]. Consequently, the reported prevalence rates might not accurately reflect the true scope of this problem as the reported statistics revealed that 20% of women consulted healthcare professionals for urinary incontinence, while 72% of those affected had been silently battling it for over a year, citing reasons such as unawareness and societal taboos as barriers to seeking help [##REF##18339574##4##].</p>","<p>Understanding the causes of SUI reveals a complex interplay of factors. Vaginal deliveries, for instance, are associated with a heightened risk due to potential alterations in pelvic floor innervation, injury to the levator ani muscle, and damage to the endopelvic fascia from stretching or tearing. Moreover, vaginal births may lead to reduced mobility of the bladder neck, further exacerbating the risk of SUI [##REF##17304528##5##].</p>","<p>The concept of the integrated continence system, encompasses deficits in the intrinsic urethral closure system, the urethral support system, and the lumbopelvic stability system [##REF##17304528##5##]. These systems are interconnected through neural and endopelvic fascia connections and thus play a critical role in maintaining urinary continence [##REF##17304528##5##]. The lumbopelvic region's control, in turn, relies on the coordination of various muscle groups, including the diaphragm, transversus abdominis, pelvic floor musculature, and lumbar multifidus [##REF##17304528##5##]. These muscles regulate intra-abdominal pressure and the tension in the thoracolumbar fascia, influencing postural control [##UREF##1##6##].</p>","<p>However, addressing SUI is not as straightforward as concentrating solely on pelvic floor muscle strengthening. Emerging evidence suggests that the abdominal muscles also play a crucial role in achieving optimal results [##REF##11135380##7##,##REF##23439921##8##]. It has been asserted that pelvic floor muscle rehabilitation reaches its full potential when addressing the abdominal muscles in tandem [##REF##11135380##7##]. It has been reported that pelvic floor muscle contraction led to increased activity in the abdominal muscles and vice versa, highlighting the potential benefits of abdominal muscle (transverse abdominis) training in SUI rehabilitation [##REF##11135380##7##].</p>","<p>To tackle the multifaceted nature of SUI, a promising approach known as dynamic neuromuscular stabilization (DNS) may be introduced, which works on synergistic action of the entire core musculature [##REF##23439921##8##, ####REF##29254112##9##, ##REF##29184312##10##, ##UREF##2##11####2##11##]. DNS relies on developmental kinesiology, comparing the stabilizing patterns of individuals to those of healthy infants [##REF##24411146##12##]. It targets the integrated spinal stabilization system, involving deep cervical flexors, diaphragm, transversus abdominis, multifidus, and the pelvic floor [##REF##23439921##8##]. This system provides a stable foundation for purposeful activities by maintaining axial spine extension and centered extremity joint positions, thereby increasing intra-abdominal pressure sensed by the central nervous system [##REF##23439921##8##]. DNS exercises aim to activate the spinal stabilizing system effectively through repetition, helping individuals regain control during various tasks [##REF##23439921##8##].</p>","<p>Furthermore, dynamic core stability is crucial for optimal athletic performance and hinges on the precise coordination of the integrated spinal stabilization system and the regulation of intra-abdominal pressure [##REF##23439921##8##, ####REF##29254112##9##, ##REF##29184312##10##, ##UREF##2##11##, ##REF##24411146##12##, ##UREF##3##13####3##13##]. This coordination is far more vital than simply building strength in isolated muscle groups as the muscles do not work in isolation [##REF##23439921##8##, ####REF##29254112##9##, ##REF##29184312##10##, ##UREF##2##11####2##11##,##UREF##4##14##].</p>","<p>The DNS method acknowledges that the core musculature functions as a cohesive unit in which the various components work together synergistically [##UREF##5##15##]. When one part of this system is weak or dysfunctional, it can have repercussions on the functioning of other core muscles [##UREF##5##15##,##UREF##6##16##]. DNS exercises are designed to address this interconnection by fostering comprehensive activation and strengthening of the entire core musculature, rather than targeting individual muscles in isolation [##UREF##5##15##,##UREF##6##16##].</p>","<p>By engaging in DNS exercises, individuals can train their core muscles to operate in harmony, providing stability and support to the spine, pelvis, and surrounding structures. As a result, this approach improves posture, enhances control over movements, and bolsters overall body stability [##UREF##5##15##]. DNS exercises contribute to the enhancement of core muscle strength by promoting synchronized activation, improving stability, and optimizing the performance of the entire core unit. By taking a holistic approach to core training, DNS exercises offer a comprehensive strategy for boosting core strength and stability, ultimately leading to improved overall physical function and performance. While DNS has been effectively utilized in various conditions, such as sports injuries, cerebral palsy, hemiplegia, and other diverse ailments [##UREF##6##16##, ####REF##29599856##17##, ##UREF##7##18##, ##UREF##8##19##, ##REF##24411145##20##, ##REF##21943629##21##, ##UREF##9##22####9##22##], there is currently no research investigating the impact of DNS exercises on SUI.</p>","<p>Considering the intricate connections between the pelvic floor and core musculature, it becomes evident that a comprehensive approach to treating SUI is warranted. Thus, this study seeks to evaluate the efficacy of a DNS exercise program in comparison to a traditional pelvic floor exercise program for women dealing with SUI.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Stress urinary incontinence (SUI) is a prevalent (4-35% prevalence rate) and often distressing condition that affects individuals, primarily women, across various age groups [##REF##12559262##1##]. It is characterized by the involuntary leakage of urine during activities that increase intra-abdominal pressure, such as sneezing, coughing, running, or lifting heavy objects [##UREF##0##2##]. The impact of SUI extends beyond the physical symptoms, as it can significantly disrupt an individual's social life and personal well-being, ultimately compromising their overall quality of life [##REF##18755458##3##].</p>\",\n \"<p>Intriguingly, the prevalence of SUI appears to transcend age boundaries, affecting women not only as they age but also in their younger years [##REF##18339574##4##]. This widespread occurrence underscores the importance of addressing this condition comprehensively. However, in many societies, particularly in countries like India, women often endure urinary incontinence silently, with reluctance to discuss their health issues openly [##REF##18339574##4##]. Consequently, the reported prevalence rates might not accurately reflect the true scope of this problem as the reported statistics revealed that 20% of women consulted healthcare professionals for urinary incontinence, while 72% of those affected had been silently battling it for over a year, citing reasons such as unawareness and societal taboos as barriers to seeking help [##REF##18339574##4##].</p>\",\n \"<p>Understanding the causes of SUI reveals a complex interplay of factors. Vaginal deliveries, for instance, are associated with a heightened risk due to potential alterations in pelvic floor innervation, injury to the levator ani muscle, and damage to the endopelvic fascia from stretching or tearing. Moreover, vaginal births may lead to reduced mobility of the bladder neck, further exacerbating the risk of SUI [##REF##17304528##5##].</p>\",\n \"<p>The concept of the integrated continence system, encompasses deficits in the intrinsic urethral closure system, the urethral support system, and the lumbopelvic stability system [##REF##17304528##5##]. These systems are interconnected through neural and endopelvic fascia connections and thus play a critical role in maintaining urinary continence [##REF##17304528##5##]. The lumbopelvic region's control, in turn, relies on the coordination of various muscle groups, including the diaphragm, transversus abdominis, pelvic floor musculature, and lumbar multifidus [##REF##17304528##5##]. These muscles regulate intra-abdominal pressure and the tension in the thoracolumbar fascia, influencing postural control [##UREF##1##6##].</p>\",\n \"<p>However, addressing SUI is not as straightforward as concentrating solely on pelvic floor muscle strengthening. Emerging evidence suggests that the abdominal muscles also play a crucial role in achieving optimal results [##REF##11135380##7##,##REF##23439921##8##]. It has been asserted that pelvic floor muscle rehabilitation reaches its full potential when addressing the abdominal muscles in tandem [##REF##11135380##7##]. It has been reported that pelvic floor muscle contraction led to increased activity in the abdominal muscles and vice versa, highlighting the potential benefits of abdominal muscle (transverse abdominis) training in SUI rehabilitation [##REF##11135380##7##].</p>\",\n \"<p>To tackle the multifaceted nature of SUI, a promising approach known as dynamic neuromuscular stabilization (DNS) may be introduced, which works on synergistic action of the entire core musculature [##REF##23439921##8##, ####REF##29254112##9##, ##REF##29184312##10##, ##UREF##2##11####2##11##]. DNS relies on developmental kinesiology, comparing the stabilizing patterns of individuals to those of healthy infants [##REF##24411146##12##]. It targets the integrated spinal stabilization system, involving deep cervical flexors, diaphragm, transversus abdominis, multifidus, and the pelvic floor [##REF##23439921##8##]. This system provides a stable foundation for purposeful activities by maintaining axial spine extension and centered extremity joint positions, thereby increasing intra-abdominal pressure sensed by the central nervous system [##REF##23439921##8##]. DNS exercises aim to activate the spinal stabilizing system effectively through repetition, helping individuals regain control during various tasks [##REF##23439921##8##].</p>\",\n \"<p>Furthermore, dynamic core stability is crucial for optimal athletic performance and hinges on the precise coordination of the integrated spinal stabilization system and the regulation of intra-abdominal pressure [##REF##23439921##8##, ####REF##29254112##9##, ##REF##29184312##10##, ##UREF##2##11##, ##REF##24411146##12##, ##UREF##3##13####3##13##]. This coordination is far more vital than simply building strength in isolated muscle groups as the muscles do not work in isolation [##REF##23439921##8##, ####REF##29254112##9##, ##REF##29184312##10##, ##UREF##2##11####2##11##,##UREF##4##14##].</p>\",\n \"<p>The DNS method acknowledges that the core musculature functions as a cohesive unit in which the various components work together synergistically [##UREF##5##15##]. When one part of this system is weak or dysfunctional, it can have repercussions on the functioning of other core muscles [##UREF##5##15##,##UREF##6##16##]. DNS exercises are designed to address this interconnection by fostering comprehensive activation and strengthening of the entire core musculature, rather than targeting individual muscles in isolation [##UREF##5##15##,##UREF##6##16##].</p>\",\n \"<p>By engaging in DNS exercises, individuals can train their core muscles to operate in harmony, providing stability and support to the spine, pelvis, and surrounding structures. As a result, this approach improves posture, enhances control over movements, and bolsters overall body stability [##UREF##5##15##]. DNS exercises contribute to the enhancement of core muscle strength by promoting synchronized activation, improving stability, and optimizing the performance of the entire core unit. By taking a holistic approach to core training, DNS exercises offer a comprehensive strategy for boosting core strength and stability, ultimately leading to improved overall physical function and performance. While DNS has been effectively utilized in various conditions, such as sports injuries, cerebral palsy, hemiplegia, and other diverse ailments [##UREF##6##16##, ####REF##29599856##17##, ##UREF##7##18##, ##UREF##8##19##, ##REF##24411145##20##, ##REF##21943629##21##, ##UREF##9##22####9##22##], there is currently no research investigating the impact of DNS exercises on SUI.</p>\",\n \"<p>Considering the intricate connections between the pelvic floor and core musculature, it becomes evident that a comprehensive approach to treating SUI is warranted. Thus, this study seeks to evaluate the efficacy of a DNS exercise program in comparison to a traditional pelvic floor exercise program for women dealing with SUI.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":["<title>Materials and methods</title>","<p>The study commenced after obtaining approval from the Institutional Review Board of Amity Institute of Health Allied Sciences (AUUP/IEC/2021-Jan/03) and was registered in the Clinical Trial Registry, India under the reference CTRI/2021/09/036247. It was designed as a single-blinded (participants were blinded), single-center randomized controlled trial. We recruited a convenient sample of 24 female participants (12 in each group) [##UREF##10##23##]. Participants were eligible if they were females aged 18-40 years, married, diagnosed with mild to moderate SUI, at least one-year post delivery, and medically and physically fit for assessment and physiotherapy. Exclusion criteria included continuous urinary leakage, current urinary incontinence drug therapy, pelvic prolapse > stage I, pregnancy, vaginal/urinary tract infections, menstruation during the examination, presence of tumors/fractures/acute inflammatory diseases, current estrogen treatment, and use of anticholinergics, antidepressants, or serotonin-affecting substances [##REF##20185357##24##]. All the participants were recruited by convenience sampling after being referred and diagnosed by a gynecologist.</p>","<p>Randomization and allocation of participants</p>","<p>Participants who met the inclusion criteria and were screened for exclusion were assigned to one of the treatment groups in a 1:1 ratio. A random number table, generated by a statistician, was used to allocate participants to their respective groups. Subsequently, an allocation plan was meticulously documented in sequential order and securely sealed within opaque envelopes. During the allocation process, an independent individual, unrelated to the study, was responsible for opening the envelope to disclose the assigned group. This unbiased procedure determined whether participants were allotted to either the DNS or Kegel exercise group.</p>","<p>Procedure</p>","<p>Baseline measurements for outcome measures included pelvic floor muscle (PFM) strength (perineometer), electromyography (EMG) of PFM, and quality of life (Urogenital Distress Inventory-6 (UDI-6)). These measurements were repeated after a 12-week intervention period. The Consolidated Standards of Reporting Trials (CONSORT) flowchart, presented in Figure ##FIG##0##1##, provides a visual representation of the participant enrollment and progression throughout the study, illustrating their inclusion, allocation, follow-up, and analysis stages.</p>","<p>The experimental group followed a four-phase exercise protocol (detailed in Table ##TAB##0##1##), while the control group performed PFM contractions, holding each contraction for 10 seconds, with 10 repetitions per set and three sets in total, with one minute of rest between sets [##REF##20185357##24##]. According to the DNS principles, for correct activation of the integrated spinal stabilization system, the abdominal muscles should not only expand in the caudal direction but should expand in all directions, i.e., the posterior and lateral as well. Thus, the investigator should palpate the medial to anterior superior iliac spine anteriorly and the posterolateral aspect of the abdominal wall below the lower ribs from behind to assess the correct expansion of the entire abdominal wall [##UREF##6##16##]. The exercises for both groups were carried out under the supervision of the investigator in the clinical setting. They were instructed not to perform the exercises at home.</p>","<p>Outcome measures</p>","<p>In the completed study, the outcome measures encompassed changes in perineometer values, average, peak, and maximum voluntary contraction (MVC) of PFM as measured by EMG, and the impact of urinary incontinence on quality of life by using UDI-6 after the 12-week treatment period.</p>","<p>Perineometer</p>","<p>The device (Bionics Perineometer Analogue, Mumbai, India) consisted of a conical vaginal insert linked to a handheld microprocessor for measuring pressure in mmHg upon compression of the insert. Participants assumed a crook-lying position, and the perineometer was inserted into the vaginal canal until the compressible portion extended beyond the hymeneal ring. Baseline pressure readings were recorded, and participants were instructed to exert maximum effort to contract their PFM for two to three seconds, completing three consecutive squeezes with a one-minute rest interval. The peak of these three contractions represented their maximum perceived strength. Careful observations and a pressure biofeedback device were employed to ensure a neutral spine position and prevent elevated perineometer readings due to excessive intra-abdominal pressure. The vaginal insert was covered with a condom to ensure safe and hygienic use by multiple participants. The perineometer demonstrated high reliability, indicated by an intraclass correlation coefficient (ICC) of 0.95 [##REF##21419362##25##].</p>","<p>EMG</p>","<p>EMG data were gathered using the NeuroTrac MyoPlus2A version 11.1 device (Hampshire, UK). A pear-shaped intravaginal sensor with stainless steel electrodes was gently inserted into the vagina while patients were in a supine lithotomy position. A reference electrode was placed on the right anterior superior iliac spine. Prior to examination, participants received training to ensure accurate contraction of their PFM. PFM strength was evaluated by recording the average scores from three maximum contractions. The EMG values were documented in terms of average (EMG average), peak (EMG peak), and maximum voluntary contractions (EMG MVC), with measurements in microvolts for EMG average and EMG peak (µV) and in percentage (%) for EMG MVC. An automated protocol software offered on-screen instructions and voice guidance, indicating when to contract and relax the PFM, following a pattern of five-second work and rest intervals [##REF##29635954##26##]. The data were displayed after getting filtered by the inbuilt software.</p>","<p>Urogenital Distress Inventory-6 (UDI-6)</p>","<p>This inventory featured six questions, with responses categorized as \"not at all\" (0), \"a little bit\" (1), \"moderately\" (2), and \"greatly\" (3). Scores were obtained by summing the responses, dividing by six, and multiplying by 25 to yield the Urogenital Distress Inventory (UDI) score. Scores exceeding 33.33 indicated higher distress [##REF##33726776##27##]. The kappa statistics vary from 0.699 to 0.350 for each question [##REF##33726776##27##]. The sensitivity for UDI-6 is 97% [##REF##12382243##28##].</p>","<p>Statistical analysis</p>","<p>The analysis was carried out after the completion of the 12-week treatment period for all participants. We adhered to the Enhancing the Quality and Transparency of Health Research (EQUATOR) network guidelines for reporting descriptive statistics and tests. This approach accounted for the possibility that dropouts might have returned to their initial readings, and any missing data were replaced with the readings and responses from the first day of assessment. To enhance sensitivity, data available after the 12-week period were also analyzed separately. However, there were no dropouts in the current study.</p>","<p>To explore the differences between the two groups, the Mann-Whitney U-test was utilized. Mean differences at a 95% confidence interval were reported to convey the data. However, for within-group comparison, the Wilcoxon sign rank test was used. All data analyses were conducted by a biostatistician who was blinded to the treatment group assignments. The analysis was performed using IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, NY).</p>","<p>Additionally, we calculated the rank-biserial correlation coefficient (r) as a measure of effect size in our study. “r\" quantifies the strength and direction of the relationship between groups in a non-parametric context. The interpretation of r is as follows: 0 indicates no relationship, 1 indicates a perfect positive relationship, and -1 indicates a perfect negative relationship. Traditionally, the magnitude of r is considered small if around 0.1, medium if around 0.3, and large if around 0.5 or above. This provides insight into the practical significance of the observed differences between groups. A higher absolute value of r suggests a more substantial effect, and it complements the p-value by quantifying the magnitude of the observed differences [##UREF##11##29##]. The formula used for the calculation of “r” was r = Z / √N, where, r represents the rank biserial correlation coefficient, and Z is the Z-score obtained from the Mann-Whitney U test. The Z-score is a measure of how many standard deviations an observation or data point is from the mean of a distribution. √N is the square root of the total sample size [##UREF##11##29##].</p>"],"string":"[\n \"<title>Materials and methods</title>\",\n \"<p>The study commenced after obtaining approval from the Institutional Review Board of Amity Institute of Health Allied Sciences (AUUP/IEC/2021-Jan/03) and was registered in the Clinical Trial Registry, India under the reference CTRI/2021/09/036247. It was designed as a single-blinded (participants were blinded), single-center randomized controlled trial. We recruited a convenient sample of 24 female participants (12 in each group) [##UREF##10##23##]. Participants were eligible if they were females aged 18-40 years, married, diagnosed with mild to moderate SUI, at least one-year post delivery, and medically and physically fit for assessment and physiotherapy. Exclusion criteria included continuous urinary leakage, current urinary incontinence drug therapy, pelvic prolapse > stage I, pregnancy, vaginal/urinary tract infections, menstruation during the examination, presence of tumors/fractures/acute inflammatory diseases, current estrogen treatment, and use of anticholinergics, antidepressants, or serotonin-affecting substances [##REF##20185357##24##]. All the participants were recruited by convenience sampling after being referred and diagnosed by a gynecologist.</p>\",\n \"<p>Randomization and allocation of participants</p>\",\n \"<p>Participants who met the inclusion criteria and were screened for exclusion were assigned to one of the treatment groups in a 1:1 ratio. A random number table, generated by a statistician, was used to allocate participants to their respective groups. Subsequently, an allocation plan was meticulously documented in sequential order and securely sealed within opaque envelopes. During the allocation process, an independent individual, unrelated to the study, was responsible for opening the envelope to disclose the assigned group. This unbiased procedure determined whether participants were allotted to either the DNS or Kegel exercise group.</p>\",\n \"<p>Procedure</p>\",\n \"<p>Baseline measurements for outcome measures included pelvic floor muscle (PFM) strength (perineometer), electromyography (EMG) of PFM, and quality of life (Urogenital Distress Inventory-6 (UDI-6)). These measurements were repeated after a 12-week intervention period. The Consolidated Standards of Reporting Trials (CONSORT) flowchart, presented in Figure ##FIG##0##1##, provides a visual representation of the participant enrollment and progression throughout the study, illustrating their inclusion, allocation, follow-up, and analysis stages.</p>\",\n \"<p>The experimental group followed a four-phase exercise protocol (detailed in Table ##TAB##0##1##), while the control group performed PFM contractions, holding each contraction for 10 seconds, with 10 repetitions per set and three sets in total, with one minute of rest between sets [##REF##20185357##24##]. According to the DNS principles, for correct activation of the integrated spinal stabilization system, the abdominal muscles should not only expand in the caudal direction but should expand in all directions, i.e., the posterior and lateral as well. Thus, the investigator should palpate the medial to anterior superior iliac spine anteriorly and the posterolateral aspect of the abdominal wall below the lower ribs from behind to assess the correct expansion of the entire abdominal wall [##UREF##6##16##]. The exercises for both groups were carried out under the supervision of the investigator in the clinical setting. They were instructed not to perform the exercises at home.</p>\",\n \"<p>Outcome measures</p>\",\n \"<p>In the completed study, the outcome measures encompassed changes in perineometer values, average, peak, and maximum voluntary contraction (MVC) of PFM as measured by EMG, and the impact of urinary incontinence on quality of life by using UDI-6 after the 12-week treatment period.</p>\",\n \"<p>Perineometer</p>\",\n \"<p>The device (Bionics Perineometer Analogue, Mumbai, India) consisted of a conical vaginal insert linked to a handheld microprocessor for measuring pressure in mmHg upon compression of the insert. Participants assumed a crook-lying position, and the perineometer was inserted into the vaginal canal until the compressible portion extended beyond the hymeneal ring. Baseline pressure readings were recorded, and participants were instructed to exert maximum effort to contract their PFM for two to three seconds, completing three consecutive squeezes with a one-minute rest interval. The peak of these three contractions represented their maximum perceived strength. Careful observations and a pressure biofeedback device were employed to ensure a neutral spine position and prevent elevated perineometer readings due to excessive intra-abdominal pressure. The vaginal insert was covered with a condom to ensure safe and hygienic use by multiple participants. The perineometer demonstrated high reliability, indicated by an intraclass correlation coefficient (ICC) of 0.95 [##REF##21419362##25##].</p>\",\n \"<p>EMG</p>\",\n \"<p>EMG data were gathered using the NeuroTrac MyoPlus2A version 11.1 device (Hampshire, UK). A pear-shaped intravaginal sensor with stainless steel electrodes was gently inserted into the vagina while patients were in a supine lithotomy position. A reference electrode was placed on the right anterior superior iliac spine. Prior to examination, participants received training to ensure accurate contraction of their PFM. PFM strength was evaluated by recording the average scores from three maximum contractions. The EMG values were documented in terms of average (EMG average), peak (EMG peak), and maximum voluntary contractions (EMG MVC), with measurements in microvolts for EMG average and EMG peak (µV) and in percentage (%) for EMG MVC. An automated protocol software offered on-screen instructions and voice guidance, indicating when to contract and relax the PFM, following a pattern of five-second work and rest intervals [##REF##29635954##26##]. The data were displayed after getting filtered by the inbuilt software.</p>\",\n \"<p>Urogenital Distress Inventory-6 (UDI-6)</p>\",\n \"<p>This inventory featured six questions, with responses categorized as \\\"not at all\\\" (0), \\\"a little bit\\\" (1), \\\"moderately\\\" (2), and \\\"greatly\\\" (3). Scores were obtained by summing the responses, dividing by six, and multiplying by 25 to yield the Urogenital Distress Inventory (UDI) score. Scores exceeding 33.33 indicated higher distress [##REF##33726776##27##]. The kappa statistics vary from 0.699 to 0.350 for each question [##REF##33726776##27##]. The sensitivity for UDI-6 is 97% [##REF##12382243##28##].</p>\",\n \"<p>Statistical analysis</p>\",\n \"<p>The analysis was carried out after the completion of the 12-week treatment period for all participants. We adhered to the Enhancing the Quality and Transparency of Health Research (EQUATOR) network guidelines for reporting descriptive statistics and tests. This approach accounted for the possibility that dropouts might have returned to their initial readings, and any missing data were replaced with the readings and responses from the first day of assessment. To enhance sensitivity, data available after the 12-week period were also analyzed separately. However, there were no dropouts in the current study.</p>\",\n \"<p>To explore the differences between the two groups, the Mann-Whitney U-test was utilized. Mean differences at a 95% confidence interval were reported to convey the data. However, for within-group comparison, the Wilcoxon sign rank test was used. All data analyses were conducted by a biostatistician who was blinded to the treatment group assignments. The analysis was performed using IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, NY).</p>\",\n \"<p>Additionally, we calculated the rank-biserial correlation coefficient (r) as a measure of effect size in our study. “r\\\" quantifies the strength and direction of the relationship between groups in a non-parametric context. The interpretation of r is as follows: 0 indicates no relationship, 1 indicates a perfect positive relationship, and -1 indicates a perfect negative relationship. Traditionally, the magnitude of r is considered small if around 0.1, medium if around 0.3, and large if around 0.5 or above. This provides insight into the practical significance of the observed differences between groups. A higher absolute value of r suggests a more substantial effect, and it complements the p-value by quantifying the magnitude of the observed differences [##UREF##11##29##]. The formula used for the calculation of “r” was r = Z / √N, where, r represents the rank biserial correlation coefficient, and Z is the Z-score obtained from the Mann-Whitney U test. The Z-score is a measure of how many standard deviations an observation or data point is from the mean of a distribution. √N is the square root of the total sample size [##UREF##11##29##].</p>\"\n]","isConversational":false},"results":{"kind":"list like","value":["<title>Results</title>","<p>Demographic data analysis conducted with Mann-Whitney U-tests revealed no statistically significant differences between the DNS and Kegel exercise groups in terms of age, height, weight, BMI, number of children, and duration of symptoms (as presented in Table ##TAB##1##2##).</p>","<p>Strength of the pelvic floor by using a perineometer</p>","<p>Initially, there was no significant difference between the two groups at baseline (p = 0.075). Median ± SD values for DNS and Kegel groups at baseline were 10 ± 2.71 and 10 ± 4.58, respectively. However, at 12 weeks, a statistically significant difference was observed (p = 0.005). Notably, the DNS group exhibited a significant increase in pelvic floor muscle (PFM) strength compared to the Kegel exercise group, i.e., median ± SD values were found to be 24 ± 4.30 and 14 ± 4.60, respectively (Table ##TAB##2##3##). When comparing each group individually from baseline to 12 weeks, significant improvements were observed in both groups (DNS group: p = 0.005; Kegel exercise group: p = 0.020), as presented in Table ##TAB##3##4##.</p>","<p>Electromyography of the pelvic floor muscles</p>","<p>All EMG data were automatically filtered by the inbuilt software of the NeuroTrac MyoPlus2A version 11.1. At baseline, no statistically significant difference was found between the groups for EMG average, EMG peak, and EMG MVC with p-values of 0.545, 0.696, and 0.807, respectively. Median ± SD values for EMG average, EMG peak, and EMG MVC for DNS and Kegel exercise groups at baseline were 41.1 ± 19.08, 78.1 ± 43.11, 100.30 ± 90.7 and 37.4 ± 16.06, 60.2 ± 17.2, 45.2 ± 21.72, respectively. However, a statistically significant difference was observed at 12 weeks for EMG average, EMG peak, and EMG MVC (average p = 0.005; peak p = 0.001; MVC p = 0.009). Significant improvements were noted in both groups for all three components (EMG average, EMG peak, and MVC) when compared from baseline to 12 weeks (DNS group: average p = 0.001; peak p = 0.001; MVC p = 0.030; Kegel exercise group: average p = 0.005; peak p = 0.001; MVC p = 0.005), as presented in Tables ##TAB##2##3##, ##TAB##3##4## for between and within-group analyses, respectively.</p>","<p>UDI-6</p>","<p>No statistically significant difference was found between the two groups at baseline (p = 0.481). Median ± SD values for DNS and Kegel exercise group at baseline were 42 ± 4.95 and 41 ± 4.48, respectively. However, a statistically significant difference was observed between the two groups at 12 weeks (p = 0.001) with median ± SD values for DNS and Kegel exercise groups at 12 weeks as 14 ± 5.45 and 21 ± 7.21, respectively. When evaluating improvements in both groups individually from baseline to 12 weeks, statistically significant improvements were seen in both groups (DNS group: p = 0.032; Kegel exercise group: p = 0.046), as detailed in Tables ##TAB##2##3##, ##TAB##3##4## for between and within-group analyses.</p>","<p>Calculation of “r” effect size</p>","<p>In our study, the \"r\" values were as follows: 0.72 for perineometer, 0.70 for EMG average, 0.74 for EMG peak, 0.8 for EMG MVC, and 0.89 for UDI-6.</p>","<p>These values indicate that all variables had \"r\" values greater than 0.7. An effect size of \"r\" value above 0.7 signifies a medium to large effect size [##REF##12382243##28##]. This suggests that women in the DNS group experienced a more effective treatment compared to the Kegel exercise group for all the variables (Table ##TAB##4##5##).</p>"],"string":"[\n \"<title>Results</title>\",\n \"<p>Demographic data analysis conducted with Mann-Whitney U-tests revealed no statistically significant differences between the DNS and Kegel exercise groups in terms of age, height, weight, BMI, number of children, and duration of symptoms (as presented in Table ##TAB##1##2##).</p>\",\n \"<p>Strength of the pelvic floor by using a perineometer</p>\",\n \"<p>Initially, there was no significant difference between the two groups at baseline (p = 0.075). Median ± SD values for DNS and Kegel groups at baseline were 10 ± 2.71 and 10 ± 4.58, respectively. However, at 12 weeks, a statistically significant difference was observed (p = 0.005). Notably, the DNS group exhibited a significant increase in pelvic floor muscle (PFM) strength compared to the Kegel exercise group, i.e., median ± SD values were found to be 24 ± 4.30 and 14 ± 4.60, respectively (Table ##TAB##2##3##). When comparing each group individually from baseline to 12 weeks, significant improvements were observed in both groups (DNS group: p = 0.005; Kegel exercise group: p = 0.020), as presented in Table ##TAB##3##4##.</p>\",\n \"<p>Electromyography of the pelvic floor muscles</p>\",\n \"<p>All EMG data were automatically filtered by the inbuilt software of the NeuroTrac MyoPlus2A version 11.1. At baseline, no statistically significant difference was found between the groups for EMG average, EMG peak, and EMG MVC with p-values of 0.545, 0.696, and 0.807, respectively. Median ± SD values for EMG average, EMG peak, and EMG MVC for DNS and Kegel exercise groups at baseline were 41.1 ± 19.08, 78.1 ± 43.11, 100.30 ± 90.7 and 37.4 ± 16.06, 60.2 ± 17.2, 45.2 ± 21.72, respectively. However, a statistically significant difference was observed at 12 weeks for EMG average, EMG peak, and EMG MVC (average p = 0.005; peak p = 0.001; MVC p = 0.009). Significant improvements were noted in both groups for all three components (EMG average, EMG peak, and MVC) when compared from baseline to 12 weeks (DNS group: average p = 0.001; peak p = 0.001; MVC p = 0.030; Kegel exercise group: average p = 0.005; peak p = 0.001; MVC p = 0.005), as presented in Tables ##TAB##2##3##, ##TAB##3##4## for between and within-group analyses, respectively.</p>\",\n \"<p>UDI-6</p>\",\n \"<p>No statistically significant difference was found between the two groups at baseline (p = 0.481). Median ± SD values for DNS and Kegel exercise group at baseline were 42 ± 4.95 and 41 ± 4.48, respectively. However, a statistically significant difference was observed between the two groups at 12 weeks (p = 0.001) with median ± SD values for DNS and Kegel exercise groups at 12 weeks as 14 ± 5.45 and 21 ± 7.21, respectively. When evaluating improvements in both groups individually from baseline to 12 weeks, statistically significant improvements were seen in both groups (DNS group: p = 0.032; Kegel exercise group: p = 0.046), as detailed in Tables ##TAB##2##3##, ##TAB##3##4## for between and within-group analyses.</p>\",\n \"<p>Calculation of “r” effect size</p>\",\n \"<p>In our study, the \\\"r\\\" values were as follows: 0.72 for perineometer, 0.70 for EMG average, 0.74 for EMG peak, 0.8 for EMG MVC, and 0.89 for UDI-6.</p>\",\n \"<p>These values indicate that all variables had \\\"r\\\" values greater than 0.7. An effect size of \\\"r\\\" value above 0.7 signifies a medium to large effect size [##REF##12382243##28##]. This suggests that women in the DNS group experienced a more effective treatment compared to the Kegel exercise group for all the variables (Table ##TAB##4##5##).</p>\"\n]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>In this study, we conducted a thorough evaluation to assess the relative effectiveness of DNS exercises in comparison to conventional Kegel exercises for the management of SUI in women. Our study is a significant addition to the existing body of knowledge regarding the array of treatment approaches available for addressing SUI, shedding light on potentially more efficient interventions for this widespread condition.</p>","<p>Our investigation revealed substantial improvements across various key parameters following a 12-week intervention with DNS exercises. These improvements included enhanced PFM strength, increased electrical activity in the PFM, and lower scores on the UDI-6. Conversely, the group that underwent traditional Kegel exercises did not exhibit similar significant improvements when compared to the DNS group, particularly in terms of PFM strength, pelvic floor EMG activity, and UDI-6 scores. These findings suggest that DNS exercises may hold promise as a more advantageous intervention, especially for women dealing with SUI.</p>","<p>One of the intriguing aspects of our study is the direct comparison of DNS exercises and Kegel exercises. This comparison fills a gap in the literature, as there has been limited research directly comparing these two approaches in the context of SUI. Our study provides valuable insights into whether DNS exercises, designed to target the integrated spinal stabilization system, including the pelvic floor, present a more effective alternative to the commonly recommended Kegel exercises.</p>","<p>The rationale behind the superior outcomes observed in the DNS exercise group likely stems from the biomechanical link between the diaphragm, abdominals, multifidus, and the pelvic floor [##REF##17304528##5##, ####UREF##1##6##, ##REF##11135380##7##, ##REF##23439921##8####23439921##8##]. DNS exercises are structured to activate and coordinate these muscle groups, which constitute an integrated spinal stabilization system [##REF##29254112##9##, ####REF##29184312##10##, ##UREF##2##11##, ##REF##24411146##12####24411146##12##]. This holistic approach emphasizes the importance of these muscles working together efficiently.</p>","<p>Coordination of the diaphragm, abdominal muscles (including the transverse abdominis), multifidus, and pelvic floor is central to the success of DNS exercises [##REF##29254112##9##, ####REF##29184312##10##, ##UREF##2##11##, ##REF##24411146##12####24411146##12##]. These exercises emphasize the coordinated activation of this integrated system to maintain the continence mechanism under stress. Additionally, it has been reported that if one part of the stabilizing system is affected, it impacts the other parts as well [##REF##23439921##8##]. In contrast to previous approaches that predominantly focused on strengthening individual muscles, DNS recognizes the importance of these muscles acting in unison.</p>","<p>The literature also supports the idea that treating the lumbopelvic system as a whole is crucial for effective SUI rehabilitation [##REF##18339574##4##,##UREF##1##6##,##REF##11135380##7##]. Another study illustrated that the training of the diaphragm and abdominal muscles along with the pelvic floor was superior to the isolated contraction of PFM for urinary incontinence [##REF##20185357##24##]. The DNS approach, which targets this entire system, has been shown to lead to more significant improvements in our study. It is worth noting that while some studies have focused on different conditions, such as enhancing core stability in stroke patients, they have highlighted the potential of DNS exercises to enhance core stability [##REF##29254112##9##, ####REF##29184312##10##, ##UREF##2##11####2##11##]. Core stability, in turn, plays a pivotal role in providing support to the pelvic floor [##REF##17304528##5##, ####UREF##1##6##, ##REF##11135380##7####11135380##7##].</p>","<p>The timeline for observing improvements in our study was 12 weeks, which aligns with the existing consensus in muscle physiology [##REF##20185357##24##]. Previous research has demonstrated that improvements in pelvic floor strength can be observed within a similar timeframe, validating our findings and underscoring the potential for relatively short-term interventions to yield meaningful results [##REF##20185357##24##].</p>","<p>Limitations</p>","<p>It is important to acknowledge the limitations of our study. We did not include a follow-up to assess the long-term effectiveness of the treatment. Our study was single-blinded, and it is essential to consider that the age group of participants was limited to females between 18 and 40 years old, which may affect the generalizability of our results to other age groups. Additionally, participants with severe SUI were not included in the study, restricting the use of DNS exercises to mild to moderate SUI cases. Beyond these considerations, our study did not incorporate an examination of various pertinent factors, including socioeconomic status, the presence of endometriosis, and dietary habits. These omissions represent a potential source of confounding that could influence the observed outcomes. Consequently, the broader applicability of our findings to diverse demographic and health contexts is constrained. Addressing these limitations necessitates future research endeavors incorporating a follow-up mechanism, diverse age groups, and a more inclusive participant selection process. Additionally, a comprehensive exploration of confounding factors and their potential impact on treatment outcomes will contribute to a more nuanced understanding of the intervention's efficacy.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>In this study, we conducted a thorough evaluation to assess the relative effectiveness of DNS exercises in comparison to conventional Kegel exercises for the management of SUI in women. Our study is a significant addition to the existing body of knowledge regarding the array of treatment approaches available for addressing SUI, shedding light on potentially more efficient interventions for this widespread condition.</p>\",\n \"<p>Our investigation revealed substantial improvements across various key parameters following a 12-week intervention with DNS exercises. These improvements included enhanced PFM strength, increased electrical activity in the PFM, and lower scores on the UDI-6. Conversely, the group that underwent traditional Kegel exercises did not exhibit similar significant improvements when compared to the DNS group, particularly in terms of PFM strength, pelvic floor EMG activity, and UDI-6 scores. These findings suggest that DNS exercises may hold promise as a more advantageous intervention, especially for women dealing with SUI.</p>\",\n \"<p>One of the intriguing aspects of our study is the direct comparison of DNS exercises and Kegel exercises. This comparison fills a gap in the literature, as there has been limited research directly comparing these two approaches in the context of SUI. Our study provides valuable insights into whether DNS exercises, designed to target the integrated spinal stabilization system, including the pelvic floor, present a more effective alternative to the commonly recommended Kegel exercises.</p>\",\n \"<p>The rationale behind the superior outcomes observed in the DNS exercise group likely stems from the biomechanical link between the diaphragm, abdominals, multifidus, and the pelvic floor [##REF##17304528##5##, ####UREF##1##6##, ##REF##11135380##7##, ##REF##23439921##8####23439921##8##]. DNS exercises are structured to activate and coordinate these muscle groups, which constitute an integrated spinal stabilization system [##REF##29254112##9##, ####REF##29184312##10##, ##UREF##2##11##, ##REF##24411146##12####24411146##12##]. This holistic approach emphasizes the importance of these muscles working together efficiently.</p>\",\n \"<p>Coordination of the diaphragm, abdominal muscles (including the transverse abdominis), multifidus, and pelvic floor is central to the success of DNS exercises [##REF##29254112##9##, ####REF##29184312##10##, ##UREF##2##11##, ##REF##24411146##12####24411146##12##]. These exercises emphasize the coordinated activation of this integrated system to maintain the continence mechanism under stress. Additionally, it has been reported that if one part of the stabilizing system is affected, it impacts the other parts as well [##REF##23439921##8##]. In contrast to previous approaches that predominantly focused on strengthening individual muscles, DNS recognizes the importance of these muscles acting in unison.</p>\",\n \"<p>The literature also supports the idea that treating the lumbopelvic system as a whole is crucial for effective SUI rehabilitation [##REF##18339574##4##,##UREF##1##6##,##REF##11135380##7##]. Another study illustrated that the training of the diaphragm and abdominal muscles along with the pelvic floor was superior to the isolated contraction of PFM for urinary incontinence [##REF##20185357##24##]. The DNS approach, which targets this entire system, has been shown to lead to more significant improvements in our study. It is worth noting that while some studies have focused on different conditions, such as enhancing core stability in stroke patients, they have highlighted the potential of DNS exercises to enhance core stability [##REF##29254112##9##, ####REF##29184312##10##, ##UREF##2##11####2##11##]. Core stability, in turn, plays a pivotal role in providing support to the pelvic floor [##REF##17304528##5##, ####UREF##1##6##, ##REF##11135380##7####11135380##7##].</p>\",\n \"<p>The timeline for observing improvements in our study was 12 weeks, which aligns with the existing consensus in muscle physiology [##REF##20185357##24##]. Previous research has demonstrated that improvements in pelvic floor strength can be observed within a similar timeframe, validating our findings and underscoring the potential for relatively short-term interventions to yield meaningful results [##REF##20185357##24##].</p>\",\n \"<p>Limitations</p>\",\n \"<p>It is important to acknowledge the limitations of our study. We did not include a follow-up to assess the long-term effectiveness of the treatment. Our study was single-blinded, and it is essential to consider that the age group of participants was limited to females between 18 and 40 years old, which may affect the generalizability of our results to other age groups. Additionally, participants with severe SUI were not included in the study, restricting the use of DNS exercises to mild to moderate SUI cases. Beyond these considerations, our study did not incorporate an examination of various pertinent factors, including socioeconomic status, the presence of endometriosis, and dietary habits. These omissions represent a potential source of confounding that could influence the observed outcomes. Consequently, the broader applicability of our findings to diverse demographic and health contexts is constrained. Addressing these limitations necessitates future research endeavors incorporating a follow-up mechanism, diverse age groups, and a more inclusive participant selection process. Additionally, a comprehensive exploration of confounding factors and their potential impact on treatment outcomes will contribute to a more nuanced understanding of the intervention's efficacy.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>In conclusion, our study's results suggest that DNS exercises, which emphasize the coordinated activation of the diaphragm, abdominals, multifidus, and pelvic floor, may offer a more effective approach for managing stress urinary incontinence in women compared to traditional Kegel exercises. The biomechanical synergy of these muscle groups appears to be a critical factor in the observed improvements. However, further research is warranted to explore the long-term effectiveness and specificity of this approach for different populations by incontinence type, severity, age, and other important factors. Nonetheless, our study opens new avenues for the management of SUI, offering individuals and healthcare professionals a potentially more effective intervention to consider.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>In conclusion, our study's results suggest that DNS exercises, which emphasize the coordinated activation of the diaphragm, abdominals, multifidus, and pelvic floor, may offer a more effective approach for managing stress urinary incontinence in women compared to traditional Kegel exercises. The biomechanical synergy of these muscle groups appears to be a critical factor in the observed improvements. However, further research is warranted to explore the long-term effectiveness and specificity of this approach for different populations by incontinence type, severity, age, and other important factors. Nonetheless, our study opens new avenues for the management of SUI, offering individuals and healthcare professionals a potentially more effective intervention to consider.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Background and objective</p>","<p>Stress urinary incontinence (SUI) is a prevalent condition affecting women of various age groups, significantly impacting their quality of life. To address this multifaceted issue, a comprehensive approach that goes beyond traditional pelvic floor exercises is needed. Dynamic neuromuscular stabilization (DNS) exercises, targeting the integrated spinal stabilization system, offer a promising alternative. Thus, this study aimed to compare the effectiveness of DNS exercises and Kegel exercises in managing SUI among women.</p>","<p>Methods</p>","<p>This single-blinded, pilot study involved 24 women aged 18-40 years with mild to moderate SUI. Participants were divided into DNS and Kegel exercise groups. Outcome measures included perineometer readings, electromyography (EMG) data, and the Urogenital Distress Inventory-6 (UDI-6). Statistical analysis compared baseline and 12-week data within and between groups, and rank-biserial correlation coefficient (r) as a measure of effect size in our study was calculated.</p>","<p>Results</p>","<p>At 12 weeks, the DNS group showed significant improvement in pelvic floor muscle strength compared to Kegel exercises (p = 0.005). Both groups had significantly enhanced pelvic floor muscle strength (p < 0.05). A significant change occurred for EMG average, EMG peak, and EMG maximum voluntary contraction (MVC) at 12 weeks (average p = 0.005; peak p = 0.001; MVC p = 0.009), with significant improvements in both groups (p < 0.05). For UDI-6, a significant difference emerged between the two groups at 12 weeks (p < 0.05), with significant improvements in both groups individually from baseline to 12 weeks (p < 0.05). The effect size \"r\" for all variables indicated a medium to large effect size, underscoring the substantial and significant impact of DNS exercises in managing SUI among women compared to Kegel exercises.</p>","<p>Conclusion</p>","<p>This study suggests that DNS exercises, emphasizing the coordinated activation of the diaphragm, abdominals, multifidus, and pelvic floor, may provide a more effective approach for managing SUI in women compared to traditional Kegel exercises.</p>"],"string":"[\n \"<p>Background and objective</p>\",\n \"<p>Stress urinary incontinence (SUI) is a prevalent condition affecting women of various age groups, significantly impacting their quality of life. To address this multifaceted issue, a comprehensive approach that goes beyond traditional pelvic floor exercises is needed. Dynamic neuromuscular stabilization (DNS) exercises, targeting the integrated spinal stabilization system, offer a promising alternative. Thus, this study aimed to compare the effectiveness of DNS exercises and Kegel exercises in managing SUI among women.</p>\",\n \"<p>Methods</p>\",\n \"<p>This single-blinded, pilot study involved 24 women aged 18-40 years with mild to moderate SUI. Participants were divided into DNS and Kegel exercise groups. Outcome measures included perineometer readings, electromyography (EMG) data, and the Urogenital Distress Inventory-6 (UDI-6). Statistical analysis compared baseline and 12-week data within and between groups, and rank-biserial correlation coefficient (r) as a measure of effect size in our study was calculated.</p>\",\n \"<p>Results</p>\",\n \"<p>At 12 weeks, the DNS group showed significant improvement in pelvic floor muscle strength compared to Kegel exercises (p = 0.005). Both groups had significantly enhanced pelvic floor muscle strength (p < 0.05). A significant change occurred for EMG average, EMG peak, and EMG maximum voluntary contraction (MVC) at 12 weeks (average p = 0.005; peak p = 0.001; MVC p = 0.009), with significant improvements in both groups (p < 0.05). For UDI-6, a significant difference emerged between the two groups at 12 weeks (p < 0.05), with significant improvements in both groups individually from baseline to 12 weeks (p < 0.05). The effect size \\\"r\\\" for all variables indicated a medium to large effect size, underscoring the substantial and significant impact of DNS exercises in managing SUI among women compared to Kegel exercises.</p>\",\n \"<p>Conclusion</p>\",\n \"<p>This study suggests that DNS exercises, emphasizing the coordinated activation of the diaphragm, abdominals, multifidus, and pelvic floor, may provide a more effective approach for managing SUI in women compared to traditional Kegel exercises.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":[],"string":"[]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Trial design overview - CONSORT flow chart</title><p>DNS: dynamic neuromuscular stabilization; CONSORT: Consolidated Standards of Reporting Trials.</p></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>Trial design overview - CONSORT flow chart</title><p>DNS: dynamic neuromuscular stabilization; CONSORT: Consolidated Standards of Reporting Trials.</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Dynamic neuromuscular stabilization exercise protocol for the rehabilitation of stress urinary incontinence</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">PHASE</td><td rowspan=\"1\" colspan=\"1\">DURATION (weeks)</td><td rowspan=\"1\" colspan=\"1\">PATIENT’S POSITION</td><td rowspan=\"1\" colspan=\"1\">INVESTIGATOR’S POSITION</td><td rowspan=\"1\" colspan=\"1\">INSTRUCTIONS</td><td rowspan=\"1\" colspan=\"1\">REPETITIONS</td><td rowspan=\"1\" colspan=\"1\">SETS</td></tr><tr><td rowspan=\"2\" colspan=\"1\">I</td><td rowspan=\"2\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">1. Crook lying</td><td rowspan=\"1\" colspan=\"1\">At the patient’s side, palpating the transverse abdominis muscle (medial to anterior superior iliac spine)</td><td rowspan=\"1\" colspan=\"1\">Contract the pelvic floor slightly like holding urine, and while maintaining this, inhale so that your abdominal wall expands against the therapist’s fingers, while maintaining this expansion, exhale out and then keep breathing normally</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">3</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">2. Prone</td><td rowspan=\"1\" colspan=\"1\">The posterolateral aspect of the abdominal wall below the lower ribs from behind</td><td rowspan=\"1\" colspan=\"1\">Same as the previous exercise</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td rowspan=\"2\" colspan=\"1\">IIa.</td><td rowspan=\"2\" colspan=\"1\">1.5</td><td rowspan=\"1\" colspan=\"1\">1. Supine with legs supported on a stool</td><td rowspan=\"1\" colspan=\"1\">Palpating the abdominal muscle medial to anterior superior iliac spine</td><td rowspan=\"1\" colspan=\"1\">Same as Phase I</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">3</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">2. Quadruped</td><td rowspan=\"1\" colspan=\"1\">The posterolateral aspect of the abdominal wall below the lower ribs from behind</td><td rowspan=\"1\" colspan=\"1\">Same as Phase I</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td rowspan=\"2\" colspan=\"1\">IIb</td><td rowspan=\"2\" colspan=\"1\">1.5</td><td rowspan=\"1\" colspan=\"1\">1. Supine with legs supported on gym ball</td><td rowspan=\"1\" colspan=\"1\">Palpating the abdominal muscle medial to anterior superior iliac spine</td><td rowspan=\"1\" colspan=\"1\">Same as Phase I</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">3</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">2. Quadruped with knees supported on an unstable surface</td><td rowspan=\"1\" colspan=\"1\">The posterolateral aspect of the abdominal wall below the lower ribs from behind</td><td rowspan=\"1\" colspan=\"1\">Same as Phase I</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td rowspan=\"1\" colspan=\"1\">III</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">Quadruped to heel sitting</td><td rowspan=\"1\" colspan=\"1\">Sitting behind the patient</td><td rowspan=\"1\" colspan=\"1\">Ask the patient to come in a heel sitting position and maintain the spine in neutral along with the abdominal and pelvic floor contraction while the therapist will resist the same in the first part, and in the second part, the patient will try to do heel sitting while the therapist’s force will overpower the patient’s force and movement will occur cranially in the direction of therapist’s force</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">3</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">IV</td><td rowspan=\"2\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">1. Sagittal stabilization with hip abduction</td><td rowspan=\"1\" colspan=\"1\">Palpating the abdominal muscle medial to anterior superior iliac spine</td><td rowspan=\"1\" colspan=\"1\">Contract the pelvic floor slightly like holding urine, and while maintaining this, inhale so that your abdominal wall expands against the therapist’s fingers, while maintaining this expansion, exhale out and then keep breathing normally, while maintaining this, ask the patient to take one hip in abduction while keeping the other hip in neutral (starting) position. Repeat the same on the other side</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td rowspan=\"1\" colspan=\"1\">2. Sagittal stabilization with hip flexion</td><td rowspan=\"1\" colspan=\"1\">Palpating the abdominal muscle medial to anterior superior iliac spine</td><td rowspan=\"1\" colspan=\"1\">Contract the pelvic floor slightly like holding urine and while maintaining this, inhale so that your abdominal wall expands against the therapist’s fingers, while maintaining this expansion, exhale out and then keep breathing normally, while maintaining this, ask the patient to take one hip in flexion while keeping the other hip in neutral (starting) position. Repeat the same on the other side</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">3</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Demographics of the participants</title><p>The median, SD, and range (maximum and minimum values) of the demographics (age, weight, height, BMI, number of children, and duration of symptom) of the DNS and Kegel exercise groups are presented in the table. No statistically significant difference was reported for the demographics as p-value > 0.05.</p><p>DNS: dynamic neuromuscular stabilization exercise group; Kegel: Kegel exercise group.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\"> </td><td rowspan=\"2\" colspan=\"1\">Group</td><td colspan=\"2\" rowspan=\"1\">Range</td><td rowspan=\"2\" colspan=\"1\">Median</td><td rowspan=\"2\" colspan=\"1\">Standard deviation</td><td rowspan=\"2\" colspan=\"1\">p-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Minimum</td><td rowspan=\"1\" colspan=\"1\">Maximum</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Age (years)</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">23</td><td rowspan=\"1\" colspan=\"1\">40</td><td rowspan=\"1\" colspan=\"1\">35</td><td rowspan=\"1\" colspan=\"1\">4.9</td><td rowspan=\"2\" colspan=\"1\">0.173</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">25</td><td rowspan=\"1\" colspan=\"1\">40</td><td rowspan=\"1\" colspan=\"1\">33</td><td rowspan=\"1\" colspan=\"1\">3.9</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Weight (kg)</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">42</td><td rowspan=\"1\" colspan=\"1\">77</td><td rowspan=\"1\" colspan=\"1\">60</td><td rowspan=\"1\" colspan=\"1\">8.1</td><td rowspan=\"2\" colspan=\"1\">0.713</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">44</td><td rowspan=\"1\" colspan=\"1\">75</td><td rowspan=\"1\" colspan=\"1\">60</td><td rowspan=\"1\" colspan=\"1\">7.81</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Height (cm)</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">144</td><td rowspan=\"1\" colspan=\"1\">171</td><td rowspan=\"1\" colspan=\"1\">154</td><td rowspan=\"1\" colspan=\"1\">5.7</td><td rowspan=\"2\" colspan=\"1\">0.228</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">146</td><td rowspan=\"1\" colspan=\"1\">168</td><td rowspan=\"1\" colspan=\"1\">153</td><td rowspan=\"1\" colspan=\"1\">4.67</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">BMI (kg/m<sup>2</sup>)</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">19.56</td><td rowspan=\"1\" colspan=\"1\">30.82</td><td rowspan=\"1\" colspan=\"1\">24.9</td><td rowspan=\"1\" colspan=\"1\">3.40</td><td rowspan=\"2\" colspan=\"1\">0.322</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">21.08</td><td rowspan=\"1\" colspan=\"1\">30.8</td><td rowspan=\"1\" colspan=\"1\">26.6</td><td rowspan=\"1\" colspan=\"1\">2.8</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Number of children</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">0.99</td><td rowspan=\"2\" colspan=\"1\">0.735</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">0.86</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Duration of symptom</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">48</td><td rowspan=\"1\" colspan=\"1\">18</td><td rowspan=\"1\" colspan=\"1\">10.25</td><td rowspan=\"2\" colspan=\"1\">0.971</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">60</td><td rowspan=\"1\" colspan=\"1\">19</td><td rowspan=\"1\" colspan=\"1\">12.73</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB3\"><label>Table 3</label><caption><title>Results of between-group comparison at baseline and after 12 weeks of intervention</title><p>Median ± SD values of perineometer in mmHg, EMG (average, peak, and MVC), and UDI-6 at baseline and 12 weeks for DNS and Kegel exercise groups.</p><p>EMG: electromyography; MVC: maximum voluntary contraction; UDI-6: Urogenital Distress Inventory-6; DNS: dynamic neuromuscular stabilization. * P-value < 0.05 depicts a statistically significant difference between the two groups.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"2\">Outcome</td><td rowspan=\"2\" colspan=\"1\">Measurements</td><td colspan=\"2\" rowspan=\"1\">Group</td><td rowspan=\"2\" colspan=\"1\">Median difference (DNS-Kegel)</td><td rowspan=\"2\" colspan=\"1\">95% confidence interval (lower limit, upper limit)</td><td rowspan=\"2\" colspan=\"1\">p-value for Mann-Whitney U test for between-group comparison</td><td rowspan=\"2\" colspan=\"1\">Z-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">Kegel</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"2\">Perineometer (mmHg)</td><td rowspan=\"1\" colspan=\"1\">Baseline (median ± SD)</td><td rowspan=\"1\" colspan=\"1\">10 + 2.71</td><td rowspan=\"1\" colspan=\"1\">10 + 4.58</td><td rowspan=\"1\" colspan=\"1\">-2</td><td rowspan=\"1\" colspan=\"1\">-3.35, -0.46</td><td rowspan=\"1\" colspan=\"1\">0.075</td><td rowspan=\"1\" colspan=\"1\">-1.65</td></tr><tr><td rowspan=\"1\" colspan=\"1\">12 weeks (median ± SD)</td><td rowspan=\"1\" colspan=\"1\">24 + 4.3</td><td rowspan=\"1\" colspan=\"1\">14 + 4.60</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">6.54, 10.34</td><td rowspan=\"1\" colspan=\"1\">0.005*</td><td rowspan=\"1\" colspan=\"1\">-3.56</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"6\" colspan=\"1\">Electromyography</td><td rowspan=\"2\" colspan=\"1\">Average (µV)</td><td rowspan=\"1\" colspan=\"1\">Baseline (median ± SD)</td><td rowspan=\"1\" colspan=\"1\">41.1 + 19.08</td><td rowspan=\"1\" colspan=\"1\">37.4 + 16.06</td><td rowspan=\"1\" colspan=\"1\">-2</td><td rowspan=\"1\" colspan=\"1\">-7, 9.6</td><td rowspan=\"1\" colspan=\"1\">0.545</td><td rowspan=\"1\" colspan=\"1\">-0.95</td></tr><tr><td rowspan=\"1\" colspan=\"1\">12 weeks (median ± SD)</td><td rowspan=\"1\" colspan=\"1\">77.1 + 37.4</td><td rowspan=\"1\" colspan=\"1\">55.4 + 18.72</td><td rowspan=\"1\" colspan=\"1\">17.6</td><td rowspan=\"1\" colspan=\"1\">7.2, 26.8</td><td rowspan=\"1\" colspan=\"1\">0.005*</td><td rowspan=\"1\" colspan=\"1\">-3.64</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Peak (µV)</td><td rowspan=\"1\" colspan=\"1\">Baseline (median ± SD)</td><td rowspan=\"1\" colspan=\"1\">78.1 + 43.11</td><td rowspan=\"1\" colspan=\"1\">60.2 + 17.2</td><td rowspan=\"1\" colspan=\"1\">-10.4</td><td rowspan=\"1\" colspan=\"1\">-20.53, 3.22</td><td rowspan=\"1\" colspan=\"1\">0.696</td><td rowspan=\"1\" colspan=\"1\">-0.40</td></tr><tr><td rowspan=\"1\" colspan=\"1\">12 weeks (median ± SD)</td><td rowspan=\"1\" colspan=\"1\">100.3 + 90.7</td><td rowspan=\"1\" colspan=\"1\">77.9 + 33.6</td><td rowspan=\"1\" colspan=\"1\">21.1</td><td rowspan=\"1\" colspan=\"1\">5.48, 32.74</td><td rowspan=\"1\" colspan=\"1\">0.001*</td><td rowspan=\"1\" colspan=\"1\">-3.45</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Maximum voluntary contraction (%)</td><td rowspan=\"1\" colspan=\"1\">Baseline (median ± SD)</td><td rowspan=\"1\" colspan=\"1\">50 + 20.8</td><td rowspan=\"1\" colspan=\"1\">45.2 + 21.72</td><td rowspan=\"1\" colspan=\"1\">-6.8</td><td rowspan=\"1\" colspan=\"1\">-11.43, 7.63</td><td rowspan=\"1\" colspan=\"1\">0.807</td><td rowspan=\"1\" colspan=\"1\">-0.71</td></tr><tr><td rowspan=\"1\" colspan=\"1\">12 weeks (median ± SD)</td><td rowspan=\"1\" colspan=\"1\">69.8 + 90.8</td><td rowspan=\"1\" colspan=\"1\">58.2 + 16.92</td><td rowspan=\"1\" colspan=\"1\">17.8</td><td rowspan=\"1\" colspan=\"1\">13.2, 56.18</td><td rowspan=\"1\" colspan=\"1\">0.009*</td><td rowspan=\"1\" colspan=\"1\">-0.80</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"2\">UDI-6</td><td rowspan=\"1\" colspan=\"1\">Baseline (median ± SD)</td><td rowspan=\"1\" colspan=\"1\">42 + 4.95</td><td rowspan=\"1\" colspan=\"1\">41 + 4.48</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">-1.37, 2.7</td><td rowspan=\"1\" colspan=\"1\">0.481</td><td rowspan=\"1\" colspan=\"1\">-0.38</td></tr><tr><td rowspan=\"1\" colspan=\"1\">12 weeks (median ± SD)</td><td rowspan=\"1\" colspan=\"1\">14 + 5.45</td><td rowspan=\"1\" colspan=\"1\">21 + 7.21</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">3.89, 9.17</td><td rowspan=\"1\" colspan=\"1\">0.001*</td><td rowspan=\"1\" colspan=\"1\">-0.89</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB4\"><label>Table 4</label><caption><title>Results of within-group comparison at baseline and after 12 weeks of intervention</title><p>Median ± SD of perineometer values, EMG (average, peak, and MVC), and UDI-6 for DNS and Kegel exercise groups at baseline and 12 weeks.</p><p>EMG: electromyography; MVC: maximum voluntary contraction; UDI-6: Urogenital Distress Inventory-6.</p><p>* P < 0.05 depicts a significant statistical difference between the baseline and 12 weeks' values for the various outcome measures for the two groups.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"2\">Outcome</td><td rowspan=\"2\" colspan=\"1\">Group</td><td colspan=\"2\" rowspan=\"1\">Measurements</td><td rowspan=\"2\" colspan=\"1\">Median difference (baseline-12 weeks)</td><td rowspan=\"2\" colspan=\"1\">95% confidence interval (lower limit, upper limit)</td><td rowspan=\"2\" colspan=\"1\">P-value for Wilcoxon sign rank test for within-group comparison</td><td rowspan=\"2\" colspan=\"1\">Z-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Baseline</td><td rowspan=\"1\" colspan=\"1\">12 weeks</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"2\">Perineometer (mmHg)</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">10 + 2.71</td><td rowspan=\"1\" colspan=\"1\">24 + 4.3</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">12.07, 14.9</td><td rowspan=\"1\" colspan=\"1\">0.005*</td><td rowspan=\"1\" colspan=\"1\">-5.84</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">10 + 4.58</td><td rowspan=\"1\" colspan=\"1\">14 + 4.60</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">2.68, 3.58</td><td rowspan=\"1\" colspan=\"1\">0.020*</td><td rowspan=\"1\" colspan=\"1\">-5.81</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"6\" colspan=\"1\">Electromyography</td><td rowspan=\"2\" colspan=\"1\">Average (µV)</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">41.1 + 19.08</td><td rowspan=\"1\" colspan=\"1\">77.1 + 37.4</td><td rowspan=\"1\" colspan=\"1\">27.7</td><td rowspan=\"1\" colspan=\"1\">26.62, 43.29</td><td rowspan=\"1\" colspan=\"1\">0.001*</td><td rowspan=\"1\" colspan=\"1\">-5.82</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">37.4 + 16.06</td><td rowspan=\"1\" colspan=\"1\">55.4 + 18.72</td><td rowspan=\"1\" colspan=\"1\">16.7</td><td rowspan=\"1\" colspan=\"1\">13.96, 19.32</td><td rowspan=\"1\" colspan=\"1\">0.005*</td><td rowspan=\"1\" colspan=\"1\">-5.74</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Peak (µV)</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">78.1 + 43.11</td><td rowspan=\"1\" colspan=\"1\">100.3 + 90.7</td><td rowspan=\"1\" colspan=\"1\">21.2</td><td rowspan=\"1\" colspan=\"1\">20.95, 30.65</td><td rowspan=\"1\" colspan=\"1\">0.001*</td><td rowspan=\"1\" colspan=\"1\">-5.84</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">60.2 + 17.2</td><td rowspan=\"1\" colspan=\"1\">77.9 +33.6</td><td rowspan=\"1\" colspan=\"1\">12.3</td><td rowspan=\"1\" colspan=\"1\">11.76, 18.90</td><td rowspan=\"1\" colspan=\"1\">0.001*</td><td rowspan=\"1\" colspan=\"1\">-5.87</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Maximum voluntary contraction (%)</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">50 + 20.8</td><td rowspan=\"1\" colspan=\"1\">69.8 +90.8</td><td rowspan=\"1\" colspan=\"1\">23.1</td><td rowspan=\"1\" colspan=\"1\">15.69, 28.51</td><td rowspan=\"1\" colspan=\"1\">0.030*</td><td rowspan=\"1\" colspan=\"1\">-4.88</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">45.2 + 21.72</td><td rowspan=\"1\" colspan=\"1\">58.2 + 16.92</td><td rowspan=\"1\" colspan=\"1\">13</td><td rowspan=\"1\" colspan=\"1\">6.34, 14.27</td><td rowspan=\"1\" colspan=\"1\">0.005*</td><td rowspan=\"1\" colspan=\"1\">-5.32</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"2\">UDI-6</td><td rowspan=\"1\" colspan=\"1\">DNS</td><td rowspan=\"1\" colspan=\"1\">42 + 4.95</td><td rowspan=\"1\" colspan=\"1\">14 + 5.45</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">23.2, 27.5</td><td rowspan=\"1\" colspan=\"1\">0.032*</td><td rowspan=\"1\" colspan=\"1\">-5.84</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kegel</td><td rowspan=\"1\" colspan=\"1\">41 + 4.48</td><td rowspan=\"1\" colspan=\"1\">21 + 7.21</td><td rowspan=\"1\" colspan=\"1\">19</td><td rowspan=\"1\" colspan=\"1\">15.68, 20.58</td><td rowspan=\"1\" colspan=\"1\">0.046*</td><td rowspan=\"1\" colspan=\"1\">-5.87</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB5\"><label>Table 5</label><caption><title>Values of effect size \"r\" for perineometer, EMG, and UDI-6</title><p>EMG: electromyography; MVC: maximum voluntary contraction; UDI-6: Urogenital Distress Inventory-6.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Outcome measure</td><td rowspan=\"1\" colspan=\"1\">Value of \"r\"</td></tr><tr><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">Perineometer</td><td rowspan=\"1\" colspan=\"1\">0.72</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">EMG average</td><td rowspan=\"1\" colspan=\"1\">0.70</td></tr><tr><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">EMG peak</td><td rowspan=\"1\" colspan=\"1\">0.74</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">MVC</td><td rowspan=\"1\" colspan=\"1\">0.8</td></tr><tr><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">UDI-6</td><td rowspan=\"1\" colspan=\"1\">0.89</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB1\\\"><label>Table 1</label><caption><title>Dynamic neuromuscular stabilization exercise protocol for the rehabilitation of stress urinary incontinence</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">PHASE</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DURATION (weeks)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">PATIENT’S POSITION</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">INVESTIGATOR’S POSITION</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">INSTRUCTIONS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">REPETITIONS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">SETS</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">I</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1. Crook lying</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">At the patient’s side, palpating the transverse abdominis muscle (medial to anterior superior iliac spine)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Contract the pelvic floor slightly like holding urine, and while maintaining this, inhale so that your abdominal wall expands against the therapist’s fingers, while maintaining this expansion, exhale out and then keep breathing normally</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2. Prone</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The posterolateral aspect of the abdominal wall below the lower ribs from behind</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Same as the previous exercise</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">IIa.</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">1.5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1. Supine with legs supported on a stool</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Palpating the abdominal muscle medial to anterior superior iliac spine</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Same as Phase I</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2. Quadruped</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The posterolateral aspect of the abdominal wall below the lower ribs from behind</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Same as Phase I</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td></tr><tr><td rowspan=\\\"2\\\" colspan=\\\"1\\\">IIb</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">1.5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1. Supine with legs supported on gym ball</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Palpating the abdominal muscle medial to anterior superior iliac spine</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Same as Phase I</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2. Quadruped with knees supported on an unstable surface</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">The posterolateral aspect of the abdominal wall below the lower ribs from behind</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Same as Phase I</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">III</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Quadruped to heel sitting</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Sitting behind the patient</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Ask the patient to come in a heel sitting position and maintain the spine in neutral along with the abdominal and pelvic floor contraction while the therapist will resist the same in the first part, and in the second part, the patient will try to do heel sitting while the therapist’s force will overpower the patient’s force and movement will occur cranially in the direction of therapist’s force</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">IV</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1. Sagittal stabilization with hip abduction</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Palpating the abdominal muscle medial to anterior superior iliac spine</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Contract the pelvic floor slightly like holding urine, and while maintaining this, inhale so that your abdominal wall expands against the therapist’s fingers, while maintaining this expansion, exhale out and then keep breathing normally, while maintaining this, ask the patient to take one hip in abduction while keeping the other hip in neutral (starting) position. Repeat the same on the other side</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2. Sagittal stabilization with hip flexion</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Palpating the abdominal muscle medial to anterior superior iliac spine</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Contract the pelvic floor slightly like holding urine and while maintaining this, inhale so that your abdominal wall expands against the therapist’s fingers, while maintaining this expansion, exhale out and then keep breathing normally, while maintaining this, ask the patient to take one hip in flexion while keeping the other hip in neutral (starting) position. Repeat the same on the other side</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB2\\\"><label>Table 2</label><caption><title>Demographics of the participants</title><p>The median, SD, and range (maximum and minimum values) of the demographics (age, weight, height, BMI, number of children, and duration of symptom) of the DNS and Kegel exercise groups are presented in the table. No statistically significant difference was reported for the demographics as p-value > 0.05.</p><p>DNS: dynamic neuromuscular stabilization exercise group; Kegel: Kegel exercise group.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\"> </td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Group</td><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Range</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Median</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Standard deviation</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">p-value</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Minimum</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Maximum</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Age (years)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">23</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">40</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">35</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.9</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.173</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">25</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">40</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">33</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.9</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Weight (kg)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">42</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">77</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">60</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">8.1</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.713</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">44</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">75</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">60</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.81</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Height (cm)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">144</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">171</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">154</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.7</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.228</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">146</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">168</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">153</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4.67</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">BMI (kg/m<sup>2</sup>)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">19.56</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">30.82</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">24.9</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.40</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.322</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">21.08</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">30.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">26.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.8</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Number of children</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.99</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.735</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.86</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Duration of symptom</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">48</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">18</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10.25</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">0.971</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">60</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">19</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12.73</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB3\\\"><label>Table 3</label><caption><title>Results of between-group comparison at baseline and after 12 weeks of intervention</title><p>Median ± SD values of perineometer in mmHg, EMG (average, peak, and MVC), and UDI-6 at baseline and 12 weeks for DNS and Kegel exercise groups.</p><p>EMG: electromyography; MVC: maximum voluntary contraction; UDI-6: Urogenital Distress Inventory-6; DNS: dynamic neuromuscular stabilization. * P-value < 0.05 depicts a statistically significant difference between the two groups.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"2\\\">Outcome</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Measurements</td><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Group</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Median difference (DNS-Kegel)</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">95% confidence interval (lower limit, upper limit)</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">p-value for Mann-Whitney U test for between-group comparison</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Z-value</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td></tr><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"2\\\">Perineometer (mmHg)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Baseline (median ± SD)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 + 2.71</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 + 4.58</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-3.35, -0.46</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.075</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-1.65</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12 weeks (median ± SD)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">24 + 4.3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14 + 4.60</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.54, 10.34</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.005*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-3.56</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"6\\\" colspan=\\\"1\\\">Electromyography</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Average (µV)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Baseline (median ± SD)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">41.1 + 19.08</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">37.4 + 16.06</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-7, 9.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.545</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-0.95</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12 weeks (median ± SD)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">77.1 + 37.4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">55.4 + 18.72</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">17.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7.2, 26.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.005*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-3.64</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Peak (µV)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Baseline (median ± SD)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">78.1 + 43.11</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">60.2 + 17.2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-10.4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-20.53, 3.22</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.696</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-0.40</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12 weeks (median ± SD)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">100.3 + 90.7</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">77.9 + 33.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">21.1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5.48, 32.74</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.001*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-3.45</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Maximum voluntary contraction (%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Baseline (median ± SD)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">50 + 20.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">45.2 + 21.72</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-6.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-11.43, 7.63</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.807</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-0.71</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12 weeks (median ± SD)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">69.8 + 90.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">58.2 + 16.92</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">17.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">13.2, 56.18</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.009*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-0.80</td></tr><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"2\\\">UDI-6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Baseline (median ± SD)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">42 + 4.95</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">41 + 4.48</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-1.37, 2.7</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.481</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-0.38</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12 weeks (median ± SD)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14 + 5.45</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">21 + 7.21</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">7</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3.89, 9.17</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.001*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-0.89</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB4\\\"><label>Table 4</label><caption><title>Results of within-group comparison at baseline and after 12 weeks of intervention</title><p>Median ± SD of perineometer values, EMG (average, peak, and MVC), and UDI-6 for DNS and Kegel exercise groups at baseline and 12 weeks.</p><p>EMG: electromyography; MVC: maximum voluntary contraction; UDI-6: Urogenital Distress Inventory-6.</p><p>* P < 0.05 depicts a significant statistical difference between the baseline and 12 weeks' values for the various outcome measures for the two groups.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"2\\\">Outcome</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Group</td><td colspan=\\\"2\\\" rowspan=\\\"1\\\">Measurements</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Median difference (baseline-12 weeks)</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">95% confidence interval (lower limit, upper limit)</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">P-value for Wilcoxon sign rank test for within-group comparison</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Z-value</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Baseline</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12 weeks</td></tr><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"2\\\">Perineometer (mmHg)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 + 2.71</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">24 + 4.3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12.07, 14.9</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.005*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-5.84</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">10 + 4.58</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14 + 4.60</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2.68, 3.58</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.020*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-5.81</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"6\\\" colspan=\\\"1\\\">Electromyography</td><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Average (µV)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">41.1 + 19.08</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">77.1 + 37.4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">27.7</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">26.62, 43.29</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.001*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-5.82</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">37.4 + 16.06</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">55.4 + 18.72</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">16.7</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">13.96, 19.32</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.005*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-5.74</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Peak (µV)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">78.1 + 43.11</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">100.3 + 90.7</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">21.2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">20.95, 30.65</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.001*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-5.84</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">60.2 + 17.2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">77.9 +33.6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">12.3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">11.76, 18.90</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.001*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-5.87</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"2\\\" colspan=\\\"1\\\">Maximum voluntary contraction (%)</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">50 + 20.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">69.8 +90.8</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">23.1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">15.69, 28.51</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.030*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-4.88</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">45.2 + 21.72</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">58.2 + 16.92</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">13</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">6.34, 14.27</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.005*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-5.32</td></tr><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"2\\\">UDI-6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">DNS</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">42 + 4.95</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">14 + 5.45</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">26</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">23.2, 27.5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.032*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-5.84</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Kegel</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">41 + 4.48</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">21 + 7.21</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">19</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">15.68, 20.58</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.046*</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">-5.87</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB5\\\"><label>Table 5</label><caption><title>Values of effect size \\\"r\\\" for perineometer, EMG, and UDI-6</title><p>EMG: electromyography; MVC: maximum voluntary contraction; UDI-6: Urogenital Distress Inventory-6.</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\"> </td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Outcome measure</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Value of \\\"r\\\"</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">1</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">Perineometer</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.72</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">2</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">EMG average</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.70</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">3</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">EMG peak</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.74</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">4</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">MVC</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.8</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">5</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">UDI-6</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">0.89</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Meena Gupta, Kiran Sharma, Raju K Parasher</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Meena Gupta, Kiran Sharma, Raju K Parasher, Jasmine Kaur Chawla</p><p><bold>Drafting of the manuscript:</bold> Meena Gupta, Kiran Sharma</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Meena Gupta, Kiran Sharma, Raju K Parasher, Jasmine Kaur Chawla</p><p><bold>Supervision:</bold> Meena Gupta, Raju K Parasher, Jasmine Kaur Chawla</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study. Institutional Review Board of Amity Institute of Health Allied Sciences issued approval AUUP/IEC/2021-Jan/03</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Concept and design:</bold> Meena Gupta, Kiran Sharma, Raju K Parasher</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Meena Gupta, Kiran Sharma, Raju K Parasher, Jasmine Kaur Chawla</p><p><bold>Drafting of the manuscript:</bold> Meena Gupta, Kiran Sharma</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Meena Gupta, Kiran Sharma, Raju K Parasher, Jasmine Kaur Chawla</p><p><bold>Supervision:</bold> Meena Gupta, Raju K Parasher, Jasmine Kaur Chawla</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study. Institutional Review Board of Amity Institute of Health Allied Sciences issued approval AUUP/IEC/2021-Jan/03</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Animal Ethics</title><fn fn-type=\\\"other\\\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0015-00000050551-i01\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0015-00000050551-i01\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"2\"], \"article-title\": [\"The association 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[\"\\n\"], \"surname\": [\"Berry\", \"Johnston\", \"Mielke\"], \"given-names\": [\"KJ\", \"JE\", \"PW\"], \"publisher-loc\": [\"New York, NY\"], \"publisher-name\": [\"Springer\"], \"year\": [\"2018\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":29,"string":"29"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:43:45"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50551"},"package_file":{"kind":"string","value":"oa_package/aa/f9/PMC10787939.tar.gz"}}},{"rowIdx":196,"cells":{"accession_id":{"kind":"string","value":"PMC10787940"},"pmid":{"kind":"string","value":"38222225"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Hemobilia is an infrequent cause of upper gastrointestinal bleeding, which is most often secondary to intrabiliary procedures, hepatic trauma, inflammatory diseases, biliary tumors, and vascular malformations [##REF##36156458##1##,##UREF##0##2##]. Although common in the gastrointestinal tract, angiodysplasia is extremely rare in the biliary ducts, with few cases described thus far. The resolution of these pathologies has become quicker and more precise with the advancement of cholangioscopy as a diagnostic and therapeutic tool [##UREF##1##3##]. Until now, the diagnosis of these conditions depended on radiological assistance and empirical treatments. We report a case of a patient with hemobilia due to angiodysplasia of the major biliary duct, diagnosed by cholangioscopy, and the condition was resolved after the placement of a biliary stent.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Hemobilia is an infrequent cause of upper gastrointestinal bleeding, which is most often secondary to intrabiliary procedures, hepatic trauma, inflammatory diseases, biliary tumors, and vascular malformations [##REF##36156458##1##,##UREF##0##2##]. Although common in the gastrointestinal tract, angiodysplasia is extremely rare in the biliary ducts, with few cases described thus far. The resolution of these pathologies has become quicker and more precise with the advancement of cholangioscopy as a diagnostic and therapeutic tool [##UREF##1##3##]. Until now, the diagnosis of these conditions depended on radiological assistance and empirical treatments. We report a case of a patient with hemobilia due to angiodysplasia of the major biliary duct, diagnosed by cholangioscopy, and the condition was resolved after the placement of a biliary stent.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>Hemobilia remains a challenging entity to diagnose and treat, requiring the use of various technologies and a multidisciplinary approach. Common causes include intrabiliary procedures (endoscopic or percutaneous), hepatic trauma, cholangiopathies secondary to cirrhosis, tumors, and vascular malformations. These, however, account for a minority of cases, usually occurring secondary to portobiliary fistulas, varices, hereditary telangiectasias, and rarely angiodysplasias [##REF##35937199##4##,##UREF##2##5##].</p>","<p>Angiodysplasia is a common finding in upper and lower gastrointestinal endoscopies, primarily located in the stomach, duodenum, and colon. Its occurrence in the biliary tract is extremely rare, making diagnosis challenging and requiring specific resources and trained professionals, with few cases reported to date. It is characterized by the malformation of vessels in the mucosa and submucosa, with no hereditary or racial connection, but with a higher incidence in elderly patients with aortic stenosis, chronic kidney disease, lung diseases, and von Willebrand disease. Its pathophysiology remains uncertain but may be related to common bile duct contractions causing intermittent vessel obstruction, leading to focal dilations, tortuosities, and collateral vessel formation [##UREF##3##6##,##REF##32908760##7##].</p>","<p>The advancement of endoscopic devices and technologies now allows for the determination of the causes of hemobilia. Once a diagnostic tool, ERCP has become an important therapeutic tool, evolving toward digital cholangioscopy. Adverse events, such as biliary tract perforation, air embolism, and bacteremia, are rare, though slightly higher than ERCP. Risk factors include patient age, stent placement, and, primarily, lithotripsy for biliary stones [##UREF##4##8##,##REF##27236413##9##]. In our case, cholangioscopy played a crucial role in diagnosis and treatment. After an unsuccessful attempt at endoscopic treatment and cholangiography showing no major abnormalities, cholangioscopy revealed the source of bleeding as an angiodysplasia in the distal major biliary duct. The few cases reported in the literature have had varying approaches to this finding. One patient with a history of hemobilia, but no active bleeding at the time of diagnosis, maintained stable hemoglobin levels without intervention. Another used laser therapy for both angiodysplasia and actively bleeding neoplasms, and a third underwent radiointerventional embolization [##UREF##1##3##,##UREF##5##10##,##REF##10385725##11##]. Factors influencing our treatment decision were active bleeding, a distal location near the papilla, and the unavailability of hemostasis accessories with SpyScope DS® at the time of the examination. Possible complications related to biliary stent placement include acute pancreatitis, cholangitis, migration, and acute cholecystitis. In patients eligible for laparoscopic cholecystectomy, the procedure can be performed as described in our case. Percutaneous or endoscopic ultrasound-guided gallbladder drainage is an alternative for gallbladder decompression in patients who are not surgical candidates [##UREF##4##8##,##UREF##6##12##].</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>Hemobilia remains a challenging entity to diagnose and treat, requiring the use of various technologies and a multidisciplinary approach. Common causes include intrabiliary procedures (endoscopic or percutaneous), hepatic trauma, cholangiopathies secondary to cirrhosis, tumors, and vascular malformations. These, however, account for a minority of cases, usually occurring secondary to portobiliary fistulas, varices, hereditary telangiectasias, and rarely angiodysplasias [##REF##35937199##4##,##UREF##2##5##].</p>\",\n \"<p>Angiodysplasia is a common finding in upper and lower gastrointestinal endoscopies, primarily located in the stomach, duodenum, and colon. Its occurrence in the biliary tract is extremely rare, making diagnosis challenging and requiring specific resources and trained professionals, with few cases reported to date. It is characterized by the malformation of vessels in the mucosa and submucosa, with no hereditary or racial connection, but with a higher incidence in elderly patients with aortic stenosis, chronic kidney disease, lung diseases, and von Willebrand disease. Its pathophysiology remains uncertain but may be related to common bile duct contractions causing intermittent vessel obstruction, leading to focal dilations, tortuosities, and collateral vessel formation [##UREF##3##6##,##REF##32908760##7##].</p>\",\n \"<p>The advancement of endoscopic devices and technologies now allows for the determination of the causes of hemobilia. Once a diagnostic tool, ERCP has become an important therapeutic tool, evolving toward digital cholangioscopy. Adverse events, such as biliary tract perforation, air embolism, and bacteremia, are rare, though slightly higher than ERCP. Risk factors include patient age, stent placement, and, primarily, lithotripsy for biliary stones [##UREF##4##8##,##REF##27236413##9##]. In our case, cholangioscopy played a crucial role in diagnosis and treatment. After an unsuccessful attempt at endoscopic treatment and cholangiography showing no major abnormalities, cholangioscopy revealed the source of bleeding as an angiodysplasia in the distal major biliary duct. The few cases reported in the literature have had varying approaches to this finding. One patient with a history of hemobilia, but no active bleeding at the time of diagnosis, maintained stable hemoglobin levels without intervention. Another used laser therapy for both angiodysplasia and actively bleeding neoplasms, and a third underwent radiointerventional embolization [##UREF##1##3##,##UREF##5##10##,##REF##10385725##11##]. Factors influencing our treatment decision were active bleeding, a distal location near the papilla, and the unavailability of hemostasis accessories with SpyScope DS® at the time of the examination. Possible complications related to biliary stent placement include acute pancreatitis, cholangitis, migration, and acute cholecystitis. In patients eligible for laparoscopic cholecystectomy, the procedure can be performed as described in our case. Percutaneous or endoscopic ultrasound-guided gallbladder drainage is an alternative for gallbladder decompression in patients who are not surgical candidates [##UREF##4##8##,##UREF##6##12##].</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>In conclusion, digital cholangioscopy played a crucial role in the diagnosis and treatment decision for hemobilia due to angiodysplasia of the major biliary duct. There is still a lack of data in the literature to define the gold-standard therapy, but the use of a fully covered metallic stent proved effective in achieving hemostasis.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>In conclusion, digital cholangioscopy played a crucial role in the diagnosis and treatment decision for hemobilia due to angiodysplasia of the major biliary duct. There is still a lack of data in the literature to define the gold-standard therapy, but the use of a fully covered metallic stent proved effective in achieving hemostasis.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Hemobilia is described as bleeding from the intra- or extrahepatic biliary tree expressed through the major duodenal papilla into the duodenum, with angiodysplasia of the major biliary duct as a rare etiological factor with few cases reported in the literature. Cholangioscopy plays a pivotal role in diagnosing and making therapeutic decisions regarding biliary tract lesions. We report a case of the diagnosis and treatment of hemobilia secondary to bleeding from angiodysplasia of the major biliary duct, which was resolved after the placement of a fully covered metallic stent, with a review of the literature.</p>"],"string":"[\n \"<p>Hemobilia is described as bleeding from the intra- or extrahepatic biliary tree expressed through the major duodenal papilla into the duodenum, with angiodysplasia of the major biliary duct as a rare etiological factor with few cases reported in the literature. Cholangioscopy plays a pivotal role in diagnosing and making therapeutic decisions regarding biliary tract lesions. We report a case of the diagnosis and treatment of hemobilia secondary to bleeding from angiodysplasia of the major biliary duct, which was resolved after the placement of a fully covered metallic stent, with a review of the literature.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Case presentation</title>","<p>An 87-year-old male patient with atrial fibrillation who was on apixaban 5 mg/day presented to the emergency department with exertional dyspnea and lower limb edema. He had no history of external bleeding but complained of mild abdominal pain upon examination. Laboratory tests revealed acute anemia with a hemoglobin level of 5.7 g/dL.</p>","<p>Due to the patient's age and comorbidities, the attending team opted for a capsule endoscopy to avoid a more invasive procedure like a traditional endoscopy after an inconclusive computed tomography. Capsule endoscopy revealed blood residues in the stomach and small intestine, with recent clots predominantly in the duodenal region. Upper gastrointestinal endoscopy (EGD) identified active bleeding at the major duodenal papilla with an unsuccessful attempt at hemostasis through sclerosis (Figure ##FIG##0##1##).</p>","<p>Following an investigation with magnetic resonance imaging of the biliary tract, which identified a punctate enhancement focus in the distal common bile duct (Figure ##FIG##1##2##), the patient underwent endoscopic retrograde cholangiopancreatography (ERCP). During the procedure, bleeding at the papillary orifice persisted, and cholangiography showed no abnormalities.</p>","<p>Cholangioscopy with SpyScope DS® (Boston Scientific, Massachusetts) immediately above the papilla revealed anomalous vascular formations, suggestive of angiectasias with active bleeding (Figures ##FIG##2##3##, ##FIG##3##4##).</p>","<p>A 10 mm x 60 mm fully covered self-expanding metallic stent (WallFlex® Biliary RX Stent, Boston Scientific, Massachusetts) was placed for mechanical hemostasis, resulting in a favorable outcome.</p>","<p>The patient remained hospitalized, with stabilization of hematimetric values but developed acute cholecystitis due to obstruction of the cystic duct by the stent. He underwent laparoscopic cholecystectomy and was discharged on the fourth postoperative day. The stent was removed by EGD after 90 days, with resolution of the bleeding, and anticoagulation was resumed.</p>"],"string":"[\n \"<title>Case presentation</title>\",\n \"<p>An 87-year-old male patient with atrial fibrillation who was on apixaban 5 mg/day presented to the emergency department with exertional dyspnea and lower limb edema. He had no history of external bleeding but complained of mild abdominal pain upon examination. Laboratory tests revealed acute anemia with a hemoglobin level of 5.7 g/dL.</p>\",\n \"<p>Due to the patient's age and comorbidities, the attending team opted for a capsule endoscopy to avoid a more invasive procedure like a traditional endoscopy after an inconclusive computed tomography. Capsule endoscopy revealed blood residues in the stomach and small intestine, with recent clots predominantly in the duodenal region. Upper gastrointestinal endoscopy (EGD) identified active bleeding at the major duodenal papilla with an unsuccessful attempt at hemostasis through sclerosis (Figure ##FIG##0##1##).</p>\",\n \"<p>Following an investigation with magnetic resonance imaging of the biliary tract, which identified a punctate enhancement focus in the distal common bile duct (Figure ##FIG##1##2##), the patient underwent endoscopic retrograde cholangiopancreatography (ERCP). During the procedure, bleeding at the papillary orifice persisted, and cholangiography showed no abnormalities.</p>\",\n \"<p>Cholangioscopy with SpyScope DS® (Boston Scientific, Massachusetts) immediately above the papilla revealed anomalous vascular formations, suggestive of angiectasias with active bleeding (Figures ##FIG##2##3##, ##FIG##3##4##).</p>\",\n \"<p>A 10 mm x 60 mm fully covered self-expanding metallic stent (WallFlex® Biliary RX Stent, Boston Scientific, Massachusetts) was placed for mechanical hemostasis, resulting in a favorable outcome.</p>\",\n \"<p>The patient remained hospitalized, with stabilization of hematimetric values but developed acute cholecystitis due to obstruction of the cystic duct by the stent. He underwent laparoscopic cholecystectomy and was discharged on the fourth postoperative day. The stent was removed by EGD after 90 days, with resolution of the bleeding, and anticoagulation was resumed.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Active bleeding from the major duodenal papilla</title></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Enhancement focus in the distal common bile duct (black arrow)</title></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG3\"><label>Figure 3</label><caption><title>Transition between normal biliary mucosa and angiodysplasia on cholangioscopy</title></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG4\"><label>Figure 4</label><caption><title>Abnormal vessels on cholangioscopy</title></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>Active bleeding from the major duodenal papilla</title></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG2\\\"><label>Figure 2</label><caption><title>Enhancement focus in the distal common bile duct (black arrow)</title></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG3\\\"><label>Figure 3</label><caption><title>Transition between normal biliary mucosa and angiodysplasia on cholangioscopy</title></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG4\\\"><label>Figure 4</label><caption><title>Abnormal vessels on cholangioscopy</title></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":[],"string":"[]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Acquisition, analysis, or interpretation of data:</bold> Marcus Vinícius S. Costa, Lucas V. Aragão, Tomazo P. Franzini</p><p><bold>Drafting of the manuscript:</bold> Marcus Vinícius S. Costa, Lucas V. Aragão, Julia M. Jesus, Fabio C. Mancini, Tomazo P. Franzini</p><p><bold>Concept and design:</bold> Lucas V. Aragão, Julia M. Jesus, Fabio C. Mancini, Tomazo P. Franzini</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Tomazo P. Franzini</p><p><bold>Supervision:</bold> Tomazo P. Franzini</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Acquisition, analysis, or interpretation of data:</bold> Marcus Vinícius S. Costa, Lucas V. Aragão, Tomazo P. Franzini</p><p><bold>Drafting of the manuscript:</bold> Marcus Vinícius S. Costa, Lucas V. Aragão, Julia M. Jesus, Fabio C. Mancini, Tomazo P. Franzini</p><p><bold>Concept and design:</bold> Lucas V. Aragão, Julia M. Jesus, Fabio C. Mancini, Tomazo P. Franzini</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Tomazo P. Franzini</p><p><bold>Supervision:</bold> Tomazo P. Franzini</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0015-00000050552-i01\" position=\"float\"/>","<graphic xlink:href=\"cureus-0015-00000050552-i02\" position=\"float\"/>","<graphic xlink:href=\"cureus-0015-00000050552-i03\" position=\"float\"/>","<graphic xlink:href=\"cureus-0015-00000050552-i04\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0015-00000050552-i01\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0015-00000050552-i02\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0015-00000050552-i03\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0015-00000050552-i04\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"2\"], \"article-title\": [\"Endovascular and endoscopic treatment of hemobilia: a report of two cases\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Cardona\", \"Rivero\", \"Pinto\", \"Barrag\\u00e1n\", \"Torres\"], \"given-names\": [\"JD\", \"OM\", \"R\", \"CA\", \"DF\"], \"fpage\": [\"0\"], \"volume\": [\"14\"], \"year\": [\"2022\"]}, {\"label\": [\"3\"], \"article-title\": [\"Hemobilia from biliary angiodysplasia diagnosed with cholangioscopy\"], \"source\": [\"ACG Case Rep J\"], \"person-group\": [\"\\n\"], \"surname\": [\"Foong\", \"Lee\", \"Kudakachira\", \"Ramberan\"], \"given-names\": [\"KS\", \"A\", \"S\", \"H\"], \"fpage\": [\"0\"], \"volume\": [\"3\"], \"year\": [\"2016\"]}, {\"label\": [\"5\"], \"article-title\": [\"Small arteriovenous malformation of the common bile duct causing hemobilia in a patient with hereditary hemorrhagic telangiectasia\"], \"source\": [\"Cardiovasc Intervent Radiol\"], \"person-group\": [\"\\n\"], \"surname\": [\"Hayashi\", \"Baba\", \"Ueno\", \"Nakajo\"], \"given-names\": [\"S\", \"Y\", \"K\", \"M\"], \"fpage\": [\"0\"], \"lpage\": [\"4\"], \"volume\": [\"31 Suppl 2\"], \"year\": [\"2008\"]}, {\"label\": [\"6\"], \"article-title\": [\"Angiodysplasia\"], \"person-group\": [\"\\n\"], \"surname\": [\"Aghighi\", \"Taherian\", \"Sharma\"], \"given-names\": [\"M\", \"M\", \"A\"], \"publisher-loc\": [\"Treasure Island, FL\"], \"publisher-name\": [\"StatPearls Publishing\"], \"year\": [\"2023\"], \"uri\": [\"https://www.ncbi.nlm.nih.gov/books/NBK549777/\"]}, {\"label\": [\"8\"], \"article-title\": [\"Use of fully covered self-expanding metal biliary stents for managing endoscopic biliary sphincterotomy related bleeding\"], \"source\": [\"Endosc Int Open\"], \"person-group\": [\"\\n\"], \"surname\": [\"Bilal\", \"Chandnani\", \"McDonald\"], \"given-names\": [\"M\", \"M\", \"NM\"], \"fpage\": [\"0\"], \"lpage\": [\"73\"], \"volume\": [\"9\"], \"year\": [\"2021\"]}, {\"label\": [\"10\"], \"article-title\": [\"Rare cause of obstructive haemobilia with recurrent biliopancreatic complications: a paradigmatic case\"], \"source\": [\"BMJ Case Rep\"], \"person-group\": [\"\\n\"], \"surname\": [\"Correia\", \"Almeida\", \"Gomes\", \"Figueiredo\"], \"given-names\": [\"C\", \"N\", \"D\", \"P\"], \"fpage\": [\"245303\"], \"volume\": [\"15\"], \"year\": [\"2022\"]}, {\"label\": [\"12\"], \"article-title\": [\"Hemostasis using a covered self-expandable metal stent for pseudoaneurysm bleeding from the perihilar bile duct\"], \"source\": [\"DEN Open\"], \"person-group\": [\"\\n\"], \"surname\": [\"Ishii\", \"Nakayama\", \"Kikuchi\"], \"given-names\": [\"Y\", \"A\", \"K\"], \"fpage\": [\"0\"], \"volume\": [\"3\"], \"year\": [\"2023\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":12,"string":"12"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:43:45"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50552"},"package_file":{"kind":"string","value":"oa_package/14/dd/PMC10787940.tar.gz"}}},{"rowIdx":197,"cells":{"accession_id":{"kind":"string","value":"PMC10787941"},"pmid":{"kind":"string","value":"38222993"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Primary ciliary dyskinesia (PCD), an autosomal recessive condition characterized by motile cilia malfunction, displays clinical and genetic variation. Some of the clinical symptoms of PCD include left-right lateralization, infertility, and chronic upper and lower respiratory illness [##REF##25422025##1##, ####REF##31624012##2##, ##REF##30166424##3##, ##REF##27514592##4##, ##REF##17634184##5####17634184##5##]. There is no one best way to diagnose PCD; instead, a variety of methods can be used, such as a combination of nasal nitric oxide (nNO), high-speed genetic analysis, immunological fluorescence of ciliated cells, transmission electron microscopy (TEM), high-speed video microscopy analysis, immune fluorescence of ciliated cells, and genetic analysis (gene panel analysis or extensive genetic analysis) [##REF##27836958##6##,##UREF##0##7##].</p>","<p>There is a significant correlation between phenotype and specific genetic changes. Reduced generation of multiple motile cilia (RGMC) has been linked to <italic>CCNO </italic>gene mutations, which are also more likely to have a more severe respiratory disease phenotype with pulmonary failure at a younger age [##REF##24747639##8##].</p>","<p>For the first time, we describe the <italic>CCNO NM 021147.4</italic> (c.258 262dup.p, Gln88argfs*8 homozygous) gene mutation that caused severe PCD in a consanguineous Indian family.</p>","<p>This article was previously presented as a meeting abstract at the 2022 American Thoracic Society International Conference Meeting on May 13-18, 2022.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Primary ciliary dyskinesia (PCD), an autosomal recessive condition characterized by motile cilia malfunction, displays clinical and genetic variation. Some of the clinical symptoms of PCD include left-right lateralization, infertility, and chronic upper and lower respiratory illness [##REF##25422025##1##, ####REF##31624012##2##, ##REF##30166424##3##, ##REF##27514592##4##, ##REF##17634184##5####17634184##5##]. There is no one best way to diagnose PCD; instead, a variety of methods can be used, such as a combination of nasal nitric oxide (nNO), high-speed genetic analysis, immunological fluorescence of ciliated cells, transmission electron microscopy (TEM), high-speed video microscopy analysis, immune fluorescence of ciliated cells, and genetic analysis (gene panel analysis or extensive genetic analysis) [##REF##27836958##6##,##UREF##0##7##].</p>\",\n \"<p>There is a significant correlation between phenotype and specific genetic changes. Reduced generation of multiple motile cilia (RGMC) has been linked to <italic>CCNO </italic>gene mutations, which are also more likely to have a more severe respiratory disease phenotype with pulmonary failure at a younger age [##REF##24747639##8##].</p>\",\n \"<p>For the first time, we describe the <italic>CCNO NM 021147.4</italic> (c.258 262dup.p, Gln88argfs*8 homozygous) gene mutation that caused severe PCD in a consanguineous Indian family.</p>\",\n \"<p>This article was previously presented as a meeting abstract at the 2022 American Thoracic Society International Conference Meeting on May 13-18, 2022.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>We described two Indian sisters who experienced early-onset respiratory symptoms, obstructive ventilatory dysfunction, and situs solitus. They were identified as having PCD based on the full exon of the CCNO gene and low nNO levels.</p>","<p>Patients with ciliogenesis-related mutations in the genes <italic>CCNO </italic>and <italic>MCIDAS </italic>have been reported to have a worse phenotype than those with other kinds of PCD, which is consistent with the current cases [##REF##24747639##8##]. There are currently 50 known PCD genes that cause a variety of functional defects ranging from abnormal beat patterns through impairment of dynein arms to a complete absence of cilia [##REF##25351953##9##,##REF##28481653##10##]. Although nNO showed good diagnostic accuracy as a PCD diagnostic test when compared to the extended reference standard of TEM and/or genetic testing [##REF##34353866##11##], it is crucial to confirm the genetic diagnosis, given the present evidence of links between genotype and phenotype [##REF##25638182##12##].</p>","<p>Patients with PCD, which is secondary to <italic>CCNO </italic>mutation, have early-onset respiratory symptoms, recurrent lower respiratory tract infections, and progressive loss of lung function, which is consistent with our cases. Situs solitus, like in the cases presented, has only been documented in people with <italic>CCNO </italic>mutations [##REF##24747639##8##]. The other documented symptoms are recurrent pneumonia, sinusitis, and otitis media (Table ##TAB##0##1##).</p>","<p>There are no reports of genetic PCD in India, and only one publication stated that patients with recurrent sinusitis, otitis, and pneumonia in India who also had a fractional exhaled NO level under 10 ppb and who had not undergone genetic testing were likely to have PCD [##REF##24824133##13##]. To our knowledge, this is India’s first report on PCD with <italic>CCNO </italic>which accounts for two out of 318 cases that have been published in the literature (Table ##TAB##0##1##).</p>","<p>In 16 people who had the first <italic>CCNO </italic>mutation in 2014, a malfunction in the mother centriole formation and migration at a late stage of multiple motile cilia (MMC) differentiation led to a significantly decreased number of MMCs. Congenital mucociliary clearance disorder with RGMC is used to denote this hereditary condition [##REF##24747639##8##]. Following the study, there have since been several <italic>CCNO </italic>reports (Table ##TAB##0##1##). In a study of five PCD patients from three different Irish traveler families, it was discovered that a sibling pair in Irish family B had the <italic>CCNO </italic>gene [##REF##26777464##14##]. In another investigation, researchers looked for <italic>CCNO </italic>mutations in 170 Israeli families with mucociliary clearance disorders and identified two novel variations (p.Gly56Alafs38; c.165delC, c.638T>C, p.Leu213Pro), and two known mutations were found in 15 individuals from 10 families (6% prevalence) [##REF##34102041##15##]. In Lisbon, Portugal, 12 patients underwent PCD genetic testing confirming the diagnosis, with three presenting <italic>CCNO </italic>mutations [##REF##31765523##16##]. In Turkey, out of a total of 265 patients with PCD during a five-year period, 46 had genetically determined PCD using whole-exome sequencing at a single facility, and four had <italic>CCNO </italic>[##REF##33577779##17##]. There have been multiple publications from China. One of these described 58 individuals with PCD, 51 with hereditary PCD, and three with <italic>CCNO </italic>[##REF##36157652##18##]. This presenting case study is not a meta-analysis, and its limitation arises from the small number of patients from India, which has not been previously explored in the current literature.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>We described two Indian sisters who experienced early-onset respiratory symptoms, obstructive ventilatory dysfunction, and situs solitus. They were identified as having PCD based on the full exon of the CCNO gene and low nNO levels.</p>\",\n \"<p>Patients with ciliogenesis-related mutations in the genes <italic>CCNO </italic>and <italic>MCIDAS </italic>have been reported to have a worse phenotype than those with other kinds of PCD, which is consistent with the current cases [##REF##24747639##8##]. There are currently 50 known PCD genes that cause a variety of functional defects ranging from abnormal beat patterns through impairment of dynein arms to a complete absence of cilia [##REF##25351953##9##,##REF##28481653##10##]. Although nNO showed good diagnostic accuracy as a PCD diagnostic test when compared to the extended reference standard of TEM and/or genetic testing [##REF##34353866##11##], it is crucial to confirm the genetic diagnosis, given the present evidence of links between genotype and phenotype [##REF##25638182##12##].</p>\",\n \"<p>Patients with PCD, which is secondary to <italic>CCNO </italic>mutation, have early-onset respiratory symptoms, recurrent lower respiratory tract infections, and progressive loss of lung function, which is consistent with our cases. Situs solitus, like in the cases presented, has only been documented in people with <italic>CCNO </italic>mutations [##REF##24747639##8##]. The other documented symptoms are recurrent pneumonia, sinusitis, and otitis media (Table ##TAB##0##1##).</p>\",\n \"<p>There are no reports of genetic PCD in India, and only one publication stated that patients with recurrent sinusitis, otitis, and pneumonia in India who also had a fractional exhaled NO level under 10 ppb and who had not undergone genetic testing were likely to have PCD [##REF##24824133##13##]. To our knowledge, this is India’s first report on PCD with <italic>CCNO </italic>which accounts for two out of 318 cases that have been published in the literature (Table ##TAB##0##1##).</p>\",\n \"<p>In 16 people who had the first <italic>CCNO </italic>mutation in 2014, a malfunction in the mother centriole formation and migration at a late stage of multiple motile cilia (MMC) differentiation led to a significantly decreased number of MMCs. Congenital mucociliary clearance disorder with RGMC is used to denote this hereditary condition [##REF##24747639##8##]. Following the study, there have since been several <italic>CCNO </italic>reports (Table ##TAB##0##1##). In a study of five PCD patients from three different Irish traveler families, it was discovered that a sibling pair in Irish family B had the <italic>CCNO </italic>gene [##REF##26777464##14##]. In another investigation, researchers looked for <italic>CCNO </italic>mutations in 170 Israeli families with mucociliary clearance disorders and identified two novel variations (p.Gly56Alafs38; c.165delC, c.638T>C, p.Leu213Pro), and two known mutations were found in 15 individuals from 10 families (6% prevalence) [##REF##34102041##15##]. In Lisbon, Portugal, 12 patients underwent PCD genetic testing confirming the diagnosis, with three presenting <italic>CCNO </italic>mutations [##REF##31765523##16##]. In Turkey, out of a total of 265 patients with PCD during a five-year period, 46 had genetically determined PCD using whole-exome sequencing at a single facility, and four had <italic>CCNO </italic>[##REF##33577779##17##]. There have been multiple publications from China. One of these described 58 individuals with PCD, 51 with hereditary PCD, and three with <italic>CCNO </italic>[##REF##36157652##18##]. This presenting case study is not a meta-analysis, and its limitation arises from the small number of patients from India, which has not been previously explored in the current literature.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>PCD with <italic>CCNO </italic>mutations is a relatively rare disease. Our findings highlight the significance of considering PCD based on <italic>CCNO </italic>mutations in people with situs solitus to have a more severe respiratory disease phenotype with lung failure at earlier ages. It is also critical to include individuals from various racial and ethnic origins in PCD-associated genetic <italic>CCNO </italic>mutation.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>PCD with <italic>CCNO </italic>mutations is a relatively rare disease. Our findings highlight the significance of considering PCD based on <italic>CCNO </italic>mutations in people with situs solitus to have a more severe respiratory disease phenotype with lung failure at earlier ages. It is also critical to include individuals from various racial and ethnic origins in PCD-associated genetic <italic>CCNO </italic>mutation.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease marked by organ lateralization in 50% of patients, chronic sinopulmonary disease, infertility in men, and neonatal respiratory distress.</p>","<p>Respiratory control cells contain <italic>CCNO </italic>in their apical cytoplasm, which is necessary for the development of multiciliate cells, basal body amplification, and migration. Reduced generation of multiple motile cilia, a rare form of PCD, has been linked to <italic>CCNO </italic>gene abnormalities<italic>. </italic>Individuals with <italic>CCNO</italic> mutations have been reported to suffer from severe lower respiratory infections that cause progressive impairment of lung function. For the first time, we describe the <italic>CCNO NM 021147.4</italic> (c.258 262dup.p, Gln88argfs*8 Homozygous) gene mutation in an Indian consanguineous family that resulted in severe PCD.</p>"],"string":"[\n \"<p>Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease marked by organ lateralization in 50% of patients, chronic sinopulmonary disease, infertility in men, and neonatal respiratory distress.</p>\",\n \"<p>Respiratory control cells contain <italic>CCNO </italic>in their apical cytoplasm, which is necessary for the development of multiciliate cells, basal body amplification, and migration. Reduced generation of multiple motile cilia, a rare form of PCD, has been linked to <italic>CCNO </italic>gene abnormalities<italic>. </italic>Individuals with <italic>CCNO</italic> mutations have been reported to suffer from severe lower respiratory infections that cause progressive impairment of lung function. For the first time, we describe the <italic>CCNO NM 021147.4</italic> (c.258 262dup.p, Gln88argfs*8 Homozygous) gene mutation in an Indian consanguineous family that resulted in severe PCD.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Case presentation</title>","<p>Our case study focuses on two consanguineous sisters from India who visited the Pediatric Pulmonary Department at Sidra Hospital in Qatar when they were 17 and 15 years old. They presented with chronic wet cough and were found to have progressive loss of lung function that eventually led to end-stage lung disease and the requirement for lung transplantation in the case of the elder sister. Since infancy, both siblings had suffered significant lower respiratory infections, chronic rhinorrhea, and recurrent ear infections.</p>","<p>Bronchiectasis was detected by computed tomography (CT) of the chest when the elder sister was 14 years old (Figure ##FIG##0##1##) and the younger sister was seven years old (Figures ##FIG##1##2##, ##FIG##2##3##).</p>","<p>Physical examination was pertinent for bilateral crackles and clubbing in both sisters. The elder sister was hypoxic requiring 1‐2 L of oxygen per minute via a nasal cannula. Expiratory flow volume (spirometry) revealed significant mixed restrictive and obstructive airway disease, which was worse in the elder sibling. The forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) in the elder sister were 25% and 40% predicted, respectively (Figure ##FIG##3##4##). Whereas the FEV1 and FVC in the younger sister were 39% and 57% predicted, respectively (Figure ##FIG##4##5##).</p>","<p>Immune deficiency and cystic fibrosis were ruled out by blood tests and sweat chloride measurements. Both sisters had low nNO levels. The elder sister’s nNO was 63.3 ppb (predicted 200‐1,000) and the younger sister’s nNO was 88.3 ppb. PCD was diagnosed based on clinical phenotype and low nNO levels, which were further confirmed through genetic sequence analysis and deletion/duplication for PCD, which revealed the pathogenic variant <italic>CCNO NM 021147.4</italic> (c.258 262dup.p, Gln88argfs*8 homozygous) gene mutation.</p>"],"string":"[\n \"<title>Case presentation</title>\",\n \"<p>Our case study focuses on two consanguineous sisters from India who visited the Pediatric Pulmonary Department at Sidra Hospital in Qatar when they were 17 and 15 years old. They presented with chronic wet cough and were found to have progressive loss of lung function that eventually led to end-stage lung disease and the requirement for lung transplantation in the case of the elder sister. Since infancy, both siblings had suffered significant lower respiratory infections, chronic rhinorrhea, and recurrent ear infections.</p>\",\n \"<p>Bronchiectasis was detected by computed tomography (CT) of the chest when the elder sister was 14 years old (Figure ##FIG##0##1##) and the younger sister was seven years old (Figures ##FIG##1##2##, ##FIG##2##3##).</p>\",\n \"<p>Physical examination was pertinent for bilateral crackles and clubbing in both sisters. The elder sister was hypoxic requiring 1‐2 L of oxygen per minute via a nasal cannula. Expiratory flow volume (spirometry) revealed significant mixed restrictive and obstructive airway disease, which was worse in the elder sibling. The forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) in the elder sister were 25% and 40% predicted, respectively (Figure ##FIG##3##4##). Whereas the FEV1 and FVC in the younger sister were 39% and 57% predicted, respectively (Figure ##FIG##4##5##).</p>\",\n \"<p>Immune deficiency and cystic fibrosis were ruled out by blood tests and sweat chloride measurements. Both sisters had low nNO levels. The elder sister’s nNO was 63.3 ppb (predicted 200‐1,000) and the younger sister’s nNO was 88.3 ppb. PCD was diagnosed based on clinical phenotype and low nNO levels, which were further confirmed through genetic sequence analysis and deletion/duplication for PCD, which revealed the pathogenic variant <italic>CCNO NM 021147.4</italic> (c.258 262dup.p, Gln88argfs*8 homozygous) gene mutation.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Chest X-ray and CT chest findings of the elder sister.</title><p>A: Anteroposterior chest X-ray. B: CT chest, axial view, lung window.</p><p>Chest radiographical imaging of the elder sister showing bilateral varicose bronchiectasis worse on the right side.</p></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Anteroposterior chest X-ray of the younger sister.</title><p>Chest radiography showing left lower lobe consolidation.</p></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG3\"><label>Figure 3</label><caption><title>CT chest findings of the younger sister.</title><p>A: CT chest, axial view, lung window. B: CT chest, coronal view, lung window.</p><p>CT of the chest shows decreased volume of the left upper lobe and lingula with diffuse cystic bronchiectasis. There is also bronchiectasis within the left lower lobe with a mucus plug and hyperinflation of the right lung with diffuse tree-in-bud changes of the right middle lobe likely secondary to infection.</p></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG4\"><label>Figure 4</label><caption><title>Spirometry findings of the elder sister.</title><p>Spirometric measurement of the elder sister showed mixed restrictive and obstructive airway disease.</p><p>FVC = forced vital capacity; FEV = forced expiratory volume; PEF = peak expiratory flow; PIF = peak inspiratory flow; FET = forced expiratory time</p></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG5\"><label>Figure 5</label><caption><title>Spirometry findings of the younger sister.</title><p>Spirometric measurement of the younger sister showed mixed restrictive and obstructive airway disease.</p><p>FVC = forced vital capacity; FEV = forced expiratory volume; PEF = peak expiratory flow; PIF = peak inspiratory flow; FET = forced expiratory time</p></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>Chest X-ray and CT chest findings of the elder sister.</title><p>A: Anteroposterior chest X-ray. B: CT chest, axial view, lung window.</p><p>Chest radiographical imaging of the elder sister showing bilateral varicose bronchiectasis worse on the right side.</p></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG2\\\"><label>Figure 2</label><caption><title>Anteroposterior chest X-ray of the younger sister.</title><p>Chest radiography showing left lower lobe consolidation.</p></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG3\\\"><label>Figure 3</label><caption><title>CT chest findings of the younger sister.</title><p>A: CT chest, axial view, lung window. B: CT chest, coronal view, lung window.</p><p>CT of the chest shows decreased volume of the left upper lobe and lingula with diffuse cystic bronchiectasis. There is also bronchiectasis within the left lower lobe with a mucus plug and hyperinflation of the right lung with diffuse tree-in-bud changes of the right middle lobe likely secondary to infection.</p></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG4\\\"><label>Figure 4</label><caption><title>Spirometry findings of the elder sister.</title><p>Spirometric measurement of the elder sister showed mixed restrictive and obstructive airway disease.</p><p>FVC = forced vital capacity; FEV = forced expiratory volume; PEF = peak expiratory flow; PIF = peak inspiratory flow; FET = forced expiratory time</p></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG5\\\"><label>Figure 5</label><caption><title>Spirometry findings of the younger sister.</title><p>Spirometric measurement of the younger sister showed mixed restrictive and obstructive airway disease.</p><p>FVC = forced vital capacity; FEV = forced expiratory volume; PEF = peak expiratory flow; PIF = peak inspiratory flow; FET = forced expiratory time</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>CCNO gene mutation studies.</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nStudy\n</td><td rowspan=\"1\" colspan=\"1\">\nNumber of cases\n</td><td rowspan=\"1\" colspan=\"1\">\nGenetic\n</td><td rowspan=\"1\" colspan=\"1\">\nPhenotype\n</td><td rowspan=\"1\" colspan=\"1\">\nNasal NO\n</td><td rowspan=\"1\" colspan=\"1\">\nUltrastructural defect\n</td><td rowspan=\"1\" colspan=\"1\">\nVideo microscopy\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nWallmeier et al. (2014) [##REF##24747639##8##]\n</td><td rowspan=\"1\" colspan=\"1\">\n16\n</td><td rowspan=\"1\" colspan=\"1\">\nThe study discovered homozygous loss-of-function mutations (p.Gly85Cysfs*10) in CCNO using whole-exome sequencing\n</td><td rowspan=\"1\" colspan=\"1\">\nAfter birth, 12 out of 16 babies experienced respiratory distress. One of the women was infertile. Everyone exhibited situs solitus, bronchitis, and recurrent infections of the upper and lower respiratory tracts. At the age of 34, two people who had terminal respiratory failure underwent lung transplants\n</td><td rowspan=\"1\" colspan=\"1\">\nNot done\n</td><td rowspan=\"1\" colspan=\"1\">\nAll affected people either had no cilia at all or had a significant reduction in cilia\n</td><td rowspan=\"1\" colspan=\"1\">\nRespiratory epithelial cells showed a marked reduction in the number of multiple motile cilia (MMC) covering the cell surface. The few residual cilia that correctly expressed axonemal motor proteins were motile and did not exhibit obvious beating defects\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nKumar et al. (2015) [##REF##25638182##12##]\n</td><td rowspan=\"1\" colspan=\"1\">\n80\n</td><td rowspan=\"1\" colspan=\"1\">\nUnavailable\n</td><td rowspan=\"1\" colspan=\"1\">\nThe mean age of presentation was 9.6 (range = 2–15) years. Overall, 62.5% of the population was younger than 5 years old. Clubbing was present in 58 (72.5%) children\n</td><td rowspan=\"1\" colspan=\"1\">\nNot done\n</td><td rowspan=\"1\" colspan=\"1\">\nNot done\n</td><td rowspan=\"1\" colspan=\"1\">\nNot done\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nCasey et al. 2015 [##REF##24824133##13##]\n</td><td rowspan=\"1\" colspan=\"1\">\n5\n</td><td rowspan=\"1\" colspan=\"1\">\nA novel 1 bp duplication in RSPH4A. CCNO, KCNN3, and CDKN1C.a ∼3.5-kb deletion in DYX1C1\n</td><td rowspan=\"1\" colspan=\"1\">\nRecurrent lower respiratory tract infection: 5/5. Bronchiectasis on CT of the thorax: 5/5. Hearing loss in 2/5 patients. 2/5 of the patients had early-onset severe cardiomyopathy, type III glycogen storage disease, and developmental delay. Neonatal pneumonia affected 1/5 of patients. Recurrent otitis media in 2/5\n</td><td rowspan=\"1\" colspan=\"1\">\nRepeat nasal oxide screening tests consistently yielded low results of 30 to 50 ppb\n</td><td rowspan=\"1\" colspan=\"1\">\nA displacement of one of the peripheral doublets was seen in certain cilia, with 22% of them lacking the central pair. Both the outer and inner dynein arms were normal. Ciliary aplasia was present in 2 instances. In one instance, both the inner and outer dynein arms were absent\n</td><td rowspan=\"1\" colspan=\"1\">\nAll of the cilia were abnormally static or dyskinetic. Although the pattern was incomplete and the cilia seemed stiff without cleaning any material, it was still possible to see the cilia moving when viewed from above\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nAmirav et al. 2016 [##REF##26777464##14##]\n</td><td rowspan=\"1\" colspan=\"1\">\n91\n</td><td rowspan=\"1\" colspan=\"1\">\nIn 15 people (16%), biallelic CCNO mutations were found. Three compound heterozygous mutations and seven homozygous mutations were found. Every single identified mutation was inherited autosomally recessively within the families. Three frameshift mutations (c.262263dupGGCCC, p.Gln88Argfs8; c.165delC, p.Gly56Alafs38; c.481482delCT, p.Leu161Glyfs73), one missense mutation (c.638T>C, p.Leu213Pro), and one deletion mutation were found\n</td><td rowspan=\"1\" colspan=\"1\">\nThe age of affected individuals ranged from 5 to 54 years. At a median age of 20 years, the initial genetic diagnosis was made. All situs solitus 11/14 cases (85%) of neonatal respiratory distress syndrome. Otitis media recurrent in 10/15 (67%) people, and 13/14 (93%) people had sinusitis. Documentation of bronchiectasis by radiographic imaging in 13/14 (93%). At the age of 43, lung transplantation was done. The composition of the situs was normal in all those with CCNO mutations. One affected female underwent assisted reproduction using in vitro fertilization to become pregnant. One affected male fathered a child without medical assistance. One individual with CCNO mutation had arrested hydrocephalus\n</td><td rowspan=\"1\" colspan=\"1\">\nTwo people showed nasal NO readings that were within the normal range. Mean nasal NO values of 50.32 ± 68.62 nL/minute (14 individuals)\n</td><td rowspan=\"1\" colspan=\"1\">\nThe transmission electron microscopy of three people revealed normal microvilli composition, but the basal bodies, specialized centrioles that initiate ciliary axonemes in the apical regions of respiratory epithelial cells, were absent or significantly diminished. Displaced basal bodies and rootlets (propagating from the basal body) were found in the cytoplasm of some respiratory epithelial cells\n</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nHenriques et al. 2021 [##REF##34102041##15##]\n</td><td rowspan=\"1\" colspan=\"1\">\n3\n</td><td rowspan=\"1\" colspan=\"1\">\nHeterozygous mutation, CCNO gene c.253_257GGCCC(3)(p.Gln88fs) and c.263_267dup(p.Val90fs) homozygous mutation, c.263_267dup(p.Val90fs) and c.263_267dup(p.Val90fs)\n</td><td rowspan=\"1\" colspan=\"1\">\n3/3 respiratory symptoms that started early. Situs solitus. Due to lung collapse, one required a lobectomy. One patient had two tympanostomy tube insertions due to recurrent otitis media with effusion and conductive hearing loss\n</td><td rowspan=\"1\" colspan=\"1\">\nNot done\n</td><td rowspan=\"1\" colspan=\"1\">\nReduced or absent number of cilia, and normal ultrastructure in residual cili.\n</td><td rowspan=\"1\" colspan=\"1\">\nMost epithelial cells with bald epithelium, residual cilia with uncoordinated ciliary beat frequency and pattern\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nEmiralioğlu et al. 2019 [##REF##31765523##16##]\n</td><td rowspan=\"1\" colspan=\"1\">\n46\n</td><td rowspan=\"1\" colspan=\"1\">\nDNAH5, CCDC40, RSPH4A, DNAH11, HYDIN, CCNO, DNAI1, ARMC4, TTC25, DNAH1, and CCDC39 gene\n</td><td rowspan=\"1\" colspan=\"1\">\nThe median age at diagnosis (median: 3 years; range, 6 months to 4 years). 44 patients had rhinitis, whereas 41 had newborn respiratory distress. The sinusitis returned in 36. 14 people had recurrent otitis. Six were hard of hearing. Clubbing was seen in seven. Fourteen had a total inversus situation. Six (atrial septal defect, patent ductus arteriosus, and mitral valve prolapse) had congenital cardiac defects. Four patients had Lobectomy. Four patients had undergone ear, nose, and throat surgery\n</td><td rowspan=\"1\" colspan=\"1\">\nMedian nasal NO was 8 ppb (minimum: 5, maximum: 40)\n</td><td rowspan=\"1\" colspan=\"1\">\nSeven patients had a nasal biopsy: three had outer dynein arm defect. Two microtubule disorganizations with the inner dynein arm. Two had central pair abnormality\n</td><td rowspan=\"1\" colspan=\"1\">\nCilia were hypokinetic in 28 individuals. Four exhibited hyperkinetic cilia. Twelve had stiff patterns. Two had abnormal circular movement\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nGuan et al. 2021 [##REF##33577779##17##]\n</td><td rowspan=\"1\" colspan=\"1\">\n75\n</td><td rowspan=\"1\" colspan=\"1\">\nDNAH11 variants (15 individuals). DNAH5 variants (nine individuals), CCDC39 variants (five individuals), DNAH1 variants (four individuals), CCNO variants (three individuals), DNAI1, HEATR2, RSPH9, or DNAAF3 (two individuals for each). CCDC40, LRRC6, SPAG1, ARMC4, RSPH4A, CCDC114, and DNAH14 mutated in one individual each\n</td><td rowspan=\"1\" colspan=\"1\">\nMedian age at diagnosis was 7.0 years (range = 2 months to 14 years). A chronic wet cough affected 66 out of 75 people. 58/75 people had recurrent sinusitis. 57/75 people had bronchiectasis. Respiratory distress in newborns was present in 30% of the cases. There were 6/75 cases of postinfectious bronchiolitis obliterans as the first presentation, while 21/75 patients had coexisting asthma\n</td><td rowspan=\"1\" colspan=\"1\">\nNot done\n</td><td rowspan=\"1\" colspan=\"1\">\n(8/50) outer dynein arm (ODA) defects. Inner dynein arm (IDA) defects in conjunction with central apparatus (CA) defects and microtubule disorganization (MTD) were classified as IDA/CA/MTD. (8/50) had IDA defects, CA defects, and MTD. (12/50) ODA and IDA. (10/50) CA or IDA defects. (4/50 ) Oligocilia. (5/50) Normal structure\n</td><td rowspan=\"1\" colspan=\"1\">\nNot done\n</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB1\\\"><label>Table 1</label><caption><title>CCNO gene mutation studies.</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStudy\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNumber of cases\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nGenetic\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nPhenotype\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNasal NO\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nUltrastructural defect\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nVideo microscopy\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nWallmeier et al. (2014) [##REF##24747639##8##]\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n16\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nThe study discovered homozygous loss-of-function mutations (p.Gly85Cysfs*10) in CCNO using whole-exome sequencing\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nAfter birth, 12 out of 16 babies experienced respiratory distress. One of the women was infertile. Everyone exhibited situs solitus, bronchitis, and recurrent infections of the upper and lower respiratory tracts. At the age of 34, two people who had terminal respiratory failure underwent lung transplants\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNot done\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nAll affected people either had no cilia at all or had a significant reduction in cilia\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nRespiratory epithelial cells showed a marked reduction in the number of multiple motile cilia (MMC) covering the cell surface. The few residual cilia that correctly expressed axonemal motor proteins were motile and did not exhibit obvious beating defects\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nKumar et al. (2015) [##REF##25638182##12##]\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n80\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nUnavailable\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nThe mean age of presentation was 9.6 (range = 2–15) years. Overall, 62.5% of the population was younger than 5 years old. Clubbing was present in 58 (72.5%) children\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNot done\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNot done\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNot done\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nCasey et al. 2015 [##REF##24824133##13##]\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n5\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nA novel 1 bp duplication in RSPH4A. CCNO, KCNN3, and CDKN1C.a ∼3.5-kb deletion in DYX1C1\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nRecurrent lower respiratory tract infection: 5/5. Bronchiectasis on CT of the thorax: 5/5. Hearing loss in 2/5 patients. 2/5 of the patients had early-onset severe cardiomyopathy, type III glycogen storage disease, and developmental delay. Neonatal pneumonia affected 1/5 of patients. Recurrent otitis media in 2/5\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nRepeat nasal oxide screening tests consistently yielded low results of 30 to 50 ppb\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nA displacement of one of the peripheral doublets was seen in certain cilia, with 22% of them lacking the central pair. Both the outer and inner dynein arms were normal. Ciliary aplasia was present in 2 instances. In one instance, both the inner and outer dynein arms were absent\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nAll of the cilia were abnormally static or dyskinetic. Although the pattern was incomplete and the cilia seemed stiff without cleaning any material, it was still possible to see the cilia moving when viewed from above\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nAmirav et al. 2016 [##REF##26777464##14##]\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n91\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nIn 15 people (16%), biallelic CCNO mutations were found. Three compound heterozygous mutations and seven homozygous mutations were found. Every single identified mutation was inherited autosomally recessively within the families. Three frameshift mutations (c.262263dupGGCCC, p.Gln88Argfs8; c.165delC, p.Gly56Alafs38; c.481482delCT, p.Leu161Glyfs73), one missense mutation (c.638T>C, p.Leu213Pro), and one deletion mutation were found\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nThe age of affected individuals ranged from 5 to 54 years. At a median age of 20 years, the initial genetic diagnosis was made. All situs solitus 11/14 cases (85%) of neonatal respiratory distress syndrome. Otitis media recurrent in 10/15 (67%) people, and 13/14 (93%) people had sinusitis. Documentation of bronchiectasis by radiographic imaging in 13/14 (93%). At the age of 43, lung transplantation was done. The composition of the situs was normal in all those with CCNO mutations. One affected female underwent assisted reproduction using in vitro fertilization to become pregnant. One affected male fathered a child without medical assistance. One individual with CCNO mutation had arrested hydrocephalus\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nTwo people showed nasal NO readings that were within the normal range. Mean nasal NO values of 50.32 ± 68.62 nL/minute (14 individuals)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nThe transmission electron microscopy of three people revealed normal microvilli composition, but the basal bodies, specialized centrioles that initiate ciliary axonemes in the apical regions of respiratory epithelial cells, were absent or significantly diminished. Displaced basal bodies and rootlets (propagating from the basal body) were found in the cytoplasm of some respiratory epithelial cells\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\"> </td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nHenriques et al. 2021 [##REF##34102041##15##]\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n3\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nHeterozygous mutation, CCNO gene c.253_257GGCCC(3)(p.Gln88fs) and c.263_267dup(p.Val90fs) homozygous mutation, c.263_267dup(p.Val90fs) and c.263_267dup(p.Val90fs)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n3/3 respiratory symptoms that started early. Situs solitus. Due to lung collapse, one required a lobectomy. One patient had two tympanostomy tube insertions due to recurrent otitis media with effusion and conductive hearing loss\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNot done\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nReduced or absent number of cilia, and normal ultrastructure in residual cili.\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nMost epithelial cells with bald epithelium, residual cilia with uncoordinated ciliary beat frequency and pattern\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nEmiralioğlu et al. 2019 [##REF##31765523##16##]\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n46\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nDNAH5, CCDC40, RSPH4A, DNAH11, HYDIN, CCNO, DNAI1, ARMC4, TTC25, DNAH1, and CCDC39 gene\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nThe median age at diagnosis (median: 3 years; range, 6 months to 4 years). 44 patients had rhinitis, whereas 41 had newborn respiratory distress. The sinusitis returned in 36. 14 people had recurrent otitis. Six were hard of hearing. Clubbing was seen in seven. Fourteen had a total inversus situation. Six (atrial septal defect, patent ductus arteriosus, and mitral valve prolapse) had congenital cardiac defects. Four patients had Lobectomy. Four patients had undergone ear, nose, and throat surgery\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nMedian nasal NO was 8 ppb (minimum: 5, maximum: 40)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nSeven patients had a nasal biopsy: three had outer dynein arm defect. Two microtubule disorganizations with the inner dynein arm. Two had central pair abnormality\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nCilia were hypokinetic in 28 individuals. Four exhibited hyperkinetic cilia. Twelve had stiff patterns. Two had abnormal circular movement\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nGuan et al. 2021 [##REF##33577779##17##]\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n75\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nDNAH11 variants (15 individuals). DNAH5 variants (nine individuals), CCDC39 variants (five individuals), DNAH1 variants (four individuals), CCNO variants (three individuals), DNAI1, HEATR2, RSPH9, or DNAAF3 (two individuals for each). CCDC40, LRRC6, SPAG1, ARMC4, RSPH4A, CCDC114, and DNAH14 mutated in one individual each\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nMedian age at diagnosis was 7.0 years (range = 2 months to 14 years). A chronic wet cough affected 66 out of 75 people. 58/75 people had recurrent sinusitis. 57/75 people had bronchiectasis. Respiratory distress in newborns was present in 30% of the cases. There were 6/75 cases of postinfectious bronchiolitis obliterans as the first presentation, while 21/75 patients had coexisting asthma\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNot done\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n(8/50) outer dynein arm (ODA) defects. Inner dynein arm (IDA) defects in conjunction with central apparatus (CA) defects and microtubule disorganization (MTD) were classified as IDA/CA/MTD. (8/50) had IDA defects, CA defects, and MTD. (12/50) ODA and IDA. (10/50) CA or IDA defects. (4/50 ) Oligocilia. (5/50) Normal structure\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNot done\\n</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0016-00000052237-i01\" position=\"float\"/>","<graphic xlink:href=\"cureus-0016-00000052237-i02\" position=\"float\"/>","<graphic xlink:href=\"cureus-0016-00000052237-i03\" position=\"float\"/>","<graphic xlink:href=\"cureus-0016-00000052237-i04\" position=\"float\"/>","<graphic xlink:href=\"cureus-0016-00000052237-i05\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0016-00000052237-i01\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0016-00000052237-i02\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0016-00000052237-i03\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0016-00000052237-i04\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0016-00000052237-i05\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"7\"], \"article-title\": [\"Diagnosis of primary ciliary dyskinesia. An official American Thoracic Society clinical practice guideline\"], \"source\": [\"Am J Respir Crit Care Med\"], \"person-group\": [\"\\n\"], \"surname\": [\"Shapiro\", \"Davis\", \"Polineni\"], \"given-names\": [\"AJ\", \"SD\", \"D\"], \"fpage\": [\"0\"], \"lpage\": [\"39\"], \"volume\": [\"197\"], \"year\": [\"2018\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":18,"string":"18"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:43:45"},"citation":{"kind":"string","value":"Cureus.; 16(1):e52237"},"package_file":{"kind":"string","value":"oa_package/e0/e3/PMC10787941.tar.gz"}}},{"rowIdx":198,"cells":{"accession_id":{"kind":"string","value":"PMC10787942"},"pmid":{"kind":"string","value":"38222226"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Acute appendicitis is one of the most prevalent causes of presentation to the emergency department (ED) [##REF##27826565##1##]. With the treatment being resection, whether open or laparoscopic, it is not uncommon for patients to present with complications of appendectomy. One of the delayed complications of this procedure is stump appendicitis, a once rare entity with a now increasing incidence [##UREF##0##2##]. The estimated figure in literature is one in 50,000, although this is likely underestimated and the condition underreported [##UREF##1##3##].</p>","<p>Stump appendicitis can occur as soon as two months and as late as 50 years post-appendectomy [##REF##35308435##4##]. It is often diagnosed radiologically on an abdominal CT scan performed to rule out other diagnoses [##UREF##2##5##]. A lack of awareness by the ED physician can lead to delayed diagnosis, which inadvertently leads to delayed treatment and an increased complication rate [##REF##35195078##6##]. We report a case of a young male presenting with acute abdominal pain and a history of appendectomy. Stump appendicitis was diagnosed incidentally on CT and the patient was managed conservatively.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Acute appendicitis is one of the most prevalent causes of presentation to the emergency department (ED) [##REF##27826565##1##]. With the treatment being resection, whether open or laparoscopic, it is not uncommon for patients to present with complications of appendectomy. One of the delayed complications of this procedure is stump appendicitis, a once rare entity with a now increasing incidence [##UREF##0##2##]. The estimated figure in literature is one in 50,000, although this is likely underestimated and the condition underreported [##UREF##1##3##].</p>\",\n \"<p>Stump appendicitis can occur as soon as two months and as late as 50 years post-appendectomy [##REF##35308435##4##]. It is often diagnosed radiologically on an abdominal CT scan performed to rule out other diagnoses [##UREF##2##5##]. A lack of awareness by the ED physician can lead to delayed diagnosis, which inadvertently leads to delayed treatment and an increased complication rate [##REF##35195078##6##]. We report a case of a young male presenting with acute abdominal pain and a history of appendectomy. Stump appendicitis was diagnosed incidentally on CT and the patient was managed conservatively.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>Stump appendicitis (SA) is defined as interval inflammation of residual appendicular tissue after appendectomy [##UREF##2##5##]. First described in 1945 by Rose, its incidence has since been slowly rising [##UREF##0##2##]. This has been attributed to laparoscopic surgery by some authors but refuted by others; an alternative explanation is the increasing awareness to this clinical entity in recent years [##UREF##3##8##]. However, a low index of suspicion remains one of the principal reasons behind delayed diagnosis and treatment [##REF##29849750##9##]. A 2020 literature review by Enzerra et al. counted 160 cases reported in published literature; another review in the same year by Burbano et al. concluded that “the incidence of stump appendicitis seems to be higher than the one reported of 1 in 50,000” [##REF##33021832##10##,##REF##32126353##11##]. </p>","<p>Several anatomic and surgical risk factors have been suggested to increase the risk of SA; the most consistent seem to be inappropriate identification of the base of the appendix at the time of surgery and length of the residual stump [##REF##35308435##4##]. Although there is no clinical consensus on the exact measurement, the general recommendation is that the stump be less than 5 mm long [##REF##10966030##12##]. A disputed risk factor is undergoing laparoscopic as opposed to open appendectomy. Theoretically, “the lack of a three-dimensional approach and the absence of a tactile return” increase the risk of SA post-laparoscopic appendectomy by increasing the length of tissue left behind [##UREF##0##2##]. Interestingly, one review found the incidence post-laparoscopy to be less than half of that post open appendectomy [##REF##22153086##13##]. Ultimately, surgical technique and operator experience play a role in the complication rate of any procedure. Other risk factors include a retro-colic position of the appendix, poor blood supply, and appendicolith formation [##UREF##2##5##]. </p>","<p>Clinical presentation of SA is similar to that of acute appendicitis, further confounding the diagnostic process. Although no one symptom or sign is specific, the most common findings are RLQ pain, leukocytosis, peritonism, fever, nausea and vomiting [##REF##32126353##11##]. Epidemiologically, the diagnosis is made in patients ranging from eight to 80 years, with a median age of 33 years, and a male-to-female ratio of 1.1:1 [##REF##16536249##14##]. Abdominal imaging is often needed to confirm the diagnosis, with computerized tomography (CT) being the gold standard [##REF##34229211##15##]. Compared to ultrasound scans, CT is more sensitive and specific and can simultaneously exclude other abdominopelvic disease entities [##UREF##3##8##]. Findings are similar to those of acute appendicitis and can include cecal wall thickening, free fluid, abdominal collections or abscesses- as well as identification of the appendicular stump [##UREF##3##8##]. Less frequently, barium enema and colonoscopy can aid in the diagnosis [##UREF##4##16##]. In cases of ambiguity or diagnostic uncertainty, a decision to proceed to diagnostic laparoscopy can be made [##REF##33088403##17##]. </p>","<p>The vast majority of reported cases have cited a completion appendectomy as the treatment of choice for SA, with the open approach being preferred to laparoscopic surgery [##REF##35195078##6##]. This may be due to the fact that a delayed diagnosis leads to more complicated presentations, such as stump gangrene, perforation, and peritonitis [##UREF##2##5##]. One review cited a perforation rate as high as 70% [##REF##16536249##14##]. Conservative management with parenteral antibiotics and analgesia with or without colonoscopy may be appropriate for some patients, such as the case reported above; although it is less commonly described in the literature [##REF##35308435##4##]. Potential uses of colonoscopy are washout, pus drainage and clearance, and appendicolith removal with a snare [##UREF##3##8##]. An interval appendectomy has also been suggested by some authors after the acute inflammatory phase subsides with medical management to improve intra-operative visualization of the stump and prevent recurrence [##REF##35308435##4##]. </p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>Stump appendicitis (SA) is defined as interval inflammation of residual appendicular tissue after appendectomy [##UREF##2##5##]. First described in 1945 by Rose, its incidence has since been slowly rising [##UREF##0##2##]. This has been attributed to laparoscopic surgery by some authors but refuted by others; an alternative explanation is the increasing awareness to this clinical entity in recent years [##UREF##3##8##]. However, a low index of suspicion remains one of the principal reasons behind delayed diagnosis and treatment [##REF##29849750##9##]. A 2020 literature review by Enzerra et al. counted 160 cases reported in published literature; another review in the same year by Burbano et al. concluded that “the incidence of stump appendicitis seems to be higher than the one reported of 1 in 50,000” [##REF##33021832##10##,##REF##32126353##11##]. </p>\",\n \"<p>Several anatomic and surgical risk factors have been suggested to increase the risk of SA; the most consistent seem to be inappropriate identification of the base of the appendix at the time of surgery and length of the residual stump [##REF##35308435##4##]. Although there is no clinical consensus on the exact measurement, the general recommendation is that the stump be less than 5 mm long [##REF##10966030##12##]. A disputed risk factor is undergoing laparoscopic as opposed to open appendectomy. Theoretically, “the lack of a three-dimensional approach and the absence of a tactile return” increase the risk of SA post-laparoscopic appendectomy by increasing the length of tissue left behind [##UREF##0##2##]. Interestingly, one review found the incidence post-laparoscopy to be less than half of that post open appendectomy [##REF##22153086##13##]. Ultimately, surgical technique and operator experience play a role in the complication rate of any procedure. Other risk factors include a retro-colic position of the appendix, poor blood supply, and appendicolith formation [##UREF##2##5##]. </p>\",\n \"<p>Clinical presentation of SA is similar to that of acute appendicitis, further confounding the diagnostic process. Although no one symptom or sign is specific, the most common findings are RLQ pain, leukocytosis, peritonism, fever, nausea and vomiting [##REF##32126353##11##]. Epidemiologically, the diagnosis is made in patients ranging from eight to 80 years, with a median age of 33 years, and a male-to-female ratio of 1.1:1 [##REF##16536249##14##]. Abdominal imaging is often needed to confirm the diagnosis, with computerized tomography (CT) being the gold standard [##REF##34229211##15##]. Compared to ultrasound scans, CT is more sensitive and specific and can simultaneously exclude other abdominopelvic disease entities [##UREF##3##8##]. Findings are similar to those of acute appendicitis and can include cecal wall thickening, free fluid, abdominal collections or abscesses- as well as identification of the appendicular stump [##UREF##3##8##]. Less frequently, barium enema and colonoscopy can aid in the diagnosis [##UREF##4##16##]. In cases of ambiguity or diagnostic uncertainty, a decision to proceed to diagnostic laparoscopy can be made [##REF##33088403##17##]. </p>\",\n \"<p>The vast majority of reported cases have cited a completion appendectomy as the treatment of choice for SA, with the open approach being preferred to laparoscopic surgery [##REF##35195078##6##]. This may be due to the fact that a delayed diagnosis leads to more complicated presentations, such as stump gangrene, perforation, and peritonitis [##UREF##2##5##]. One review cited a perforation rate as high as 70% [##REF##16536249##14##]. Conservative management with parenteral antibiotics and analgesia with or without colonoscopy may be appropriate for some patients, such as the case reported above; although it is less commonly described in the literature [##REF##35308435##4##]. Potential uses of colonoscopy are washout, pus drainage and clearance, and appendicolith removal with a snare [##UREF##3##8##]. An interval appendectomy has also been suggested by some authors after the acute inflammatory phase subsides with medical management to improve intra-operative visualization of the stump and prevent recurrence [##REF##35308435##4##]. </p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>Right lower quadrant abdominal pain comes with a wide range of differential diagnoses. In patients with a clinical picture of acute appendicitis and history of appendectomy, it is the emergency physician’s responsibility to maintain a high index of suspicion for stump appendicitis. The gold standard for diagnosis is an abdominal CT. Prompt identification and treatment- primarily with a completion appendectomy- is key to avoid complications such as perforation.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>Right lower quadrant abdominal pain comes with a wide range of differential diagnoses. In patients with a clinical picture of acute appendicitis and history of appendectomy, it is the emergency physician’s responsibility to maintain a high index of suspicion for stump appendicitis. The gold standard for diagnosis is an abdominal CT. Prompt identification and treatment- primarily with a completion appendectomy- is key to avoid complications such as perforation.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Acute appendicitis is one of the most common diagnoses in the emergency department. As with other surgical procedures, post-appendectomy complications are numerous and can be either immediate or delayed. Stump appendicitis is an underreported and underrecognized complication that is often diagnosed radiologically while ruling out other diagnoses.</p>","<p>We report a case of a 26-year-old male presenting with acute right lower quadrant abdominal pain. Although he initially denied any surgical history, a focused abdominal exam revealed an incisional scar which turned out to be the result of an appendectomy nine years ago. The patient was worked up for alternate causes of right lower quadrant pain.</p>","<p>Investigations revealed high inflammatory markers and hematuria. We proceeded with a non-contrast CT scan to rule out vesicoureteric junction stone. Instead, the scan was suggestive of stump appendicitis. The patient was admitted and treated conservatively.</p>","<p>Maintaining a high index of suspicion for stump appendicitis, especially in patients with a clinical picture typical of appendicitis but a history of appendectomy, is key to making an early diagnosis and avoiding further complications.</p>"],"string":"[\n \"<p>Acute appendicitis is one of the most common diagnoses in the emergency department. As with other surgical procedures, post-appendectomy complications are numerous and can be either immediate or delayed. Stump appendicitis is an underreported and underrecognized complication that is often diagnosed radiologically while ruling out other diagnoses.</p>\",\n \"<p>We report a case of a 26-year-old male presenting with acute right lower quadrant abdominal pain. Although he initially denied any surgical history, a focused abdominal exam revealed an incisional scar which turned out to be the result of an appendectomy nine years ago. The patient was worked up for alternate causes of right lower quadrant pain.</p>\",\n \"<p>Investigations revealed high inflammatory markers and hematuria. We proceeded with a non-contrast CT scan to rule out vesicoureteric junction stone. Instead, the scan was suggestive of stump appendicitis. The patient was admitted and treated conservatively.</p>\",\n \"<p>Maintaining a high index of suspicion for stump appendicitis, especially in patients with a clinical picture typical of appendicitis but a history of appendectomy, is key to making an early diagnosis and avoiding further complications.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Case presentation</title>","<p>A 26-year-old male presented to ED with a one-day history of right lower abdominal pain associated with nausea and one episode of vomiting. The pain was of gradual onset, dull achy character, mild to moderate intensity, and intermittent course. He denied fever, altered bowel habit, or dysuria; review of systems was otherwise unremarkable. During the initial interview, he denied any past medical or surgical history. His only medications were simple analgesics, he was a non-smoker and did not consume alcohol, and family history was non-contributory. </p>","<p>Vitally, the patient was stable and afebrile. Focused abdominal examination revealed an incisional appendectomy scar; when asked about this finding, the patient then added that he underwent an open appendectomy nine years ago. He also had moderate right lower quadrant (RLQ) tenderness with rebound and mild guarding. No other peritoneal signs were present, and the systemic examination was otherwise normal. At this point, appendicitis was ruled out and the work-up was aimed at identifying alternate causes for his RLQ pain.</p>","<p>Investigations</p>","<p>Laboratory investigations showed mild leukocytosis (12.6 x 109/L) with neutrophilia (8.6 x 109/L) and an elevated C-reactive protein (CRP) (140.4mg/L). Urinalysis was positive for erythrocytes. The high inflammatory markers combined with hematuria led to the consideration of vesicoureteric junction stone as a possible diagnosis; thus, we proceeded to a non-contrast CT scan of the abdomen and pelvis, which revealed diffuse wall thickening of a blind-ended, tubular structure arising from the cecal pole, suggestive of inflamed appendicular stump (Figure ##FIG##0##1##). A repeat CT with contrast further delineated the inflammatory changes to include the cecum and proximal ascending colon with surrounding fat stranding and multiple prominent reactive lymph nodes. </p>","<p>Differential diagnosis</p>","<p>Although the clinical picture of RLQ pain and tenderness allows for a wide range of differential diagnoses, it is confounded by the history of appendectomy. One literature review concluded that the most common misdiagnoses were constipation, gastroenteritis and right-sided diverticulitis [##REF##26255005##7##]. </p>","<p>Treatment</p>","<p>General surgery was consulted, and no acute surgical intervention was deemed necessary. The patient was then admitted as a case of stump appendicitis and managed conservatively. He was kept nothing by mouth (NPO) and started on intravenous analgesia, anti-emetics and antibiotics (cefuroxime, metronidazole). During admission, the gastroenterology service was consulted and the patient underwent colonoscopy which was grossly normal. Random biopsies were taken for pathology examination. </p>","<p>Outcome and follow-up</p>","<p>The patient was discharged three days later on an oral course of the same antibiotics for a total of seven days. On a follow-up appointment with gastroenterology, he reported improved symptoms and pathology reports showed no significant histological abnormality. Repeat labs showed a downward trend of inflammatory markers. The final diagnosis was stump appendicitis.</p>"],"string":"[\n \"<title>Case presentation</title>\",\n \"<p>A 26-year-old male presented to ED with a one-day history of right lower abdominal pain associated with nausea and one episode of vomiting. The pain was of gradual onset, dull achy character, mild to moderate intensity, and intermittent course. He denied fever, altered bowel habit, or dysuria; review of systems was otherwise unremarkable. During the initial interview, he denied any past medical or surgical history. His only medications were simple analgesics, he was a non-smoker and did not consume alcohol, and family history was non-contributory. </p>\",\n \"<p>Vitally, the patient was stable and afebrile. Focused abdominal examination revealed an incisional appendectomy scar; when asked about this finding, the patient then added that he underwent an open appendectomy nine years ago. He also had moderate right lower quadrant (RLQ) tenderness with rebound and mild guarding. No other peritoneal signs were present, and the systemic examination was otherwise normal. At this point, appendicitis was ruled out and the work-up was aimed at identifying alternate causes for his RLQ pain.</p>\",\n \"<p>Investigations</p>\",\n \"<p>Laboratory investigations showed mild leukocytosis (12.6 x 109/L) with neutrophilia (8.6 x 109/L) and an elevated C-reactive protein (CRP) (140.4mg/L). Urinalysis was positive for erythrocytes. The high inflammatory markers combined with hematuria led to the consideration of vesicoureteric junction stone as a possible diagnosis; thus, we proceeded to a non-contrast CT scan of the abdomen and pelvis, which revealed diffuse wall thickening of a blind-ended, tubular structure arising from the cecal pole, suggestive of inflamed appendicular stump (Figure ##FIG##0##1##). A repeat CT with contrast further delineated the inflammatory changes to include the cecum and proximal ascending colon with surrounding fat stranding and multiple prominent reactive lymph nodes. </p>\",\n \"<p>Differential diagnosis</p>\",\n \"<p>Although the clinical picture of RLQ pain and tenderness allows for a wide range of differential diagnoses, it is confounded by the history of appendectomy. One literature review concluded that the most common misdiagnoses were constipation, gastroenteritis and right-sided diverticulitis [##REF##26255005##7##]. </p>\",\n \"<p>Treatment</p>\",\n \"<p>General surgery was consulted, and no acute surgical intervention was deemed necessary. The patient was then admitted as a case of stump appendicitis and managed conservatively. He was kept nothing by mouth (NPO) and started on intravenous analgesia, anti-emetics and antibiotics (cefuroxime, metronidazole). During admission, the gastroenterology service was consulted and the patient underwent colonoscopy which was grossly normal. Random biopsies were taken for pathology examination. </p>\",\n \"<p>Outcome and follow-up</p>\",\n \"<p>The patient was discharged three days later on an oral course of the same antibiotics for a total of seven days. On a follow-up appointment with gastroenterology, he reported improved symptoms and pathology reports showed no significant histological abnormality. Repeat labs showed a downward trend of inflammatory markers. The final diagnosis was stump appendicitis.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Non-contrast abdominal CT image showing wall thickening of a blind-ended tubular structure arising from the cecal pole (yellow arrow), suggestive of inflamed appendiceal stump.</title></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>Non-contrast abdominal CT image showing wall thickening of a blind-ended tubular structure arising from the cecal pole (yellow arrow), suggestive of inflamed appendiceal stump.</title></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":[],"string":"[]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Nada A. Mohammed, Mustak Dukandar</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Nada A. Mohammed</p><p><bold>Drafting of the manuscript:</bold> Nada A. Mohammed</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Mustak Dukandar</p><p><bold>Supervision:</bold> Mustak Dukandar</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Concept and design:</bold> Nada A. Mohammed, Mustak Dukandar</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Nada A. Mohammed</p><p><bold>Drafting of the manuscript:</bold> Nada A. Mohammed</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Mustak Dukandar</p><p><bold>Supervision:</bold> Mustak Dukandar</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0015-00000050557-i01\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0015-00000050557-i01\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"2\"], \"article-title\": [\"Stump appendicitis, a rare but serious complication of appendectomy: a case report\"], \"source\": [\"Clin Case Rep\"], \"person-group\": [\"\\n\"], \"surname\": [\"Hadrich\", \"Mroua\", \"Zribi\", \"Bouassida\", \"Touinssi\"], \"given-names\": [\"Z\", \"B\", \"S\", \"M\", \"H\"], \"fpage\": [\"0\"], \"volume\": [\"9\"], \"year\": [\"2021\"]}, {\"label\": [\"3\"], \"article-title\": [\"Stump appendicitis: a rare and unusual complication after appendectomy. Case report and review of the literature\"], \"source\": [\"Ann Ital Chir\"], \"person-group\": [\"\\n\"], \"surname\": [\"Papi\", \"Pecchini\", \"Gelmini\"], \"given-names\": [\"S\", \"F\", \"R\"], \"volume\": [\"85\"], \"year\": [\"2014\"], \"uri\": [\"https://pubmed.ncbi.nlm.nih.gov/25027013/\"]}, {\"label\": [\"5\"], \"article-title\": [\"A case of stump appendicitis secondary to appendicular fecolith\"], \"source\": [\"Cureus\"], \"person-group\": [\"\\n\"], \"surname\": [\"Khazindar\"], \"given-names\": [\"AR\"], \"fpage\": [\"0\"], \"volume\": [\"14\"], \"year\": [\"2022\"]}, {\"label\": [\"8\"], \"article-title\": [\"A rare clinical entity: stump appendicitis. Case report and complete review of literature\"], \"source\": [\"Clin Ter\"], \"person-group\": [\"\\n\"], \"surname\": [\"Geraci\", \"Lena\", \"D'Orazio\", \"Cudia\", \"Rizzuto\", \"Modica\"], \"given-names\": [\"G\", \"A\", \"B\", \"B\", \"S\", \"G\"], \"fpage\": [\"0\"], \"lpage\": [\"17\"], \"volume\": [\"170\"], \"year\": [\"2019\"]}, {\"label\": [\"16\"], \"article-title\": [\"Recurrent (stump) appendicitis: a case series\"], \"source\": [\"Am J Emerg Med\"], \"person-group\": [\"\\n\"], \"surname\": [\"Rios\", \"Villanueva\", \"Stirparo\", \"Kane\"], \"given-names\": [\"RE\", \"KM\", \"JJ\", \"KE\"], \"fpage\": [\"480\"], \"lpage\": [\"482\"], \"volume\": [\"33\"], \"year\": [\"2015\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":17,"string":"17"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:43:45"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50557"},"package_file":{"kind":"string","value":"oa_package/4a/d6/PMC10787942.tar.gz"}}},{"rowIdx":199,"cells":{"accession_id":{"kind":"string","value":"PMC10787943"},"pmid":{"kind":"string","value":"38222158"},"introduction":{"kind":"list like","value":["<title>Introduction</title>","<p>Common variable immunodeficiency (CVID) is one of the prevalent primary immunodeficiencies, characterized by a wide range of symptoms and recurrent bacterial infections. Patients with CVID are at increased risk of infections, including gastrointestinal infections. Chronic diarrhea is one of the presentations of CVID, and it is often the initial symptom that leads to diagnosis. Recognizing the link between CVID and chronic diarrhea is vital. Early diagnosis can guide appropriate treatments, including immunoglobulin therapy, improving patients' quality of life and preventing complications. This case report underscores the significance of considering CVID in patients with chronic diarrhea, promoting prompt recognition and intervention.</p>"],"string":"[\n \"<title>Introduction</title>\",\n \"<p>Common variable immunodeficiency (CVID) is one of the prevalent primary immunodeficiencies, characterized by a wide range of symptoms and recurrent bacterial infections. Patients with CVID are at increased risk of infections, including gastrointestinal infections. Chronic diarrhea is one of the presentations of CVID, and it is often the initial symptom that leads to diagnosis. Recognizing the link between CVID and chronic diarrhea is vital. Early diagnosis can guide appropriate treatments, including immunoglobulin therapy, improving patients' quality of life and preventing complications. This case report underscores the significance of considering CVID in patients with chronic diarrhea, promoting prompt recognition and intervention.</p>\"\n]","isConversational":false},"methods":{"kind":"list like","value":[],"string":"[]","isConversational":false},"results":{"kind":"list like","value":[],"string":"[]","isConversational":false},"discussion":{"kind":"list like","value":["<title>Discussion</title>","<p>The patient had chronic diarrhea with recurrent sinopulmonary infections, hypogammaglobinemia, hypoproteinemia, and vitamin deficiency, along with lymphoid nodular hyperplasia of the duodenum, suggesting the diagnosis of CVID (primary immunodeficiency disorder). Other differential diagnoses that were kept were: HIV-associated enteropathy (acquired immunodeficiency disorder) as the patient had fever, recurrent respiratory tract infections, chronic diarrhea, weight loss, and a history of blood transfusion, however, HIV antibodies were negative; microscopic colitis as the patient had chronic watery diarrhea and weight loss favored the diagnosis, but the normal findings of a colon biopsy favored against it; bile salt malabsorption as the patient had chronic diarrhea, weight loss, and vitamin deficiencies favored the diagnosis; however, there was no history of abdominal pain, cramping, bloating, urgency, difficulty controlling bowel movements, or steatorrhea, which was against the diagnosis of bile salt malabsorption. Inflammatory bowel disease (IBD) was our fifth differential as the patient had chronic diarrhea, anemia, and hypoalbuminemia favored the diagnosis; however, other clinical features of IBD (blood in stools, joint pain, or other extraintestinal manifestations), histopathological evidence on biopsy, or biochemical evidence (raised ESR, CRP, and fecal calprotectin levels) were absent. Lastly, our sixth differential diagnosis was disseminated tuberculosis (pulmonary and gastrointestinal tuberculosis) as the patient had a fever with an evening rise in temperature and a chronic cough. The patient was residing in a tuberculosis-endemic region, and the patient had significant weight loss. However, there was no evidence of tuberculosis based on histopathological examination of abdominal lymph nodes and sputum, and there was no history of pain in the abdomen or alteration of bowel habits or ascites.</p>","<p>Chronic diarrhea is defined as the passage of abnormally liquid or unformed stools at an increased frequency with a duration of more than 4 weeks [##REF##29653941##1##, ####REF##31302098##2####31302098##2##]. The approach to chronic diarrhea is different from acute diarrhea and requires complex evaluation [##REF##29653941##1##]. Primary clinical assessment is done to distinguish common possibilities with further diagnostic approach requires differentiating osmotic diarrhea from secretory diarrhea by stool osmotic gap and based on this approach the study patient had secretory diarrhea [##REF##22677080##3##]. Microscopic colitis presents with similar clinical features but was ruled out due to lack of histopathological evidence, similarly, bile salt diarrhea also has similar presentation but due to lack of availability of its diagnostic tests (75SeHCAT scan) in our setup and other clinical features it was not approached further. Since the patient was asymptomatic before the onset of her symptoms 9 years back, reduced immunoglobulin levels observed were likely due to acquired hypogammaglobinemia. Various causes of acquired hypogammaglobinemia are defined in the literature, namely drug-induced, infections, malignancy, and excess losses of immunoglobulins [##UREF##0##4##].</p>","<p>CVID is one of the primary immunodeficiency syndromes which is defined by The European Society for Immunodeficiencies (ESID) as hypogammaglobulinemia with IgG levels two standard deviations below the mean; poor vaccination responses or no isohemagglutinins; and ruling out alternative causes of hypogammaglobulinemia [##REF##30776527##5##]. There is no single definitive test for CVID, and diagnosis can sometimes be challenging. The study patient was diagnosed with CVID due to her recurrent respiratory tract infection, chronic diarrhea, reduced serum IgA and IgG levels, reduced anti-HBS antibody titers, no other causes of hypogammaglobinemia were found and the disease started in the patient's second decade of life. Whole exome sequencing (WES) was performed to detect known phenotypic gene variants causing immunodeficiency and no pathognomonic or likely pathognomonic variants causative of the phenotype were detected. Prominent respiratory symptoms were also observed in study patients which could be due to recurrent respiratory tract infection or in rare cases granulomatous-lymphocytic interstitial lung disease (GLILD), which is reported in around 8-20% of cases [##REF##28351785##6##]. In the large single-center prospective study done by Resnick et al. conducted in 2012, 94% of their study patients diagnosed with CVID had a history of infections; 68% of the study patients developed non-infectious complications, 29% of study patients had chronic lung disease and 15% of study patients had gastrointestinal inflammatory disease [##REF##22180439##7##].</p>","<p>The most common infections reported in patients with CVID are bacterial infections causing sinopulmonary infections and gastrointestinal infections [##REF##18419489##8##]. Apart from infections, 10-20% of patients with CVID are reported to have gastrointestinal manifestations with diarrhea being the most common symptom. These manifestations include inflammatory bowel-like disease, nodular lymphoid hyperplasia, bacterial overgrowth, nonspecific malabsorption, and gastrointestinal lymphoma [##REF##10413651##9##, ####REF##19665769##10##, ##REF##26951230##11##, ##REF##30747770##12####30747770##12##]. Our patient had diffuse nodular lymphoid hyperplasia which can occur in up to 20% of CVID patients. It occurs due to chronic antigenic stimulation, these can be asymptomatic or present with pain in the abdomen, chronic diarrhea, intestinal obstruction, and, very rarely, a massive GI bleed.</p>","<p>Immune globulin replacement, which is the cornerstone of therapy, has significantly changed the clinical course of CVID by lowering the burden of recurrent infections and subsequent complications [##REF##20332369##13##]. Early recognition and appropriate treatment not only alleviate symptoms but also enhance the overall well-being of affected individuals.</p>"],"string":"[\n \"<title>Discussion</title>\",\n \"<p>The patient had chronic diarrhea with recurrent sinopulmonary infections, hypogammaglobinemia, hypoproteinemia, and vitamin deficiency, along with lymphoid nodular hyperplasia of the duodenum, suggesting the diagnosis of CVID (primary immunodeficiency disorder). Other differential diagnoses that were kept were: HIV-associated enteropathy (acquired immunodeficiency disorder) as the patient had fever, recurrent respiratory tract infections, chronic diarrhea, weight loss, and a history of blood transfusion, however, HIV antibodies were negative; microscopic colitis as the patient had chronic watery diarrhea and weight loss favored the diagnosis, but the normal findings of a colon biopsy favored against it; bile salt malabsorption as the patient had chronic diarrhea, weight loss, and vitamin deficiencies favored the diagnosis; however, there was no history of abdominal pain, cramping, bloating, urgency, difficulty controlling bowel movements, or steatorrhea, which was against the diagnosis of bile salt malabsorption. Inflammatory bowel disease (IBD) was our fifth differential as the patient had chronic diarrhea, anemia, and hypoalbuminemia favored the diagnosis; however, other clinical features of IBD (blood in stools, joint pain, or other extraintestinal manifestations), histopathological evidence on biopsy, or biochemical evidence (raised ESR, CRP, and fecal calprotectin levels) were absent. Lastly, our sixth differential diagnosis was disseminated tuberculosis (pulmonary and gastrointestinal tuberculosis) as the patient had a fever with an evening rise in temperature and a chronic cough. The patient was residing in a tuberculosis-endemic region, and the patient had significant weight loss. However, there was no evidence of tuberculosis based on histopathological examination of abdominal lymph nodes and sputum, and there was no history of pain in the abdomen or alteration of bowel habits or ascites.</p>\",\n \"<p>Chronic diarrhea is defined as the passage of abnormally liquid or unformed stools at an increased frequency with a duration of more than 4 weeks [##REF##29653941##1##, ####REF##31302098##2####31302098##2##]. The approach to chronic diarrhea is different from acute diarrhea and requires complex evaluation [##REF##29653941##1##]. Primary clinical assessment is done to distinguish common possibilities with further diagnostic approach requires differentiating osmotic diarrhea from secretory diarrhea by stool osmotic gap and based on this approach the study patient had secretory diarrhea [##REF##22677080##3##]. Microscopic colitis presents with similar clinical features but was ruled out due to lack of histopathological evidence, similarly, bile salt diarrhea also has similar presentation but due to lack of availability of its diagnostic tests (75SeHCAT scan) in our setup and other clinical features it was not approached further. Since the patient was asymptomatic before the onset of her symptoms 9 years back, reduced immunoglobulin levels observed were likely due to acquired hypogammaglobinemia. Various causes of acquired hypogammaglobinemia are defined in the literature, namely drug-induced, infections, malignancy, and excess losses of immunoglobulins [##UREF##0##4##].</p>\",\n \"<p>CVID is one of the primary immunodeficiency syndromes which is defined by The European Society for Immunodeficiencies (ESID) as hypogammaglobulinemia with IgG levels two standard deviations below the mean; poor vaccination responses or no isohemagglutinins; and ruling out alternative causes of hypogammaglobulinemia [##REF##30776527##5##]. There is no single definitive test for CVID, and diagnosis can sometimes be challenging. The study patient was diagnosed with CVID due to her recurrent respiratory tract infection, chronic diarrhea, reduced serum IgA and IgG levels, reduced anti-HBS antibody titers, no other causes of hypogammaglobinemia were found and the disease started in the patient's second decade of life. Whole exome sequencing (WES) was performed to detect known phenotypic gene variants causing immunodeficiency and no pathognomonic or likely pathognomonic variants causative of the phenotype were detected. Prominent respiratory symptoms were also observed in study patients which could be due to recurrent respiratory tract infection or in rare cases granulomatous-lymphocytic interstitial lung disease (GLILD), which is reported in around 8-20% of cases [##REF##28351785##6##]. In the large single-center prospective study done by Resnick et al. conducted in 2012, 94% of their study patients diagnosed with CVID had a history of infections; 68% of the study patients developed non-infectious complications, 29% of study patients had chronic lung disease and 15% of study patients had gastrointestinal inflammatory disease [##REF##22180439##7##].</p>\",\n \"<p>The most common infections reported in patients with CVID are bacterial infections causing sinopulmonary infections and gastrointestinal infections [##REF##18419489##8##]. Apart from infections, 10-20% of patients with CVID are reported to have gastrointestinal manifestations with diarrhea being the most common symptom. These manifestations include inflammatory bowel-like disease, nodular lymphoid hyperplasia, bacterial overgrowth, nonspecific malabsorption, and gastrointestinal lymphoma [##REF##10413651##9##, ####REF##19665769##10##, ##REF##26951230##11##, ##REF##30747770##12####30747770##12##]. Our patient had diffuse nodular lymphoid hyperplasia which can occur in up to 20% of CVID patients. It occurs due to chronic antigenic stimulation, these can be asymptomatic or present with pain in the abdomen, chronic diarrhea, intestinal obstruction, and, very rarely, a massive GI bleed.</p>\",\n \"<p>Immune globulin replacement, which is the cornerstone of therapy, has significantly changed the clinical course of CVID by lowering the burden of recurrent infections and subsequent complications [##REF##20332369##13##]. Early recognition and appropriate treatment not only alleviate symptoms but also enhance the overall well-being of affected individuals.</p>\"\n]","isConversational":false},"conclusion":{"kind":"list like","value":["<title>Conclusions</title>","<p>Evaluation of chronic diarrhea requires thorough clinical evaluation and should be evaluated with an open mind. A review of a patient’s investigations should always be considered when clinical judgment regarding a case is not satisfied. Although common diseases should be considered first in differential diagnosis points favoring the diagnosis and points against diagnosis should be compared. CVID is a primary immunodeficiency disorder that can cause a wide range of clinical manifestations, including chronic diarrhea. Early diagnosis and treatment with immunoglobulins are essential to improve the quality of life and reduce the risk of complications in patients with CVID.</p>"],"string":"[\n \"<title>Conclusions</title>\",\n \"<p>Evaluation of chronic diarrhea requires thorough clinical evaluation and should be evaluated with an open mind. A review of a patient’s investigations should always be considered when clinical judgment regarding a case is not satisfied. Although common diseases should be considered first in differential diagnosis points favoring the diagnosis and points against diagnosis should be compared. CVID is a primary immunodeficiency disorder that can cause a wide range of clinical manifestations, including chronic diarrhea. Early diagnosis and treatment with immunoglobulins are essential to improve the quality of life and reduce the risk of complications in patients with CVID.</p>\"\n]","isConversational":false},"front":{"kind":"list like","value":["<p>Chronic diarrhea poses a diagnostic challenge due to its diverse etiology, encompassing various gastrointestinal disorders. This case report emphasizes the clinical significance of considering common variable immunodeficiency (CVID) as a potential underlying cause in a patient presenting with chronic diarrhea. In this case study, we describe a 36-year-old female with a 9-year history of chronic diarrhea, recurrent sinopulmonary infections, and weight loss for 3 years, where previous evaluations failed to yield a diagnosis. This case underscores the diagnostic hurdles faced by healthcare professionals, often causing a delay in identifying fewer common conditions like immunodeficiency syndromes. Early recognition of CVID is crucial, enabling timely intervention with immunoglobulin replacement therapy, markedly enhancing patients' quality of life and averting complications. This report highlights the necessity for a comprehensive evaluation of non-responsive chronic diarrhea cases and raises awareness about CVID as an essential consideration, facilitating precise diagnoses and tailored treatments.</p>"],"string":"[\n \"<p>Chronic diarrhea poses a diagnostic challenge due to its diverse etiology, encompassing various gastrointestinal disorders. This case report emphasizes the clinical significance of considering common variable immunodeficiency (CVID) as a potential underlying cause in a patient presenting with chronic diarrhea. In this case study, we describe a 36-year-old female with a 9-year history of chronic diarrhea, recurrent sinopulmonary infections, and weight loss for 3 years, where previous evaluations failed to yield a diagnosis. This case underscores the diagnostic hurdles faced by healthcare professionals, often causing a delay in identifying fewer common conditions like immunodeficiency syndromes. Early recognition of CVID is crucial, enabling timely intervention with immunoglobulin replacement therapy, markedly enhancing patients' quality of life and averting complications. This report highlights the necessity for a comprehensive evaluation of non-responsive chronic diarrhea cases and raises awareness about CVID as an essential consideration, facilitating precise diagnoses and tailored treatments.</p>\"\n]","isConversational":false},"body":{"kind":"list like","value":["<title>Case presentation</title>","<p>A 36-year-old female presented to our emergency department with complaints of watery stools for 9 years, recurrent respiratory tract infections for 9 years, and weight loss for 3 years. Initially, the patient developed loose motions that were watery in consistency, with a stool output of about 150 mL per episode and a frequency of four to five episodes of loose motions per day, which were associated with nocturnal diarrhea. The patient also complained of coughing and nasal stuffiness for 9 years, which was associated with expectoration for the past 4 years. The patient had also complained of fever for 9 years. The frequency of the fever varied, ranging from occurring three to four times a week to sometimes once a month. The fever was mostly associated with coughing, and there was no notable association between the fever and diarrhea. The maximum temperature recorded by the patient was 100°F sublingually. The fever was associated with an evening rise in temperature, which subsided after taking over-the-counter medications. For the past 4 to 5 years, the patient has also complained of fatigue and unintentional weight loss of 8 kg. There was a history of blood transfusions in the past 10 years due to blood loss during delivery, and she was vaccinated for hepatitis B and COVID-19 in the past. There was no significant family history, or history suggestive of pulmonary tuberculosis in any family members. There was no significant obstetrical or gynecological history.</p>","<p>At presentation, the patient’s blood pressure was 106/60 mmHg, pulse rate was 92 per minute, respiratory rate was 14 per minute, and the temperature recorded was 98.6°F sublingually. On a general physical examination, mild pallor was observed. There were no signs of icterus, cyanosis, clubbing, pedal edema, or lymphadenopathy. The patient’s systemic examination there was nonremarkable. On anthropometric examination, her body mass index (BMI) was 18.5 kg/m<sup>2</sup>, and mid-upper arm circumference was 23 cm.</p>","<p>Initial investigations, which were performed and are shown in Table ##TAB##0##1##, revealed mild macrocytic anemia, thrombocytopenia, hypoproteinemia with hypogammaglobinemia, vitamin B12 deficiency, folate deficiency, and vitamin D deficiency. The stool examinations were also performed with negative results for stool microscopy (for ova, cysts, and parasites), stool culture and sensitivity for pathogenic organisms, and fecal occult blood test. The patient’s calculated stool osmotic gap suggested secretory diarrhea, which corresponded with a normal 72-hour fecal fat estimation test that ruled out malabsorption syndrome and functional diarrhea (Table ##TAB##1##2##). Upper gastrointestinal endoscopy was performed, which revealed atrophic gastric mucosa and nodular lymphoid hyperplasia of the D2 part of the duodenum (Figures ##FIG##0##1a##, ##FIG##0##1b##) (Table ##TAB##2##3##). Histopathological examination of these lesions revealed chronic active duodenitis with nodular lymphoid hyperplasia with no evidence of dysplasia or malignancy. Similarly, a lower gastrointestinal endoscopy was also performed, but it was unremarkable (Table ##TAB##3##4##). Biopsies were taken from the transverse and descending colons and were also unremarkable(Table ##TAB##4##5##). An x-ray of the chest was also done and showed bilateral calcified and fibrotic lesions (Figure ##FIG##1##2##), and the sputum examination (for biochemical tests, microscopic examination for gram stain and acid-fast stain, and culture for pathogenic organisms) was nonremarkable. A Triple-Phase Contrast-Enhanced CT A whole abdomen was performed to rule out any structural disease and showed mild splenomegaly with variable-sized, homogenously enhancing mesenteric lymph nodes. A CT-guided abdominal lymph node biopsy was then performed, which showed reactive lymphocytosis with no evidence of granulomatous changes or malignancy (Table ##TAB##4##5##).</p>","<p>A secondary clinical assessment was done to assess for chronic diarrhea associated with immunodeficiency. A serological test was conducted to detect viral markers for human immunodeficiency virus (HIV), and the result was negative. Since hypogammaglobinemia was already documented during the initial investigations, the patient was then evaluated for a poor response to immunization. The patient's anti-HBs titer was ordered, and despite receiving a booster dose of the hepatitis B vaccine in the past, low levels of anti-HBs antibodies were observed. (Table ##TAB##5##6##).</p>","<p>Initially, the patient was given a course of injectable antibiotics for 5 days. Ceftriaxone (Gram-positive and Gram-negative coverage) and metronidazole (anaerobic coverage) were given along with supportive treatment of antisecretory agents and encouraged oral fluid intake. The patient was relieved for a few days, but her symptoms began again. After the diagnosis of CVID was considered, the patient was started on intravenous immunoglobulin (IVIG) at 500 mg/kg body weight every four weeks with premedication under supervision. The patient was discharged in satisfactory condition and was in follow-up. Serum immunoglobulin levels were done in follow-up, and they showed rising titers of serum IgA, IgG, and IgM.</p>","<p>During follow-up, the patient was asked about her general well-being, appetite, symptoms of cough, water stools, fever, and any complications related to IVIG administration. Two weeks after receiving the third dose of IVIG, the patient’s watery stools improved and her fever subsided. After the sixth dose, the patient's bowel movements returned to normal, her cough subsided, her appetite increased, and her BMI increased from an initial 18.5 to 21.2. The patient did not suffer from any complications from IVIG, and after the eighth dose of IVIG, the patient became asymptomatic. One year after her first dose of IVIG, the patient is still asymptomatic.</p>"],"string":"[\n \"<title>Case presentation</title>\",\n \"<p>A 36-year-old female presented to our emergency department with complaints of watery stools for 9 years, recurrent respiratory tract infections for 9 years, and weight loss for 3 years. Initially, the patient developed loose motions that were watery in consistency, with a stool output of about 150 mL per episode and a frequency of four to five episodes of loose motions per day, which were associated with nocturnal diarrhea. The patient also complained of coughing and nasal stuffiness for 9 years, which was associated with expectoration for the past 4 years. The patient had also complained of fever for 9 years. The frequency of the fever varied, ranging from occurring three to four times a week to sometimes once a month. The fever was mostly associated with coughing, and there was no notable association between the fever and diarrhea. The maximum temperature recorded by the patient was 100°F sublingually. The fever was associated with an evening rise in temperature, which subsided after taking over-the-counter medications. For the past 4 to 5 years, the patient has also complained of fatigue and unintentional weight loss of 8 kg. There was a history of blood transfusions in the past 10 years due to blood loss during delivery, and she was vaccinated for hepatitis B and COVID-19 in the past. There was no significant family history, or history suggestive of pulmonary tuberculosis in any family members. There was no significant obstetrical or gynecological history.</p>\",\n \"<p>At presentation, the patient’s blood pressure was 106/60 mmHg, pulse rate was 92 per minute, respiratory rate was 14 per minute, and the temperature recorded was 98.6°F sublingually. On a general physical examination, mild pallor was observed. There were no signs of icterus, cyanosis, clubbing, pedal edema, or lymphadenopathy. The patient’s systemic examination there was nonremarkable. On anthropometric examination, her body mass index (BMI) was 18.5 kg/m<sup>2</sup>, and mid-upper arm circumference was 23 cm.</p>\",\n \"<p>Initial investigations, which were performed and are shown in Table ##TAB##0##1##, revealed mild macrocytic anemia, thrombocytopenia, hypoproteinemia with hypogammaglobinemia, vitamin B12 deficiency, folate deficiency, and vitamin D deficiency. The stool examinations were also performed with negative results for stool microscopy (for ova, cysts, and parasites), stool culture and sensitivity for pathogenic organisms, and fecal occult blood test. The patient’s calculated stool osmotic gap suggested secretory diarrhea, which corresponded with a normal 72-hour fecal fat estimation test that ruled out malabsorption syndrome and functional diarrhea (Table ##TAB##1##2##). Upper gastrointestinal endoscopy was performed, which revealed atrophic gastric mucosa and nodular lymphoid hyperplasia of the D2 part of the duodenum (Figures ##FIG##0##1a##, ##FIG##0##1b##) (Table ##TAB##2##3##). Histopathological examination of these lesions revealed chronic active duodenitis with nodular lymphoid hyperplasia with no evidence of dysplasia or malignancy. Similarly, a lower gastrointestinal endoscopy was also performed, but it was unremarkable (Table ##TAB##3##4##). Biopsies were taken from the transverse and descending colons and were also unremarkable(Table ##TAB##4##5##). An x-ray of the chest was also done and showed bilateral calcified and fibrotic lesions (Figure ##FIG##1##2##), and the sputum examination (for biochemical tests, microscopic examination for gram stain and acid-fast stain, and culture for pathogenic organisms) was nonremarkable. A Triple-Phase Contrast-Enhanced CT A whole abdomen was performed to rule out any structural disease and showed mild splenomegaly with variable-sized, homogenously enhancing mesenteric lymph nodes. A CT-guided abdominal lymph node biopsy was then performed, which showed reactive lymphocytosis with no evidence of granulomatous changes or malignancy (Table ##TAB##4##5##).</p>\",\n \"<p>A secondary clinical assessment was done to assess for chronic diarrhea associated with immunodeficiency. A serological test was conducted to detect viral markers for human immunodeficiency virus (HIV), and the result was negative. Since hypogammaglobinemia was already documented during the initial investigations, the patient was then evaluated for a poor response to immunization. The patient's anti-HBs titer was ordered, and despite receiving a booster dose of the hepatitis B vaccine in the past, low levels of anti-HBs antibodies were observed. (Table ##TAB##5##6##).</p>\",\n \"<p>Initially, the patient was given a course of injectable antibiotics for 5 days. Ceftriaxone (Gram-positive and Gram-negative coverage) and metronidazole (anaerobic coverage) were given along with supportive treatment of antisecretory agents and encouraged oral fluid intake. The patient was relieved for a few days, but her symptoms began again. After the diagnosis of CVID was considered, the patient was started on intravenous immunoglobulin (IVIG) at 500 mg/kg body weight every four weeks with premedication under supervision. The patient was discharged in satisfactory condition and was in follow-up. Serum immunoglobulin levels were done in follow-up, and they showed rising titers of serum IgA, IgG, and IgM.</p>\",\n \"<p>During follow-up, the patient was asked about her general well-being, appetite, symptoms of cough, water stools, fever, and any complications related to IVIG administration. Two weeks after receiving the third dose of IVIG, the patient’s watery stools improved and her fever subsided. After the sixth dose, the patient's bowel movements returned to normal, her cough subsided, her appetite increased, and her BMI increased from an initial 18.5 to 21.2. The patient did not suffer from any complications from IVIG, and after the eighth dose of IVIG, the patient became asymptomatic. One year after her first dose of IVIG, the patient is still asymptomatic.</p>\"\n]","isConversational":false},"back":{"kind":"list like","value":[],"string":"[]","isConversational":false},"figure":{"kind":"list like","value":["<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Upper GI endoscopy showing nodular lymphoid hyperplasia of the duodenum</title></caption></fig>","<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>: Chest X-ray</title><p>X-ray shows bilateral calcified and fibrotic lesions involving right upper, middle, and lower zones and in left upper and middle zones with evidence of hyperinflation, bronchial wall thickening in right lower zone.</p></caption></fig>"],"string":"[\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG1\\\"><label>Figure 1</label><caption><title>Upper GI endoscopy showing nodular lymphoid hyperplasia of the duodenum</title></caption></fig>\",\n \"<fig position=\\\"anchor\\\" fig-type=\\\"figure\\\" id=\\\"FIG2\\\"><label>Figure 2</label><caption><title>: Chest X-ray</title><p>X-ray shows bilateral calcified and fibrotic lesions involving right upper, middle, and lower zones and in left upper and middle zones with evidence of hyperinflation, bronchial wall thickening in right lower zone.</p></caption></fig>\"\n]","isConversational":false},"table":{"kind":"list like","value":["<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Initial Investigations performed</title><p>ALT: alanine aminotransferase; AST: aspartate aminotransferase</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nInvestigation\n</td><td rowspan=\"1\" colspan=\"1\">\nValue\n</td><td rowspan=\"1\" colspan=\"1\">\nNormal Range\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nHemoglobin (g/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n10.1\n</td><td rowspan=\"1\" colspan=\"1\">\n12.1-15.1\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nTotal leucocyte count (per mL)\n</td><td rowspan=\"1\" colspan=\"1\">\n6200\n</td><td rowspan=\"1\" colspan=\"1\">\n4000-11,000\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nDifferential leucocyte count\n</td><td rowspan=\"1\" colspan=\"1\">\nN65% L30% M5%\n</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nPlatelet-count(per mL)\n</td><td rowspan=\"1\" colspan=\"1\">\n87000\n</td><td rowspan=\"1\" colspan=\"1\">\n150,000-450,000\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nMean corpuscular volume (femtoliters/cell)\n</td><td rowspan=\"1\" colspan=\"1\">\n106\n</td><td rowspan=\"1\" colspan=\"1\">\n80-100\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nMean corpuscular hemoglobin (picogram/cell)\n</td><td rowspan=\"1\" colspan=\"1\">\n30\n</td><td rowspan=\"1\" colspan=\"1\">\n27-31\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nMean corpuscular hemoglobin concentration (g/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n34.6\n</td><td rowspan=\"1\" colspan=\"1\">\n32-36\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nProthrombin time (seconds)\n</td><td rowspan=\"1\" colspan=\"1\">\n15\n</td><td rowspan=\"1\" colspan=\"1\">\n11-13.5\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nINR\n</td><td rowspan=\"1\" colspan=\"1\">\n1.1\n</td><td rowspan=\"1\" colspan=\"1\">\n<1.1\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nBlood urea nitrogen (mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n12\n</td><td rowspan=\"1\" colspan=\"1\">\n6 to 24\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nCreatinine(mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.6\n</td><td rowspan=\"1\" colspan=\"1\">\n0.6-1.1\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nSerum sodium (mEq/L)\n</td><td rowspan=\"1\" colspan=\"1\">\n136\n</td><td rowspan=\"1\" colspan=\"1\">\n135-145\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nSerum potassium (mEq/L)\n</td><td rowspan=\"1\" colspan=\"1\">\n4.4\n</td><td rowspan=\"1\" colspan=\"1\">\n3.5-5.2\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nAST(IU/L)\n</td><td rowspan=\"1\" colspan=\"1\">\n20\n</td><td rowspan=\"1\" colspan=\"1\">\n10 to 36\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nALT(IU/L)\n</td><td rowspan=\"1\" colspan=\"1\">\n22\n</td><td rowspan=\"1\" colspan=\"1\">\n19-25\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nAlkaline phosphatase (IU/L)\n</td><td rowspan=\"1\" colspan=\"1\">\n113\n</td><td rowspan=\"1\" colspan=\"1\">\n44-147\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nTotal bilirubin (mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.63\n</td><td rowspan=\"1\" colspan=\"1\">\n0.1-1.2\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nDirect bilirubin (mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n0.23\n</td><td rowspan=\"1\" colspan=\"1\">\n<0.3\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nTotal serum protein\n</td><td rowspan=\"1\" colspan=\"1\">\n5.2\n</td><td rowspan=\"1\" colspan=\"1\">\n6.7-8.6 g/dL\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nSerum albumin\n</td><td rowspan=\"1\" colspan=\"1\">\n3.2\n</td><td rowspan=\"1\" colspan=\"1\">\n3.5-5.5 g/dL\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nSerum globulin\n</td><td rowspan=\"1\" colspan=\"1\">\n1.2\n</td><td rowspan=\"1\" colspan=\"1\">\n2.0-3.5 g/dL\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nA/G ratio\n</td><td rowspan=\"1\" colspan=\"1\">\n2.6\n</td><td rowspan=\"1\" colspan=\"1\">\n1.5-2.5:1\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nSerum ferritin(ng/mL)\n</td><td rowspan=\"1\" colspan=\"1\">\n280\n</td><td rowspan=\"1\" colspan=\"1\">\n11-307\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nSerum iron(mcg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n65\n</td><td rowspan=\"1\" colspan=\"1\">\n60-160\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nT3\n</td><td rowspan=\"1\" colspan=\"1\">\n152\n</td><td rowspan=\"1\" colspan=\"1\">\n60-215 ng/dL\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nT4\n</td><td rowspan=\"1\" colspan=\"1\">\n9.52\n</td><td rowspan=\"1\" colspan=\"1\">\n5.2-12.7 µg/dL\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nTSH\n</td><td rowspan=\"1\" colspan=\"1\">\n3\n</td><td rowspan=\"1\" colspan=\"1\">\n0.35-5.50 µIU/mL\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nVitamin B12 levels (pg/mL)\n</td><td rowspan=\"1\" colspan=\"1\">\n170\n</td><td rowspan=\"1\" colspan=\"1\">\n200-900\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nSerum folate levels (ng/mL)\n</td><td rowspan=\"1\" colspan=\"1\">\n5.1\n</td><td rowspan=\"1\" colspan=\"1\">\n>5.38\n</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB2\"><label>Table 2</label><caption><title>Stool examination</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nStool Examination\n</td><td rowspan=\"1\" colspan=\"1\">\nReport\n</td><td rowspan=\"1\" colspan=\"1\">\nNormal Range\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nMicroscopy for ova, cyst, and parasite\n</td><td rowspan=\"1\" colspan=\"1\">\nNegative\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nStool culture and sensitivity for pathogenic organisms (aerobic and anaerobic bacterial pathogens)\n</td><td rowspan=\"1\" colspan=\"1\">\nNegative\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nFecal occult blood test\n</td><td rowspan=\"1\" colspan=\"1\">\nNegative\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nFecal calprotectin levels (mcg/g)\n</td><td rowspan=\"1\" colspan=\"1\">\n32\n</td><td rowspan=\"1\" colspan=\"1\">\n<50 µg/g\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nFecal fat estimation (72 hours)\n</td><td rowspan=\"1\" colspan=\"1\">\n5.1 g/day\n</td><td rowspan=\"1\" colspan=\"1\">\n<7.0 g/day\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nStool Giardia antigen test (Indirect Coombs test)\n</td><td rowspan=\"1\" colspan=\"1\">\nNegative\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nStool Cryptosporidium antigen test (Indirect Coombs test)\n</td><td rowspan=\"1\" colspan=\"1\">\nNegative\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nStool osmolality (mOsm/kg)\n</td><td rowspan=\"1\" colspan=\"1\">\n171\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nStool sodium (mmol/L)\n</td><td rowspan=\"1\" colspan=\"1\">\n43\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nStool potassium (mmol/L)\n</td><td rowspan=\"1\" colspan=\"1\">\n20\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nStool osmotic gap (mOsm/kg)\n</td><td rowspan=\"1\" colspan=\"1\">\n45 mOsm/kg\n</td><td rowspan=\"1\" colspan=\"1\">\nStool Osmotic Gap (Stool Osmolality - [2 * (Stool Sodium + Stool Potassium)] mOsm/kg\n</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB3\"><label>Table 3</label><caption><title>Upper GI Endoscopy of the study patient</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"1\">\nUpper GI Endoscopy (Figures ##FIG##0##1a##-##FIG##0##1b##)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nEsophagus\n</td><td rowspan=\"1\" colspan=\"1\">\nNormal\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nStomach\n</td><td rowspan=\"1\" colspan=\"1\">\nAtrophic Gastric Mucosa\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nDuodenum\n</td><td rowspan=\"1\" colspan=\"1\">\nD1: normal; D2: multiple small, discrete, nodules scattered throughout the duodenal mucosa. The nodules are approximately 2-3 mm in diameter.\n</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB4\"><label>Table 4</label><caption><title>Colonoscopy Report of the study patient</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nProcedure\n</td><td rowspan=\"1\" colspan=\"1\">\nFindings\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nColonoscopy\n</td><td rowspan=\"1\" colspan=\"1\">\nThe scope was passed till the transverse colon. The visualized portion did not reveal any significant abnormalities or pathological findings. The mucosa appeared healthy, and there were no visible signs of inflammation, polyps, or masses.\n</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB5\"><label>Table 5</label><caption><title>Biopsy reports of the study patient</title><p>AFB: acid-fast bacilli</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nBiopsy\n</td><td rowspan=\"1\" colspan=\"1\">\nFindings\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nBiopsy from duodenum\n</td><td rowspan=\"1\" colspan=\"1\">\nMarked villous blunting with crypt hyperplasia. Lamina propria prominent lymphoid follicles with active germinal centers and acute on chronic inflammatory infiltrate with evidence of cryptitis. Chronic active duodenitis with nodular lymphoid hyperplasia and no evidence of dysplasia/malignancy.\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nBiopsy from colonoscopy\n</td><td rowspan=\"1\" colspan=\"1\">\nBased on the histological findings, there is no evidence of pathological changes or significant abnormalities in the colonic mucosa.\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nBiopsy from abdominal lymph nodes including ZN (Ziehl-Neelsen) staining for AFB\n</td><td rowspan=\"1\" colspan=\"1\">\nThe histopathological examination of the abdominal lymph node biopsy demonstrates reactive changes within the lymph nodes, characterized by enlarged germinal centers and increased lymphocyte populations with no findings suggesting any underlying granulomatous inflammation, caseation necrosis, or multinucleated giant cells and is negative on ZN staining.\n</td></tr></tbody></table></table-wrap>","<table-wrap position=\"float\" id=\"TAB6\"><label>Table 6</label><caption><title>Investigations performed for final diagnosis</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nInvestigation\n</td><td rowspan=\"1\" colspan=\"1\">\nValue\n</td><td rowspan=\"1\" colspan=\"1\">\nNormal Range\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nHIV 1&2 antibodies(ELISA)\n</td><td rowspan=\"1\" colspan=\"1\">\nNegative\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nanti-HBs titers (mIU/mL)\n</td><td rowspan=\"1\" colspan=\"1\">\n1\n</td><td rowspan=\"1\" colspan=\"1\">\n>10 post-vaccination\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nESR (mm/hour)\n</td><td rowspan=\"1\" colspan=\"1\">\n10\n</td><td rowspan=\"1\" colspan=\"1\">\n<20\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nCRP(mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n3.8\n</td><td rowspan=\"1\" colspan=\"1\">\n<3\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nProcalcitonin levels (ng/mL)\n</td><td rowspan=\"1\" colspan=\"1\">\n<0.1\n</td><td rowspan=\"1\" colspan=\"1\">\n<0.1\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nImmunoglobulins (mg/dL)\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td><td rowspan=\"1\" colspan=\"1\">\n-\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nIgA\n</td><td rowspan=\"1\" colspan=\"1\">\n<33\n</td><td rowspan=\"1\" colspan=\"1\">\n40-350\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nIgG\n</td><td rowspan=\"1\" colspan=\"1\">\n352\n</td><td rowspan=\"1\" colspan=\"1\">\n650-1600\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nIgM\n</td><td rowspan=\"1\" colspan=\"1\">\n<21\n</td><td rowspan=\"1\" colspan=\"1\">\n50-300\n</td></tr></tbody></table></table-wrap>"],"string":"[\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB1\\\"><label>Table 1</label><caption><title>Initial Investigations performed</title><p>ALT: alanine aminotransferase; AST: aspartate aminotransferase</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nInvestigation\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nValue\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNormal Range\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nHemoglobin (g/dL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n10.1\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n12.1-15.1\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nTotal leucocyte count (per mL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n6200\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n4000-11,000\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nDifferential leucocyte count\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nN65% L30% M5%\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\"> </td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nPlatelet-count(per mL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n87000\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n150,000-450,000\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nMean corpuscular volume (femtoliters/cell)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n106\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n80-100\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nMean corpuscular hemoglobin (picogram/cell)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n30\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n27-31\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nMean corpuscular hemoglobin concentration (g/dL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n34.6\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n32-36\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nProthrombin time (seconds)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n15\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n11-13.5\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nINR\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n1.1\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<1.1\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nBlood urea nitrogen (mg/dL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n12\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n6 to 24\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nCreatinine(mg/dL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n0.6\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n0.6-1.1\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nSerum sodium (mEq/L)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n136\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n135-145\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nSerum potassium (mEq/L)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n4.4\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n3.5-5.2\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nAST(IU/L)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n20\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n10 to 36\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nALT(IU/L)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n22\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n19-25\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nAlkaline phosphatase (IU/L)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n113\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n44-147\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nTotal bilirubin (mg/dL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n0.63\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n0.1-1.2\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nDirect bilirubin (mg/dL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n0.23\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<0.3\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nTotal serum protein\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n5.2\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n6.7-8.6 g/dL\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nSerum albumin\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n3.2\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n3.5-5.5 g/dL\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nSerum globulin\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n1.2\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n2.0-3.5 g/dL\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nA/G ratio\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n2.6\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n1.5-2.5:1\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nSerum ferritin(ng/mL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n280\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n11-307\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nSerum iron(mcg/dL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n65\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n60-160\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nT3\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n152\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n60-215 ng/dL\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nT4\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n9.52\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n5.2-12.7 µg/dL\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nTSH\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n3\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n0.35-5.50 µIU/mL\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nVitamin B12 levels (pg/mL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n170\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n200-900\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nSerum folate levels (ng/mL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n5.1\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n>5.38\\n</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB2\\\"><label>Table 2</label><caption><title>Stool examination</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStool Examination\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nReport\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNormal Range\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nMicroscopy for ova, cyst, and parasite\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNegative\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStool culture and sensitivity for pathogenic organisms (aerobic and anaerobic bacterial pathogens)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNegative\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nFecal occult blood test\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNegative\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nFecal calprotectin levels (mcg/g)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n32\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<50 µg/g\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nFecal fat estimation (72 hours)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n5.1 g/day\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<7.0 g/day\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStool Giardia antigen test (Indirect Coombs test)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNegative\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStool Cryptosporidium antigen test (Indirect Coombs test)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNegative\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStool osmolality (mOsm/kg)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n171\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStool sodium (mmol/L)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n43\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStool potassium (mmol/L)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n20\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStool osmotic gap (mOsm/kg)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n45 mOsm/kg\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStool Osmotic Gap (Stool Osmolality - [2 * (Stool Sodium + Stool Potassium)] mOsm/kg\\n</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB3\\\"><label>Table 3</label><caption><title>Upper GI Endoscopy of the study patient</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td colspan=\\\"2\\\" rowspan=\\\"1\\\">\\nUpper GI Endoscopy (Figures ##FIG##0##1a##-##FIG##0##1b##)\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nEsophagus\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNormal\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nStomach\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nAtrophic Gastric Mucosa\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nDuodenum\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nD1: normal; D2: multiple small, discrete, nodules scattered throughout the duodenal mucosa. The nodules are approximately 2-3 mm in diameter.\\n</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB4\\\"><label>Table 4</label><caption><title>Colonoscopy Report of the study patient</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nProcedure\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nFindings\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nColonoscopy\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nThe scope was passed till the transverse colon. The visualized portion did not reveal any significant abnormalities or pathological findings. The mucosa appeared healthy, and there were no visible signs of inflammation, polyps, or masses.\\n</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB5\\\"><label>Table 5</label><caption><title>Biopsy reports of the study patient</title><p>AFB: acid-fast bacilli</p></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nBiopsy\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nFindings\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nBiopsy from duodenum\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nMarked villous blunting with crypt hyperplasia. Lamina propria prominent lymphoid follicles with active germinal centers and acute on chronic inflammatory infiltrate with evidence of cryptitis. Chronic active duodenitis with nodular lymphoid hyperplasia and no evidence of dysplasia/malignancy.\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nBiopsy from colonoscopy\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nBased on the histological findings, there is no evidence of pathological changes or significant abnormalities in the colonic mucosa.\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nBiopsy from abdominal lymph nodes including ZN (Ziehl-Neelsen) staining for AFB\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nThe histopathological examination of the abdominal lymph node biopsy demonstrates reactive changes within the lymph nodes, characterized by enlarged germinal centers and increased lymphocyte populations with no findings suggesting any underlying granulomatous inflammation, caseation necrosis, or multinucleated giant cells and is negative on ZN staining.\\n</td></tr></tbody></table></table-wrap>\",\n \"<table-wrap position=\\\"float\\\" id=\\\"TAB6\\\"><label>Table 6</label><caption><title>Investigations performed for final diagnosis</title></caption><table frame=\\\"hsides\\\" rules=\\\"groups\\\"><tbody><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nInvestigation\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nValue\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNormal Range\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nHIV 1&2 antibodies(ELISA)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nNegative\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nanti-HBs titers (mIU/mL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n1\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n>10 post-vaccination\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nESR (mm/hour)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n10\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<20\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nCRP(mg/dL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n3.8\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<3\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nProcalcitonin levels (ng/mL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<0.1\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<0.1\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nImmunoglobulins (mg/dL)\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n-\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nIgA\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<33\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n40-350\\n</td></tr><tr style=\\\"background-color:#ccc\\\"><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nIgG\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n352\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n650-1600\\n</td></tr><tr><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\nIgM\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n<21\\n</td><td rowspan=\\\"1\\\" colspan=\\\"1\\\">\\n50-300\\n</td></tr></tbody></table></table-wrap>\"\n]","isConversational":false},"formula":{"kind":"list like","value":[],"string":"[]","isConversational":false},"box":{"kind":"list like","value":[],"string":"[]","isConversational":false},"code":{"kind":"list like","value":[],"string":"[]","isConversational":false},"quote":{"kind":"list like","value":[],"string":"[]","isConversational":false},"chemical":{"kind":"list like","value":[],"string":"[]","isConversational":false},"supplementary":{"kind":"list like","value":[],"string":"[]","isConversational":false},"footnote":{"kind":"list like","value":["<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Ahmad G. Ansari , Husaini S. Haider Mehdi, Ariba Nasar</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Ahmad G. Ansari , Husaini S. Haider Mehdi, Ariba Nasar</p><p><bold>Drafting of the manuscript:</bold> Ahmad G. Ansari , Husaini S. Haider Mehdi, Ariba Nasar</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Ahmad G. Ansari , Husaini S. Haider Mehdi, Ariba Nasar</p><p><bold>Supervision:</bold> Husaini S. Haider Mehdi, Ariba Nasar</p></fn></fn-group>","<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>","<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"],"string":"[\n \"<fn-group content-type=\\\"other\\\"><title>Author Contributions</title><fn fn-type=\\\"other\\\"><p><bold>Concept and design:</bold> Ahmad G. Ansari , Husaini S. Haider Mehdi, Ariba Nasar</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Ahmad G. Ansari , Husaini S. Haider Mehdi, Ariba Nasar</p><p><bold>Drafting of the manuscript:</bold> Ahmad G. Ansari , Husaini S. Haider Mehdi, Ariba Nasar</p><p><bold>Critical review of the manuscript for important intellectual content:</bold> Ahmad G. Ansari , Husaini S. Haider Mehdi, Ariba Nasar</p><p><bold>Supervision:</bold> Husaini S. Haider Mehdi, Ariba Nasar</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"other\\\"><title>Human Ethics</title><fn fn-type=\\\"other\\\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>\",\n \"<fn-group content-type=\\\"competing-interests\\\"><fn fn-type=\\\"COI-statement\\\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>\"\n]","isConversational":false},"graphic":{"kind":"list like","value":["<graphic xlink:href=\"cureus-0015-00000050556-i01\" position=\"float\"/>","<graphic xlink:href=\"cureus-0015-00000050556-i02\" position=\"float\"/>"],"string":"[\n \"<graphic xlink:href=\\\"cureus-0015-00000050556-i01\\\" position=\\\"float\\\"/>\",\n \"<graphic xlink:href=\\\"cureus-0015-00000050556-i02\\\" position=\\\"float\\\"/>\"\n]","isConversational":false},"media":{"kind":"list like","value":[],"string":"[]","isConversational":false},"unknown_pub":{"kind":"string","value":"[{\"label\": [\"4\"], \"article-title\": [\"Chapter 18 - Hypogammaglobulinemia and common variable immune deficiency\"], \"source\": [\"Stiehm's Immune Deficiencies (Second Edition)\"], \"person-group\": [\"\\n\"], \"surname\": [\"Cunningham-Rundles\", \"Warnatz\"], \"given-names\": [\"C\", \"K\"], \"fpage\": [\"467\"], \"lpage\": [\"497\"], \"publisher-name\": [\"Elsevier\"], \"year\": [\"2020\"]}]"},"glossary":{"kind":"string","value":"{\n \"acronym\": [],\n \"definition\": []\n}"},"n_references":{"kind":"number","value":13,"string":"13"},"license":{"kind":"string","value":"CC BY"},"retracted":{"kind":"string","value":"no"},"last_updated":{"kind":"string","value":"2024-01-15 23:43:45"},"citation":{"kind":"string","value":"Cureus.; 15(12):e50556"},"package_file":{"kind":"string","value":"oa_package/e1/87/PMC10787943.tar.gz"}}}],"truncated":false,"partial":true},"paginationData":{"pageIndex":1,"numItemsPerPage":100,"numTotalItems":120000,"offset":100,"length":100}},"jwt":"eyJhbGciOiJFZERTQSJ9.eyJyZWFkIjp0cnVlLCJwZXJtaXNzaW9ucyI6eyJyZXBvLmNvbnRlbnQucmVhZCI6dHJ1ZX0sImlhdCI6MTc3NjgwODYwNiwic3ViIjoiL2RhdGFzZXRzL1RvbVRCVC9wbWNfb3Blbl9hY2Nlc3NfeG1sIiwiZXhwIjoxNzc2ODEyMjA2LCJpc3MiOiJodHRwczovL2h1Z2dpbmdmYWNlLmNvIn0.qrcGbXiYdwx7SvqICCjvRaqZYkL_CEd_JYR7_2QjGEhBf4zcgGr4ZJhMuvHZpjfs8BbZCmssK1jySnssvJv0Dg","displayUrls":true,"splitSizeSummaries":[{"config":"commercial","split":"train","numRows":120000,"numBytesParquet":2058504704},{"config":"non_commercial","split":"train","numRows":167000,"numBytesParquet":2229724384},{"config":"other","split":"train","numRows":166000,"numBytesParquet":2181079699}]},"discussionsStats":{"closed":1,"open":1,"total":2},"fullWidth":true,"hasGatedAccess":true,"hasFullAccess":true,"isEmbedded":false,"savedQueries":{"community":[],"user":[]}}">
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PMC10787770 | 38222190 | [
"<title>Introduction</title>",
"<p>Acute scrotal pain is a relatively common emergency presentation, both in the primary care setting and in the emergency department, comprising approximately 0.5% of all emergency visits in the United States annually [##UREF##0##1##]. Testicular torsion is a true urological emerg... | [
"<title>Materials and methods</title>",
"<p>All patients who underwent scrotal exploration for a primary diagnosis of testicular torsion between June 1, 2016, and January 15, 2023, were reviewed. All data were recorded from the hospital’s electronic database. Patients were excluded if they underwent an orchidecto... | [
"<title>Results</title>",
"<p>A total of 109 patient records were reviewed within the specific time frame. Forty-seven patients were excluded as per the exclusion criteria mentioned, which gave us a sample size of 62 patients. The patient and surgical parameters are given in Table ##TAB##0##1##.</p>",
"<p>Our d... | [
"<title>Discussion</title>",
"<p>The management of testicular torsion is immediate surgical intervention with the aim of untwisting the spermatic cord and restoring blood supply to the affected testis as soon as possible [##UREF##3##7##]. Testicular salvageability is directly correlated to the time it takes to un... | [
"<title>Conclusions</title>",
"<p>Our study indicates that earlier surgical intervention and correction of torsion are associated with enhanced preservation of postoperative testicular volume. Both the degree of torsion and time to surgery influence mean volume loss; however, time to surgery shows a greater effec... | [
"<p>Introduction</p>",
"<p>Testicular torsion is an urological emergency. It is a time-sensitive condition in which twisting of the spermatic cord and testicular blood supply occurs, causing acute onset severe scrotal pain. The incidence of testicular torsion is highest amongst prepubertal males; however, it can ... | [] | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Mean postoperative volume loss in ml in affected testis with regard to severity of degree of torsion (blue curve) compared to that of time to surgery severity (orange curve)</title></caption></fig>"
] | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Patient and surgical parameters</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\">Mean + SD</td><td rowspan=\"1\"... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Ahmed A. Al Rashed, Nader Awad, Khalid Abdulaziz, Basma Malalla, Ali H. Al Aradi</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Ahmed A. Al Rashed, Nader Awad, Khali... | [
"<graphic xlink:href=\"cureus-0015-00000050543-i01\" position=\"float\"/>"
] | [] | [{"label": ["1"], "article-title": ["Testicular torsion"], "source": ["StatPearls [Internet]"], "date-in-citation": ["\n"], "month": ["11"], "year": ["2023", "2023"], "person-group": ["\n"], "surname": ["Schick", "Sternard"], "given-names": ["MA", "BT"], "publisher-loc": ["Treasure Island (FL)"], "publisher-name": ["St... | {
"acronym": [],
"definition": []
} | 16 | CC BY | no | 2024-01-15 23:41:58 | Cureus.; 15(12):e50543 | oa_package/d3/73/PMC10787770.tar.gz |
PMC10787771 | 38222220 | [
"<title>Introduction</title>",
"<p>Obesity, which is considered a multifactorial and complex disease that negatively affects health, is one of the most important causes of preventable deaths today. It contributes to the development of many health problems, such as type 2 diabetes mellitus (DM), cardiovascular dis... | [
"<title>Materials and methods</title>",
"<p>Study design and participants</p>",
"<p>This single-center and retrospective study was approved by the Ethics Committee of Bursa City Hospital (approval number 2023-19/2) and was performed per the Declaration of Helsinki. </p>",
"<p>The 67 participants between 18 an... | [
"<title>Results</title>",
"<p>Seventy-one patients were evaluated in the study. Due to the higher cost of LG, 10 participants could not continue the treatment for 16 weeks. Therefore, it was not included in the statistical analysis. Sixty (89.5%) women and seven (10.5%) men with a mean age of 42.8 ± 4.4 years met... | [
"<title>Discussion</title>",
"<p>This study aimed to demonstrate the effectiveness and SE profile of overweight and obese patients with LG treatment evaluated in clinical practice. The findings of this retrospective study showed that mean BW, BMI, FG, FI, HOMA-IR, HbA1c, LDL, and TG levels were significantly redu... | [
"<title>Conclusions</title>",
"<p>In recent years, medical treatments for obesity, in addition to lifelong adequate and balanced nutrition, physical activity, and behavioral therapies, have come forth. This study showed a clinically significant decrease in BW and improved cardiometabolic parameters in four and 16... | [
"<p>Background</p>",
"<p>One of the essential chronic diseases is obesity, which negatively affects the individual and society. Liraglutide (LG) is an effective treatment for both obesity treatment and metabolic control. This study aims to show the effect of a 3.0 mg dose of LG, injected subcutaneously once a day... | [] | [] | [] | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Characteristics of the participants at baseline</title><p>BMI - body mass index; CVD - cardiovascular disease; OSAS - obstructive sleep apnea syndrome; PCOS - polycystic ovary syndrome</p></caption><table frame=\"hsides\" rules=\"grou... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Ayşen Akkurt Kocaeli</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Ayşen Akkurt Kocaeli</p><p><bold>Drafting of the manuscript:</bold> Ayşen Akkurt Kocaeli</p><p><... | [] | [] | [{"label": ["5"], "article-title": ["World Health Organization (WHO) obesity and overweight"], "date-in-citation": ["\n"], "month": ["1"], "year": ["2023", "2020"], "uri": ["http://www.who.int/mediacentre/factsheets/fs311/en/"]}, {"label": ["14"], "article-title": ["Classification and diagnosis of diabetes: standards o... | {
"acronym": [],
"definition": []
} | 27 | CC BY | no | 2024-01-15 23:41:58 | Cureus.; 15(12):e50544 | oa_package/9e/bf/PMC10787771.tar.gz |
PMC10787772 | 38222173 | [
"<title>Introduction</title>",
"<p>One of the prime reasons for patients seeking orthodontic treatment is improvement in their aesthetics or appearance. With a greater number of adult patients now opting for orthodontic treatment, the demand for aesthetic orthodontic materials has increased [##REF##25709410##1##]... | [
"<title>Materials and methods</title>",
"<p>Four brands of wires were included in this study. The wires were Teflon-, epoxy-, or ceramic-coated. Convenience sampling was done, and five samples of each brand were prepared to be tested in each solution. The samples needed to be in a tile form of 10 x 10 mm dimensio... | [
"<title>Results</title>",
"<p>Table ##TAB##1##2## provides the descriptive statistics like mean and standard deviation for color change, i.e., ΔE obtained for four brands of wires dipped in six solutions for two weeks. At two weeks, the highest ΔE value of 26.92 (0.35) was noticed in the U Orthodontics (New Delhi... | [
"<title>Discussion</title>",
"<p>Multiple studies have been conducted on the color stability of various archwires in different staining solutions such as coffee, tea, cola, wine, etc. The consumption of beverages in the Indian context is quite different and has considerably changed after the COVID pandemic. As pe... | [
"<title>Conclusions</title>",
"<p>Our study tested the staining effect of six solutions on four different brands of archwires at two, four, and eight weeks. At the end of all time intervals, none of the archwires resisted a color change irrespective of the brand or coating of archwires. With respect to the soluti... | [
"<p>Introduction</p>",
"<p>One of the prime reasons for patients seeking orthodontic treatment is improvement in their aesthetics or appearance. With a greater number of adult patients now opting for orthodontic treatment, the demand for aesthetic orthodontic materials has increased. With the background of the re... | [] | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Preparation of samples by juxtaposing 10 mm long pieces of archwires</title></caption></fig>",
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Measurement of color chan... | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Critical marks of color change according to National Bureau Standards (NBS)</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">NBS Unit</td><td colspan=... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Sharvari S. Khedkar, Usha Shenoy, Ananya Hazare, Himija Karia, Pritam Khorgade, Nivedita Nandeshwar, Sangeeta Bhattacharya</p><p><bold>Acquisition, analysis, or interpretation of data:</bo... | [
"<graphic xlink:href=\"cureus-0015-00000050542-i01\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050542-i02\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050542-i03\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050542-i04\" position=\"float\"/>",
"<gr... | [] | [{"label": ["6"], "article-title": ["Contemporary Orthodontics"], "source": ["Contemporary orthodontics"], "person-group": ["\n"], "surname": ["Proffit", "Fields", "Sarver", "Ackerman"], "given-names": ["WR", "HW", "DM", "JL"], "fpage": ["2013"], "publisher-loc": ["Amsterdam, Netherlands"], "publisher-name": ["Elsevier... | {
"acronym": [],
"definition": []
} | 18 | CC BY | no | 2024-01-15 23:41:58 | Cureus.; 15(12):e50542 | oa_package/db/e5/PMC10787772.tar.gz |
PMC10787773 | 38218868 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Fluid simulations enable the investigation of blood flow distribution in the cardiovascular system to better understand disease progression, inform surgical procedures and evaluate responses to internal and external conditions affecting the body. These simulations ca... | [
"<title>Methods</title>",
"<title>Imaging methods and 3D reconstruction</title>",
"<p id=\"Par7\">This study was approved by the University of Western Australia (2022/ET000688). We sourced 3D arterial vasculature models from numerous past studies<sup>##REF##31140054##18##–##UREF##5##22##</sup>. We imported the ... | [
"<title>Results</title>",
"<title>Haemodynamic responses to simulated microgravity</title>",
"<p id=\"Par21\">Qualitative distributions of the relative change in TAWSS and OSI between control and simulated microgravity conditions across the continuous arterial geometry as well as detailed views of regions of in... | [
"<title>Discussion</title>",
"<p id=\"Par24\">Since the beginning of spaceflight, there has been interest in understanding the effects of space travel on the human body. While the implications of microgravity on muscle mass and strength have been investigated in both animal models<sup>##REF##33911096##49##</sup> ... | [] | [
"<p id=\"Par1\">We investigated variations in haemodynamics in response to simulated microgravity across a semi-subject-specific three-dimensional (3D) continuous arterial network connecting the heart to the eye using computational fluid dynamics (CFD) simulations. Using this model we simulated pulsatile blood flow... | [
"<title>Supplementary information</title>",
"<p>\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41526-024-00348-w.</p>",
"<title>Acknowledgements</title>",
"<p>This work was supported by resources provided by the Pawsey Supercomputing Centre with funding from the Australian Government... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Continuous arterial network model.</title><p>Examples of types of corresponding image data are provided alongside detailed views of the 3D geometry. This model was comprised of four 3D reconstructions associated with separate image datasets, consisting of the r... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Assumed flow distribution to different arterial networks expressed as a percentage of cardiac output.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Artery region (<italic>N</italic>)</th><th>Cardiac output</th><th>Refs.</th></tr></the... | [
"<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepack... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>The number of vessels associated with each arterial network is provided in brackets (<italic>N</italic>). Paired arteries are assumed to have symmetrical flow distribution to each side. Flow to the subclavian arteries was assumed to be the residual of cardiac output.</p></table-wrap-foot>",
"... | [
"<graphic xlink:href=\"41526_2024_348_Fig1_HTML\" id=\"d32e340\"/>",
"<graphic xlink:href=\"41526_2024_348_Article_Equ1.gif\" position=\"anchor\"/>",
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"<media xlink:href=\"41526_2024_348_MOESM1_ESM.pdf\"><caption><p>Supplementary Material</p></caption></media>"
] | [{"label": ["1."], "surname": ["Norsk"], "given-names": ["P"], "article-title": ["Adaptation of the cardiovascular system to weightlessness: surprises, paradoxes and implications for deep space missions"], "source": ["Acta Physiol."], "year": ["2020"], "volume": ["228"], "fpage": ["e13434"], "pub-id": ["10.1111/apha.13... | {
"acronym": [],
"definition": []
} | 102 | CC BY | no | 2024-01-15 23:41:58 | NPJ Microgravity. 2024 Jan 13; 10:7 | oa_package/12/4a/PMC10787773.tar.gz |
PMC10787774 | 38218995 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Millimeter wave (mmWave) has attracted huge interest in wireless technologies, in particular for the fifth generation of mobile communication (5G) applications in recent years. The demand for massive data rates and low latency in 5G systems can be addressed with the ... | [] | [
"<title>Fabrication and measurement results</title>",
"<p id=\"Par95\">The designed Rotman lens beamformer including 8 beam ports and integrated 8 Vivaldi antennas is fabricated and measured as shown in Fig. ##FIG##10##11## to validate the design and simulation results.</p>",
"<p id=\"Par96\">The SMPM cables ar... | [] | [
"<title>Conclusion</title>",
"<p id=\"Par109\">Beamforming is an important part of mmWave technologies to improve the link budget and spectral efficiency and abilities of MIMO and SDMA. The beamforming requirements for 5G mmWave applications indicate the wide-angular coverage of ± 60° and SLL > 10 dB. A wide-a... | [
"<p id=\"Par1\">The attachment of 5G with millimeter wave (mmWave) frequencies offers massive capacity and low latency to reveal the full 5G experiences. High directive gain and beamforming are considered essential for mmWave 5G systems. The main requirements of the beamforming network for 5G mmWave applications ar... | [
"<title>Rotman lens beamformer design</title>",
"<p id=\"Par28\">The Rotman lens is a wide-angle lens that can be utilized as a wideband beamformer. The schematic of a conventional Rotman lens is shown in Fig. ##FIG##0##1##, which consists of a parallel-plate contour surrounded by <italic>M</italic>-beam ports an... | [
"<title>Author contributions</title>",
"<p>A.A.; Formal analysis, Resources, Writing-original draft, Writing-review & editing, A.S.; Supervision, H.A.; Supervision.</p>",
"<title>Data availability</title>",
"<p>All the data required to evaluate the findings of this work is available in the manuscript. Any... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Schematic of a conventional Rotman-lens parameters.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Limiting values of versus for some values.</p></caption></fig>",
"<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Fabricated ... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Main requirements of AAS<sup>##UREF##0##1##</sup>.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\">Operating frequency range</td><td align=\"left\"><p>26.5–29.5 GHz</p><p>24.25–27.5 GHz</p><p>27.5–28.35 GHz</p></td></tr><... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41598_2024_51733_Fig1_HTML\" id=\"MO1\"/>",
"<inline-graphic xlink:href=\"41598_2024_51733_Article_IEq1.gif\"/>",
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"<inline-graphic xlink:href=\"41598_2024_51733_Article_IEq3.gif\"/>",
"<inline-graphic xlink:href=\"4... | [] | [{"label": ["1."], "surname": ["Azari", "Aliakbarian"], "given-names": ["A", "H"], "article-title": ["Requirements for 5G applications: A strategic approach"], "source": ["Microw. J."], "year": ["2022"], "volume": ["65"], "fpage": ["1"]}, {"label": ["2."], "surname": ["Asplund", "Astely", "Butovitsch", "Chapman", "Fren... | {
"acronym": [],
"definition": []
} | 29 | CC BY | no | 2024-01-15 23:41:58 | Sci Rep. 2024 Jan 13; 14:1245 | oa_package/08/dc/PMC10787774.tar.gz |
PMC10787775 | 38218748 | [
"<title>Introduction</title>",
"<p id=\"Par12\">The cilium, a highly conserved organelle, extends from the cell surface and serves a variety of functions. Structurally, it consists of the ciliary membrane, axoneme, and basal body [##REF##30601682##1##, ##REF##32989303##2##]. As an essential organelle, the cilium ... | [] | [] | [] | [] | [
"<p id=\"Par1\">Cilia are highly conserved eukaryotic organelles that protrude from the cell surface and are involved in sensory perception, motility, and signaling. Their proper assembly and function rely on the bidirectional intraflagellar transport (IFT) system, which involves motor proteins, including antegrade... | [
"<title>Facts</title>",
"<p id=\"Par2\">\n<list list-type=\"bullet\"><list-item><p id=\"Par3\">Cilium assembly involves a specialized protein transport mechanism known as intraflagellar transport (IFT), which is characterized by the bidirectional trafficking of a large protein complex along the microtubules withi... | [
"<title>Author contributions</title>",
"<p>JR and LL drafted the original manuscript and prepared the figures, JR revised the manuscript. All authors have read and agreed the final version of the manuscript.</p>",
"<title>Funding</title>",
"<p id=\"Par50\">This work was supported by grants from the National N... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Structures of motile and primary cilia.</title><p>Cilia typically have three main components: the ciliary membrane, the axoneme, and the basal body. The ciliary membrane contains numerous signaling receptors, endowing cilia with signaling functions. Axonemes ar... | [] | [] | [] | [] | [] | [] | [] | [
"<fn-group><fn><p>Edited by Professor Anastasis Stephanou</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41419_2024_6428_Fig1_HTML\" id=\"d32e287\"/>",
"<graphic xlink:href=\"41419_2024_6428_Fig2_HTML\" id=\"d32e377\"/>",
"<graphic xlink:href=\"41419_2024_6428_Fig3_HTML\" id=\"d32e423\"/>",
"<graphic xlink:href=\"41419_2024_6428_Fig4_HTML\" id=\"d32e508\"/>"
] | [] | [{"label": ["11."], "surname": ["Ran", "Zhou"], "given-names": ["J", "J"], "article-title": ["Targeting the photoreceptor cilium for the treatment of retinal diseases"], "source": ["Acta Pharm Sin"], "year": ["2020"], "volume": ["41"], "fpage": ["1410"], "lpage": ["5"], "pub-id": ["10.1038/s41401-020-0486-3"]}, {"label... | {
"acronym": [],
"definition": []
} | 99 | CC BY | no | 2024-01-15 23:41:58 | Cell Death Dis. 2024 Jan 13; 15(1):47 | oa_package/ab/2b/PMC10787775.tar.gz |
PMC10787776 | 38218731 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Understanding the changing characteristics of floods<sup>##UREF##0##1##</sup> and their relationship with the physical causative mechanisms is a prerequisite for developing effective flood management strategies<sup>##UREF##1##2##,##UREF##2##3##</sup>. Physical causes... | [
"<title>Methods</title>",
"<title>Datasets</title>",
"<p id=\"Par17\">Daily streamflow time series for 70 catchments in six major river basins of Peninsular India (Fig. ##FIG##0##1##a) are obtained from India Water Resources Information System (<ext-link ext-link-type=\"uri\" xlink:href=\"https://indiawris.gov.... | [
"<title>Results</title>",
"<title>Trend analysis</title>",
"<p id=\"Par8\">Annual flood magnitudes have decreased in Peninsular catchments over the period 1979–2018 (Fig. ##FIG##1##2##a). An increase in flood magnitudes is observed only in two catchments (Kurubhata and Kantamal) of Mahanadi river basin and one ... | [] | [
"<title>Conclusions</title>",
"<p id=\"Par14\">The active role of groundwater in storm runoff in streams is discovered decades ago<sup>##UREF##29##38##,##UREF##30##39##</sup>. Baseflow also exerts significant influence over the entire flood frequency curves<sup>##UREF##31##40##</sup>. Despite the significant role... | [
"<p id=\"Par1\">Extreme rainfall prior to a flood event is often a necessary condition for its occurrence; however, rainfall alone is not always an indicator of flood severity. Antecedent wetness condition of a catchment is another important factor which strongly influences the flood magnitudes. The key role of soi... | [
"<title>Study area</title>",
"<p id=\"Par7\">Narmada, Tapi, Mahanadi, Godavari, Krishna and Cauvery are six major river basins of Peninsular India. Tapi is the smallest river basin with an area of 65,145 km<sup>2</sup> and Godavari is the largest which covers an area of 312,812 km<sup>2</sup>. Narmada and Tapi ar... | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51850-w.</p>",
"<title>Acknowledgements</title>",
"<p>Funding received from the Ministry of Earth Sciences (MoES), Government of India, through the project “Advanced Research in H... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>(<bold>a</bold>) Locations of streamflow gauges with catchment boundaries in six major river basins of Peninsular India, (<bold>b</bold>) elevation map and mean annual maximum runoff rate (streamflow per unit catchment area), (<bold>c</bold>) Aridity Index (AI) a... | [] | [
"<inline-formula id=\"IEq3\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41598_2024_51850_Fig1_HTML\" id=\"MO1\"/>",
"<inline-graphic xlink:href=\"41598_2024_51850_Article_IEq3.gif\"/>",
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"<inline-graphic xlink:href=\"4... | [
"<media xlink:href=\"41598_2024_51850_MOESM1_ESM.pdf\"><caption><p>Supplementary Information.</p></caption></media>"
] | [{"label": ["1."], "surname": ["Winsemius"], "given-names": ["HC"], "article-title": ["Global drivers of future river flood risk"], "source": ["Nat. Clim. Change"], "year": ["2016"], "volume": ["6"], "fpage": ["381"], "lpage": ["385"], "pub-id": ["10.1038/nclimate2893"]}, {"label": ["2."], "surname": ["Berghuijs", "Woo... | {
"acronym": [],
"definition": []
} | 69 | CC BY | no | 2024-01-15 23:41:58 | Sci Rep. 2024 Jan 13; 14:1251 | oa_package/79/5e/PMC10787776.tar.gz |
PMC10787777 | 38218922 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Cancer cells face heightened proteotoxic stress compared to their normal counterparts due to various factors, including the production of mutated proteins, the upregulation of multiprotein complex components induced by aneuploidy [##REF##26144554##1##], and increased... | [
"<title>Materials and methods</title>",
"<title>Chemicals and antibodies</title>",
"<p id=\"Par25\">Chemicals were purchased from various sources: eeyarestatin-1 (Eer1), LY294002, PD98059, U0126, SP600125, and SB203580 from Calbiochem (EDM Millipore Corp., Billerica, MA, USA); CB-5083 from Biovision (Milpitas, ... | [
"<title>Results</title>",
"<title>VCP is a molecular target of paraptosis</title>",
"<p id=\"Par6\">While various natural products and chemicals have been shown to induce paraptosis [##REF##26802901##19##, ##REF##31306970##20##], the molecular mechanisms underlying this process remain unclear. To identify poten... | [
"<title>Discussion</title>",
"<p id=\"Par17\">Identifying cancer-selective targets and understanding their underlying mechanisms are pivotal in developing effective cancer therapies. Among these potential targets, VCP has emerged as both a prognostic biomarker and a prospective therapeutic target in cancer [##REF... | [] | [
"<p id=\"Par1\">Valosin-containing protein (VCP)/p97, an AAA+ ATPase critical for maintaining proteostasis, emerges as a promising target for cancer therapy. This study reveals that targeting VCP selectively eliminates breast cancer cells while sparing non-transformed cells by inducing paraptosis, a non-apoptotic c... | [
"<title>Supplementary information</title>",
"<p>\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41419-024-06434-x.</p>",
"<title>Author contributions</title>",
"<p>DML, IYK, HJL, MJS, HIL, MYC, JHJ, YHC, SSP, and MY performed experiments and analyse the data. GY, SYJ, EKC, and COY pro... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>VCP impairment induces paraptotic cell death in MDA-MB 435 S cells.</title><p><bold>a</bold>, <bold>b</bold> MDA-MB 435 S cells were transfected with either a negative control siRNA (siNC) or a VCP-targeting siRNA (siVCP) and incubated with fresh medium for 48 ... | [] | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>"
] | [
"<fn-group><fn><p>Edited by Professor Boris Zhivotovsky</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Dong Min Lee, In Young Kim.</p></fn></fn-group... | [
"<graphic xlink:href=\"41419_2024_6434_Fig1_HTML\" id=\"d32e641\"/>",
"<graphic xlink:href=\"41419_2024_6434_Fig2_HTML\" id=\"d32e767\"/>",
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"<graphic xlink:href=\"41419_2024_6434_Fig4_HTML\" id=\"d32e1058\"/>",
"<graphic xlink:href=\"4141... | [
"<media xlink:href=\"41419_2024_6434_MOESM1_ESM.docx\"><caption><p>Supplementary Information</p></caption></media>",
"<media xlink:href=\"41419_2024_6434_MOESM2_ESM.tif\"><caption><p>Uncropped Western Blot</p></caption></media>"
] | [{"label": ["2."], "mixed-citation": ["Guang MHZ, Kavanagh EL, Dunne LP, Dowling P, Zhang L, Lindsay S, et al. Targeting proteotoxic stress in cancer: a review of the role that protein quality control pathways play in oncogenesis. Cancers (Basel). 2019;11."]}, {"label": ["3."], "mixed-citation": ["Brancolini C, Iuliano... | {
"acronym": [],
"definition": []
} | 76 | CC BY | no | 2024-01-15 23:41:59 | Cell Death Dis. 2024 Jan 13; 15(1):48 | oa_package/9b/cc/PMC10787777.tar.gz |
PMC10787778 | 38218979 | [
"<title>Introduction</title>",
"<p id=\"Par7\">Despite an increased understanding of the physiological processes involved in tumor metastasis, there are limited therapies that have proven clinical efficacy in advanced metastatic cancers such as glioblastoma, ovarian, prostate, pancreatic, and triple-negative brea... | [
"<title>Methods</title>",
"<title>Clinical study design</title>",
"<p id=\"Par10\">NCT03364400 was a phase 1, first-in-human, multicenter, open-label, dose escalation, and expansion study of VT1021 designed and sponsored by Vigeo Therapeutics, Inc. Data for the dose escalation phase are reported here. The data ... | [
"<title>Results</title>",
"<p id=\"Par24\">The RP2D for VT1021 was determined to be 11.8 mg/kg, twice weekly dosing. The consideration was based on the combination of assessment of safety, tolerability, as well as PK exposure across all dose levels. The individual assessments of these parameters are described in ... | [
"<title>Discussion</title>",
"<p id=\"Par38\">We report here the first-in-human experience of VT1021 in patients with advanced solid tumors who were refractory to multiple lines and various classes of systemic therapies. The study rigorously assessed the safety, PK/PD, and clinical activities of this first-in-cla... | [] | [
"<title>Background</title>",
"<p id=\"Par1\">VT1021 is a cyclic peptide that induces the expression of thrombospondin-1 (TSP-1) in myeloid-derived suppressor cells (MDSCs) recruited to the tumor microenvironment (TME). TSP-1 reprograms the TME via binding to CD36 and CD47 to induce tumor and endothelial cell apop... | [
"<title>Supplementary information</title>",
"<p>\n\n\n\n\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s43856-024-00433-x.</p>",
"<title>Author contributions</title>",
"<p>Patient enrollment and study monitoring: D.M., W.H., A.P., A.B. Development of methodology for biomarker studies: S.W., M... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Study schema and participants of the phase 1 clinical trial of VT1021.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Concentration-time profiles for VT1021, by dose cohort.</title><p><bold>a</bold> Cycle 1 day 1. <bold>b</bold> Cycl... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Baseline patient demographics and characteristics, by treatment group.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>0.5 mg/kg <italic>n</italic> = 3</th><th>1.0 mg/kg <italic>n</italic> = 6</th><th>2.0 mg/kg <italic>n</italic> =... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>",
"<supplementary-material ... | [
"<table-wrap-foot><p><italic>ECOG PS</italic> Eastern Cooperative Oncology Group performance status.</p></table-wrap-foot>",
"<table-wrap-foot><p>Treatment-Emergent Adverse Events is defined as any event that occurs on or after the first dose of study drug administration or any pre-existing event which worsened i... | [
"<graphic xlink:href=\"43856_2024_433_Fig1_HTML\" id=\"d32e355\"/>",
"<graphic xlink:href=\"43856_2024_433_Fig2_HTML\" id=\"d32e382\"/>",
"<graphic xlink:href=\"43856_2024_433_Fig3_HTML\" id=\"d32e417\"/>",
"<graphic xlink:href=\"43856_2024_433_Fig4_HTML\" id=\"d32e431\"/>",
"<graphic xlink:href=\"43856_202... | [
"<media xlink:href=\"43856_2024_433_MOESM1_ESM.docx\"><caption><p>Description of Additional Supplementary Files</p></caption></media>",
"<media xlink:href=\"43856_2024_433_MOESM2_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media>",
"<media xlink:href=\"43856_2024_433_MOESM3_ESM.xlsx\"><captio... | [{"label": ["1."], "surname": ["Chen"], "given-names": ["F"], "article-title": ["New horizons in tumor microenvironment biology: challenges and opportunities"], "source": ["BMC Med."], "year": ["2015"], "volume": ["5"], "fpage": ["45"], "pub-id": ["10.1186/s12916-015-0278-7"]}, {"label": ["14."], "mixed-citation": ["Sc... | {
"acronym": [],
"definition": []
} | 20 | CC BY | no | 2024-01-15 23:41:59 | Commun Med (Lond). 2024 Jan 13; 4:10 | oa_package/9b/02/PMC10787778.tar.gz |
PMC10787779 | 38218994 | [
"<title>Introduction</title>",
"<p id=\"Par2\">In humans, the superfamily of Cytochrome P450 (CYP) enzymes comprises 18 families of heme-containing proteins that belong to the group of oxidoreductases. Enzymes of only three CYP families are involved in hepatic drug metabolism. The name CYP450 derives from its cha... | [
"<title>Methods</title>",
"<title>Template generation</title>",
"<p id=\"Par8\">Templates for the synthesis of CYPs in cell-free systems were generated by Biocat GmbH. The protein encoding sequence and further regulatory factors for CAP-independent protein synthesis by using a Cricket paralysis virus-IRES<sup>#... | [
"<title>Results</title>",
"<p id=\"Par20\">CYP3A4 is known to be involved in the metabolism of most approved medications. Consequently, it was selected as a model protein for initiating cell-free synthesis of cytochrome P450s in eukaryotic lysates.</p>",
"<title>Generation of a modified CHO-CPR Lysate</title>",... | [
"<title>Discussion</title>",
"<p id=\"Par34\">Recombinant expression of membrane proteins has been challenging for many years<sup>##REF##29702227##36##,##UREF##3##37##</sup>. More than half of all pharmacologically relevant proteins are membrane-bound<sup>##REF##29075003##38##</sup>. Therefore, an outstanding int... | [
"<title>Conclusion</title>",
"<p id=\"Par41\">The high demand of active CYPs requires a straightforward method for the synthesis of members of this enzyme superfamily. Cell-free protein synthesis enables the synthesis of specific active CYPs using a timesaving procedure. By creating a vesicle containing protein p... | [
"<p id=\"Par1\">Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1... | [
"<title>Aim of the work</title>",
"<p id=\"Par7\">Cytochrome P450 enzymes are one of the best-studied classes of enzymes, but recombinant production is challenging due to their membrane localization and enzymatic coupling. The use of vesicle-based cell-free protein synthesis, which enables the fast and efficient ... | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51781-6.</p>",
"<title>Acknowledgements</title>",
"<p>For the lysate preparation, the authors would like to thank D. Wenzel (Fraunhofer IZI-BB, Potsdam-Golm, Germany).</p>",
"<t... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Validation of the CPR activity in the generated lysates by Cytochrome P450 reductase activity assay kit (colorimetric) (ab204704). The assay was performed according to the manufactures protocol. Diphenyleneiodonium chloride was used as inhibitor control. (<bold>A... | [] | [
"<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepack... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41598_2024_51781_Article_Equ1.gif\" position=\"anchor\"/>",
"<graphic xlink:href=\"41598_2024_51781_Article_Equ2.gif\" position=\"anchor\"/>",
"<graphic xlink:href=\"41598_2024_51781_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51781_Fig2_HTML\" id=\"MO2\"/>",
"<graphi... | [
"<media xlink:href=\"41598_2024_51781_MOESM1_ESM.pdf\"><caption><p>Supplementary Figures.</p></caption></media>"
] | [{"label": ["4."], "surname": ["Ogu", "Maxa"], "given-names": ["CC", "JL"], "article-title": ["Drug interactions due to cytochrome P450"], "source": ["Proceedings"], "year": ["2000"], "volume": ["13"], "issue": ["4"], "fpage": ["421"], "lpage": ["3"]}, {"label": ["15."], "surname": ["Walsh", "Miwa"], "given-names": ["J... | {
"acronym": [],
"definition": []
} | 65 | CC BY | no | 2024-01-15 23:41:59 | Sci Rep. 2024 Jan 13; 14:1271 | oa_package/81/35/PMC10787779.tar.gz |
PMC10787780 | 38218972 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Dye-contaminated wastewater affects to be toxic to aquatic organisms because it has an aromatic structure difficult to degrade, and the colored particles may block the transmission of light into the water body. As a result, aquatic plants and algae are unable to phot... | [
"<title>Material and method</title>",
"<title>Raw material and preparation</title>",
"<p id=\"Par6\">Sugarcane bagasse was taken from the local market in Khon Kaen province, Thailand. Before use, it was washed with tap water to remove contaminations, and then it was dried in a hot air oven (Binder, FED 53, Germ... | [
"<title>Result and discussion</title>",
"<title>BET</title>",
"<p id=\"Par29\">The specific surface area, pore volumes, and pore sizes of SBB, SBBT, SBBM, SBBA, and SBBZ are illustrated in Table ##TAB##1##2##. Their specific surface area and pore volume could be arranged from high to low of SBBM > SBBT > ... | [
"<title>Result and discussion</title>",
"<title>BET</title>",
"<p id=\"Par29\">The specific surface area, pore volumes, and pore sizes of SBB, SBBT, SBBM, SBBA, and SBBZ are illustrated in Table ##TAB##1##2##. Their specific surface area and pore volume could be arranged from high to low of SBBM > SBBT > ... | [
"<title>Conclusion</title>",
"<p id=\"Par48\">Five adsorbent materials of sugarcane bagasse beads (SBB), sugarcane bagasse modified with titanium dioxide beads (SBBT), sugarcane bagasse modified with magnesium oxide beads (SBBM), sugarcane bagasse modified with aluminum oxide beads (SBBA), and sugarcane bagasse m... | [
"<p id=\"Par1\">The direct red 28 (DR28) dye contamination in wastewater blocks the transmission of light into the water body resulting in the inability to photosynthesize by aquatic life. In addition, it is difficult to break down and persist in the environment, and it is also harmful to aquatic life and water qua... | [
"<title>The possible mechanisms for DR28 dye adsorptions</title>",
"<p id=\"Par47\">The possible mechanisms for DR28 dye adsorptions of SBB, SBBT, SBBM, SBBA, and SBBZ are demonstrated in Fig. ##FIG##8##9## which modified the idea from the study of Ngamsurach et al.<sup>##REF##36211078##8##</sup> and Praipipat et... | [
"<title>Author contributions</title>",
"<p>P.P.: Supervision, Project administration, Conceptualization, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing—Original Draft, Writing-Review and Editing. P.N.: Visualization, Writing—Original Draft. N.L.: Investigation. C.K.: Investiga... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>The synthesis of SBB, SBBT, SBBM, SBBA, and SBBZ.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>The surface morphologies and the chemical distributions by EDX mapping of (<bold>a</bold>, <bold>f</bold>) SBB, (<bold>b</bold>, <bold>g... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>The agricultural wastes with or without modifications for removing various dyes<bold>.</bold></p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Materials</th><th align=\"left\">Dyes</th><th align=\"left\">Dose (g)</th><th a... | [
"<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepack... | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>*BET specific surface area.</p><p>**Barrett–Joyner–Halenda (BJH) method.</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41598_2024_51934_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51934_Article_Equ1.gif\" position=\"anchor\"/>",
"<graphic xlink:href=\"41598_2024_51934_Article_Equ2.gif\" position=\"anchor\"/>",
"<graphic xlink:href=\"41598_2024_51934_Article_Equ3.gif\" position=\"anchor\... | [] | [{"label": ["2."], "mixed-citation": ["Sekar, N. "], "italic": ["Direct Dyes", "Handbook of Textile and Industrial Dyeing: Principles, Processes and Types of Dyes"]}, {"label": ["3."], "surname": ["Katheresan", "Kansedo", "Lau"], "given-names": ["V", "J", "SY"], "article-title": ["Efficiency of various recent wastewate... | {
"acronym": [],
"definition": []
} | 51 | CC BY | no | 2024-01-15 23:41:59 | Sci Rep. 2024 Jan 13; 14:1278 | oa_package/f5/a4/PMC10787780.tar.gz |
PMC10787781 | 38218964 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Nearly half of China’s coal resource reserves and output are attributed to thick coal seams<sup>##UREF##0##1##–##UREF##2##3##</sup>. The fully mechanized longwall top coal caving (LTCC) mining technology is one of the main technologies used for mining thick seams<sup... | [
"<title>Methods</title>",
"<title>Distribution characteristics of natural radionuclides in thick coal seams</title>",
"<title>Natural radionuclide</title>",
"<p id=\"Par6\">Natural radionuclides are formed during interstellar processes, such as the Big Bang, and continue to exist. They were transported to Ear... | [
"<title>Result</title>",
"<title>Application of coal gangue identification system in simple structure thick coal seam fully mechanized caving face</title>",
"<p id=\"Par51\">Working face 1303 (coal seam 3) of Lilou Coal Mine is located in the middle and lower part of Shanxi Formation. Most of the coal seams (co... | [
"<title>Discussion</title>",
"<p id=\"Par68\">LTCC mining technology is one of the main technologies for safe and efficient mining of extra-thick coal seams in China. At present, LTCC mining technology has been applied in most of the extra-thick coal seams in China, and has made breakthroughs and development in t... | [
"<title>Conclusions</title>",
"<p id=\"Par71\">\n<list list-type=\"order\"><list-item><p id=\"Par72\">Among the common gangue and roof rocks, the radioactive intensity of carbonaceous mudstone is the highest, followed by sandy mudstone and kaolin mudstone, while the radioactive intensity of lamprophyre is relativ... | [
"<p id=\"Par1\">To address the technical limitations of automatic coal and gangue detection technology in fully mechanized top coal caving mining operations, the low radiation level radioactivity measurement method is utilized to assess the degree of coal-gangue mixture in top coal caving process. This approach is ... | [] | [
"<title>Acknowledgements</title>",
"<p>We would like to thank all the authors of this article for their work and the relevant leaders and technicians of Tashan, Xiaoyu and Lilou Coal Mines for their support and help.</p>",
"<title>Author contributions</title>",
"<p>C.L. put forward the research ideas and meth... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Comparison chart of sedimentary radioactive content.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Immediate roof lithologic distribution of full-mechanized caving mining face.</p></caption></fig>",
"<fig id=\"Fig3\"><label>Figure... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>The characteristics of three kinds of natural radioactive substances.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Nuclide</th><th align=\"left\">Number of nuclide decays/g s</th><th align=\"left\">Number of photons pr... | [] | [] | [] | [] | [] | [] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41598_2024_51424_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51424_Fig2_HTML\" id=\"MO2\"/>",
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"<graphic xlink:href=\"41598_2024_51424_Fig4_HTML\" id=\"MO4\"/>",
"<graphic xlink:href=\"41598_2024_51424_... | [] | [{"label": ["1."], "surname": ["Chi", "Zhang", "Fan", "Zhang", "Liu"], "given-names": ["MB", "DS", "GW", "W", "HL"], "article-title": ["Prediction of top-coal caving and drawing characteristics by the analytic hierarchy process-fuzzy discrimination method in extra-thick coal seams"], "source": ["J. Intell. Fuzzy Syst."... | {
"acronym": [],
"definition": []
} | 37 | CC BY | no | 2024-01-15 23:41:59 | Sci Rep. 2024 Jan 13; 14:1276 | oa_package/25/aa/PMC10787781.tar.gz |
PMC10787782 | 38218912 | [
"<title>Introduction</title>",
"<p id=\"Par2\">For many years, laser surgery has been an accepted tool in various surgical fields<sup>##REF##21500720##1##</sup>. The use of lasers in hospitals is also increasing<sup>##UREF##0##2##</sup>. The advantage of lasers is that they can achieve results similar to those of... | [
"<title>Methods</title>",
"<p id=\"Par10\">The method section consists of four sections. In the first two sections, experimental procedure including the origin of the tissue and the statistical analysis is explained. In the following two sections, the varied parameters are discussed in detail.</p>",
"<title>Exp... | [
"<title>Results</title>",
"<p id=\"Par23\">Each section of the results is divided into two parts. The first part presents the means and compares the significance of the data sets. The second part presents the results of the ANOVA analysis.</p>",
"<title>Influence non-laser parameters</title>",
"<title>Experim... | [] | [
"<title>Conclusion</title>",
"<p id=\"Par43\">In this study, the parameters of pulse duration, laser power, laser scan speed, line energy, air cooling, exhaust system and number of breaks between cuts were investigated. Among these parameters, line energy is the most important. It determines the maximum cutting q... | [
"<p id=\"Par1\">In recent years, the laser has become an important tool in hospitals. Laser surgery in particular has many advantages. However, there is still a lack of the understanding of the influence of the relevant parameters for laser surgery. In order to fill this gap, the parameters pulse frequency, use of ... | [
"<title>Supplementary Information</title>",
"<p>\n</p>"
] | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51449-1.</p>",
"<title>Acknowledgements</title>",
"<p>The authors gratefully acknowledge funding of the Erlangen Graduate School in Advanced Optical Technologies (SAOT) by the Bav... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Setup with tissue, the compressed air supply, the exhaustion system and the laser source.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Exemplary scoring for the five different cuts (taken from our previous publication<sup>##REF##36... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Constant parameters in all experiments.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Constant parameter</th><th align=\"left\">Value</th></tr></thead><tbody><tr><td align=\"left\">Laser wavelength</td><td align=\"left\... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>For experiment B, 540 cuts are evaluated. From these, 180 cuts are the same as in experiment A.</p></table-wrap-foot>",
"<table-wrap-foot><p>Three line energies are taken and for each line energy three scan speeds and corresponding laser powers are selected.</p></table-wrap-foot>",
"<fn-gro... | [
"<inline-graphic xlink:href=\"41598_2024_51449_Article_IEq1.gif\"/>",
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"<inline-graphic xlink:href=... | [
"<media xlink:href=\"41598_2024_51449_MOESM1_ESM.zip\"><caption><p>Supplementary Information.</p></caption></media>"
] | [{"label": ["2."], "surname": ["Sp\u00e4th", "Kl\u00e4mpfl", "Stelzle", "Hohmann", "Lengenfelder", "Schmidt"], "given-names": ["M", "F", "F", "M", "B", "M"], "article-title": ["A quantitative evaluation of the use of medical lasers in German hospitals"], "source": ["J. Biophoton."], "year": ["2019"], "volume": ["20"], ... | {
"acronym": [],
"definition": []
} | 37 | CC BY | no | 2024-01-15 23:41:59 | Sci Rep. 2024 Jan 13; 14:1263 | oa_package/d5/11/PMC10787782.tar.gz |
PMC10787783 | 38218953 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Pilot-wave theory (also called de Broglie-Bohm theory or Bohmian mechanics) is a realist, nonlocal formulation of quantum mechanics originally presented in the 1927 Solvay conference by de Brogile<sup>##UREF##0##1##,##UREF##1##2##</sup>. In 1952, Bohm showed how the ... | [] | [] | [
"<title>Discussion</title>",
"<p id=\"Par54\">We have discussed some of the implications of our work in the previous section. However, the list of implications is necessarily inexhaustive as the normalizability constraint is ubiquitous in orthodox quantum mechanics. It would, for example, be interesting to study ... | [] | [
"<p id=\"Par1\">Non-normalizable states are difficult to interpret in the orthodox quantum formalism but often occur as solutions to physical constraints in quantum gravity. We argue that pilot-wave theory gives a straightforward physical interpretation of non-normalizable quantum states, as the theory requires onl... | [
"<title>Non-normalizable solutions of the harmonic oscillator</title>",
"<p id=\"Par6\">We start by noting that several elementary theorems in orthodox quantum mechanics are no longer applicable once the normalizability constraint on quantum state is dropped. In the non-normalizable scenario, eigenstates in one d... | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-023-50814-w.</p>",
"<title>Acknowledgements</title>",
"<p>I am thankful to Matt Leifer for encouragement and several helpful discussions. I am also thankful to Siddhant Das and Tathag... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Schematic illustration of (<bold>a</bold>) and (<bold>b</bold>) <italic>v</italic>(<italic>y</italic>) for the sample non-normalizable eigenstate . Note that at large .</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Schematic illus... | [] | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<inline-graphic xlink:href=\"41598_2023_50814_Article_IEq1.gif\"/>",
"<inline-graphic xlink:href=\"41598_2023_50814_Article_IEq2.gif\"/>",
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"<inline-graphic xlink:href=... | [
"<media xlink:href=\"41598_2023_50814_MOESM1_ESM.pdf\"><caption><p>Supplementary Information 1.</p></caption></media>"
] | [{"label": ["1."], "surname": ["Bell"], "given-names": ["JS"], "source": ["Speakable and Unspeakable in Quantum Mechanics: Collected Papers on Quantum Philosophy"], "year": ["2004"], "publisher-loc": ["Press"], "publisher-name": ["Cambridge Univ"]}, {"label": ["2."], "surname": ["Bacciagaluppi", "Valentini"], "given-na... | {
"acronym": [],
"definition": []
} | 40 | CC BY | no | 2024-01-15 23:41:59 | Sci Rep. 2024 Jan 13; 14:669 | oa_package/dc/54/PMC10787783.tar.gz |
PMC10787784 | 38218738 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Urinary Tract Infections (UTIs) are one of the most common bacterial infections in older adults, constituting around 25% of all infections<sup>##REF##12848468##1##–##UREF##1##5##</sup>. Clinical presentation ranges from self-limited illness to severe sepsis. UTIs acc... | [
"<title>Methods</title>",
"<title>Study design and population</title>",
"<p id=\"Par18\">This study was performed in collaboration with Imperial College London and Surrey and Borders Partnership NHS Trust. Participants were recruited from the following: (1) health and social care partners within the primary car... | [
"<title>Results</title>",
"<title>Model performance</title>",
"<p id=\"Par6\">We examined Logistic Regression (LR), Extreme Gradient Boosting Decision Trees (XGBoost)<sup>##UREF##15##27##</sup>, Multilayer Perceptron (MLP), Self-Attention<sup>##UREF##16##28##</sup>, Random Forest (RF)<sup>##UREF##17##29##</sup>... | [
"<title>Discussion</title>",
"<p id=\"Par11\">The 2020 report of the Lancet Commission on dementia prevention, treatment, and care emphasises the significance of individualised interventions to address complex medical problems in dementia, which result in unnecessary hospital admissions, accelerated functional de... | [] | [
"<p id=\"Par1\">Urinary Tract Infections (UTIs) are one of the most prevalent bacterial infections in older adults and a significant contributor to unplanned hospital admissions in People Living with Dementia (PLWD), with early detection being crucial due to the predicament of reporting symptoms and limited help-se... | [
"<title>Supplementary information</title>",
"<p>\n\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41746-023-00995-5.</p>",
"<title>Acknowledgements</title>",
"<p>This study is funded by the UK Dementia Research Institute (UKDRI) Care Research and Technology Centre funded by the Medical ... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>The dataset.</title><p><bold>a</bold> All PIR activations measured and the corresponding time of day. <bold>b</bold> The average proportions of PIR activations in a time period for days containing a verified positive and negative UTI label for a selected partic... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Mean (95% CI) % of sensitivity, specificity, and area under the precision-recall curve of the UTI prediction model on the different data splits with 10 bootstrap repeats.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"5\">Bef... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>"
] | [
"<table-wrap-foot><p>These results are reported before and after risk stratification is performed.</p></table-wrap-foot>",
"<table-wrap-foot><p>Some participants requested not to share their information outside the study and correspond to the Not Available information.</p></table-wrap-foot>",
"<fn-group><fn><p>... | [
"<graphic xlink:href=\"41746_2023_995_Fig1_HTML\" id=\"d32e649\"/>",
"<inline-graphic xlink:href=\"41746_2023_995_Article_IEq1.gif\"/>",
"<inline-graphic xlink:href=\"41746_2023_995_Article_IEq2.gif\"/>",
"<graphic xlink:href=\"41746_2023_995_Fig2_HTML\" id=\"d32e874\"/>",
"<graphic xlink:href=\"41746_2023_... | [
"<media xlink:href=\"41746_2023_995_MOESM1_ESM.pdf\"><caption><p>CR&T Group Members</p></caption></media>",
"<media xlink:href=\"41746_2023_995_MOESM2_ESM.pdf\"><caption><p>Supplementary Materials</p></caption></media>",
"<media xlink:href=\"41746_2023_995_MOESM3_ESM.pdf\"><caption><p>Reporting Summary</p><... | [{"label": ["4."], "mixed-citation": ["Gharbi, M. et al. Antibiotic management of urinary tract infection in elderly patients in primary care and its association with bloodstream infections and all cause mortality: population based cohort study. "], "italic": ["BMJ"], "bold": ["364"]}, {"label": ["5."], "surname": ["Go... | {
"acronym": [],
"definition": []
} | 46 | CC BY | no | 2024-01-15 23:41:59 | NPJ Digit Med. 2024 Jan 13; 7:11 | oa_package/9e/78/PMC10787784.tar.gz |
PMC10787785 | 38218742 | [
"<title>Introduction</title>",
"<p id=\"Par2\">In the current era, ionizing radiation has been employed worldwide in many fields, such as medical diagnostics and treatments<sup>##UREF##0##1##</sup>, nuclear facilities, agricultural and industrial applications, mining areas, and research<sup>##REF##35683923##2##</... | [
"<title>Materials and methods</title>",
"<title>Materials</title>",
"<p id=\"Par9\">Self-cured acrylic resin (Acrostone Cold Cure Acrylic Resin), a commercial product with a density of 1.18 g/cm<sup>3</sup>, comes in two bottles containing powder (Poly methyl methacrylate, prepolymer (–(–CH2¼C(CH3) COOCH3-)n-PM... | [
"<title>Result and discussion</title>",
"<title>Characterization</title>",
"<title>Transmission electron microscope (TEM) analysis</title>",
"<p id=\"Par28\">The TEM micrographs of ZrO<sub>2</sub> MPs and NPs are depicted in Fig. ##FIG##4##5##. Figure ##FIG##4##5##a shows that ZrO<sub>2</sub> MPs are nearly s... | [
"<title>Result and discussion</title>",
"<title>Characterization</title>",
"<title>Transmission electron microscope (TEM) analysis</title>",
"<p id=\"Par28\">The TEM micrographs of ZrO<sub>2</sub> MPs and NPs are depicted in Fig. ##FIG##4##5##. Figure ##FIG##4##5##a shows that ZrO<sub>2</sub> MPs are nearly s... | [
"<title>Conclusion</title>",
"<p id=\"Par45\">In the current study, seven PMMA-based polymer samples were prepared and reinforced with ZrO<sub>2</sub> MPs and NPs at concentrations of 15, 30, and 45 wt% to examine their radiation shielding capabilities for diverse purposes. The investigated composites are coded a... | [
"<p id=\"Par1\">This research aimed to examine the radiation shielding properties of unique polymer composites for medical and non-medical applications. For this purpose, polymer composites, based on poly methyl methacrylate (PMMA) as a matrix, were prepared and reinforced with micro- and nanoparticles of ZrO<sub>2... | [] | [
"<title>Author contributions</title>",
"<p>A.M.E.-K. proposed the main aim and the methodology of the research. A.Y.E. and M.T.A. prepared the composite materials. Mahmoud I. Abbas depicted all the figures. M.T.A. and A.Y. E. wrote the main manuscript text. All authors reviewed and revised the manuscript.</p>",
... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Silicon rubber mold.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>The prepared specimen’s final shape.</p></caption></fig>",
"<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Experimental arrangement for the γ-rays measuring s... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>The physical properties of MMA<sup>##UREF##31##46##</sup>.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Physical properties</th><th align=\"left\">Boiling point</th><th align=\"left\">Density</th><th align=\"left\">Vap... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<inline-graphic xlink:href=\"41598_2024_51551_Article_IEq1.gif\"/>",
"<graphic xlink:href=\"41598_2024_51551_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51551_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"41598_2024_51551_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"41598_2024_515... | [] | [{"label": ["1."], "surname": ["Almatari"], "given-names": ["M"], "article-title": ["Photon and neutron shielding characteristics of samarium doped lead alumino borate glasses containing barium, lithium and zinc oxides determined at medical diagnostic energies"], "source": ["Results Phys."], "year": ["2019"], "volume":... | {
"acronym": [],
"definition": []
} | 67 | CC BY | no | 2024-01-15 23:41:59 | Sci Rep. 2024 Jan 13; 14:1279 | oa_package/c1/8e/PMC10787785.tar.gz |
PMC10787786 | 38218885 | [
"<title>Introduction</title>",
"<p id=\"Par3\">Antimicrobial stewardship aims to optimise drug use to prolong current therapeutic effectiveness and combat antimicrobial resistance (AMR)<sup>##REF##28882725##1##</sup>. One key aspect of antimicrobial stewardship is the route of administration. It is common for cri... | [
"<title>Methods</title>",
"<title>Datasets</title>",
"<p id=\"Par24\">Two publicly available large de-identified real-world clinical datasets containing routinely collected EHR information were used within this research. MIMIC-IV (4th version of the Medical Information Mart for Intensive Care database) which co... | [
"<title>Results</title>",
"<title>Data</title>",
"<p id=\"Par5\">8694 unique intensive care unit (ICU) stays, were extracted from the MIMIC dataset<sup>##UREF##7##21##,##REF##10851218##22##</sup>, with 1,668 from eICU<sup>##REF##10851218##22##–##UREF##8##24##</sup>. 10 clinical features were selected based on t... | [
"<title>Discussion</title>",
"<p id=\"Par15\">To maximise clinical utility we aimed to minimise complexity during feature selection and model development. Through the genetic algorithm, two feature sets of interest were identified. The short set utilised only 5 features but maintained performance, while the long ... | [] | [
"<p id=\"Par1\">Antimicrobial resistance (AMR) and healthcare associated infections pose a significant threat globally. One key prevention strategy is to follow antimicrobial stewardship practices, in particular, to maximise targeted oral therapy and reduce the use of indwelling vascular devices for intravenous (IV... | [
"<title>Supplementary information</title>",
"<p>\n\n\n\n</p>",
"<title>Source data</title>",
"<p>\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41467-024-44740-2.</p>",
"<title>Acknowledgements</title>",
"<p>William Bolton was supported by the UKRI CDT in AI for Healthcare <ext-link ext-link-type=\"uri\" xlink:href=\"http://ai4heal... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Overview of the steps taken in this research study to develop fair and interpretable machine learning models for antimicrobial switch decision making.</title><p>MIMIC Medical Information Mart for Intensive Care, ICU Intensive care unit, IV Intravenous, IVOS Int... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Dataset demographics and statistics</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th/><th colspan=\"3\">Dataset</th></tr><tr><th colspan=\"2\">Statistic</th><th>MIMIC preprocessing</th><th>MIMIC hold-out</th><th>eICU</th></tr></thea... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>",
"<supplementary-material ... | [
"<table-wrap-foot><p>Note that infection types in MIMIC are determined through ‘hadm_id’ and are not definitively linked to the antimicrobial switch of interest as diagnoses are only coded for billing purposes upon hospital discharge. This results in totals over 100% as patients have multiple infection episodes. Se... | [
"<graphic xlink:href=\"41467_2024_44740_Fig1_HTML\" id=\"d32e321\"/>",
"<graphic xlink:href=\"41467_2024_44740_Fig2_HTML\" id=\"d32e1421\"/>",
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"<graphic xlink:href=\"41467_2024_44740_Fig4_HTML\" id=\"d32e1492\"/>"
] | [
"<media xlink:href=\"41467_2024_44740_MOESM1_ESM.pdf\"><caption><p>Supplementary information</p></caption></media>",
"<media xlink:href=\"41467_2024_44740_MOESM2_ESM.pdf\"><caption><p>Reporting Summary</p></caption></media>",
"<media xlink:href=\"41467_2024_44740_MOESM3_ESM.pdf\"><caption><p>Peer Review File</p... | [{"label": ["3."], "mixed-citation": ["Agency, U. H. S. Antimicrobial stewardship: Start smart - then focus "], "ext-link": ["https://www.gov.uk/government/publications/antimicrobial-stewardship-start-smart-then-focus"]}, {"label": ["4."], "mixed-citation": ["Davar, K. et al. Can the Future of ID Escape the Inertial Do... | {
"acronym": [],
"definition": []
} | 55 | CC BY | no | 2024-01-15 23:41:59 | Nat Commun. 2024 Jan 13; 15:506 | oa_package/6c/b5/PMC10787786.tar.gz |
PMC10787787 | 38218745 | [
"<title>Introduction</title>",
"<p id=\"Par2\">In recent years, intelligent transportation have become increasingly complex due to rapid urbanization and population growth. There is a growing need for abnormal event detection in transportation networks. The Shanghai Bund trampling incident that occurred on Decemb... | [
"<title>Methodology</title>",
"<p id=\"Par14\">Although many traffic anomaly detection methods have achieved optimal performance, they often overlook the hidden relationships between nodes during the detection process. For instance, traffic congestion during peak periods upstream can impact downstream traffic. Th... | [
"<title>Result and analysis</title>",
"<p id=\"Par43\"><bold>(1) Comparison with state-of-the-art work</bold> Initially, we compared our MTGAE model with some baseline models using the AUC as evaluation metric. The calculation of AUC considers both the classification ability of the classifier for positive and neg... | [] | [
"<title>Conclusions</title>",
"<p id=\"Par49\">This paper proposes MTGAN, a spatio-temporal anomaly detection framework for traffic. In the encoder, we propose two modules: the Mirror TCN (MTCM) and a variant of GCGRU, namely the GCGRU CELL that captures correlations in spatial and temporal dimensions, and a prac... | [
"<p id=\"Par1\">Traffic time series anomaly detection has been intensively studied for years because of its potential applications in intelligent transportation. However, classical traffic anomaly detection methods often overlook the evolving dynamic associations between road network nodes, which leads to challenge... | [
"<title>Related work</title>",
"<p id=\"Par9\">In this section, we introduce the graph convolution networks, temporal convolutional networks, and autoencoder-based anomaly detection.</p>",
"<title>Graph convolution networks</title>",
"<p id=\"Par10\">Recently, Graph Neural Network (GNN) variants, such as Grap... | [
"<title>Acknowledgements</title>",
"<p>This work was supported by the National Key Research and Development Program of China (2022ZD0115604) and National Natural Science Foundation of China (Grant Nos. 42130608, 42075142) and the Sichuan Science and Technology program (Grant Nos. 2023ZHCG0018, 2023NSFSC0470, 2021... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Illustration of two parts of NYC dataset graph (first column) and corresponding weighted adjacency matrix (second column) corresponding to scaled inverse travel times between points on the graph. Note that the mobility data is modeled as a series of time-dependen... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Given fixed and , the AUC scores of different models with the fraction of the time slices chosen to be polluted.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">anomaly rate </th><th align=\"left\">5%</th><th align=\"le... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Zhiyu Ren and Xiaojie Li.</p></fn></fn-group>"
] | [
"<inline-graphic xlink:href=\"41598_2024_51374_Article_IEq1.gif\"/>",
"<inline-graphic xlink:href=\"41598_2024_51374_Article_IEq2.gif\"/>",
"<inline-graphic xlink:href=\"41598_2024_51374_Article_IEq3.gif\"/>",
"<inline-graphic xlink:href=\"41598_2024_51374_Article_IEq4.gif\"/>",
"<inline-graphic xlink:href=... | [] | [{"label": ["1."], "surname": ["Gao", "Zheng", "Yang"], "given-names": ["J", "D", "S"], "article-title": ["Perceiving spatiotemporal traffic anomalies from sparse representation-modeled city dynamics"], "source": ["Pers. Ubiquit. Comput."], "year": ["2020"], "volume": ["27"], "fpage": ["1"], "lpage": ["14"]}, {"label":... | {
"acronym": [],
"definition": []
} | 60 | CC BY | no | 2024-01-15 23:41:59 | Sci Rep. 2024 Jan 13; 14:1247 | oa_package/55/7b/PMC10787787.tar.gz |
PMC10787788 | 38218939 | [
"<title>Introduction</title>",
"<p id=\"Par3\">High-throughput spatial omics technologies are at the forefront of modern molecular biology, and promise to provide topographic context to the wealth of available transcriptomic data. Recent breakthroughs in profiling technology have revolutionised our understanding ... | [
"<title>Methods</title>",
"<title>Datasets and preprocessing</title>",
"<p id=\"Par31\">We used publicly available data resources from three different SST commercial platforms (10 × Genomics Xenium, NanoString CosMx, and Vizgen MERSCOPE), and sequencing data from Human Cell Atlas.</p>",
"<title>Subcellular sp... | [
"<title>Results</title>",
"<title>BIDCell: Incorporating biological insights using deep learning to improve cell shape representation</title>",
"<p id=\"Par9\">BIDCell is a DL-based cell segmentation method that identifies each individual cell and all its pixels as a cohesive mask. BIDCell uses subcellular spat... | [
"<title>Discussion</title>",
"<p id=\"Par25\">Here we presented BIDCell, a method for cell segmentation in subcellular spatially resolved transcriptomics data. BIDCell leverages DL with its biologically-informed loss functions that allow the model to self-learn and capture both cell type and cell shape informatio... | [] | [
"<p id=\"Par1\">Recent advances in subcellular imaging transcriptomics platforms have enabled high-resolution spatial mapping of gene expression, while also introducing significant analytical challenges in accurately identifying cells and assigning transcripts. Existing methods grapple with cell segmentation, frequ... | [
"<title>Supplementary information</title>",
"<p>\n\n\n\n</p>",
"<title>Source data</title>",
"<p>\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41467-023-44560-w.</p>",
"<title>Acknowledgements</title>",
"<p>The authors thank all their colleagues, particularly at the Sydney Precision Data Science Centre and Charles Perkins Centre f... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>BIDCell framework.</title><p><bold>a</bold> Schematic illustration of the BIDCell framework and the loss functions used for training. In the deep learning model, E1 to E5 and D1 to D4 are respectively the encoding and decoding layers, while the connectivity bet... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Single-cell RNA-seq references used in this study</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Data collection</th><th>Data</th><th># of cell types</th><th>Source</th></tr></thead><tbody><tr><td rowspan=\"10\">TISCH-BRCA</td><td><ext... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>",
"<supplementary-material ... | [
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Xiaohang Fu, Yingxin Lin.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41467_2023_44560_Fig1_HTML\" id=\"d32e388\"/>",
"<graphic xlink:href=\"41467_2023_44560_Fig2_HTML\" id=\"d32e1007\"/>",
"<graphic xlink:href=\"41467_2023_44560_Fig3_HTML\" id=\"d32e1146\"/>",
"<graphic xlink:href=\"41467_2023_44560_Fig4_HTML\" id=\"d32e1242\"/>",
"<graphic xlink:href=... | [
"<media xlink:href=\"41467_2023_44560_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media>",
"<media xlink:href=\"41467_2023_44560_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media>",
"<media xlink:href=\"41467_2023_44560_MOESM3_ESM.pdf\"><caption><p>Reporting Summary</p... | [{"label": ["4."], "mixed-citation": ["Moen, E. et al. Accurate cell tracking and lineage construction in live-cell imaging experiments with deep learning. Biorxiv, 803205. 10.1101/803205 (2019)."]}, {"label": ["8."], "mixed-citation": ["Li, M. et al. StereoCell enables highly accurate single-cell segmentation for spat... | {
"acronym": [],
"definition": []
} | 38 | CC BY | no | 2024-01-15 23:41:59 | Nat Commun. 2024 Jan 13; 15:509 | oa_package/8d/28/PMC10787788.tar.gz |
PMC10787789 | 38218986 | [
"<title>Introduction</title>",
"<p id=\"Par2\">The surface faulting rupture site will rupture under the appropriate bedrock dislocation and damage the tunnel structure on it. With the building and operation of the cross-tunnel structure in an active fault-intensive area, tunnel designs that avoid or only conceptu... | [] | [] | [] | [
"<title>Conclusion</title>",
"<p id=\"Par42\">This article adopts the design concept of \"fuse\", which is to use segmented flexible joint tunnel segments during fault displacement, based on the principle of \"sacrificing small for large\", to reduce the overall damage of soil and surrounding rock to the length o... | [
"<p id=\"Par1\">The fortification system of the tunnel structure spanning the active fault, such as the failure mechanism and fault-resistant design (measures), has not been thoroughly established. In this study, the self-developed cross-fault large-scale bedrock dislocation loading device platform is utilized to c... | [
"<title>Design of a large-scale box test apparatus and scheme for cross-fault rupture tunnel models</title>",
"<title>Large-scale model box test apparatus and instrumentation arrangement</title>",
"<p id=\"Par7\">The device adopts a self-developed large-scale experimental box platform, including a fault disloca... | [
"<title>Acknowledgements</title>",
"<p>The authors would like to express their profound gratitude to my supervisor, Jian Yi Zhang, for his invaluable direction and counsel during the study process. My supervisor gave me tolerance and support when I ran into problems, as well as insightful inquiries regarding the ... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>The actual platform of a model test apparatus for cross-fault tunnels.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Three-dimensional diagram of the platform for the cross-fault tunnel model test apparatus.</p></caption></fig>",
... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Model test working condition parameter table.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Test number</th><th align=\"left\">Soil cover thickness (mm)</th><th align=\"left\">Soil type</th><th align... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41598_2024_51830_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51830_Fig2_HTML\" id=\"MO2\"/>",
"<inline-graphic xlink:href=\"41598_2024_51830_Article_IEq1.gif\"/>",
"<inline-graphic xlink:href=\"41598_2024_51830_Article_IEq2.gif\"/>",
"<inline-graphic xlink:href=\"4159... | [] | [{"label": ["1."], "surname": ["He", "Li", "Zhang", "Gen", "Yan"], "given-names": ["C", "L", "J", "P", "QX"], "article-title": ["Seismic damage mechanism of tunnels through fault zones"], "source": ["Chin. J. Geotech. Eng."], "year": ["2014"], "volume": ["36"], "issue": ["03"], "fpage": ["427"], "lpage": ["434"]}, {"la... | {
"acronym": [],
"definition": []
} | 20 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1281 | oa_package/d0/58/PMC10787789.tar.gz |
PMC10787790 | 38218887 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Neurological illnesses are characterized by the progressive and permanent loss of neurons in particular brain areas<sup>##REF##34716723##1##</sup>. Many neurodegenerative disorders are the result of several causes, most likely involving a variety of mechanistic pathw... | [
"<title>Materials and methods</title>",
"<title>Chemicals</title>",
"<p id=\"Par6\">For synthesis, isatin derivatives, hydrazine hydrate, and benzoic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA) and TCI Chemical (Toshima, Tokyo, Japan). Substrates (benzylamine and kynuramine) and reference inhibi... | [
"<title>Results and discussion</title>",
"<title>Synthesis</title>",
"<p id=\"Par26\">The target molecules were synthesized in two steps. In the first step, an intermediate <italic>acylhydrazide</italic> molecule was synthesized by reacting benzoic acid with hydrazine hydrate. This intermediate was then reacted... | [
"<title>Results and discussion</title>",
"<title>Synthesis</title>",
"<p id=\"Par26\">The target molecules were synthesized in two steps. In the first step, an intermediate <italic>acylhydrazide</italic> molecule was synthesized by reacting benzoic acid with hydrazine hydrate. This intermediate was then reacted... | [
"<title>Conclusion</title>",
"<p id=\"Par43\">We synthesized acylhydrazone-based isatin compounds and evaluated their ability to inhibit MAOs. <bold>IS15</bold> was a potent competitive reversible MAO-A inhibitor, whereas <bold>IS6</bold>, <bold>IS7</bold>, and <bold>IS13</bold> were potent competitive reversible... | [
"<p id=\"Par1\">Sixteen isatin-based hydrazone derivatives (<bold>IS1</bold>–<bold>IS16</bold>) were synthesized and assessed for their ability to inhibit monoamine oxidases (MAOs). All the molecules showed improved inhibitory MAO-B activity compared to MAO-A. Compound <bold>IS7</bold> most potently inhibited MAO-B... | [
"<title>Supplementary Information</title>",
"<p>\n</p>"
] | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51728-x.</p>",
"<title>Acknowledgements</title>",
"<p>The authors thank the Indian Council of Social Science Research under the Grant number (File No. 02/90/2022-23/RP/MN).</p>",
... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Base structure of isatin and their derivatives for FDA-approved drugs.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Design strategy of acylhydrazone-based isatin derivatives.</p></caption></fig>",
"<fig id=\"Sch1\"><label>Scheme ... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Monoamine oxidase (MAO)-A and MAO-B inhibition by the 16 compounds of the IS series<sup>a</sup>.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Compound</th><th align=\"left\" colspan=\"2\">Residual activit... | [
"<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><sup>a</sup>Results are the means ± standard errors of duplicate or triplicate experiments.</p><p><sup>b</sup>Selectivity index (SI) values are expressed for MAO-B compared to MAO-A.</p></table-wrap-foot>",
"<table-wrap-foot><p><italic>Pe</italic> (10<sup>−6</sup> cm/s) > 4.00: CNS + (high... | [
"<graphic xlink:href=\"41598_2024_51728_Fig1_HTML\" id=\"MO1\"/>",
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"<media xlink:href=\"41598_2024_51728_MOESM1_ESM.docx\"><caption><p>Supplementary Information.</p></caption></media>"
] | [{"label": ["17."], "surname": ["Chajkowski-Scarry", "Rimoldi"], "given-names": ["S", "JM"], "article-title": ["Monoamine oxidase A and B substrates: Probing the pathway for drug development, Future"], "source": ["Med. Chem."], "year": ["2014"], "volume": ["6"], "fpage": ["697"], "lpage": ["717"]}, {"label": ["21."], "... | {
"acronym": [],
"definition": []
} | 64 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1264 | oa_package/c8/1a/PMC10787790.tar.gz |
PMC10787791 | 38218976 | [
"<title>Introduction</title>",
"<p id=\"Par3\">Mucosal pentraxin 2 (<italic>Mptx2</italic>) has been proposed to be a member of the pentraxin family due to the high homology of its sequences ( ~ 83% identity in amino acid sequences) to C-reactive protein (<italic>CRP</italic>) and serum amyloid P component protei... | [
"<title>Materials and methods</title>",
"<title>Generation of <italic>Mptx2</italic> knockout mice</title>",
"<p id=\"Par18\"><italic>Mptx2</italic><sup><italic>−/−</italic></sup> mice (Δexon 1–2) were generated as in our previous study<sup>##REF##35609377##18##</sup> via genome engineering mediated by clustere... | [
"<title>Results</title>",
"<title><italic>Mptx2</italic> was specifically expressed in Paneth cells</title>",
"<p id=\"Par6\">As shown in Fig. ##FIG##0##1a##, <italic>Mptx2</italic> messenger RNA (mRNA) was synthesized in the intestines of <italic>Wt</italic> mice from embryonic day 12.5 (E12.5). The levels <it... | [
"<title>Discussion</title>",
"<p id=\"Par14\">To the best of our knowledge, this study confirms <italic>Mptx2</italic> acts as a novel marker of Paneth cells, implying that it might play important roles in intestinal inflammation and homeostasis. We firstly showed that <italic>Mptx2</italic> protein was selective... | [
"<title>Conclusions</title>",
"<p id=\"Par17\">Our findings revealed that <italic>Mptx2</italic> was a novel marker of Paneth cells. <italic>Mptx2</italic> deficiency triggered microbiota dysbiosis and increased epithelial invasion by bacteria, leading to greater susceptibility to intestinal inflammation with red... | [
"<p id=\"Par1\">A recent single-cell survey of the small-intestinal epithelium suggests that mucosal pentraxin 2 (<italic>Mptx2</italic>) is a new Paneth cell marker, but its function and involved mechanism in the Paneth cell are still unknown. Therefore, we create <italic>Mptx2</italic> knockout (<italic>Mptx2</it... | [
"<title>Supplementary information</title>",
"<p>\n\n\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s42003-024-05785-7.</p>",
"<title>Acknowledgements</title>",
"<p>This work was supported by the Natural Science Foundation of Shanghai (22ZR1480600), Shanghai Key Laboratory of Pediatric Gas... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title><italic>Mptx2</italic> was selectively expressed in the Paneth cells.</title><p><bold>a</bold> Alteration of <italic>Mptx2</italic> mRNA and Lysozyme (<italic>Lyz1</italic>) mRNA from the embryonic stages (E12.5 – 17.5) to the postnatal time (P0 – 13.5) (each g... | [] | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>",
"<supplementary-material ... | [
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Weihui Yan, Shanshan Chen.</p></fn></fn-group>"
] | [
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"<media xlink:href=\"42003_2024_5785_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media>",
"<media xlink:href=\"42003_2024_5785_MOESM2_ESM.pdf\"><caption><p>Description of Additional Supplementary Files</p></caption></media>",
"<media xlink:href=\"42003_2024_5785_MOESM3_ESM.xlsx\"><capt... | [{"label": ["41."], "mixed-citation": ["Ranjan, K., Hedl, M., Sinha, S., Zhang, X. & Abraham, C. Ubiquitination of ATF6 by disease-associated RNF186 promotes the innate receptor-induced unfolded protein response. "], "italic": ["J. Clin. Invest."], "bold": ["131"]}] | {
"acronym": [],
"definition": []
} | 43 | CC BY | no | 2024-01-15 23:42:00 | Commun Biol. 2024 Jan 13; 7:94 | oa_package/e2/64/PMC10787791.tar.gz |
PMC10787792 | 38218905 | [
"<title>Introduction</title>",
"<p id=\"Par3\">The recent development of mapping technologies such as Hi–C<sup>##REF##19815776##1##</sup> that probes the 3D genome organization reveals that a chromosome is divided into topologically associating domains (TADs)<sup>##REF##22495300##2##,##REF##22495304##3##</sup>. T... | [
"<title>Methods</title>",
"<p id=\"Par30\">In this section, we first introduce the first part of DiffDomain: a model-based method to identify reorganized TADs. We state the model assumptions and their verification using real Hi–C data before reporting the second part of DiffDomain: classification of reorganized T... | [
"<title>Results</title>",
"<title>Overview of DiffDomain</title>",
"<p id=\"Par6\">The workflow of DiffDomain is illustrated in Fig. ##FIG##0##1##. Its input is a set of TADs called in biological condition 1 and their corresponding Hi–C contact matrices from biological conditions 1 and 2 (Fig. ##FIG##0##1##a). ... | [
"<title>Discussion</title>",
"<p id=\"Par25\">In this work, we present a statistical method, DiffDomain, for comparative analysis of TADs using a pair of Hi–C datasets. Extensive evaluation using real Hi–C datasets demonstrates clear advantages of DiffDomain over alternative methods for controlling false positive... | [] | [
"<p id=\"Par1\">Topologically associating domains (TADs) are critical structural units in three-dimensional genome organization of mammalian genome. Dynamic reorganizations of TADs between health and disease states are associated with essential genome functions. However, computational methods for identifying reorga... | [
"<title>Supplementary information</title>",
"<p>\n\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41467-024-44782-6.</p>",
"<title>Acknowledgements</title>",
"<p>This work was supported by the National Natural Science Foundation of China grant 12271536 (D.T. and Z.B.), National Key Rese... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>DiffDomain workflow and example outputs.</title><p><bold>a</bold> Input are a TAD in condition 1 and its two Hi–C contact matrices (<bold><italic>A</italic></bold><sub>1</sub> and <bold><italic>A</italic></bold><sub>2</sub>) in two biological conditions 1 and 2... | [] | [
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"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Dunming Hua, Ming Gu, Xiao Zhang, Yanyi Du.</p></fn></fn-group>"
] | [
"<inline-graphic xlink:href=\"41467_2024_44782_Article_IEq1.gif\"/>",
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"<media xlink:href=\"41467_2024_44782_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media>",
"<media xlink:href=\"41467_2024_44782_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media>",
"<media xlink:href=\"41467_2024_44782_MOESM3_ESM.pdf\"><caption><p>Reporting Summary</p... | [{"label": ["27."], "surname": ["Mourad"], "given-names": ["R"], "article-title": ["TADreg: a versatile regression framework for TAD identification, differential analysis and rearranged 3D genome prediction"], "source": ["BMC Bioinform."], "year": ["2022"], "volume": ["23"], "fpage": ["1"], "lpage": ["14"], "pub-id": [... | {
"acronym": [],
"definition": []
} | 75 | CC BY | no | 2024-01-15 23:42:00 | Nat Commun. 2024 Jan 13; 15:502 | oa_package/30/94/PMC10787792.tar.gz |
PMC10787793 | 38218975 | [
"<title>Background & Summary</title>",
"<p id=\"Par2\">GRACE (Gravity Recovery And Climate Experiment) satellites are designed to monitor spatiotemporal variations of the Earth’s gravitational field to improve our understanding of the changes in the global climate system, with the primary goal of properly map... | [
"<title>Methods</title>",
"<title>Residual deep convolutional autoencoders</title>",
"<p id=\"Par5\">Convolutional Neural Networks (CNNs) are special types of neural networks and are useful for processing data with a grid-like architecture, such as images or time series data<sup>##REF##7370364##12##,##UREF##11#... | [] | [] | [] | [
"<p id=\"Par1\">Since April 2002, Gravity Recovery and Climate Experiment (GRACE) and GRACE-FO (FollowOn) satellite gravimetry missions have provided precious data for monitoring mass variations within the hydrosphere, cryosphere, and oceans with unprecedented accuracy and resolution. However, the long-term product... | [
"<title>Data Records</title>",
"<p id=\"Par22\">The simulated monthly dataset is released both in the form of gridded mass anomalies with and spherical harmonic coefficients in accordance with the ICGEM (International Center for Global Earth Models) format. These datasets are available in figshare repository<sup>... | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41597-023-02887-5.</p>",
"<title>Acknowledgements</title>",
"<p>This work is partially supported by Scientific and Technological Research Council of Turkey - TÜBİTAK (Contract number 119Y17... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Comparison of overall RMSE and NSE values computed over (<bold>a</bold>) land + ocean (global), (<bold>b</bold>) land-only and (<bold>c</bold>) ocean-only areas on Earth from 57 test months.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Fig. 2</label><caption><p... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Overall monthly RMSE (cm) and NSE metrics for 57 test months.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Region</th><th>Sol1</th><th>Sol2</th><th>Sol3</th><th>Sol4</th></tr></thead><tbody><tr><td rowspan=\"3\"><bold>RMSE</bold... | [
"<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepack... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>(Note that the pre-GRACE era is not shown for comparisons to CSRM because there are no CSRM solutions available for that period.).</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and... | [
"<graphic xlink:href=\"41597_2023_2887_Article_Equ1.gif\" position=\"anchor\"/>",
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"<media xlink:href=\"41597_2023_2887_MOESM1_ESM.pdf\"><caption><p>41597_2024_2887_MOESM1_ESM</p></caption></media>"
] | [{"label": ["1."], "surname": ["Tapley", "Bettadpur", "Watkins", "Reigber"], "given-names": ["BD", "S", "M", "C"], "article-title": ["The gravity recovery and climate experiment: Mission overview and early results"], "source": ["Geophys. Res. Lett."], "year": ["2004"], "volume": ["31"], "fpage": ["L09607"], "pub-id": [... | {
"acronym": [],
"definition": []
} | 99 | CC BY | no | 2024-01-15 23:42:00 | Sci Data. 2024 Jan 13; 11:71 | oa_package/a2/10/PMC10787793.tar.gz |
PMC10787794 | 38218858 | [
"<title>Introduction</title>",
"<p id=\"Par3\">Neural networks have made significant contributions to the field of Artificial Intelligence, serving as both a tool for mathematical modeling and a means to understand brain function. The Hopfield paradigm<sup>##REF##6953413##1##,##REF##10031847##2##</sup> has played... | [
"<title>Methods</title>",
"<title>Background</title>",
"<p id=\"Par36\">In our experiment (similarly to a typical wavefront shaping experiment), light from a coherent source is controlled by a spatial light modulator and transmitted after propagation through a disordered medium into the mode <italic>ν</italic>.... | [
"<title>Results</title>",
"<p id=\"Par10\">The idea stems from the fact that intensity scattered by a disordered medium into a mode <italic>ν</italic> resulting from an input pattern <bold><italic>ϕ</italic></bold>) may be written as:with the scattering process driven by the matrix <bold>V</bold> :generated from ... | [
"<title>Discussion</title>",
"<p id=\"Par32\">In summary, the Stochastic Emergent Storage (SES) paradigm enables classification with a significantly smaller number of sensors/pixels/modes compared to the elements composing the pattern. This opens up the possibility of fabricating complex pattern classifiers with ... | [] | [
"<p id=\"Par1\">Disorder is a pervasive characteristic of natural systems, offering a wealth of non-repeating patterns. In this study, we present a novel storage method that harnesses naturally-occurring random structures to store an arbitrary pattern in a memory device. This method, the Stochastic Emergent Storage... | [
"<title>Supplementary information</title>",
"<p>\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41467-023-44498-z.</p>",
"<title>Acknowledgements</title>",
"<p>This research was funded by: Regione Lazio, Project LOCALSCENT, Grant PROT. A0375-2020-36549, Call POR-FESR “Gruppi di Ricerc... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Emergent memory storage scheme.</title><p>The sketch describes both the input query <bold><italic>ϕ</italic></bold> presentation and the measurement of the optical synaptic matrix <bold>V</bold><sup><italic>ν</italic></sup> (details in the Methods section). The... | [] | [
"<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepack... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41467_2023_44498_Article_Equ1.gif\" position=\"anchor\"/>",
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"<media xlink:href=\"41467_2023_44498_MOESM1_ESM.pdf\"><caption><p>Supplementary informations</p></caption></media>",
"<media xlink:href=\"41467_2023_44498_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media>"
] | [{"label": ["3."], "surname": ["Farhat", "Psaltis", "Prata", "Paek"], "given-names": ["NH", "D", "A", "E"], "article-title": ["Optical implementation of the hopfield model"], "source": ["Appl. Optics"], "year": ["1985"], "volume": ["24"], "fpage": ["1469"], "pub-id": ["10.1364/AO.24.001469"]}, {"label": ["4."], "surnam... | {
"acronym": [],
"definition": []
} | 38 | CC BY | no | 2024-01-15 23:42:00 | Nat Commun. 2024 Jan 13; 15:505 | oa_package/fc/45/PMC10787794.tar.gz |
PMC10787795 | 38218740 | [
"<title>Background & Summary</title>",
"<p id=\"Par2\"><italic>Decapterus maruadsi</italic>, a pelagic fish in the family Carangidae, lives widely in distributed warm offshore waters of East and Southeast Asia<sup>##REF##32744461##1##</sup>. And it is especially abundant along the coasts of the South China Se... | [
"<title>Methods</title>",
"<title>Ethics statement</title>",
"<p id=\"Par4\">The <italic>D. maruadsi</italic> in our experiments were collected from Dongshan, Zhangzhou City, Fujian Province, China. Furthermore, the methods used in this work are strictly in accordance with the Guidelines for The Care and Use of... | [] | [] | [] | [
"<p id=\"Par1\"><italic>Decapterus maruadsi</italic> is one of the representative offshore fish in the Western Pacific. Since the last century, it has become a commercially valuable marine fishery species in the Western Pacific region. Despite its high economic value, there is still a lack of high-quality reference... | [
"<title>Data Records</title>",
"<p id=\"Par14\">The raw sequencing reads of all libraries have been deposited into NCBI SRA database via the accession number of SRP408505<sup>##UREF##11##43##</sup>. The assembled genome has been deposited at Genbank under the accession number GCA_030347415.2<sup>##UREF##12##44##<... | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41597-024-02912-1.</p>",
"<title>Acknowledgements</title>",
"<p>We acknowledge financial support from the Fundamental Research Funds for the Central Universities (No.20720200119).</p>",
"... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Characteristics of <italic>Decapterus maruadsi</italic> genome assembly. (<bold>A</bold>) Contact map of chromosomal interactions in the <italic>D. maruadsi</italic> genome using Hi-C data. (<bold>B</bold>) A circos plot of 23 chromosomes in <italic>D. maruadsi</it... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Statistics for the sequencing data of the <italic>D. maruadsi</italic> genome.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Platform</th><th>Insert size (bp)</th><th>Raw data (Gb)</th><th>Clean data (Gb)</th><th>Average Read Length (... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>"
] | [
"<table-wrap-foot><p>Note: Genome size estimated by genome survey (720.70 Mb) were used for sequencing coverage calculation</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.... | [
"<graphic xlink:href=\"41597_2024_2912_Fig1_HTML\" id=\"d32e621\"/>",
"<graphic xlink:href=\"41597_2024_2912_Fig2_HTML\" id=\"d32e900\"/>"
] | [
"<media xlink:href=\"41597_2024_2912_MOESM1_ESM.pdf\"><caption><p>Supplement Figure</p></caption></media>",
"<media xlink:href=\"41597_2024_2912_MOESM2_ESM.xlsx\"><caption><p>Supplement Table</p></caption></media>"
] | [{"label": ["2."], "surname": ["Chen", "Li"], "given-names": ["G", "Y"], "article-title": ["Distribution of the Carangidae fishes in the continental shelf waters of northern South China Sea"], "source": ["J. Shanghai Ocean Univ."], "year": ["2003"], "volume": ["12"], "fpage": ["146"], "lpage": ["151"]}, {"label": ["3."... | {
"acronym": [],
"definition": []
} | 48 | CC BY | no | 2024-01-15 23:42:00 | Sci Data. 2024 Jan 13; 11:69 | oa_package/4c/cb/PMC10787795.tar.gz |
PMC10787796 | 38218961 | [
"<title>Introduction</title>",
"<p id=\"Par3\">All-carbon quaternary stereocenters are an important synthetic motif found in natural products and bioactive molecules (Fig. ##FIG##0##1##) that are especially difficult to synthesize enantioselectively<sup>##REF##31515549##1##–##UREF##2##4##</sup>. Successful strate... | [] | [
"<title>Results and discussion</title>",
"<p id=\"Par5\">To overcome steric deactivation via increased electrophilic activation, we looked to enones bearing additional electron-withdrawing groups. In particular, the use of 2–ene–1,4–diones (1) could allow an approach to often difficult-to-access chiral 1,4-diketo... | [
"<title>Results and discussion</title>",
"<p id=\"Par5\">To overcome steric deactivation via increased electrophilic activation, we looked to enones bearing additional electron-withdrawing groups. In particular, the use of 2–ene–1,4–diones (1) could allow an approach to often difficult-to-access chiral 1,4-diketo... | [] | [
"<p id=\"Par1\">The chemical synthesis of molecules with closely packed atoms having their bond coordination saturated is a challenge to synthetic chemists, especially when three-dimensional control is required. The organocatalyzed asymmetric synthesis of acyclic alkenylated, alkynylated and heteroarylated quaterna... | [
"<title>Supplementary information</title>",
"<p>\n\n\n</p>",
"<title>Source data</title>",
"<p>\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41467-024-44744-y.</p>",
"<title>Acknowledgements</title>",
"<p>The authors are grateful for generous financial support from the NSF (grant CHE-2102282, JAM) and the Welch Foundation (grant... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Representative natural products bearing alkenylated quaternary carbons.</title><p>Inset shows how quaternary carbon formation applies to a synthesis<sup>##UREF##39##86##</sup>.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Enant... | [] | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41467_2024_44744_Fig1_HTML\" id=\"d32e263\"/>",
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"<media xlink:href=\"41467_2024_44744_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media>",
"<media xlink:href=\"41467_2024_44744_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media>",
"<media xlink:href=\"41467_2024_44744_MOESM3_ESM.xlsx\"><caption><p>Source Data</p></ca... | [{"label": ["2."], "surname": ["Ling", "Rivas"], "given-names": ["T", "F"], "article-title": ["All-carbon quaternary centers in natural products and medicinal chemistry: recent advances"], "source": ["Tetrahedron"], "year": ["2016"], "volume": ["72"], "fpage": ["6729"], "lpage": ["6777"], "pub-id": ["10.1016/j.tet.2016... | {
"acronym": [],
"definition": []
} | 86 | CC BY | no | 2024-01-15 23:42:00 | Nat Commun. 2024 Jan 13; 15:504 | oa_package/11/e7/PMC10787796.tar.gz |
PMC10787799 | 38218996 | [
"<title>Introduction</title>",
"<p id=\"Par2\">The axillary nerve is a branch of the posterior cord of the brachial plexus and contains nerve components from the fifth and sixth cervical spinal segments<sup>##UREF##0##1##</sup>. It passes through the posterior wall of the axilla via the quadrangular space with th... | [
"<title>Materials and methods</title>",
"<p id=\"Par23\">Fifty upper extremities, 24 right and 26 left, from 29 formalin-embalmed cadavers (mean age, 75.8 years; range 52–95 years) with no pathologies or surgical history in the upper extremity were used in this study. All study procedures approved by the Surgical... | [
"<title>Results</title>",
"<title>Branching patterns of the axillary nerve</title>",
"<p id=\"Par6\">The clavicular and acromial parts of the deltoid muscle were constantly innervated by the anterior division of the axillary nerve. The teres minor muscle was constantly innervated by the posterior division of th... | [
"<title>Discussion</title>",
"<p id=\"Par20\">The axillary nerve ran a mean distance of 44.0 mm in the axilla and divided into anterior and posterior divisions before passing through the quadrangular space in every specimen we examined. Early bifurcation of the axillary nerve before passing through the quadrangul... | [
"<title>Conclusions</title>",
"<p id=\"Par22\">The significant role of the axillary nerve in maintaining deltoid muscle functionality and in preventing palsy or malfunction highlights its vital importance in clinical and surgical procedures. Functional loss of the anterior division may be more critical because it... | [
"<p id=\"Par1\">This study investigated the anatomical details of the axillary and radial nerves in 50 upper limbs from 29 adult formalin-embalmed cadavers, and ten fresh upper limbs. The focus was on understanding the course, division, and ramifications of these nerves to improve treatment of shoulder dysfunction ... | [] | [
"<title>Acknowledgements</title>",
"<p>The authors sincerely thank those who donated their bodies to science so that anatomical research could be performed. Results from such research can potentially increase mankind's overall knowledge that can then improve patient care. Therefore, these donors and their familie... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>The three types of branching patterns of the axillary nerve to the deltoid and teres minor muscles. (<bold>A,D</bold>) Mixed type: the anterior division innervates all parts of the deltoid and the posterior division innervates both the spinous part of the deltoid... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Number and lengths of each part of the axillary nerve.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Spinous part</th><th align=\"left\">Acromial part</th><th align=\"left\">Clavicular part</th></tr>... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p><italic>p</italic> < 0.05.</p></table-wrap-foot>",
"<table-wrap-foot><p>Data presented as mean ± SD, unit area: 0.04 mm<sup>2</sup>.</p><p><italic>p</italic> < 0.05.</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with rega... | [
"<graphic xlink:href=\"41598_2024_51923_Fig1_HTML\" id=\"MO1\"/>",
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"<graphic xlink:href=\"41598_2024_51923_Fig4_HTML\" id=\"MO4\"/>"
] | [] | [{"label": ["1."], "surname": ["Moser"], "given-names": ["T"], "article-title": ["The deltoid, a forgotten muscle of the shoulder"], "source": ["Skelet. Radiol."], "year": ["2013"], "volume": ["42"], "fpage": ["1361"], "lpage": ["1375"], "pub-id": ["10.1007/s00256-013-1667-7"]}, {"label": ["2."], "surname": ["Standring... | {
"acronym": [],
"definition": []
} | 28 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1262 | oa_package/d0/4a/PMC10787799.tar.gz |
PMC10787801 | 38218873 | [
"<title>Introduction</title>",
"<p id=\"Par3\">Reducing fossil fuel use and global climate change requires a fast energy transition, and nations across the globe have successively set out their own targets and pathways to carbon neutrality<sup>##UREF##0##1##</sup>. Since 2009, as the fastest-growing renewable pow... | [
"<title>Methods</title>",
"<p id=\"Par14\">The newly developed EPM and fast cloud mask algorithm in the NCP_CF system are applied to predict CF (or CSR) at the leading time of 0–4 h at two test PV plants. In this system, the −4–0 h geostationary satellite radiance data is used as input to dynamically provide 0–4 ... | [
"<title>Results</title>",
"<title>Cloud fraction nowcasting and validations</title>",
"<p id=\"Par6\">In order to better simulate real application scenarios, a quasi-operational and near real-time (NRT) and cyclically updated prediction system is newly developed for 0–4 h CF nowcasting at solar PV plants (herea... | [
"<title>Discussion</title>",
"<p id=\"Par11\">Our study demonstrates that the NCP_CF system can provide high-efficiency, high-quality and adaptable 0–4 h CF nowcasting data for PV plants. As shown in Figs. ##FIG##1##2##a–##FIG##1##f##, the mean RMSE (MBE) values are 0.21 (−0.09), 0.25 (−0.08), 0.3 (−0.07), and 0.... | [] | [
"<p id=\"Par1\">Accurate nowcasting for cloud fraction is still intractable challenge for stable solar photovoltaic electricity generation. By combining continuous radiance images measured by geostationary satellite and an advanced recurrent neural network, we develop a nowcasting algorithm for predicting cloud fra... | [
"<title>Supplementary information</title>",
"<p>\n\n\n\n</p>",
"<title>Source data</title>",
"<p>\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41467-023-44666-1.</p>",
"<title>Acknowledgements</title>",
"<p>We appreciate the Facebook Inc. for freely providing the Pytorch toolkit (<ext-link ext-link-type=\"uri\" xlink:href=\"https:... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Geographical distributions and photographs of ground-based sites.</title><p>PV plants (dark green small solid circles), 12 manual observation stations (red solid circles), 3 all-sky imager stations (blue solid circles), and 5 PV power test plants (yellow solid ... | [] | [
"<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepack... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>",
"<supplementary-material ... | [
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41467_2023_44666_Fig1_HTML\" id=\"d32e376\"/>",
"<graphic xlink:href=\"41467_2023_44666_Fig2_HTML\" id=\"d32e407\"/>",
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"<graphic xlink:href=\"4... | [
"<media xlink:href=\"41467_2023_44666_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media>",
"<media xlink:href=\"41467_2023_44666_MOESM2_ESM.pdf\"><caption><p>Peer review file</p></caption></media>",
"<media xlink:href=\"41467_2023_44666_MOESM3_ESM.pdf\"><caption><p>Reporting Summary</p... | [{"label": ["1."], "surname": ["Hofmann"], "given-names": ["M"], "article-title": ["A sustainable development pathway for climate action within the UN 2030 Agenda"], "source": ["Nat. Clim. Change"], "year": ["2021"], "volume": ["11"], "fpage": ["656"], "lpage": ["664"], "pub-id": ["10.1038/s41558-021-01098-3"]}, {"labe... | {
"acronym": [],
"definition": []
} | 37 | CC BY | no | 2024-01-15 23:42:00 | Nat Commun. 2024 Jan 13; 15:510 | oa_package/ba/ab/PMC10787801.tar.gz |
PMC10787822 | 38218883 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Long fusion surgery for adult spinal deformity (ASD), performed only in a limited number of centers more than a decade ago, has rapidly spread and is now a standard and widely performed procedure<sup>##REF##31305254##1##</sup>. ASD surgery was primarily performed for... | [
"<title>Methods</title>",
"<title>Patients</title>",
"<p id=\"Par5\">This study was a multicenter, self-report questionnaire survey conducted at two spine centers. In total, 106 patients were included: 97 patients who underwent long fusion surgery between 2007 and 2020 and nine patients who were undergoing cons... | [
"<title>Results</title>",
"<title>Demographics of the patients</title>",
"<p id=\"Par16\">Of a total of 98 patients, 88 were women. The mean age of the 89 operative patients was 68 ± 7 years, and the mean time since the last surgery was 56 ± 35 months (Table ##TAB##2##3##). The mean number of fixed vertebral se... | [
"<title>Discussion</title>",
"<p id=\"Par32\">This study is the first to use factor analysis to create a disease-specific scale for ASD. The most important point in a scale is to be able to measure the construct it is trying to measure<sup>##REF##29435801##10##,##UREF##2##31##</sup>. Factor analysis is a techniqu... | [
"<title>Conclusion</title>",
"<p id=\"Par46\">We developed a disease-specific outcome for ASD using factor analysis. This analysis is the first scientifically validated measure that could simultaneously assess the benefits and limitations of ASD surgery. This tool can complement existing outcomes and will be usef... | [
"<p id=\"Par1\">Adult spinal deformity (ASD) is a complex condition that combines scoliosis, kyphosis, pain, and postoperative range of motion limitation. The lack of a scale that can successfully capture this complex condition is a clinical challenge. We aimed to develop a disease-specific scale for ASD. The study... | [
"<title>Supplementary Information</title>",
"<p>\n</p>"
] | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51783-4.</p>",
"<title>Acknowledgements</title>",
"<p>This work was supported by a Grant from JSPS KAKENHI No. JP21K20966.</p>",
"<title>Author contributions</title>",
"<p>T.F... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Schematic of changes in a typical long fusion surgery. Preoperatively, the patient cannot maintain posture due to kyphotic deformity. Postoperatively, the patient can maintain posture, but has limited range of motion.</p></caption></fig>",
"<fig id=\"Fig2\"><la... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Review list of the questionnaires.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Abbreviation</th><th align=\"left\">Number of question items</th></tr></thead><tbody><tr><td align=\"left\">The 36-ite... | [
"<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepack... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><italic>SD</italic> standard deviation.</p></table-wrap-foot>",
"<table-wrap-foot><p>Raw data means the score points of the answer options. For raw data, higher numbers indicate more activity restrictions.</p><p><italic>SD</italic> standard deviation.</p></table-wrap-foot>",
"<table-wrap-fo... | [
"<graphic xlink:href=\"41598_2024_51783_Fig1_HTML\" id=\"MO1\"/>",
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"<graphic xlink:href=\"4... | [
"<media xlink:href=\"41598_2024_51783_MOESM1_ESM.xlsx\"><caption><p>Supplementary Information.</p></caption></media>"
] | [{"label": ["14."], "mixed-citation": ["Ware, J. E. "], "italic": ["SF-36 Physical and Mental Health Summary Scales: A User\u2019s Manual"]}, {"label": ["30."], "surname": ["Cohen"], "given-names": ["J"], "article-title": ["Statistical power analysis for the behavioral-sciences"], "source": ["Percept. Motor Skill"], "y... | {
"acronym": [],
"definition": []
} | 36 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 14; 14:1286 | oa_package/7e/00/PMC10787822.tar.gz |
PMC10787823 | 38218984 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Sugarcane juice is a nutritious and energetic green brownish beverage with low acidity (pH 4.8-5.5) and high water activity (Aw~0.99). The juice’s composition depends on the variety, cultivar, maturation stage, soil, climatic and agricultural conditions. Cane juice h... | [
"<title>Methods</title>",
"<p id=\"Par37\">The cane juice was procured from a local vendor, in the city of Pirassununga/SP-Brazil. The freshly extracted juice was collected in a plastic bottle by the vendor, kept on ice in an isothermal container, and rapidly transported to the laboratory in the Food Engineering ... | [
"<title>Results and discussion</title>",
"<title>Physicochemical tests</title>",
"<p id=\"Par6\">Table ##TAB##0##1## exhibits the pH and soluble solids values determined in raw and processed cane juice.</p>",
"<p id=\"Par7\">The pH values ranged from 4.6 to 6.0 in the raw juice and between 4.4 and 6.3 for the... | [
"<title>Results and discussion</title>",
"<title>Physicochemical tests</title>",
"<p id=\"Par6\">Table ##TAB##0##1## exhibits the pH and soluble solids values determined in raw and processed cane juice.</p>",
"<p id=\"Par7\">The pH values ranged from 4.6 to 6.0 in the raw juice and between 4.4 and 6.3 for the... | [] | [
"<p id=\"Par1\">Sugarcane juice is a nutritious and energetic drink. For its processing, the use of supercritical carbon dioxide (SC-CO<sub>2</sub>) technology as an intervention potentially capable of rendering a high quality product can be considered. This study evaluated the combined effect of SC-CO<sub>2</sub> ... | [
"<title>Supplementary information</title>",
"<p>\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41538-023-00242-x.</p>",
"<title>Acknowledgements</title>",
"<p>Grateful acknowledgment is made for the grant (2020/08011-5) provided by the Sao Paulo Research Foundation/Brazil (Fundação d... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Total color difference (TCD) between raw and SC-CO<sub>2</sub>-processed cane juice.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Pareto diagrams for cane juice treated with SC-CO<sub>2</sub>.</title><p>Pareto diagrams for mesophil... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Physicochemical parameters of raw and SC-CO<sub>2</sub>-treated cane juice.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Trial</th><th>Treatment</th><th>pH</th><th>Soluble solids (°Brix)</th></tr></thead><tbody><tr><td rowspan=\"3\">... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>Mean values of 3 replicates ± standard deviation.</p><p>Δ - variation.</p></table-wrap-foot>",
"<table-wrap-foot><p>Mean values of 3 replicates ± standard deviation.</p><p>est – estimate count (under detection limit).</p></table-wrap-foot>",
"<table-wrap-foot><p>Mean values of 3 replicates ... | [
"<inline-graphic xlink:href=\"41538_2023_242_Article_IEq1.gif\"/>",
"<graphic xlink:href=\"41538_2023_242_Fig1_HTML\" id=\"d32e2698\"/>",
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"<graphic xlink:hre... | [
"<media xlink:href=\"41538_2023_242_MOESM1_ESM.pdf\"><caption><p>Reporting summary</p></caption></media>"
] | [{"label": ["1."], "surname": ["Oliveira", "Spoto", "Canniatti-Brazaca", "Souza", "Sousa"], "given-names": ["ACG", "MHF", "SG", "CWO", "CP"], "article-title": ["Percep\u00e7\u00e3o dos consumidores sobre o com\u00e9rcio de alimentos de rua e avalia\u00e7\u00e3o do teste de mercado do caldo de cana processado e embalado... | {
"acronym": [],
"definition": []
} | 34 | CC BY | no | 2024-01-15 23:42:00 | NPJ Sci Food. 2024 Jan 13; 8:6 | oa_package/9f/0e/PMC10787823.tar.gz |
PMC10787824 | 38218946 | [
"<title>Introduction</title>",
"<p id=\"Par3\">Proton exchange membrane fuel cells (PEMFCs) have shown great potential as power sources for electric vehicle applications owing to their high energy conversion efficiency and environmental benign characteristics<sup>##REF##34677946##1##–##REF##33479539##4##</sup>. H... | [
"<title>Methods</title>",
"<title>Chemicals</title>",
"<p id=\"Par20\">Platinum (II) acetylacetonate (Pt(acac)<sub>2</sub>, 97%), nickel (II) acetylacetonate (Ni(acac)<sub>2</sub>, 97%), molybdenum (III) acetylacetonate (Mo(acac)<sub>3</sub>, 97%), gold (III) chloride trihydrate (HAuCl<sub>4</sub>·3H<sub>2</sub... | [
"<title>Results</title>",
"<title>Synthesis and characterizations of PtNi-based model catalysts</title>",
"<p id=\"Par6\">To explore the effect of Mo and Au dopants on the ORR performance of the PtNi catalyst, we synthesized a series of PtNi-based NWs, containing PtNi NWs, PtNiMo NWs, and PtNiAu NWs with the sa... | [
"<title>Discussion</title>",
"<p id=\"Par19\">To sum up, we have identified the distinct roles of Mo and Au dopants in inhibiting the dissolution of Ni and Pt metal, respectively, in terms of PtNi-based NWs/C as model catalysts, by combining the experimental evidences and DFT calculations. Insightful studies indi... | [] | [
"<p id=\"Par1\">Stabilizing active PtNi alloy catalyst toward oxygen reduction reaction is essential for fuel cell. Doping of specific metals is an empirical strategy, however, the atomistic insight into how dopant boosts the stability of PtNi catalyst still remains elusive. Here, with typical examples of Mo and Au... | [
"<title>Supplementary information</title>",
"<p>\n\n\n</p>",
"<title>Source data</title>",
"<p>\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41467-024-44788-0.</p>",
"<title>Acknowledgements</title>",
"<p>This work was supported by the National Key Research and Development Program of China (No. 2021YFA1502000 to H.H.), NSFC (Nos... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Structural characterizations and ORR performance.</title><p>HAADF-STEM images and EDS EDS line-scanning profiles of <bold>a</bold> PtNi NWs, <bold>b</bold> PtNiMo NWs, and <bold>c</bold> PtNiAu NWs. <bold>d</bold> XRD patterns of PtNi-bassed NWs. <bold>e</bold>... | [] | [
"<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepack... | [] | [] | [] | [] | [
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"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Lei Gao, Tulai Sun, Xuli Chen.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41467_2024_44788_Fig1_HTML\" id=\"d32e364\"/>",
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"<graphic xlink:href=\"4... | [
"<media xlink:href=\"41467_2024_44788_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media>",
"<media xlink:href=\"41467_2024_44788_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media>",
"<media xlink:href=\"41467_2024_44788_MOESM3_ESM.xlsx\"><caption><p>Source Data</p></ca... | [{"label": ["2."], "surname": ["Wang", "Swihart", "Wu"], "given-names": ["XX", "MT", "G"], "article-title": ["Achievements, challenges and perspectives on cathode catalysts in proton exchange membrane fuel cells for transportation"], "source": ["Nat. Catal."], "year": ["2019"], "volume": ["2"], "fpage": ["578"], "lpage... | {
"acronym": [],
"definition": []
} | 47 | CC BY | no | 2024-01-15 23:42:00 | Nat Commun. 2024 Jan 13; 15:508 | oa_package/49/d6/PMC10787824.tar.gz |
PMC10787825 | 38218897 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Abiotic factors play a pivotal role in the decline of agricultural and allied sector production. Climate change, pollution, and degraded water quality stand out as major abiotic influencers shaping the life patterns of aquatic organisms, including fish<sup>##UREF##0#... | [
"<title>Materials and methods</title>",
"<title>Ethics statement</title>",
"<p id=\"Par9\">The Research Advisory Committee (RAC) of the Institute (ICAR-National Institute of Abiotic Stress Management, Baramati, Pune) has approved the experimental procedures, and this study adheres to the Animal Research: Report... | [
"<title>Results</title>",
"<p id=\"Par28\">3.1. Effect of Mn on cortisol levels.</p>",
"<p id=\"Par29\">In the present investigation, cortisol levels were assessed in response to dietary manganese (Mn) at 4, 8, and 12 mg kg<sup>−1</sup> diet fed to <italic>P. hypophthalmus</italic>. The fish were reared under n... | [
"<title>Discussion</title>",
"<p id=\"Par45\">Cortisol is secreted from the inter-renal cell of head kidney released directly into the blood<sup>##UREF##30##55##</sup>. It is an important stress hormone plays a role in growth, reproduction, and osmoregulation<sup>##REF##30667059##56##</sup>. The multiple stressor... | [
"<title>Conclusion</title>",
"<p id=\"Par60\">The present study is the first report on the role of manganese (Mn) on gene regulations and biochemical regulators in response to arsenic and ammonia toxicity and high-temperature stress in <italic>P. hypophthalmus</italic>. The immunity, anti-oxidative status, growth... | [
"<p id=\"Par1\">The ongoing challenges of climate change and pollution are major factors disturbing ecosystems, including aquatic systems. They also have an impact on gene regulation and biochemical changes in aquatic animals, including fish. Understanding the mechanisms of gene regulation and biochemical changes d... | [] | [
"<title>Acknowledgements</title>",
"<p>The present work was supported by Indian Council of Agricultural Research (ICAR), New Delhi, India under Project “Lal bahadur Shastri Young Scientist Award (Project code: OXX5181). Authors also sincerely acknowledged to Director ICAR-NIASM for providing research facilities f... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Manganese diets control the cortisol and gene expression of HSP 70, CYP 450, DNA damage inducible protein (DDIP) and metallothionine (MT) against multiple stressors in fish. Within endpoints and groups, bars with different superscripts differ significantly (a–h) ... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>experimental design of present investigation.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">S. No</th><th align=\"left\">Details of the treatments</th><th align=\"left\">Notation</th></tr></thead><tbody><tr><td align=\"... | [
"<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t... | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p><sup>a</sup>Procured from local market, <sup>b</sup>Himedia Ltd, Himedia Ltd, <sup>c</sup>SD Fine Chemicals Ltd., India.</p><p>*Manual prepared Vitamin mineral mixture; Composition of vitamin mineral mix (quantity/250 g starch powder): vitamin A 55,00,00 IU; vitamin D3 11,00,00 IU; vitamin B1:2... | [
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"<graphic xlink:href=\"41598_2024_51740_Article_Equd.gif\" po... | [] | [{"label": ["1."], "surname": ["Kumar", "Gupta", "Bhushan", "Singh"], "given-names": ["N", "SK", "S", "NP"], "article-title": ["Impacts of acute toxicity of arsenic (III) alone and with high temperature on stress biomarkers, immunological status and cellular metabolism in fish"], "source": ["Aquat. Toxicol."], "year": ... | {
"acronym": [],
"definition": []
} | 105 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1273 | oa_package/a7/4f/PMC10787825.tar.gz |
PMC10787826 | 38218973 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Ki-67 immunohistochemistry (IHC) serves as a reliable marker of cell proliferation and is widely used to evaluate the aggressiveness and prognosis of human tumors. Notably, Ki-67 has been adopted for prognostication in breast cancer, with elevated Ki-67 expression co... | [
"<title>Materials and methods</title>",
"<p id=\"Par7\">Ethics approval for the study was obtained from Toronto Metropolitan University (REB: 2022-154). All experiments were performed in accordance with the Tri-Council policy statement 2 for the ethical conduct of research involving humans.</p>",
"<title>Case s... | [
"<title>Results</title>",
"<title>Scoring accuracy</title>",
"<p id=\"Par22\">The respondents' PI scores and PI errors per case and within ranges are shown in Table ##TAB##0##1##. Responses including outliers are shown in Supplementary Table ##SUPPL##0##2##. The overall mean PI error was found to be 2.1 (2.2) u... | [
"<title>Discussion</title>",
"<p id=\"Par33\">Ki-67 serves as a crucial indicator for predicting cancer recurrence and survival among early-stage high-risk breast cancer patients<sup>##REF##28154782##1##,##REF##20152769##2##</sup>. It informs decisions regarding adjuvant chemotherapy<sup>##UREF##0##12##</sup> and... | [] | [
"<p id=\"Par1\">The Ki-67 proliferation index (PI) guides treatment decisions in breast cancer but suffers from poor inter-rater reproducibility. Although AI tools have been designed for Ki-67 assessment, their impact on pathologists' work remains understudied. 90 international pathologists were recruited to assess... | [
"<title>Supplementary Information</title>",
"<p>\n</p>"
] | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51723-2.</p>",
"<title>Acknowledgements</title>",
"<p>This research acknowledges the Canadian Cancer Society and Mitacs.</p>",
"<title>Author contributions</title>",
"<p>A.D. ... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Examples of TMA with no AI aid (left) and TMA with AI tool overlay and calculated proliferation index (PI) (right). The TMA shown is case 7.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Graphs of absolute PI error. (<bold>A</bold>)... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>PI scores, PI error and ground truth are shown as mean (SD) per case and within PI ranges. </p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Case</th><th align=\"left\" rowspan=\"2\">Ground truth</th><th alig... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>*The p values were computed for paired comparisons between pathologists and pathologists with AI with the paired Wilcoxon signed-rank test.</p><p>Significant values are in bold.</p></table-wrap-foot>",
"<table-wrap-foot><p>The * highlights situations where the consensus misaligns with the est... | [
"<graphic xlink:href=\"41598_2024_51723_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51723_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"41598_2024_51723_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"41598_2024_51723_Fig4_HTML\" id=\"MO4\"/>",
"<graphic xlink:href=\"41598_2024_51723_... | [
"<media xlink:href=\"41598_2024_51723_MOESM1_ESM.docx\"><caption><p>Supplementary Information.</p></caption></media>"
] | [{"label": ["12."], "surname": ["Faragalla"], "given-names": ["H"], "article-title": ["Ki67 in breast cancer assay: An ad hoc testing recommendation from the Canadian Association of Pathologists Task Force"], "source": ["Curr. Oncol. (Tor. Ont.)"], "year": ["2023"], "volume": ["30"], "fpage": ["3079"], "lpage": ["3090"... | {
"acronym": [],
"definition": []
} | 45 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1283 | oa_package/90/05/PMC10787826.tar.gz |
PMC10787827 | 38218952 | [
"<title>Introduction</title>",
"<p id=\"Par2\">In schizophrenia, only ~70% of affected individuals respond to antipsychotic drug treatment, and even fewer go into remission [##REF##26925705##1##, ##REF##22834451##2##]. Pre-treatment prediction of the subsequent response could reduce the time spent on ineffective ... | [
"<title>Methods</title>",
"<title>Participants</title>",
"<p id=\"Par8\">A total of 68 drug-naive patients between 18 and 45 years old were diagnosed with schizophrenia based on the Structured Clinical Interview for the DSM-IV Axis I disorder and had no previous psychiatric treatment. Patients underwent MRI sca... | [
"<title>Results</title>",
"<title>12-Week treatment outcome</title>",
"<p id=\"Par15\">By the end of the 12th week, 30 patients met the remission criteria as responders, and 20 patients were classified as nonresponders. Demographic and clinical variables at baseline were not significantly different between resp... | [
"<title>Discussion</title>",
"<p id=\"Par18\">Here, we provide evidence for a baseline psychotic symptom-related white matter tract biomarker that potentially predicts response to antipsychotic treatment in first-episode schizophrenia. Importantly, the study sample was treatment-naive to ensure that the findings ... | [] | [
"<p id=\"Par1\">There is significant heterogeneity in individual responses to antipsychotic drugs, but there is no reliable predictor of antipsychotics response in first-episode psychosis. This study aimed to investigate whether psychotic symptom-related alterations in fractional anisotropy (FA) and mean diffusivit... | [
"<title>Supplementary information</title>",
"<p>\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41398-023-02714-w.</p>",
"<title>Acknowledgements</title>",
"<p>This study was supported by the National Major Project of Scientific and Technical Supporting Programs of China during the 11... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Relationship between diffusion properties and the severity of core symptoms at baseline in first-episode schizophrenia.</title><p><bold>A</bold> Fiber tracts significantly associated with symptoms. <bold>B</bold> The red lines indicate MD of fiber tracts were p... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Clinical and demographic information for the first-episode schizophrenia patients.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Characteristic</th><th>Responders, <italic>N</italic> = 30</th><th>Nonresponders, <italic>N</italic> = 20... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><italic>ILF</italic> longitudinal fasciculi, <italic>IFOF</italic> inferior fronto-occipital fasciculi, <italic>SLF</italic> superior longitudinal fasciculus, <italic>FA</italic> fractional anisotropy, <italic>MD</italic> mean diffusivity.</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publi... | [
"<graphic xlink:href=\"41398_2023_2714_Fig1_HTML\" id=\"d32e605\"/>",
"<graphic xlink:href=\"41398_2023_2714_Fig2_HTML\" id=\"d32e760\"/>"
] | [
"<media xlink:href=\"41398_2023_2714_MOESM1_ESM.docx\"><caption><p>Supplement materials</p></caption></media>"
] | [{"label": ["4."], "surname": ["Conley", "Kelly"], "given-names": ["R", "D"], "article-title": ["Current status of antipsychotic treatment"], "source": ["Curr Drug Targets CNS Neurol Disord"], "year": ["2022"], "volume": ["1"], "fpage": ["123"], "lpage": ["8"], "pub-id": ["10.2174/1568007024606221"]}, {"label": ["14."]... | {
"acronym": [],
"definition": []
} | 49 | CC BY | no | 2024-01-15 23:42:00 | Transl Psychiatry. 2024 Jan 13; 14:23 | oa_package/51/c8/PMC10787827.tar.gz |
PMC10787828 | 38218965 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Modern science has become increasingly collaborative over the past decades<sup>##REF##17431139##1##</sup>. Large teams have become almost necessary to tackle complex problems in various disciplines, requiring a large pool of knowledge and skills. On the other hand, s... | [
"<title>Methods</title>",
"<p id=\"Par31\">Data We analyze papers from the February 2023 snapshot of the bibliometric dataset OpenAlex: the successor to Microsoft Academic Graph (MAG). We found incomplete citation coverage for papers published before 1990. So, we only consider papers published between 1990 and 20... | [
"<title>Results</title>",
"<p id=\"Par7\">We use the scientific publication dataset OpenAlex<sup>##UREF##9##23##</sup>. We present the results for twenty topics belonging to three disciplines: Physics, Computer Science, and Biology & Medicine. See “<xref rid=\"Sec6\" ref-type=\"sec\">Methods</xref>” for detai... | [
"<title>Discussion</title>",
"<p id=\"Par24\">Collaboration allows scholars to deepen existing knowledge and be exposed to new ideas. In this paper, we assessed if and how collaboration patterns affect the probability of switching research topics. We determined that the probability for a scholar to start working ... | [] | [
"<p id=\"Par1\">Collaboration is a key driver of science and innovation. Mainly motivated by the need to leverage different capacities and expertise to solve a scientific problem, collaboration is also an excellent source of information about the future behavior of scholars. In particular, it allows us to infer the... | [
"<title>Supplementary Information</title>",
"<p>\n</p>"
] | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51606-6.</p>",
"<title>Acknowledgements</title>",
"<p>We acknowledge the support of the AccelNet-MultiNet program, a project of the National Science Foundation (Award #1927425 and... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Schematic setup for our analysis. (<bold>a</bold>) Stream of papers across interaction (IW) and activation (AW) windows. Papers tagged with the focal topic <italic>t</italic> are marked in red. (<bold>b</bold>) Author collaboration graph at the end of IW. Authors... | [] | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Sara Venturini and Satyaki Sikdar.</p></fn></fn-group>"
] | [
"<inline-graphic xlink:href=\"41598_2024_51606_Article_IEq1.gif\"/>",
"<inline-graphic xlink:href=\"41598_2024_51606_Article_IEq2.gif\"/>",
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"<inline-graphic xlink:href=\"41598_2024_51606_Article_IEq4.gif\"/>",
"<inline-graphic xlink:href=... | [
"<media xlink:href=\"41598_2024_51606_MOESM1_ESM.pdf\"><caption><p>Supplementary Information.</p></caption></media>"
] | [{"label": ["5."], "surname": ["Pan", "Kaski", "Fortunato"], "given-names": ["RK", "K", "S"], "article-title": ["World citation and collaboration networks: Uncovering the role of geography in science"], "source": ["Sci. Rep."], "year": ["2012"], "volume": ["2"], "fpage": ["1"], "lpage": ["7"], "pub-id": ["10.1038/srep0... | {
"acronym": [],
"definition": []
} | 34 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1258 | oa_package/58/e7/PMC10787828.tar.gz |
PMC10787829 | 38218954 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Coronary artery disease (CAD) is the leading cause of mortality around the globe. In developed and developing countries, CAD is the foremost cause of death. According to a previous study, people in the Middle East demonstrate heart disease at younger ages<sup>##REF##... | [
"<title>Methods</title>",
"<title>Sample collection</title>",
"<p id=\"Par39\">The present study recruited 93 premature CAD and 87 non-CAD age-matched subjects hospitalized at Rajaie Cardiovascular Medical and Research Center, Tehran, Iran, between 2017 and 2019. All patients were diagnosed by angiography. A qu... | [
"<title>Results</title>",
"<title>Clinical and demographic features represented an association with premature CAD</title>",
"<p id=\"Par13\">The results of clinical and demographic characteristic analysis are included in Table ##TAB##0##1##. The levels of triglyceride, total cholesterol, low-density lipoprotein... | [
"<title>Discussion</title>",
"<p id=\"Par23\">Atherosclerosis normally progresses in the form of late plaques in the fourth decade of life and involves older people. Nonetheless, in some cases, women aged below 55 and men younger than 45 experience CAD symptoms<sup>##REF##31601367##3##</sup>. The principal cause ... | [] | [
"<p id=\"Par1\">Coronary artery disease (CAD) is the major cause of mortality in the world. Premature development of CAD can be attributed to women under 55 and men under 45. Many genetic factors play a part in premature CAD. Among them, <italic>ANRIL</italic>, a long noncoding RNA is located at the 9p21 risk locus... | [
"<title>Limitations of this study</title>",
"<p id=\"Par38\">It is necessary to consider the limitations of the current study when interpreting its outcomes. Firstly, our small sample size precludes the generalizability of our results concerning the association of rs10757274, rs2383206, rs2383207, rs496892, rs107... | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51715-2.</p>",
"<title>Author contributions</title>",
"<p>E.T.B.: experiment design, lab work, data production, data interpretation, writing first draft of manuscript; A.Z., F.S.,... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Genomic location of <italic>ANRIL</italic> (<italic>CDKN2B-AS1</italic>) and its splicing variants. <italic>ANRIL</italic> resides in 9p21 locus and spans approximately 126 kb of genomic DNA, comprising 20 exons in total (black rectangles) and 14 splicing isoform... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Clinical and demographic characteristics of the included patients.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Parameters</th><th align=\"left\">CAD (n = 93)</th><th align=\"left\">Non-CAD (n = 87)</th><th align=\"lef... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><italic>BMI</italic> body mass index, <italic>CAD</italic> Coronary Artery Disease, <italic>DBP</italic> diastolic blood pressure, <italic>FBS</italic> fasting blood sugar, <italic>HDL-C</italic> high-density lipoprotein-cholesterol, <italic>LDL-C</italic> low-density lipoprotein-cholesterol, <... | [
"<graphic xlink:href=\"41598_2024_51715_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51715_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"41598_2024_51715_Fig3_HTML\" id=\"MO3\"/>"
] | [
"<media xlink:href=\"41598_2024_51715_MOESM1_ESM.pdf\"><caption><p>Supplementary Information.</p></caption></media>"
] | [{"label": ["6."], "mixed-citation": ["Lawford, P.R.H.A.P.V. Atherosclerosis. in "], "italic": ["Cardiovascular Biomechanics"]}, {"label": ["8."], "mixed-citation": ["Hu, L., G. Su, & X. Wang. The roles of ANRIL polymorphisms in coronary artery disease: A meta-analysis. "], "italic": ["Biosci. Rep"], "bold": ["39"]}, {... | {
"acronym": [],
"definition": []
} | 43 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1244 | oa_package/2d/75/PMC10787829.tar.gz |
PMC10787830 | 38218945 | [
"<title>Introduction</title>",
"<p id=\"Par2\">The presence of cell-in-cell (CIC) structures has been observed in a wide range of human cancers and is mainly associated with poor prognosis [##REF##26504802##1##–##REF##34178655##7##]. CIC formation involves a dynamic interaction between an outer and an inner cell.... | [
"<title>Materials/subjects and methods</title>",
"<title>Cell culture</title>",
"<p id=\"Par18\">The liver cancer cell lines (Hep3B, Huh7, HepG2, Huh6) and the breast adenocarcinoma cell line MCF-7 were cultured in Dulbecco modified Eagle’s medium (DMEM 1x glutamax, Fisher Scientific) supplemented with 10% heat... | [
"<title>Results</title>",
"<title>Liver cancer cells are prone to perform entosis</title>",
"<p id=\"Par4\">In order to investigate entosis in liver cancer, cultured cells (Hep3B, Huh7, Huh6, or HepG2) were subjected to stress conditions known to favor entosis in breast cancer cells such as nutrient deprivation... | [
"<title>Discussion</title>",
"<p id=\"Par13\">In this study, we characterized entosis in liver cancer cells, which resembles to that largely described in the literature in breast cancer cells [##REF##27048820##23##]. We demonstrated that entosis could be induced by matrix detachment and nutrient deprivation in HC... | [] | [
"<p id=\"Par1\">Entosis is a process that leads to the formation of cell-in-cell structures commonly found in cancers. Here, we identified entosis in hepatocellular carcinoma and the loss of Rnd3 (also known as RhoE) as an efficient inducer of this mechanism. We characterized the different stages and the molecular ... | [
"<title>Supplementary information</title>",
"<p>\n\n\n\n\n\n\n\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41419-024-06420-3.</p>",
"<title>Acknowledgements</title>",
"<p>We thank N. Dugot-Senant from the Histopathology platform (TBMCore, UMS005, Bordeaux). Flow cytometry, hypoxia, and qRT-PCR e... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Liver cancer cells perform entosis upon starvation and matrix detachment.</title><p><bold>A</bold> Representative Hep3B entotic cell characterized using confocal microscopy by the nucleus deformation showed with the DAPI staining (blue) and high concentration o... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Clinical and tumoral characteristics of patients.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Clinical characteristics</th><th>Value</th></tr></thead><tbody><tr><td>Age (mean ± standard deviation)</td><td>69.5 ± 9.1 years</td></tr><... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>",
"<supplementary-material ... | [
"<fn-group><fn><p>Edited by Professor Stephen Tait</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41419_2024_6420_Fig1_HTML\" id=\"d32e374\"/>",
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"<graphic xlink:href=\"41419... | [
"<media xlink:href=\"41419_2024_6420_MOESM1_ESM.pdf\"><caption><p>Supplemental Figures</p></caption></media>",
"<media xlink:href=\"41419_2024_6420_MOESM2_ESM.xlsx\"><caption><p>Supplemental Table 1</p></caption></media>",
"<media xlink:href=\"41419_2024_6420_MOESM3_ESM.xlsx\"><caption><p>Supplemental Table 2</... | [{"label": ["13."], "mixed-citation": ["Durgan J, Tseng YY, Hamann JC, Domart MC, Collinson L, Hall A, et al. Mitosis can drive cell cannibalism through entosis. Elife [Internet]. 2017;6. Disponible sur: "], "ext-link": ["https://www.ncbi.nlm.nih.gov/pubmed/28693721"]}, {"label": ["16."], "surname": ["Basbous", "Azzare... | {
"acronym": [],
"definition": []
} | 33 | CC BY | no | 2024-01-15 23:42:00 | Cell Death Dis. 2024 Jan 13; 15(1):46 | oa_package/9b/d5/PMC10787830.tar.gz |
PMC10787831 | 38218871 | [
"<title>Introduction</title>",
"<p id=\"Par3\">Ferroelectricity, characterized by a remanent and switchable polarization, just experiences the 100th anniversary of its first discovery in Rochelle salt in 1920<sup>##UREF##0##1##,##UREF##1##2##</sup>. The research and application of ferroelectrics were first limite... | [
"<title>Methods</title>",
"<title>P(VDF-TrFE) based FFD</title>",
"<p id=\"Par25\">The structure and fabrication process of the designed FFD are orderly presented in Fig. ##FIG##1##2a##. First of all, the striped 100-nm Pt films sputtered by DC magnetron sputtering instrument on the cleaned SiO<sub>2</sub>/Si w... | [
"<title>Results</title>",
"<title>Structure of two-terminal FFD</title>",
"<p id=\"Par6\">Ferroelectric thin films sandwiched between a top electrode and a bottom electrode are used to support a sidewall semiconductor, forming a vertical fin-like structure where the channel length defined by the thickness of th... | [
"<title>Discussion</title>",
"<p id=\"Par24\">A robust ferroelectric-based non-volatile memory with a novel FFD structure is proposed as a new building block for future electronic circuit architectures. The device absorbs merits of non-destructive read mode with resistive switching as in FTJs and long endurance a... | [] | [
"<p id=\"Par1\">Among today’s nonvolatile memories, ferroelectric-based capacitors, tunnel junctions and field-effect transistors (FET) are already industrially integrated and/or intensively investigated to improve their performances. Concurrently, because of the tremendous development of artificial intelligence an... | [
"<title>Supplementary information</title>",
"<p>\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41467-024-44759-5.</p>",
"<title>Acknowledgements</title>",
"<p>B.T. would like to thank fundings of National Key Research and Development Program of China (No. 2021YFA1200700), National Na... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Comparison of ferroelectric memory performances.</title><p><bold>a</bold>–<bold>d</bold> Memory performances in the ferroelectric random access memory capacitor (FeRAM) (<bold>a</bold>), ferroelectric tunnel junctions (FTJ) (<bold>b</bold>), ferroelectric field... | [] | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Guangdi Feng, Qiuxiang Zhu.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41467_2024_44759_Fig1_HTML\" id=\"d32e530\"/>",
"<graphic xlink:href=\"41467_2024_44759_Fig2_HTML\" id=\"d32e643\"/>",
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"<inline-graphic xlink:... | [
"<media xlink:href=\"41467_2024_44759_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media>",
"<media xlink:href=\"41467_2024_44759_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media>"
] | [{"label": ["1."], "mixed-citation": ["Valasek, J. A century of ferroelectricity. "], "italic": ["Nat. Mater."], "bold": ["19"]}, {"label": ["2."], "surname": ["Valasek"], "given-names": ["J"], "article-title": ["Piezo-electric and allied phenomena in Rochelle salt"], "source": ["Phys. Rev."], "year": ["1921"], "volume... | {
"acronym": [],
"definition": []
} | 53 | CC BY | no | 2024-01-15 23:42:00 | Nat Commun. 2024 Jan 13; 15:513 | oa_package/09/5c/PMC10787831.tar.gz |
PMC10787832 | 38218926 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Industries that work with dyestuff, textiles, leather, paper, plastics, etc., typically drain synthetic dyes along with their effluent. The synthetic dye wastewater is mainly composed of organic components, which are complex and easy to show color in water, and usual... | [
"<title>Materials and methods</title>",
"<title>Materials</title>",
"<p id=\"Par5\">Chitosan medium molecular weight (≥ 75% DD) and methyl orange dye (MO; 85%) were procured from Sigma-Aldrich (Germany). The compound 2-Chloro ethyl amine hydrochloride (99%) was acquired from Sigma-Aldrich (Germany). The other c... | [
"<title>Results and discussion</title>",
"<title>Characterization</title>",
"<title>Physicochemical characterization</title>",
"<p id=\"Par14\">The confirmation of the immobilisation of excess amine into the chitosan backbone is achieved through the measurement of ion exchange capacity. Figure ##FIG##0##1## i... | [
"<title>Results and discussion</title>",
"<title>Characterization</title>",
"<title>Physicochemical characterization</title>",
"<p id=\"Par14\">The confirmation of the immobilisation of excess amine into the chitosan backbone is achieved through the measurement of ion exchange capacity. Figure ##FIG##0##1## i... | [
"<title>Conclusion</title>",
"<p id=\"Par33\">Novel amino-ethyl Chitosan derivatives (CS-ENH2) with varied and higher amine group content than Chitosan have been developed using one step click chemistry reaction of Chitosan (CS) with 2-chloroethylamine (ENH2) amination reagent, then crosslinked using Glutaraldehy... | [
"<p id=\"Par1\">The present study introduces a new and straightforward method for the amination of Chitosan. This method involves coupling Chitosan (CS) with 2-chloroethylamine (ENH2) in a single step to produce an amino-ethyl Chitosan derivatives with increased amine group content (CS-ENH2) using click chemistry. ... | [] | [
"<title>Author contributions</title>",
"<p>Prof. Dr. A.M.O. and Prof. Dr. T.M.T. have proposed the point of research, design the experimental work, and follow the executive of the work. Prof. Dr. W.A.S. and Prof. Dr. R.A. follow the executive of the work, and supervise the writing of the first draft of the manusc... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Water uptake and ion exchange capacity of Chitosan and amino-ethyl Chitosan hydrogels.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>FTIR of Chitosan and amino-ethyl Chitosan hydrogels.</p></caption></fig>",
"<fig id=\"Fig3\"><lab... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparison of the Adsorption Capacity of CS-NH2-4 adsorbent with Other Adsorbents.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Adsorbent</th><th align=\"left\">Adsorption capacity (mg/g)</th><th align=\"left\">Referen... | [
"<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepack... | [] | [] | [] | [] | [] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41598_2024_51538_Article_Equ1.gif\" position=\"anchor\"/>",
"<graphic xlink:href=\"41598_2024_51538_Article_Equ2.gif\" position=\"anchor\"/>",
"<graphic xlink:href=\"41598_2024_51538_Article_Equ3.gif\" position=\"anchor\"/>",
"<graphic xlink:href=\"41598_2024_51538_Fig1_HTML\" id=\"MO1\... | [] | [{"label": ["3."], "surname": ["Chang", "Lin"], "given-names": ["JS", "CY"], "article-title": ["Decolorization kinetics of a recombinant "], "italic": ["Escherichia coli"], "source": ["Biotechnol. Lett."], "year": ["2001"], "volume": ["23"], "fpage": ["631"], "lpage": ["636"], "pub-id": ["10.1023/A:1010306114286"]}, {"... | {
"acronym": [],
"definition": []
} | 56 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1284 | oa_package/71/63/PMC10787832.tar.gz |
PMC10787833 | 38218895 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Atrial fibrillation (AF) is the most common sustained arrhythmia and it is a major public health problem worldwide associated with substantial morbidity and mortality<sup>##REF##8114238##1##,##REF##27057292##2##</sup>. Risk factors for atrial fibrillation include age... | [
"<title>Methods</title>",
"<title>Study design</title>",
"<p id=\"Par5\">This was a retrospective observational cross – sectional study that reviewed records of patients diagnosed with hypertension and admitted to the cardiology department of King Abdullah university hospital (KAUH) in Irbid, Jordan from 1/1/20... | [
"<title>Results</title>",
"<p id=\"Par12\">There was a total of 958 hypertensive patients included in the study. The mean age of study patients was 61.40 (± 11.46) years, most of them were males (65.4%), and 40.3% were current smokers. There was a high prevalence of coronary artery disease (CAD) and diabetes mell... | [
"<title>Discussion</title>",
"<p id=\"Par17\">Atrial fibrillation is associated with several risk factors and predisposing conditions that increases its occurrence, including hypertension that accelerates the development and progression of AF<sup>##REF##18060995##18##</sup>. The prevalence of AF in this current s... | [
"<title>Conclusion</title>",
"<p id=\"Par25\">Findings suggest that LA enlargement in hypertensive patients play a major role in the development of AF through structural and electrophysiological changes to the atrium and other possible mechanisms<sup>##REF##28592961##21##</sup>. Although there are several risk fa... | [
"<p id=\"Par1\">Atrial fibrillation (AF) is the most common sustained arrhythmia and it is a major public health problem worldwide. Hypertension is one of the major risk factors for the development of AF. This study is carried out to determine the prevalence and independent risk factors for atrial fibrillation (AF)... | [] | [
"<title>Author contributions</title>",
"<p>A.S. and R.S. conceptualization and study design. A.S. and B.J. methodology and supervised data collection. T.Z., S.G. and T.A. data collection and validation. R.S. analysis. A.S. and R.S. writing the manuscript.</p>",
"<title>Data availability</title>",
"<p>The data... | [] | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Demographic and other characteristics of patients.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Characteristics</th><th align=\"left\">Atrial fibrillation</th><th align=\"left\">No atrial fibrillation</th><th align=\"l... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>Categorical variables were analyzed using chi – square test of independence.</p><p>Continuous variables were analyzed using student t-test.</p><p>*The percentage in total column represents total percentage of each row.</p><p>Significant values are in bold.</p></table-wrap-foot>",
"<table-wrap... | [] | [] | [{"label": ["10."], "surname": ["Widimsk\u00fd"], "given-names": ["J"], "article-title": ["Arterial hypertension and atrial fibrillation: Selecting antihypertensive therapy"], "source": ["Cor et Vasa"], "year": ["2012"], "volume": ["54"], "fpage": ["e248"], "lpage": ["e252"], "pub-id": ["10.1016/j.crvasa.2012.08.004"]}... | {
"acronym": [],
"definition": []
} | 29 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1250 | oa_package/2e/61/PMC10787833.tar.gz |
PMC10787834 | 38218739 | [] | [] | [] | [] | [
"<title>Conclusion and outlook</title>",
"<p id=\"Par20\">There is ample evidence that efferocytosis by neutrophils plays an important role in the response to dead cell accumulation. During inflammation, they contribute to the clearance of aggregates of apoptotic neutrophils. In cancer, they participate in the re... | [
"<p id=\"Par1\">When a cell dies of apoptosis, it is eliminated either by neighbouring cells or by attracted professional phagocytes. Although it was generally believed that neutrophils also have the ability to perform efferocytosis, their contribution to the clearance of apoptotic cells was considered less importa... | [
"<title>FACTS</title>",
"<p id=\"Par2\">\n<list list-type=\"bullet\"><list-item><p id=\"Par3\">Apoptotic cells release “find-me” signals which attract predominantly neutrophils.</p></list-item><list-item><p id=\"Par4\">Neutrophils accumulate in areas of tissue damage and apoptotic tumour cells.</p></list-item><li... | [
"<title>Acknowledgements</title>",
"<p>This study was supported by the Medical University of Vienna.</p>",
"<title>Author contributions</title>",
"<p>CR and RO performed writing, review and revision of the paper. Both authors read and approved the final paper.</p>",
"<title>Data availability</title>",
"<p... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Efferocytosis by neutrophils.</title><p>In response to sterile injury or local infection, neutrophils migrate to the site of tissue damage in a multistep process termed “neutrophils swarming”. Neutrophils undergo apoptosis after phagocytosis of invading pathoge... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Correlative assessment of spontaneous apoptosis in situ with cancer patients’ outcome in several tumour types.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Tumour type</th><th>Method to detect apoptosis</th><th>OS<sup>a</sup></th><th... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>Patient-derived histological samples were analyzed by using either DNA end-labelling techniques (TUNEL), plain morphology combined with hematoxylin and eosin (H&E), immunohistochemistry (IHC), flow cytometry (FC), or enzyme-linked immunosorbent assay (ELISA). Clinical studies in which patie... | [
"<graphic xlink:href=\"41420_2024_1809_Fig1_HTML\" id=\"d32e269\"/>",
"<graphic xlink:href=\"41420_2024_1809_Fig2_HTML\" id=\"d32e402\"/>"
] | [] | [{"label": ["5."], "mixed-citation": ["Kirschnek S, Ying S, Fischer SF, H\u00e4cker H, Villunger A, Hochrein H, et al. Phagocytosis-induced apoptosis in macrophages is mediated by up-regulation and activation of the Bcl-2 homology domain 3-only protein bim. J Immunol. 2005. 10.4049/jimmunol.174.2.671."]}, {"label": ["2... | {
"acronym": [],
"definition": []
} | 89 | CC BY | no | 2024-01-15 23:42:00 | Cell Death Discov. 2024 Jan 13; 10:26 | oa_package/5b/91/PMC10787834.tar.gz |
PMC10787835 | 38218730 | [
"<title>Introduction</title>",
"<p id=\"Par3\">In a non-centrosymmetric material, light-matter interactions can generate a finite DC photocurrent under homogeneous illumination in absence of external bias and spatial inhomogeneity. This photovoltaic effect, governed by the intrinsic symmetry properties of materia... | [
"<title>Methods</title>",
"<title>Sample preparation</title>",
"<p id=\"Par16\">ReS<sub>2</sub> samples were prepared on silicon substrate covered with 300 nm SiO<sub>2</sub> through standard mechanical exfoliation method. Angle-resolved polarized optical microscope is used to identify GBs and domains in ReS<su... | [
"<title>Results</title>",
"<title>Characterization of 1D vdW GBs in ReS<sub>2</sub></title>",
"<p id=\"Par5\">Bulk 1 T′-ReS<sub>2</sub> is a vdW semiconductor with a centrosymmetric crystalline structure under the inversion symmetric space group of <italic>P</italic><sup>##REF##26280493##35##–##REF##26390381##3... | [
"<title>Discussion</title>",
"<p id=\"Par13\">To better understand the giant IPVE photocurrent densities in ReS<sub>2</sub> GBs and its underlying physical mechanism, the first-principles calculations of band properties are performed. Detailed information about calculations can be found in the Method Section and ... | [] | [
"<p id=\"Par1\">The photovoltaic effect lies at the heart of eco-friendly energy harvesting. However, the conversion efficiency of traditional photovoltaic effect utilizing the built-in electric effect in p-n junctions is restricted by the Shockley-Queisser limit. Alternatively, intrinsic/bulk photovoltaic effect (... | [
"<title>Supplementary information</title>",
"<p>\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41467-024-44792-4.</p>",
"<title>Acknowledgements</title>",
"<p>The work was financially supported by the National Natural Science Foundation of China (62275117, X.C.; 62261136552, J.M.; 52... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Characterization of 1D van der Waals (vdW) grain boundaries (GBs) in ReS2.</title><p><bold>a</bold> Atomic-resolution scanning transmission electron microscope (STEM)/high angle annular dark field (HAADF) image of bulk ReS<sub>2</sub>, accompanied with a lattic... | [] | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Yongheng Zhou, Xin Zhou.</p></fn></fn-group>"
] | [
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"<media xlink:href=\"41467_2024_44792_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media>",
"<media xlink:href=\"41467_2024_44792_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media>"
] | [{"label": ["2."], "surname": ["Glass", "von der Linde", "Negran"], "given-names": ["AM", "D", "TJ"], "article-title": ["High\u2010voltage bulk photovoltaic effect and the photorefractive process in LiNbO"], "sub": ["3"], "source": ["Appl. Phys. Lett."], "year": ["1974"], "volume": ["25"], "fpage": ["233"], "lpage": ["... | {
"acronym": [],
"definition": []
} | 50 | CC BY | no | 2024-01-15 23:42:00 | Nat Commun. 2024 Jan 13; 15:501 | oa_package/69/a9/PMC10787835.tar.gz |
PMC10787836 | 38218955 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Diabetes mellitus (DM) is a major public health problem worldwide and one of the major diseases threatening human health and survival. Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in pe... | [
"<title>Methods</title>",
"<title>Sample and overall</title>",
"<p id=\"Par5\">The National Center for Health Statistics (NCHS) conducted a population-based cross-sectional NHANES to collect data on the nutritional and health conditions of children and adults in the United States of America via interviews, phys... | [
"<title>Results</title>",
"<title>Participant characteristics</title>",
"<p id=\"Par13\">This study included 1583 T2DM patients, of which 331 had concomitant DR and 1252 had no concomitant DR. The demographic details of participants with or without DR are shown in Table ##TAB##0##1##. DR participants were older... | [
"<title>Discussion</title>",
"<p id=\"Par17\">The present study aimed to elucidate whether there was an association between serum levels of heavy metals and DR among individuals with T2DM in the United States of America. We found a substantial negative relationship between blood Mn and DR, which was partially med... | [
"<title>Conclusions</title>",
"<p id=\"Par23\">In summary, the present study found a significant negative association between blood Mn levels and DR, suggesting that an appropriate increase in Mn intake may delay the onset and development of DR.</p>"
] | [
"<p id=\"Par1\">The present study utilized the National Health and Nutrition Examination Survey (NHANES) database to examine the relationship between serum levels of heavy metals and Diabetic retinopathy (DR) in individuals aged over 30 years with type 2 diabetes mellitus (T2DM) in the United States. A cross-sectio... | [] | [
"<title>Author contributions</title>",
"<p>This study’s data gathering, curation, analysis of statistics, and writing of the publication were all jointly completed by Y.Z. Input from X.K.L. was used in the statistical analysis. W.X. oversaw the research and helped with the editing and revision of the manuscript. ... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Flowchart of study target population NHANES (2011–2020).</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Effect of the serum albumin (mediator) on the relationship between blood manganese (exposure) and diabetic retinopathy (DR outcom... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Characteristics of study sample with and without diabetic retinopathy.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Non-diabetic retinopathy (n = 1252)</th><th align=\"left\">Diabetic retinopathy (n... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>Continuous variables are presented as mean ± SD, p-value was calculated by a linear regression model. Categorical variables are presented as %, p-value was calculated by Chi-square test.</p></table-wrap-foot>",
"<table-wrap-foot><p>Model 1: no modification variables; Model 2: only adjusts for... | [
"<graphic xlink:href=\"41598_2024_51749_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51749_Fig2_HTML\" id=\"MO2\"/>"
] | [] | [{"label": ["27."], "surname": ["Kaur", "Kumar", "Arora", "Tomar", "Ashish", "Sur", "Dutta"], "given-names": ["G", "V", "A", "A", "R", "D"], "article-title": ["Affected energy metabolism under manganese stress governs cellular toxicity"], "source": ["Sci. Rep."], "year": ["2013"], "volume": ["7"], "issue": ["1"], "fpag... | {
"acronym": [],
"definition": []
} | 33 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1268 | oa_package/b6/a5/PMC10787836.tar.gz |
PMC10787837 | 38218935 | [
"<title>Introduction</title>",
"<p id=\"Par3\">Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are common, life-threatening critical illnesses that lead to significant morbidity and mortality [##REF##34217425##1##]. Despite prominent breakthroughs in the pathophysiolog... | [
"<title>Materials and methods</title>",
"<title>Animals</title>",
"<p id=\"Par25\">Male specific-pathogen-free C57/BL6 mice (8–10 weeks old) were obtained from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). All mice were housed under controlled, pathogen-free conditions at the Laboratory Ani... | [
"<title>Results</title>",
"<title>Global m<sup>6</sup>A levels and METTL14 expression are increased in ALI mice</title>",
"<p id=\"Par8\">To confirm the role of m<sup>6</sup>A modification in ALI/ARDS, we evaluated the global m<sup>6</sup>A levels in the lung tissues of control and ALI mice. Both dot blot assay... | [
"<title>Discussion</title>",
"<p id=\"Par17\">In this study, we discovered that the contents of m<sup>6</sup>A and METTL14 in lung tissues of ALI mice subjected to LPS were enhanced. METTL14-mediated NLRP3 mRNA m<sup>6</sup>A modification increases NLRP3 mRNA stability in injured lungs in an IGF2BP2-dependent man... | [] | [
"<p id=\"Par1\">Acute lung injury (ALI) as well as its more severe form, acute respiratory distress syndrome (ARDS), frequently leads to an uncontrolled inflammatory response. N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) modification was associated with the progression of several inflammatory diseases. However, t... | [
"<title>Supplementary information</title>",
"<p>\n\n\n\n\n\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41419-023-06407-6.</p>",
"<title>Acknowledgements</title>",
"<p>We thank our colleagues for technical help and stimulating discussion. This work was supported by grants from the National Na... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Global m<sup>6</sup>A levels and METTL14 expression are increased in ALI mice.</title><p>Mice were injected intraperitoneally with LPS (15 mg/kg) or an equal volume of saline. <bold>A</bold>, <bold>B</bold> m<sup>6</sup>A dot-blot assays of lung tissues from Ct... | [] | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>",
"<supplementary-material ... | [
"<fn-group><fn><p>Edited by Sudan He</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Fei Cao, Guojun Chen, Yixin Xu.</p></fn></fn-group>"
] | [
"<graphic position=\"anchor\" xlink:href=\"41419_2023_6407_Figa_HTML\" id=\"d32e234\"/>",
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"<media xlink:href=\"41419_2023_6407_MOESM1_ESM.docx\"><caption><p>Supplementary Table 1</p></caption></media>",
"<media xlink:href=\"41419_2023_6407_MOESM2_ESM.docx\"><caption><p>Supplementary Table 2</p></caption></media>",
"<media xlink:href=\"41419_2023_6407_MOESM3_ESM.docx\"><caption><p>Supplementary Table... | [{"label": ["8."], "mixed-citation": ["Kelley N, Jeltema D, Duan Y, He Y. The NLRP3 inflammasome: an overview of mechanisms of activation and regulation. Int J Mol Sci. 2019;20:3328."]}, {"label": ["11."], "mixed-citation": ["Cornut M, Bourdonnay E, Henry T. Transcriptional regulation of inflammasomes. Int J Mol Sci. 2... | {
"acronym": [],
"definition": []
} | 47 | CC BY | no | 2024-01-15 23:42:00 | Cell Death Dis. 2024 Jan 13; 15(1):43 | oa_package/52/7f/PMC10787837.tar.gz |
PMC10787838 | 38219004 | [
"<title>Introduction</title>",
"<p id=\"Par2\">One of the most important evolutionary characteristics of human beings is their social nature. The ability to develop social bonds in the environment in which we live largely depends on the ability of individuals of the same species to recognize and accurately interp... | [
"<title>Methods</title>",
"<title>Participants</title>",
"<p id=\"Par26\">The cohort comprised 85 individuals (66 women; 77%) aged between 18 and 35 years (average = 23.9, SD = 3.7). The exclusion criteria were: a medical diagnosis of psychiatric diseases; history of peripheral or central facial paralysis; and ... | [
"<title>Results</title>",
"<title>Cohort characterization</title>",
"<p id=\"Par6\">The participants were divided into the non-depressive (n = 69; 55 women; mean age = 24.29; SD = 3.4) and depressive (n = 16; 11 women; mean age = 25.6; SD = 4.08) groups. The groups were similar in age (t = 0.69; p = 0.48) and s... | [
"<title>Discussion</title>",
"<p id=\"Par15\">The present study evaluated the differences in EMG reactivity of the zygomatic major muscle and emotional states in depressive and non-depressive individuals upon exposure to visual stimuli that were neutral, with social interaction (affiliative), and without social i... | [] | [
"<p id=\"Par1\">Individuals with severe depressive symptoms present diminished facial expressions compared to healthy individuals. This reduced facial expression, which occurs in most depressive patients could impair social relationships. The current study sought to investigate whether pictures with social interact... | [] | [
"<title>Acknowledgements</title>",
"<p>This work was supported by the National Council for Scientific and Technological Development, Brazil (<italic>Conselho Nacional de Desenvolvimento Científico e Tecnológico</italic>—CNPq); Coordination for the Improvement of Higher Education Personnel, Brazil (<italic>Coorden... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Least square means of electromyographic activation of the zygomatic major muscle in the non-depressive (white circles) and depressive (black squares) groups during the visualization of neutral, affiliative and control blocks. The points represent the mean value o... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Sample characterization.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Non-depressive, n = 69</th><th align=\"left\">Depressive, n = 16</th><th align=\"left\">p value*</th></tr></thead><tbody><tr><td... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>Data expressed as mean (SD).</p><p>*Statistically significant difference (p < 0.05).</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn>... | [
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"<graphic xlink:href=\"41598_2024_51813_... | [] | [{"label": ["1."], "surname": ["Delle-Vigne", "Wang", "Kornreich", "Verbanck", "Campanella"], "given-names": ["D", "W", "C", "P", "S"], "article-title": ["Emotional facial expression processing in depression: Data from behavioral and event-related potential studies"], "source": ["Neurophysiol. Clin. Clin. Neurophysiol.... | {
"acronym": [],
"definition": []
} | 67 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1266 | oa_package/d2/d2/PMC10787838.tar.gz |
PMC10787839 | 38218918 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Bacterial biofilms pose a formidable global health challenge, exhibiting resilience against antibiotics, phagocytosis, and environmental stressors, perpetuating chronic infections, particularly by Staphylococcal and Pseudomonas strains<sup>##REF##23880136##1##</sup>.... | [
"<title>Materials and methods</title>",
"<title>Chemicals and materials</title>",
"<p id=\"Par7\">Silver nitrate and copper sulfate (analytical grade) were obtained from Sigma Aldrich Bangalore, India. All bacteriological media components such as crystal violet, tryptone, yeast extract, glucose, and sodium chlo... | [
"<title>Results and discussions</title>",
"<title>Synthesis of Ag–Cu NPs</title>",
"<p id=\"Par20\">The plant chosen for this research belongs to the <italic>Amaranthaceae</italic> family and is found extensively in tropical and subtropical regions around the world. Due to its rich composition of phytochemicals... | [
"<title>Results and discussions</title>",
"<title>Synthesis of Ag–Cu NPs</title>",
"<p id=\"Par20\">The plant chosen for this research belongs to the <italic>Amaranthaceae</italic> family and is found extensively in tropical and subtropical regions around the world. Due to its rich composition of phytochemicals... | [
"<title>Conclusion</title>",
"<p id=\"Par31\">The present study successfully demonstrates the development of an eco-friendly, rapid, and cost-efficient method for synthesizing Ag–Cu NPs using <italic>Aerva lanata</italic> extract, fabricated through a phytofabrication process, exhibit great potential for applicat... | [
"<p id=\"Par1\">In this study, we demonstrate the green synthesis of bimetallic silver-copper nanoparticles (Ag–Cu NPs) using <italic>Aerva lanata</italic> plant extract. These NPs possess diverse biological properties, including in vitro antioxidant, antibiofilm, and cytotoxic activities. The synthesis involves th... | [
"<title>Supplementary Information</title>",
"<p>\n</p>"
] | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51647-x.</p>",
"<title>Acknowledgements</title>",
"<p>All the authors are thankful to their respective universities and institutes for their support. The work was supported by the... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Schematic representation of the study.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>The XRD pattern of green synthesized bimetallic Ag–Cu nanoparticles.</p></caption></fig>",
"<fig id=\"Fig3\"><label>Figure 3</label><caption><p>T... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Summary of the anti-bacterial activity of Ag–Cu NPs.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"3\">Organisms</th><th align=\"left\" colspan=\"4\">Zone of inhibition (mm)</th></tr><tr><th align=\"left\" col... | [
"<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepack... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><italic>C</italic> control.</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Gopishankar Thirum... | [
"<graphic xlink:href=\"41598_2024_51647_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51647_Article_Equ1.gif\" position=\"anchor\"/>",
"<graphic xlink:href=\"41598_2024_51647_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"41598_2024_51647_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"4... | [
"<media xlink:href=\"41598_2024_51647_MOESM1_ESM.docx\"><caption><p>Supplementary Figures.</p></caption></media>"
] | [{"label": ["10."], "surname": ["Sharma"], "given-names": ["G"], "article-title": ["Novel development of nanoparticles to bimetallic nanoparticles and their composites: A review"], "source": ["J. King Saud Univ. Sci."], "year": ["2019"], "volume": ["31"], "fpage": ["257"], "lpage": ["269"], "pub-id": ["10.1016/j.jksus.... | {
"acronym": [],
"definition": []
} | 54 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1270 | oa_package/62/2f/PMC10787839.tar.gz |
PMC10787840 | 38218875 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Glioblastoma (GBM) is the most aggressive brain tumor with extensive angiogenesis and poor clinical outcome [##REF##34185076##1##]. Abnormally increased vascularization results in tumor progression and recurrence. Abundant pro-angiogenic factors in the tumor environm... | [
"<title>Materials and methods</title>",
"<title>Glioblastoma specimen and cell culture</title>",
"<p id=\"Par6\">Twenty-eight paraffin-embedded samples from human glioma patients (WHO I-IV) with corresponding clinic-pathological data were collected in the Department of Neurosurgery at Zhujiang Hospital from 201... | [
"<title>Results</title>",
"<title>IFITM3 expression is elevated in human GBM tissue</title>",
"<p id=\"Par19\">To examine the expression of IFITM3 in GBM, we investigated IFITM3 gene expression level in glioma datasets from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA). Results showed a ... | [
"<title>Discussion</title>",
"<p id=\"Par24\">In the present study, we describe a novel mechanism of GSCs-induced GBM angiogenesis through expression of IFITM3 and bFGF (Fig. ##FIG##4##5J##). IFITM3 is a interferon-induced protein mediating antiviral activity via suppressing the entry of viruses [##REF##30951861#... | [] | [
"<p id=\"Par1\">Interferon-induced transmembrane protein 3 (IFITM3) has been previously verified to be an endosomal protein that prevents viral infection. Recent findings suggested IFITM3 as a key factor in tumor invasion and progression. To clarify the role and molecular mechanism of IFITM3 in Glioblastoma multifo... | [
"<title>Supplementary information</title>",
"<p>\n\n\n\n\n\n\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41419-023-06416-5.</p>",
"<title>Acknowledgements</title>",
"<p>This work was supported by the National Natural Science Foundation of China (No. 82002631, No. 82072762, and No. 82203135) an... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>IFITM3 correlates with glioma grade and prognosis of GBM patients.</title><p><bold>A</bold> Gene expression profile of IGFBP4 in TCGA/CGGA glioma datasets stratified by tumor grade. <bold>B</bold> Gene expression analysis of IFITM3 in GBM (TCGA) and normal brai... | [] | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>",
"<supplementary-material ... | [
"<fn-group><fn><p>Edited by Dr. Maria Victoria Niklison Chirou</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Zhangsheng Xiong, Xiangdong Xu.</p></fn... | [
"<graphic xlink:href=\"41419_2023_6416_Fig1_HTML\" id=\"d32e529\"/>",
"<graphic xlink:href=\"41419_2023_6416_Fig2_HTML\" id=\"d32e628\"/>",
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"<graphic xlink:href=\"41419_2023_6416_Fig4_HTML\" id=\"d32e756\"/>",
"<graphic xlink:href=\"41419... | [
"<media xlink:href=\"41419_2023_6416_MOESM1_ESM.docx\"><caption><p>Supplementary figures and legends</p></caption></media>",
"<media xlink:href=\"41419_2023_6416_MOESM2_ESM.pdf\"><caption><p>Checklist</p></caption></media>",
"<media xlink:href=\"41419_2023_6416_MOESM3_ESM.tif\"><caption><p>Supplementary Fig.1</... | [{"label": ["2."], "surname": ["Braghiroli", "Sabbaga", "Hoff", "Ignez", "Sabbaga"], "given-names": ["MI", "J", "PM", "M", "J"], "article-title": ["Bevacizumab: overview of the literature"], "source": ["Expert Rev Anticancer Ther"], "year": ["2017"], "volume": ["12"], "fpage": ["567"], "lpage": ["80"], "pub-id": ["10.1... | {
"acronym": [],
"definition": []
} | 55 | CC BY | no | 2024-01-15 23:42:00 | Cell Death Dis. 2024 Jan 13; 15(1):45 | oa_package/b5/05/PMC10787840.tar.gz |
PMC10787841 | 38218746 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Since the beginning of the twenty-first century, industrial big data has experienced rapid development with the improvement of data collection and processing capabilities<sup>##UREF##0##1##</sup>. The centralized big data processing model centered around cloud comput... | [
"<title>Active control method for quality prediction</title>",
"<p id=\"Par26\">This section first analyzed the product’s quality characteristics and selected criticalquality-related parameters with correlation coefficients greater than the set threshold based on the correlation coefficients of industrial product... | [
"<title>Experiments and results</title>",
"<title>Qualitys correlation parameter selection</title>",
"<p id=\"Par67\">Some studies<sup>##UREF##41##44##,##UREF##42##45##</sup> have pointed out that the equipment process data obtained from the processing equipment, including spindle power (P), spindle current (I)... | [
"<title>Discussion</title>",
"<p id=\"Par79\">In terms of imbalanced data, Table ##TAB##5##6## and Fig. ##FIG##8##9## demonstrate that the SMOTE and XGBoost combination outperforms the combination of SMOTE with other classification algorithms Fig. ##FIG##6##7## displays the importance of quality features; selecti... | [
"<title>Conclusion</title>",
"<p id=\"Par82\">This article proposes an Edge computing-based proactive control method for industrial product manufacturing quality prediction, addressing the issue of imbalanced data in the manufacturing process. Firstly, an edge computing-based framework for quality prediction in i... | [
"<p id=\"Par1\">With the emergence of intelligent manufacturing, new-generation information technologies such as big data and artificial intelligence are rapidly integrating with the manufacturing industry. One of the primary applications is to assist manufacturing plants in predicting product quality. Traditional ... | [
"<title>Related work</title>",
"<p id=\"Par6\">The current research on quality prediction methods is mainly divided into two categories: model-based prediction methods and data-driven prediction methods. The main difference between the two methods lies in whether the design of the controller is based on the syste... | [
"<title>Author contributions</title>",
"<p>Formal analysis, L.L.; data curation, M.C.; writing original draftpreparat-ion, M.C.; supervision, Z.W.; format adjustment K.Z. All authors have read and agreed to the publi-shed version of the manuscript.</p>",
"<title>Funding</title>",
"<p>The support of this work ... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Research directions for equipment management.</p></caption></fig>",
"<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Industrial internet of things architecture for industrial production line.</p></caption></fig>",
"<fig id=\"Fig3\"><label>Figure 3</label>... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Research of related papers.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Type</th><th align=\"left\">Problem-oriented</th><th align=\"left\">Author</th><th align=\"left\">Main work</th></tr></thead><tbody><tr><td align... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [] | [
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41598_2024_51974_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51974_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"41598_2024_51974_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"41598_2024_51974_Fig4_HTML\" id=\"MO4\"/>",
"<inline-graphic xlink:href=\"41598_2024... | [] | [{"label": ["1."], "surname": ["Tao", "Q"], "given-names": ["F", "QL"], "article-title": ["New IT driven service-oriented smart manufacturing: Framework and characteristics"], "source": ["IEEE Trans. Syst. Man. Cybern. Syst."], "year": ["2019"], "volume": ["49"], "issue": ["1"], "fpage": ["81"], "lpage": ["91"], "pub-i... | {
"acronym": [],
"definition": []
} | 46 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 14; 14:1288 | oa_package/9d/db/PMC10787841.tar.gz |
PMC10787842 | 38219006 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Online health consultation is a novel form of doctor-patient communication that has emerged alongside the rapid advancements in Internet technology<sup>##UREF##0##1##</sup>. Patients can employ mobile phones, computers and other terminal devices to engage in various ... | [
"<title>Methods</title>",
"<title>Manipulation check</title>",
"<p id=\"Par18\">To divide the abundant physician information provided on the online health consultation platform into two types: cognitive stimulation information and affective stimulation information, this paper referred to the physician-related i... | [
"<title>Results</title>",
"<title>Manipulation check results</title>",
"<p id=\"Par8\">Through the manipulation checks, this paper selected four types of physician information from the seven types of physician information extracted on the online health consultation platform for formal experiment. The cognitive ... | [
"<title>Discussion</title>",
"<p id=\"Par12\">The results of behavioral experiment indicate that the preferences of patients in choosing physician online varied based on their levels of health involvement. When the health involvement was low, physicians with professionalism or good attitude can be preferred by pa... | [] | [
"<p id=\"Par1\">In traditional offline health-seeking behavior, patients consistently exhibit a preference for similar types of physicians due to limited access to physicians’ information. Nevertheless, with the advent of online health consultation platforms offering comprehensive physicians’ information for patien... | [
"<title>Supplementary Information</title>",
"<p>\n</p>"
] | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51519-4.</p>",
"<title>Acknowledgements</title>",
"<p>This work was supported by the Open Project Funding from the Shanghai Key Laboratory of Brain-Machine Intelligence for Inform... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Simple effect analysis results of patients' scores of willingness to choose with different levels of health involvement. Note: (<bold>a</bold>) low level health involvement; (<bold>b</bold>) high level health involvement; H, high level of cognitive trust; L, low ... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Physician information manipulation checks results.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\" colspan=\"2\">Physician information</th><th align=\"left\">CT</th><th align=\"left\" rowspan=\"2\">Sig</th><... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>Note: CT, cognitive trust; AT, affective trust; all the cognitive and affective trust scores ranged between 1 to 9.</p></table-wrap-foot>",
"<table-wrap-foot><p>Note: CT, cognitive trust; AT, affective trust.</p></table-wrap-foot>",
"<table-wrap-foot><p>Note: H, high level of cognitive trus... | [
"<graphic xlink:href=\"41598_2024_51519_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"41598_2024_51519_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"41598_2024_51519_Fig3_HTML\" id=\"MO3\"/>"
] | [
"<media xlink:href=\"41598_2024_51519_MOESM1_ESM.pdf\"><caption><p>Supplementary Information.</p></caption></media>"
] | [{"label": ["1."], "mixed-citation": ["Xiong, D., Zhao, L. & Iop. In "], "italic": ["2017 International Conference on Cloud Technology And Communication Engineering"]}, {"label": ["2."], "surname": ["Wan", "Zhang", "Yan"], "given-names": ["Y", "Y", "M"], "article-title": ["What influences patients' willingness to choos... | {
"acronym": [],
"definition": []
} | 36 | CC BY | no | 2024-01-15 23:42:00 | Sci Rep. 2024 Jan 13; 14:1269 | oa_package/f5/55/PMC10787842.tar.gz |
PMC10787843 | 38218957 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Advances in computational materials science have enabled the accurate prediction of novel materials possessing exceptional properties. Remarkably, these computational advancements have facilitated the successful experimental synthesis of materials that exhibit the an... | [
"<title>Methods</title>",
"<title>Diffusion probabilistic model</title>",
"<p id=\"Par23\">In the diffusion probabilistic model, the data distribution is gradually perturbed by noise in the forward process until it becomes a normal distribution at late times. In this study, the distribution of the fractional co... | [
"<title>Results</title>",
"<p id=\"Par8\">The performances of DP-CDVAE models are herein presented. There are four DP-CDVAE models, differed by the choice of the encoder (see Fig. ##SUPPL##0##S1## in Supplemental Information (SI)). DimeNet has been employed for the main encoder for every DP-CDVAE models<sup>##URE... | [
"<title>Discussion</title>",
"<p id=\"Par21\">The DP-CDVAE models significantly enhance the generation performance, particularly in terms of property statistics, while maintaining comparable COVs to those of CDVAE. Specifically, for Carbon-24 and MP-20 datasets, the density distributions between the generated and... | [] | [
"<p id=\"Par1\">The crystal diffusion variational autoencoder (CDVAE) is a machine learning model that leverages score matching to generate realistic crystal structures that preserve crystal symmetry. In this study, we leverage novel diffusion probabilistic (DP) models to denoise atomic coordinates rather than adop... | [
"<title>Supplementary Information</title>",
"<p>\n</p>"
] | [
"<title>Supplementary Information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41598-024-51400-4.</p>",
"<title>Acknowledgements</title>",
"<p>This research project is supported by the Second Century Fund (C2F), Chulalongkorn University. This Research is funded by Tha... | [
"<fig id=\"Fig1\"><label>Figure 1</label><caption><p>The schematic summarizing the architecture for training the DP-CDVAE model. Multiple sub-networks are trained to minimize the total loss function. The encoder () compresses input pristine crystal structures into the latent feature (). The predicted lattice parame... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Reconstruction performance.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Models</th><th align=\"left\" colspan=\"3\">Match rate (%) </th><th align=\"left\" colspan=\"3\">\n</th></tr><tr><th align=\"left\"... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>Significant values are in [bold].</p></table-wrap-foot>",
"<table-wrap-foot><p>Significant values are in [bold].</p></table-wrap-foot>",
"<table-wrap-foot><p>Significant values are in [bold].</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature rem... | [
"<inline-graphic xlink:href=\"41598_2024_51400_Article_IEq1.gif\"/>",
"<inline-graphic xlink:href=\"41598_2024_51400_Article_IEq2.gif\"/>",
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"<inline-graphic xlink:href=... | [
"<media xlink:href=\"41598_2024_51400_MOESM1_ESM.pdf\"><caption><p>Supplementary Information.</p></caption></media>"
] | [{"label": ["1."], "surname": ["Needs", "Pickard"], "given-names": ["RJ", "CJ"], "article-title": ["Perspective: Role of structure prediction in materials discovery and design"], "source": ["APL Mater."], "year": ["2016"], "volume": ["4"], "fpage": ["053210"], "pub-id": ["10.1063/1.4949361"]}, {"label": ["2."], "mixed-... | {
"acronym": [],
"definition": []
} | 52 | CC BY | no | 2024-01-15 23:42:01 | Sci Rep. 2024 Jan 13; 14:1275 | oa_package/f6/42/PMC10787843.tar.gz |
PMC10787844 | 38218925 | [
"<title>Introduction</title>",
"<p id=\"Par2\">With the continuous improvement of the global economy, the prevalence of chronic kidney disease (CKD) continues to elevate which has emerged as a severe public health problem, ≈15% of people suffer from CKD<sup>##REF##34571064##1##,##REF##31205024##2##</sup>. CKD is ... | [
"<title>Methods</title>",
"<title>Ethics statement for human samples and animal models</title>",
"<p id=\"Par23\">Written informed consent was obtained from healthy donors, and discarded human umbilical cord tissues were acquired according to the approved protocol of the Institutional Review Board (IRB) at the ... | [
"<title>Results</title>",
"<title>Characteristics of MSC derived extracellular vesicles with CHIP high expression</title>",
"<p id=\"Par6\">Umbilical derived MSCs with high purity were isolated from rat according to previous protocols (Supplementary Fig. ##SUPPL##0##1##). We next infected MSCs with lentivirus e... | [
"<title>Discussion</title>",
"<p id=\"Par17\">In this study, we reported a method for manufacturing engineered SPION-EVs and evaluated the feasibility, safety, and effectiveness of MSC-EVs with CHIP overexpressed in treating renal interstitial fibrosis in UUO and DKD models. Impressively, provide packed CHIP prot... | [] | [
"<p id=\"Par1\">Renal interstitial fibrosis (RIF) is a fundamental pathological feature of chronic kidney disease (CKD). However, toxicity and poor renal enrichment of fibrosis inhibitors limit their further applications. In this study, a platform for CKD therapy is developed using superparamagnetic iron oxide nano... | [
"<title>Supplementary information</title>",
"<p>\n\n\n</p>"
] | [
"<title>Supplementary information</title>",
"<p>The online version contains supplementary material available at 10.1038/s41536-024-00348-0.</p>",
"<title>Acknowledgements</title>",
"<p>C.J. and J.Z. contributed equally to this work. The authors thank the members of Qian lab for helpful discussion and paper pr... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Characteristics of MSC-derived extracellular vesicles with CHIP high-expression.</title><p><bold>a</bold> MSCs were transfected with lentivirus encoding the CHIP gene. The expression of CHIP (green) with GFP-tagged in MSCs was determined by immunofluorescence (... | [] | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Cheng Ji, Jiahui Zhang, Linru Shi.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"41536_2024_348_Fig1_HTML\" id=\"d32e388\"/>",
"<graphic xlink:href=\"41536_2024_348_Fig2_HTML\" id=\"d32e452\"/>",
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"<graphic xlink:href=\"41536_2024_348_Fig4_HTML\" id=\"d32e658\"/>",
"<graphic xlink:href=\"41536_202... | [
"<media xlink:href=\"41536_2024_348_MOESM1_ESM.pdf\"><caption><p>Supplementary material</p></caption></media>",
"<media xlink:href=\"41536_2024_348_MOESM2_ESM.pdf\"><caption><p>Reporting-summary</p></caption></media>"
] | [{"label": ["9."], "surname": ["Jin"], "given-names": ["Q"], "article-title": ["Therapeutic potential of artemisinin and its derivatives in managing kidney diseases"], "source": ["Front Pharm."], "year": ["2023"], "volume": ["14"], "fpage": ["1097206"], "pub-id": ["10.3389/fphar.2023.1097206"]}, {"label": ["13."], "sur... | {
"acronym": [],
"definition": []
} | 33 | CC BY | no | 2024-01-15 23:42:01 | NPJ Regen Med. 2024 Jan 13; 9:3 | oa_package/0b/12/PMC10787844.tar.gz |
PMC10787845 | 38222223 | [
"<title>Introduction</title>",
"<p>Diarrhea is one of the top three infectious diseases responsible for ~8%-10% of total deaths among children below the age of five years; 90% of these deaths are estimated to occur in South Asia and sub-Saharan Africa [##UREF##0##1##, ##UREF##1##2##]. Rotavirus and diarrheagenic ... | [
"<title>Materials and methods</title>",
"<p>This hospital-based prospective observational study was conducted between November 2017 and April 2019 at the University College of Medical Sciences, Delhi, an urban hospital catering mainly to the low-income population of eastern Delhi and adjoining areas of the state ... | [
"<title>Results</title>",
"<p>Enrollment and baseline characteristics</p>",
"<p>During the study period, 209 children aged between one month and five years with acute diarrhea were hospitalized, out of which 150 were included in the present study. Figure ##FIG##0##1## depicts the flow of participants in the stu... | [
"<title>Discussion</title>",
"<p>In this hospital-based study on under-five children with acute diarrhea, we documented that stool culture's diagnostic yield was very low compared to stool PCR amplification using a specific primer for the ipaH gene for the diagnosis of <italic>Shigella </italic>infection. The maj... | [
"<title>Conclusions</title>",
"<p>The present study concludes that <italic>Shigella </italic>is an important cause of diarrhea in children under five, often missed by conventional laboratory methods. Blood in stools as a syndromic indicator for <italic>Shigella </italic>infection needs to be reconsidered as the m... | [
"<p>Background and objectives: <italic>Shigella </italic>is an important cause of diarrhea in children under five, often missed by conventional laboratory methods. Blood in stools has always been a syndromic indicator for <italic>Shigella </italic>diarrhea, but most cases present with watery diarrhea without blood.... | [] | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Flowchart depicting the selection of participants for the study</title><p>mo: month; y: year; d: days; TB: tuberculosis</p></caption></fig>"
] | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Comparison of clinical and anthropometric parameters at presentation between children with or without stool PCR Shigella positivity</title><p><sup>a</sup>Data in number (%) unless stated otherwise; <sup>b</sup>n=7 missing cases as the... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study. Institutional Ethics Committee for Human Research, University College of Medical Sciences, Delhi issued approval IEC-HR/2017/32/88</p></fn></fn-group>",
"<fn-grou... | [
"<graphic xlink:href=\"cureus-0015-00000050546-i01\" position=\"float\"/>"
] | [] | [{"label": ["1"], "article-title": ["Diarrhoeal disease"], "date-in-citation": ["\n"], "month": ["11"], "year": ["2022", "2017"], "uri": ["https://www.who.int/news-room/fact-sheets/detail/diarrhoeal-disease"]}, {"label": ["2"], "article-title": ["Levels and trends in child mortality 2019"], "date-in-citation": ["\n"], ... | {
"acronym": [],
"definition": []
} | 22 | CC BY | no | 2024-01-15 23:42:01 | Cureus.; 15(12):e50546 | oa_package/45/9d/PMC10787845.tar.gz |
PMC10787846 | 38222208 | [
"<title>Introduction</title>",
"<p>Mucoid degeneration (MD) of the anterior cruciate ligament (ACL) is a rare condition, with a prevalence rate ranging from 0.2% to 1.2% [##REF##11372618##1##]. The cause of this condition is not well understood and has been the subject of vigorous debate, with only a limited numb... | [] | [] | [
"<title>Discussion</title>",
"<p>MD of ACL is an uncommon condition that leads to knee pain and discomfort, primarily impacting individuals in their fourth decade of life [##REF##18997652##6##]. Kumar et al. [##REF##10204939##7##] initially coined the term mucoid cystic degeneration to describe this condition aff... | [
"<title>Conclusions</title>",
"<p>The clinical and radiological characteristics of MD of ACL can be often inconclusive, potentially leading to a misdiagnosis of a torn ACL. Arthroscopists must be cognizant of this uncommon condition, as they may encounter a mucoid ACL during surgical procedures. This awareness is... | [
"<p>Mucoid degeneration (MD) is an uncommon pathological phenomenon that specifically affects the anterior cruciate ligament (ACL). This condition arises from the infiltration of yellowish material within the fibers of the ACL, contributing to the clinical presentation characterized by discomfort and limited mobili... | [
"<title>Case presentation</title>",
"<p>The patient was a 57-year-old female who presented to our hospital with a two-year history of right knee pain and limited flexion following a minor injury. A thorough examination was conducted. Notably, there was no apparent swelling or instability observed. The knee exhibi... | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Examination of the patient at presentation</title><p>A: Range of flexion of the right knee 90°. B: Range of extension of the right knee 0°</p></caption></fig>",
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\... | [] | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Oussama El Alaoui, Adnane Lachkar, Najib Abdeljaouad, Hicham Yacoubi</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Oussama El Alaoui, Ousama Jelti, Adnane Lachkar</... | [
"<graphic xlink:href=\"cureus-0015-00000050545-i01\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050545-i02\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050545-i03\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050545-i04\" position=\"float\"/>"
] | [] | [{"label": ["8"], "article-title": ["Mucoid degeneration of the anterior cruciate ligament"], "source": ["Arthroscopy"], "person-group": ["\n"], "surname": ["Fealy", "Kenter", "Dines", "Warren"], "given-names": ["S", "K", "JS", "RF"], "fpage": ["0"], "volume": ["17"], "year": ["2001"]}] | {
"acronym": [],
"definition": []
} | 16 | CC BY | no | 2024-01-15 23:42:01 | Cureus.; 15(12):e50545 | oa_package/c8/bb/PMC10787846.tar.gz |
PMC10787847 | 38222263 | [] | [] | [] | [] | [] | [
"<p>Approved by: Agustin Ibanez, Latin American Brain Health Institute (BrainLat), Chile</p>"
] | [
"<p>The journal retracts the 18 June 2021 article cited above.</p>",
"<p>Following publication, the publisher uncovered evidence that false identities were used in the peer-review process. The assignment of fake reviewers was confirmed by an investigation, conducted in accordance with Frontiers' policies and the ... | [] | [] | [] | [] | [] | [] | [] | [] | [] | [] | [] | [] | [] | {
"acronym": [],
"definition": []
} | 0 | CC BY | no | 2024-01-15 23:42:01 | Front Aging Neurosci. 2023 Dec 28; 15:1358292 | oa_package/c4/9e/PMC10787847.tar.gz |
PMC10787848 | 38222136 | [
"<title>Introduction and background</title>",
"<p>The development of more efficient, well-tolerated, and easy combination antiretroviral treatment (cART) has significantly enhanced HIV-positive people's (PLWH) life expectancy, even exceeding that of the general population [##REF##27831953##1##]. Their extended su... | [] | [] | [] | [
"<title>Conclusions</title>",
"<p>As OPLWH lives longer, healthcare for them is changing. However, there are issues with the growth in frailty and other geriatric problems among OPLWH. Once associated with elderly people, fragility is now a critical factor in determining susceptibility to unfavorable health outco... | [
"<p>The life expectancy of people living with HIV (PLWH) has greatly increased due to advancements in combination antiretroviral treatment (cART). However, this longer life has also increased the prevalence of age-related comorbidities, such as frailty, which now manifest sooner in this group. Frailty, a term coine... | [
"<title>Review</title>",
"<p>Frailty: concept and evolution</p>",
"<p>Derived from the Latin word \"fragilis\" (meaning \"breakable\"), the term frailty indicates a condition of increased sensitivity. Fried devised the Frailty Phenotype (FP) and the Frailty Index (FI) to emphasize the complexity of the idea in ... | [] | [] | [] | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Mohammad Mansour, Monisha Augustine, Mahendra Kumar, Amna Naveed Butt, Thanmai Reddy Thugu, Parvinder Kaur, Nipakumari J. Patel, Ankit Gaudani, M. Bilal Jahania, Elhama Jami, Mouhammad Sha... | [] | [] | [{"label": ["6"], "article-title": ["Frailty and pre-frailty in middle-aged and older adults and its association with multimorbidity and mortality: a prospective analysis of 493\u2008737 UK Biobank participants"], "source": ["Lancet Public Health"], "person-group": ["\n"], "surname": ["Hanlon", "Nicholl", "Jani", "Lee"... | {
"acronym": [],
"definition": []
} | 58 | CC BY | no | 2024-01-15 23:42:01 | Cureus.; 15(12):e50539 | oa_package/79/92/PMC10787848.tar.gz |
PMC10787849 | 38222186 | [
"<title>Introduction</title>",
"<p>Autism is a disorder distinguished by significant challenges in social interaction and communication coupled with repetitive and stereotypical patterns of behavior and activities. Deficits in social interaction and language development become apparent before the age of three. In... | [] | [] | [
"<title>Discussion</title>",
"<p>This study presents intriguing findings that warrant careful consideration and interpretation. The observed improvements in speech abilities and attention deficits following the implementation of the multimodal physiotherapy intervention suggest the potential efficacy of this comp... | [
"<title>Conclusions</title>",
"<p>In conclusion, this case report provides valuable insights into a holistic intervention strategy for individuals with ASD facing concurrent challenges of speech impairments and attention deficits. The observed improvements in speech abilities and attention following the multimoda... | [
"<p>Autism is a disorder distinguished by significant challenges in social interaction and communication coupled with repetitive and stereotypical patterns of behavior and activities. Deficits in social interaction and language development become apparent before age three. In children, this condition is referred to... | [
"<title>Case presentation</title>",
"<p>Patient information</p>",
"<p>We present a case involving a three-year-old male child characterized by a spectrum of developmental anomalies. Predominant concerns encompass intellectual disability, hyperactivity, speech dysfluency, and attentional deficits. The patient wa... | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Patient undergoing holding therapy.</title></caption></fig>",
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Picture Exchange Communication System (PECS).</title></cap... | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Physical examination findings.</title><p>cm: Centimeter; kg: Kilogram.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Anthropometric measures</td><td ro... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vaishnavi M. Thakre</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Pradhyum D. Kolhe, H V Sharath, Prajyot Ankar, Vais... | [
"<graphic xlink:href=\"cureus-0015-00000050547-i01\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050547-i02\" position=\"float\"/>"
] | [] | [{"label": ["1"], "article-title": ["Addressing health inequities in the united states: a case report on autism spectrum disorder (ASD) and social determinants of health"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Fox", "Maribona", "Quintero", "Lange", "Semidey"], "given-names": ["KE", "AS", "J", "C",... | {
"acronym": [],
"definition": []
} | 20 | CC BY | no | 2024-01-15 23:42:01 | Cureus.; 15(12):e50547 | oa_package/f8/cf/PMC10787849.tar.gz |
PMC10787871 | 38221988 | [
"<title>Introduction</title>",
"<p>Road accidents are a significant public health concern worldwide, with an estimated 1.35 million deaths caused by road traffic accidents each year.\n<sup>\n##UREF##0##1##\n</sup> Developing countries, such as India, are disproportionately affected, with over 150,000 fatalities r... | [
"<title>Methods</title>",
"<p>The proposed methodology for this research involves the following steps for implementing a RF model machine learning technique for accident severity prediction.</p>",
"<p>Data Preparation: The first step in implementing a RF model for accident severity prediction is to collect and ... | [
"<title>Result and Discussion</title>",
"<title>Model Performance</title>",
"<p>The classification model used three hyperparameters -’max_depth’: 10,’max_features’:’sqrt’, and’n_estimators’: 100, and the results generated a confusion matrix for the training set. The matrix indicated the number of correctly and ... | [
"<title>Result and Discussion</title>",
"<title>Model Performance</title>",
"<p>The classification model used three hyperparameters -’max_depth’: 10,’max_features’:’sqrt’, and’n_estimators’: 100, and the results generated a confusion matrix for the training set. The matrix indicated the number of correctly and ... | [
"<title>Conclusion</title>",
"<p>The RF classifier model predicted the severity of traffic accidents with an overall accuracy of 67% on the training set and approximately 41.47% on the test set. Indicating possible bias or imbalance in the training dataset, the model tended to predict a lower severity index than ... | [
"<p>No competing interests were disclosed.</p>",
"<p>\n<bold>Background:</bold> Road accidents claim around 1.35 million lives annually, with countries like India facing a significant impact. In 2019, India reported 449,002 road accidents, causing 151,113 deaths and 451,361 injuries. Accident severity modeling he... | [
"<title>Future Scope</title>",
"<p>The study presented provides a good starting point for future research in the field of road safety modeling and accident prevention for Indian highways. However, with the limitations of the present study there opens potential areas for future research as mentioned below which wi... | [
"<title>Acknowledgement</title>",
"<p>We are grateful to National Highways Authority of India and IL&FS Engineering and Construction Company for making the raw accident data available.</p>",
"<title>Data availability</title>",
"<p>Zenodo. Data for Accident Severity Prediction Modelling for Indian Highways... | [
"<fig position=\"float\" fig-type=\"figure\" id=\"f1\"><label>Figure 1. </label><caption><title>Pune-Sholapur Section of NH-9 in km.144/400 to Km. 249/000 in the State of Maharashtra.</title></caption></fig>",
"<fig position=\"float\" fig-type=\"figure\" id=\"f2\"><label>Figure 2. </label><caption><title>Six-Lani... | [
"<table-wrap position=\"float\" id=\"T1\"><label>Table 1. </label><caption><title>Dataset Attributes and Parameters Mapping.</title></caption><table frame=\"hsides\" rules=\"groups\" content-type=\"article-table\"><thead><tr><th align=\"left\" colspan=\"1\" rowspan=\"1\" valign=\"top\">Sl No</th><th align=\"left\" ... | [
"<disp-formula id=\"e5\">\n<mml:math id=\"M1\" display=\"block\" overflow=\"scroll\"><mml:mo>[</mml:mo><mml:mi mathvariant=\"normal\">RF</mml:mi><mml:mo>(</mml:mo><mml:mi mathvariant=\"normal\">X</mml:mi><mml:mo>)</mml:mo><mml:mo>=</mml:mo><mml:mo>(</mml:mo><mml:mi mathvariant=\"normal\">1/B</mml:mi><mml:mo>)</mml:... | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"pub-status\"><fn><p>[version 2; peer review: 1 approved</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"f1000research-12-157426-g0000\" id=\"gr1\" position=\"float\"/>",
"<graphic xlink:href=\"f1000research-12-157426-g0001\" id=\"gr2\" position=\"float\"/>",
"<graphic xlink:href=\"f1000research-12-157426-g0002\" id=\"gr3\" position=\"float\"/>",
"<graphic xlink:href=\"f1000research-12-157... | [] | [{"label": ["1"], "mixed-citation": ["\n"], "collab": ["World Health Organization"], "article-title": ["Global Status Report on Road Safety 2018."], "ext-link": ["Reference Source"]}, {"label": ["2"], "mixed-citation": ["\n"], "collab": ["Ministry of Road Transport & Highways"], "article-title": ["Road Accidents in Ind... | {
"acronym": [],
"definition": []
} | 18 | CC BY | no | 2024-01-15 23:43:45 | F1000Res. 2023 Oct 20; 12:494 | oa_package/95/3e/PMC10787871.tar.gz |
PMC10787873 | 37815684 | [
"<title>Introduction</title>",
"<p id=\"Par5\">Breast cancer is the most common malignancy affecting women worldwide [##REF##30207593##1##]. In the United States, approximately 5% of patients with breast cancer have de novo metastatic disease at diagnosis, and at least 20% of those initially diagnosed with early-... | [
"<title>Methods</title>",
"<title>Patients</title>",
"<p id=\"Par8\">Male or female patients ≥ 18 years who had histologically confirmed locally advanced, unresectable or metastatic carcinoma of the breast of all subtypes were eligible to enroll with no limitation on prior lines of therapy. Eligibility included... | [
"<title>Results</title>",
"<title>Patient characteristics</title>",
"<p id=\"Par16\">Patients with histologically confirmed metastatic or ABC with any number of prior lines of therapy were eligible to enroll in this study, and 44 patients enrolled in total. Baseline patient characteristics are summarized in Tab... | [
"<title>Discussion</title>",
"<p id=\"Par24\">This trial was conducted to assess the safety, efficacy, and tolerability of EC for the treatment of patients with metastatic or ABC. CBR and PFS were 79.5% and 16.4 weeks respectively, comparing favorably to historic data of single agent eribulin for ABC (PFS 14.8 we... | [] | [
"<title>Purpose</title>",
"<p id=\"Par1\">We hypothesized that eribulin combined with cyclophosphamide (EC) would be an effective combination with tolerable toxicity for the treatment of advanced breast cancer (ABC).</p>",
"<title>Methods</title>",
"<p id=\"Par2\">Patients with histologically confirmed metast... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Funding</title>",
"<p>This work was supported in part by funding to the UCSF Regents from Eisai, Inc.</p>",
"<title>Data availability</title>",
"<p>Enquiries about data availability should be directed to the authors.</p>",
"<title>Declarations</title>",
"<title>Conflict of interest</title>",
"<p... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Kaplan–Meier plot of progression-free survival (PFS). <bold>a</bold> Shown is the Kaplan–Meier plot for PFS for all patients in the study. The median PFS was 16.4 weeks (95% CI: 13.8–21.1 weeks) in all patients. <bold>b</bold> Shown is the Kaplan–Meier plot for PFS... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Patient characteristics at baseline</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Characteristics</th><th align=\"left\">EC (n = 44)</th><th align=\"left\">%</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"3\">... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><italic>CNS</italic> central nervous system, <italic>ECOG</italic> Eastern Cooperative Oncology Group</p></table-wrap-foot>",
"<table-wrap-foot><p><italic>CBR</italic> clinical benefit rate, <italic>CI</italic> confidence interval, <italic>CR</italic> complete response, <italic>n</italic> num... | [
"<inline-graphic xlink:href=\"10549_2023_7073_Article_IEq1.gif\"/>",
"<inline-graphic xlink:href=\"10549_2023_7073_Article_IEq2.gif\"/>",
"<inline-graphic xlink:href=\"10549_2023_7073_Article_IEq10.gif\"/>",
"<inline-graphic xlink:href=\"10549_2023_7073_Article_IEq11.gif\"/>",
"<inline-graphic xlink:href=\"... | [
"<media xlink:href=\"10549_2023_7073_MOESM1_ESM.docx\"><caption><p>Supplementary file1 (DOCX 30 KB)</p></caption></media>"
] | [{"label": ["4."], "surname": ["Hortobagyi"], "given-names": ["G"], "article-title": ["LBA17 overall survival (OS) results from the phase III MONALEESA-2 (ML-2) trial of postmenopausal patients (pts) with hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2\u2212) advanced breast cancer... | {
"acronym": [],
"definition": []
} | 30 | CC BY | no | 2024-01-15 23:42:01 | Breast Cancer Res Treat. 2024 Oct 10; 203(2):197-204 | oa_package/13/b8/PMC10787873.tar.gz |
PMC10787874 | 37204642 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Many studies support the notion that when parents are consistent in their responses to their child’s misbehavior, children show less externalizing behavior (Barry et al., ##UREF##3##2009##; Gardner, ##REF##2745902##1989##; Gryczkowski et al., ##UREF##8##2010##; Halgu... | [
"<title>Method</title>",
"<title>Sample</title>",
"<p id=\"Par11\">We included two samples, to allow for conceptual replication: Sample 1 is a community sample of 134 mothers of 1.5–3.5 year old children (<italic>M</italic> = 30 months, 44.3% girls), who reported on their child’s temper tantrums (frequency and ... | [
"<title>Results</title>",
"<p id=\"Par28\">On average, children in Sample 1 had an average 5.90 tantrums during the 7-day period (<italic>SD</italic> = 4.21, range 2–20), and for children in Sample 2 the mean level of disruptive behavior was rated 3.32 on the 10 point scale across the 14 days (<italic>SD</italic>... | [
"<title>Discussion</title>",
"<p id=\"Par34\">Aim of this study was to investigate how within- and across-episode parental consistency in responding to misbehavior are associated to externalizing problem behavior in children, using a daily diary approach. Within- and across-episode consistency were moderately str... | [
"<title>Conclusion</title>",
"<p id=\"Par43\">Results of this study show that it is meaningful to separate parental consistency within- from consistency across-episodes of misbehavior as they are correlated, but not strongly so. Furthermore, the aspects of consistency may be differentially important for the sever... | [
"<p id=\"Par1\">Consistent discipline is thought to reduce early child externalizing behavior. It is unclear, however, whether consistency is important mainly within episodes of misbehavior (e.g., threatening with discipline but then giving in) or across episodes of misbehavior (e.g., disciplining each instance of ... | [] | [
"<title>Appendix I: Questions as included in the daily diaries</title>",
"<title>Parental Reactions</title>",
"<title>Sample 1</title>",
"<p id=\"Par46\">For each tantrum during that day in daily diary, question:</p>",
"<title>Sample 2</title>",
"<p id=\"Par48\">For the most difficult to manage disruptive... | [] | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Descriptives and Intercorrelations for the Study Variables</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Measures</th><th align=\"left\">1.</th><th align=\"left\">2.</th><th align=\"left\">3.</th><th align=\"left\">4.</... | [
"<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t... | [] | [] | [
"<disp-quote><p id=\"Par47\"><italic>‘How did you react to tantrum x?’ (x = 1–7 max)</italic></p></disp-quote>",
"<disp-quote><p id=\"Par49\"><italic>‘How did you react to this disruptive behavior?’</italic></p></disp-quote>",
"<disp-quote><p id=\"Par52\">‘<italic>Which of the following behaviors did your child... | [] | [] | [
"<table-wrap-foot><p>Estimates for Sample 1 are provided below the diagonal, and for Sample 2 above the diagonal</p><p>*<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdic... | [
"<graphic xlink:href=\"10802_2023_1073_Article_Equa.gif\" position=\"anchor\"/>"
] | [] | [{"mixed-citation": ["Achenbach, T. M., & Rescorla, L. A. (2000). "], "italic": ["Manual for the ASEBA preschool forms & profiles"]}, {"surname": ["Ainsworth", "Blehar", "Waters", "Wall"], "given-names": ["MDS", "MC", "E", "SN"], "article-title": ["Patterns of attachment: a psychological study of the strange situation"... | {
"acronym": [],
"definition": []
} | 39 | CC BY | no | 2024-01-15 23:42:01 | Res Child Adolesc Psychopathol. 2024 May 19; 52(1):79-92 | oa_package/7e/9a/PMC10787874.tar.gz |
PMC10787875 | 38217693 | [
"<title>Introduction</title>",
"<p id=\"Par5\">Multiparametric MRI (mpMRI) of the prostate is nowadays considered the key tool for the diagnosis of clinically significant prostate cancer (csPCa) [##UREF##0##1##].</p>",
"<p id=\"Par6\">The quality of MRI has progressively increased in the last years and standard... | [
"<title>Patients and methods</title>",
"<title>Study design and population</title>",
"<p id=\"Par11\">This is a retrospective analysis of patients who had undergone mpMRI before RP at Fundació Puigvert – Barcelona (ES), between April 2013 (date of the implementation of mpMRI driven pathway) and December 2017. T... | [
"<title>Results</title>",
"<p id=\"Par19\">The clinical, bioptic and MRI features are summarised in Table ##TAB##0##1##. The median age at prostate biopsy, PSA and PSA-density were 66.6 years, 7.2 ng/ml and 0.2 ng/ml<sup>2</sup>, respectively.</p>",
"<p id=\"Par20\">Overall, 41 patients (32.5%) had T2 or T3 cli... | [
"<title>Discussion</title>",
"<p id=\"Par29\">The prognosis of PCa is highly related to tumour stage, and ECE is the most common adverse feature found in histopathology after RP [##REF##23083655##8##]. Inadequate selection of patients with ECE to nerve-sparing RP increases the risk of positive surgical margins wi... | [
"<title>Conclusions</title>",
"<p id=\"Par46\">The mpMRI confirms limitations in predicting the ECE at the RP specimen, even when involving tailored scoring systems. On the other side, MRI-LCI is the most robust factor associated to ECE; in our series, we found a strong predictive accuracy when combined with the ... | [
"<title>Objectives</title>",
"<p id=\"Par1\">To identify the predictive factors of prostate cancer extracapsular extension (ECE) in an institutional cohort of patients who underwent multiparametric MRI of the prostate prior to radical prostatectomy (RP).</p>",
"<title>Patients and methods</title>",
"<p id=\"P... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Acknowledgements</title>",
"<p>We are thankful to Cristina Esquinas Lopez, Professor of Statistics at at Vall d’Hebron Research Institute, Universitat Autonoma de Barcelona (Spain) for undertaking the statistical analysis on the manuscript. <underline>crise4@hotmail.com</underline></p>",
"<title>Funding... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>ROC curve analysis for predictive model</p></caption></fig>"
] | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Patients’ clinical data</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Variables</th><th align=\"left\">Median (IQR)</th><th align=\"left\"><italic>N</italic> (%)</th></tr></thead><tbody><tr><td align=\"left\">Age at bio... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><italic>yr</italic> years, <italic>ng</italic> nanograms, <italic>ml</italic> millilitres, <italic>mm</italic> millimetres, <italic>cc</italic> cubic centimetres</p></table-wrap-foot>",
"<table-wrap-foot><p><italic>mm</italic> millimetres</p></table-wrap-foot>",
"<table-wrap-foot><p><bold>M... | [
"<graphic xlink:href=\"345_2023_4720_Fig1_HTML\" id=\"MO1\"/>"
] | [
"<media xlink:href=\"345_2023_4720_MOESM1_ESM.docx\"><caption><p>Supplementary file1 (DOCX 129 KB)</p></caption></media>"
] | [{"label": ["1."], "mixed-citation": ["Mottet N, B J, Briers E, Bolla M, Bourke L, Cornford P, De Santis M, Henry A, Joniau S, Lam T, Mason MD, Van den Poel H, Van den Kwast TH, Rouvi\u00e8re O, Wiegel T, members of the EAU \u2013 ESTRO \u2013 ESUR \u2013SIOG Prostate Cancer Guidelines Panel (2021) EAU \u2013 ESTRO \u2... | {
"acronym": [],
"definition": []
} | 31 | CC BY | no | 2024-01-15 23:42:01 | World J Urol. 2024 Jan 13; 42(1):37 | oa_package/5e/6f/PMC10787875.tar.gz |
PMC10787876 | 37833450 | [
"<title>Introduction</title>",
"<p id=\"Par16\">Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer after invasive ductal carcinoma (IDC), representing 10–15% of all cases [##REF##25848941##1##]. In the metastatic setting, ILC differs in its pattern of metastatic sites... | [
"<title>Methods</title>",
"<title>Data source and study cohort</title>",
"<p id=\"Par20\">The NCDB is a national comprehensive clinical surveillance resource representing over 70% of all newly diagnosed cancer cases in the United States and includes patient demographics, clinical information, and survival outco... | [
"<title>Results</title>",
"<title>Study cohort</title>",
"<p id=\"Par27\">There were 100,147 patients with stage IV breast cancer, of whom 25,294 had HR-positive, HER2-negative invasive lobular or ductal histology, and met study criteria (Fig. ##FIG##0##1##). Of these patients, 19,171 (75.8%) had IDC and 6,123 ... | [
"<title>Discussion</title>",
"<p id=\"Par43\">Recent randomized trial data suggest that the role of primary-site surgery in the management of patients with metastatic breast cancer is limited to local control in select cases, with no evidence of impact on OS [##REF##34995128##9##–##REF##26363985##11##]. However, ... | [] | [
"<title>Purpose</title>",
"<p id=\"Par1\">Primary site surgery for metastatic breast cancer improves local control but does not impact overall survival. Whether histologic subtype influences patient selection for surgery is unknown. Given differences in surgical management between early-stage lobular versus ducta... | [] | [
"<title>Author contributions</title>",
"<p>Supervision, study concept and design, and interpretation of data were provided by Rita A Mukhtar. The first draft of the manuscript was written by Harriet Rothschild and all authors commented on previous versions of the manuscript. Amy Shui performed statistical analysi... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>CONSORT flow diagram for study population of stage IV, hormone receptor positive, HER2-negative breast cancer patients from the National Cancer Database (PUF 2010–2016). <italic>ER</italic>, estrogen receptor; <italic>PR</italic>, progesterone receptor; <italic>HER... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparison of patient characteristics between invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) cohorts in unmatched population</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Patient Characteristics</t... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p><sup>a</sup>Community treatment facility includes Community Cancer Programs, Comprehensive Community Cancer Programs, and Integrated Network Cancer Programs</p><p><sup>b</sup>Median Income Quartiles from 2012–2016</p></table-wrap-foot>",
"<table-wrap-foot><p><sup>a</sup> Treatment facility da... | [
"<graphic xlink:href=\"10549_2023_7125_Fig1_HTML\" id=\"d32e462\"/>",
"<graphic xlink:href=\"10549_2023_7125_Fig2_HTML\" id=\"d32e1318\"/>",
"<graphic xlink:href=\"10549_2023_7125_Fig3_HTML\" id=\"d32e2276\"/>"
] | [] | [] | {
"acronym": [
"CDCI",
"ER",
"HER2",
"HR",
"IDC",
"ILC",
"NCDB",
"OS",
"PR"
],
"definition": [
"Charlson-Deyo Co-Morbidity Index",
"Estrogen receptor",
"Human epidermal growth factor receptor 2",
"Hormone receptor",
"Invasive ductal carcinoma",
"Invasive... | 23 | CC BY | no | 2024-01-15 23:42:01 | Breast Cancer Res Treat. 2024 Oct 13; 203(2):245-256 | oa_package/92/6e/PMC10787876.tar.gz |
PMC10787878 | 0 | [
"<title>Introduction</title>",
"<p id=\"Par5\">Terpenoids are a large group of natural compounds constructed from isoprene units (C5). Many different terpenoids are used in the food, beverage, cosmetic, and pharmaceutical industries (Tetali ##REF##30467631##2019##). The current industrial supply of terpenoids rel... | [
"<title>Materials and methods</title>",
"<title>Genes for yeast strain construction</title>",
"<p id=\"Par9\">The genes used for the construction of the yeast strains were prepared as described in the Supplementary Information. The plasmids, primers, and DNA fragments used are listed in Tables ##SUPPL##0##S1##,... | [
"<title>Results</title>",
"<title>The strain expressing the MVA pathway in mitochondria</title>",
"<p id=\"Par19\">Proteins can be delivered to the mitochondria by fusion with a mitochondrial localization signal (MLS); however, their enzymatic activities are sometimes interrupted in the mitochondria if the MLS ... | [
"<title>Discussion</title>",
"<p id=\"Par23\">In this study, we demonstrated the beneficial effect of the MVA pathway introduced into the mitochondria for terpenoid production by measuring IPP/DMAPP, an essential precursor of terpenoids. Furthermore, using mutants with defective mitochondrial morphology, we found... | [] | [
"<title>Abstract</title>",
"<p id=\"Par1\">Terpenoids are widely used in the food, beverage, cosmetics, and pharmaceutical industries. Microorganisms have been extensively studied for terpenoid production. In yeast, the introduction of the mevalonate (MVA) pathway in organelles in addition to the augmentation of ... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Acknowledgements</title>",
"<p>The authors are grateful for the support from NEDO Projects No. P16009 (Development of production techniques for highly functional biomaterials using plant and other organism smart cells).</p>",
"<title>Author contribution</title>",
"<p>SY designed and performed the expe... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Introduction of the MVA pathway into mitochondria. <bold>a</bold> Introduction of the MVA pathway into mitochondria. Native and engineered pathways are shown in black and blue, respectively. <bold>b</bold> The localization of the MLS-fused GFP. The left and right p... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Strains used or constructed in this study</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Strain</th><th align=\"left\">Genotype</th></tr></thead><tbody><tr><td align=\"left\">BY4741</td><td align=\"left\">MATa, <italic>h... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"253_2023_12922_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"253_2023_12922_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"253_2023_12922_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"253_2023_12922_Fig4_HTML\" id=\"MO4\"/>"
] | [
"<media xlink:href=\"253_2023_12922_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 1399 KB)</p></caption></media>"
] | [{"mixed-citation": ["Lv X, Wang F, Zhou P, Ye L, Xie W, Xu H, Yu H (2016) Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in "], "italic": ["Saccharomyces cerevisiae"]}, {"mixed-citation": ["Ohsumi Y, Shimoda T (2007) \u201cAll about yeast\u201d [Translated from... | {
"acronym": [],
"definition": []
} | 29 | CC BY | no | 2024-01-15 23:42:01 | Appl Microbiol Biotechnol. 2024 Jan 13; 108(1):1-9 | oa_package/7d/53/PMC10787878.tar.gz |
PMC10787880 | 0 | [
"<title>Introduction</title>",
"<p id=\"Par6\">The asymmetric reduction of C = C double bonds is widely applied in the pharmaceutical, fine chemical, and agrochemical industries, as up to two stereogenic centers can be created in those processes. Unfortunately, the chemo-catalyzed reduction of olefins using hydro... | [
"<title>Materials and methods</title>",
"<title>Reagents</title>",
"<p id=\"Par11\">Alkene substrates and racemic reduction products were purchased from MilliporeSigma (MA, USA), Aladdin (Shanghai, China), or TCI (Shanghai, China). Glucose dehydrogenase from <italic>Bacillus</italic> was purchased from Aladdin ... | [
"<title>Results</title>",
"<title>Mining OYEs from A. flavus and sequence analysis</title>",
"<p id=\"Par27\">To identify new OYEs from <italic>A. flavus</italic>, the thermophilic-like OYE YqjM from <italic>Bacillus subtilis</italic> was used as a reference to search the <italic>A. flavus</italic> proteins in ... | [
"<title>Discussion</title>",
"<p id=\"Par40\">The <italic>Af</italic>OYE1 was previously named afvA, which was annotated as an NADPH dehydrogenase. The proposed function of afvA was to hydroxylate siderin and 7-demethyl siderin in the biosynthesis of aflavarin (Cary et al. ##REF##26209694##2015##). Here, the reco... | [] | [
"<title>Abstract</title>",
"<p id=\"Par1\">Old yellow enzymes (OYEs) have been proven as powerful biocatalysts for the asymmetric reduction of activated alkenes. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. To expand the OYEs toolbox, a new thermophilic-like OYE (<itali... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Acknowledgements</title>",
"<p>We thank SSRF 18U1 beamline scientists for their help with data collection and the NMR center in the College of Science at Henan Agricultural University for taking NMR spectra. We acknowledge English editorial assistance by Maria Ajmal (Henan Agricultural University, China).... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Phylogenetic analysis of <italic>Af</italic>OYE1 and previously described OYEs. The phylogenetic analysis was conducted using the Maximum Likelihood statistical method based on the Jones-Taylar-Thornton (JTT) model in MEGAX. The corresponding alignment was done by ... | [] | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>Na Li and Yuan Wang contributed equally to the work.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"253_2023_12963_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"253_2023_12963_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"253_2023_12963_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"253_2023_12963_Fig4_HTML\" id=\"MO4\"/>",
"<graphic xlink:href=\"253_2023_12963_Fig5_HTML\... | [
"<media xlink:href=\"253_2023_12963_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 22613 KB)</p></caption></media>"
] | [{"surname": ["Adams", "Afonine", "Bunkoczi", "Chen", "Davis", "Echols", "Headd", "Hung", "Kapral", "Grosse-Kunstleve", "McCoy", "Moriarty", "Oeffner", "Read", "Richardson", "Richardson", "Terwilliger", "Zwart"], "given-names": ["PD", "PV", "G", "VB", "IW", "N", "JJ", "L-W", "GJ", "RW", "AJ", "NW", "R", "RJ", "DC", "JS... | {
"acronym": [],
"definition": []
} | 42 | CC BY | no | 2024-01-15 23:42:01 | Appl Microbiol Biotechnol. 2024 Jan 13; 108(1):1-11 | oa_package/54/2b/PMC10787880.tar.gz |
PMC10787881 | 0 | [
"<title>Introduction</title>",
"<p id=\"Par5\">The model yeast species <italic>Saccharomyces cerevisiae</italic> is used in many fundamental and applied research applications, such as production of many industrially relevant compounds (Krivoruchko and Nielsen ##REF##25544013##2015##) and as biosensors to various ... | [
"<title>Materials and methods</title>",
"<title>Yeast cultivation</title>",
"<p id=\"Par8\">The yeast strain used for this work was BY4742 (<italic>MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0</italic>; Baker Brachmann et al. ##REF##9483801##1998##). For the experimental exposures, overnight suspension cultures were inocul... | [
"<title>Results</title>",
"<title>Overview of transcriptional response to lactic acid enantiomers</title>",
"<p id=\"Par14\">The performed analysis revealed differentially expressed genes (hereafter DEGs; absolute log2 fold change > 1 and adjusted <italic>p</italic>-value < 0.05) only in the case of the t... | [
"<title>Discussion</title>",
"<p id=\"Par23\">In this study, we explored the transcriptional response of <italic>S. cerevisiae</italic> to LA enantiomers. One of the goals of this work was to find genes that would specifically and quantitatively respond to DLA but not to LLA. We did not find such genes. In genera... | [] | [
"<title>Abstract</title>",
"<p id=\"Par1\">The model yeast, <italic>Saccharomyces cerevisiae</italic>, is a popular object for both fundamental and applied research, including the development of biosensors and industrial production of pharmaceutical compounds. However, despite multiple studies exploring <italic>S... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Author contribution</title>",
"<p>PD, AG, MT, and EB designed the research. PD, AS, AV, and EI performed the experiments. PD, AG, AV, and EZ analyzed the data and prepared text and figures. MT and EB supervised the project. All authors read and approved the final manuscript.</p>",
"<title>Funding</title... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Overview of gene expression changes in all comparisons. <bold>a</bold>–<bold>f</bold> show volcano plots for major comparisons with differentially expressed genes (absolute log<sub>2</sub> fold change > 1 and adjusted <italic>p</italic>-value < 0.05) are indi... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Significantly (<italic>p</italic> < 0.05) overrepresented GO terms</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\" colspan=\"2\">45 mM DLA vs. control</td></tr><tr><td align=\"left\">Upregulated (82 genes)</td><td alig... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>",
"<supplementary-material ... | [
"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"253_2023_12863_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"253_2023_12863_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"253_2023_12863_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"253_2023_12863_Fig4_HTML\" id=\"MO4\"/>"
] | [
"<media xlink:href=\"253_2023_12863_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 649 KB)</p></caption></media>",
"<media xlink:href=\"253_2023_12863_MOESM2_ESM.xlsx\"><caption><p>Supplementary file2 (XLSX 14 KB)</p></caption></media>",
"<media xlink:href=\"253_2023_12863_MOESM3_ESM.xlsx\"><caption><p>S... | [{"mixed-citation": ["Blighe K, Rana S, Lewis M (2023) EnhancedVolcano: publication-ready volcano plots with enhanced colouring and labeling. "], "ext-link": ["https://github.com/kevinblighe/EnhancedVolcano"]}, {"mixed-citation": ["Drozdova P (2023) "], "italic": ["S. cerevisiae"], "ext-link": ["https://github.com/droz... | {
"acronym": [],
"definition": []
} | 68 | CC BY | no | 2024-01-15 23:42:01 | Appl Microbiol Biotechnol. 2024 Jan 13; 108(1):1-12 | oa_package/b8/86/PMC10787881.tar.gz |
PMC10787882 | 37861889 | [
"<title>Introduction</title>",
"<p id=\"Par5\">A substantial proportion of cancers are associated with pathogenic variants (PV) in hereditary cancer genes [##REF##35971132##1##, ##REF##27978560##2##], including an estimated 10% of breast, 10% of colon, and 20% of ovarian cancers. Approximately, 1 in 300 to 500 pe... | [
"<title>Materials and methods</title>",
"<title>Study population</title>",
"<p id=\"Par8\">This retrospective cohort study included unselected female patients who were offered and underwent genetic testing at the time of breast imaging at three imaging centers (Memorial MRI and Diagnostics, Texas) from November... | [
"<title>Results</title>",
"<title>Study population</title>",
"<p id=\"Par18\">A total of 1,943 women undergoing breast imaging elected to have hereditary cancer genetic testing during the study period (Table ##TAB##1##2##). Median age was 66 years (range 18–89 years). Self-reported race and ethnicity were Asian... | [
"<title>Discussion</title>",
"<p id=\"Par26\">We found that 2% (39/1943) of women undergoing hereditary cancer gene testing as part of their clinical care at the time of breast imaging had PVs in hereditary cancer genes that had implications for cancer surveillance and clinical management. Importantly, the popula... | [] | [
"<title>Purpose</title>",
"<p id=\"Par1\">Up to 10% of all breast cancers (BC) are attributed to inherited pathogenic variants (PV) in BC susceptibility genes; however, most carriers of PVs remain unidentified. Here, we sought to determine the yield of hereditary cancer gene PVs among diverse women attending brea... | [] | [
"<title>Acknowledgements</title>",
"<p>We would like to acknowledge Sofia Hurtado, BS and Mayra Rodas, BS from Natera, Inc. for data acquisition and Urmi Sengupta, PhD, from Natera, Inc., for assistance with the development of the manuscript.</p>",
"<title>Author contributions</title>",
"<p>LW, VS, and JW hav... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Personal cancer history in the full cohort (<italic>N</italic> = 1943)</p></caption></fig>",
"<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Number of clinically actionable PVs identified in high- or moderate-risk genes in patients who either met or did not me... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>List of 25 actionable genes reported for all patients</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">High-risk Genes</th><th align=\"left\">Moderate-risk Genes</th></tr></thead><tbody><tr><td align=\"left\"><italic>APC</... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>HBOC, Hereditary Breast and Ovarian Cancer-related genes <italic>(BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, NF1, BRIP1)</italic></p><p>LS, Lynch syndrome genes <italic>(MSH2, PMS2)</italic></p></table-wrap-foot>",
"<table-wrap-foot><p>*Pre-test Tyrer–Cuzick score was calculated for the patients... | [
"<graphic xlink:href=\"10549_2023_7137_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"10549_2023_7137_Fig2_HTML\" id=\"MO2\"/>"
] | [] | [{"label": ["24."], "mixed-citation": ["Network NCC (2023) Genetic/familial high-risk assessment: breast, ovarian, and pancreatic. "], "ext-link": ["https://www.nccn.org/professionals/physician_gls/pdf/genetics_bop.pdf"]}, {"label": ["25."], "mixed-citation": ["NCCN.org. (2023). "], "ext-link": ["https://www.nccn.org/p... | {
"acronym": [],
"definition": []
} | 46 | CC BY | no | 2024-01-15 23:42:01 | Breast Cancer Res Treat. 2024 Oct 20; 203(2):365-372 | oa_package/86/b6/PMC10787882.tar.gz |
PMC10787883 | 38217724 | [
"<title>Introduction</title>",
"<p id=\"Par5\">Robotic surgery has revolutionized the treatment of several diseases given the improved clinical outcomes, such as less blood loss, shorter length of hospital stay and fewer complications compared with laparoscopy or open approach. The main limitation has been the hi... | [
"<title>Methods</title>",
"<p id=\"Par10\">This is a retrospective observational study involving two Institutions, ASST Santi Paolo and Carlo, Milan, and at the Apuane Hospital, Massa, Italy.</p>",
"<p id=\"Par11\">The Versius CMR was installed by September, 2022, at the former institution, and by 2021 in the l... | [
"<title>Results</title>",
"<p id=\"Par21\">Overall, a total of 171 interventions were performed with the Versius CMR at the ASST Santi Paolo and Carlo (120) and at the Apuane Hospital (51). Forty-two of them involved pelvic procedures. A full list of interventions stratified by center and specialty is reported in... | [
"<title>Discussion</title>",
"<p id=\"Par23\">The current series represents the first one completely focused on pelvic surgery with the new Versius surgical system.</p>",
"<p id=\"Par24\">Sparse reports evaluating single indications with Versius [##REF##37221826##7##–##UREF##0##9##] are available yet; similarly... | [
"<title>Conclusions</title>",
"<p id=\"Par38\">From the present series, pelvic surgery with the Versius system is feasible without severe intra- or peri-operative complications; long-term oncological and functional outcomes are yet to be defined. All steps of pelvic surgery are reproducible with the Versius, prov... | [
"<title>Introduction</title>",
"<p id=\"Par1\">Versius CMR is a novel robotic system characterized by an open surgical console and independent bedside units. The system has potentials of flexibility and versatility, and has been used in urological, gynecological, and general surgical procedure. The aim is to depi... | [] | [
"<title>Author contributions</title>",
"<p>MCS: protocol and project development, manuscript writing. MDM: project development, data management. JM: data collection, data analysis. MF: project development, data management. APC: project development, data management, manuscript editing. LM: data management. CC: dat... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>OR setup for prostatectomy (<bold>a</bold>), gynecologic surgery (<bold>b</bold>), rectopexy (<bold>c</bold>), signoidectomy (<bold>d</bold>)</p></caption></fig>"
] | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Procedures stratified by Center</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Procedure</th><th align=\"left\">Number</th><th align=\"left\">Center</th><th align=\"left\">Details</th></tr></thead><tbody><tr><td align=\"... | [] | [] | [] | [] | [] | [] | [
"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>Maria Chiara Sighinolfi and Maurizio De Maria shared the first-authorship.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"345_2023_4730_Fig1_HTML\" id=\"MO1\"/>"
] | [] | [{"label": ["9."], "surname": ["El Dahdah", "Halabi", "Kamal", "Zenilman", "Moussa"], "given-names": ["J", "M", "J", "ME", "HJ"], "article-title": ["Initial experience with a novel robotic surgical system in abdominal surgery"], "source": ["Robot Surg"], "year": ["2023"], "volume": ["17"], "issue": ["3"], "fpage": ["84... | {
"acronym": [],
"definition": []
} | 17 | CC BY | no | 2024-01-15 23:42:01 | World J Urol. 2024 Jan 13; 42(1):31 | oa_package/36/d9/PMC10787883.tar.gz |
PMC10787884 | 37812362 | [
"<title>Introduction</title>",
"<p id=\"Par5\">Breast cancer in men (male breast cancer; MBC) is a rare entity accounting for 0.5–1% of all breast cancer diagnoses and an estimated lifetime risk of 1:1000 [##REF##30620402##1##]. Due to its rarity and lack of prospective dedicated MBC trials, the treatment guideli... | [
"<title>Patients and methods</title>",
"<title>Study design, data sources, participants and data collection</title>",
"<p id=\"Par8\">For this nationwide, register-based retrospective <italic>cohort study</italic>, all patients with stage I–III invasive breast cancer diagnosed in Sweden between January 1, 2008 ... | [
"<title>Results</title>",
"<title>Characteristics of study cohort</title>",
"<p id=\"Par15\">In total, 114,290 patients with breast cancer were registered in NKBC between 2008 and 2020. Applying the inclusion and exclusion criteria, 82,888 patients (82,401 women and 428 men) with stage I-III breast cancer who u... | [
"<title>Discussion</title>",
"<p id=\"Par19\">Using nationwide, register-based data from Sweden, we found no evidence of sex disparities regarding utilization of NAC in breast cancer patients when analyses were adjusted for patient- and tumor characteristics. In addition, the effectiveness of NAC in terms of pCR ... | [] | [
"<title>Purpose</title>",
"<p id=\"Par1\">Evidence supporting the use of neoadjuvant chemotherapy (NAC) in early breast cancer is based on studies mainly including women, whereas the utilization and effectiveness of NAC in men is less studied. The present study aimed to investigate the utilization and effectivene... | [] | [
"<title>Author contributions</title>",
"<p>AV conceived the original idea about the study and study’s design. Material preparation, data collection and analysis were performed by AV. The first draft of the manuscript was written by AS and all authors commented on previous versions of the manuscript. All authors r... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>CONSORT diagram of study population. Cohort I: All patients registered in NKBC with stage I-III breast cancer who underwent surgery and had adequate information for IHC-subtyping. Cohort II: restricted only to patients who received neoadjuvant chemotherapy (NAC) (B... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Summary of demographics, tumor-related variables, and treatment patterns for the utilization cohort; women and men with stage I-III breast cancer who underwent surgery and had adequate information for IHC-subtyping (BCBaSe 3.0/NKBC, 2008–2019)</p></caption>... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>Bold text indicates a statistically significant difference with a p-value less than 0.05</p></table-wrap-foot>",
"<table-wrap-foot><p>Bold text indicates a statistically difference with a p-value less than 0.05</p></table-wrap-foot>",
"<table-wrap-foot><p>The multivariate models were comple... | [
"<graphic xlink:href=\"10549_2023_7129_Fig1_HTML\" id=\"MO1\"/>"
] | [] | [] | {
"acronym": [],
"definition": []
} | 16 | CC BY | no | 2024-01-15 23:42:01 | Breast Cancer Res Treat. 2024 Oct 9; 203(2):235-243 | oa_package/e1/ed/PMC10787884.tar.gz |
PMC10787885 | 38217834 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Robotic-assisted surgery (RAS) has gained widespread diffusion over the last years, demonstrating the ability to overcome the technical limitations of conventional laparoscopy. Enhancements provided by robotic assistance include three-dimensional view and magnificati... | [
"<title>Materials and methods</title>",
"<p id=\"Par5\">All children and adolescents up to 18 years of age, operated using RAS for gynecological indications in 4 different institutions over a 3-year period, were included. The exclusion criteria were patients over 18 years old as well as all gynecological surgical... | [
"<title>Results</title>",
"<title>Patient baseline</title>",
"<p id=\"Par12\">Twenty-three girls, with median age at surgery of 12.3 years (range 0.6–17.8) and median weight of 47.2 kg (range 9–73), received RAS for gynecological indication in the study period and were included. Associated comorbidities were re... | [
"<title>Discussion</title>",
"<p id=\"Par20\">Despite the growing number of indications in pediatric urology, the application of RAS remains still limited in other fields of pediatric surgery. Analyzing the pediatric literature, very few reports on RAS application in gynecology are available, with limited case se... | [
"<title>Conclusion</title>",
"<p id=\"Par28\">This preliminary experience showed that RAS is safe and feasible for surgical treatment of pediatric gynecological pathology, although no conclusive data are available to confirm its superiority over traditional laparoscopy. Case–control and comparative prospective st... | [
"<p id=\"Par1\">Robotic-assisted surgery (RAS) is increasingly adopted in the pediatric population. This retrospective multicenter study aimed to report application of RAS for gynecological indications in pediatric patients. The medical records of all girls with gynecological pathology, operated in 4 different inst... | [] | [
"<title>Author contributions</title>",
"<p>All authors contributed to the study conception and design. All authors performed material preparation, data collection, and data analysis. M.E. and C.E. wrote the first draft of the manuscript. G.E. and C.D.M. prepared Fig. ##FIG##0##1##. A.C. and M.E. prepared video ##... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>After removal of giant paratubal cyst (<bold>a</bold>, <bold>b</bold>), ICG-guided fluorescence was helpful to check the vascular permeability of fallopian tube (<bold>c</bold>)</p></caption></fig>",
"<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Pre-operativ... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Outcomes of RAS in pediatric gynecological indications</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Patient</th><th align=\"left\">Age (years)</th><th align=\"left\">Side</th><th align=\"left\">Comorbidity</th><th alig... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p><italic>RAS</italic> robotic-assisted surgery, <italic>OT</italic> operative time, <italic>LOS</italic> length of stay, <italic>DSD</italic> disorder of sex development</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard ... | [
"<graphic xlink:href=\"11701_2023_1767_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"11701_2023_1767_Fig2_HTML\" id=\"MO2\"/>"
] | [
"<media xlink:href=\"11701_2023_1767_MOESM1_ESM.mp4\" id=\"MOESM1\"><caption><p>Supplementary file1 (MP4 46006KB)</p></caption></media>"
] | [{"label": ["4."], "surname": ["Andolfi", "Kumar", "Boysen", "Gundeti"], "given-names": ["C", "R", "WR", "MS"], "article-title": ["Current status of robotic surgery in pediatric urology"], "source": ["J Laparoend Adv Surg Tech Part A"], "year": ["2019"], "volume": ["29"], "issue": ["2"], "fpage": ["159"], "lpage": ["16... | {
"acronym": [],
"definition": []
} | 20 | CC BY | no | 2024-01-15 23:42:01 | J Robot Surg. 2024 Jan 13; 18(1):20 | oa_package/d2/98/PMC10787885.tar.gz |
PMC10787886 | 37249705 | [
"<title>Introduction</title>",
"<p id=\"Par2\">In the DSM-5, a “with Limited Prosocial Emotions” specifier was added to the Conduct Disorder diagnostic criteria to differentiate those children and adolescents who also display a callous absence of empathy and remorse, shallow or narrow affect, and deficient concer... | [
"<title>Method</title>",
"<title>Participants and Procedure</title>",
"<p id=\"Par9\">Adolescents living in a western Canadian province were recruited between April 2019 and October 2020 to participate in an online study examining their psychological and behavioral adjustment (see Xu & Zheng, ##REF##3558379... | [
"<title>Results</title>",
"<title>Callous-unemotional Traits at the Item Level</title>",
"<p id=\"Par15\">Descriptive statistics, ICCs, and within-level and between-level CU item correlations are shown in ##SUPPL##0##Supplementary Table S1##. ICC is the proportion of the total variance that is accounted for by ... | [
"<title>Discussion</title>",
"<p id=\"Par23\">By collecting intensive longitudinal data and applying analytic approaches that elucidate temporal dynamics, we can expand our understanding of CU traits in a more ecologically valid way. Accordingly, this exploratory study examined whether adolescent CU traits manife... | [
"<title>Conclusion</title>",
"<p id=\"Par34\">In this exploratory study, we observed meaningful fluctuations of CU traits at both item- and subscale-levels. These findings suggest that CU traits may be more susceptible to change and malleable than previously thought. In addition, callousness and uncaring subscale... | [
"<p id=\"Par1\">Intensive longitudinal methods (e.g., daily diary) inform understanding of dynamic processes by parsing <italic>within-person</italic> state-like fluctuations from stable <italic>between-person</italic> trait-like differences. In this exploratory study, we investigated whether self-reported callous-... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Acknowledgements</title>",
"<p>We thank all the participating parents, adolescents, Elk Island and St. Albert public schools, and our research assistants. Study data were collected and managed using REDCap electronic data capture tools hosted and supported by the Women and Children’s Health Research Insti... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Schematic of DSEM. Note. Observed items are decomposed into time-varying within-level (w) and time-invariant between-level (b) components. CAL = callousness, UNC = uncaring, POS = positive affect, NEG = negative affect, CON = conduct problems, EMO = emotional probl... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Callous-Unemotional Traits Items and Subscales</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"><italic>Item Description</italic></th><th align=\"left\"><italic>Subscale</italic></th></tr></thead><tbody><tr><td align=\"lef... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><italic>r</italic> reverse coded items</p></table-wrap-foot>",
"<table-wrap-foot><p>Correlations estimated using the Bayes estimator. Within-day residual correlations are shown above the diagonal. Between-level correlations, means (<italic>M</italic>), and variances (σ<sup>2</sup><bold>)</bol... | [
"<graphic xlink:href=\"10802_2023_1077_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"10802_2023_1077_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"10802_2023_1077_Fig3_HTML\" id=\"MO3\"/>"
] | [
"<media xlink:href=\"10802_2023_1077_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 306 KB)</p></caption></media>"
] | [{"collab": ["American Psychiatric Association"], "source": ["Diagnostic and statistical manual of mental disorders"], "year": ["2013"], "edition": ["5"], "publisher-name": ["American Psychiatric Publishing"]}, {"surname": ["Asparouhov", "Hamaker", "Muth\u00e9n"], "given-names": ["T", "EL", "B"], "article-title": ["Dyn... | {
"acronym": [],
"definition": []
} | 58 | CC BY | no | 2024-01-15 23:42:02 | Res Child Adolesc Psychopathol. 2024 May 30; 52(1):51-63 | oa_package/3c/1a/PMC10787886.tar.gz |
PMC10787887 | 0 | [
"<title>Introduction</title>",
"<p id=\"Par3\">Social learning can be defined “as a change in behavior that follows the observation of another (typically a conspecific) perform a similar behavior, the products of the behavior, or even the products alone” (Zentall ##REF##21895354##2012##: 114). Which type of infor... | [
"<title>Method</title>",
"<title>Subjects</title>",
"<p id=\"Par7\">Eighteen kea from the Haidlhof Research Station (Bad Vöslau) participated in this study. All individuals were group-housed in an outdoor aviary equipped with perches, nesting areas, ponds, and various enrichment. All birds were fed three times ... | [
"<title>Results</title>",
"<p id=\"Par18\">After all subjects had experienced a non-functional apparatus (phase 1), they were allowed to engage in the task with or without prior demonstration of a skilled conspecific (phase 2). In sum, four individuals from the test group (out of a total of 8) successfully solved... | [
"<title>Discussion</title>",
"<p id=\"Par20\">We show that kea were able to solve a rather difficult two-step sequence task and that receiving a demonstration of a skilled conspecific had a positive effect on solving success. However, successful birds showed a high variation in their response topography and often... | [] | [
"<p>Communicated by F. Bairlein.</p>",
"<p id=\"Par1\">Social learning is an important aspect of dealing with the complexity of life. The transmission of information via the observation of other individuals is a cost-effective way of acquiring information. It is widespread within the animal kingdom but may differ... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Acknowledgements</title>",
"<p>We are thankful to Andràs Pèter for his help with devising the test box, Louise Mackie for her help with interobserver reliability check, Remco Folkertsma for his help with data analysis, the Austrian Science Fund (FWF) for funding the project (P 33507-B) and to the entire s... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Test box <bold>a</bold> front view of two pins <bold>b</bold> side view of green and blue ring respectively</p></caption></fig>"
] | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Individuals that successfully solved the task per experimental group</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Name</th><th align=\"left\">Sex</th><th align=\"left\">Age</th><th align=\"left\">Raised</th><th align=\... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM2\"></supplementary-material>",
"<supplementary-material content-type=\"local-data\" id=\"MOESM3\"></supplementary-material>",
"<supplementary-material ... | [
"<table-wrap-foot><p>Table indicates name, sex, age, rearing, and experimental group. Number of successful sessions out of three test sessions, assigned demonstrator, application of demonstrated sequence and according session information, application of non-demonstrated sequence and according session information, c... | [
"<graphic xlink:href=\"10336_2023_2127_Fig1_HTML\" id=\"MO1\"/>"
] | [
"<media xlink:href=\"10336_2023_2127_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 574 KB)</p></caption></media>",
"<media xlink:href=\"10336_2023_2127_MOESM2_ESM.xlsx\"><caption><p>Supplementary file2 (XLSX 60 KB)</p></caption></media>",
"<media xlink:href=\"10336_2023_2127_MOESM3_ESM.mov\"><caption><p... | [{"surname": ["Akins", "Zentall"], "given-names": ["CK", "TR"], "article-title": ["Imitation in Japanese quail: the role of reinforcement of demonstrator responding"], "source": ["Psychon Bull Rev"], "year": ["1998"], "volume": ["5"], "fpage": ["694"], "lpage": ["697"], "pub-id": ["10.3758/BF03208847"]}, {"surname": ["... | {
"acronym": [],
"definition": []
} | 56 | CC BY | no | 2024-01-15 23:42:02 | J Ornithol. 2024 Dec 16; 165(1):49-55 | oa_package/79/d5/PMC10787887.tar.gz |
PMC10787888 | 38150097 | [
"<title>Introduction</title>",
"<p id=\"Par4\">Glucuronoyl esterases cleave ester-linked lignin-carbohydrate complexes (LCCs) in lignocellulosic biomass (Mosbech et al. ##REF##29321810##2018##). In this way, their catalytic action assists in decomposing the plant cell wall matrix by removing some of the covalent ... | [
"<title>LC–MS method</title>",
"<p id=\"Par17\">Reaction product profiles were analyzed by LC–MS. 5 µL of reaction mixture was injected onto a Hypercarb column (150 mm × 2.1 mm; 3μm, Thermo Fischer Scientific, Sunnyvale, CA, USA). The chromatography was performed on a Dionex UltiMate 3000 UPLC (Thermo Fischer Sci... | [
"<title>Results and discussion</title>",
"<p id=\"Par26\">Initial hydrolytic experiments with either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA as substrate and in the absence of alcohol acceptors show substrate conversion as expected (Fig. ##FIG##1##2##), with a significantly higher level of conversion for the 4-... | [
"<title>Results and discussion</title>",
"<p id=\"Par26\">Initial hydrolytic experiments with either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA as substrate and in the absence of alcohol acceptors show substrate conversion as expected (Fig. ##FIG##1##2##), with a significantly higher level of conversion for the 4-... | [
"<title>Conclusion</title>",
"<p id=\"Par33\"><italic>Cu</italic>GE prefers methyl-4-<italic>O</italic>-methyl-glucuronate as donor substrate and the γ-positioned acceptor according to the experiments conducted here, and evaluated based on yield and rate of product formation. It is important to emphasize that the... | [
"<title>Purpose</title>",
"<p id=\"Par1\">Glucuronoyl esterases (GE, family CE15) catalyse the cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), and this study demonstrate how transesterification reactions with a fungal GE from <italic>Cerrena unicolor</italic> (<italic>Cu</italic>GE) can reveal... | [
"<title>Materials and enzyme preparation</title>",
"<p id=\"Par8\">Methyl 4-<italic>O</italic>-methyl-D-glucopyranosyluronate (Me-4-O-MeGlcA) was purchased from Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia. Glucuronic acid methyl ester (Me-GlcA) and benzyl D-glucuronate (Bnz-GlcA) were... | [
"<title>Author contributions</title>",
"<p>Author VP performed the experiments and wrote parts of the paper. Author JWA designed the experiments and wrote the paper.</p>",
"<title>Funding</title>",
"<p>Open access funding provided by Technical University of Denmark. The work was funded by the Novo Nordisk Fou... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Four transesterification reactions catalyzed by <italic>Cu</italic>GE performed by combining either one of the two different donor substrates, methyl-4-<italic>O</italic>-methyl-glucuronate (1 and 2) and methyl-glucuronate (3 and 4) with either 3-phenyl-1-propanol ... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparison of substrate conversion of either Me-4-<italic>O</italic>-MeGlcA or Me-GlcA relative to enzyme dosage within 100 min reaction time</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Reaction time</th... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>Accumulated substrate conversion signifies how much substrate is converted at a given time point relative to the enzyme concentration. Substrate conversion within each time interval describes how much additional substrate is converted at a given time point since the previous, relative to the en... | [
"<graphic xlink:href=\"10529_2023_3456_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"10529_2023_3456_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"10529_2023_3456_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"10529_2023_3456_Fig4_HTML\" id=\"MO4\"/>"
] | [
"<media xlink:href=\"10529_2023_3456_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 3147 KB)</p></caption></media>"
] | [{"surname": ["Biely"], "given-names": ["P"], "article-title": ["Microbial glucuronoyl esterases \u2013 ten years after discovery"], "source": ["Appl\u00a0environ Microbiol"], "year": ["2016"], "volume": ["82"], "issue": ["24"], "fpage": ["02396"], "lpage": ["2416"], "pub-id": ["10.1128/AEM.02396-16"]}, {"surname": ["L... | {
"acronym": [],
"definition": []
} | 19 | CC BY | no | 2024-01-15 23:42:02 | Biotechnol Lett. 2024 Dec 27; 46(1):107-114 | oa_package/ad/28/PMC10787888.tar.gz |
PMC10787889 | 38217712 | [
"<title>Introduction</title>",
"<p id=\"Par5\">Adolescent and young adult cancer survivors (AYAs), those aged 15–39 years at initial cancer diagnosis, are considered a unique group with age-specific challenges, from cancer diagnosis until end of life [##REF##25228567##1##, ##UREF##0##2##]. This age range is, howe... | [
"<title>Methods</title>",
"<title>Study population and data collection</title>",
"<p id=\"Par9\">Data of the population-based, cross-sectional SURVAYA study was used, which was approved by the Institutional Review Board (IRBd18122) and registered within clinical trial registration (NCT05379387). The study popul... | [
"<title>Results</title>",
"<title>Characteristics AYA cancer survivors</title>",
"<p id=\"Par20\">In total, 11296 AYAs were invited to participate in the study, of whom 4010 (36%) responded. After excluding 414 records with missing data, we included 3596 AYAs in our final analysis; their sociodemographic and cl... | [
"<title>Discussion</title>",
"<p id=\"Par26\">This study shows the results of a stable and reliable network analysis based on HRQoL data of 3596 long-term AYAs, as the first to our knowledge. Although the overall global quality of life score was 77.3 on average, the lowest functioning scale score was sexual funct... | [
"<title>Conclusions</title>",
"<p id=\"Par33\">This innovative network analysis provides insight in the nodes, correlations, and clusters that could be targeted to improve the HRQoL outcomes of AYAs. Future studies with longitudinal data and subgroup analyses can tailor the interventions and provided healthcare e... | [
"<title>Purpose</title>",
"<p id=\"Par1\">Adolescent and young adult cancer survivors (AYAs) are at increased risk of long-term and late effects, and experience unmet needs, impacting their health-related quality of life (HRQoL). In order to provide and optimize supportive care and targeted interventions for this... | [
"<title>Strengths and limitations</title>",
"<p id=\"Par32\">This explorative study represents the very first network analysis using data on a range of HRQoL outcomes of long-term AYAs to our knowledge. Strengths include the large sample size, the establishment of a stable and reliable network, and the inclusion ... | [
"<title>Acknowledgements</title>",
"<p>The authors wish to thank all the patients for their participation in the study and the registration team of the Netherlands Comprehensive Cancer Organisation (IKNL) for the collection of data for the Netherlands Cancer Registry.</p>",
"<title>Author contribution</title>",... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>The (cluster) network of HRQoL outcomes of long-term AYA cancer survivors. In this partial correlation network model, the nodes (PF: physical functioning, CF: cognitive functioning, EF: emotional functioning, RF: role functioning, BI: body image, SA: symptom awaren... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Sociodemographic and clinical characteristics of the study cohort</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\" colspan=\"2\"/><th colspan=\"2\">Respondents N = 3596</th></tr><tr><th><italic>n</italic></th><th>%</th></tr... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>*Other includes urinary tract, respiratory tract, male genitalia, neuroblastoma, adrenal, paraganglioma, and eyes</p></table-wrap-foot>",
"<fn-group><fn><p>Tom I. Bootsma and Deborah van de Wal share first authorship.</p></fn><fn><p><bold>Publisher’s note</bold></p><p>Springer Nature remains ... | [
"<graphic xlink:href=\"520_2023_8295_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"520_2023_8295_Fig2_HTML\" id=\"MO2\"/>",
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"<graphic xlink:href=\"520_2023_8295_Fig4_HTML\" id=\"MO4\"/>"
] | [
"<media xlink:href=\"520_2023_8295_MOESM1_ESM.docx\"><label>ESM 1</label><caption><p>(DOCX 98 kb)</p></caption></media>"
] | [{"label": ["2."], "mixed-citation": ["Adolescent and Young Adult Oncology Progress Review Group, \"Closing the gap: research and care imperatives for adolescents and young adults with cancer,\" 2006."]}, {"label": ["7."], "mixed-citation": ["Janssen SHM, Vlooswijk C, Manten-Horst E, Sleeman SHE, Bijlsma RM, Kaal SEJ, ... | {
"acronym": [],
"definition": []
} | 50 | CC BY | no | 2024-01-15 23:42:02 | Support Care Cancer. 2024 Jan 13; 32(2):104 | oa_package/4c/bd/PMC10787889.tar.gz |
PMC10787890 | 37794263 | [
"<title>Introduction</title>",
"<p id=\"Par11\">Bioactive lipids are important for brain function. In particular, the homeostasis of the sphingolipids ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate (S1P) has been described to regulate proliferation, differentiation, cell growth and inflammation in... | [
"<title>Methods</title>",
"<title>Animals</title>",
"<p id=\"Par16\">Experiments were performed according to EU Directive 2010/63/EU under approval of the Malmö/Lund Committee for Animal Experiment Ethics (permit numbers 994/2018 and 9987/2020) and are reported following the ARRIVE guidelines (Animal Research: ... | [
"<title>Results</title>",
"<p id=\"Par23\">We have previously detailed metabolic phenotypes of insulin-resistant GK rats [##REF##30686981##29##] and HFD-induced obese mice [##REF##29027041##40##]. In the present study, we have confirmed that GK rats displayed lower body weight, increased glycemia, and similar fed... | [
"<title>Discussion</title>",
"<p id=\"Par26\">This study demonstrates for the first time that S1PR density in cortical nerve terminals is reduced in lean and obese models of insulin resistance, along with possible increases of S1P concentrations in the cortex, implying a role for S1PR signaling in the neuropathol... | [] | [
"<p id=\"Par1\">Sphingosine-1-phosphate (S1P) is a phosphosphingolipid with pleiotropic biological functions. S1P acts as an intracellular second messenger, as well as extracellular ligand to five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation, apoptosis, synaptic activity... | [] | [
"<title>Acknowledgements</title>",
"<p>The authors thank Nadiia Kravchenko for technical assistance with insulin determination, and Eugenia Cordero Concha for handling the GK rats.</p>",
"<title>Author Contributions</title>",
"<p>AM, JMND designed the study. CS, HE, LV, JPPV, FM conducted experiments, CS, JMN... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Study design. Samples from Wistar and GK rats were collected at 6 months of age (<bold>A</bold>). Mice were kept on CD or HFD for 1, 4 or 8 weeks before tissue sampling (<bold>B</bold>)</p></caption></fig>",
"<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>S1P ... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Antibodies used for Western blot (WB) and molecular weight of the analyzed band</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Antibody</th><th align=\"left\">Dilution</th><th align=\"left\">Source</th><th align=\"left\"... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>Data is mean ± SD (<italic>n.s.</italic> non-significant difference, <italic>P</italic> > 0.05)</p></table-wrap-foot>",
"<table-wrap-foot><p>Data is mean ± SD (<italic>n.s.</italic> non-significant difference, <italic>P</italic> > 0.05)</p><p>*Homeostatic model assessment for insulin re... | [
"<graphic xlink:href=\"11064_2023_4033_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"11064_2023_4033_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"11064_2023_4033_Fig3_HTML\" id=\"MO3\"/>"
] | [] | [{"label": ["10."], "surname": ["Choi", "Chun"], "given-names": ["JW", "J"], "article-title": ["Lysophospholipids and their receptors in the central nervous system"], "source": ["Biochim Biophys Acta"], "year": ["1831"], "volume": ["1"], "fpage": ["20"], "lpage": ["32"], "pub-id": ["10.1016/j.bbalip.2012.07.015"]}] | {
"acronym": [
"CNS",
"HFD",
"GK",
"GTT",
"S1P",
"S1PR",
"Sph",
"SphK",
"T2D"
],
"definition": [
"Central nervous system",
"High-fat diet",
"Goto-Kakizaki",
"Glucose tolerance test",
"Sphingosine 1-phosphate",
"Sphingosine-1-phosphate receptors",
"Sp... | 53 | CC BY | no | 2024-01-15 23:42:02 | Neurochem Res. 2024 Oct 4; 49(2):338-347 | oa_package/c1/70/PMC10787890.tar.gz |
PMC10787891 | 0 | [
"<title>Introduction</title>",
"<p id=\"Par5\">Antibiotic resistance, a globalized public health peril in this new era of modern medicine, prevailed from the pathogenic microbial development of novel resistance mechanisms of genetic and epigenetic origins against the available antibiotics by circumventing the the... | [
"<title>Materials and methods</title>",
"<title>Reagents, plasmid, enzymes, and bacteria strains</title>",
"<p id=\"Par8\">\n<italic>Escherichia coli</italic> DH5α cells employed for routine plasmid propagation and subcloning experiments were supplied by New England Biolabs (NEB) (Ipswich MA, USA). Protease-def... | [
"<title>Results</title>",
"<title>Design of hybrid peptide and construction of recombinant plasmids</title>",
"<p id=\"Par18\">In designing the hybrid peptide with the molecular gene map shown (Fig. ##FIG##1##2##A), we employed the mature amino acid sequences that encode for peptides LL-37 and Renalexin retriev... | [
"<title>Discussion</title>",
"<p id=\"Par25\">Antimicrobial peptides (AMPs) as drug candidates are being considered the new hope for the biomedical and pharmaceutical industries in conjunction with their multifunctional antibacterial pharmacological actions against infectious agents (Montfort-Gardeazabal et al. #... | [] | [
"<title>Abstract</title>",
"<p id=\"Par1\">An alarming global public health and economic peril has been the emergence of antibiotic resistance resulting from clinically relevant bacteria pathogens, including <italic>Enterococcus faecium</italic>, <italic>Staphylococcus aureus</italic>, <italic>Klebsiella pneumoni... | [
"<title>Supplementary Information</title>",
"<p>\n</p>"
] | [
"<title>Acknowledgements</title>",
"<p>We thank Mexico’s Consejo Nacional de Humanidades Ciencias y Tecnologias (CONAHCYT) for the financial support to the graduate student JKN.</p>",
"<title>Author contribution</title>",
"<p>JKN made the DNA constructs, performed the experiments, and wrote the manuscript. NG... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Amino acid sequence flowchart representation of the expression cassette encoding for the recombinant fusion peptides. <bold>A</bold> Expression cassette encoding for CusF3H+_LL-37_Renalexin. <bold>B</bold> Expression cassette encoding for SmbP_LL-37_Renalexin</p></... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>MICs of LL-37_Renalexin expressed in BL21(DE3) and SHuffle T7(DE3) <italic>E. coli</italic> strains</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\">Pathogens</th><th colspan=\"2\">MICs: LL-37_Renalexin (μM)</th></tr><tr><t... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><italic>N/A</italic> Not Analyzed (peptide expressed in <italic>E. coli</italic> SHuffle T7(DE3) was used for subsequent bioactivity)</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher’s note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published... | [
"<graphic xlink:href=\"253_2023_12887_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"253_2023_12887_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"253_2023_12887_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"253_2023_12887_Fig4_HTML\" id=\"MO4\"/>",
"<graphic xlink:href=\"253_2023_12887_Fig5_HTML\... | [
"<media xlink:href=\"253_2023_12887_MOESM1_ESM.pdf\"><label>ESM 1</label><caption><p>(PDF 2236 kb)</p></caption></media>"
] | [{"surname": ["Bayarbat", "Jae-Hag", "Soon-Youl"], "given-names": ["I", "L", "L"], "article-title": ["Expression of recombinant hybrid peptide Gaegurin4 and LL37 using fusion expression in "], "italic": ["E. coli"], "source": ["Miocrob Biotechnol Lett"], "year": ["2016"], "volume": ["40"], "issue": ["2"], "fpage": ["92... | {
"acronym": [],
"definition": []
} | 45 | CC BY | no | 2024-01-15 23:42:02 | Appl Microbiol Biotechnol. 2024 Jan 13; 108(1):1-15 | oa_package/f9/42/PMC10787891.tar.gz |
PMC10787892 | 37679534 | [
"<title>Background</title>",
"<p id=\"Par5\">According to the National Cancer Institute (NCI), an individual is considered a cancer survivor from diagnosis to the end of life [##UREF##0##1##]. In the United States (US), it is estimated that by 2026, the number of cancer survivors will surpass 20 million, which ca... | [
"<title>Methods</title>",
"<title>Data collection and sample</title>",
"<p id=\"Par11\">Cross-sectional data for this study were obtained from the “All of Us” research program collected by online survey between May 2018 and April 2021. Briefly, this program is open to individuals who are 18 and over and are liv... | [
"<title>Results</title>",
"<p id=\"Par18\">The median age of the study population (<italic>n</italic> = 10,020) was approximately 64 (interquartile range [IQR Q1, Q3] 55.5, 71.8) years. The majority of participants were female (66.1%), US-born (92%), and self-identified as White (82.3%). There was a higher distri... | [
"<title>Discussion</title>",
"<p id=\"Par21\">Using data from the All of Us research cohort, we aimed to investigate the associations between SES barriers and health literacy with healthcare delays. We also explored whether there were any differences in these associations by nativity. We found that among all canc... | [
"<title>Conclusion</title>",
"<p id=\"Par31\">Using data from the All of Us research program, we found that SES-related barriers are significantly associated with healthcare delays in cancer survivors in our study. However, a greater impact was observed among those who were foreign-born. Similarly, we observed a ... | [
"<title>Purpose</title>",
"<p id=\"Par1\">We aimed to assess whether nativity differences in socioeconomic (SES) barriers and health literacy were associated with healthcare delays among US cancer survivors.</p>",
"<title>Methods</title>",
"<p id=\"Par2\">“All of Us” survey data were analyzed among adult part... | [
"<title>Measures</title>",
"<title>Demographics</title>",
"<p id=\"Par13\">Personal level characteristics accounted for in this study were age (at survey completion), sex (male vs female), race (Asian, Black, Hispanic, White, multiracial/biracial, other [includes those who selected: none of this, another popula... | [
"<p>The authors would like to thank all of the participants of the All of Us Research Program, Drs. Cecilia Patino-Sutton, and Elizabeth Burner. The All of Us Research Program is supported by the National Institutes of Health, Office of the Director: Regional Medical Centers: 1 OT2 OD026549; 1 OT2 OD026554; 1 OT2 O... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Theoretical framework of the impact nativity has on healthcare delays</p></caption></fig>"
] | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Descriptive characteristics of the sample and their association with SES barriers (<italic>n</italic> = 10,020)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Variables</th><th align=\"left\" colspan=\"4\">... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>Single = includes those who reported: divorced, widowed, separated, and never married. p-values were obtained from chi-square and Kruskal–Wallis tests. Per “All of Us” data use agreement policy, groups < 20 participants are shown as ≤ 20 (%) with a corresponding > (%) category to prevent ... | [
"<graphic xlink:href=\"10552_2023_1782_Fig1_HTML\" id=\"MO1\"/>"
] | [
"<media xlink:href=\"10552_2023_1782_MOESM1_ESM.docx\"><caption><p>Supplementary file1 (DOCX 19 KB)</p></caption></media>"
] | [{"label": ["1."], "mixed-citation": ["National Cancer Institute (2023) NCI dictionary of cancer terms [definition of survivor]. "], "ext-link": ["https://www.cancer.gov/publications/dictionaries/cancer-terms/def/survivor"]}, {"label": ["27."], "ext-link": ["https://www.researchallofus.org/wp-content/themes/research-hu... | {
"acronym": [],
"definition": []
} | 49 | CC BY | no | 2024-01-15 23:42:02 | Cancer Causes Control. 2024 Sep 7; 35(2):203-214 | oa_package/b4/1d/PMC10787892.tar.gz |
PMC10787893 | 0 | [
"<title>Introduction</title>",
"<p id=\"Par5\">As a major discovery in the twentieth century, antibiotics play an irreplaceable role in human health, animal epidemic prevention, and disease treatment (Coleman et al. ##REF##26196923##2015##). However, due to the overuse of antibiotics, a variety of bacteria have d... | [
"<title>Materials and methods</title>",
"<title>Strains and cell lines</title>",
"<p id=\"Par8\">Gram-positive bacteria: <italic>S. aureus</italic> (ATCC 25923, 43300), <italic>Staphylococcus epidermidis</italic> (<italic>S. epidermidis</italic>) (ATCC 12228, 35984), and <italic>Streptococcus agalactiae</italic... | [
"<title>Results</title>",
"<title>Analysis of AP138 and designation of AP138L-arg26</title>",
"<p id=\"Par34\">Plectasin was searched for as a keyword in the DBAASP database, and 12 sequences were retrieved; these derived peptides, AP138, NZ2114, NZX, MP1102, and MP1106, and other reported peptides DLP4, ID3, a... | [
"<title>Discussion</title>",
"<p id=\"Par46\"><italic>S</italic>. <italic>aureus</italic> is a pathogenic bacterium that greatly threatens human health and the production efficiency of livestock and poultry. Many bacteria have developed drug resistance, particularly MRSA (Pinto et al. ##REF##31127270##2019##). Th... | [] | [
"<title>Abstract</title>",
"<p id=\"Par1\">The low activity and yield of antimicrobial peptides (AMPs) are pressing problems. The improvement of activity and yield through modification and heterologous expression, a potential way to solve the problem, is a research hot-pot. In this work, a new plectasin-derived v... | [
"<title>Biological information analysis of AP138L-arg26</title>",
"<p id=\"Par11\">AP138L-arg26 and plectasin derived AMP sequences were obtained from DBAASP database (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.dbaasp.org/home\">https://www.dbaasp.org/home</ext-link>) (Pirtskhalava et al. ##REF##331... | [
"<title>Acknowledgements</title>",
"<p>We acknowledge Chunli Li from the Core Facility at the Institute of Microbiology at the Chinese Academy of Sciences (CAS) for the technical support with SEM and Tong Zhao for her technical support with FACS analysis.</p>",
"<title>Author contribution</title>",
"<p>KZ and... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>The 3D structure analysis of plectasin (left) and AP138L-arg26 (right). <bold>a</bold> Molecular modeling of plectasin and AP138L-arg26. <bold>b</bold> Electrostatic surface of plectasin (left) and AP138L-arg26 (right). Blue and red represent positive and negative ... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Physicochemical properties and likelihood prediction of antimicrobial peptide</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Peptide</th><th align=\"left\">Sequence</th><th align=\"left\">AMP probability</th><th align=\"... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>The line means no results; all data in this table were predicted by online software (APD, DBAASP, and CAMP)</p></table-wrap-foot>",
"<table-wrap-foot><p>MICa, the MIC of peptide/antibiotic alone; MICc, the MIC of the peptide and antibiotic in combination</p></table-wrap-foot>",
"<fn-group><... | [
"<graphic xlink:href=\"253_2023_12947_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"253_2023_12947_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"253_2023_12947_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"253_2023_12947_Fig4_HTML\" id=\"MO4\"/>",
"<graphic xlink:href=\"253_2023_12947_Fig5_HTML\... | [
"<media xlink:href=\"253_2023_12947_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 272 KB)</p></caption></media>"
] | [{"surname": ["Delpech", "Lissarrague", "Ceci", "Allende", "Lallee", "Baldaccini", "Sparo"], "given-names": ["G", "S", "M", "GL", "A", "B", "DM"], "article-title": ["Enterocin AP-7121: combination with colistin against human multi-drug resistant Gram-negative pathogens"], "source": ["J Integr OMICS"], "year": ["2019"],... | {
"acronym": [],
"definition": []
} | 63 | CC BY | no | 2024-01-15 23:42:02 | Appl Microbiol Biotechnol. 2024 Jan 13; 108(1):1-18 | oa_package/45/d3/PMC10787893.tar.gz |
PMC10787894 | 38217709 | [
"<title>Introduction</title>",
"<p id=\"Par26\">Neprilysin (NEP) is a ubiquitous membrane-bound zinc-dependent metalloprotease that hydrolyzes numerous regulatory peptides including vasoactive peptides such as natriuretic peptides, adrenomedullin, angiotensins I and II, bradykinin, and endothelin-1 as well as non... | [
"<title>Materials and methods</title>",
"<title>Peptide design and purification</title>",
"<p id=\"Par29\">The protein sequence of human NEP (GenBank Accession no. NP000893) was analyzed using the B Cell Linear Epitope Prediction tool on the Immune Epitope Database Analysis Resource server (<ext-link ext-link-t... | [
"<title>Results</title>",
"<title>Recombinant protein design and preparation</title>",
"<p id=\"Par37\">Sixteen antigenic sites (8–24 amino acids long) on human NEP were identified using the BepiPred B-cell linear epitope prediction tool [##REF##16635264##18##]. Four of these (designated AgNEP-1, -2, -3, and -4... | [
"<title>Discussion</title>",
"<p id=\"Par47\">Careful selection of peptide immunogens is an important first step in the entire antibody production workflow from antigen preparation to antibody characterization and validation. The B-cell linear epitope prediction tool used in this study is trained to locate antige... | [
"<title>Conclusion</title>",
"<p id=\"Par54\">We show that the thioredoxin scaffold constitutes an effective strategy for surface presentation of <italic>in silic</italic>o predicted peptides as immunogens to generate high-affinity mAbs with well-defined epitope binding footprint. The validated antibody pair comp... | [
"<p id=\"Par1\">Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly ... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Acknowledgements</title>",
"<p>Funding support for this work was provided under the Centre Grant (NMRC/CG21APR1008) awarded to A. Mark Richards by the National Medical Research Council, Singapore.</p>",
"<title>Author contribution</title>",
"<p>OWL and SSML designed and developed the antibody producti... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Properties of selected antigen peptides. <bold>a</bold> 3D-ribbon structure of the NEP extracellular domain using PDB entry 6suk.1.A as template. The locations of the antigen sequences with their start residues in bold, AgNEP-1 (V<sub>169</sub>ATENWEQKYGASW)<sub>,<... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>MRM<sup>HR</sup> product ion scan parameters of neprilysin peptides</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Neprilysin peptide sequences</th><th align=\"left\">Precursor charge</th><th align=\"left\">Precursor the... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>Kinetic rate constants were obtained from the interaction between recombinant sNEP and the respective mAb. Critical residues (highlighted in enlarged bold typeset in red) are defined as those that reduce the ELISA colorimetric response to < 50% of the wild-type sequence (Fig. S3)</p><p><ital... | [
"<graphic xlink:href=\"18_2023_5083_Fig1_HTML\" id=\"MO1\"/>",
"<graphic position=\"anchor\" xlink:href=\"18_2023_5083_Tab2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"18_2023_5083_Fig2_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"18_2023_5083_Fig3_HTML\" id=\"MO4\"/>",
"<graphic xlink:href=\"18_2023_5083_... | [
"<media xlink:href=\"18_2023_5083_MOESM1_ESM.docx\"><caption><p>Supplementary file1 (DOCX 7385 KB)</p></caption></media>"
] | [{"label": ["1."], "surname": ["Bayes-Genis", "Barallat", "Richards"], "given-names": ["A", "J", "AM"], "article-title": ["A test in context: neprilysin: function, inhibition, and biomarker"], "source": ["J Am Coll Cardiol"], "year": ["2006"], "volume": ["68"], "issue": ["6"], "fpage": ["639"], "lpage": ["653"], "pub-i... | {
"acronym": [
"NEP",
"HF",
"RUO",
"ELISA",
"MAb",
"pAb",
"Trx",
"IMAC",
"SPR",
"MRMHR",
"BSA",
"PBS",
"ECE-1",
"ECE-2",
"PHEX",
"ECEL-1",
"LOD",
"LLOQ",
"CV",
"rEF",
"pEF",
"CTRL",
"CE"
],
"definition": [
"Neprilysin"... | 42 | CC BY | no | 2024-01-15 23:42:02 | Cell Mol Life Sci. 2024 Jan 13; 81(1):42 | oa_package/2d/2b/PMC10787894.tar.gz |
PMC10787895 | 38051494 | [
"<title>Introduction</title>",
"<p id=\"Par4\">Interferons (IFN) are a widely expressed family of cytokines. They are categorised, based on their receptor signalling, into types I, II, and III [<xref ref-type=\"bibr\" rid=\"CR1\">1</xref>]. IFN-I signal via a heterodimeric receptor composed of two distinct multi-... | [] | [] | [] | [
"<title>Conclusions</title>",
"<p id=\"Par34\">There is growing evidence that IFN-α plays an important role in early RA pathophysiology and Fig. ##FIG##4##5## summarises a working paradigm on IFN-α influencing RA progression. However, the triggers of IFN-α production and its timing in relation to early immune dys... | [
"<title>Purpose of Review</title>",
"<p id=\"Par1\">Type 1 interferons (IFN-I) are of increasing interest across a wide range of autoimmune rheumatic diseases. Historically, research into their role in rheumatoid arthritis (RA) has been relatively neglected, but recent work continues to highlight a potential cont... | [
"<title>The Interferon Gene Signature (IGS)</title>",
"<p id=\"Par5\">Measuring IFN-α protein <italic>in vivo</italic> has been historically challenging due to low circulating levels being frequently below the detection thresholds of standard assays. A solution was to infer IFN-I exposure, and hence levels, by me... | [
"<title>Funding</title>",
"<p>This work was supported by the Research into Inflammatory Arthritis Centre Versus Arthritis (RACE) (grant number 22072) and the National Institute for Health and Care Research (NIHR) Newcastle Biomedical Research Centre for Ageing and Long-Term Conditions; views expressed are the aut... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Schematic of interferon (IFN) triggers and downstream signalling pathways. <bold>A</bold> The production of IFN-I can occur following recognition of pathogen-associated molecular patterns (PAMPs), often associated with foreign bacteria or viruses, such as cytosolic... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Known single nucleotide polymorphisms (SNPs) associated with RA genetic risk and how their function may affect IFN-I biology</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Gene with known RA risk variant</th><th>Role in IFN biology</th... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p>*Separate reference list in supplementary file ##SUPPL##0##1##.</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"11926_2023_1125_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"11926_2023_1125_Fig2_HTML\" id=\"MO2\"/>",
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"<graphic xlink:href=\"11926_2023_1125_Fig4_HTML\" id=\"MO4\"/>",
"<graphic xlink:href=\"11926_2023_1125_Fig5_... | [
"<media xlink:href=\"11926_2023_1125_MOESM1_ESM.docx\"><caption><p>ESM 1</p></caption></media>"
] | [] | {
"acronym": [],
"definition": []
} | 0 | CC BY | no | 2024-01-15 23:42:02 | Curr Rheumatol Rep. 2024 Dec 5; 26(2):37-52 | oa_package/5b/23/PMC10787895.tar.gz |
PMC10787896 | 38217570 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Various technical and procedural modifications have been implemented intraoperatively to reduce fluoroscopy exposure to both urologists and patients, including proper shielding, low power fluoroscopy settings, and conservative use of fluoroscopy [##REF##26025493##1##... | [
"<title>Methods</title>",
"<title>Study design and set up</title>",
"<p id=\"Par5\">After approval from the Loma Linda University’s Department of Pathology and Human Anatomy, and in compliance with institutional policies for use of anatomical specimens in research, a simulated percutaneous renal access for PCNL... | [
"<title>Results</title>",
"<p id=\"Par13\">During the 300 s of fluoroscopy with AEC settings, the surgeon’s hand in the control group received an average equivalent dose of 3.92 mSv (Table ##TAB##0##1##). Compared to the control, both the radiation-attenuating glove (2.48 mSv) and needle holder (1.37 mSv) reduced... | [
"<title>Discussion</title>",
"<p id=\"Par17\">The existing literature extensively covers the various impacts of ionizing radiation such as DNA damage, malignancy, cataracts, dermatologic changes, and delayed wound healing [##REF##18046031##2##–##REF##18406966##6##, ##REF##31402748##21##]. Despite this research ev... | [
"<title>Conclusion</title>",
"<p id=\"Par24\">Protective measures during percutaneous renal access for PCNL can effectively reduce radiation exposure to a surgeon’s hand. Radiation-attenuating gloves showed a significant reduction in hand exposure but increased patient dose, while needle holders resulted in decre... | [
"<p id=\"Par1\">Percutaneous nephrolithotomy confers the highest radiation to the urologist’s hands compared to other urologic procedures. This study compares radiation exposure to the surgeon’s hand and patient’s body when utilizing three different techniques for needle insertion during renal access. Simulated per... | [] | [
"<title>Author contributions</title>",
"<p>RC: Methodology, Formal Analysis, Investigation, Writing—Original Draft, Writing—Review and Editing. EJ: Methodology, Formal Analysis, Investigation, Writing—Original Draft, Project Administration. CB: Methodology, Investigation. JH: Investigation. ASA: Conceptualization... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Cadaver hand representing a surgeon’s hand positioning and respective fluoroscopic images obtained during simulated percutaneous renal access on a cadaver patient model using <bold>A</bold> a surgical glove, <bold>B</bold> radiation-attenuating glove, and <bold>C</... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Mean equivalent radiation dose and percent change versus control for the surgeon hand and patient between the control and radiation reduction techniques</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th a... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>Bold indicates statsitically significant <italic>p</italic>-value</p><p><italic>n</italic> number of patients, <italic>mSv</italic> millisievert, <italic>SD</italic> standard deviation</p><p>*<italic>p</italic> < 0.05</p></table-wrap-foot>",
"<table-wrap-foot><p>Bold indicates statsiticall... | [
"<graphic xlink:href=\"240_2023_1510_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"240_2023_1510_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"240_2023_1510_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"240_2023_1510_Fig4_HTML\" id=\"MO4\"/>",
"<graphic xlink:href=\"240_2023_1510_Fig5_HTML\" id=... | [] | [{"label": ["19."], "surname": ["Protection"], "given-names": ["R"], "article-title": ["ICRP publication 103"], "source": ["Ann ICRP"], "year": ["2007"], "volume": ["37"], "issue": ["2.4"], "fpage": ["2"]}, {"label": ["20."], "mixed-citation": ["AliMed"], "sup": ["\u00ae"], "ext-link": ["https://www.alimed.com/alimed-o... | {
"acronym": [],
"definition": []
} | 29 | CC BY | no | 2024-01-15 23:42:02 | Urolithiasis. 2024 Jan 13; 52(1):27 | oa_package/3b/6b/PMC10787896.tar.gz |
PMC10787897 | 38217735 | [
"<title>Introduction</title>",
"<p id=\"Par3\">The genome integrity of all living organisms is constantly threatened by endogenous and exogenous sources of DNA damage (Lindahl ##REF##8469282##1993##). Endogenous sources include oxidation by reactive oxygen species (ROS), alkylation, mismatch of DNA bases, topoiso... | [
"<title>Material and methods</title>",
"<title>DNA substrates</title>",
"<p id=\"Par8\">Oligonucleotides used (Supplementary Table ##SUPPL##0##S1##) were synthesized by Integrated DNA Technologies (IDT) and purified by PAGE or dual HPLC before use. Double-stranded DNA substrates were prepared by mixing a 5 µM s... | [
"<title>Results</title>",
"<title>Characterization of AtPol λ gap filling activity</title>",
"<p id=\"Par15\">In order to characterize the catalytic activity of <italic>Arabidopsis thaliana</italic> Pol λ, a recombinant protein (AtPol λ) was expressed in <italic>E. coli</italic> and purified. Subsequently, opti... | [
"<title>Discussion</title>",
"<p id=\"Par25\">In this work we have performed a biochemical characterization of <italic>A. thaliana</italic> DNA polymerase λ and explored its possible role in BER. Firstly, we have shown that, as the rice and human orthologs (Garcia-Diaz et al. ##REF##11821417##2002##; Uchiyama et ... | [] | [
"<p id=\"Par1\">Base excision repair (BER) generates gapped DNA intermediates containing a 5′-terminal 2-deoxyribose-5-phosphate (5′-dRP) group. In mammalian cells, gap filling and dRP removal are catalyzed by Pol β, which belongs to the X family of DNA polymerases. In higher plants, the only member of the X family... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Acknowledgements</title>",
"<p>We are grateful to members of our lab for helpful criticism and advice. We thank Dr. Luis Blanco (Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain) for his advice on amino acid conservation analysis in Pol λ proteins.</p>",
"<title>Author contributions</t... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Arabidopsis Pol λ gap filling activity. <bold>a</bold>\n<italic>Schematic diagram of substrate and product</italic>. <bold>b</bold>\n<italic>AtPol λ</italic>\n<italic>gap filling assay</italic>. DNA substrates (100 nM) were incubated at 30 °C with AtPol <italic>λ</... | [] | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"11103_2023_1407_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"11103_2023_1407_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"11103_2023_1407_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"11103_2023_1407_Fig4_HTML\" id=\"MO4\"/>",
"<graphic xlink:href=\"11103_2023_1407_Fig5_... | [
"<media xlink:href=\"11103_2023_1407_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 1763 KB)</p></caption></media>"
] | [{"surname": ["Cavanaugh", "Beard", "Wilson"], "given-names": ["NA", "WA", "SHJJOBC"], "article-title": ["DNA polymerase \u03b2 ribonucleotide discrimination: insertion, misinsertion, extension, and coding"], "source": ["Nat Rev Genet"], "year": ["2010"], "volume": ["285"], "fpage": ["24457"], "lpage": ["24465"], "pub-... | {
"acronym": [],
"definition": []
} | 52 | CC BY | no | 2024-01-15 23:42:02 | Plant Mol Biol. 2024 Jan 13; 114(1):3 | oa_package/d6/08/PMC10787897.tar.gz |
PMC10787898 | 38217571 | [
"<title>Introduction</title>",
"<p id=\"Par2\">Sensing and regulating the tension of the plasma membrane is crucial for cells to function effectively, especially when challenged by changes in the local micromechanical environment. Whether during malignant transformation, cytokinesis or morphogenesis, altered cell... | [
"<title>Methods</title>",
"<title>Cell line</title>",
"<p id=\"Par52\">HeLa cell line (CCL-2, ATCC) was used to perform all the experimental studies.</p>",
"<title>Cell culture</title>",
"<p id=\"Par53\">HeLa cells were grown in Dulbecco’s Modified Essential Medium (DMEM, Gibco, Life Technologies, USA) with... | [
"<title>Results</title>",
"<title>De-adhesion-mediated increase in membrane fluctuations is actin-dependent</title>",
"<p id=\"Par9\">IRM images reflect the distance of the cellular basal membrane from the glass coverslip and, thus, can be used to study the spatiotemporal basal PM height fluctuations in adheren... | [
"<title>Discussion</title>",
"<p id=\"Par43\">Loss or remodelling of the adhesion machinery are known determinants of malignant transformation [##REF##19909018##42##, ##REF##26919980##47##], while regulated de-adhesion has been shown to aid tumour invasion [##REF##35838828##26##]. In this study, we aimed to under... | [] | [
"<p id=\"Par1\">Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms, including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis are poorly understood. To inve... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Acknowledgements</title>",
"<p>BS acknowledges support from Wellcome Trust/DBT India Alliance fellowship (Grant number IA/I/13/1/500885), SERB (Grant number SERB_CRG_2458) and CEFIPRA (Grant number 6303-1). The authors are grateful to CSIR and IISER Kolkata for providing scholarships to TM and AB, respect... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Phases and variability in de-adhesion. <bold>a</bold> IRM images of a representative cell before and after Trypsin–EDTA addition at 0.25% (faster) concentrations and corresponding temporal fluctuation maps. The time after de-adhering solution addition is mentioned.... | [
"<table-wrap id=\"Taba\"><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Resource availability reagent or resource</th><th align=\"left\">Source</th><th align=\"left\">Identifier</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"3\">Pharmacological reagents</td></tr><tr><td align=\"lef... | [
"<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n... | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"18_2023_5072_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"18_2023_5072_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"18_2023_5072_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"18_2023_5072_Fig4_HTML\" id=\"MO4\"/>",
"<graphic xlink:href=\"18_2023_5072_Fig5_HTML\" id=\"MO5... | [
"<media xlink:href=\"18_2023_5072_MOESM1_ESM.pdf\"><caption><p>Supplementary file1 (PDF 3906 KB)</p></caption></media>"
] | [{"label": ["16."], "mixed-citation": ["Deserno M (2007) Fluid lipid membranes\u2014a primer, pp 1\u201329. See "], "ext-link": ["https://www.cmu.edu/biolphys/deserno/pdf/membrane_theory.pdf"]}, {"label": ["23."], "surname": ["Helfrich"], "given-names": ["W"], "article-title": ["Elastic properties of lipid bilayers ela... | {
"acronym": [],
"definition": []
} | 80 | CC BY | no | 2024-01-15 23:42:02 | Cell Mol Life Sci. 2024 Jan 13; 81(1):43 | oa_package/7f/66/PMC10787898.tar.gz |
PMC10787899 | 38222211 | [] | [] | [] | [] | [] | [
"<p>Tuberculosis is one of the oldest known infective diseases. It continues to pose a major threat to global health. In low- and middle-income countries, drug-resistant strains of tuberculosis have made the disease increasingly dangerous. In high-burden countries like India, people with tuberculosis may not always... | [
"<title>Editorial</title>",
"<p>Tuberculosis (TB) is still an immense threat to health in endemic nations. Drug-resistant strains of mycobacteria have made matters worse, mandating creative approaches for efficient treatment [##UREF##0##1##]. At the national level, according to the National TB Prevalence Survey (... | [
"<p>Acknowledge the Tamil Nadu Kasanoi Erappila Thittam (TN-KET) team.</p>"
] | [] | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Five indicators for assessment of the severity of illness.</title><p>BMI: Body mass index.</p><p>Reference [##UREF##3##4##].</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Sankalp Yadav</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Sankalp Yadav</p><p><bold>Drafting of the manuscript:</bold> Sankalp Yadav</p><p><bold>Critical review ... | [] | [] | [{"label": ["1"], "article-title": ["India\u2019s decision to deny an extension of patent for bedaquiline: a public health imperative"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Yadav", "Rawal", "Jeyaraman"], "given-names": ["S", "G", "M"], "fpage": ["0"], "volume": ["15"], "year": ["2023"]}, {"label"... | {
"acronym": [],
"definition": []
} | 7 | CC BY | no | 2024-01-15 23:42:02 | Cureus.; 15(12):e50550 | oa_package/4e/0e/PMC10787899.tar.gz |
PMC10787900 | 38222222 | [
"<title>Introduction</title>",
"<p>A ureterocele is a cystic dilatation of the distal submucosal ureter, frequently located within the bladder [##REF##29043421##1##]. The development of tumors within the ureterocele is very rare; there are few reports in the literature, and the majority are of urothelial carcinom... | [] | [] | [
"<title>Discussion</title>",
"<p>A ureterocele is a congenital abnormality with an incidence as high as 1 in 500 in autopsy and a female preponderance four to six times greater than in men [##REF##12406111##5##]. It can appear in single or duplex systems associated with the upper pole system, as in the Weigert-Me... | [
"<title>Conclusions</title>",
"<p>Tumors in ureteroceles are very rare, and there are no guidelines for diagnosis, management, and follow-up. The diagnosis is challenging with the help of imaging studies and cystoscopy, although a normal exam may not exclude this diagnosis. Management varies according to histolog... | [
"<p>Urothelial carcinoma on a ureterocele is extremely rare in the literature, and few case reports have been reported. There are no guidelines for diagnosis and management, and current practice is extrapolated from bladder and upper urothelial tract carcinoma. We present a case from a 61-year-old man with urotheli... | [
"<title>Case presentation</title>",
"<p>A 61-year-old caucasian man presented in the outpatient clinic with episodes of macroscopic hematuria for one year. The patient had no relevant past medical history and had no other complaints, and the physical examination was innocent. On blood analyses, there were no rele... | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Axial (A) and sagittal (B) contrast-enhanced CT on the excretory phase with a right parietal thickening of a ureterocele (yellow circle) with hydroureter associated (yellow arrows)</title></caption></fig>",
"<fig posi... | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>List of reports of urothelial carcinomas on ureteroceles</title><p>CT - computed tomography, DUR - distal ureter resection, F - female, IVU/P - intravenous urography/pyelography, L - left, M - male, NMI - non-muscle invasive, MI - mus... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Antonio M. Pinheiro, Pedro Bargão, Fernando Ribeiro, Filipa Galante Pereira, Ana Germano</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Antonio M. Pinheiro, Pedro Ba... | [
"<graphic xlink:href=\"cureus-0015-00000050549-i01\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050549-i02\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050549-i03\" position=\"float\"/>"
] | [] | [{"label": ["7"], "article-title": ["Carcinoma in ureterocele: caso clinico"], "source": ["Urologia"], "person-group": ["\n"], "surname": ["Perego", "Marini"], "given-names": ["S", "F"], "fpage": ["117"], "lpage": ["119"], "volume": ["41"], "year": ["1974"]}, {"label": ["14"], "article-title": ["Transitional cell carci... | {
"acronym": [],
"definition": []
} | 19 | CC BY | no | 2024-01-15 23:42:02 | Cureus.; 15(12):e50549 | oa_package/d9/20/PMC10787900.tar.gz |
PMC10787901 | 38222195 | [
"<title>Introduction</title>",
"<p>Iron accumulation in the brain, especially in the basal ganglia, frequently leads to neurodegeneration [##REF##26136767##1##]. Mutations in C19orf12 have recently been identified in individuals with a diagnosis of neurodegeneration with brain iron accumulation (NBIA) [##REF##232... | [] | [] | [
"<title>Discussion</title>",
"<p>This is the first clinical case report to investigate the effects of a physiotherapy and rehabilitation program on body structure, function, and levels of activity and participation in a child with MPAN. The syndrome presents a variety of symptoms, ranging from gait disturbances t... | [
"<title>Conclusions</title>",
"<p>The purpose of the investigation was to evaluate the impact of an 18-week rehabilitation program, comprising balance and coordination exercises, on two siblings diagnosed with neurodegeneration caused by iron accumulation in the brain. Comprehensive understanding of these conditi... | [
"<p>This case report reports the effects of an 18-week physiotherapy program in children with mitochondrial membrane protein-associated neurodegeneration (MPAN). The study involved two brothers, aged 11 and 12, who had been diagnosed with MPAN. The physiotherapy program was divided into three phases and consisted o... | [
"<title>Case presentation</title>",
"<p>Two brothers with MPAN, aged 11 and 12, who were born at term with birth weights of 2,000 g (36 weeks) and 3,500 g (40 weeks), respectively, presented to the clinic with a complaint of falling. Muscle weakness, loss of coordination and balance, and gait disturbance were obs... | [] | [] | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Timeline of events related to patient treatment</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Events</td><td rowspan=\"1\" colspan=\"1\">Date</td><... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Ömer Faruk Özçelep, Atahan Turhan</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Ömer Faruk Özçelep, Sibel Fi̇dan, Safiye Kandemir</p><p><bold>Drafting of the manusc... | [] | [] | [{"label": ["8"], "article-title": ["Daniels and Worthingham\u2019s Muscle Testing-Techniques of Manual Examination and Performance Testing"], "person-group": ["\n"], "surname": ["Brown", "Avers"], "given-names": ["M", "D"], "publisher-loc": ["Saunders"], "year": ["2018"], "uri": ["https://shop.elsevier.com/books/danie... | {
"acronym": [],
"definition": []
} | 20 | CC BY | no | 2024-01-15 23:42:02 | Cureus.; 15(12):e50540 | oa_package/c5/4c/PMC10787901.tar.gz |
PMC10787902 | 38222127 | [
"<title>Introduction and background</title>",
"<p>Helicobacter pylori peptic ulcers may be treated with one of less than ten medications, including metronidazole, amoxicillin, clarithromycin, furazolidone, levofloxacin, and tetracycline. Antimicrobial resistance may be avoided and effective eradication is achieve... | [] | [] | [] | [
"<title>Conclusions</title>",
"<p>In conclusion, therapies that can successfully eliminate this infection in the first attempt must be considered the gold standard when prescribing medications. However, the rising global resistance to antibiotics like amoxicillin, metronidazole, clarithromycin, tetracycline, and ... | [
"<p>Helicobacter pylori has been reported as a health problem worldwide, affecting a sizable portion of people. Peptic ulcers, gastric cancer, and various extra gastric conditions are associated with this bacterium. The rampant overprescribing of antibiotics has led to the emergence of H. pylori strains resistant t... | [
"<title>Review</title>",
"<p>Methods</p>",
"<p>The systematic review was done in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A rigorous search strategy was developed to test this hypothesis and identify relevant literature. A comprehensive search o... | [
"<p>We would like to express our gratitude to all those who contributed to the successful completion of this systematic review article. It is through the collective efforts and dedication of each author that this work has come to fruition.</p>"
] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>PRISMA chart</title><p>PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses</p></caption></fig>"
] | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Data extraction using the MeSH strategy</title><p>MeSH, Medical Subject Headings</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">MeSH Strategy Used</td><... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"cureus-0015-00000050541-i01\" position=\"float\"/>"
] | [] | [{"label": ["3"], "article-title": ["List of Bacteria for Which New Antibiotics Are Urgently Needed"], "source": ["WHO"], "date-in-citation": ["\n"], "month": ["11"], "year": ["2022", "2017"], "person-group": ["\n"], "surname": ["WHO"], "given-names": ["(2017)"], "fpage": ["4"], "volume": ["38"], "uri": ["https://www.w... | {
"acronym": [],
"definition": []
} | 29 | CC BY | no | 2024-01-15 23:42:02 | Cureus.; 15(12):e50541 | oa_package/28/bf/PMC10787902.tar.gz |
PMC10787903 | 38217706 | [
"<title>Introduction</title>",
"<p id=\"Par5\">Successful treatment of urethral stricture disease requires not only adequate surgical experience but also appropriate preoperative diagnosis. The basic tools widely used for the initial evaluation of patients with suspicion of urethral stricture (US) are uroflowmetr... | [
"<title>Materials and methods</title>",
"<p id=\"Par9\">A comprehensive literature search was performed using the Medline and Cochrane databases in October 2022. Studies that evaluated the use of SUG in the diagnosis of urethral stricture disease were included in the analysis. Prospective studies were selected fo... | [
"<title>Results</title>",
"<p id=\"Par10\">Seventeen papers were selected as a result of our literature review on the use of SUG in assessing urethral strictures. Final analysis is based on prospective studies, the majority of which are limited by a small patient population (number of patients varied from 28 to 1... | [] | [
"<title>Conclusion</title>",
"<p id=\"Par27\">Sonourethrography assessment of the male anterior urethra in patients with anterior urethral strictures is a safe, well-tolerated, minimally invasive and cost-effective diagnostic modality. For the posterior urethra, this technique cannot be recommended, based on the ... | [
"<title>Purpose</title>",
"<p id=\"Par1\">To synthetize the current scientific knowledge on the use of ultrasound of the male urethra for evaluation of urethral stricture disease. This review aims to provide a detailed description of the technical aspects of ultrasonography, and provides some indications on clini... | [
"<title>Highlights and clinical indications</title>",
"<p id=\"Par23\">Retrograde urethrography has historically been the gold standard for identifying urethral strictures; however, because of its certain drawbacks, novel imaging techniques have been investigated and evaluated. Before deciding on surgical interve... | [
"<title>Author’s contribution</title>",
"<p>MF: protocol/project development, data Collection, manuscript writing/editing. MV: protocol/project development, data Collection, manuscript writing/editing. KM: data Collection, manuscript writing/editing. JA: data Collection, manuscript writing/editing. FC-J: protocol... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>PRISMA flowchart</p></caption></fig>",
"<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Ultrasound image of bulbar urethra in longitudinal scan shown within the white box (Figure provided by the authors) <italic>U</italic> urethra. <italic>CS</italic> corpus sp... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Diagnostic accuracy of sonourethrography compared to other modalities and surgical findings if applicable</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Study</th><th align=\"left\" rowspan=\"2\">Study type... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>CEUS—contrast-enhanced ultrasonography, SUG—sonourethrography, RUG—retrograde urethrography, VCUG—voiding cystourethrography, MRU—magnetic resonance urethrography, SE—sonoelastography, SWE—shear wave elastography</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Sp... | [
"<graphic xlink:href=\"345_2023_4760_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"345_2023_4760_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"345_2023_4760_Fig3_HTML\" id=\"MO3\"/>",
"<graphic xlink:href=\"345_2023_4760_Fig4_HTML\" id=\"MO4\"/>"
] | [] | [{"label": ["2."], "surname": ["Eaton", "Richenberg"], "given-names": ["J", "J"], "article-title": ["Imaging of the urethra: current status"], "source": ["Imaging"], "year": ["2005"], "volume": ["17"], "issue": ["2"], "fpage": ["139"], "lpage": ["149"], "pub-id": ["10.1259/imaging/98659318"]}, {"label": ["5."], "surnam... | {
"acronym": [],
"definition": []
} | 44 | CC BY | no | 2024-01-15 23:42:02 | World J Urol. 2024 Jan 13; 42(1):32 | oa_package/ec/0f/PMC10787903.tar.gz |
PMC10787904 | 38112900 | [
"<title>Introduction</title>",
"<p id=\"Par4\">Urinary tract infections (UTIs) are the most prevalent outpatient illnesses and affect about 50% of the population at some point in their lives [<xref ref-type=\"bibr\" rid=\"CR1\">1</xref>]. The incidence of UTIs is increasing with age, and close to 10% of postmenop... | [
"<title>Material and Methods</title>",
"<title>Search Strategy</title>",
"<p id=\"Par6\">In July 2023, the systematic publication search was done in several databases, including ACM Digital Library, CINAHL, IEEE Xplore, PubMed, and Google Scholar via Boolean operators with the use of the following terms: “AI,””... | [
"<title>Results</title>",
"<p id=\"Par11\">Out of the 782 papers that were considered, only 14 studies on AI models in the area of UTIs met the criteria for inclusion (Fig. ##FIG##0##1##). These can be grouped according to the scenarios the AI models were developed for, namely, (1) diagnosis of uncomplicated UTI ... | [] | [
"<title>Conclusion</title>",
"<p id=\"Par25\">AI-driven UTI detection is a promising new area of healthcare research; however, it is still in the exploratory rather than implementation phase. To fully realize AI’s potential for enhancing UTI diagnosis, more study is needed, ideally guided by larger, more diversif... | [
"<title>Purpose of Review</title>",
"<p id=\"Par1\">Artificial intelligence (AI) can significantly improve physicians’ workflow when examining patients with UTI. However, most contemporary reviews are focused on examining the usage of AI with a restricted quantity of data, analyzing only a subset of AI algorithms... | [
"<title>Limitations and Future Directions</title>",
"<p id=\"Par21\">AI algorithms can identify unique correlations between symptoms, urinalysis results, and inflammatory processes in the urinary tract, as well as concise variable sets that are accurate in predicting urinary tract infections. Unquestionably, arti... | [
"<title>Author Contribution</title>",
"<p>NN—conception, data collection, data analysis, writing the main manuscript; AT- conception, data collection, data analysis, writing the main manuscript; DRS—data analysis, writing, and editing; BZH—conception, data collection, data analysis, writing main manuscript; RZ—da... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Artificial intelligence (AI)–based treatment and diagnosis of UTIs</p></caption></fig>",
"<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>PRISMA flowchart: AI-based approaches for UTI diagnosis</p></caption></fig>"
] | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Uncomplicated UTI AI-based diagnosis and symptom checkers</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Author</th><th align=\"left\">Objective</th><th align=\"left\">Dataset (<italic>n</italic>)</th><th align=\"left\">... | [] | [] | [] | [] | [] | [] | [
"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic xlink:href=\"11934_2023_1192_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"11934_2023_1192_Fig2_HTML\" id=\"MO2\"/>"
] | [] | [] | {
"acronym": [],
"definition": []
} | 0 | CC BY | no | 2024-01-15 23:42:02 | Curr Urol Rep. 2024 Dec 19; 25(1):37-47 | oa_package/c0/67/PMC10787904.tar.gz |
PMC10787905 | 38217694 | [
"<title>Introduction</title>",
"<p id=\"Par6\">Schwannomas are peripheral nerve sheath tumors (PNST) found in cranial and spinal nerves throughout the body. Schwannomas of the orbit are rare, and there are less than 500 cases reported in the literature [##REF##33397169##16##]. It is estimated that these tumors co... | [
"<title>Methods</title>",
"<title>Admission routine</title>",
"<p id=\"Par9\">The study center is the primary Swedish referral center for multidisciplinary management of orbital tumors, including schwannomas. During the period of 2005 to 2021, 16 patients with a new diagnosis of OS were managed. Other neurosurg... | [
"<title>Results</title>",
"<title>Incidence</title>",
"<p id=\"Par19\">Considering the 16 documented cases of OS between the years of 2005 and 2021, the apparent incidence of OS in Sweden was estimated at 0.1 per million and year. However, the true incidence may be higher.</p>",
"<title>Baseline characteristi... | [
"<title>Discussion</title>",
"<p id=\"Par25\">This study reports on a population-based cohort of orbital schwannomas. Of the 16 patients included, four (25%) underwent early surgery at the time of diagnosis, while three were surgically treated later, after initial conservative management (19%), and nine (56%) did... | [
"<title>Conclusion</title>",
"<p id=\"Par33\">There were no differences in long term outcome between patients who had been managed with early surgery and those operated later after an initially conservative management. Conservatively treated patients had minimal to no symptoms and remained clinically stable throu... | [
"<title>Abstract</title>",
"<title>Introduction</title>",
"<p id=\"Par1\">Orbital schwannomas (OS) are rare occurrences with no more than 500 cases reported in the literature. The tumor’s potential to compromise the delicate neuro-ophthalmic structures within the orbit prompts surgical removal. Tumor removal is... | [
"<title>Supplementary information</title>",
"<p>\n</p>"
] | [
"<title>Author contribution</title>",
"<p>All authors approved of the submitted manuscript. All authors qualify for authorship according to the ICJME guidelines, as all have participated in the conceptualization, design, data extraction, writing, and drafting, as well as reviewing the manuscript. AET, EE, and EB ... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p><bold>a</bold>) MRI 5 years after initial diagnosis of a large orbital schwannoma in a patient who had refused surgery (Patient 8), <bold>b</bold>) MRI of the same patient 10 years later showing further growth during the observation period</p></caption></fig>",
"... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Baseline characteristics of patients included in the study</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>ID</th><th>Sex, age, and chief complaint</th><th>Referral differential diagnoses</th><th>Tumor size (cm)</th><th>Location and ext... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><sup>*</sup>Including patients surgically treated after initially being managed conservatively</p></table-wrap-foot>",
"<table-wrap-foot><p>FU = Follow-up, n/a = Not Applicable</p></table-wrap-foot>",
"<fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with... | [
"<graphic xlink:href=\"701_2024_5899_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"701_2024_5899_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"701_2024_5899_Fig3_HTML\" id=\"MO3\"/>"
] | [
"<media xlink:href=\"701_2024_5899_MOESM1_ESM.docx\"><label>ESM 1</label><caption><p>Supplementary file 1: STROBE guidelines checklist. (DOCX 32 kb)</p></caption></media>"
] | [{"label": ["8."], "mixed-citation": ["Ghaith AK, Johnson SE, El-Hajj VG et al (2023) Surgical management of malignant melanotic nerve sheath tumors: an institutional experience and systematic review of the literature. J Neurosurg Spine:1\u201310"]}, {"label": ["9."], "surname": ["Grover", "Rastogi", "Chaturvedi", "Gup... | {
"acronym": [],
"definition": []
} | 21 | CC BY | no | 2024-01-15 23:42:02 | Acta Neurochir (Wien). 2024 Jan 13; 166(1):9 | oa_package/a7/d3/PMC10787905.tar.gz |
PMC10787907 | 38217801 | [
"<title>Introduction</title>",
"<p id=\"Par6\">Periodontal disease is a chronic, multifactorial, and infectious disease caused by bacteria. It is characterized by the formation of an inflammatory response in the supporting bone and connective tissue against microbial dental plaque, and the nature of the resulting... | [
"<title>Methods</title>",
"<p id=\"Par9\">Sixty-five systemically healthy individuals were recruited from the Clinic of Periodontology, Faculty of Dentistry, Atatürk University, between October 2021 and July 2022. The study population was designated into two groups: 1. Thirty-five patients with periodontitis (per... | [
"<title>Results</title>",
"<p id=\"Par20\">Sixty-five patients were included in this study, and there was no statistically significant difference between the groups in terms of gender (<italic>p</italic>=0.97). Asprosin levels and other factors were compared between participants with periodontitis and those with ... | [
"<title>Discussion</title>",
"<p id=\"Par25\">Asprosin was first identified by Romere et al., who also verified that patients with neonatal progeroid syndrome have mutations at the 3' terminus of the fibrillin-1 (FBN-1) gene that cause asprosin to be absent near the C-terminal cleavage site [##REF##27087445##8##]... | [
"<title>Conclusion</title>",
"<p id=\"Par31\">As a result of our study, asprosin may be a useful parameter as a biomarker in the course of periodontal diseases. However; BMI status should be considered when evaluating asprosin levels in patients with periodontitis. It is necessary to conduct molecular research to... | [
"<title>Objectives</title>",
"<p id=\"Par1\">A newly discovered adipokine known asprosin in serum and saliva in patients with periodontitis has not been explored. The aim of this study was to determine the relationship between serum and saliva asprosin levels and periodontitis by grouping it according to body mas... | [] | [
"<title>Author contributions</title>",
"<p>SNSG worked on the conception and design of the study, interpretation of data, and drafted the manuscript. AD worked on the conception and design of the study and critically revised the manuscript. EL worked on the execution of biochemical analysis, interpretation of dat... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Determination of the diagnostic sensitivity and specificity of serum and saliva asprosin levels in periodontitis and control groups by ROC curve analysis. ROC: receiver-operating characteristic curve</p></caption></fig>"
] | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparison between control and periodontitis groups</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Control Group<break/>(<italic>n</italic>=30)<break/>Mean ± SD</th><th>Periodontitis Group<break/>(<italic>n</italic>=35)<break/>Mea... | [] | [] | [] | [] | [] | [] | [
"<table-wrap-foot><p>Abbreviations: <italic>BMI</italic> body mass index, <italic>PI</italic> plaque index, <italic>GI</italic> gingival index, <italic>BOP</italic> bleeding on probing, <italic>CAL</italic> clinical attachment level, <italic>PD</italic> pocket depth, <italic>SD</italic> standart deviation</p></tabl... | [
"<graphic xlink:href=\"784_2024_5494_Fig1_HTML\" id=\"MO1\"/>"
] | [] | [{"label": ["6."], "surname": ["Zhu"], "given-names": ["J"], "article-title": ["Association of circulating leptin and adiponectin with periodontitis: A systematic review and meta-analysis"], "source": ["BMC Oral Health"], "year": ["2017"], "volume": ["17"], "issue": ["1"], "fpage": ["1"], "lpage": ["14"], "pub-id": ["1... | {
"acronym": [],
"definition": []
} | 37 | CC BY | no | 2024-01-15 23:42:02 | Clin Oral Investig. 2024 Jan 13; 28(1):91 | oa_package/dc/a1/PMC10787907.tar.gz |
PMC10787908 | 38217553 | [
"<title>Introduction</title>",
"<p id=\"Par6\">Eating disorders (ED) are defined, according to the current edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5-TR) [##UREF##0##1##], by specific disturbances in eating behaviors, and by a persistent and undue influence of body weight or shape... | [] | [] | [] | [
"<title>Conclusions</title>",
"<p id=\"Par36\">As long as the “feminine” is lived and conceptualized as the “Other”, the feminine gender will be associated with a higher risk to adopt a dysfunctional identification of the self through the “gaze of the Other”, especially in an ocular-centric society. Current theor... | [
"<title>Background</title>",
"<p id=\"Par1\">Phenomenological research has enriched the scientific and clinical understanding of Eating Disorders (ED), describing the significant role played by disorders of embodiment in shaping the lived experience of patients with ED. According to the phenomenological perspecti... | [
"<title>Lived experience and sociocultural factors</title>",
"<p id=\"Par14\">The present narrative review suggests a possible integration of the phenomenological approach with some elements of other available theoretical perspectives (e.g., cognitive–behavioral, psycho-dynamic, psychophysiological findings). Phe... | [
"<title>Author contributions</title>",
"<p>All authors equally contributed. L.T. wrote the first draft of the manuscript, under the supervision of G.S., V.R. and G.C.; all authors reviewed the manuscript and agree with its content.</p>",
"<title>Funding</title>",
"<p>Open access funding provided by Università... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Conceptual summary of key phenomenological concepts. The “physical body” (<italic>Koerper</italic>) is the material, third-person view of one’s own body. The “lived body” (<italic>Leib</italic>) represents the lived experience of it. A third dimension can also be d... | [] | [] | [] | [] | [
"<disp-quote><p id=\"Par19\">“(...) humanity is male, and man defines woman not herself, but as relative to him.\"</p></disp-quote>"
] | [] | [] | [
"<fn-group><fn id=\"Fn1\"><label>1</label><p id=\"Par40\">One’s body experienced <italic>from within</italic>, i.e. from a first-person perspective. Also called the <italic>body-I-am</italic> or body-subject.</p></fn><fn id=\"Fn2\"><label>2</label><p id=\"Par41\">One’s body apprehended <italic>from without</italic>... | [
"<graphic xlink:href=\"40519_2023_1632_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"40519_2023_1632_Fig2_HTML\" id=\"MO2\"/>"
] | [] | [{"label": ["1."], "mixed-citation": ["American Psychiatric Association (2022) Diagnostic and Statistical Manual of Mental Disorders\u202f: Fifth Edition Text Revision DSM-5-TR"], "sup": ["TM"]}, {"label": ["2."], "surname": ["Fairburn", "Fairburn"], "given-names": ["CG", "CG"], "article-title": ["Eating disorders: the... | {
"acronym": [],
"definition": []
} | 115 | CC BY | no | 2024-01-15 23:42:02 | Eat Weight Disord. 2024 Jan 13; 29(1):8 | oa_package/b1/93/PMC10787908.tar.gz |
PMC10787909 | 38222998 | [
"<title>Introduction</title>",
"<p>Constipation can notably affect children's health and their parents' quality of life. Functional constipation (FC) in children is defined as irregular or complex bowel movements without underlying systemic or anatomical causes [##UREF##0##1##]. Although the prevalence of constip... | [
"<title>Materials and methods</title>",
"<p>Study design and participants</p>",
"<p>This web-based descriptive cross-sectional study evaluated parents' knowledge, attitude, and practice toward childhood constipation. The data were obtained through an online questionnaire directed to parents in Makkah, Saudi Ara... | [
"<title>Results</title>",
"<p>Among 796 participants, a notable majority were female, accounting for 74.2% of the sample, while the remaining 25.8% were male. Each participant willingly agreed to partake in the survey. The age distribution varied, with the largest group falling in the 35-60 years category at 62.8... | [
"<title>Discussion</title>",
"<p>Our study aimed to determine parents’ knowledge, attitudes, and practices toward childhood constipation in a major city in Saudi Arabia. The dominance of female participants, constituting 74.2% of the sample, was a noteworthy observation.</p>",
"<p>Moreover, the high percentage ... | [
"<title>Conclusions</title>",
"<p>This study highlights the variability in knowledge levels among parents, with a moderate overall understanding of childhood constipation. It emphasizes a moderate level of adherence to recommended practices related to childhood constipation, with some room for improvement in cert... | [
"<p>Introduction</p>",
"<p>Functional constipation in children is described as irregular or difficult bowel motions without underlying systemic or anatomical causes. Although constipation can have a serious negative impact on a child's health and the lives of their parents. This study aimed to assess the knowledg... | [] | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Source of information of the participants.</title></caption></fig>",
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Correlation of knowledge and practice scores of the... | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Sociodemographic characteristics of the participants.</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"1\">Characteristics</td><td rowspan=\"1\" colspan=\... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Albraa J. Khayyat, Moath A. Khayat, Refal T. Abumansour, Nada O. Almalayo, Raghad E. Saleh, Doaa S. Baashar, Musaad M. Almhmadi , Rayan O. Almalki, Mohammed Ageel</p><p><bold>Drafting of t... | [
"<graphic xlink:href=\"cureus-0016-00000052236-i01\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0016-00000052236-i02\" position=\"float\"/>"
] | [] | [{"label": ["1"], "article-title": ["Functional constipation in children: a cross-sectional study"], "source": ["Int J Sci Res"], "person-group": ["\n"], "surname": ["Singh", "Singh"], "given-names": ["RD", "SK"], "volume": ["7"], "year": ["2018"], "uri": ["https://www.worldwidejournals.com/international-journal-of-sci... | {
"acronym": [],
"definition": []
} | 23 | CC BY | no | 2024-01-15 23:42:02 | Cureus.; 16(1):e52236 | oa_package/56/1d/PMC10787909.tar.gz |
PMC10787910 | 38217744 | [
"<title>Background</title>",
"<p id=\"Par6\">Colorectal cancer (CRC) is more common among men than women, and as of 2020, CRC had a 5-year prevalence rate of 51.9 per 100,000 people in Asia and an incidence rate of 68 per 100,000 in Hong Kong [##REF##31085968##1##, ##UREF##0##2##]. Surgical methods are commonly a... | [
"<title>Methods</title>",
"<p id=\"Par8\">This systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [##REF##19621070##14##] and was registered in the International Prospective Register of Systematic Reviews (PROSP... | [
"<title>Results</title>",
"<p id=\"Par15\">The study selection process, which followed the PRISMA approach, is summarized in Fig. ##FIG##0##1##. The studies that were excluded at the full-text screening stage and the reasons for exclusion are listed in Supplementary Appendix ##SUPPL##0##2##. A total of 4413 artic... | [
"<title>Discussion</title>",
"<p id=\"Par22\">This systematic review and meta-analysis investigated the effectiveness of physiotherapy interventions on FI and QoL following colorectal surgery. The literature searches identified 4413 potentially relevant articles indexed in both English- and Chinese-language datab... | [
"<title>Conclusion</title>",
"<p id=\"Par30\">The current systematic review and meta-analysis identified significant improvements in the lifestyle, coping behavior, and embarrassment components of the FIQL among patients who received PFMT compared with those who received UC. Considering the non-invasive nature of... | [
"<title>Purpose</title>",
"<p id=\"Par1\">To investigate the effectiveness of physiotherapy interventions compared to control conditions on fecal incontinence (FI) and quality of life (QoL) following colorectal surgery.</p>",
"<title>Methods</title>",
"<p id=\"Par2\">Electronic searches in English-language (S... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Acknowledgements</title>",
"<p>The research team acknowledges Mr. Muhammad Usman Ali for his assistance and support.</p>",
"<title>Author contributions</title>",
"<p>P.K. contributed to the study’s conception and design.</p>",
"<p>P.C. and C.T.C performed electronic and hand searches.</p>",
"<p>P.... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Flow diagram of the study selection process applied for this review</p></caption></fig>",
"<fig id=\"Fig2\"><label>Fig. 2</label><caption><p><bold>A</bold> Assessments made by review authors for each risk-of-bias domain are presented as percentages across all inc... | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Characteristics of included trials (<italic>n</italic> = 10)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">First author, year, country of study</th><th align=\"left\">Mean age of participants (SD); sample size of each g... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<table-wrap-foot><p><italic>ARM</italic>, anorectal manometry; <italic>ARP</italic>, anal resting pressure; <italic>BFT</italic>, biofeedback therapy; <italic>CCIS</italic>, Cleveland Clinic Incontinence Score; <italic>Con</italic>, control group; <italic>EMG</italic>, electromyography; <italic>ES</italic>, electr... | [
"<graphic xlink:href=\"520_2023_8294_Fig1_HTML\" id=\"MO1\"/>",
"<graphic xlink:href=\"520_2023_8294_Fig2_HTML\" id=\"MO2\"/>",
"<graphic xlink:href=\"520_2023_8294_Fig3a_HTML\" id=\"d32e1281\"/>",
"<graphic xlink:href=\"520_2023_8294_Fig3b_HTML\" id=\"d32e1282\"/>",
"<graphic xlink:href=\"520_2023_8294_Fig... | [
"<media xlink:href=\"520_2023_8294_MOESM1_ESM.docx\"><caption><p>Supplementary file1 (DOCX 27 KB)</p></caption></media>"
] | [{"label": ["2."], "mixed-citation": ["Hong Kong Cancer Registry, Hospital Authority (2021) Top ten common cancers in Hong Kong in 2021. Retrieved Oct 2021, from "], "ext-link": ["https://www3.ha.org.hk/cancereg/tc/topten.html"]}, {"label": ["6."], "mixed-citation": ["CJH cjh van_de_velde@ lumc. nl cciotDTMEtLMdDMBEMCP... | {
"acronym": [],
"definition": []
} | 38 | CC BY | no | 2024-01-15 23:42:02 | Support Care Cancer. 2024 Jan 13; 32(2):103 | oa_package/88/1d/PMC10787910.tar.gz |
PMC10787911 | 37405589 | [] | [
"<title>Method</title>",
"<p id=\"Par18\">Data come from a 100-day intensive longitudinal study of adolescent daily experiences. The study protocol was approved by University of Michigan Institutional Review Board for Health Sciences and Behavioral Sciences (IRB-HSBS). No data have been previously reported.</p>",... | [
"<title>Results</title>",
"<title>Reliability and Validity of Daily IC</title>",
"<p id=\"Par44\">The interindividual means and <italic>SD</italic>s of IC across 100 days are shown in Fig. ##FIG##1##2##. Each bar represents <italic>that day’s</italic> IC averaged across all adolescents. The thick black line rep... | [
"<title>Discussion</title>",
"<p id=\"Par54\">IC is often conceptualized as a relatively stable construct, but growing evidence suggests that cognition varies across contexts and time (Brose et al., ##REF##24364855##2014##; Sliwinski et al., ##REF##16953716##2006##). Although this has significant implications for... | [
"<title>Conclusions</title>",
"<p id=\"Par71\">Externalizing behaviors normatively increase during adolescence in ways that accentuate individual differences and implicate IC (Young et al., ##REF##19222319##2009##). Yet, IC – and its expression across different contexts and times – is thought to be stable even th... | [
"<p id=\"Par1\">Inhibitory control is a transdiagnostic risk factor for externalizing behaviors, particularly during adolescence. Despite advances in understanding links between inhibitory control and externalizing behaviors across youth <italic>on average</italic>, significant questions remain about how these link... | [
"<p id=\"Par6\">Behaviors on the externalizing spectrum, such as impulsivity, substance use, and conduct problems, increase during adolescence. Although this increase is generally considered normative, individual differences in externalizing behaviors can result in social difficulties, school failure, and persisten... | [
"<title>Acknowledgements</title>",
"<p>The authors thank members of the Methods, Sex differences, and Development – M(SD) – Lab at the University of Michigan, especially Amy Loviska who assisted with study implementation, Kaitlyn Zhao and Emma Donnelly-Sironen who assisted with participant testing, Jennifer Clear... | [
"<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Mathematical equation, model fitting procedure, and graphical depiction of standard GIMME, which contains group- and individual-level relations; the extension of GIMME used in this paper also included confirmatory subgroup-level relations (see Henry et al., ##REF##... | [] | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<fn-group><fn id=\"Fn1\"><label>1</label><p id=\"Par20\"> This included adolescents with transgender, gender non-conforming, and genderfluid identities.</p></fn><fn id=\"Fn2\"><label>2</label><p id=\"Par23\"> Adolescents who were at least 18 years old could opt to receive their emails directly.</p></fn><fn id=\"Fn... | [
"<graphic xlink:href=\"10802_2023_1071_Fig1_HTML\" id=\"d32e781\"/>",
"<graphic xlink:href=\"10802_2023_1071_Fig2_HTML\" id=\"d32e816\"/>",
"<graphic xlink:href=\"10802_2023_1071_Fig3_HTML\" id=\"d32e908\"/>",
"<graphic xlink:href=\"10802_2023_1071_Fig4_HTML\" id=\"d32e1005\"/>",
"<graphic xlink:href=\"1080... | [
"<media xlink:href=\"10802_2023_1071_MOESM1_ESM.docx\"><caption><p>Supplementary Material 1</p></caption></media>"
] | [{"mixed-citation": ["Bolger, N., & Laurenceau, J. P. (2013). "], "italic": ["Intensive longitudinal methods: An introduction to diary and experience sampling research"]}, {"mixed-citation": ["Chaku, N., & Beltz, A. M. (2022). Using temporal network methods to reveal the idiographic nature of development. In J. J. Lock... | {
"acronym": [],
"definition": []
} | 87 | CC BY | no | 2024-01-15 23:42:02 | Res Child Adolesc Psychopathol. 2024 Jul 5; 52(1):93-110 | oa_package/6c/cb/PMC10787911.tar.gz |
PMC10787912 | 37878150 | [
"<title>Background</title>",
"<p id=\"Par5\">Breast cancer affects almost 56,000 women every year in the United Kingdom (UK) [##UREF##0##1##] and despite improvements in treatment, approximately 40% of these will require mastectomy [##REF##11091232##2##]. Seroma formation following mastectomy and/or axillary clea... | [
"<title>Methods</title>",
"<p id=\"Par8\">An online national practice survey was developed in REDCap<sup>®</sup> to capture current UK practice regarding the use of strategies to reduce seroma formation following mastectomy and axillary surgery including the use of drains and flap fixation techniques, and details... | [
"<title>Results</title>",
"<title>Respondent demographics</title>",
"<p id=\"Par10\">A total of 147 responses were received of which 97 were completed with data that could be included in the analysis. The partially complete responses were excluded from further analysis where they had very limited, or no data en... | [
"<title>Discussion</title>",
"<p id=\"Par20\">This survey has demonstrated considerable variation in the management of seromas following mastectomy and axillary surgery in the UK and the need for and potential feasibility of a large-scale pragmatic RCT to establish best practice. It is likely that a future trial ... | [] | [
"<title>Purpose</title>",
"<p id=\"Par1\">Up to 40% of the 56,000 women diagnosed with breast cancer each year in the UK undergo mastectomy. Seroma formation following surgery is common, may delay wound healing, and be uncomfortable or delay the start of adjuvant treatment. Multiple strategies to reduce seroma fo... | [
"<title>Supplementary Information</title>",
"<p>Below is the link to the electronic supplementary material.</p>"
] | [
"<title>Acknowledgements</title>",
"<p>List of PUBMed citable collaborators (names as given in the online survey; some who completed did not leave a name). Nick Abbott, Raj Achuthan, Goran Ahmed, Rachel Ainsworth, Laura Arthur, Salena Bains, Zoe Barber, Jeremy Batt, Ashleigh Bell, Jane Carter, Alice Chambers, Ann... | [] | [
"<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Respondent demographics</p></caption></table-wrap>",
"<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Operative practice regarding drain use following mastectomy and axillary surgery</p></caption></table-wrap>",
"<table-wrap id=\"Tab3\"><label... | [] | [] | [] | [] | [] | [
"<supplementary-material content-type=\"local-data\" id=\"MOESM1\"></supplementary-material>"
] | [
"<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group>"
] | [
"<graphic position=\"anchor\" xlink:href=\"10549_2023_7042_Tab1_HTML\" id=\"MO1\"/>",
"<graphic position=\"anchor\" xlink:href=\"10549_2023_7042_Tab2_HTML\" id=\"MO2\"/>",
"<graphic position=\"anchor\" xlink:href=\"10549_2023_7042_Tab3_HTML\" id=\"MO3\"/>",
"<graphic position=\"anchor\" xlink:href=\"10549_202... | [
"<media xlink:href=\"10549_2023_7042_MOESM1_ESM.docx\"><caption><p>Supplementary file1 (DOCX 215 KB)</p></caption></media>"
] | [{"label": ["1."], "mixed-citation": ["Cancer Research U.K. (2019). "], "ext-link": ["https://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/breast-cancer"]}, {"label": ["9."], "surname": ["Thomson", "Sadideen", "Furniss"], "given-names": ["DR", "H", "D"], "article-title": ["Wo... | {
"acronym": [],
"definition": []
} | 31 | CC BY | no | 2024-01-15 23:43:45 | Breast Cancer Res Treat. 2024 Oct 25; 203(2):187-196 | oa_package/1d/dd/PMC10787912.tar.gz |
PMC10787939 | 38222147 | [
"<title>Introduction</title>",
"<p>Stress urinary incontinence (SUI) is a prevalent (4-35% prevalence rate) and often distressing condition that affects individuals, primarily women, across various age groups [##REF##12559262##1##]. It is characterized by the involuntary leakage of urine during activities that in... | [
"<title>Materials and methods</title>",
"<p>The study commenced after obtaining approval from the Institutional Review Board of Amity Institute of Health Allied Sciences (AUUP/IEC/2021-Jan/03) and was registered in the Clinical Trial Registry, India under the reference CTRI/2021/09/036247. It was designed as a si... | [
"<title>Results</title>",
"<p>Demographic data analysis conducted with Mann-Whitney U-tests revealed no statistically significant differences between the DNS and Kegel exercise groups in terms of age, height, weight, BMI, number of children, and duration of symptoms (as presented in Table ##TAB##1##2##).</p>",
... | [
"<title>Discussion</title>",
"<p>In this study, we conducted a thorough evaluation to assess the relative effectiveness of DNS exercises in comparison to conventional Kegel exercises for the management of SUI in women. Our study is a significant addition to the existing body of knowledge regarding the array of tr... | [
"<title>Conclusions</title>",
"<p>In conclusion, our study's results suggest that DNS exercises, which emphasize the coordinated activation of the diaphragm, abdominals, multifidus, and pelvic floor, may offer a more effective approach for managing stress urinary incontinence in women compared to traditional Kege... | [
"<p>Background and objective</p>",
"<p>Stress urinary incontinence (SUI) is a prevalent condition affecting women of various age groups, significantly impacting their quality of life. To address this multifaceted issue, a comprehensive approach that goes beyond traditional pelvic floor exercises is needed. Dynami... | [] | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Trial design overview - CONSORT flow chart</title><p>DNS: dynamic neuromuscular stabilization; CONSORT: Consolidated Standards of Reporting Trials.</p></caption></fig>"
] | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Dynamic neuromuscular stabilization exercise protocol for the rehabilitation of stress urinary incontinence</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Meena Gupta, Kiran Sharma, Raju K Parasher</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Meena Gupta, Kiran Sharma, Raju K Parasher, Jasmine Kaur Chawla</p><p><bold... | [
"<graphic xlink:href=\"cureus-0015-00000050551-i01\" position=\"float\"/>"
] | [] | [{"label": ["2"], "article-title": ["The association between urinary and fecal incontinence and social isolation in older women"], "source": ["Am J Obstet Gynecol"], "person-group": ["\n"], "surname": ["Yip", "Dick", "McPencow", "Martin", "Ciarleglio", "Erekson"], "given-names": ["SO", "MA", "AM", "DK", "MM", "EA"], "f... | {
"acronym": [],
"definition": []
} | 29 | CC BY | no | 2024-01-15 23:43:45 | Cureus.; 15(12):e50551 | oa_package/aa/f9/PMC10787939.tar.gz |
PMC10787940 | 38222225 | [
"<title>Introduction</title>",
"<p>Hemobilia is an infrequent cause of upper gastrointestinal bleeding, which is most often secondary to intrabiliary procedures, hepatic trauma, inflammatory diseases, biliary tumors, and vascular malformations [##REF##36156458##1##,##UREF##0##2##]. Although common in the gastroin... | [] | [] | [
"<title>Discussion</title>",
"<p>Hemobilia remains a challenging entity to diagnose and treat, requiring the use of various technologies and a multidisciplinary approach. Common causes include intrabiliary procedures (endoscopic or percutaneous), hepatic trauma, cholangiopathies secondary to cirrhosis, tumors, an... | [
"<title>Conclusions</title>",
"<p>In conclusion, digital cholangioscopy played a crucial role in the diagnosis and treatment decision for hemobilia due to angiodysplasia of the major biliary duct. There is still a lack of data in the literature to define the gold-standard therapy, but the use of a fully covered m... | [
"<p>Hemobilia is described as bleeding from the intra- or extrahepatic biliary tree expressed through the major duodenal papilla into the duodenum, with angiodysplasia of the major biliary duct as a rare etiological factor with few cases reported in the literature. Cholangioscopy plays a pivotal role in diagnosing ... | [
"<title>Case presentation</title>",
"<p>An 87-year-old male patient with atrial fibrillation who was on apixaban 5 mg/day presented to the emergency department with exertional dyspnea and lower limb edema. He had no history of external bleeding but complained of mild abdominal pain upon examination. Laboratory te... | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Active bleeding from the major duodenal papilla</title></caption></fig>",
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>Enhancement focus in the distal common bile du... | [] | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Acquisition, analysis, or interpretation of data:</bold> Marcus Vinícius S. Costa, Lucas V. Aragão, Tomazo P. Franzini</p><p><bold>Drafting of the manuscript:</bold> Marcus Vinícius S. Costa, Lucas V. Aragão, Julia... | [
"<graphic xlink:href=\"cureus-0015-00000050552-i01\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050552-i02\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050552-i03\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050552-i04\" position=\"float\"/>"
] | [] | [{"label": ["2"], "article-title": ["Endovascular and endoscopic treatment of hemobilia: a report of two cases"], "source": ["Cureus"], "person-group": ["\n"], "surname": ["Cardona", "Rivero", "Pinto", "Barrag\u00e1n", "Torres"], "given-names": ["JD", "OM", "R", "CA", "DF"], "fpage": ["0"], "volume": ["14"], "year": ["... | {
"acronym": [],
"definition": []
} | 12 | CC BY | no | 2024-01-15 23:43:45 | Cureus.; 15(12):e50552 | oa_package/14/dd/PMC10787940.tar.gz |
PMC10787941 | 38222993 | [
"<title>Introduction</title>",
"<p>Primary ciliary dyskinesia (PCD), an autosomal recessive condition characterized by motile cilia malfunction, displays clinical and genetic variation. Some of the clinical symptoms of PCD include left-right lateralization, infertility, and chronic upper and lower respiratory ill... | [] | [] | [
"<title>Discussion</title>",
"<p>We described two Indian sisters who experienced early-onset respiratory symptoms, obstructive ventilatory dysfunction, and situs solitus. They were identified as having PCD based on the full exon of the CCNO gene and low nNO levels.</p>",
"<p>Patients with ciliogenesis-related m... | [
"<title>Conclusions</title>",
"<p>PCD with <italic>CCNO </italic>mutations is a relatively rare disease. Our findings highlight the significance of considering PCD based on <italic>CCNO </italic>mutations in people with situs solitus to have a more severe respiratory disease phenotype with lung failure at earlier... | [
"<p>Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease marked by organ lateralization in 50% of patients, chronic sinopulmonary disease, infertility in men, and neonatal respiratory distress.</p>",
"<p>Respiratory control cells contain <italic>CCNO </italic>in their apical cytoplasm, ... | [
"<title>Case presentation</title>",
"<p>Our case study focuses on two consanguineous sisters from India who visited the Pediatric Pulmonary Department at Sidra Hospital in Qatar when they were 17 and 15 years old. They presented with chronic wet cough and were found to have progressive loss of lung function that ... | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Chest X-ray and CT chest findings of the elder sister.</title><p>A: Anteroposterior chest X-ray. B: CT chest, axial view, lung window.</p><p>Chest radiographical imaging of the elder sister showing bilateral varicose br... | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>CCNO gene mutation studies.</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nStudy\n</td><td rowspan=\"1\" colspan=\"1\">\nNumber of cases\n</td><td... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained or waived by all participants in this study</p></fn></fn-group>",
"<fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.... | [
"<graphic xlink:href=\"cureus-0016-00000052237-i01\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0016-00000052237-i02\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0016-00000052237-i03\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0016-00000052237-i04\" position=\"float\"/>",
"<gr... | [] | [{"label": ["7"], "article-title": ["Diagnosis of primary ciliary dyskinesia. An official American Thoracic Society clinical practice guideline"], "source": ["Am J Respir Crit Care Med"], "person-group": ["\n"], "surname": ["Shapiro", "Davis", "Polineni"], "given-names": ["AJ", "SD", "D"], "fpage": ["0"], "lpage": ["39... | {
"acronym": [],
"definition": []
} | 18 | CC BY | no | 2024-01-15 23:43:45 | Cureus.; 16(1):e52237 | oa_package/e0/e3/PMC10787941.tar.gz |
PMC10787942 | 38222226 | [
"<title>Introduction</title>",
"<p>Acute appendicitis is one of the most prevalent causes of presentation to the emergency department (ED) [##REF##27826565##1##]. With the treatment being resection, whether open or laparoscopic, it is not uncommon for patients to present with complications of appendectomy. One of... | [] | [] | [
"<title>Discussion</title>",
"<p>Stump appendicitis (SA) is defined as interval inflammation of residual appendicular tissue after appendectomy [##UREF##2##5##]. First described in 1945 by Rose, its incidence has since been slowly rising [##UREF##0##2##]. This has been attributed to laparoscopic surgery by some a... | [
"<title>Conclusions</title>",
"<p>Right lower quadrant abdominal pain comes with a wide range of differential diagnoses. In patients with a clinical picture of acute appendicitis and history of appendectomy, it is the emergency physician’s responsibility to maintain a high index of suspicion for stump appendiciti... | [
"<p>Acute appendicitis is one of the most common diagnoses in the emergency department. As with other surgical procedures, post-appendectomy complications are numerous and can be either immediate or delayed. Stump appendicitis is an underreported and underrecognized complication that is often diagnosed radiological... | [
"<title>Case presentation</title>",
"<p>A 26-year-old male presented to ED with a one-day history of right lower abdominal pain associated with nausea and one episode of vomiting. The pain was of gradual onset, dull achy character, mild to moderate intensity, and intermittent course. He denied fever, altered bowe... | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Non-contrast abdominal CT image showing wall thickening of a blind-ended tubular structure arising from the cecal pole (yellow arrow), suggestive of inflamed appendiceal stump.</title></caption></fig>"
] | [] | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Nada A. Mohammed, Mustak Dukandar</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Nada A. Mohammed</p><p><bold>Drafting of the manuscript:</bold> Nada A. Mohammed</p... | [
"<graphic xlink:href=\"cureus-0015-00000050557-i01\" position=\"float\"/>"
] | [] | [{"label": ["2"], "article-title": ["Stump appendicitis, a rare but serious complication of appendectomy: a case report"], "source": ["Clin Case Rep"], "person-group": ["\n"], "surname": ["Hadrich", "Mroua", "Zribi", "Bouassida", "Touinssi"], "given-names": ["Z", "B", "S", "M", "H"], "fpage": ["0"], "volume": ["9"], "y... | {
"acronym": [],
"definition": []
} | 17 | CC BY | no | 2024-01-15 23:43:45 | Cureus.; 15(12):e50557 | oa_package/4a/d6/PMC10787942.tar.gz |
PMC10787943 | 38222158 | [
"<title>Introduction</title>",
"<p>Common variable immunodeficiency (CVID) is one of the prevalent primary immunodeficiencies, characterized by a wide range of symptoms and recurrent bacterial infections. Patients with CVID are at increased risk of infections, including gastrointestinal infections. Chronic diarrh... | [] | [] | [
"<title>Discussion</title>",
"<p>The patient had chronic diarrhea with recurrent sinopulmonary infections, hypogammaglobinemia, hypoproteinemia, and vitamin deficiency, along with lymphoid nodular hyperplasia of the duodenum, suggesting the diagnosis of CVID (primary immunodeficiency disorder). Other differential... | [
"<title>Conclusions</title>",
"<p>Evaluation of chronic diarrhea requires thorough clinical evaluation and should be evaluated with an open mind. A review of a patient’s investigations should always be considered when clinical judgment regarding a case is not satisfied. Although common diseases should be consider... | [
"<p>Chronic diarrhea poses a diagnostic challenge due to its diverse etiology, encompassing various gastrointestinal disorders. This case report emphasizes the clinical significance of considering common variable immunodeficiency (CVID) as a potential underlying cause in a patient presenting with chronic diarrhea. ... | [
"<title>Case presentation</title>",
"<p>A 36-year-old female presented to our emergency department with complaints of watery stools for 9 years, recurrent respiratory tract infections for 9 years, and weight loss for 3 years. Initially, the patient developed loose motions that were watery in consistency, with a s... | [] | [
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG1\"><label>Figure 1</label><caption><title>Upper GI endoscopy showing nodular lymphoid hyperplasia of the duodenum</title></caption></fig>",
"<fig position=\"anchor\" fig-type=\"figure\" id=\"FIG2\"><label>Figure 2</label><caption><title>: Chest X-ray</title><... | [
"<table-wrap position=\"float\" id=\"TAB1\"><label>Table 1</label><caption><title>Initial Investigations performed</title><p>ALT: alanine aminotransferase; AST: aspartate aminotransferase</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\"... | [] | [] | [] | [] | [] | [] | [
"<fn-group content-type=\"other\"><title>Author Contributions</title><fn fn-type=\"other\"><p><bold>Concept and design:</bold> Ahmad G. Ansari , Husaini S. Haider Mehdi, Ariba Nasar</p><p><bold>Acquisition, analysis, or interpretation of data:</bold> Ahmad G. Ansari , Husaini S. Haider Mehdi, Ariba Nasar</p><p><b... | [
"<graphic xlink:href=\"cureus-0015-00000050556-i01\" position=\"float\"/>",
"<graphic xlink:href=\"cureus-0015-00000050556-i02\" position=\"float\"/>"
] | [] | [{"label": ["4"], "article-title": ["Chapter 18 - Hypogammaglobulinemia and common variable immune deficiency"], "source": ["Stiehm's Immune Deficiencies (Second Edition)"], "person-group": ["\n"], "surname": ["Cunningham-Rundles", "Warnatz"], "given-names": ["C", "K"], "fpage": ["467"], "lpage": ["497"], "publisher-na... | {
"acronym": [],
"definition": []
} | 13 | CC BY | no | 2024-01-15 23:43:45 | Cureus.; 15(12):e50556 | oa_package/e1/87/PMC10787943.tar.gz |
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