DESCRIPTION: Induced pluripotent stem cell technology, which allows reprogramming of cell fate through ectopic expression of selected transcription factors, has opened the possibility to reprogram fibroblasts or other somatic cells directly into specific cell types. This direct reprogramming offers an alternative approach for generating lineages of interest without passing through a progenitor state. Similar to the pluripotent reprogramming by the Yamanaka factors, the process to convert fibroblasts directly to neuronal cells, hepatocyte-like cells, insuin producing-cells or cardiomyocyte-like cells requires the delivery of multiple transcription factors into the cells. Several vectors, such as retroviral-, lentiviral-, AAV-vectors or plasmids, have been used for this purpose with different levels of success, but with the potential of integration f genetic material in the host genome. Thus, there is a need for the development of a new viral platform to generate transgene-free direct reprogrammed cells. Here, we propose to develop a new RNA vector system based on a non-integrating human Paramyxovirus, measles virus (MV), to deliver the transcription factors needed for the direct reprogramming of fibroblast into insulin producing cells. We have developed a reverse genetic system allowing the production of a recombinant virus equivalent to the Moraten vaccine strain, which is currently used for vaccination in the US. From this viral genome, we have produced a one cycle vector and used it to express the Yamanaka factors for iPSCs reprogramming. We propose to modify these vectors and use them to express the factors required for direct reprogramming. We will test the hypothesis that our replication-deficient measles vector can be modified to express the transcription factors shown to be necessary for the reprogramming of adult somatic cells into functional insulin-producing cells. The cells will be then tested in a diabetic mouse model that will validate the functionality of the reprogrammed insulin producing cells. Achieving the proposed studies outlined here will lead to the development of a new polycistronic vector platform allowing direct trans-differentiation without genomic modification. These studies will open the perspective of using these vectors for future direct reprogramming in vivo.


Dietary restriction extends lifespan, improves mid-life vigor, and retards age-related disease in many species. Mechanisms underlying these benefits are not well understood. Defining the cellular pathways by which dietary restriction is initiated and maintained holds potential for combating obesity, diseases of old age such as cancer, and the general functional decline that accompanies aging. We have begun to investigate how microRNAs (miRNAs), small molecules that target partially homologous transcripts to block their translational expression, impact the "quality of aging" (healthspan) in the powerful experimental model C. elegans. In this animal, conserved genes can be readily manipulated to address specific hypotheses regarding their activities. Moreover, nearly all the C. elegans microRNA genes are likely to have been identified, deletions of most of these genes have been recently generated, and our preliminary work suggests several miRNAs impact lifespan and and/or healthspan phenotypes. In mammalian systems, miRNA expression profiles change with different metabolic conditions, although no studies to date have addressed changes in dietary restriction. The working hypothesis we propose to test here is that specific microRNAs exert significant effects on the initiation and/or maintenance of dietary restriction metabolism. If we identify these miRNAs and figure out how they work, we could suggest manipulation of their mammalian counterparts to improve healthy mid- and late-life. We will therefore exploit the considerable experimental advantages of C. elegans model to identify miRNAs that are differentially expressed in dietary restricted animals, and we will genetically test candidates for causative action in DR induction and / or maintenance. Because dietary restriction appears to transpire by a fundamental mechanism conserved from nematodes to humans and because virtually nothing is known yet of microRNA modulation of dietary restriction, our planned work should produce data relevant to human biology that pioneers a new area in dietary restriction research with implications for therapeutic development.


The broad objective of this interdisciplinary project is to elucidate the pathogenesis of human neuromuscular diseases. The Center comprises 17 independent investigators in 13 laboratories. In many projects basic scientists and clinical investigators work together. Basic research includes the dynamics of muscle activation and contraction, calcium regulation in muscle, physiology of human muscle fibers, ultrastructure of muscle, development of muscle, muscle culture immunocytochemistry, glycoconjugates, structure and function of acetylcholine receptor. Clinical investigation includes study of muscular dystrophy, myasthenia gravis, malignant hyperthermia, disorders of glycogen and lipid metabolism, hexosaminidase deficiency, and peripheral neuropathy.


The proposed CRECD Program at Charles R. Drew University (CDU), Mentored Postdoctoral Training in Translational Research, is a Phase II mentored clinical research and career development program designed to develop a diverse cadre of clinical and translational investigators who conduct innovative research on the underlying causes of diseases, in particular those diseases that disproportionately impact minority populations in the United States (e.g., HIV/AIDS, cancer, cardiovascular disease, diabetes, and mental health/psychiatric disorders), and to foster and facilitate professional development activities in clinical and translational sciences. Over the initial 5-year program period, the CRECD Program will accept a total of eight postdoctoral trainees at the junior faculty level at CDU, each for 3-year appointments, staggered in appointment periods. The CRECD Program will be embedded within the existing, extensive clinical research and training infrastructure at CDU, and thus will mutually leverage resources with other CDU research education and career development programs so as to achieve maximal cross-program synergies as well as efficiencies wrought from utilizing already-in-place education and training curricula and related resources germane to the CRECD agenda. However, the CRECD Program will also carve out a unique niche of training and career development opportunities within the broader CDU science-generating critical mass by (1) focusing exclusively on health disparities and mastery of community-partnered participatory methods in the research and research training agenda and (2) providing to each trainee a first-of-its-kind (at CDU and, by our estimate, nationwide) intensive and community-immersive mentoring configuration that includes CDU faculty from both the standard or conventional Academic Career track and the newly emerging Community Faculty track, called the Partnership for Equity and Equality in Health and Wellness. This intensive mentoring innovation directly emerges from, and formalizes and systematizes into a signature trainee mentoring configuration, a long and fruitful history of community-engaged research, training, and educational curricula at CDU. As an integrated research training and career development package, this approach is designed to bring social determinants of health to the forefront of molecular and clinical research. Ultimately, the goal is to increase the impact of community- academic partnered research and adoption of its evidence-based best practices through comprehensive dissemination of research findings so as to encourage and facilitate implementation of evidence-based treatment and prevention practices within health care organizations and the communities most in need of reliable access to high-quality care.


Mast cells and basophils, the central effector cells of immediate type hypersensitivity, will be purified from animals and humans. The mast cell and basophil content of preformed chemotactic mediators and enzymes and their release by immunologic method will be studied. The effect upon mediator release of permeant anions will be assessed and the regulatory neurohormone receptors identified and characterized. Chemotactic mediators released in vitro from isolated cells or in vivo in human disease with known mast cell involvement will be purified by physiochemical technique and characterized functionally with regard to target cell specificity for chemotaxis, chemotactic deactivation, and cell membrane receptor alteration. The effect of chemotactic factors and cells will be extended to in vivo models in animals. Taken together, these studies will expand the understanding of the biologic importance of chemotactic mediators in regulation of inflammatory events in human disease.


A diethylenetriamine functional stationary phase, capable of complexing transition metal ions, was developed and evaluated for use in HPLC separations. Synthesis involved evaporation of the trimethoxysilylpropyl- monomer from acetate buffered aqueous solution and derivatization at 200 degree C 0.55 torr. Reproducible surface coverages of 3.0 microgram moles/m-squared were obtained on 100-angstrom, 550 m-squared/g silica. Analytical columns were loaded with Cu(II), Ni(II) by frontal elution process; breakthrough volumes corresponded well with metal uptake determined by atomic absorption spectrophotometry. Buffered aqueous- organic mobil phases were used for separations of sulfonamide antimicrobials that included sulfa-diazine, sulfathiazole, sulfa-merazine and sulfa-pyridine. An empirical retention expression was developed that related chromatographic retention to: a)electrostatic interaction of the acidic sulfonamide function with the cationic stationary phase, b) secondary metal-ligand complexations involving the heterocyclic aromatic substituents of the solutes and c) solubilization of the relatively hydrophobic solutes in the mobile phase. Retentions were inversely proportional to concentrations of organic modifier and buffer and were generally directly proportional to eluent pH. An eluent of pH 7.3 phosphate buffer and 50% methanol was effective in minimizing electrostatic interactions while allowing retention based on metal-ligand interactions. This work was the topic of a Ph.D dissertation, entitled "Electrostatic and Metal Interaction Chromatography of Sulfonamides" that was defended and accepted pending editorial revisions on April 30, 1991.


Idiopathic pulmonary fibrosis a fibrotic interstitial lung disease characterized by a median survival of 3-5 years post-diagnosis but exhibits heterogeneous longitudinal disease progression. Recent studies of novel agents confirm beneficial effects on longitudinal change in forced vital capacity but inconsistent benefits on clinical endpoints or health status. Both agents are difficult to tolerate and are likely to be prohibitively expensive. Conducting clinical trials to assess clinical endpoints requires that larg numbers of patients are enrolled and followed for a sufficient period of time. The inability to rapidly recruit sufficient numbers of IPF patients means that many key clinical questions have not been addressed. Restrictive inclusion criteria ensure that patients enrolled in clinical trials often differ from those seen in clinical practice. There remains a critical need to use innovative, pragmatic study designs to identify well tolerated and inexpensive therapies which improve clinical outcomes in patients with IPF. Our group was the first to identify that an abnormal lung microbial community is independently associated with disease progression in IPF subjects. Additional preliminary data link this to a circulating gene expression signature of altered host response. Intriguingly, one investigative group has suggested improved clinical outcomes in IPF patients treated with trimethoprim/ sulfamethoxazole compared to a matched placebo. The totality of these data suggests that an abnormal lung microbiome interacting with genetic susceptibility in host response may be associated with impaired clinical outcomes in IPF. Our principal hypothesis is that antimicrobial therapy in IPF patients will improve clinical outcomes. Our long-term goal is to define patient-specific therapy in IPF. Using a pragmatic trial design CleanUP-IPF will remove many of the known obstacles to clinical trial enrollment in order to recruit a patient population that is highly representative of those seen in clinical practice. We anticipate demonstrating that: 1) a large, pragmatic study in IPF is feasible and will identify clinically meaningful endpoints beyond FVC; 2) anti- microbial therapy will improve clinical outcomes; and 3) genetically predisposed patients will experience differential response to therapy. CleanUP-IPF will revolutionize future studies in IPF and provide data that will alter therapeutic guidelines.


: The revised application focuses on complement activation induced by heparin-protamine complexes. A specific aim of the proposal is to study the relationship of complement activation induced by protamine neutralization of heparin with pulmonary dysfunction. The study will be conducted in patients undergoing first-time cardiopulmonary bypass for coronary artery surgery. A second specific aim is to correlate preoperative complement activation in the presence of heparin and protamine with the actual measurements of in vivo complement activation induced by protamine neutralization of heparin. A third specific aim is to determine whether excessive in vivo complement activation by heparin-protamine is dependent upon the phenotypic or functional differences in C4. Two hundred patients will be entered into the study. Pulmonary shunt fraction will be determined as will pulmonary vascular resistance. Red cell bound C3b/C3d will be determined. IgE and IgG antiprotamine antibodies will be detected using ELISA. Blood samples will be studied at four time points: 1. prior to sternotomy, 2. prior to bypass, 3. before protamine, 4. 10 minutes after protamine. Leukotriene B4 and thromboxane B2 will be determined on samples drawn from pulmonary artery and radial artery lines to represent pre-and post-pulmonary circulation samples. Other studies will be directed at determining the relationship between C3d binding to red cells and subsequent heparin-protamine induced complement activation. Preoperative blood samples will be assessed for C3d binding to red cells. These samples will be used in a series of in vitro assays to determine whether or not the differences in sera with respect to C3d binding are due to differences in the C4b binding to red cells. Statistical analysis will include a number of independent variables such as gender, body surface area, age, number of diseased vessels, number of grafts, heparin dose, time on pump, diabetes, Leukotriene B4 levels, thromboxane B2 levels, changes in C3a during bypass, changes in C3a and C4a during protamine infusion, etc.


This study is a phase II, placebo-controlled clinical trial to evaluate the safety and efficacy of orally administered SP-303, an inhibitor of ileal chloride transport, in patients with HIV-associated disease. Male and female patients with AIDS between 18-60 will be studied. Subjects will qualify for the study of they have a confirmed stool volume >200 gm and >3 liquid bowel movements during the 24-hour screening visit. Qualified subjects will next be randomized and orally adminstered either SP-303 or placebo for the next 96 hours (4 days), ruding which they will remain hospitalized in the GCRC.


The overarching aims of this Project are to synthesize the microtubule stabilizing natural product dictyostatin-1 and its analogs, and to evaluate the potential of these compounds as anticancer agents. In addition to the tight collaborative medicinal chemistry aspects of the work, the use of the new technique of fluorous mixture synthesis is also featured. The specific aims are: 1) Assignment of the structure of dictyostatin by total synthesis. Aim 1 is well advanced. We have recently completed the total synthesis of dictyostatin 1, and we now know its full structure, including absolute and relative configurations. It turns out that dictyostatin and discodermolide have the same configurations at all ten shared stereocenters. This structure determination has removed a major roadblock to development of dictyostatin as a potential anti-cancer agent. However, we still would like to learn how similar (spectroscopically, biologically) the isomers are with the same relative configurations at the three main fragments but coupled together in different pairings. This question will be answered by making multiple isomers by fluorous mixture synthesis. We have also nearly completed synthesis of the original Pettit structures, so this work will be finalized. 2) Synthesis of 0.35-1.0 g of dictyostatin for in vivo characterization. To accomplish this goal, the current synthesis must be improved, and a plan to streamline it by increasing convergency is outlined. 3) Synthesis of stereoisomers and analogs of dictyostatin for SAR studies. We will use the recently established synthetic route to make analogs by fluorous mixture synthesis. We plan to make stereoisomers, and to make simplified analogs with the goal of beginning to elucidate the structure/activity relationship. 4) Conformational modeling of dictyostatin and key stereoisomers. Multiconformational searching coupled with MM3 calculations in Macromodel will be used to predict conformational minima of key isomers. Initially, this data will be used to help interpret NMR experiments, and will serve as a basis for analog design as well as for more sophisticated modeling and docking experiments.


Hearing impairment/deafness is the most common sensory limitation in the U.S. An estimated 11 million individuals in the US are deaf or hard-of-hearing. It has been estimated that approximately 1 million Americans use American Sign Language (ASL) as their primary means of communication making it a distinct linguistic minority. This proposal seeks to improve clinical practice by creating a computerized, self-administered depression screener in ASL that is culturally and linguistically accessible to deaf individuals, an at-risk and traditionally underserved population. No current depression screeners have been shown to be valid for the majority of prelingually deaf persons who use ASL as their main communication mode. Studies have shown that depression occurs in higher rates among deaf persons than hearing persons. While the U.S. Preventive Services Task Force recommended systematic screening for depression in primary care clinical settings in 2002, deaf persons have not yet routinely had access to this preventive service. Providing primary care physicians with a depression screener that is culturally and linguistically accurate and can be self-administered via computer and can be used on iPads and iPhones with no further development, can greatly increase the chance that deaf persons with depression will receive proper diagnostic assessment and treatment which can substantially improve their quality of life. The Deaf Depression Screener will also improve scientific knowledge by providing a valid method for estimating the prevalence of depression among deaf persons in primary care which cannot now be accomplished due to the lack of a valid screening instrument.


This is a request for renewal of a Program-Project Grant to support research on the neural and humoral mechanisms regulating endocrine function. It also requests funds for continuation of the research previously supported by a grant to Drs. J. de Groot and M. Dallman (NS09538) for the study of brain-pituitary-adrenal interrelations. The research, which is carried out by the Program Director and faculty associates, faculty collaborators in other departments, postdoctoral fellows, graduate students, medical students and visiting scientists, is aimed at elucidation of the fundamental mechanisms that regulate the secretion of the endrocrine glands. Attention will be focused on extending current research on the regulation of renin secretion, with particular emphasis on the role of sympathetic nervous system in this process. The mechanism by which norepinephrine stimulates renin secretion will be studied in vitro. The factors affecting angiotensinogen secretion will also be studied. The relation of growth hormone to the maintenance of the secretory capacity of the zona glomerulosa of the adrenal cortex will be investigated. Research on the regulation of ACTH secretion will include studies to determine the site at which glucocorticoid hormones act to inhibit ACTH secretion. The inhibitory influence of norepinephrine-secreting neurons in the hypothalamus on ACTH secretion will be investigated further, along with the influence of these neurons and dopamine-secreting neurons on the secretion of growth hormone and prolactin in the dog. The effects of electrical stimulation of these aminergic neurons on the secretion of anterior pituitary hormones will be investigated. In addition, research on the neural mechanisms regulating the onset of puberty will be continued and experiments are planned to elucidate the effect of changes in endocrine environment on responses of single neurons in the hypothalamus and related parts of the brain.


Autoimmunity is caused by conspiring effects of genetic predisposition and environmental factors such as injury, infection and microbiome constitution. While multiple genetic loci affect susceptibility, in most cases each in isolation has only a small effect, suggesting that disease develops only when multiple risk-conferring alleles that function in concert are inherited by a single individual. We hypothesize that such a situation exists in autoreactive B cells where multiple SLE risk alleles encode molecules that appear to function in signaling pathways that function normally to limit/terminate antigen receptor signaling. In this application we propose to test this hypothesis, analyzing the functional interplay of this set of genes/protein and their risk conferring alleles. Future development and implementation of ?precision? medical approaches for treatment of autoimmunity will require an understanding of the mechanisms by which genetic variations conspire to increase disease risk, and research proposed here represents a critical first step to enable these efforts. A number of autoimmunity risk alleles encode molecules previously proposed to function as intermediaries in signaling pathways involved in regulation of B cell activation. As such they may be important in keeping autoreactive B cells from becoming activated and contributing to autoimmunity. In this application we request support to define the functions and functional interactions of proteins encoded by six genes, variants of which confer increased risk of autoimmunity. Previous reports indicate that B cell-targeted deletion of genes encoding SHIP-1, PTEN, SHP-1 or LYN, expression of PTPN22 (PEP-R619W), or increased expression of CSK, promote the development of autoimmunity. However the mechanism by which this occurs is unknown. We hypothesize that these proteins function as intermediaries in a bifurcating pathway in which final effectors are the inositol lipid phosphatases SHIP-1 and the tyrosine phosphatase SHP-1. Further, we suggest that both terminal effectors are required for maintenance of antigen unresponsiveness of anergic B cells. The studies will employ reductionist genetic models in which risk allele mimetic changes in expression/function of the proteins can be induced acutely in anergic B cells, and subsequent cell activation, proliferation, differentiation and autoantibody production monitored. Aim 1 will test the hypothesis that PTPN22, CSK and LYN act in linear pathways upstream of SHIP-1 and SHP-1, and that genetic variations that confer risk compromise anergy by undermining their regulatory function. Aim 2 will define the downstream consequences of acute introduction of risk allele mimetic conditions in terms of development of autoimmune disease, and will test candidate therapeutic kinase inhibitors. Aim 3 will translate findings, examining the role of SHIP-1 and SHP-1 phosphatases in maintenance anergy of human B cells. Proposed studies will provide important new insight regarding the in vivo lifestyle of autoreactive B cells whose anergy is compromised by autoimmunity risk alleles.


Although more than 200,000 children are treated annually in hospital emergency rooms for injuries incurred on the playground, our review of current teacher training materials reveals no interactive multimedia program targeting effective playground supervision. This project will develop a comprehensive set of multimedia materials to guide elementary school faculty in implementing a school-wide approach to positive and well-disciplined playground supervision. The Phase I prototype will teach playground supervisors effective behavior management techniques. PROPOSED COMMERCIAL APPLICATION: Multimedia educational software is a growing field, with schools comprising a large customer base. The individualization afforded by multimedia, combined with superior instructions design and remediation as needed, make this product a simple cost-effective way for schools to design and implement safe playground policies.


During the current period of funding, we have shown that the framework of the equilibrium-point hypothesis can be used to address issues of synergy organization. A novel method for analysis of the structure of variability of multi-element systems has been developed and tested experimentally. The next challenges are to generalize the applicability of these tools and approaches to different effector systems and tasks and to link them to physiological processes within the human body. If this is accomplished successfully, our understanding of the human motor function will reach a level when direct application of the presently theoretical and metaphorical notions to practical issue of motor disorders and rehabilitation becomes possible. Hence, our next plans include: (1) Testing the new method of analysis of the structure of variability as a tool for discovering performance variables that are selectively stabilized during a variety of motor tasks; (2) Studying the process of synergy evolution with practice; and (3) Approaching the central mechanisms of synergy organization using the method of transcranial magnetic stimulation. We plan to address the following specific hypothesis: 1). Within a synergy, variability of individual elements is structured so as to selectively stabilize functionally important variables; 2). Learning a novel multi-element task leads to the creation of a synergy reflected in changes of the structure of variability within the state space of elements and responses of the elements to stimulation of the corticospinal tract; 3). Organization of elements into synergies with practice leads to plastic changes reflected in task-specific patterns of finger force responses to transcranial magnetic stimulation; and 4). Quick corrections to perturbations are organized in a synergy-specific way, so as to minimize deviations of functionally important variables. Their qualitative pattern (timing of muscle activation) may be largely independent of the local joint kinematics while quantitative features reflect joint kinematics in accordance with the EP-hypothesis. Seven experiments with oscillatory and discrete force production are suggested to test the hypotheses using tasks of multi-finger coordination within a hand and multi-finger coordination between the two hands. We also plan to monitor changes in indices of synergy organization with practice of an unusual multi-finger coordination task.


Summary Chikungunya virus (CHIKV) is an emerging arthropod-borne virus for which there are no vaccines or therapeutics. Infection with CHIKV can lead to severe and chronic arthralgia. CHIKV displays wide tropism targeting the epithelial, endothelial, and myeloid compartments. The innate immune system is the first line of defense. Viral nucleic acids are sensed, leading to the activation of classical type I interferons (IFN) which induces hundreds of interferon stimulate genes (ISGs). In response, viruses including CHIKV, encode antagonists of interferon signaling to attenuate these antiviral activities. In addition, there is a growing list of basally expressed, effector proteins known as intrinsic factors, which play roles in controlling infection. Altogether, it is clear that there are additional antiviral regulators that play important roles in controlling infection, and in particular, there is less known about antiviral responses in non-hematopoietic cells which are the major targets of CHIKV. Moreover, while much of our understanding of antiviral effectors has been focused on antiviral proteins, there is an emerging literature that suggests that noncoding (nc)RNAs can also play an important role in controlling infection. Long noncoding RNAs (lncRNAs) are an emerging and abundant subclass of ncRNAs that are known to dynamically regulate transcription and translation and can control innate immune responses. The contribution of lncRNAs to innate defense downstream of viral infection is poorly understood, and there is nothing known during CHIKV infection. We hypothesize that lncRNAs play a regulatory role in the control of innate antiviral immunity against CHIKV. Therefore, we will characterize the role of lncRNAs in the innate immune response to CHIKV infection. We are beginning our studies in endothelial cells, as these are a target during infection and little is known about antiviral defenses in this tissue. In addition, we are particularly interested in cytoplasmic lncRNAs as there is less known about their functions, and CHIKV replication occurs exclusively in the cytoplasm. To achieve these goals, our specific aims will (1) Characterize lncRNAs that control CHIKV infection and (2) define the mechanism of action of one antiviral lncRNA we have validated (chikvLNC)


We are studying the biological mechanism of action of hepatocyte growth factor (HGF). u(In vivo), HGF exists in both a single chain form and as a heterodimer. u(In vitro), HGF has been shown to be multifunctional since the protein can promote DNA synthesis as well as the ``scattering" of cells. Experiments are under way to determine which parts of the protein are important for the biological effects of HGF. Programs available through the supercomputing facility will initially be used to develop subcloning strategies for the HGF cDNA and later used to assist in analysis of protein structure and function.


Central leptin resistance results in decreased downstream signaling to brain circuits that regulate food intake and energy expenditure (EE), thereby promoting hyperphagia and obesity. We and others have shown that the nonapeptide, oxytocin (OT), circumvents leptin resistance and elicits body weight (BW) loss in diet-induced obese (DIO) rodents, nonhuman primates and obese humans, by reducing both food intake and increasing EE. The discovery of recruitable brown adipose tissue (BAT) in humans has renewed interest in targeting BAT to elicit weight loss by increasing EE. OT neurons that project directly from the parvocellular paraventricular nucleus (pPVN) to the hindbrain nucleus of the solitary tract (NTS) are positioned to regulate energy homeostasis by reducing food intake and increasing BAT thermogenesis. In Specific Aim 1 we will test the hypothesis that OT-induced stimulation of sympathetic nervous system (SNS) outflow to interscapular (IBAT) contributes to its ability to elicit weight loss in DIO rodents. To test this, we will determine whether disrupting sympathetic activation of IBAT blocks the ability of fourth ventricular (4V) OT administration to increase EE and elicit weight loss in DIO rats. We will also determine if sympathetic outflow to both IBAT and white adipose tissue (WAT) mediates the effects of OT on EE by testing the extent to which pharmacological blockade of beta-3 adrenergic receptors (?-3-AR) impairs the ability of 4V OT administration to increase EE in DIO rats. In addition, we will determine if OT can be combined with low doses of the ?-3-AR agonist, CL316243, to increase EE and promote weight loss in DIO rats. Endpoints will include EE, IBAT temperature, norepinephrine turnover (NETO; marker of sympathetic activity), food intake, body composition (total and relative fat mass, lean mass), and BW. We anticipate these studies to establish a key role for SNS outflow to IBAT in the mechanism by which OT increases EE and elicits BW loss in DIO rats. In Specific Aim 2 we will test the hypothesis that in DIO rhesus monkeys, intranasal OT reduces BW and improves glucose tolerance and other metabolic parameters, in part, by stimulating SNS outflow to BAT. To accomplish this, we will determine if intranasal OT increases the temperature of axillary BAT (ABAT, the predominant BAT depot found in rhesus monkeys) at a dose that elicits weight loss in DIO NHPs. We will also determine the extent to which intranasal OT elicits weight loss by reducing body adiposity while sparing lean mass and identify if these effects are associated with improvements in glucose tolerance. We will further identify if intranasal OT may also increase SNS outflow to WAT by measuring changes in subcutaneous WAT temperature and uncoupling protein-1 protein expression in subcutaneous WAT relative to ABAT (pre- and post-intervention). If we find that intranasal OT increases EE and BAT thermogenesis in the NHP model, these studies will provide evidence for a role for BAT activation in how OT elicits weight loss in a translational model of diet-induced obesity. Moreover, these studies will identify intranasal OT as a potential treatment option to evoke weight loss and improve glucose tolerance in humans and direct future studies to address the extent to which intranasal OT may reverse obesity in humans by stimulating BAT thermogenesis.


Resource Core PROJECT SUMMARY / ABSTRACT The overarching goal of the Resource Core is to provide nonhuman primates in the quantity necessary to meet the needs of Somatic Cell Genome Editing (SCGE) awardees. The Resource Core is embedded within the established, comprehensive infrastructure provided by the California National Primate Research Center. Rhesus macaques and marmosets will be available for SCGE investigator studies to be conducted in the Genome Editing Testing Core and in order to generate data on efficiency, specificity, and safety of the technologies developed. The Resource Core will accelerate translation of genome editing technologies from studies in nonhuman primates into treatments for human diseases through the following Specific Aims: (1) Provide nonhuman primates and related capabilities for investigator projects, and (2) Receive, breed, and maintain reporter nonhuman primates. The Resource Core will provide at least 100 animals per year, which will include wild-type and reporter rhesus and marmosets. The Resource Core will ensure investigators have the quality and quantity of nonhuman primates necessary to meet their needs both through the provision of currently available animals (wild-type), and through procurement and expansion of reporter strains that will be provided to the Nonhuman Primate Testing Center from the SCGE Large Animal Production investigators. Paired with a robust Coordination Component and the Genome Editing Testing Core, and within the context of the infrastructure of the UC Davis California National Primate Research Center, the Resource Core will deliver the animals needed to comprehensively assess the promise of new genome editing technologies developed by SCGE awardees.


Short bowel syndrome (SBS) is characterized by severe malabsorption following massive small intestinal resection. Intestinal Lengthening Procedures (ILP) have been performed in children who fail to wean from Parenteral Nutrition (PN) within one year. Although ILP is associated with increased intestinal nutrient tolerance, most patients remain PN- dependent. Intestinal absorption has improved in adults with refractory SBS utilizing growth hormone (GH) and glutamine (GLN). Some adults have discontinued PN with this therapy, and the cost of medical care has been reduced. GH may act via insulin-like growth factor-I (IGF-I) to stimulate morphologic and functional adaptation. GLN is the primary fuel for small intestinal cells and may become depleted in catabolic states. GLN supplementation attenuates PN-induced mucosal atrophy in rats. The purpose of this protocol is to assess whether a 3-month course of treatment with GH and GLN modifies nutrient absorption in children who underwent intestinal resections.


Our understanding of the transcriptional regulation of cardiac development has improved greatly over the last decade, as many transcriptional regulators important for heart morphogenesis have been identified and characterized. It is also becoming clear that many of these regulators recruit a host of other protein cofactors that modulate gene expression in a cardiac specific fashion. One such family of transcriptional modulators important for cardiac development is the Friend of GATA (FOG) family. Both members of the FOG family have been shown to be expressed in the heart, interact with GATA factors, and be required for normal heart development. In this proposal, we demonstrate that both members of the FOG transcriptional modulator family contain a repression domain localized to their N-terminus that physically interacts with subunits of a large chromatin remodeling complex called the NuRD complex. Given their important role in gene regulation, it is not surprising that chromatin remodeling complexes might also be very important in regulating events during embryonic development. In this proposal, we outline research to further characterize these FOG/NuRD interactions and demonstrate their importance for mammalian cardiac development. Specifically, we propose to (1) characterize the subunits of the NuRD complex that interact with the FOG repression motif during cardiac development, (2) determine the functional significance of FOG-2/NuRD interactions during cardiac development, and (3) determine the functional significance of FOG-I/NURD interactions during cardiac development. The results of this work will lead to new insights into the transcriptional regulation of heart development, and may lead to a better understanding of the molecular basis of congenital and acquired heart disease.


The sodium/potassium adenosine triphosphatase (Na+,K+-ATPase) is the membrane protein that maintains the cell's Na+/K+ electrochemical gradient via the active transport of Na+ and K+ across the plasma membrane. Through coupling with other cation transport mechanisms, it is involved in the regulation of cell volume, differentiation and proliferation; ion/solute uptake in the kidney, liver, intestine; propagation of action potential of nerve, skeletal and cardiac muscles, modulation of synaptic action, and cardiac glycoside inotropy. It has also been implicated in cardiovascular hypertension, cardiac hypertrophy, brain edema, cystic fibrosis, and autohemolytic red cell membrane diseases. Its study is, therefore, important in basic and medical sciences. Molecular biology coupled with cell and protein biochemistry provides an incisive tool to the analysis of structure-function relationships and gene regulation of the Na+,K+-ATPase alpha- subunit. Recently we have characterized cDNA clones coding for three rat alpha-subunit isoforms alpha 1, alpha 2, and alpha 3). They are encoded by a multigene family that is differentially expressed in a tissue-specific and developmental manner. Of interest is their unique developmental expression in the heart. The alpha 1 isoform is constitutive; alpha 2 is feta/neonatal predominant; and alpha 3 is adult predominant. We have obtained the complete primary structure of alpha 1 and alpha 2 isoforms. This research proposes to study the molecular genetics of these cardiac Na+,K=-ATPase alpha-subunit isoforms, thus gaining insight into their role(s) in the heart during development and in pathology by aiming to: 1) complete the structural analysis of alpha 3 determining its complete primary structure, 2) establish a human tissue culture system to study the synthesis of the rat alpha-subunit isoforms, 3) define the functional differences of the presently isolated alpha 1, alpha 2, and alpha 3 isoforms, 4) assess the structure-functional significance of the putative ouabain- binding sites I and II via site-directed mutagenesis, and 5) study the Na+-pump's role(s) in cardiac development and cardiac hypertrophy by assessing the modulation of expression of the Na+- pump isoforms in rat by Northern blot, in-situ hybridization, and S1 nuclease mapping analyses. This research study will provide insight into the mechanisms of the Na+-pump's general and highly specialized functions. It will also pave the way for future research on precise mechanisms of Na+-,K+-ATPase structure- function relationships and gene regulation in cardiac development, hypertrophy, and cardiac-glycoside induced inotropy.


The Career Developmental Research Program exists to identify, support, and mentor candidates with promising independent careers in translational research. The Career Development Research Program also represents an opportunity to encourage new and established non-translational investigators, including women and minorities, to consider careers in translational research. In this manner the program ultimately serves as a means to expand translational research, as well as a source of translational projects and investigators for the SPORE Program itself.


DESCRIPTION: This application proposes the development of an interactive, video-based program targeted to children who do not engage in enough physical activity and their families. The program will help children learn how to be physically active, teach them basic behavioral skills needed to develop and maintain a physically active lifestyle and actively involve parents in the process. The program will be delivered by primary care pediatricians and will capitalize on their authority to motivate patient behavioral change. The combination of these elements represents a true innovation in physical activity promotion and primary care prevention of illnesses related to sedentary behavior. During Phase I, a prototype program will be developed to demonstrate the nature and quality of the materials, consisting of a rapid, self-administered screening measure for use by physicians, and videotape, parent guidebook and child- parent workbook materials for home use by families. The prototype will be tested with families and physicians for user satisfaction. Phase II will include a randomized clinical field trial of the programs efficacy. If effective in improving levels of child physical activity and teaches parents to support their children in this manner, it would represent a significant advance in child health promotion. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE


As the HIV epidemic moves into its third decade it is increasingly a disease of older women who have age-related co-morbidities, and concomitant prolonged exposure to the virus, to ART, and to the metabolic consequences of both infection and therapy. The WIHS in general, and the Brooklyn site in particular, are ideally suited to address the most salient questions related to an aging female cohort. We propose three projects that capture important aspects of the HIV epidemic in aging women. Each project contains specific studies with their own sets of aims and hypotheses. Project 1 focuses on neurocognition, metabolic, and vascular factors in HIV and includes three studies: 1. Adipose tissue, genetic susceptibility, and neurocognition; 2. The HIV-Neuroimaging Initiative; 3. Vascular factors and neurocognition. Project 2 focuses on aging biomarkers in HIV and includes two studies: 1. Frailty-Physical, functional and neurocognitive aspects of aging; 2. Reproductive aging and telomeres. Project 3 focuses on behaviors and implementation science approaches to understanding and promoting successful aging in HIV. It includes three studies: 1. Life course transitions and care engagement; 2. An intervention to increase adherence among women who have experienced childhood sexual abuse; 3. An intervention to enhance smoking cessation. The Brooklyn site, the largest WIHS site, has an exceptional record in cohort retention as well as participation in WIHS leadership and substudies. We further energize our activities for WIHS-V by expanding our cadre of co-investigators and biostatisticians to include those with expertise matched to the aforementioned projects and studies. Dr. Minkoff, who has been chair of the WIHS EC for over a decade, will be joined in a dual-PI leadership role by Dr. Deborah Gustafson, a neuroepidemiologist who has worked with aging cohorts in Sweden and Argentina. Among the many new collaborators are Dr. David Keefe, Chair of OB/GYN at NYU and one of the nation's leaders in reproductive aging, and Dr. Richard Havlik who has served at the NIH in research related to aging. We will also collaborate with the Alzheimer's Disease Neuroimaging Initiative, led by Dr. Michael Weiner. These and other new investigators will join an array of established site investigators, including Dr. Tracey Wilson who heads the WIHS behavioral working group, Dr. Howard Crystal, a neurologist who has a WIHS-linked R01 and many others, to assure the scientific productivity of the site. We have also established a biostatistical core, with members whose expertise corresponds to our project needs, including Drs. Lanza and Yang (Penn State) and Dr. Wilson (SUNY) for analysis of the behavioral projects; Drs. Gustafson (SUNY), Nalls (Molecular Genetics Section, NIA, NIH), Donahue (UCSD) and Weedon (SUNY) for neurocognitive, neuroimaging, and genetic analyses; and Dr. Wu (Penn State) for telomere analysis. Given our exemplary track record to date, in conjunction with an innovative fusion of new and seasoned investigators, and an invigorated statistical core, we look forward to a continued productive relationship with our colleagues in WIHS.


Despite recent advances in biomedical sciences, treatment options remain limited for patients suffering from stroke or other forms of brain injuries. Endogenous regenerative capacities in the brain hold therapeutic promise for nervous system repair after disease and injury. Throughout embryonic and postnatal development, neural progenitors/stem cells give rise to differentiated neurons, astrocytes, and oligodendrocytes. While these progenitors are relatively abundant during embryogenesis, they become restricted to specialized regions in the adult brain. In contrast to the well-investigated area of adult neural stem cell biology, relatively little is known about the cellular and molecular mechanisms controlling multiciliated ependymal cell (EC) function. As a primary cell type lining the adult brain ventricles, ECs form an important part of the lateral ventricular neurogenic niche, but their roles in postnatal/adult neurogenesis have long been controversial: whether ECs represent a neurogenic source remains unclear. Beyond ciliary movements, we know relatively little about basic ependymal biology. Using a combination of mouse genetics, biochemistry, and multiphoton live-imaging, we plan to elucidate the steps necessary to induce and control ependymal cellular plasticity. Our proposal is centered on basic molecular discoveries: we found that expression of the Foxj1 transcription factor, necessary for EC development from radial glial progenitors, is kept inherently unstable in mature ECs. Moreover, continued Foxj1 expression is required to prevent mature ECs from de-differentiating back into a progenitor-like state. We found that this surprising intrinsic cellular instability of mature ECs can be controlled by inhibitor of NF-?B kinase (IKK) signaling, a novel pathway distinct from cannonical NF-?B control. We plan to further explore these unexpected observations by determining the following: 1) whether the non- canonical IKK signal transduction pathway in ECs integrates extracellular signals to determine EC phenotypic stability; 2) what are the molecular mechanisms regulating Foxj1 transcription factor stability and proteolytic degradation - signaling cascades required to initiate EC de-differentiation; and 3) whether molecular events initiating de-differentiation of ECs represent the first steps toward their neurogenic transformation. Tackling the poorly-understood basic molecular and cellular mechanisms regulating EC biology, and using them as a basis for solving a long-standing problem on ependymal contributions to postnatal/adult neurogenesis, should further our understanding of CNS regenerative capacities in health and disease.


Oxygenated derivatives of arachidonic acid (AA) produced by inflammatory cells act as potent mediators of inflammation and allergy. This study proposes: 1) that vegetarians who consume little preformed AA have lower AA in serum and cellular complex lipids; and 2) dramatic reductions in serum and cellular AA levels as well as eicosanoid biosynthesis can be achieved when AA and its metabolic precursors, such as linoleic acid, are largely removed from diets of omnivore volunteers.


Inhibitory interneurons crucially control information processing in neuronal networks, but their vast molecular and anatomical diversity has made it difficult to dissect their functional roles. The cerebellar cortex with its relatively simple architecture and few neuron subtypes constitutes an ideal model circuit to study interneuron function. In the cerebellar cortex, mossy fibers (MFs) relay sensory information to granule cells (GCs) that send their axons to the molecular layer to excite Purkinje cells (PCs). As the only interneurons of the cerebellar input layer, Golgi cells (GoCs) are strategically positioned to control the propagation of sensory information to the cerebellar output layer. Spontaneously active GoCs inhibit GCs with two distinct time courses: rapid phasic inhibition that narrows the time window for excitatory input integration, and persistent tonic inhibition that controls the gai of incoming signals. Moreover, GoCs are thought to mediate the slow oscillations observed in the GC layer prior to the onset of motor behaviors. Strong electrical coupling between GoCs permits these oscillations that coordinate large assemblies of GCs. Although it is well established that GoCs crucially determine the flow of information within the cerebellar cortex, less is known about the mechanisms that orchestrate GoC firing, regulate GoC activity, and dynamically control GC excitability. Contrary to current beliefs in the field, preliminary data supports the hypothesis that active GoC dendrites enhance electric coupling. This proposal thus seeks to determine the cellular mechanisms that enable GoCs to fire synchronously using two-photon calcium imaging, patch clamp electrophysiology, voltage imaging and array tomography. Preliminary results also suggest that GoC activity dictates dendritic calcium concentration. The planned experiments will therefore examine the functional relationship between GoC activity and dendritic calcium dynamics, and determine the consequences for synaptic plasticity of excitatory PF and MF input. Preliminary data indicates that activation of metabotropic receptors on GoCs suppresses firing, and that this suppression is accompanied by a decrease in tonic inhibition of GCs. With the help of electrophysiology and optogenetics, this proposal will therefore test the hypothesis that dynamic modulation of GoC firing rate controls GC excitability and MF input integration. Completion of the outlined work will elucidate the mechanisms that control integration of sensory information by varying the activity of a single interneuron subtype in the cerebellar cortex. It will also extend our general understanding of how inhibition governs computational processes in neural networks.


The presence of food in the gut produces both direct and indirect effects on the growth of the gastrointestinal mucosa. Becasue ingestion of foodstuffs leads to increased desquamation of the epithelium and changes in luminal or local nutrition, direct effects on growth are produced when food moves through the gut after a meal. Indirect effects of a meal include release of GI hormones, increased secretions and increased activity of the autonomic nervous system. There may be a common mechanism which mediates both types of stimulation. The polyamines are a group of ubiquitously distributed polycations which are required for normal cell growth and proliferation. Preliminary studies have shown that they are involved in the regulation of mucosal growth. These data demonstrate that 1) there is a proximal to distal gut gradient of polyamine metabolism, 2) the aliphatic amine, ethylamine, has direct trophic effects in the gut, 3) the trophic response to intestinal obstruction has, as a required event, the induction of polyamine metabolism. These observation may explain 1) the proximal to distal gut gradient of villus height and crypt depth, 2) the role of amines, both aliphatic and polyamines, as regulators of GI mucosal growth, 3) the mechanism whereby the hormones of the gut exert their trophic in GI tissues, and 4) the as yet unexplained adaptive response of the gut to intestinal surgery. It is proposed to study and define the role of amines as regulators of mucosal growth. Initial experiments are designed to localize the areas of the gut in which amine metabolism is important to growth. It will also be necessary to study amine metabolism in the bacteria of the intestinal microflora as amines secreted from these cells may have direct effects on mucosal growth. It will be determined whether the hormones of the GI tract induce polyamine metabolism and whether this step is required for expression of their trophic effects. Finally, the role of amines as mediators of the adaptive response to intestinal surgery will be defined in various animal models of postsurgical hyperplasia and hypertrophy. The importance of a thorough understanding of GI growth is underscored by the realization that many diseases of the bowel, e.g., Zollinger-Ellison syndrome, chronic gastritis, pernicious anemia and the growth of cancers of the alimentary canal, are accompanied by disorders of gastrointestinal growth.


DESCRIPTION (Principal Investigator's Abstract): The O-alkyl ether linkage in glycerolipids was originally discovered as a significant component of fish oils. As with the n-3 fatty acids, fish from the ocean are thought to be highly enriched in ether lipids. The aim of this proposal is to develop a better understanding of the nutritional significance and possible health-related benefits of ether-linked lipids in the diet. In one portion of the planned work, the investigators propose to conduct a systematic quantitative analysis of the amount, type, and acyl composition of ether-linked glycerolipids in fish, meat, and poultry products consumed in the American diet. The second aim of this investigation is to evaluate the influence of dietary supplements of ether lipids and its fatty alcohol precursor on: a) the levels of ether lipids and their n-3/n-6 acyl composition in rat tissues, b) various biological parameters (growth, blood pressure, and cellular responses of neutrophils and alveolar macrophages to proinflammatory agents), and c) de novo enzyme activities for the synthesis of tissue ether lipids involved as cellular membrane components or as bioactive mediators. The experiments described in this application would be the first comprehensive dietary study ever conducted with the glyceryl ethers. Such information is important to obtain in view of the important role that potent alkyl type glycerolipids such as PAF (e.g., 10 -11M PAF can aggregate platelets), alkylglycerols, alkylacetylglycerols, and alkylacylglycerols play as modifiers of signal transduction events and as factors that can affect cellular differentiation and the activation of macrophages.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Previous workers have proposed high resolution models for the docking of the myosin heads on actin on the basis of combined crystallographic and electron microscopy data from isolated proteins (Mendelson and Morris, 1997 PNAS 94:8533;Holmes et al. 2003 Nature 425:423). Another group also claims that the first step in the force generation is associated with a rearrangement of the myosin-actin interface, followed by the lever arm tilt, and that it is temperature-dependent (Ferenczi et al. 2005 Structure 13:131). The goal of this work is to collect small angle X-ray scattering (SAXS) data from muscle that may be used to check in vivo the prediction of the models for the acto-myosin docking and whether there is a temperature-dependent rearrangement of the myosin-actin interface. For this purpose, the most sensitive reflection in the pattern is the 2.73nm meridional reflection arising from the regular repeat of the actin monomers along the actin filament, which changes its intensity upon myosin attachment to actin. Preliminary modelling has shown that the reflection intensity is little influenced by the lever arm tilt but it is highly sensitive to the relative axial position of actin and catalytic domain of myosin. 2D patterns will be taken from muscle at rest and during isometric contraction at different temperatures (4 to 17[unreadable]C) up to 0.5 nm-1 in reciprocal space, in order to collect the actin-based 2.73nm meridional reflection and the 5.9nm and 5.1nm layer lines, also influenced by myosin attachment to actin.


Alcohol (EtOH) abuse and dependence continue to be significant public health problems. Thus, a better understanding of their neurobiology will facilitate the development of interventions targeting prevention and/or treatment of these major health issues. Emerging evidence indicates that many aspects of EtOH and drug dependence involve changes in glutamate transmission. Because glutamate transporter 1 (GLT1) is responsible for the uptake of the majority of extracellular glutamate, we tested the hypothesis that increased GLT1 function would attenuate EtOH consumption in alcohol-preferring rats (P rats). After P rats had been chronically exposed to a free choice of EtOH (15 and 30%) for five weeks, they were administered ceftriaxone (i.p.), a beta-lactam antibiotic known to elevate GLT1 expression, for five consecutive days. We found that ceftriaxone-treated P rats showed a reduction in EtOH intake for the duration of treatment as compared to rats that received a saline vehicle. The long-term effects of ceftriaxone, however, are unknown. Here, we will test the long-term effects of ceftriaxone and other drugs (GPI-1046 and MS-153) known to activate GLT1 in the attenuation of EtOH intake at two different time points of chronic EtOH exposure in P rats. We will also investigate the effects of GLT1 activation in EtOH-drinking behavior in Wistar rats as comparison control groups. Our working hypothesis in aim 1 is that an increase in GLT1 function, via up- regulation or activation, attenuates EtOH consumption in both P and Wistar rats. The EtOH deprivation effect, which we will employ, has been used to assess relapse-like behavior in P rats. In aim 2, our working hypothesis is that an increase in GLT1 function during withdrawal periods will reduce relapse-like behavior when animals are re-exposed to EtOH. The lowest tested dose of ceftriaxone (25 mg/kg), which did not show an increase in GLT1 expression, was also effective in reducing EtOH intake. These findings suggest that ceftriaxone may have other pharmacological effects on GLT1 or may act by another mechanism (a functional increase may involve a change in one of several mechanisms). Thus, we propose in aim 3 to determine the signaling pathways involving ceftriaxone or GPI-1046 in up-regulation of GLT1 expression. Moreover, we aim to determine the molecular mechanisms of action of ceftriaxone, GPI-1046 and MS-153, which may involve activation of GLT1 function through phosphorylation of key proteins. The findings generated from this proposal will provide ample information about the role of GLT1 in the regulation of EtOH consumption and will pave the path toward finding a potential therapeutic target for alcohol addiction.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Structural studies of bacterial signal-transduction protein complexes Histidine kinases have been attractive new targets for drug design to circumvent drug resistance in Gram-positive pathogenic bacteria. Our research is aimed at structures and functions of a multi-domainal, membrane-spanning histidine kinase and its response regulator from an essential two-component signal transduction system. Proposal: Histidine kinase of Gram-positive pathogenic bacteria Resistance to common antimicrobial drugs is a global phenomenon, and an increase in resistance has been observed for every major group of bacterial pathogens. There is an obvious need for identification of novel targets for development of new antimicrobials. Two-component systems are ubiquitous in bacteria and are central elements of the intricate regulatory pathways utilized by pathogenic bacteria to control a wide range of cellular processes including expression of virulence factors and resistance to certain antimicrobial. A paradigmatic two-component system consists of two proteins: sensor kinase and response regulator. The sensor kinases monitor environmental signals, and modulate functions of response regulators through phosphotransfer reactions. The response regulators usually contain an N-terminal receiver domain and a C-terminal DNA-binding domain. The receiver domain receives signals from histidine kinase through phosphorylation, which enables the DNA-binding domain to act as a transcription factor to regulate expression of certain genes. YycF (response regulator):YycG (histidine kinase) system is highly conserved in the low G+C Gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and Listeria monocytogenis, many of which are human pathogens and pose significant threat on public health in the U.S. and worldwide. The YycF:G system is the only two-component system essential for cell viability known to date, and has been suggested as a prime antimicrobial target and subjected to small-molecule inhibitor developments. The YycF:G system has been implied in cell wall biogenesis. However, it is not clear what signal YycG senses and how the signal is relayed in the YycF:G system.


The use of antiepileptic drugs (AEDs) in pregnancy and early infancy poses special challenges and concerns, as even transient exposure to certain drugs during CNS development may impede subsequent neuronal and synaptic development. Several AEDs, including phenobarbital and phenytoin, when given in therapeutically relevant doses to rats during the early postnatal period, cause a pronounced increase in apoptotic cell death in several brain regions. Other drugs such as lamotrigine (in low or moderate doses) and levetiracetam are devoid of this effect. We have recently discovered a single exposure to phenobarbital, at the time of peak vulnerability to the propapoptotic action (postnatal day 7 in the rat), resulted In the suppression of the normal, developmental increase in the frequency of inhibitory post-synaptic currents (IPSCs) recorded via patch clamp from striatal medium spiny neurons (MSNs) in slices taken between postnatal day 10 and 14. We have also identified impairments in adult rotorod performance in rats treated at P7 with a proapoptotic dose of phenytoin, a reduction in prepulse inhibition (a measure of sensory-motor gating) and a reduction in seizure threshold to pentylenetetrazol In animals treated in the first postnatal week with phenobarbital. Each of these behavioral assays are sensitive to damage or altered function in the striatum. The proposed experiments will follow up on these exciting preliminary findings to determine if there is a consistent and predictive (potentially causative) relationship between AED-induced neuronal apoptosis and impaired maturation of I PSC frequency in the striatum and/or adult behavioral toxicitiy. This will be assessed by comparing AED treatments that are proapoptotic with those that avoid this toxicity. We hypothesize that AEDs that do not cause cell death will not cause impaired maturation of IPSC frequency in striatal MSNs, nor will they result in impaired rotorod performance, reduced seizure threshold, and impaired prepulse inhibition ofthe acoustic starle response. Moreover, the extent to which a neuroprotective treatment can prevent impaired maturation of IPSC frequency in striatal MSNs and/or behavior will be evaluated to test the hypothesis that neuronal death is necessary for this adverse functional outcome. The results ofthe proposed experiments will allow us to better understand the potential functional outcomes of exposure to AEDs in late gestation or early infancy, and the extent to which induction of excessive neuronal death is a valid marker of subsequent functional impairment. Furthermore, it will identify strategies to avoid deleterious functional sequelae of AED therapy during critical developmental periods. GOALS FOR KIRSCHSTEIN-NRSA FELLOWSHIP TRAINING AND CAREER I am seeking this fellowship tp support my dissertation research in Dr. Karen Gale and Dr. Stefano Vicini's laboratories, investigating the impact of neonatal anticonvulsant drug exposure. My goal for dissertation research is to develop a broad skill set of experimental techniques that will allow me to address scientific questions at the level of molecules, cells, networks and behavior. The complementary expertise of my mentors provides such exposure. A limited portion of this support is also requested to allow me to continue to teach (present lectures to graduate and undergraduate students, and continue to direct a course entitled, "Diseases and Disorders of the Brain". A key goal of my training is to complete and publish a series of focused studies on outcomes following drug exposure during development. My long-term goals are predicated on the broad training I am receiving at Georgetown. Following Georgetown I will seek post-doctoral training and then I hope to develop an independent, productive research program at an academic institution where I can ask and answer questions of interest at multiple levels of function, to maintain an active Involvement in teaching and mentoring, and to contribute to scientific discourse.


The broad objectives of this proposal are to understand the metabolic role and enzymatic mechanism of one of the most abundant folate enzymes, 10-formyltetrahydrofolate dehydrogenase (FDH). FDH converts 10-formyltetrahydrofolate to tetrahydrofolate in an NADP-dependent dehydrogenase reaction or in an NADP-independent hydrolase reaction thus regulating two of the major folate pools. It has been also proposed that the enzyme serves as an intracellular folate depot protecting folate coenzymes from oxidative degradation. The enzyme is a natural fusion of two unrelated proteins. The amino-terminal domain bears the folate-binding site and functions as a hydrolase. The aldehyde dehydrogenase like carboxyl-terminal domain works as the catalytic tool in the dehydrogenase reaction when the two domains are combined in one polypeptide. A hundred residue intermediate domain is a linker between the two functional domains required to bring them together to catalyze the dehydrogenase reaction. It is hypothesized that the hydrolase reaction of FDH although by itself is not of physiological significance, is an important and essential part of the FDH dehydrogenase mechanism. The FDH dehydrogenase mechanism is a combination of two sequential reactions, the hydrolase and aldehyde dehydrogenase. During the dehydrogenase reaction transfer of an intermediate product from the hydrolase domain of FDH to the aldehyde dehydrogenase domain takes place. The intermediate domain is crucial to bring two functional domains in correct orientation to allow the transfer. Another part of this project is based on the hypothesis that one of the major roles of FDH is to regulate de novo purine biosynthesis by controlling 10-formyltetrahydrofolate levels. The recent findings that FDH is highly down-regulated in carcinogenesis, apparently due to increased demand of cancer cells for purines, make the protein an important potential target in anticancer chemotherapy. The following specific aims are proposed to test the hypotheses. (1) To determine the role of the intermediate domain in the enzyme mechanism. (2) To characterize the folate binding site and to evaluate the hydrolase mechanism of FDH. (3) To crystallize and to resolve the crystal structure of the FDH individual domains and the full- length protein. (4) To elucidate the role of FDH in cellular metabolism. Site-directed mutagenesis and protein design approaches, enzyme activity assays, binding studies, crystallographic and immunochemical methods, mammalian cell expression, antisense oligonucleotide techniques, purine and folate assays will be used to achieve the goals of the project. The well known role of folate in prevention of megaloblastic anemia, vascular disease, neural tube birth defects and cancer make these studies particularly relevant.


The search for biological correlates of endogenous depression (ED) has been on for several years. Anomalous regulation of the hypothalamo-pituitary-adrenal (HPA) axis has been implicated using the Dexamethasone Suppression Test (DST). While there is active debate concerning the clinical usefulness of the DST, there is general agreement that some ED patients show defective HPA regulation, especially as seen in their baseline plasma cortisol and the lack of suppression after dexamethasone (DEX). In our previous funding period, we studied the pituitary component of the HPA axis in ED patients by measuring the peptide products from the ACTH/endorphin precursor. We have shown substantial pituitary regulation defects as reflected by plasma beta-endorphin (BE) assays in ED patients when compared to psychiatric controls. These studies revealed a partial concordance between BE and cortisol dysregulation. The combination of both measures after DEX reveals abnormalities in 70% of the ED patients. Based on the peptide and steroid measures we are proposing the possible existence of 4 subgroups of ED patients with different patterns of HPA dysfunction. The present proposal is aimed at testing these notions and further exploring the nature of the HPA defect. Studies on the effect of age, sex and time of testing in normals and psychiatric subjects will further explore variables contributing to the different patterns of response post DEX. Studies with CRF-induced release of ACTH and BE, metyrapone, cortisol fast feedback and biochemical characterization of the released peptides will be conducted to elucidate the locus and nature of the dysregulation. Finally, we will study patients in remission whose DST has normalized to determine if other HPA correlates reveal continuing abnormalities.


The Northeast Center for Research to Evaluate & Eliminate Dental Disparities (CREEDD) is committed to the elimination of oral health disparities through research, training and action. Our efforts will build on a strong foundation of early and continuous community engagement, community-based research interventions, integrated training and career development, broad dissemination of research findings, and targeted health policy initiatives. Based at Boston University, the CREEDD will maintain its current regional approach, with partnering institutions in Massachusetts, Maryland and Ohio. A diverse, multidisciplinary and multi-institutional team will implement community-based intervention research projects aimed at reducing early childhood caries (ECC). Our organizing theme is engaging non-dental care providers in oral health promotion, and extending the venues for oral health promotion to non-clinical care settings directly in health disparity communities. The two major community-based intervention research projects will be: Project #1 (D&IR Project) - Partnering with Community Health Centers to Prevent ECC (D&IR Project) Physician-delivered interventions, targeted at children aged 1 to 3 years old, with include fluoride varnish applications, patient-centered counseling, and systems-level changes to clinical information systems and clinical prompts used in order to include age-appropriate, oral health-specific anticipatory guidance items. Project #2 - Oral Health Advocates in Public Housing Resident Health Advocates (RHA), trained peer health advocates, will incorporate motivational interviewing and community oral health promotion activities into their ongoing health promotion efforts targeting caregivers with children from birth to 5 yrs old, aimed at caries risk reduction and lowering incidence of ECC. We will use a community-based cluster randomized study design, in Project #1 with community health centers (CHC) as the unit of randomization, with CHC located in Boston, Maryland, and southeastern Ohio; and in Project #2 with individual public housing developments in Boston as the unit of randomization. [unreadable] [unreadable] [unreadable]


Targeted dendrimer-based nanodevices have shown excellent promise in both in vitro cell culture and in vivo animal studies as cancer therapeutics. However, each device must be custom synthesized for a particular set of targeting molecules, imaging agents, and desired therapeutics. We propose a unique solution to this limitation by developing single function dendrimer modules linked by complementary oligonucleotides. This allows targeting, imaging, and therapeutic dendrimers to be combined into multifunctional therapeutics simply by heating mixtures of these agents above the annealing temperature of the oligonucleotide duplex. The project will consist of five specific aims. Specific Aim 1 will involve the design and synthesis of complementary oligonucleotides conjugated to poly(amido)amine dendrimers or dendrons to achieve the desired structural topologies. The ability to construct complex devices will be assessed using well-defined targeting molecules (folic acid, her2 antibodies and RGD peptides), drugs (methotrexate, Taxol, cis-platin and doxorubicin), imaging agents (Gadolinium chelators and fluorescent dyes). Specific Aim 2 will characterize the self-assembled nanodevices using techniques including PAGE, HPLC, CE, Mass Spectroscopy, NMR, AFM, and NSOM. Specific Aim 3 involves testing the DNA-linked nanodevices for binding and internalization in vitro;the avidity and specificity of binding will be examined using CD, differential calorimetry and Biacore analyses. Devices carrying therapeutics will be tested for effectiveness at inducing cell death, and all devices will also be tested for inherent cytotoxicity. Specific Aim 4 employs animal models to assess the effectiveness of the dendrimer linked therapeutics to treat tumors in vivo. In addition, the biodistribution of the therapeutics will be assessed using radiolabeled material and a novel fiber optic probe that uses two-photon excitation with femto-second pulses. Finally, under Specific Aim 5 we will work with the NCI nanoparticle characterization lab in Frederick to make the materials developed in this program available to other investigators. This platform has the potential to revolutionize cancer therapeutics and facilitate "personalized medicine." Lay description: We are designing a method and the tools for developing targeted cancer drugs that can be tailored to the needs of an individual patient. The physician can select various components and the components are then linked together like "tinker toys" to make a personalized medicine. This medicine would selectively target only the cancer, thereby avoiding the nausea, hair loss and illness caused by regular cancer chemotherapy.


We request continued support for a NINR-funded study on the effectiveness of an innovative parent training program (The Chicago Parent Program; CPP) developed in collaboration with a parent advisory council and used in childcare centers serving low-income families of color. The current study has been affected by issues common in prevention trials: low enrollment and attendance rates and the inability to include Spanish- speaking Latino parents. Historically, scientists have addressed these challenges by excluding non-English- speaking Latinos and instituting costly and unrealistic subject incentives, strategies that hinder the growth of prevention science for people of color and its translation to health care policy. The primary goal of the proposed study is to build upon the current grant by testing the effectiveness of two strategies for increasing participation rates among Latinos and maximizing the effects of preventive parent training for low-income families of color using methods that can be generalized beyond the research context. Using a randomized experimental design (n=450), we will test the effectiveness of (1) a Spanish-language version of the CPP and (2) discounting parents' childcare co-pay costs contingent upon attendance. Eight day care centers will be matched and randomly assigned to control and experimental conditions: (1) a 12-week intervention condition in which barriers to attendance are reduced but no financial incentives for attendance are offered (Standard), (2) a discount co-pay incentive condition in which barriers to participation are reduced and parents receive a discount on their weekly childcare co-pay bill contingent upon attendance (Discount Co- pay), and (3) a no-intervention control condition. Participation will be defined by enrollment rate, attendance, and parent engagement in the intervention. CPP effectiveness will be examined using multiple parent and child outcomes assessed up to 1 year post-intervention. The cost-effectiveness of using a discount co-pay strategy compared to the standard condition will also be examined. Relevance: The goals of this study are to use a real world approach to increase participation rates in a prevention intervention designed to promote positive parenting and mental health among low-income ethnic minority families of 2-4 year old children. Data from this study would lead to improved outcomes for parents and children during the first 5 years of life. [unreadable] [unreadable] [unreadable] [unreadable]


Histone deacetylases (HDACs) are vital regulators of fundamental cellular events, including cell cycle progression, stem cell functions, cell fate determination, cell differentiation, and the pathogenesis of many diseases. As such, it is not surprising that protein acetylation is central to human diseases, as diverse as neurodegenerative disorders, cardiac hypertrophy, cancer, HIV infection, and more generally the process of aging. More importantly, small molecule HDAC inhibitors and activators are currently in clinical trials for the treatment of leukemia and lymphoma, solid tumors, neuromuscular disorders, metabolic disorders and other diseases. In addition, the Sirtuins, a subclass of HDACs, were fingered in yeast genetic studies as regulating lifespan, and these enzymes might be targeted by resveratrol, one of the components in red wine that has been linked to increased lifespan in humans. Therefore, a thorough understanding of HDACs is required, not merely for understanding the regulation of chromatin structure, gene regulation and protein function, but also because HDACs are intimately involved in normal and abnormal cellular processes that greatly impact human health. With the identification, isolation, cloning and functional characterization of 18 human HDACs (HDAC1- 11 and SIRT1-7) and many acetyltransferases in the past decade, the coming years will see a continued dramatic expansion in our knowledge of the biological roles of HDACs and protein acetylation. As the only conference dedicated to this field, this biannual meeting plays an essential role in bringing together approximately 44 basic and clinical scientist speakers to exchange information and develop new therapeutic avenues with approximately 120 participants from around the world. A primary objective is to transfer knowledge between basic academic researchers, clinical scientists, and pharmaceutical scientists to create efficiencies in understanding how they control human health and how these activities can be harnessed to fight a diverse slate of diseases. A second objective is to foster the development and interests of younger investigators to help support their career development. To accomplish these objectives, the meeting venue houses meeting space, dining, and rooms so that participants have ample time to move from formal presentations to informal brainstorming and collaborative discussions. In addition, the morning and evening oral presentation sessions include 2-3 talks from junior scientists selected from the submitted abstracts, which is important for career development and to encourage the next generation of HDAC scientists. There will be an emphasis through both the scientific and social programs on creating a global HDAC community. This meeting will be particularly timely because data from on a new generation of HDAC and Bromodomain drugs will be presented. Therefore, support is requested for the 4th biannual conference on the biology and therapeutic targeting of HDACs and Sirtuins, and their role in aging and disease to be held at Il Ciocco, Lucca, Italy, August 18-23, 2013 in conjunction with FASEB.


The molecules that control Retinoblastoma (Rb) phosphorylation called the Rb pathway consisting of D-type cyclins, cyclin dependent kinases (cdks) and cdk inhibitors (cdkis) as well as Rb itself are regularly disrupted in most cancer types including breast cancer leading to excessive phosphorylation (hyperphosphorylation) of the Rb protein. Hyperphosphorylation of Rb is known to promote proliferation and block apoptosis. Treatments that inhibit cdk activity have begun to exhibit promise in the clinic in several cancer types. Although Rb can be phosphorylated on 15 amino acid residues, very little is known about the contribution any individual phosphorylated amino acid has on the function of Rb in proliferation and apoptosis. In this project we propose to undertake the first investigation of Rb phosphorylation in proliferation and apoptosis utilizing a model of breast epithelial cells using three-dimensional cultures. This model system is employed to recapitulate the physiological context in which breast epithelial cells regulate cellular processes. We propose to target Rb hyper- phosphorylation by using a novel method of activating Rb-specific phosphatase activity in breast epithelial cells grown in 3D cultures. In this way we can determine the effect of dephosphorylation of Rb on proliferation and apoptosis in these cells. In addition, we will perform site directed mutagenesis of each Rb phosphorylation site to either alanine or glutamic acid to block phosphorylation or mimic phosphorylation at each site and evaluate the effects on proliferation and apoptosis in breast epithelial cells grown in the 3D culture model. Finally, we will clarify the functional significance of complex formation between Rb and the pro-apoptotic protein Bak that is regulated by Rb dephosphorylation in apoptosis. Thus, the proposed studies will elucidate the role of specific Rb phosphorylation sites involved in the regulation of proliferation and apoptosis in breast cancer and will yield useful information that could inform the development of future therapies that target Rb phosphorylation in cancer.


SUMMARY This study focuses on understanding the psychosocial impacts of genetic causal attribution in the epilepsies, a set of common neurologic disorders with significant psychosocial dimensions, including stigma, discrimination, reduced rates of marriage and reproduction, and reduced quality of life. Genetic research and clinical genetic testing in the epilepsies are advancing rapidly, and plans are underway to develop precision medicine approaches for clinical care. These developments reflect a strong emphasis on genetic causes of epilepsy, which is being communicated to patients in multiple ways. For some people with epilepsy, this emphasis will be welcome; for others, psychological and behavioral responses may be more complex. However, little is known about the psychosocial impact of genetic causal attribution on people with epilepsy. In previous research, we studied a unique set of >100 families containing multiple individuals with epilepsy, to assess their preferences for genetic testing, their beliefs about genetic causes, and the associations of these beliefs with psychosocial outcomes. We now propose to build on these studies to include a more diverse and representative sample of ~600 adults treated for epilepsy at our institution. These patients are being offered whole exome sequencing (WES) as part of a research program sponsored by Columbia's Institute for Genomic Medicine (IGM), and we will take advantage of this opportunity to study patients' decisions to participate or not participate in WES and the impact or receiving or not receiving genomic results. In Aim 1, we will assess the relations of genetic causal attributions to other illness perceptions (e.g., severity, persistence, treatability), and psychosocial (e.g., felt stigma, depression, anxiety) and behavioral (medication self-management) outcomes. Data analyses will test consistency with theoretical expectations under models of attribution theory, ?genetic optimism,? and genetic essentialism. In Aim 2, we will investigate the ways in which receiving or not receiving genomic results may influence illness perceptions, psychological well-being, and health-related behavior, through in-depth qualitative interviews with patients who receive epilepsy-related findings, receive secondary findings, have WES but do not receive findings, and elect not to have WES. In Aim 3, we will develop mechanisms to translate our findings into improvements in education about the role of genetics in the epilepsies. The results will clarify the beliefs and attitudes about genetics that underlie any adverse impacts of geneticization in epilepsy and provide a framework for education programs designed to ameliorate them, in order to maximize the benefits of genomic medicine.


Purification of individual chromosomes by flow sorting is often a first step in defining the structure of normal and abnormal human genes. The polymerase chain reaction drastically reduces the amount of sample required for isolation of chromosomal DNA, making it feasible to develop simpler, less expensive chromosome sorters. The performance of an instrument using air-cooled ultraviolet (UV) and blue helium-cadmium (He-Cd) laser light sources in flow cytometry of chromosomes stained with DAPl/olivomycin or Hoechst 33258/chromomycin A3 will be evaluated to determine whether the system can resolve the human karyotype as well as can existing commercial apparatus. As an alternative, several nucleic acid fluorochromes not previously applied to chromosome analysis and potentially usable in a large number of existing flow sorters will be examined as adjuncts or alternatives to stains presently used for bivariate flow karyotyping.


Retinoic acid (RA) is a metabolic derivative of vitamin A (retinol) that functions as a signaling molecule. RA is essential for eye development, but its action is poorly understood. RA signaling occurs when retinol is metabolized to RA which serves as a ligand for nuclear RA receptors that regulate gene expression. The enzymes controlling synthesis of RA during embryogenesis are now under investigation and such studies are providing new information on the mechanism of RA action during eye development. Studies on mouse embryos have demonstrated the existence of three retinaldehyde dehydrogenases differentially expressed in the eye that synthesize RA, i.e. RALDH1, RALDH2, and RALDH3. Investigations of Raldh1, Raldh2, and Raldh3 null mutant mice have uncovered eye defects, and further studies of these mice are beginning to reveal the mechanism of RA action. As the three Raldh genes are conserved in mice and humans, the null mutants we have developed are excellent mouse models for understanding the mechanism of RA action during human eye development. Evidence exists suggesting that RA deficiency caused by dietary vitamin A deficiency may be linked to the human eye defect known as ocular coloboma. The genetic studies proposed will provide information relevant to treatment of human eye diseases whose etiology involves genetic deficiency in RA synthesis and/or dietary vitamin A deficiency: We have found that the location of RA synthesis undergoes dynamic spatiotemporal changes during eye development, and that the location of RA action changes in synchrony. Raldh2/Raldh3 double mutant mouse embryos develop an optic vesicle, but this structure lacks RA synthesis and fails to invaginate ventrally to form the optic cup. Raldh3 null mutant embryos develop an optic cup but they display defects in closure of the optic fissure (coloboma). Raldhl null mutant embryos lack RA synthesis in the dorsal retina, but eye defects are not observed. However, Raldh1/Raldh3 double mutants display excessive invasion of perioptic mesenchyme anterior to the retina, thus revealing a function for Raldhl that is normally compensated by Raldh3 (and vice-versa). These findings have led to the hypothesis that RA controls eye morphogenetic movements rather than dorsoventral patterning of the retina as previously thought. The overall goal of this project is to determine the mechanism of RA signaling during eye development, particularly the gene networks regulated by RA in the eye. We will test the hypothesis that RA regulates eye morphogenetic movements of both the retina and the surrounding perioptic mesenchyme. These studies will be performed genetically using Raldh compound null mutant mice that are unrescued or rescued by various genetic or pharmacological methods. Specific investigations will focus upon: (1) RA control of cell shape and cell adhesion during optic cup formation; (2) RA-FGF antagonism during optic cup formation; (3) RA control of perioptic mesenchyme invasion following optic cup formation. [unreadable] [unreadable] [unreadable]


Polymorphonuclear neutrophils must respond to multiple exogenous stimuli to proceed through the complex transformation from quiescence to activated phenotypes. The plasma membrane is the interface where this transformation must be initiated. Recent work has shown that plasma membranes are heterogeneous structures containing discrete regions, "lipid raft microdomains", enriched in glycosphingolipids, cholesterol, lipid-anchored receptors, and signaling molecules. Selective partitioning into lipid rafts appears to be a key mechanism for imposing organization on the distribution of proteins in the plasma membrane, thereby compartmentalizing discrete functions within the plasma membrane and, in essence, creating a syntax that translates otherwise dissociated signaling effectors into a meaningful language of signal transduction. The central hypothesis underlying this proposal is that the composition, structural integrity, and spatial distribution of lipid raft microdomains critically regulate key processes in neutrophil activation. Our preliminary studies of human neutrophils have shown that lipid rafts: i) spatially compartmentalize and selectively regulate neutrophil activation signaling, ii) selectively influence cellular responsiveness to proinflammatory agonists, and iii) regulate signal propagation during cell migration. The long-term objective of this project is to determine how the function and composition of lipid raft microdomains regulate the antimicrobial and proinflammatory functions of neutrophils. The short-term objectives are to: i) determine the mechanisms by which lipid rafts influence agonist-specific signal transduction, ii) determine how lipid rafts regulate the production and release of reactive oxygen intermediates, iii) determine how lipid rafts influence the expression, distribution, and function of beta2 integrins, and iv) determine how lipid rafts control cellular polarity and calcium signaling during non-directional and chemotaxin-driven migration. Hopefully, targeting the function of lipid raft microdomains will engender new therapeutic strategies for enhancing antimicrobial defenses and suppressing the deleterious effects of acute inflammation.


Project Summary Most people living with HIV (PLWH) in Malaysia also have an opioid use disorder (OUD), yet only a minority receive ART and OUD treatment, resulting in increased mortality and HIV incidence. The proposed pragmatic, multisite, implementation and effectiveness research will evaluate a strategy to improve HIV treatment outcomes (increased rates of patients on ART with virological suppression, improved treatment retention and ART adherence) for PLWH with OUD. Engaging 4 large regional HIV/AIDS treatment centers in Malaysia, with a stepped rollout of the study protocol across the study sites, offset by one year with a random order of the sites initiation, the study will evaluate barriers and facilitators for implementation of improved care model and will evaluate the comparative effectiveness of the model in a clinical trial. At each of the study sites, individuals testing HIV positive who also have OUD (n=4x70) will be enrolled to receive concurrent ART and MMT based on the usual care standards. Their patient level outcomes will be compared with individuals meeting the same inclusion criteria (n=4x70) and treated under the proposed improved model (post implementation evaluation). The usual care standard will consist of provision of ART and medical care for HIV and other medical HIV co- morbidities provided at the HIV/AIDS treatment center with an expedited and facilitated referral to a methadone maintenance treatment (MMT). The improved care model will include the usual care supplemented by continuing education and coaching of medical staff at HIV/AIDS and MMT clinics and by provision of additional peer-based counseling intervention focused on behavioral skills and strategies that patients can learn and master to achieve uninterrupted, long-term ART treatment participation while continuing OUD recovery through MMT. The primary outcome measure, rates of patients with virologic suppression (< 20 copies/mL) in the two care models will be assessed at 24 weeks. The secondary outcomes, also followed for 24 weeks, will include ART adherence measured by objective measures (tenofovir dried blood spots, clinic records) and self-report; decreased illicit opioid use measured by rates of opioid negative urine toxicology results and self-report; and improvements on other health-related and functional status outcomes. A statistically significant effect on the primary outcome and clinically meaningful effects on secondary outcomes favoring the improved care model are hypothesized. Concurrently at each study site, using implementation science mixed methods research tools and engaging key local stakeholders (treatment providers, patients, their families, and community activists), and evaluating clinical and healthcare data, the study will assess existing barriers (organizational, personnel, and community level factors) and uncover available resources and facilitators for a successful implementation of the improved care model. The research will provide critically important evidence for implementation of effective Seek-Test-Treat, and Retain models for PLWH and OUD throughout Malaysia and inform healthcare policy in other low to middle income countries and regions with limited healthcare resources.


The proposed studies are designed to define the alterations in tubular sodium, chloride and bicarbonate reabsorption that occur when the number of functioning nephrons is reduced. Using micropuncture and microanalytic techniques in the rat, we will determine the relative contributions of changes in proximal and distal reabsorption in maintaining sodium balance and extracellular fluid composition in this model of renal insufficiency. These studies will be carried out for the first time in animals in which the surgically induced plasma losses are replaced to maintain volume status and renal function at levels seen in awake animals. The relative roles of alterations in single nephron GFR, aldosterone, peritibular physical factors, and "natriuretic hormone" in producing the alterations sodium transport will be systematically examined. With respect to tubular bicarbonate transport in the remnant kidney, experiments will be conducted to examine the role of changes in filtered load chronic acid-base disturbances, and parathyroid hormone in producing the observed changes in reabsorption. These studies should provide new insights into the nature of the adaptive response of the kidney to nephron loss.