Treatment for head and neck cancer (HNC) is arduous and debilitating. As a result, up to 40% of patients develop depression which is rarely recognized or treated. Untreated depression in cancer patients lengthens hospital stays, and reduces adherence with medical treatment, quality of life, and survival. Patients with HNC typically develop depression within the first 3 months of their diagnosis. Developing depression can lead to interruptions or delays in cancer treatment which in turn may diminish the overall prognosis. Depression can also lead to suicide;patients with cancers of the larynx and tongue account for 2 of the 3 highest suicide rates in cancer patients. We are proposing a groundbreaking approach to the problem of depression in HNC. Patients with HNC have the greatest risk for depression in the several months after diagnosis and during treatment for their cancer. We seek to prevent major depression before it can develop by initiating prophylactic treatment with the antidepressant citalopram soon after the diagnosis of HNC is made, thus potentially avoiding many serious sequelae including treatment interruption or delays. Depression also has an enormous impact on quality of life in patients with HNC. Additionally, an overwhelming majority of HNC patients use alcohol and tobacco, and despite adverse effects on prognosis, it is often difficult for them to decrease use of these substances. We will therefore evaluate whether prophylactic use of citalopram can preserve quality of life during and after treatment for HNC and reduce alcohol and tobacco use by these patients. We are therefore proposing a randomized, double blind, placebo-controlled trial investigating the prophylactic utility of the antidepressant citalopram in nondepressed patients who are about to begin treatment for newly diagnosed or recurrent head and neck cancer. We will evaluate whether such prophylactic treatment can significantly reduce the development of major depression, improve adherence to cancer treatment, preserve quality of life and reduce tobacco and alcohol use. This proposal has considerable relevance to public health in that it not only tests whether depression can be prevented in patients with HNC during treatment but also examines whether this intervention can also improve timely completion of the cancer therapy. Additionally, we will examine whether the quality of life of such patients can be better preserved during treatment and whether alcohol and tobacco use can be diminished. If successful, this approach may be applicable to other medical illnesses that have high rates of comorbid depression.


DESCRIPTION: (Applicant's abstract) Long-term potentiation of synaptic transmission in the hippocampus is a widely used model for studying the synaptic plasticity which underlies learning and memory. Most previous studies have focused on a form of LTP which requires the NMDA type of glutamate receptor. This proposal is to study a form of LTP which does not require NMDA-Rs, but instead requires voltage-dependent calcium channels (VDCCs). In both forms of LTP, calcium entry into postsynaptic neurons, through either NMDA-Rs or VDCCs, is required for induction of the potentiation. While much is known about the subsequent cellular events underlying maintenance of NMDA-R dependent LTP, little is known about the maintenance of NMDA-R independent LTP. It is unclear if cellular processes following induction are shared by the two forms of LTP, or if a distinct set of processes underlies each form of LTP. The major aim of this proposal is to define the signaling pathway leading from the induction of NMDA-R independent LTP, through the maintenance of this LTP. Multiple components of potential signaling pathways will be investigated. These include the ion channels., neurotransmitter receptors, second messengers, and the protein kinase targets of these second messengers. This proposal also aims to determine the roles played by metabotropic glutamate receptors in NMDA-R independent LTP. These roles may include: modulation of LTP induction by regulation of postsynaptic membrane potential, inhibitory interneuronal circuitry, or presynaptic neurotransmitter release, as well as regulation of second messenger systems. These experiments will be done using an in vitro brain slice preparation of rat hippocampus (area CA1). Synaptic transmission and LTP will be monitored by electrophysiological measures (extracellular field potential and whole cell/patch clamp recordings of evoked synaptic responses). Electrophysiological and pharmacological manipulations will be used to test possible components of the signaling pathway for NMDA-R independent LTP.


An important limitation in animal models of cognitive learning has been the difficultly in specifying how animals code (or represent) events. Coding processes are particularly important in the development of stimulus classes. Recent evidence suggests that pigeons (and other animals) can form stimulus classes from originally unrelated stimuli. The characteristic of members of such stimulus classes is that they are able to substitute for one another in much the same way that arbitrarily-designated spoken and written words can be substituted for objects and actions in human language. The proposed research with pigeons will use delayed-matching procedures in which pigeons have been shown to develop single-code-default strategies (one of two samples is coded - the other comparison is chosen by default). Evidence for single-code-default strategies restricts the class of possible codes and allows one to gain access to the underlying codes. The immediate goals of the present research are (a) to examine the coding processes used by pigeons, (b) to determine the conditions under which these coding processes are used, and (c) to identify the nature of the underlying codes. In the first section of this proposal we will examine commonalities in the coding of present/absent samples. In the second section we will apply similar strategies to the analysis of duration sample coding. Finally, we will examine the similar coding of samples that are related only through their association with a common comparison. The long-term goal of this research is to be able to specify the bases of stimulus class formation by animals so one can determine which components might be missing from humans who fail to acquire the stimulus classes needed for language development.


This is a request for support of a Gordon Research Conference on Proteolytic Enzymes and Their Inhibitors. The objective of this conference will be to bring together thirty-two international authorities in this field and to have them discuss current work, new directions and controversial issues before a critical audience of 100 or more working experts from academic, industrial, and governmental institutions throughout the U.S. The subject is of great importance for widely diversified groups of scientists ranging from protein chemists and crystallographers to immunologists, hematologists, rheumatologists and biomedical investigators interested in inflammation, connective tissue disease, emphysema and cancer. Proteases and their endogenous inhibitors play a central role in the control of immune responses (complement pathway), blood clotting and fibrinolysis, and the generation of peptide mediators of inflammation and blood pressure regulation. Proteases are also key regulators of sperm penetration, ovulation and embryo-implantation and perhaps of cancer cell invasiveness and metastasis. They are also instrumental in the post-translational processing of peptide hormones, including neuropeptides, and of collagen and other connective tissue macromolecules. All of these aspects, from molecular structure and catalytic mechanism to physiological and pathological functions of proteases and their inhibitors, will be covered in this five-day conference (31 major speakers plus two poster sessions). The subject matter of the Conference is of considerable importance to human health; PHS support would be consonant with the mission of the DHEW.


Clostridium difficile infections (CDI) have become more difficult to treat due to the rise of hypervirulent strains that have increased morbidity as well as mortality and the likelihood of persistence and relapse in infected patients. To address the dire need for new anti-difficile agents, we explored the premise that nature utilizes good bacteria (i.e. probiotics), including Lactobacillus spp., to suppress gut pathogens such as C. difficile by producing novel antimicrobials, which were optimized by evolution to the microenvironment of the gut. In this regard, we explored the potential for developing reutericyclin from Lactobacillus reuteri as a natural anti- difficile agent. Probiotics are emerging as alternate treatments for CDI. However, there is ongoing debate on whether live probiotics may be used treat severe CDI, especially in immunocompromised patients. We therefore hypothesize that developing the antimicrobial produced by the probiotic specie would harness one of its natural therapeutic properties for localized killing of C. difficile in the gut, providing a more reliable and efficacious treatment strategy. Application of this concept to our studies on reutericyclin revealed that it has impressive antimicrobial and pharmacological properties for treating CDI. These include: rapid killing of nongrowing, toxin- producing stationary phase C. difficile; a novel mechanism of action specific to the bacterial membrane; a narrow spectrum of activity; lack of cytotoxicity against gut epithelia; stability to proteolysis; ability to achieve high non- absorbed concentrations in gut for killing; a low molecular weight and ease of synthesis that will allow advanced chemical optimization. Importantly, the killing of toxin-producing stationary phase cells is not shown by currently prescribed antibiotics vancomycin and metronidazole, which only kill actively growing C. difficile. We believe that this proposal for developing probiotic-derived reutericylcin derivatives to treat CDI is highly innovative and will be achieved through three iterative aims: (i) Synthesis of an expanded sets of reutericyclin analogs to optimize anti-difficile activity and increase affinity for the membrane target site ; (ii) Lead development and characterization involving the stepwise progression of compounds through three stages of tests that include antimicrobial assessment, pharmacokinetic testing, toxicologic and in vivo efficacy experiments. Compounds meeting the selection criteria of the tests will move onto the next stage such that a lead candidate is obtained with potent in vivo efficacy and excellent safety profile; (iii) Mode of action studies to explore the fundamental antibacterial effects of targeting the clostridial membrane. The long-term goal of this project is to develop optimized lead analogs of probiotic derived reutericyclin that exhibit novel modes of action at the membrane target and have characterized antibiotic properties that would allow their progression into advanced preclinical studies as candidates for treating CDI.


Transcription and regulation of gene expression are temporally mediated by modification of the C terminal domain (CTD) of subunit 1 of RNA polymerase II. The combinatorial phosphorylation pattern of the CTD, a 52 consensus repeat of a YSPTSPS heptad, coordinates RNA biogenesis in the transcription cycle. Progress in resolving the regulatory roles of the CTD has been impeded by lack of suitable methods for site-specific and quantitative mapping of CTD phosphorylation, an issue that directly ties structure to function. This proposal addresses this challenge by the development of ultraviolet photodissociation (UVPD) mass spectrometry to map the phosphorylation pattern and occupancy of the CTD in conjunction with label-free quantitation. The three proposed aims include: (1) Analysis of the combinatorial phosphorylation pattern of the CTD using 193 nm UVPD. The ~26 kDa CTD peptide of RNA pol II will be isolated, digested by Proteinase K, and the resulting peptides analyzed by nanoLC-UVPD-MS in the negative mode. Both the phosphorylation sites and occupancies will be characterized. (2) Quantitative analysis of global CTD phosphoryl occupancy and exposure to cell stressors. Changes in the CTD code as a function of particular cell stressors, including heat shock, exposure to salt, dithiothreitol, lipopolysaccharides (LPS), sorbitol or hydrogen peroxide, will be elucidated by label-free quantitation. (3) Quantitative analysis of global CTD phosphoryl occupancy between a control and a yeast strain with a functionally deficient Ssu72 phosphatase. Ssu72 displays specific phosphatase activity toward Ser2 and Ser5 of the CTD heptad repeat, two sites that play a critical role in the temporal regulation of transcription. RNA pol II from Ssu72-deficient and control yeast strains will be harvested, digested with trypsin, and processed to isolate the CTD peptides. The resulting phosphorylated peptides, which will differ in occupancy and abundance for each cell state, will be evaluated by label-free quantitation. This application of innovative UVPD technology will provide critical insight into the role of CTD phosphorylation in transcriptional processing.


Proprotein convertase subtilisin kexin type 9 (PCSK9) regulates plasma cholesterol levels by enhancing the intracellular degradation of the LDL receptor (LDLR), the primary driver of LDL clearance from plasma. Human PCSK9 is composed of 692 amino-acids. After removal of the signal peptide (residues 1-30), the secreted PCSK9 presents three domains: 1. The N-terminal pro-domain (PD, residues 31-152), which is cleaved but remains associated with the rest of the protein, and displays a highly disordered N-terminus (residues 31-60); 2. The catalytic domain (CA, residues 153-449), which contains a proteolytic site masked and neutralized by the associated PD; 3. The C-terminal domain (CD, residues 453-692), which consists of three tightly packed modules, CM1 (residues 457-527), CM2 (residues 534-601) and CM3 (residues 608-679). The consensus view is that PCSK9 binds to the epidermal growth factor type A (EGF-A) repeat of the LDLR via its CA, but does not cause direct proteolysis of the LDLR. Understanding the function of PSCK9 is central to the development of novel therapeutic agents to reduce cholesterol levels and cardiovascular risk. Although PCSK9 primarily targets the hepatic LDLR, it may also function on other lipoprotein receptors, such as apoER2, LRP1, and VLDLR, as well as on LDLR in extra-hepatic tissues. Our published work and preliminary studies have addressed the role of different PCSK9 domains in the secretion and function of the protein. We have found that: 1. Deletion of CM2 produces a protein that is secreted and functional; 2. Deletion of either CM1 or CM3 produces a protein that is neither secreted nor functional; 3. Deletion of the entire CD produces a protein that is secreted but not functional; 4. The PD of a PCSK9 fragment lacking CD facilitates the secretion of a PCSK9 fragment lacking PD; 5. The discrete CD of PCSK9 binds the ectodomain of the LDLR in solution and dose- dependently inhibits the LDLR-degrading effect of full-length PCSK9 cells; 6. PCSK9 undergoes pH-dependent self-association, which is mediated by the CA and increases the LDLR-reducing functionality; 7. Over- expression of PCSK9 reduces LDLR, apoER2, and LRP1 in HEK293T cells, and reduces LDLR levels in macrophages; 8. Transgenic expression of human PCSK9 in macrophages reduces liver LDLR and raises plasma cholesterol levels in mice. The central hypothesis of this proposal is that PCSK9 uses multiple domain interactions for secretion and function, and contributes to atherogenesis by targeting multiple lipoprotein receptors. We plan to: 1. Examine the role of the C-terminal domain in PCSK9 secretion and LDLR binding; 2. Test the hypothesis that domain interaction and self-association modulate PCSK9 secretion and function; 3. Test the hypothesis that PCSK9 secreted by macrophages modifies the biology of the atheroma through multiple effects on lipoprotein receptors. Investigating these novel roles of PCSK9's C-terminal domain and pro-domain may provide new leads in the development of inhibitors and advance our understanding of plasma cholesterol regulation.


Ross Products Division has developed a complete pediatric elemental formula (PEF) suitable for feeding to children on allergen free diets. The product is based on free amino acids and meets the recommended dietary allowances (RDA) for the 1 to 3 year old child in 10000 kcal and for the 4 to 6 year old child in 1500 kcal. The purpose of this study is to assess whether PEF will support adequate growth, tolerance and biochemical response in children requiring an elemental formula as their primary source of nutrition for 4 months while maintaining the appropriate allergen avodance diets. The primary indicators used to test the research hypothesis will be weight, height, intake, stool patterns, and blood biochemistries (serum albumin, ferritin, transthyretin, retinol binding protein [RBP], retinol and urea nitrogen, and plasma amino acids). Secondary variables will include allergic and gastrointestinal symptoms. Subjects will be children with documented AEG currently maintained on NeoCate or NeoCate 1 as their primary feeding.


Dengue virus is a major human pathogen that presents a daunting challenge to treatment design and implementation. No antivirals are currently available and, as for all positive-strand RNA viruses, it is anticipated that multi-drug therapy will be needed due to the high error rate ofthe viral polymerases which can lead to the rapid selection of drug-resistant viruses. However, we have developed a strategy to identify dominant drug targets' in RNA viruses and are testing this strategy with Dengue viruses. Specifically, when the target of a drug is an oligomeric protein or is involved in the generation of an oligomeric species, it is often true that drug-sensitive viruses are genetically dominant over the inevitable drug-resistant ones. Thus, the drug-resistant viruses are not selected from the cells in which they were generated. For Dengue virus, candidate proteins are the core protein C, the envelope protein E, the protease NS2/3, and certain inhibitors ofthe polymerase NSS. We have obtained inhibitors of E, NS2/3 and NS5 and selected and partially sequenced drug-resistant variants of each. For these and other inhibitors of Dengue virus, we will test in coinfections whether drug susceptibility or drug resistance is dominant. Then, we will test the frequency of the emergence of drug resistant viruses in murine infections. We anticipate that, for some drug targets and not others, drug resistance will be dramatically suppressed, paving the way for the use of fewer compounds to treat Dengue virus infection, and possibly even monotherapy. The development of logical strategies to counteract drug resistance is crucial to combatting many pathogens, with Dengue virus as a currently pressing example.


Recent advances in biotechnology and molecular biology have brought about new concerns in protein purification. The proposed protein purification knowledge base addresses some of these issues by providing new computer- readable protein purification databases coupled with an expert system to suggest protocols for purifying new proteins. Experimental data will be scanned, digitized and analyzed for relevant information that the expert system can use to update the working purification strategy. This knowledge base system can decrease the costs of developing new biotechnology products by optimizing the development of large-scale protein purification processes and by using the expertise of key protein purification personnel more efficiently. In addition, a subset of the knowledge base can be used to train students in protein purification techniques. The purification databases will also be distributed independently of the expert system and will be updated on a regular basis.


Many studies suggest that overexpression of DNA repair factors could contribute to resistance to radiochemotherapy. Therefore, studying this pathway has important implications in cancer pathogenesis and cancer therapy. We found that UCHL3, a deubiquitinase, is overexpressed in breast cancer. In addition, increased expression of UCHL3 correlates with poor prognosis for breast cancer patients. However, whether or not UCHL3 casually contributes to clinical phenotypes is unclear, because the cellular function of UCHL3 remains unclear. Here we show for the first time that UCHL3 is involved in DNA repair. We found that UCHL3 interacts Rad51 and deubiquitinates Rad51 without affecting Rad51 levels. UCHL3 itself is phosphorylated following DNA damage and is recruited to double strand breaks. In addition, UCHL3 is important for the recruitment of Rad51 to double strand breaks and homologous recombination. Downregulating UCHL3 sensitizes breast cancer cells to radiation and PARP inhibitors, while overexpressing UCHL3 renders breast cancer cells resistant to these treatments. Based on these Preliminary Data, we hypothesize that UCHL3 is a new factor in DNA repair. UCHL3 deubiquitinates Rad51 and promotes Rad51 function and homologous recombination. Increased UCHL3 contributes to resistance to radiation and chemotherapy by enhancing DNA repair. In this application, we will further explore how UCHL3 regulates Rad51 and how UCHL3 itself is regulated. We will also test the role of UCHL3 in radiochemoresistance using clinically relevant models. Our Specific Aims are: 1. Investigate the regulation of Rad51 by UCHL3; 2. Study the regulation of UCHL3; 3. Investigate the role of UCHL3 in breast cancer therapy. Our studies will reveal a novel function of UCHL3 in DNA repair and response to radiation and chemotherapy. In addition, it will reveal a new therapeutic target in sensitizing breast cancer cells, especially those overexpressing UCHL3, by targeting the UCHL3-Rad51 pathway.


It has recently been established that certain benzodiazepine (BZ) receptors are closely associated with receptors for gamma-aminobutyric acid (GABA) in (neuronal) membranes. The physiological significance of this close association is at present entirely unknown, but may be the first example of a more general phenomenon, and could be related to the co-release of two neurotransmitters from the same neuron. I propose to investigate this close association using two general methods: 1) the ability of type-1 GABA mimetics (e.g., GABA, muscimol) to protect associated BZ receptors from heat inactivation, and 2) the ability of type-2 GABA mimetics (e.g., THIP, isoguvacine, piperidine-4-sulphonic acid, 3-amino-propane sulfonic acid) to prevent binding of 3H-BZ ligands to GABA-associated BZ receptors. Type-1 and type-2 GABA mimetics are mutually antagonistic in a competitive way. Preliminary results indicate that some but not all BZ receptors are associated with GABA receptors, and that these GABA receptors are of two types. First we will do time courses of heat inactivation of BZ receptors in the presence of GABA mimetics of both types. We will then do dose-response experiments with all active GABA mimetics using a single-time (usually 30 min.) at 60 degrees C to determine whether one or more GABA receptors are involved. The ability of type-2 mimetics to antagonize the stabilizing effects of type-1 mimetics will be determined. The inhibitory effects of THIP and isoguvacine as a function of heat inactivation in the presence of GABA will be investigated. Several new potential GABA mimetics will be synthesized and tested in the above systems.


TORSIN A AND THE PATHOPHYSIOLOGY OF DYSTONIA Mutations of the DYT1 gene cause early onset torsion dystonia. This gene encodes torsinA, a member of a novel family of proteins with homology to the HSP 100/CLP family of heat shock proteins. In preliminary studies, we have found that the gene for torsin A is active in nerve cells in several regions of normal adult human brain, including the dopamine neurons of the substantia nigra pars compacta. We have also found that the gene is always active in the brains of patients who are ill for more than a day but not in the brains of those who die suddenly. This finding is consistent with, but does not prove, that torsinA is a stress response protein. In this application we seek funding to obtain detailed information about the localization and regulation of the torsinA mRNA and protein in both human brain and genetically engineered rodent models. We will determine exactly which cells in human and mouse brain contain the mRNA and protein, where in the cells the protein is located, whether mRNA expression is regulated by stress and whether the mutation effects the location or amount of mRNA expression, the subcellular location of the protein or the function of dopamine neurons. These data are essential to the construction of mechanistic models of the neural dysfunction which produces dystonia.


PROJECT SUMMARY Chemo-radiation therapy is a standard treatment regimen for locally advanced head and neck squamous cell carcinoma (HNSCC). The treatment regimen, however, is difficult for patients as they experience high rates of grade 3 or higher toxicities including leukopenia (42%) and the need for feeding tube (52%). Recent studies showed that a subgroup of HNSCC patients with human-papilloma virus (HPV)-positive oropharyngeal (OP) SCC have significantly better prognosis. These clinical data lead to important considerations to de-intensify treatment for this low-risk, younger population in order to reduce acute and chronic toxicity without compromising disease control. It has been suggested that the adaptive de-escalation of treatment can be tailored for individual patients based on the early tumor volume change. However, volumetric assessment is often inadequate because the treatment response of a tumor can be heterogeneous in terms of (i) cell viability, (ii) cellular metabolism, and (iii) perfusion that are relevant to the success of any chemoradiation therapy. These complex changes may not be adequately represented by tumor volume change at the early stage. The proposed study is based on a combination of the quantitative diffusion MRI (dMRI) methods with their own technical innovations that can also be applied to other clinical studies. dMRI is a unique in vivo imaging technique sensitive to cellular microstructures at the scale of water diffusion length on the order of a few microns. However, quantitative dMRI remains challenging as dMRI data represent different biophysical properties of tissue depending on diffusion weighting strength (q) and diffusion time (t) used for the measurement. The scientific premise of the proposal is that this study will establish a quantitative way to utilize both q- and t-dependent dMRI data as a tailored approach to quantify cell viability, cellular metabolism and perfusion from this non-contrast MRI method. We demonstrated that both diffusion coefficient D and diffusional kurtosis coefficient K are promising imaging markers for cell viability. Cellular metabolism can be evaluated in terms of the water exchange ?ex, measured by the diffusion time-dependent K, that is regulated by the ATP- dependent trans-membrane ion channels co-transporting water molecules. Intravoxel incoherent motion MRI metrics (pseudo diffusivity, Dp; perfusion fraction, fp) can provide information about perfusion flow. Ultimately, these dMRI measures will better identify patients who have the potential to benefit from adaptive de-escalation or escalation of therapy. In this proposal, we will further optimize and establish a set of quantitative non-contrast imaging markers of cell viability (D and K), cellular metabolism (?ex), and perfusion (fp?Dp) as a clinical tool for assessment of treatment response and validate it in a clinical trial. The data acquisition and analysis software tools to be developed in this study will enable comprehensive and quantitative assessment of cancer treatment response to tailor chemoradiation therapies for individual patients.


The Genomics Core will facilitate transcriptional profiling of cells and vascular tissues obtained in the studies of the individual projects of the SCOR using high-throughput technologies. Services provided include: 1) nucleic acid (RNA or cDNA) hybridization to commercial arrays purchased by the individual projects and 2) array printing and analyses of subsets of genes pertinent to the specific focus of each project to provide maximum flexibility in customization/high throughput of cell and tissue samples. Core services will include optimization studies, array imaging, and analytical assistance. Procedures for assuring equitable sharing of the instruments and coordinated scheduling will be developed. Activities will be facilitated by a GeneMachines OmniGrid Sample Microarraying Robot, and a Packard Biochip Scanarray 4000 Scanner, together with a dedicated computer. Additional components of the system, underwritten by other resources and at no cost to this grant are a Biomek 2000 Laboratory Automated Workstation for multipurpose fluid handling, and array software licenses. An in-house microarray printer and scanner are central to the Core's function and will permit the SCOR investigators great flexibility in high throughput genomics capability. The arrayer will provide customized printing of selected cDNAs or oligomers prepared by the investigators' own labs (or outsourced synthesis) to facilitate detailed analysis of gene expression relationships based on specific project-focused questions. The instruments are also central to the use of commercial microarrays for whole genome screening. The Associate Director and Director have 3 years experience with microarray analyses. The Genomics Core will coordinate activities with the Proteomics Core (Core B) to seek concordant and discriminant patterns of mRNA vs protein expression in response to pro- and antioxidant interventions by providing genomic profiling complementary to proteomic analyses. This is an important and necessary approach for investigations of frequently divergent gene and protein expressions in the same cells/tissues. A similar pattern of paired analysis will be applied to studies of circulating cells, initially polymorphonuclear leucocytes, as potential markers of gene and protein expression in the vasculature.


Disorganization (Ds), a single gene mouse mutation, has profound effects on mouse development. Unlike most mutations where particular traits with diagnostic features are inherited among generations, the heritable trait in mice with the Ds mutation is the propensity for the body to be formed in an unpredictable manner. No two mice are affected in identical ways. Affected mice may show agenesis, duplication or fusion of structures, and all morphological structures seem prone to anomaly. Remarkably, these mice are not prone to cancer, spotting, behavior, and longevity or fertility problems. Ds is one of the few examples in mammals of a true dominant, gain-of-function mutation. Determining the identity of the Ds gene is important because deep insights into the regulation of pattern formation during development can be gained. In addition, insights into the molecular nature of mutations that show variable expression and low penetrance may be forthcoming. Ds has been located to a 0.2 cM segment of mouse Chr 14, cloned in a 360 kb BAC contig and 80 kb of sequence has been obtained. Cloning of the mouse Ds gene is proposed. The genomic sequence of the Ds locus will be determined in wild-type mice in order to identify genes and markers, evidence for rearrangement in Ds will be sought, cDNA expression and sequence will be assessed in wild type and Ds mice, and the genomic sequence of the Ds locus will be determined. In addition, it is proposed that the identity of Ds will be verified by generation and characterization of transgenic mice with the candidate Ds mutation.


This 3-generation study of families at high and low risk for Major Depressive Disorder (MDD) has documented the strong familial transmission of mood disorders across generations. We have conducted 5 waves of assessments in this cohort over 25 years. In the 5th wave, we added Magnetic Resonance Imaging (MRI) measures and found a remarkably robust association of familial risk for MDD with asymmetries in cortical thickness, with a nearly 30% reduction in thickness observed in the lateral parietal, temporal, and frontal cortices of the right hemisphere of the high risk group. These MRI findings are consistent with EEG findings from the 4th wave of assessments that demonstrated reduced activity over the posterior cortices of the right cerebral hemisphere. Both the MRI and EEG findings were present in high-risk individuals who never had MDD in their lifetimes, suggesting that these abnormalities were not simply a consequence of previously having been depressed or having been treated for depression. Thinning of the cortical mantle and reduced electrophysiological activity in the right hemisphere therefore may constitute related endophenotypes for familial vulnerability to developing MDD. We are proposing a 6th wave of study to gain a deeper understanding of the right hemisphere abnormalities in familial MDD in the 216 individuals imaged thus far. This represents the largest MRI study published for MDD to date, and it is the only sample studying 3 generations of individuals at high or low risk for MDD. We are proposing to collect additional MRI and EEG measures, as well as clinical and cognitive neuroscience data, that will inform us about the neural bases of the right hemisphere thinning and their consequences for brain function and emotional processing. We will also determine whether additional cortical thinning in the left cerebral hemisphere predicts new or recurrent MDD in those people who were imaged in Wave 5. Our cohort has exceptional attributes that will aid our search for detecting MDD endophenotypes, including (1) an elevated clinical risk for developing MDD in the high risk group that has been amply demonstrated; (2) multiple prospective assessments, conducted blind to risk status, provide great confidence in the accuracy of the clinical diagnoses; and (3) the sample contains individuals who have not yet become ill but who are nevertheless at elevated risk of becoming ill, thereby allowing us to disentangle the neurobiological determinants of vulnerability from the compensatory responses that would be present in already-affected individuals. MDD is an early-onset, highly prevalent disorder with significant morbidity, and it afflicts people in their most productive years. Identifying the biological vulnerability for depression before the onset of illness has important implications for prevention and public health.


In the past year, effort in nuclear magnetic resonance imaging has been expanded on several research fronts. A. Following the failure of the magnet manufacturer to install successfully the clinical NMR imaging system in the Department of Radiology, Clinical Center, the research group took over the homogenizing of the magnetic field necessary for clinical imaging, and, using a new technique, achieved the specified homogeneity, thereby enabling the Radiology Department to produce high quality images. B. Considerable controversy exists within the NMR image community over the choice of an optimal field strength for imaging. Bench methods were developed for accurately assessing the signal-to-noise ratio from, and radio-frequency power deposition in, the human body at any frequency used for imaging. It is hoped these results will help resolve the matter. C. A pulse for highly selective spin population inversion has been discovered. The result is of considerable experimental and theoretical importance for it represents only the second known analytical solution of the non-linear differential equations governing the motion of an NMR spin system. Further, above a critical threshold, the inversion is independent of applied power. D. A so-called "quadrature" probe system has been invented which reduced radio-frequency power dissipation in the body by almost a factor of 2 while improving signal-to-noise ratio by almost 40%. E. The electronic upper frequency limit for adult head imaging has been pushed from 84 MHz to 130 MHz with the aid of a novel phased-array receiving coil.


The Operations Core (OC) supports OAIC investigations of multifactorial geriatric conditions by recruiting and retaining diverse populations of older persons, seeking input from the local community in research planning and dissemination, monitoring participant safety, ensuring regulatory compliance, developing surveys and instruments, designing Information Technology (IT) systems to implement research, collecting and preparing data for statistical analysis, and providing continuity and shared knowledge across projects. The overall goal of the OC (RC1) is to ensure the successful implementation of research focused on multifactorial geriatric conditions. This goal will be accomplished by leading, managing, and coordinating the effective, efficient and innovative use of facilities, data, staff, resources, and space. There is a consistent demand for experienced personnel with the ability to quickly execute aging-focused research and an increasing need for informatics skills and technology to streamline work. The Specific Aims of the OC are: 1) to provide an infrastructure suitable to support Yale OAIC-funded projects and External Projects. This infrastructure includes experienced personnel, staff training modules, standard operating procedures for conducting field studies, and comprehensive clinical research informatics (CRI) services; 2) to collaborate with the REC, PESC, and Biostatistics Core (RC2) to develop specific CRI and field operations systems for Yale OAIC-funded projects and External Projects; and 3) to conduct innovative OC Development Projects that advance the mission of the Yale OAIC. During the current funding cycle (2013-present), the Operations Core was integrally involved in research support of 80 OAIC-funded projects and External Projects, numerous collaborations within Yale, and engagement with several other Pepper Centers. Altogether, these efforts have resulted in 114 peer-reviewed publications and 81 extramurally funded grants (>$145 million total costs) of which 25 involved 18 junior faculty specializing in aging/geriatrics research. During the next funding cycle, the OC will: (1) collaborate on 4 of the 5 proposed REC and PESC projects, 24 of the 29 funded External Projects, and 7 pending projects from grant submissions involving more than $13 million in total costs; (2) substantially expand and enhance the clinical research informatics infrastructure at Yale School of Medicine, in partnership with leaders from each clinical research support center; and (3) lead an innovative Development Project, the Clinical Decision Support System (CDSS) Toolkit, that will make it possible for any research team having access to REDCap to implement a sophisticated, HIPAA-secure CDSS. The CDSS toolkit will support the growth of projects related to the OAIC subtheme of individualized decision making for older persons with multiple chronic conditions.


Muscles must be able to relax as well as contract. Contraction and relaxation are regulated by molecular switches on the thick (myosin-containing) and thin (actin-containing) filaments, which together make up the contractile machinery. Defects in regulation can lead to muscle disease. Our long term goal is to understand the structural basis of muscle regulation; our focus in this application is the relaxed state. During the current grant period we achieved two key breakthroughs in defining the molecular structure and regulatory mechanism of myosin filaments, resolving basic questions dating back more than 40 years. We also gained new insights into troponin-tropomyosin regulation of actin filaments. Our insights raise key new mechanistic questions, which we propose to investigate in this renewal. To understand how muscles relax, the structures of the actin and myosin filaments in the relaxed state must be determined. To achieve this, state-of-the-art electron microscopic and 3D image reconstruction techniques will be applied to native filaments and their isolated component molecules. Using these approaches: (1) The molecular interactions underlying the relaxed state of striated and smooth muscle myosin filaments will be defined in detail. (2) The specific amino acid residues and protein domains essential to these relaxed-state interactions will be determined by analyzing the effects of targeted mutations on the structure of expressed single myosin molecules. (3) The structural basis of thin filament relaxation will be defined by analyzing the native, relaxed-state organization of the regulatory components troponin and tropomyosin, and the thin filament template protein, nebulin. In each study, reconstructions will be fitted to atomic resolution crystal structures of thick and thin filament subunits, defining molecular contacts at near-atomic resolution. Our studies of myosin molecules and filaments will provide new insights into general mechanisms of thick filament regulation, and especially into the nature of the relaxed state in vertebrate striated muscle. Further advances in our studies of troponin-tropomyosin regulated thin filaments will provide new information on native thin filament structure, the 3D organization of troponin and tropomyosin, and the structural contribution of nebulin to the relaxed state. Preliminary data demonstrate the feasibility of our aims. The new insights arising from these proposals will provide a deeper understanding of the structural basis of muscle relaxation. Defining molecular mechanisms in healthy muscle is essential to understanding structural defects that underlie muscle disorders.


The broad goal of the proposed research is to further understand the role of intrinsic brain networks in cognition. Intrinsic networks (INs) are collections of disparate brain regions that are consistently identified in task-free functional magnetic resonance imaging (fMRI), and are presumed to underlie sensory, motor, and cognitive functions. Further understanding requires detailed knowledge about the dynamics of the networks, both in task-free "resting state" as well as during cognitive function. What are the interactions between Ins and the traditional "task-active" regions? Is there an integration of information along the networks that may [unreadable] subserve task responses? The current proposal focuses on using fMRI to elucidate dynamic properties of a particular IN known as the default-mode network (DMN). [unreadable] In Specific Aim 1, we model directions of task-evoked information flow among the DMN and task activated regions. The chronometry of initial responses to a brief, attentionally demanding task will be obtained using onset latency analysis, and the evolution of temporal dependences in the (long) poststimulus interval will be quantified using time-varying Granger causality. Specific Aims 2 and 3 pertain to methodological issues in studying INs using fMRI. In Aim 2, we quantify the stationarity of coupling strength and phase differences among regions of the DMN in task-free resting state. Currently, the majority of studies define INs using algorithms that assume that the coupling of IN regions remains constant over time. We employ time-varying functional connectivity and ICA over a long resting-state scan to examine the degree of change overtime, and relate changes in connectivity strength to physiological variables that may signify changes in arousal or awareness level. In Aim 3, we correct for regional differences in hemodynamic latency due to vascular reactivity, so that the relative timing of BOLD signals reflects underlying neural interactions more closely. Our approach is to measure vascular response latencies across the brain using a breath holding task, and develop methods for correcting for these non-neural latency differences. [unreadable] Investigating the temporal behavior of the DMN will provide valuable insight into how the network underlies cognitive dysfunction, as well as function. While the DMN is receiving widespread attention for its purported role in episodic memory and task performance, it is also proving effective as a biomarker for disorders such as Alzheimer's Disease, major depression, ADHD, and schizophrenia. The current proposal provides a framework for studying the temporal behavior of the DMN (and other INs), which will serve as a basis for further investigation of network responses and connectivity in patient populations. [unreadable] [unreadable] [unreadable]


RNA editing via the ADAR enzymes (adenosine deaminases that act on RNA) has emerged as a novel and significant post- transcriptional mechanism for altering protein products encoded by a single genetic locus. ADAR activity has been detected across phyletically distant organisms and within a variety of tissues within an organism. Paradoxically, the number of mRNA targets of ADARs remains small and limited primarily to ligand- gated ion channels of the mammalian nervous system. A limited number of well characterized mRNA substrates for ADARs have been characterized and a general requirement for a dsRNA intermediate in pre-mRNA has been proven. Still, central questions with no consistent answers remain such as; What determines ADAR specificity? What comprises an RNA editing site and can we predict their existence rather than relying on serendipity? What are the phenotypic consequences for the organism of editing of a particular RNA editing site? What is the global role of RNA editing in gene regulation? In this proposal, these major areas are addressed using Drosophila as a model genetic system. First, known RNA editing sites in several Drosophila voltage- and ligand-gated ion channels as well as one in an enzyme will be utilized to elucidate the cis-determinants of pre-mRNA editing sites via in vivo transgenic and in vitro methods. Second, a genetic and molecular approach will determine the effects of knocking out or reducing RNA editing of a specific RNA editing site in a ion-channel gene known to have effects on nervous system function and behavior. Lastly, a recently isolated knock- out mutation of the Drosophila ADAR, dADAR, will be characterized at the molecular, genetic and behavioral level to assess the global significance of specific RNA editing.


The long-term objective of this proposal is to understand in molecular terms the mechanism by which Agrobacterium tumefaciens interacts with its host resulting in Crown Gall tumor formation in a wide variety of plants. This proposal follows the same general approach that we have used in previous years to gain insight into these mechanisms, namely, to analyze mutants altered in tumor formation. However, the experiments described are much more focused because we now know the sequence of the Agrobacterium genome and we have access to microarray chip technology. With this new information and technology we can carry out directed mutant studies and types of analyses that were not possible previously. The information revealed by the sequence of the genome has raised questions but also the means to answer these questions which we could only speculate on previously. Thus, we now know that far more genes are activated in Agrobacterium by interaction with host plants than was recognized previously. Using microarray chip technology we will identify and characterize global regulatory circuits that are directly activated or suppressed by plant signal molecules. Using microarray technology, we will expand on previous observations by determining the alterations in the global expression of genes in Agrobacterium grown with plant cells at a temperature optimum for the growth of Agrobacterium but inhibitory for the transfer of T-DNA (28[unreadable]). We will also continue our studies on the mechanism by which T-DNA is transferred from Agrobacterium into host cells, focusing on the role of VirJ in this process. Finally, the genome sequence of Agrobacterium has revealed many genes that serve pathogenicity functions in related bacteria. We will mutate these genes to determine if they are also required for pathogenicity of Agrobacterium. The studies proposed in this application should open up new vistas and provide an increased understanding of the many levels of interaction of Agrobacterium with its variety of eukaryotic hosts.


The impact of recent legislative changes focused on improving our knowledge of drugs used in the treatment !of children is slowly being realized, but current studies have not investigated the differences in drug disposition afforded by disease state. This is most notable for critically ill children, in who changes in organ function, total body water, tissue and plasma proteins, can all affect the disposition and action of therapeutic drugs. The long-term research goal of this proposal is to optimize the safety and efficacy of drugs used in the treatment of critically ill children by bridging our gap in knowledge of the clinical pharmacology of drugs frequently used in these patients. We will use sophisticated pharmacokinetic-pharmacodynamic (PK-PD) modeling techniques and clinical trial simulation to streamline the development and performance of clinical studies. Our central hypothesis is that informative clinical trials can be designed more efficiently when well characterized models that account for sources of variation in pharmacokinetics and pharmacodynamics are utilized. We plan to initially construct select models based on published clinical pharmacologic data, and then perform clinical trials simulations for the design of prospective studies of drugs frequently used in the care of critically ill children. Based on the results of our models and trial simulations, we will then carry forward and perform clinical studies of two frequently used drugs (pentobarbital and dopamine) in critically ill children. lastly, we will integrate results from these clinical studies back into our models to allow for pharmacologically more rational dosing in this difficult to study pediatric population. For this mentored training, two complementary approaches will be pursued. The first will be formal coursework covering a range of topics iintegral to the field of clinical pharmacology. The second approach will be through a mentored clinical and laboratory research program. This intensive didactic and mentored training will allow for the development of a future independent pediatric clinical investigator.


Actinobacillus actinomycetemcomitans(Aa), a major pathogen in certain human periodontal diseases, elaborates a potent leukotoxin (Ltx). This polypeptide kills human neutrophils, monocytes and T lymphocytes and may impair host antibacterial resistance by destroying or perturbing these cells in infected tissues. Results of current studies indicate that the toxin disrupts the permeability barrier of the plasma membrane of susceptible cells. This results in a 2+rapid rise in Ca (and probably other ions) levels within the cell which ultimately leads to cell death. This proposal will determine the molecular mechanism(s) for this observation. Two separate lines of experimentation form the basis for this proposal: l) Ltx-treated lymphocytes and NK cells exhibit many of the morphological characteristics of apoptosis; and 2) the Ltx receptor is a beta2 integrin, a molecule which when stimulated is capable of elevating cytosolic 2+Ca levels. In Specific Aim #1, Ltx-treated cells will be evaluated by DNA degradation, macromolecular synthesis, and FACS-associated morphologic alterations to determine if programmed cell death is occurring. Specific Aim 2 will focus on the expression of genes known to be involved in programmed cell death such as Bc1-2, Bc1-x, ICE, Bax, CPP32 and p53 to determine a molecular mechanism for cell death. Specific Aim 3 proposes experiments to define the nature of the toxin/receptor interaction. We envision that the first contact that the Ltx contact on a cell is a (32 integrin cell surface molecule. It is our goal to define these earliest consequences of this interaction such as determine the signal 2+pathway that is activated and the relationship to elevated cytosolic Ca +2 levels. Finally Specific Aim 4 will identify genes which are unique to Ltx- mediated apoptosis. It now emerging that the process of apoptosis involves different pathways when different agents are used to initiate the process. From these experiments we will have a better understanding of the pathway that is utilized by the toxin to kill cells. These studies provide fundamental insights into the pathobiology of the Aa Ltx and will bring us closer to understanding of its role in human periodontal infections on a molecular level. In addition, detailed studies on the action of the Aa Ltx will contribute to a better understanding of the biology of other membrane-active bacterial cytolynsins.


We are trying to understand how mRNA polyadenylation occurs in male germ cells in the absence of the canonical AAUAAA polyadenylation signal. We discovered and characterized a variant form of the CstF-64 polyadenylation protein that is expressed at high levels in meiotic and postmeiotic germ cells, and in no other tissue except the brain. This new protein, called "(CstF-64," is the leading candidate to account for AAUAAA-independent polyadenylation in germ cells. Our overall laboratory hypothesis is that (1) the somatic CstF-64 gene (Cstf2) is on the X chromosome in mice and humans, (2) the X-chromosomal CstF-64 is inactivated during male meiosis, (3) since CstF-64 function is essential, an auxiliary CstF-64 gene must be expressed from an autosome, and (4) the auxiliary CstF-64 protein is responsible for AAUAAA-independent polyadenylation of mRNAs in male germ cells and is essential for spermatogenesis. In the previous grant period, we addressed items 1-3 of our hypothesis. The goals of this grant proposal are to address item 4. This Independent Scientist Award (K02) will assist me in accomplishing those goals. Specifically, I am seeking to receive salary support to release me from some or all of my teaching and administrative duties so that I can devote a greater portion of my time to research and career development. For the research portion, I will be able to spend more time in the laboratory myself, and to direct the research of my workers more effectively. For the career development portion, I will be able to attend national and international meetings, and spend short periods visiting laboratories of collaborators to learn new techniques relevant to our work. These activities will be especially helpful, because my background is in biochemistry and not in reproductive biology. The Department of Cell Biology & Biochemistry is an excellent place from which to achieve these goals: the major strength of the Department is in many aspects of reproductive biology, and my colleagues have been exceedingly generous in sharing their ideas and expertise. This environment, supplemented with trips to meetings and visits to collaborators, will provide excellent support for my endeavors.


More than 20 million people in the US have chronic kidney disease, and the cost of end stage renal disease management is over $40 billion annually in public and private funding. For renal failure patients on hemodialysis, broad consensus is that native arteriovenous fistulas (AVF) are the preferred access type, with better survival, fewer complications, longer patency, and reduced costs relative to other options. However, because up to 60% of attempted AVF never mature to the point of usability for dialysis, early AVF failure is a key barrier to maximizing health outcomes in these patients. The high AVF failure rate contributes to initiation of catheter-based dialysis in a large percentage of patients, with increased risks of infection and premature death. Maturation failure is thought to be related to peri-anastomotic stenoses resulting from intimal hyperplasia, inflammation, and negative wall remodeling. These factors occur in the presence of metabolically active perivascular adipose tissue which we theorize impacts overall remodeling. Chronic systemic inflammation is linked to the uremic milieu of patients with advanced kidney disease, and emerging evidence directly links elevation of pro-inflammatory mediators to adipose tissue. Furthermore, our pilot studies on AVF patients suggest clear links between base adipose phenotype and eventual fistula maturation. In addition, a genetic contributor to the process of atherogenesis may also lead to high rates of AVF failure. Polymorphisms associated with the ApoL1 gene on chromosome 22 have been shown to cause kidney failure in African Americans. The gene codes for a protein whose function incompletely understood, but is a component of HDL in circulation and is expressed in higher levels in patients with known comorbid predictors of atherogenesis. This indicates that the protein itself may be either associated with or a marker for development of atherogenesis and therefore may also contribute to increased rates of AVF failure. Thus for the current project we hypothesize that peri-anastomotic AVF adipose inflammation and APOL1 SNP status/gene expression patterns drive early AVF negative wall remodeling, contributing to failure to mature. These hypotheses will be tested via the following Specific Aims: 1) Identify adipose tissue expression profiles and clinical determinants of AVF failure. 2) Determine relationships between circulating and tissue levels of adipocyte-related mediators. 3) Understand the impact of ApoL1 gene status and protein expression on AVF remodeling and maturation. Completion of these aims will provide insights into the mechanisms of fistula maturation which may ultimately translate into novel clinical and therapeutic approaches in this extremely ill patient population. Furthermore, this work will be executed in a setting where I will gain enormous personal training experience by combining didactic coursework with clinical and basic research in a highly structured, actively mentored environment.


Two population genetic features of the human histocompatibility (HLA) system make it ideally suited for the study of evolutionary forces acting on a population. These are the high level of polymorphism exhibited by many of the loci of the HLA system, and the existence of significant linkage disequilibrium (non-random association) between certain pairs of antigens at different loci. The distribution of allele frequencies at the HLA loci, and the patterns of linkage disequilibria, are consistent with selective events acting in the region. This proposal involves: 1) population data analysis of a number of very large data sets, including the Caucasian and Japanese data from the 8th (1980) and 9th (1984) International Histocompatibility Workshops, Japanese data from the All Japanese HLA Workshops of 1983 and 1985, Chinese data from Singapore, Indian data from New Delhi, extension of our previous analysis of a large Danish study to include additional HLA loci, and the data that will be available from the Third Asia Oceania Histocompatibility Workshop (Sapporo, Japan, 1986); 2) simulation of two and three locus neutrality models, including migration, bottleneck and founder effects; 3) statistical analysis of the distributional properties of population wide measures of disequilibrium and application of bootstrap and jackknife techniques to determine confidence limits of the measures; 4) deterministic analysis of selection models; 5) comparison of generated population genetic parameters under neutrality and selection models with the HLA data; 6) correlation of HLA antigen disease association patterns with linkage disequilibrium patterns, including comparison of patterns in different racial groups, to study the evolution of disease predisposing genes. The nature of selective events acting in the HLA region will be delineated, and the mechanism by which disease predisposing genes become common in a population investigated. The methods to be developed have applicability to other multigene families, marker trait associations and restriction fragment length polymorphism data, in all organisms.


This research uses mathematical and computational techniques to determine the functional properties of local-circuit neurons, based on the well-founded results of cable theory, compartmental analysis, and biophysis of ionic fluxes across nerve-cell membranes. Five specific problem areas are addressed: (1) interaction of chemical and electrical synapses in neuronal circuits, (2) integrative properties of nonspiking neurons, (3) dynamics of reciprocal synapses, (4) functional roles of dendritic spines, and (5) properties of neurons exhibiting dendritic spikes. The aim of the studies is to provide realistic, quantitative, and consistent data for the behavior of these systems, so that both experimental and theoretical investigations can be rigorously based on solid theory, rather than on qualitative estimates.


The Epidemiology Branch is conducting a number of birth defect studies in collaboration with the Health Research Board and Trinity College, Dublin, Ireland. The main objective of these studies is to determine the relationship between folate and birth defects. The birth defects studied to date are neural tube defects (NTDs), oral clefts, congenital heart defects, Down syndrome and omphalocele. These studies focus on biochemical factors in the area of folate metabolism, and on genetic mutations in folate related genes associated with birth defects. In the past we have shown that elevated homocysteine is a risk factor for NTDs, that a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene 677C->T is a risk factor for NTDs, and that a small dose of folic acid (100-200 micrograms) can raise red cell folate to levels that can prevent a fifth to almost a half of NTDs. We have shown that methylenetetrahydrofolate reductase (MTHFD), an important gene in the production of purine and pyrimidine for DNA synthesis is a risk factor for NTDs. Mothers who have the R653Q variant of this gene are at increased risk of having a child with an NTD. [unreadable] [unreadable] We have expanded our work on MTHFD, confirming in a second population that the MTHFD R653Q variant is a maternal risk factor for NTDs. We have examined other polymorphisms and variants related to folate metabolism as potential NTD risks. The reduced folate carrier is essential for transfer of folate into cells. A variant of the enzyme gene, H27R, has been reported to be a risk factor for NTDs, but this is uncertain. We stuied this variant in a large Irish population and showed that it was unrelated to NTDs in cases, their mothers and their fathers. [unreadable] [unreadable] Both the MTHFR C677T and A1298C variants have been reported to reduce the risk for some cancers. The biochemical effect of the MTHFR C677T variant is clear--it reduces folate stores; however, the biochemical effect of the MTHFR A1298C variant is unclear, perhaps because it is in linkage dysequilibrium with the MTFHR 677C wild type SNP. We were able to measure the effect of MTHFR A1298C independently of the MTHFR 677 effect, showing the the A1298C variant produces increased cellular (red cell) folate levels. This is a rare example of SNPs on the same exon producing opposite effects and it suggests that MTHFR 1298C influences cancer risk by a different mechanism than MTHFR 677T. We continue to investigate the role of other genes as NTD risk factors.[unreadable] [unreadable] We recently explored the role of another critical folate enzyme gene, dihydrofolate reductase as a risk factor for NTDs. Previous work did not show a protective effect of a 19bp intron deletion in mothers of NTD cases. Our study demonstrated that in the Irish population either one or two copies of the allele was significantly protective (p=.01) in mothers. There was a small increase in expression, a 1.5 fold increase in mRNA, as well. The difference did not reach statistical significance. Thus, the presence of the 19bp intron deletion may be protective against NTDs when present in mothers.


Amphotericin B plays a critical role in the clinical treatment of life threatening systemic fungal infections. It is often considered the gold standard drug of choice in these instances due to its broad spectrum efficacy and the lack of alternatives. However, amphotericin B is also well known for its severe and potentially lethal side effects, including severe nephrotoxicity. Doctors are often left with the difficult choice of balancing the risk of infection with the risk of treatment. A number of strategies have been employed to decrease nephrotoxicity of amphotericin B including, but not limited to, generating glycosylated derivatives at a fixed position to improve solubility, purifying the analogs more stringently, and creating liposomal formulations. These efforts have met with limited success, decreasing toxicity to allow for longer treatment regimens, but also showing considerable different pharmokinetic characteristics compared to standard amphotericin B. Strikingly, medicinal chemistry has not had much success altering the characteristics of amphtotericin B. This is largely true because there are few positions in polyene natural products that are accessible to traditional medicinal chemistry. Still, the few published studies that generate even minor chemical modifications of the core scaffold demonstrate that it is possible to dramatically alter the activity and binding specificity of the compound, supporting the possibility that the antifungal activity of amphoterici B and its toxicity to humans can be decoupled. This would be of enormous value to the infectious disease field. Radiant Genomics believes that the enormous diversity of natural product gene clusters that can be identified through a combination of bioinformatics and next generation sequencing of metagenomes and individual organisms can now be mined to provide candidate natural amphotericin analogs that may have decreased toxicity while maintaining its antifungal properties. These natural analogs will have modifications and structural changes in the core amphotericin B scaffold that are impossible to duplicate through synthetic medical chemistry. Successful production and characterization of a range of natural amphotericin analogs will provide strong proof of concept that the Radiant Genomics natural product discovery platform can allow scientists to quickly interrogate the natural diversity surrounding a targeted natural product, an impossibility using traditional natural product discovery methods, and would revolutionize the field of natural product discovery. A less toxic version of amphotericin B would be primed to become the standard treatment for all systemic fungal infections.


The Illinois Center for Health Statistics, Illinois Department of Public Health, has submitted a grant application to CDC to continue its longstanding PRAMS survey as part of Illinois' effort to reduce infant mortality and morbidity, and improve maternal outcomes of its citizens. These survey data will guide maternal and child health policy and resource allocation, direct immunization strategies for reaching pregnant women, help determine the effectiveness of Medicaid and other health insurance funding related to deliveries, be used to promote breast- feeding, broaden our understanding of postpartum depression and strategies to address it, and be made available for a myriad of related research. Illinois has a proven record of achieving high response rates, especially for a larger state with a diverse population, and thus ensuring the usefulness of PRAMS data for an array of health programs, their policies and related research.


The epithelial barrier presents a significant obstacle to the delivery of macromolecules in the size range of 20 - 150 kDa. In particular, the tight junctional complex, which links adjacent cells and occludes the paracellular space, presents a significant obstacle to delivery of macromolecules. To improve the transport of macromolecular biologics across epithelia, new approaches need to be developed that enhance paracellular drug transport by specifically and reversibly modulating tight junctions. In this proposal, we investigate the effect of nanostructured surfaces on the modulation of tight junction permeability and transport of key therapeutic molecules in vitro. We seek to determine the mechanisms through which epithelial permeability is enhanced by nanotopography and optimize nanostructured materials to broaden the types of drugs that can be delivered paracellularly. It is expected that the fundamental knowledge gained in these studies will enhance the development of new epithelial drug delivery systems.


Perturbation of the metabolism of the brain has serious negative effects on the well-being and survival of the organism. The contribution of the various cell types in the brain to overall metabolism cannot be deter- mined by in vivo experiments. Growth of these sub-populations separately in tissue culture permits examination of their metabolism independently. Furthermore, the effect of varying the extracellular concentration of glucose to mimic conditions seen in vivo in pathologic conditions can be assessed on the separate cell populations. Results from such studies demonstrate that neurons and astroglia handle 2-[14C]DG in a similar fashion under normoglycemic (1.0 - 2.0 mM glucose), slightly hyperglyce- mic (5.0 mM glucose) and hypoglycemic (0.1 - 0.5 mM glucose) conditions; the intracellular:extracellular distribution of 2-[14C]DG is similar in both cell types. Results from studies on glucose were the same as that for 2-[14C]DG at normoglycemic and hyperglycemic levels of glucose. However, results with glucose in the hypoglycemic range were impossible to interpret due to the presence of an intracellular pool of glucose apparently separate from the metabolically active pool. Since the 2- [14C]DG appears to be in equilibrium with the metabolically active glucose pool , movement of glucose at the hypoglycemic levels will have to be inferred from the 2-[14C]DG data. Thus, the ability of neurons and astroglia to transport glucose into the cell's metabolically active pool does not appear to differ.


Pain is one of the most devastating, persistent and incapacitating symptoms in patients with cancer. More than eighty percent of patients with advanced cancer develop pain before death. The great majority of these patients will require opioid analgesics for appropriate pain control. Morphine is recommended as a first line opioid by the World Health Organization Cancer Pain Relief Guidelines. However, the active metabolites of morphine can accumulate and result in opioid induced neurotoxicity, and repeated administration will lead to the need for higher dose or opioid rotation. In addition, the price of morphine can be prohibitively expensive for developing countries, and therefore, most patients with cancer elsewhere in the world die without having ever received a single dose of strong opioid analgesic. Methadone has demonstrated its excellent oral bioavailability, rapid onset of analgesic action, a long life associated with prolonged analgesic effect requiring infrequent administration, lack of known active metabolites. Further, methadone is not opioid derivative, and is much less expensive. Our pilot randomized controlled trial demonstrates similar levels of pain relief in patients receiving methadone or morphine in a 4-week long trial. This leads us to hypothesize that over time the maintenance of pain control in patients receiving methadone due to its absence of NMDA production and opioid metabolite accumulation will result in less frequent opioid induced toxicity, dose escalation and treatment failure, and therefore, improved quality of life. Our long term goal of this study is to provide superior analgesic control for patients with advanced cancer. In this randomized, double blind, parallel study, we aim to determine the clinical efficacy (measuring pain intensity, toxicity, dose escalation, treatment failure and quality of life), to perform economic evaluation (comparing the costs and clinical benefits). 250 patients will be enrolled in the study, half of the patients will receive slow release morphine every 12 hours plus rescue opioid dose, and half will receive methadone every 12 hours plus rescue opioid dose for a total of 12 weeks. All patients will be titrated up to their optimal opioid dose level. Validated instruments will be used to assess pain, symptoms, toxicities and quality of life. Cost-effectiveness analysis will be performed using resource utilization, and medical and non-medical costs. Relevance to Public Health: In this study, we will test if methadone is better than morphine in cancer pain control, quality of life, and is more cost-effective.


The purpose of this study is to investigate by sonography the impact of intracatheter nitroglycerin infusion upon the incidence of vascular thrombosis following percutaneous placement of femoral venous catheters in critically ill children. We hypothesize that intracatheter nitroglycerin infusion will be associated with a lower incidence of thrombosis when compared to control catheters. The study will include a total of 62 children ages birth to 6 years of age. Two groups will be studied. Group 1 (nitroglycerin; n=31) will include critically ill children admitted to the intensive care unit in whom the primary care team has elected to place a double or triple lumen femoral venous catheter. Group 2 (D5W; n=31) will also include critically ill children admitted to the intensive care unit in whom the primary care team has elected to place a double or triple lumen femoral venous catheter. Ultrasound examinations of the catheterized femoral vein will be performed and analyzed by the Radiology investigator within 2 days of catheter insertion and a final study will be performed prior to discharge from the hospital. The primary outcome variable is the difference in the incidence of ultrasound-diagnosed thrombosis between the nitroglycerin and D5W groups. Secondary outcome variables include difference in clinical evidence of thrombosis between the two groups, difference in the incidence of catheter-related infection between the two groups, impact of TPN, intralipid, and heparin infusions on thrombosis in each group, and impact of catheter duration on thrombosis in each group.


This project involves the development of new methods for estimating dose-response relationships between environmental carcinogens and cancer morbidity, and the application of these methods to new and existing datasets. The high resolving power of the new nonparametric procedures will be utilized to better visualize the characteristcs of environmental exposure and cancer morbidity data. For purposes of validation, results obtained through use of these new procedures will be compared to previously completed analyses of existing data sets. New data from several morbidity studies will also be analyzed in order to further fieldtest, and ascertain the properties of, the new techniques.


Socioeconomic and other factors such as race, ethnicity, income, education, and place of residence are associated with reduced access to and poorer quality healthcare. Clinicians serving disadvantaged populations are often overloaded, and have little chance to gather a comprehensive biomedical and psychosocial (biopsychosocial, BPS) evaluation of patients. This situation is unfortunate because many physical symptoms are medically unexplained, reflecting a pattern of high symptom reporting or underlying psychosocial issues, such as depression, trauma, or poorly-tolerated stress. These BPS factors are particularly problematic for patients from disadvantaged populations. Lastly, severe, medically unexplained symptoms, psychosocial issues, and poor quality of life (QOL) occur together in a subset of at-risk individuals (ARI), which comprises about 20% of typical clinic populations. ARI are at risk for poor outcomes and high costs from inappropriate care, a problem that is compounded in disadvantaged and minority populations where access to primary care is limited and urgent care is more prevalent than preventive care. SOLUTION: Our objective is to deploy a clinical methodology and infrastructure to ensure delivery of patient-centered care to minority and disadvantaged patients. Our work builds on an online, patient self-assessment system called CarePrep, now operational in clinic. CarePrep allows patients to easily enter and track BPS and QOL data over the Internet from home or clinic. However, just handing a diagnosis of depression, for example, to a clinician does not change outcomes. Therefore, we will also deploy an integrated intervention to support BPS care. APPROACH: 1) We will adapt, enhance cultural sensitivity, deploy, refine, and initially validate CarePrep in a primary care setting. Our goals are to meet the needs of patients and clinicians for enhancing communication and patient- centered care, ensuring that BPS issues are uncovered. Patients will be asked to do CarePrep before clinic. We will assess validity, feasibility, and CarePrep's ability to accurately identify patients who warrant extra attention (ARI) by findings such as severe symptoms, psychosocial issues, impaired QOL, or substance abuse. 2) We will analyze and develop a plan for supporting culturally competent, BPS care using some combination of automated CarePrep functionality, training and supporting care managers or existing clinic staff in delivering simple, brief BPS interventions, and telehealth support. IMPACT stems from 1) creating a system that generates sufficient value to patients, clinicians, and administrators to warrant routine use; 2) creating a streamlined, branching assessment that minimizes redundancy and maximizes relevance, thereby supporting collection of the full spectrum of relevant data and delivery of context-relevant education and guidance; 3) using this technology and clinical methodology to transcend barriers to care for minorities and disadvantaged populations, and 4) identifying, characterizing, and targeting care to the patients with the greatest need and highest utilization within such populations, thereby facilitating cost-effective use of limited healthcare dollars.


In the United States there are approximately 10 million diabetics; over 75,000 diabetic cataracts are surgically removed annually. Lens aldose reductase has been implicated in the pathogenesis of sugar cataracts; therefore, inhibition of this enzyme may represent a therapeutic approach for the prevention or possible delay of cataractogenesis. Considerable work sponsored by NEI has established the in vitro activity of potential aldose reductase inhibitors. However, little is known about their effect in vivo. In our proposed investigation, we will employ an in vivo system to establish the concomitant protective effect of 3 types of aldose reductase inhibitors on both morphological and biochemical changes during sugar cataractogenesis. The 4 lenticular parameters are: lens growth, fiber structure, protein components (alpha, beta and gamma crystallins) and sugar levels. These parameters can be quantitated; they provide a comprehensive index of lens integrity. These parameters will be correlated with clinical findings (slit lamp and blood sugar levels). Then the effect of aldose reductase inhibitors on normal lens physiology will be analyzed in a similar manner. In order to determine which structure provides maximal therapeutic activity in vivo, dose response studies will be performed on a flavonoid, quercetin, a sphirohydantoin, CP, 45, 634, and a third structure ALO-1349. Our in vivo system is the rat neonate. The fused eyelids provide an orbital pouch for the topical administration of aldose reductase inhibitors. The reasons for selecting this model are explained on page 18. In addition to the clinical relevance, this work will test the hypothesis of lenticular aldose reductase in vivo and provide a model for further investigation of lens therapy.


The Department of Obstetrics and Gynecology at the University of Tennessee, Memphis has been part of the Maternal-Fetal Medicine Network since April 1986. With this application, we hope to participate in a large multicenter network designed to develop and conduct clinical trials in the field of Maternal-Fetal Medicine, which could not be undertaken in a single center. In comparison to our previous application, we have recently expanded our resources and facilities to include patients at all major hospitals in the city. The obstetric population will now total approximately 13,000 women of various ethnic and economic groups. We are particularly interested in pursuing trials which require large sample size in order to adequately address specific questions and those regarding rare events of obstetric interest. The Principal Investigator, Dr. Baha Sibai, and the Alternate Principal Investigator, Dr. Brian Mercer, as well as the faculty in the Division of Maternal-Fetal Medicine continue to be active in Network administrative activities, and the design and conduct of protocols. The Principal Investigator currently participates on the Concurrent Research Committee, the Ad Hoc Committee on Preterm Studies, and the "High-Risk" Aspirin Protocol, the Interim Progesterone, and the Preliminary Terbutaline subcommittees. The Alternate Principal Investigator chairs the "Premature Rupture of Membranes" protocol and the Chart Review Subcommittee. He serves on the Capitation Subcommittee, Preterm PROM Pathology Subcommittee, Preterm Prediction Protocol Subcommittee, Obstetrical Determinants of Neonatal Survival Protocol Subcommittee, Neural Tube Defect Protocol Subcommittee and the Preliminary MgSO4 Subcommittee. He has recently submitted for consideration a clinical trial regarding tocolytic, corticosteroid and antimicrobial therapy after PROM. We are applying to continue as a clinical research center within the Network and agree to join protocols in existence and participate in the design of protocols in cooperation with other centers selected by the NICHD. The University of Tennessee, Memphis, and the Department of Obstetrics and Gynecology are committed to collaborative Maternal-Fetal research as documented by listed publications and the enclosed letters of commitment. The Division of Maternal-Fetal Medicine agrees to cooperate with the policy of capitation of research costs for each individual protocol, in addition to a base budget.


This proposal seeks to determine the role of the secondary heart field in the morphogenesis of the outflow tract during cardiogenesis in the mouse. The specific aims are 1) To establish the basal proliferation and apoptosis rates in different areas of the outflow tract and secondary heart field during normal mouse development. 2) To establish the fate of labeled cells that originate in the secondary heart field in a mouse model in which there is loss of Fgf8 activity in the secondary heart field. 3) To determine the effect that loss of FGF8 signal in the secondary heart field derived outflow tract cells has on apoptosis and proliferation rates in the outflow tract. We hypothesize that the secondary heart field cells have a unique and previously unrecognized role in the morphogenesis of the outflow tract. The fates of cells derived from the secondary heart field during normal heart development will be determined and compared with those in a model of conotruncal defects. This will help elucidate the role of the secondary heart field in the remodeling and septation of the outflow tract. [unreadable] [unreadable]


Project Summary: The goals of this application are to understand to what extent replication fitness influences the emergence of HIV variants resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs), and to delineate the underlying biochemical mechanisms for how drug resistance mutations interact to influence replication fitness. NNRTI-resistant variants of HIV, usually cross-resistant to other NNRTIs, develop rapidly if viral replication is not suppressed. We have shown that HIV replication fitness, as measured in cell culture, is reduced in NNRTI-resistant mutants that are infrequent clinically, even though they confer higher levels of resistance than the most common mutant, K103N. These poorly replicating mutants also reduce RNase H cleavage by reverse transcriptase. Two mutants, G190S and A, also reduce priming from tRNA(Lys,3), raising the question of whether defects in RNA priming also contribute to the reduced fitness of NNRTI- resistant mutants. We also have shown that the nucleoside (nRTI) resistance mutation L74V compensates for the reduced fitness of K103N+L100I, but does not improve its reduced RNase H cleavage rates. Published studies by others have shown that L74V reduces RNA priming. We therefore propose that L74V can compensate for the reduced RNase H activity of poorly replicating, highly NNRTI-resistant variants by improving steps in reverse transcription, such as strand transfer and strand displacement synthesis, that compensate for RNase H cleavage defects. We postulate that selection for highly NNRTI-resistant variants that have improved fitness from compensatory nRTI resistance mutations can lead to early virologic failure. These studies have important implications for the more rational design of nRTI-NNRTI combination regimens. During the next funding period, we plan to: 1. Evaluate the association of nRTI and NNRTI resistance mutations in clinical samples. 2. Determine the effects of nRTI resistance mutations on the replication fitness and drug resistance of NNRTI-resistant mutants. 3. Characterize the biochemical basis for how L74V improves the fitness of NNRTI-resistant variants. Relevance: The proposed studies will evaluate what factors lead to the development of drug resistant strains of HIV, how changes in reverse transcriptase function affect replication of HIV, and whether combinations of drugs can be chosen to influence which drug resistant strains develop.


Literacy requires a prior set of emergent literacy skills that, in turn, require both print-related experiences and a foundation of early oral language competence. For children from Spanish-speaking homes, limited exposure to English and/or limited oral language competence in either Spanish or English may limit the development of their emergent English literacy both directly and indirectly. The eventual results of these limitations contribute to academic underachievement (Goldenberg, 1987), a persistent problem in the US Hispamc population in which only 63% of students graduate from high school (Healthy People 2000). These limitations may be ameliorated by early exposure to English language in addition to an enriched home language environment. The Bilingual Early Language and Literacy Support (BELLS) project was funded by NIH and OERI (Contract# 5-R01- HD39501) to test the language and emergent literacy outcomes of Spanish-speaking/bilingual children who either were enrolled in an early childhood program that included English exposure/immersion and home language and literacy support or were in a community, where limited early childhood experiences were available. BELLS was funded to assess children up to Kindergarten entry. However, many of the children enrolled in BELLS will be entering kindergarten prior to the end of the BELLS project and many will be in first grade when the project ends. The literature supports longitudinal research that follows children from early childhood into the school setting (Dickinson & McCabe, 2001). The purpose of this supplemental study will be to follow BELLS children into Kindergarten and first grade. This will allow us to examine school and home pathways to reading achievement.


The objectives of the project remain to study the concept of collagenolytic activity as a proximal event in the acquisition of an invasive potential by tumor cells and to define inter-relationships between tumor protease (collagenase) activities, host protease (collagenase) inhibitor(s) and tumor growth characteristics (non-invasive vs. invasive growth) in cell lines derived from chemical carcinogen (FANFT)-induced transitional cell carcinomas arising in the urinary bladder of Fischer rats. Normal epithelial cells, microvascular endothelial cells, and fibroblasts will be studied for their involvement in the regulation of the connective tissue turnover at the epithelial-stromal interphase (protease (collagenase) activities; protease (collagenase) inhibitors) and invasive carcinoma cells analyzed for mechanisms involved in the degradation of connective tissue barriers.


The objective of this proposal is to develop ultra-sensitive ELISA (Enzyme-Linked ImmunoSorbent Assay) methods for quantification of HIV p24 antigen. At present, the p24 antigen test is relatively insensitive, being able to detect only 5- 10 pg/ml. This quantity of antigen may not be present in the serum of infected individuals, even when the virus is actively replicating. In fact, only about 50-60% of AIDS patients, 30-40% of ARC patients, and 10% of asymptomatic patients will have p24 antigenemia that is detectable. Therefore, the development of more sensitive p24 antigen tests is of great importance. It is in this context that we are proposing to develop a highly sensitive p24 antigen assays that will be less expensive than RNA viral load tests and appropriate for use both in developed and developing countries in which the limitations of infrastructure and laboratory capability prohibit nucleic acid testing. The specific aims of the Phase I and Phase II, in part, include 1) analyzing and optimizing of all variables affecting the accuracy and functionality of the p24 tests;and 2) validation of the HIV-1 p24 detection assay. Advantages of the proposed assays such as the increased sensitivity, signal-to- noise ratio, and dynamic range, reduced time and low cost of the analysis substantiates our belief that the proposed amplification method has a prominent commercial and scientific potential. PUBLIC HEALTH RELEVANCE: The development of the proposed method will allow using HIV p24 antigen tests for monitoring HIV infection, blood screening, identification of acute infection, to assist in the diagnosis of infection in the newborn, and in detecting antigen in supernatants from cultures. The performance of the developed tests will be improved significantly as compared with that for conventional assays. Successful completion of these studies will be beneficial for public health, and make available all of the technology needed for a substantial business opportunity to license the technology and manufacture commercial products.


Recent work from several laboratories has shown that murine gamma interferon induces the biosynthesis and cell surface expression of immune-associated (Ia) glycoprotein determinants on both normal and neoplastic mouse mononuclear phagocyte lineage cells. Experiments carried out by the principal investigator demonstrate that gamma interferon activates a murine macrophage tumor cell line, (P-388.D1), to release a second, noninterferon factor activity which, in turn, stimulates Ia expression on mouse macrophage tumor cells. The major goal of this research proposal is to test the hypothesis that gamma interferon regulates the positive expression of macrophage Ia determinants by stimulating the induction of a second group of Ia-inducing factors (IaIF) by the macrophage which subsequently control the cell surface expression of individual Ia subregion determinants. Thus, a variety of murine macrophage tumor cell lines and normal mononuclear phagocyte subpopulations will be cultured in vitro using different Ia induction procedures, and conditioned medium from these cells will be tested against neoplastic and normal Ia negative macrophage target cells for the ability to induce Ia expression. Quantitation of Ia induction will be carried out using direct and indirect immunofluorescence with anti-Ia monoclonal antibodies and flow cytometry analysis (FcM). Where active IaIF supernatants are found, they will be examined using standard biochemical analysis techniques in an effort to characterize the material and show if there are multiple IaIF activities which control the expression of discrete Ia subregion glycoproteins. Normal or neoplastic macrophages which are induced by IaIF activity for one or more Ia subregion antigens (i.e., I-A, I-E/C, or I-J), will be studied for functional activity as antigen-presenting cells in T-cell proliferation and activation assays using MHC-restricted, antigen-specific, continuous helper T-cell lines, or T hybridomas as effector cells. I-J-inducible macrophages will be tested as accessory cells in the induction of a primary in vitro burro red blood cell IgM response, and as antigen-presenting cells in the activation of third-order suppressor T cells (Ts 3) in the azobenzenearsonate (ABA) system. This study will provide basic information describing the mechanism by which gamma interferon may regulate the cell surface expression of individual Ia subregion antigens on phenotypically and functionally unique subsets of mononuclear phagocytes. (SR)


This is an application requesting support for the 6th Gordon Research Conference (GRC) on: In Vivo Magnetic Resonance (MR). This meeting will cover an array of topics spanning the use of MR imaging and spectroscopy in cells, animals, and humans;addressing both basic science and clinical research issues. Particular emphases will be placed on emerging technological, biological, and health related matters that will significantly impact the growth of this remarkable imaging field. The 6th Conference will be held July 25 - 30, 2010, at Proctor Academy, Andover, NH. The Chair of the 6th Conference is Risto A Kauppinen, MD PhD, Director of the Biomedical NMR Research Center and Research Professor of Radiology, Dartmouth College, Hanover, NH;the vice-Chair is Craig Stanisz, PhD, Associate Professor, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. The 5th In Vivo MR GRC was held in 2008 in the same location. The previous Conference was extremely successful. Significant advances in instrumentation, imaging speed, and in a wide array of advancements in coil and gradient technologies continue to create a rich environment for rapid growth of MR and its in vivo applications. It is both timely and necessary to gather together the leading individuals and future investigators to address, in depth, as is done at no other scientific venue, the next ventures of this important imaging science field. The In Vivo MR GRC will include scientific sessions, each with two to four speakers and a discussion leader. There will also be four poster sessions (from experience gathered in previous GRCs most participants will present a poster) and discussions designed to facilitate close interaction between the participants. By design, GRC meetings are small by conventional standards (approximately 140-150 participants) but participants will be invited/accepted in a manner to ensure attendance by a diverse group of junior and senior investigators representing academic and government institutions and individuals from corporate research programs. Students and postdoctoral fellows will be encouraged to attend. Junior investigators will be given opportunity for oral presentations. PUBLIC HEALTH RELEVANCE: Support is requested for the 6th Gordon Research Conference (GRC) on :In Vivo Magnetic Resonance: (July 25-30, 2010). Magnetic resonance (MR) techniques play a critical role in the delivery of high quality health care and MR is an essential player in the modern molecular medicine. The GRC meeting will emphasize emerging and cutting-edge technological, biological, and health related issues that will significantly impact the growth and application of this remarkable imaging field.


Type 1 pili of Escherichia coli and other gram negative enteric bacteria are filamentous, proteinaceous appendages composed primarily of a subunit called pilin. Type 1 pili mediate the mannose-sensitive adhesion of E. coli to a variety of eucaryotic cells and are a factor in promoting colonization in the lower urinary tract. The long term goal of this project is to establish the molecular nature of the genetic control, assembly and receptor binding activity of type 1 pili and in doing so, contribute to the understanding of the control and assembly of supramolecular structures and receptor-ligand interactions in general. Also, it is hoped that this research will lead to a better understanding of the role of bacterial adhesion in the pathogenesis of infectious disease. The research herein proposed uses a variety of genetic and biochemical techniques to: 1) Discern the factors effecting pilA expression. The pilA gene encodes the major subunit of pili and is subject to at least two types of transcriptional control. One type is a repression effected by the product of an adjacent gene, the other type is a metastable (ON or OFF) control effected by the inversion of the pilA promotor. Ways are proposed to create and analyze lesions affecting these two types of transcriptional control. 2) Discern the molecular interactions required for pilus assembly. Methods are suggested for the analysis of mutants having point mutations in pilE, a gene encoding a minor component of pili that is required for receptor binding. Projects include the isolation of extrageneic compensatory mutations, analysis of conditional pilE mutations and localization of the pilE protein. In addition, methods for isolating and examining mutants having lesions affecting two other assembly related genes, one of them affecting pilus length, are proposed. 3) Discern the molecular interactions required for receptor binding. Methods are outlined for isolating and analyzing mutants with an altered receptor specificity. Also, ways for isolating the pilE gene product and assessing the role of the pilE protein in receptor binding are outlined. In addition, the role of receptor binding and pilus length on the interaction of piliated E. coli with macrophages is examined.


Investigators who need to use morphological techniques frequently do not have the facilities in their own laboratories. In addition, project personnel, especially young investigators, often come to the Council without training in these techniques. The Morphology Core provides a number of avenues which enable investigators to produce high quality work involving light and electron microscopy, immunocytochemistry, in situ hybridization, and photography.