This application proposes to continue the participation of investigators at Thomas Jefferson University in an interactive network of six centers (the Asthma Clinical Research Network or ACRN ) in conducting studies of novel therapeutic approaches to asthma and in disseminating findings to the practicing community. The need for such a network was suggested by epidemiological data showing increases in mortality, morbidity, prevalence, and costs of asthma, by clinical and basic research studies showing that asthma is linked to inflammation in the airways, and by the accelerating rate of development of potentially effective, but also potentially costly treatments for asthma. Defining the place of these therapies was seen as requiring collaborative, multi-center studies examining large numbers of subjects reflecting the diversity of the U.S. population. In its first 5 years, the ACRN established an infrastructure to meet this need and has added another center at Harlem Hospital in New York, which serves a predominantly minority population. The ACRN has completed studies of the effects of regular beta-agonist use in mild asthma ( BAGS ) and of the efficacy of the anti-inflammatory agent, colchicine, as an alternative to an inhaled corticosteroid in moderate asthma. Now underway are two additional trials comparing the effects of a long-acting beta-agonist, an inhaled corticosteriod, and the combination of the two in altering clinical and physiologic outcomes, and airway inflammation in moderate or severe asthma. A fifth study, establishing doses of different inhaled corticosteroids with equivalent effects on cortisol secretion, is imminent. Completed trials as well as 10-12 ancillary studies designed to improve the performance of clinical research have been presented at meetings of the ATS, ACCP, and AAAAI. Three of these studies have thus far been published in peer- reviewed journals. The ACRN has also reported the results of a subgroup analysis of subjects in the BAGS study showing that subjects with different genotypes for the beta-adrenergic receptor are differently affected by regular use of albuterol. This application proposes continued participation of the Jefferson Center in the multicentered, collaborative trials of the ACRN. Proposed studies include a comparison of the efficacy of doses of different inhaled corticosteriods with equal systemic effects (as estimated from the study described above), a prospective study of the effects of regular use of an inhaled beta-agonist in subjects stratified by genotype for the beta-adrenergic receptor, and a study of the efficacy of a leukotriene pathway antagonist in enabling reduction or elimination of inhaled corticosteriods in subjects with mild or moderate persistent asthma. Other planned studies are also briefly discussed, with the understanding that they could be modified or replaced in response to new information, new therapies, or changing clinical research priorities.


In Orientals, ALDH2 deficiency due to a common polymorphism frequently causes a flushing reaction after alcohol consumption and this aversive reaction is responsible for lower rates of alcoholism in individuals with the inactive ALDH2-2 allele. ALDH2 deficiency was detected in South American Indian populations; however, these findings have never been confirmed and a previous search for the Oriental ALDH2-2 allele in South American Indians was negative. Recently R. Peterson has identified a series of additional markers at ALDH2. By restriction enzyme analysis, SSCP and sequencing enabled him to identify haplotypes characteristic for the ALDH2-2 and ALDH2-1 alleles in different populations. This analysis is shedding light on the origins and functional role of the variant ALDH2-2 allele, which appears to have appeared on a single haplotype lineage and spread among East Asian populations. Although ALDH22R [Glu487Lys] probably originated on a single genetic background, haplotype analysis reveals that it is sufficiently ancient for additional mutations to have occurred subsequently. This result is most compatible with an effect of selection to maintain the Oriental ALDH2 variant, Glu487Lys.


Converging lines of research indicate significant sex differences in schizophrenia. Females have a more benign course, typified by later age of onset, better premorbid adjustment, more affective symptoms, fewer cognitive deficits, better neuroleptic response, shorter hospital stay, fewer relapses, lower risk for suicide, lower family Expressed Emotion (EE) and better social and work adjustment. Nevertheless, evidence of sex differences tends to be underemphasized and hypotheses concerning sex differences have rarely been investigated in a systematic, prospective study. AIMS: This study will assess: (1) sex-related variation in the clinical symptom pattern of schizophrenia; (2) sex differences in premorbid factors and precipitants of onset; (3) sex-related parameters of posthospital recovery and relationship to family environment factors; (4) prominence of sex-related differences at early and later phases of the illness; and (5) the relative influence of neurocognitive, neuroanatomic and family environment factors in prediction of two-year recovery/relapse rates for first- admission, and more chronic, males and females. METHODS: Study 1. From a pool of approximately 1100 annual inpatient admissions, a sample of approximately 700 consecutive admission psychotic patients will be identified over a 30-month period and given a standard (SCID) interview. Patients who meet broad-band (New Haven) criteria for schizophrenia will be cross-classified on more narrow-band (RDC and DSM-III) criteria and the sex ratio will be compared across diagnostic systems to determine the extent of differential exclusion by sex. Study 2. 300 (150 early- and 150 later-phase) patients who meet DSM-III criteria for schizophrenia spectrum disorders will be assessed on premorbid, neurocognitive, neuroanatomic (VBR), and family environment measures to identify sex-related parameters of psychopathology. Study 3. The same 300 patients will be studied longitudinally in order to determine sex differences in neurocognitive, neuroanatomic and family environment factors and their relative importance in predicting recover/relapse and functioning for males and females over the two-year post-admission phase. SIGNIFICANCE: This study will contribute to (1) general knowledge and practice in the diagnosis and classification of schizophrenia; (2) understanding of the pathogenesis and course of illness; (3) increased accuracy of prognosis and selection of treatment strategies for males and females.


CORE 2: DRIVING BIOLOGICAL PROBLEMS Three driving biological problems will focus our biocomputation research. A macroscale DBP focused on neuroprosthetic dynamics, a mesoscale DBP focused on viral and cellular dynamics, and a molecular scale DBP focused on drug target dynamics. We use seven criteria to select our Driving Biological Problems (DBPs): Canonical: The problem should be an archetype of problems in an entire field of inquiry to guarantee broad applicability of the tools we develop. [unreadable] Collectively cover a range of scales: It is critical that activities of the center cover scales from molecular through cellular to organismal levels. [unreadable] Physics-based: The DBP should present a problem that can be addressed by representing and analyzing the geometry and physics of the biological system. [unreadable] Data rich: Biology is dominated by experimental data. These data provide the real-world constraints that drive and validate models and simulations. [unreadable] World-class, engaged experimentalists: We seek close integration and deep interactions among the biological and computational participants. [unreadable] Have important implications for disease: We ensure that our DBPs are related to disease processes or treatments to ensure that they contribute to the advancement of human health. Our past DBPs met these criteria and enabled us to develop software that is now used by a very broad community of researchers. Our next three DBPs also satisfy these criteria and are briefly reviewed below.


This project is aimed at studying renal lymph in experimental hypertension and in experimental renal failure. Essential hypertension in man frequently leads to a gradual deterioration of renal function which is a consequence of both functional and structural vascular damage in the kidney. Similarly, circulatory factors have been shown to play an important role in the pathomechanism of acute and chronic renal failure. In this research alterations of the renal microcirculation in experimental hypertension and renal failure will be investigated. This will be accomplished by studying the renal lymph and obtaining information on: 1. capillary permeability to macromolecules, primarily albumin, 2. total lymph drainage of the kidney, and 3. abnormal constituents in lymph that may reveal the extent of cellular injury. In addition, structural alterations of the renal microcirculation as seen by light and electron microscopy will be correlated with the functional studies. The morphological investigation will be carried out by the Department of Pathology of this school.


The objective of this laboratory is to develop techniques for quantitative 3D light and electron microscopy and to make these available to the scientific community for research and education. A 400 kV transmission electron microscope allows examination of specimens 10-20 times thicker than usual and has been modified for an unlimited range of specimen tilt angles for 3D imaging. A laser scanning confocal light microscope provides optical sectioning in thick specimens, including wet and living specimens, superior to that obtainable using ordinary light microscopes. The foci in the core research are quantitation, comparison of images using different imaging modalities, and the evaluation of the effects of preparation methods and other potential sources of artifacts. Images are processed using image analysis programs imported from other sources or developed here. Programs are available for reconstruction from serial section images and from images taken at various tilts (including stereo images). These programs provide 3D localization of particulate markers (gold particles), reconstruction of membranes and other surfaces, and quantitative analysis of networks. Initially, manual processes are implemented. We plan in the longer term to automate as many processes as possible. Current collaborative research projects are focussed on the localization of surfaces and surface receptor molecules in motile cells, the organization of the cytoplasmic matrix, the structure of protein networks, the structure of the transverse tubular system in skeletal muscle, and the development of the fibrillar system in differentiating cardiac and skeletal muscle cells. Service projects cover a broad range of topics. Users have come from several surrounding states and from Japan. Training is done on-site at the facility, and through workshops presented at the facility and elsewhere. Dissemination of information on the facility is done through publications, lectures and, posters presented at national and international meetings, and direct mailing to biomedical scientists.


This Project will characterize the role of nitric oxide (NO) and NO-derived species in mouse models of inflammatory bowel disease (IBD) and cancer. These studies will use 129/SvEv Recombinase-activating gene 2 (Rag-2) knockout (KO) mice and C57BL/6 T cell receptor (TCR) alphabeta KO mice. Rag-2 KO mice on a 129/SvEv background develop severe IBD and cancer when infected with H. hepaticus. Histopathologic lesions and the pattern of macrophage and neutrophil infiltration will be correlated with biomarkers for nitration, oxidation, and halogenation of DNA and proteins in Rag-2 KO mice. These biomarkers will be further validated by comparing disease in naive and effector CD45RB high T cell-bearing Rag-2 KO mice, and by modulating NO production with N-methylarginine (NMA). TCR alphabeta KO mice develop severe IBD when infected with H. hepaticus. IBD in TCRbeta KO mice, like in Rag-2 KO mice, is a model for Crohn's disease (CD) and features activated macrophages and abundant interferon-gamma (IFN) and tumor necrosis factor-alpha (TNF). TCR alpha KO mice are a model for ulcerative colitis (UC), and are dependent on interleukin-4 (K,-4) but not IFN or TNF for the development of disease. Histopathologic lesions and cytokine levels will be correlated with biomarkers in TCR alpha and TCR beta KO mice. To further characterize the role of NO and NO-derived species in IBD, we will compare pharmacologic inhibition of inducible nitric oxide synthase (iNOS) with genetic inactivation of the enzyme by generating iNOS-deficient TCR alphabeta KO mice. In vitro analysis of somatic mutations arising in vivo in the presence and in the absence of NO and NO-derived species will also be performed. By using a novel IBD associated cancer model and models for UC and CD, we will be able to validate new biomarkers for nitration, oxidation, and halogenation of DNA and proteins. We will also gain a better understanding of the role of NO and NO-derived species across a broad range of carcinogenic events, including genotoxicity, cellular proliferation, cytotoxicity, and angiogenesis. The Specific Aims are: Specific Aim #1. Characterize the role of NO an oxidative stress in IBD and cancer in Rag-2 KO mice Specific Aim #2. Characterize the role of NO and oxidative stress in IBD in TCR alphabeta KO mice


Transcription will be studied in intact nuclei isolated from tissue culture cells. Products are characterized by size, rapidly hybridizing content, and sensitivity to selective inhibitors. Polymerase systems will be further characterized by their response to prior treatment of cells with specific inhibitors. Nuclei will be sub-fractionated to identify the location of the different polymerase systems in specific structures. The organization of transcription in adenovirus infected cells will be compared to normal cells. The location of adenovirus genes in different polymerase systems in rat embryo transformed cells will be measured. Additional specific inhibitors of heterogeneous RNA synthesizing systems in vivo and in vitro will be sought. The regulation of translation of mRNA at the level of initiation will be studied further. Initiation of adenovirus mRNA will be studied, both in vivo and in vitro. The nature of histone synthesis regulation of DNA synthesis will be studied in synchronized cultures. The mechanism of inhibitors of initiation, such as poliovirus and 5- azacytidine will be studied in adenovirus infected cells, where specific mRNA is identifiable and in vitro studies of initiation may be possible, and in immunoglobulin and growth hormone producing cells. The effect of the inhibitors and such clinically significant compounds as BCNU on the initiation factors will be studied in the adenovirus in vitro system. The messenger RNA lifetime for general protein synthesis and for specialized proteins will be studied using cordycepin, which gives significantly different results than actinomycin. The effects on RNA, protein and DNA metabolism of nucleoside analogues and compounds used in clinical cancer chemotherapy will be examined in detail. Both base and sugar substituted analogues of adenosine and uridine will be studied for their selectivity amongst different polymerase systems and for their effect on RNA metabolism. The inhibition of DNA synthesis and of the initiation of translation of the alkylating agent, BCNU will be studied further.


Research on cannabinoids has progressed tremendously since the discovery of specific recognition sites (receptors) for chemicals like 9-tetrahydrocannabinol (THC), the main active ingredient in marijuana. The endocannabinoid signaling system (ECS) thus likely serves as the biological substrate for the marijuana high. This subjective state presumably underlies human marijuana consumption that may lead to compulsive cannabis intake and dependence disorders. Drug discrimination is a powerful, pharmacologically selective model for assessing subjectively experienced drug effects in animals (and man) and is the major in vivo behavioral technique in this application. Cannabinoid receptor CB1 (CB1R) agonists and antagonists will be trained in drug discrimination using different doses, providing for in vivo assays with different sensitivity levels. This is complemented by observational studies and schedule controlled responding allowing for an in depth characterization of ligands. A major aim of this research is to identify new medications that will translate into better pharmacotherapies for combating marijuana addiction. Alleviation of withdrawal-related effects in physically dependent individuals likely is an important motivational factor in addiction processes. New molecules are designed and synthesized by on site expertise. One focus is to identify and refine in vivo neutral CB1R antagonists. Most current CB1R antagonists also display intrinsic activity (inverse agonism) that may hamper patient compliance in treatment settings. Thus, the studies will expand our understanding of the ECS, comprised of two major endogenous signaling molecules, anandamide (AEA) and 2-arachidonoylglycerol (2-AG) acting on two known receptors, CB1 and CB2, in normal body function and pathophysiology. Additional therapeutic targets for the application concern ligands selectively affecting the enzymes involved in the deactivation of the endocannabinoids, thus potentially avoiding direct receptor activation. The behavioral studies are aided by neuro/biochemical procedures provided by collaborating faculty in pursuing these goals. In addition to AEA, recent developments regarding 2-AG allow for a much more comprehensive understanding of this major endocannabinoid in ECS signaling. By obtaining information on the functions of the endogenous substances (AEA and 2-AG) and the exogenous THC, as well as in vivo neutral blocking agents, these studies will not only further our understanding of ECS signaling in the behavioral neurobiology of cannabis abuse / dependence, but may also lead to the development of effective medications for treating disorders involving cannabis / marijuana, and designer THC-like cannabimimetics.


Activation of cellular tumor suppressor pathways is the cell's major defense against cancer induced by activated oncogenes. The ARF-p53 tumor suppressor pathway, which is one of the most important in mammalian cells, can be activated by a number of viral and cellular oncogenes. Activated ARF induces a p53-mediated block to cell division via a cell cycle arrest or apoptotic cell death. How oncogenic stress activates ARF remains to be elucidated. Polyoma virus middle T-antigen (PyMT) is a potent oncogene able to bind a number of key regulatory cellular proteins and activate a number of important cellular signaling pathways including the ARF-p53 tumor suppressor pathway. We will use PyMT as a model oncogene in order to better understand how ARF is being activated. We hypothesize that PyMT induces ARF by the inappropriate activation of one or more cellular signaling pathways that also mediate the ability of PyMT to transform cells. Our plan is to identify the cellular signaling pathways induced by PyMT that results in activation of ARF. REF52 cells differ from most other established cell lines in containing an intact ARF-p53 tumor suppressor pathway and are distinct in resembling primary cells in their requirement for oncogene cooperation for their transformation. PyMT activates the ARF-p53 pathway, blocking REF52 cell division and will not transform REF52 cells in the absence of a co-operating oncogene. We plan to take advantage of these unique properties of REF52 cells to isolate PyMT and cell mutants that are involved in the activation of ARF. Three interrelated aims will be pursued. Aim-1 will be to use previously isolated PyMT mutants to identify which domains of PyMT are required to activate ARF. Aim-2 will be to use mutagenized PyMT to identify sequences in both defined and undefined regions of PyMT that are required for the activation of the ARF-p53 pathway in REF52 cells. Aim-3 will be to isolate and define REF52 cellular mutants in which PyMT signaling fails to activate the ARF-p53 tumor suppressor surveillance pathway. We believe that such cell mutants will help to differentiate between normal and oncogene activation of important cellular signal transduction pathways. Understanding the mechanism(s) of oncogenic activation of the ARF-p53 tumor pathway will help in designing better drugs and therapies for the treatment of cancer.


People living with HIV/AIDS (PLWH/A) disproportionately smoke cigarettes, putting them at risk for many adverse health outcomes, yet little is known about how to tailor cessation interventions for PLWH/A. Oregon's AIDS Drug Assistance Program (ADAP), known as CAREAssist, serves more than 2,100 low-income PLWH/A by assisting them with health insurance coverage and providing co-pays and prescription drugs. Survey data from 2006 indicated that the smoking prevalence among CAREAssist clients was high (42%), but that most smokers wanted to quit (72%) and were interested in using medicines for stopping smoking if available at no cost (83%). Recognizing an opportunity, CAREAssist in 2008 integrated cessation pharmacotherapy into its drug formulary, making it available at no cost to clients whose insurance plans would not pay for them, and the program started paying for counseling through the Oregon Tobacco Quit Line. Despite the removal of these financial barriers, preliminary data from November 2009 suggests the smoking prevalence among CAREAssist clients is virtually unchanged from 2006. The overall goal of the proposed project is to improve understanding of barriers to CAREAssist clients using free cessation pharmacotherapy and counseling services in order to inform the development of an intervention that would integrate additional cessation support into the integrated HIV system of care in Oregon. Existing survey data on over 1200 CAREAssist clients will be analyzed and in-depth interviews will be conducted with 15 HIV case managers, 15 medical providers, and 45 CAREAssist clients to describe:1) current smoking, quit behaviors, and utilization of prescribed pharmacotherapies and the Quit Line among CAREAssist clients by demographics, housing instability, substance abuse, overall physical health, overall mental health, co-morbidities, adherence to HIV treatment, visits to medical providers, practical and social support, and smoking behaviors among their social networks; 2) client-level barriers to utilizing cessation services, taking pharmacotherapies, and successfully quitting smoking; 3) current HIV medical provider and HIV case manager knowledge, attitude, and practices regarding tobacco cessation; 4) HIV medical provider, HIV case manager, and system-level barriers to screening for smoking behaviors among PLWH/A, providing cessation counseling and referrals, prescribing cessation pharmacotherapies, and integrating these pharmacotherapies into HIV treatment regimens. Project relevance: PLWH/A disproportionately smoke cigarettes, putting them at risk for many adverse health outcomes, yet little is known about how to tailor cessation interventions for PLWH/A. The goal of the proposed project is to improve understanding of the barriers to Oregon AIDS Drug Assistance Program (ADAP) clients using free cessation pharmacotherapy and counseling services. This information will be used to inform the development of an intervention that would integrate additional cessation support into Oregon's HIV system of care and could be implemented with ADAP populations across the nation.


The aim of this research training program is to contribute to a knowledge base about health behavior that: (a) promotes maximal well-being; (b) facilitates early detection of disorder to minimize its effect; and (c) enhances quality of life in illness situations. Research training will be provided annually to three predoctoral and three postdoctoral trainees that develops the following research skills: (a) analysis of concepts and theories from the behavioral and biological sciences that are relevant to health behavior, health behavior change, and quality of life in chronic illness; (b) synthesis of concepts, theories, and research findings from the behavioral and biological sciences to frame research concerned with enhancing quality of life in persons with chronic illness; (c) developing and testing instruments that measure concepts related to health behavior and quality of life in chronic illness; (d) design and conduct of research to advance knowledge related to the development and testing of tailored health behavior interventions; (e) interdisciplinary research collaboration; and (f) scientific writing, research application preparation, scientific presentations, and publications. To provide trainees with such an experience, Indiana University School of Nursing will: (a) recruit diverse and qualified trainees; (b) provide a high quality research training program, with student access to accomplished interdisciplinary mentors; and (c) provide a research-intensive environment, with an expanding portfolio of faculty health behavior research. Important advancements of this research training program for the next five years are: a greater interdisciplinary focus with a larger number of interdisciplinary faculty available as research mentors; a sharpened focus on chronic illness across the lifespan; and an increased emphasis on developing and testing of tailored behavioral interventions, particularly those informed by information technology. Research training includes bi-weekly Health Behavior Research Seminars and Research In Progress meetings, Behavioral Intervention Workshops, and experience on interdisciplinary research teams and similar intensive mentoring experiences, in addition to coursework as appropriate. Predoctoral trainees are supported for 2-3 years and expected to apply for individual NRSA funding; postdoctoral trainees are supported for 2-3 years and expected to submit at least one grant application for external funding. Trainees have access to resources within three research programs embedded within the School of Nursing: the Center for Enhancing Quality of Life in Chronic Illness, the Mary Margaret Walther Program for Cancer Care Research, and the Behavioral Cooperative Oncology Group. Trainees also have access to Clarian Health Partners, the third largest hospital network in the United States [unreadable] [unreadable] [unreadable]


Project Summary: Prepulse inhibition (PPI) represents a powerful method for understanding a fundamental component of information-processing, sensorimotor gating. PPI refers to the diminution of the startle response when a sudden startling stimulus is preceded 30-500 ms by a barely detectable prestimulus. This basic form of startle plasticity, seen across species from rodents to primates, has been studied extensively as an exemplar of preattentional sensory 'filters'that defend cognitive processes against the information inundation of our sensory world. A prominent theory holds that dysfunction in these filtering systems represents a core endophenotype manifested in schizophrenia, obsessive compulsive disorder, tic disorders, and possibly attention deficit hyperactivity disorder and post traumatic stress disorder. Patients suffering from this diverse range of illnesses share the common feature of an inability to screen out sensory, motor, or cognitive information, and display markedly deficient PPI. Because PPI deficits can be studied across species, PPI has become well-established as a crucial paradigm with which to study deficient sensorimotor gating in animal models of these psychiatric disorders. Much information has accrued regarding the neurochemical systems and circuits underlying PPI, particularly regarding the role of the dopamine and serotonin systems. Nevertheless, there is a striking gap in knowledge about the role of the norepinephrine (NE) system in regulating PPI. Our preliminary evidence indicates that this catecholamine plays a fundamental role in the ability of phencyclidine-like drugs (potent psychotomimetics in humans) to disrupt PPI in rodent models and the unique ability of atypical antipsychotic medications to reverse these PPI deficits. In addition, we have recently discovered that pharmacologically activating the locus coeruleus (LC), the major source of forebrain norepinephrine, markedly disrupts PPI. These data indicate that the LC-NE system represents a critical, yet almost completely overlooked central modulator of PPI. The experiments outlined in this proposal are designed to systematically characterize the role of the LC and its NE-innervated forebrain targets in regulating PPI, and in mediating the actions of psychotomimetic and antipsychotic drugs on PPI. These data may provide insights into novel circuitries underlying the regulation of PPI, and be of great relevance to understanding the neurobiology of the large set of psychiatric disorders of which dysfunctional sensorimotor gating is a central feature. Relevance to Public Health: Information-processing deficits are a major part of several psychiatric illnesses such as schizophrenia. When totaled, these illnesses represent a staggering 9-10% of the United States population. This proposal will study the neurobiology of the information-processing deficits seen in illnesses like schizophrenia in order to ultimately develop new treatments for these diseases.


PROJECT SUMMARY The objective of this SBIR Commercialization Readiness Program (CRP) project is to evaluate chronic toxicity of a new therapeutic for diabetic neuropathy in support of an IND submission. Of the 25 million Americans who suffer from diabetes, approximately 50% will be diagnosed with neuropathy, which is characterized by nerve degeneration. Despite the high prevalence of the disease, there is currently no FDA-approved treatment to either prevent diabetes-induced nerve degeneration or promote nerve regeneration. Thus, there is a substantial unmet need to develop more effective treatments for diabetic neuropathy. The founders of WinSanTor have identified a promising candidate which both prevents and reverses neuropathy in rodent models of the disease. The candidate molecule, pirenzepine, was identified using a novel screening methodology developed in the labs of the company?s founders. Pirenzepine has subsequently been evaluated in over a dozen in vivo tests, and has demonstrated the unique ability to ameliorate both epidermal fiber loss and thermal hypoalgesia. Pirenzepine is an approved drug for other indications in non-US countries, and so it is substantially de-risked as a drug development candidate. In a SBIR Fast-track program, WinSanTor successfully executed an expedited pre-clinical program that included: 1) development and validation of bioanalytical methods; 2) pharmacokinetic analyses; 3) optimization of formulation to enhance delivery; 4) generation of a safety profile; and 5) GMP manufacturing of pirenzepine. These efforts fully support the continued execution of the pre-clinical program through the evaluation of chronic toxicity assessments. The focus of this CRP program will be to evaluate the toxicity of pirenzepine when applied topically for 9 months. A 9-month study was chosen to fulfill the 9-month chronic toxicity study requirement for an NDA submission and to support the duration of the anticipated Phase 2 clinical trial protocol that will involve administration for at least 5 months. This study will be executed in mini-pigs and will use a number of toxicity end points, such as mortality observations, clinical observations, Draize scoring, clinical pathology, and histopathology, to fully define a toxicological profile of pirenzepine. The metric of success for this Aim is to achieve to achieve a NOAEL at an exposure level such that there is up to a 10x safety margin for human studies. The completion of this study is critical to an IND submission to the FDA to support subsequent clinical trials.


[unreadable] We aim to develop novel non-invasive and reiterative tests of nerve (small) fibre damage and repair in human diabetic neuropathy. We will quantify corneal nerve fibre degeneration and regeneration in diabetic patients compared with control subjects. We will assess circulating levels of the neurotrophin receptor p75 (p75NTR) as an index of small fibre stress/damage. Both measures will be validated against gold standard measures of neuropathic severity. A cross-sectional study will assess corneal nerve morphology and the p75NTR titre in diabetic subjects with a varying severity of neuropathy and non-diabetic control subjects. Moreover, subjects who have either recently undergone or are planned to undergo fascicular sural nerve biopsy will also have assessment of corneal nerve morphology and the p75NTR titre to define their exact relationship with peripheral nerve fibre degeneration and regeneration. Corneal nerve morphology and the p75NTR titre will be assessed in Type 1 diabetic subjects with painful neuropathy in a trial of strict glycaemic control using continuous subcutaneous insulin infusion, in order to assess the effect of near-normoglycemia and reduced glucose flux on these parameters. To further understand the relevant biology of the p75mR, and place its clinical assessment into a scientific frame of reference, we will undertake in vitro and animal studies. Tissue culture studies of neurones and Schwann cells will be employed to assess the effects of hyperglycemia, oxidative stress and NGF deficiency on expression, turnover and shedding of p75NTR. The role of these regulators specifically NGF therapy will be assessed longitudinally in STZ-rats in both peripheral nerve and in sensory ganglia to help further refine our understanding of the site of expression and shedding of p75NTR. The results of these studies will enable the introduction of two complimentary, non-invasive, and reiterative measures of small fibre damage and repair, thus enabling efficient, but accurate, assessment of treatment efficacy in human diabetic neuropathy. [unreadable] [unreadable]


The objectives of this grant are to characterize the cellular immune responses to Epstein-Barr virus (EBV) and to define the immunoregulatory events important in successful control of EBV infection during acute infectious mononucleosis (IM). We will continue our studies to further characterize the in vivo EBV induced MHC-nonrestricted cytotoxic T-cells (anomalous T-killer cells). The T cell lineage of these anomalous T-killer cells will be established by looking for molecular rearrangement of the T cell receptor genes. We will determine whether these T-killer cells are directed against EBV antigens and test the hypothesis that they are in fact alloreactive T-cells directed at altered class I molecules. Anomalous T-killer cells will be cloned to further prove polyclonality and determine target cell specificities. We plan to investigate the immunoregulatory role of in vivo EBV induced activated helper/inducer T cells. We will use CD4 helper/inducer T cell monoclonals to identify functionally distinct subpopulations. These subpopulations will be functionally studied in assays to evaluate help and suppression of B cell and T cell function. Cloning studies will be attempted to look at function at the clonal level. The long-term objective of our laboratory is to better understand the immunoregulatory events which occur during acute EBV infection. This knowledge will be used to understand immune defects which predispose individuals to fatal infection and lymphoproliferative disorders.


The availability of the Transgenic Mouse Shared Resource enables our CCSG Research Base Investigators to conduct versatile, cutting-edge research with a battery of sophisticated genetic techniques for manipulation of the mouse genome to create models for studies of cancer for incisive in vivo mechanistic investigations of the fundamental processes involved in the etiology of tumor development. Transgenic mice carrying new or novel genes are created by microinjection of DNA into the pronuclei of fertilized eggs and knock-out mice lacking specific genes of interest are created by homologous recombination in embryonic stem cells followed by injection into blastocysts to create chimeric mice for breeding to homozygosity. The high degree of conservation of most sequences in genomes of humans and mice makes using mouse genetic manipulation technology to create models of human cancer pathogenesis extremely useful. These approaches are remarkably powerful in cancer research particularly in the analysis of oncogenes, metastasis, cell-cycle control, tumor suppressor genes, and in the crafting of cancer model systems for developing new treatment regimens, and methods for drug testing and tumor imaging. The mission of this Shared Resource is to provide the highly technical aspects of manipulation of genes in embryos and mouse embryonic stem cells as a service to our Center members, allowing them to create the genetically manipulated mouse models they need. This is an outstanding example of specialized techniques, highly trained dedicated personnel, and expensive equipment can be accessed by researchers who could not reasonably expect to develop or obtain them on an individual basis. The UCSD CCSG Shared Resource provides cutting-edge, rapidly evolving services that adapt and drive the field of mouse genetics and continue to be very responsive to the vast creativity of the CCSG membership, fulfilling their needs for the most cutting-edge embryology and molecular genetics in the mouse.


Children with a parent diagnosed with substance abuse disorder (SUD) show increased risk for greater drug use, delinquency, and their engagement in health-risking sexual behavior, suggesting maladaptive choice processes with great social and public-health costs (e.g., Kirisci et al., 2007; Reynolds et al., 2007; Tarter et l., 2002). Little is known, however, about the mechanisms that underlie the decisions to engage in these behaviors. We propose to conduct hypothesis-driven secondary data analyses to test a conceptual model that links parental SUD and childhood dysregulation to the development of poor decision-making skills and increased incidence of health-risking behaviors later in life. We will leverage data collected through the Center for Education and Drug Abuse Research (CEDAR; NIH# 5P50 DA05605), using its unique longitudinal dataset that includes validated measures of transmissible liability for SUD risk (Kirisci et al., 2009; Vanyukov et al., 2009), a battery of laboratory-based measures of decision-making competence (DMC; Parker & Fischhoff, 2005), and adult assessments of substance abuse, antisocial behaviors, and sexual behavior. We intend to address five main research questions in the proposed project. First, we plan to identify classes of developmental trajectories for psychological regulation. We will conduct analyses to characterize different patterns of intra-individual change in psychological regulation from preadolescence to emerging adulthood. Second, we plan to refine the measurement of DMC, which can be defined as adherence to normative, rational standards when making decisions (Parker & Fischhoff, 2005) by expanding the construct to encompass excessive risk-taking and insensitivity to expected value. Third, we will examine the associations between family and parenting variables on subsequent DMC and health-risking outcomes later in life. Fourth, we will examine the effects of developmental trajectories of psychological regulation on DMC. While research has linked self-regulative processes to advantageous decision making, such studies have largely relied on cross-sectional designs, and are thus silent on the progression from development of self-regulation to adolescent decision making. Finally, we will test the extent to which poor DMC can predict future health-risking behaviors, such as drug use, criminal behavior, high-risk sexual behavior, and inter-partner violence.


The Vision Science Research Center of the School of Optometry at the University of Alabama in Birmingham has now been in existence for nine months. During the first three months, a great deal of time and effort was spent recruiting and hiring the various technical and administrative support personnel, purchasing supplies and equipment and establishing the procedures and guidelines under which the Center would operate. During the next six months, the technical staff was trained, most of the equipment and supply purchases were received and the different CORE Support Modules began to meet the needs of the participants. Establishment of Vision Science Research Center at the University of Alabama in Birmingham has had an interesting effect. Given the concentration of vision scientists here, other departments on campus are becoming increasingly interested in hiring new faculty with vision research interests. A perfect example of this is the recent hiring of Dr. Michael Friedlander by the Department of Physiology. The Department of Physiology and others are finding that high quality vision scientists are attracted to Birmingham by the existence of the Vision Science Research Center and the individual investigators that are presently here. Given that several departments on campus are in the process of increasing their faculty, we are hopeful that even more vision scientists will be attracted to Birmingham and the Vision Science Research Center.


The International Association of Fire Fighters (IAFF) is requesting 664,757 dollars during the initial budget period and 3,529,286 dollars for the total project period of the DOE Nuclear Weapons Complex Cooperative Agreement. Emergency personnel responding to incidents related to the DOE complex face health and safety challenges involving radioactive and other hazardous materials. Since 1994, an average of 2,200 responders have been injured at hazardous materials incidents annually. Many more suffer serious health effects from toxic exposure associated with fire fighting and EMS response. The effective remedy to combat these health effects is a flexible training program that emphasizes occupational safety and health and OSHA defined responder training as a key to effective emergency response. The IAFF proposes to continue to implement such a proven training plan. This effort relies heavily on an efficient Train-the-Trainer approach. It offers three new course formats which can be customized to the specific hazards faced by a given target audience; uses a combination of the Internet, advanced training technologies and regional programs; and emphasizes Integrated Safety Management (ISM). The estimated 425 annual attendees leave the course with the knowledge and the tools needed to implement this program in local fire/rescue departments in and around ten specified DOE sites, as well as other regions upon request. The IAFF is the only national organization serving professional fire fighters and enjoys longstanding training partnerships and access with fire/rescue departments across the US. Our training curricula is current, focused, and ready to be delivered. In addition, we have a highly regarded 100-member professional fire fighter/paramedic instructor team trained in using facilitation techniques and problem-based learning to reinforce responder safety and health. It is a state-of-the-art program with a focused safety and health message provided by experienced, committed instructors.


The focus of this research will be important neurobiological substrates of aggressive behavior in mice, and how genetic, developmental, and experiential determinants of aggression are mediated by specific neuronal systems. To study this problem systematically, an integrated multidisciplinary approach will be used to examine the behavior and neurobiology of lines of mice, derived from ICR foundational stock, which have been selected over 21 generations for high and low levels of aggression. Alterations in aggression induced by genetic, ontogenetic, and experiential factors will be related to specific neurobiological mechanisms. Preliminary data support the hypothesis that an important mechanism for genetically-induced behavioral differences is an alteration in dopaminergic activity in specific terminal regions. Thus, the aims of the proposed studies are: (1) To determine whether alterations in brain monoamine, particularly dopamine, activity in specific terminal regions mediate behavioral differences observed in mice selectively bred for high and low levels of aggression; (2) to determine whether developmental and genetic differences in aggression are due to alterations in the magnitude or temporal development of innervation of discrete catecholamine terminal fields; (3) to determine whether social experience alters the neurobiological mechanisms mediating genetic and developmental differences in aggressive behavior. Because there are qualitative and quantitative changes in aggressive behavior changes as a function of developmental period, and as a function of social experience, the activity of dopamine and other monoamine (serotonin and norepinephrine) systems in both high and low aggressive mice will be studied during ontogeny, using behavioral, pharmacological, neurochemical, receptor, and immunocytochemical techniques. A cosibial\longitudinal design will be used that allows genetic, developmental, and experiential factors to be disentangled. Computer-supported observational methods (interactional analyses) will be used to quantify behavior, while high performance liquid chromatography (HPLC) and quantitative receptor autoradiography will be used to measure neurochemical and receptor changes in monoamine systems. Immunocytochemical techniques, using antisera to tyrosine hydroxylase (TH) and dopamine-beta-- hydroxylase (DBH), will be used to characterize the distribution of catecholaminergic fibers in specific brain regions of high and low aggressive mice. The proposed studies will provide important, new information about the neurobiology of aggression and timidity, and the brain mechanisms that mediate genetic, developmental, and experiential effects on social behavior.


KKHKHKHKHK FFFFF LLLLLLL


The goal of this research is to develop a culture dish (OxyDish R) that, together with oxyrase (tm), will support growth of clinically significant, anaerobic microorganisms on an agar-medium surface without using anaerobic bags, jars, or chambers. The use of oxyrase and this dish to grow anaerobes is intended to be transparent to the microbiologist and to the microorganisms. The microbiologist is intended to be able to work with most anaerobic microbes. The combination of oxyrase with the dish is intended to provide an environment that will reliably support the rapid growth of anaerobic microorganisms. No other environmental intervention or special equipment will be needed to grow clinically important microorganisms. The simplicity of the OxyDish is intended to enhance the efficiency of the clinical microbiologist working with anaerobic pathogens. This increased efficiency, together with elimination of special equipment supplies, will reduce the cost of working with anaerobic pathogens.


This application requests support for 2 physician training positions for 3rd and 4th year neonatology fellows, which will allow them to develop the knowledge, understanding and skills needed to pursue independent research careers in academic neonatology. Two major academic research paths will be available to trainees, and each track will be associated with a specific curriculum containing required course work: Track I. Basic Research, Physician-Scientist (laboratory research mentorship), and Track II. Clinical Research, Physician-Scientist (clinical research mentorship) which includes Health Care Policy and Medical Informatics mentorship components. This program with its two research tracks resides with the Division of Neonatology which includes: five clinical research physician-scientists (two of whom are Masters trained); two basic research physician-scientists; ten Ph.D.s; and the Neonatal-Perinatal Research Institute (NPRI). The NPRI includes sixty-five senior investigators, forty of whom are NIH funded, and draws upon the academic strength and depth found across Duke University needed to support the formal training for the program in developmental biology, clinical sciences and health care research. Extramurally funded research within the NPRI currently supports 4 broad areas of investigation: 1) early cardio/cranio/facia development; 2) neonatal lung development/repair; 3) neural injury and repair in the fetus and neonate; and 4) clinical neonatal research in conjunction with the NICHD Neonatal Network and the Duke Clinical Research Institute. In addition, this grant proposes a unique program with curricula for the study of Health Care Policy and Medical Informatics in the context of a medical research career. The requested funds will provide critical support for the research years of the neonatology fellowship, which currently trains six to seven fellows at Duke University Medical Center. The unique research training program with its separate Tracks will provide a structured learning experience tailored to the needs and interests of the individual fellow, with each fellow working with a senior scientist who will serve as mentor. Upon completion of the training program, the young physician-scientist will have the tools, knowledge, experience and confidence to compete successfully as an independent investigator.


The long-range goals of this proposal are to better understand the molecular neurobiological events that underlie the development of excessive alcohol drinking, and contribute to the long-range consequences of excessive alcohol drinking. The overall hypothesis to be tested is that changes in the expression of genes involved in neurotransmission, neuroplasticity, and intracellular signaling pathways within discrete regions of the extended amydala (E-AMYG) contribute to the development of excessive alcohol drinking. Excessive drinking is defined as sustainable blood alcohol concentrations (BACs) in the range of 100-150 mg% that are repeatedly attained over a chronic period. The overall hypothesis will be tested using selectively bred alcohol-preferring (P) and high-alcohol-drinking (HAD) rats. Micro-punch techniques will be used to obtain samples containing the nucleus accumbens shell (ACB-sh) and central nucleus of the amygdala (CeA). Changes in gene expression will be determined using Affymetrix microarrays. RT-PCR and in situ hybridization will be used to verify key findings from the microarray experiments. The excessive alcoholdrinking paradigm to be used will be the 'drinking in the dark multiple scheduled access' (DID-MSA) procedure. Time-course changes in gene expression will be determined following a drinking episode, and during the development of excessive alcohol drinking. The specific aims will be designed to determine changes in gene expression within the ACB-sh and CeA of P and HAD rats prior to, during initiation of, and following development of excessive alcohol drinking. The effects of chronic alcohol exposure and the development of excessive alcohol drinking are influenced by multiple genetic and environmental factors. This project will provide important molecular neurobiological information in discrete regions of the E-AMYG of genetically vulnerable subjects that could more comprehensively describe the complex inter- and intracellular events leading to the development, maintenance, and consequences of excessive alcohol drinking. Such information would be critically important for developing pharmacotherapeutic strategies to treat alcoholism and alcohol abuse. [unreadable] [unreadable] [unreadable]


Continued research is proposed toward the development of a chemical mediated artificial pancreas. This system will deliver glycosylated insulin (G-Ins) in response to glucose concentrations, based on the competitive binding between G-Ins and glucose to a saccharide binding substrate. This application will focus on optimizing the components of the system and comprehensive in vitro and in vivo animal tests. The synthesis, characterization, and isolation of glucosyl, as well as fucosyl, mannosyl, and galactosyl saccharride derivatives of insulin will be performed. By expanding the range of insulin derivatives, improved physical-chemical properties (binding kinetics, glucose exchange rates, and permeation) may be achieved. The interaction of G-Ins with surface bound insulin receptors on an insulinoma cell will be evaluated. This in vitro model will assess the conformation or bioactivity associated with G- Ins through changes in cell growth rates and the amount of insulin secreted by the cell. The immunogenicity of the G-Ins will be investigated. Mice will be injected with G-Ins and the antibody titer to G-Ins will be determined. The central component of the self-regulated insulin delivery system is a substrate that binds both G-Ins and glucose. ConA was evaluated as a soluble monomeric protein, enclosed within microcapsules, and formed into microspheres. This proposal focuses on coupling ConA as a soluble high molecular weight oligomer. This new form will maintain the binding characteristics and not leak or permeate through the device membrane. Other saccharride binding substrates will be investigated particularly, hexokinase and glucokinase. These proteins strongly bind sugars and may achieve a greater sensitivity in G-Ins release to glucose levels. A composite membrane 'pouch' will be used to house the G-Ins/binding substrate complex. A heat sealable, 40K MWCO membrane coated with a thin dense biocompatible material will prevent the permeation of immunoglobulins from the peritoneum into the device and maintain suitable glucose and G-Ins diffusion rates. A vascular graft delivery device will also be developed. G-Ins and binding substrate will he housed in a casing surrounding a vascular graft fabricated from the same membrane as the pouch. Glucose will rapidly equilibrate into the casing and release bound G-Ins. In vitro tests of both systems will evaluate the release of G-Ins in response to glucose concentrations, binding capacity, and effective duration of the device. Both the pouch and the vascular graft systems (anastomosed to the common illiac vein) will be implanted in the peritoneum of pancreatectomized dogs for short and long term in vivo evaluations including: G-Ins levels in response to blood glucose, lowering of blood glucose, biocompatibility, in vivo duration, and decreased long term complication (glycosylated hemoglobin) by the self-regulated insulin delivery device. It is anticipated that by the completion of this application, a final prototype device aimed toward human clinical uses will be realized.


Recurrent interactions between neurons generate dynamic patterns of activity that serve as a substrate for behaviorally relevant computations. However, we do not yet have a principled framework for relating neural dynamics to neural computations. We have recently synthesized a theory that explains how low-rank recurrent neural networks may serve as a building block for computations. Our overarching goal is to integrate insights from this theory with behavior and electrophysiology in awake, behaving monkeys to establish a principled framework relating neural dynamics to neural computations. The project will start with reverse engineering low-rank network models that capture cortical dynamics in simple timing tasks. We then move systematically toward progressively higher rank network models that can perform timing tasks with progressively more sophisticated computational demands such as probabilistic inference of time intervals. We aim to create models that simultaneously succeed in performing task-relevant computations (i.e., behavior) and emulate cortical dynamics recorded in monkeys performing those tasks. We will use this iterative process to establish a principled framework relating neural dynamics to neural computations underlying inference. Finally, we will put this framework to test using a novel task that demands an unprecedented level of computational flexibility. RELEVANCE (See instructions): It has become increasingly apparent that the neurobiology of behavior in health and disease has to be probed at the level of populations of neurons. However, we do not yet have a rigorous and quantitative language for linking population neural activity to behavior. Our work combines primate electrophysiology with neural network modeling and aims to develop such a language through the mathematics of dynamical systems. The results hold promise for future translational research to diagnose behavioral symptoms of brain dysfunction in terms of their computational modules and the dynamic patterns of activity that support those modules.


Our study of the neurochemical mediation of amphetamine response is continuing with the use of the serotonin receptor blocker metergoline in conjunction with amphetamine. Five persons have participated in pilot studies related to this project. We have undertaken to use several agents that are reported to cause the release of adreno-corticotrophic hormone (ACTH) because of reported abnormalities of this hormone in affective illness. Thirteen subjects have participated in a pilot study of the opiate antagonist naloxone and five persons in a pilot study of the serotonin precursor tryptophan. A replication study of cholinergic REM induction is in progress. Four out of six bipolar patients have shown high sensitivity so far, as compared with 1 out of 7 controls. This is consistent with our previous findings, and with our hypothesis of increased cholinergic sensitivity associated with vulnerability to affective illness. We plan to extend this study to offspring of patients in the high risk study. A study of the effect of physostigmine on melatonin is underway. Four persons have participated in a pilot study.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This research is studying a dietary supplement called Inositol, which is part of the Vitamin B complex and is important for the development of the eyes. Specifically, this study is looking for the best dose of Inositol to give babies before they are ready to start taking feedings. Previous studies in premature infants have shown that blood levels of Inositol drop quickly in the first week after premature birth. Inositol is important for normal cell functioning and to help body organs grow and mature. Infants receive Inositol from their mother before they are born [through their bloodstream] and from their mother's milk after they are born. Inositol is now added to the formula that is fed to premature infants, because studies have shown that the Inositol levels fall gradually after the first week of birth if the infant is not eating, or is fed formula that does not have Inositol added. Studies have also shown that keeping steady levels of Inositol in premature infants may decrease certain eye and lung diseases. This study is looking at the level of Inositol in the blood after giving inositol through a vein when a baby is not ready to be fed breast milk or formula, because it is not known how much Inositol is the best dose. When the best dose of Inositol to give to premature babies is known, the investigators plan to do a large study to give Inositol from birth until a baby is full term. The FDA has not yet approved for Inositol to be given through a vein to infants. This study, and the large study to follow, will give information to the FDA to decide if they should approve Inositol to be given through a vein.


Voltage-gated sodium (NaV) channels are fundamental signaling molecules in excitable cells that determine the rapid influx of Na+ ions. Perturbation to the function of human cardiac NaV1.5 channels (SCN5A gene) can result in arrhythmias and in some cases death. Genetic defects in the SCN5A gene are a known cause of long QT type 3 syndromes, and are associated with other life threatening conditions such as Brugada syndrome and sudden infant death syndrome. For normal functioning NaV channels, changes in intracellular calcium ([Ca2+]i) lead to fast inactivation of NaV1.5. This process is known to involve the intracellular calcium sensing protein calmodulin (CaM), specific elements of the channel located near the C terminus, and the linker region between domains 3 and 4 (D3D4 linker). Recent studies have confirmed the D3D4 linker functions as the fast inactivation gate (FIG). In several instances, the above-mentioned mutations occur in, or are likely to alter interactions and functioning of CaM and the FIG, which is the focus of this work. Recent advances in the characterization of CaM-FIG interactions have identified mutations to specific residues that uncouple fast inactivation gating from [Ca2+]i dependence. However, the molecular basis for the action of the Ca2+ sensing apparatus on the FIG is currently debated and largely unknown. Several models have been proposed in the literature based on conflicting views, but no functional model has been generated that is able to reconcile all the available data. I hypothesize that changes in [Ca2+]i lead to remodeling of CaM and specific elements in the C-terminus of NaV1.5, which result in CaM interacting with the FIG (D3D4 linker) in a manner that is NOT described by previous models. Understanding the molecular details of CaM-FIG interactions are essential to the development of an accurate model that describes the Ca2+ and CaM-dependent modulation of NaV1.5 fast inactivation. Completion of this project will provide a detailed description of the highly complex mechanism that involves CaM and the D3D4 linker translating changes in [Ca2+]i into modulation of fast inactivation gating of the human cardiac sodium channel. This investigation will utilize a complement of biophysical, structural, and electrophysiological techniques to characterize interactions between CaM and the FIG that are essential to this regulatory mechanism. The results will be used to determine the relative extent of perturbation to NaV1.5 fast inactivation caused by mutations to the CaM-FIG complex. This information will aid in understanding the molecular basis of specific cardiac life threatening arrhythmias as well as lay a broad foundation for evaluating the potential use of small molecules to target the NaV1.5 [Ca2+]i sensing apparatus for the treatment of certain cardiac arrhythmia syndromes.


The role of RNA polymerase in the regulation of gene expression during growth and sporulation of Bacillus subtilis will be investigated. Since the sigma-43 form of RNA polymerase plays a major role during growth, early stationary phase, and catabolite repression, the following aspects of this enzyme will be investigated in detail: (1) the genetic organization and regulation of the recently cloned and sequenced sigma-43 operon will be analyzed by determining its promoter sites and RNA polymerase specificities, by testing various environmental and physiological conditions that alter transcription from these promoters, by determining the function of the first gene of the operon, and by modifying the promoter regions by site directed mutagenesis; (2) the role of sigma-43 enzyme in catabolite resistant sporulation and catabolite repression will be investigated by studying the relationship between the crsA mutation in the rpoD (sigma-43) gene and the spoO and non-sporulation related suppressor genes for crsA. The studies will involve the isolation of additional suppressor genes for crsA including those which are not related to sporulation. Glucose sensitive promoters will be isolated by a newly developed promoter expression probe plasmid and the effect of the crsA mutation will be tested on the promoters by in vivo and in vitro transcription studies. Those spoO genes which suppress crsA and which can be suppressed by crsA will be cloned and characterized. The effect of spoO gene products will be examined on transcription carried out by the wild type sigma-43 enzyme and the crsA mutated sigma-43 enzyme.


The use of cardiopulmonary bypass (CPB) in cardiothoracic surgery is associated with significant postoperative bleeding and pulmonary injury due to massive activation of plasma protease systems during extracorporeal circulation. Contact activation of the kallikrein- kinin system, intrinsic coagulation, the complement cascade and fibrinolysis during CPB all contribute to excessive post-bypass blood loss and an inflammatory response manifested by pulmonary dysfunction. The goal of this research is to discover a potent protein inhibitor of human factor XIIa and plasma kallikrein which can be used to prevent activation of the contact system during CPB in order to reduce blood loss and attenuate parameters of pulmonary dysfunction. Protein engineering techniques have been used to identify variants of the Kunitz protease inhibitor domain (KPI) of the human amyloid beta-protein precursor which are potent inhibitors of factor XIIa, plasma kallikrein and plasmin. In the Phase II portion of this research, a high yield recombinant expression system will be established to generate preclinical supplies of one KPI variant. The human KPI variant will be tested in a sheep model of cardiopulmonary bypass for its ability to significantly reduce post-operative blood loss and lung injury. PROPOSED COMMERCIAL APPLICATIONS A potent recombinant human protein inhibitor of factor XIIa, plasma kallikrein and plasmin would be administered prophylactically during surgeries requiring cardiopulmonary bypass to reduce postoperative blood transfusions and improve recovery times.


We propose to undertake a fundamental research program in mass spectrometry and combined gas chromatography-mass spectrometry (GC-MS) dealing with the analysis of selected classes of biologically significant compounds. Primary emphasis will be placed on the analysis of dopaminergic agonists such as apomorphine and related compounds, selenium amino acids and natural products isolated from plants exhibiting antitumor activity. We expect to develop correlations between fragmentation pattern and structure, and apply this information to the identification of metabolic products and novel compounds in naturally derived mixtures. Close interaction with clinicians in Boston area hospitals and other applied scientists at Northeastern University will ensure the relevance of the basic research effort and applicability of information obtained from it. Derivatization techniques will be examined for the vapor phase (GC-MS) analysis of the various types of compounds indicated above. In addition, we expect that derivative formation will be useful for the mass spectrometric differentiation of positional or stereochemical isomers, based on ionically induced functional group interaction. Chemical ionization (CI) mass spectrometry will also be employed in the structural analysis of selected classes of natural products (e.g., sesquiterpene lactones). We expect to utilize intermediates, available through synthetic programs currently in progress in the university, for the preparation of isotopically labeled compounds, to assist both in the interpretation of fragmentation patterns and for use as internal standards for quantitation by mass fragmentography.


3,4-methylenedioxymethamphetamine (MDMA/ 'ecstasy') is a popular "recreational" drug with considerable abuse liability including potential neurotoxicities directed toward the central serotonergic system. Although much attention has focused on MDMA's neurotoxic effects and its' ability to promote release of monoamine transmitters, little is known about how the drug affects the operation of neural circuits and brain function either at high neurotoxic doses or doses considered to be in the "recreational" range. For example, MDMA users report that one of the major pleasurable outcomes of ecstasy self-administration is enhanced tactile sensation, but there is no explanation for how the drug produces this desirable and much sought after sensory experience. Because of the potential for long term damage to the nervous system after MDMA ingestion, there is a pressing need to understand the neural substrates underlying the motivation for human self-administration of this agent. The long-term goal of the proposed research is to better understand the neurophysiology underlying ecstasy's effects. The immediate goal of the present proposal is to develop and validate procedures for evaluating the impact of MDMA on sensory signal processing in the VPM thalamus of intact rats. The project employs plasma level analysis of drug concentrations, and electrophysiological determination of VPM cellular function in intact anesthetized or waking rats. Multi-channel, multi-neuron extracellular recording, systemic drug administration, activation of afferent trigeminal somatosensory pathways, and computer based analysis of spike train data are used to assess the impact of MDMA on sensory signal processing. A significant feature of this multi-dimensional approach is that drug effects will be determined at acute and chronic doses that: 1) approximate human self-administration regimens, 2) produce known plasma levels of the drug, and 3) elicit measurable changes in monoamine efflux within sensory circuits of the brain. Understanding the relationship between MDMA administration, monoamine transmitter efflux, and the operation of the somatosensory system will provide a basis for understanding the neurophysiological mechanisms underlying the drugs' effects on tactile sensory perception, in particular; and neural circuit functions, in general. A detailed knowledge of MDMA's effects on cellular and neural circuit function including its effects on sensory neurophysiology is essential in order to provide the public with accurate information regarding the risks associated with recreational use of this popular compound and its derivatives. Furthermore, once established, these methods will be used in future studies to characterize MDMA actions in other brain networks (prefrontal cortex, limbic) and more sophisticated behavioral assays (sensory discrimination, self-administration, craving and re-instatement) as a means of further clarifying its abuse liability. Waterhouse, Barry D. PROJECT NARRATIVE ()3,4-methylenedioxymethamphetamine (MDMA/ 'ecstasy') is an increasingly popular "recreational" drug that poses a significant threat to the nation's health because of its: 1) adverse acute and chronic effects on behavior and physiological functions, 2) neurotoxicity toward selected neurotransmitter systems in the brain, and 3) overall addictive potential. Although much attention has focused on MDMA's neurotoxic effects and its' ability to promote release of endogenous transmitters, little is known about how the drug affects the operation of neural circuits and brain function either at high neurotoxic doses or doses considered to be in the "recreational" range. Because of the potential for addiction and long term damage to the nervous system after MDMA ingestion, there is a pressing need to understand the neural substrates underlying the motivation for human self-administration of this agent. The goal of the proposed project is to develop and validate procedures that will help us better comprehend the neurophysiological basis for ecstasy's effects in human drug users. [unreadable] [unreadable] [unreadable]


Xenopus Nucleoplasmin (Np) and N1 are equally abundant chaperones found in oocytes and eggs. Np participates in nucleosome assembly and exchange reactions involving histone H2A/H2B dimers and DNA; whilst N1 functions as a chaperone specific for the H3/H4 tetramer. Therefore, N1 and Np may function together to promote nucleosome assembly in oocytes. DNA replication and transcription are sensitive to the local packing of nucleosomes, which may be modulated by histone acetylation/deacetylation, phosphorylation, DNA methylation, or a combination thereof. Hence, nucleosome assembly and exchange factors may play a critical role in these cellular processes. We propose to study the roles of Np and N1 in nucleosome assembly and in the decompaction of sperm chromatin after fertilization, by obtaining the first high resolution crystal structures of these chaperones, alone and complexed with their cognate histories. This will reveal the molecular architecture and function of the Np and N1-families, while providing insights into the extreme thermal and chemical stability of the Np pentamer. It was suggested previously that N1 and Np function sequentially to deliver core histones to DNA during nucleosome assembly. We have demonstrated that Np forms a decamer in solution which can be reconstituted with separately purified histories H2A/H2B and H3/H4, to form stable Np decamer-octamer complexes in the absence of DNA. Hence, the Np decamer with 522 point group symmetry may provide a docking ring for 10 H2A/H2B dimers and 5 H3/H4 tetramers, to form an Np decamer-octamer complex which utilizes their common 2-fold symmetry. We further suggest that N1 may establish an N1 dimeric cap on H3/H4 tetramers in cells, which then docks with Np decamer-dimer complexes to form a larger Histone Octamer co-Chaperone (HOC) complex. Overall, our data suggest a new paradigm for nucleosome assembly, in which pre-formed histone octamer-chaperone complexes and DNA are brought together in a concerted mechanism. To understand this process, the structures of the Np decamer-octamer and putative HOC complexes will be determined by electron cryo-microscopy and X-ray crystallography, in combination with biochemical studies of nucleosome assembly.


APOBECSG (A3G) and APOBECSF (A3F) are involved in various anti-viral protein and nucleic acid transactions. Important primary interactions include interprotein interactions, binding single-stranded nucleic acids (RNA and ssDNA), and neutralization by HIV Vif. The full elucidation of these intermolecular interactions is necessary to understand the mechanism by which these cellular proteins inhibit HIV replication. The role of oligomerization in their activity is an issue of great importance. These are interactions at the nanoscale level, and AFM is the method of choice capable to addressing problems listed above. AFM currently reached a level of development that enables this unique instrument to provide a wealth of important information to the characterization of biomolecular complexes at nanoscale. The topographic analysis of molecular system is the initial area of the AFM application for characterization of the structure and specificity of protein-DNA systems of a broad complexity. In addition to imaging, AFM is capable of direct measurements of molecular interaction within the system and research during this decade led to a dramatic progress in this area of the AFM applications. Our Preliminary Studies have already enabled us to provide the first images of A3G bound to ssDNA substrates. We further anticipate that the use of all modalities of AFM technology will help us detail these complexes, dissect the protein and chemical requirements, and extend key observations to ASF. To help achieve these overarching objectives, we will pursue the following three Specific Aims: Aim 1. Image A3 complexes at nanoscale resolution using AFM (Program Objectives 1 &2);Aim 2. Measure interactions within A3-ssDNA complexes using force spectroscopy (Program Objectives 1 &2) and Aim 3. Characterize A3-Vif interactions using AFM imaging and probing modalities (Program Objectives 3). The studies under this project are an integral component of our overall Program Project aiming to provide a comprehensive understanding of A3-AS3 A3-nucleic acid, and A3-Vif interactions. The nanoscale studies will provide a platform for evaluating any future therapeutics that function by leveraging the AS-Vif interaction.


ADMINISTRATIVE CORE SUMMARY/ABSTRACT The mission of Mayo Translational PKD Center (MTPC) is to advance the understanding and reduce the individual and societal burden of PKD. The overall goal of MTPC is to develop Core facilities to support existing and stimulate new PKD research by Mayo and non-Mayo investigators and to facilitate the translation of basic research breakthroughs into improvements in clinical practice through the implementation of clinical trials. Since July 2010, the Administrative Core of the MTPC has: i) provided strong strategic and administrative oversight in the newly established PKD Research and Translation Core Center; ii) fostered the interaction and synergism of three, heavily used MTPC Biomedical Research Cores (Molecular Genetics and Biomarker Core, Model Systems Core, and Human Imaging Core), chosen as the most effective to achieve the Center's goals and building on the strength of the Mayo investigators; iii) strengthened the Internal Research Base and added a strong External Research Base of PKD investigators; iv) attracted and nurtured new PKD investigators through a dynamic Pilot and Feasibility Program; v) adopted software tools and developed metrics to monitor the performance of the Biomedical Research Cores and the Pilot and Feasibility Program; vi) implemented a vigorous Scientific Enrichment Program; and v) established relationships with internal and external partners to facilitate MTPC member access to resources. The Administrative Core leadership, provided by the Director and Co-Director, in conjunction with the Operations Committee, Executive Committee, Internal Advisory Committee, and External Advisory Committee, will continue to 1) Promote cooperative, successful scientific interactions among the multidisciplinary internal faculty and an extended faculty from 39 other institutions; 2) conduct periodic membership surveys to determine the needs of the internal and external members; 3) conduct periodic reviews of the Services provided by the Biomedical Research Cores of the MTPC to ensure that cutting edge technologies replace those no longer needed; 4) promote the communication and coordinate the activities of the three Biomedical Research Cores of the MTPC to ensure that they synergistically enhance the research capabilities of the members; 5) conduct periodic reviews of Core Use and involvement in Center Activities to modify the Research Base membership as needed; 6) provide budgetary and administrative management necessary for efficient, cost-effective accomplishment of Center goals and allocate resources commensurate with member needs; 7) ensure a successful Scientific Enrichment Program including weekly seminars, monthly conferences, periodic workshops, annual mini-symposia, visiting faculty program, mini-sabbaticals and communications via electronic media; 8) continue a Pilot and Feasibility Program funded equally by this grant and by the Mayo Foundation under the direction of the Pilot and Feasibility Program Director and the External Advisory Committee; 9) stimulate interest in PKD and provide career development opportunities for junior investigators through the NIH Summer Undergraduate Research Fellowship, NIH T32 training grant (T32) and other funding sources and by creating a forum for their research updates; 10) continue and expand existing cost-effective, scientific interactions between MTPC and the NIDDK-funded C-SiG, the Mayo CCaTS, and other NIH funded Centers.


The human body contains a diverse community of bacteria, a majority of which is found in the gastrointestinal (GI) tract. Alterations in the GI microbiota have been correlated to diseases such as obesity and irritable bowel syndrome. Secretory diarrhea exhibits alterations on the GI community, and up to 60% of people traveling from developed to developing countries acquire a form of secretory diarrhea know as travelers' diarrhea (TD). Enterotoxigenic E. coli (ETEC) and Norovirus (NV) are the leading cause of bacterial and viral TD, respectively. ETEC expresses two toxins, heat labile toxin (LT) and heat stable toxin (ST), which ultimately lead to secretory diarrhea. The mechanism of noroviral diarrhea is not well understood. Previous studies have shown that an etiologic agent is not identified in as many as 50% of TD cases, suggesting that travel alone may play a role in TD. Presumably, TD causes a shift in bacterial GI communities; however, this has not been studied. The goal of this proposal is to use 16S rDNA metagenomic sequencing and analysis methods to investigate how TD caused by ETEC and NV affects the gut microbiome and to identify shifts in the gut microbiome associated with foreign travel. In Aim 1 I will complete a retrospective study to examine how ETEC-TD alters the gut microbiome by comparing the GI microbial populations in stool samples from healthy travelers to samples from individuals with ETEC-TD and to samples from individuals with unidentified pathogen-TD. I will also develop a PCR assay to rapidly and reliably detect ETEC in human stool samples. Aim 2 will investigate how the microbial populations in the gut shift during NV-TD. In this retrospective study I will compare paired samples where the first sample was collected during an acute diarrheal episode and the second sample, collected from the same individual, was collected after amelioration of diarrheal symptoms. This will allow me to determine how the gut microbiota shifts after an acute episode of NV-TD. In my third Aim, I will complete a prospective study, which seeks to determine the effect of travel on the gut microbiome. I will compare bacterial populations from stool samples, collected at various time points, from healthy individuals traveling to Guadalajara, Mexico. Stool samples will be collected prior to travel, at 7, 14, and 21 days after arrival in Mexico, and upon return to the United States, which will permit me to identify specific changes within the gut microbiome associated with travel to a developing country. This proposal outlines the necessary first steps in understanding how TD affects the gut microbiome. Results from my research could potentially lead to development of preventative treatments and novel therapeutics against TD. )


Respiratory syncytial virus (RSV) infection is the most common cause of respiratory disease leading to hospitalization in children. Preterm infants are especially susceptible to severe RSV infection. Respiratory epithelium is an initial site of RSV infection and epithelial cells along with alveolar macrophages (AM) and dendritic cells (DC) are vital to innate and adaptive immune responses. However, the extent of innate immune gene expression by epithelia and AM-DC in preterm infants can be variable/limited. The hypothesis is that: Reduced innate immunity by respiratory epithelia and AM-DC preterm enhances susceptibility to RSV infection This hypothesis is based on the facts above and our preliminary data in lambs demonstrating limited expression of surfactant proteins A and D (SP-AD), sheep beta-defensin-1 (SBD-1), and Toll-like receptor 4 (TLR4) preterm. It will be tested in preterm lambs which have close similarities with human disease including susceptibility, lesions, and innate immunity. Specific Aim 1 compares expression of key innate immune genes (SBD-1, SP-AD, TLR4) and protein/peptide production in vivo as well as AM-DC cytokine expression in pre- and full-term lung. It also tests the hypothesis that limited epithelial cell proliferation and/or differentiation pre-term underlie(s) the mechanistic basis for limited SP-AD, SDB-1 expression and tests this by comparing transcriptional activity, protein/peptide production in pre- and full term cultured cells with or without cell proliferation and differentiation. Specific Aim 2 tests the hypothesis that SP-AD, SBD-1 and AM-DC responses to RSV are less at pre- than full-term in the lamb model and in vitro with cultured epithelial cells. A second hypothesis, that increased cell proliferation and/or differentiation of epithelia protects against RSV infection, will be tested in vitro with treatments from Aim 1. The extent to which SP-AD, SBD-1 directly prevent RSV infection will be tested with RNAi assays. Specific Aim 3 tests the hypothesis that yet other innate immune genes expressed with cell proliferation/differentiation prevent RSV infection. This Aim uses gene expression profiling of primary polarized human lung cells and a respiratory epithelia-specific probe set (Unigene) that is the most well-annotated and defined gene target set to date. It also identifies ovine orthologs of human genes and test for impaired/reduced expression preterm. This team combines veterinary and human medical expertise to attain the goal of this project: to discover the reason(s) for inadequate expression of SP-AD, SBD-1 and other innate immune genes as well as AMDC responses at preterm that predispose to RSV infection. The work is significant because it discovers the underlying basis for RSV susceptibility preterm and mechanistic approaches to enhance innate immunity. [unreadable] [unreadable] [unreadable]


Fatty acid amide hydrolase (FAAH) is the enzyme predominantly responsible for the catabolism of several fatty acid amides (FAA), including the endogenous cannabinoid N-arachidonoyI ethanolamine (anandamide), the sleep-inducing agent oleamide, the food-suppressing compound N-oleoylethanolamine (OEA), and the peripheral pain-suppressing agent N-palmitoylethanolamine (PEA). The creation of genetically engineered mice that lack this enzyme (i.e., FAAH(-/-) mice) has provided a powerful model to evaluate the function of this enzyme. These mice exhibit a CBI-mediated reduction in pain sensitivity, accompanied by substantial increases in endogenous anandamide levels compared to wild type mice. In the studies proposed in this application, we will evaluate the in vivo effects of a series of highly selective and reversible FAAH inhibitors. These FAAH inhibitors provide complementary tools to further our understanding of the physiological functions of the FAAH/FAA system. This project will employ pharmacological and behavioral methods to address three Specific Aims: 1) To determine the role of endocannabinoid-metabolizing enzymes in acute and chronic pain; 2) To determine the role of endocannabinoid-metabolizing enzymes in cognition and emotion; and 3) To determine the role of endocannabinoid-metabolizing enzymes in morphine reward and withdrawal. RELEVANCE (See instructions): The endogenous cannabinoid system regulates a broad range of neurophysiological processes. Elucidation of the enzymes that regulate endogenous cannabinoids and their mechanisms of action may lead to the identification of new therapeutic targets for the treatment of human disorders such as chronic pain, depression, and anxiety.


The postnatal development of both the optical and neural visual systems is dependent on visual experience. Visual experience is defined by the information available in the retinal images in the two eyes. The goal of the proposed research is to extend our previous examination of retinal image quality in one eye during infancy to full binocular viewing conditions. We will examine human infants' visual experience in the context of image clarity and image alignment, which are primarily defined by accommodation and vergence responses and their interaction. These studies will document the emergence of the interaction between accommodation and vergence and their role in the development of refractive and accommodative strabismus. There are three specific aims: i) To understand the normal maturation of the relationship between accommodation and vergence with emmetropisation and growth of the distance between the eyes. ii) To determine the relative bias towards accommodation or vergence accuracy during the critical period of human development. iii) To understand the effects of accommodation and vergence behavior on visual experience of infants and children with high hyperopia or strabismus. PUBLIC HEALTH RELEVANCE: This project will determine how young infants and children manage apparently conflicting focusing and eye alignment demands during typical development. It will also investigate why some children develop refractive or accommodative strabismus while others, with apparently matching visual systems, do not. The goal is to develop intervention strategies to prevent this strabismus and associated amblyopia.


Nira Pollock, MD, PhD, is currently an instructor of medicine and infectious diseases (ID) at Beth Israel Deaconess Medical Center (BIDMC). The candidate's long-term goal is to develop an independent patient- oriented research career focused on the development and evaluation of new diagnostic tests for infectious diseases. Dr. Pollock's interest in diagnostics emerged from her strong combined basic science and clinical training and was focused by her ID specialization. Recognizing the rise of global tuberculosis (TB) case rates in the era of HIV and the limitations placed on TB control efforts by the weaknesses of currently available TB diagnostics, the candidate has chosen to focus her research and clinical efforts on TB. Under the guidance of two senior Harvard-affiliated TB researchers, Dr. Antonio Campos-Neto and Dr. Edward Nardell, she has initiated a promising TB diagnostic development project and has prepared a multi- disciplinary career development plan which will allow her to develop into an independent translational researcher with an exceptionally strong grounding in both ID and clinical microbiology. For her project, Dr. Pollock will work to develop a new urine antigen detection test for diagnosis of active TB disease. Her goal is to create a fully non-invasive, point-of-care, rapid test which would be useful in resource-limited settings, thereby making a substantial contribution to patient care and TB control. After completing pre-clinical development, she will evaluate the new test by integrating testing into an established clinical study of TB in Peru, run by Partners in Health. This will allow her to assess test performance for routine diagnosis of active pulmonary TB in adults, as well as to gain preliminary data about test utility for diagnosis of TB in children and for monitoring treatment efficacy. Dr. Pollock will combine her research program with a structured, comprehensive didactic curriculum in clinical research methodology at the Harvard School of Public Health. Finally, she will continue to develop her expertise in clinical microbiology through further practical experience in the BIDMC microbiology laboratory, recognizing that the clinical microbiology laboratory provides an ideal setting for applied infectious diseases diagnostics research. RELEVANCE (See instructions): The goal of this project is to develop a new, rapid, simple, and highly accurate urine test for diagnosis of active tuberculosis (TB). This test could dramatically change how TB is diagnosed world-wide, and thus could help prevent the spread of this highly contagious and often deadly disease.


Recurrent HSV-2 shedding is a major risk factor for HIV-1 transmission. Recent data indicate that HSV-2 is present up to 40% of days in the periphery. The factors that control HSV-2 shedding in the skin are incompletely understood. One contributor is likely HSV-2-specific CDS T-cells, which will be studied in Project 1. This Project will focus on the expression of antiviral genes in the skin and correlating this with shedding of HSV-2. We hypothesize that a net antiviral state, as measured by the mRNA levels of interferon-stimulated genes (ISG), will correlate with the re-appearance of HSV-2 DMA after the healing of symptomatic HSV-2 lesions. Because circulating HSV-2-specific CDS T-cells over-express CXCR3, and mice with lesions in the CXCR3 axis have accelerated HSV-2 pathogenesis, we further hypothesize that the CXCR3 ligand chemokines, CXCL9, 10, and 11, will correlate with the infiltration of HSV-2-specific CDS cells (Project 1) and pDC, and negatively with detection of HSV-2 DMA and mRNA. Plasmacytoid dendritic cells (pDC) infiltrate HSV-2 lesions in animals and humans (our data), react strongly with HSV-2 via TLR9 to secrete IFN-a, and are required for defense against HSV-2 in mice. Substantial heterogeneity in pDC reactivity to HSV-2 has been observed, as have rare patients with pDC numerical or functional deficiencies and severe HSV infections. pDC likely contribute to the net IFN-driven antiviral state in lesions and the anti- HSV drug action of resiquimod. We propose blood-based studies to investigate the hypothesis that pDC number of function correlates with HSV-2 shedding rates, using subjects from the Clinical Core who are studied in Project I. Aim 1 will correlate ISG and interferon-producing cells with HSV-2 DNA/mRNA detection in human skin biopsies. Aim 2 will focus on the chemokine receptor utilization of HSV-2-specific CDS T-cells. Aim 3 will correlate pDC number and function in blood with HSV-2 shedding and clearance rates. The results may assist in designing vaccines for HSV-2 that elicit cells with the appropriate homing strategies, and clarify the mechanism of action of innate immunity modifiers as local therapy for HSV-2.


Technological developments in the field of genomics now afford the opportunity to define the complete sequence of an individual's genome in a rapid and affordable manner. Such whole genome sequencing and its simpler corollary, whole exome sequencing (WES), have already established themselves as powerful research tools. The natural next step in the evolution of this technology is its direct application in the clinical arena. However, while such technology holds considerable clinical promise, tremendous challenges exist in applying it and deriving practical benefit to patients. In this proposal we outline a highly interdisciplinary approach to identifying, confronting and overcoming the major challenges which must be met in order to implement deep sequencing technology in clinical medicine. Aim 1 will explore the use of WES as a diagnostic tool in the care of a broad array of patients, evaluate its performance, identify critical clinical characteristics which can guide its application and measure the impact of such information on patients and providers. Aim 2 will tackle one of the most pressing challenges in the clinical application of WES: the inevitable generation of collateral or bystander information. Educational materials will be developed to enable patients to make decisions about appropriate return of results and the impact of collateral information will be assessed at the level of the provider, laboratory and patient. The third major challenge in clinical implementation of genomic medicine, how do deal with vast amounts of information, will be addressed by Aim 3. A clinically oriented binning structure will be created and refined for classifying, storing and transmitting data within practical categories so as to make sense of the large amounts of data generated. Finally, as we stand on the cusp of genomic medicine, we must ensure that all have access to its benefits. Thus, Aim 4 will pursue clinical WES in traditionally underrepresented populations to identify special opportunities and challenges in the clinical translation of this new tool to the broadest possible population. Our ultimate aim is to establish a set of best practices to guide future implementation of robust genomic technologies for the real and practical betterment of human health.


A notable determinant of human intellectual capacity is the enormous size and complexity of our neocortex. The neocortex forms during embryogenesis and then expands during fetal development when progenitors differentiate to populate the cortical plate. Defects in brain growth and morphogenesis result in a host of neurodevelopmental disorders, neuropsychiatric diseases, and intellectual disabilities. A key step towards understanding the normal and abnormal functions of the brain thus lies in defining the mechanisms driving neocortical growth. Progress towards this goal has been made though the identification of functionally distinct neural progenitor populations, most prominently ventricular radial glia (vRG), intermediate progenitor (IP), and basal/outer radial glia (bRG) cell. These classes of progenitors are common to both rodents and humans. However, recent studies have proposed that the neocortical enlargement and complexity seen in humans may result in part from a substantial increase in the genesis of bRG and IP cells that is not seen in rodents. Remarkably little is known about the mechanisms behind this human-specific expansion. Our preliminary experiments implicate Foxp transcription factors as important components to this process. Foxp1 and Foxp4 are expressed in the human neocortex as vRG cells transform into bRG and IP cells, and altering Foxp1 and Foxp4 functions in mouse changes cortical development in a manner suggesting that they play pivotal roles controlling the production of bRG and IP cells respectively. In this proposal, we will determine the function of Foxp proteins in both mouse and human cortical development. In Aim 1 we will characterize the expression of Foxp proteins in the developing mouse and human neocortex. In Aim 2, we will determine how manipulation of Foxp functions alters the generation of bRG and IP cells, and the overall size and structure of the cerebral cortex. Lastly, in Aim 3 we will define the genomic targets of Foxp proteins mediating their contributions to neocortical growth.


The purpose of the NHANES (National Health and Nutrition Examination Survey) Initial Epideomiologic Follow-up Survey was to conduct a longitudinal study of 14,407 adults originally surveyed in 1971-1975, to investigate subsequent health and mortality outcomes. Respondents have been traced and re-examined. Additional information was obtained from hospital records, the National Death Index and death certificates. The NHANES Initial Follow-up Survey is essentially complete. The purpose of this intramural project is to examine the relationship of chemopreventive and nutritional factors and cancer in this representative population. This study provides an opportunity to examine these factors and potentially confounding or modifying factors in a prospective fashion, and to examine the effectiveness or toxicity of some dietary agents which are currently of great interest for cancer prevention. The relationship of baseline vitamin use, biochemical or nutritional measures and subsequent health status will be examined. In addition, descriptive data and trends in potential risk factors or protective factors over time will be examined. A continued follow-up for this cohort is expected to begin in 1985.