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Timaeus 12

The long-term objective is to define the role of MHC class I transplantation antigens in tumor progression, in order to identify immunological and molecular interactions which might be relevant to the origins and treatment of human cancer. These studies are intended to address the molecular origin and functional significance of novel class I products on murine tumors. The experiments are intended to probe the generality and significance of the phenomena defined by the UV-induced C3H fibrosarcoma, 1591. This tumor expresses at least three novel class I products, encoded by genes which appear to have been derived by recombination. Two complementary sets of studies are proposed. First, DNA from a large variety of tumors will be examined by genomic Southern blot analysis for evidence of class I alterations comparable to those observed in 1591. A variety of chemotherapeutic and mutagenic treatments will be tested for their potential to elicit class I alterations in vitro. These studies will address the etiologic component of class I alterations. The second set of experiments involve the characterization of the novel class I product expressed on the C3Hf adenocarcinoma, LT85. Peptide mapping studies suggest that, as in the case of 1591, the gene encoding the LT85 molecule was generated by recombination from an endogenous H-2 gene. Furthermore, 40% of the C3Hf tumors tested express immunologically cross-reactive molecules suggesting that the LT85 mutational event occurs reproducibly. The gene encoding this novel antigen will be cloned, and its origin identified. Specific molecular probes will be generated, and the generality of the LT85 event will be analyzed in other C3Hf tumors. The potential reproducibility of the class I alterations in the C3Hf system might afford histological identification of the activation of altered class I molecules in nascent C3Hf tumors and to help elucidate the subtle consequences of their expression on the process of tumorigenesis. Given the complexity of the human HLA system as well as the absence of appropriate transplant hosts to determine the immunogenicity of human tumors, immunobiological studies of genetically defined models, such as those described here, are essential in providing fundamental biological information applicable to the molecular characterization of comparable phenomena in human disorders.

DESCRIPTION (adapted from the application): Genetic evidence has implicated three proteins, the beta-amyloid precursor protein (beta-APP) and the two homologous presenilins (PS-1 and PS-2), in the etiology of Alzheimer's disease (AD). The investigator had previously proposed based on precedents in other developmental systems, that one or more forms of beta-APP and PS-1 (or PS-2) may be components of an intercellular signaling system in normal physiology. beta-APP and PS-1 or PS-2 on the surfaces of neighboring cells would bind to one another specifically through their extra-cellular domains protruding from the cell surface membranes and generate a signal. beta-amyloid (Abeta) would be a proteolytic by-product of other events following this interaction. The present proposal is based on evidence that the PI and colleagues have shown for this proposed beta-APP: PS-mediated intercellular signaling event. Cultured cells, transiently transfected with either beta-APP, PS-1 or PS-2, were appropriately mixed, and were analyzed for protein tyrosine kinase activity, and for net protein tyrosine phosphorylation, with antibodies specific for phosphotyrosine (Ptyr). Within several minutes after mixing the beta-APP transfected cells with either the PS-1 or PS-2 transfected cells, the cell extracts showed significant transient increases in protein tyrosine kinase activity, and in Ptyr modification of protein substrates, that did not appear in controls. Furthermore, the spectrum of proteins modified by tyrosine phosphorylation differs depending on whether PS-1 or PS-2 is involved in the specific intercellular binding to beta-APP, which implies that PS-1 and PS-2 have distinct, rather than redundant, functions in normal physiology. The specific aims are to study the intercellular interactions between beta-APP and the PS, to determine the direction of signal, to identify the non-receptor tyrosine kinases that mediate it and to characterize the role of src kinases in the process.

The effects of lead (Pb) on the developing brain have been studied for decades but there are still gaps in our understanding of how this environmental toxicant influences brain development and function. The modification of Pb's influences on nervous system development and function by genetic background and the manner in which Pb interacts with the genome to produce long-lasting behavioral and other effects are mostly unknown. These research questions are the basis of the parent grant associated with this competitive revision application. However, to more fully understand Pb-genome interactions, effects on the epigenome also need to be studied. In particular, this competitive revision application will focus in general on genome-wide Pb-induced alterations in methylation and in particular, on changes in methylation state and expression of MeCP2 (a DNA binding protein involved in transcriptional regulation of a multitude of genes) and critically involved in neuronal maturation and plasticity as well as a variety of cognitive and behavioral disorders including Rett syndrome, autism, mental retardation, ADHD, and learning disabilities. Since recent studies suggest that environmental toxicants can affect the integrity of the genome through effects on epigenetic mechanisms, the extent to which this occurs with different levels and types of Pb exposure need to be studied. Thus, the research proposed in this application has the following specific aim: Specific Aim. Assess the extent to which different types and levels of developmental lead exposure result in epigenetic influences on DNA methylation and MeCP2 expression/methylation in particular and the extent to which these effects correlate with gender and behavioral outcome. These studies will examine the extent to which different types and levels of lead exposure in male and female animals influence DNA methylation on a genome-wide basis and the extent to which there is aberrant MeCP2 promoter methylation and MeCP2 protein expression in the hippocampus, a brain regions known to be functionally affected by developmental lead exposure. We will then correlate these findings with behavioral outcomes. Our hypothesis is that lead exposure leads to a neurodevelopmental disorder, fundamentally, a disorder of plasticity, related to altered epigenetic gene regulation (i.e., alterations in DNA methylation) and that these effects may vary with gender. PUBLIC HEALTH RELEVANCE: The proposed research will provide new data on the effects of developmental lead exposure on epigenetic modifications (i.e., modifications to genes that do not involve changes in the DNA sequence). Recent reports suggest that environmental toxicants may affect the integrity of the genome and can do so through epigenetic mechanisms. Understanding effects of developmental lead exposure on the epigenome may help to tie together basic, clinical and epidemiological data showing effects on a multitude of diverse physiological processes and outcomes including impairments in neuronal structure and functioning and impairments in cognitive, social, language and motor skills that persist into adulthood.

Uterine fibroids affect 77% of women by menopause in the U.S. and account for $2.1 billion in healthcare costs each year. Until recently, tumor tissu and cell culture studies investigating fibroid growth have been the primary sources for understanding fibroid pathophysiology. Genetic analysis provides a powerful and cost effective tool to identify etiological and causal factors, especially since a genetic predisposition to fibrods has already been shown from twin studies. As much as 69% of risk is explained by genetic factors. Racial disparities also support a role for genetics in fibroid risk. African American (AA) women have earlier age-of-onset, more numerous and larger fibroids with a greater lifetime incidence compared to European Americans (EAs). Among existing genetic analysis approaches, whole exome genotyping (WEG) is the most cost-effective and efficient compared to genome-wide association studies (GWAS) that focuses on common variants that may themselves not be causal. In this study, we will take advantage of a unique Vanderbilt resource, the BioVU DNA databank. BioVU has over 141,221 adult DNA samples linked to electronic medical records (EMR). From BioVU we have identified 3,535 AA subjects (612 cases and 2,923 controls) who meet our stringent inclusion criteria. Studies have shown that many women with fibroids are asymptomatic and without imaging as many as 51% of women may be misclassified. As a result, we have required pelvic imaging for both cases and controls. 50,000 BioVU subjects are currently being genotyped using Illumina's Exome Chip as part of an institutional initiative, including all AAs (completion summer of 2013). Our first aim is to conduc a WEG study of fibroids using AA BioVU subjects (n = 3,535) using logistic regression (common variants) and gene-based allele collapsing approaches (rare variants) to evaluate associations of SNPs with fibroids risk. Our second aim is to resequence exomes at 50X using AA BioVU extreme fibroid cases (n = 75) and controls (n = 75) to discover novel coding variants associated with fibroid risk. Extreme cases are those with youngest fibroid onset and largest number and size of fibroid. Extreme controls are the oldest subjects with no recorded history of fibroids. Aim 2 will allow us to validate variants discovered in Aim 1, as well as to identify nove variants not included on the genotyping chip. The NIH, NICHD, AHRQ, and the OWHR have made understanding the mechanisms underlying fibroid risk research priorities. This study is the largest and first whole exome experiment of fibroid among AAs. Immediate availability of samples and resources allow us to accomplish these R01 scope Aims within the budgetary and time constraints of an R03. These aims will allow us to identify novel variants associated with fibroids using two distinct approaches (WEG and exome resequencing using extreme phenotypes) and will lay the ground work for a future R01 examining rare variants associated with fibroids across racial groups. Our proposed study will fundamentally change knowledge about fibroids and lead to breakthroughs in understanding mechanisms in fibroid formation.

A novel computational analysis platform is proposed, which enables collaboration in the discovery and characterization of proteomics biomarkers. The proposed system is a broad academic-commercial collaboration, integrates several prominent proteomics efforts, and marks first major use in proteomics of virtual cluster technology. Consequently, the proposed platform offers new solutions to the security, scalability, and ease of use problems that have hampered proteomics collaborations to date. The proposed software brings together several popular open-source proteomics tools projects, together with experts in the development of scientific computing solutions. The resulting system is validated by applying it to the study the human plasma proteome, in conjunction with the international collaborative HUPO Human Plasma Proteome Project. This research builds on a successful collaboration of experts in proteomics and software development. The impressive research team includes pioneers in proteomics, public health, and bioinformatics. The proposed software platform will be widely applicable to the study of biological mechanisms and measurement of complex protein systems. to public health: The aim of this research is to enable a new proteomics technology that will both lower the cost of proteomics experiments and make the resulting data more biologically relevant. A successful outcome would increase the potential health benefit of a broad array of current and future research. The HUPO Human Plasma Proteome Project is the largest and broadest proteomics collaboration ever attempted. The goals of this project are comprehensive analysis of plasma and serum protein constituents in people, and identification of biological sources of variation within individuals over time, with validation of biomarkers. By working closely with this project, the proposed research has the potential for a substantial positive influence on human health.

The goal of the proposed research is the development of novel approaches to ameliorate erythrocyte sickling in individuals with sickle cell disease (SCD). Although this disease was discovered more than a century ago, we still lack effective mechanism-based therapies. This disease is the most prevalent inherited genetic disorder and affects millions worldwide. As an important mission of NIH is to develop highly innovative treatments for this disease, the goal of the proposed work should be of high priority. Research proposed here is based on unexpected discoveries resulting from an unbiased, high-throughput metabolomic screen that identified several metabolites including adenosine (Ado), sphingosine-1-phosphate (S1P) and 2,3- diphosphoglycerate (2,3-DPG) that are highly elevated in the blood of mice and humans with SCD. Follow-up studies showed that these three elevated metabolites collaboratively work together to promote sickling, the central pathogenic process of the disease. The project has interrelated goals to translate our findings into innovative therapeutics for SCD by providing new insight into disease pathogenesis. In AIM I, we propose to use both pharmacological and genetic approaches to determine whether excess Ado signaling via A2BR induces 2,3-DPG production by protein kinase A (PKA)-dependent phosphorylation of key enzymes involved in 2,3-DPG generation. These studies will provide novel mechanism underlying Ado signaling-mediated 2,3-DPG induction and add a new chapter to erythrocyte physiology and pathology. In AIM II, we will explore different approaches including genetic, pharmacological, cellular, biochemical, and high throughput robotic co- crystallography coupled with spectral functional analysis to determine how S1P: 1) functions as an intracellular allosteric modulator to regulate Hb-O2 affinity and 2) functions via extracellular S1P receptors to induce inflammation. Additional experiments encompass a set of preclinical studies to test the efficacy and safety of lowering S1P by specific antagonists in SCD mice. These studies are expected to provide the foundation for appropriate clinical trials in humans with SCD. Third, the molecular basis underlying elevated S1P inside RBCs and in plasma will be investigated. Briefly, we propose to test a novel hypothesis that A2BR functions upstream to induce S1P production within RBCs and that activated platelets and complement activation-induced intravascular hemolysis underlie the further elevation of S1P in plasma. In AIM IV, we propose translational studies to conduct an analysis of approximately 235 adult blood samples obtained from a large cohort of SCD patients by collaborating with the NHLBI to determine whether these newly identified elevated metabolites are pathogenic biomarkers correlating to disease severity and phenotypic variation. Overall, the proposed preclinical animal studies and translational human studies are expected to reveal important therapeutic targets in the medical management of SCD patients.

Alcohol use Is a significant behavioral cofactor for HIV transmission. The association of alcohol with sexual risk behavior appears especially strong among high-risk adolescents, including adjudicated adolescents, who are at particular risk due to high rates of alcohol use and comorbid psychiatric disorders. Theoretical models of sexual risk behavior rely almost exclusively on social-cognitive perspectives. Basic knowledge about biological influences on HIV risk behavior, including alcohol-related risk taking, is virtually non-existent. Externalizing behaviors in adolescence and young adulthood show a common genetic diathesis, suggesting common biological and/or temperamental influences. In particular, shared genetic risk for a spectrum of externalizing behaviors, including sexual behavior, is attributed to a highly heritable factor reflecting behavioral disinhibition. Recent research has linked specific genetic variants to cognitive, behavioral and neurobiological phenotypes associated with behavioral disinhibition and externalizing disorders. Given the importance of the mesolimbic dopamine system in reward-related behavior and risk-based decision-making, genes with implications for dopaminergic function are high-priority candidates. The goal of the proposed research is to evaluate genetic factors that may a) predict common risk for alcohol use, sexual risk behavior and other externalizing behaviors, and/or b) moderate the association of alcohol use and HIV risk behavior. This research will take place in the context of a prospective investigation of genetic and neurobiological correlates of alcohol and HIV risk-taking among adjudicated adolescents (R01AA017390-01). Analyses will examine genetic influences on alcohol use, HIV risk behaviors and externalizing behaviors using impulsivity/disinhibition as a plausible endophenotype. Specific hypotheses concerning the moderating effect of the DRD4 VNTR polymorphism on alcohol-related sexual risk behavior will be evaluated. This project, which responds to the need for integrative and translational methods in studies of alcohol and HiV risk, will contribute basic information about genetic factors and related behavioral characteristics that may place some individuals at elevated risk for HIV via the propensity for externalizing behaviors, including alcohol use.

This is an integrated clinical and laboratory program which will examine the determinants of human cell mediated immunity in the context of AIDS. Model studies of Leprosy and Leishmaniasis are evaluating the factors responsible for the emigratory, vascular and maturational responses of human skin to delayed type antigens. Cellular dynamics, structural and functional phenotypes of dermal and epidermal populations are considered. The role of the recombinant lymphokines gamma- IFN and IL-2 in this milieu as well as the resulting monokines IL- 1, TNF, arachidonate metabolites and O2 intermediates are under investigation. The selective binding, emigration and biochemical dialogues between mononuclear cells and endothelial cells (EC), interacting on defined substrates leads to a mutually cooperative state. In contrast, PMN when stimulated with TNF and LT while attached to EC, lead to a massive respiratory burst and target cell damage. The alpha-keto acid pyruvate is an important, secretory scavenger of H2O2, protecting targets from oxidative attack. The role of these reactions in TNF induced hemorrhagic necrosis and lethality is under study. Dendritic cells (DC) are central, accessory cells for primary T cell responses. They are particularly enriched in the joint fluids of patients with rheumatoid arthritis (RA) where they cluster with T cells. These T cells will be cloned and their restriction to disease associated HLA-DR4 antigens studied. The secretory activity of monocytes and DC obtained from joints of patients with RA is being examined. The role of LFA associated adhesion promoting receptors during monocyte, T cells and DC interactions is under study at the biochemical and ultrastructural level (immunogold labeling). The natural ligands of these important receptors include surface LPS of gram negative bacteria. Purification and cloning of a novel lymphokine required for human CTL differentiation is underway. The preparation of specific antibodies will allow sensitive bioassays and blocking experiments. CTL granulogenesis will be followed by quantitating the biosynthesis of perforin 1 and the serine esterase. A new clinical program at the Rockefeller University Hospital will evaluate the basic immunological defects in patients with AIDS. Longitudinal analysis of the functions of mononuclear phagocytes, DC, T cell subsets, Langerhans cells will include surface receptors, endocytosis, intracellular killing, secretory repertoire, accessory function, tissue emigration and DTH reactivity. Experiments will characterize the presence of the viral genome in patients' cells and establish their permissiveness to infection in vitro.

DESCRIPTION (Scanned from the applicant's description): In the current funding period, we have demonstrated that persistent exposure of the intestine to lipid hydroperoxides (LOOH) induces an imbalance in tissue glutathione (GSH) and glutathione disulfide (GSSG) redox status, impairs peroxide detoxication, and disrupts enterocyte turnover, independently of cell injury. GSH supplementation restores cellular redox balance and maintains normal turnover kinetics. These findings suggest that oxidative stress and the accompanying redox imbalance are important mediators of specific cellular and molecular responses in intestinal cell growth and death. We currently propose to define the role of cellular redox in intestinal apoptosis and proliferation and the mechanisms by which redox mediates these responses. Our central hypothesis is that LOOH-induced redox imbalance mediates transition of intestinal cells from a quiescent state to that of a prohferative or apoptotic state by (differential activation of cell signaling pathways. We further hypothesize that upregulation of mitochondrial MnSOD abrogates mitochondria ROS generation, restores matrix redox balance and attenuates cell apoptosis. The aims address 4 specific hypotheses. Aim 1. To test the hypothesis that LOOH-induced redox imbalance differentially mediates intestinal cell proliferation or apoptosis depending on the severity and duration of the redox shift. Aim 2. To test the hypothesis that redox-mediated transition of intestinal cells to the proliferative or apoptotic states is associated with activation of proliferative or apoptotic signaling. Aim 3. To test the hypothesis that redox-induced apoptotic or proliferative signaling is mediated through differential activation of nitrogen activated protein kinases (MAPKs), Akt, or NFkB. Aim 4. To test the hypothesis that upregulation of mitochondrial MnSOD abrogates mitochondrial ROS generation, restores matrix redox balance and attenuates cell apoptosis. The studies will provide (a) important new information on the impact of oxidant challenge and loss of redox balance on regulation of intestinal apoptotic and proliferative responses, and (b) new insights into the potential use of antioxidant enzyme overexpression in the maintenance of metabolic integrity and turnover homeostasis of the intestinal epithelium.

A radiometric technic has been developed for measuring gastric mucosal blood flow using C14-labelled aminopyrine. Close agreement between radiometric and colorimetric technics was found. Using this method an attempt is being made to separate histamine H1 and H2 receptor regulation of mucosal blood flow. Using C14-metiamide we have studied kinetics of mucosal membrane binding of the histamine H2-receptor antagonist to define an apparent receptor. We have found that the membrane fraction which contains the histamine H2-receptor contains an adenylyl cyclase which is unresponsive to histamine but is activated by prostaglandins which also increase the mucosal active transport of sodium. BIBLIOGRAPHIC REFERENCES: Chou, A.C., Jacobson, E.D. and Shanbour, L.L. Electrophysiological responses of the canine gastric mucosa to endotoxin, epinephrine and hemorrhage. Circ. Shock 2:13-20, 1975. Bowen, J.W., Kuo, Y-J., Pawlik, W., Williams, D., Shanbour, L.L. and Jacobson, E.D. The electrophysiological effects of burimamide and 16,16 dimethyl prostaglandin E2 on the canine gastric mucosa. Gastroenterology 68:1480-1484, 1975.

Restoration of dental, oral and craniofacial (DOC) tissues lost to disease, injury or congenital disorders is a major goal of oral reconstructive therapy. Tissue engineering (TE) and regenerative medicine (RM) offer strong potential to yield more predictable, less invasive and personalized treatments for patients. The goal of this Resource Center (RC) is to transform the treatment of DOC deformities due to disease or injury through the development and translation of clinically-relevant TE/RM technologies. Our vision to achieve this goal is to integrate advances in life science and engineering technologies to solve clinically-defined problems in an environment having a translational infrastructure. Fulfillment of this vision will enable dentistry to be transformed, will establish Michigan as a national leader i regenerative DOC therapies, and will serve as an economic engine for the region. To advance translation of TE/RM technologies to the clinical arena and fulfill the goals of this RFA, a shift n thought regarding the practice of TE/RM is required. Towards this end, we will establish an interactive, multidisciplinary team comprised of investigators at Michigan, the Clinical Practice Community and Harvard/Wyss Institute. This team will develop an organizational structure and operational procedures for establishing a centralized RC for identifying the most promising TE/RM technologies for clinical application. By establishing multidisciplinary teams working toward a common vision, our RC will enable technologies to progress through the translational pipeline. To achieve this vision, in this planning grant, we will: 1) Develop an organizational structure to select regenerative strategies to translate to clinical practice. The RC will leverage existing excellence at the University of Michigan, Clinical Practice Key Opinion Leaders (KOLs) and Harvard/Wyss Institute, along with Michigan's translational infrastructure in TE/RM and industrial partners to form a coordinated team. This team will be dedicated to selecting the most scientifically sound, clinically and commercially applicable strategies for the regeneration of DOC tissues. 2) Develop a support and infrastructure team for technical advancement of selected strategies. The RC will orchestrate high quality technical support and a research infrastructure dedicated to guiding TE/RM investigators at Michigan and other RCs on how to test and validate technologies, standardize procedures and compare to existing technologies, and define appropriate endpoints and functional outcomes. 3) Develop teams and operating procedures to support the clinical translation of selected TE/RM technologies. We will organize: a) the application of clinically relevant pre-clinical animal models that recapitulate human disease and injury conditions; b) manufacturing protocols; c) FDA regulatory expertise and protocol development for Phase I/II trials and d) clinical DOC expertise for clinical practice translation.

[unreadable] [unreadable] The current application is aimed at providing the Principal Investigator with in-depth training in the area of host-microbial interactions along with formal courses at the graduate level to enhance her research and laboratory skills. The expertise of the sponsor and co-sponsor combined with a highly interactive basic-research environment at the Department of Medicine at University of Illinois at Chicago, offer a great training opportunity for the Principal Investigator to achieve her long-term goals in becoming an independent research investigator in the area of host- microbial interactions. The proposed studies are focused to examine the role of anion transport in the pathophysiology of diarrhea associated with infection by an important food-borne pathogen, Enteropathogenic E. coli (EPEC). To date, the mechanism(s) underlying EPEC associated diarrhea are not clear. Diarrhea results from increased secretion or decreased absorption or both. Earlier studies have failed to conclusively show an increase in host secretory responses by EPEC infection. We hypothesized that EPEC-induced diarrhea might involve a decrease in intestinal ion absorptive processes. Our preliminary data showed a decrease in CI'-OH' exchange activity, butyrate transport and Na+ absorbing isoform, NHE3 in response to EPEC infection. The present studies will explore the effects of EPEC infection on Cl'-OH' exchange activity in both in vitro and in vivo models and elucidate the contribution of luminal anion exchangers, DRA (SLC26A3) and PAT-1 (SLC26A6). Studies in Specific Aim 1 will determine the effects of EPEC on Cl' transport in model human small intestinal (Caco-2) and colonic (T84 and non-transformed NCM460) epithelia; along with elucidation of signaling mechanism(s), role of EPEC virulence genes and kinetic parameters of EPEC mediated effects on Cl' transport. Specific Aim 2 will utilize siRNA technology to define the role of DRA and PAT-1 in modulation of CI'-OH' exchange in response to EPEC infection. Specific Aim 3 will critically examine the effects of EPEC on CI' transport in ileum and colon utilizing the in vivo murine model of EPEC infection. The results from these studies will not only increase our understanding of the mechanisms of regulation of human intestinal Cl'-OH' exchangers and their modulation by pathogenic organisms, but will also provide basis for the pathogenesis of EPEC-induced diarrhea which might aid in the development of improved therapeutic modalities in future. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

The processing of gustatory information by neurons in the hamster central nervous system will be studied using both electrophysiological and behavioral techniques. Neurons in the nucleus tractus solitarius, parabrachial nucleus, and thalamus that respond to gustatory stimuli will be examined for their responsiveness to a variety of stimuli and stimulus mixtures. Concurrently with this electrophysiological recording, behavioral exeriments will be conducted to determine how hamsters categorize these gustatory stimuli so that the neurophysiological data can be interpreted within this psychophysical framework. An attempt will also be made to obtain electrophysiological data from taste-responsive neurons in a chronic, behaving hamster preparation. The time course of the neural response to taste stimulation will be of particular interest, particularly in the behaving preparation.

Project Summary The Connecticut Veterinary Medical Diagnostic Laboratory at the University of Connecticut (CVMDL) is fully accredited by AAVLD and a member of the FDA CVM Vet-LRN and USDA's NAHLN. Our participation in the organizations listed above demonstrates our keen interest in investigating emerging animal and zoonotic diseases and disease surveillance, as well as our commitment to shared goals of quality veterinary diagnostics and networking to improve the scientific knowledge base and response capabilities. The CVMDL offers a full service veterinary diagnostic laboratory with qualified faculty and staff, modern facilities and up to date equipment. Co-PI Risatti has significant expertise in development of molecular based diagnostic assays for federal (USDA) laboratory network use; this experience could be helpful when developing standardized protocols for Vet-LRN use. CVMDL has long standing expertise in veterinary pathology, as well as microbiology, mastitis and molecular based detection of pathogens, including those in environmental samples (animal housing and transport vehicles) and food products (primarily eggs and milk). We currently detect chemical toxins via pathology, histopathological lesions (melamine, ethylene glycol), special histochemical stains (lead, iron, calcium, copper) and immunohistochemical stains (algal toxins, fungal toxins), but are very willing to expand testing for chemical analytes based on Vet-LRN needs. CVMDL is willing participate in Vet-LRN testing, method development and standardization, provide surge capacity for testing if needed, and to expand its testing modalities as resources allow to more fully participate in this Vet LRN cooperative agreement. We understand the need for timely and appropriate response, reporting, and communication with Vet-LRN, and have already established reporting via eLEXNET. CVMDL is the only remaining full service state veterinary diagnostic laboratory in the 6 state New England region, and provides critical diagnostic and surveillance services for animal and zoonotic diseases in the region. We have established linkages with the CT Department of Public Health (particularly in areas of zoonotic diseases and emergency response), as well as the CT Department of Agriculture and USDA APHIS Veterinary Services. CVMDL's participation in this joint endeavor will enhance the linkages between veterinary and public health, providing early diagnosis and response to food-borne and other pathogens that can impact both animal and human populations.

Platelet aggregation has been shown to be a consistent manifestation of endotoxin shock. It is possible that prostaglandin metabolism is altered by endotoxin as a mechanism for its effect on aggregation. Thromboxane A2, a potent aggregant, is synthesized in the platelet. In the blood vessel wall, a potent anti-aggregant, Prostaglandin I2, is formed. Both sites will be studied in order to determine the specific effect of the endotoxin molecule as well as that of endotoxemia on prostaglandin metabolism. A dose-response relationship will be established between endotoxin and platelet aggregation in dogs. Platelets from endotoxemic dogs will be studied to reveal their aggregation characteristics and prostaglandin (PG) synthesis. Synthesis of anti-aggregant PGI in endotoxemic vascular tissue will be assayed against aggregating platelets. In vitro studies will also be carried out to pharmacologically determine the site of action for endotoxin on Thromboxane synthesis in aggregating platelets and on Prostaglandin I synthesis in normal vascular tissue.

Project Summary The coronary vasculature plays essential roles in maintaining a continuous supply of oxygen and nutrients to the heart. Coronary heart disease is a major cause of myocardial infarction and heart failure, which continues to be the leading cause of mortality worldwide. Despite its importance, the mechanisms that regulate coronary vascularization of the myocardium remain a major gap in our knowledge. We observed that during zebrafish heart development, coronary vessels form a close association with a specific subpopulation of cortical cardiomyocytes marked by the transcription factor Gata4. gata4+ cardiomyocytes follow the tracks of coronary endothelial cells when they emerge onto the surface of the juvenile zebrafish hearts. Furthermore, our preliminary data suggest that expression of the Cxcl12b chemokine, a critical angiogenic factor during zebrafish coronary vessel development, is increased in expanding gata4+ cardiomyocytes. Moreover, gata4+ cardiomyocytes fail to associate with coronary vessels in hearts lacking Cxcr4a, the receptor for Cxcl12b. Importantly, adult cxcr4a mutant zebrafish fail to regenerate after heart injury. We hypothesize that heart development occurs in two phases that are regulated by temporally distinct mechanisms: newly formed coronary endothelial cells provide instructive cues, such as paracrine factors, during the emerging phase to guide gata4+ cardiomyocytes when they emerge, while during the expanding phase, cxcl12b expression increases in gata4+ cardiomyocytes to attract coronary vessels that are essential for their morphogenesis/development and regeneration. We propose to 1) To determine how new coronary vessels guide gata4+ cardiomyocytes to populate the heart ventricle by analyzing differentially expressed candidate genes identified from RNAseq encoding secreted molecules (e.g. cxcr4, erbb4) using CRISPR mutant fish lines and a novel explant culture system; 2) To determine how coronary vessels affect morphogenesis and the regenerative capacity of gata4+ cardiomyocytes during myocardial expansion using multicolor clonal analysis and by manipulating Cxcl12-Cxcr4 chemokine signaling. We will further determine how cxcl12b expression is regulated by hypoxia and Gata4. Our proposed study will reveal potential developmental causes of coronary heart diseases. Furthermore, elucidation of the mechanisms underlying myocardial vascularization in zebrafish will shed light on potential therapeutic approaches for humans.

LRRK2 and Parkinson's disease cell biology Parkinson's disease is a disorder of movement, cognition and emotion, characterized neuropathologically by neuronal degeneration and deposits of protein aggregates termed Lewy bodies. While most cases are sporadic, rare genetic forms of the disease, caused by mutations in alpha-synuclein, parkin, DJ-1 and PINK1, are helping to elucidate pathogenesis. In previous studies, we have defined the role of alpha- synuclein protein interactions (including interactions with synphilin-1 and parkin) in PD-related cell biology. Mutations in leucine-rich repeat kinase 2 (LRRK2) have recently been found to cause autosomal dominant PD. Our overall hypothesis is that identifications of LRRK2 protein interactions will help elucidate pathogeneses of LRRK2 related PD, and possibly sporadic PD. We have identified interactions between LRRK2 and several other proteins, including parkin, synphilin-1, WSB-1 and CARD7. We have found that that mutant LRRK2 causes direct cellular toxicity, and have initial data that for at least some of the LRRK2 mutations, GTP binding and kinase activity are necessary for toxicity. In Specific Aim 1 we will identify LRRK2 interacting proteins using the yeast two-hybrid system and co-immunopreciptation from transfected cells, and define interaction domains of these proteins. In Specific Aim 2 we will study the LRRK2 interactions in expression studies in cells in culture, and in mouse and human tissue, including postmortem human sporadic and mutant LRRK2 PD tissue. We will study the role of these interactors in LRRK2 cellular toxicity, by using siRNA, and by modifying the interaction domains. In Specific Aim 3 we will determine whether LRRK2 kinase activity is critical for cell toxicity, and determine whether LRRK2 can phosphorylate the interactors-and if so, we will determine whether this has a role in toxicity. These studies will help define the role of LRRK2 and its interacting proteins in cellular pathogenesis related to PD, and potentially identify targets for future therapeutic interventions. [unreadable] [unreadable] [unreadable]

Theoretical studies are made of various molecular isoelectronic changes.

For women after menopause and men during hormone therapy in the treatment of prostate cancer, the neurologic disorder known as a hot flash can become a frequent occurrence. This transient hyperthermic shift in body temperature has been linked to the neuropeptide calcitonin gene-related peptide (CGRP), which acts peripherally to increase vasodilation and centrally to increase sympathetic activation, including an increase in metabolic heat production. Recent studies in rodents have demonstrated that these centrally mediated responses may be the result of CGRP dependent changes in the activity of thermoregulatory neurons in the preoptic and anterior regions of the hypothalamus (PO/AH). Using an isolated tissue slice preparation, the electrophysiologic responses and local network properties of PO/AH neurons will be characterized. Initial studies will record the extracellular single-unit activity of these neurons in response to temperature and CGRP. Neurons that demonstrate CGRP dependent changes in firing rate will also be tested for responses in the presence of CGRP antagonists or selective inhibitors of the cAMP signaling pathway. This will be followed by whole-cell recordings to characterize the cellular conductance(s), responsible for CGRP dependent changes in the firing rates of these thermoregulatory neurons. Additional retrograde labeling and histochemistry will be done to determine if CGRP responsive neurons are inhibitory, and project to either the dorsomedial hypothalamus or the raphe pallidus, two regions of the brain directly involved with the sympathetic activation of hyperthermic mechanisms, leading to greater heat retention and increased thermogenesis. We expect that our studies will lead to the characterization of the effects of CGRP on the activity of thermoregulatory neurons in the PO/AH and provide information on the efferent pathways which lead to this hyperthermic response. Clinically, this research may identify new sites of therapeutic intervention in the treatment of this transient hyperthermic shift in body temperature, known as a hot flash. [unreadable] [unreadable]

Epithelial planar cell polarity (PCP) is present when the cells of a tissue are all polarized along a uniform axis lying in the plane of the epithelium. Physiologically important examples of PCP are found in many vertebrate tissues including the respiratory system, where ciliated cells must be polarized in a uniform direction for their beating to drive mucus from the lungs, and the ear, where the sensory hair cells must be polarized in a uniform direction so that their stereocilia are correctly arrayed to respond to sound. Among the most important questions regarding PCP is how individual cells sense the proper direction in which to polarize. Previous studies in Drosophila have provided a partial answer to this question by showing that cells respond to Frizzled, a transmembrane receptor protein, whose activity is present in a gradient across the tissue. However, Frizzled is not itself expressed in a graded fashion and the mechanisms used to establish the Frizzled signaling gradients are poorly understood. In a recent study of PCP in the Drosophila eye, we demonstrated that Fat, a protocadherin protein, is essential for establishing the Frizzled activity gradient that directs PCP. We also showed that Fat is regulated during this process by Dachsous (another protocadherin) and Four-jointed (a transmembrane protein). We further suggested that Fat, Dachsous and Four-jointed may form an evolutionarily conserved signaling cassette that is used to control PCP in many tissues. Fat is also a negative regulator of epithelial cell proliferation. Cells lacking Fat exhibit hyperplasic growth. Despite this critical role, little is known about the Fat regulation or function during growth control. We propose to study the role of Fat by: 1) examining the roles of graded Four-jointed, Dachsous and Fat activity in specifying the direction of PCP, 2) examining the ability of Four-jointed and Dachsous to regulate Fat during the control of cell proliferation, 3) identifying the downstream pathways used by Fat to control PCP and proliferation and 4) determining how Fat is regulated by Four-jointed and Dachsous. These experiments should yield new insights into PCP and growth control as well as into the function of protocadherins, which have been implicated in the Usher Syndrome hearing disorders and as tumor suppressors in liver and colon carcinomas. [unreadable] [unreadable]

With the majority of persons with intellectual disabilities now living in the community instead of institutions, functional use of money has become a particularly important and challenging special education priority. To address this challenge, Praxis, Inc. has undertaken development of a suite of computer-based software products designed to establish the behavioral prerequisites for functional use of money by persons with intellectual disabilities. The first product of the suite, MERIT-1, is a money skills readiness product that assesses and teaches identification of all coins and bills up to $20. To do develop this essential product, Praxis, Inc. has taken advantage of its recent experience in developing other teaching programs for establishing basic discrimination performances and equivalence classes. The programs have been supported by an extensive body of scientifically-validated procedures, all of which have been developed in NICHD-supported projects aimed at improving instructional technology for persons with intellectual disabilities. In Phase 1 of the MERIT-1 project, the Company developed prototype software that implemented the essential features of the MERIT-1 readiness curriculum. The software and supporting items were successfully field-tested, and critical milestones were achieved. The Specific Aims of this Phase-2 project are to: (1) transform the prototype software into a high quality turn-key application for teaching money discrimination skills that are prerequisite for learning the values of coin, bills, and their combinations; and (2) field-test the application to verify its effectiveness with teachers and students who are representative of those who will be the ultimate consumers of the product. The objective of the MERIT suite is to offer a comprehensive curriculum that can potentially prepare all children who have the requisite cognitive capacity to use money independently. The development of MERIT-1 (Phase 1 completed) was initiated to support special education teachers whose students have IEP (i.e., individualized educational plans) objectives relating to money skills, but who had yet to achieve success in assessing and teaching the prerequisite basic discrimination skills. [unreadable] [unreadable] [unreadable]

The Duke Older Americans Independence Center (OAIC) is structured to (1) enhance and support research and (2) research career development in aging research through its Core resources. The central theme of our OAIC is to understand and modify the multiple pathways of functional decline. The OAIC is based in the Duke Center for the Study of Aging and Human Development, an all-university program with strong multidisciplinary affiliated programs such as the Durham VA GRECC, the RAND/Hartford Interdisciplinary Geriatric Research Center, the Duke Institute for Genomic Sciences and Policy, the Duke Clinical Research Institute, the Duke Center for Living, Trajectories of Aging and Care Center, and the Stedman Nutrition and Metabolism Center. This rich mileu includes 126 faculty as Senior Fellows of the Aging Center and over 21 million dollars of research germane to the goals of the OAIC. The Duke OAIC will support two research cores which have evolved from prior OAIC support: (1) an Analysis Core and (2) a Biological Studies Core. Six externally funded NIH/VA grants, with study aims and study populations that integrate into our thematic focus, will receive support from these cores; with new specific research aims relevant to our Center. The Research Career Development Core will facilitate career development with established post-doctoral Research and Geriatric Training Programs. Subsequent support for career development and pilot projects will be selected on a competitive basis using criteria clearly defined in the OAIC guidelines. The public impact will be a significant advancement in the basic understanding of the mulitple factors that contribute to functional decline, and the enhancement of interventions directed at improving the overall quality of life of older adults. [unreadable] [unreadable] [unreadable]

The rise in antibiotic resistance among bacterial pathogens has rekindled the threat of infectious disease and accompanying morbidity and mortality. Thus, it is imperative that we develop new strategies to combat bacterial infections. Further, new understanding of the beneficial roles of normal host microbiota in human nutrition, immunology and health suggests that targeted therapies that eliminate harmful bacteria from sites of infection without affecting beneficial microbes are needed. One promising strategy for developing such treatments is to intervene in the mechanisms by which pathogens colonize the particular niches in which they cause infection. An important mechanism affecting the tropisms of a wide range of Gram-negative bacteria is the elaboration of adhesive extracellular fibers called chaperone-usher pathway (CUP) pili. One of the best studied of these systems is type 1 pili and its mannose binding adhesin, FimH, which is critical for host pathogen interactions throughout the course of urinary tract infections (UTIs). UTI: i) is one of the most common bacterial infections; ii) primarily affects otherwise healthy females (50% of women will have a UTI) and; iii) is highly recurrent and is associated with significant morbidity and economic impact, with over $2.5 billion spent annually on treatment. Uropathogenic E. coli (UPEC), the causative agent of ~80% of UTIs, is becoming increasingly antibiotic-resistant. Structural, genetic and biophysical studies have defined the binding interactions between mannose and FimH and indicate that conformational equilibria within FimH regulate its function and role in UTI pathogenesis. Surprisingly, little to nothing is known regarding the structure, function and mechanism of action of the majority of common UPEC CUP adhesins. Therefore, this proposal will elucidate the structures and functions of the two-domain CUP adhesins from two prototypical UPEC strains, used widely to study UTIs: i) UTI89 (a human clinical cystitis isolate) and ii) CFT073 (a human clinical pyelonephritis/urosepsis isolate. Each of these strains encodes complete CUP pilus assembly operons for type 1, Yqi, Yfc1, P, F1C/S, Yeh, Yad3, and Mat pili. In addition, UTI89 encodes a complete F17-like CUP operon and CFT073 encodes complete F9 (Fml) and Auf CUP operons. This proposal will study how conformational equilibria of the FimH adhesin and the FimA rod of the type 1 pilus impact host-pathogen interactions critical in pathogenesis (Aim 1) and determine the function of common UPEC two-domain CUP adhesins, determining UPEC adhesin tissue tropisms, ligand specificity, structure-function correlates and conformational dynamics (Aim 2). This proposal will also investigate structure/function correlates within the family of FimH-like adhesins, revealing likely evolutionary steps leading from one binding specificity to another within the family of FimH-like adhesins and characterizing the probable influence of host innate immunity on natural selection in adhesins (Aim 3). The successful completion of this proposal will give a holistic view into the functional CUP piliome of UPEC, providing new insights into the dynamic function of pili and development of novel therapeutics.

A self-administered dental health program was initiated in Nelson County, Va., a fluoride-deficient community in October 1972. Children in the County's schools, under teacher supervision, chewed and ingested daily a 1 mg F tablet and rinsed weekly with a 0.2% NaF solution. A fluoride dentifrice was provided for ad libitum use at home. Baseline DMFS examinations were made of 2,138 children in the County's elementary, junior high, and senior high schools. Follow-up DMFS examinations were conducted at two-to-three year intervals. Final examinations were conducted in 1983 when the full effectiveness of the fluoride program could be assessed. In the fall of 1983, a sealant program was added to the ongoing fluoride program. Children who were 6, 7, 12, and 13 were eligible to have pit-and-fissure sealants applied. An initial screening to identify those tooth surfaces to be sealed was made in December 1983. Caries data (DMFS) from the September 1983 dental examination served as a baseline for those children who participated in the sealant phase of the study. In succeeding years, new groups of 6 and 12 year olds were enrolled. Treatments continued for four years. Interim dental examinations took place at the start of the third year of the study (September 1985) and final examinations were made in September 1987. Data analysis is nearly complete and preparation of final reports has been initiated.

Effects of the anesthetics isoflurane, halothane, enflurane, and nitrous oxide upon epicardial coronary dimensions and coronary arteriolar tone will be studied in dogs and humans. The coronary vasculature consists of proximal epicardial coronary arteries that conduct blood from the aorta to myocardium and smaller distal intramyocardial arteries and arterioles that regulate flow. Coronary artery disease affects only the proximal epicardial vessels, not the arterioles. Atherosclerotic lesions are responsible for blood flow restriction. Epicardial lesions are not static and fixed but are-capable of considerable constriction and dilatation, depending upon underlying coronary vascular tone. Episodic changes in coronary tone occur in both variant and typical coronary disease. Endothelial damage may be responsible for increased sensitivity to endogenous vasconstrictor substances. Drugs that reduce elevated epicardial tone are therapeutic in both conditions while drugs that increase large vessel tone may further impair blood supply to the heart. Volatile anesthetics have definite effects on peripheral vasculature tone. Preliminary evidence suggests they also may dilate or attenuate constriction of epicardial coronary arteries. Nitrous oxide increases pulmonary and systemic vascular resistance. Preliminary data suggests it may also constrict epicardial coronary arteries. Little is currently known concerning the effects of anesthetics on epicardial coronary arteries. Effects on resistance arterioles have been reported but results are controversial. In intact dogs, anesthetic actions will be determined by measuring epicardial artery crosssectional dimensions using high resolution computer assisted coronary angiography. Effects on arteriolar vessels will be assessed by measuring coronary blood flow using both 133Xenon washout and coronary sinus thermodilution. In order to mimic coronary artery disease, in some experiments vascular endothelium will be removed and in others vasoconstrictors associated with vasospasm in man will be administered. Using isolated canine epicardial coronary rings, the ability of anesthetics to inhibit vasoconstriction will be tested. In patients undergoing coronary angiography, effects of nitrous oxide on coronary dimensions and myocardial blood flow will be determined. An understanding of the relationship anesthetic drugs have to coronary vasomotion is likely to help in the anesthetic management of patients with coronary artery disease undergoing both cardiac and non-cardiac surgery.

DESCRIPTION: The overall purpose of this project is to develop, evaluate, and market a more realistic, computer-based driving advisement system. The proposed system will incorporate a number of enhancements into an existing advisement system, the Elemental Driving Simulators (EDS) developed by the applicant organization for assessment of the driving skills of persons with disabilities. The EDS system yields reliable, psychometrically valid, norm-referenced measures. However, its simple graphics and lack of realism limit its marketability and credibility for consumers. With the widespread availability of graphically powerful multimedia computers, it is now possible to address this issue and produce a better simulator. The simulator will also be used as a tool for training and practice in addition to the evaluation of individuals who may pose a safety risk due to cognitive or behavioral impairments. PROPOSED COMMERCIAL APPLICATION: With improved graphics and the addition of a speed control module, the More Realistic Driving Simulator (MRDS) will have widespread commercial applications. there is an increasing need to evaluate elderly and disabled drivers including those with head injuries, CVA, and cognitive deficits. Development of the MRDS is the next logical step in the evolution of driving simulators. Our organization has a strong foundation on which to build, based on the past success, proven utility and reliability of both the Driving Advisement System (DAS) and Elemental Driving Simulator (EDS) in rehabilitation settings in the United States and foreign countries. We have 255 trained administrators of these systems and they will be the first commercial audience for the system under development. In addition to the well-established market represented by the rehabilitation facilities, we intend to offer a driving advisement procedure that is practical and economical to all professionals called upon to make judgments regarding an individual's ability to continued to drive. Estimates indicate that the number of older drivers is increasing dramatically and that there is an increase in the proportion of older drivers within the entire U.S. driving population. The MRDS would be an effective and helpful tool for older drivers, who may be concerned about their driving skills but reluctant to consult their physician of state DMV, fearing they may have their driving privileges summarily revoked. Based on the popularity AARP's defensive driving courses for seniors, it is reasonable to assume that many older drivers have concerns about their driving abilities and are willing to take steps to improve those abilities. Thus, the commercial application is an affordable assessment tool that will be marketed to physicians, occupational therapists, driver rehabilitation specialists and other counselors and professionals.

Blood flow and rate of oxygen consumption are closely matched in vivo under normal conditions. Coronary flow is regulated through the combined action of metabolic, adrenergic, and mechanical (myogenic and shear) mechanisms. Since vascular mechanical processes are linked to the mechanics of cardiac contraction, understanding flow regulation requires a model that accounts for mechanics from the microvessel to the whole-organ level. Accordingly, the overall objective of this grant is to develop a validated multi-scale model of coronary autoregulation that accounts for the various major determinants of coronary flow regulation. To accomplish this goal, we set the following three Specific Aims: Aim 1: To construct a multi-scale mechanistic model of coronary flow regulation integrating cell-level models of endothelial and smooth-muscle function, single-vessel mechanics of coronary resistance arteries, autonomic function, network-level myocardium- coronary vessel interaction and conducted metabolic response; Aim 2: To validate the ability of the model of Aim 1 to predict physiological dynamics observed in the awake exercising pig; and Aim 3: To use the models from Aim 1, refined by data from Aim 2 to understand the multiscale effects of stenosis on pulsatile flow in coronary arteries. We will test the hypothesis that the multiple parallel control mechanisms fail when coronary arteries become stenotic because they act out of sync and interfere. The model predictions will be compared to data from three complimentary protocols: (1) dynamic measurements of flow, pressure, and diameter in coronary arteries ranging from 50 mm to the large epicardial vessels; (2) steady-state measurements of venous (coronary sinus) pO2 versus left-ventricular oxygen consumption in different exercise states; and (3) measurement of the dynamic reactive hyperemic response flow following acute transient occlusion of the left anterior descending coronary artery. Protocols will be conducted with and without specific pharmacological interventions to inhibit receptors and channels represented in the cell-level models. The validated model will be used to investigate critical questions, including: What is the principle mechanism coupling coronary blood flow to metabolism in vivo? How is high resting oxygen extraction functionally linked to the ability of the system to effectively respond to increased demand in exercise?

Glioblastoma (GBM) is the most common primary brain tumor in adults and one of the most lethal of all cancers, with a median patient survival of 12-15 months despite advanced treatment. There is no effective therapeutic strategy to antagonize GBM malignant growth. Therefore, identification of new molecular targets and development of innovative treatment strategies are desperately needed. EGFR/PI3K/Akt signaling has been shown to be activated in around 88% of GBM patients, which suggests that it could be a promising therapeutic strategy to target this pathway to treat GBM. Unfortunately, targeting EGFR, PI3K and mTOR using its small molecular inhibitors has shown no or very short-term response. To significantly improve the efficacy of GBM treatment, it is essential to better understand the underlying molecular mechanisms of GBM pathogenesis and its biologic characteristics. Metabolism reprogramming has been shown to coordinate with oncogenic growth signaling and promote rapid tumor growth. However, the detailed mechanisms of metabolic changes and their molecular links with oncogenic signaling are still unclear. Our previous study was the first to demonstrate that fatty acid synthesis is highly elevated in GBM and is upregulated by EGFR/PI3K/Akt signaling through activation of sterol regulatory element-binding protein-1 (SREBP-1), a master lipogenesis transcriptional factor. In addition to fatty acid cholesterol is also important for cells as it is an essential component of cell membranes. However, whether cholesterol metabolism is altered in cancers remains unknown. Our preliminary data shows that cholesteryl esters and cholesterol-rich low density lipoprotein (LDL) receptor (LDLR) are both highly elevated in GBM cell lines and patient tissues, particularly in EGFRvIII- expressing cells. Our data further demonstrate that GBM cell growth is highly dependent on LDL uptake, and activating nuclear receptor liver X receptor (LXR) significantly inhibits GBM cell growth. The hypothesis of this application is that GBM cells are dependent on cholesterol uptake for rapid growth, and its high levels are maintained by EGFR/PI3K/Akt signaling through upregulation of the SREBP-1/LDLR pathway to promote LDL uptake. We predict that LDLR and LXR are novel molecular targets in GBM and depriving cells of cholesterol alone or in combination with inhibition of fatty acid synthesis will significantly inhiit GBM growth. In this study, we aim to identify a novel therapeutically targetable tumor survival pathway, and investigate the efficacy of targeting LDLR or activating LXR by its synthetic agonists GW3965 and T9091317, separately or in combination with the FASN inhibitor C75 on GBM xenograft tumor growth. We will: 1) determine the molecular mechanism by which EGFR/PI3K signaling upregulates LDLR and LDL uptake, and test atorvastatin treatment in GBM cells in Aim 1; 2) investigate the role of LDLR on GBM tumor growth in Aim 2; 3) determine the mechanism and efficacy of activating LXR by GW3965, T9091317 alone or in combination with FASN inhibitor C75 on GBM tumor growth, and evaluate the translational potential of these drugs to treat GBM in Aim 3.

A central activity of the ongoing Human Genome Project is the mapping and sequencing of mammalian genomes. The major aims of the Physical Mapping Section are to construct detailed physical maps of vertebrate chromosomes, to facilitate the sequencing of the corresponding DNA, and to utilize the resulting information for studying important biological problems. We initially focused our attention on the ~170-megabase human chromosome 7, first mapping this chromosome and then facilitating its sequencing. Indeed, the sequencing of human chromosome 7 is now complete. In parallel, we are actively using this sequence data to construct physical maps of the corresponding regions of other vertebrate genomes, en route to their sequencing. The latter includes a large program of multi-species comparative sequencing being performed in collaboration with the NIH Intramural Sequencing Center (NISC). Finally, there are several ongoing projects aiming to study regions of chromosome 7 associated with human genetic disease. These efforts have resulted in our identification of the Pendred syndrome gene, a gene responsible for cerebral cavernous malformations, a long sought-after tumor suppressor gene, and a gene defective in one form of Charcot-Marie-Tooth syndrome (CMT2D). These findings have opened up numerous new avenues of biological study relating to the structure and function of the genes and their encoded proteins, including the development of mouse models for these genetics disorders. Searches continue for the genes implicated in a number of other important diseases, including Williams syndrome, childhood-onset schizophrenia, and some forms of cancer.

Our long-term goal is to test the hypothesis that growth factors such as the glial cell line-derived neurotrophic factor (GDNF) counteract the adverse actions of alcohol and thus prevent or delay the development of alcohol addiction. In the first round of funding we found that the activation of the GDNF pathway in the midbrain dopaminergic ventral tegmental area (VTA) rapidly attenuates the motivation for, and relapse to, ethanol consumption. Using a multidisciplinary approach, we plan to elucidate the mechanism underlying the actions of GDNF to reduce consumption of ethanol. In Aim 1, we will examine whether GDNF in the VTA possesses intrinsic rewarding or stimulant properties. In Aim 2, we will determine whether GDNF adjusts the excitability of VTA neurons in a rat model of long-term excessive ethanol consumption. In Aim 3 we will test if GDNF in the VTA modifies the neuroadaptations in dopamine levels in the nucleus accumbens resulting from excessive ethanol consumption. In Aim 4, we will elucidate the molecular mechanism responsible for GDNF's action to attenuate ethanol-drinking behaviors. Alcoholism is a devastating disease that manifests itself in uncontrolled consumption. Identifying signaling pathways that inhibit this phenotype, such as the ones activated by GDNF, are therefore of great interest, as this will likely lead to the identification of new targets for medication development to treat alcoholism, and may also lead to the identification of genetic risk factors for the disease. PUBLIC HEALTH RELEVANCE: Alcoholism is a devastating disease that affects approximately 14 million people per year in the USA alone. Unfortunately, only limited numbers of drugs are currently approved by the FDA to treat adverse phenotypes associated with the disease. Therefore, there is a great need to identify novel targets for medication development. We found that the growth factor GDNF is a negative regulator of alcohol-drinking behaviors and relapse. Therefore, GDNF and its downstream effector proteins are potential drug targets to treat alcoholism. Here, we plan to elucidate the mechanism by which GDNF regulates alcohol's actions. Results generated from these studies may lead to development of novel, selective agents to treat alcohol abuse.

Over 150 million Americans spend - $15 billion/yr for corrective eyewear to compensate for refractive error. Presbyopia, the age related loss of accommodation, affects everyone by about age 50. One quarter of US - sold lenses in 1999 were Progressive Addition Lenses (PALs), used to correct Presbyopia. This fraction will increase as the population ages. Commercially available pre-fabricated PALs require a design compromise for each patient and pattern of usage. All PAL wearers must accept the tradeoffs selected by the lens manufacturer - for some lens wearers these tradeoffs may be nearly optimal, but for others they may be so uncomfortable that the lenses are rejected. The alternative, custom ground PALs, is prohibitively expensive. Squid Labs is developing a novel, programmable molding technology, based on our existing prototype for fabrication of simple lenses. 'Mass customization' of corrective lenses will be achieved using a variable, programmable, and thus reusable molding membrane. Phase I proposes 1) computer modeling of the novel membrane, based on analysis of commercially prefabricated PALs, to determine elastic requirements for PAL production; 2) fabrication of prototype membrane(s) with the minimum necessary properties, using several different processes; and 3) casting and analysis of lenses made using these membrane(s). Confocal microscopy and Shack Hartmann wavefront sensing will be the principal methods for evaluating lens surface topologies. The fabrication method producing the best membrane, judged by the caliber of lenses produced, will be utilized for Phase II activities: 1) construction and testing of a more powerful membrane able to produce arbitrary lens surfaces and 2) development of a production level prototype lens fabrication machine utilizing the membrane

By combining an Electrospray ionization source and one or more Time of Flight mass filters, one may begin to exploit an important biomedical Mass Spectrometry technique on an instrument platform which is inherently simple in both design and operation, inexpensive and capable of performing the most rigorous time resolved analyses of chromatography effluents. The overall goals of the subject study are two-fold. First, to identify and characterize the operating behavior of such an ESPI-TOF instrument, seeking to optimize both sensitivity and resolution. Specifically, this will entail studies of ion packet formation strategies, comparisons of performance between linear and reflection TOF machines, tandem TOF analyses using both surface and channel induced dissociation, and duty cycle optimization via ion storage. Secondly, implementing this technique, to begin developing a commercially viable instrument by constructing a prototype complete with both data acquisition and data analysis hardware and software. As a complete, turn-key "add-on" to existing LC or CZE devices, this instrument would offer unprecedented scanning speed and mass range, while also providing very high sensitivity and adequately high resolution to perform both molecular weight and structured analyses of biologically important molecules.

The objective of the proposed research is to study the organization of immunogobulin(Ig) genes, their regulated expression through transcription, translation and the synthesis of Ig precursor to provide the two final products - circulating antibodies and the antigen-recognizing receptor on the surface of lymphoid cells. The L-chain precursor, which is the immediate product of mRNA translation, contains an N-terminal extra piece (19-22 residues long) which precedes the N-terminus of the mature protein. Our studies of Ig precursors have already provided new information on the structure of Ig-genes. These include: 1) the V-gene may be larger than hitherto realized; 2) Ig secretion may be regulated by the cleavage process in which the extra piece is removed to yield the mature L-chain; 3) the marked hydrophobicity of the extra piece may favor interaction of the precursor with cell membranes (endoplasmic membranes and cell-surface membrane); 4) independent expression of the C-gene, associated with translocation of the extra piece DNA from the V-gene to the C-gene. We plan to continue investigation of the structure-function relationship of the Ig precursor, and to extend the studies done on mouse myeloma L-chains to L- and H-chain of normal lymphocytes from mouse and other species. Another major objective is to study V- and C-genes in nuclear DNA, in order to decide between the germ line and somatic mutation hypothesis, provide direct evidence for possible translocation of V- and C-genes, and identify the location of the DNA coding for the extra piece. To these aims we shall prepare distinct nucleic acid probes for the V- and C-regions, by using the C-mRNA (recently purified in our laboratory) as well as whole L-chain mRNA, cDNAs, and appropriate nuclease-digests of hybrids formed between cDNAs and mRNAs of different subgroups. The distinct V- and C-region probes will be used to enumerate the V- and C-genes, and to localize these genes in restriction enzyme fragments of nuclear DNA from Ig producer and non producer cells.

Chemical and immunologic studies are in progress involving the cell walls of Histoplasma capsulatum and Blastomyces dermatitidis. Both phases of these two fungi are being included but the primary emphasis is on the yeast phase. On the basis of chitin and monosaccharide analyses, as well as ethylenediamine extraction, we described two chemotypes of H. capsulatum yeast phase cell walls, one of which was similar to B. dermatitidis. These three groups, i.e., H. capsulatum chemotype I and II and B. dermatitidis, are now being examined for possible differences in the types of glycosidic bonds present in the wall, and for the presence of unique or similar proteinaceous components. The immunologic studies involve both humoral and cell-mediated responses elicited in guinea pigs by immunization with cell walls or fractions thereof. Complement fixation, the fluorescent antibody test and immunodiffusion are being used to evaluate the sera generated in the immunizations, while skin testing and migration inhibitory factor assays are being employed for the cell-mediated response.

This is an application is for a proposal for curriculum development of Trauma-focused CBT for children is submitted in response to MH PA-08-080, inviting applications for projects focused on training in validated behavioral treatments. Social workers provide by far the most mental health services in the community. Social work practitioners are dedicated to improving services for low income and underserved minority populations. However, few programs currently teach well evaluated interventions. Clinicians must be retrained after graduation, resulting in considerable workforce inefficiencies. Our goal is to target masters' level education to teach proven efficacious mental health interventions. Childhood trauma is all too common with potentially serious sequelae. Epidemiologic studies indicate up to 68% of children in the US experience traumatic events. Children with trauma histories are disproportionately represented in programs that treat substance use disorders (SUD) and juvenile delinquency. Early recognition and treatment of PTSD symptoms could potentially prevent more severe behavioral problems in youth. Trauma-focused CBT (TF-CBT) is a well evaluated behavioral treatment shown to be effective. Dr. Kimberly Hoagwood, Co-I on this project, has trained over 800 clinicians in TF-CBT across NY state. In this application we propose to extend this training to masters level social work students. Columbia University School of Social Work (CUSSW) enrolls 350 masters' students each year. Many students work in field agencies supervising clinicians have been trained in TF-CBT and we will collaborate with these agencies. Working with Dr. Hoagwood and Dr. Judith Cohen, co-developer of TF-CBT, we plan to 1) develop and implement a curriculum for teaching TF-CBT to social work students, 2) develop and implement an evaluation procedure to test effectiveness of our curriculum, and 3) develop and implement a dissemination program. CUSSW has a strong academic research faculty and a commitment to bringing research tested treatments into the masters' students' classrooms. Successful development of TF- CBT will be a major step in this effort and a model for other social work educational programs. [unreadable] [unreadable] [unreadable]

The transformation of liver cells to the neoplastic state is accompanied by changes in the cell phenotype. These changes presumably result from modifications in normal control processes. A large body of evidence has accumulated which demonstrates the importance of messenger RNA (m-RNA) in regulating the phenotypic expression of cells. The object of this proposal is to analyze polysomal m-RNAs from normal tissue and hepatocellular carcinomas to determine if differences exist between these RNAs. Polysomal m-RNA will be isolated by affinity chromatography of poly (U) Sepharose and hybridized to labeled single copy DNA and c-DNA synthesized from isolated m-RNA. Procedures will be developed to purify alpha-fetoprotein m-RNA, a protein produced in large amounts by many hepatocellular carcinomas but not by normal tissue. This messenger will be isolated by a combination of cell fractionation, antibody precipitation, and affinity chromatography. Once alpha-fetoprotein m-RNA has been purified, it will be used to synthesize a complementary DNA c-DNA probe, and the probe used to analyze the modifications in alpha-fetoprotein m-RNA related to the malignant state.

Diabetic retinopathy is serious, costly and prevalent, especially among minority populations. Annual ophthalmic examinations and appropriate follow-up care would result in substantial savings in preventable vision loss, health care costs, and lost productivity. However, most people do not receive these services. In our previous study, we doubled the rate of ophthalmic examinations in a low-income African-American population using a multi-component intervention. We now propose to evaluate the incremental effects and costs of components of that intervention in a broader diabetes population, including Spanish-speaking patients The specific aims of the proposed study are to: 1) test the hypothesis that a tailored telephone intervention will result in a higher rate of ophthalmic examinations than a standard print intervention; 2) improve understanding about reasons why people obtain an ophthalmic examination and assess differences in subgroups (gender and Spanish language preference); 3) improve understanding about reasons why people who are diagnosed with diabetic eye disease do or do not receive recommended follow-up treatment; and 4) conduct cost-effectiveness analyses. The study design is a randomized, controlled intervention trial with masking; the individual is the unit of sampling, assignment, and analyses. A total of 800 patients with type 1 or type 2 diabetes mellitus who have not had a dilated eye examination in the last year will be sampled from the databases of two large urban health systems. After eligibility is ascertained and written informed consent is obtained, patients will be randomized within site by gender and preferred language (Spanish or English) to either the tailored telephone group or the standard print group. Sample size determinations are based on having sufficient power to detect differences between groups at the p<0.05 level of significance. The main study outcome will be receipt of a dilated eye exam at 6 months post-randomization as ascertained by medical record abstraction, with a second outcome assessment conducted at 18 months post randomization. Receipt of follow-up care for diagnosed eye disease will also be assessed. Pre- and post-intervention telephone interviews will provide data on changes in knowledge, beliefs, risk perceptions and behavior regarding ophthalmic exams. Cost data will be collected using standardized methods. Study results will inform implementation and dissemination of practical, low-cost interventions to increase ophthalmic examinations and follow-up care, and thereby contribute to a decrease in vision loss and health care costs in diabetes populations.

This grant is aimed at facilitating the continued involvement of investigators at the University of minnesota in the multi- institutional cooperative therapeutic and non-therapeutic studies conducted by the Childrens Cancer Study Group (CCSG). During the past five years, the University of Minnesota has maintained its role as a leading institution in CCSG, both logistically and scientifically. Since 1983, there has been a 50% increase in the number of patients placed on study by the University of Minnesota such that by 1987 over 125 patients are expected on study, placing the University of Minnesota second among CCSG's 32 principal institutions. Scientifically, the University of Minnesota investigators have been leaders in various aspects of CCSG studies, including treatment of acute lymphocytic leukemia, acute non- lymphocytic leukemia, and neuroblastoma; investigation of new agents and pharmacokinetics of anti-cancer drugs; bone marrow transplantation for leukemia, neuroblastoma, and other childhood solid tumors; radiation therapy including total body irradiation: characterization of cancer cells, including cytogenetics and cell surface antigen determinations; cancer epidemiology; and studies investigating the late effects of childhood cancer and its treatment. Thirteen University of Minnesota investigators hold 87 positions on CCSG study committees, strategy groups, scientific committees, discipline committees, or administrative committees. Furthermore, these investigators occupy 27 chairpersonships of study committees and strategy groups, placing the University of Minnesota first among the CCSG institutions in scientific leadership. CCSG studies are conducted using a "team approach" which involves individuals from a wide range of disciplines including oncologists, radiation therapists, surgeons, immunologists, pathologists, cytogeneticists, pharmacologists, and epidemiologists. University of Minnesota investigators are involved in virtually all areas of CCSG interest and our involvement in future CCSG studies will continue to grow. This grant requests personnel support for University of Minnesota investigators and personnel key to the maintenance of effective data management and quality control for CCSG studies; travel for University of Minnesota CCSG investigators; and miscellaneous expenses for administering the University of Minnesota's continued involvement in both scientific input and the direction, planning, development, and conduct of clinical investigation and ancillary studies to be conducted by the Childrens Cancer Study Group.

The use of bone morphogenic proteins (BMPs) shows promise as therapeutics for improving bone regeneration; however, high supraphysiological concentrations required for desired osteoinductive effect, costs, and patient variability have prevented the full advantages of BMP-based therapeutics from being realized. Thus, there is a clinical need to develop growth factor delivery strategies that will optimize release kinetics and overcome the need for non-physiologic high concentrations to promote bone repair. The proposed approach utilizes a matrix metalloprotease (MMP)-sensitive hyaluronic acid (HA)-based hydrogel scaffold as a cell-mediated delivery vehicle, where the remodeling of the gel releases the molecules. Scaffold degradation kinetics can be controlled through varying the MMP sensitivity of the proteolytic peptide and through cross-link density. Canonical Wnt signaling is known to promote osteoblastogenesis, osteoblast function, and bone regeneration and can be promoted using the R-spondin (Rspo) family of secreted molecules. Enhancing canonical Wnt signaling through the synergistic delivery of BMP2 and Rspo2 using an engineered synthetic HA-based hydrogel should allow for improved bone tissue repair. With these features in mind, the objective of this work is two-fold: (1) evaluate the influence of hydrogel remodeling and BMP2 growth factor release kinetics on in vivo tissue repair and (2) determine if the synergistic delivery of Rspo2 and BMP2 improves BMP2 induced osteogenesis. Growth factor release kinetics and bioactivity will be evaluated using in vitro characterization techniques. A cranial defect rat model will be used to determine the influence of both growth factor release profiles and the combined delivery of Rspo2 and BMP2 on bone tissue formation. Thus, the specific aims of the work are: (1) Determine the effect of BMP2 release kinetics on osteogenesis (a) in vitro and (b) in vivo using MMP-sensitive hydrogels of varying sensitivity and (2) Synergistic delivery of Rspo2 and BMP2 for improved bone tissue repair. Upon completion of this work, a more thorough understanding of BMP-based therapies will be obtained, as well as work towards a clinically translatable approach to improve the treatment of patients with bone loss.

To further evaluate the use of methotrexate and citrovorum factor rescue in women as a non-surgical treatment for unruptured ectopic pregnancy. Response to treatment will be monitored by serial serum hCG measurement, clinical signs and symptoms and hysterosalpingography 2-3 months later.

Large-amplitude conformational changes in proteins, including loop motions, relative motions between domains, collective breathing of protein cores, ligand-binding or oligomerization reactions, and overall folding-unfolding events, may be closely coupled, and in some instances rate-limiting, to biological functions such as molecular recognition, transitions along the catalytic cycle of enzymes, and inhibition or activation of proteins through intra-or inter-molecular protein-protein interactions. Mutations that perturb dynamical processes and conformational equilibria are associated with significant pathology, including loss or gain of function and misfolding. Recent developments, including those from the applicant laboratory, have opened new opportunities for investigation of large amplitude conformational dynamic processes on microsecond-millisecond time scales using NMR spin relaxation measurements at equilibrium in solution and with atomic site resolution, without potential complications introduced by non-native modifications necessary for other solution-state spectroscopic techniques. The proposed research has four primary objectives: (1) identification of the mechanistic basis for cell adhesion mediated by domain (strand) swapping in the cadherin superfamily, (2) assessment of the role of conformational mobility in catalysis by the essential enzyme ornithine 5'-monophosphate decarboxylase, (3) elucidation of the folding mechanism and description of the unfolded-state ensemble for the villin headpiece domain HP67, and (4) development of novel experimental and theoretical methods for characterizing protein dynamics on s-ms time scales. Time-dependent structural changes underlie the normal function of proteins, and misfunction in genetic diseases, cancer, and other pathologies; the proposed research will quantify this linkage for three protein systems involved in cellular structure and basic metabolism. Completion of these goals will enable additional future applications to a wide range of protein systems of biological importance. PUBLIC HEALTH RELEVANCE: The present proposal addresses the coupling between structure, dynamics, and function in cell adhesion mediated by cadherins, catalysis by the essential enzyme orotidine 5'-monophosphate decarboxylase, and folding of the villin headpiece domain. Elaboration of the roles of conformational dynamics in actuating and regulating these processes is essential for understanding the fundamental functions of proteins in human health and disease.

The objective of this study is to determine thresholds and dose-response relationships for the effects of 27-MHz radiofrequency rediation (RF) on mammalian somatic and germ cells in vitro under accurately measured and precisely controlled exposure conditions. The extent to which said effects are dependent upon RF frequency will be determined by comparision with the effects of other RF frequencies, specifically 1-, 10-, 50- and 100-MHz. The rationale for this study is to provide data to be used in the assessment of potential health risks associated with occupational exposure to 27-MHz RF radiation generated by RF heat sealers, diathermy equipment and other devices that are known sources of human exposure. Since such devices are generally operated intermittently, comparative studies will be conducted of continuous wave and amplitude modulated 27-MHz RF. Well-characterized somatic cells (erythrocytes, neutrophils, glioma [C5 and LN-18], fibroblasts and HeLa) and germ cells (mouse ova and sperm) will be exposed under conditions of accurately measured electric and magnetic field strength in a temperature-controlled coaxial line RF cell exposure system. The dependent variables will include: 1) cell viability and morphology, 2) phagocytosis and chemotaxis, 3) plasmalemma cation permeability 4) cell cycle regulation, 5) DNA and protein synthesis, 6) sperm viability and motility, and 7) fertilization (in vitro).

We seek five more years of support for the popular Biotechnology Training Program (BTP) at UVA In the first 9 years of funding we built the BTP into a highly interactive community of PhD trainees drawn selectively from an annual Spring competition open to PhD students from all science and engineering departments university-wide. BTP students in training (17) or graduated (23) entered with an average undergraduate GPA of 3.53, currently include 4 minorities, and are hosted by 6 different departments. Trainees have received multiple awards, have published 47 first author articles in journals with impact factors as high as 19, experienced 32 different externships from 21 different companies, and after graduation are now employed in industry (11), academia (7; including 3 Ass't Professors) or government (3). Two are completing med school. Trainees have taken direct responsibility for programmatic features of the BTP including: editorship ofthe BTP newsletter, organization of BTP Minority Day, organization of BTP seminars, social activities, new student dinner, and BTP Symposia. Mentoring our trainees is a highly engaged, collaborative and well-funded faculty of 50 individuals from 13 departments, 3 of which are ranked in the top 10 in NIH funding; another #15 by USN&WR. Institutional support has been essential for our success, particularly the important role of the Graduate Programs Office and more recently the Molec. Cell and Develop. Biology Office in day-to-day administration of the Program. Also important has been the enthusiastic joint organization of externships by Engineering Associate Dean and Health Science Translational Offices. We view the BTP's mission as threefold: (i) most importantly to train our students to do the best science possible, (ii) to provide a meaningful experience in industrial science via our externship program, and (iii) to promote scientific synergy among BTP students of different disciplines through regular BTP Journal Clubs/Data Sessions, Seminars and Symposia. We argue in the following competitive renewal that success in each of these areas has been achieved, thereby helping to develop a new generation of young scientists who approach new scientific challenges with analytical minds and an extensive array of

The current "gold-standard" for quantitative assessment of steatorrhea, 72-h stool fat, is universally recommended in evaluation of malabsorption but vastly under-utilized because it is tedious and difficult to perform. We propose a novel approach for direct measurement of steatorrhea from all causes which eliminates the limitations of the 72-h stool test while retaining its important features. Our approach is based on administration of 1,3--[13C]distearyl, 2-oleolyl glycerol and the nonabsorbable marker DyCI3 with a high fat meal followed by analysis of a single stool. The test is expected to be sensitive to steatorrhea of maldigestion and fatty acid malabsorption, cost effective, and to provide results that are highly correlated with 72-h stool fat in individual patients. During Phase I, we demonstrated the feasibility of the approach by showing that Dy is quantitatively excreted and follows the same excretion kinetics as 13C- labeled triglyceride. During Phase II, we will study details of the approach leading to a practical test for clinical use. We will demonstrate that labeled triglyceride absorption is reproducible, accurately reflects the degree of steatorrhea in individual patients, and has excellent diagnostic sensitivity/specificity. The developed test kit and analytical services will be marketed through BioChemAnalysis Corporation. PROPOSED COMMERCIAL APPLICATION: This research will lead to the availability of a test kit and procedure for accurate quantitation of steatorrhea from all causes and requires collection of only a single stool. Because the procedure highly reproducible, accurate and relatively simple to use it will be applicable to clinical diagnostic use as well as use in research.

The most worrisome infectious agents of bioterrorism are those that would be artificially disseminated as aerosols to the lungs where the immune responses are unique, primarily driven by an immunoregulatory program. Critical mediators of the immune response in this environment are alveolar macrophages (AM) and epithelial cells. Francisella tularensis (Ft), a targeted infectious agent of bioterrorism, is an intracellular pathogen which causes acute life-threatening disease, particularly when transmitted via aerosols. The eukaryotic and microbial factors mediating host cell recognition and response for Ft remain poorly defined and their definition will be required for the rational development of new therapies and vaccines. This competitive renewal of RP9 unites an established group of collaborative scientists from the Ohio State University, Medical College of Wisconsin and University of Cincinnati to focus on the molecular pathogenesis of pneumonic tularemia. Based on recently published work and preliminary data, our central hypothesis is that Ft's success as a human pathogen is linked to its ability to suppress and/or subvert important elements of the protective innate immune response during the early phase of infection. To test this hypothesis we will 1) further define the molecular mechanisms that mediate Ft entry, intracellular trafficking, antimicrobial responses and initiation of the inflammatory program both in vitro and in vivo;2) construct, validate, and probe global microbial and host genetic screening platforms to identify key determinants mediating Ftinduced immune suppression and/or subversion;and 3) use targeted genetic approaches to identify novel Ft virulence determinants focusing on bacterial transcriptional regulators and Ft and host factors involved in Fe metabolism, including those of the bacterial oxidative stress response. Collectively, these studies will provide important new insights into host-pathogen interactions by the most virulent Ft subspecies, and will use novel mutant derivatives and relevant model systems of infection. This RP will synergize the talents of its investigators to place itself in the most competitive position to make fundamental discoveries related to pathogenic mechanisms for Ft, and begin to translate these discoveries into product development.

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. At the Emory Vaccine Center, our continuing mission is to improve human health by conducting fundamental and clinical research that leads to the development of effective vaccines against infectious diseases of global importance. The Emory Vaccine Center represents one of the largest academic vaccine centers in the world, and is renowned for its expertise in cellular immunity and immune memory. This expertise has lead to the creation of new technologies for the prevention of emerging infectious diseases. Established in 1996 with support from Emory University and the Georgia Research Alliance, the Emory Vaccine Center has over 25 faculty who continue to study AIDS, malaria, and other global infectious disease threats. In the past year we have been joined by new faculty who have brought additional expertise and have expanded our areas of investigation to include TB and the Francisella virus.

Tools to monitor immune cells interactions and their consequences in vitro and in vivo are needed to accelerate development of successful immunotherapies for cancer, HIV disease, and autoimmune disorders. Stable fluorescent labels for cell proteins (CFSE) or membranes (PKH dyes) are such tools. However, high levels of autofluorescence and spectral overlap in the visible region limit their use in vitro, and real time optical imaging in intact animals is precluded by high levels of tissue absorption and scattering. We hypothesize that stable membrane labels that fluoresce in the far red (FR) or near infrared (NIR) will: a) enable more sensitive cell tracking using flow cytometry and confocal microscopy, b) provide novel non-radioactive contrast agents for optical imaging of immune cell trafficking in whole animals, and c) have potential as future photodiagnostic and/or phototherapeutic agents. In Phase I we will synthesize two classes of probes, one with FR (600-700 nm) excitation/emission and the other with MR (700-900 nm) excitation/emission. Each probe's cytotoxicity, membrane retention, signal:noise, and spectral overlap with commonly used antibody labels will be characterized using flow cytometry and confocal microscopy. Potential utility for macroscopic imaging will be assessed by determining image quality vs. depth in tissue "phantoms" containing varying admixtures of labeled and unlabeled cells. PROPOSED COMMERCIAL APPLICATIONS: Membrane labeling dyes emitting in the far red or near infrared have significant commercial potential in three markets: l) a research reagent market serving both basic and clinical investigators using flow cytometers; 2) a similar but growing market in optical imaging, especially macroscopic or whole body imaging; and 3) either photodiagnosis or phototherapy.

The potential utility of adult stem or progenitor cells for repair of radiation-damaged salivary glands is high, but is currently only a theoretical solutin for patients suffering from xerostomia. There remain several critical obstacles that must be resolved before cell-based therapy for dysfunctional salivary glands can be moved into the clinical arena. These include the identification of appropriate donor cells, the technology for promoting implantation, and direct functional assays to assess the outcomes. We propose to address these issues, using the powerful and well-defined genetic tools available in mice for tracing cell lineages, and cell types. In combination with tunable hydrogel scaffolds, we will use known salivary gland progenitor cells to determine environmental and cellular cues required for their differentiation in vitro and in vivo. The hydrogels will be used to transplant cells into two opposite environments: regenerating atrophic glands, and damaged irradiated glands. The goal is to determine if the use of hydrogels can promote in vivo differentiation of transplanted progenitor cells. To resolve whether the transplanted cells can produce saliva, we propose to generate a mouse model expressing a tagged secretory protein. Because this tool identifies the cellular source of the secretion, it may also prove to be a critical model for evaluating ways to stimulate endogenous regeneration of damaged salivary glands. The successful completion of this project will establish a foundation for subsequent translational research to progress the technology into clinical applications. PUBLIC HEALTH RELEVANCE: This application combines the tools of mouse genetics with recent advances in biomaterial engineering to ask two questions: 1) Can isolated progenitor cell populations be used to reconstitute functional secretory structures in a damaged salivary gland? 2) Does the introduction of progenitor cells into a dysfunctional gland induce endogenous repair and/or secretion? Until these critical questions are resolved, progress toward cell- based replacement therapy in human salivary glands cannot occur. The successful completion of this project may lay the foundation for subsequent translational research to take the developed technology into clinical applications.

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 project is about studying the structural and dynamic properties of chaperone proteins involved in type three secretion systems.The chaperone itself as well as various complexes with substrate proteins will be investigated.

The goal of this project is to determine the mechanisms by which the endothelial cell protein C receptor (EPCR) functions. The aims grow out of our observations which follow. EPCR is expressed preferentially on the large vessel endothelium and enhances protein C activation. It is found on the cell surface and in discrete organelles include caveolae and is shed in a highly regulated fashion induced by cytokines and thrombin. Thrombin also up-regulates EPCR mRNA levels in vivo. Blocking EPCR-protein C interactions leads to a dramatic exacerbation of the primate response to E. coli resulting in a elevated cytokine levels, DIC, capillary leak, and thrombosis at sites where little EPCR is expressed. EPCR can also undergo nuclear translocation form the plasma membrane with our without carrying activated protein C as cargo. In cultured 293 cells, EPCR transfection results in an altered expression of a limited number of genes without changing the expression of the vast majority of genes. The aims of the present project are to determine whether lateral mobility of EPCR of EPCR in the membrane is important for EPCR acceleration of protein C activation and the EPCR concentration dependence of protein C activation: to determine the sites on EPCR and protein C required for interaction; to determine the topography of the complex by measuring the distance from the active site of APC to the membrane surface when APC is bound to EPCR vs. directly to phospholipid; and to determine the impact of EPCR promoter mutations identified in thrombotic patients on expression in cultured cells. The structural requirements within EPCR and the cellular mechanisms involved in EPCR mediated alteration of gene expression will be investigated. These studies are designed to improve our understanding of the mechanisms by which EPCR functions, provide insights into how EPCR mutations might influence thrombotic risk, and improve our understanding of the role of EPCR in the pathophysiology of thrombotic disease.

Using the SIVmac251-rhesus macaque model of HIV-1 infection, the Animal Core will provide the long-term ART-suppressed SIV/macaque platform for all projects of this proposal. The Animal Core will 1) coordinate all macaque studies across all research projects, and 2) facilitate acquisition and distribution of valuable animal cells and tissue samples among all research teams in the Program Project. This Core will provide the in vivo validation platform and the blood, CSF, and tissue samples and primary cells necessary to achieve the Program Project?s goals through the following Specific Aims: Aim 1. To coordinate and perform all macaque studies to include animal acquisition and MHC screening, SIV inoculation, ART drug administration, longitudinal sampling of blood (for plasma, serum and PBMCs) and CSF, and biopsy sampling of peripheral lymph nodes. Aim 2. To perform comprehensive necropsies to harvest macaque tissues and fluids at study end points and to perform complete pathology evaluation for all animals. Aim 3. To administer anti-rhesus CD4 antibody to deplete the latent resting CD4+ T cell reservoir in SIV-infected ART treated macaques and monitor for evidence of opportunistic infections.

The general purpose and intent of this project is to conduct test, research, and experiments directed toward the use, improvement, and development of clinical procedures and instruments for measuring functions or properties relating to vision and the eyes. This has consisted primarily of subjective measurements of visibility and chromaticity thresholds. Electroretinography, which is an objective measurement of electrophysiological activity in the retina, is used only in selected cases because of its limited usefulness. The most significant work of the year was the measurment and analysis of defects in color vision with the chromagraph. This is our new instrument which permits the measurement of any type of color defect, many of which are too subtle or too bizarre to be shown with the conventional tests.

Uncovering molecular mechanisms that underlie pain hypersensitivity in neuropathic pain may allow development of novel therapeutic strategies for treating this disorder. Non-coding RNAs that have been identified in mammalian cells regulate gene expression. Their expression is associated with the development of neurological diseases, but how non-coding RNA is causally linked to the diseases is unknown. We recently indentified a large, native, full-length non-coding RNA (2,574 nt) that is complementary to voltage-gated K+ channel (Kv) 1.2 mRNA in the dorsal root ganglion (DRG). Our data indicate that this Kv1.2 antisense (AS) RNA might be up-regulated via the activation of MZF-1 transcription factor in the injured DRG after peripheral nerve injury. Blocking this up-regulation might attenuate the induction of neuropathic pain. These preliminary findings suggest that DRG Kv1.2 AS RNA participates in the molecular mechanisms that underlie neuropathic pain. This proposal will further characterize native Kv1.2 AS RNA in the DRG and determine whether and how this AS RNA contributes to neuropathic pain. In Specific Aim 1, we will examine the expression and distribution of Kv1.2 AS RNA in the DRG and define cytochemical characteristics of Kv1.2 AS RNA-containing DRG neurons in normal rats. In Specific Aim 2, we will examine whether peripheral nerve injury produces an increase in expression of Kv1.2 AS RNA and its transcription factor MZF-1 in the DRG. Furthermore, we will examine whether Kv1.2 AS RNA is up- regulated as a result of MZF-1 binding to the consensus sequence on the Kv1.2 AS gene promoter in the injured DRG after peripheral nerve injury. In Specific Aim 3, we will use a virally mediated gene transfer strategy to determine whether over-expression of Kv1.2 AS RNA specifically and selectively reduces expression of Kv1.2 mRNA and protein and total Kv current density in DRG neurons, increases DRG neuronal excitability, and leads to major symptoms of neuropathic pain in rats. We will also examine whether blocking nerve injury-induced up-regulation of Kv1.2 AS RNA in rats reverses nerve injury-induced reductions in DRG Kv1.2 expression and total Kv current density and attenuates nerve injury-induced abnormal DRG neuronal spontaneous activity and pain hypersensitivity. The proposed studies will provide major conceptual advances to our understanding of the molecular mechanism of neuropathic pain and might open a door for developing new strategies for treating neuropathic pain. PUBLIC HEALTH RELEVANCE: Neuropathic pain is poorly managed by standard drug therapy. Understanding mechanisms of pain hypersensitivity in neuropathic pain is important to improving clinical treatment and developing novel therapeutic strategies. The proposed studies will test novel hypothesis that peripheral nerve injury up- regulates the endogenous antisense RNA transcript of the Kv1.2 potassium channel in the injured dorsal root ganglion and that this up-regulation may contribute to the development and maintenance of neuropathic pain. The proposed studies may provide new strategies for clinical intervention of neuropathic pain.

One of the major causes for deafness is the loss of inner ear hair cells, the sensory cells that detect sounds. In mammals, hair cells are born during embryonic development and are maintained in quiescence throughout life. Mammalian inner ear, unlike the counterpart in lower vertebrates such as chick or fish, does not regenerate hair cells after damage. Deletion of negative growth genes (Rb1 and p27kip1) led to cell cycle re-entry in embryonic and neonatal inner ear. However, we have shown in adult inner ear, Rb1 deletion is not sufficient to induce proliferation. Further, the proliferating hair cells and supporting cells will ultimately die. Thus the inability to re-enter cell cycle by mature inner ear and apoptosis of proliferating cells present two main challenges to hair cell regeneration. This proposal is designed to specifically address the two issues. We showed that FGF signaling is necessary for hair cell regeneration in zebrafish neuromasts. For the specific aim 1, we will test the hypothesis that, with an inducible mouse model, FGF activation with Rb1 deletion could lead to cell cycle re-entry in adult inner ear in vivo. We have observed such events in vitro. Correlation of FGF activity in the proliferating cells by immunostaining will support the crucial role of FGF in cell cycle re-entry. Using an inducible mouse model to mark supporting cells genetically, we will identify hair cells derived from supporting cell transdifferentiation. In the aim 1b, we will characterize regenerated hair cells in differentiation, synapse formation and function by immunostaining, FM1-43 uptake and transduction current recording. In the second aim, we will evaluate two pathways, IGF1 and p53, for their roles in survival and apoptosis of proliferating hair cells. We have the evidence that IGF1 activation or p53 blockade protect Rb1-/- cochlear hair cells from apoptosis. In the aim 2a, we will determine the necessity of IGF1 in Rb1-/- cochlear hair cell survival by blocking IGF1 function to induce apoptosis. Further, we will specifically block two IGF1 signaling pathways: PI3K/Pdk1/Akt and Raf/Mek/Erk, to assess their respective role in the survival of Rb1-/- cochlear hair cells. In the aim 2b, we will block p53 function by a specific inhibitor and correlate p53 inactivation with Rb1-/- cochlear hair cell survival. We will further study the effects on the p53 pathway after IGF1 activation or inhibition. Inactivation of the p53 pathway after IGF1 activation, or vice versa, is an indication that IGF1 antagonizes p53 function to promote survival. Finally we will induce cell cycle re-entry in the adult inner ear on the p53-null background, and study long-term survival of proliferating hair cells. Cell cycle re-entry in mature inner ear and the survival of proliferating hair cells will make it possible to regenerate functional hair cells. PUBLIC HEALTH RELEVANCE: The proposal is designed to demonstrate that FGF activation and deletion of retinoblastoma gene can lead to cell cycle re-entry and regeneration of hair cells, the sensory cells that detect sounds, in adult mouse inner ear in vivo. It will demonstrate that manipulation of IGF1 (insulin-like growth factor) and p53 could promote survival of regenerated hair cells. Success of the proposal will lay the foundation for the study of functional recovery of hearing by regenerated hair cells.

DESCRIPTION (From the Applicant's Abstract): The capability of tandem spectrometry to detect single and multiple SNPs in a single stretch of DNA will be evaluated. The p53 gene contains polymorphisms in codon 47 (TCG and CCG) and codon 47 (CGC and CCC). This will be studied as a model system. In initial work a 67 bp PCR product containing only the C to T switch will be analyzed by mass spectrometry (MS). In subsequent work 120 bp PCR products containing both alleles will be analyzed by MS2. This is pushing the envelope for mass discrimination using triple quadrupole or ion trap tandem mass spectrometry. The use of stable isotope labeled nucleotides increases the mass differences on C to T substitution (from 15 to 27 mass) units. This and the use of MSn (by sequential fragmentation of the molecule into smaller pieces) may be necessary to analyze larger PCR products.

The Newsletter is published quarterly and distributed to more than 600 investigators world-wide. The format of the newsletter has been changed to highlight NFCR activities and advise the scientific community of workshops or other noteworthy developments at the NFCR. During the year we conducted our triannual subscription renewal during which we requested that subscribers indicate whether they would prefer to receive the Newsletter in paper for or be notified of the avialability of new issues on the World Wide Web. Approximately 20% of the subscribers are now receiving electronic notification of new issues.

Therapeutic strategies for reducing serum cholesterol have been aimed at decreasing the intake or absorption of dietary cholesterol and at inhibiting the rate-limiting enzyme of de novo cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl Coenzyme A reductase (HMGR). HMGR is extremely sensitive to oxidative inactivation and intermolecular disulfide crosslinking in glutathione redox buffers (mixtures of glutathione and glutathione disulfide) that are comparable to the highly reducing intracellular environment. This leads to the hypothesis that the reversible formation of disulfide bonds is an important component of the regulation of this enzyme. The major form of HMGR found in vivo using methods to freeze the thiol/disulfide redox state of the enzyme is a disulfide-linked dimer between a full-length 97 kDa species and a partially proteolyzed fragment. Mevinolin, an HMGR inhibitor used clinically, alters the redox behavior of HMGR significantly. The long-range goal of this research program is to understand the molecular details and regulatory consequences of the thiol/disulfide redox state changes that are observed for HMGR in vivo. The coupling between the thiol/disulfide redox state of HMGR and the intracellular glutathione redox state will be investigated by manipulating the intracellular glutathione redox buffer of hepatocytes by 2-oxothiazolidine-4-carboxylate, glutathione monoethyl ester, and buthionine sulfoximine and observing the effects on cholesterol biosynthesis and the redox state and turnover rate of HMGR. The structural basis for the oxidative inactivation and disulfide crosslinking of HMGR in the presence and absence of mevinolin will be studied by specific labeling, peptide mapping, and sitedirected mutagenesis of a 58 kDa soluble, active fragment of HMGR expressed in E coli. Experiments will probe several potential mechanisms that could account for the formation of the disulfide dimers of HMGR that are observed in vivo including the effects of phosphorylation and proteolysis on the redox behavior of HMGR. The structural basis for the effect of mevinolin on the redox behavior of the enzyme will be investigated with mevinolin analogs, and experiments will search for interactions between thiol/disulfide redox state changes and other mechanisms of HMGR regulation including phosphorylation and proteolysis.

Our laboratory was the first to identify VCP mutations as a cause of hereditary inclusion body myopathy, Paget's disease of bone, frontotemporal dementia and amyotrophic lateral sclerosis, and has made progress in identifying the underlying molecular pathogenesis of these diseases. Studies of patients' myoblasts and the heterozygous R155H mouse have placed VCP at the intersection of the ubiquitin-proteasome signaling and autophagy pathways, both mechanisms being considered responsible for the intracellular protein degradation and abnormal pathology seen in muscle and brain. We have developed the first knock-in VCP mouse model carrying the common R155H mutation, which has many clinical features typical of the human disease (1). Unlike disease progression in the R155H heterozygous (VCPR155H/+) mouse model, homozygotes (VCPR155H/R155H) have a more severe muscle, brain and spinal cord pathology, with most surviving no more than 21 days. Homozygotes demonstrate an abnormal autophagic pathway and mitochondrial proliferation, as evidenced by structural and functional studies. Recent preliminary studies have shown that feeding pregnant heterozygous dams a diet with a 3% increase in fat results in a dramatic improvement in the survival of their homozygous offspring. These offspring live longer than 21 days and are successfully weaned, demonstrating that an increased fat diet ameliorates the lethal phenotype. In this application, we propose to investigate the optimum percentage of fat content on further rescuing the lethal phenotype. We will also study the important altered mitochondrial, autophagy and ubiquitin-proteasome molecular pathways in the homozygous VCP knock-in mice treated with normal versus high-fat diets. Studying this observation affords the opportunity to develop a promising therapeutic strategy for patients with VCP and related diseases.

Widespread incidence of Leishmania infections, increased global AIDS problems, with an increased incidence of co-infections, has made Leishmaniasis the world's seventh largest public health problem. Current diagnostic procedures require either long-term cultures or serological assays which, at times, are not reliable. Consequently, a DNA probe-based assay is a preferable choice. In Phase l, we developed a simple Specimen preparation procedure which renders minicircle kDNA more available for PCR amplification, and provides a 10,000-fold increase in sensitivity. This was established with consensus primers and probes to detect all Leishmania species, albeit with different levels of sensitivity. This increase in sensitivity from our lysis buffer should allow the development of a direct hybridization kit to test the majority of field.specimens, In Phase II, we propose to develop a PCR-based test kit for detection and species identification of Leishmania from clinical specimens, containing an extremely small number of parasites. A less expensive direct hybridization-based kit is proposed for the majority of the specimens. We will incorporate a simplified specimen handling system to meet the needs of the endemic regions of the world. In addition to clinical diagnostics, the test kits will be designed for large throughput, so that they can be used by public health officials of various government agencies for eradication purposes. Proposed commercial applications: We proposed to develop a rapid, economical, and reliable DNA probe test for the detection of Leishmania, both Old and New World species, in clinical samples. The test will rely on methodologies which are practical for application in developing nations, and will provide significant improvement In relIability over existing tests.

Project Summary Lipid transport is integral to lipid metabolism and stress response in the central nervous system. Lipoproteins are the major intercellular lipid carrier between neurons and glia. The lipid composition, trafficking, and metabolic fate of lipoproteins are largely dependent on the associated apolipoproteins. Disrupted function of apolipoproteins result in neurodegenerative phenotypes in animal models of neurodegeneration. Furthermore, allelic variation of the Apolipoprotein E (APOE) gene is the major risk factor of Alzheimer's Disease (AD). Glial cells such as astrocytes and microglia are the major cell types that process and secret ApoE in the nervous system. Glia also serve a protective role by mitigating neuronal oxidative stress, a common hallmark of AD and other neurodegenerative diseases. While it is well established that apolipoproteins play critical roles in lipid metabolism, our understanding of apolipoprotein transport in the nervous system is still limited. In addition, whether glial cells couple oxidative stress response with apolipoprotein trafficking remains to be tested. Since intracellular vesicular compartments are required for the trafficking of apolipoproteins, we hypothesize that endolysosomal proteins functionally interact with ApoE in brain. By examining the expression profile of putative endolysosomal genes, we identify Tweety Homolog 1 (TTYH1) that co-expresses with APOE in human brain. Preliminary studies performed in mammalian cells and Drosophila mutant of tty, homolog of TTYH1, suggest that tty and TTYH1 are required for the secretion of apolipoproteins, such as ApoD and ApoE, from glia and regulating oxidative stress in the nervous system. tty mutant flies show shortened lifespan and locomotor phenotypes. Our central hypothesis is that tty and TTYH1 regulate apolipoprotein trafficking to mitigate oxidative stress in glial cells. In this proposal, we aim to address the roles of tty and TTYH1 in: 1) the secretion of apolipoprotein from glia to circulation; and 2) mitigating glial oxidative stress via apolipoprotein secretion. Our long-term goal is to delineate specific pathway that regulate ApoE/D processing and secretion in glial cells and reveal new therapeutic targets for AD and related dementias.

Metastasis of aggressive breast cancer is a devastating and ultimately fatal condition. The current drugs which are used to treat breast cancer focus on the reduction of the tumor size and have proven to be successful. However, currently there are no available treatment options which can effectively inhibit tumor invasion and metastasis. Novel drug strategies therefore are needed to treat this chronic, fatal and incurable condition. Our therapeutic strategy for the treatment of aggressive breast cancer is based on reducing Id-1 expression. In the Phase 1 segment of this Fast-Track proposal our focus will be on developing lead compounds that display a favorable in vivo profile. In Phase II segment of this proposal our lead compounds will be evaluated in multiple in vivo models. We will also develop leads that display a favorable ADME-Tox profile. The ultimate goal of this proposal is to develop a novel therapeutic approach for the treatment of metastatic human breast cancers by down-regulation of Id-1.

This proposal is concerned with a detailed investigation on the mechanism of microbial cleavage of branched hydrocarbon side chains of phytosterols, with special reference to those of Beta-sitosterol and Campesterol. These latter sterols will be the ultimate sources of raw materials for steroid hormone synthesis by microbial fermentation proceses.

The overall objective of the proposed work is to understand how the Dmrt1 gene controls development and function of the testis. The testis has two essential functions: production of sperm, the cells that serve as vehicles for the immortality of male germ line DNA; and production of hormones that direct other parts of the body to develop in a male-specific manner. Dmrt1 belongs to family of conserved transcriptional regulators and controls multiple critical processes in the mammalian testis. This work has direct human health relevance: loss of DMRT1 in humans is associated with male-to-female sex reversal, disorders of sexual differentiation (DSD), infertility, and testicular germ cell tumors (TGCTs). In mice Dmrt1 controls germ cell pluripotency in the fetal gonad and controls the mitosis/meiosis decision in adults. A new discovery is that testis determination must be actively maintained postnatally by Dmrt1 and that Dmrt1 mutant testes undergo massive postnatal reprogramming to become ovary-like organs. This proposal has three aims focused on deepening our understanding of how DMRT1 controls key processes in the testis. Aim 1 asks how DMRT1 prevents transdifferentiation in the postnatal testis. This is a newly discovered and unstudied biological process. The proposed experiments use conditional gene targeting approaches to ask whether DMRT1 prevents reactivation of the fetal sex determination network postnatally, identify new regulators of postnatal sex maintenance, and use ChIP-seq to identify genes that are bound by DMRT1 in the mouse and human testis. Aim 2 tests the hypothesis that DMRT1 is critical for the transition from spermatogonia stem cell to committed progenitor cell, using precisely controlled loss- and gain-of-function approaches. Aim 3 will determine how DMRT1 is inactivated as spermatogonia transition from mitosis to meiosis and will test the consequences when this fails to occur. The results of this study should aid in treatment of gonadal cancer and infertility, and will inform studies of cell fate reprogramming and design of novel male contraceptives. PUBLIC HEALTH RELEVANCE: The proposed work is highly relevant to human health. DMRT1 is implicated in human infertility, testicular dysgenesis, and testicular cancer and the proposed studies may permit better diagnosis and treatment of these conditions. Furthermore, because this work focuses on cell fate reprogramming, regulation of germ line stem cells, and control of the mitosis/meiosis decision, the resulting data may aid in stem cell therapy, direct reprogramming of cell fate, infertility treatment, and contraception.

This K01 Mentored Research Scientist Award will provide the mentorship and support necessary to establish the Candidate as an independent researcher in translational psychiatric neuroscience, with a focus on bridging neuropsychopharmacology, brain imaging, and treatment studies in anxiety disorders through an integrative approach. The training component of this application builds on the Candidate's expertise in basic science research investigating the neural circuits involved in regulating extinction of fear memories, as well as her skills in basic neuroimaging methods/analysis and implementation of pharmaco-fMRI studies in healthy controls. Her advanced training will be focused on three key objectives: 1) enhanced knowledge of neuropsychopharmacology; 2) advanced training in neuroimaging (fMRI) research methodology, analyses and implementation of pharmaco-fMRI in post-traumatic stress disorder (PTSD) patients; and 3) advanced understanding of the clinical aspects (etiology, assessment, and treatment) of PTSD. The career development activities will be mentored by Dr. Israel Liberzon (Primary Mentor), Dr. Sheila Rauch (Co-Mentor), Dr. Scott Peltier (Co-Mentor), Dr. K. Luan Phan (Co-Mentor), and Dr. Mohammed Milad (Consultant); a team of researchers that collectively possess non-overlapping, but highly related expertise and exceptional records of mentoring junior faculty. The proposed K01 project will take place within the Department of Psychiatry at the University of Michigan. The University of Michigan is noted for its interdisciplinary research initiatives and the Department of Psychiatry is exceptional with its subspecialty Clinics and laboratories dedicated to translational research. It is an intellectually stimulating yet supportive environment that promotes numerous opportunities for scholarly interactions, which foster collaborations, and has an established track record for mentoring junior investigators. The research component will investigate the cannabinoid system as a potential pharmacological target for improving the retention of fear extinction and its effect on the underlying neural circuits in patients with PTSD A common, empirically-validated approach to treat PTSD is Prolonged Exposure Therapy (PE), one component of which involves repeated exposure to fear-linked cues to produce extinction of fear. PE is generally effective, but a significant number of patients have incomplete responses or fail to sustain improvements over time. New strategies to improve efficacy and sustainability could significantly enhance remission rates and reduce the public health burden of this common disorder. Limited efficacy and lack of sustainability could be due to the fact that extinction learning, which is the active ingredient of exposure-based therapy, is vulnerable to the return of fear. Prior studies have shown that retention of extinction learning depends upon limbic-frontal brain networks (hippocampus [HPC], ventromedial prefrontal cortex [vmPFC]). PTSD patients show deficits in these regions, and in fact exhibit poor extinction retention. Adjunct interventions that address vmPFC-HPC dysfunction and redress extinction retention deficits could be particularly potent in enhancing both the efficacy and sustainability of exposure therapy for PTSD. Compelling new evidence from work by the Candidate has shown that an acute oral dose of 9- tetrahydrocannibinol (THC), a type 1 cannabinoid receptor (CB1) agonist, given prior to extinction learning in healthy volunteers, facilitates the ability to maintain and successfully retrieve extinction memory via increased activation and functional connectivity of the vmPFC and HPC. Given that extinction retention deficits and vmPFC-HPC dysfunction have been observed in patients with PTSD, and that enhancing cannabinoid transmission helps extinction recall, the cannabinoid system is a promising target for improving the learning that goes on in therapy and perhaps increasing efficacy and durability of PE in treating PTSD (e.g., shortening treatment while strengthening and prolonging gains). However, direct tests of cannabinoid effects on extinction recall and associated neural circuits have not yet been conducted in PTSD patients. The objective of the proposed project is to test the hypotheses that administration of THC will enhance recall of fear extinction in patients with PTSD and that these effects will be mediated via increased activation and functional connectivity of the vmPFC and HPC. The Candidate will couple a standard Pavlovian fear extinction paradigm in fMRI and skin conductance response (SCR) recordings to compare the effects of THC (vs. placebo [PBO]) administered prior to extinction learning in 80 trauma-exposed individuals with (n=40) and without (n =40) PTSD and 40 healthy adult volunteers, testing extinction recall 24 hours after extinction learning. This randomized, double- blind, placebo-controlled study will provide the most direct translational and critical test of THC effects in PTSD, advancing our understanding of the neurobiology of extinction learning and potentially hastening the development of novel pharmacological modulators of the cannabinoid system to maximize the efficacy of exposure therapy for anxiety disorders. The knowledge, skills, and data obtained during this career development period will be synthesized into a R01 award application to test whether cannabinoid agonists can be used as an 'extinction recall enhancer' when coupled with conventional, validated exposure therapies to enhance the efficacy, improve extinction retention, and/or expedite the pace of treatment response in PTSD and other anxiety disorders.

The basic purpose of this research is to provide information on how the primate brain comes to be assembled, so as to provide a basis for understanding and treating the diseases of the nervous system that arise before birth. Work on the embryonic and fetal brain directly in monkey is essential for structures which have reached the peak of development in primates. The fetal brains are processed by several specialized cytological methods including autoradiography, Golgi staining, electronmicroscopy, histochemistry and manipulation of development by intrauterine neurosurgery. Attention is given to the interactions of brain cells as they proliferate and migrate relative to one another at early stages of development and as post-migratory neurons develop their dendritic and axonal processes and establish synaptic connections that ultimately attain "wiring" arrangements of the mature brain. Consequences of selective destruction of various brain centers and/or pathways sustained prenatally are evaluated in postnatal monkeys to determine the extent of neuronal plasticity. The projects ccurrently under study concern: (1) Histogenesis and synaptogenesis of the hippocampal formation; (2) time of neuron origin, neuronal differentiation including synaptogenesis of the neostriatum; (3) kinetics of proliferation of radial glial cells and their transformation into astrocytes; (4) neurogenesis of the spinal cord with particular emphasis on synaptogenesis of the substantia Rolandi; (5) development of deep cerebellar nuclei; (6) Dynamics and mechanism of cell migration in the primate telencephalon.

Abstract. Cytosolic and endosomal DNA sensing pathways are known to play a critical role in host defense against microbial pathogens. The same pattern recognition receptors also detect endogenous ligands and thereby promote the onset and progression of autoimmune and autoinflammatory diseases. For example, endosomal TLRs contribute to the pathogenesis of Systemic Lupus Erythematosis (SLE), in part by promoting the production of type I IFNs. Cytosol DNA can be detected by a variety of receptors, including cGAS, which in turn generates an unusual cyclic dinucleotide that activates pathways downstream of STING. Overactivation of STING, either by loss of nuclease activity or STING gain-of-function mutations, also drives a strong type I IFN response now associated with diseases such as Aicardi-Goutieres Syndrome. We have explored the potential crosstalk between cytosolic and endosomal sensors in murine SLE. Quite unexpectedly, we found that STING-deficient SLE-prone mice developed more severe, not less severe, clinical disease. These obser- vations point to a novel role for STING in the negative regulation of TLR-driven systemic autoimmunity. Our preliminary studies have led us to propose that constitutive activation of the STING pathway in hemato- poietic cells limits the inflammatory response of myeloid cells to TLR ligands, promotes the production of negative regulators of immune activation such as A20 and immunomodulatory enzymes such as Indoleamine-pyrrole 2,3-dioxygenase (IDO), and contributes to B cell tolerance induction; the nucleotidyl transferase DNA sensor cGAS acts upstream to recognize host DNA and regulate these STING-mediated effects. We will validate and explore this hypothesis through the following specific aims: (1) identify the cell types directly activated as a result of STING-deficiency; (2) explore the molecular mechanisms responsible for STING-mediated suppression of TLR-driven inflammation; and (3) determine the role of cGAS in the negative regulatory role of STING. To precisely compare STING-sufficient and STING-deficient macrophages, dendritic cells and B cells in models of SLE, we will utilize autoimmune-prone CD45 and Igh allelically distinct mixed bone marrow chimeras.This strategy will preclude any potential confounding factors arising from subclinical infection, changes in microbiome, or effects of disease driven inflammation. SLE is a complex chronic systemic autoimmune disease that afflicts over 1.5 million Americans. Current treatments involve immuno-suppressive regimens associated with debilitating adverse side effects. Attempts to develop safe and efficient therapies that block TLR activation have been stymied by the relative short in vivo half lives of known inhibitors and the potential dangerous outcome of complete MyD88 blockade. Better understanding of the natural regulators of the disease process will provide major insights toward the design of more disease-specific therapeutic options and also avoid the use of STING antagonists that could trigger unanticipated dangerous outcomes. Therefore, the mechanisms we are exploring are highly relevant to human disease and key to future targeted therapies.

About a quarter of patients with severe aplastic anemia remain pancytopenic despite immunosuppressive therapy, and others have suboptimal responses in at least one lineage. We have developed a program to ask whether use of a drug stimulating the c-mpl receptor, which binds the endogenous hormone thrombopoietin, could stimulate human hematopoietic stem cells in vivo. Murine, our own rhesus, and human data all suggest that thrombopoietin can stimulate primitive hematopoietic stem and progenitor cells to expand. The small molecule oral thrombopoietin mimetic eltrombopag was initially developed to overstimulate platelet production from marrow megakaryocytes to compensate for immune platelet destruction. We developed a protocol utilizing eltrombopag in a phase 1/2 clinical trial for patients with refractory severe aplastic anemia. We initially reported that eltrombopag had efficacy in this setting with 44% (11/25) of patients having clinically significant hematologic responses (Olnes et al, NEJM 2012). We then reported additional safety and efficacy data on an expanded cohort of 43 patients, confirming an overall response rate or 40% with 3 to 4 months of treatment, including tri- and bilinear responses (Desmond et al, Blood, 2014). The majority of patients who remained on eltrombopag in an extension study (14/17) continued to show improvement, and 7 eventually had significant increases in neutrophil, red cell, and platelet lineages. Five patients with robust near-normalization of blood counts had drug discontinued at a median of 28.5 months after entry (range, 9-37 months), and all maintained stable counts a median of 13 months (range, 1-15 months) off eltrombopag. Eight patients, including 6 nonresponders and 2 responders, developed new cytogenetic abnormalities on eltrombopag, including 5 with chromosome 7 loss or partial deletion. During the current reporting period, we focused on an additional study, asking whether more prolonged administration of eltrombopag to patients with refractory SAA, for 6 months instead of 3 months, would improve response rate and rescue a larger fraction of refractory patients. This protocol is ongoing and has accrued 37 patients. To date, response rates appear higher than in the initial trial. Samples from these patients are being utilized to investigate pharmacokinetics of the drug in this patient population, and in collaboration with Dr. Neal Young's and Dr. Andre Larochelle's research groups, to investigate the impact of this treatment on genetic abnormalities in marrow failure, and on in vivo and in vitro stem cell expansion. This program resulted in FDA approval of a new labeled indication for eltrombopag in August 2014, and during this reporting period, approval was also granted in Europe for eltrombopag treatment of refractory aplastic anemia. This is the first new drug approved for aplastic anemia in decades and the first drug approved specifically for the refractory aplastic anemia patient population. These trials have now also expanded to treating a number of other bone marrow failure patient populations, as detailed in Dr. Neal Young's report.

The object of this grant is to study a number of problems in infants which may be related to 25-hydroxyvitamin D (25-OHD) deficiency: 1) early neonatal hypocalcemia, 2) late neonatal hypocalcemia, 3) rickets and severe osteopenia, 4) Sudden Infant Death Syndrome (S.I.D.S.) and 5) dental hypoplasia. Laboratory determinations and x-rays obtained during the first 3 months of life are to be further correlated with long term growth and development. 73 premature infants, 34 SGA infants, and 8 IDM infants have been enrolled in the study and been prospectively followed. Additional post-mortem samples have been obtained from 23 SIDS and 7 control infants. 8 near-missinfants and 6 infants with late neonatal hypocalcemia seizures have been studied. Analysis of 25-OHD concentrations has been completed in 59 cases and calcium, magnesium, phosphorous and copper have been completed in 100 cases. During the third year, we will concentrate on getting good follow-up of those infants enrolled and obtaining complete dental examinations. We hope to complete analyses of all samples early in the year and begin to correlate laboratory, x-ray and clinical data within the first 3 months. We will also set up a system for analysis of follow-up data related to these earlier findings.

Project Summary/Abstract The brain can be viewed as an extremely complex and high-dimensional dynamical system. Despite its complexity, only very limited measures of brain activity are generally accessible to recording?e.g. the electroencephalogram (EEG). Nonlinear dynamics provides the tools to extract information from a limited measurement to determine the invariant nonlinear properties of the underlying dynamical system. In Delay Differential Analysis (DDA), a low-dimensional nonlinear functional embedding is built from the dynamical structure of the data; this serves as a basis onto which the data can be mapped. By constraining the models used to low dimensionality, we ensure that DDA is immune to over?tting, insensitive to noise, and generalizes well to new data. DDA has already been applied to human intracranial recordings of sleep to detect sleep spindles and characterize their spatiotemporal development. In the proposed project, this method will also be applied to EEG data from a large study of schizophrenia. In both of these datasets, distinct observed phenomena can be linked to different underlying cortical states. By ?nding DDA models which detect sleep spindles, insights can be gained into their dynamics, and this information can be used to re?ne sophisticated circuit models for their generation. Likewise, by ?nding models which reliably distinguish schizophrenia patients from control subjects, we can develop a better understanding of the dynamical differences that might give rise to sensory processing de?cits and other symptoms of schizophrenia. Further extensions of this work could help to address aditional questions related to functionally distinct states of the brain including in additional neurological and psychiatric disorders.

This proposal is designed to strengthen the biomedical research preparation of Benedict Students for professional careers in biomedical science and biomedical research capability of the faculty for the mainstream of biomedical research competition. These overlapping goals will be achieved through six seperate but interrelated biomedical research projects, involving collaborative efforts by faculty and students. All research will focus on pollution analysis: the effects of metals on cardiovascular disease in humans and rats, on regeneration in the frog, and developmeent and behavior in the rodent Peromyscus will bee assessed; also the effects of ozone on rodents will be investigated; and analytical studies on air, soil and water samples will be conducted using neutron activation and chromatography. The research thrust will build on and enhance the base of faculty expertise, so that the participating faculty will be better qualified to compete in biomedical research. Collaborative efforts with outstanding faculty members at other institutions will enhance the research projects. Also, a Biomedical Seminar will bring eminent scientists to Benedict to present seminars, advise the research projects and provide role models. All MBS students will participate in this seminar and in the informal MBS seminar for research presentations. The implementation of the grant will be coordinated by the Program Director, in association with the MBS Advisory Committee.

The central aim of this longitudinal study, funded by the NHLBI (1-R01-HL53391) is to clarify the extent to which and the mechanisms whereby specific family processes may be protective or deleterious to adherence behaviors and treatment outcome in 3 groups of asthmatic children, aged 7-12 years: 80 children participating in the Denver site of the Childhood Asthma Management Program (CAMP), 80 children receiving asthma-related care in a large, state-of-the-art, health maintenance organization, and 60 children recruited by using school records to identify children with asthma receiving their care in a variety of different health care systems. The three primary specific aims are: (1) to examine the extent to which family process variables are cross-sectionally associated with and longitudinally influence adherence to asthma treatment, as well as the consistency of this relationship across the three sites - a clinical trial sample, a managed care sample, and a community treatment sample; (2) to examine the extent to which family process variables are cross-sectionally associated with and longitudinally influence asthma treatment outcome, as well as the consistency of this relationship across the three sites; (3) to determine whether adherence with treatment mediates the relationship between specific family processes and asthma treatment outcome, as well as the consistency of this relationship across the three sites.

Somatosensory neurons project peripheral axons to the skin early in development to detect touch stimuli. Although the cutaneous terminals of these axons are often a proportionally small component of the total peripheral axon length, they are critical for function, since they are the sites where touch stimuli are first detected. Cutaneous axon endings are particularly vulnerable to damage by injury, diabetes, and inherited syndromes, notably Charcot-Marie-Tooth diseases. All of these conditions cause debilitating peripheral neuropathies characterized by chronic pain or the inability to sense touch. Characterizing how cutaneous sensory endings are formed, maintained, and respond to injury is thus essential for understanding these conditions and developing effective treatments. We have developed a larval zebrafish model to study the development of somatosensory peripheral axons and the skin cells that they innervate. Because zebrafish larvae are fertilized externally, develop rapidly, and are optically clear, the zebrafish somatosensory system offers unparalleled experimental access to the early stages of skin innervation. By contrast, studying these cutaneous sensory terminals in mammals is challenging, since they develop in utero and their complete three-dimensional structures are difficult to visualize. Most anatomical and molecular features of somatosensory axon territories in the skin are well conserved from fish to mammals, making zebrafish a relevant model for uncovering potential disease mechanisms. Our studies of the past few years have revealed that skin cells play several critical roles in the development, repair and function of somatosensory axon terminals in the skin. The goal of this proposal is to identify and characterize the molecular dialogues between axons and skin cells that regulate the establishment and maintenance of somatosensory axon territories. Using a unique and powerful set of molecular techniques and transgenic tools that we have developed in recent years, we will investigate three questions about the nature of axon/skin interactions during specific stages of somatosensory neuron ontogeny. First, how are sensory axons guided to the skin? Second, once in the skin how do axons become structurally associated with skin cells? And third, how does the skin respond to axon damage and contribute to repair? We will address these questions with a powerful combination of live imaging, embryology and molecular perturbations. Collectively, these studies will provide the first molecular insight into the regulation of several newly discovered functions of skin cells in the development and maintenance of the somatosensory system.

The long-term goals of this project represent the use of cloned Ia+, IL-1 inducible and Ia+, IL-1 tumor cell clones to determine the molecular events in antigen presentation accessory cell function and the induction of a syngeneic mixed lymphocyte reaction (SMLR). We will continue to use these cell lines as probes to dissect the signals involved in accessory cell function with a particular emphasis on determining the targets of accessory cell function. We will also continue an analysis of antigen presentation of these cell lines with the long-term goal of determining the nature of selective Ia-antigen fragment interaction on the antigen-presenting cell surface. Of particular interest this coming year will be an analysis of the role of the SMLR in the induction of antigen-specific T-cell proliferation. Moreover, our success in establishing a series of T-cell clones and T-cell lines that recognize self-Ia will enable us to approach several other questions concerning the SMLR in a more direct fashion than we had anticipated. In particular we will be asking the following questions: (1)\Is the universe of antigen reactive T cells located with the synreactive population of T cells? (2)\Can synreactive T cells serve as helper cells in a cognitive recognition system with antigen coupled either to purified Ia or to the surface of an Ia+ cell? (3)\Do synreactive T cells provide nonspecific amplification during the induction of what appear to be unrelated immune responses? (4)\Is the T-cell receptor that recognizes self-Ia the same as the antigen-specific T-cell receptor that recognizes antigen in association with Ia? (MI)

The overall objectives of the research are directed toward establishing the hierarchical role endogenous opioid peptides may have on specific central nervous system neurophysiological processes, and to apply knowledge of these mechanisms to better understand the potential neurochemical substrates underlying reinforcing properties of opiate drugs of abuse. These studies will be focused on the unique opioid-containing circuits within a major limbic-cortical network containing several loci of prodynorphin- and proenkephalin-derived peptides. Both extracellular and intracellular recording techniques will be employed in investigate selected portions of an established entorhinal cortical- hippocampal-accumbens network in both anesthetized, freely- moving, and in vitro preparations. In this fashion, more precise neuropharmacological knowledge will be gained regarding areas established as important in both self-stimulation and drug-seeking behavior. The objectives of these studies will be to: 1) attempt to determine whether opioid peptides contained within specific circuits of this system function as neurotransmitters; 2) whether there are unique cellular correlates of opiate seeking behavior; and 3) whether the opioid peptides and relevant opiate receptors in this telencenphalic anatomical system are critically involved in this behavior. Persuant to my experimental goals, in vivo studies will employ the use of opiate alkaloids, opioid peptides and specific opiate receptor agonists (and antagonists) applied by local iontophoresis and micropneumatic methods, as well as by i.v. or i.p. systemic routes. In vitro studies will employ drug superfusion as well as local application techniques. The role of endogenous opioids in normal synaptic processes will be investigated in vivo and in vitro by electrical stimulation of opioid-containing pathways. Single-electrode voltage clamp methods will be used for analysis of the more covert voltage-dependent opioid effects. In a major committment to studies in freely moving animals, recordings will be made from neurons in the circuits described above (i.e. hippocampus and nucleus accumbens) in awake animals involved in a heroin self-administration protocol. Using this paradigm the cellular correlates of opiate reward phenomena will be investigated in a more relevant preparation.

The outer layer of the corpus cavernosum, the tunica albuginea, is rich in elastic fibers and has the capacity to expand in response to the force of blood pressure, resulting in an increased length and diameter of the penis. However, the expandability of the tunica is finite, and ultimately the initial rise in intracavernosal pressure (ICP) along with the opposing force of the tunica albuginea activates a mechanical occlusion or sandwiching of the venous outflow. Thus, the combined inflow of blood following penile arteriole and sinusoidal dilation, as well as subsequent veno-occlusion, result in maintained elevation of ICP and erection. We recently found that Db/db mice, a common mouse model of type II diabetes, have a veno-occlusive disorder that stems from a lack of tissue filling due, in part, to altered vasoreactivity consistent with impaired cavernosal relaxant ability. We also showed that these mice have may impairment in tissue distensability resulting from altered deposition of fibrillar collagen and elastin. Elastic fibers are assembled extracellularly and are comprised of elastin and specific microfibrillar proteins. Unlike most proteins, elastin production is limited to a brief period of development, beginning during fetal growth and peaking during early neonatal periods. Thereafter, elastin production declines rapidly. By maturity, assembly of elastic fibers is complete, and synthesis of new tropoelastin, the soluble precursor, has declined. Certain diseases, however, such as pulmonary hypertension and emphysema, are characterized by an abnormal accumulation of elastin or by an inability to re-initiate elastin production in response to injury. Still other diseases, such as Marfan's syndrome and cutis laxa, arise from mutations in genes that support elastin organization. In this proposal, we hypothesize that elastin deposition and fiber formation is critical for proper erectile function and that this process is altered in type II diabetic mice, resulting in impaired veno-occlusive function and erectile dysfunction. As elastin degradation is also known to occur in tissue following smoking and/or aging, this knowledge gained from this proposal may also have implications for erectile dysfunction associated with these risk factors as well. PUBLIC HEALTH RELEVANCE: Erection is dependent on distensability of the penis which is enabled by the abundance of elastin in the tunica albuginea and sinusoids. We hypothesize that disruptions in elastin content and elastic fiber organization contribute to erectile dysfunction associated with type II diabetes.

: This application provides pre and postdoctoral training in Drug Abuse Research with a multidisciplinary and triinstitutional faculty that is proficient in the Molecular, Structural, Biochemical, Behavioral and Clinical aspects of Drug Abuse Research. The faculty includes members from the Departments of Pharmacology, Neuroscience, Biochemistry and Public Health at Weill Medical College of Cornell University and from the Memorial Sloan-Kettering Cancer Center (MSKCC) and the Rockefeller University (RU). Faculty research interests include: the phenotypic consequences for pain states, opioid tolerance, reward, anxiety or gaseous anesthetic action of the mutation or deletion of opioid, glutamatergic, GABA or serotonergic receptors, the role soluble and transmembrane adenylyl cyclases in opioid signal transduction, in vivo receptor and drug imaging techniques, anatomical and ultrastructure characterization of the dopaminergic, glutamatergic and opioid peptides systems as they relate to the effects of drugs of abuse, the biological basis of opioid, cocaine and alcohol tolerance and dependency, maternal-fetal pharmacodynamics of drugs of abuse, drug abuse prevention research and the clinical pharmacokinetics and pharmacodynamics of opioid and nonopioid analgesics. Predoctoral trainees will have a choice of a major concentration in either Pharmacology or Neuroscience. In addition to lectures and seminars devoted to topics in drug abuse, both the 5 pre and the 5 postdoctoral trainees will attend a biweekly Pain Conference at MSKCC where patient presentations are followed by a discussion with a multidisciplinary Pain Research Team of pain management, drug abuse and related issues. The program provides training in study design, biostatistics and the ethics of scientific research. Special programs are available to identify minority trainees. The Pharmacology-Neuroscience Drug Abuse Training Program provides an opportunity for young investigators to participate in basic and clinical research in drug abuse. An important aspect of this program is that the trainees will have the opportunity to learn directly about the clinical issues and opportunities in drug abuse research.

It is well established that new episodic and contextual memories are stored in the hippocampus. Over time, these memories are transferred to the cortex through a process called systems consolidation. This process is assumed to occur during periods of inactivity and sleep when the hippocampus replays newly acquired information. Replay is thought to drive the formation of intra-cortical connections that eventually allow memory to be retrieved without input from the hippocampus. Although these assumptions are widely accepted in the field, there is little direct evidence to support them. To address this significant gap in our knowledge, we will use recently developed genetic tools to: 1) identify and control hippocampal neurons that are active during learning and 2) determine if reactivation of these cells is necessary and sufficient for memory retrieval and long-term storage in the cortex. We will accomplish these goals by using newly generated transgenic mice to permanently label neurons that are active during learning. Tagging these cells will allow us to identify networks in the hippocampus and cortex that encode memory and follow their activity during the consolidation period. Next, we will use optogenetic and pharmacogenetic tools to control the activity of labeled hippocampal neurons and determine the effects on long-term memory storage in the cortex. Standard models of consolidation predict that hippocampal stimulation will reactivate cortical neurons that were tagged during learning and induce long-term storage. In contrast, silencing hippocampal ensembles after learning should prevent consolidation and induce amnesia. Our experiments will either: a) substantiate these long-held assumptions and provide mechanistic insight or b) refute these assumptions and provide a new framework for understanding the contributions of the hippocampus to memory consolidation.

Abstract Bacteriophages are viruses that specifically and solely infect bacteria and are a natural platform delivery system of genetic information, encoded by nucleic acids, to bacteria. Lytic bacteriophages do not integrate their DNA into the bacterial chromosome, but replicate independently and ultimately kill their host bacteria. In the early 1900's bacteriophages were used to fight infections in humans, but largely fell to the wayside after the discovery of antibiotics in the 1920's. Only in Eastern Europe and the former Soviet Republics are bacteriophages still considered as effective therapeutic agents. With the antibiotic armamentarium currently challenged by the combination of increasing antibiotic resistance and a dearth of new investment and discovery in new classes of antibiotics, natural bacteriophage therapy is experiencing a renaissance. Along with this renaissance in bacteriophage therapy, advances in systems and synthetic biology now enable exquisite engineering of bacteriophage genomes to introduce nucleic acid therapeutics to alter the physiology, pathogenicity, virulence and antibiotic sensitivity of targeted bacterial species. Bacteriophage can thus be designed to deliver genetic payloads to achieve specific desired effects, such as enhancing antibiotic killing by genetic repression of DNA repair mechanisms, the self-generation of antimicrobial peptides and proteins and expression of enzymes to degrade bacterial biofilms. Biofilm-associated infections are particularly difficult to treat due to the physical and physiological barriers biofilms pose to antimicrobial agents and host defenses. Biofilms are central to the pathogenesis of many serious clinical infections, and often colonize foreign-body surfaces, such as in prosthetic joint infections (PJI). The annual cost of PJI to US hospitals in 2009 was $566M and is projected to increase to $1.62B by 2020. Much of this cost is for surgical replacement of infected prostheses due to the failure of medical treatment. Failure of medical treatment is most problematic in device infections caused by S. aureus, due to its virulence and rapid biofilm formation. This proposal seeks to improve medical treatment for PJI, caused by S. aureus, by utilizing engineered bacteriophage to deliver nucleic acid therapeutics to the bacteria encoding biofilm-degrading enzymes to disperse the protective biofilm. Removal of the biofilm will reduce the inherent tolerance of bacteria to antimicrobial therapy, improve medical treatment outcomes and thereby reduce surgical intervention, prosthesis replacement and the associated health costs and patient burden.

PROJECT SUMMARY The importance of coronary atherosclerosis and its clinical manifestations cannot be overstated; in this country, half a million people die from acute myocardial infarction every year and an equal number of patients survive with substantial morbidity. Coronary atherosclerosis is an inflammatory process governed by dysregulated endothelium, leukocytes, macrophages, and smooth muscle cells. These cells conspire to form lesions within the artery wall, effectuate plaque progression or regression, and play a critical role in precipitating thrombosis, which can block blood flow to the heart. Progress towards improving the understanding, diagnosis, and treatment of coronary artery disease has been slowed by our inability to observe these cells in the coronary arteries of living human patients. Our laboratory has been on the forefront of pushing the resolution limits of coronary imaging in vivo. In the parent R01, we developed an intracoronary imaging technology, optical frequency domain imaging (OFDI), that makes it practical to obtain 10-m resolution, three-dimensional images of the coronary wall in the clinical setting. Now that OFDI has been transferred to the commercial sector, we will focus our efforts on developing the next generation coronary imaging technology, one that will provide images at a resolution of 1-m. This advance will enable the investigation of macromolecules and cells involved in the pathophysiology of CAD and will allow clinicians to diagnose and treat this disease based on cellular information obtained from their patients' coronary arteries. The research strategy of this project parallels that of our previous development and translation of intracoronary OFDI. First, new OCT technology will be designed and fabricated, focusing on solving catheter and system- level challenges. Alongside this development process, we will validate the accuracy of OCT ex vivo for identifying relevant cellular and subcellular features of the coronary wall, using histopathology as a gold standard. The safety, feasibility, and efficacy of OCT will then be evaluated in 25 patients undergoing coronary intervention. !

These proposed studies in ewes are designed to obtain information on the role of the hormones responsible for control of oviductal muscle activity and the resultant effect of muscle contractions on gamete transport. The serum hormone concentrations of LH, estrogen and progesterone as determined by validated radioimmunoassays will be compared quantitatively with: 1) the rate and amplitude of oviductal contractions; 2) the time of ovulation; and 3) the rate of cumulus mass transport in the intact unanesthetized ewe. Instrumental techniques using optoelectronic transducers will be employed for the chronic monitoring of oviduct contractions and for detecting the presence and transport of the cumulus mass. Emphasis will be placed on observing and measuring the oviduct contractile rate, the propagation velocity of the peristaltic (anti-peristalic) contractile wave and the segmental transport velocity and ampullary transport time of the cumulus mass. The physiological experiments, to be conducted in three phases, include: 1) relating the circulating hormone levels of LH, estrogen and progesterone to the time of ovulation and oviduct muscle activity in the normal cycling ewe; 2) determining the effect of a synthetic LH-RH/FSH-RH analog on the time of ovulation, oviduct motility and serum LH, estrogen and progesterone concentrations in anestrous ewes, and 3) determining the effects of exogenous 17-beta estradiol and progesterone on oviduct motility in anestrous ewes. Increasing our knowledge of the reproductive process at the oviduct level may provide valuable information for explaining how steroid contraceptives which do not block ovulation are effective in preventing pregnancy.

Abstract Klebsiella pneumoniae (Kpn) is a leading cause of Gram-negative nosocomial infections and is associated with a high mortality rate. Antibiotic resistance is a growing issue among the Enterobacteriaceae and of the Enterobacteriaceae Kpn is the most prevalent extended spectrum ?-lactamase and carbapenem resistant Enterobacteriaceae isolate. The increasing prevalence of antibiotic-resistant Kpn only serves to compound its clinical importance and to complicate treatment options. Capsule has been established as a key virulence factor and is this species best studied virulence factor. Kpn strains are broadly classified as hypervirulent (hv) or classical, with hv strains typically causing community acquired liver abscess and invasive infections. Classical strains are more typically associated with nosocomial infections. The hv strains have a hypermucoviscous (HMV) phenotype thought to be due to over-production of capsule and have acquired rmpA that contributes to increased capsule (cps) gene expression. Most classical strains, including recent clinical isolates associated with carbapenem resistance (ST258) are not HMV, do not have the rmpA gene and are not virulent in mouse models of infection. Recent reports of these multidrug resistant strains acquiring the HMV phenotype is of significant concern and amplifies the need to understand what HMV is, how it is produced and how it contributes to virulence. Despite the apparent association between HMV and over-production of capsule, what causes the HMV phenotype and its relationship to capsule production is not known. Recent work from our lab using the HMV strain KPPR1S has shown that deletion of rmpA causes decreases in expression from cps promoters, and reduction (but not complete loss) in both capsule production and HMV. In addition, we found that rmpA is the first gene of an operon (rmpADC) and that RmpA positively regulates expression of the operon. Results from analysis of a complete deletion of the operon with the individual genes indicates that from this locus, RmpA primarily is required for expression of the operon, that only RmpC is necessary for expression of cps genes, and that only RmpD is necessary for HMV. Consistent with this, a mutant lacking only rmpD retains WT levels of cps gene expression, capsule production and is HMV negative. The rmpD mutant is the first mutant identified that separates HMV from the over- production of capsule, and the phenotype of the strain only expressing rmpC indicates that over- production of capsule alone is not sufficient for HMV. Due to the importance of HMV to the virulence of hv Kpn strains, the potential for classical strains to acquire HMV, the incomplete picture of what constitutes HMV and what is required to produce HMV, we propose to a) gain a better understanding of RmpD, (b) characterize the interacting partners of RmpD and how they affect HMV, and determine the composition of the HMV exopolysaccharide, and (c) examine how HMV contributes to virulence.

Self-injuring behavior (SIB) surprisingly prevalent among neurodevelopmentally delayed and mentally retarded individuals, has no known cause and no agreed upon treatment. Expression of SIB may persist throughout the lifespan and into later stages of life, with little change in frequency or quality. Two distinct patterns of SIB have been proposed. One pattern consists of short bouts that may be maintained by environmental contingencies. A second pattern involves protracted periods of SIB that probably is motivated by biological factors. Recent studies from our laboratory indicated that self-injuring behavior was predicted best (and in some cases only) by its own recent history. In a significant majority of individuals, SIB episodes were self-perpetuating and not related to antecedent conditions. We report in this application that individuals exhibiting "contagious" (i.e. sequentially dependent) patterns of SIB also have a disregulated hypothalamic- pituitary-adrenal (HPA) stress axis. We have concluded that there is both a biological predisposition to, and a biological consequence for, the maintenance of SIB. These conclusions may be premature and a more extensive evaluation of the environmental context is warranted. Moreover, there are no data about what initiates a contagious cycle. The purpose of this proposal is to examine the behavioral, environmental and biological circumstances that initiate and maintain SIB episodes. Thirty individuals exhibiting SIB and thirty control subjects, all residing in a large residential facility will be observed in vivo and during an experimental functional analysis of behavior (FAB). Their behavior, interactions and the environmental conditions will be recorded with computer-assisted procedures. Blood will be collected for measures of HPA activity in the morning on a separate day. The Specific Aims are: (i) Determine how the patterns of SIB are affected by environmental context in both in vivo and FAB conditions using sequentially- and temporally-dependent time series analysis, (ii) Determine if there are consistent patterns of environmental events or relations among behaviors that are antecedent to the first or isolated SIB episodes;(Hi)Determine the relation between the HPA stress axis the initiation of SIB, (iv) Apply and evaluate a rational pharmacological treatment to a selected cohort of subjects who present clear evidence of biological (or internal) motivation for SIB. Findings from this project will contribute to the understanding of the emergence and maintenance of SIB.

Substance abuse is one of the most common health disorders in US veterans. Of the over million opiate- dependent subjects today, less than a quarter of such individuals receive treatment. Pharmacotherapeutic approaches have traditionally targeted 5 opioid receptors since heroin and its metabolites bind with highest affinity to this receptor subtype. Treatment strategies such as methadone have improved substance abuse outcomes, but they do not effectively block opiate craving and thus are still associated with high rates of relapse. Using a strategy of indirectly regulating neural systems to modulate opioid-related behavior, our preclinical rat pilot studies (using a drug self-administration paradigm) consistently demonstrated that cannabidiol (CBD), a nonpsychoactive component of cannabis, specifically inhibits cue-induced heroin-seeking behavior. CBD's selective effect on cue-induced heroin- seeking behavior was pronounced after 24 hours and endured even two weeks after the last drug administration following short-term CBD exposure. The fact that drug seeking is generally triggered by exposure to conditioned cues suggests that CBD might be an effective treatment for heroin relapse, specially given its protracted impact on behavior. The overall aim of this project is to understand the potential role of CBD as a treatment for drug craving. To this end, the experiments outlined in this application will: (1) Identify the specific relapse condition sensitive to CBD. Although our preliminary findings showed that CBD specifically attenuated drug-seeking behavior associated with environmental cues, but not heroin prime, factors such as stress can also trigger relapse. We will evaluate the effect of CBD on drug-seeking induced by mild footshock stress in animals with moderate and extended access heroin self-administration history. (2) Examine the effects of CBD in methadone-maintained subjects in relation to heroin-seeking behavior. Methadone is the most common pharmacological treatment for opioid abuse available today for US veterans, but it does not effectively reduce craving. It is thus important to evaluate whether CBD would be an effective adjunct treatment in methadone-maintained subjects. (3) Obtain insight as to the neurobiology underlying CBD effects on heroin-seeking behavior. Postmortem molecular and biochemical studies will be conducted on markers linked to cannabinoid, glutamate and dopamine systems in mesocorticolimbic brain regions of animals in which drug relapse behavior has been characterized. Given the important role of glutamatergic and dopaminergic transmission in drug addiction vulnerability, in vivo microdialysis will also be used to directly monitor dynamic fluctuations of glutamate and dopamine levels during active drug seeking behavior. This investigation, together with complementary clinical investigation on the role of CBD on drug craving, has the potential to significantly impact the development of a novel agent for relapse prevention that is critical for preventing the continued cycle of drug abuse. PUBLIC HEALTH RELEVANCE: Drug abuse is of major concern in the US veteran population. Despite the current available therapies for opioid-dependent patients, most patients relapse. This research project focuses on the development of a novel compound, cannabidiol, to inhibit drug-seeking behavior. Preliminary results have already shown that cannadibiol effectively inhibits cue-induced drug seeking behavior in animal models. Various factors can, however, induce relapse, thus it is critical to evaluate whether cannabidiol can also inhibit drug seeking triggered by stress which is known to induce drug craving in humans. Studying the impact of cannabidiol on molecular and chemical alterations in the brain will help us identify the neurobiological mechanisms underlying relapse vulnerability. Overall, the development of a targeted treatment for opioid relapse would be of tremendous medical value to our veteran population.

Project Summary/Abstract: African Americans have the highest death rates from cervical and colorectal cancers than any other racial or ethnic group. Effective screening and treatment exist for the pre-cancerous conditions that lead to the development of these deadly cancers, but there is low adherence among African Americans. Public Health Television (PHTV), in partnership with the Case Comprehensive Cancer Center, proposes to increase screening and treatment adherence for pre-cancerous among low-income African Americans for these 2 cancers by developing video-based training for patient navigators. Patient navigation refers to using lay members of the community to guide and support underserved populations through the process of obtaining screening and medical care for chronic diseases. NCI endorses patient navigation as a means to help underserved populations obtain more timely cancer treatment. PHTV will combine the concept of patient navigation with its proven process of culturally specific health communication to engage low-income African Americans in the secondary prevention of colorectal and cervical cancers. 2 specific aims will accomplish this objective. First, PHTV plans to produce a video/DVD series to train patient navigators to guide low-income African American patients through participating in colorectal cancer screening and adhering to follow up treatment for pre-cancerous polyps. Second, PHTV will develop content for a video/DVD series to train patient navigators to assist low-income African American women with cervical cancer screening algorithms and compliance with treatment for human papillomavirus (HPV) associated pre-cancerous cervical lesions. PHTV will identify and meet target group information needs with our proven culturally specific production process that uses members of the target population to design messages. Focus groups of low-income African Americans and review meetings comprised of navigators currently working with minority patients in urban settings will be conducted to identify barriers and solutions for increasing participation in secondary prevention activities. An Advisory Panel comprised of representatives from comprehensive cancer centers, public health agencies, and the American Cancer Society will validate feasibility of plans to produce and evaluate the videos among patient navigators working across Ohio and in Harlem, New York, using a web-based tracking system. These efforts will enable PHTV to successfully launch and test a video based navigator-training program in Phase II. PATIENT NAVIGATOR VIDEO TRAINING FOR CANCER IN LOW-INCOME AFRICAN AMERICANS Project Narrative Relevance: Public health relevance lies in the potential to develop a transferable instrument designed to increase participation in screening procedures and treatments to prevent invasive cervical and colorectal cancers among a population dying from these diseases at disproportionate rates. This will result in reduced overall health care costs to society. [unreadable] [unreadable] [unreadable]

Koronis Biomedical Technology Corporation (KBT) proposes to develop a navigation system for ultra-thin bronchoscopes using advanced thin-film magnetic sensor technology. Recent technological innovations in the semiconductor processing industry make this program technically achievable at this time. The use of advanced magnetic-based localization technology to provide real-time navigation imaging without fluoroscopic guidance is highly desirable. Such a localization system in an ultra-thin bronchoscope allows an operator to navigate within the periphery of the lung and track the path of a biopsy tool as it approaches a peripheral lesion. PUBLIC HEALTH RELEVANCE: The proposed project relates to the navigation of ultra-thin bronchoscopes. More than 150,000 solitary pulmonary nodules are reported annually in the US, each of which may indicate the presence of lung cancer. Bronchoscopy is used to locate anomalies in a patient's bronchus and if necessary, take a biopsy. Over 3,000,000 bronchoscopies are performed annually worldwide. The majority of target lung lesions are located in the peripheral areas of the lungs where large bronchoscopes cannot reach.

OVERALL SUMMARY/ABSTRACT This Case GI SPORE renewal application provides for a cutting edged Specialized Program of Research Excellence in gastrointestinal malignancies with emphasis on colorectal cancers and adenocarcinoma of the esophagus. This comprehensive program builds on the resources of the Case Comprehensive Cancer Center to propose 4 translational Research Projects to bring new molecular advances to patients with GI Cancers. A series of 3 core resources support these projects and also establish a strong programmatic infrastructure for translational research in GI cancers. We have further developed a comprehensive infrastructure for identifying new Developmental Research Projects from basic science and clinical investigators from across the Case Cancer Center. Drawing on our strong track record of developing new faculty who emphasize translational research in GI cancers, we have also developed a targeted Career Enhancement Program to further advance and recruit to the translational research cadre of SPORE faculty. The 4 SPORE translational research projects constitute novel and cutting edge approaches to GI cancers and include studies of: i) Targeting the 15-PGDH colon cancer tumor suppressor pathway for prediction of cancer risk, prediction of response to chemoprevention with aspirin, and development of new colon cancer prevention and treatment strategies (Project 1, Drs. Markowitz, Li, and Berger); ii) Elucidating the basis for and the clinical significance of a mutational signature of African American colon cancer (Project 2, Drs. Willis, Li, and Wang); iii) Development of molecular markers of and non-endoscopic detection of Barrett's esophagus and early esophageal adenocarcinomas (Project 3, Drs. Chak, Guda, and Markowitz); and iv) Development of personalized treatment of PIK3CA mutant colon cancers using targeted inhibitors of glutamine metabolism (Project 4, Drs. Wang and Meropol). These projects are built on major advances from the SPORE's first funding period that include: i) publication in Science that 15-PGDH regulates colon crypt stem cells; ii) publication in Science Translational Medicine that individual's 15-PGDH levels regulate whether taking aspirin will or will not reduce their colon cancer risk; iii) publication in Proceedings of the National Academy of Sciences of genes preferentially targeted for somatic mutations in colorectal cancers of African Americans; iv) publication on the cover of Cancer Epidemiology, Biomarkers and Prevention that testing for methylated vimentin DNA in esophageal brushings could detect over 90% of Barrett's esophagus and esophageal adenocarcinomas; v) publication in Nature Communications that PIK3CA mutant cancers are addicted to glutamine and sensitive to inhibitors of glutamine metabolism. These projects are advantaged by special populations and scientific resources developed for evaluation of GI cancer biomarkers, new drug discovery, Next-Generation sequencing of formalin fixed tumor samples, and studies of Barrett's esophagus. The strength of these investigators and the resources at their disposal will insure this program leads to significant important translational advances.

Photosystem II (PSII) is a membrane protein complex that uses light to catalyze water oxidation and quinone reduction. The oxygen-evolving complex (OEC) of PSII is the source of the atmospheric oxygen that we breathe. Because atmospheric oxygen is essential to sustain all aerobic life on Earth, the function of the OEC is of major importance to human health. The proposed studies will provide new information on how oxygen is produced by the OEC. The recent 3.5 [unreadable] crystal structure of PSII provides the first structural model of the OEC. The revealed geometry of the OEC fits well with the water-splitting mechanism advanced by this group in which, a nucleophilic Ca-bound water attacks the oxygen of a Mn(V)=O species in the O?O bond- forming step. Following from the new structure, the OEC will be characterized by using a combination of biophysical and computational studies of PSII and bioinorganic Mn model chemistry. The long-term objectives are to develop an understanding at the molecular level of the structure and assembly of the OEC, the mechanism of water oxidation, the electron-transfer properties of PSII and the damaging side reactions that occur in the process of water oxidation. This project is divided into two specific aims: (1) to characterize the structure and function of the OEC, and (2) to use Mn coordination complexes to aid in the interpretation of biophysical studies of the OEC. In aim 1, studies are proposed to characterize the function of Ca in the OEC by using cation- and/or an ion-substituted PSII, to test the proposal that CP43-Arg357 functions as a catalytic base in the O-O bond-forming step by study of CP43-Arg357 mutants, and to use computational modeling to gain insight into the structure and function of the OEC. In aim 2, Mn coordination complexes will be used to gain insight into how the substrate waters are bound in the OEC by measuring the exchange of 18-O-labeled water with water/hydroxide/mu-oxo species in a series of Mn coordination complexes that model the structural properties of the OEC. In addition to providing a clearer picture of the water-oxidation chemistry of PSII, the proposed studies will provide insight into the mechanisms of other metalloenzymes that are essential to human health such as those that use oxygen or hydrogen peroxide as a substrate.

The goal of this project is to evaluate the chronic toxicity and carcinogenicity of alpha-pinene in rats and mice, following whole body inhalation exposure. Alpha-pinene is found in turpentine and in flavorings and fragrances. Humans may be exposed through use of personal care and cleaning products as well as in lumber processing and building activities. 2-Year chronic toxicity and carcinogenicity studies of alpha-pinene are the in life phase. These studies include an assessment of fertility and sperm count and motility, as male reproductive effects are anticipated. Keywords: toxicity, carcinogenicity, inhalation, alpha-pinene

The inverse correlation between exposure to helminths and the incidence of certain immune-mediated diseases, including inflammatory bowel diseases (IBD), evidenced from epidemiological studies (i.e. the hygiene hypothesis), has provided the framework for the pioneering studies of Weinstock et al. and shows promise of using helminths to treat Crohn's disease patients. It has become clear that the chronic inflammation observed in the intestines of IBD patients is due to an aberrant immune response to enteric bacteria. Our understanding of the exact mechanism by which helminths modulate the mucosal response to enteric bacteria is incomplete. Further, the distribution of several pathogenic helminth infections coincides geographically with many devastating microbial diseases, such as HIV, malaria, tuberculosis and infantile diarrhea (caused by enteropathogenic Escherichia coll, EPEC). In many parts of the world, coinfections are also very common. In order to dissect the mechanisms by which helminths modulate the host's response to concurrent pathogens and enteric bacteria, we have established a co-infection model, which involves two murine enteric infectious agents that induce distinct Th responses. We have found that co-infection of mice with the helminth (Heligmosomoides polygyrus) and the enteric bacteria (Citrobacter rodentium) promotes bacterial infection and enhances bacteria-induced colitis. We also observed that dendritic cells (DCs, the most potent antigen-presenting cells), which are primed by the helminth are capable of altering the host's response to the bacteria. Moreover, our preliminary results suggest a role for Th2 cytokine-stimulated macrophages in impairing host defense and promoting TNF-a production, Our overall hypothesis is that intestinal helminth infection can act as a risk factor for enteric bacterial infection and bacteria-associated intestinal inflammation by affecting innate immune cells, resulting in impaired protective immunity and increased tissue injury. Therefore, the objective of this proposal is to explore the mechanisms by which intestinal helminths modulate bacteria-associated intestinal inflammation. Specific Aim 1 is designed to characterize the helminth-induced alterations in DC phenotype and function that are responsible for impairing the host defense and modulating the pathogenesisi of bacteria-mediated intestinal injury. Specific Aim 2 is designed to determine the role of helminth-primed DCs in the modulation of mucosal T responses to C. rodentium and the contribution of Th2 cytokine-stimulated macrophages to the alterations in host response to concurrently exposed bacterial pathogens. A better understanding of immunomodulatory effects of helminth will provide information for establishing novel and more effective treatments for immune- mediated diseases, such as IBD, and for the design of effective intestinal vaccines for the prevention and treatment of microbial diseases in the areas where multiple infections exist. [unreadable] [unreadable] [unreadable]

Dynorphins are a family of opioid peptides derived from a single precursor and known to have a wide profile of pharmacological actions. Since the pharmakinetics vary with peptide size, the catabolism of dynorphin A (the most active kappa-selective fragment) will be emphasized to determine the nature of enzyme(s) present in different brain fractions (SPM, Golgi fractions, etc.) responsible for C-terminal shortening, and hence alteration of the address signals directed towards the different opioid receptor types. To confirm observations made in preliminary studies with brain fractions, a number of key C- terminally active enzymes will be purified and used in a coordinate manner to demonstrate production of active dynorphin intermedicates. Such enzymes will include one or more membrane bound carboxypeptidases active at pH 5-6.5, angiotension coverting enzyme, cysteine proteinases with peptidyl dipeptidase actions (cathepsin B), and prolyl endopeptidase. With the aim of developing new classes of inhibitor active in vivo, a number of known synthetic compounds will be compared to ones recently synthesized in the laboratory, or purified from brain fractions themselves. Such materials will include a new class of potent cysteine proteinase inhibitors shown to suppress conversion of prodynorphins or proenkephalins.

The primary rationale for the Duke Aging Center Postdoctoral Research Training Program (RTP) proposed here is to continue to train highly skilled research scientists who have strong backgrounds in substantive areas related to aging and who also have the potential for leadership in gerontological research. Each fellow is assigned a mentor or mentors and works within that person's research program;we also have an interdisciplinary seminar which all fellows must attend. Fellows typically spend two years in the RTP. The RTP faculty and their facilities enable outstanding research training in the following areas related to aging: biomedical (biochemistry, cardiology, endocrinology, epidemiology, immunology, neurology, neuroscience, neuroendocrinology, nutrition, oncology, physiology, pharmacology, psychiatry, and radiology);behavioral (behavioral medicine, neuropsychology, neuroeconomics, psychophysiology, clinical, developmental, cognitive, experimental, personality and social psychology): and social science (anthropology, economics, sociology, health services research). Fellows spend a significant portion of their time on research. They are integrated into their mentors'research programs and mentored through collaborative and independent research projects. All fellows and some faculty attend our weekly RTP seminar on research in aging. This seminar follows a structured curriculum on the biopsychosocial aspects of aging and the interdisciplinary nature of aging research, as well as professional development, research design and analysis, and research ethics. Potential fellows submit a detailed NIH-format research proposal (with input from their mentors), a curriculum vita, graduate transcripts, career and training plans, and letters of recommendation. Ultimately, most fellows either join university faculties or research organizations when they complete the RTP. We request support for 6 postdoctoral fellows. The Duke Center for the Study of Aging and Human Development is the primary research training facility for fellows supported by this grant. Associated faculty members are located throughout the University and Medical Center. PUBLIC HEALTH RELEVANCE: The aging research completed by the fellows in the Duke Postdoctoral Research Training Program is highly relevant to public health concerns. The research covers aging from basic science (laboratory work) to clinical (in a wide variety of therapeutic areas) to epidemiological (population level databases). This translational approach to research in aging drives the application of new knowledge to clinical interventions that promote quality of life of older adults.

Project Summary Intermediate filament (IF) proteins are encoded by ~65 functionally distinct genes, placing them among the 100 largest gene families in humans. The remarkable discoveries of hundreds of mutations in IF genes causing a plethora of human diseases have attracted the attention of basic scientists and physicians. The etiology and clinical manifestations of abnormal intermediate filament proteins are wide-ranging, including premature aging, cardiomyopathies, neurodegenerative disorders, myopathies and muscular dystrophies, as well as skin blistering diseases, amongst many others. Abnormal IF aggregates are now recognized factors in motor neuron diseases, Alzheimer?s disease, Parkinson?s disease and giant axonal neuropathy. The first therapeutic interventions based on this primary research are now benefiting patients, and it is therefore an exciting time and a new phase for the field. Furthermore, intermediate filament proteins, which serve as markers of the tissue origin of poorly differentiated tumors, as tumor markers in serum, and as a means of detecting micro-metastases, are now recognized as key initiating points in the metastatic cascade. The 2018 Gordon Research Conference (GRC) IF Meeting aims to unite researchers and clinicians who are contributing distinct perspectives and experience, but who together reveal insights relevant to intermediate filament biology, such as their structural and functional relationships to other established or emerging areas, including clinical medicine. Our objective is to promote a stimulating series of scientific sessions and an environment attractive to young investigators/trainees from diverse backgrounds while encouraging established scientists to embark on novel questions and deliver new therapeutic interventions. In addition to the use of human tissue and database resources, model systems represented will include Drosophila, Xenopus, zebrafish, C. elegans, and mouse. The approaches represented will include molecular/biochemical, cellular and structural/biophysical, genetic/developmental, grouped into nine sessions that represent the broad range of physiological and pathological processes in which IF proteins play a central role. The GRC IF meetings, which have been held continuously since 1990, bring together individuals that would not otherwise gather in a similarly intimate scientific forum. Thus, both established investigators and trainees look-forward to this opportunity once every two years. As represented in our speaker/chair list, we have been attentive to gender diversity, early-career investigators, and researchers new to the IF field. All applicants are invited to present posters describing their latest research; all of these will be at least briefly introduced orally. In addition, a number of platform presentations will be chosen from the submitted abstracts in order to ensure inclusion of the latest breakthroughs during the formal sessions. The attendance of young scientists has always been encouraged and in recognition of this, the meeting will include the fourth IF Gordon Research Seminar (GRS), which will precede the GRC and which will be organized by graduate students and postdoctoral fellows.

Successful development of a vaccine against HIV will likely require the induction of both antibody and/or cellular immune responses sufficient to prevent infection or disease respectively following infectious challenge. While the induction of antibody responses for a variety of other infectious pathogens is readily achieved by a variety of vaccine formulations, live attenuated, recombinant viral vaccines or plasmid DNA vaccines only induce the induction of long-lived cellular immune responses, particularly CD8+ T cell responses. Moreover, since live attenuated HIV vaccines might be precluded from use due to safety concerns and DNA vaccines at present only induce modest CD8+ T cell responses in humans, there is an urgent need to develop ways to enhance the generation and maintenance of CD8+ T cell responses in humans in following immunization. This study focuses on how to optimize the magnitude and duration of CD8+ T cell responses following vaccination in rodents and primates using a variety of vaccine formulations. The data obtained over this past year have shown the following; 1. A comparative analysis of single or combination TLR ligands was done in NHP using SIV Gag protein and an oil/water emulsion. Amongst the adjuvants used, Poly IC and a TLR 7/8 agonist were the most potent for inducing SIV Gag specific CD4 and CD8+ T cell responses. 2. A comparative analysis of adjuvants and formulation was recently completed in NHP using HIV Env protein. This study focused on two clinically approved adjuvants ( alum and MF-59) with or without TLR 4 or TLR 7 ligands as additional immune stimulators. The data generated so far show that MF-59 induces more potent humoral and cellular immunity than alum when given with HIV Env protein. Combining alum with a TLR 7 ligand strongly enhanced immunity compared to alum alone. By contrast, adding TLR 4 or TLR 7 ligands to MF 59 did increase immunity compared to alum alone. In terms of T cell immunity, MF 59 induced a mixed Th1 and Th2 cytokine response. More recent analysis has been done to determine the extent of affinity maturation of the HIV Env B cell response using deep sequencing. 4. A comparative analysis of adenoviral or pox viral vectors encoding SIV Gag was initiated this year. The data show that the amount and duration of antigen has a dramatic influence on the strong CD8+ T cell immunity with rAd5 compared to other Ad serotypes. By contrast, there is minimal innate immunity induced by Ad5. The current work focuses on the mechanisms by which innate immune controls antigen expression in antigen presenting cells and determining how specific innate pathways enhance or inhibit the CD8+ T cell response.

Periodontitis is the leading cause of tooth loss in the U.S. It is thought to be mediated by the interaction of bacteria with host cells leading to the production of destructive proteases. While many different bacteria have been isolated from the tissue of patients with periodontitis, no single bacterial species has emerged as essential to the development of periodontitis and it remains unclear how different bacteria contribute to the pathogenesis of the disease. The major proteases found to be involved in periodontitis come from a class of enzymes called matrix metalloproteinases (MMPs). MMPs all share a function in degradation of extracellular matrix (ECM) proteins. Multiple MMPs have been found to be elevated in the gingival space of patients with periodontitis and therapy with MMP inhibitors has been shown to improve outcomes in patients with periodontitis. Individual bacteria have been shown to induce MMPs using in vitro systems and other bacteria have been shown to produce enzymes, which can activate MMPs or inactivate their inhibitors. Nevertheless, while the role of bacterially induced MMPs in the pathogenesis of periodontitis has become clearer, the mechanisms by which MMP induction may benefit the bacteria remain unknown. Recently, it has been shown that MMPs have substrates beyond ECM proteins and may be involved in immunomodulation through cleavage of cell surface receptors or inactivation of inflammatory cytokines and chemokines. We will outline our systematic plans to examine the association of individual and combinations of bacterial species with pathologic increases in MMPs. Patients with periodontitis and healthy controls will have gingival crevicular fluid examined for the presence of different MMPs and will also undergo identification of bacterial present in the diseased crevices. Based on these results, we will then choose bacterial combinations to test a novel hypothesis: that different microbial communities, causing periodontitis, share the ability to induce and activate MMPs, which in turn protects the community by modulating the host immune response through cleavage of chemotactic chemokines. These studies will be performed using in vitro cell culture and animal models. Results of this application will lead to a better understanding of the complexities of the host-pathogen and pathogen-pathogen interactions and could potentially result in the development new interventions in periodontitis. [unreadable] [unreadable] [unreadable]

The mammalian kidney is susceptible to injury by ischemia/reperfusion and toxins, and regeneration following injury is characterized by hyperplasia and recovery of the damaged epithelial cells lining the tubules. Recent studies have indicated that in response to acute injury, epithelial cells dedifferentiate and become capable of proliferating, and all renal epithelial cells appear to retain the capacity to serve as progenitor cells. Because the response to acute renal failure recapitulates certain aspects of renal ontogeny, investigation of the mechanisms underlying regeneration from acute renal injury may also lead to a better understanding of mechanisms of renal development. Studies have suggested that epidermal growth factor (EGF) or EGF family members may be mediators of renal regeneration following acute injury After either ischemic injury or folic acid nephropathy, 1251-EGF binding increases and administration EGF or TGF-alpha accelerates [3H] thymidine incorporation and recovery of tubular function. However, both preproEGF mRNA levels and urinary EGF levels decrease and remain significantly depressed for up to seven days. We have recently found that acute renal injury significantly increases expression of mRNA and bioactive protein of another member of the BGF-like family of growth factors, heparin-binding epidermal growth factor-like growth factor (HB-EGF). The present studies will continue investigation into characterization of HB-EGF expression in renal injury and will explore potential roles of the growth factor in regeneration of renal epithelia. The specific aims are: l) to localize time-dependent expression of HB-EGF mRNA and bioactive HB-EGF in acute renal injury and characterize the relationship of HB-EGF expression to specific sites of injury; 2) to examine mechanisms regulating HB-EGF mRNA expression in acute renal injury; 3) to determine mechanisms by which HB-EGF may prevent or minimize renal injury and/or mediate recovery; and 4) to determine whether altering renal HB-EGF levels influences renal response to acute injury. In vivo studies will concentrate upon two models of acute renal injury, ischemia/reperfusion and aminoglycoside nephrotoxicity. Parallel and complementary studies will be performed in cultured renal tubular epithelial cells to explore regulation of expression and actions of HB-EGF in injury. These studies will examine the roles that mediators of acute injury play in expression of HB-EGF mRNA and processing of the precursor peptide. They will also examine potential roles of HB-EGF in recovery from injury both by blocking expression or action of the peptide and by administering exogenous HB-EGF or overexpressing endogenous peptide. Therefore, the overall goal of these studies is to elucidate further the role of EGF-like growth factors in the regulation of regeneration following acute renal injury.

The ability of low concentrations of fluoride ions to protect teeth against the development of carious lesions is well known. However, the mechanism by which the protective effect is exerted still is only poorly understood. Two hypotheses have been put forward to explain the findings. The first related the protective action to the increased stability of the enamel to acid as a result of the incorporation of fluoride into the apatite lattice. The second deals with the ability of fluoride to interfere with the acid-producing capacities of plaque organisms. Our studies have confirmed the inhibitory capacity of even 1 ppm of fluorine. However, different plaque-derived organisms exhibit widely divergent sensitivities to the anion. One such microorganism has been found to take up fluoride intracellularly and to concentrate it in two distinct compartments. The goals of the proposed investigation are to examine fluoride uptake in order to determine which of the compartments contains metabolically active fluoride and to try to correlate uptake and binding processes with susceptibility to fluoride in different microorganisms. An appreciation of these mechanisms may lead to improvements in the effective delivery of fluoride for therapeutic purposes.

The central neuroregulation of peripheral events by peptide hormones is a relatively new field. The intracerebral ventricular (icv) administration of bombesin, dermorphin (a frog-skin derived opiate heptapeptide), calcitonin (salmon) and lithium (the drug of choice for manic depression) effectively decreased all parameters of gastric secretion in pylorus-ligated fasted rats: neutralized acidic pH, reduced hydrogen ion concentration, and decreased volume of stomach secretions. However, subcutaneous (sc) injections of these peptides yielded differential effects relative to icv: bombesin was very weakly active (without a dose-response curve), while dermorphin was 0.0003 as potent; calcitonin, on the other hand, exhibited twice the activity sc as icv. Lithium chloride was effective both icv and sc, although the reduced response by sc is in keeping with the dilution of lithium throughout the body. Both bombesin and lithium appear to act centrally, perhaps through a common mechanism, on gastric secretion via products of arachidonate metabolism (e.g., prostaglandins) since their effects can be blocked by several cyclooxygenase inhibitors (indomethacin, diclofenac, naproxen, ketoprofen). Lithium and bombesin, which produce a similar effect and involve prostaglandins, do so through different routes; bombesin levels in the hypothalamus are unaffected by icv administered lithium. Several dimeric analogues of dermorphin (2 chains of dermorphin linked though the C-terminal amide nitrogen either directly or through methylene bridges) were tested for their ability to suppress gastric secretion and correlate that effect with an analysis of delta and mu receptors in brain synaptosomes. Only those analogues exhibiting mu-selectivity were active in vivo and their spectrum of bioactivity on gastric secretion were distinct from dermorphin. Although dermorphin requires central mu-type opiate receptors for gastric secretion, the role of calcitonin is less clear and may involve multiple interacting mechanisms.

Project 1: Abstract Rice (Oryza sativa), a staple food for over half the world's population, represents a significant dietary source of inorganic arsenic (As), a non-threshold, class 1 human carcinogen. It is imperative that strategies to reduce grain As are developed, and establishing the mechanisms that enable As to reach and accumulate within the rice grain is key to this endeavor. This project will elucidate the genetic control of As homeostasis in plants to enable the development of plants that do not accumulate As. The first specific aim tests the hypothesis that genetic diversity in rice can be exploited to lower grain As levels by mapping genes in accessions known to vary in grain As content. The second aim asks more generally what genes are involved in transferring As to the grain and the third aim tests strategies to limit the movement of As to the shoot/grain based on information gained from study of rice, Arabidopsis and the As accumulating fern Pteris vittata. The long-term goal is to prevent As accumulation in the edible portion of rice grain, but the work will also potentially provide information on genes responsible for transporting As and other contaminant metal(loid)s into the tissues of other edible plant organs. In addition to identifying the causal loci, synchrotron X-ray fluorescence microanalysis (SXRF) will be used to precisely localize and speciate As in plants, an innovative approach that has been used successfully to examine seed loading of As.

Neurodegenerative diseases such as Alzheimer's Disease (AD) and Multiple Sclerosis (MS) are exacerbated as a result of damaging neuroinflammation. A common theme observed with these neurodegenerative diseases is the activation of normally quiescent microglia to become potent antigen presenting ceils. Once activated, microglia elaborate a variety of cytokines, chemokines and neurotoxins that contribute to neuroinflammation. One of the mechanisms to activate microglia is the expression of CD40, a member of the TNF receptor superfamily. Interaction between CD40 and its ligand, CD154, is critical for a productive immune response. Activation of microglia also results in secretion of many chemokines, including monocyte chemo attractant protein (MCP-1) that causes the recruitment of new microglia J monocytes to the site of lesions in MS and senile plaques in AD. Ultimately, increased CD40 expression coupled with secretion of MCP-1 results in exacerbation of neuroinflammation. The development of drugs targeted to specifically inhibit or down modulate the production of CD40 and MCP-1 would have great utility in reducing neuroinflammation in the CNS. The goal of the experiments in this proposal is to develop drug targets specific for CD40 and MCP-1. These targets will be used to develop assays to identify drugs that can be utilized to specifically inhibit the expression of CD40 and MCP-1. To accomplish this goal, we will focus on the design and development of drugs targeted to specifically hind to mRNAs of MCP-1 and CD40 as a means to inhibit or down modulate expression. In this regard, small molecule agents that target RNA, notably the aminoglycoside antibiotics, are now actively being pursued as RNA targeted therapeutics to inhibit expression of specific proteins. The following Specific Aims are proposed: Specific Aim 1: To develop ligands that target MCP-1 and CD40 mRNAs. Specific Aim 2: To establish that the ligands inhibit expression of MCP-I and CD40 proteins. Once we have established and characterized the RNA targets in the mRNAs of MCP-1 and CD40, we will use this information to establish a high throughput screening assay for drugs that will specifically bind to these RNAs; for these studies, we will collaborate with the centralized high throughput screening facility that will be developed at NINDS. The best candidates can be screened for their capacity to down modulate MCP-1 and CD40 expression in in vitro glial cell culture systems.

Protonated species are often postulated as key intermediates in enzyme-induced as well as simple chemical reactions. In this research proposal a simple method of some generality is utilized; which when applied to biological reactions, can be used to establish not only the protonated species, but also the specific site of protonation. Irreversibly alkylated, charged analogs of the suspected protonated intermediates are synthesized, and then tested for the desired reactivity. During the past year we have concentrated our efforts toward the elucidation of the mechanism of the coenzyme, biotin. We have synthesized and tested an "irreversibly acidified" model of N-carboxybiotin in which "protonation" (in the form of an ethyl group) has occurred at the ureido oxygen atom. Treatment of this model compound with a series of surrogates for the biological acceptor molecule (i.e. water, tert-butanol, hydroxide, tert-butoxide, triethylamine, morpholine, potassium cyanide, phenyl lithium) did not lead to the transfer of the carbon dioxide moiety, instead dealkylation was observed in each case. The mechanisms of these reactions have been elucidated. We have also continued our studies on our proposed intramolecular mechanism reported last year. In this hypothesis, biotin acts as a template for the two reactants. Treatment of a series of acyl substituted imidazolidones and imidazolidinethiones with ketenes (ketene, diphenylketene, dichloroketene, and bis(trifluoromethyl)ketene) did not lead to the transfer of the acyl group. Notably, however, in the case of disubstituted imidazolidinethiones an isosteric S yields O substitution reaction occurred at the thione position with bis(trifluoromethyl)ketene and diphenylketene. The generality of this unique reaction has been investigated. BIBLIOGRAPHIC REFERENCES: Harold Kohn, Barbara Kohn, Marie Louise Steenberg, and Joseph P. Buckley, Syntheses and Pharmacological Activity of Substituted Imidazolidinethiones and Thiomidazolines, J. Med. Chem. 1977 (January issue), in press. Harold Kohn and Raymond E. Davis, Syntheses and Chemistry of N-Acyl Substituted Dihydroimidazo(2,1-b)thiazolium Salts, J. Org. Chem., 1977 (February issue), in press.