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This proposal presents coordinated efforts of more than 30 investigators from several different disciplines to provide new approaches to research, education, and community efforts. During the first two years of MAC funding (10/1/77-8/30/79) fruitful collaboration in all of these areas has occurred and has promoted ever-increasing interactions between rheumatologists, endocrinologists, radiologists, biochemists, orthopedists, immunologists, geneticists, economists, educators, allied health professionals and community leaders. As a result, we are proposing eight new development-and-feasibility studies in research, 3 in education and 2 in community programs. The remainder of the projects described under education and community sections expand upon our initial efforts. The programs are designed to emphasize the assets of this particular center, which include a large patient population (2100 hospital beds, 6 rheumatic disease clinics) drawn from 8 midwestern states, strong research divisions in rheumatology, immunology, genetics and metabolism, a large demographically defined prepaid medical care group which is part of the university, and active community groups associated with the Arthritis Foundation and the Lupus Foundation.
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We are using the SP to fit kinetic state models to our experimental data. Such kinetic models allow individual folding events, such as the isomerization of prolines or the collapse of a hydrophobic core, to be investigated; such information is very hard to obtain otherwise. The fitting is normally accomplished using simulated annealing Monte Carlo methods. This paper fit experimental data exhaustively to all possible models of proline isomerization in ribonuclease A. This modelling identified regions of the protein which are essential in its conformational folding, and analyzed the state of the protein's internal hydrogen bonds. We are actively pursuing this approach of making localized changes in ribonuclease A, and studying their effects on its conformational folding. Four additional papers citing the Theory Center are being prepared at present. We are developing a molecular model of its conformational folding, to serve as a quantitative benchmark against which computer simulations will be compared.
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{
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O-glycosylation is one type of important protein modification and plays crucial roles in many cellular processes. Currently, there is limited commercial source for the glycosyl amino acid building blocks that can be utilized in glycopeptide synthesis. In this Phase I project, Life BioScience Inc. (LBSI) proposed to utilize its established glycan technologies and extensive research experience of its employees in carbohydrate chemistry to synthesize a chemical library of 36 O-linked core structures on amino acids (Ser/Thr) that have been protected by Fmoc for further glycopepetide synthesis. The proposed glycan compounds will be well purified (>98% purity) and structurally identified by NMR and Mass Spectrometry. New carbohydrate and peptide synthesis strategies including microwave-assisted synthesis, formation of alpha-O-GaINAc conformation, and [2+1] synthetic approach, will be empolyed in the sythetic routes leading to O-glycosyl amino acid building blocks. In addition, LBSI will collaborate with Dr. David Live at CCRC in Phase I to assemble the glycosyl building blocks to glycopeptides for proof-of-concept demonstration. At the end of the Phase I project, LBSI will provie the purified glycan samples to a NIGMS-designated screen center for the use as analytical standards in structural studies and carbohydrate-based high throughput assays.
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{
"pile_set_name": "NIH ExPorter"
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At present, significant resources are committed by both academic research and industry to deliver groundbreaking therapeutic and diagnostic strategies aimed at curbing cancer occurrence. Despite the critical mass of available knowledge and technology platforms in the anti-cancer drug development field providing for the development of pathway-targeted therapies, only a few efficacious treatments have been developed. The major preclinical point of the government-regulated process for extensive animal testing of potential anti-cancer therapeutic compounds is directed towards safety assessment prior to the clinical introduction of any new anti-cancer drug. This analytical path of demonstrating the desired drug efficacy while proving it to be non-toxic has been implemented in a number of countries as multiphase clinical trial procedures. The initial stage in drug candidate evaluation demands routine investigation of future therapeutic compounds in animal and cell culture models, primarily to obtain knowledge of non-toxicity ranges, interaction with metabolic pathways, and systemic pharmacological behavior. This phase, termed preclinical characterization, may also provide a fair estimate point for the compounds therapeutic efficacy given the availability of appropriate disease model(s). In general, considering the extremely long (routinely >10 years) and expensive (often in excess of $700 million per drug lead) process to obtain the regulatory approval to market therapeutic compounds, the quality and the scope of efficacy data obtained during the preclinical stages may expedite clinical testing, dramatically increase affordability of downstream drug development steps, and advance the identification of effective cancer therapies.Currently, drug efficacy studies are conducted almost exclusively in xenograft models that employ transformed human cell lines to initiate tumor growth upon injection into immunocompromised animals. Though easily derived, the xenograft models feature multiple intrinsic limitations that jeopardize the predictability of drug testing output data. Xenograft tumors are developed from a genetically heterogeneous cell population that has been maintained in vitro for multiple passages. Moreover, the tumor growth occurs in an ectopic, non-physiological environment in the absence of immune surveillance and systemic interactions with the vascular system. As an alternative source of experimental tumors, animal models of spontaneous carcinogenesis may be also employed, but these models generally lack the reproducibility in timing of tumor onset and feature by heterogeneous tumor characteristics/drug response due to a considerable genetic noise caused by non-inbred strain background.The drawbacks of the xenograft and the spontaneous tumorigenesis models are largely ameliorated in genetically engineered mouse (GEM) tumor lines, which provide preclinical researchers with the ability to study naturally occurring tumors featuring pathway aberrations typical for similar human cancer types in the context of an appropriate tissue environment in immunocompetent animals. This approach finds ground in the rapidly expanding knowledge base for molecular mechanisms underlying carcinogenesis in human patients and is further fueled by the recent and rapid progress in the methodology and availability of resources to design and construct sophisticated animal models for a broad spectrum of human malignancies. At present, the GEM strategy not only provides an opportunity to combine in transgenic animals multiple genetic aberrations closely matching those detected in human patients, but also the potential to interfere with cancer-related molecular pathways in both a tissue-restricted and time-specific manner, providing genetic evidence for drug target legitimacy. This translates into a more accurate prediction of the dynamics of tumor progression while minimizing individual variations. In contrast to xenograft models, the cancerous lesions that occur in GEM animals exhibit a high degree of genetic similarity due to the availability of inbred and congenic lines for GEM generation. Among other benefits, this genetic similarity allows the decoding of individualized tumor molecular signatures that may be applicable for identifying cancer prognostic markers and developing personalized anti-tumor therapies based on predicting an individuals response to drug treatment known as the patient stratification principle. The CCR Center for Applied Preclinical Research (CAPR), will develop and implement a comprehensive preclinical trial framework for evaluating the anti-tumor efficacy and selectivity, safety, biodistribution, and metabolism of early stage candidate drugs using GEM models. CAPR will establish the infrastructure required for preclinical evaluation of anti-cancer drug leads in a recently derived collection of GEM models for high-occurrence cancers such as lung, ovary, and prostate gland tumors, as well as more rare cancer types that fall under the unmet demand category such as high-grade astrocytomas, hepatocellular carcinomas, and pancreatic cancers. CAPR will also be responsible for continuous sampling of the dynamic knowledge base on the mechanisms of carcinogenesis to seek additional molecular targets related to the tumor formation process. The appropriate GEM strains will be designed, derived, or adapted to address the CAPRs internal demand for novel mouse tumor models; however, these resources will also be shared with other scientific and industrial communities engaged in anti-cancer drug discovery and development.
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{
"pile_set_name": "NIH ExPorter"
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Despite the substantial morbidity and mortality associated with bipolar disorder in adolescence, there are no empirically validated psychosocial interventions for this population to date. The purpose of this Mentored Patient-Oriented Research Career Development Award is to enable the candidate to develop a program of research that integrates clinical, developmental, and neurobiological approaches to the assessment and treatment of affective dysregulation for adolescents with bipolar disorder. The proposed research plan focuses on treatment development and preliminary investigation of a family-based psychosocial intervention targeting affective dysregulation for adolescents with bipolar disorder. The intervention will include age- and [unreadable] illness-related modifications to Dialectical Behavior Therapy (DBT), an evidence-based treatment for adults focused on enhancing emotion regulation skills. Feasibility and efficacy of Family-Based DBT will be examined in the proposed open pilot trial. The study will be conducted at the Child and Adolescent Bipolar Services clinic at Western Psychiatric Institute and Clinic. Sixty adolescents (age 12-18) with bipolar disorder will receive Family-Based DBT. It is hypothesized that DBT will be feasible to deliver and acceptable to this population, and will be associated with improvements in affect regulation, mood [unreadable] symptomatology, and psychosocial functioning over one-year follow-up. Exploratory analyses will examine predictors of treatment response. Training will be sought in: 1) adolescent development, 2) the neurobiology of affect regulation, 3) pharmacological management of pediatric bipolar disorder, and 4) statistical methods for analyzing longitudinal treatment-outcome data. Such training activities will support the research plan by enhancing the candidate's ability to develop, deliver, and assess outcomes for the intervention that incorporate a multidisciplinary approach to emotion regulation. Together, the training and research plans support the candidate's long-term goal of establishing a multidisciplinary program of innovative treatment research targeting affective dysregulation among adolescents with bipolar disorder. Further research in this area is of significant public health importance, as it has the potential to decrease poor outcomes associated with early-onset bipolar disorder, including a deteriorative course into adulthood, chronic psychosocial impairment, treatment resistance, and suicidality. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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The long-range objectives of this proposal are to identify the structural and biochemical properties of specific membrane domains and membrane-associated assemblies of mammalian spermatozoa and to determine how these membrane domains and membrane-associated assemblies function during the fertilization process. In the present proposal attention will be focused upon the membranes participating in the acrosome reaction, in order to identify mechanisms contributing to the membrane fusion process, and upon the plasma membrane of the postacrosomal segment, in order to characterize its role in gamete fusion. The specific objectives of this proposal are 1) to characterize the macromolecular complex (fuzzy-coat) which is exclusively localized on the luminal face of the outer acrosomal membrane; 2) to determine how the fuzzy-coat complex associates with integral membrane proteins of the outer acrosomal membrane; 3) to define the role of the fuzzy-coat complex in the membrane vesiculation process of the acrosome reaction; 4) to utilize antibodies against the fuzzy-coat complex to identify acrosome reacting sperm; 5) to determine if the fuzzy-coat assembly is modified during sperm maturation in the epididymis; 6) to identify enzyme activities associated with membrane domains participating in the acrosome reaction; 7) to characterize the postacrosomal sheath; and 8) to identify the intracellular distribution of cytoskeletal proteins in mammalian spermatozoa. Nitrogen cavitation and selective extraction procedures will be employed to disrupt spermatozoa and purified subcellular fractions consisting of specific membrane domains or membrane-associated assemblies will be isolated by a variety of centrifugation protocols. A combination of ultrastructural, biochemical and immunological techniques, which have previously been utilized to identify the functional properties of membrane-associated assemblies in other cell types, will be employed to pursue these specific aims. A resolution of these specific aims will contribute to our understanding of membrane physiology, to our understanding of major events in the fertilization process and to male reproductive biology.
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"pile_set_name": "NIH ExPorter"
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Amyloids fall into two broad categories: infectious and non-infectious. Infectious amyloids are called prions. Our first studies were with yeast prions, focusing on Ure2p, a negative regulator of nitrogen catabolism. Its N-terminal domain (NTD) is responsible for prionogenesis, while its C-terminal domain (CTD) performs the regulatory function of the wild-type protein. The NTD is unfolded in the wild-type protein and amyloid in the prion fibril. The CTD remains folded in the fibril but is inactivated by a steric mechanism. NTDs form the fibril backbone that is surrounded by CTDs. In 2005, we published the parallel superpleated beta-structure model for amyloid fibril backbones. The model envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Structures of this kind are good candidates for some other amyloid fibrils, including amylin. Our ongoing work is aimed at testing and refining this model and investigating fibril polymorphism. In FY17 we continued to focus on amyloid fibrils of alfa-synuclein (aS). Parkinson disease (PD) is a chronic and progressive neurodegenerative disease characterized by the intra-cerebral presence of Lewy bodies- intracerebral deposits whose principal components are amyloid fibrils of aS. Normally, the 140 aa-long protein has a membrane remodeling function that we have also researched, as reported in project AR027015-19. aS is alfa-helical when associated with lipid and a random coil in solution. In fibril formation, the protein polymerizes into a cross-beta structure. We expressed recombinant aS in E. coli, purified the protein and assembled it into fibrils, which were observed by cryo-EM in our laboratory and by dark-field STEM at Brookhaven National Laboratory. Our fibrils are markedly polymorphic, as in previous reports by others. Our analysis focused on a twisting fibril with an axial repeat length of 77 nm between crossovers and an average diameter of 8.6 nm. Their reconstructed cross-section resolved this fibril into two asymmetrically associated protofibrils. Mass-per-length measurements made from the STEM data gave a unimodal distribution with a mean density equivalent to two subunits per 0.47 nm axial rise, i.e. one subunit per axial step per protofibril, consistent with a superpleated structure. The STEM images of unstained freeze-dried specimens showed two thread-like densities running along each fibril that we interpret as metal ions. These observations support the idea that metal binding promotes fibrillation and hence Lewy Body formation in PD. A paper reporting this analysis was published towards the end of the previous reporting period (A. D. Dearborn et al., J. Biol. Chem. 291:2310-8, 2016). Our continuing efforts have aimed at extending the resolution of this analysis. In particular, we have sought to identify the metal ion(s) that form the high density threads mentioned above. A priori, copper was a plausible candidate as it has been implicated in other studies in the onset or progression of PD, and, in vitro, copper ions had been shown to bind with aS with high affinity and to accelerate the production of aS amyloid fibrils. We found that our dense threads represented the binding of metal ions scavenged from the water used to prepare them, as newly assembled preparations of aS fibrils made using highly purified water showed no threads. On the other hand, when copper was applied in defined amounts to pre-assembled thread-free fibrils, dense threads were detected on some but not all of the fibril morphotypes present. The presence of copper and the absence of other metals was confirmed by ICP-MS. The observed threading implies that the copper binding sites are axially stacked. A paper reporting this work is in preparation.
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"pile_set_name": "NIH ExPorter"
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The goal of the proposed research is to develop instruments for the rapid detection and quantitation of bilirubin, a metabolite which is an indicator for a variety of metabolic disorders, including hyperbilirubinemia in infants. The instrument will also detect and quantify levels of acetaminophen and salicylic acid, common sources of childhood poisonings. These goals will be accomplished by the use of specialized coatings sensitive to these compounds in conjunction with surface enhanced Raman scattering (SERS) spectroscopy. The technology differs from that commonly employed because the selective interactions between the coatings and the target analytes should make the need for sample pre- processing or separation unnecessary. The high information content in Raman spectroscopy will mean that the likelihood of sample misidentification is much smaller than for methods which utilize less content rich forms of spectroscopy. A laboratory grade instrument should be capable of reading tens of samples per minute. A second instrument will be sufficiently small and portable that it will be appropriate in bed-to- bed measurements of these compounds in maternity wards and emergency rooms. PROPOSED COMMERCIAL APPLICATION: The portable instfl1ments developed from this work will find wide applicability in emergency rooms and maternity wards due to their accuracy, rapidity and ease of use. The laboratory grade instrument will be useful in hospitals in which large numbers of samples must be processed
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{
"pile_set_name": "NIH ExPorter"
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Intimate Partner Violence in Rural Aging Women: Promoting Community Knowledge Intimate partner violence (IPV), which includes physical, sexual, or psychological abuse, is a major public health concern. National age-aggregated data available suggest that a significant number of mid-life and older women experience IPV. With the aging of the baby boom generation, the incidence and prevalence of IPV in aging women is expected to increase significantly. The purpose of this collaborative partnership between the Center for Gerontology at Virginia Tech and the Women's Resource Center of the New River Valley is to enhance service professionals'knowledge of IPV of aging women as they engage in a process for developing a strategic model of intervention to address the needs of women in their community. The specific aims of the collaborative effort include: identifying existing NIH supported and other available empirical research and program descriptions addressing issues of IPV in aging women (e.g., individual outcomes, community interventions);projecting the future prevalence of aging women who are victims of IPV in the study area;obtaining the perspectives of community leaders (e.g., service professionals, healthcare providers, law enforcement, clergy) about IPV of aging women as well acquiring input from the women themselves;and developing a intervention model to address the needs aging victims of IPV living in rural communities. To achieve these aims, the project relies on community-based participatory research practices to address issues facing rural aging women who wish to lead safe or violence-free lives in their later years and the community support to help them safely and successfully rebuild their lives. Data collection strategies include a review of the scientific literature and community programs designed to address the needs of aging women who experience IPV, focus groups with professional service providers, and interviews with women who experienced IPV in mid and late life. Data will be used to develop a strategic planning model that will serve as the framework from which to construct prevention and intervention programs to accommodate the needs of rural aging older women seeking assistance to end violent relationships. The results of this collaborative project are likely to improve public understanding of federally funded biomedical and behavioral research related to IPV of aging women. Specifically, the project products (i.e., white papers/research briefs) will be distributed to policymakers, law enforcement agencies, community services, and faith-based organizations to further their understanding of the issues and challenges facing rural older rural women who experience IPV. Intimate partner violence, which includes physical, sexual, or psychological abuse, is a major public health concern. This collaborative partnership between the Center for Gerontology at Virginia Tech and the Women's Resource Center of the New River Valley will enhance service professionals'knowledge of IPV of aging women as they engage in a process for developing a strategic model of intervention to address the needs of women in their community.
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"pile_set_name": "NIH ExPorter"
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Research on the effects of amphetamine in schezophrenia is conflicting. It is known that amphetamine may cause paranoid psychosis. On the other hand, transitory, improvement in some patients given acute doses of amphetamine has been reported. It is reasonable to speculate that negative symptoms of schizophrenia, such as anergia, lack of motivation, flattened affect, and poor planning may be amenable to such treatment. In addition, patients with schizophrenia generally do poorly on neuropsychological tests though to assess frontal lobe functioning. We therefore propose a pharmacological strategy involving the administration of amphetamine in the hope that this drug may reverse some of the abnormalities found in chronic schizophrenia, whether they be symptomatic or cognitive.
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{
"pile_set_name": "NIH ExPorter"
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The mission of the Gene Expression and Genotyping Facility (GEGF) is to facilitate implementation of high throughput genetic technologies for the research conducted by members of the Case community. The GEGF serves as a technical and consulting resource for Cancer Center members, as well as other investigators who have laboratories at Case Western Reserve University, University Hospitals of Cleveland, the Cleveland Clinic Foundation, the VA Hospital, and MetroHealth Hospital in the areas of high throughput expression analysis and genotyping. It is the only facility of its capability in Cleveland. This facility utilizes Affymetrix and Applied Biosystems (ABI) technologies to provide global and quantitative (on user-identified genes) gene expression analysis services. A diverse array of Affymetrix and ABI technologies are available to address a spectrum of genotyping needs, including whole genome scans, custom designed fine-mapping studies involving 3,000-10,000 SNPs and interrogation of small numbers (<100) of specific SNPs in thousands of samples. The core operates in a full-service mode with staff providing initial consultation, executing the chemistry, carrying out the analysis, and offering data trimming and data interpretation services. To carry out these technologies, the GEGF has 14 computers, an Affymetrix Workstation dedicated to data generation, 3 fluidics stations, 2 hybridization ovens and a GC3000 scanner with autoloader. ABI equipment includes a 7,900 Sequence Detection System, with a barcode reader, robotics allowing the capability to process 5,000 sample wells per day, and the modules for processing Low Density Arrays, 96- and 384-well plates. The GEGF also has 5 GeneAMP ABI 9700 PCR machines, with 96- & 384-well block capability and an ABI 6100 Nucleic Acid Prep Station. For PCR-based assays, the GEGF established an amplicon-free room, containing 2 PCR hoods, dedicated pipettors, small microfuges and minithermocyclers. Additional equipment includes: 2 Matrix robotic liquid handling systems, the PlateMate 2x2 (X-Y stage movement) and the PlateMate Plus (allows rapid reconfiguration from 96- to 384-well format), dedicated centrifuges, an Agilent Bioanalyzer and a NanoDrop Spectrophotometer, which quantifies nucleic acids in 1-2 ul samples. Turn-around time for sample preparation and analysis is <10 days for average size projects and satisfactory timetables are established for large projects during initial consultations. Cancer Center members receive a 35% discount on all GEGF provided services. The core has steadily grown from its inception in 2000, when we had 22 users and a single full-time staff member to its current staff of 51/2 and 142 registered users. Approximately 60 investigators use the GEGF every year and 30-50% of these are first time users, documenting that the GEGF continues to enjoy a healthy level of growth and a robust level of sustained work with repeat users. Finally, the GEGF has provided services for members from 8 of the Cancer Center's programs, and has played a role in securing more than 33 grants awarded to Cancer Center members.
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"pile_set_name": "NIH ExPorter"
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The long-term goal of our research is to elucidate the molecular cascades of LH-induced signals within preovulatory follicles, leading to ovulation. The LH surge stimulates the synthesis of progesterone and its intracellular receptors, progesterone receptors (PRs), in the granulosa cells of preovulatory follicles. Interaction between progesterone and PRs in an autocrine/paracrine fashion is essential for ovulation. However, the exact mechanisms by which ligand-dependent activation of PRs controls ovulation and thus normal reproductive cyclicity and fecundity are unknown. To gain insight into the molecular mechanisms underlying PR-mediated ovarian functions, we initiated cloning of PR downstream genes in luteinizing granulosa cells. The two genes we have characterized as PR-downstream are the ligand-receptor system for pituitary adenylate cyclase activating polypeptide (PACAP): PACAP and its receptor type 1 (PAC1). The temporal and spatial pattern of expression and secretion of the ligand PACAP along with the cellular localization of the receptor PAC1 in the ovary advocates the potential significance of this ligand-receptor system for ovulatory processes. Indeed, pharmacological blockade of ligand-dependent activation of PAC1 appears to interfere with the efficacy of LH and progesterone in bringing about ovulatory processes. Thus, our working hypothesis is that PACAP-induced activation of PAC1 mediates, at least in part, PR function critical for follicular rupture with release of a meiotically mature oocyte. The immediate goal of this application is to determine the functional importance of PACAP within preovulatory follicles during the periovulatory period, using in vivo and in vitro approaches. In Aim 1, we will test whether PR-induced PACAP is critical for follicular rupture and for expression of ovulation-related genes, including proteolytic enzymes. In addition, we will identify PAC1-downstream genes that may play an important role in follicular rupture. In Aim 2, we will determine the initial death/survival pathway(s) that is modulated by PR-induced PACAP in luteinizing granulosa cells. In Aim 3, we will test whether PR-induced PACAP regulates the polyadenylation/translation capacity of meiotically maturing oocytes. The proposed studies are designed to provide functional endpoint(s) of interaction between PR-induced PACAP and PAC1 in preovulatory follicles during the periovulatory period. Information derived from our results will allow us to better manage fertility, infertility, and endocrine-based disorders.
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{
"pile_set_name": "NIH ExPorter"
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Our studies in 1981 are a) on alanine turnover in vivo. b) The regulation of phosphofructokinase and futile cycling in rat liver cells and c) Gluconeogenesis and lipogenesis in avian (quail) hepatocytes. A. Alanine turnover and its contribution to gluconeogenesis in starved rats. 3-3H and U-14C alanine will be injected or infused. From the tracer kinetics the replacement rate of alanine and alanine mass, recycling of alanine carbon, and its role in glucose synthesis will be obtained. B. Hepatocytes from streptozotacin diabetic and diabetic insulin treated rats will be prepared. The randomization of 14C from 1-14C galactose in glucose will be determined. From this the rate of phosphofructokinase, and the effect of insulin treatment on cycling between Fructose-6P and Fructose 1,6 diP will be estimated. C. Gluconeogenesis and lipogenesis by hepatocytes from Japanese Quail (Coutournix coutournix). We have developed a preparation of liver cells from these birds. The rates of glucose synthesis and fat synthesis is several times higher than that in isolated rat liver cells. We have observed a large stimulation of glycolysis and lipogenesis from glucose in the presence of alanine. The effect of alanine on key glycolytic and lipogenic enzymes will be studied. The role of malic enzyme and isocitrate dehydrogenase in providing NADPH for fatty acid synthesis will be explored.
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{
"pile_set_name": "NIH ExPorter"
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The proposed study will focus on bases for decision making among women with unplanned problem pregnancies. It will compare: (1) those who have decided on abortion with those who have chosen some other solution, (2) those who sought help early with those who delayed, an (3) those going to varying types of agency or facility. The major variables on which they will be compared include: (1) anticipated consequences of alternative actions and their relative importance, (2) concepton of feminine role, (3) sense of competency in dealing with one's own problems, and (4) self report on past contraceptive usage. Data will be gathered throughout the state of Michigan by means of a questionnaire which includes background material, a Consequence Model instrument, and four scales measuring selected aspects of self concept. The study should provide guidance, fromthe women involved, for developmeft or modification of abortion services so as to minimize difficulties. It will provide information for counselling, provide a basis for social and educational policies aimed at stimulating selection of certain options, and if the Michigan referendum passes, it will help in assessing its impact.
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{
"pile_set_name": "NIH ExPorter"
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The proposed studies focus on the development of a new paradigm for targeted cancer therapy. Our approach is based on the development of small molecules that possess the functional properties of antibodies; these functions include the ability to bind antigens with high affinity and selectivity, and the ability to trigger the action of professional phagocytic and killer cells (e.g., natural killer cells, macrophages, and dendritic cells). Such materials would combine the many advantages of antibody-based therapeutics with those of traditional small-molecule-based approaches. Here we propose to develop small molecule antibody mimics (SAMs) that bind selectively to cancer cells that overexpress the prostate-specific membrane antigen (PSMA), and induce immune-mediated cytolysis through the Fc-gamma receptor type 1 (Fc?RI). Fc?RI triggers immune cells to release cytotoxic contents only upon multivalent binding to an appropriate cell-surface-immobilized ligand. PSMA is a cell surface enzyme that is overexpressed both in prostate cancer cells and in tumor neovasculature for a wide variety of cancers (including pancreatic ductal carcinoma, colon adenocarcinoma, glioblastoma multiforme, and non-small cell lung carcinoma). Preliminary data suggests that formation of complexes between the small molecule, cancer cells, and immune cells will lead to highly selective immune-mediated cancer cell destruction. The strategy proposed here is not limited any one particular disease - the therapeutic action is determined simply by which cell type is targeted by antibody mimics. As such, we believe that the proposed research is highly significant, and constitutes a critical step toward improving the lives of patients suffering with cancer and other diseases.
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{
"pile_set_name": "NIH ExPorter"
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Our long-term objective in this investigation is to delineate the critical three-dimensional structural features which are required for the control of specificity and the generation of reactivity in both co- and post-translational modification reaction. We plan to initiate our investigations with the in-depth examination of the following enzyme catalyzed derivatization reactions; the N- asparagine-linked glycosylation, mediated by glycosyl transferases, and the cAMP-dependent phosphorylation mediated by a protein kinase. The first process is co-translational and the second post- translational. Since enzyme-cataly derivatization of proteins is central to innumerable biological control processes, an understanding of the manner in which specificity is achieved in these reactions is of clear importance both to fundamental biochemistry and in a practical consideration of the mechanisms of homeostatic control. The experimental approach in the investigation is a multidisciplinary one and incorporates both the design and synthesis of substrate analogues of well-defined three-dimensional structure, as well as the investigation of these analogues using biochemical techniques. The substrate analogues would, in the initial phase be based on peptidyl structures, which incorporate features such as conformational restrictions, reporter groups or affinity labels. From the information obtained from this phase of the work we could then design and synthesize non-peptidyl mimics of the modified sequences. In these compounds we could truly identify the precise features controlling recognition independent of the supporting peptide framework.
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{
"pile_set_name": "NIH ExPorter"
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Studying circadian (~24hr) rhythms offers an excellent opportunity to understand a key brain function at molecular, cellular and circuit levels as well as possibly identifying novel targets for therapies for sleep disorders and jetlag. Studies in Drosophila identified the first circadian clock gene, which is conserved in humans and linked to an inherited human sleep disorder. Clock genes function in central brain pacemaker neurons to control whole animal behavioral rhythms. The endogenous molecular and neural rhythms of pacemaker neurons provide a unique model to study how gene expression controls daily changes in neuronal signaling which, at least in Drosophila, includes rhythms in structural plasticity. To identify novel regulators of circadian rhythms, we generated whole genome expression profiles from a group of purified master pacemaker neurons, the Drosophila LNvs. We identified a set of 10 genes that are expressed with a daily rhythm in a clock-dependent manner and which are much more highly expressed in LNvs than in other differentiated neurons. Of these 10 genes, four encode previously identified core clock genes such as period. Here, we propose to study CG33275, one of the other 6 genes. CG33275 is an unstudied gene which likely encodes a Guanine nucleotide Exchange Factor (GEF) that activates a Rho family GTPase. CG33725 is the Drosophila ortholog of human Puratrophin, which has been linked with a hereditary form of spinocerebellar ataxia, but is unstudied at the molecular level. We refer to CG33275 as dPuratrophin (dPura) and believe that basic studies of this gene in Drosophila could help explain the disease-association of human Puratrophin. Rhythmic dPura expression could impose circadian rhythms on the activity of a Rho family GTPase, which have also not yet been implicated in circadian rhythms. Here, we first aim to determine if dPura is indeed a GEF using genetics and biochemistry. Using genetics, we will test which of the 6 Drosophila Rho GTPase family members genetically interact with dPura in clock neurons to regulate circadian behavior. We will complement these in vivo experiments with in vitro biochemical experiments that directly measure dPura GEF activity. Our second aim is to identify the role of dPura in LNvs by characterizing dPura mutants we have identified that strongly alter circadian behavioral rhythms. Specifically, we will ask if dPura mutants show altered circadian gene expression, structural plasticity and/or intracellular trafficking in LNvs that could underlie the behavioral defects. Since GEFs are often activated by extracellular signals, dPura could help pacemaker neurons integrate internal clock time (via rhythmic expression) with external signals. Given that mouse Puratrophin also shows circadian expression in the brain, our studies should give insight into both fly and mammalian circadian rhythms as well as helping understand Puratrophin function in general.
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{
"pile_set_name": "NIH ExPorter"
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The interplay between environmental exposures, respiratory tract microbiome, and immune responses related to asthma and other respiratory diseases is not well understood. High levels of traffic-related air pollutants (TRAP) have been associated with children's asthma. TRAP can increase adherence of microorganisms to the epithelial cells of the respiratory tract and damage the epithelial layers resulting in increased susceptibility to microbial growth. Many studies suggest a role for altered human microbiota in the etiology of asthma. Furthermore, circumstantial evidence indicates that bacterial infections in the respiratory tract may play a role in asthma development. The airway microbiota may interact with the innate and adaptive arms of the children's developing mucosal immune system in the respiratory tract, which can be critically important in maintaining tolerance against allergc immune responses. Our recent data show that increased exposure to traffic-related particles at birth is associated with longitudinal childhood wheezing. We hypothesize that exposure to TRAP early in life significantly alters the diversity of microorganisms in the respiratory tract in chilren and this effect persists to early adolescence. In Specific Aim 1, we will characterize the respiratory tract microbiome of adolescent children exposed to high and low levels of traffic related air pollution during childhood. Children from the existing cohort of the NIEHS-funded Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS) will be recruited for this purpose. This cohort is well characterized regarding childhood exposure to TRAP and indoor aeroallergens as well respiratory health of children from birth to age 12. TRAP exposure at ages 12-15 will be estimated by a land use regression (LUR) model of exposure to truck and bus traffic. Bacterial composition, operational taxonomic units (OTUs), and diversity indices in the respiratory tract of children will be characterized by collecting induced sputum samples, extracting DNA, amplifying bacteria-specific PCR products (using 16S rRNA primers), analyzing DNA sequences by deep sequencing, clustering and assignment of Illumina MiSeq reads into Operational Taxonomic Units (OTUs), analysis of OTUs, and determination of bacterial diversity by RDP database and pipeline, as well as MG-RAST and Qiime software packages. In Specific Aim 2, we will assess associations between bacterial OTUs, diversity indices, and TRAP. To our knowledge, there are no previous reports on the effects of air pollutants on the human respiratory tract microbiome, particularly among children. This information is critically important to understand the interaction between air pollution, human microbiome, and respiratory health among children.
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{
"pile_set_name": "NIH ExPorter"
}
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The New York State Department of Health (NYSDOH) Emerging Infections Program is an integrated collaboration among the Department's Division of Epidemiology, Wadsworth Center Laboratories, Office of Science, Environmental Health, and Bureau of Healthcom Systems Network as well as partner agencies such as the University of Rochester's Center for Community Health and the New York City Department of Health and Mental Hygiene. This group of collaborators support the program in efforts to conduct active population-based surveillance activities, applied public health epidemiologic and laboratory projects, and the implementation and evaluation of prevention/intervention projects for emerging infectious diseases. The proposed objectives will strengthen and enhance the epidemiology and laboratory infrastructure by recruiting additional staff, conducting training and providing educational materials, and enhancing information technology and exchange efforts. Additional objectives will focus on increasing capacity for influenza surveillance and diagnostic testing, evaluating the burden of influenza on public health and to evaluate healthcare-associated infections in dialysis centers.
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{
"pile_set_name": "NIH ExPorter"
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We will use an extracorporeal silicone membrane lung for prolonged partial veno-venous and veno-arterial respiratory blood gas exchange of potentially salvageable patients dying of acute respiratory failure. Perfusion can rapidly correct hypoxia and hypercapnea, eliminate the need for dangerously high inspired oxygen concentrations and inflation pressures, and allow pulmonary repair without additional physiologic insult. We hope to identify types of acute pulmonary failure responsive to extracorporeal perfusion and to determine criteria for the optimum time to initiate extracorporeal support. Perfusion will be carried out in a fully equippped and staffed unit specializing in the therapy of acute respiratory failure. The disposable fixed priming volume, spiral coil silicone membrane lung will be used. This membrane lung has been uniquely benign and reliable when used for animal and clinical perfusions lasting more than one week. Prior to clinical use, new models of the spiral coil membrane lung will be tested in sheep. However, both heparin bonding to silicone rubber and minimal systemic doses of heparin are potentially valuable techniques that need to be tested in an animal whose coagulation system more closely resembles man than that of sheep. We will therefore use a primate model for our basic investigations of the natural history of pulmonary recovery through veno-venous and veno-arterial perfusions. These proposed studies should allow a more rational and safer choice of perfusion techniques that may save patients with acute respiratory failure.
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{
"pile_set_name": "NIH ExPorter"
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A novel orally administered macrophage delivered gene therapy is being developed to address the present therapeutic limitations in treating Gaucher Disease. The skeletal and central nervous system complications of Gaucher disease still present an enormous challenge for current enzyme and gene replacement therapies. Despite the successes of gene therapy strategies in animal models, the clinical trials conducted to date have generally resulted in either low levels of gene expression or a failure of engraftment. In this study, an orally administered formulation of yeast cell wall particles (YCWP) containing DMA encoding human glucocerebrosidase is used to restore human glucocerebrosidase activity in macrophages of Gaucher mice. To accomplish this goal we will: 1) orally administer formulations of YCWP containing DNA encoding human glucocerebrosidase to long-lived L444P Gaucher mice that have clinical manifestations similar to those observed in patients with Gaucher disease;2) determine the extent of improvement in enzyme levels in macrophages and tissues of treated Gaucher mice;3) determine the extent of reversal of lipid storage and tissue pathology, and impact on survival;and, 4) identify the location and degree that macrophages in bone and brain are expressing human glucocerebrosidase. Through this study we will determine parameters that will be used for in-vivo optimization of human glucocerebrosidase expression by this orally administered macrophage delivered gene therapy. In addition to improved delivery of human glucocerebrosidase to many tissues, including bone and brain, we expect that this approach will achieve significant reversal of tissue pathology in treated L444P Gaucher mice. If macrophages containing human glucocerebrosidase migrate into brain, the resulting increased enzyme levels could provide clinical benefit for the neurological manifestations of Gaucher disease. The successful development of this innovative therapeutic strategy should provide a safer, more efficient and cost effective treatment for patients with Gaucher disease, as well as providing a prototype of therapy to benefit those having a wide range of other lysosomal diseases.
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{
"pile_set_name": "NIH ExPorter"
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The objective of this contract is to test chemicals for mutagenicity in Salmonella. The chemicals are tested, under code, in a series of Salmonella tester strains, both with and without metabolic activation. The results of this testing are used, in combination with other information, to evaluate the toxicity, and potential carcinogenicity, of the chemicals. The results of the testing are distributed throughout the Program, to other Federal agencies, and to other public and private organizations, and are published in the peer-reviewed scientific literature.
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{
"pile_set_name": "NIH ExPorter"
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This project will study complex signal processing in the cochlear nucleus where the first steps of a cascade of parallel and hierarchical auditory signal processing events take place. Distinct anatomical cell types, each with different targets, form the substrate for parallel pathways and along with a rich network of intranuclear circuits shape the outputs of projecting cells. The present aims are: to make structure/function correlations that allow physiological identification of anatomical cell types and to explore how these types process complex sounds including amplitude modulation, N-harmonic component, and speech stimuli. Extracellular-and intracellular-recording will be combined with neurobiotin labeling to establish structure- function relations for five groups of cells that are not well understood: (1) Onset cells in the PVCN: What cells group(s) are responsible for O/I and O/L patterns and what is their relation to O/C cells? (2) The primary-like responses, PL, PL/N, and O/L, in the AVCN arise from bushy cells but distinguishing between them is often difficult based on responses to tones alone. Do these patterns arise from bushy-cells exclusively? (3) Establish the morphological basis for sustained and transient choppers in the AVCN. (4) Establish whether onset-graded response patterns, units with type II receptive fields and tuberculoventral cells in the DCN are one and the same cell group? (5) Establish whether cartwheel cells in the DCN respond to tones clicks, AM, noise, noise with notches, and/or contra-lateral acoustic stimulation. A new initiative will investigate neurophysiological responses to a diverse set of speech sounds including sounds that have not been used in previous neurophysiological studies. Examining a large array of speed sounds will make possible the extrapolation of results to speech perception. Psychophysical capabilities will be studied in the chinchilla as a behavioral adjunct to investigations of neural responses to speech using the same speech sounds that will be presented in the neurophysiological studies. In many cases, neural responses to stimulus pairs, drawn from series of speech stimuli varying from one phoneme to another, that bracket the animal's behaviorally derived thresholds will be most intensely studied. Information-theoretic computational analyses of neural responses will be used to enable direct inference from neural encoding to sensory capacity.
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{
"pile_set_name": "NIH ExPorter"
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DESCRIPTION: (after application) There is an urgent need in the post-acute care/rehabilitation community for a more practical yet precise system to measure disability outcomes across a continuum of care settings and services. The long-term objective of the proposed project is to achieve a major transformation in the technology used to evaluate disability outcomes for post-acute care. This project amplifies work underway in the Boston University (BU) Center for Measuring Outcomes funded by the National Institute of Disability and Rehabilitation Research (NIDRR). The specific aims of this project are: 1) to build a computer adaptive testing (CAT) system and to determine the accuracy of CAT algorithms in measuring activity outcomes (execution of tasks during daily routines) and participation outcomes (involvement in life situations) by comparing computer-simulated CAT scores with full-length fixed forms; and 2) to test the comparability, validity and responsiveness of CAT disability scores. The project aims will be addressed by means of two major field studies. In Field Study #1, activity and participation data will be collected from 600 patients/instrument in four post-acute care settings, and these data will be combined with item pool data currently being collected by the BU Center for Measuring Outcomes. Item response theory and other statistical methods will be used to evaluate the structure of the activity and participation item pools, establish item calibrations, and create algorithms for a CAT system for disability outcomes assessment. The accuracy of CAT estimates will then be tested using computer simulations. In a subsequent field study, 100 patients/instrument selected from the original sample will be resurveyed to evaluate the comparability of CAT scores to fixed forms, and to determine discriminant validity (across severity groups), and CAT responsiveness to change. The CAT system for post-acute care will radically transform outcome measurement breadth and achieve the practical requirements for routine use in post-acute care settings. Standardizing the instruments used to assess disability outcomes will eliminate the current fragmentation of functional instruments used across post-acute care settings and create an integrated system of outcomes monitoring.
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{
"pile_set_name": "NIH ExPorter"
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Epidemiological studies have shown that women and men differ in their patterns of substance abuse and dependence. Sex differences in the effects of D9-tetrahydrocannabinol (THC), the primary psychoactive substituent of marijuana, have been reported in humans and in rodent models; however, the mechanisms responsible for these differences are unclear and are the focus of the proposed research. Aims 1 and 2 will determine whether sex differences in the antinociceptive and dependence-related effects of THC, respectively, are modulated by gonadal hormones, estradiol, and progesterone in females and testosterone in males. Emphasis will be on the effects of repeated THC administration on behavior, as substance abuse, by definition, requires chronic use. Dependence-related behaviors include precipitated withdrawal (somatic and affective signs), THC discrimination (a rodent model of marijuana intoxication), and conditioned place preference/aversion (CPP/CPA), a rodent model to assess the degree to which drug effects are associated with pleasure or aversion. Sex differences in tolerance to the antinociceptive effects of THC, one of the potential therapeutic indications of cannabinoid-based medications, will also be evaluated. Aim 3 will characterize the pharmacokinetics of THC and will identify regional alterations in central CB1 and CB2 receptor densities and functioning and endocannabinoid levels that are associated with behavioral changes. Understanding these basic mechanisms underlying sex differences in antinociceptive and dependence-related effects of cannabinoids will facilitate development of sex-specific approaches to treat marijuana dependence and to use cannabinoid-based medications therapeutically, specifically in the treatment of pain and spasticity. In addition, increasing scientific knowledge of endocrinological mechanisms that affect endocannabinoid system functioning could enhance understanding and treatment of a wide variety of disorders, in which dysregulation of the endocannabinoid system has been implicated, including substance abuse, psychiatric disorders, obesity, and various neurological disorders.
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{
"pile_set_name": "NIH ExPorter"
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DNA topoisomerases catalyze changes in DNA topology through a concerted mechanism of DNA strand breakage and rejoining. The transient cleavage of the DNA backbone is accompanied by the formation of a covalent enzyme- DNA intermediate. These intermediates, termed cleavable complexes, are reversibly stabilized by a number of therapeutically important drugs, including camptothecin, a potent neoplastic agent, that specifically targets eukaryotic DNA topoisomerase I. This enzyme plays an important role in DNA replication, RNA transcription and DNA recombination and is highly conserved among eukaryotes. This is reflected in similarities in enzyme structure, function and sensitivity to camptothecin. The cytotoxic activity of camptothecin is S-phase specific, presumably resulting from the interaction of DNA replication forks with the drug- stabilized enzyme-DNA cleavable complex. However, the exact nature of the molecular interactions required for the formation of these cleavable complexes and their subsequent conversion into lethal events remains unknown. This application proposes to define the specific molecular interactions required for camptothecin-induced cell lethality in the yeast Saccharomyces cerevisiae. Earlier reports of the restoration of camptothecin sensitivity to yeast cells expressing either the yeast or human DNA topoisomerase I gene, suggest that the processes involved in converting the drug stabilized complex into a lethal lesion could be experimentally addressed in this genetically tractable system. specifically, the particular enzyme domains and amino acid residues required for a productive interaction with camptothecin and DNA will be defined, as will the effects of camptothecin on enzyme structure and DNA binding. Two classes of DNA topoisomerase I mutants, one exhibiting resistance to camptothecin and the other exhibiting a camptothecin- independent lethal phenotype, will similarly be characterized. In addition, a genetic screen will be used to identify second site suppressors of camptothecin sensitivity. Subsequent identification of these genes and their in vivo function(s) will elucidate the molecular interactions required for drug-induced cell death. These studies, as well as those involving second site suppressors of camptothecin- independent lethal mutants, will not only further our understanding of the mechanism of drug-induced lethality, but will also lead to greater understanding of how normal cellular functions can be perturbed to cause cell death. As camptothecin analogues are currently being developed for clinical application to cancers including ovarian and lung, these results will have much broader application in the design and development of new therapeutics.
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{
"pile_set_name": "NIH ExPorter"
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The global objective of this proposal is to understand the molecular process by which normal epithelial cells polarize and assemble into a three dimensional (3D) tissue and how this process goes awry in cancers. Using the mammalian epidermis as a model, I aim to (1) determine which polarity gene homologues are expressed in mouse epidermis. (2) Develop a system with which to temporally induce the expression of siRNAs specifically in skin keratinocytes. (3) Target key polarity molecules for knock down by siRNA in primary keratinocytes in vitro and determine the consequences to the organization of cellular architecture in epithelial sheets with particular focus on the structure, composition and dynamics of adherens junctions, desmosomes, actin and microtubule-based cytoskeletons and apical and basal membrane domains. I will also determine the effects of loss of polarity on the balance between keratinocyte proliferation and differentiation. (4) Graft keratinocytes expressing inducible siRNA transgenes on the back skin of a nude mouse and determine how impaired polarity affects proliferation, morphogenesis and migration in the epidermis in vivo.
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{
"pile_set_name": "NIH ExPorter"
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Capturing nature's statistical structure in the neural coding is essential for optimal adaptation to the environment. This proposal investigates this issue by asking how the brain can approach statistical optimality in the sound localization system of barn owls. A Bayesian theoretical framework will be used to describe how sensory and a priori information can be combined optimally to guide orienting behavior. Specifically, we seek to demonstrate that sensory reliability and a priori information are represented in the response properties and topography of the neural population that represents auditory space. The first aim studies how sensory cue reliability is represented in the brain. Optimal use of sensory information requires that the statistical reliability of sensory cues is accessible from neural responses. Previous theories have suggested that cue reliability is encoded in the gain of neural responses or alternatively the selectivity of neural responses but how reliability is represented is not known. In the owl, changes in the statistical reliability of spatial cues resultin changes in sound localization behavior consistent with a Bayesian model. Our model predicts that the reliability is encoded in the tuning curve widths of space-specific neurons located in the owl's midbrain. We will manipulate tuning-curve widths and firing rates independently to test this hypothesis and test the model with behavior. The second aim will study whether the integration of spatial cues for sound localization follows the rules of statistical optimality. Perception in natural environments often depends on the integration of multiple cues, both within modalities and across modalities. Here, whether the integration is linear or nonlinear is crucial, as extending a Bayesian model from one to two dimensions indicates that optimal combination of conditionally independent sensory cues should be nonlinear. In the owl's brain, the spatial cues used to determine elevation and azimuth are processed independently and combined nonlinearly in the midbrain to form spatial receptive fields. However, whether or not sound localization cues are conditionally independent is unknown. This aim will demonstrate why nonlinear operations are essential for optimal cue combination and how they arise. We will perform in vivo intracellular recording and behavioral tests to address these questions. This will provide an experimental test of the prediction that optimal combination of conditionally independent cues is nonlinear. The third aim will extend the model to coding dynamic auditory scenes; the time dimension will be incorporated into the Bayesian model of sound localization. We will use a population vector model to determine how a neural system can achieve predictive power in auditory space through Bayesian inference. We will measure receptive fields of midbrain neurons in space and time to test the hypothesis that the owl has a bias for sources moving toward the center of gaze. We will use behavioral tests to measure detection thresholds for moving sound sources. Finally, we will study whether a dynamic gain control in a non-uniform network can account for Bayesian predictive coding of sound motion with a bias for sources moving toward the center of gaze. Broader Impacts: Outstanding open questions of how statistics of natural scenes are captured by neural coding include how reliability of sensory information is represented and combined with prior probabilistic knowledge, and how sensory cues are integrated to optimally guide behavior. This project addresses these questions in the heterogeneous representation of space of the owl's auditory midbrain. Whether non-uniform representations can be decoded using a population vector to perform Bayesian inference and that this mechanism works in multiple dimensions transcends sound localization in barn owls, becoming of general interest to neural coding. The PIs involved in this project, one of them a junior researcher, gather complementary expertise in modeling, physiology and behavioral approaches allowing for a truly interdisciplinary approach. This project will thus consolidate a powerful collaboration while providing groundbreaking information on outstanding questions in Neuroscience. The three institutions involved are committed to the training of underrepresented groups. The location of the Albert Einstein College of Medicine in the Bronx, makes it a pole of development in one of the most diverse and poor counties in the country and provides the potential for direct access to translational research. The inclusion of the Department of Mathematics at Seattle University, ranked among the top ten universities in the West for undergraduate programs, and the University of Oregon will ensure that this project will enhance training from the undergraduate to postdoctoral levels.
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{
"pile_set_name": "NIH ExPorter"
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The topic of this proposal is applicable to Research Objectives 7 and 23 in PAR-03-056. The goal of this NIA pilot grant is to develop and characterize transgenic mouse lines that constitutively overexpress either wildtype or mutant ubiquitin in the aging central nervous system (CNS). We recently found that a reduction in the level of free ubiquitin following various neurotoxic insults leads to an accumulation of the pro-apoptotic protein, p53, and selective neurodegeneration. This novel cell death pathway is also associated with increased expression of a mutant form of ubiquitin, termed Ub+l, caused by a post-transcriptional frameshift in ubiquitin mRNA. By impairing ubiquitination and disrupting proteasome function Ub+l promotes additional protein accumulation and cell death. Both impaired ubiquitin-proteasome function and protein aggregates containing Ub+l have been demonstrated in Alzheimer's and other neurodegenerative diseases. However, the exact nature of the relationships between formation of Ub+l by "molecular misreading", abnormal ubiquitin-proteasome function and neuronal cell death remain poorly understood. We therefore propose to develop transgenic mouse lines that overexpress either free wild-type ubiquitin or mutant Ub+l in CNS neurons. Transgene expression will be directed by a Thyl promoter which is neuron-specific. An additional and highly beneficial feature of this expression system is that the Thyl promoter is inactive during embryonic and early postnatal life, thereby avoiding potentially adverse or confounding effects of transgene expression during early stages of development. Phenotypes will be characterized according to gross and microscopic neuropathology, apoptotic gene expression and the degree of cell death at different ages and in different brain regions. The immediate benefits provided by these mice will include establishing clinical relevance for the accumulation of Ub+l in age-related neurodegenerative diseases, and determining whether increasing the available pool of free ubiquitin is neuroprotective following adverse stimuli. Future applications will employ ubiquitin transgenic mice in high throughput screening studies to identify ubiquitin-modulating pharmacological agents for age-related neurodegenerative disorders such as Alzheimer's disease.
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{
"pile_set_name": "NIH ExPorter"
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This project studies the ability of rhesus monkeys and humans to detect small changes in innocuous warm pulses and noxious heat pulses applied to the face and evaluates the ability of medullary dorsal horn thermally sensitive neurons in the monkey to detect the same small temperature changes. This project also investigates the influence of attention on thermal discriminative capacity. A signal correctly indicating the location or modality of a subsequent thermal change improves detection performance while an incorrect signal worsens performance. Consequently, attentional factors may influence the perception of and response to oro-facial pain.
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{
"pile_set_name": "NIH ExPorter"
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A pseudodipeptide is a dipeptide in which the amide linkage has been substituted with a thioether linkage. We have made several of these compounds in order to determine to what extent the molecular structure of these thioethers mimics that of a normal peptide. We are incorporating one of these derivatives into a tetrapeptide, and this tetrapeptide (AlaPhe-pseudo-Phe Ala) will be submitted for x-ray analysis. In addition, the same pseudodipeptide [S,S(-)-Phe-pseudo-Phe] has been incorporated into a pentapeptide which is related to the C-terminus of EGF (epidermal growth factor). This pentapeptide will then be incorporated into a larger EGF fragment either chemically or enzymatically, and the biological activity of the resulting material will be assayed.
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{
"pile_set_name": "NIH ExPorter"
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Abstract This application is being submitted to PA-18-591 in accordance with NOT-OD-18-195. The goal of this administrative supplement is to expand our currently funded research involving Down syndrome (DS) as a model of neurodegeneration and to better understand potential mechanisms for enhanced xenobiotic toxicity observed in this population. Our research (R01 ES027593) is aimed at testing the working hypothesis that environmental exposures contribute to cognitive phenotype variability in DS via disrupted thiol redox signaling and control due to enhanced basal levels of cellular stress and mitochondrial dysfunction. In addition to the cellular stress that we have observed in DS using patient-derived cell lines, our laboratory and others have found that cells from DS individuals display enhanced toxicity to a variety of agents. As DS affects the entire body, cellular stress impacting hepatocytes could alter efficiency and specificity of metabolic enzymes within this cell type, providing mechanistic insight into the sensitivity of the DS population to xenobiotics and pharmaceuticals. Therefore, an additional factor that may contribute to enhanced toxicity in DS is the distinct possibility that xenobiotic biotransformation systems in DS may differ considerably compared to euploid individuals. Furthermore, xenobiotic biotransformation processes can both eliminate or enhance the toxicity of xenobiotics and pharmaceutical agents; thus, it is vital to understand any differences in biotransformation in the DS population, which will better inform the scientific community as to potential adverse events pertaining to both xenobiotic and pharmaceutical agent exposure. Due to these observations it is hypothesized that, because of alterations in cellular proteostasis and redox status, both of which can impact protein expression and function, DS individuals will display altered hepatic xenobiotic biotransformation compared to euploid controls. A single aim has been proposed that will investigate this innovative concept and will determine alterations in basal expression, protein abundance and activity of a variety of xenobiotic biotransformation enzymes. Also, the four xenosensor transcription factors will also be assessed for any differences in activation, gene inducibility, and the impact that the fungicide maneb has on induction of the xenobiotic response. Finally, as per the Notice, NOT-OD-18-195, the research proposed here and within our currently funded R01 (R01 ES027593) are completely appropriate and data generated are likely to translate into new therapeutic approaches (component 1 of INCLUDE project objectives). Given that 100% of DS patients have cognitive deficits, understanding the mechanistic differences in cell death in this population is important. Also, since the phenotype of DS individuals at birth is a predictor of the severity of pathologies in later adult years, this research will begin to answer the question that pertains to whether phenotypic variations at birth and later life prognosis can be predicted from cell viability and vulnerability.
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{
"pile_set_name": "NIH ExPorter"
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Studies elucidate cause and pathogenesis of chronic degenerative CNS disorders with emphasis on MS, ALS, Parkinsonism-dementia, Parkinson's, Pick's, and Alzheimer's disease, Huntington's chorea, supranuclear palsy, other presenile dementias, spinocerebellar ataxias, epilepsy, chronic encephalitis with focal epilepsy, muscular dystrophies, chronic schizophrenia, autism, SSPE, PML, dialysis encephalopathy, and intracranial neoplasm. Even familial, apparently hereditary diseases may be slow virus infections. Subacute spongiform virus encephalopathies: kuru and Creutzfeldt-Jakob disease (CJD) of man; scrapie and mink encephalopathy are caused by unconventional viruses with unique properties posing important theoretical problems to microbiology and molecular biology; a major goal is elucidation of their structure and mechanisms of replication. Transmissible virus dementias are increasingly recognized worldwide causes of death: high incidence foci, transmission by corneal transplant or brain surgery, and occupational hazards from exposure to diseased or infectious brain. In order to determine the usual mode of infection with the virus, a worldwide epidemiological study of transmissible virus dementia (CJD) cases is underway with special attention to familial clusters of cases and with a quest for possible relationship of scrapie of sheep to the human disease. Familial and nonfamilial dementia and the dementias of senility are studied. The autoimmune responses to specific brain antigens in CNS diseases are under intensive investigation. DNA in situ hybridization and electrophoretic focusing partition of proteins along with enzymatic and hybridoma immunofluorescence and many other techniques are used to try to identify viral subunits and partial genomes in tissues in chronic diseases.
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{
"pile_set_name": "NIH ExPorter"
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New causal methods are proposed for understanding randomized behavioral interventions in randomized mental health trials with mediation and interaction analyses. Motivated by specific hypotheses from four mental health studies, the two primary aims are: 1) to aid the explanation of the mechanism of action of these interventions through post-randomization factors such as provider adherence to treatment guidelines and social support (i.e., mediation with possible interactions); and 2) to help identify subgroups of patients based on post-randomization factors such as medical comorbidities and non-study treatments across which randomized intervention effects vary. An additional third aim addresses dissemination. The proposed methods relax a strong assumption of current mediation/interaction methods that assumes no unmeasured confounding for the mediating or interaction factors (sequential ignorability). We make other assumptions involving more parametric models with sensitivity analyses based on different modeling approaches. For the mechanism of action goal, we propose extensions of structural mean models (SMM) for estimating prescriptive and natural direct effects and natural indirect effects. Natural effects are appropriate for the effectiveness research represented by the studies of interest as they provide a theoretical basis for indirect effects and accommodate interactions between baseline interventions and post-baseline behavioral and process factors. We also will focus on developing optimally efficient weights that improve precision without assuming sequential ignorability. For the post-randomization stratification goal, latent principal strata or subgroups are identified under the principal stratification (PS) approach, using post-randomization factors and randomized baseline intervention information. The baseline intervention effect in strata corresponding to constant post-randomization factor levels regardless of randomization arm represent prescribed direct effects. We extend both the SMM and PS approaches to multiple nested post-randomization factors (e.g., physician prescription and patient medication behavior), longitudinal outcomes, binary outcomes, and cases where post-randomization outcomes influence post-randomization adherence behavior. The above methods and standard mediation/interaction procedures and their assumptions will be evaluated and compared with simulations and analyses to answer the specific hypotheses from four studies of interest. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Calcium oscillations in airway smooth muscle using fluo 3
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{
"pile_set_name": "NIH ExPorter"
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The Institute of Laboratory Animal Resources of the National Research Council, National Academy of Sciences proposes to develop guidelines for the recognition and alleviation of pain and distress in laboratory animals. The program goal is to assist institutions, scientists, and animal-care personnel in accomplishing their research objectives, while meeting their legal obligations under the amendments to the Animal Welfare Act (7 U.S.C. 2143) and portions of the Health Research Extension Act of 1985 (42 U.S.C. 289d). To conduct the proposed project, ILAR will establish an eight-member committee, comprised of recognized biomedical investigators and experts in laboratory animal science and medicine. The guidelines are expected to address definitions of pain, distress, anxiety, discomfort, and suffering in animals; clinical signs; assessment of clinical signs; and nonpharmacologic and pharmacologic methods of alleviating pain. It is anticipated that the report, which will require 18- months to complete, will be in the form of a handbook to provide a ready source of information for laboratory animal veterinarians; animal caretakers; and scientists, laboratory technicians, and students who work with experimental animals.
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{
"pile_set_name": "NIH ExPorter"
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Accomplishments for FY2010: 1) We have extended our experimental support for the conclusion that mammalian prions that cause a transmissible spongiform encephalopathy (TSE or prion disease) can be generated solely from bacterially expressed recombinant prion protein. This discovery provides the most compelling evidence so far for the potential protein-only prion hypothesis for the nature of the TSE infectious agent. However, the data leave open the likelihood that other molecules strongly enhance the infectious titers of pathological forms of prion protein. 2) We have studied the strain-dependent structures of prion seeded recombinant PrP fibrils. 3) We have extended our characterization of TSE strain-dependent differences in the interactions between prion protein and complement factors. 4) We have probed the effects of glycosylation and GPI-anchoring on the conformations of different strains of scrapie-prion protein by infrared spectroscopy. 5) We have studied cellular effects of nucleic acid binding to normal prion protein.
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{
"pile_set_name": "NIH ExPorter"
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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The major goals of this project are to develop applications of paramagnetic chemical exchange saturation transfer (PARACEST) imaging agents when bound to antibody surfaces and as small molecule sensors of hypoxia. MRI is the imaging modality of choice for soft tissue imaging but in general lacks sufficient sensitivity for molecular imaging of biological processes associated with cancer. Although Gd-based contrast agents are widely used in clinical MRI as non-specific extracellular agents, new approaches need to be developed to bring MRI into competition with optical and nuclear molecular imaging modalities. Paramagnetic complexes based on chemical exchange saturation transfer (PARACEST) offer a new mechanism for MRI contrast that could potentially improve sensitivity substantially and at the same time offer the unique ability to modulate imaging contrast (on/off) plus reflect specific tissue environments or physiology (pH, redox state, glucose levels). The first aim is to develop bifunctional ligands based on PARACEST for attachment to protein surface residues. These will be attached to model proteins and the water exchange characteristics of the resulting products will be evaluated and the lower detection limit of these systems evaluated by MRI. Two specific targeting systems will be evaluated, a phosphatidyl serine antibody and adenovirus particles with modified knob domain proteins, with the goal of creating activatible PARACEST systems that are completely "off" unless bound to their intended targets in vivo. A PARACEST agent will be developed that is trapped only in hypoxic tumor cells. The overriding goal of this project is to develop a new paradigm of molecular imaging agents for anatomical MR imaging of cancer based upon high sensitivity PARACEST agents.
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{
"pile_set_name": "NIH ExPorter"
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At approximately half the normal life span of a number of mammals, including man, we have demonstrated an altered histochemistry of the media of the arteriolar wall; we have termed these "old" arterioles. However, in old animals with revascularized tissue due to injury, the newly formed arterioles are "young" but age subsequently. The aims of this proposal are: (1) to define and quantitate the chemical (histochemical) and morphological changes in the smallest arteries and arterioles which occur simultaneously in aging; (2) to determine the time course (weeks/months) of the conversion of "young' arterioles to "old" arterioles in the revascularized tissue of injury of old animals "accelerated aging"; (3) to evaluate the relative roles of local environmental (tissue fluid) factors and/or genetic factors in the development of microvascular aging in such revascularized tissues; and (4) to quantify similarly the basal lamina thickening of capillaries and altered adjacent interstitium which are part of the microvascular aging complex. Tissues from various organs will be analyzed to obtain the necessary data from the various ages spanning the natural lifetime of the inbred albino rat. Studies of the revascularized (injured by incision) skin tissue will be in both the (old) donor and (young) recipient animal and the converse. The methods to be used are morphologic and physiologic: the former include histological, histochemical and electron microscopical, and employ quantitative analytic methods to obtain the various data by image scopical, and employ quantitative analytic methods to obtain the various data by image analysis techniques; the latter include microhemodynamics, capillary permeability and vasoactive responses. From these studies it is anticipated that this knowledge of the aging of the microvasculature may lead to further understanding of the alterations in blood flow, diffusion and exchange which may occur in aging. These studies should also provide a precisely defined control model for future research in microvascular aging.
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{
"pile_set_name": "NIH ExPorter"
}
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This aspect of the program is concerned with charcterization of gonadotropin and prolactin receptors of the gonads and of the physical and functional relationships of the LH receptor site and adenylate cyclase. We have devised a procedure for purificatin of microgram quantities of active lactogen receptors from rat ovaries. The receptor is composed of two dissimilar subunits of Mr 88,000 and 40,000, the latter being probably an integral part of the larger form. Aggregation of receptor subunits and/or holereceptor has been suggested by FLPC fractionation of the free receptor in the presence of non-ionic detergents. Free receptors showed binding activity of Mrs 150,000 and 250,000, and aggregates dissociateid upon SDs/mercaptoethanol tratment into the lower molecular forms. These could represent dimeric and trimeric forms of the holoreceptror (80,000). A method for iodination of purified prolactin receptor with preservation of activity was used to corroborate the Mr/s of the free receptor and hormone-receptor complexes after crosslinking the 125-I-receptor with unlabeled hormone. Results confirmed values initially obtained by crosslinking experiments using labeled hormone and unlabeled receptor. When radiolabeled receptor was used for studies on receptor subunit aggregation, dimeric and trimeric forms were observed. Our results indicate that the detergent soluble receptor appeared to be aggregated forms of holoreceptors. The potential for aggregation of subunits and/or holoreceptors is suggested by these findings, and the mechanism by which aggregation or clustering of prolactin receptors would trigger the biological response remains to be elucidated. Chromatofocusing of free receptors showed three isoforms of pIU, 4.0 5.0 and 5.3 indicating glycoprotein and or phosphorylation heterogeneity. We have purified the LH/hCG receptor on wheat germ lectin-Sepharose and hCG Sepharose, which also allows purification of lactogen receptor from the initial starting material. The LH receptor was identified as a single protein Mr 70,000. The technique is simple and allows purification of microgram amounts of active receptor suitable for structural studies microsequencing, and functional reconstitution.
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{
"pile_set_name": "NIH ExPorter"
}
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Our central hypothesis is that glycoproteins and proteoglycans in the pericellular matrix of human gliomas mediate tumor-host cellular interactions and play a central role in tumor growth and invasion. The objective is to identify and characterize these macromolecules by monoclonal antibody analysis of the pericellular matrix of cultured human glioma cells. Antibodies will be raised against two human glioma-derived cell lines, U- 251MG and N-31MG. Both lines express glial fibrillary acidic protein. Secreting hybridomas will be screened by enzyme-linked immunoassay on intact cells and isolated glioma cell matrix and by avidin-biotin immunohistochemistry on frozen sections of a human glioma. Antibodies showing specificity for glioma pericellular matrix will be used in the purification of antigen from cell cultures by immunoaffinity chromatography or immunoprecipitation. Radiolabelled antigen will be isolated from conditioned medium or solubilized matrix and analyzed by SDS- polyacrylamide gel electrophoresis and autofluorography. The ability of isolated antigen to enhance or inhibit glioma cell adhesion in vitro will be determined. In vivo localization of pericellular matrix antigens will be determined by immunohistochemical analysis of frozen sections of a broad spectrum of primary human neuropithelial tumors, normal tissue, and extra-neural neoplasms. Formalin-fixed, paraffin-embedded tumors will also be examined if the epitopes are resistant to such treatment. Human glioma xenografts will be produced in athymic (nude) mice by stereotaxic intracerebral tumor cell transplantation. The localization of pericellular matrix antigens in glioma xenografts will be determined immunohistochemically. Ultrastructural localization of antigens in both human brain tumors and intracerebral xenografts will be determined by immunoelectron microscopy. A specific monoclonal antibody- defined glioma-mesenchymal extracellular matrix antigen (2A6) will be studied in detail. Correlations between antigen expression, tumor morphology, and tumor invasiveness will be assessed with the goal of identifying the biologic and potential diagnostic or prognostic significance of antigen expression.
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{
"pile_set_name": "NIH ExPorter"
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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. Recent developments in applied mathematics have yielded an analytic Green's function to the Boltmann Transport Equation (BTE) which provides an exact description for photon migration in turbid media. The use of a Green's function rather than Monte Carlo simulations promises to model important problems with much less computational expense. We are investigating approaches to utilizing this solutions to address important forward and inverse problems of photon migration in homogeneous and heterogeneous tissues using this approach. We are also working on incorporating the Green's function based ERT kernel into the Solver module of the Virtual Tissue Simulator.
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{
"pile_set_name": "NIH ExPorter"
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A major scientific challenge in neuroscience is the identification and characterization at molecular level of the cellular changes in various physiological conditions and pathological neurological disorders. Critical to the success of this endeavor is the development and implementation of new tools for rapid and sensitive protein identification and characterization of their post-translational modifications. High sensitivity of mass spectrometry has become an invaluable tool for such studies. The major goal of the proposed "MINDS Mass Spectrometry Proteomics Core Facility for Neurosciences at MSSM" is to establish a core facility with dedicated personnel in supporting NINDS-funded neuroscience research programs, to expand and upgrade the capacity and sensitivity of our mass spectrometry facility, to apply state-of-the-art mass spectrometry technologies to critical protein and neuropeptide analysis involved in neuroscience and neurological disease research, and to enable and strengthen neuroscience research program at Mount Sinai. The specific aims of this core facility are: (1) To provide rapid, cost-effective, and high-quality mass spectrometry proteomics service for NINDS and other neuroscience-oriented investigators at Mount Sinai and other NINDS-funded investigators in other institute in order to perform cutting-edge research in which new mass spectrometric technologies are used to solve important biochemical and biomedical problems. (2) To transfer newly developed mass spectrometry technology from my research laboratory to the NINDS Core Facility and expand the capacity and upgrade the sensitivity of mass spectrometry techniques for assisting NINDS-funded and other neuroscience-oriented investigators. (3) To educate NINDS-funded investigators, other neuroscience-oriented scientists, and their staffs on using mass spectrometry for protein analysis in neuroscience. (4) To stimulate new research initiatives and facilitate collaborations in neuroscience.
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{
"pile_set_name": "NIH ExPorter"
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Abstract - Small Animal Imaging Core The Small Animal Imaging Core (SAIC) established under the Phase I COBRE at the University of Oklahoma Health Sciences Center (OUSHC) provides access to state of the art small animal imaging modalities for evaluating tumor growth, progression, metastasis, and response to therapy. The functions of the SAIC include providing assistance, and consultation in designing in vitro and in vivo studies that involve molecular imaging. The SAIC staff's provide their expertise to the COBRE principal junior investigators (PJIs), pilot project junior investigators (PPIs), and other investigators from OUHSC and neighboring institutions with animal studies that require non-invasive molecular imaging; provide training in the use of the imaging equipment's; and subsequent collection and analysis of data. SAIC also provides assistance in optimizing imaging and other pre- and post-imaging surgical procedures. In addition, SAIC provides advanced high-content live cell imaging. Further, SAIC has developed a cell bank of human and murine cancer cell lines with molecular tags that are annotated and characterized. Using these cell lines eliminates variability in the experimental results and maintain rigor. Finally, SAIC staff's undergo annual training for software updates and other imaging-related techniques to keep them abreast of advances in imaging. During the Phase I COBRE funding period, 4 PJIs, 2 PPIs, and 89 other investigators have received service and/or training in using various equipment's and have accessed the SAIC facility for conducting their research. This has led in obtaining significant scientific data and advancing research projects resulting in conference presentations, peer-reviewed publications, and grant application submissions. Finally, SAIC has continued to expand the facility by adding new equipment's thus enabling investigators in conducting cutting-edge research. In the Phase II period, the SAIC plans continuing to train and offer assistance to new PJIs, PPISs, and other investigators in successfully conducting animal studies with ease and have rigor and reproducibility. For achieving these objectives, SAIC has identified three specific aims ? 1) provide service, assistance, and consultation in designing in vitro and in vivo studies related to molecular imaging; 2) provide labeled cells for monitoring gene expression, cell trafficking, and disease progression in in vitro and in vivo models; 3) Assist in high-throughput and high- content imaging of live cells and in imaging of tumor xenografts and other tissues. Continued availability of SAIC services will allow PJIs, PPIs, and other investigators in generating preliminary data for submitting grant applications and obtaining research funding.
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{
"pile_set_name": "NIH ExPorter"
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Recently a pattern of altered growth, physical abnormalities and intellectual impairment has been described in children of chronic alcoholic women. Animal models of this Fetal Alcohol Syndrome (FAS) have indicated that a mild form of the disorder involving only neural anomalies may exist, but go completely unrecognized. The proposed research is primarily directed at using an animal model to determine the behavioral consequnces of prenatal exposure to alcohol as well as the physiological outcomes. Female rats will be fed a liquid diet throughout gestation in which 0%, 8%, 16%, or 24% of the calories will be derived from ethanol. The offspring of these females will be assessed for evidence of FAS and be tested on a variety of behavioral tasks. We see the contributions from this research as follows: (1) very little data now exists documenting the effects of prenatal exposure to alcohol on later behavior. (2) Those behavioral studies that have been done usually employed only a single concentration of alcohol. Our research will specifically examine dose-response relations. (3) Again, those studies that have been performed examined behavior on only one or two specific tasks, making generalizations difficult at best. (4) The resemblance between the physical anomalies noted in rodents following prenatal alcohol exposure and FAS, indicate a common pathogenesis. Thus, behavioral consequences noted in rodents should indicate the general types of problems children exposed to alcohol parentally may face.
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{
"pile_set_name": "NIH ExPorter"
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The overall objectives of this research project are: (1) determination of the cell type which expresses the Ir-1 gene product; (2) genetic mapping of immune response genes with respect to the H-2 locus; (3) attempts to characterize and isolate the Ir-1 gene product; (4) search for histocompatibility-linked immune response genes associated with susceptibility or resistance to infectious, neoplastic, and autoimmune diseases. For the study of these four problems, the following experimental approaches are used: (1) the development of an in vitro secondary, and hopefully primary, antibody-forming system in which immunized or normal lymphocytes from high responder and low responder mice can be stimulated with (T,G)-A--L, (H,G)-A--L, and (Phe,G)-A--L in vitro. Once such a system has been developed, the role of hapten-specific B cells and carrier-specific T cells will be analyzed using (T,G)-A--L as the immunogen and the stimulus for the production of specific antibodies, as well as DNP-(T,G)-A--L as the immunogen, in which case (T,G)-A--L will function as the carrier and DNP as the hapten. The inhibitory effect of anti-Ig, anti-H-2K, anti-H-2D, and anti-Ia antisera will be studied. We hope to pinpoint the expression of Ir genes in T cells. In vivo experiments on the interaction of T and B cells in the immune response to (T,G)-A--L, when these cells differ for a major histocompatibility complex, will also be studied. (2) Mapping studies on natural antigens, such as ovomucoid and BGG, and on synthetic antigens of specified sequence. The latter will be used to elucidate the structure of the antigenic determinant recognized in Ir-l regulated immune reactions. (3) Attempts will be made to isolate T antigen binding cells for (T,G)-A--L. Once this has been done, a variety of techniques will be used to attempt to produce anti-T-cell receptor antibody. (4) We are continuing to develop the necessary cell lines and congenic inbred strains for the study of Rav-1, and H-2-linked murine virus-leukemia resistance gene. The possible role of Ir-l in the Rgv-l effect will be studied. Similar studies will be carried out on the genetic control of susceptibility to murine lymphocytic choriomeningitis.
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{
"pile_set_name": "NIH ExPorter"
}
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Results of standard treatment for squamous cell carcinoma (SCC) of the head and neck or esophagus are poor. New approaches employing combined modality treatment, new combination chemotherapy regimens and new agents are needed. In Project A, newly diagnosed pts with esophageal SCC receive 2 cycles of cisplatinum, bleomycin and VP 16-213 prior to local treatment (surgery or radiotherapy). Those with recurrent disease receive the 3 drug combination until disease progression. To date, among 6 pts there are 2 partial responses (PR), 1 minor response, 1 early death, and 2 are too early to evaluate. In Project B, a combined modality approach is employed for previously untreated, pts with unresectable head and neck SCC. Two cycles of cyclophosphamide and cis-platinum are given prior to radiotherapy and then continued during radiotherapy. One pt has been entered. In Project C, advanced, recurrent head and neck cancer is treated with the combination of cis-platinum and cyclophosphamide. To date, responses in 12 evaluable pts are: 2 CR and 6 PR (66%). Project D evaluates new agents (see also report Z01 CM 09117 03 COB). A phase II trial for recurrent head and neck cancer with aziridinylbenzoquinone (AZQ) has accrued 4 pts.
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{
"pile_set_name": "NIH ExPorter"
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The overall objectives are to discover how to attach substitution-inert metal centers to specific sites on molecules of biochemical interest, and to study the changes in reactivity and in other properties, which attend the coordination. When a solution of a complex of ethylglycinate N-bound to pentaammineruthenium(III) is acidified, rapid isomerization ensues with carboxylate replacing the amine group leading to ester hydrolysis. With a dipeptide, rearrangement to an amide bound form takes place, but peptide hydrolysis is not rapid. The chemistry is being explored in the hope that it may lead to a new facile method of end-specific peptide hydrolysis. Mixed valence molecules containing ruthenium(II) and ruthenium(III) bound to dipeptides and more complex peptide units are being prepared, in an effort to assess the extent of electron delocalization by studying the intervalence absorption. Dimethyl sulfide bound to ruthenium is readily oxidized in the presence of nucleophiles, in the simplest case yielding the dimethylsulfoxide complex of ruthenium(II). This chemistry is being explored as is the possibility that it will provide a means of producing energy rich phosphate bonds. The chemistry of osmium ammines coordinated to imidazole and other polar groups featured by biologically significant molecules is being explored.
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{
"pile_set_name": "NIH ExPorter"
}
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The erythropoietin receptor (EPOR) is a transmembrane protein that mediates the transmission of a signal from the hormone erythropoietin (EPO) to a blood cell protenitor; this signal induces the maturation of the cell to an erythrocyte. Like many transmebrane receptor proteins, EPOR transmits the signal across the cell membrane by dimerizing in response to divalent binding by the EPO protein. A small organic molecule that can dimerize EPOR in the absence of EPO would be highly valued as a therapeutic treatment for severely anemic patients. A small-petide EPOR mimetic has been developed that can dimerize EPOR, although peptide.based drugs suffer from poor bioavailability and rapid breakdown in vivo. A combinatorial chemistry-based drug discovery program has been developed at NeoGenesis that is capable of synthesizing and rapidly screening millions of drug-like molecules to identify small-molecule ligands for any protein target. This project proposes the synthesis of new combinatorial libraries of small organic molecules and the development of novel screening protocols which will identify compounds that dimerize or can be modified to dimerize human EPOR. If this strategy is successful, it will be applied to other high-value drug targets which work by similar mechanisms, such as the receptors for growth hormone and insulin. PROPOSED COMMERCIAL APPLICATIONS: This project will develop a small-molecule mimetic of erythropoietin, and the technology developed here will be applicable to the development of small-molecule mimetics of growth hormone, insulin, and other hormones that function through dimerization of membrane-spanning receptors.
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{
"pile_set_name": "NIH ExPorter"
}
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Basement membranes are thin extracellular matrices that form an interface between endothelial, epithelial, muscular, or neural cells and their adjacent stroma. Developmentally, basement membranes are the first extracellular matrix to appear, and they are degraded and regenerated during development and wound repair. They not only support cells and cell layers, but they also have an essential role in tissue organization that affects cell adhesion, migration, proliferation, and differentiation. Additionally, basement membranes provide major barriers to invasion by metastatic tumor cells. Thickness and composition of basement membranes are different in various tissues, suggesting tissue-specific functions. Thus, extracellular matrices have both functional and structural roles. The major molecules in basement membranes are collagen IV, laminin, perlecan, and nidogen/entactin. Recently, diversity has been discovered in the subunits that make up the trimers of collagen IV and laminin molecules. Our primary objectives have been to identify the specific functions of basement membrane components, to elucidate the mechanisms by which they are regulated, and to describe related protein interactions in development and diseases. Our effort has also focused on establishing animal models for basement membrane development and disease and on creating therapeutic reagents for diseases associated with basement membranes. We are also identifying bioactive sites on laminin that have a number of biological activities such as promoting cell adhesion, migration, and neurite outgrowth, and affecting metastatic activity of tumor cells. These studies are aimed towards developing reagents useful for diagnostic and therapeutic applications.
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{
"pile_set_name": "NIH ExPorter"
}
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1. Elucidation of the involvement of the second extracellular loop (ECL2) and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion.We examined the interactions between CCR5 and novel CCR5 inhibitors containing the SDP scaffold, AK530 and AK317, both of which were docked into the hydrophobic cavity located between the upper transmembrane domain and ECL2 of CCR5 (67). Figure 11 shows the lipophilic potential mapped on the binding cavity of CCR5 and relative binding modes of AK530 and AK317. Molecular dynamics simulations for inhibitor-unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants. We continued the design, synthesis, and evaluation of different CCR5 inhibitors in collaboration with Professor Ghosh of Purdue University. We initially started from a published CCR5 antagonist from the literature, and built structural models of its interaction with CCR5. Over the last four years, the design, synthesis and biological evaluation of more than 100 inhibitors has been carried out and several novel and potent inhibitors of CCR5 have been discovered in our study. Identified CCR5 antagonists include GRL-117, GRL-10007, and GRL-10018, which proved to be active against R5-HIV-1 with antiretroviral IC50 values of 0.6 nM, 1.4 nM and 2.9 nM, respectively. The IC50 value of MVC, the only approved CCR5 inhibitor at present, was 0.7 nM in the same assay. These newly identified compounds were also potent against vicriviroc (VCV)-resistant R5-HIV-1. The IC50 value of GRL-10007 decreased by 5.3-fold against VCV-resistant virus whereas the activity of MVC decreased by 10.6-fold. Experiments using CCR5 antibody revealed that these compounds were bound to ECL2 of CCR5 as other reported CCR5 antagonists do. These compounds also effectively blocked the interactions between CC-chemokines and CCR5, but GRL-10007 only partially blocked RANTES binding to CCR5 and RANTES-induced Ca2+ flux (Nakata, Das, Ghosh and Mitsuya: manuscript in preparation). Of note, the RANTES binding and Ca2+ flux profile of GRL-10007 were similar to those of APL, suggesting that, as was in the case of APL, GRL-10007 may have highly specific binding to CCR5 without overly inhibiting CCR5-chemokine interactions. 2. Determination of binding mode of the new CCR5 inhibitors and elucidation of structure-activity relationshipsWe built structural models of newly identified CCR5 inhibitors to gain insight to the mechanism of potency of such inhibitors. A model of the interaction of GRL117 is shown in Figure 13. GRL117 binds in a cavity formed within the transmembrane helices and ECL2. The models suggest that GRL117 has polar interactions with Y37, C178, K191, and T195. Y37 is located in transmembrane-1 (TM-1), K191 and T195 are located in TM-5. C178 is located in ECL2 and is highly conserved amongst class-A GPCRs. These residues are also important for gp120 fusion, and for the binding of APL and other CCR5 inhibitors.　We postulated that interactions of inhibitors with CCR5 residues that are important for gp120 fusion may cause conformation change in CCR5, and may represent a salient molecular mechanism enabling allosteric inhibition. The interactions of GRL117 with such residues might be responsible for its potent antiretroviral IC50 of 0.6 nM. 3. Study of impacts of amino acid substitutions on CD4/gp120-CXCR4-induced fusion and identification of lead CXCR4 inhibitors.We first attempted to examine the impacts of amino acid substitution on CD4/gp120-CXCR4-induced fusion by introducing amino acid substitutions into human wild-type CXCR4 CXCR4WT)and determined the changes in fusion levels, compared to the fusion activity of CXCR4WT (referenced as 100%). We found that amino acid substitutions D97A, D262A, and E288A located in different transmembrane domains resulted in substantial loss of fusion activity. These residues have also been shown to be important for the binding of different CXCR4 inhibitors. As expected, various amino acid substitutions in ECL2, such as A175F, D182A, D187A, R188A and Y190A also resulted in compromised fusion (Figure 14).The availability of the crystal structure of CXCR4 with a small molecule inhibitor greatly aided our efforts to identify lead CXCR4 inhibitors. Out of an initial selection of 16 molecules with virtual screening of 622,000 different molecules, we identified cyclopentane-piperidine analogues as having anti-HIV activity (Das, Maeda, Hayashi, and Mitsuya: manuscript in preparation). Subsequently, other molecules containing the cyclopentane-piperidine scaffold were selected from the general screening library. Such molecules were found to have IC50 values ranging 400 nM to 9 microM in MTT assay. CX-6 and CX-20 were identified as lead molecules, and our data warrants further optimization of the candidates by increasing interactions with other key residues in the active site. We have started collaboration with Professor Ghosh of Purdue University in optimizing the scaffold and the newly identified CXCR4 inhibitors
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{
"pile_set_name": "NIH ExPorter"
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The major aims of this project have been accomplished through establishment of a large community based family study of adult probands who participate in comprehensive clinical and biologic assessments followed by comparable evaluations of their adult and child relatives. Recruitment of probands and evaluation of relatives are now near completion and analyses of the findings are underway. To date, we have enrolled 344 participants into the study from the community, and 238 from clinical sources. Our total enrollment from all sources is 786 probands, of which almost 500 have completed the study, and 1,283 relatives, including 175 children. Over the past year, 11 probands and 49 relatives have been recruited. Probands represent a range of disorders including: 30% with bipolar, 63% with depression, 60% with anxiety, 50% with migraine, and 22% without any of these conditions. During the next 3 months we plan to complete recruitment of probands and evaluation of relatives and will commence data analysis. In order to identify the core features of mood and anxiety disorders and their overlap with cardiovascular diseases and migraine, we have established a set of clinical, psychophysiological and neuropsychological measures. The comprehensive set of measures in the study are divided into three components: (1) clinical information that includes a semi-structured diagnostic interview for mood and anxiety disorders, structured diagnostic interviews for major headache syndromes and for sleep disorders, family history of psychiatric disorders, sleep disorders and headache syndromes, and a series of self-reported measures of symptoms, sleep patterns and personality, medical history, family structure, history and function, and major life and family events; (2) in person evaluation at the NIH Clinical Center that includes physical and neurological examination; autonomic nervous system evaluation using tilt and valsalva maneuver; laboratory measures of metabolic, cardiovascular, and immunologic function; psychophysiologic evaluation including startle reflex and eyeblink reflex; structural magnetic resonance imaging; a computerized cognitive assessment battery; and olfactory function; and (3) two-week assessment of daily activities including an electronic diary that assesses daily rhythms including mood, anxiety, sleep, stress, activity, diet, and social activities; heart rate reactivity using a holter monitor; activity and sleep using an actiwatch; and saliva hormones collected four times per day. In the past year, we have been running preliminary analyses on several of these clinical components and working with external collaborators to design and implement analyses of all of the measures in the study. This study has yielded substantial methodological developments including the development and validation of a structured diagnostic interview for headache syndromes; systematic assessment of the validity of the NIMH Family Study Diagnostic Interview for Affective Spectrum Disorders that was developed for this study compared to the Structure Clinical Interview for DSM-IV (SCID); and the validity of the NIMH Sleep Diagnostic Interview also developed for this study compared with sleep disorder diagnoses made by sleep experts. We have completed the primary analyses of the familial aggregation of mood disorder subtypes, their patterns of comorbidity, and their co-aggregation in relatives, and have submitted the principal paper that reports these findings. Likewise, we have also completed analyses of the familial aggregation of migraine and comorbid conditions. The major findings confirm the familial aggregation of bipolar disorder and major depression that has been found in earlier family studies of mood disorders but in a community based sample that suspended diagnostic hierarchies of earlier diagnostic systems. The most important finding that emerged from this study is the independent familial transmission of mania and depression suggesting that these two mood states may represent distinct underlying diatheses. The lack of familial aggregation of hypomania suggests that the bipolar spectrum concept may not be a valid entity. We also found strong evidence for familial specificity of atypical depression in probands and interviewed relatives, thereby confirming the validity of this subtype of depression. We also have investigated the familial aggregation of migraine and its links with mood disorders. We found a 2.6-fold increased risk of migraine in the relatives of probands with migraine, primarily attributable to migraine with aura. Sex of the proband was not associated with differential risk of migraine in relatives, thereby indicating that the sex difference in migraine cannot be attributed to genetic factors. Additional analyses that are now being investigated include the familial aggregation of subtypes of sleep disorders, early manifestations of mood and anxiety disorders in offspring of parents with mood spectrum disorders, and heritability analyses of both disorders and underlying traits. During the past several months, we have begun to examine the non-clinical measures as well, with substantial progress in analysis of the eyeblink reflex, actigraphy, olfactory measures and attention using the Attention Network Test. We have also conducted extensive analyses of links between cardiovascular disease and risk factors, migraine and depression in other data sets to develop specific hypotheses for the analyses of the family study data. Some intriguing findings that have emerged include: greater reactivity of the eyeblink reflex among those with migraine with aura compared to those without aura and non-headache controls; greater variability in activity among those with major depression; and increased orienting to cues in those with migraine with aura, and decreased speed of attention among those with depression. Public Health Impact: Integration of the clinical, neuropsychological and psychophysiological measures within families will allow us an in-depth analysis of the biological mechanisms crucial for mood and anxiety disorders and their underlying diatheses. This will not only lead to a better understanding of these conditions, and assist in identifying common genetic mechanisms, but may also lead to the development of novel treatment options and possible strategies for prevention and early intervention in those with elevated risk for these conditions. Future Plans: During the next year, we plan to follow up the probands and relatives to update the diagnostic status, and repeat the measures of circadian rhythms of activity, sleep and cognitive and mood states to test their stability and seasonal changes. We will complete enrollment of children under age 18 and follow up the 150 youth who have already participated in the study. Finally, we will examine common measures to match the NIMH local community to the supplemental study that we are now conducting in the Neurodevelopmental Genomics Study at the University of Pennsylvania.
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{
"pile_set_name": "NIH ExPorter"
}
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Generation of cell polarity is a key biological process that defines multiple aspects of normal cell growth, and differentiation. The major goal of this proposal is to understand how microtubules and microtubule motors contribute to generation of cell polarity and organization of the cytoskeleton. During the previous funding period our group discovered that microtubules in the cell are actively transported in the cytoplasm, with one microtubule serving as a track and another as a cargo for a motor protein. This movement is powered by conventional kinesin (kinesin-1), a major microtubule motor that has previously only been implicated in movement of organelles along microtubules. Microtubule movement by kinesin is required for formation of processes by neurons and non-neuronal cells. Here we propose to study the role of microtubule movements by kinesin and other microtubule motors in generation of cell polarity and specifically in formation of processes by neuronal and non-neuronal cells. We will initially use Drosophila S2 cells for identification of molecular components involved in microtubule sliding and formation of processes because these cells are highly sensitive to protein knock-down by RNAi and because microtubule movement and process formation can easily be visualized, tracked, quantified and manipulated. Once the components are identified, we will use Drosophila neurons to study how these components contribute to formation of neurites in culture and neurogenesis in vivo. We will use tools of Drosophila genetics combined with imaging of live neurons and micromanipulation to address these questions. The three overlapping areas to be investigated in this project are: (i) How does motor-driven microtubule sliding organize polarized microtubule arrays? (ii) How is motor-driven microtubule sliding regulated in a cell? (iii) What is the role of microtubule sliding in axon regeneration after injury? We believe that proteins driving and regulating microtubule sliding in neurons could serve as excellent new therapeutic targets for pharmacological stimulation of neurite outgrowth required for successful treatment of neuronal injuries and neurodegenerative diseases.
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{
"pile_set_name": "NIH ExPorter"
}
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This revised application is using the NIMH "R-34" treatment development award mechanism and is entitled "treatment of PTSD in cardiac patients". The goal of this application is to develop a treatment for posttraumatic stress disorder (PTSD) symptoms that are caused by the emotionally traumatic experience of having had a myocardial infarction (Ml). Ml is an acute, life-threatening event that may be accompanied by a feeling of helplessness or fear. Therefore, it can qualify as emotionally traumatic according to the DSM-IV-TR definition, and may elicit symptoms of PTSD. Indeed, recent studies report that significant PTSD symptoms exist in 10-20% of patients with cardiovascular illnesses, and that these symptoms are associated with nonadherence to medications and poor medical as well as psychiatric outcome. PTSD symptoms are highly comorbid with depression post-MI. Yet, PTSD symptoms are not usually targeted in efforts to treat patients post-MI, and classic depression treatments are not as helpful in patients who had an Ml as compared with depressed patients without Ml. The treatment of PTSD may therefore be a promising new approach to improve psychiatric as well as medical outcome in patients who had a myocardial infarction. Effective treatment options for PTSD do exist, but they were not studied in Ml survivors. Existing treatments need to be substantially modified in order to be used in this population of medically ill individuals who are adjusting to a new post-MI reality (as opposed to a single-incident trauma survivor who is expected to adjust back to his or her pre-trauma functioning). The plan of this proposal is to develop a modified trauma-focused cognitive-behavioral treatment for PTSD symptoms that occur in Ml survivors and test it in a randomized, controlled study. We will evaluate the safety as well as the potential benefits of this approach. A full efficacy trial is expected to follow this pilot study if the results are encouraging.
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{
"pile_set_name": "NIH ExPorter"
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What is known: Disulfide reduction-fueled enzymes s!upport homeostasis and combat oxidative damage that contributes to neurodegeneration, inflammatory diseases, and cancer. NADPH provides the reducing power for most anabolic and cytoprotective reduction reactions, yet only two enzymes can use NADPH to reduce cytosolic disulfides: thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr) 1. Both TrxR1 and Gsr have active sites that are dominantly inhibited by electrophilic toxins and oxidants 2, 3. In Co-PI Schmidt's lab, mice with TrxR1/Gsr-null livers uncovered unexpected robustness in the disulfide reductase systems, including an NADPH-independent pathway that uses catabolism of methionine (Met) to sustain redox homeostasis 4. Importantly, this pathway is also thought to sustain normal cells under oxidative or electrophilic stress 5. Met and Cys are the 2 sulfur (S)-amino acids found in proteins, but S-containing molecules synthesized from Met or Cys, including S-adenosyl-Met (SAM), glutathione (GSH), CoA, and others, are also important in redox, detox, energetics, biosynthesis, regulation, and other processes. Co-PI DeNicola has been studying the roles of altered S-amino acid metabolism in sustaining some cancers 6. These studies are revealing how some cancers use altered S-amino acid redox metabolism, which could uncover targetable cancer-specific susceptibilities. Unresolved questions: It remains unknown how other metabolic activities, including those that directly utilize Met or Cys, as well as more peripheral systems that either (i) supply resources to these pathways; (ii) depend upon these pathways; or (iii) might, in some conditions, compete with these pathways for substrates, are realigned to help cells survive stress. We hypothesize that conversion to Met-dependence involves realignment of diverse metabolic pathways. A better understanding of these processes will uncover processes that can be therapeutically targeted to either specifically increase the robustness of critical cells under oxidative or toxic stress, or specifically increase the vulnerability of pathogenic cells in cancer or inflammatory diseases. What is proposed: In this collaborative project, we will define the metabolic pathway realignments that occur when hepatocytes switch from NADPH-dependent to -independent disulfide reduction. We propose 3 Specific Aims: Aim 1, Define how NADPH- versus Met-fueled disulfide reductase homeostasis influences S-metabolism prioritization. Aim 2, Define how re-wiring of serine metabolism supports Met-fueled disulfide reductase homeostasis. Aim 3, Test whether Met-dependent survival increases the activity and dependence on liver methyltransferases. Anticipated outcomes, value: This project will help us understand how global shifts in hepatic metabolism occurs in response to severe oxidative or electrophilic stress in liver, and how this helps sustain health.
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{
"pile_set_name": "NIH ExPorter"
}
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PROJECT SUMMARY/ ABSTRACT Central nervous system (CNS) infections, including meningitis, encephalitis, and myelitis cause significant long-term morbidity, mortality and health care costs, with an increased incidence in children. Rapid identification of an infectious agent in the cerebrospinal fluid (CSF) is essential to guiding the targeted use of effective antimicrobials in children with suspected CNS infection. Currently utilized diagnostic techniques are low-yield, slow, and expensive leading to widespread use of unnecessary empiric antimicrobial therapies and increased health care costs. Newly developed multiplex PCR panels allow for rapid identification of the most common organisms to cause CNS infections within one hour. However, optimal implementation strategies to translate these more rapid test results into more rapid, appropriate clinical action are unknown. This project seeks to optimize implementation of new rapid molecular CSF diagnostic technology in a tertiary care children?s hospital in conjunction with real-time antimicrobial stewardship decision support using RE-AIM methodology for implementation and evaluation. The research detailed in this proposal meets the critical unmet need for clinical outcomes and cost-effectiveness data for new rapid diagnostic technologies. This research aims to develop reproducible and sustainable strategies for implementing rapid diagnostics to optimally impact the clinical care of children through the following specific aims: (1) To optimize a reproducible implementation strategy for rapid cerebrospinal fluid diagnostics in children with suspected central nervous system infection, (2)To measure the impact of a rapid cerebrospinal fluid diagnostics program on the clinical care and health care costs of children with suspected central nervous system infection. This project is the culmination of the candidate?s interest and experience in CNS infections and rapid molecular diagnostic technologies. The overall objective of this career development award is to develop the candidate into an independent principal investigator in clinical research with expertise in the implementation and evaluation of infectious disease diagnostics to facilitate timely and efficient clinical care of children. Through experiential mentored research training and didactic coursework in a doctoral clinical sciences program, essential skills in clinical research, implementation and dissemination science, clinical outcomes, and cost-effectiveness analysis will be developed. The candidate has assembled a multidisciplinary team of mentors and advisors with extensive clinical research experience and topical expertise in the above realms to ensure his success in achieving the stated specific aims and career goals.
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{
"pile_set_name": "NIH ExPorter"
}
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The oligosaccharides on soluble and membrane glycoproteins exhibit tremendous structural diversity, suggesting they encode highly specific information. For this to be the case there must exist: l) Unique oligosaccharide structures, 2) Highly specific glycosyltransferases, and 3) Receptors capable of recognizing these structures and utilizing their unique information. Biologic functions making use of information encoded in oligosaccharides are likely to be manifest primarily in multicellular organisms and include such phenomena as cell:cell recognition, cell:matrix recognition, and both intra- and extra-cellular trafficking of glycoproteins. The challenge has been to identify systems which utilize the structural information encoded in oligosaccharides for a biologic purpose. We have identified a major new carbohydrate-specific receptor system in liver which regulates the circulatory half life and, thereby, the biologic activity of the glycoprotein hormone lutropin and other glycoproteins bearing terminal SO4-4GalNAc-beta, 14GlcNAc-beta1, 2Man- alpha (S4GGnM) on their oligosaccharides. The synthesis of S4GGnM-terminal structures is highly regulated and they are found on only a limited number of glycoproteins. Hepatic endothelial cell express >500,000 S4GGnM-binding sites/cell at their surface. The S4OGnM-receptor has been isolated from rat liver and will be extensively characterized using approaches we have developed for other carbohydrate-specific systems. The properties of the receptor will be defined in isolated hepatic endothelial cells. The primary structure will be determined by cDNA analysis. Proteolytic fragmentation and mutagenesis will be used to define the regions involved in ligand binding and targeting. Additional potential functions will be established by using the receptor to identify other glycoproteins bearing S4GGnM-terminal structures. Cells expressing the receptor in other tissues will be identified and characterized to establish if the receptor is part of a multigene family. Immunolocalization and in situ hybridization will be used to examine the regulation of S4GGnM-receptor expression during fetal development, pregnancy, and gain of sexual maturity in newborn rats. Since many glycoproteins including membrane glycoproteins on normal and malignant cells, surface glycoproteins on viruses, growth factor precursors, coagulation factors, complement components, and hormones may interact with the receptor system it has tremendous potential relevance to both pathologic and normal states.
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{
"pile_set_name": "NIH ExPorter"
}
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Inflammation is a foundational cause of brain injury during central nervous system (CNS) infections and the reason why anti-inflammatory therapy is used along with antibiotics during infections such as meningitis. Corticosteroids are the only anti-inflammatory treatment currently approved for bacterial infections of the CNS. However, corticosteroids are sub-optimal as their benefit is restricted to individuals in high-income countries, to infections with certain bacteria, and ultimately do not improve long-term neurological outcomes. Thus, new therapies are needed that will ameliorate inflammatory pathology and deliver more positive patient outcomes. MicroRNA (miR) are short, non-coding RNA expressed in myeloid cells that have key roles as modulators of innate and adaptive immune responses. Our data suggest several miR, e.g. miR-155, are up-regulated in cerebrospinal fluid (CSF) of patients with bacterial meningitis as well as in the brains of mice infected with the neurotropic bacterium, L. monocytogenes (Lm). miR-155 up-regulation coincides with up-regulation of pro-inflammatory cytokines and recruitment of inflammatory leukocytes into the brain. Notably, preliminary data presented here show miR-155-/- mice recruit more Ly-6Chigh monocytes and neutrophils to the brain than do normal mice suggesting miR-155 limits inflammation during infection. The goals of this proposal are to establish the novel finding that miR-155 has a role in reducing brain inflammation during infection and to identify the mechanisms by which infection induces brain expression of miR that are found both in patients with meningitis and in experimental mouse infection. Specific Aims of this proposal include: Aim 1. Elucidate the mechanisms leading to increased inflammatory cell influxes in the brains of miR-155-/- mice during Lm infection. Our working hypothesis is that loss of miR-155 leads to increased production of monocyte and neutrophil-attracting chemokines in the brain during CNS infection. Aim 2. To identify key biological stimuli that up-regulate miR expression in the brain during CNS infection. Our working hypothesis is that the initial miR up-regulation in the brain is driven by cytokines produced during Lm infection. Experiments will be performed in part using the mouse model of Lm infection as well as with novel techniques including bone marrow chimeras and organotypic brain slice cultures. Successful completion of these aims will establish for the first time that a specific miR influences brain inflammation during bacterial infection and will gauge the potential benefit of targeting miRs or specific miR-expressing inflammatory cells as novel adjunctive treatment for CNS infections. This is a necessary first step towards discovering new molecular targets for modulating CNS inflammation.
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{
"pile_set_name": "NIH ExPorter"
}
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The overall purpose of this project is to extend a series of highly successful experiments testing the hypothesis that the reflexive startle response is modulated by emotional state. The new experiments will critically examine the biphasic model of emotion. In this view, emotions are motivationally driven by reciprocal brain states, either appetitive (favoring approach, attachment, and consummatory behavior) or aversive (favoring avoidance, escape, and defense). Furthermore, the response system as a whole (from cognitions to exteroceptive reflexes) is tuned according to the current status of this central affect-motivational organization. Thus, reflexes associated with an appetitive set should be enhanced, if activated when the subject is already engaged in a positive emotional response; conversely, the startle reflex to a sudden noise is an aversive, defensive response, and would be augmented if it occurred in the context of an ongoing aversive emotion. Inhibition of startle in an appetitive context is also postulated. Specific experiments testing this view manipulate parameters of probe startle stimuli, and examine variations in emotional foregrounds and subject populations. The research is intended to determine: (1) if reflex modulation occurs for both visual and auditory startle probes, in both pictorial and auditory emotional foregrounds; (2) if these effects are in any way constrained by probe intensity or aversiveness; and, further, this work will (3) pursue the significance of lateralized probes for theories of the hemispheric processing of emotion; and (4) begin to determine if the affect-startle effect is unique to startle probes, or is a general phenomena of defensive reflexes. The research will manipulate characteristics of the emotional foregrounds tested by the probe methodology: to determine (1) if the affect-startle effect is consistent at different intensities of foreground emotion and (2) if reflex augmentation is consistent across different negatively valent emotional states; and, further, (3) to explore the effect during emotional memory imagery; and (4) to begin an examination of individual temperament differences in emotional state modulation of the startle reflex.
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{
"pile_set_name": "NIH ExPorter"
}
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Vascular disease is the leading cause of death in the United States. Neural-tube defects are a major contributor to perinatal morbidity and mortality. Elevated levels of plasma homocysteine (a non-protein amino acid) have been found to correlate with an increased risk of NTDs and vascular disease. Methionine synthase (MS) catalyzes the vitamin B12 dependent, interconversion of homocysteine and 5-methyltetrahydrofolate to methionine and tetrahydrofolate. This is one of the few homocysteine consuming reactions in the body and reduced MS activity is predicted to lead to increased plasma homocysteine. We have cloned the human MS gene and demonstrated that hyperhomocysteinemia in a subset of patients is in fact due to mutations in the MS gene. Most of the genomic structure of the MS gene has been determined. Once all MS exons have been identified, mutation screening assays will be developed. Families with children affected with NTDs have been screened to determine if MS plays a role in NTD. Initial data has does not show a connection between MS and NTD. We are now testing to see if the enzyme that acts with MS, MS- reductase plays a role in NTD. In addition to the human genetic studies, we have used homologous recombination techniques to create a mouse model of MS deficiency. The phenotype of this mouse is currently being investigated.
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{
"pile_set_name": "NIH ExPorter"
}
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We propose to begin to create links between Williams syndrome (WMS), a rare genetic disorder that typically results in mental retardation, a distinctive facies and a heart defect, its cognitive sequellae and its genetic basis. In most cases, WMS is associated with a hemizygous deletion around the elastin gene on chromosome band 7q11.2. To ultimately identify the pathway from genes to cognition in WMS, we will carefully define the genetic regions deleted in the patients studied in projects I-III. Because the expression of genes located near the deletion may also be affected and contribute to the phenotype, the genetic structure of the flanking regions will also be determined. To obviate the deficiencies of the current physical map of the WMS region in yeast artificial chromosomes (YACs), an independent approach employing bacterial artificial chromosomes (BACs) will be used. Established as an ideal tool for molecular cytogenetics, genome mapping and sequencing, an array of 50 BACs has been defined that map within and flanking the WMS region. Using these, a portion of the WMS deleted region has now been cloned, the approximate size of the common deletions has been estimated, BACs closely flanking the deletion have been identified; and 4) A novel family of repeated sequences mapping only in this chromosome band has been identified that may be ultimately responsible for causing the WMS deletion. The project is organized into four aims. Aim 1: A physical map of the WMS region will be constructed in BACs and PACs using end clone walking, PCR, and clone to clone Southern analysis. Aim 2: A critical region likely to contain the genes responsible for the WMS cognitive phenotype will be defined by using fluorescence in situ hybridization of BACs (bacterial artificial chromosomes) and PACs (PI artificial chromosomes), Southern blot dosage analysis of single copy DNA markers, and PCR analyses of polymorphic markers. Aim 3: The molecular data will then be combined with the clinical and neurocognitive data from projects I-IV to generate a Phenotypic Map of WMS, to define molecularly, the regions of chromosome 7q11.23 that are likely to contain the genes for some of the physical and metabolic features, and a part of the mental retardation and cognitive features. Aim 4: The genes mapping in these regions will be isolated by cDNA selection and characterized. The results of this work will lay the groundwork for elucidating the common genetic origins of cognition.
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{
"pile_set_name": "NIH ExPorter"
}
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The overall objective of the proposed study is to develop and validate an in vitro multicellular layer (MCL) model that can be used to gain a better understanding of transport mechanisms and to facilitate testing a large number of experimental parameters that are pertinent to in vivo applications of electric field-mediated gene delivery. As a technique, electric field-mediated gene delivery is likely to be far from optimal since DNA transport in tumors is a complicated process. It is unpractical to optimize the technique using tumor tissues nor can it be optimized by using polymeric gels because of the absence of cells in gels. Therefore, I propose to develop and validate the MCL model (Specific Aim 1) since it is less expensive than tumor tissues and more relevant than polymeric gels. Using this model, I will identify effective pulse sequences forDNA transport (Specific Aim 1) and investigate effects of extracellular matrix (ECM) and cells on DNA mobility (Specific Aim 2). In addition, I will investigate the impact of DNA electrophoresis on transfection efficiency in electric field-mediated gene transfer (Specific Aim 3). Results from this study may lead to the identification of the most effective procedures for electric field-mediated gene transfer in solid tumors.
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{
"pile_set_name": "NIH ExPorter"
}
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Medical Oncology Community: We feel that it is vital to maintain a cohesive medical oncology community for the following reasons. 1. For quality of clinical care. 2. To share resources (patients, research nurses, fellows etc.). 3. To learn from each other. 4. To leverage new collaborative opportunities. 5. To optimize our clinical trials and correlative science. Thus we have liaisons with each branch, program or laboratory (Chief or Clinical Director) with whom we hold regularly scheduled meetings and / or provide service related information for dissemination. This core group also provides feedback on policies and procedures and alerts the MOS when there are issues regarding the efficiency or quality of clinical care or clinical research. The medical oncology service is responsible for maintaining the quality and efficiency of medical oncology care across multiple branches and laboratories that have clinical programs. This includes all medical oncology licensed personnel (Nurse Practitioners, Physician Assistants, Fellows, Volunteers, Staff Clinicians, Investigators and Senior Investigators). We have monthly didactic meetings with the branches involved in the Solid Tumor Service (DTB, GMB, TGIB, WMB), monthly didactic clinical walk rounds and there are weekly didactic meetings with the medical oncology fellows that all MOS staff are strongly encouraged to attend. The MOS is also involved in all credentialing activities for medical oncology staff. Medical Oncology Clinical Service: We maintain efficient quality care within the in-patient service including around-the-clock scheduling of licensed independent practitioners and attending level personnel. We also maintain efficient quality care in the outpatient service addressing regulatory, procedural and logistical issues to assure optimal care and optimal clinical research. Medical Oncology Fellowship: While this falls under the Medical Oncology Service, it has a separate annual report.
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{
"pile_set_name": "NIH ExPorter"
}
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This proposal focuses on biological markers in cerebrospinal fluid (CSF) related to Alzheimer's Disease (AD). Biomarkers that are clearly related to the pathology of AD, namely A-beta42 (the major protein in plaques) and tau (found in tangles) can discriminate patients with AD from controls. Less is known about these and other biomarkers in mild cognitive impairment (MCI), which is often a prodromal stage of mild AD, and in relation to aging and to genetic and other risk factors for AD. In this proposal, 4 AD Research Centers will collaborate to obtain CSF and plasma samples from well-characterized subjects with AD, MCI and healthy controls spanning the age range 20-80. About 50% of subjects will contribute a set of serial CSF and plasma samples at 12 month follow-up. This project builds on an existing collaborative CSF and plasma bank, and aims to accrue and bank samples from over 500 subjects. A-beta processing, production, deposition and clearance are important factors in AD. These will be investigated by measuring levels of species of A-beta (A-beta 38, 40 and 42) and of secreted, cleaved forms of beta-amyloid precursor protein (APP), the parent molecule of A-beta, in CSF. Indices of neurodegeneration and tangle formation will be studied by quantifying CSF levels of tau and phospho-tau, and tau will be purified from CSF and sequenced. As indices of oxidative damage and inflammation, mechanisms implicated in neuronal damage in AD, biomarkers such as F-2 isoprostanes, S100B and alpha1ACT will be measured. The relationship between these biomarkers and age, sex, diagnosis (normal, MCI, mild AD), degree of cognitive impairment, and genetic risk factors for AD (apolipoprotein E [ApoE] and CYP46 genotypes) will be examined, and the extent of change in biomarkers over 12 months will be analyzed. Banked CSF and plasma will be available for further research into novel biomarkers, including broad-based proteomic studies.
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{
"pile_set_name": "NIH ExPorter"
}
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Evidence is accumulating that supports the concept that environmental carcinogenesis in rodents and humans is a time-dependent multi-genic process. Also, the importance of the inheritance of specific genetic susceptibility as a critical determinant of the disease process is being recognized. The primary assay for the identification of potential human carcinogens is the two year rodent bioassay, where inbred mice and rats are used as surrogates. We have proposed to supplement the two year cancer bioassays with rapid identification bioassays using transgenic mouse models. Two different groups of NTP chemicals are currently under evaluation in the p53-deficient (+/-) and Tg.AC mice. The first group chosen by NIEHS/NTP scientists contain both human carcinogens as well as rodent mutagenic and nonmutagenic carcinogens and noncarcinogens. The results from this group should provide information on the ability of these mouse lines to identify as well as discriminate between carcinogens of differing potency. A second group of chemicals represents another approach for validation of these models: the prospective testing by the rapid bioassays of chemicals that are currently being tested in the NTP cancer two year bioassay. Prospective analysis provides the opportunity to remove the potential bias of selection of chemicals for validation. A high correspondence between this short-term cancer bioassay and the long-term bioassay (of both groups) would provide assurance that mutagenic and nonmutagenic carcinogens and noncarcinogens may be identified using these mouse models. However, many critical questions remain to be answered in order to promote a scientific consensus on the use of these transgenic lines for identification of potential human carcinogens.
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{
"pile_set_name": "NIH ExPorter"
}
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Ambulatory children with cerebral palsy (CP) have diminished muscle power and rate of force production, which limits their walking activity and ability to physically participate in day to day life. Locomotor treadmill training (LTT) to enhance walking in children with CP has been shown to be no more effective than traditional rehabilitation strategies. The objective of this application is to determine the effect of short-bust interval LTT without body weight support on the primary outcomes of walking performance and capacity, and secondary outcome of daily participation in children with CP. To identify key muscular mechanisms with this type of training, we will examine quadriceps muscle performance and architecture. In addition, we will collect preliminary data on two LTT dosing schedules. The rationale for this proposed research is that short-burst interval LTT will enhance muscle power production and subsequently walking performance and capacity via training protocols that are motivating and mirror the walking and physical activity patterns of typically developing (TD) youth. We hypothesize that LTT strategies for children with CP modeled on activity patterns in TD children, rather than activity patterns of adults, will positively affect muscle power generation and subsequently improve community-based walking activity with a positive trend in participation. This hypothesis will be tested by the following specific aims. Aim #1: examine the effect of short-burst interval LTT on walking performance, capacity, and day-to-day participation in ambulatory children with CP. Short-burst interval LTT will consist of a 30-sec burst of high speed walking alternating with a 30-sec interval of slow to moderate speed walking, working towards a total of 30 minutes. Twelve ambulatory children, 6-12 years old with bilateral spastic CP will be enrolled. Sub-aim #1a: preliminary comparison of two dosing schedules of short-burst interval LTT. All participants will be randomized to one of two dosing schedules of 20 total sessions, 5x/week for 4 weeks or 2x/week for 10 weeks. Aim #2: quantify the effects of short-burst interval LTT on in vivo muscle architecture and muscle performance. Muscle architecture will be measured using ultrasound imaging and muscle power, strength, and rate of force development will be measured with isokinetic dynamometry. This project is innovative because it focuses on a unique approach for addressing walking limitations in children with CP and implements the StepWatch accelerometer for community-based walking activity outcomes. The proposed research is significant because it will be the first step in a continuum of research that is expected to direct locomotor training protocols and rehab strategies across pediatric disabilities and positively effect changes in community-based walking activity for children with CP. Advances in the understanding of the muscular mechanisms by which children with CP respond to high speed short-burst training are expected. Such knowledge also has relevance to the health and functional benefits of enhanced mobility and physical activity across the life span.
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{
"pile_set_name": "NIH ExPorter"
}
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The objective of this study is to analyze the structural characteristics of the retinal pigment epithelium (RPE) and retina of pearl mutant mice that could relate to the subnormal retinal sensitivity of the mutant. The immunolocalization of specific, well-characterized antisera directed against cytoskeletal components will be determined in the RPE of mutant and wild-type mice in order to identify components of the cytoskeleton associated with the basal surface of the RPE. The basement membrane (BM) of the RPE will be characterized by the immunolocalization of BM components and by the application of cationic dyes to assess the density and distribution of anionic binding sites. The chemical nature of the anionic binding sites will be determined by applying glucosaminoglycan degradative enzymes prior to the application of cationic dyes. The simultaneous application of two antisera will be used to determine cytoskeletal and basement membrane contacts. Cytoskeleton and basement membrane alterations are suggested as the cause of the structural anomalies of the basal surface of the mutant RPE, i.e., lack of basal infoldings and increased amount of basement membrane. These structural anomalies could affect RPE transport essential for the normal function of the retina. The immunolocalization of cytoskeletal components will also be determined in rod photoreceptor terminals of pearl mutant and wild-type mice. Cytoskeletal alterations in the mutant terminal could mediate alterations of synaptic lamellae structure. The modified synaptic lamellae of rod photoreceptors implicate the rod synaptic terminal as a possible site of altered synaptic transmission in the pearl mutant retina. Experiments will test whether the pearl mutant RPE influences the morphology of synaptic lamellae by observing lamellae in retinas of chimeric mice (+/+ leads to pe/pe) and pearl mutant mice which exhibit regions of reverse mutation (pe+/pe) in their RPE. An RPE influence would be demonstrated if modified synaptic lamellae were located solely in retinal areas underlying the pearl mutant RPE. The structural anomalies of the pearl RPE and retina are not unique characteristics of the pearl mutant but also are present in diseased and senescent human retinas, in diabetic BB rats and in rats with inherited retinal dystrophy. Thus, these investigations should give a better understanding of potential malfunctions of mammalian neural retina and pigment epithelium in general.
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{
"pile_set_name": "NIH ExPorter"
}
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Acute lymphoblastic leukemia (ALL) and advanced-stage lymphoblastic lymphoma (LL) respond well to remission induction therapy, but about 40% of children and 90% of adults eventually relapse and die of the diseases. The major reason for treatment failure is resistance of leukemic cells to cytotoxic drugs. The likelihood of development of resistance increases with the mass of the leukemia. Therefore, it is plausible that the smaller the burden of leukemic cells, the more likely that any given combination of drugs will successfully control the disease. Since therapy is toxic, it should be given only to those who will benefit from it. These considerations lead to the conclusion that a sensitive assay for minimal residual disease is an important requirement for improving therapy in these lymphoblastic neoplasms. In the proposed study, sensitive assays for minimal residual lymphoblastic disease will be developed. These assays are based upon the expression of terminal deoxynucleotidyl transferase (TdT) by more than 90% of these neoplasms, while less than 10% of normal marrow and less than 0.1% of normal blood cells contain the enzyme. Fluorchrome-conjugated monoclonal antibodies to TdT will be used to identify this population of cells, which will then be analyzed for a variety of markers designed to distinguish leukemic from normal TdT+ subsets in blood and marrow samples from patients who are receiving continuation therapy. The DNA content and state of activation of the TdT+ subset will also be analyzed. The frequencies of appropriate TdT+ subsets will be correlated with clinical events including relapse. The sensitivity and specificity of elevations in certain TdT+ subsets for the prediction of relapse will be calculated. If prediciton of relapse is sufficiently powerful, future trials will be designed to test whether therapeutic intervention based on specific assay results can change the outcome for patients with lymphoblastic neoplasms.
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{
"pile_set_name": "NIH ExPorter"
}
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The overall objective of this proposal is to explore the powers of the ocular proteomics approach for obtaining novel information on protein composition of individual cell types and their individual subcellular compartments. The Specific Aim is to generate a proteome map of the photoreceptor cell layer of the retina where a large number of individual proteins will be assigned to their relative subcellular locations. This will be achieved by conducting comprehensive mass spectrometry protein sequencing in progressive tangential sections throughout the photoreceptor layer. The data will be summarized in a database where all identified proteins will be grouped according to their subcellular localization, as well as their potential involvement in various functional groups. Obtaining such a proteome map would enable us and many other investigators to refine existing hypotheses and to formulate novel hypotheses regarding a broad array of photoreceptor functions and diseases. The technologies developed in this study will also serve as a template for similar investigations addressing protein distributions in other layers of the retina as well as other layered tissues. The project described in this application fulfills the goals of this Program Announcement (PAR-03-107) for two major reasons. First, it has a potential to lead to a significant technological breakthrough by establishing the methodologies for conducting large scale proteomics analyses of ocular tissues. As we are entering the post-genomics era, there is a pressing need to developing advanced proteomics approaches in many areas of ophthalmology and visual sciences. To our knowledge, several key aspects of the proposed studies (such as the analysis of serial tissue sections and the use of "two-dimensional chromatography" peptide separation protocols) have not been used in application to any ocular tissue. Second, this project aims to acquire a body of data that has a potentially high-impact on vision research because knowledge of the protein composition of individual photoreceptor compartments will benefit many scientists studying a broad array of problems in photoreceptor cell biology and elucidating molecular bases of eye diseases. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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Stool specimens submitted to Charity Hospital and the HIV Outpatient Clinic, New Orleans, LA from HIV-infected individuals with abdominal pain and/or chronic diarrhea were examined for the presence of microsporidia by histochemistry (modified trichrome staining and calcofluor staining) and PCR-based methods. In addition, survey questionaire results and clinical chart data were evaluated to assess demographics, risk factors, and natural history of microsporidiosis. Of 265 stool specimens examined, 41 (15.5%) were positive for microsporidia and 78 % of the positive specimens were collected during the summer months (May - August). Those persons with < 100 CD4+ T cells/fl blood were more likely to continue shedding microsporidia and experiencing clinical manifestations at the six-month follow-up examination, than persons with higher CD4+ T cell levels who were more likely to resolve their microsporidial infections. One risk factor identified for microsporidiosis was exposure to freshwater or seawater fish. Microsporidiosis is believed to be zoonotic since several species of microsporidia identified in humans also are known to infect other animals. Additional evidence to support this hypothesis includes the identification of Encephalitozoon cuniculi strain III (originally identified in domestic dogs) in AIDS patients in the U.S.A., and Encephalitozoon hellem, which had only been identified in AIDS patients, was recently identified in psittacine birds in an aviary in Mississippi and from archival avian tissues in Texas. A new Encephalitozoon species is being characterized in African lizards (skinks), and E. cuniculi strain I has been identified in the lens of a rabbits, suggesting that congenital transmission of microsporidiosis can occur in this host. Studies will continue to characterize new species of microsporidia that may infect humans and to identify zoonotic risks of microsporidiosis in humans.
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{
"pile_set_name": "NIH ExPorter"
}
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The DNA binding factor Ikaros and its chromatin remodeling associates of the Nucleosome Remodeling and Deacetylase (NuRD) complex control many key aspects of T cell differentiation and function providing us with a formidable entry point into the epigenetic regulation of this developmental process. Breakdown in this Ikaros-based epigenetic machinery interferes with T cell maturation and result in rapid development of T cell leukemia demarcated by activation of Notch signaling. This process and the mechanisms involved are the focus of our current investigation. In aim 1, we test the role of Ikaros in setting chromatin environments by examining the cause-effect relationship between Ikaros-loss-of-function and the epigenetic changes manifested in its immediate neighborhood as well as the transcriptional changes that follow. Several important insights are to be gleaned here, such as the role of Ikaros in setting epigenetic code, in regulating gene networks and signaling pathways that control normal development and how these are subverted for leukemia development. We also test whether a change in the potential for leukemia development seen during T cell maturation is due to a change in Ikaros gene targets or in their epigenetic state and mode of regulation. In aim 2, we go deeper into the central mechanism by which Ikaros regulates chromatin accessibility. We test Ikaros' antagonism with its chromatin remodeling associate, Mi-22, in dictating local nucleosome dynamics and histone modifications at their sites of action and the role of Ikaros' DNA binding in this process. We examine whether Ikaros promotes or inhibits access to other factors that also target its immediate neighborhood. The role of signaling pathways activated in T-ALL in targeting Ikaros' DNA binding through post-translational modifications is investigated as a potential key to altering the chromatin remodeling output of the NuRD complex to achieve rapid changes in gene expression during development. The Ikaros-based epigenetic mechanisms and the functional gene networks they control, deduced from our proposed studies will provide the means to manipulate both normal and aberrant stages of T cell differentiation. Importantly, these studies may in the future empower the design of intelligent/tailored therapies for T-ALL treatment.
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{
"pile_set_name": "NIH ExPorter"
}
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DESCRIPTION (Applicant's Description): The TEL-PDGFBR fusion protein was identified as the protein product of a t(5;12) translocation in a patient with chronic myelomonocytic leukemia (CMML). The protein fuses the amino portion of TEL with the transmembrane and cytoplasmic domains of the PDGFBR. TEL, a member of the ETS family of transcription factors, has subsequently been described as a common site of rearrangement in multiple forms of leukemia. This is not yet the case for the PDGFBR. The applicants find, however, in the Preliminary Results, that another patient with CMML has a novel t(5;7) translocation. Southern blotting analysis has identified a breakpoint in this patient at the same genomic localization in the PDGFBR as the t(5;12) translocation. Their hypothesis is, that in this patient, as for the t(5;12) TEL-PDGFR patients, PDGFBR is constitutively activated by fusion with a 7q24 partner. Although rare (as for identification of TEL), the PDGFBR fusion partner at 7q24 may identify a gene involved in a broader group of malignancies. Also, in light of the facts that other patients with CMML have PDGFR containing fusions and the region of 7q24 is frequently deleted in MDS, the cloning, characterization and manipulation of this fusion protein is paramount. Hence, in Specific Aim 1, they will use anchored PCR to clone the breakpoint. Their specific oligos will come from the known PDGFBR sequence. In Specific Aim 2, they will obtain a full length cDNA and determine the relevance of this by performing ribonuclease protection assays and mapping back to chromosome 7. Finally, for Specific Aim 3 they propose to characterize the fusion protein by determining its transforming, activity(s) and biological properties using mutational and biochemical analyses.
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{
"pile_set_name": "NIH ExPorter"
}
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The overall goal of the Tetramer Core is to develop and provide HLA Class I/peptide tetramers for use by Program Project Investigators in Projects 1, 2, 3 that examine the induction and maintenance of virus- specific CD8+ T cell-mediated immunity in humans across the age spectrum. Recently, the laboratories of Davis and McMichael have pioneered technological advances that allow the direct detection, quantitation, and isolation of antigen-specific T cells in the peripheral blood by using a fluorescent-labeled tetrameric complex of components consisting of MHC Class I alpha chain, beta2-microgolubin, and an antigenic peptide (HLA Class I/Peptide Tetramers). Individual HLA Class I-peptide tetramers must be generated for each antigen peptide to be studied at present, the availability of HLA Class I-peptide tetramers is extremely limited We therefore propose to apply or experience in the construction of the HLA Class I/HIV-1 peptidramers to the construction of tetramers for use by Program Project investigators in their studies of virus-specific CD8+ T cell immunity in human viral infections. Tetramer Core personnel will be responsible for the generation and distribution of tetramers. The generation of tetramers for Program Project Investigators will be prioritized based on identified epitopes and the distribution of HLA Class I alleles in the populations under study. The availability of this new technology to Program Project investigators should greatly enhance their ability to enumerate, isolate, and characterize virus-specific CD8+ T cells in human viral infections.
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{
"pile_set_name": "NIH ExPorter"
}
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The UCLA project develops and applies structural and bioinformatic tools for the discovery of protein drug targets and[unreadable] drugs against tuberculosis. We have developed the database for the TB Structural Genomics Consortium. This[unreadable] contains a wealth of information on the genome and proteome of Mycobacterium tuberculosis, which is open to[unreadable] researchers world wide to aid them in their structural studies of MTb proteins. Improvements to the database will[unreadable] enhance the ease with which scientists can view and update information about each protein. The information will[unreadable] include the complete history of experiments on each protein, as the project moves from cloning and expression, through[unreadable] purification, crystallization, structure determination, and drug discovery. The database will also incorporate new[unreadable] bioinformatics methods being developed at UCLA. These include methods for inferring the biological function of[unreadable] proteins from their structures by the server ProKnow and for inferring biological function from genome sequences from[unreadable] the database ProLinks. A new method for inferring biological function from combined genome sequences and[unreadable] microarray data will be developed to aid consortium members in selecting the most effective targets for structural[unreadable] studies in the effort to determine drugs.[unreadable] Structural studies of Mtb proteins at UCLA will focus on secreted, regulatory, and metabolic proteins. These proteins[unreadable] are involved in the invasion of host cells by Mtb, in the protection of Mtb against host cell defenses, in survival of Mtb[unreadable] and are thus potential protein drug targets. Secreted proteins of Mtb in particular are excellent anti- Mtb drug targets[unreadable] because drugs inhibiting these proteins do not have to penetrate the waxy cell wall of Mtb. Structures of each of these[unreadable] protein targets can then be screened in silico against ligands, in collaboration with Dr. James Sacchettini's laboratory.[unreadable] Predicted ligands will be co-crystallized for further structural studies. Potential inhibitors will be studied in collaboration[unreadable] with Dr. William Jacobs' laboratory to see if they prevent growth of Mtb within a mouse model.[unreadable] Each protein whose structure we determine, we will also characterize biochemically. Developing an in vitro assay will[unreadable] allow high-throughput in vitro drug screening, continuing our collaboration with the Southern Research Institute. All lead[unreadable] compounds will be cocrystallized with its target protein for structural studies. Where functions of proteins are unknown,[unreadable] our bioinformatics tools may predict a function, which will then be verified experimentally, to move the protein into the[unreadable] drug discovery pipeline. Dozens of MTb proteins are in the UCLA pipeline, with some half dozen at the stage of[unreadable] crystals.[unreadable]
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{
"pile_set_name": "NIH ExPorter"
}
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The proposed experiments are addressed to the question, "How do specific sites on the genome interact with those proteins which are involved in mediating and regulating the expression of the genome?" The three regions which are being studied are the promoters of bacteriophage lambda, the regulatory sequences of the lac operon, and the promoters of the tyrosine tRNA genes. Site-specific endodeoxyribonucleases are used to obtain specific DNA fragments which are fractionated by polyacrylamide-agarose gel electrophoresis. The desired fragments are identified by their ability to interact with the appropriate protein or RNA molecules and/or by their location in a reconstructed "genetic" map of the individual fragments. The desired fragments will be isolated in preparative quantities and the nucleotides sequence of the relevant regions determined. By a comparison of different but analogous regions, by the sequencing of selected mutants, and by in vitro biochemical analysis of the isolated fragments, attempts will be made to correlate the structure of these genomic regions with their known biological properties. It is also proposed to continue present experiments on the enzymology of site-specific deoxyribonucleases, procedures for fractionating specific DNA fragments, and the application of these techniques to eucaryote DNAs.
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{
"pile_set_name": "NIH ExPorter"
}
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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. Effective strategies for HIV prevention are urgently needed, but recent failures in key vaccine and 'microbicide'clinical trials highlight the need for new approaches validated in relevant animal models. In the continued absence of an effective vaccine, a microbicide that can be topically applied to the vagina may be the best hope for slowing vaginal HIV-1 transmission. However, such a microbicide must be highly effective and also inexpensive, so that it may be distributed to economically challenged areas where the epidemic is spreading most rapidly. We have previously shown that the CCR5 inhibitor PSC-RANTES can completely prevent vaginal transmission of R5 SHIV to macaques, but the cost of production limits its potential for a practical microbicide. We have thus developed two novel RANTES analogs that promise low cost recombinant production by fermentation. Here we show that these two new CCR5 inhibitors, 5P12-RANTES and 6P4-RANTES, protect fully against infection in the rhesus vaginal challenge model. These highly potent molecules, which are amenable to low-cost production, represent promising new additions to the microbicides pipeline.
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{
"pile_set_name": "NIH ExPorter"
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The development of novel and effective treatments for viral infections requires a fundamental understanding of how viruses recruit the host cell translational machinery for viral protein synthesis during the early stages of infection. Understanding how large multi-subunit translation initiation factors and viral RNAs control the binding and activity of ribosomes poses a formidable challenge that forms the basis of this grant proposal. To tackle the difficult mechanistic and structural problems inherent in studying translation, we propose to launch a coordinated and interdisciplinary effort to determine the compositions, intermolecular interactions and structures of key initiation factor complexes and unravel the mechanisms that regulate protein synthesis in human cells and viruses. A key aspect of this P01 Program Project Grant will be to establish highly interactive collaborations across disciplines and research institutions to study the chemical, structural and mechanistic properties of key complexes controlling translation initiation. By combining the expertise of several investigators we propose to implement a battery of complementary biophysical and biochemical approaches including: Projects by Doudna and Cate, cryo-electron microscopy and X-ray crystallography of translation initiation factors and complexes;Project by Hershey, in vitro and in vivo translation assays with viral and cellular translation components;and Project by Sarnow, purification and analysis of viral translation complexes. A major component of this proposal will be the establishment of a core laboratory for mass spectrometry of large multi-subunit complexes that will serve as the nerve center and clearinghouse for analyzing the many protein and protein-RNA complexes involved in translation initiation. This core laboratory will be used extensively by all five Research Projects to identify the macromolecules and post-translational modifications that, respectively, comprise and control translation activity in human cells and viruses. Thus, our goal is to develop a highly synergistic and concerted effort to carry out a pioneering set of interdependent experiments by using overlapping molecular reagents but applying distinct and complementary research strategies to dissect the core machinery responsible for protein synthesis in normal and virally-infected mammalian cells.
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{
"pile_set_name": "NIH ExPorter"
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Liver cancer is the 5th most common cancer (748,000 cases a year) and the 3rd most common cause of cancer death (695,000 deaths a year) in the world. In the United States, while the incidence of many major cancers is declining, the incidence of hepatocellular carcinoma (HCC), the most common type of liver cancer, has almost tripled over the past two decades to ~20,000 per year. Despite the success of HBV vaccines to prevent new HBV infection, the large pool of existing HBV (350 million) and HCV (170 million) patients and the recent recognition that obesity/diabetes become a major risk factor of HCC in US suggest that HCC incidence will continue increasing over next few decades. Unfortunately, treatment options for HCC are limited, contributing to the dismal 17% 3-year survival rate. Liver resection and transplantation are curative for small tumors. But, the lac of adjuvant treatments after surgery is a critical barrier to the success of liver resection, which results in ~70% 5-year recurrence rate. Thus, there is an urgent need to develop novel approaches for HCC. Our long-term goal is to develop HCC vaccines to activate patient's immune system to conduct immune surveillance and thereby prevent HCC relapse or de novo development in high risk population. Approximately 84% of HCC (but not normal hepatocytes) express alpha fetoprotein (AFP), glypican 3 (GPC3), or both, making them excellent immune targets. However, they are self/tumor antigens and weakly immunogenic, and thus cannot effectively activate immune cells to generate significant antitumor effect. In this application, we propose to use murine autochthonous models to test the hypotheses that innovative approach of antigen engineering will increase the immunogenicity of HCC-associated self/tumor antigens of AFP and GPC3 to create effective HCC vaccines and that novel immunization strategy with the engineered HCC vaccines will induce potent and highly responsive memory T cells that conduct immune surveillance to prevent autochthonous HCC. The objectives are 1) to create immunogenic HCC vaccines, 2) to establish effective immunization strategy, and 3) to prevent autochthonous HCC. The outcomes will be the effective activation of immune system to prevent autochthonous HCC and generation of critical preclinical data that will help development of human HCC vaccine, which eventually lead to new therapy to fill the gap of HCC management after surgery. This will greatly benefit HCC patients who have undergone liver resection by preventing relapse and also offer an attractive option to prevent HCC de novo development in high risk populations who are living in the fear of developing HCC. This will bring about a radical change of HCC management, shifting the focus of cancer vaccine research from therapy to prevention, which is what a vaccine does best.
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{
"pile_set_name": "NIH ExPorter"
}
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CD36 is a broadly expressed 88 kD transmembrane glycoprotein which functions as a scavenger receptor, adhesion and signaling molecule and a facilitator of fatty acid transport. Previous work by our group and others determined that CD36 was a major mediator of macrophage foam cell formation and atherogenesis. Recently, a paradox has emerged: significant protection against lesion development and progression was observed in experimental conditions where CD36 was rendered absent genetically and when CD36 was upregulated by pharmacological means. Using stem cell transfer of CD36/apoE bone marrow into apoE null animals, we found that the atheroprotective effect afforded by complete absence of CD36 was not fully realized. Thus, a major hypothesis of this application is that CD36 has atheroprotective properties independent of its role as a scavenger receptor. Based on emerging data, we focus on the role of CD36 in efflux pathways and in modulating insulin-resistance, both of which are stimulated by drugs which activate PPARs, transcription factors which regulate CD36 expression. We hypothesize that CD36 is a critical player in maintenance of vessel wall homeostasis, and that entry of cholesterol into macrophages via CD36 facilitates removal of pro-atherogenic modified LDL from the intima, and ultimately allows lipid clearance through efflux pathways. Elucidation of the regulation of CD36 in relationship to the major efflux pathways is a major goal of our studies. Insulin resistance is a separate risk factor for atherogenesis and results in its premature development. We hypothesize that a portion of the protective role of CD36 in atherogenesis is related to its effect on glucose and fatty acid metabolism. We will utilize unique animal models we have on hand to explore the differential impact of CD36 on atherosclerosis based on scavenger receptor function or its role as a facilitator of fatty acid transport and determinant of insulin sensitivity. These approaches will enable us to design more specific treatment regimens which will ultimately impact on human health and disease.
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{
"pile_set_name": "NIH ExPorter"
}
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The overall goal of this research proposal is for Stanford University, the University of Arizona, and the Kaiser Permanente Medical Centers of the South San Francisco Bay Area to continue their active involvement in Pediatric Oncology Group research activities. Stanford faculty and the University of Arizona faculty have already assumed key leadership positions in POG and have or have had major roles in the scientific and administrative aspects of the Group. Further, Stanford, the University of Arizona, and Kaiser have maintained excellent performance ratings in their participation in POG studies and have received commendations for the large numbers of evaluable patients placed on therapeutic protocols. Specifically: l) We plan to continue to enter patients on appropriate POG studies where they exist. The number of patient entries from Stanford has increased each year as appropriate POG studies become available. We anticipate that between 65 and 80 patients will be entered on front-line therapeutic studies each year from Stanford in addition to patients who will be entered from the affiliates; in addition, 40-50 patients or more will be entered on POG non-therapeutic studies. 2) We anticipate that the activities of individual investigators from Stanford and the University of Arizona will continue and increase during the period of this research proposal. Currently, our faculty serve as study coordinators for front- line therapeutic studies in lymphoma and leukemia, and they have coordinated and analyzed data from recently closed protocols in osteosarcoma, lymphoma, leukemia, and Ewing's sarcoma. Our faculty also serve key scientific and administrative roles as Group Vice Chair, Disease and Discipline Committee Chairmen and Co-Chairmen, as members of Disease and Discipline Core Committees, and as members of the Executive Committee. Thus, our faculty are in position to influence the future direction of the scientific activities of POG. 3) We anticipate that involvement of Stanford faculty in the laboratory scientific activities of POG will continue. The laboratories of Drs. Link and Cleary have served as immunology reference laboratories and molecular biologic reference laboratories for leukemia studies of POG. 4) We anticipate that non-POG related laboratory and clinical research conducted at Stanford University and its affiliates will become increasingly relevant to POG activities. Some of these activities have already been incorporated into POG laboratory and therapeutic studies and others are targeted for incorporation into future POG studies.
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{
"pile_set_name": "NIH ExPorter"
}
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Across developmental transitions, relationships with family, peers, and intimate partners can be contexts of risk or resilience. Ideally, these relationships protect and support positive adaptation to new roles, but sometimes they expose the individual to risk that is predictive of later problem behaviors, such as substance abuse. Our research team has developed an effective intervention that targets these key relationships in order to reduce substance use and other risk behaviors while simultaneously enhancing protective factors that lead to healthy, adaptive behavior. This intervention, called the Family Check-Up (FCU), has been studied across various ages and samples. We propose to now focus on the transition to adulthood, the period associated with the highest rates of substance use and life-threatening risk behaviors (e.g., high-risk sexual behavior, drinking and driving). This study will extend our previous work by offering the FCU as youth transition out of high school. It will examine how parent-child relationships in late adolescence may be protective or may contribute to escalating substance use and abuse typically observed during the transition to adulthood. We propose to follow up an existing community sample of 593 ethnically and socioeconomically diverse youth and their families who were originally recruited at age 11 as part of a school-based, family-centered intervention for substance use prevention during the middle school years. Families assigned to the original treatment condition will be offered an additional intervention that would target parent-youth relationships during this transition, and would provide critical information about developmental changes in family processes that protect youth from substance abuse during this period. The late-adolescence version of the FCU intervention is designed to (a) prevent escalation of substance use, (b) focus on parent-youth relationships that foster independent living, (c) discourage unhealthy peer relationships and activities that promote drug use, and (d) provide support to enhance adaptive behavior and healthy adult outcomes during the transition to adulthood. We will also evaluate the enduring efficacy of the FCU and the preliminary efficacy of the FCU for late adolescence in terms of reducing growth in substance use and problem behaviors and supporting healthy adaptation. The study will significantly contribute to our understanding of parenting in late adolescence and risk factors associated with substance use, as well as protective factors that lead to health and adaptive behavior in early adulthood. If successful, this intervention will be a cost-effective means to reduce substance use and other risk behavior that can be embedded into high schools as youth transition to independent living. PUBLIC HEALTH RELEVANCE: The focus of this study is to examine how parent-youth relationships in late adolescence may be protective or may contribute to escalating substance use and abuse during the transition to adulthood. We propose to follow up an existing community sample in order to understand unique aspects of parenting during this period, and to administer a family-based intervention designed to (a) prevent escalation in late adolescence substance use, and (b) promote protective factors that lead to health and adaptive behavior in emerging adulthood. It is anticipated that this intervention will be a cost-effective means to reduce substance use and other risk behavior that could be easily implemented in a variety of service settings across the United States.
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{
"pile_set_name": "NIH ExPorter"
}
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The goal of this proposal is to gather preliminary data to test the feasibility and calculate the sample size for a larger study on the effects of age on skin protein turnover and wound healing in humans. [unreadable] [unreadable] Our general hypothesis is that dermal collagen turnover changes with aging and these changes are [unreadable] responsible for many of the alterations in skin function found in older adults, which may increase the risk of mechanical injury and delayed wound healing. Therefore, the measurement of dermal collagen turnover may be used as an objective tool to determine the effects of age on the skin, quantify the rate of wound healing, and test the effects of specific interventions aimed at stimulating skin regeneration and repair. Since collagen makes up the bulk of skin proteins, we further hypothesize that mixed skin protein kinetics can reliably reflect the turnover rate of skin collagen, and thus be used as a surrogate measure of skin collagen turnover. [unreadable] [unreadable] The specific aims of this proposal will be (1) to determine whether mixed skin protein turnover can be used as a surrogate measure of skin collagen turnover in human subjects, and (2) to determine whether skin protein synthesis is slower and/or breakdown faster in older subjects as compared to younger controls. [unreadable] [unreadable] To accomplish our aims we will simultaneously measure the synthesis and breakdown rates of mixed skin proteins, and the skin collagen synthesis rate in healthy human subjects using a new stable isotope method. [unreadable] [unreadable] The data obtained with this pilot project will be used as a basis for further studies (to pursue using the [unreadable] R01 mechanism) on the effects of aging on skin metabolism and wound healing. [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
}
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Age Related Macular Degeneration (AMD) is the leading cause of blindness in adults over 50 years. Choroidal neovascularization (CNV), the pathologic creation of new blood vessels in the choroid layer of the eye, is a principal cause of blindness due to AMD. Current therapeutic management of AMD is far from optimal and requires repeated injections in the vitreous cavity of the eye. Such repeated administrations, besides being inconvenient for the patient, might lead to retinal detachment and endophthalmitis. Thus, there is an unmet need to develop delivery systems which can be administered by relatively safer routes of administration such as topical dosing. Therefore, this project is aimed at investigating the feasibility of using a nanoparticle technology of NanoTrans for topical administration of a model drug diclofenac, for the management of choroidal neovascularization. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) that is off patent, allowing its use in conjunction with proprietary NanoTrans nanoparticle technologies for product development. Further, diclofenac eye drops are currently approved for human use, especially for treating inflammation of the eye surface and the anterior eye segment, minimizing the regulatory hurdles in the development nanoparticle eye drops of diclofenac. Diclofenac inhibits cyclooxygenase (COX) enzymes and hence inflammatory mediators including vascular endothelial growth factor (VEGF), a key growth factor underlying the pathology of choroidal neovascularization. However, currently available eye drop formulations of diclofenac do not deliver the drug to the back of the eye in adequate amounts for the drug to be effective in treating choroidal neovascularization. The purpose of this study is to test the hypothesis that nanoparticles surface functionalized with a permeability enhancing ligand can increase diclofenac delivery from an eye drop to the back of the eye, resulting in improved efficacy in a laser induced choroid neovascularization model. Besides CNV treatment, the proposed diclofenac nanoparticles are of potential value in treating other inflammatory disorders of the back of the eye. The findings and technologies developed in this study can potentially be extrapolated to other therapeutic agents intended for treating disorders of the back of the eye. PUBLIC HEALTH RELEVANCE: Age related macular degeneration (AMD) is a disease of the back of the eye that can cause blindness. The available treatments require frequent injections into the eye, which is painful, inconvenient, and potentially damaging to the eye. The proposed project is aimed at the development of an eye drop dosage form based on nanoparticle technologies of NanoTrans for treating AMD. Use of nanoparticle technologies of NanoTrans are expected to improve drug delivery to the back of the from an eye drop. Such an approach avoids the pitfalls associated with invasive drug administrations. The proposed technologies are likely to benefit various drugs intended for treating diseases of the back of the eye.
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{
"pile_set_name": "NIH ExPorter"
}
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This proposal outlines a plan to investigate the mechanism of assembly of E. coli ribosomes both in vitro and in vivo. The ribosome is the macromolecular machine responsible fbr all protein synthesis in cells, and it is efficiently assembled from over 50 components. Using a combination of biophysical methods, we will Investigate the order of events in assembly and the kinetics of assembly in vitro. We will examine the role of assembly cofactors in E. coli, and examine the effects of antibiotics on the assembly process. We have developed a novel isotope pulse-chase assay that enables measurement of binding kinetics for ribosomal proteins using mass spectrometry. We are developing a two-photon fluorescence correlation microscope to monitor the assembly of fluorescently labeled ribosomal proteins in real time. Finally, we are extending our mass spectrometry analysis to isotope pulse experiments in living bacteria, which allows us to directly monitor the biogenesis of ribosomes. Taken together, these studies will provide new mechanistic insights into the critical process of ribosome assembly.
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{
"pile_set_name": "NIH ExPorter"
}
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PROJECT SUMMARY Uterine vascular adaptation with the striking increase of uterine blood flow during gestation is essential for fetal development as well as for cardiovascular well-being of the mother. Although vasodilatation is of critical importance in the adaptation of uterine circulation to pregnancy, the underlying mechanisms are not fully understood. Spontaneous transient outward currents (STOCs) at physiological membrane potentials (? 30-40 mV) of vascular smooth muscle cells fundamentally regulate vascular myogenic tone and blood flow in an organ, as well as arterial pressure. Our preliminary studies in sheep revealed that pregnancy significantly increased STOCs in uterine arterial smooth muscle cells, identifing an exciting regulatory target in uterine vascular adaptation in gestation. STOCs are mainly mediated by large-conductance Ca2+- activated K (BKca) channels. The BKca channel in vascular smooth muscle contains the channel-forming ? + subunit and up to four accessory ?1 subunits that sense Ca2+ sparks via the opening of ryanodine receptors. Our previous studies demonstrated a significant upregulation of BK?1 subunit expression, resulting in increased ?1:? subunit stoichiometry in uterine arteries of pregnant animals. This resulted from a decrease in promoter methylation of the BK?1 subunit gene in uterine arteries. Dynamic regulation of DNA methylation and demethylation is a chief mechanism in epigenetic modifications of gene expression pattern and biological function. Recent studies have suggested a robust mechanism of ten-eleven translocation 1-3 (TET1-3) proteins in active DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Our preliminary studies showed that pregnancy and steroid hormones significantly increased TET1 and TET2 expression and 5hmC abundance at the BK?1 promoter in uterine arteries, suggesting a highly novel mechanism of hormone-mediated active DNA demethylation in uterine vascular adaptation to pregnancy. In addition, the preliminary data suggested that pregnancy resulted in a significant increase in Ca2+ sparks mediated by ryanodine receptors in uterine arteries. With these exciting findings and many highly novel leads, we are now positioned to move the field forward significantly in a manner by launching a new focus of research aiming at the understanding of molecular and epigenetic mechanisms in the regulation of STOCs that are of fundamental importance in uterine arterial vasodilatation during gestation. Thus, the proposed study will test the hypothesis that pregnancy upregulates BK?1 subunit expression via TET-mediated DNA demethylation and increases ryanodine receptor-induced Ca2+ sparks, resulting in heightened STOCs in uterine vascular adaptation to pregnancy. The outcome of proposed study will provide novel insights in molecular mechanisms of uteroplacental adaptation to pregnancy, and will have a major impact in our understanding of pathophysiologic mechanisms underlying pregnancy complications including preeclampsia and fetal growth restriction, caused by maladaptation of uteroplacental circulation. Given that STOCs are fundamentally important in regulating vascular tone and pressure in virtually all vascular beds, revealing epigenetic regulation of STOCs function in vascular smooth muscle will indeed have a much broader impact in the comprehensive understanding of molecular mechanisms in vascular physiology and pathophysiology.
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{
"pile_set_name": "NIH ExPorter"
}
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Antigen mediated histamine release from cultures of RBL-2H3 cells is associated with increase in cytosol Ca++ levels (Ca signal) and substantial hydrolysis of membrane inositol phospholipids. Several clones of the RBL-2H3 cell line showed varied responses to antigen that ranged in extent from undetectable (BUDR 1A3, 2B1 and 1B3) to about 80% of those in 2H3 cells (TG 2B6). The initial rate of response in the partially responsive clones was similar to that of 2H3 cells but the maximal responses were blunted. In most of these clones, as in the 2H3 cells, the Ca signal (as measured by quin 2 fluorescence) and hydrolysis of the phospholipids were correlated. However, TG 1B3, which showed very little Ca signal, still showed modest phospholipid hydrolysis and histamine release. Phospholipase C activity towards all inositol phospholipids was present in extracts and membranes of all the clones tested. Moreover, activity in the nonresponsive clones was 3 to 5 times higher than that in 2H3 cells. Studies with phorbol ester and Ca2+ ionophore also indicated the presence of protein kinase C activity in the 1A3 and 1B3 clones. These data point to no obvious defect in the genetic expression of enzymes involved in the inositol phospholipid cascade system in the nonresponsive clones. Our preliminary indications are that these clones have defective coupling of IgE receptors to phospholipase C through G-protein(s).
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{
"pile_set_name": "NIH ExPorter"
}
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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. The development of cutting edge technologies for imaging the mitochondrial metabolic function including the redox state of biological tissues are in great need, since mitochondria and its functional/genetic abnormalities have been connected to a number of diseases including cancer, diabetes, cardiovascular diseases, and neurodegenerative diseases. Low temperature NADH/Fp (reduced nicotinamide adenine dinucleotide/oxidized flavoproteins) fluorescence imaging or "redox scanning" has been developed to image the in vivo mitochondrial redox states of tissues on the basis of the redox ratios (Fp/NADH or Fp/(NADH+Fp)). A CCD-based redox imager has also been built to acquire images much faster with higher spatial resolution (~10 [unreadable]m). However, redox ratio is based on the ratio of the relative intensities of the fluorescence signals of NADH and Fp, which usually depend on instrument settings such as filters and lamp conditions. In this subproject we aim to 1) perform calibrations of the redox scanner and the CCD imager to obtain absolute NADH, Fp and redox ratios to facilitate the comparison of results obtained at different time with different instrument settings;2) application of redox imaging to differentiate breast tumor mouse xenografts with different metastatic potentials.
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{
"pile_set_name": "NIH ExPorter"
}
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Pregnant guinea pigs will be exposed to tape recorded textile mill (weaving room) noise. The hearing level of offspring will be measured and compared with that of offspring of otherwise identical mothers. Study of the effects of high level sounds on fetuses in utero is important because: 1. Many women are moving into very noisy occupations, some as jet engine mechanics where whole body exposures exceed 120 dBA, 2. a period of enhanced susceptibility was recently demonstrated to occur in hamsters beginning at the point of final maturation of the ear, i.e., about 20 days post partum. 3. the equivalent period on humans and guinea pigs occurs near the end of the last trimester, 4. a Polish study recently found elevated hearing thresholds in guinea pigs exposed in utero to weaving room noise.
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{
"pile_set_name": "NIH ExPorter"
}
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The development of neuromuscular junctions will be studied in vitro where the process can be disescted by the methods of biochemistry, electrophysiology and electron microscopy. Clonal cell lines of nerve and muscle and primary cultures derived from Xenopus embryos will be used in these studies. The mechanism of acetylcholine receptor localization and other trophic effects of innervation will be investigated. The biochemical requirements for synaptogenesis will be examined using specific probes for membrane components. The defect in myasthenia gravis will be investigated. The mechanism by which antibody against the acetylcholine receptor increases receptor degradation will be studied.
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{
"pile_set_name": "NIH ExPorter"
}
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Primorigen Biosciences LLC SBIR Project Abstract Primorigen will use SBIR funds to develop and validate a microcarrier-based cell culture system for selection, scale-up and feeder-free mass production of iPS cells, based on Primorigen's proprietary pluripotent stem cell attachment factor (StemCadhere") for direct physical selection and propagation of iPSC. Phase I studies will define coupling chemistry that optimizes E-cadherin mediated cell adhesion to StemCadhere"-coated microcarriers, analyze iPSC attachment efficiency and propagation on the StemCadhere"-coated microcarriers, analyze the utility of StemCadhere-coated microcarriers for direct physical selection of newly induced iPS cells, and compare performance of cryopreserved iPSC on StemCadhere"-coated microcarriers with Matrigel-coated microcarriers. This work will provide the foundation for Phase II studies, which will investigate production of differentiated cells directly on the microcarriers. Similar methods will be used to test and select cellular adhesion molecules specific for each germ layer (ectoderm, mesoderm and endoderm), and for a limited number of terminally differentiated lineages such as hepatocytes, cardiomyocytes, and hematopoietic cells. The initial commercialized products will be a) ready-to-use (cryopreserved) iPS cells attached to the microcarriers, ready for addition of differentiation factors and downstream protocols;and b) a new application for attachment factors coupled to microcarriers that Primorigen can market at large to pharmaceutical and biotechnology firms engaged in differentiated cell production from iPS. PUBLIC HEALTH RELEVANCE: Primorigen Biosciences LLC SBIR Project Narrative Current methods for production of iPS cells require labor intensive manual culturing steps, frequent feeding of cultures, and inclusion of drug-selectable resistance markers to achieve purification of iPS from non-induced parental cells (such as foreskin fibroblasts). To fulfill the promise of iPS cells as a source for cellular therapies and differentiated cells for pharmaceutical development, it is mandatory to avoid the use of lentiviral vectors and other agents capable of genomic integration, which precludes the use of integrated drug-resistance markers for iPS selection. In addition, to decrease variability in production lots, it is desirable to automate as much of the culture as possible, while maintaining the proliferation and pluripotency of the iPS cells. Primorigen's SBIR proposal will address this need by developing a comprehensive, integrated solution for selection and mass production of iPS cells by taking advantage of Primorigen's proprietary stem cell attachment factor for feeder-free propagation of iPS cells in microcarrier suspension culture.
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{
"pile_set_name": "NIH ExPorter"
}
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The nicotinic Acetylcholine (ACh) receptor mediates fast synaptic transmission by converting transiently to an open-channel form when activated by ACh released into the synaptic cleft. Our goal is to understand the structural transition underlying this event, i.e. to provide a definitive molecular picture of how the channel works. The ACh receptor-rich membranes for our studies come from the electric organ of the Torpedo ray, and we analyze tubular membrane crystals grown from them by cryo-electron microscopy. The proposed research builds on a long-term programmed of technical development, which recently yielded a refined atomic model of the membrane-bound receptor in the closed-channel form. To investigate the open-channel structure we will propel acetylcholine-containing spray droplets onto the tubular crystals lying within a thin aqueous film on the e.m. grid and then trap the reaction (within ~2ms) by plunge-freezing. By this means we recapitulate the brief reaction of neurotransmitter with receptors at the synapse and capture the opened channels in both a physiological ionic environment and a native membrane setting. The electron images will be recorded with an ultra- stable liquid-helium cooled stage to minimize radiation damage and to optimize electron- optical performance. Distortion correction of the tube images by alignment of successive segments will incorporate a newly developed approach to optimize the extraction of high resolution information, and the computer programs will be automated/made-more-general in collaboration with colleagues working on related specimens at Scripps. Our electron crystallographic methodology provides a unique opportunity to analyze the open ACh receptor channels because it is the only structural method devised so far that can examine this short- lived transition under essentially the same conditions as exist in vivo. It is unlikely that x-ray structures of purified ACh (or other related) receptors, if they are obtained in the future, would be able to deliver such an unambiguous result. PUBLIC HEALTH RELEVANCE: The nicotinic acetylcholine receptor is the best understood member of a family of synaptic ion channels which function in the central and peripheral nervous system, and are pharmaceutical targets for numerous human diseases and psychiatric disorders, including myasthenia gravis, neuromuscular degeneration, epilepsy, depression, nicotine addiction, schizophrenia and Alzheimer's disease. Our structural studies are providing three-dimensional information about the binding sites for anesthetics and other drugs which affect the brain by modulating the function of these receptors. By understanding better the biological mechanisms of these receptors, we contribute basic insight into the nature of the disorders themselves.
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{
"pile_set_name": "NIH ExPorter"
}
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The objectives of this grant continuation are: 1) To define the regulatory sequences in the gene of the bovine 9-kilodalton vitamin D-dependent calcium-binding protein (9-kD CaBP), 2) To sequence the human 9-kD CaBP gene and determine its tissue specific expression and, 3) To determine the amino-acid sequences that are critical to the binding of calcium (Ca) in the 9-kD CaBP and the related 28-kD Ca binding protein. We hypothesize that: 1) Because the synthesis of CaBP is under transcriptional control, it is likely that 1,25(OH)2D3-regulated promoter or enhancer elements are present proximal (5') to the transcription start site or in the gene itself; 2) The structure of the 9-kD CaBP gene is conserved through evolution, suggesting an important functional role for certain substructures in the gene; 3) The tissue specific expression of human 9-kD CaBP is similar to that found in other species; 4) Ca binding by the protein is mediated by critical residues or regions in the protein. The specific aims of our proposal are as follows: 1. a) To sequence the bovine 9-kD CaBP gene and, b) to identify promoter and/or enhancer motifs adjacent to or in the gene of the 9-kD CaBP that are regulated by 1,25(OH)2D3. 2. a) To isolate and determine the sequence of the human 9- kD CaBP gene. b) Examine various tissues for the expression of the mRNA for this protein. 3. a) To examine the Ca and fluorescent lanthanide binding properties of full-length recombinant 28-kD CaBP and its mutants and to b) to express full-length 9-kD CaBP and its mutants in order to examine the Ca and fluorescent lanthanide binding properties of the proteins. Our methods will include the sequencing of genomic clones specific for the bovine and human 9-kD CaBP, construction and testing of reporter plasmids with appropriate putative promoter regions of the gene(s) and the synthesis and expression of appropriate plasmids containing the cDNAs for the 9-kD and 28-kD CaBP. The expressed products will be tested for Ca and lanthanide binding. An understanding of the mechanism(s) governing the synthesis of CaBP (structure of the 1,25(OH)2D3-regulated promoter and its function in the presence or absence of receptor) and the stereochemistry of Ca2+ binding by the protein which binds calcium are important in our understanding of calcium transport in man and experimental animals.
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{
"pile_set_name": "NIH ExPorter"
}
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The experiments described in this proposal examine a newly defined regulatory mechanism, a developmental checkpoint - the developmental analogue of a cell-cycle checkpoint - that is activated by tissue damage during larval growth of the fruit fly, Drosophila melanogaster. This developmental checkpoint regulates systemic endocrine and metabolic signals to delay development and preserve the regenerative capacity of damaged tissues. The systemic influences on the regenerative capacity of tissues are poorly understood. Therefore, a mechanistic understanding of this developmental checkpoint will provide an important, new insight into the regulation of the systemic signals that control regenerative growth. The aims of the research described in this proposal are to build on our initial observations and further define each of the key steps within this developmental checkpoint. First, this proposal explores how retinoid signaling, which participates in checkpoint activation, functions to regulate neuroendocrine activity in the larval brain and produce developmental delay. The relevant functional localization of retinoid biosynthesis during damage will be examined using transgenic constructs, in situ hybridization, and tissue-targeted inhibition and rescue of retinoid biosynthesis. Molecular and genetic experiments will be used to determine the role of retinoids in inhibiting a neuroendocrine positive feedback loop that regulates developmental progression. Second, experiments in this proposal are designed to identify the damage signal that regulates the systemic responses to localized tissue damage. Genetic epistasis experiments will be used to test the role of Eiger, the Drosophila TNF homologue, in producing the systemic damage signal that mediates the developmental checkpoint activation. At the same time, unbiased genetic approaches will also be used to establish the identity of the damage signal. Third, this proposal examines the systemic metabolic effects produced by localized tissue damage. The effect of local tissue damage on systemic insulin signaling will be examined using several tissue-specific and systemic reporters, and the role of altered insulin signaling in checkpoint delay will be examined using tissue-targeted inhibition of the forkhead-transcription factor, dFoxo. Completion of the experiments described in this proposal will provide valuable new insights into the regulation of the systemic signals that control tissue regeneration. In addition, these experiments will reveal the mechanisms by which persistent tissue damage and inflammation can produce systemic effects on growth and endocrine control of development, which are each observed in clinical examples of persistent inflammation such as obesity-related insulin resistance and cancer cachexia. PUBLIC HEALTH RELEVANCE: The capacity of tissues to regenerate is widely varied across evolution. However, little is understood about the systemic developmental signals that govern regenerative capacity. This proposal examines a molecular mechanism by which damaged tissues regulate systemic metabolic and endocrine signals to preserve regenerative competence. Describing this mechanism will provide a greater understanding of the systemic signals that govern regenerative growth and should also provide new insights into the systemic responses to chronic tissue damage and inflammation (e.g. impaired growth, developmental delay, and cachexia) that have a profound impact on patients in a clinical setting.
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{
"pile_set_name": "NIH ExPorter"
}
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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our goal is to determine intermolecular interactions that regulate the concerted assembly of Venezuelan equine encephalitis virus (VEEV), an alphavirus that causes epidemics in Central and South America, and a highly developed biological weapons agent without effective therapy. Aim 1. To determine interactions between the VEE virus capsid protein and RNA that promote viral nucleocapsid assembly, using complementary biochemical, biophysical, molecular and structural biology approaches. Aim 2. To determine if intermolecular capsid protein interactions within the nucleocapsid undergo large-scale rearrangements during VEE virus maturation by determining the structure of pre-viral, viral, and post-viral nucleocapsids.
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{
"pile_set_name": "NIH ExPorter"
}
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This core serves to develop and make accessible technology for stochastic optical reconstruction microscopy (STORM), a combination of selective chromophore activation and computational reconstruction that has resolution in the 20-30 nm range. We begin with an instrument that was almost fully constructed, donated to the Core by the Corey Lab, but we needed to complete construction and develop a use-friendly interface appropriate for general use by a core user base. An additional goal was to continue to develop the instrument to reflect technological advances in STORM in the field.
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{
"pile_set_name": "NIH ExPorter"
}
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Project Summary/Abstract. There are 3.5 million annual cases of Traumatic Brain Injury (TBI)1 in which an individual receives an injury to the head that results in a temporary loss of consciousness (LoC) or alteration of consciousness (AoC).2 By age 25, nearly one-third of young adults will experience repeated TBI (rTBI; i.e., 2 or more injuries resulting in LoC or AoC),3 thereby exacerbating the potential for persistent cognitive and emotional dysfunction that is linked to a variety of psychopathological conditions,4,5 including problematic cannabis use.6-8 Given recent changes to cannabis legality, and associated increases in cannabis use,9 it is critical to understand the apparent link between rTBI and cannabis problems, defined as impairment in social (e.g., difficulty maintaining relationships), occupational (e.g., decreased performance at work or school), or financial (e.g., being unable to pay bills due to substance use) domains as a result of cannabis use. Although several epidemiological surveys have demonstrated that those with a TBI history have increased rates of problematic substance use relative to non-TBI controls little is known about the potential mechanisms explaining this link due to the systematic exclusion of individuals with a rTBI history from nearly all prospective investigations of cannabis use and the exclusion of cannabis users from nearly all prospective studies of rTBI (see Bjork and Grant, 2009 for a review).10 This oversight has left inadequate answers to two concerning questions: Does rTBI history predict greater cannabis-related problems and, if so, what are the explanatory mechanisms of this vulnerability? Consistent with a central focus of NIDA on identifying explanatory processes of vulnerability in high-risk populations, the present study proposes a novel prospective investigation that tests whether rTBI is associated with cannabis-related problems. We will then test putative indirect pathways via which rTBI may increase cannabis problems by utilizing event-related potential (ERP) methodology, notably the Late Positive Potential (LPP) amplitude while viewing and down-regulating responses to emotional images and the N2/P3 component amplitudes during a response inhibition task. The first specific aim of the proposed study is to test if rTBI has a direct relationship with cannabis problems. This will be accomplished through both cross-sectional (i.e., baseline) and prospective cannabis problems, as assessed via self-report and clinical interview. The second specific aim is to test the indirect effect of rTBI on cannabis problems through heightened emotion reactivity (i.e., greater LPP while viewing cannabis cues and threatening images). The third specific aim is to test the indirect effect of rTBI on cannabis problems through emotion regulation difficulties (i.e., greater LPP while volitionally regulating cannabis craving, greater LPP while cognitively re-appraising threatening images). Lastly, the fourth specific aim is to test the indirect effect of rTBI on cannabis problems through deficits in inhibitory control (i.e., blunted N2 and P3 component amplitudes during response inhibition).
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{
"pile_set_name": "NIH ExPorter"
}
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The long-term goal of this research is the investigation of the cellular physiology of the placental transfer of important nutrients. These investigations have been extended from original studies of amino acid transport. During the forthcoming project period they will focus on the transfer of both amino acids and calcium and investigation of the cellular and biochemical processes that may be expected to underlie their transport in the human placental trophoblast. Amino acids are utilized by the fetus for growth and catabolism and calcium for skeletal mineralization. The importance of these processes, the large quantities required by the fetus and the recognition of clinical disorders of fetal growth and neonatal calcium metabolism all point to the importance of understanding the placental transfer of these important nutrients. The strengths of this project are: (1) its use of well-characterized specific preparations, some of which were developed specifically for this project, of each of the two plasma membranes and of subcellular material from the syncytiotrophoblast; and (2) its relatively unique application to the placenta of established methods for the elucidation of cellular transport mechanisms whose utility has been proven in widely accepted reports from many laboratories studying a variety of cellular and subcellular systems. Specific aims include the investigation of calcium transport methanisms of the syncytial microvillous and basal plasma membranes and cytoplasm, the characterization of amino acid transport systems of the two plasma membranes, and the investigation of the regulation of placental cellular pathways for calcium and amino acid transfer.
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{
"pile_set_name": "NIH ExPorter"
}
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In mammalian development, the formation of a tissue and its specialized cell types is often guided by a genetic hierarchy involving the successive activation of key developmental control genes. These genes may encode transcription factors that prompt an immature, non-specialized cell mass to become a mature, functioning organ composed of appropriate proportions of specific cell types. Nuclear receptors are ligand-regulated transcription factors that respond to hormones or other ligands. Orphan receptors are a special group of nuclear receptors that lack known physiological ligands but play critical functions in tissue development. The retinoid-related orphan nuclear receptor b gene (Rorb), is prominently expressed in the brain and retina. It has recently been reported that mutations in the human RORB gene are associated with cases of intellectual disability and epilepsy, thus implicating this gene with an important role in the human nervous system. However, the functions of this orphan receptor gene and how defects in the gene result in disease are poorly understood. The goal of this project is to elucidate the tissue-specific functions of the Rorb gene in development in a mammalian model and to indicate how dysfunction of this gene causes disease. The study of the Rorb gene offers the opportunity to elucidate novel functions for an orphan nuclear receptor in a defined neurodevelopmental system. Progress: 1. The Rorb gene in neurodevelopment. We have investigated in detail the expression and function of the Rorb gene in the nervous system and have extended these studies to different sensory systems and the brain in the mouse, a mammalian model species. In the retina, undifferentiated progenitor cells generate a range of specific cell types including photoreceptors and different types of interneurons and ganglion cells. The expression pattern of the Rorb gene suggests functions in the cell fate choice between rod and cone photoreceptors and in the early steps in the generation of horizontal and amacrine interneurons. We have established a key role for Rorb in the differentiation of rods, the photoreceptors that mediate vision in dim light. The Rorb gene is essential for the induction of the key rod-determining gene, Nrl. In the absence of Rorb, there is a nearly complete loss of rods and an excess of cone-like cells, consistent with the Rorb acting in the same pathway as the Nrl gene. 2. The Rorb gene is remarkable for the variety of functions it controls in retinal development. To investigate how a single gene can control diverse developmental functions, we have determined the roles of two N-terminal products encoded by the Rorb gene. These Rorb1 and Rorb2 isoforms are differentially expressed in the nervous system. Targeted deletions of RORb1 and RORb2 have indicated that each isoform controls the differentiation of distinct cell types. Our studies have established that Rorb1 directs the generation of two classes of retinal interneurons, horizontal cells and amacrine cells, that modify visual information as it is relayed from the photoreceptor layer through the inner nuclear layer of the retina to the ganglion cells that form the optic nerve. 3. The Rorb gene is also a valuable marker for cell types in other regions of the brain, including the superior colliculus, a region that receives different inputs and responds by influencing head and eye movements. Studies in collaboration with Dr. In-Jung Kim (Yale University), showed that the Rorb1 isoform marks a layer of neurons in the superficial region of the superior colliculus, which has been useful in defining these neuronal sub-types. We continue to build on these findings by pursuing studies to investigate critical downstream target genes that underlie the functions of these orphan receptor isoforms, using a range of tissue and cell-isolation procedures and next generation sequencing approaches.
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{
"pile_set_name": "NIH ExPorter"
}
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The proposed studies focus on the polymerase and RNase H functions of HIV-1 reverse transcriptase (RT) in viral replication and recombination. In HIV infected individuals, 5 to 10 percent of HIV clinical isolates are identified as recombinant. This recombining of sequences contributes to viral adaptation. Examining recombination mechanisms using purified components, we found that the hairpin structures at the 5'-ends of retroviral genomes, such as the HIV-1 TAR, facilitate homologous recombination through template exchange by the DNA primer. We have proposed a mechanism whereby hairpin interactions can bring homologous regions of the RNA genomes together, promoting crossovers. Recent cell culture studies using MLV highlight the kissing loop, a well characterized stem loop structure within the dimerization domain of the retroviral genome, as a hot spot for recombination. In kissing loops, the loop sequence allows base pairing between two identical copies of the stem loops. We now suspect that there is a hierarchy in the ability to promote crossovers in natural stem-loops, based on specific structural features that enhance template interactions, and create a favorable geometry for primer transfer. We propose to determine how template switches are facilitated by interacting hairpins, and whether structural features of the hairpin and interaction stability correlate directly with crossover frequency. Changes in sequence that alter stem and loop size, complementarity, and kissing stability will be analyzed. The RNase H function of RT cuts the genomic RNA into oligomers in the course of minus strand synthesis. Non-PPT RNA oligomers are degraded by RT through an ordered series of primary and secondary cuts made from the RNA 5'-end. From clinical isolates, we found non-nucleoside drug resistant RT mutants, e.g. P237L, that are defective in this mode of RNA 5'-end directed cleavage. We propose an analysis of the mechanism of 5'-end cleavage, and experiments to determine how defects in this process could impair viral replication.
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{
"pile_set_name": "NIH ExPorter"
}
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. Late-onset breast cancer is probably caused by the interaction of multiple genes, endogenous environments, and exogenous exposures. One consequence of this complex, multifactorial aetiology of breast cancer is that aetiologic heterogeneity may exist. Aetiologic heterogeneity implies that two or more groups of breast cancer cases in the general population are caused by different sets of aetiological events. The ability to define aetiologically-distinct (i.e., homogeneous) subgroups in the population may facilitate: 1) epidemiological studies to identify causative agents in breast cancer aetiology; 2) identification of optimal breast cancer diagnosis or treatment regimens; and 3) the targeted application of cancer detection and prevention strategies. A case-series study is proposed, that will focus on aetiologic heterogeneity in late onset breast cancer. This study will address three specific hypotheses. First, candidate susceptibility genotypes at the cytochromes P450 and glutathione-S-transferase loci will be evaluated for their capacity to define aetiologically heterogeneous case groups with respect to age at breast cancer diagnosis. Second, candidate genotypes will be evaluated for their capacity to define aetiologically heterogeneous case groups with respect to somatic genetic damage. Third, interactions of candidate susceptibility genotypes and somatic genetic mutation will be evaluated for their capacity to define aetiologically heterogeneous case groups. In order to address these hypotheses, a study will be conducted, using an existing subject accrual system to identify a sample of 600 incident, invasive breast cancer cases at the Hospital of the University of Pennsylvania (HUP). Risk factor information will be obtained from a questionnaire interview, a biosample containing DNA will be collected using a non-invasive cheek swab method, and pathology information will be collected using a standardized medical record abstraction approach. In addition, breast tumor and normal tissue will be obtained from half of these subjects for loss of constitutional heterozygosity analysis. The results of these analyses will be used to attempt to elucidate the complex, multifactorial aetiology of late onset breast cancer.
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{
"pile_set_name": "NIH ExPorter"
}
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