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-{"text": "The objectives of this proposal are to develop an understanding of the mechanisms by which protease activity in coagulation, fibrinolysis, and inflammation is regulated. To accomplish these objectives the interaction of proteases with the plasma protease inhibitor, a2-Macroglobulin (a2M), and the interaction of the resultant complexes with cell surface receptors will be characterized. Major areas of focus will be to elucidate the mechanism by which proteinases react with this inhibitor, to access the importance and role of a2M as a regulator of protease activity in vivo, and to isolate and characterize the a2M receptor from fibroblasts. Conformational changes occurring in a2M are central to the function of the inhibitor, and not only result in inhibition of protease activity, but also generate receptor determinants on the molecule. Studies are proposed to determine the relationship between conformational changes, protease inhibition, and the generation of the receptor binding sites on the inhibitor-protease complex. Specific probes for monitoring each process have been developed, and will be extensively utilized in these studies. The specific in vivo processes in which a2M plays a key role will be addressed by isolating complexes formed under physiological conditions and identifying which specific enzymes associate with the inhibitor. Complexes will be isolated by immunoaffinity chromatography using a monoclonal antibody that is specific for a2M-protease complexes. Studies are also proposed to characterize the interaction of the inhibitor-protease complex with specific receptors. To accomplish this goal the receptor determinants on the complex will be identified by preparing anti- bodies against synthetic peptides representing regions of the molecule. The receptor will also be isolated from fibroblasts, and the properties characterized. Studies will be initiated on the amino acid sequence of regions of the receptor. Monoclonal antibodies prepared against the purified receptor will assist in characterizing subunits with the goal of identifying regions that participate in receptor function, and will be useful for comparing the structure of the receptor from various cell types and species."}
-{"text": "The proposal aims to define the molecular boundaries of size, shape, structure, and relevant physicochemical properties for a volatile organic compound (VOC) to act as an effective trigeminal (i.e., chemesthetic) stimulus in humans. Chemesthetic responses in the mucosae of the face to airborne chemicals include nasal pungency (i.e., irritation) and eye irritation, two frequently mentioned adverse symptoms arising from indoor (e.g., sick building syndrome) and occupational environments. This proposal is a follow-up of our previous work exploring the physicochemical basis for the production of these chemesthetic responses by VOCs. Systematic chemosensory testing of members of homologous chemical series has revealed that a homolog can be reached where the ability to evoke these trigeminal responses begins to fade, and finally disappears for all ensuing homologs, constituting a \"cut off\" effect. The proposal focuses on the two or three homologs from each series at the boundary of the cut off effect, and will measure the sensory responses of nasal pungency, nasal localization, and eye irritation. From each substance selected to be tested, we will obtain stimulus-response (psychometric) functions spanning the range from chance detection to virtually perfect detection in order to identify the precise homolog reaching the cut off point in each homologous series. By means of chemical modeling and by additional sensory testing of rigid chemical structures molded on the molecular parameters of the cut off homologs, we will define the molecular and physicochemical boundaries for any volatile compound to be able to act as an effective trigeminal chemosensory stimulus. Knowledge of such boundaries has important implications for both basic and applied aspects of trigeminal chemoreception. From a basic perspective it will contribute to the chemical characterization of the trigeminal reception process(es), from an applied perspective it will allow to understand and prevent adverse sensory reactions from air pollutants."}
-{"text": "This proposal is concerned with mutagenesis and carcinogenesis induced by aromatic polycyclic hydrocarbons which are environmental pollutants. The experiments described will show whether, and how, DNA adducts formed from the proximate carcinogens produce mutations. Our study offers a new approach: we will use the mammalian virus SV40, with its DNA modified in vitro by treatment with the proximate hydrocarbon carcinogen. The modified and characterized SV40 DNA vector will then be studied in infected cells from which we will learn the molecular nature (base change, insertion, deletion) and location of mutations caused by the hydrocarbon adducts. Our studies will also help to elucidate the role of DNA repair in mutagenesis. We will study both pre- and post-replicative repair and will determine whether the repair process itself is error-prone. A close correlation between mutagenesis and carcinogenesis will be made possible by examining the extent of mutagenesis of SV40 in the same cells in which transformation by the hydrocarbons will be studied. This last approach will also provide a basis for the screening of potential carcinogens."}
-{"text": "Recent advances in biotechnology and molecular biology have brought about new concerns in protein purification. The proposed protein purification knowledge base addresses some of these issues by providing new computer- readable protein purification databases coupled with an expert system to suggest protocols for purifying new proteins. Experimental data will be scanned, digitized and analyzed for relevant information that the expert system can use to update the working purification strategy. This knowledge base system can decrease the costs of developing new biotechnology products by optimizing the development of large-scale protein purification processes and by using the expertise of key protein purification personnel more efficiently. In addition, a subset of the knowledge base can be used to train students in protein purification techniques. The purification databases will also be distributed independently of the expert system and will be updated on a regular basis."}
-{"text": "This 3-generation study of families at high and low risk for Major Depressive Disorder (MDD) has documented the strong familial transmission of mood disorders across generations. We have conducted 5 waves of assessments in this cohort over 25 years. In the 5th wave, we added Magnetic Resonance Imaging (MRI) measures and found a remarkably robust association of familial risk for MDD with asymmetries in cortical thickness, with a nearly 30% reduction in thickness observed in the lateral parietal, temporal, and frontal cortices of the right hemisphere of the high risk group. These MRI findings are consistent with EEG findings from the 4th wave of assessments that demonstrated reduced activity over the posterior cortices of the right cerebral hemisphere. Both the MRI and EEG findings were present in high-risk individuals who never had MDD in their lifetimes, suggesting that these abnormalities were not simply a consequence of previously having been depressed or having been treated for depression. Thinning of the cortical mantle and reduced electrophysiological activity in the right hemisphere therefore may constitute related endophenotypes for familial vulnerability to developing MDD. We are proposing a 6th wave of study to gain a deeper understanding of the right hemisphere abnormalities in familial MDD in the 216 individuals imaged thus far. This represents the largest MRI study published for MDD to date, and it is the only sample studying 3 generations of individuals at high or low risk for MDD. We are proposing to collect additional MRI and EEG measures, as well as clinical and cognitive neuroscience data, that will inform us about the neural bases of the right hemisphere thinning and their consequences for brain function and emotional processing. We will also determine whether additional cortical thinning in the left cerebral hemisphere predicts new or recurrent MDD in those people who were imaged in Wave 5. Our cohort has exceptional attributes that will aid our search for detecting MDD endophenotypes, including (1) an elevated clinical risk for developing MDD in the high risk group that has been amply demonstrated; (2) multiple prospective assessments, conducted blind to risk status, provide great confidence in the accuracy of the clinical diagnoses; and (3) the sample contains individuals who have not yet become ill but who are nevertheless at elevated risk of becoming ill, thereby allowing us to disentangle the neurobiological determinants of vulnerability from the compensatory responses that would be present in already-affected individuals. MDD is an early-onset, highly prevalent disorder with significant morbidity, and it afflicts people in their most productive years. Identifying the biological vulnerability for depression before the onset of illness has important implications for prevention and public health."}
-{"text": "The purpose of this study is to investigate by sonography the impact of intracatheter nitroglycerin infusion upon the incidence of vascular thrombosis following percutaneous placement of femoral venous catheters in critically ill children. We hypothesize that intracatheter nitroglycerin infusion will be associated with a lower incidence of thrombosis when compared to control catheters. The study will include a total of 62 children ages birth to 6 years of age. Two groups will be studied. Group 1 (nitroglycerin; n=31) will include critically ill children admitted to the intensive care unit in whom the primary care team has elected to place a double or triple lumen femoral venous catheter. Group 2 (D5W; n=31) will also include critically ill children admitted to the intensive care unit in whom the primary care team has elected to place a double or triple lumen femoral venous catheter. Ultrasound examinations of the catheterized femoral vein will be performed and analyzed by the Radiology investigator within 2 days of catheter insertion and a final study will be performed prior to discharge from the hospital. The primary outcome variable is the difference in the incidence of ultrasound-diagnosed thrombosis between the nitroglycerin and D5W groups. Secondary outcome variables include difference in clinical evidence of thrombosis between the two groups, difference in the incidence of catheter-related infection between the two groups, impact of TPN, intralipid, and heparin infusions on thrombosis in each group, and impact of catheter duration on thrombosis in each group."}
-{"text": "Human studies show that cocaine dependence affects the microstructure of white matter, probably as a result of vasoconstrictive effects of the drug. Little is known, however, about the temporal development of the white matter injury and the changes in cerebral vasculature during the dependence period, or about the recovery of both with cessation of the drug use. Magnetic resonance imaging (MRI) provides tools that are highly suitable for investigating in a non-invasive manner the anatomical, structural and functional and chemical characteristics of the brain. In this application we propose to implement a set of MRI techniques for investigating the effects of the use of cocaine on monkey brain, in particular its effects on white matter microstructure on gray/white matter volumes, on the vascularity and perfusion of gray and white matter and on the neurochemical profile of the monkey brain. These techniques will be used to perform a preliminary study on a small and well-controlled population of monkeys. Aim 1. To develop a robust methodology for investigating structural, anatomical and functional measures in monkey brains. Aim 2. To collect preliminary data on the effects of drug use and its cessation on monkey brain. We hypothesize that: 1. Long term cocaine self-administration will result in brain changes in the monkeys similar to those seen in human studies. 2. Cessation of drug use will result in a normalization of brain measures. This will address the important issue of whether or not there is significant brain recovery following cessation of drug use."}
-{"text": "The Sickle Cell Scholar training program is an integral part of the education component of the Comprehensive Sickle Cell Center grant. The Sickle Cell Scholar will be chosen according to the guidelines in the RFA. Under the auspices of the Los Angeles Sickle Cell Center, the scholar will have the opportunity to do clinical and translational research related to the efficacy of L-glutamine in ameliorating the symptoms of sickle cell disease, the genesis of cardiac dysfunction in transfused sickle cell patients, the health services research related to provision of adult care, or translational research related to the role of viscosity in alteration of blood flow and oxygen delivery in humans and the use of engineering techniques to detect evidence of sickling. Unique to this program, the scholar will be supervised by the Center education director who is a cell biologist and doctoral level educator. This individual will be responsible for setting educational milestones and systematically monitoring the progress of the scholar to the training program. Also described here is a plan for the management of the summer high school student program that will also be run by the Center education director."}
-{"text": "Activation of cellular tumor suppressor pathways is the cell's major defense against cancer induced by activated oncogenes. The ARF-p53 tumor suppressor pathway, which is one of the most important in mammalian cells, can be activated by a number of viral and cellular oncogenes. Activated ARF induces a p53-mediated block to cell division via a cell cycle arrest or apoptotic cell death. How oncogenic stress activates ARF remains to be elucidated. Polyoma virus middle T-antigen (PyMT) is a potent oncogene able to bind a number of key regulatory cellular proteins and activate a number of important cellular signaling pathways including the ARF-p53 tumor suppressor pathway. We will use PyMT as a model oncogene in order to better understand how ARF is being activated. We hypothesize that PyMT induces ARF by the inappropriate activation of one or more cellular signaling pathways that also mediate the ability of PyMT to transform cells. Our plan is to identify the cellular signaling pathways induced by PyMT that results in activation of ARF. REF52 cells differ from most other established cell lines in containing an intact ARF-p53 tumor suppressor pathway and are distinct in resembling primary cells in their requirement for oncogene cooperation for their transformation. PyMT activates the ARF-p53 pathway, blocking REF52 cell division and will not transform REF52 cells in the absence of a co-operating oncogene. We plan to take advantage of these unique properties of REF52 cells to isolate PyMT and cell mutants that are involved in the activation of ARF. Three interrelated aims will be pursued. Aim-1 will be to use previously isolated PyMT mutants to identify which domains of PyMT are required to activate ARF. Aim-2 will be to use mutagenized PyMT to identify sequences in both defined and undefined regions of PyMT that are required for the activation of the ARF-p53 pathway in REF52 cells. Aim-3 will be to isolate and define REF52 cellular mutants in which PyMT signaling fails to activate the ARF-p53 tumor suppressor surveillance pathway. We believe that such cell mutants will help to differentiate between normal and oncogene activation of important cellular signal transduction pathways. Understanding the mechanism(s) of oncogenic activation of the ARF-p53 tumor pathway will help in designing better drugs and therapies for the treatment of cancer."}
-{"text": "APOBECSG (A3G) and APOBECSF (A3F) are involved in various anti-viral protein and nucleic acid transactions. Important primary interactions include interprotein interactions, binding single-stranded nucleic acids (RNA and ssDNA), and neutralization by HIV Vif. The full elucidation of these intermolecular interactions is necessary to understand the mechanism by which these cellular proteins inhibit HIV replication. The role of oligomerization in their activity is an issue of great importance. These are interactions at the nanoscale level, and AFM is the method of choice capable to addressing problems listed above. AFM currently reached a level of development that enables this unique instrument to provide a wealth of important information to the characterization of biomolecular complexes at nanoscale. The topographic analysis of molecular system is the initial area of the AFM application for characterization of the structure and specificity of protein-DNA systems of a broad complexity. In addition to imaging, AFM is capable of direct measurements of molecular interaction within the system and research during this decade led to a dramatic progress in this area of the AFM applications. Our Preliminary Studies have already enabled us to provide the first images of A3G bound to ssDNA substrates. We further anticipate that the use of all modalities of AFM technology will help us detail these complexes, dissect the protein and chemical requirements, and extend key observations to ASF. To help achieve these overarching objectives, we will pursue the following three Specific Aims: Aim 1. Image A3 complexes at nanoscale resolution using AFM (Program Objectives 1 &2);Aim 2. Measure interactions within A3-ssDNA complexes using force spectroscopy (Program Objectives 1 &2) and Aim 3. Characterize A3-Vif interactions using AFM imaging and probing modalities (Program Objectives 3). The studies under this project are an integral component of our overall Program Project aiming to provide a comprehensive understanding of A3-AS3 A3-nucleic acid, and A3-Vif interactions. The nanoscale studies will provide a platform for evaluating any future therapeutics that function by leveraging the AS-Vif interaction."}
-{"text": "Human retinal pigment epithelial (RPE) cells accumulate fluorescent lipofuscin granules with age and this phenomenon has been implicated in several age-related degenerative retinopathies. The standing hypothesis of lipofuscin accumulation implicates lipid peroxidation processes in the formation of indigestable fluorescent (460 nm peak emission) residues which are sequestered and concentrated in tertiary lysosomes of the cellular phagolysosomal system. In preliminary studies utilizing human RPE, blue-emitting flurophores were not found within the lipofuscin granules as this hypothesis predicts. Rather, at least two yellow-emitting compounds of unknown identity have been extracted and isolated from purified lipofuscin granules. Yellow-emitting fluorophores are not normally considered indicative of lipid peroxidation byproducts, but their presence is more in keeping with subjective descriptions of yellow fluorescent emissions observed by fluorescence microscopy. I have further isolated at least two age-related blue-emitting fluorophores from cellular sources outside the lipofuscin compartment, and another compount which is present only in aged human RPE and which produces an intensely fluorescent compount upon reaction with ninhydrin. The aims of the present proposal are to utilize thin layer chromatography (TLC) to isolate and purify these compounds from human RPE extracts and to characterize and classify or identify them by means of corrected spectrofluorometry and uv-vis, infrared, nuclear magnetic resonance and mass spectrometry. Standardized high performance TLC and quantitative densitometry will be used to track the age-related appearnce and accumulation of these compounds in relation to one another in RPE from a wide age-range of human donors, and to survey a range of species in search of appropriate animal models. Fluorescence assays will be developed and deployed in further subcellular fractionation studies on the acute effects of the lipofuscinolytic drug, centrophenoxin, on these compounds. These studies will serve to focus the direction of future research and will yield new information essential for the reformulation of the hypothesis of lipofuscin formation and accumulation, and of our understanding of aging phenomena in postmitotic cells in general."}
-{"text": "Patients with systemic lupus erythematosus have been found to have disturbances of cell-mediated immune responses. Alterations of B and T lymphocytes as well as monocyte function, natural killer cell and interferon responses, have been observed both in patients with SLE and certain of their relatives. The major goal of these studies is to further characterize the types of immunological distrubances in SLE and to elucidate the mechanisms which lead to alterations of immune regulation and are responsible for the pathogenesis of this disease. The ability of immunosuppressive drugs to alter cell mediated immune function and possibly to re-establish more nearly normal immunoregulation is being actively studied."}
-{"text": "Most oral leukoplakic lesions represent hyperkeratotic epithelium (benign), dysplasia, or squamous cell carcinoma. The lesions must be biopsied and examined microscopically for diagnosis. But, morphology alone does not answer the question about which non-invasive lesions will progress to cancer. The uncertainty of whether a given lesion will progress to cancer or not often compromises the use of the best therapeutic option which is complete excision because of the complications of surgery. So, the question to be answered by the proposed research is, \"Will a given leukoplakic oral lesion likely progress to cancer?\" The research will begin to answer the question by examining the genomic sequences of pp32r1 (GenBank AF008216) present within existing biopsy specimens. In a pilot sequencing survey of the pp32r1 gene in tobacco-associated, oral, leukoplakic lesions, the principal investigator found a mutant, growth-accelerating pp32r1 gene in an oral squamous cell carcinoma. The first specific aim of the project is to amplify and sequence the pp32r1 gene within formalin-fixed, paraffin embedded specimens of oral leukoplakia. The basic hypothesis is that certain neoplastic cells within those lesions contain mutations encoding non-conservative amino acid substitutions within residues 136-172. These mutations define malignant cells long before they clonally enlarge and form a clinical cancer. Another question concerns the discrete molecular activities of mutant, carcinoma-associated PP32R1 proteins as they accelerate cellular growth. The pp32r1 gene is a member of a family of genes that encode proteins with myriad activities. All the family members contain N-terminal domains with leucine-rich repeats that form adapter sites for protein-protein interactions. And, all the family members contain extremely acidic C-terminals that form alternative adapter sites for protein-protein interactions by ionic forces. Another common physical characteristic is that other family members, particularly PP32, often appear in nature bound to other intracellular proteins. It is thought that mutant, carcinoma-associated, growth-accelerating PP32R1s differentially bind intracellular proteins in comparison to wild-type PP32R1s and other members of the PP32 family. The resultant abnormal macromolecular complexes change cellular homeostasis in ways that accelerate growth. The second specific aim of the project is co-immunoprecipitation of PP32R1 binding proteins, separation of the binding partners by 2-dimensional (2D) gel electrophoresis, and identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Identification of the intracellular protein ligands will provide a specific functional context for mutant PP32R1S."}
-{"text": "Arsenic is a carcinogen, a Superfund toxic compound, and a common drinking-water contaminant. The Environmental Protection Agency (EPA) has identified 1,300 sites on its National Priorities List (NPL) and arsenic has been found in at least 781 of these sites in the USA. Exposure may occur by a variety of pathways including inhalation of dusts in air, ingestion of contaminated soil or water, or through the food chain. The primary mode of exposure in non-occupationally exposed populations is drinking-water contamination from either alluvial deposits or industrial contamination. The precise relation of arsenic to cancer has not been established at low exposure levels. In addition, arsenic toxicokinetics in humans remains poorly understood. Thus, we propose to assess exposure and skin-lesion risk, as well as arsenic dosimetrics in a population with a wide range of arsenic exposure via drinking-water contamination. The proposal builds upon a small study in which a case-control study has been initiated. This proposal will use the necessary resources to analyze biomarkers of exposure and susceptibility and for statistical analyses of both a case-control study of skin lesions and a repeated-measures biomarker dosimetry study. The proposed studies in Bangladesh will assess this risk in a population with a wide range of exposures - from low to very high. Together, these data will add substantially to the existing risk assessment information by elucidating both early and late outcomes alter arsenic exposure, and the influence of susceptibility traits at all levels of exposure. This project is relevant to the overall aims of NIEHS in several ways: First, we will examine a range of health effects of an important environmental carcinogen. Second, we will define human biomarkers of exposure, early effects, and genetic susceptibility to arsenic exposure. Third, we will examine exposure-response relationships for arsenic-induced non-malignant skin lesions as well as for skin cancers."}
-{"text": "Attended NCRR/NIH session to edit a workshop report on ~Technologies for the Future~, May 15, 1996. Consulting with various personnel on the progress/nuances of 2~PE scanning microscopy."}
-{"text": "Catecholamine systems, operating at the interface between the mind and the body, exemplify three ways the brain regulates homeostasis -- via neurotransmitters (norepinephrine, NE), hormones (epinephrine, EPI), and autocrine/paracrine factors (dopamine, DA). We conducted investigations in the areas of clinical neurocardiology, catecholamine neurochemistry, and novel catecholaminergic systems. 6-[18F]fluorodopamine ([18F]-6F-DA) positron-emission tomographic (PET) scanning led to a new pathophysiological classification of primary chronic autonomic failure. Patients with pure autonomic failure (PAF) or parkinsonism and autonomic failure had cardiac sympathetic denervation, whereas patients with the Shy-Drager syndrome had decreased or absent post-ganglionic sympathetic neural outflows to intact nerve terminals. 13N-ammonia and [18F]-6F-DA PET scanning detected perfusion or sympathoneural abnormalities in the affected limbs of patients with sympathetic dystrophy (RSD). Patients prone to neurocardiogenic syncope had decreased cardiac NE spillover, and patients with hypertrophic cardiomyopathy (HCM) had intact sympathetic innervation of the hypertrophic myocardium. Clinical neurochemical studies linked specific catecholaminergic phenotypes with genotypic abnormalities in Menkes' disease, familial dysautonomia (FD), and L-aromatic-amino-acid decarboxylase (LAAAD) deficiency. Treatment with L-DOPA as a dopaminergic pro-drug produced beneficial natriuretic responses in patients with congestive heart failure. Plasma DA-sulfate levels were found to depend on dietary factors and on production of endogenous DA in the gastrointestinal tract."}
-{"text": "The goal of this project is to map and identify genes for herditary deafness. Linkage analyses are being conducted in several large pedigrees segregating non-syndromic and syndromic forms of deafness. If linkage to the known syndromic, DFNA (dominant) and DFNB (recessive) loci in large families are excluded, we initiate genome-wide screens. This strategy has allowed us to map new deafness loci such as DFNA20, DFNA27, DFNA28 and DFNA36. The chromosomal map locations of these novel deafness genes are then refined prior to initiating positional cloning strategies to identify the genes responsible for the hearing loss. Recently we have identified genes for DFNB12, DFNA28, Usher 1D and usher 1F. Additional families with dominant and recessive modes of inheritance with profound congenital or progressive hearing loss are being ascertained with the goal of mapping and cloning additional novel genes that are necessary for hearing and/or maintenace of the auditory system. We are also asscertaining families, mapping loci and identifying genes for Usher syndrome. The defining clinical features of Usher syndrome are hearing loss and progressive retinopathy."}
-{"text": "I am a surgical oncologist dedicated to caring for patients with complex malignancies and to developing novel immunotherapeutic approaches for metastatic liver cancer. Early in my medical training, I developed a passion for immunology and host-tumor interactions. After graduating from the New York University (NYU) School of Medicine with the top academic record in my class, I trained in general surgery at NYU and Bellevue. I spent two years as a research fellow with Dr. Ronald DeMatteo at Memorial Sloan-Kettering (MSK), where I gained valuable experience in immunologic research and published papers on liver immune cells in Hepatology and the Journal of Immunology. During my clinical surgical oncology fellowship at MSK, I studied immune infiltrates in liver metastases, work published in the Annals of Surgical Oncology and featured in Nature Reviews Clinical Oncology. I was recruited to Roger Williams to join a strong immunotherapy program and build a lab focused on investigating how suppressive liver immune cells may contribute to the development and progression of liver metastases. My K08 proposal is a critical step in my career development as the project represents a fusion of my basic liver immunology background with a therapeutic platform, genetically modified or designer T cells (dTc), we are presently using in clinical trials at Roger Williams. The associated training plan, as noted below, will provide me with important educational opportunities to facilitate my transition toward independent funding. Environment - The Roger Williams Medical Center (RWMC) is an ideal environment for my academic growth and development. Dr. Richard Junghans, my mentor, is a well established immunologist with a rich experience with immunotherapy trials and dTc. I also work with Dr. Vincent Falanga, the RWMC Chief of Dermatology, as he shares valuable resources obtained through their COBRE grant. As the RWMC is of modest size, my laboratory and career development are top priorities for the institution. I receive a tremendous level of support from th administration and my Division Chief and Cancer Center Director, Dr. N. Joseph Espat. This enables me to have the necessary protected time and maintain a high level of focus on my laboratory work. Development and Training Plan - I have assembled a mentoring team of well respected experts to monitor my progress, support my research, and promote my career development. Each member of the team has successfully competed for NIH funding and offers a particular area of expertise that meshes well with my proposal. Dr. Junghans will be the primary mentor, and his experience with immunotherapy and T cell biology will be invaluable. The three co- mentors will make important contributions as well. Dr. Espat, an authority on liver metastases, will ensure that the work and publications maintain a translational focus. My mentor from MSKCC, Dr. DeMatteo, will provide important insight and critique from the vantage point of an expert in liver immune cell biology. Dr. Alfred Ayala at Brown, with whom I have recently begun to collaborate, is another well respected expert in liver immunobiology. I have also included translational medicine and immunology course-work at Brown to expand my knowledge base in critical areas. Research Plan - Based upon the work I have published to date, I hypothesize that suppressive liver immune cells limit the effectiveness of T cell based therapies for eradicating liver metastases. T regulatory cells (Treg) and myeloid derived suppressor cells (MDSC) are likely contributors to liver tolerance. Our lab has spent over one year optimizing our murine model of CEA+ colorectal liver metastases and the immunologic assays described in the proposal. My first specific aim focuses on elucidating the mechanisms by which Treg and MDSC may limit anti-CEA dTc function in the liver. I have designed the experiments to focus on each component of the model separately, including the Treg or MDSC, dTC, and immune receptor express by dTc. In addition, contact-dependent mechanisms such as the programmed death-1 pathway and secreted factors will be studied. For the second specific aim, specific strategies for blocking the suppressive interactions between Treg or MDSC an dTc will be tested in vivo. We hope some of our immunologic manipulations will be translatable into the clinical arena."}
-{"text": "PROJECT SUMMARY The overarching hypothesis of this proposal is that kisspeptin may explain many of the changes in glucose metabolism in pregnancy: high levels of kisspeptin during normal pregnancy amplify insulin release to maintain normoglycemia, and relative hypokisspeptinemia may underlie the pathophysiologic defects in gestational diabetes mellitus (GDM). The initial steps to examining this overarching hypothesis are to 1) define the effect of kisspeptin (at pregnancy levels) on insulin physiology in non-pregnant women and 2) to determine if kisspeptin levels predict the development of GDM. Given the complexity of factors in pregnancy that regulate metabolism and insulin physiology, these initial studies are designed to isolate the effect of one factor: kisspeptin. This proposal utilizes gold standard (hyperglycemic and hyperinsulinemic euglycemic clamps) and physiologic (mixed meal tolerance test) methods in randomized, placebo-controlled and blinded clinical trials to assess kisspeptin?s impact on insulin secretion and insulin sensitivity in non-pregnant women. Going further, the grant utilizes comprehensive specimen banks with longitudinal pregnancy samples and patient outcome data to measure kisspeptin levels across pregnancy and to determine if kisspeptin levels early in pregnancy can predict the development of GDM. This grant will refine our understanding of the impact of hyperkisspeptinemia of pregnancy on insulin and incretin physiology and development of GDM. This application details a comprehensive five-year training program for mentored career development in patient- oriented research. The Applicant proposes research, including independent clinical trials, specifically constructed to provide focused training pregnancy physiology and in the mechanisms of insulin physiology. To achieve this goal, she has chosen mentors with complementary expertise: Dr. Stephanie Seminara is an expert in kisspeptin physiology with a strong background in human physiology research, and she is Chief of the Reproductive Endocrine Unit at Massachusetts General Hospital (MGH); Dr. Patrick Catalano is an expert in pregnancy physiology and gestational diabetes, and he is a Professor of Obstetrics and Gynecology at Tufts University School of Medicine and Principal Investigator in the Mother Infant Research Institute; Dr. Jose Florez is an expert in physiological mechanisms in the development of diabetes, and he is Chief of the Diabetes Unit at MGH. This mentoring team will position the Applicant well to launch a successful independent investigative career in metabolism, with a focus on pregnancy and the influence of reproductive hormones, a key NICHD research priority area. The Applicant's career development plan entails rigorous coursework and seminars, hands-on practical experience, and close guidance from scientific advisors with diverse scientific expertise. Collectively, the experience gained during this award will serve as the foundation for the Applicant's independent, academic career as a physician-scientist with expertise in translational research in human metabolic disease."}
-{"text": "This is a multi-institutional, Intergroup III trial examining the potential chemopreventative effect of Finasteride for prostate cancer. Study subjects who accrue can have no evidence of prostate cancer clinically. Enrolled participants randomize to either placebo or the study drug (proscar 5 mg daily), which is administered in a double-blind fashion. Participants are followed with yearly digital rectal exams and serum PSA values. All PSA samples are sent to a central lab for processing, and elevated values are evaluated with biopsy. any cancer detected during the study results in patients being taken off study. The follow-up period will continue for 7 years, after which all study participants will undergo trasnrectal unltrasound-guided biopsy, and tiddues form the biopsy will be processed at a central pathology site. Differential rates, if any in the prevalence of prostate cancer between the two study groups will be recorded."}
-{"text": "This project involves the measurement of prostaglandins and phospholipids in adipose tissue and liver, with a special regard to the interrelationship of these compounds in lipolysis and antilipolysis. Studies are progress, studying the relationship of the levels of the prostaglandin E series (measured by thin-layer chromatography and radioimmunoassay) and the different classes of phospholipids (measured by thin-layer chromatography) in situations within the cells of increased and decreased cyclic AMP levels. Levels of cyclic AMP are increased by exposing the isolated cells to epinephrine or norepinephrine, and cyclic AMP levels are lowered by the use of the hormone, insulin. It is hoped by these maneuvers to better understand the role and interrelationship of these compounds to this metabolic process. In addition, it is hoped that a better understanding of the physiology of these compounds will also be forthcoming."}
-{"text": "Mycobacterium avium-intracellulare complex organisms are the most common cause of mycobacterial lung disease other than tuberculosis and are the leading cause of morbidity and mortality in the HIV-infected AIDS patients. Infections caused by M. avium patients with AIDS often go untreated as existing antibiotics are ineffective in adequately controlling these infections. This necessitates the need for novel molecular targets for chemotherapy. Replication of the bacterium leading to its multiplication is one of the necessary steps to establish an infection. Initiation is the first committed step in the replication, and the replication process is believed to be regulated at the level of initiation. Thus, understanding the basic steps involved in initiation of DNA replication in M. avium will help define important molecular targets against which new generation drugs can be developed. Initiation of replication occurs once per cell cycle at a specific site on the chromosome called oriC. Initiation is believed to be triggered by the interactions of dnaA with its recognition sequences present in oriC called the dnaA-boxes. These interactions are thought to facilitate recruiting of other proteins resulting in the completion of initiation. This research proposal focuses on identification and characterization of the oriC and Dna protein of M. avium. To obtain oriC, chromosomal DNA fragments of M. avium that support autonomous replication when present in nonreplicative plasmids will be identified, cloned and their nucleotide sequence will be determined. Sequential deletions from both the 5' and 3' regions will be carried out to identify the minimal DNA region that is essential for oriC activity. Site directed mutagenesis will be carried out to identify the putative DnaA boxes that are essential for oriC activity. The ability of M. avium oriC to function as autonomously replicating sequences in other bacteria will be determined. The dnaA gene will be over-expressed, the gene product will be purified and its interaction with the wild type and mutant sequences will be investigated. Antisense dnaA oligonucleotides that target to the dnaA mRNA to affect the expression of M. avium dnaA gene will be explored in an effort to evaluate the role of the M. avium gene. Using oriC plasmids and cell free extracts, an in vitro replication system will be established. The ultimate long-term goal of these experiments is to utilize the knowledge thus gained to develop rational drugs that affect the initiation of replication thereby preventing growth and resulting M. avium infections."}
-{"text": "The threat of terrorist attacks involving radioactive material and the potential for radiation accidents require the development of improved treatment strategies for victims of radiation exposure. Hematopoietic cells are highly sensitive to radiation damage, and their loss after radiation exposure results in lethal infections. Development of treatment that prevents immune damage from radiation and reconstitutes immune function after radiation exposure would be a significant advance. The aim of this project is to study four promising, clinical-grade cytokines that are likely to significantly improve immune reconstitution after lethal dose irradiation in the well-established dog model. The four cytokines are fms-like tyrosine kinase-3 ligand (Flt3 ligand [FL]), keratinocyte growth factor (KGF), interleukin (IL)-7, and transferrin (Tf) given alone or in combination either before or after exposure to lethal doses of total body irradiation (TBI). All four cytokines have anti-apoptotic activity after gamma irradiation and have direct or indirect beneficial effects on lymphocytes and other immune cells. We will study these cytokines in the dog since (1) the dog model of radiation exposure and hematopoiesis has been highly predictive of human clinical outcomes, (2) there is extensive preliminary data with cytokines for radioprotection of the dog after acute radiation, (3) all four of the human cytokines we have proposed are cross-reactive in the dog, and (4) these cytokines have been studied in humans and are in various stages of clinical development. The goal is to achieve survival of dogs after an otherwise lethal dose of irradiation with sustained immune reconstitution without hematopoietic stem cell (HSC) transplantation. In Specific Aim 1, cytokines will be given after TBI and in Specific Aim 2, cytokines will be given before and after TBI. In Aim 1, we will give 500 cGy TBI and treat dogs with G-CSF plus each study cytokine. In this model, a radioprotective cytokine is defined as achieving significantly improved survival compared to G-CSF alone. In the subsequent experiments, the TBI dose will be successively increased by 100 cGy increments and dogs will be treated with a combination of radioprotective cytokines. The primary endpoint is recovery of hematopoiesis and survival beyond day 30. The secondary endpoint is immune reconstitution. Upon study completion, we will have identified the optimal cytokine treatment and the highest dose of TBI that can be reliably survived without HSC support."}
-{"text": "Epithelial ovarian cancer is the leading cause of death in women with gynecologic cancer. Although it represents only one-fourth of all new cases of gynecologic cancer, it causes over one-half of all deaths due to these diseases. Memorial Sloan-Kettering Cancer Center (MSKCC) is an institution with extensive clinical and laboratory research facilities. The Gynecology Service of the Department of Surgery in collaboration with the Breast/Gynecology Service of the Division of Solid Tumor Oncology of the Department of Neurology, the Department of Pathology, the Department of Epidemiology and Biostatistics and the Immunology Program of the Sloan- Kettering Institute with their principal interest being directed towards ovarian cancer. Since approximately 140 new cases (both untreated and previously treated) of ovarian cancer are seen annually, an adequate patient population exists. For these reasons, a program project to develop new therapeutic strategies for the treatment of epithelial ovarian cancer has been developed. The aim of this program project is 1) to conduct phase I and II clinical trials of innovative chemotherapy regimens 2) to collect a bank of ovarian cancer tissues and body fluids, 3) to evaluate the symptomatology of cancer pain and to assess quality of life as related to therapy, 4) to perform pharmacokinetic studies of new monoclonal antibodies against ovarian cancer antigens in animal models and humans with the aim of being able to target ovarian cancers for diagnostic imaging and radioisotope therapy, and 5) to study in a systematic fashion the immunology and immunochemistry of ovarian cancer in order to characterize malignant and normal ovarian and mesothelial tissues and develop a better group of monoclonal antibodies for clinical use. Based on initial collaboration, the working structure of this program project has been developed. Progress to date warrants funding of a program project to expand this research and carry it forward."}
-{"text": "RNA-directed DNA polymerase (reverse transcriptase) is found in animal oncornaviruses and some human cancer cells. The avian myeloblastosis virus (AMV) DNA polymerase is the most readily available and best studied such enzyme and has been shown to have an unusually high degree of infidelity of transcription. Examples of this infidelity include random initiation, premature termination, a double strand reaction, incorporation of noncomplementary bases, a slippage reaction and inability to transcribe certain homopolymers. These phenomenon have not yet been studied with the human enzyme, in part because of the limited amounts of enzyme purified. By doing a large scale purification of RNA-directed DNA polymerases from human malignant tissues, it will be possible to do a number of studies on these enzymes which have already been done by us with the AMV DNA polymerase, compare the human enzyme to the AMV DNA polymerase, and compare enzymes from patients with different cancers. If the human enzyme behaves qualitatively the same as AMV DNA polymerase, studies with AMV DNA polymerase can be extrapolated to the human enzyme. If there are differences, these studies will be the start of detailed investigations of the human enzyme which may lead to more specific therapy of cancer."}
-{"text": "The process of cytoskeleton assembly and disassembly is essential for many critical phagocyte functions, including locomotion, phagocytosis, adhesion, egress from the vasculature, and cytokine production. Understanding the mechanisms by which cytoskeleton assembly is regulated and acts in turn to direct the functions of the phagocytic cell is at the core of understanding the mechanisms of augmentation of host defense at sites of inflammation. The molecular details of this regulation and it's influence on leukocyte activation has been difficult to study. This laboratory has developed a system which has significantly advanced the understanding of the role of the cytoskeleton in the mechanism of leukocyte activation. This lab has found that the pathway initiated by ligation of FcR by Ig6 in phagocytes increases [Ca2+]i by stimulating release of Ca2+ from intracellular stores through a novel IP3-independent pathway that requires the actin cytoskeleton. A specific actin-binding protein called l-plastin is required for this release of Ca2+. Our hypothesis is that l-plastin is an important link between cell surface receptors, such as FcR, and the cytoskeleton in phagocytes. In order to test this hypothesis, we will study the cell biology and biochemistry of l-plastin in phagocytes that naturally express the protein, as well as develop an in vitro transfection system to allow us to study the effects of altering or mutating l-plastin on l-plastin cell biology and biochemistry as well as IgG and integrin-mediated signal transduction. Actin binding, phosphorylation, and immunolocalization of l- plastin and IgG-mediated calcium responses and phagocytosis in phagocytic cells will be studied as models of normal l-plastin function. L-plastin function will be defined further by applying these assays to cell transfection systems using mutated forms of l-plastin. Cells that do not normally express l-plastin as well as mouse cell lines that express endogenous l-plastin will be transfected with the human form of l-plastin. The function of wild type human l-plastin in these cell lines will be validated by comparing l-plastin function and IgG-mediated responses in cells transfected with normal l-plastin and normal phagocytes."}
-{"text": "One aspect of infant functioning, effectance motivation, the motivation to master problems and to have an impact on his environment, has received considerable theoretical attention, but little empirical study. In this study we have developed methods to measure infants' efforts to secure feedback from the environment and their motivation to solve problems. Data have been collected on 44 one year old infants, 23 boys and 21 girls. We are analyzing the relationships between scores on our effectance motivation tasks and contemporaneous measures of cognitive development and exploratory behavior. We are also looking at the early antecedents of effectance motivation by analyzing relationships between specific components of the environment at 6 months and scores on effectance motivation at one year."}
-{"text": "It is proposed to investigate pitch recognition judgments where other tones are interpolated during the retention interval. Certain systematic interactive effects which appear to be based on lateral inhibitory interactions will be studied as a function of serial position. These specific effects will also be investigated as a function of ear of input. The influence of concurrent relational information will be studied as a function of retention interval. In sequences where the tones to be compared are simultaneously accompanied by other tones of lower pitch, the effects will be investigated of interpolating simultaneous tonal combinations as a function of their relationship to the standard and comparison combinations. The effects on pitch recognition of varying other tonal attributes (timbre, duration) will also be explored."}
-{"text": "The ultimate goal of this project is to provide insight into the functioning of cellular machines composed of RNA and protein subunits. As a model system, we are studying the introns of the yeast mitochondrial cytochrome b pre-mRNA and the proteins that assist in their splicing. In these simple systems, the RNA components contain the active site for the splicing reaction while the protein components enhance the rate of splicing by holding the RNA in its active conformation. As a first step toward understanding the functioning of this system, we have determined the crystal structure of the bI3 maturase, an intron-encoded protein that facilitates splicing of the intron that encodes it. This crystal structure revealed a conserved nucleic acid binding surface with other members of the LAGLIDADG endonuclease family. Furthermore it revealed how this protein has been inactivated as a functioning DNA endonuclease while maintaining the core structure. Biochemical experiments performed in our collaborator's laboratory showed that this protein binds to the folded self-splicing RNA intron in a peripheral region distant from the splicing active site. Thus, the protein acts at a distance to facilitate folding of the self-splicing RNA. The protein also was shown to bind to the minor groove of the RNA versus typical binding of the LAGLIDADG endonucleases to the major groove of DNA substrates. This suggests that the protein is not changed structurally from DNA-binding homologues, but instead takes advantage of the RNA's wider and more accessible minor groove."}
-{"text": "During terminal differentiation epidermal keratinocytes manifest a programmed set of morphological and biochemical changes that result in the production of two major structures: 1) an envelope of covalently linked protein enclosing 2) a constellation of keratin intermediate filaments. A major precursor of the envelope is a soluble protein called involucrin (hINV) which is incorporated into the envelope by a calcium- dependent transglutaminase. hINV is likely to account for the majority of glutamyl-lysine linkages that hold the envelope together. In spite of its importance, inadequate information is available regarding which glutamines within involucrin are targeted for crosslinking by transglutaminase or which sections of the hINV molecule are essential for high strength envelope formation. Active envelope formation is essential for survival and abnormal envelope formation is a feature of several epidermal diseases. The ultimate aim of the experiments described in this proposal is to understand the role of hINV in the envelope assembly process and how this impacts on the disease state. To provide tools for these studies, we cloned and sequenced the complete hINV gene, structurally characterized the protein and produced the normal and mutant hINV proteins in bacteria. Our results show that the molecule is an extended alpha-helix composed of highly similar, tandemly linked repeats of ten amino acids. Each repeat contains three glutamine residues, each of which is a potential crosslink site. The proposed studies are designed to gain a better understanding of cornified envelope structure and the role of hINV in envelope formation and are a logical extension of the studies completed during the initial two years of grant support. In the present experiments we propose to 1) identify which proteins become crosslinked to hINV during cornified envelope formation, 2) construct a series of hINV mutants to determine why GLN496, among the more than 100 glutamine residues present in the hINV protein, is the preferred site for initial crosslink formation and 3) determine the effects of expression of selected mutant hINV proteins on epidermal function in transgenic mice."}
-{"text": "Mast cells and basophils, the central effector cells of immediate type hypersensitivity, will be purified from animals and humans. The mast cell and basophil content of preformed chemotactic mediators and enzymes and their release by immunologic method will be studied. The effect upon mediator release of permeant anions will be assessed and the regulatory neurohormone receptors identified and characterized. Chemotactic mediators released in vitro from isolated cells or in vivo in human disease with known mast cell involvement will be purified by physiochemical technique and characterized functionally with regard to target cell specificity for chemotaxis, chemotactic deactivation, and cell membrane receptor alteration. The effect of chemotactic factors and cells will be extended to in vivo models in animals. Taken together, these studies will expand the understanding of the biologic importance of chemotactic mediators in regulation of inflammatory events in human disease."}
-{"text": "The overarching aims of this Project are to synthesize the microtubule stabilizing natural product dictyostatin-1 and its analogs, and to evaluate the potential of these compounds as anticancer agents. In addition to the tight collaborative medicinal chemistry aspects of the work, the use of the new technique of fluorous mixture synthesis is also featured. The specific aims are: 1) Assignment of the structure of dictyostatin by total synthesis. Aim 1 is well advanced. We have recently completed the total synthesis of dictyostatin 1, and we now know its full structure, including absolute and relative configurations. It turns out that dictyostatin and discodermolide have the same configurations at all ten shared stereocenters. This structure determination has removed a major roadblock to development of dictyostatin as a potential anti-cancer agent. However, we still would like to learn how similar (spectroscopically, biologically) the isomers are with the same relative configurations at the three main fragments but coupled together in different pairings. This question will be answered by making multiple isomers by fluorous mixture synthesis. We have also nearly completed synthesis of the original Pettit structures, so this work will be finalized. 2) Synthesis of 0.35-1.0 g of dictyostatin for in vivo characterization. To accomplish this goal, the current synthesis must be improved, and a plan to streamline it by increasing convergency is outlined. 3) Synthesis of stereoisomers and analogs of dictyostatin for SAR studies. We will use the recently established synthetic route to make analogs by fluorous mixture synthesis. We plan to make stereoisomers, and to make simplified analogs with the goal of beginning to elucidate the structure/activity relationship. 4) Conformational modeling of dictyostatin and key stereoisomers. Multiconformational searching coupled with MM3 calculations in Macromodel will be used to predict conformational minima of key isomers. Initially, this data will be used to help interpret NMR experiments, and will serve as a basis for analog design as well as for more sophisticated modeling and docking experiments."}
-{"text": "Hearing impairment/deafness is the most common sensory limitation in the U.S. An estimated 11 million individuals in the US are deaf or hard-of-hearing. It has been estimated that approximately 1 million Americans use American Sign Language (ASL) as their primary means of communication making it a distinct linguistic minority. This proposal seeks to improve clinical practice by creating a computerized, self-administered depression screener in ASL that is culturally and linguistically accessible to deaf individuals, an at-risk and traditionally underserved population. No current depression screeners have been shown to be valid for the majority of prelingually deaf persons who use ASL as their main communication mode. Studies have shown that depression occurs in higher rates among deaf persons than hearing persons. While the U.S. Preventive Services Task Force recommended systematic screening for depression in primary care clinical settings in 2002, deaf persons have not yet routinely had access to this preventive service. Providing primary care physicians with a depression screener that is culturally and linguistically accurate and can be self-administered via computer and can be used on iPads and iPhones with no further development, can greatly increase the chance that deaf persons with depression will receive proper diagnostic assessment and treatment which can substantially improve their quality of life. The Deaf Depression Screener will also improve scientific knowledge by providing a valid method for estimating the prevalence of depression among deaf persons in primary care which cannot now be accomplished due to the lack of a valid screening instrument."}
-{"text": "Inhibitory interneurons crucially control information processing in neuronal networks, but their vast molecular and anatomical diversity has made it difficult to dissect their functional roles. The cerebellar cortex with its relatively simple architecture and few neuron subtypes constitutes an ideal model circuit to study interneuron function. In the cerebellar cortex, mossy fibers (MFs) relay sensory information to granule cells (GCs) that send their axons to the molecular layer to excite Purkinje cells (PCs). As the only interneurons of the cerebellar input layer, Golgi cells (GoCs) are strategically positioned to control the propagation of sensory information to the cerebellar output layer. Spontaneously active GoCs inhibit GCs with two distinct time courses: rapid phasic inhibition that narrows the time window for excitatory input integration, and persistent tonic inhibition that controls the gai of incoming signals. Moreover, GoCs are thought to mediate the slow oscillations observed in the GC layer prior to the onset of motor behaviors. Strong electrical coupling between GoCs permits these oscillations that coordinate large assemblies of GCs. Although it is well established that GoCs crucially determine the flow of information within the cerebellar cortex, less is known about the mechanisms that orchestrate GoC firing, regulate GoC activity, and dynamically control GC excitability. Contrary to current beliefs in the field, preliminary data supports the hypothesis that active GoC dendrites enhance electric coupling. This proposal thus seeks to determine the cellular mechanisms that enable GoCs to fire synchronously using two-photon calcium imaging, patch clamp electrophysiology, voltage imaging and array tomography. Preliminary results also suggest that GoC activity dictates dendritic calcium concentration. The planned experiments will therefore examine the functional relationship between GoC activity and dendritic calcium dynamics, and determine the consequences for synaptic plasticity of excitatory PF and MF input. Preliminary data indicates that activation of metabotropic receptors on GoCs suppresses firing, and that this suppression is accompanied by a decrease in tonic inhibition of GCs. With the help of electrophysiology and optogenetics, this proposal will therefore test the hypothesis that dynamic modulation of GoC firing rate controls GC excitability and MF input integration. Completion of the outlined work will elucidate the mechanisms that control integration of sensory information by varying the activity of a single interneuron subtype in the cerebellar cortex. It will also extend our general understanding of how inhibition governs computational processes in neural networks."}
-{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Previous workers have proposed high resolution models for the docking of the myosin heads on actin on the basis of combined crystallographic and electron microscopy data from isolated proteins (Mendelson and Morris, 1997 PNAS 94:8533;Holmes et al. 2003 Nature 425:423). Another group also claims that the first step in the force generation is associated with a rearrangement of the myosin-actin interface, followed by the lever arm tilt, and that it is temperature-dependent (Ferenczi et al. 2005 Structure 13:131). The goal of this work is to collect small angle X-ray scattering (SAXS) data from muscle that may be used to check in vivo the prediction of the models for the acto-myosin docking and whether there is a temperature-dependent rearrangement of the myosin-actin interface. For this purpose, the most sensitive reflection in the pattern is the 2.73nm meridional reflection arising from the regular repeat of the actin monomers along the actin filament, which changes its intensity upon myosin attachment to actin. Preliminary modelling has shown that the reflection intensity is little influenced by the lever arm tilt but it is highly sensitive to the relative axial position of actin and catalytic domain of myosin. 2D patterns will be taken from muscle at rest and during isometric contraction at different temperatures (4 to 17[unreadable]C) up to 0.5 nm-1 in reciprocal space, in order to collect the actin-based 2.73nm meridional reflection and the 5.9nm and 5.1nm layer lines, also influenced by myosin attachment to actin."}
-{"text": "The use of antiepileptic drugs (AEDs) in pregnancy and early infancy poses special challenges and concerns, as even transient exposure to certain drugs during CNS development may impede subsequent neuronal and synaptic development. Several AEDs, including phenobarbital and phenytoin, when given in therapeutically relevant doses to rats during the early postnatal period, cause a pronounced increase in apoptotic cell death in several brain regions. Other drugs such as lamotrigine (in low or moderate doses) and levetiracetam are devoid of this effect. We have recently discovered a single exposure to phenobarbital, at the time of peak vulnerability to the propapoptotic action (postnatal day 7 in the rat), resulted In the suppression of the normal, developmental increase in the frequency of inhibitory post-synaptic currents (IPSCs) recorded via patch clamp from striatal medium spiny neurons (MSNs) in slices taken between postnatal day 10 and 14. We have also identified impairments in adult rotorod performance in rats treated at P7 with a proapoptotic dose of phenytoin, a reduction in prepulse inhibition (a measure of sensory-motor gating) and a reduction in seizure threshold to pentylenetetrazol In animals treated in the first postnatal week with phenobarbital. Each of these behavioral assays are sensitive to damage or altered function in the striatum. The proposed experiments will follow up on these exciting preliminary findings to determine if there is a consistent and predictive (potentially causative) relationship between AED-induced neuronal apoptosis and impaired maturation of I PSC frequency in the striatum and/or adult behavioral toxicitiy. This will be assessed by comparing AED treatments that are proapoptotic with those that avoid this toxicity. We hypothesize that AEDs that do not cause cell death will not cause impaired maturation of IPSC frequency in striatal MSNs, nor will they result in impaired rotorod performance, reduced seizure threshold, and impaired prepulse inhibition ofthe acoustic starle response. Moreover, the extent to which a neuroprotective treatment can prevent impaired maturation of IPSC frequency in striatal MSNs and/or behavior will be evaluated to test the hypothesis that neuronal death is necessary for this adverse functional outcome. The results ofthe proposed experiments will allow us to better understand the potential functional outcomes of exposure to AEDs in late gestation or early infancy, and the extent to which induction of excessive neuronal death is a valid marker of subsequent functional impairment. Furthermore, it will identify strategies to avoid deleterious functional sequelae of AED therapy during critical developmental periods. GOALS FOR KIRSCHSTEIN-NRSA FELLOWSHIP TRAINING AND CAREER I am seeking this fellowship tp support my dissertation research in Dr. Karen Gale and Dr. Stefano Vicini's laboratories, investigating the impact of neonatal anticonvulsant drug exposure. My goal for dissertation research is to develop a broad skill set of experimental techniques that will allow me to address scientific questions at the level of molecules, cells, networks and behavior. The complementary expertise of my mentors provides such exposure. A limited portion of this support is also requested to allow me to continue to teach (present lectures to graduate and undergraduate students, and continue to direct a course entitled, \"Diseases and Disorders of the Brain\". A key goal of my training is to complete and publish a series of focused studies on outcomes following drug exposure during development. My long-term goals are predicated on the broad training I am receiving at Georgetown. Following Georgetown I will seek post-doctoral training and then I hope to develop an independent, productive research program at an academic institution where I can ask and answer questions of interest at multiple levels of function, to maintain an active Involvement in teaching and mentoring, and to contribute to scientific discourse."}
-{"text": "Histone deacetylases (HDACs) are vital regulators of fundamental cellular events, including cell cycle progression, stem cell functions, cell fate determination, cell differentiation, and the pathogenesis of many diseases. As such, it is not surprising that protein acetylation is central to human diseases, as diverse as neurodegenerative disorders, cardiac hypertrophy, cancer, HIV infection, and more generally the process of aging. More importantly, small molecule HDAC inhibitors and activators are currently in clinical trials for the treatment of leukemia and lymphoma, solid tumors, neuromuscular disorders, metabolic disorders and other diseases. In addition, the Sirtuins, a subclass of HDACs, were fingered in yeast genetic studies as regulating lifespan, and these enzymes might be targeted by resveratrol, one of the components in red wine that has been linked to increased lifespan in humans. Therefore, a thorough understanding of HDACs is required, not merely for understanding the regulation of chromatin structure, gene regulation and protein function, but also because HDACs are intimately involved in normal and abnormal cellular processes that greatly impact human health. With the identification, isolation, cloning and functional characterization of 18 human HDACs (HDAC1- 11 and SIRT1-7) and many acetyltransferases in the past decade, the coming years will see a continued dramatic expansion in our knowledge of the biological roles of HDACs and protein acetylation. As the only conference dedicated to this field, this biannual meeting plays an essential role in bringing together approximately 44 basic and clinical scientist speakers to exchange information and develop new therapeutic avenues with approximately 120 participants from around the world. A primary objective is to transfer knowledge between basic academic researchers, clinical scientists, and pharmaceutical scientists to create efficiencies in understanding how they control human health and how these activities can be harnessed to fight a diverse slate of diseases. A second objective is to foster the development and interests of younger investigators to help support their career development. To accomplish these objectives, the meeting venue houses meeting space, dining, and rooms so that participants have ample time to move from formal presentations to informal brainstorming and collaborative discussions. In addition, the morning and evening oral presentation sessions include 2-3 talks from junior scientists selected from the submitted abstracts, which is important for career development and to encourage the next generation of HDAC scientists. There will be an emphasis through both the scientific and social programs on creating a global HDAC community. This meeting will be particularly timely because data from on a new generation of HDAC and Bromodomain drugs will be presented. Therefore, support is requested for the 4th biannual conference on the biology and therapeutic targeting of HDACs and Sirtuins, and their role in aging and disease to be held at Il Ciocco, Lucca, Italy, August 18-23, 2013 in conjunction with FASEB."}
-{"text": "[unreadable] [unreadable] Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder for which there are currently no established effective treatments. The identification of the disease gene in 1996 and the subsequent elucidation of the function of the encoded protein, frataxin, have opened the door to possible therapeutic approaches, including conventional high-throughput drug screening. This proposal describes a novel, complementary approach to the development of therapeutics for FRDA. We devised a method to construct a library that encodes random, short-hairpin-loop RNAs (shRNAs). This library can be used to identify shRNA sequences of therapeutic, therapeutic-targeting, and/or biological interest. The identification is based on functional selection, with effective sequences retrieved by PCR from cells that survive a particular condition or exhibit a predetermined phenotype. The design allows for hit-optimization of effective sequences, with re-selection for sequences with improved effects. Using primary FRDA fibroblasts, we developed screening and selection assays based on the critical role of mitochondrial dysfunction in the signs and symptoms of FRDA and on the sensitivity of FRDA cells to oxidative stress. With our live/dead selection assays, we can use our random shRNA-encoding library to identify shRNA sequences of benefit to FRDA cells. With our screening assays of mitochondrial function, we can confirm and prioritize these sequences. The primary objective of this proposal is to identify potential shRNA therapeutics. A secondary objective is to begin to understand the mechanisms of action of the shRNAs we identify. The Specific Aims are: 1. To identify shRNA sequences that allow survival under conditions lethal to primary FRDA fibroblasts but non-lethal to normal control cells. 2. To optimize the shRNA sequences identified in Aim 1. 3. To confirm and prioritize optimized shRNAs and begin to understand mechanisms of action. This proposal describes an approach to develop novel therapeutics for Friedreich ataxia using a random shRNA library. This approach has potential implications for the development of therapeutics for other genetic diseases, as well as for infectious diseases, and is therefore highly relevant to public health. [unreadable] [unreadable] [unreadable] [unreadable]"}
-{"text": "Conventional methods for treating chronic diseases such as HIV/AIDS have proven to be relatively inefficient both in terms of administration of therapeutic molecules and eradication of the underlying diseases themselves; while conventional antiretroviral therapy (ART) suppresses plasma viremia, low-level viral replication persist. Our recent findings have indicated that pigtailed macaques transplanted with gene-modified CD34+ cells expressing a membrane-anchored fusion inhibitor (mC46) develop infection-resistant CD4+ T-cells, maintain normal CD4+ T-cell levels, and develop an enhanced immune response against the SHIV-challenge virus resulting in a 300- to 1400-fold decrease in plasma viremia. Despite these very encouraging results, additional methods to further reduce plasma viremia to undetectable levels and/or potentially eliminate viral reservoirs will be required. Hence, we propose the development of a novel delivery system based on modifying hematopoietic stem cells (HSCs) or CD4+ T-cells in order to directly target viral reservoirs in vivo. The use of primary cells to deliver therapeutic peptides represents an innovative and ideal mode for systemically delivering therapeutic molecules throughout a patient's body. Furthermore, the ability of HSC-derived lineages to transverse both physiological and anatomical barriers represents a unique potential of this therapy. Low levels of engraftment would likely yield sufficient quantities of secreted antiretroviral peptides, while the efficiency f uptake would be greatly enhanced as there is a continuous source of the therapeutic molecules at sites typically thought to maintain persistent viral reservoirs. In order to deliver proviral targeting endonucleases to latent reservoirs, terminally differentiated primary cell such as CD4+ T-cells that migrate throughout lymphoid tissue represents the ideal vehicle. Hence, the goal of this study is to examine the feasibility of using genetically modified HSCs and CD4+ T-cells to secrete antiretroviral molecules to control viral replication and directly target latent reservoirsin vivo."}
-{"text": "Breast cancer is the second most prevalent cancer in women, with a mortality rate of 40,000 per year in the US. However, if detected and treated early, more than 95% of breast cancer patients will survive. Current method for breast cancer detection relies heavily on X-ray mammography, which produces many false positive findings. These false positive readings cause substantial mental anguish in patients and in many cases, costly and painful biopsies. A full 80% of all biopies uncover only benign masses and calcifications at an estimated annual cost of 2.5 billion dollars. To improve breast cancer detection, we propose a highly innovative design for a dedicated Positron Emission Mammography (PEM) camera utilizing long, linear, lead-walled straw (LWS) detectors that provide high spatial resolution and rigorously accurate depth of interaction determination. Thus detectors with ample field of view to contain the entire breast without translation can be employed and very close detector pair spacing can be used without resolution degradation. Our proposed camera offers markedly increased sensitivity and resolution with substantially reduced cost compared to crystal-based PEM cameras under development. Phase I of the project will achieve proof of concept by constructing and testing two substantial submodules of a full scale camera. In Phase II, a full scale high sensitivity prototype camera will be constructed and tested."}
-{"text": "The goal of our proposed research is to improve diagnosis of patients presenting to the Emergency Department [ED] with dizziness, some of whom are misdiagnosed with potentiallygrave medical consequences. The prevailing diagnostic paradigm for the evaluation of the dizzy patient is based upon a 'pathophysiologic' approach. This approach begins a search for etiology with the assumption that the quality of symptoms (vertigo, presyncope, imbalance, or non-specific dizziness) reflects the underlying pathophysiologic mechanism (vertigo = vestibular, presyncope = cardiovascular, imbalance = neurologic, and non-specific = psychiatric). Although this assumption often holds true, the 'pathophysiologic' approach mandates a thorough etiologic search in each organ system, not only the one suggested by symptom quality. This strategy is well suited to the referral clinic setting where it was developed, but poorly suited to the time-pressured environment of the ED, where the high index of illness severity demands effective triage rather than diagnostic certainty. We hypothesize that (a) potentially serious misdiagnoses of dizzy patients are uncommon but not rare events in the ED and may result from an over-reliance on the diagnostic importance on symptom quality; (b) a novel 'triage' approach to diagnosis would reduce misdiagnoses and improve outcomes in an 'in vitro' computer model of the diagnostic approach to dizziness; and (c) a clinical decision-support system based on this approach would reduce misdiagnoses under simulated patient conditions. To test our hypotheses, we have designed three specific aims to: (1) measure the frequency, potential severity, and possible cause of misdiagnosis of dizzy patients in the ED (by gathering extensive case data on each ED dizzy patient and referencing ED physician [EP] diagnoses against those of a multidisciplinary expert panel); (2) design a computerized decision model to test a new 'triage' approach to diagnosis (by comparing 'in vitro' simulations of the two diagnostic approaches using hypothetical case scenarios); and (3) 'pilot' a web-based decision support system to reduce misdiagnosis of simulated ED dizzy patients (by comparing EP performance on a video-case-based examination with or without the use of the decision support system, using a randomized trial design). Results of this study will form the foundation for subsequent research into the effectiveness of error-reduction interventions among dizzy patients. The research career award candidate has devoted himself to acquiring the clinical and research skills required to complete this project and launch a successful career as an independent investigator. He has garnered the support and enthusiasm of both his clinical department and a large, multidisciplinary team that will enable him to complete the stated objectives. This research project and the research paradigms derived from it will form the nucleus of a career devoted to research in medical decision-making, causes of diagnostic errors, and methods to preventthem."}
-{"text": "Traumatic brain injury (TBI) is a significant problem in the pediatric population. Ninety per cent of pediatric TBIs present to an emergency department, but only 8% are hospitalized. Since the majority of pediatric patients with mild TBI are therefore discharged home with the diagnosis of concussion, accurate assessment of the severity if concussion and consequent outpatient management and instructions are critical for ensuring safe recovery from injury. Without state-of-the-art knowledge and clinical tools, mild TBI (mTBI) may go undiagnosed and untreated, leaving individuals who have sustained a mTBI with an increased risk for functional problems. The ACE and ACE care plan were developed as part of the CDC's \"Heads Up: Brain Injury in your Practice\" toolkit for physicians to manage mTBI. Adapting the ACE for the ED and implementing a standardized clinical protocol by ED physicians systematically should improve management by ensuring accurate diagnosis and improving patient education and adherence with discharge recommendations. The goal of this research is to demonstrate the capacity to improve diagnosis and management of mTBI presenting to the Emergency Department (ED) by the feasible application of systematic procedures in the form of the ACE and the ACE Care Plan. This study will be conducted collaboratively by Children's National Medical Center and UPMC/ Children's Hospital of Pittsburgh with the specific aims to: (1) evaluate the feasibility of the ACE and ACE Care Plan for standardized implementation in the ED setting (2) determine if the ACE and ACE-ED Care Plan can be implemented by the ED staff and disseminated to the Primary Care Providers and (3) determine if routine use of the ACE-ED and ACE-ED Care Plan will improve patient/family follow-up behavior and patient recovery. We have designed the study to progress in two stages: Stage 1 proposes to develop expert consensus agreement regarding the importance and feasibility of using the ACE and ACE Care Plan in the ED setting, including an understanding of current concussion management care pathways. An outcome of Stage 1 will be the consensus-based adaptation of the ACE and ACE Care Plan for the ED, referred to as the ACE-ED and the ACE-ED Care Plan. Stage 2 applies these revised tools via a pilot implementation study for patients age 5-22 years old presenting with mTBI. The primary outcome will be patient/family follow-up behavior with the primary care/specialist. As secondary outcomes, we will examine clinician adherence to use of the ACE-ED and ACE-ED Care Plan, and its dissemination to primary care providers. Feasibility of implementation will be further evaluated by identifying the actual facilitative and barrier conditions to ACE-ED/ Care Plan use within the ED setting. We will also develop estimates of effect of this implementation on patient recovery. Upon study completion, key data will be available to support the development of"}
-{"text": "HIV-positive adult male and female study subjects will be recruited from Moore Clinic patients who have positive HCV antibodies and positive HCV RNA on stable antiretroviral regimens (>4week). Thirty (30) subjects will be selected for study participation according to inclusion and exclusion criteria in the clinical protocol."}
-{"text": "The overall objective of our study is to understand the manner in which the genetic information of mammalian cells is expressed and regulated and to determine how these processes are altered in cancerous cells. Our immediate goal is to describe the steps involved in the synthesis of mRNA in the nucleus, any modifications that it may undergo before being expressed in the cytoplasm, and finally its fate during and after translation. In addition we wish to characterize the early events associated with globin mRNA synthesis in erythroleukemia cells. These studies are in extension of our earlier work concerned with the identification, isolation and characterization of the rabbit and mouse globin mRNAs. During this time we have successfully translated these mRNAs in a variety of cell-free systems, described their translation efficiency, purification, poly(A) content, molecular weight, methylated base content, presence in ribonucleoprotein particles and their distribution between membrane bound and free polysomes. We have initiated studies concerning the synthesis of globin mRNA in the nucleus and described a specific poly(A) shortening mechanism. Our immediate aims are: (1) to continue our studies on the shortening of the poly(A) sequence of mouse globin mRNA, (2) to describe the location of the methylated nucleosides in globin mRNA, (3) to isolate and characterize the globin mRNA precursor, (4) to measure the synthesis and turnover of globin mRNA following the committed division in erythroleukemia cells and (5) to ascertain if post-transcriptional regulation occurs during differentiation of these cells. The ability to isolate molecules containing globin mRNA sequences using mRNA specific affinity column prepared by attaching globin cDNA to cellulose is an important technical advance which will aid these studies. We have prepared such a column and found it successfully isolates globin mRNA. The column will now be used to prepare precursors to globin mRNA."}
-{"text": "This second revision of an R29 application seeks to advance our understanding of the molecular mechanisms by which the movement of water across the human cervical epithelial barrier. The applicant will use cultured human epithelial cell models developed in his laboratory to explore three hypotheses: 1) that estrogen increases transcervical permeability by means of an effect on cervical cell tight junctions, 2) that estrogen increases calcium mobilization in the cells, which in turn increases the permeability of the intercellular space, and 3) that the mechanism of Ca++ mobilization in the human cervical cell is a Ca++-dependent stimulation of cellular KCl transport, which results in a reduction in cell volume. It is proposed that the results of these studies will increase both our understanding of the basic biology of cervical fluid homeostasis, but also a better understanding of clinically relevant settings in which the cervical fluid status is abnormal."}
-{"text": "Alcohol abuse can have devastating impacts on the brain and behavior and is a big burden on public health. However, the molecular underpinnings of alcohol induced neural cell injury are not fully understood. Recent advances in functional genomics suggest that long non-coding RNAs (lncRNAs) may play critical roles in alcohol- induced neural cell death. The recent work from our laboratory support this hypothesis. Therefore, a comprehensive screening system of lncRNAs related to alcohol-induced neural cell death is of significant benefits for identifying potential therapeutic targets. Genetic editing tools such as Zinc Finger Nuclease (ZFN) and Transcription Activation-Like Element Nuclease (TALEN) have great potential for the functional study of genes or the application of gene therapy through knockout or knockin techniques. A new type of genetic editing tool based on bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR?Associated System [Cas]) has been successfully used in various organisms from bacteria and yeast to mammals. Relative to ZFN and TALEN, CRISPR/Cas is advantageous because it only requires changing the sequence of the guide RNA (gRNA) and it can also be directly delivered into embryos to generate gene-modified organisms. Furthermore, the multiplexing capability of CRISPR/Cas makes it possible to target multiple genes simultaneously. In this study we will use our recently developed dual guide CRISPR/Cas approach to generate an lncRNA knockout (gRNA) library in SH-SY5Y cell model. We will then perform a genome-wide screening for lncRNAs involved in alcohol-induced cell death pathways. Our preliminary data indicated that alcohol increased the expression of a nuclear paraspeckle lncRNA in SH-SY5Y cells. Our previous work have demonstrated that alcohol activates the oxidative stress-apoptotic cell death pathway in a dose dependent manner to reduce neuronal cell viability and increase cellular oxidative stress. We expect that the application of the high throughput screening platform in alcohol-induced neuronal death system will allow for the accurate identification of all other specific lncRNAs as well as their potential roles in mediating alcohol-induced neurodegeneration. The generated lncRNA gRNA library in this study can also be available for the characterization of cell toxicity induced by other neurotoxins or other substance abuse. Thus this research has a potential to reduce the health impacts of alcohol abuse and also has benefits for other substances abuse related public heath burdens."}
-{"text": "There has been a huge increase in demand for comprehensive quantitative analysis of neurovascular imaging data produced in the clinical setting for diseases such as multiple sclerosis, traumatic brain injury, stroke and dementia. Our objective in this project is to design and develop advanced image processing software that can rapidly and accurately analyze such data. To achieve this objective, we propose a range of novel algorithms to process data from the following MR imaging sequences widely used in the aforementioned applications: time resolved 3D contrast enhanced MR angiography (CE-MRA) for the assessment of vascular anatomy, time resolved 2D phase contrast flow imaging (PC-MRI) for the evaluation of vascular hemodynamics, susceptibility weighted imaging (SWI) for quantifying iron deposition in the brain, and fluid attenuated inversion recovery (FLAIR) imaging for the detection of white matter hyperintensities (WMH) and lesions. A variety of tools will be designed and implemented to tackle these problems including: tissue similarity mapping and active shape models to segment the vasculature in both CE-MRA and PC-MRI images; automatic tissue segmentation in the basal ganglia and thalamus for a two-region of interest analysis for iron quantification with SWI; and finally adaptive approaches incorporating fuzzy C-means, shape factor analysis, compactness and fractional anisotropy to quantify lesions and WMHs. To exploit the advantages provided by different imaging sequences, co-registration algorithms will be used to improve segmentation of vessels between CE-MRA and PC-MRI, and between 3D T1 weighted imaging and SWI. Upon finishing this project, we expect a multi-fold increase in processing efficiency and a significant increase in accuracy will be achieved. The resulting software will not only help the growth of our company, but also improve the diagnosis and treatment of neurovascular diseases. PUBLIC HEALTH RELEVANCE: The huge increase in demand for a more comprehensive and accurate analysis of the vast amount of clinical MR imaging data for neurovascular diseases such as multiple sclerosis, traumatic brain injury, stroke and dementia is the driving force for th development of more advanced image processing software in our company. In this project, we propose an integrated approach to develop a set of processing software for imaging sequences that target the assessment of both anatomy and function of the neurovasculature system. The results will lead to a better access to quantitative data about the brain's vasculature, flow, hemodynamics and iron content present in neurovascular diseases. The completion of this project will not only help the growth of our company by increasing processing throughput and accuracy, but also improve the diagnosis and treatment of patients with neurovascular disease."}
-{"text": "Nocturnal cyclic intermittent hypoxia, such as experienced by patients with obstructive sleep apnea (OSA) is thought to alter vascular tone and function leading to peripheral vasoconstriction and consequent arterial hypertension. Studies in patients indicate that sleep apnea results in diminished reactivity to endogenous vasodilators, and altered sensitivity to some endogenous vasoconstrictors. Furthermore, OSA patients demonstrate an augmented pressor response when exposed to hypoxia and fail to decrease forearm vascular resistance (FVR) when exposed acutely to progressive isocapnic hypoxia as do non-apneic volunteers. Our own preliminary data, collected in normal volunteers and OSA patients, suggests, however, that intermittent hypoxic exposure may lead to vasodilation rather than vasoconstriction. Normal volunteers exposed to intermittent hypoxia for 14 nights have no change in vascular resistance despite increased sympathetic activity and also fail to vasodilate when acutely exposed to isocapnic hypoxia after the repetitive exposure. Based on these and other observations we propose three hypotheses. First, we hypothesize that hypoxic exposure causes either sustained vasodilator release (e.g., epinephrine, NO) that persists during normoxia or altered sympathetic transduction with diminished vasoconstriction (e.g., altered receptor density or transmitter release), or both. Second, we speculate that an acute re-exposure to hypoxia after a prior intermittent exposure results in an abrupt increase in sympathetic nervous system activity but little further increase in vasodilator release, thus resulting in impaired vasodilation. Finally, we hypothesize that maximum vasodilator release declines over time with continued hypoxic exposure, resulting in a gradual increase in arterial pressure. To test these hypotheses we plan a series of investigations in normal volunteers before and after an exposure to cyclic nocturnal hypoxia for 28 nights, and in OSA patients before and after 30 days of monitored therapy with nasal CPAP. These studies will use selective intra-arterial infusions of specific pharmacological agonists and antagonists to assess the roles of endogenous vasodilators and vasoconstrictors in altering vascular tone following an exposure to hypoxia. We anticipate that these studies will significantly enhance our understanding of how intermittent hypoxia and obstructive sleep apnea influence vascular tone and function."}
-{"text": "Examination of the potential therapeutic effect of 4-ASA on patients with ulcerative colitis. This is an FDA \"Orphan Drug\" sponsored double blind randomized 6 week controlled trial that includes state of the art assessmen of the severity of the disease."}
-{"text": "Cellular immunity (CI) to tumor-antigens (TA) was evaluated and monitored using microassays of direct and indirect migration inhibition (MI), isotopic footpad and Winn neutralization tests as a function of growth of mouse mammary tumor-virus (MuMTV)-induced tumors, SV-40 virus-induced sarcomas, chemically induced sarcomas and plasmacytomas, and guinea pig hepatocarcinoma. CI to TA and antigens of MuMTV was observed in tumor bearers. CI decreased as magnitude of tumor-burden increased. Immunological \"eclipse\" with plasmacytomas was associated with Sephadex G10 adherent cells and the effect was reversed by levamisole therapy. Direct capillary tube and indirect MI microassays demonstrated production of leukocyte inhibitory factor (LIF) of human breast and lung carcinoma, Ewing's sarcoma and melanoma patients in response to tissue culture tumor extracts, and of breast cancer patients to MuMTV. High titered LIF supernatants were generated with small numbers of mononuclear cells by PPD and lung and breast cancer extracts and MuMTV. Physicochemical separation of supernatants was successful in partially purifying LIF. Evaluation of indicator cells for indirect human and mouse MI demonstrated that some humans and mouse strains were insensitive to lymphokine. In mice this was due to suppressor cells."}
-{"text": "Breast cancer susceptibility gene, BRCA1, has been implicated in the maintenance of genomic integrity. However, the molecular mechanism(s) by which BRCA1 plays a role in maintenance of genomic fidelity remains to be defined. Interestingly, recent studies in our group and others have demonstrated that BRCA1 can regulate the GADD45, a p53-regulated stress inducible gene that plays an important role in cellular response to DNA damage. These results have evidently linked GADD45 to BRCA1 and raised the possibility that GADD45 might be a BRCA1-downstream effector and mediate BRCA1's role in maintenance of genomic stability. Therefore, this proposal seeks to define the biochemical mechanism(s) by which BRCA1 transactivates the GADD45 gene, and to define the role of the BRCA1-GADD45 pathway in the induction of apoptosis following genotoxic stress. The long-term objective described in this application will specifically focus on three key issues: (1). To characterize the GADD45 gene as a BRCA1's downstream effector. We will define the BRCA1-regulatory elements in the GADD45 promoter and identify the proteins that modulate the BRCA1 transactivation of the GADD45 promoter. (2). To define whether the GADD45 is an essential player in the BRCA1-induced apoptosis. We will analyze the induction of apoptosis following expression of GADD45 and BRCA1. We will also examine the alterations of the BRCA1-activated apoptosis and BRCA1-induced growth suppression in GADD45- deficient cells. (3). Our most recent studies demonstrated that BRCA1 is cleaved by caspase-3 during apoptosis induced by DNA damage. This DNA damage-activated cleavage results in an accumulated 90-kDa band of the BRCA1 C-terminus. Therefore, we will first determine whether the BRCA1 cleavage is required for BRCA1-activated apoptosis following DNA damaging agents. We will also determine the functional role of the cleaved 90-kDa band of the BRCA1 C-terminus in activation of apoptosis. Finally, we will analyze the role of this cleaved product in the BRCA1 transactivation of the GADD45 promoter. The studies proposed in this application would define a novel pathway (BRCA1-GADD45) controlling apoptosis following DNA damage and provide information regarding the biochemical mechanism by which BRCA1 regulates its targeted genes. Since apoptosis is closely associated with the therapeutic sensitivity, the perspective outcome will also provide insight into the development of therapeutic agents."}
-{"text": "[unreadable] [unreadable] Epilepsy affects over 2.5 million people in the US, and over 180,000 new cases of the disease are diagnosed each year. Nearly half of the people suffering from epilepsy are not effectively treated. Moreover, currently used anticonvulsants can cause significant side effects, which often interfere with compliance. Clearly, there is a need for new, safer drugs to treat epilepsy. Glutamate, by stimulating NMDA receptors, has been implicated in the neuropathology and clinical symptoms of epilepsy, and NMDA receptor antagonists are potent anticonvulsants. Antagonists at the GlyB co-agonist site inhibit NMDA receptor function and are also anticonvulsant. Importantly, GlyB antagonists have fewer side effects than classic NMDA receptor antagonists and other antiepileptic agents, making them a safer alternative to available anticonvulsant medications. 7-Chlorokynurenic acid (7-C1-KYNA) is one of the most potent and specific GlyB antagonists currently known and is a powerful anticonvulsant when injected into the brain. However, like almost all GlyB antagonists developed so far, 7-C1-KYNA crosses the blood-brain barrier very poorly and is therefore ineffective following peripheral administration. Its pro-drug, L-4-chlorokynurenine (4-C1-KYN), on the other hand, readily gains access to the brain. Following systemic administration, 4-C1-KYN is efficiently converted to 7-C1-KYNA and prevents seizures in animal models of epilepsy. 7-C1-KYNA formation occurs preferentially in brain areas that suffer seizure-related brain injury, thereby reducing the risk for side effects with chronic use. Furthermore, 4-C1-KYN forms a second metabolite, which blocks the synthesis of the proconvulsant quinolinic acid in brain. These unique properties make 4-C1-KYN a highly innovative candidate for the treatment of epilepsy. The project proposed here by Vistagen will advance the preclinical development of 4-C1-KYN as a treatment for adult and childhood epilepsy. We will conduct extensive pharmacokinetic and ADME analyses to further delineate the oral bioavailability of the drug observed in pilot studies, will subject 4-C1-KYN to a battery of safety and toxicology studies and will test anticonvulsant efficacy of the compound in two chronic animal models of epilepsy, CLE and kindling. Finally, we will begin to develop the protocols needed to submit an IND for FDA approval to conduct clinical testing of the drug in humans as a new and improved anti-epileptic agent. [unreadable] [unreadable] [unreadable]"}
-{"text": "Abstract for Coordinating Center Core (CCC) The UPCI-CMCR-CCC will provide three critical services to NIAID and the other components of the 5 ? 7 CMCR Programs. The CCC will cooperatively develop a program for archiving and distributing data from 5 ? 7 CMCR sites and will maintain electronic data storage and develop security passcodes and validations for retrieval of data. The CCC will develop a program for timely submission of data and data analysis at NIAID/NIH and maintain rapid availability of electronic exchange of records to CMCR Programs as well as the general scientific community. The CCC will coordinate administrative activities for the scientists organizing national meetings, coordinating conference calls, establishing three levels of firewalls to distribute information at levels of CMCR Centers locally, CMCR Programmatic Centers for all 5 ? 7 programs, and to the general scientific community. Finally, the CCC will administer funds for the development of web-based training programs in four key areas: 1) Principles of Radiobiology, 2) Use of assays, methods, reagents, and animal models, 3) Technologies to study radiobiology and develop new product regulatory process, and, finally, 4) A course on triaging radiation casualties in the event of a radiation terrorist event."}
-{"text": "This research proposal tests the hypothesis that identification of the requirements for in situ activation of tumor-infiltrating lymphocytes (TIL) will provide the biological basis for immunotherapy of human solid tumors without infusion of ex vivo- activated lymphocytes. Specific aims are as follows: 1. Characterization of TIL from melanoma, renal carcinoma and sarcoma. There are at least six different TIL subsets as follows: CD4-CD8+ T cells with (i) autologous tumor-specific or (ii) MHC- nonrestricted (LAK) cytotoxicity; CD4+CD8- T cells with (iii) autologous tumor-specific The activity or (iv) LAK activity, (v) CD3-CD16+ NK cells with LAK activity and (vi) CD4-CD8- T cells. We will (i) purify each of these subsets before and after incubation with rIL2; (ii) establish cloned cells; (iii) investigate their functions; (iv) elucidate requirements for activation of each TIL subset (IL-2,other cytokines, autologous tumor cells or autologous macrophages), and (v) investigate molecules involved in the TIL activation (TCR, CD3, CD2, CD4, CD8, CD16 and LFA antigens on TIL). 2. MHC-restriction of antitumor activity. We will investigate if autologous tumor-specific CTL or The activity in melanoma TIL is restricted by MHC-Class I or Class II antigen expression on tumor targets respectively by determining MHC-haplotypes of tumors. 3 and 4. Determination of requirements for CD4 CD8- TIL to produce IL-2, and differentiation of resting blood lymphocytes into TIL. Role of macrophages. Requirements tested include: IL-l, IL-2, IL2R-inducing factor, anti-CD3 mAb., hybridomas expressing anti-CD3 mAb, autologous monocytes, tumor cells, CD4+CD8- or CD4-CD8+ cloned TIL. 5. Antitumor activity of lymphocytes from LN with melanoma metastasis. LN-lymphocyte subsets will be incubated in the presence of IL-2, anti-CD3 mAb, IL-l and/or autologous monocytes, followed by testing their proli- feration and antitumor activity. 6. Identification and purification of IL-2 receptor- inducing factor (IL2R-IF). Culture supernatants from the established CD4+ TIL line (TILh-Ll) and PBMC stimulated with PHA and PMA will be used for this study. Knowledge of immunological properties of TIL and understanding the mechanisms involved in TIL activation and the biology of host-tumor relationships will provide important findings for development of in situ lymphocyte activation and new strategies in the immunotherapy of human solid cancers."}
-{"text": "Our studies on novel small molecules inhibitors of HIV integrase have led to the identification of new potent phenanthrene diketoacid (DKA) HIV integrase inhibitors that also inhibit HIV replication in cell culture, with a selectivity index up to 10. In light of the need for more effective and less toxic anti- HIV drugs, these results appear promising and warrant follow-up optimization and preclinical studies. However, since phenanthrenes are polyaromatic hydrocarbons (PAHs) with potential carcinogenicity we propose to investigate these novel phenanthrene diketoacids for carcinogenicity potential. Our hypothesis going forward is that appropriate substitution with electron withdrawing groups can minimize mutagenicity and produce useful anti-HIV agents. This is supported by the example of the new anti-malarial drug halofantrine, a substituted phenanthrene that is effective against multidrug resistant malaria, and is neither mutagenic nor teratogenic. We thus believe that appropriate substitution of our HIV integrase inhibitory phenanthrene DKAs will make them non-mutagenic and non-carcinogenic. Thus the specific aims of this proposal are: 1) to synthesize and test the IN inhibitory activity of new phenanthrene diketoacids with multiple electron withdrawing substituents and 2) to determine the carcinogenicity potential of the phenanthrene DKAs synthesized in Specific Aim 1. We will use the Ames test for in vitro carcinogenicity testing. The success of this project will provide vital information to decide whether or not to go ahead with optimization of the potent phenanthrene DKA HIV integrase inhibitors that we discovered recently and shown to selectively inhibit HIV replication in human peripheral blood mononuclear cells. If this approach to reducing carcinogenicity is successful it will not only provide a means of eliminating carcinogenicity of the phenanthrene DKAs, but may also provide a general method for reducing the carcinogenicity of PAH containing drugs. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: This project is aimed at investigating whether or not a novel class of potent phenanthrene diketo acid HIV integrase inhibitors that have been shown to suppress HIV viral replication in cell culture, are carcinogenic. The results of the research will determine whether to carry on with further work on optimizing this class of new anti-HIV agents towards AIDS therapeutics development. [unreadable] [unreadable] [unreadable] [unreadable]"}
-{"text": "For the past three years, the Depression Unit and the Clinical Research Unit have collaborated in a study which surveys a population of individuals maintained on methadone to determine the prevalence of depression. The group studied included about 100 volunteers, and was generally representative of the entire clinic population in terms of such features as age, race and sex. In this research participants were evaluated based on several standardized rating scales measuring depression as characterized by verbal self-report, behavior, and symptoms. On the basis of the survey result, over 30 percent of these individuals were considered to be significantly depressed, including some patients with rather severe degrees of depressive symptoms. In most clinical settings, the degree of depression as shown by members of this group is considered of sufficient intensity to require antidepressant medication. We therefore propose a further study which assesses the efficacy of an antidepressant medication in treating depression in individuals maintained on methadone. The study will include 50 patients who exhibit significant depression at the time of admission to the Methadone Program. The selected individuals will be randomly assigned to receive dosages of the antidepressant Tofranil (imipramine) or a placebo on a daily basis for eight weeks. All medication will be dispensed on a double-blind basis and will be combined with daily methadone dosages. Throughout this drug trial, depression will be measured by self-reports, and by clinical assessment of behavior and symptoms. It is expected that the study will take two years, and will provide a sufficient sample to assess the efficacy of antidepressant medication in treating depression in this population."}
-{"text": "The in vitro carcinogenesis model derived from organ culture of the whole mammary glands of BALB/c female mice will be used to study the transforming action of the environmental carcinogens, benzo[a]pyrene (B[a]P) and N-diethylnitrosamine (DENA). Appearance of the potentially neoplastic nodule-like alveolar lesions (NLAL) in the mammary glands in vitro after exposure to B[a]P or DENA will be used as a morphological marker for preneoplastic transformation. Carcinogenecity of the mammary epithelial cells transformed in vitro will be confirmed by the ability of cells to produce mammary carcinomas after transplantation into gland-free mammary fat pads of syngeneic virgin mice. The unique organ culture model of mammary cell transformation also will be used to study the mechanics of chemopreventive action of the dietary compounds, a new retinoid, Beta-carotene and selenium, during the neoplastic process induced by DMBA, B[a]P or DENA. Influence of the chemopreventive agents on the molecular events such as cellular accumulation of the electrophilic reactants, DNA alkylation and DNA repair activity initiated by the carcinogenic chemicals will be examined. Attempts will be made to ascertain whether influence of the chemopreventive agents on the molecular events of \"initiation\" may alter morphological expression of the transformed cells as NLAL in the glands in vitro and/or in the mammary tumors in the mammary fat pads in vivo. Studies also will include determination of the influence of the chemopreventive agents during hormone mediated \"promotion\" of the initiated (transformed) cells, both in vitro and in vivo. These studies will examine influence of chemopreventive agents at the specific stages of transformation between normal -- preneoplasia and preneoplasia -- neoplasia. The results should provide elucidation o the mechanics of chemopreventive action of these dietary compounds."}
-{"text": "The feasibility of a device to enable precise and repeatable calibration of single-breath lung diffusion (DLCO) instruments will be investigated. The calibrator will enable the accuracy of DLCO instruments to be verified between pulmonary laboratories, allowing greater confidence in a diagnosis based on the single breath DLCO test. This study will verify the performance of a unique gas collection and mixing system which will allow generation of precise carbon monoxide (CO) and Helium (HE) gas fractions without using gas analyzers. This will allow production of a DLCO calibrator which will not require any calibration of its own."}
-{"text": "Continued study of human fetal hemoglobin in normal and abnormal hematological conditions will be made to increase insight into the genetics and control of the linked beta, gamma, and delta structural genes. In particular, examination of cord bloods will be made to gain more information about a newly detected but poorly understood anomaly in the regulation of the gamma genes. These studies will be made in collaboration with Prof. T.H.J. Huisman of the Medical College of Georgia. It is hoped to complete the sequence studies on the catalases. If possible, studies of sequence in lactoperoxidase will be carried further."}
-{"text": "Project Summary Every year, a substantial number of children injure a growth plate, a cartilaginous region found at the end of all long bones in children that provides signals for the bones to lengthen. The growth plate is the most fragile structure in a child?s developing bones, making it prone to injury. Damaged cartilage within the growth plate is often replaced by unwanted bone, forming a ?bony bar?, which can lead to angular deformities or halt bone growth completely. Current surgical methods to correct bone growth defects are invasive, prone to infections and have low success rates. There is no current treatment that leads to complete repair of an injured growth plate. Innovative treatment strategies that prevent bony bar formation, restore functional growth plate cartilage, and permit normal longitudinal bone growth in affected individuals are greatly needed. This proposal seeks to develop an injectable hydrogel biomaterial system that could prevent bony bar formation and promote the formation of cartilage tissue, and which could ultimately be examined for its ability to heal growth plate injuries in children. It has been shown that mesenchymal stem cells (MSCs) infiltrate the injured growth plate and undergo osteogenic differentiation. Here, two important areas for inhibiting the osteogenesis of MSCs via a biomaterial system will be studied: (1) the role of biopolymer hydrogel substrate mechanics in preventing osteogenesis, and (2) the delivery of short interfering RNA (siRNA) from biopolymer hydrogels that can block osteogenic differentiation. We hypothesize that hydrogel systems that are less stiff and that provide sustained exposure of MSCs to p38 MAPK siRNA will inhibit osteogenesis. This proposal seeks to engineer hydrogel systems with these characteristics as a first step towards creating new technologies for helping to heal growth plate injuries in children. This will be accomplished as by two Aims: Aim 1 - To engineer a hydrogel system with mechanical cues that prevent osteogenic differentiation of MSCs. Aim 2 - To design a hydrogel that would provide sustained release of siRNA targeting p38 MAPK to prevent osteogenesis of MSCs. The p38 MAPK pathway has been linked to osteogenesis in various cell types, including MSCs. Local inhibition of this pathway by siRNA could prevent MSC osteogenesis from occurring after growth plate injury. For both Aims, these studies will be conducted in vitro and also in vivo in a rat growth plate injury model. This project is a first step towards successful development of a biomaterial system that can prevent MSC osteogenesis, which could ultimately aid in growth plate tissue repair."}
-{"text": "Through the proposed training in his K01, I will pursue the additional mentorship that I need and the additional research time to enhance my passion for intestinal epithelial biology. During this award period I will have the opportunity to acquire and refine fundamental skills of becoming a developed scientist. Career development will be fostered through twice-weekly meetings with Dr. Rustgi, convening an expert interdisciplinary advisory committee twice annually, and taking advantage of opportunities in career and professional development through UPenn, the NIH and the AGA. These skills include (but are not limited to) manuscript and grant preparation, lab meeting presentations, participation in the seminars and courses described herein, and presentations at international research conferences (DDW, Keystone). Training will also entail course-work in biostatistics (two semesters), short courses in RNA biology and basic bioinformatics, ongoing training in bioethics, weekly participation in the seminar series (where I will meet visiting professors), journal clubs (presentations semi-annually), and twice yearly floo lab meetings through the NIDDK P30 Center for Molecular Studies in Digestive and Liver Diseases and Division of Gastroenterology. The overarching hypothesis of this project is the following: LIN28B plays a critical role in regulating growth and proliferation in the intestinal epithelium via cooperation with c-MYC, with key roles for intestine stem cell (ISC) maintenance. Two potential interrelated mechanisms will be explored: 1) LIN28B binds to target mRNAs associated with cellular growth and cooperates with the transcription factor c-MYC, which is a master regulator of cell growth. and 2) LIN28B affects the maintenance of the ISC by promoting stem cell identity and growth. LIN28B is normally expressed in the intestinal epithelial crypt where cell division occurs. LIN28B represses the maturation of miRNAs such as Let-7, which is restricted to villus epithelium, where post- mitotic differentiated cells are located. The spatial restriction of LIN28B and Let-7 expression in the intestinal epithelium is likely crucial for maintaining sufficient but limited compartments of differentiation and proliferation. C-MYC is also restricted to the crypt epithelium and is required for epithelial proliferation and growth. W have evidence to support a Let-7-independent function of LIN28B entailing intimate cooperation with c-MYC. The Specific Aims of this proposal to test the hypothesis are the followin: 1) We will explore the functional relationship between LIN28B and c-MYC, in vitro. 2) We will assess how LIN28B cooperates with c-MYC through in vivo studies of Lin28b in the intestinal epithelium. 3) We will examine the potential of LIN28B to modulate the intestinal stem cell compartment. LIN28B function may be relevant to ISC homeostasis and epithelial proliferation. Determining how LIN28B functions in the intestinal crypt will likely provide an important link among the cellular mechanisms governing epithelial proliferation, differentiation, and/or mucosal regeneration. PUBLIC HEALTH RELEVANCE: This project will explore the regulation of cell growth in the lining of the intestine in stem cells and other dividing cells. Stem cells are required to constanly replenish cells of the intestinal lining throughout the entire life of an individual. This study wll provide key insights into how intestinal cell growth is maintained, controlled, and perturbed in disease states."}
-{"text": "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 ARF-p53 tumor suppressor pathway is one of the cell's major defenses against the stimulation of uncontrolled cell division induced by activated cellular and viral oncogene. ARF and/or p53 are mutated in over 70% of human cancers. The inappropriate activation of growth promoting cellular signaling pathways by oncogenes can result in the induction of ARF. The expression of ARF can activate p53 leading to apoptotic cell death or cell cycle arrest. The mechanisms by which the ARF-p53 pathway is regulated remains to be precisely elucidated. The expression of ARF can activate p53 leading to apoptotic cell death or cell cycle arrest. We have shown that the polyoma virus oncogene, PYMT, activates an ARF-induced p53 mediated block. We find that the polyoma virus small T-antigen, PYST, via its ability to bind to cellular protein phosphatase 2A (PP2A), can negate the ARF-induced block to cell division induced by PYMT. We intend to use the PY induction and inhibition of ARF signaling to p53 to better define this important tumor suppressor pathway. Our hypothesis is that the polyoma virus proteins are revealing an important new aspect of the ARF-p53 tumor suppressor signaling circuit, and we plan to use these viral proteins as tools to study its molecular basis. To better define the role of PYMT in activating ARF and as an oncogene, and to define the role of PYST in blocking ARF signaling to p53 we plan to characterize the proteins complexed to PYMT, PYST and members of the ARF-p53 signaling pathway. In the first instance proteins bound to TAP fusion constructs would be identified by Mass Spectrometry."}
-{"text": "The field of dermatology is riddled with many problems that expect solutions from modern biomedicine. Several biologicals/biotherapeutical approaches such as anti-TNFalpha antibodies have already made their impact in the daily treatment of dermatology patients. However, many challenges are still ahead. We still have to fill many gaps in our understanding of biological processes in the skin. For example until recently, an entire class of small regulatory RNA molecules, microRNAs, had remained undetected. Their emergence as important players in virtually all tissues and signaling pathways has led us to explore their significance for skin biology. In our proposal we will focus on one microRNA, miR-31, with an exceptional expression pattern in many skin diseases. This finding prompted us to address the functional significance of miR-31 in epidermal homeostasis, aberrant growth control, wound healing, stress response and hair growth. Its involvement in the regulation of major skin signaling pathways such as TNFa, TGFb and BMP signaling makes it a central node for the control of epithelial-mesenchymal transitions. We have identified a crucial role of miR-31 in hair folliculogenesis, hair growth, nail growth, and the response to wounding and the phorbol ester, TPA. TNFa, EGF and FGF7 can induce miR-31 expression and TGFb stimulates the expression of miR-31 precursors. Based on our existing mouse model of miR-31 overexpression and our preliminary data, we have developed a research plan aimed to establish miR-31 as an important regulator of such processes as EMT, proliferation and motility in keratinocytes. Thereby, we will be able to judge the potential of miR-31 as a therapeutic target for skin diseases with defects in these processes. We plan to use our existing mouse model and establish novel tools in this proposal to apply to our career-long goal to translate this knowledge into therapeutic potential of miR-31 using miR-31 inhibitors in vivo. In Specific Aim 1 of this current research plan, we will test our hypothesis that miR-31 is a key regulator of hair growth and the response of the skin to challenges such as wounding. In Specific Aim 2, we will address the important issue of identifying miR-31 target genes to understand miR-31 mediated processes. And in Specific Aim 3, we will test our hypothesis that miR-31 regulates epithelial-mesenchymal interactions and transitions, EMI and EMT. Furthermore, we will test whether miR-31 buffers certain expression patterns and signaling pathways against unwanted fluctuations and define which pathways exactly are influenced by miR-31 in addition to EMT and hair growth regulating signaling networks."}
-{"text": "It has been shown that T lymphocytes are primarily responsible for the cell-mediated immunity of the host and the most important cells in the body to eliminate the neoplastic cells. The development of these cells depends upon the thymus gland. We have shown that there is a decrease of T cells in patients whose thymus glands were irradiated during early infancy. This population of patients also has a higher incidence of cancer. It is possible the decreased T lymphocytes may account for the increased incidence of cancer. Our present proposal is to extend our studies to include other populations of thymus-irradiated patients, especially the prenatal fetus, the premature infants and adult patients treated with radiation to their lung or breast. We hope these studies will determine the contributions of the thymus gland upon the T cell development during the different periods of life and the radiation effect to this gland at various thymus gland maturation stages as expressed by the proportion of T cells in the peripheral blood. Also, we will determine the radiation-induced chromosomal abnormalities in the peripheral blood lymphocytes in these patients."}
-{"text": "This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project focuses upon Infrastructure development in Computational Biology and Bioinformatics as part of the Core Resources at the Center for Biomedical Research (CBR) at Tuskegee University. Research involving advanced information technologies, the Internet and databases is crucial in examining and analyzing patterns of disease occurrence, genetic traits and molecular level dynamics including AIDS research."}
-{"text": "This study examines whether reductions in air pollution are associated with reductions in the prevailing mortality rates. The areas considered are census tracts that have \"high\" residential stability; this restriction reduces the effect of migration into and out of the area. Analysis of data from Buffalo will be completed by the end of the first year of the project. Next year, data from Detroit, Seattle, and the major cities of Ohio will be analyzed. Results to date from Buffalo support the hypothesis of an association between reduced air pollution levels and a reduction in the prevailing mortality rate; however, these results are not statistically significant. Consideration of the larger data sets will clarify this point. In addition to time trend analyses, the study is examining whether there are any unusual geographical clusterings of mortality rate changes and performing other exploratory analyses. The deaths are analyzed by cause of death to specify the nature of the putative air pollution and mortality association. Primary attention, of course, is given to lung and chronic heart disease."}
-{"text": "The Cell Deposition System (CDS) deposits biological cells on predetermined sites on a microscope slide. With this improvement in flow microfluorometry (FMF) and cell sorting technology, it will be possible to associate an arbitrary cell on the microscope slide with its FMF data. Such correlation is possible by using a computer to store a cell's FMF data and concomitantly determine a cell's location on the slide. An initial study (Tyrer et al., Proc. IEEE 67:10, 1979) demonstrated the feasibility of this device. This proposal seeks to develop an improved CDS prototype which can be used in our laboratory for slide preparation of diagnostic samples. This requires a straight-forward instrumentation development task for a laboratory prototype; studies of the interaction between slide substrate, cell fixative, cell stain and cellular morphology; and studies of the interaction between substrate, cell nutrients and cells when dealing with viable cell systems. This will result in the reliable preparation of slides so that human visualization as well as automated cell localization and analysis can be accomplished with confidence. The prototype will be used in two separate applications to determine a wide range of utility in fixed or living cells. Applications in cancer detection include preparation of microscope slides by placement of cells in a highly ordered fashion for human or machine analysis. The slides will have only cells of interest and be relatively free of debris. The other application involves depositing viable cells in a mechanically stable cell culture medium. Individual cells can then be assessed for their tissue culture character. The CDS will permit true single cell analysis since the computerized determination of cell address and storage of FMF values can be used to drive a Scanning Transmission Microscope (STOM). The STOM, a computerized image analysis microscope system, can use the CDS-derived data and microscope slide preparations to locate and further analyze interesting cells."}
-{"text": "We propose to develop an integrated microchip platform for rapid, reliable and comprehensive diagnosis of cancer by examining the pathophysiological mechanism of tumor-immune interaction. This will be done at both molecular and cellular level. By applying this platform to analyze a large number of clinical samples, we expect to gain new insights about cancer immunobiology that point to new approaches for cancer prevention and treatment. During the K99 phase, I have demonstrated an Integrated barcode platform that can measure a large number of protein markers from small quantities of whole blood or from single cells. A panel of signaling proteins that have important implication in tumor inflammation has been integrated Into the microchip platform. In the ROO phase, I will further utilize this technology to Investigate the fundamental biology of tumor-immune interaction via measuring the paracrine signaling pathways between tumor and immune cells. I will also study the heterogeneity of tumor microenvironment by combining population dynamics modeling and the microchip-based molecular analysis. Once this model is trained with experimental or clinical data, it will become a useful tool to predict the outcomes or therapeutic responses of cancer patients. In addition, an on-chip culture of tumor cells and immune cells will be developed to emulate tumor microenvironment and then used for rapid, effective anti-cancer drug screening. To accomplish these goals, I will (1) use the Integrated barcode chip platform to study chronic Inflammation as a common mechanism in various human diseases and to reveal their correlations; (2) apply this tool to monitor the Immunological responses of cancer treatment; and (3) design a Tumor-on-a-Chip to re-engineer the tumorimmune Interactions ex vivo and use this platform for effective anti-cancer drug screening with rich feedback."}
-{"text": "Purkinje neurons and granule neurons are two major cell populations in the cerebellar cortex. Purkinje neurons are well recognized by the size and complexity of their dentritic arbors. The numerous spines on the arbors serve as postsynaptic sites for reception of afferent input from the parallel fiber axons of granule neurons. The presynaptic axonal membranes, associated vesicles containing neurotransmitters and the thickened postsynaptic spinous membranes form structural complexes called synapses. The total number of synapses on Purkinje dendritic arbors of normally aging rats was known to be significantly reduced after chronic ethanol treatment, an effect that was reversed after recovery from the ethanol treatment. There were no ethanol-induced decreases in the number of parallel fibers because the number of granule neurons did not change. The overall size of the dendritic arbors was unchanged, but dendritic segments within the arbors were longer than normal following ethanol treatme nt, suggesting that some segments and their branch points had been deleted. The purpose of this study was to determine whether the loss of synapses represented a direct effect of ethanol on the density of spines (spines/nm) on the dendritic segments or an indirect effect of segment deletion. Spine densities on terminal dentrites were determined in Purkinje neurons from 40 old male Fischer 344 rats that were assigned randomly to one of three treatments groups, a chow-fed control group (n=8), an isocaloric and isovolumetric (pair-fed) control group (n=16), or an ethanol-fed group (n=16). Brain tissues were prepared by the Golgi-Cox procedure for light microscopy and coded to avoid investigator bias. 14 Purkinje neurons from each rat (a total of 560 cells) were randomly selected for quantitation of spines. In each neuron, spine counts and terminal segment lengths were determined for 14 paired and 14 umpaired terminal dendritic segments (a total of 15,680 measurements). Group averages tested by ANOVA showed no statistically significant decreases in spine density on spiny branchlets of Purkinje ne urons after chronic ethanol consumption. It was concluded that ethanol did not reduce the density of spines on dendritic segments of Purkinje neurons directly and that the decrease in synaptic density after ethanol treatment represented an indirect effect of segment deletion. (Funded by NIH/NIAAA grant AA05592) Keywords: Cerebellar Cortex, Ethanol, Golgi Cox, Purkinje Neurons, Synaptic Spine"}
-{"text": "Moderate alcohol intake is thought to be cardioprotective. The postulated mechanism has always been the elevated high-density lipoprotein cholesterol concentration seen in alcohol consumers compared to nondrinkers. Recent studies have raised the possibility that insulin sensitivity may be mediating the effect between moderate alcohol consumption and lowered cardiovascular disease (CVD) risk. This study therefore has the following aims: 1.To assess whether the daily consumption of a moderate amount of alcohol can improve insulin sensitivity in a volunteer group of nondrinkers predefined as insulin resistant. 2. To determine if moderate alcohol intake by improving insulin sensitivity will have the beneficial metabolic outcomes in HDL, insulin and glucose to lower CVD risk. 3. To investigate if measures of endothelial function (asymmetric dimethylarginine and soluble cellular adhesion molecules) as risk factors for CVD are affected by moderate alcohol consumption and insulin sensitivity. Insulin sensitivity/resistance will be measured by the insulin suppression test. Given the morbidity and mortality of insulin resistance and CVD, it becomes important to clarify the role of moderate alcohol consumption to both."}
-{"text": "Selenium has a role in preventing heart disease and is rapidly becoming recognized as a chemopreventive agent against cancer. The beneficial affects of this element are due, at least in part, to its presence in selenoproteins as the amino acid, selenocysteine (Sec). Our program, therefore, focuses on the means by which Sec is incorporated into protein and the role of specific selenoproteins in human health. As Sec tRNA is regarded as the key molecule in selenoprotein biosynthesis, we have produced transgenic mice encoding as few as 2 and as many as 20 extra copies (transgenes) of the Sec tRNA gene to examine effects of its overexpression. We introduced a silent mutation in the transgene that enables us to distinguish its expression from that of the corresponding wild type gene by primer extension. The transgenes and the wild type genes contribute equally to the total Sec tRNA population, but the level of expression is not linear with gene copy number, strongly suggesting that gene expression is regulated by a feedback mechanism. Overexpression does not appear to affect selenoprotein synthesis or protein synthesis as a whole. This observation, coupled with our earlier findings of normal selenoprotein synthesis in cells containing only ? the Sec tRNA population, demonstrate that Sec tRNA is not a limiting component in selenoprotein translation. We have also sequenced the Sec tRNA gene in zebrafish and Chinese hamsters. Zebrafish contains two functional copies of this gene whereas all other higher and lower animals examined to date contain only one functional gene. Chinese hamsters contain three pseudogenes whereas all other organisms examined to date contain only one or no pseudogenes and since the Chinese hamster gene encodes two pyrimidine transtions compared to all other mammals, it seems likely that these transitions occurred by editing of a transcript and re-insertion into its genome. In our studies on examining the role of specific selenoproteins in human health, we have focused on a recently discovered selenoprotein, designated as the 15 kDa protein. We have shown that its levels are normally elevated in prostate as compared to other tissues, but are reduced substantially in prostate cancer. Since selenium is known to have a chemopreventive role in prostate cancer, it would seem that the 15 kDa protein may have a role in preventing malignancy in this tissue. We, therefore, have made the appropriate constructs encoding the 15 kDa protein to examine the affects of its overexpression in mammalian cells in culture and in transgenic mice. These studies are in progress. We have confirmed the occurrence of two polymorphisms in the 3-untranslated regions of the 15 kDa protein gene and 1 of the polymorphisms may be involved in regulating protein expression. There was statistically significant differences in allele distribution between head and neck cancer tissues and controls. In a related study on the effects of selenium on human health, a previous member of our group (V.N.G., see collaborators below), had shown earlier that the incorporation of selenium into protein is diminished in HIV infected cells. We have subsequently shown that the HIV encoded TAT protein binds to the SECIS element which is a specific stem-loop structure in all selenoprotein mRNAs required for insertion of Sec into protein; but TAT also binds to most double stranded RNAs examined and thus shows no specificity. We are presently using in vivo experiments to determine if specificity occurs in selenoprotein synthesis. We have discovered two new human selenoenzyme thioredoxin reductases and characterized the role of a previously identified thioredoxin reductase in redox regulation of cell signaling. In addition, we identified a thioredoxin reductase in C. elegans and this appears to be the only selenoprotein in this organism. Our study on examining the Sec tRNA population in Drosophila has been completed as well as our study on examining the role of hypermodified bases in the anticodon loop of specific tRNAs on ribosomal frameshifting. - Genes, Gene Targeting, Gene expression, Post-transcription regulation, Selenium, Selenocysteine, Transgenic mice, Translation, - Neither Human Subjects nor Human Tissues"}
-{"text": "Synaptic connections are formed during development but are continually modified through experience and learning and memory. Any alteration to the normal connectivity leads to behavioral and cognitive changes that underlies psychological and neurodegenerative disorders. Therefore, understanding how functional networks of synaptic connections are established is critical for gaining insights into normal brain function. A mutant screen for defects in synapse in Drosophila has led to the discovery of a mutant line that shows aberrant synapse formation. Named as baha (bad hair day), homozygous mutant photoreceptors and motor neurons project to their correct target area in early developmental stages but subsequently fail to maintain contacts or form mature and functional synapses. Preliminary work thus suggests that baha may function as a critical factor in synapse formation. To investigate baha's role in synaptogenesis, I propose to perform further characterization of its synaptic phenotype, to identify and clone the baha gene, and to investigate the molecular mechanisms of its action. Cloning and characterization of baha gene may thus lead to identification of components of synapse formation, a process that remains poorly understood."}
-{"text": "Obstetric practices, evaluative tools, and treatments have been sometimes passed down by tradition or introduced without rigorous study to evaluate their impact on maternal, fetal, and/or neonatal outcomes. Long-term maternal and infant outcomes are particularly understudied, and surrogate markers of morbidity are commonly substituted in studies of smaller size. The impact of pregnancy complications can be devastating, affecting decades of life. The health and financial impacts of adverse maternal and infant outcomes on the family are potentially staggering. With millions of births in the United States each year, even uncommon complications can have a profound societal impact. Since its inception, the Eunice Kennedy Shriver NICHD Maternal-Fetal Medicine Units (MFMU) research network has conducted clinical trials and observational studies that have brought important understanding regarding prediction and prevention of preterm birth (Preterm Prediction Study, 17-OH progesterone in singletons, twins, and triplets, omega-3 supplements, antibiotics for asymptomatic bacterial vaginosis and T. Vaginalis), treatments for those at imminent risk (antibiotics for preterm labor and premature rupture of the membranes, repeated antenatal corticosteroids, magnesium sulfate neuroprotection), cesarean delivery and VBAC, prevention of preeclampsia, and treatment of gestational diabetes, among others. This network played a key role in evaluating technologies used in obstetric practice (ultrasound cervical length, fetal fibronectin, home uterine monitoring, fetal pulse oximetry). In this application, we demonstrate that the Department of Reproductive Biology at Case Western Reserve University (CASE) investigators have the ability to conduct collaborative research, and have successfully participated in and are qualified to continue in the MFMU network. Our investigators have strong backgrounds in clinical and multicenter studies, those involving neonatal and long term infant follow-up, and in the above listed network studies. CASE investigators provide leadership in network study development and administrative committees necessary to study completion and dissemination of their results. There is a strong administrative and research infrastructure to support the MFMU network, including the CASE-Clinical & Translational Science Collaborative. CASE offers the strengths needed to successfully lead and participate in NICHD-MFMU network research which will directly influence obstetric practice and improve pregnancy outcomes nationally and internationally. PUBLIC HEALTH RELEVANCE: Research performed by the CASE Department of Reproductive Biology, within the Eunice Kennedy Shriver NICHD-MFMU network, will inform and change obstetric practice. This research will identify interventions and tools to predict, prevent or mitigate pregnancy complications such as preterm birth, preeclampsia, fetal growth restriction and perinatal infections, among others. Focusing on significant perinatal and long-term outcomes, we will bring to an end procedures and practices that are ineffective or incur undue risks."}
-{"text": "The project's objective is to develop enabling technology for a co-robotic navigation aid, called a Co-Robotic Cane (CRC), for the visually impaired. The CRC is able to collaborate with its user via intuitive Human-Device Interaction (HDl) mechanisms for effective navigation in 3D environments. The CRCs navigational functions include device position estimation, wayfinding, obstacle detection/avoidance, and object recognition. The use of the CRC will improve the visually impaired's independent mobility and thus their quality of life. The proposal's educational plan is to involve graduate, undergraduate and high school students in the project, and use the project's activities to recruit and retain engineering students. The proposal's Intellectual Merit is the development of new computer vision methods that support accurate blind navigation in 3D environments and intuitive HDl interfaces for effective use of device. These methods include: (1) a new robotic pose estimation method that provides accurate device pose by integrating egomotion estimation and visual feature tracking; (2) a pattern recognition method based on the Gaussian Mixture Model that may recognize indoor structures and objects for wayfinding and obstacle manipulation/ avoidance; (3) an innovative mechanism for intuitive conveying of the desired travel direction; and (4) a human intent detection interface for automatic device mode switching. The GPU implementation of the computer vision methods will make real-time execution possible. The proposed blind navigation solution will endow the CRC with advanced navigational functions that are currently unavailable in the existing blind navigation aids. The PI's team has performed proof of concept studies for the computer vision methods and the results are promising. The broader impacts include: (1) the methods' near term applications will impact the large visually impaired community; (2) the methods will improve the autonomy of small robots and portable robotic devices that have a myriad of applications in military surveillance, law enforcement, and search and rescue; and (3) the project will improve the research infrastructure of the Pi's university and train graduate and undergraduate students for their future careers in science and engineering."}
-{"text": "The long range goal of this research is to understand the roles played by the superior colliculus (SC) in visual function. This requires a full accounting of the functional properties of the retinal ganglion cells innervating the SC. In the cat, almost all of these cells belong to the W-cell class. W-cells comprise 50 percent of cat ganglion cells and have obvious equivalents in primates and other mammals. In addition to dominating the retinocollicular pathway, W-cells provide most or all of the retinal input to a host of other visual nuclei that subserve visuomotor reflexes. Despite the ubiquity and importance of W-cells, study of their structure and function has been severely limited by technical factors. This project will overcome these limitations by means of a novel in vitro approach, thereby shedding new light on the organization of the retinocollicular pathway. W-cells are extremely heterogeneous in morphology and function. Rather than representing a true class, W-cells apparently constitute a loose grouping of many distinct ganglion-cell types, each as different from the others as it is from the X-and Y-cell types. Work from this laboratory in past and current project periods indicates that individual W-cell subtypes may exhibit distinctive patterns of collicular projection. The retinocollicular pathway is thus far more complex that has been generally appreciated. Functionally distinct W-cell channels may be processed independently by distinct collicular microcircuits, or may undergo a highly orderly integrative recombination by collicular neurons. Clearly, a prerequisite for understanding the functional meaning of this parallel retinocollicular organization is a detailed understanding of individual W-cell types. The proposed project begins this process through an in depth analysis of a single W-cell type -- the zeta cell -- which is a dominant contributor to the retinocollicular pathway. These W-cells will be fluorescently tagged, either by retrograde transport of tracers from the colliculus or by uptake of a vital dye. The retina will then be maintained in vitro and electrodes advanced toward tagged zeta cells under visual control. The visual response properties of these cells will be studied by extracellular and intracellular recording and their morphology revealed in detail by intracellular dye injection. This approach provides the first practical method for making direct correlations between the morphology, physiology and central projections of single ganglion cells. The method will be used to analyze the stimulus selectivities of these cells, to explore the intraretinal circuitry that produces these response properties, and to characterize the anatomical mosaic through which these cells sample the visual scene. The study will expand our understanding of diversity among the output cells of the retina and the parallel channels of visual information they emit to the SC and other brain centers. It will also establish a powerful new experimental paradigm with extremely broad applicability to the study of retinal ganglion cells."}
-{"text": "This project is concerned with the investigation of the mechanism of action of ACTH on the adrenal cortex. Specifically, it relates to the effect of this trophic hormone on the cytochrome P-450 systems of adrenal cortical mitochondria. Methods have been developed for measuring the binding of cholesterol to side chain cleavage cytochrome P-450 in the adrenal cortex and the studies in this project involve measuring how this binding is affected by agents such as ACTH and cyclic AMP. Also under study is the early and late pathway of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. Agents to be studied in that project include potassium, ACTH, sodium and angiotensin II. Methods are being developed for the measurement of the late pathway of aldosterone biosynthesis utilizing high pressure liquid chromatography of extracts following incubation of corticosterone with zona glomerulosa mitochondria. In addition, corticosterone binding to the 18-hydroxylase system of zona glomerulosa will be studied by spectrophotometry and electron spin resonance assays. BIBLIOGRAPHIC REFERENCE: Effect of Hypophysectomy on the Volume and Ultrastructure of Zona Glomerulosa in Rat Adrenal. Peter A. Nickerson and Alexander C. Brownie. Endocrin. Exper. 9, 187, 1975."}
-{"text": "The research described in this proposal aims to increase our knowledge concerning the fever response in animals other than humans; specifically relating to the occurrence of the fever response in one species of amphibian and two species of mammals living in their natural environment, the generality of the fever response in reptiles, and the energetic cost of the fever response in a free-living reptile. All of the studies concerning the fever response has never been demonstrated or investigated under natural field conditions where the greater heterogeneity of biotic and abiotic factors may lead to a more complex response. Also, although the fever response has been demonstrated in a number of ectothermic vertebrates under laboratory conditions, no systematic study of the fever response has been undertaken with any specific taxonomic group of ectothermic vertebrates to demonstrate the generality of the response and its relationship to life history characteristic of a species. Data on the fever response of the bullfrog, the California Ground squirrel, and the desert cottontail rabbit will first be gathered in the laboratory and than the laboratory baseline data will be applied to experiments on free-living animals. Body temperatures will be determined with the use of temperature sensitive radio transmitters. In the survey of the Class Reptilia for the fever response, II species representing 7 different Families will be given injections of heat-killed Aeromonas hydrophila, a gram negative bacterium known to be pathogenic to reptiles. The reptilian species to be used represent animals from different habitats (aquatic, mesic, desert), with different food habits (herbivorous, insectivorous, carnivorous), and with different activity patterns (diurnal, nocturnal). In the section that pertains to the energetic cost of the fever response, time budget analysis and oxygen consumption measurements will be combined to yield an estimate of the energetic cost of the fever response in a free-living reptile; the chuckwalla."}
-{"text": "Molecular signatures collected from intact tissue sections by MALDI imaging mass spectrometry (MALDI IMS) have shown high potential for use as a prognostic or diagnostic pathology tool in the clinical setting. A major obstacle to the widespread deployment of MALDI IMS, however, is the difficulty of identifying the proteins contributing to the signatures. Researchers have tried a number of approaches, including in situ digestion, MALDI TOF/TOF tandem mass spectrometry, and top-down proteomics on specific image regions. In preliminary work, we have obtained promising experimental results using top-down proteomics on intact proteins in the 2 - 20 kDa range. However, the lack of successful algorithms and software to identify the proteins in IMS mass signatures poses a major bottleneck. In particular, available top-down proteomics software relies heavily on high-accuracy mass spectrometry. The requirement for high accuracy precludes the use of some of the most sensitive mass analyzers such as linear ion traps, especially useful for these very small and complex samples. Protein Metrics Inc. is a new software company building on six years of algorithms and software research at Palo Alto Research Center. We plan to extend Byonic, our next- generation proteomics search engine, to intact proteins up to about 20 kDa. For proteins larger than 20 kDa, we will also build software for middle-down proteomics, specifically for assembling large peptides (2 - 20 kDa) produced by limited digestion to recover the identity of the intact proteins observed in IMS. The proposed Phase I feasibility study will allow us to perform controlled studies to determine the best experimental and bioinformatics approaches. Phase II will then build commercial-grade software. The proposed project will advance the state of the art in imaging mass spectrometry. Translation of imaging mass spectrometry to routine clinical pathology use will advance the state-of-the-art in disease diagnosis and treatment, and advance medical imaging and public health. PUBLIC HEALTH RELEVANCE: The project will develop commercial software that will improve our ability to identify the proteins and modifications represented in imaging mass spectrometry molecular signatures. Project success will make imaging mass spectrometry much more useful as a clinical pathology tool."}
-{"text": "The overall goal is to study the licensing policies and practices of 12 academic institutions regarding their DNA-based patents. The proposed project is conceived as a pilot study that will test the feasibility of conducting a more comprehensive follow-up study of such policies and practices. Specific aims are as follows: (1) To provide a clear, concise definition of the phrase \"DNA-based patents\"; (2) to analyze DNA-based patents into subtypes, using categories that are useful for understanding the policies and practices under which they are commercialized; (3) to gather and publish precise, up-to-date information on the number of U.S. DNA-based patents held by all U.S. and Canadian academic institutions; (4) to invite the technology transfer offices of all institutions holding at least 25 such patents to participate in a pilot study of their patenting and licensing policies regarding DNA-based patents; (5) with the aid of a project advisory board, to select a representative group of 12 positive respondents for more detailed study of their licensing policies; (6) to provide the technology transfer offices of the 12 participating institutions with categorized lists of their DNA-based patents, and to solicit input on categories; (7) to gather detailed information about the licensing of DNA-based patents at these institutions through a questionnaire and follow-up interviews; (8) to analyze and publish the data that have been gathered, paying special attention to policies and practices regarding the licensing of DNA-based patents that were based, at least in part, on research supported by federal funding; (9) during the second year of the project, to select 10 patents or clusters of patents and to develop case studies that illustrate technology transfer based on DNA-based patents. At least one of these case studies will be focused on a research tool. (10) In light of the results from the pilot study, to consider the feasibility and utility of conducting a more comprehensive follow-up study of licensing policies and practices regarding DNA-based patents at U.S. and Canadian academic institutions. [unreadable] [unreadable]"}
-{"text": "The N.C.I. Biologic Response Modifer Program has undertaken poly ICLC as a drug to study intensively. In preclinical testing a variety of augmentary actions on several immune functions have been noted, some of which do not appear to be interferon mediated. A Phase I study of biological response modifications in humans is under way in two institutions--N.C.I. and the Naval Hospital in Portsmouth, VA. Upon completion a Phase II study is planned. In addition the N.C.I. has agreed to undertake the monitoring of all cancer protocols with poly ICLC. Six clinical therapy protocols are currently ongoing, involving neuroblastoma, breast cancer, renal cell carcinoma, malignant melanoma, dysimmune peripheral paralytic neuropathies, and multiple sclerosis. There were no beneficial effects in neuroblastoma and breast cancer. There was a partial response in 2 of 5 patients with renal cell carcinoma. It is too early to evaluate, even in a preliminary way the malignant melanoma study. In 14 patients with peripheral neuropathy, there were at least 9 with moderate to marked improvement. In M.S. there are indications of positive effects, but it is too early to have much confidence in the results."}
-{"text": "Ribonucleotide reductase (RR) is an important therapeutic target. Therefore, understanding the function and regulation of the RR gene, and development of new RR inhibitors may have a direct impact on cancer treatment. Our long-term goal is to better understand the molecular mechanism of RR gene regulation, and the role of RR in chemotherapeutic drug resistance. We have shown that gemcitabine resistant as well as hydroxyurea resistant clones both overexpress the RR small subunit, hRRM2, mRNA and RR protein. Further studies confirmed that these drug-resistant tumor cell clones are associated with nucleus factor Y (NFY) binding to the RR promoter. Therefore, in Specific Aim I we will investigate whether hRRM2 overexpression in drug resistant cells is directed through transcription factor regulation o: the hRRM2 promoter. Promoter binding of NPY and a new transcription factor, band c, will be examined by gel-shift analysis and specificity will be confirmed by affinity chromatography. The functional consequences of NFY and band c in the regulation of hRRM2 expression will be examined in transfection assays. Since p53R2 is an integral part of the RR gene family, we are also intrigued by the possibility that interactions between different subunits of RR are important. In Specific Aim II, we will examine the interactions of p53R2, hRRM2, and hRRM1 (the RR large subunit), identify interacting domains, and examine the biochemical characteristics of such subunit interactions. A mammalian expression system will be employed to examine in vitro recombination of each subunit. Deletion analysis and mutagenesis will be performed to examine interacting domains and results will be confirmed by affinity chromatography. The plasmid vector containing p53R2 will be transfected into cloned drug-resistant cells for study in vivo. Once we clarify the interaction among RR subunits, we will explore the role of p53 in RR regulation. Our immunoprecipitation results have shown that both p53R2 and hRRM2 can bind to wild-type p53. Our hypothesis is that RR subunit composition may be regulated by p53. In specific aim ifi, we will confirm the protein interaction between p53R2 and wild-type p53 using a yeast two hybrid system. These findings will be confirmed by immunoprecipitation and Western blotting. The effect o wild-type p53 on RR activity and subunit composition will also be examined by transfecting wild-type p53 into cell lines expressing mutant p53. These experiments will help to confirm the interactions between p53R2, hRRM2, and p53. Finally, in Specific Aim IV, we will investigate the molecular pharmacology of a new RR inhibitor, HSC 1 that inhibits human cancer cells through an interaction with hRRM2. Taken together, our proposed studies will provide an integrated evaluation of the molecular control of RR-mediated chemotherapeutic drug resistance."}
-{"text": "The Cystic fibrosis transmembrane conductance regulator (CFTR) is the membrane protein product of the gene known to be defective in cystic fibrosis patients. CFTR functions by transporting chloride ions through a transmembrane (TM) domain believed to consist of two sets of six tightly packed and assembled membrane spanning helical segments (TM1-6, TM7- 12, ca, 20 amino acids each). However, channel remain to be deduced in molecular terms. (1) Since packing of TM domains appears to be directed by specific recognition elements contained within the individual TM segments, and the N-terminal portion of CFTR (TM1-6) is known to form a functional channel, we propose to prepare a series of peptides based on the native sequences of the CFTR TM domain segments 1-6 by solid-phase synthesis, and to determine the affinities of the individual TM segments for each other. Individual TM segments will be combined in combinations (e.g., TM5-6, TM6-1, TM1-2, etc.).Affinities would be quantitated by fluorescence energy transfer, circular dichroism spectroscopy, and gel electrophoresis. These experiments will be supported by molecular modeling to identify favorably- packed helical dimers of CFTR helices, and by channel conductance measurements performed on CFTR TM peptides individually and in combinations. Pairs found to have affinity of dimerization would likely be in direct 3-D contact in the native channel. (2) An increasing number o missense mutations within the CFTR TM domain are now being associated with CF disease. We reason that certain 'high-information' residues impair channel function by interfering with helix-helix packing. Peptides prioritized to key TM-domain mutations observed in genotyped CF patients from the in-house database at the Hospital for Sick Children (e.g., G85E, R347H, R347W) ill be prepared and studied as above in combinations with wild type partners. From the combined information thus obtained, we will construct a working model for the molecular geometry of the CFTR channel, and use the epidemiological data to generate hypotheses concerning possible relationships between severity of CF disease and molecular defects in the corresponding mutant CFTR's."}
-{"text": "The psychoactive component of marijuana, ?9-tetrahydrocannabinol, binds to and activates cell surface receptors called cannabinoid receptors. Activation of these receptors produces intense responses in humans, which suggest that endogenous cannabinoid (endocannabinoid) substances may contribute in important ways to brain functions as diverse as appetite, mood and pain. Two such substances are anandamide and 2- arachidonoylglycerol (2-AG), lipid-derived compounds that are released from neural cells and activate cannabinoid receptors with high affinity. Anandamide and 2-AG undergo a rapid deactivation process, which contributes to arrest of their biological actions. In the case of anandamide, this process is thought to consist of two steps, transport into cells and intracellular hydrolysis by the enzyme fatty-acid amide hydrolase (FAAH). By contrast, much less is known about 2-AG deactivation. Previous work in our lab has provided evidence that the enzyme monoacylglycerol lipase (MGL) - a presynaptic serine hydrolase that converts 2-AG into arachidonic acid and glycerol - participates in 2-AG hydrolysis. Based on these results, we hypothesize that MGL is an essential component of the physiological mechanism by which 2-AG is deactivated at brain synapses. Our first objective is to discover potent and selective inhibitors of MGL activity. In our initial studies, we have identified two promising leads, which will be optimized through a series of experimental and computational approaches, including structure-activity relationships, site-directed mutagenesis, mass spectrometry and molecular modeling. Our second objective is to characterize 2-AG transport into neurons and define the role of MGL in this process. We will combine pharmacological and genetic strategies to test the hypothesis that MGL- mediated hydrolysis provides the driving force for neuronal 2-AG internalization. Finally, we will use MGL inhibitors and mutant mice that overexpress MGL in the forebrain to determine whether 2-AG hydrolysis terminates the behavioral effects of endogenous 2-AG. We will focus our attention on two behaviors - feeding and stress-coping responses - in which as-yet-unidentified endocannabinoid signals are thought to play a crucial role. These studies will help uncover the functions served by the endocannabinoid system in the brain and may open innovative avenues for the treatment of neuropsychiatric and substance abuse disorders. PUBLIC HEALTH RELEVANCE: Marijuana works by mimicking important transmitter substances in the brain, called endocannabinoids. The objective of our research is to understand how these transmitters are produced and eliminated, and discover innovative medicines that target these processes."}
-{"text": "Several hypotheses have been created to account for the neurodegeneration and subsequent cognitive deficits observed in Alzheimer's disease. One hypothesis, in particular, has focused on the effects of inflammation as a mediator of neurodegeneration. To address this possibility, we have initiated studies examining the direct and indirect effects that endotoxin and inflammatory cytokines may have on neural tissue and neuronal cell signaling. Initial studies have demonstrated that the intracerebral infusion of endotoxin produces a significant age-related increase in brain tumor necrosis factor- alpha (TNFa) levels, but does not effect the production of a number of other inflammatory cytokines such as interleukin-1 or interleukin-12. We are currently examining young and old rodent brain homogenates and plasma post endotoxin treatment for the presence of various inflammatory cytokines (e.g., interleukin-6, interleukin-4, interleukin-10) and chemokines (e.g., interleukin- 8, MCP-1). Moreover,animals are also being examined for the effects of direct cytokine administration on inducing brain inflammation, permeabilizing of the blood-brain barrier, and effects on leukocyte trafficking, CNS surface markers, neurodegeneration, and cognitive behavior. We believe that cytokine and chemokine infusion (icv and iv) in rodent brains will have significant biological and physiological effects on neural tissue and will ultimately reveal a relationship between neuroinflammation, age, and cognitive behavior. These studies have been initiated using intracerebral TNF-a infusion and we hope to soon follow these efforts with chemokine and interferon infusions. In addition, additional studies will also be performed to determine whether administration of signaling inhibitors and antagonists and/or through in vivo leukocyte depletion can ameliorate the biological and physiological effects of administered cytokines. It is our hope that these efforts will assist in our understanding of the contribution of cytokines and chemokines in the neuroinflammatory and neurodegenerative effects observed in various neurotrauma models and age-related disease states."}
-{"text": "The overall objective of this project is to understand the role of E2F transcription factor in regulation of cell proliferation and how a loss of this regulation can lead to oncogenesis. Indications that the cellular transcription factor E2F plays a role in cell proliferation control was obtained from recent analyses which demonstrated a physical interaction between the tumor suppressor protein Rb and E2F. Further, the loss of Rb function is closely associated with a loss of the Rb-E2F interaction, suggesting that E2F is a functionally relevant target for Rb. Additional proteins involved in cell cycle regulation like p 107, cyclins A and E, and the kinase cdk2 are also found to be associated with E2F, indicating that E2F could be playing a major role in regulating cell proliferation. This project aims at studying the role of E2F in tumorigenesis, differentiation and cell cycle regulation. It will be assessed whether there is correlation between the loss of the Rb-E2F interaction and the generation of a wide variety of human tumors. In addition, the possibility that a mutation of E2F can lead to oncogenesis will be examined. To further understand the mechanism of E2F action, additional proteins that interact with E2F will be identified and cloned. Experiments will also be conducted on the role of E2F and associated proteins in the differentiation process of cells. Finally, attempts will be made to identify the cellular promoters that are targeted by E2F an E2F associated proteins."}
-{"text": "Tolerance to nicotine develops with chronic smoking and may be associated with the onset of tobacco dependence. It is unclear how quickly this tolerance dissipates after terminating exposure to nicotine. We propose to longitudinally assess responses to measure doses of nicotine by nasal spray in 30 male and female smokers as they smoke ad lib for two days (baseline), quit for six days, and then resume smoking for two days on the GCRC. We hypothesize that responses to nicotine will become larger over the six days of cessation, showing dissipation of tolerance and then be smaller during the final two days of ad lib smoking, showing reinstatement of tolerance with return to smoking."}
-{"text": "Achieving long-term allograft survival in the absence of ongoing immunosuppressive treatment is an important goal in clinical organ transplantation. Homeostatic mechanisms that regulate T-lymphocyte survival during alloimmune responses are probably central to determining the fate of a transplanted organ. There is evidence to show that T-lymphocytes are susceptible to activation-induced apoptotic death following exposure to high antigen dose or persistent antigenic stimulation. The extent to which activation-induced T-cell death contributes to long-term allograft survival and transplantation tolerance is not known. The underlying thesis in this proposal is that activation-induced death of alloreactive T-cells is necessary but not sufficient for induction of long-term allograft survival and transplantation tolerance; suppression of factors that rescue T-cells from death is required to achieve these outcomes. Specific aim 1 is to study the role of activation-induced T-cell death in the induction of long-term allograft survival and transplantation tolerance to vascularized allografts. Heart transplant acceptance will be examined in gene-knockout mice deficient in membrane molecules or cytokines that mediate activation-induced cell death. Biochemical, histochemical, and flow cytometric techniques will be used to study apoptosis of alloreactive T-cells in vivo and in vitro. Specific aim 2 is to study the role of activated antigen presenting cells in rescuing T-lymphocytes from cell death, thus precluding long term-allograft survival and transplantation tolerance. In vivo and in vitro systems will be used to determine which antigen presenting cell cytokines or membrane molecules can rescue activated, alloreactive T-lymphocytes from apoptosis. An in vivo system consisting of scid mice transfused with T-cell receptor-transgenic lymphocytes will be utilized to study intragraft factors that prevent activation-induced T-cell death. The proposed studies will enhance our understanding of T-cell survival following allostimulation and refine our abilities to design and implement new therapies that induce tolerance to transplanted organs."}
-{"text": "When presented with food or cocaine, dopamine (DA) in the nucleus accumbens (NAc) initially signals that the item is rewarding. When behaviors, such as drug taking, become compulsive we hypothesize that this reflects a shift in neural systems maintaining the behavior. There is enhanced DA release in the dorsolateral striatum (DLS) and attenuation of NAc DA release. Furthermore, within DLS, activity in the Direct Pathway from the striatum to the substantia nigra, that is important for initiation of appetitive behaviors is enhanced, while activity in the Indirect Pathway, from the striatum to the globus pallidus, that inhibits competing repetitive or stereotyped behaviors is attenuated. Females (women and laboratory rats) exhibit more rapid escalation of drug taking than do males and estradiol (E2) enhances acquisition of drug taking and motivation for drugs of abuse. Experiments from the Becker laboratory have demonstrated that E2 inhibits GABA release, down-regulates DA D2 receptors (D2DR), and enhances cocaine- or amphetamine-stimulated DA release in DLS but not NAc. The overarching hypotheses for this proposal are: 1) An attenuated cocaine-induced DA increase in NAc combined with an enhanced DA increase to cocaine in DLS is related to the propensity to develop a preference for cocaine over palatable food pellets in both males and females; 2) Estradiol's action in DLS of females enhances the cocaine-induced dopamine (DA) increase, inhibits GABA release, and inhibits D2 DA receptors. This combined effect enhances the rate of preference formation and motivation for cocaine over pellets in females; and 3) Decreased inhibition of the indirect pathway in DLS contributes to enhanced motivation for cocaine over pellets in both males and females. In females D2DRs are down-regulated in this pathway by E2, and this contributes to the more rapid preference formation in females and greater motivation for cocaine over pellets. Determining the mechanism(s) mediating formation of preference for cocaine over a highly palatable food reward, and how individual differences and sex differences contribute to this, are important for our understanding of and treatment of addiction in both men and women."}
-{"text": "Developmental exposure to bisphenol-A (BPA) at doses within the range of human exposure causes a complex array of adverse effects in animals. These outcomes are also known to be present in human populations and the rise in their occurrence coincides with the massive use of BPA and other endocrine disrupting chemicals in consumer goods. The main hormonal activity of BPA is as an estrogen mimic. Exposure to estrogens throughout a woman's life, including the period of fetal development, is considered a main risk factor for breast cancer. Developmental exposure to BPA altered mammary gland morphogenesis in rodents during the period of exposure and led to the development of pre-neoplastic and neoplastic lesions appearing in adulthood. The goal of this proposal is to identify the molecular, cellular and morphogenetic mechanisms underlying BPA-driven altered mammogenesis that predisposes to neoplastic transformation. To achieve this goal, we will use innovative tools such as a fetal mammary gland explant culture model that allows testing for direct effects of hormones and real-time observation of organogenesis. The Specific Aims of this proposal are to test three hypotheses, namely, 1: that the direct effect BPA on mammary gland development is mediated by ER1 and/or 2. 2: that BPA causes altered ductal morphogenesis i) by altering the composition and physical properties of the ECM and ii) by inducing adipogenesis. 3: that the different mammary gland phenotypes resulting from gestational and gestational plus lactational BPA exposure are due to alterations at the hypothalamic level."}
-{"text": "We will develop a novel approach to detecting transgene activity in tumors based on proton MRI of enzyme activated reporter molecules. Specifically, we will synthesize, evaluate, optimize, and apply novel agents to detect (3-galactosidase activity generated by the lacZ gene. Gene therapy holds great promise for treatment of cancer. However, major problems involve assessing delivery to target tissue, the uniformity (versus heterogeneity) of bio distribution and determining whether the genes are expressed. We propose a novel approach for evaluating gene expression using MRI. This research draws on foundations in optical detection of reporter genes, specifically P- galactosidase from the lacZ gene. Recently, 3,4-cyclohexenoesculetin-p-galactopyranoside, marketed commercially as S-Gal(tm), has been shown to generate an intense black color as a precipitate upon activation by p-gal in the presence of Fe3+ ions. This prompted us to consider the paramagnetic properties of the precipitate as a potential 1H MRI contrast agent. Preliminary studies have demonstrated feasibility and we now wish to translate and develop the concept to tumor cells for detection in vivo. Specific aim 1 will explore the utility of S-gal, with investigations to optimize and validate the technique. MRI parameters will be varied to maximize contrast with appropriate spatial and temporal resolution. Agent administration will be evaluated to maximize contrast and minimize toxicity. Specific aim 2 will focus on translation to tumors in living mice. MRI will be validated by comparison with optical methods in vivo and traditional methods such as histology and RT-PCR. Specific aim 3 focuses on the synthesis and development of alternate optimized substrates. We believe this approach can both become a valuable tool for detecting p-gal activity in vivo and serve as a proof of principle for a novel platform technology adaptable to other reporter genes and endogenous enzymes such as proteases. [unreadable] [unreadable] [unreadable]"}
-{"text": "The beta thalassemias are characterized by a deficiency of adult (beta) globin chains of adult hemoglobin (Hb), an excess of toxic, unmatched alpha globin chains, and intramedullary hemolysis. The resulting anemia develops only after fetal (gamma) globin synthesis and Hb F is suppressed in infancy. Induction of fetal (gamma) globin to levels which improve globin chain balance by even 10 percent can prolong red blood cell survival and diminish clinical morbidity. 5-Azacytidine has increased hemoglobin (Hb) levels by 1.8-3 gmd/d1 in thalassemia, but also causes general cytopenias and carries carcinogenicity risks. Fatty acids induce (gamma) globin experimentally. Arginine Butyrate, a prototype fatty acid, has been most effective when given intermittently or Pulsed, inducing Hb F to a mean level of 22 percent in 7/9 adults with sickle cell disease and increasing total hemoglobin by 3 gm/dl over baseline levels in 5/6 beta thalassemia patients. Two clinical pilot studies are proposed to test the hypotheses that therapy with Pulsed Butyrate, or rhu-EP0 + Pulsed Butyrate, will induce gamma globin chain synthesis sufficiently to improve non alpha: alpha globin chain balance and red blood cell survival, and increase total Hb in a significant proportion of patients with beta thalassemia intermedia. Baseline hematologic levels will be assayed four times over a two-month period. Butyrate will then be administered during an Induction Phase, to determine a patient's optimal dose, followed by a \"Maintenance Phase\" of therapy for 3 months. Pulsed Butyrate will also be tested with rhu-EPO. The proportions of patients on each study in whom the following endpoints are achieved, compared to baseline levels, will be analyzed: 1) an increase in total Hb of at least 2.0 grams/dl, 2) an increase in hematocrit of at least 5 percent, 3) a decrease in hemolysis, measured by LDH and bilirubin, 4) improvement in globin chain synthesis by 10 percent. Whether specific genotypes and in vitro response to Butyrate correlate with clinical responses will also be analyzed. These studies should determine the proportion and some genotypes of beta thalassemia patients which can benefit from Pulsed Butyrate plus/minus rhu-EP0 therapy."}
-{"text": "The Duke Department of Pediatrics'training program is based on developing pediatric Junior Faculty into physician-scientists who are skilled in cutting-edge methods of laboratory research and who will pursue independent academic careers investigating important issues related to childhood diseases. Our program is based on our pool of outstanding candidates, a strong curriculum of didactic courses, experienced mentors who perform state-of-the-art research, and an excellent research environment. Our commitment to develop future academic pediatricians is evidenced by the academic success of our Junior Faculty. Our Principal Investigator is Dr. Joseph St. Geme, III, Chairman of the Department who is assisted by Dr. Page Anderson, Program Director, Dr. Delbert Wigfall, Minority Recruitment Advisor, and an Internal and External Advisory Committee. Four young scholars will be supported each year. They will be drawn primarily from our fellows and Junior Faculty. Recruitment of underrepresented minorities is a focus of the program. Our faculty, which includes many mentors from other Departments, has strong track records in research, funding, and mentoring. Our research training centers are in the areas of Developmental Biology, Cell Biology and Cell Signaling, Microbiology and Immunology, and Genetics, Genomics, and Proteomics. Didactic courses, including a multi-lecture course on writing, will complement the laboratory research experiences, enabling the trainees to write and submit grant applications to support their career. The trainees will be required to take a five lecture course in Responsible Conduct of Research. The young Scholars will have access to all the research resources of the NCI-funded Comprehensive Cancer Center, the shared facilities at Duke University, the Institute for Genome Sciences and Policy, and the Center for Human Genetics."}
-{"text": "It is the hypothesis of this grant application that the pathogenesis of rheumatoid arthritis (RA) depends not upon the absolute numbers of T cells in the blood or the joints, but rather upon the relative percentages of auto-antigen specific T cells that produce and respond to pro-inflammatory and anti-inflammatory signals, including cytokines and chemokines. These T cell populations may be the target for therapeutic intervention. In preliminary experiments that prompted this grant application, we have shown that: i) peptides of bacterial origin (E. coli dnaJ heat shock protein) are strong immunogens in RA patients. dnaJ shares with HLA DRB1*0401 the susceptibility sequence to RA (shared epitope); ii) T cells in the blood RA patients that react with the shared epitope can be identified and isolated, using a novel technical approach; iii) T cell receptors usage and patterns of cytokine production by these shared epitope specific T cells can be analyzed. Based upon these initial results, we now need to test our hypothesis in a larger cohort of RA patients with different disease courses, and responses to treatment. Thus, the aims of this project are: 1. To characterize frequencies, function and phenotypes of T cells in RA patients reactive with the shared epitope. Parameters to be studied will include cytokine production, T cell receptor usage and membrane markers of activation and memory. Chemokine receptors will also be studied. 2. To determine the effects of disease activity and severity, and of slow acting anti rheumatic drugs, on function and phenotype of the shared epitope specific and bystander T cells. 3. To develop methods to modulate the Th1-type phenotype of shared epitope specific T cells, using in vitro immune manipulation with altered peptides. The long term objectives of this project are: i) to contribute to a better understanding of T cell-mediated events in the pathogenesis of rheumatoid arthritis; (ii) to develop paradigms for the prediction of disease outcome, and for evaluation rapidly new models of therapy."}
-{"text": "The Administrative Core will facilitate and coordinate communications between the members of the Consortium themselves and between them and the 4 members of the Scientific Advisory Board (SAB), all of whom will be Consultants to the Consortium. The major thrust of the proposals made in this application is to investigate and hopefully resolve the problems associated with coagulation dysregulation following organ xenotransplantation in pig-to-primate models. Frequent communication, most likely in the form of conference calls, is likely to be required between members of the Consortium and Consultants, all of whom are experts in the field of xenotransplantation and/or coagulation. The Core will help with travel arrangements and hotel accommodation for Consultants visiting Pittsburgh for the annual workshops at which the results of the Consortium's efforts will be discussed and plans made for the future 12-month period. The Core will also arrange travel and hotels for the members of the Consortium who will be participating in the annual meeting with other scientists funded through this RFA. The immunologic and coagulation assays to be used in the studies in Projects 1 and 2 will, as far as possible, be standardized between the two centers, and this will require exchange of samples, tissues, and reagents. This will be organized and coordinated through the Administrative Core. Data from the various experiments and assays will be collected and collated in the Administrative Core. The Core will also be responsible for assisting with the preparation of manuscripts reporting the scientific work of the consortium to be submitted for publication. The Core will do what it can to ensure timely reporting in peer-reviewed journals of the results obtained by the Consortium. It is the Consortium's plan to invite other scientists who are working in this field to the annual workshops that will take place in Pittsburgh, even if those scientists are neither Consultants to the Consortium nor funded by the RFA (although these scientists will have to fund their own travel expenses). The members of the Consortium believe that it is only through full collaboration and exchange of ideas and information that the problems of xenotransplantation will be resolved in a timely manner. The Administrative Core will provide assistance to visiting scientists with regard to reserving hotel accommodation, etc."}
-{"text": "Innovative and culturally appropriate multilevel health communications interventions are desperately needed to address the chronic disease epidemic in high-risk populations, such as low-income urban African Americans (AA). However, the vast majority of communications strategies have focused on educating individual consumers about healthy food choices, while in poor urban settings the lower availability of affordable healthy food choices greatly limits the impact of these messages. We will work with 3 wholesalers and 24 small retail food stores to develop and test novel strategies in Baltimore, Maryland, including: 1) multilevel health communications alone directed at wholesalers, retailers and low-income AA consumers intended to enhance willingness to stock and/or purchase healthy foods; 2) pricing strategies (performance based allowances) directed at wholesalers and retailers to increase their stocking of healthy foods at reduced prices; and 3) combined health communications and pricing strategies. Intervention strategies will be tailored to meet the needs of the target populations based on formative research and stakeholder input. Our proposed work is based on significant field experience in this setting, including the development of evaluation tools to assess change in stocking and pricing of key foods (at the store level), and psychosocial factors, dietary intake, and food purchasing behaviors (at the consumer level). Our study has the following aims: 1) Conduct formative research with representatives of multiple levels of the Baltimore food environment (i.e., local wholesalers and retail food store owners) in order to select key foods for promotion, determine appropriate communication strategies (e.g., messages, channels, materials) for each level, and select the most appropriate pricing approach (i.e., performance based allowance structure and stipulations). 2) Pilot the multi-level program with three wholesalers and 24 food stores (6 control, 6 health communications only, 6 pricing only, 6 combined), and assess program implementation through detailed process evaluation. 3) Assess impact of the pilot program on a) the stocking, pricing, marketing, and sales volume of promoted foods at wholesale and retail levels, and b) food purchasing behaviors and associated psychosocial variables (i.e., self-efficacy, intentions, perceived cost) at the consumer level (final sample n=12 consumers/store, 288 total). The proposed research directly addresses the objectives of the PA by seeking to develop effective, multilevel communication strategies to improve diet and reduce risk for diet-related chronic diseases. We anticipate this design will demonstrate the value of a multipronged and multilevel health communications approach to obesity and chronic disease prevention, and will lead to a large-scale trial and informed policies designed to improve food availability and affordability in low-income urban settings."}
-{"text": "Mosquitoes are insect vectors responsible for the transmission of many infectious diseases to hundreds of millions of people worldwide. Females of most mosquito species require blood from vertebrate animals for their egg development. Multiple bloodfeedings enable mosquitoes to transmit disease pathogens, including malaria parasites and dengue virus, from one person to another. Our long term goal of this project is to elucidate the molecular mechanisms that regulate mosquito egg production and identify target molecules that can be utilized for mosquito control. Mosquito egg development is governed by alternating peaks of two major insect hormones - juvenile hormone (JH) and 20-hydroxyecdysone (20E). Deprivation of JH in newly emerged adult female mosquitoes will halt egg maturation. On the other hand, topical application of JH to mosquitoes shortly after blood feeding interferes with the normal responses to 20E and impairs egg production. We have recently demonstrated that the mosquito Methoprene-tolerant (AaMet) protein is a key player in the juvenile hormone signaling pathway in the newly emerged adult female mosquitoes. AaMet protein binds to JH and forms a complex with AaFISC protein, a coactivator of the 20E receptor. AaMet and AaFISC are found to be associated with the promoters of JH target genes and activate their expression. In addition, our preliminary studies imply that AaMet mediates the inhibitory effects of JH on 20E-induced gene expression. Taken together, the results suggest that AaMet and AaFISC are components of a juvenile hormone receptor. The objective of this project is to elucidate the molecular details of how AaMet functions in juvenile hormone signaling that regulates egg maturation in mosquitoes. In Aim 1, we will perform structure-function studies of the juvenile hormone binding domain in AaMet and define the structural determinants required for high affinity binding to JH. In Aim 2, we will investigate how AaMet and AaFISC proteins are recruited to juvenile hormone response elements in the JH target genes. In Aim 3, we will test the hypothesis that AaMet is involved in the crosstalk between juvenile hormone and 20E signals in blood-fed female mosquitoes. The study will significantly advance our understanding of the molecular action of juvenile hormone in mosquitoes, and provide a structural basis for designing new pesticides that specifically target the mosquito JH signaling pathway."}
-{"text": "Autonomic dysfunction including orthostatic hypertension (OH) is a major health problem, causing significant morbidity and mortality. Its pathophysiology remains poorly understood and hence its management lacks a solid scientific base. The PPG focuses on the pathophysiology and treatment of autonomic failure. Project 1 (Low) incorporates a novel strategy of cholinesterase inhibition in the treatment of OH, an approach that promises to improve OH without supine hypertension. A second blinded treatment trial will evaluate if sodium chloride will expand plasma volume and if urinary sodium excretion is a suitable surrogate measure of plasma volume status. A series of studies, including the use of microneurography to measure sympathetic impuls3es, will evaluate the pathophysiology of postural tachycardia syndrome (POTS). A novel approach of amplitude modulation of the EEG in POTS shows a selective reduction of a frequency band of 0.02-0.05 Hz; this component is of particular interest since it may have a brainstem origin. The venous capacitance bed will be evaluated (Projects) to determine if there is excessive transcapillary efflux and changes in compliance in POTS and the effects of aging. The relative importance of the mesenteric, systematic and cerebrovascular circulations in OH will be evaluated. Project (Benarroch) will expand its studies on the neurochemical organization of autonomic control regions of the medulla in multiple system atrophy (MSA) and the parkinsonian syndromes. These include quantitative evaluates of new cellular groups (nucleus ambiguus, nuclease retroambiguus) and new receptors (including angiotensin II) that are likely to provide insights into the pathophysiology of autonomic failure in MSA. Project (Joyner) will undertake a detailed evaluation of the effects of denervation (mild in POTS and severe in neurogenic OH) and aging on the venous capacity and compliance. Project (Brimijoin) will focus on the response of the pre-ganglionic neuron to denervation and will study the mechanism of spinal intermediolateral column cell loss, using he model of immune-mediated pre-ganglionic autonomic neuropathy. The roles of apoptosis, excitotoxicity, growth factors, and aging will be evaluated and related to MSA."}
-{"text": "Immune responses are dependent on the coordinated movement of leukocytes from sites of antigen deposition, to secondary lymphoid organs and to sites of infection or inflammation. Chemokines play a critical role in this process by directing leukocyte trafficking throughout the body. Although it is well known that leukocytes express chemokine receptors and can migrate directionally in response to chemokine gradients, the molecular mechanism(s) that control chemokine receptor responses are still largely unknown. CD38, an ADP-ribosyl cyclase, that catalyzes the production of the calcium-mobilizing metabolite cyclic ADP-ribose (cADPR), appears to be a critical regulator of chemokine receptor signaling and leukocyte trafficking. We observed that neutrophil migration is impaired in CD38-deficientmice resulting in attenuated and reduced inflammatory responses. We also found that the cADPPR produced byCD38 modulates calcium mobilization in neutrophils that have been activated with inflammatory chemoattractants such as peptides derived from bacteria and viruses. Furthermore, we showed that cADPR-specific antagonists block the migratory response of neutrophils to these peptides. The data suggest that small molecule inhibitors of CD38could potentially be used to block neutrophil-dependent inflammatory responses. Recently, we observed that the migratory response of dendritic cells is also impaired in CD38 deficient mice. Specifically, we found that CD38-deficient dendritic cells cannot migrate in response to ELC or SLC, chemokines that direct dendritic cells to migrate from sites of damage or injury to lymphoid tissues. This impaired chemotactic response observed inCD38-deficient dendritic cells results in inefficient T cell priming and significantly reduced T cell-dependent immune responses. Based on our previous data, we now hypothesize that CD38, through its production of cADPR, regulates cell-dependent immune responses by modulating the migration of dendritic cells. To test this hypothesis we have proposed the following Specific Aims: (1) we will determine whether CD38 regulates migration of all mature dendritic cell subsets to ELC or SLC, (2) we will determine whether CD38 regulates the migration of dendritic cells to inflammatory chemoattractants and (3) we will determine whether cADPR production by CD38 controls dendritic cell trafficking in vivo. These experiments will validate whether CD38 antagonists have the potential to be used as immunosuppressive agents that attenuate immune responses by modulating leukocyte trafficking."}
-{"text": "This project aims at the visualization via MRI of freezinginterfaces in ice-water mixtures. Specifically, the in-situvisualization of frost and of freezing in porous media will bepursued. MRI is the only solution possible in such systems thatare strongly refracting to light. Attendant MRI thermometrystudies will also be pursued."}
-{"text": "The major objective of the proposed research is the development of an in vitro model for exocytosis. Preliminary investigations indicate that the ways in which Ca2 ion and ATP are involved in this cellular process can be determined from study of the interactions of these molecules with isolated secretion granules. Submillimolar concentrations of Ca2 ion induce total lysis of suspensions of secretion granules isolated from acinar cells of the rabbit parotid. I have identified two processes which precede lysis: 1) the Ca2 ion-specific activation of a granule-associated phospholipase A2 and 2) the large-scale rearrangement of granule membrane proteins (viewed as intramembranous particles in freeze fracture preparations) into interconnecting linear assays. I intend to evaluate the extent to which granule lysis in vitro correlates with exocytosis in vivo and to investigate the relationship of phospholipase activity to granule lysis in a series of comparative kinetic studies. Lysis will be determined by the extent of release of alpha-amylase, a secretory protein found in the granule content. Phospholipase activity will be measured by quantitating the hydrolyzed phospholipids resolved by thin layer chromatography. Membrane protein rearrangements induced by Ca2 ion will be characterized by electron microscopy of freeze fractured preparations. Provided the linear arrays can be stabilized, the molecular species involved will be identified by techniques employing covalent crosslinking and spin labeling."}
-{"text": "This proposal for a Clinical and Translational Science Center at the University of New Mexico Health Sciences Center (HSC) will catalyze the emergence of a transformative, novel, and integrated academic home for clinical and translational science at a flagship institution that serves the entire state of New Mexico and the Southwestern region ofthe U.S. This center will have the infrastructure and consolidated resources to: 1) synergize multi- and interdisciplinary clinical and translational research and researchers to catalyze the application of new knowledge and techniques to clinical practice at the front lines of patient care;2) recruit, train, and advance well-trained interdisciplinary investigators and research teams with strength in cultural sensitivity, health disparity, and biotechnology;3) create an incubator for innovative research and information technologies;and 4) expand existing partnerships between UNM Health Sciences Center (HSC) researchers, practicing clinicians, and communities to speed the development of research. Our vision of the CTSC links and focuses the efforts of our basic, clinical, and translational investigators, community clinicians, clinical practices, health care and research collaborators, and industry partners. Since New Mexico has a high proportion of ethnically diverse (i.e., Hispanic and Native American), rural, medically disenfranchised, and health-disparate populations, a CTSC in New Mexico can uniquely address these problems. The UNM CTSC will draw upon outstanding institutional commitment (at least $57,802,262 ) to achieve its goals. The UNM CTSC possesses authority over the recruiting, hiring, protected time, expectations, promotion and tenure, and evaluation of its faculty members. Evidence of the commitment and effectiveness of the UNM CTSC in these pursuits are apparent from the following achievements made during the planning process: (1) reorganization of the UNM HSC research mission under a new Vice President for Translational Research with authority over research education, compliance and research functions, (2) establishment of an institutionally-funded K12-like program, and recruitment of 5 junior faculty, (3) development of innovative new research education programs, (4) operation of a functionally expanded Participant and Clinical Interactive Resources component that is serving the needs of several community-based studies, including the landmark, new National Children's Study, (5) the redesign of our institutional approach to Biomedical Informatics and development of a regional clinical data warehouse that will be available to investigators using a $5.8 M investment, and (6) construction and renovation of new buildings for the CTSC. Additionally, UNM's commitment includes $14.5 M in new institutional funds committed to CTSC programs, and $8.5 M in matching funds to construct new space to create a physical home for the CTSC. Funding of this proposal will assure sustainability and growth of these transformations that is critically needed to support clinical and translational research at UNM. RELEVANCE (See instructions): The UNM Clinical and Translation Science Center will rapidly escalate transformative scientific discovery into improved human health outcome."}
-{"text": "Ubiquitin (Ub) is an essential signal molecule regulating protein degradation, localization and other activities. Generally the C-terminus of the Ub molecule is covalently conjugated to lysine residues of protein substrates by the E1/E2/E3 reaction cascade, and the modification is reversed by the action of deubiquitinating enzymes (DUBs). PolyUb chains can be further assembled on the substrates by the linkage between lysine groups of the first Ub molecule and the C-terminus of the next. Our current proteomic analysis revealed that all seven lysine groups on the Ub molecule can be utilized for the formation of polyUb chains, including Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63. We propose that the formation of functionally distinct polyUb chains is a regulatory step for ubiquitination and deubiquitination. To examine the function of these polyUb linkages, we will first develop a mass spectrometry technology for quantifying the abundance of all seven polyUb linkages, and then study the catalytic specificity between DUBs and polyUb chain topology. Moreover, we will focus on defining the function of Lys11 polyUb chains. Our studies will lead to the development of general proteomic tools for polyUb chain topology and better understanding of the diversity and function of polyUb chains."}
-{"text": "The purpose of this proposal is to develop an Implementation Science Resource Core to support the research efforts of several CHAART consortia, building upon existing infrastructure within these consortia. The current proposal would create the infrastructure to close the loop of the research cycle for NIAAA-funded research. ORCAAA (The Operations Research Collaboration for Alcohol Abuse and AIDS) would be a resource core using implementation science to inform decisions important to investigators, public health authorities, and clinician/patient dyads. In particular, ORCAAA will create 3 different PODS (Portals Of Decision Support) that will function as catalysts of collaboration, providing CHAART investigators with tailored decision support in order assure that research will be harnessed most effectively to improve population health. In particular the ORCAAA resource core will implement the following aims: Aim 1: Facilitate implementation of CHAART HIV/AIDS interventions targeting highest risk populations; Aim 2: Increase capacity for implementation science to impact CHAART study design; Aim 3: Increase capacity for evaluating effect of CHAART alcohol interventions on HIV progression"}
-{"text": "Broad, long-term objectives and career goals: To acquire the knowledge and skills needed for independent patient-oriented research concentrated on improving the diagnosis and management of pulmonary malignancy through imaging and image analysis techniques. Health relatedness: About 1.2 million cases of invasive cancer are diagnosed annually in the United States. About 500,000 Americans die annually from cancer, and bronchogenic cancer is the leading cause of cancer deaths. Early diagnosis of pulmonary malignancy, comprised primarily of bronchogenic cancer and metastatic disease, is important for early treatment and patient survival. Early primary lung cancer and metastatic disease to the lung parenchyma commonly manifest as indeterminate nodules, meaning nodules that are too small to be characterized as benign or malignant by other methods, on diagnostic and, more recently, screening computer tomography (CT). Other than calcification, morphologic characteristics have not been shown to differentiate benign from malignant nodules. Change in volume over time has been used as a marker of malignancy; however, for small nodules in particular, subtle volume changes are difficult to detect and quantify consistently using current techniques. Specific aims: The immediate career goal is to obtain knowledge and research skills through a structured career development program in a mentored research environment. This entails multidisciplinary didactic training in digital imaging, image segmentation, image analysis, epidemiology and mechanisms of tumorigenesis, and clinical study design, and conducting the proposed research in a mentored environment. The overall aim of the proposed research is to facilitate the CT diagnosis of malignant nodules by the development of precise quantitative techniques for the measurement of pulmonary nodule volumes. Our central hypothesis is that early nodule growth can be detected on CT using computer-assisted techniques and be correlated with biologic indicators of preneoplasia and neoplasia. The proposed method will enable early recognition of nodule growth and, therefore, the early diagnosis of malignancy."}
-{"text": "Factors and processes influencing entry into alcohol treatment programs will be investigated among population-based samples of alcoholics and first admissions to alcoholism treatment agencies. The research will be guided by a comprehensive model of factors predicting entry into an alcohol treatment program. The model includes state-of-the-art conceptualization of biopsychological factors thought to influence the severity and chronicity of alcohol abuse and dependence; a comprehensive assessment of the severity and chronicity of alcohol problems, including blood and urine tests; informal pressure by family and friends and formal pressure by employers and the legal system to seek alcohol treatment; health beliefs; and stage of change in addictive behavior. In addition, factors associated with unassisted or natural recoveries will be investigated among population-based alcoholics. Population-based alcoholics will be recruited from two prospective alcohol epidemiologic studies and from the household sample employed as controls for cases of coronary heart disease and lung cancer in Research Components 2 and 3. It is estimated that 795 individuals having a lifetime DSM-III diagnosis of alcohol abuse/dependence will be available for study; a subset of 398 will have had symptoms in the past year. Samples will be available for study; a subset of 398 will have had symptoms in the past year. Samples will be merged and post-stratified on sex, race, education, and heavy drinking. A representative samples of Erie County residents entering alcohol treatment programs for the first time(N-616) will be recruited using data on number of previous admissions to alcohol treatment, county of residence, and key study factors. These data are available from the NYS Office of Substance Abuse Service Information System for clients seen in all 50 alcohol treatment agencies in Erie County, making the recruitment of this sample feasible and cost-efficient. Analyses will employ simple and multivariate logistic regression and structural equation modelling techniques, where appropriate. A better understanding of alcohol treatment seeking behavior and unassisted recovery is critical to the improvement of current efforts to intervene more effectively and efficiently at earlier stages of alcohol abuse and dependence."}
-{"text": "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 LTQ-Velos ion trap mass spectrometer has recently been modified to allow performing higher-energy collision-induced dissociation (HCD) within the first quadrupole (Q00) in the intermediate vacuum region of the instrument. The new instrument is called the LTQ-Velos-Pro. HCD is a collision-based fragmentation technique which uses electric potentials to drive ions through a collision gas, in this case air. This beam-like fragmentation technique produces fragmentation spectra that are qualitatively similar to fragmentation patterns observed in triple-quadrupole instruments. We investigate the utility of performing HCD on the LTQ-Velos Pro for proteomics. In particular, we compare HCD and resonant CID, using several different experimental configurations, comparing the methods in the same LC/MS run and between different runs. We also evaluate the utility of HCD in producing library spectra that are well-matched to triple-quadrupole data and are thus well-suited to providing a transition from discovery-based proteomics to a more targeted approach."}
-{"text": "The long-term objective of the research described in this application is to develop new stereocontrolled methods for the synthesis of a variety of unsaturated heterocyclic systems. The vast majority of pharaceuticals used in medical practice are heterocycles. We believe by developing efficient synthetic entries to heterocyclic ring systems, we will allow promising pharacological leads in the future to be rapidly developed with potential therapeutic implications. Heterocyclization reactions are the focus of the studies we propose for the 04 to 07 project years. Cyclizations of this type are much less well developed than the corresponding preparation of carbocycles by polyene cyclizations. Our attention will continue to focus on heterocyclizations which are terminated by vinylsilanes. Since our introduction of these cyclization terminators in 1980, vinylsilanes have proven useful in a number of laboratories to terminate a wide variety of cyclization reactions. Because of the pivotal role that new initiators plan in expanding the horizons of heterocyclization chemistry, we also aim to develop new azacyclic and oxacyclic cyclization initiators. Tetrahydropridines related to the Parkinson's disease stimulaten, MPTP, will be prepared. The new antibiotic streptazolin will be prepared in enantioselective form, as will the powerful \"uteroevacuant\" diterpene zoapatanol. Intermediates and natural product targets will be broadly sdcrened for biological activity at the Biological Sciences Research Center of the Shell Development Co., and at NCI."}
-{"text": "DESCRIPTION (Adapted from applicant's description): This proposal is requesting funds for a very promising and exciting new direction of research that may shed light on a controversial issue in the scientific community. The proposes to isolate and characterize a novel form of gonadotropin-releasing hormone (GnRH), that is lamprey-like, in the primate hypothalamus. She proposes to test the hypothesis that the primate brain has two hypothalamic GnRHs. This hypothesis is based on recent data from preliminary and collaborative studies that support her earlier claims of a novel ir-GnRI-I (lamprey GnRH-like) form in humans. The applicant proposes to isolate and characterize this novel form of GnRH in the hypothalamus of primates by screening the cDNA libraries to hypothalamus using oligonucleotide probes against lamprey GnRH as well as other related probes. She theorizes that an ancient form of GnRH (lamprey) as well as the more modem mammalian form of GnRH function as hypothalamic neurohormones stimulating gonadotropin release in the primate. In mammals, it has not been fully explained how one GnRH hormone differentially stimulates the release of two gonadotropins (FSH and LH). She is in a unique position to accomplish this proposed project because she has recently identified a full length cDNA encoding the lamprey GnRH-I and has an established collaboration with research in humans. The presence of multiple GnRHs in the hypothalamus could provide the answer to this question. Her specific aims include 1) screening and isolating cDNA clones encoding the precursor forms of brain GnRH(S) in primates and (2) screening genomic libraries using the newly identified cDNAs as probes to isolate the complete GnRH gene(s). She predicts that findings from these studies will contribute to our understanding of reproductive physiology and provide further information for developing medical therapies using GnRH analogs. A new hypothalamic GnRH in mammals could shed light on the formation of new GnRH analogs that may not have some of the side effects noted with the GnRH analogs currently used."}
-{"text": "A new electrophoretic method is proposed for efficiently separating membrane proteins while retaining function. Fluid, smooth, electrically neutral lipid bilayers are proposed as the electrophoretic medium. The proposed separation would maintain the membrane proteins in their natural type of environment to produce separated zones of membrane proteins, which are then displayed as functional arrays. The separation would support such applications as membrane proteomics, drug screening and development of cancer diagnostics. Membrane proteins play key roles in human health, since they direct cellular signaling, recognition and transport, yet the steps for isolating membrane proteins for research are expensive, laborintensive and inefficient. Today's methods for separating proteins rely on water solubility. The proposed work will test the feasibility of separating membrane proteins in lipid bilayers by investigating 3 essential properties. First, the fluidity and uniformity of the lipid bilayer will be studied on ultrasmooth polyacrylamide films, grown by atom-transfer radical polymerization. The polyacrylamide film provides a uniform hydrophilic boundary on the underside of the bilayer, an aqueous buffer will be used on the upper side. Second, the electrophoretic mobilities of 5 well characterized receptors will be studied: the 3 opioid receptors and 2 melanocortin receptors. All 5 are medically important G-protein coupled receptors. Their ligands are peptides, which will be singly labeled with rhodamine. Third, the function of these membrane proteins will be studied by single-molecule fluorescence spectroscopy of the rhodamine labeled ligands. The R21 research will critically test the idea of whether the electrophoretic separation of functional membrane proteins is feasible in supported lipid bilayers. If feasible, an R01 proposal would address separations of real mixtures of membrane proteins from cell lysates. The work could have enormous impact on drug discovery, allowing inexpensive screening that encompasses many receptors."}
-{"text": "The primary objective of this study is to compare the long-term safety of a hydrofluroalbane propellent (non-ozone depleting) with that of the standard chloroflurocarbon based propellant used in metered-dose inhalers. The secondary objective of this study is to evaluate the safety and efficacy of salbutamol sulfate in those subjects who were randomized to receive Ventolin (trade name for salbutamol) in a previous study. This study has been completed."}
-{"text": "Ovulation is essential for successful reproduction. The ovulatory luteinizing hormone (LH) surge stimulates periovulatory follicles to produce prostaglandin E2 (PGE2). Blockade of PGE2 production causes ovulation failure. LH-stimulated neovascularization of the follicle is also essential for ovulation as inhibition of follicular angiogenesis prevents ovulation. This proposal puts for the overall hypothesis that PGE2 mediates the ability of the ovulatory LH surge to regulate angiogenesis within the ovulatory follicle. Angiogenesis is regulated via vascular growth factors. In Aim 1, we will determine if LH acts at follicular granulosa cells to regulate production of vascular growth factors in a PGE2-dependent manner. Monkey ovaries with multiple follicles will be obtained before and after administration of an ovulatory dose of the LH-like hormone hCG; some monkeys will also receive concomitant administration of a prostaglandin (PG) synthesis inhibitor. Granulosa cells, follicular fluid, and whole ovaries will be assessed for vascular growth factor mRNAs by qPCR and proteins by western blotting, ELISA, and immunofiuorescent detection on tissue sections. In vitro studies will be performed to determine if PGE2 acts directiy at granulosa cells to regulate vascular growth factor mRNA and proteins. Vascular growth factors act at endothelial cells to promote the formation of new blood vessels. In Aim 2, we will determine if PGE2 and vascular growth factors act at endothelial cells of the follicle to stimulate angiogenesis. Using a novel proliferating population of monkey ovarian endothelial cells, we will determine if PGE2 stimulates endothelial cell proliferation, migration, and formation of new capillaries. Similarly, vascular growth factors produced by granulosa cells, granulosa cell-conditioned media, and coculture with granulosa cells will be used to determine if vascular growth factors stimulate endothelial cell functions necessary for angiogenesis. Finally, monkey ovaries obtained after treatment in vivo with hCG, hCG+PG synthesis inhibitor, or hCG+PG synthesis inhibitor+PGE2 will be assessed histologically for neovascularization of the ovulatory follicle. The proposed studies will likely demonstrate that PGE2 and vascular growth factors act directly at endothelial cells to promote the neovascularization of the primate ovulatory follicle. Modulation of follicular PGE2 levels or PGE2 receptor activity may provide effective treatments for infertility or suggest targets for the development of novel contraceptives."}
-{"text": "A number of self-management procedures will be systematically implemented in representative middle school classes (5th grade through 8th). The basic self-management procedures to be used in the project are self-evaluation, self-determined work requirements, self-recording, and self-scheduling. In addition, a self-control science course for middle school students is being developed. The effects of the self-management procedures on classroom academic performance will be evaluated by both within group designs and comparisons with peer control subject. Student attitudes towards school will be carefully monitored using multiple administrations of likert scales. Finally, the Peers Harris Children's Self-concept Scale. The Self-esteem Invetory, and the Intellectual Achievement Responsiblity Questionaire will be given to participating students and peer controls pre and post treatment. The scores on these scales will be analyzed to determine if the self-management procedures have any desirable effects on student self-concepts."}
-{"text": "The programs of the SERCEB have immediate and projected requirements for monoclonal antibodies (mAbs) for studies of pathogenesis, immune response, vaccine development, therapeutics and diagnostics. These reagents and bio-recognition elements will be vital to the success of proposed research projects. The nature and source of the monoclonal antibodies required will depend on the objective of the research program, ranging from murine monoclonal antibodies for immunohistochemistry and detection to human or humanized mouse antibodies for development of therapeutics. Broad expertise exists within the institutions of the SERCEB in mAb development, testing and use. The spectrum of expertise in mAb development includes human, mouse, humanized mouse, rhesus, and phage display human and mouse F abs and ScFv. Finally, the antibodies will be deliverables that may have immediate application in therapeutics and diagnostics. The proposed Core C is a distributed core that uses established labs at several institutions to provide each of the needed services. The Core will provide all of the biorecognition elements needed in the SERCEB and will develop them into new assays and biosensors. The core has four Specific Aims. Aim 1. Generate human mAbs to poxvirus neutralization targets and anthrax protective antigen. Aim 2. Generate high-quality mAb reagents for SERCEB research programs. Aim 3. Develop biosensors for diagnostics or detection of select agents using novel mAbs available in the SERCEB Aim 4. To develop a database and repository for mAbs within the SERCEB that can be shared across institutions, to facilitate the other scientific programs."}
-{"text": "Population-based surveys estimate that the prevalence of methamphetamine (meth) use is 20 times higher among men who have sex with men (MSM) compared to the general population. Meth-associated sexual risk behavior is also a driving force in the MSM HIV epidemic: meth use is consistently associated with high-risk sexual behavior and sexually transmitted diseases, including HIV. Despite these alarming data, relatively few interventions have been tested among meth-using MSM, and no studies have tested the efficacy of pharmacologic interventions in reducing meth use in this population. Pilot studies demonstrate that aripiprazole (Abilify), an FDA-approved, well-tolerated antipsychotic and partial dopamine agonist, reduces the effects of meth in humans, decreases meth craving, and exhibits an excellent safety profile. Partial agonists - - ligands with target receptor affinity but low intrinsic activity - - have already been shown to be effective in treating opiate and nicotine dependence. In response to the compelling evidence supporting aripiprazole and the partial agonist approach, we propose conducting a randomized, double-blind, placebo- controlled trial of intermediate size (60 participants) and length (90 days of follow-up) to assess the efficacy of aripiprazole in reducing meth use among meth-dependent, sexually active MSM. Our specific aims are: 1) To test the hypothesis that aripiprazole 20 mg daily will reduce meth use significantly more than placebo among meth-dependent MSM, as determined by the proportion of meth-negative urines and by self- report of meth use in the aripiprazole versus placebo group. 2) To measure the acceptability of aripiprazole and placebo among meth-dependent MSM, by determining (via electronic pill caps and self-report) medication adherence to aripiprazole and placebo. 3) To measure the safety and tolerability of aripiprazole and placebo among meth-dependent MSM, as determined by the number of adverse clinical events in the aripiprazole and placebo arms. All participants will receive HIV risk-reduction counseling and brief substance use counseling. If promising, we anticipate that study results will be used to design a phase III study to determine if aripiprazole's effects on reducing meth use lead to significant reductions in meth-associated sexual risk and, if the trial sample size is appropriate, HIV incidence. [unreadable] [unreadable] [unreadable]"}
-{"text": "This modified grant proposal develops methods to address the complexity of modern Pharmacokinetics/Pharmacodynamics (PKPD) population studies focusing on computational PKPD and modeling of complex PKPD systems. AIM 1, Statistical Estimation Methods for Population PK/PD Data, develops improved estimation methods for population PKPD models, and general methods to incorporate patients compliance behavior and uncertain dosing history into PKPD models. AIM 2, Modeling System- PKPD, proposes two specific projects modeling system level PKPD across multiple organs, in particular for Cocaine/Nicotine PKPD and addiction dynamics modeling, and drug absorption physiological modeling. AIM 3, Population Stochastic Differential Equations (SDE), investigates novel approaches to population model building that are based on the use of stochastic differential equation, in particular investigating methodology to include covariates into population PKPD modelsm, and apply SDE to drug-absortion modeling."}
-{"text": "The Gordon Research Conference (GRC) on Cell-Cell Fusion will provide scientists from a wide range of disciplines with the newest research findings on the functions of cell fusion in development in a number of model organisms and in a variety of diseases. The emphasis will be on emerging universal mechanisms and future applications to cancer and stem cell research. This research conference will be the third Cell-Cell Fusion GRC and it is part of over 150 conferences that will be organized in 2011 by the GRC, an organization internationally known for the cutting edge nature of its meetings. Attendance at the meeting has grown steadily since its inception five years ago, and in 2011 the organizers expect to have 140 participants. The 2011 GRC on Cell-Cell Fusion will be held August 7-12, 2011 at the University of New England in Biddeford, ME. The program for the meeting described in this application has been designed around the theme of \"Cell Fusion in Sex, Life, Development, and Disease\". The organizer will deal mostly on the fundamental process of cell fusion during health and disease as well as the relationship between cell fusion and the larger fields of membrane fusion and fission during exocytosis, endocytosis, viral entry, and cytokinesis. The cell fusion community is entering a particularly fascinating phase as the fundamental principles of fusion emerging from theoretical analyses and studies on viral entry and intracellular trafficking start to be applied to understanding the functions and mechanisms of cell-cell fusion in development as well as disease. Important processes such as membrane fusion and fission, egg-sperm fusion, formation of bones, muscles, eye lens, stem cells, placenta, rare genetic diseases, and the invasion of cells by pathogens will be covered in eight sessions. This progress is being helped by classic and new methodology, including high throughput screening, developmental genetics in model organisms (e.g., fungi, flies, worms, and mice), and single-molecule biophysical methods. Exciting developments will be covered by the invited speakers, along with the latest progress on the basic mechanisms and applications of cell membrane fusion in vitro and in vivo. In addition there will be one round table discussion on myoblast fusion, a hotly debated topic that will encourage wide participation and scholarly organized discussions. The first Gordon-Kenan Research Seminar (GRS) on Cell-Cell fusion will be held August 6-7, 2011. This new GRS is organized by graduate students and postdoctoral fellows, and the activities during the GRS will be oriented to junior investigators and are intended to: (1) provide them with the basic background on mechanisms involved in membrane fusion in health and disease necessary to maximize their understanding of the science which will be discussed in the subsequent conference, (2) receive feedback on their ongoing research projects from experts in the field, and (3) facilitate their interaction with senior members of this scientific community to promote networking between junior and more senior researchers in the field. It is fully anticipate that the scientific discussions, research talks, poster sessions, and other informal interactions among the participants of this conference will contribute to advancing our understanding of novel molecular mechanisms involved in cell fusion during normal development as well as in human diseases, and will set the basis for the development of multidisciplinary projects aimed at discovering new treatments for these disorders. NARRATIVE: The Cell-Cell Fusion Gordon Research Conference (GRC) together with the first sister Gordon-Kenan Research Seminar (GRS) will bring leaders in diverse scientific disciplines together with graduate students and postdoctoral fellows constituting the future generation of researchers in the emerging field of cell fusion. The scientific presentations, discussions, round table discussions, as well as workshops during the GRC and GRS conferences on cell-cell fusion are tailored to expand our knowledge on the mechanisms by which membranes fuse in model organisms, and may contribute to our understanding of the first events in sexual development, the formation of many organs, cancer, stem cell biology, and rare diseases. The exciting, friendly, and multidisciplinary atmosphere that has traditionally characterized the Cell-Cell Fusion GRC will provide the ideal setting for the intellectual development and implementation of groundbreaking approaches to treat some cancers, as well as use stem cells to address fundamental biological questions and diseases."}
-{"text": "This project addresses the NHLBI RC2 GO application entitled \"Characterizing Differentiated Heart, Lung, and Blood Cells Derived by Reprogramming Human Embryonic and Induced Pluripotent Stem Cells.\" Emerging technologies to generate induced pluripotent stem cells (iPSCs) by reprogramming human somatic cells promises to revolutionize biomedical research and clinical medicine. Through in vitro culture methods, iPSCs can be differentiated into numerous cells types derived from all three germ layers. This raises the possibility that patient-derived iPSCs can be used to create relevant tissues for the study of many human disorders. In addition, iPSCs may provide starting material to manufacture transplantable cells for transfusion and regenerative therapies. However, the field is in its infancy and many core questions must be solved in order to realize these exciting long-term prospects. This proposal seeks to advance the use of iPSCs for the study of normal and pathological hematopoiesis. Multiple investigators with broad areas of expertise in hematopoiesis, embryonic stem cell/iPSC biology, chromatin biology, clinical hematology, bioinformatics, cell banking and bioethics/regulatory affairs will work together to pursue the following global issues 1) Mechanisms by which hematopoietic developmental potential might vary between different normal iPSC clones;2) The extent to which iPSC-derived hematopoietic precursors resemble normal ones with respect to cellular phenotypes, gene expression and epigenetic signatures;3) Whether hematopoietic disease phenotypes can be recapitulated by in vitro manipulation of patient-derived iPSCs. We will execute these studies using novel methods to create and culture iPSCs and state-of-the art tools to analyze and manipulate their resident genomes. Pursuit of these problems will serve as a framework in which to develop a facile infrastructure where investigators at our large pediatric institution can create, analyze, bank and distribute iPSCs from any patient of interest. If successful, this work will help to accelerate practical applications of iPSCs for the study and treatment of human diseases. This work will be based at Children's Hospital of Philadelphia with subcontracts to The Pennsylvania State University (State College, PA) and The Coriell Institute for Medical Research (Camden, NJ). The project will create 6 new jobs, thereby stimulating the economy in three different regions of the Northeastern United States. PUBLIC HEALTH RELEVANCE: Efforts to better understand blood production from patient-derived induced pluripotent stem cells (iPSCs) will enhance our understanding of blood disorders and generate new therapeutic approaches. Additionally, this work could create new general paradigms for studying the genesis of many normal tissues and their associated diseases."}
-{"text": "General rationale and organization of the Cores The main potential technical barriers to efficient execution of the biology are (1) the quality control and execution of the 3-D culture system, and (2) the requirement for expertise and ready access to state of the art confocal microscopy. To make these essential technologies readily available to everybody, and to coordinate the efforts of the various groups, we propose two core facilities, one to coordinate the 3-D culture methodology (3-D Core), and another to manage the confocal microscope (Confocal Core). Although the 3- D Cell Culture and Microscope Cores will be described separately, they will be located in adjacent labs and are designed to work together to standardize data collection procedures and provide a seamless transition from the 3-D culture phase to the confocal analysis phase of each study."}
-{"text": "Oxidative stress is thought to be a major contributor to the development of retinal degenerative disorders such as macular degeneration, retinopathy of prematurity and diabetic retinopathy. In order to understand the mechanism(s) of oxidative damage to the retina, we have investigated the gene expression in the retina of rats exposed to intense visible light. The expression of heme oxygenase-1 (HO-1), a marker for oxidative stress, in the retina is highly increased following light exposure. The expression of HO-1 mRNA in the retina in response to acute intense light was investigated as a function of age in dim cyclic light reared and dark reared rats. The HO-1 mRNA in the retina of treated rats was analyzed by Northern blotting. The intense light exposure markedly increased the HO-1 mRNA expression in the retina irrespective of the rearing environment. However, the response was more pronounced in the dark reared rats. The HO-1 induction increased with age in both dim cyclic light reared and dark reared rats. The increase in HO-1 mRNA is accompanied by apoptotic photoreceptor cell loss. DMTU, an antioxidant, was highly effective in blocking HO-1 induction and photoreceptor cell loss resulting from the intense light exposure. The age or rearing light conditions did not influence the DMTU effect. Thus, age, light history, and antioxidant status are important factors affecting the retinal gene expression in response to stress. Activin, a member of transforming growth factor-beta superfamily, is known to be expressed in the vertebrate retina. We have identified the first invertebrate activin gene from Drosophila. A genomic clone representing 102 F region of Drosophila chromosome 4 was found to encode a putative activin b. The predicted protein sequence showed a multibasic protease site that would generate a mature 113 amino acid C-terminal peptide having more than 60% similarity to vertebrate activin bB. A TGF-b family signature as well as 9 cysteine residues conserved in the vertebrate activins were also present in the mature peptide sequence. The gene was expressed in embryo, larva and adult stages of Drosophila."}
-{"text": "Ionizing radiation is widely utilized in cancer therapy. While some cancers have been treated successfully, an unacceptable fraction of treatments still fail. The ensuing recurrences and secondary tumors are often more aggressive than the original, possibly because of treatment-induced mutations. These mutations are not well defined due to the varied types of DNA damage ionizing radiation, the randomness of the damage, and its relative infrequency in the genome. Recent advances in nucleic acid chemistry make it possible to synthesize many of the types of damage detected in DNA subjected to ionizing radiation. These modified nucleotides can be incorporated into specific sites in cloned genes by standard molecular biology techniques. The proposed work will utilize this combined approach to place types of DNA damage, characteristic of ionizing radiation, at designated sites in a cloned DNA fragment. We will then determine how such damage affects eukaryotic gene expression and gene integrity. DNA damage will be positioned in synthetic oligonucleotides corresponding to transcription factor binding sites. A gel shift assay will be utilized to determine the binding and stability of transcription complexes containing either normal or damaged DNA elements. Damage will also be incorporated into specific sites in a synthetic DNA template and transcribed in vitro with a nuclear extract. The effect of specific types of DNA damage of gene transcription and fidelity will be determined. Related experiments will also determine if transcription transforms two opposed single-strand breaks into a more lethal double-strand break. Finally, we will measure the relative stability of duplex oligonucleotides containing base damage sites using the competitive mobility shift assay and determine whether a stability correlates with the polymerase by-pass or mutagenicity results obtained from the in vitro transcription studies."}
-{"text": "Retrotransposons comprise almost half of the human genome and substantial fractions of other metazoan genomes. Nonetheless, the mechanisms and localization of retrotransposon assembly are poorly understood. Ty3 is a long terminal repeat retrotransposon in budding yeast containing GAG3 and POL3 ORFs encoding structural and catalytic proteins, respectively. Because Ty3 exists in a simple eukaryote that facilitates molecular, genetic, biochemical, and even cytological approaches, it provides a useful model for understanding retrotransposon mechanics. In the current funding period, the assembling Ty3 VLP was characterized by density sedimentation, RNA protection, transmission EM and atomic force microscopy performed on Ty3 VLPs blocked at specific stages and also on mature VLPs. In addition, sixty Ala substitution mutants were analyzed to dissect the functions of Gag3 (capsid, spacer, and nucleocapsid) subdomains. These studies showed that the amino-terminal domain is critical for assembly and nuclear pore association of VLPs and that the nucleocapsid domain is important for targeting Ty3 RNA and Gag3 protein to the P body for assembly. A set of Ty3 RNA variants was developed and used to show that the Ty3 5'and 3'untranslated regions (UTRs) and POL3 contain independent cis-acting P-body targeting activities, but that only the UTR mediates packaging of Ty3 genomic RNA. The finding that the RNA processing body (P body) previously described as a site where nontranslating RNAs are sequestered or degraded, is the site of Ty3 assembly was particularly significant as assembly sites have not been characterized for retrotransposons and how retroviruses preassemble RNA and protein at a perinuclear site is not well understood, but appears to involve packaging signals. In Aim A of the proposed work, we will use mass spectroscopy to identify host proteins which are associated with Ty3 RNA and structural protein. Our collection Ty3 wt and variant RNAs tagged with the MS2- binding site coupled with a novel high affinity TAP-tagged MS2 binding protein and our antibodies against Ty3 proteins will be used to isolate proteins directly involved with Ty3 components. Strains with attenuated functions of identified genes will be tested for defects in Ty3 VLP assembly and proteins identified will be investigated in Aims B-D. A key step in assembly occurs when the mRNA transitions from translation to packaging. Based on the retrovirus model, this is associated with structural rearrangements in the UTRs of the RNA which are facilitated by nucleocapsid binding. In Aim B we will define at higher resolution the packaging site of Ty3 RNA and its interaction with the reverse transcription primer initiator tRNAMet. Previous work in the Darlix laboratory, with whom we collaborated, showed that initiator tRNAMet has a bipartite primer binding site in Ty3 5'and 3'UTRs. Retroviruses have dimeric genomes and we have shown that Ty3 is similar. Darlix proposed a novel model for Ty3 genomic RNA dimerization mediated by two itRNAMet molecules annealing at the 5'ends. We will specifically test whether the PBS, and by implication, the putative dimerization interface, is required for packaging of Ty3 RNA into VLPs. If so, that would suggest that initiator tRNAMet, which has unique roles in translation initiation, and priming Ty3 reverse transcription, is a central player in the key transition from translation to packaging. SELEX and in vitro RNA-protein binding assays will be used to identify important nucleocapsid binding contacts in the packaging site of the Ty3 UTR which could mediate primer annealing and dimerization. In Aim C we will test our hypothesis that the Ty3 UTR structure antagonizes translation initiation and imposes requirements for specialized translation helicases. In addition, we propose that reduced numbers of translating ribosomes downstream of the frameshift site attenuates 60S subunit delivery to the 5'end of the RNA and explains the sensitivity of Ty3 and other retrotransposons to imbalances in translation initiation factors and 60S ribosomal subunits. This hypothesis will be tested using host mutants and variant Ty3 RNAs. We will test whether translation is ultimately repressed by Gag3 binding in cis to the Ty3 UTR or POL3. In Aim D we will characterize the contribution of P bodies to Ty3 assembly. We hypothesize that P-body proteins cooperate with Gag3 to repress Ty3 translation, allow the concentration of assembly factors, and may promote unwinding of the RNA to allow packaging. Nonetheless, isolation of an unusual mutant which does not form large P body clusters suggests that P bodies might repress transposition at a post-assembly stage. Factors identified in genetic and mass spectroscopy screens will be tested for direct interactions with Ty3 components and for their roles in clustering P body proteins and assembling Ty3 viruslike particles. In summary, Ty3 is a well-characterized retrotransposon in a model eukaryote. It offers a unique opportunity to understand the retrotransposon particle assembly process and how P bodies might chaperone this process."}
-{"text": "This interagency agreement has the following scope: Clinical research on HIV and AIDS associated complications and co-infections with emphasis on optimizing therapeutic approaches to the treatment of HIV and related co-infections an co-morbidities in resource poor developing countries, including phase I-4 therapeutic clinical trials Clinical research on HIV prevention strategies ranging from novel pre-exposure prevention modalities such as Prevention of Mother to Child Transmission (PMTCT) and between discordant couples, treatment of sexually transmitted diseases, post exposure prophylaxis, and microbicides"}
-{"text": "Focal neuropathies of the hand are among the most common diseases treated by neurologists. The two most common are median neuropathy at the wrist (carpal tunnel syndrome) and ulnar neuropathy at the elbow. The current electrophysiological methods of assessment of focal neuropathies can be painful, and include nerve conduction studies and electromyography. Electrical impedance myography (EIM) is a new technique that can be used to assess neuromuscular disease. We have had success in using EIM in small muscles in animals, and limited experience using EIM in small muscles in human studies. We plan to refine these techniques in this proposal, while studying focal neuropathies of the hand. The long-term goal of this proposal is to make EIM a valuable tool for the assessment of neuromuscular disease. Establishing standards for use in small muscles will complement the known utility of EIM in larger muscles and help make it such a tool. We have three specific aims for this study. First, we aim to establish a range of normality for EIM for the three hand muscles that are tested most commonly in standard neurophysiologic assessment: the abductor digiti minimi, first dorsal interosseous, and abductor pollicis brevis. Second, we will evaluate EIM parameters in a group of patients with median or ulnar neuropathy, predicting that EIM parameters recorded from affected muscles in patients with median and ulnar neuropathy will differ from normal subjects due to the underlying architectural alterations in the muscle caused by neurogenic injury. Third, in order to determine the ability of EIM to judge lesion severity, we seek to determine the correlation between EIM parameters with standard electromyography (EMG) parameters in patients with focal neuropathy. Relevance to public health: Focal neuropathies of the hand, which include median neuropathy at the wrist (carpal tunnel syndrome) and ulnar neuropathy at the elbow, occur in approximately 10% of people. The standard neurophysiologic assessment of these problems includes electromyography and nerve conduction studies, both of which may be poorly tolerated. We are developing electrical impedance myography as a painless, noninvasive, quantifiable technique to assess focal neuropathies of the hand, and neuromuscular diseases in general. [unreadable] [unreadable] [unreadable]"}
-{"text": "A combination of various pancreatic, morphologic and functional assessments, along with gut hormone release, serves more accurately to gauge severity of disease, and these criteria may then be used to trace, in a longitudinal fashion, the course of the disease and to assess the efficacy of various treatment options. In addition, on the basis of the observation that chronic pancreatitis is biochemically similar to Type I diabetes mellitus, the investigators will compare the complications of these two diseases."}
-{"text": "Characterization of complications arising from cardiovascular invasive treatment procedures were analyzed using CDMAS."}
-{"text": "The U.S. appears to have worse health than people in a number of European countries. Because health reflects an accumulation of processes over a lifetime, we will study how stressful events during the working years may affect pre-retirement health and ultimately longevity. The broad aims of this application are to find whether in panel data the health gap between the U.S. and Europe increases during the working years, but not during the post-retirement years, and to explain these broad facts by differences in the stress induced in the labor market and by differences in the ameliorating effects of economic and social policy (including health insurance). Our analysis will use data on 15 countries, which provides a wealth of variation in institutions in time as well as in space. The principal outcomes will be fourfold. First, this study will clarify at what point in the life-cycle health differences emerge and chart their path thereafter. Second, this study will propose a formal economic and biological model linking indicators of physiological functioning to other health and economic outcomes clarifying some of the concepts measured in the literature and guide empirical work on the topic. Third, this study will provide micro as well as cross-country empirical evidence on the relationship between economic stressful events and physiological dysregulation using biomarkers but also reported health measures. Fourth, because of its cross-country design, this study will exploit differences in social policy across countries and over time to see estimate how these policies contribute to the international differences in health through the effect of physiological dysregulation on health. PUBLIC HEALTH RELEVANCE: The U.S. has fallen behind in terms of life expectancy in the pre-retirement years and large differences in health have emerged between the U.S. and Europe. This project aims to test the hypothesis that differences in physiological dysregulation due to a more stressful working life in the U.S. are responsible for those differences. This project will use a large array of longitudinal datasets in 15 countries to link health outcomes in the pre-retirement years to life histories and exploit differences in social policy across countries."}
-{"text": "We are concerned with the intracellular expression of the murine thymus-leukemia antigens in normal cells and tumor cells together with changes that follow modulation of surface antigen by specific antibody. The work focuses on mitochondria because they are relatively easy to isolate and have a characteristic morphology. TL antigens are demonstrated by immunoelectron microscopy. We are also characterizing plasma membrane differences on subsets of murine T cells by electrophoretic methods after iodination of intact cells."}
-{"text": "Understanding the genetic and environmental mechanisms that underlie the development of reading ability and disability has important implications for literacy education and the prevention of learning disabilities in reading. To that end, the goal of the proposed competing continuation of HD38075 is to conduct a systematic developmental genetic examination of reading comprehension. The Western Reserve Reading Project (HD38075 \"Environmental Influences on Reading: A Twin Study\") includes a representative sample of 350 same-sex twin pairs born between 1996 and 1998 who, by the proposed start date, will have been assessed longitudinally across four measurement occasions from kindergarten through third grade on a broad battery of measures of reading skills, oral language skills, cognitive skills, mathematics, and family environmental factors. The proposed continuation will extend testing via three additional annual home visits spanning middle childhood to early adolescence, with a focus on emerging reading comprehension skills. We propose to examine the univariate genetic and environmental influences upon reading comprehension, the covariance between reading comprehension and oral language skills, decoding skills, and other related skills, as well as the relationship between proximal and distal measures of the home and school environments that are associated with reading comprehension and related skills. In doing so, the proposed research will offer a unique opportunity to examine the genetic and environmental etiology of the longitudinal development of reading comprehension and related skills during the critical transition from when children are \"learning to read\" to when they are \"reading to learn.\" Extending the unique resources of the Western Reserve Reading Project is an important opportunity for a highly cost-effective and powerful genetically-sensitive investigation of the etiology of reading ability within its various developmental, cognitive, behavioral, and environmental contexts. Developing a more informative model of how genes and environments work together to affect the development of reading comprehension has important implications fostering reading development as well as to detect, ameliorate and prevent reading problems."}
-{"text": "Long term objectives: To continue to offer a Master's degree in International Research Bioethics of the highest quality to developing country academics, researchers, health professionals, and members of non government organizations from countries in the Asia Pacific Region with the poorest match of ethics capability and research activity (with a special focus on China, Vietnam and Thailand, where increasing amounts of NIH- supported research are being conducted, and where we have existing institutional linkages). To thus foster the development of national capacity for the ethical design, review and analysis of both clinical and public health research in countries in the Asia Pacific Region, with a special focus on China, Vietnam and Thailand. Specific aims: i) Through the Master of International Research Bioethics, provide a comprehensive program consisting of 12 core units that encompass bioethics, research bioethics in an international setting, research methodology in international health, relevant law and practicums;ii) Continue to offer, evaluate, refine and improve the existing Master of International Research Bioethics;iii) Select a cohort of participants from the above-mentioned priority countries who have demonstrated capacity to benefit from the program and to take up leadership positions in research bioethics, including the provision of education in research ethics, in ethical review and in providing guidance to their institutions, governments and other relevant bodies in their home countries, to undertake the Master of International Research Bioethics;iv) Continue to provide ongoing support to participants subsequent to the program."}
-{"text": "The Department of Urology at the University of Oklahoma has a 7-year history of collaborative multicenter research on the correlation of biochemical and clinical features of interstitial cystitis and the testing of new drug therapies. Within the Department are strong programs in all areas of clinical urology, including oncology, andrology, female urology and urodynamics, the basic sciences, emphasizing immunology, molecular oncology and biology, and cellular disease markers. Strong links with epidemiology and pathology have allowed us to be a leader in several areas of research. We maintain an active specimen collection and storage system for urines and other specimens collected from interstitial cystitis patients using standardized, quality-controlled procedures and a patient database and have a followup mechanism for tracking patients. The Department has a comprehensive residency training program. An informal regional network of community and academic urologists has also been developed to help meet patient needs for improved diagnostic and clinical care and for support as well as to recruit patients for specialized studies and experimental treatment protocols. Routine care is provided by community urologists or other physicians while Dr. Roy provides certain specialized services and experimental treatment protocols. The Department has drawn its patient base predominately from Oklahoma and neighboring states, a population base of 32.2 million, containing an estimated 5-6,000 undiagnosed interstitial cystitis patients with an estimated yearly incidence of roughly 200 patients. The largest out-of-state component being from Texas. Patients have come from as far as Michigan and Montana. The University of Oklahoma Department of Urology clearly meets the criteria for a clinical center. In order to meet the overall programmatic objectives, our specific objectives for the overall collaborative program: (1). Expand and further formalize the current informal network of community and academic urologists in the region to ensure standardized collection of data and specimens as well as long-term followup and to aid collaborating urologists who are responsible for routine care of patients to provide the best care based upon the latest findings of the clinical centers. (2) Expand current database and sample collection and storage programs in conjunction with other centers. (3) Expand clinical services offered to interstitial cystitis patients to include bladder retraining and reference to a university pain clinic, both to provide better services to patients while facilitating patient participation in research studies. (4) Recruit 50 patients the first year and 75 per year thereafter. (5) Present an annual seminal for physicians and workshop for patients as a means of educating physicians and the public about interstitial cystitis and reaching patients."}
-{"text": "The rapidly growing database of completely and nearly completely sequenced genomes of bacteria, archaea, eukaryotes and viruses (several thousand genomes already available and many more in progress) creates both extensive new opportunities and major new challenges for genome research. During the last year, we performed a variety of studies that took advantage of the genomic information to establish fundamental principles of genome evolution. We combined mathematical modeling of genome evolution with comparative analysis of prokaryotic genomes to estimate the relative contributions of selection and intrinsic loss bias to the evolution of different functional classes of genes and mobile genetic elements (MGE). An exact solution for the dynamics of gene family size was obtained under a linear duplication-transfer-loss model with selection. With the exception of genes involved in information processing, particularly translation, which are maintained by strong selection, the average selection coefficient for most nonparasitic genes is low albeit positive, compatible with observed positive correlation between genome size and effective population size. Free-living microbes evolve under stronger selection for gene retention than parasites. Different classes of MGE show a broad range of fitness effects, from the nearly neutral transposons to prophages, which are actively eliminated by selection. Genes involved in antiparasite defense, on average, incur a fitness cost to the host that is at least as high as the cost of plasmids. This cost is probably due to the adverse effects of autoimmunity and curtailment of horizontal gene transfer caused by the defense systems and selfish behavior of some of these systems, such as toxin-antitoxin and restriction modification modules. Transposons follow a biphasic dynamics, with bursts of gene proliferation followed by decay in the copy number that is quantitatively captured by the model. The horizontal gene transfer to loss ratio, but not duplication to loss ratio, correlates with genome size, potentially explaining increased abundance of neutral and costly elements in larger genomes. The evolution of bacterial and archaeal genomes is highly dynamic and involves extensive horizontal gene transfer and gene loss. Furthermore, many microbial species appear to have open pangenomes, where each newly sequenced genome contains more than 10% ORFans, that is, genes without detectable homologues in other species5,6. Here, we report a quantitative analysis of microbial genome evolution by fitting the parameters of a simple, steady-state evolutionary model to the comparative genomic data on the gene content and gene order similarity between archaeal genomes. The results reveal two sharply distinct classes of microbial genes, one of which is characterized by effectively instantaneous gene replacement, and the other consists of genes with finite, distributed replacement rates. These findings imply a conservative estimate of the size of the prokaryotic genomic universe, which appears to consist of at least a billion distinct genes. Furthermore, the same distribution of constraints is shown to govern the evolution of gene complement and gene order, without the need to invoke long-range conservation or the selfish operon concept. Much of our work aimed at understanding evolution of viruses and mobile elements. Among other developments in this area, a survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We showed that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesized that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element. The rapidly growing metagenomic databases have become a rich information source for discovery of new groups of viruses and microbes. We have performed several projects in this direction. One of these involved the discovery of a previously unknown but apparently abundant and ecologically important group of viruses. Marine group II Euryarchaeota (MG-II) are among the most abundant microbes in oceanic surface waters. So far, however, representatives of MG-II have not been cultivated, and no viruses infecting these organisms have been described. Here, we present complete genomes for three distinct groups of viruses assembled from metagenomic sequence datasets highly enriched for MG-II. These novel viruses, which we denote magroviruses, possess double-stranded DNA genomes of 65 to 100 kilobases in size that encode a structural module characteristic of head-tailed viruses and, unusually for archaeal and bacterial viruses, a nearly complete replication apparatus of apparent archaeal origin. The newly identified magroviruses are widespread and abundant and therefore are likely to be major ecological agents. Taken together, these studies advance the existing understanding of the general principles and specific aspects of genome evolution in diverse life forms, in particular viruses and mobile elements, and provide new insights into general principles of genome evolution."}
-{"text": "The postnatal development of both the optical and neural visual systems is dependent on visual experience. Visual experience is defined by the information available in the retinal images in the two eyes. The goal of the proposed research is to extend our previous examination of retinal image quality in one eye during infancy to full binocular viewing conditions. We will examine human infants' visual experience in the context of image clarity and image alignment, which are primarily defined by accommodation and vergence responses and their interaction. These studies will document the emergence of the interaction between accommodation and vergence and their role in the development of refractive and accommodative strabismus. There are three specific aims: i) To understand the normal maturation of the relationship between accommodation and vergence with emmetropisation and growth of the distance between the eyes. ii) To determine the relative bias towards accommodation or vergence accuracy during the critical period of human development. iii) To understand the effects of accommodation and vergence behavior on visual experience of infants and children with high hyperopia or strabismus. PUBLIC HEALTH RELEVANCE: This project will determine how young infants and children manage apparently conflicting focusing and eye alignment demands during typical development. It will also investigate why some children develop refractive or accommodative strabismus while others, with apparently matching visual systems, do not. The goal is to develop intervention strategies to prevent this strabismus and associated amblyopia."}
-{"text": "Our microchemical facility at Caltech has exceeded all expectations in being able to sequence small quantities of protein and synthesize small or large fragments of genes. Several gas phase microsequenators are now functional and each has the capacity to sequence 20 or so residues a day. We have a DNA synthesizer with a three column system that has the capacity to synthesize simultaneously three oligonucleotides with a 14 min. cycle time. Thus, the microchemical facility has the considerable ability to sequence proteins and synthesize genes. These abilities have been turned to a variety of projects that are directly related to the cancer problem. Microsequencing done in this facility on platelet derived growth factor demonstrated for the first time the serum polypeptide hormone appears to be strikingly homologous to the primate oncogene. We are now in the process of analyzing the sequences of a series of other growth hormones and will have the opportunity to determine whether additional similar correlations can be made. We have also started to analyze the gene structure organization and rearrangements of genetic elements in coding the T cell receptor. Once again this receptor molecule plays a central role in facilitating the immune response against foreign invaders including tumors. The microchemical facility has been used to synthesize a wide range of DNA probes that have been used to clone the corresponding genes (partially documented in the Progress Report). Indeed, the DNA synthesizer has been used to synthesize an entire functional E. coli serine tRNA gene. The computer facility has added valuable backup and data analysis capacity to these and many other projects. The cell sorter is functional and has provided valuable data for a number of different laboratories. Within the next year we expect to have a fully functional automated peptide synthesizer and be well along in developing an automated DNA sequenator. Thus, this complement of instruments will allow us to both sequence and synthesize genes and DNA in a sensitive and rapid fashion. This year the microchemical facility has been used by more than 15 professors at Caltech and a much larger number of investigators outside the Institute."}
-{"text": "Practice-based research networks (PBRNs) are organizations of clinicians working in practices that engage in research. PBRNs have emerged as a vital lynchpin in efforts to translate research into practice by translating practice into research. However, the development and maintenance and the continued engagement of busy community practices in cancer research requires specialized expertise and ongoing support. Therefore, the mission of the PBRN Core Facility is: [unreadable] To develop and sustain practice-based research networks that engage in cancer research; [unreadable] To advise and assist Case Comprehensive Cancer Center (Case CCC) investigators in developing community-based cancer research studies for implementation in PBRNs; [unreadable] To provide infrastructure that supports the implementation of Case CCC investigators' research studies in PBRNs, and that supports network practice-initiated research. The Practice-Based Research Network Core Facility supports the engagement of patients, clinicians, practices, and health systems in cancer research by: 1. Starting new PBRNs; 2. Developing the infrastructure of existing PBRNs; 3. Providing PBRN methods consultations to Cancer Center investigators and participating practices; 4. Advising and assisting investigators on research project implementation in PBRNs. The PBRN Core has started and sustained 8 successful PBRNs and is developing 5 new PBRNs. The established PBRNs represent major healthcare systems and include family medicine practices, federallyqualified health centers, community oncology practices, pediatrics practices, and general dentistry practices. The PBRN Core has facilitated the development and implementation of 23 funded research projects within the PBRNs that it has developed and sustained. Fourteen of these projects have been funded by NCI, and 6 supported by ACS. Grant funding for these projects totals over $15 million. The demand for the PBRN Core's services is strong and growing, and the potential for continued and expanded use of the facility is excellent. At present, 19 members of the Case CCC are pursuing 15 different lines of inquiry likely to result in funded studies to be implemented in Core-supported PBRNs."}
-{"text": "The low rate of cure of certain cancers such as lung cancer, the most common causes of cancer death, is an important health problem. Early detection appears currently to be the only way of improving the high mortality rate, but is quite difficult because of the lack of symptoms in early disease. Moreover, lung cancer is mimicked in its in vivo image appearance by benign lesions and processes that lower the specificity of detection. Current imaging methods include chest x-ray, CT, and MRI. While these current methods are able to identify curable lung cancer they also result in many false positives. They are also limited in the size of the lung nodules they can detect. Using available criteria, sensitivity for lung cancer detection is high, but specificity and positive predictive value are only moderate. Thus there is a need for enhanced sensitivity and specificity for cancer cells. Our Anti-transferrin Receptor scFv-antibody fragment (TfRscFv) immunoliposome complex (scL) is a nanoconstruct (~100 nm) for delivery of gene therapy to tumors. It has been shown to target various types of human tumor cells in vivo when implanted as xenografts in mice and is now in Phase I clinical trials for delivery of wtp53. What we are proposing in this application is a quantum jump in diagnostic accuracy, an approach specific to cancer and best for small cancers such as lung cancer. The method we are developing is a nano- sized immunoliposome complex delivering superparamagnetic iron oxide particles (SPIO). Iron Oxide particles are both paramagnetic and super-paramagnetic, giving a biphasic response with both T1 and T2* features. This complex targets cancer cells with high selectivity. Thus the efficient delivery of SPIO directly into the tumor cells by the scL-SPIO complex of this application can increase the conspicuity of the lung tumor cells. Moreover, based on previous studies, the nanocomplex delivered contrast agent which is the focus of this application should accumulate within the cancer cells themselves remaining for an extended period (hours) allowing the contrast in non-cancer areas to wash out, further enhancing cancer conspicuity. In preliminary studies using an as yet unoptimized scL-SPIO complex, we demonstrated tumor cell specificity as compared to free SPIO, and enhanced image intensity. More importantly, in earlier studies with scL complexed with another imaging agent, gadopentate dimeglumine (gad-d), in a lung tumor model, the scL-gad-d (and not free gad-d) was able to enhance and identify lung tumors as small as 1-4 pixels (0.1-0.4mm), a size smaller than possible with current technology. No toxicity was found with this complex. In this application we will optimize the scL- SPIO complex and fully characterize its capabilities for use in early detection of lung cancer in a mouse model of human lung cancer and extend our studies to a mouse model of primary lung cancer. In collaboration with investigators at NIST and NCI we will also asses the magnetic properties of the complex and determine it sub cellular localization and trafficking through the cell. Our goal is to perform the majority of the studies necessary for filing an IND as we aim to move rapidly towards clinical trials. If cancer is detected early (e.g. Stage I), it can in many instances be cured (lower mortality). The challenge is to be able to find and positively identify the cancer at this early stage, particularly lung cancer. While the current methods of detection are good, they can only detect tumors of a certain size. Moreover, lung cancer is often mimicked during imaging by non-cancerous lesions, resulting in uncertainty and many false positives, which are often resolved only by following growth of the tumor. Thus there is a pressing need for imaging agents that increase sensitivity and specificity. Our tumor-specific nano complex delivery of an MR imaging agent, e.g. gad-d and iron oxide, has shown great promise in our preliminary studies in this regard, demonstrating that its high affinity for cancer cells in the lung can result in improved sensitivity in detecting tumors and in overall specificity. The development of an imaging agent that can lead to earlier detection is a high priority in the war on cancer and could lead to increased survival."}
-{"text": "The proposed study is a third follow-up of 581 male narcotic addicts admitted to the California Civil Addict program (CAP) during 1962-1964. The subjects were interviewed once in 1974/75 (DA01146), and again ten years later in 1985/86 (DA03425). The combined prospective and retrospective follow-up interviews provide a unique natural history database that can address issues specifically related to long-term narcotics addiction, patterns and consequence of use. Similar interview instruments will be used to collect self-reported measures about narcotics use, legal supervision status, criminal involvement, drug trafficking, employment, marital status, and treatment episodes over the entire addiction career. Corroborative data will be obtained from official arrest records for each interviewed subject, from a voluntarily provided urine specimen to be taken at interview, and from official death certificates for identified decedents. Specific aims of the study are: (1) to provide an additional ten years of data to allow a detailed natural history description over an almost 40-year addiction career of a sample of narcotics addicts; (2) to assess addiction patterns over time identifying factors which influence the divergence of outcomes, in particular, relapse, cessation of use, the process of \"maturing-out \", and mortality; (3) to analyze and describe morbidity and death among this aging addict sample; (4) to evaluate the extensiveness of criminal activity and to identify specific criminal career patterns in relation to narcotics use; (5) to conduct a treatment intervention history analysis, examining successive and cumulative treatment effects and the extent to which these individual and cumulative treatment episodes have reduced narcotics use; and (6) to assess long-term costs and consequences of prolonged careers of addiction. Augmentation of the existing addict career database provides a unique opportunity for investigation into the current status of addicts now between 47 and 73 years old."}
-{"text": "A central issue in neuroscience is to understand how experience shapes brain function. Recent studies in visual cortex suggest that the maturation of intracortical inhibitory circuits is necessary to initiate and drive ocular dominance plasticity during the critical period. However, the maturation and regulation of the specific type of inhibitory circuit involved is not understood. Critical period plasticity is also modulated by visual deprivation and BDNF over-expression, although the cellular mechanisms remain unknown. Here we hypothesize that the functional maturation of a specific subtype of GABAergic neurons, parvalbumin (Pv) basket interneurons, are a component of the inhibitory mechanism underlying critical period plasticity, and a cellular target of experience deprivation and BDNF regulation. Using bacterial artificial chromosome transgenic (BAC) mice expressing GFP in Pv-interneurons, we will characterize the morphological and physiological development of Pv-interneurons using two photon laser scanning microscopy and electrophysiology in brain slices. We will then examine the effects of dark rearing on the maturation of Pv-interneurons. Finally, we will test the role of BDNF in experience-dependent maturation of Pv-interneurons by blocking trkB signaling using BAC transgenic mice expressing a dominant negative form of trkB receptor specifically in Pv-intemeurons. These results will reveal a mechanism by which experience shapes function of an identified inhibitory network and provide strong evidence that maturation of the Pv-interneuron circuit facilitates the coding of visual information necessary to drive ocular dominance plasticity. The knowledge gained will aid in the design of drug treatments in diseased states such as epilepsy and schizophrenia."}
-{"text": "Complex cellular networks underlie the functional foundation of the mammalian central nervous system (CMS). Understanding the physiological dynamics of these networks, in other words understanding how signaling between interacting groups of cells produce and modulate meaningful physiological information, will directly contribute to our understanding of how the CMS functions in health and how it fails in disease. At present, our mechanistic understanding of the dynamics of neuronal and glial networks is very limited, even though we understand the molecular functional unit that underlies it (i.e. the synapse). One approach is to apply network theory to characterize neuronal and glial networks. Network theory is a branch of statistical mechanics that classifiescomplex networks independent of the physical details of the network and provides an understanding of its dynamical behavior. Applying network theory to neuronal and glial networks requires knowing their structure or topology. However, high throughput computationally intensive measurementsof molecular signaling between neurons and glia, and the extraction of quantitative information about their underlying network structure is not possible given current techniques. What is needed therefore, are algorithms and softwarethat will allow the high throughput characterization and analysis of physiological neuronal and glial networks. Here, we propose to develop computational tools that will allow us to map the spatial and temporal topology of functional neuronal and glial signaling networks, and classify and analyze them within the context of network theory. We present a detailed discussion on the algorithms and programming required to do so, and illustrate the operation and validation of a beta version of such a program. We propose that using this approach, neuronal and glial networks can be classified within known mathematical networktypes and behave as dictated by the quantitative properties of the network types they are classified into. We also present preliminary experimental data showing for the first time that calcium signaling in astrocyte networks, mapped using our software tools, have a previously unidentified toplogy. We propose that the networktopologies of healthy neurons and glia remodel following injury and underlie the induction and maintenance of neuropathological disease states, making the clinical significance of these findings and the development of the computational tools required to investigate them very important."}
-{"text": "Need for new lead compounds against Leishmania and Trypanosoma intensifies the desirability of improved screening adapted to large-scale work. We wish to exploit a hardy Leptomonas of proven high sensitivity to several standard antitrypanosomatid agents; Crithidia fasciculata; and T. mega of amphibia -- a trypanosome easily cultivable on autoclaved media, and whose position hints it may share chemotherapeutic responses with stercorarians, e.g., T. cruzi and salivarians. Minimal media for the Leptomonas and T. mega would be applied to broaden the tolerances --at present apparently narrow -- in the published medium for T. brucei. Detection of novel agents acting as antimetabolites, and mode-of-action studies of likely targets of antimetabolites, make development of physiologically lean, highly reproducible media, desirable. By playing one flagellate against the other we hope: a) to detect lead compounds, and how best to deploy the few available effective drugs; b) use of these drugs to uncover new targets for antimetabolite therapy, especially polyamines, for in related work; the Leptomonas has already served to chart cross-resistance and collateral sensitivities to several established antitrypanosomatid agents. The organisms would be grown in physiologically minimal defined media at blood pH. They would be used to explore interrelationships among polyamines, their precursors, and Mg2 ion; the former compounds are likely targets, along with nucleic acids, of the strongly cationic trypanocides. These organisms would be used also to chart the effects of other agents on trypanosomatid growth, applied singly and in combination with cationic trypanocides. Such agents might significantly inhibit: a) biosynthesis of polyamines; 2) methylation of tRNA's; 3) heme metabolism. Work would focus on ethidium, crystal violet, Antrycide, pararosaniline, methylglyoxal-bis(guanylhydrazone) (a potent inhibitor of polyamine biosynthesis), and transmethylase inhibitors such as 6-dimethylaminonicotinamide. Effective drugs and combinations would be tested against our trypanocide-resistant Leptomonas strains. Inhibition analysis would be directed at identifying metabolites (e.g., polyamines) counteracting these drugs, then assembly of these metabolites into \"rescue\" cocktails to be tested on all 3 organisms in the presence of drugs."}
-{"text": "This is an open label Phase I study. At least 10 HIV-infected pregnant women between 14 and 28 weeks gestation will be enrolled. The women will receive triple combination therapy with indinavir, 3TC, and ZDV during the antepartum period, discontinue indinavir at the start of the intrapartum period while continuing 3TC, and ZDV, and restart indinavir, 3TC and ZDV during the post partum period thru the first 12 postpartum weeks. The mother and her primary care physicians will decide if the mother will continue the same regimen after 12 weeks post partum. The infant will receive 3TC and ZDV at birth and for six weeks following birth. Pharmacokinetic evaluations on cervical secretions and plasma will be performed at the antepartum, intrapartum, and postpartum visits in the women and postnatally in the infants. Baseline and study safety evaluations will include the monitoring of adverse experiences, clinical laboratory tests, physical examinations and vital signs. Temporarialy closed to enrollment awaiting FDA approval for modification."}
-{"text": "7. PROJECT SUMMARY/ABSTRACT High-reliability organizations (HROs) such as aircraft carrier flight decks and nuclear power plants are organizations that operate hazardous technologies in a nearly error-free manner under trying conditions rife with complexity, interdependence, and time pressure. Case studies of HROs as well as in a few healthcare organizations that follow the principles of HROs suggest that a robust safety culture enables more reliable work processes, and thus safer performance. More tangibly, safety culture can be seen ?coming to life? in HROs through specific behavioral processes observed in front-line employees collectively termed collective mindfulness (CM). These five interrelated behavioral processes (also called safety organizing behaviors) are: (1) preoccupation with failure; (2) reluctance to simplify interpretations; (3) sensitivity to operations; (4) commitment to resilience, and (5) deference to expertise. Healthcare is increasingly examining and adopting the principles of CM as a means to improve quality and safety of care delivery. The critical need for achieving HRO status in healthcare is no more apparent than in neonatal perioperative care, which is among the highest risk services hospitals may provide. Neonates are highly vulnerable to iatrogenic events due to their small size, fragility, and exceptional sensitivity to environmental stressors. The objective of this pilot study is to measure the prevalence of CM in neonatal intensive care unit (NICU) and operating room (OR) teams and its impact on non-routine events (NREs) ? defined as any event that is perceived by care providers or skilled observers as a deviation from optimal care based on the clinical situation - during neonatal perioperative care. The project will leverage an active large prospective observational study (IRB-approved NICHD R01) that is defining the epidemiology of risk for neonates in the perioperative environment through an innovative analysis of NREs and contributory factors. We propose a two-year pilot study to characterize CM behaviors in NICU and OR teams and to measure their impact on patient safety as measured by the incidence and severity of NREs during NICU-to-OR handovers and subsequent care. Our Specific Aims are to: 1) Conduct a prospective observational pilot study of NICU and OR teams to: a) estimate the prevalence of perceived CM (i.e., self-reported using the SOS) during the perioperative period and b) delineate the relationship(s) between team attributes, case attributes, and perceived CM scores; 2) Determine the impact of perceived CM on the incidence and severity of NREs occurring during and across phases of neonatal perioperative care; and 3) Conduct a preliminary validation of a provisional behavioral marker system, by assessing the concordance of observed (expert ratings of A-V recordings) and perceived CM (self-reported SOS scores) in the same perioperative teams. We anticipate that knowledge gained from this study will lay the groundwork for a multi-center study on the impact of team-based HRO interventions on neonatal safety in the perioperative environment."}
-{"text": "The major focus of research in my laboratory is transcriptional control of the pro-opiomelanocortin (POMC) gene. In particular we are interested in transcriptional factors that mediate the effects of stress and neuroimmune modulation on the POMC gene. We have previously described a transcriptional activator of POMC, designated PO-B, that interacts with the POMC promoter. PO-B represents the first defined transcriptional activator of POMC whose function has been verified by mutational analysis. Mutation of the PO-B binding site decreases basal transcription by 70% indicating a major role for this protein in POMC regulatory mechanisms. However, we do not know the physiological or pharmacological agents that regulate signal transduction pathways involving PO-B. Towards this goal we have recently purified PO-B to more than 90% homogeneity from HeLa cells. Interestingly deophosphorylation of the purified protein leads to a more 30-fold increase in DNA binding affinity. In this proposal we wish to rapidly extend these early observations by obtaining a full length cDNA clone of the PO-B gene. This is a defined project of limited duration that will enhance our knowledge of POMC transcriptional regulation and be a useful resource for other work ongoing in the laboratory. Since this laboratory was the first to identify and purify PO-B we would also like to obtain the full length clone in a timely fashion. The specific aims are: 1) To obtain reliable N-terminal and internal amino acid sequence data from purified PO-B. \"Best-guess' oligonucleotide probes will be designed from these sequences. Specific PO-B cDNA sequences will be amplified with PCR from a human cDNA library using pairs of these oligonucleotides representing N-terminal and internal sequence. Amplified fragments will be recovered and sequenced to determine if they code for PO-B amino acid sequence. If this approach is unsuccessful, or only N-terminal sequence is obtained, we will use conventional screening of a HeLa lambda gt10 cDNA library identifying positive PO-B cDNA clones by hybridization with the 'best- guess' oligonucleotide probes. 2) PCR and conventional screening methods are not always successful approaches to identifying correct cDNA clones. Therefore, in parallel with the above experiments, we will also use expression screening. This method does not rely on obtaining any amino acid sequence from PO-B. For these studies we will screen a human fibroblast plasmid cDNA library that can be transiently expressed in mammalian cells. The identity of the full length clone will be confirmed by specific binding of the in vitro translated product to the PO-B cognate DNA binding element. The in vivo activity of the full length clone will be confirmed by the ability of transfected PO-B expression vectors to induce transcription from promoters harboring the PO-B binding site. If progress on PO-B cloning is rapid, we will examine its precise role in POMC transcription. We will perform mutational analysis of the protein to determine which domains are required for DNA binding, phosphorylation status and transcriptional activation. It is expected that these experiments will yield valuable information on the signal transduction pathways in which PO-B is involved."}
-{"text": "The purpose of the proposed project is to provide minority (Black) undergraduate students in the Department of Biology, a carefully designed biomedical research that focuses on the use of video microscopy and digitized image analysis to monitor morphological changes in normal and malignantly transformed cells and to assess the impact of morphological modulations on tumorigenesis. The initial investigation will attempt to initiate morphologically variant cell types from transformed hepatocytes and a tumor cell line derived from mouse adenocarcinoma. Clonal derivatives from these cells will be examined, through the use of video counting and microdensitometry, for differences in their uptake and retention of fluorescent dyes (cell density), total cell area, shape factor (fraction for estimating the amount by which a cell varies from a circle), parameter, and nuclear/cytoplasmic ratio of normal and malignantly transformed cells. The clonal isolates will be used to determine the histological types of tumors arising from implantation of specific phenotypically variant cell lines. Karyomorphology of phenotypically variant cell lines will be determined to monitor possible chromosomal changes that may attend in vitro cytodifferentiation. It is felt that morphological and karyomorphological analysis of cells from a single tumor but variant phenotypes will provide valuable information on the series of cytological changes associated with cellular differentiation."}
-{"text": "Production of candidate chikungunya vaccines for human clinical trials will be done in compliance with current Good Manufacturing Practices (cGMP) and released for use in human clinical trials. This will necessitate developing production methods based upon novel cell substrates and If these trials demonstrate safety and immunogenicity of this vaccine in human trials, further evaluation may take place in larger (Phase 2) trials which will necessitate production of the clinical trial materials (CTM) at large scale."}
-{"text": "The proposed research is designed as an analytical and manipulative investigation of mechanisms critical to cell migration in the patterning of the developing nervous system. The studies will focus on two developing cranial nerve nuclei in the chick embryo: The oculomotor ventromedial subnucleus and the trigeminal motor nucleus. In studying the pattern of migration and possible mechanisms important for the proper execution of these patterns, a combination of approaches will be used, including interspecific embryonic transplantation, sequential electron microscopic analysis of intercellular interactions during disparate migration phases, analysis of cellular-substrate interactions, and dual embryonic microsurgical-autoradiographic study. Within the oculomotor ventromedial subnucleus, the experiments will determine the pattern of migration whereby the primordial cells attain their definitive position, the nature and sequence of intercellular contacts formed during migration, (both within and between populations), and will examine pinocytosis as a possible mechanism for mediation of guided cell movement. Within the trigeminal motor nucleus, the proposed studies will examine the nature and sequence of intercellular contacts formed prior to, during and after migration and will experimentally analyze the importance of extracellular influences (i.e., ingrowing ganglionic afferents) to migration of the nucleus."}
-{"text": "Cell signaling mediated by the Hedgehog (Hh) family of secreted proteins plays crucial roles in animal development and human diseases. The Hh pathway is operating in a similar way among organisms ranging from insects to human. Drosophila has been a powerful model organism to study Hh signaling mechanisms, as sophisticated genetic, molecular, and biochemical tools are available to dissect this important pathway in whole organisms as well as in cultured cells. The long-term goal of my laboratory is to delineate the complex regulatory network that governs Hh signal transduction in order to understand how graded Hh signal is transduced to generate multiple developmental outputs. Hh exerts its biological influence through a conserved signaling cascade that culminates in controlling the balance between the activator and repressor forms of the transcription factor Ci/Gli (CiA/GliA and CiR/GliR). The goal of this research is to investigate the multifaceted regulatory mechanisms that control Ci activity. Our recent study has uncovered a dual role of the Ser/Thr kinase Fused (Fu) in the regulation of both the production of CiR and the activity of CIA, and revealed that Fu is activated through dimerization-mediated phosphorylation of its activation loop. These findings provide a critical inroad into a mechanistic dissection of Ci activation. We will explore the mechanism by which Fu promotes the maturation of Ci into CiA and investigate how the Hh gradient is translated into a Ci activity gradient (Aim 1). In a genetic modifier screen, we have discovered that the SUMO pathway can modulate Hh signaling activity and identified Ci as a SUMO substrate. We will further characterize this new post-translational modification of Ci to explore its role and mechanism of action in Hh signaling (Aim 2). The molecular mechanism by which Sufu inhibits Ci is still poorly understood. We have uncovered a previously unidentified nuclear localization signal (NLS) that overlaps with the Sufu binding domain in Ci. We will further study the function of this NLS and its regulation (Aim 3). Finally, how Ci functions in the nucleus to control Hh target gene expression has not been fully explored. We have identified multiple domains required for CiR-mediated repression. Identifying cofactors that interact with these domains and investigating their roles in Hh signaling should shed important lights into how Ci regulates its target gene expression. Therefore, we will carry out protein- protein interaction screen and in vivo RNAi screen to identify Ci co-repressors (Aim 4). The proposed study should provide a much deeper understanding of the Hh signal transduction mechanism and shed new light into how graded Hh signals are translated into different developmental outcomes. PUBLIC HEALTH RELEVANCE: The Hh pathway is a major signaling pathway that controls embryonic development and adult tissue homeostasis. Deregulation of Hh signaling has been attributed to many human disorders including birth defects and cancers. Investigation of the multifaceted and conserved mechanisms that regulate Hh signaling activity will not only provide insights into fundamental developmental problems such as how cells interpret different levels of spatial signals but may also provide new avenues for developing diagnostic tools and therapeutic treatments for cancers caused by deregulation of Hh signaling activity."}
-{"text": "Activities in this area are focused on the development of agents with novel mechanisms of action, and the development of novel combinations. A range of compounds are under study. Currently, phase II studies being conducted in ovarian cancer include: 9-AC, a topoisomerase I inhibitor; MGI-114, a novel DNA damaging agent; and, CAI, an anti-angiogenesis agent. A phase I study is beginning, using SU5416 with carboplatin in recurrent ovarian cancer. SU5416 is an anti-VEGF agent with molecular properties that inhibit angiogenesis, and may impair platinum-DNA adduct repair. Preclinically, gene therapy approaches in ovarian cancer are being explored, using dominant negative constructs to AP1 that have been developed by Dr. Charles Vinson of DBS. Also, proteasome inhibition is being explored on the preclinical level in human ovarian cancer cells. In prostate cancer, a range of agents have also been studied. Such agents include gallium nitrate (a heavy metal with a nubmer of activities), high dose tamoxifen (PKC inhibition), thalidomide (anti-angiogenesis) and thalidomide combinations, CAI (anti-angiogenesis), and ketoconazole (anti-androgenic agent) and ketoconazole combinations. - angiogenesis, ovarian cancer, pharmacology, platinum compounds, prostate cancer, - Human Subjects & Human Tissues, Fluids, Cells, etc."}
-{"text": "PROJECT ABSTRACT Most neurodegenerative disorders exhibit highly heterogeneous genetic underpinnings. For example, with Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and Parkinson's disease (PD), mutations in different genes are causal in distinct subsets of families. In addition to this genetic heterogeneity among inherited cases, the majority of individuals exhibit sporadic forms of these disorders in which there are no known causal mutations. In part because of this sporadic onset, it is widely accepted that (largely unknown) environmental forces are at play in the initiation and/or progression of each of the above disorders. This proposal will use a systems approach in Drosophila to identify forces that drive initiation, as well as common cellular responses that may modulate progression. This will be accomplished by three scientific aims. First, we will test a series of cellular stressors, behavioral stressors, and models of injury/inflammation. The effects of these manipulations will be assayed by following 7 different neurodegenerative phenotypes and biomarkers, including a novel assay of endogenous retrovirus replication. Second, we will use a relatively new approach to purify the population of cells that are most impacted, and profile active transcription within the nuclei. This experiment will identify common downstream cellular responses. Finally, we take advantage of high throughput genetic approaches in Drosophila to systematically test for functional impact of identified gene targets."}
-{"text": "A third of the world's population is infected with Mycobacterium tuberculosis (Mtb). Tubercle bacilli can remain inactive in lung lesions only to emerge decades later to seed new outbreaks of tuberculosis. In addition, tuberculosis is one of the most difficult bacterial infections to treat and continues to cause more deaths than any other bacterial infection. Bacilli exist in replicating and non-replicating states in a range of microenvironments that vary in oxygen concentration and nutrient availability. The bacilli that survive during latent infection likely exist in a non-replicating state and antimicrobials, effectie against actively growing bacteria, are often not effective against non-replicating bacteria. Population heterogeneity, the presence of more than one phenotypic variant in a clonal population, provides bacteria diverse mechanisms to endure environmental challenges. We have demonstrated that Mtb exists in two semi-stable states that appear to be epigenetically controlled. When mycobacteria were grown as a dense population, either by pellicle or settled growth, the vast majority of the population took on characteristics of a form termed pellicle. If bacilli were passaged starting with a solitary bacillus or just a few cells, however, the ensuing population shifted to a form termed solitary. One of the most striking contrasts between the solitary- and pellicle-prepared Mtb was the solitary form adapted to hypoxic and anaerobic conditions by maintaining high transcriptional activity, while the pellicle form failed to adjust t hypoxia and became truly dormant under anaerobic conditions. We hypothesize that populations of Mtb contain at least two phenotypic variants that allow for divergent responses to imbalances in proton homeostasis, particularly resulting from hypoxia. To test this hypothesis we will determine the differential abilities of the solitary and pellicle variants in adjusting to conditions that exert pressure on internal pH homeostasis. We will also identify regulatory elements that control the shift between the variants. Pellicle growth has historically been used to increase or maintain Mtb virulence during in vitro growth. Therefore, we will investigate if the pellicle-passaged variant is more virulent than the solitary form by using a mouse model of TB that reproduces hypoxic necrotic granulomas."}
-{"text": "One in nine women suffer from pelvic floor dysfunction, including urinary incontinence and vaginal wall or uterine prolapse (VUP). Stress urinary incontinence (SUI) affects 38% of women over the age of 65 years and over 13 million women in the United States. Pelvic muscle strength is commonly assessed in these patients. However, current measurement techniques are either subjective or produce artifact, due to their non-isometric nature or contamination by intraabdominal pressure. During Phase I, we developed a second generation system that measures the isometric strength and contractile properties of female pelvic floor muscles. The system centerpiece is a novel intravaginal transducer that differentiates between intraabdominal pressure and levator ani force. During Phase II, system mechanics, electronics and software will be refined to improve system sensitivity, accuracy, and ease-of-use. Laptop- and Personal Data Assistant-based systems will be developed and validated. Clinical device performance will be confirmed by testing the null hypothesis in 120 women (40 healthy continent, 40 with VUP, 40 with SUI) that localized pelvic floor muscle defects visible on MR scans will correlate with pelvic muscle weakness. The system allows assessment of pelvic floor function and exercise intervention efficacy, and can provide biofeedback and adherence data during training."}
-{"text": "The incidence of infertility has increased 4% since the 1980s, with up to 20% cases having no known cause. One of the prevailing hypotheses is incompatibility between cognate egg and sperm proteins; however, very few pairs of interacting reproductive proteins have been identified in any organism. The best model for studying fertilization remains the marine gastropod abalone, where one of the first steps in fertilization involves the interaction between the sperm protein lysin and its egg coat receptor VERL. As a major component of the abalone egg coat, VERL is a giant, fibrous glycoprotein composed of ~22 ZP-N repeats that form intermolecular hydrogen bonds to create the highly stabilized and protective egg coat. Lysin creates a hole in the egg coat by competing for the hydrogen bonds, allowing sperm to pass and fuse with the oocyte. The VERL repeats are homologous to human egg coat proteins, and likely share a similar protein topology. However, the precise structural mechanisms that drive egg coat dissolution remains to be determined. To address this fundamental question in fertilization, two specific aims are proposed using state-of-the-art structural and proteomic approaches. In aim 1, multidimensional NMR will be used to characterize the structural basis of lysin-VERL interactions for three species of abalone. In aim 2, deep mutational scanning will be utilized to explore the specific adaptations acquired by lysin and VERL that permit species specific interactions, and simultaneously test theoretical models of sexual selection. The proposed research is innovative for its combined use of proteomic and structural techniques to characterize the evolutionary history of rapidly evolving reproductive proteins. The results are expected to shed insight into the core mechanisms that mediate egg-sperm interactions in abalone, and provide foundational information towards understanding the more complex mammalian system."}
-{"text": "Leukotriene B4 (LTB4) is a potent lipid chemo attractant that classically has been associated with myeloid cell chemotaxis. We isolated a novel murine seven transmembrane spanning G protein-coupled receptor, which we identified as BLTR1, an LTB4 receptor, and generated BLTR1-deficient (BLTR1-/-) mice by targeted gene disruption. Characterization of this mouse revealed that BLTR1 is responsible for LTB4- mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium. Further, despite functional redundancy with other chemo attractant-receptor pairs in vitro, loss of BLTR1 function significantly reduced eosinophil recruitment into the peritoneum in a model of peritonitis. Follow-up studies of BLTR1-/- mice have allowed us to make several novel observations about the roles of LTB4 and BLTR1 in leukocyte recruitment that will be the focus of this grant proposal. We have found that loss of BLTR1 function significantly and specifically impairs neutrophil transendothelial migration (TEM) in vitro and in vivo. Secondly, we have found that BLTR1 is highly expressed, functional on certain subsets of activated T cells, and important for their trafficking in vivo. Additionally, we have found that recruitment of BLTR1-/- T cells into the airways early in allergic pulmonary inflammation is profoundly impaired, despite preserved trafficking of these cells into the lung parenchyma. Based on these novel preliminary findings, the central hypothesis of this proposal is that LTB4 and BLTR1 play a critical role in facilitating the ability of leukocytes to traverse endothelial, interstitial and epithelial barriers. We hypothesize that interactions between leukocytes and barriers to migration they encounter, e.g. endothelial cells, tissue interstitium, and epithelial cells, induces the production of LTB4. We hypothesize that the LTB4 produced by these interactions in turn activates leukocyte BLTR1, which facilitates penetration of these barriers by the migrating leukocytes. We believe that the LTB4 biosynthetic pathway uniquely positions this chemo attractant-receptor pair for this role. LTB4 is produced by serial enzymatically catalyzed reactions, and hence can be produced much more rapidly than peptide chemokines, which require transcription and translation. The rapid kinetics of LTB4 biosynthesis enables leukocytes, endothelial cells, and epithelial cells to produce LTB4 contemporaneously with leukocyte migration across cellular and extracellular barriers. In this grant, we propose to investigate this hypothesis as it relates to neutrophil and lymphocyte recruitment. Specifically we propose to: (1) determine to cellular source of the LTB4 that facilitates neutrophil transendothelial migration; (2) define the roles of BLTR1 and CXCR2 in neutrophil recruitment into the airways; (3) determine the role of LTB4 and BLTR1 in antigen specific T cell trafficking; (4) determine the role of LTB4 and BLTR1 in T cell recruitment into the airways of the lung. [unreadable] [unreadable]"}
-{"text": "Many patients with hypertrophic cardiomyopathy have severe symptoms in spite of medical therapy with beta adrenergic blocking agents and/or calcium channel blocking agents. Recently we have been investigating the use of amiodarone, a benzofuran derivative with potent hemodynamic and antiarrhythmic properties in this same subgroup of patients and have noted an improvement in cardiac symptoms and an increase in exercise capacity. However, there remains a subgroup of patients who are intolerant of amiodarone or who do not improve on amiodarone and continue to have marked symptomatology. In response to a compelling clinical need in this subgroup of refractory patients, we felt it appropriate to explore other potential pharmacologic modalities. We have hypothesized that the functional and structural abnormalities in HCM are related to a primary membrane disorder leading to increased cytosolic calcium levels as a result of altered calcium fluxes involving both the myocardium and the vascular smooth muscle of the small intramural coronary arteries. Lidoflazine has been shown to be a potent calcium entry blocker, and has a cellular protective effect against calcium overload in vascular smooth muscle and cardiac muscle during ischemia, preventing ischemic contraction and myonecrosis. These properties of the drug afford an ideal mechanism for testing the above hypotheses, as well as offering a potentially important therapeutic alternative. The study will consist of 3 phases. The first phase to assess the clinical efficacy of the drug; the second to characterize the hemodynamic/metabolic correlates of the drug that may determine its efficacy; and the third to compare in a double blind fashion lidoflazine versus standard therapy. We have enrolled thus far 3 patients in phase I, two of whom have had symptomatic and exercise improvements."}
-{"text": "Nearly 5 million people in the US alone are afflicted with Alzheimer's disease, and there is currently no means to prevent or treat the disease. Though the molecular basis of the disease is unclear, the deposition of amyloid plaques in the brain is a key hallmark of the disease. These plaques are widely believed to be pathogenic, and tremendous efforts have focused on the mechanism governing their formation. Amyloid plaques are composed of the 42-residue amyloid ? peptide (A?), which is generated through proteolytic cleavage of the amyloid precursor protein (APP) by ?-secretase. This enzyme is localized within cholesterol-rich lipid raft membrane domains, while the APP substrate exists in both the fluid-phase and lipid raft domains of cellular membranes. Therefore, the efficiency with which the A? peptide is generated may be governed by the distribution of APP between the raft and non-raft membrane domains. Recent work in the Sanders lab on the 99-residue C-terminal fragment of APP (C99) has revealed a cholesterol binding pocket within the TM domain (present in APP). These studies have confirmed that C99 binds cholesterol with high affinity in bilayers, which suggests that the localization of C99 (and APP) may depend on the distribution of cholesterol in biological membranes. We recently tested this hypothesis by characterizing the localization of C99 within phase-separated giant unilamellar vesicles (GUVs), which contain both fluid phase (L?) and liquid-ordered (Lo) domains. The results show that C99 is specifically localized within raft-like Lo domains. However, C99 variants carrying mutations that abolish cholesterol binding strongly prefer the non-raft L? phase. This confirms cholesterol binding directly affects the distribution of C99 within the membrane. The most intuitive explanation for this phenomenon is that C99 is driven into the Lo domain due to the high concentration of the cholesterol ligand, which leads to favorable binding energetics. However, thermodynamic evaluations of this partitioning suggest that binding energetics cannot account for the observed differences. Thus, the physical mechanism for this coupled binding and partitioning remains unclear. In the following, I propose a series of experiments aimed at dissecting the energetic contributions of both the membrane and the protein in the coupled binding and partitioning of C99. I will first use EPR spectroscopy to assess the binding energetics of cholesterol in Lo and L? like membranes. The results will suggest whether the cholesterol binding energetics are sensitive to changes in the bilayer. Next, I will use protein engineering and confocal fluorescence microscopy to determine how differences in the length and rigidity of the TM domain affect its partitioning. These studies will reveal the structural features of C99 that are critical for its sorting within te membrane. Finally, I will examine the structural dynamics of free and cholesterol-bound C99s using solution NMR in both Lo and L? like bicelles in order to determine how bilayers affect its binding mode. Together, the results will provide novel insights into the molecular basis of Alzheimer's disease and elucidate the molecular determinants of protein sorting within the membrane."}
-{"text": "The North Central Cancer Treatment Group (NCCTG) is a regional organization made up of 10 community cancer centers with the Mayo Comprehensive Cancer Center serving as Operations Office and Statistical Center. It is designed to conduct cancer treatment research at the level of the community clinic where most cancer care is delivered. It is the intent of the NCCTG to produce the most complimentary clinical research interaction of community clinic, comprehensive cancer center, and national cooperative group. By bringing cancer patients into national research programs at an early stage of their disease, the NCCTG meets an expressed need of the National Cancer Program. Particular emphasis had been placed on multidisciplinary programs with strong participation of medical oncology, surgical oncology, radiation therapy, and pathology. We believe extraordinary high quality of research data has been achieved and we will continue our efforts to maintain and enhance quality control procedures. Significant and timely clinical research protocols are being conducted in colorectal carcinoma, lung cancer, breast cancer, pancreatic cancer, gastric cancer, gynecologic cancer, and brain tumors. Protocols for urologic cancer are under active planning. Whenever pertinent, protocols are randomized in design and concurrently controlled with untreated controls used when justified. A unique characteristic of clinical research within the NCCTG is unparalleled cost effectiveness. In spite of the severe handicap of grossly inadequate funding the NCCTG has proven itself to be a strong, viable, high quality, and productive clinical research organization."}
-{"text": "The goal of this proposal is to increase the understanding of the mechanisms contributing the exacerbation of neurological complications in Human Immunodeficiency Virus (HIV) infected drug abusers. The use of dopaminergic drugs, such as cocaine and methamphetamine, increases both the incidence and severity of HIV-induced neurological disease in HIV infected individuals. These drugs act by increasing extracellular dopamine levels in the central nervous system (CMS). Studies show that increased extracellular dopamine exacerbates HIV induced CMS pathology, but the mechanisms that mediate this effect are not fully characterized. Macrophages, the major target for HIV in the CMS, also play a central role in the neuropathology of HIV infection. The preliminary data in this proposal show that dopamine treatment increases HIV replication in macrophages, and demonstrate the presence of dopamine receptors on the surface of the eels. These data indicate that dopamine may play a direct role in the development of HIV induced CMS pathology through dopamine mediated modulation of HIV infection of macropahges. To determine the mechanism mediating this effect, studies in this proposal will fully characterize the pharmacology and kinetics of dopamine-mediated increase in HIV replication in macrophages. HIV infection in the presence of dopamine receptor agonists and antagonists will determine which dopamine receptors mediate the increase in HIV replication in macrophages. To examine the mechanism mediating this effect, studies will examine crosstalk between the active dopamine receptors and the HIV receptor CD4, and coreceptors CXCR4 and CCR5 as well as dopamine-induced alterations in the viral entry, integration, and budding processes. Experiments will also use protein kinase inhibitors during HIV infection to examine the signaling pathways involved in dopamine enhanced HIV replication. Defining the mechanisms by which dopamine increases HIV replication in macrophages will contribute to the understanding and treatment of the heightened incidence and severity of neurological disease among HIV infected drug users. PUBLIC HEALTH RELEVANCE: HIV infection can result in a variety of neurological complications that are exacerbated by drugs of abuse. Defining the mechanisms by which drug abuse increases the incidence and severity of HIV induced neurological complications will increase the understanding of HIV infection in the brain and help to develop intervention strategies to limit the devastating consequences of neurologic impairment in HIV infected drug users."}
-{"text": "Dystonia is a neurological disorder characterized by involuntary twisting movements or abnormal postures caused by excessive activation of specific muscle groups or abnormal co-activation of agonist and antagonist muscles. The neurobiological basis for this disorder is not well understood, and even the neuroanatomical substrates remain uncertain. While clinical studies of affected patients have traditionally associated dystonia with damage or dysfunction of the basal ganglia and its connections, laboratory studies of rodents have more consistently associated the disorder with dysfunction of the cerebellum and its connections. The overall goal of this proposal is to bridge our understanding of the gaps between the basal ganglia and the cerebellum in the expression of dystonia. Our hypothesis is that dystonia is not simply the reflection of dysfunction of a single motor control system, but may result from dysfunction in either the basal ganglia or cerebellum, or abnormal interactions between these two regions. In Aim 1 we will investigate the contributions of the basal ganglia in two well-characterized mouse models where dystonia is known to be triggered by dysfunction of the cerebellum. These studies will be valuable for demonstrating the involvement of the basal ganglia in these models as well as important interactions between these two motor control systems in the expression of rodent dystonia. In Aim 2 we will perform manipulations of the cerebellum in rhesus monkeys analogous to those that have been demonstrated to provoke dystonia in rodents. These studies will establish a role for abnormal cerebellar output in the genesis of dystonia in monkeys and have the potential to result in a novel monkey model for dystonia. Aim 3 is devoted to a careful reassessment of autopsy material from the cerebellum of dystonia patients. These studies will be valuable because prior autopsy studies, which traditionally focused almost entirely on the basal ganglia, have generally failed to disclose any consistent neuropathological changes in most forms of idiopathic dystonia. Overall, the studies described in this proposal have the potential to stimulate a major revision of current concepts concerning the neuroanatomical basis for dystonia. A more complete understanding of the neuroanatomical substrates for dystonia is of elemental importance for any future studies of dystonia addressing relevant pathogenetic changes at the molecular, cellular, and physiological levels. In addition, the studies will have direct relevance for modern neurosurgical approaches to the management of dystonia that involve focal brain stimulation or lesion techniques. [unreadable] [unreadable]"}
-{"text": "This project is concerned with (a) the genetic diversity of Trypanosoma cruzi and its implications to the epidemiology, course and diagnosis of Chagas' disease, (b) the development of high resolution flow cytometry instrumentation for analyses of cell populations and, (c) the utilization of flow cytometry and low light level video microscopy for the analyses of infectious agents. Flow cytometry was used to analyze the population dynamics of T. cruzi clone mixtures. The relative numbers of each clone in the population changed rapidly and the results are in quantitative agreement with mathematical models of competitive population growth; no dynamic equilibrium was found. These data emphasize the importance of working with well-defined clones in the laboratory and stress the importance of rapid isolation of single clones from clinical specimens. A similar situation occurs in Dipetalogaster maximus except that clones can coexist for longer periods of time in the bug. Electrocardiographic analyses of inbred mice infected with T. cruzi clones demonstrate that several known aspects of chronic Chagas' disease can be mimiced with the model. Electron bean x-ray microchemical analyses of T. cruzi clones demonstrate marked inter-clonal differences in Fe, Zn, S, Mg, K, Ca and P."}
-{"text": "The murine lymphocyte receptor for IgE, with regard to both structure and function is the focus of this application. Intact FceR levels increase during B cell activation in the presence of interleukin 4 (IL-4) suggesting a role for the FceR in B cell activation. Anti-FceR both in the soluble and sepharose bound form will be tested for the capacity to either directly activate B lymphocytes or to influence B cell activation by anti- Immunoqlobulin. Parameters to be measured include calcium influx, cell size changes, phosphatidyl inositol levels and Ig production. The FceR spontaneously releases lower molecular fragments via proteolysis at the cell surface and preliminary evidence suggests that these fragments enhance the level of IgE synthesis in the presence of IL-4. This phenomena will be further studied with regard to isotype specificity, role of FceR carbohydrate in the biologic activity and molecular size of the FceR fragment involved. In addition, it will be determined if the fragment interacts with a specific component on the B cell membrane. The site of interaction of IgE with the FceR is important with regard to receptor degradation and the location of this site will be explored in detail, both by using anti-peptide antibodies, mimicking specific areas of the FceR and by using crosslinking reagents that will label the site(s) of close proximity with IgE. Antibodies against the site of the FceR that interacts with IgE will also be tested for their capacity to induce FceR upregulation, analogous to IgE and their effect on IgE synthesis will be determined. The low affinity FceR on macrophages and T cells will be further investigated with regard to physiochemical relationship to the B cell FceR, association with other membrane components and regulation at the cell surface. The overall aim is to gain further insight into the structure and regulation of the low affinity FceR and to determine if FceR/anti-FceR related components are useful in the regulation of IgE synthesis."}
-{"text": "Mitochondria from young and mature BHE and Wistar rats were studied. Strain and age affected respiration, shuttel activity, ATP production, ATPase activity, and adenylate kinase activity. Mitochondria were less active in BHE rats than in Wistar rats and with age became even less active. The slower shuttle activities and Ca ions Mg ions ATPase activity in BHE rats could be made more active with thyroid hormone treatment suggesting that the genetic aberration in metabolic control which results in maturity onset lipemia and glycemia, and in a shortened lifespan, is not due to an aberration in the mitochondrial shuttle systems or of the ATPases. Studies of glucose production by isolated hepatocytes showed that gluconeogenesis was more active in BHE rats than in Wistar rats but that in BHE rats, thyroid treatment was without effect as a stimulant of glucose production."}
-{"text": "Abstract LSUHSC CARC Administrative Core The purpose of the Louisiana State University Health Sciences Center (LSUHSC) New Orleans Comprehensive Alcohol-HIV/AIDS Research Center (CARC) is to conduct cutting-edge translational research on the causes and biomedical consequences of alcohol use and abuse and their impact on biomedical and psychosocial comorbid conditions of persons living with HIV/AIDS. The goal of the CARC is to generate evidence-based knowledge on the interaction of alcohol and HIV disease that will inform health care providers and will lead to effective primary care-based interventions to decrease risky alcohol use, HIV transmission and improve health outcomes. The Administrative Core of the CARC will provide oversight and the organizational framework for the direction, management, and coordination of all activities. Specifically, the Administrative Core will (1) monitor the performance and productivity of the individual research components and cores; (2) manage the budget and fiscal activities of the CARC; (3) supervise the CARC personnel; (4) provide data collection, management, and analysis support, and (5) provide oversight for the educational, enrichment and dissemination functions of the CARC. In essence, the Administrative Core is responsible for centralizing, coordinating, and integrating research and administrative components of the CARC. Dr. Molina, Principal Investigator and Director, and Dr. Nelson, Co- Director, will direct all CARC-related activities in consultation with the Intramural Center Committee. The Administrative Core will facilitate logistics for synergistic, productive, and seamless interaction among the participants to substantially enhance the success of CARC activities. The Administrative Core will foster formal and informal exchanges among members of the CARC and the intra- and extramural scientific community to allow for peer-review, idea cross-fertilization, and development of new collaborative projects focused on the biomedical health consequences of alcohol abuse. The Administrative Core is a mature, integrated component of our CARC with a successful record in supervising, promoting, and developing comprehensive, thematically focused research programs that are both productive and cost-effective in an area of great clinical relevance."}
-{"text": "The ocular lens provides an ideal system to study cell cycle regulation during terminal differentiation. The lens is composed of two cell types: the epithelial cells, which are capable of cellular proliferation, and the fiber cells, which are post-mitotic. I hypothesize that the differentiation of epithelial cells into fiber cells will cause alterations in activity of the genes that regulate the cell cycle. In order to help test this hypothesis, the alphaA-crystallin promoter will be used to direct lens-specific alterations in gene expression in transgenic mice. Transgenic and non-transgenic mice will be used to study two general classes of genes that are likely to be critical for cell cycle control during differentiation: tumor suppressors and cyclin-dependent kinases. The Specific Aims of this grant application are: l) to assess the role of the retinoblastoma (rb) protein in cell cycle control and terminal differentiation of lens fiber cells; 2) to assess the role of p53 in the induction of lens tumors by SV4O large T antigen; 3) to assay for changes in cyclin dependent kinases that accompany fiber cell differentiation; 4) to characterize the cell death that is induced by rb inactivation in fiber cells; and 5) to genetically reverse lens tumorigenesis induced by full-length T antigen. Our preliminary experiments have shown that expression of viral proteins that bind to rb and/or p53 can induce lens cell tumorigenesis or programmed cell death. Therefore, the cell cycle can be altered in lens cells with fascinating and unexpected consequences. The proposed studies should provide insights into cell cycle regulation in vivo, not only for the lens, but for other cells undergoing terminal differentiation or neoplastic transformation."}
-{"text": "The human peptidase clan CE is defined by seven proteases with a common fold and catalytic mechanism, commonly known as SENPs (sentrin specific proteases - sentrin being an alternative name for SUMO). Current information suggests that most SENPs are specific for processing of SUMO precursors and removing SUMO conjugated to protein substrates. Small-molecule compounds that target SENPs in a selective manner will provide an important toolset to study the biology of these enzymes, to aid in the discovery of their role in disease etiology and progression, and to facilitate the subsequent development of therapeutics against disease-relevant targets. The availability of simple kinetic assays that are applicable the SENPs would significantly enhance the discovery of chemical probes for members of the family, and for .orphan. SENPs with lesser known natural substrate specificity. Our application squarely addresses this [unreadable] important unmet need for a development of high-throughput substrate-based assays. We propose to develop small peptide based substrates with broad specificity for all seven human members of the SENP family, and generate simple assay procedures. The assays are expected to provide a tool for the rapid development of chemical probes for members of the family. [unreadable] [unreadable] [unreadable]"}
-{"text": "Functional inactivation of menin, encoded by the MEN1 gene, causes the inherited multiple endocrine neoplasia type 1 (MEN1) syndrome and some but not all sporadic pancreatic endocrine tumors. Therefore, unraveling molecular events upstream or downstream of menin could point to other causative genes and/or regulatory events responsible for such tumor types. Menin resides in a histone methylating protein complex that trimethylates histone H3 at lysine-4 (H3K4me3), an epigenetic mark for active gene expression. Therefore, we have determined a genome-wide map of menin-dependent H3K4me3 (using ChIP-Seq) and menin-dependent gene-expression program in wild-type (WT) and menin-null mouse embryonic stem cells (ESCs) and in pancreatic islet-like endocrine cells (PILECs), which we derived from WT and menin-null mouse ESCs through in vitro differentiation. We found menin-dependent H3K4me3 specifically targeting the Meg3 gene in mouse ESCs, and all four Hox loci in differentiated PILECs. Gene expression from the Meg3 locus and from all of the four Hox loci was abolished in menin-null cells. Both Meg3 and Hox loci have been implicated in MEN1-like sporadic tumors: MEG3 in pituitary tumors, and HOX in parathyroid tumors. Our data suggest that these genes with menin-dependent H3K4me3 could be relevant players in the tumorigenesis of endocrine cell types associated with MEN1. Furthermore, our work shows that menin-null mouse ESCs could also be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null ESC-derived specialized cell types for genome-wide analyses studies. Our current efforts are directed towards understanding the regulation and activity of genes at the MEG3 and HOX loci. We have shown that cyclin-dependent kinase inhibitors (CDKIs) of the INK4 family (4 genes) and the Cip/Kip family (3 genes) that negatively regulate cell cycle progression and cell proliferation have rare germline or somatic mutations in endocrine tumor states related to MEN1. Also, mouse models show an endocrine neoplasia phenotype in 'knock-in' mice homozygous for the CDK4-R24C mutation, or by the combined loss of two different CDKIs, p18 and p27. Therefore, understanding the molecular basis of CDK and CDKI regulation could provide insights into their contribution to endocrine tumorigenesis. We have investigated the contribution of cell cycle regulators in endocrine tumorigenesis, particularly mutations in CDKI genes. We are interested in investigating the molecular basis of cell cycle regulation in endocrine cells."}
-{"text": "The candidate has a primary interest in investigating the associations between neighborhood socioeconomic (SES) environments, behavioral and psychosocial risk factors, and cardiovascular disease (CVD). The mentored phase will include coursework in Geographic Information Systems (GIS) and complex multilevel modeling, as well as data linkages prior to the independent phase project. The independent phase will establish the candidate's independence while enabling skill development in GIS and multilevel modeling. Data on nearly 90,000 women from the Harvard-based Nurses'Health Study, a well-established cohort, and nationally-representative data on approximately 262,000 men and 314,000 women from the National Health Interview Survey, will be analyzed using multilevel discrete-time survival analysis models to estimate the relations between neighborhood SES and risks of non-fatal and fatal coronary heart disease (CHD), and to test for the presence of behavioral and psychosocial mediators. Furthermore, GIS methods will be used to assess whether some of these mediators may be determined by particular neighborhood services and amenities. By contributing to the knowledge base on the neighborhood determinants of and pathways to CHD, the project's efforts may ultimately translate through interventions into more effective reductions in CVD burden among Americans. This award should enable the candidate to pursue a successful career in the study of the neighborhood determinants of CVD."}
-{"text": "This research proposal, submitted in response to NIH RFA HG-03-004 (Technologies to Find Functional Elements in Genomic DNA), describes the development of a novel assay technology to begin to unravel the complexities of alternative RNA splicing. The proposal seeks to develop a means of measuring, in parallel, all predicted exon-specific mRNA sequences coded by the 30MB ENCODE targeted genomic sequences for their presence in a given population of mRNA and their contextual linkage to other exons. These studies are critical to understanding how the human genome is transcribed and translated into an enormous repertoire of molecular diversity from an unexpectedly small set of identified human genes. [unreadable] [unreadable] The proposed assay, termed an \"Exon-Linkage Assay\" will combine a variety of technological innovations to the high-throughput characterization of the human spliceome through the use of a strategy of dual, internal-primer mediated double-stranded cDNA synthesis on a spatially resolved solid phase. The assay leverages a highly adaptable DNA microarray synthesis technology to provide an RNA splicing analysis tool that would be labor or cost-prohibitive by other means. [unreadable] [unreadable] The described method has the potential to quickly identify differential pre-mRNA splicing patterns in different tissues as well as shifts in splicing (caused either by regulatory defects or genomic mutations) associated with human disease in a rapid, scalable assay platform. [unreadable] [unreadable]"}
-{"text": "Unlike most chronic renal diseases, in which the female gender is a protective factor, this \"female advantage\" is lost in diabetes as diabetic females develop more renal disease than non-diabetics;we propose that this is due to low levels of plasma estradiol (E2) in diabetic females. The findings suggest that estrogens (including E2 and its metabolites, such as 2-methoxyestradiol (2-ME)) contribute to the pathophysiology of diabetic renal disease. Based on our preliminary data, we hypothesize that: Specific Aim 1: E2 regulates the renal renin-angiotensin system by reducing the expression and activity of the RAS: specifically by 1. tonically downregulating AT1Rs, 2. decreasing Ang II and renin levels, 3. increasing AT2Rs and consequently attenuating the renal functional and structural changes (in particular vascular changes) associated with diabetic nephropathy. Specific Aim 2: E2 and an estrogen metabolite 2-methoxyestradiol (2-ME) attenuate oxidative stress associated with diabetic nephropathy by reducing NADPH oxidase activity and NADPH oxidase-induced O2.- generation in the diabetic kidney. Specific Aim 3: E2 and 2-ME attenuate inflammation associated with diabetic nephropathy by reducing 1. Acute inflammation and markers of the acute phase inflammatory reaction: IL-6, MCP-1 and M-CSF;2. Chronic inflammation: vascular permeability, migration and infiltration of inflammatory cells in target tissues;3. glucose and AT1R-mediated Ang ll-induced NF-kB expression in target cells and resultant 4. NF-kB- induced TGF-b protein expression and subsequent activation of the Smad signaling pathway. Specific Aim 4: E2 and 2-ME attenuate glomerulosclerosis and tubulointerstitial fibrosis associated with diabetic nephropathy by 1. Reducing hyperglycemia/Ang ll-induced cell growth and ECM protein synthesis, namely collagen type I and IV, laminin and fibronectin, 2. Increasing ECM protein degradation, by increasing the activity and expression of matrix metalloproteinases, MMP-2 and MMP-9. The findings from these studies will provide insight into the mechanisms by which estrogens contribute to the pathophysiology of diabetic nephropathy and may stimulate novel ideas for developing gender-specific treatment modalities for diabetic nephropathy."}
-{"text": "Substance abuse in adolescence is an epidemic which takes a significant toll on society and has deleterious physical, psychological, and social effects on adolescents and their families. As such, a great deal of research in recent years has focused broadly on finding patterns among social and behavioral determinants of substance abuse in adolescence, and specifically on the goal of finding clinically relevant subgroups of adolescents according to substance abuse behaviors. This goal is reflected by a recent NIDA program announcement, PA-15-003, entitled Epidemiology of Drug Abuse; this announcement lists trajectories of drug use and patterns of comorbidity with substance use disorders, two areas frequently explored using subgrouping strategies, as areas of interest. The goal of forming meaningful behavioral subtypes has largely been pursued using mixture models, a broad class of models which seeks to divide the sample into meaningful subgroups, known as latent classes, of subjects. Mixture models have seen extensive use in substance abuse studies in the past decade, with behavioral groupings being linked to imaging data, biomarkers, and genetic information. However, despite the their widespread use, virtually all current applications of mixture models have failed to take into account one critical aspect of behavioral measurement known as differential item functioning (DIF). Given an item intended to measure some underlying construct, DIF occurs when subjects with the same level of that underlying construct differ systematically in their responses to an item measuring that construct due to their gender, race, age, or any other individual characteristic. When DIF occurs and is not accounted for, it may render estimates of the level of the underlying construct biased, and inferences about differences between groups invalid. Though techniques for measuring continuous latent variables such as item response theory (IRT) and confirmatory factor analysis (CFA) have given serious attention to DIF as a fundamental threat to the validity of inferences, there has not yet been systematic study of DIF in the measurement of latent classes in mixture models, and there are no known applications of DIF analyses to mixture model results in the substance abuse literature. The proposed research seeks to rectify this shortcoming by rigorously investigating DIF in mixture models with a focus on behavioral studies of substance abuse. The four aims of the proposed project are to: (1) analytically define the ways that DIF can manifest in mixture models; (2) develop a flexible test of DIF in mixture models; (3) use a computer simulation to test (a) the consequences of un-modeled DIF in mixture models and (b) the efficacy of the test developed in Aim 2; and (4) to use DIF modeling techniques in an empirical study of substance abuse in the transition to adulthood. Through the pursuit of these aims, this project will allow fo mixture models to better consider individual differences, thereby enhancing the generalizability of these results in behavioral substance abuse research."}
-{"text": "Despite advances in intraaortic balloon pumping and emergency myocardial revascularization, salvage of ischemic myocardium has been limited in man by the lack of effective collateral circulation into an area of acute infarction. We are currently carrying out experimental trials of perfusing ischemic myocardium by retrograde diastolic pulsation of oxygenated blood into the coronary veins draining an area of ischemia via a balloon tipped catheter that can be introduced transvenously. Results to date indicate correction of EKG changes, reversal of dyskinetic areas and improvement in myocardial performance in acutely ischemic myocardium following institution of retroperfusion."}
-{"text": "Characterization of Platelet Function in Patients with Bleeding Disorders: In FY2015 (the fourth year of this project) we have studied several patients with suspected or documented disorders of platelet function and have identified patients with deficiency of dense granules (storage pool disease), and characterized platelet function in people with congenital or acquired platelet dysfunction. The ability to make a reliable, detailed characterization of platelet function may help investigators who are studying genetics of inherited platelet disorders. One family with abnormal platelet function and other abnormalities has been described and submitted to the journal Blood for consideration to be published (York Platelet Syndrome, T. Markello et al, authors and collaborators). Another patient has been identified who has a novel platelet disorder that may be a variant of Glanzmann's thrombasthenia. He has been referred to the Undiagnosed Diseases Program (NIH/NHGRI) for further analysis and possible genome sequencing to identify the basis of this disorder. Characterization of D-Dimer and Related Coagulation Proteins in HIV-Infected Research Subjects Undergoing IL-2 Therapy: During the fifth year of this project, in support of intramural NIH research protocols, we have measured D-dimer levels and numerous coagulation parameters in research subjects who are infected with HIV or who are normal volunteers. We seek to correlate the D-dimer levels with these various parameters to gain insights into the mechanism for the observed elevations in D-dimer levels in HIV infected patients that correlate with increased morbidity and mortality. Effect of Factor VIII Haplotypes on Inhibitor Development. We have identified a set of 800 DNA samples from patients with severe hemophilia A in an NHLBI repository and have IRB approval to determine their factor VIII protein-coding polymorphisms to determine the effect of factor VIII haplotype on inhibitor development. We expect to complete the DNA sequence determinations in the coming fiscal year, and will see if the effect of protein coding polymorphisms on inhibitor development that was seen in a minority population (African-Americans) can be seen in the population as a whole. We have observed two cases of acquired factor VIII inhibitor in the setting of non-myeloablative stem cell transplantation for sickle cell disease in African-Americans, a report on one of which has now been published."}
-{"text": "Almost 200,000 new cases of epilepsy are diagnosed every year in the United States. Many of these are caused by an initial precipitating injury (IPI) (e.g status epilepticus, febrile seizures or traumatic brain injury). There is a need to develop interventions that could prevent the occurrence of epilepsy in these patients. The clinical challenge for testing and applying antiepileptogenic therapy is in identifying the subset of those who eventually became epileptic out of approximately 2 million individuals experiencing an IPI each year. The NINDS, in association with the AES, recently published a report identifying the most important research directions that should be undertaken to ultimately find cures for epilepsy. One of the 3 benchmarks considered as a top priority for the near future is the identification of biomarkers for epileptogenesis. At the present time, no biomarkers predictive of the likelihood of developing epilepsy after an IPI are available and this is an important reason why no clinical trials have identified an intervention during the latent period that clearly prevents the occurrence of epilepsy. The main goal of this proposal is to determine whether a new noninvasive electrographic putative biomarker of epileptogenesis in an animal model of chronic epilepsy can be consistently recorded during the latent period, and whether it can be used to reliably identify which animals later develop recurrent spontaneous seizures. The results of the proposed research could be used to facilitate assessment of antiepileptogenic interventions in patients. The putative biomarker we wish to study is an abnormality of the UP-DOWN State (UDS) EEG pattern. The features of the normal UDS pattern consist of an UP-phase and a Down-phase as a slow oscillation with a frequency of less than 1 Hz. The UP-phase is associated with prominent beta-gamma oscillations. The features of the pathological UDS pattern, which are seen only in epileptic animals include: 1- the occurrence of epileptiform events we have termed UPspikes during the UP-phase, and 2 - a prolongation of the UP-phase duration. We hypothesize that this pathological UDS pattern could be a valuable predictor of future seizure occurrence. We also propose to evaluate mechanisms of pathological change in the UDS pattern by analyzing the activity of principal cells and interneurons identified by juxtacellular labeling. We will focus our efforts on the analysis of the UDS electrographic pattern prior to pilocarpine induced status epilepticus and compare it to activity recorded during the latent period before spontaneous seizures occur. We anticipate that the identification of pathological features in the UDS EEG pattern will make them a valuable early diagnostic biomarker of epileptogenesis and predictor of later seizure occurrence. An understanding of the neuronal mechanisms underlying the pathological UDS EEG patterns will provide novel targets for future approaches to the treatment of epilepsy in its earlier stages, and help pave new ways to prevent epilepsy. PUBLIC HEALTH RELEVANCE: At the present time, no biomarkers predictive of the likelihood of developing epilepsy after a traumatic brain injury are available and this is an important reason why no clinical trials have identified an intervention during the latent period that clearly prevents the occurrence of epilepsy. Early diagnosis of progressive epileptogenesis with early intervention is important for more effective treatment options and disease management strategies. The goal of this proposal is to identify cellular mechanisms responsible for generation of UPspikes, which is a new biomarker of epileptogenesis."}
-{"text": "In this study we will take advantage of the ease of manipulation of yeast for the study of the regulation of mitochondrial biogenesis as influenced by the determinants of lipid synthesis, especially phospholipid synthesis. (A) The purification of CTP phosphatidic acid cytidyl transferase is to be pursued with the hope of preparing specific antibodies to this enzyme, thought to be located mainly in the inner mitochondrial membrane. Such antibodies are to be used to study the synthesis of this enzyme and its regulation. (B) Attempts are being made to isolate lipid mutants, including temperature sensitive mutants for the CTP phosphatidic acid cytidyl transferase to further elucidate the role of lipids in the control of mitochondrial biogenesis. (C) We will continue to probe the mechanism that determines the differential synthesis and assembly of cytochrome oxidase in unsaturated fatty acid auxotrophs of yeast grown in cis or trans fatty acids."}
-{"text": "This program tests molecular imaging variables, how they predict response to therapy, and how they change over the course of different treatments. We propose 5 interactive projects: 1. Proliferation studies to validate how well FLT compares to TdR in patients with a variety of tumors and explores ways to simplify the PET procedure and still distinguish flux from transport. Our hypothesis is that FLT with appropriate data analysis can be as accurate as TdR for reporting tumor proliferation. We will test the hypothesis that FLT PET plus MRI can distinguish between tumor progression and pseudoprogression in patients with GBM that finish initial therapy with external beam RT plus concurrent TMZ. 2. Tumors have variable levels of multi-drug resistance that reduces effectiveness of drug treatments. We will quantify transporter activity of P-glycoprotein as a suspected cause of treatment resistance and failure by analyzing [11C]-verapamil kinetics. We will test the hypothesis that pts who up-regulate their tumor Pgp activity after neoadjuvant chemotherapy will have shorter survival and time to progression. 3. Hypoxia is an important resistance factor that is imaged using [18F]-FMISO. In pts with brain tumors we will test the hypothesis that FMISO images following initial surgery define an appropriate target for focal boost RT that will improve time to tumor progression and survival. In pts with ER-neg metastatic breast cancer, we will compare the extent of hypoxia to response to systemic therapy and progression-free survival. Other experiments will compare FMISO and [64Cu]-ATSM and we will measure temporal changes using an FMISO test-retest protocol and BOLD-MRI to look for short term changes in regional oxygenation of tumors. 4. This project will test the use of 18F-FES PET to choose therapy in pts with a history of ER+ breast cancer who have failed prior regimens and are being considered for salvage endocrine therapy and use FES PET to measure regional ER as a pharmacodynamic response to endocrine therapy and FDG PET to localize active tumor and identify early effects of drug therapy, including HER2 and ER. The project will also image androgen receptor function in prostate cancer to identify heterogeneous AR expression. 5. Preclinical radiopharmaceutical development and research. New methods will be developed to image important aspects of the tumor phenotype. Some are new molecules;some are new applications for old molecules;all to develop promising imaging agents for characterizing the tumor phenotype. These include ligands for HER2 as a factor in treating breast cancer, new agents for imaging of androgen biology, a monoamine oxidase A ligand to select patients for a new clinical trial for prostate adenocarcinoma and studies comparing Cu-ATSM and FMISO for different mechanisms of uptake."}
-{"text": "The goal of the University of Pittsburgh Global Health Research and Research Training eCapacity Initiative is to enhance evidence-based, data-driven decision-making in India through innovations in information and communication technology using modeling and simulation to assess the health and economic impacts of alternative disease control strategies. To globalize the advantages of methods and curricula developed in the Public Health Dynamics Laboratory (PHDL) at the University of Pittsburgh, we believe it is essential to have local public health experts take the lead in developing, running and analyzing the models. Our objective is to enhance the capacity of researchers to use computational modeling, not only at our Fogarty training site, SHARE INIDA (D43 TW009078), but also at multiple Fogarty training sites in India. In the introductory Phase 1, a two-day symposium (35 participants), SHARE INDIA, Hyderabad, will introduce the concept of the integrated decision support pathway, and basic modeling software and tools. This symposium will showcase research that enables data-driven decision making using cutting-edge methods including mathematical, network, and agent-based modeling. Five Indian investigators plus one modeling support staff will receive in-depth, hands- on training in computational modeling during a week-long workshop at the University of Pittsburgh PHDL. This workshop on complex agent-based modeling will use our open-source modeling platform, FRED (Framework for Reconstructing Epidemic Dynamics). This experience will foster establishment of a new collaborative network for modeling studies among Indian researchers and with PHDL researchers. Two small modeling developmental grants will be awarded. Phase 2 will focus on sustainability. Two small research grants will help sustain newly developed, collaborative modeling projects. Two PHDL modelers will return to India to visit the recipients of the small grants and solidify collaboratios. Two week-long short courses on computational modeling in public health, incorporating educational materials and free software from the Pittsburgh Workshop, will be presented by Indian faculty at CR Rao Institute targeting 25 participants each. A second introductory symposium for 35 new participants, led by newly trained Indian modelers, will stimulate a new wave of interest in modeling. Our aims address an unmet need in Indiai.e. building capacity of researchers to use cutting-edge, yet appropriate technology to address policy-relevant questions. The education program will result in increased knowledge among Fogarty-affiliated researchers in India of the kinds of data needed to inform models and basic methods in modeling. Trainees who attend the workshops in Pittsburgh and sustain their use of modeling through small research grants are expected to continue to apply computational modeling methods to address Public Health questions of importance to India. These trainees are also expected to lead computational modeling collaborations within India and with PHDL faculty. In the future, the program is expected to result in funded research that aids data-driven decision-making in Public Health in India."}
-{"text": "The Prevention & Control Research Core has been developed with Prevention & Control and Clinical Researchers of the DRTC to provide a range of consultation and direct services to support behavioral, patient centered/clinical, educational, and prevention research. Seven core services are: (1) assistance in recruitment of clinical and non-patient samples and recruitment of research settings for community oriented research; (2) assistance regarding clinical aspects of diabetes and assessment of clinical status and diabetes risk; (3) assistance regarding epidemiological aspects of research and analyses and accessing administrative databases; (4) assistance in planning, developing and evaluating health education interventions, including addressing cultural and psychological factors, diet and nutrition; (5) measuring psychological factors and quality of life among those with diabetes; (6) data gathering, administration of population-based and clinicbased surveys, and data management; and (7) statistical analysis of data, including biostatistical consultation and analysis using geographic information systems. Because the core is newly developed following NIDDK's changed DRTC guidelines, plans are in place to monitor the use and utility of the varied core services, eliminate underutilized services, add promising services, and reallocate funds accordingly. Not including research support for projects not using the Prevention & Control Core, users include six investigators with independent NIDDK funding (Brownson, Klein, Lustman, Racette, Sinacore, White), five investigators with other NIH funding for diabetes-related research (Haire-Joshu, Holloszy, Mueller, Newcomer, Wilfley), three investigators with non-NIH, externally funded diabetes-related projects (Fisher, McGill, Polonsky), and five investigators with prior or current Pilot & Feasibility funding and/or diabetesrelated projects planned (Auslander, Hams, Hershey, Perkinson, Schootman)."}
-{"text": "When challenged with inhibitors of steroidogenesis, the cultured vascular smooth muscle cell showed depressed synthesis of cholesterol and of dolichyl(pyro)phosphate and an accompanying loss of cellular cholesterol. It was deduced that a regulatory site for the synthesis of both substances might occur at the HMG-CoA reductase. As a consequence of this inhibition cellular glycoprotein synthesis was diminished, and it was concluded that the availability of dolichyl(pyro)phosphates which mediate glycoprotein synthesis may also contribute to the regulation of their assembly. Cellular response to these changes may involve redistribution of subcellular membrane systems. This redistribution, perhaps necessary as a defense response, may present the cell with a new set of problems. Such redistribution will be assessed in a variety of conditions using subcellular fractionation schemes and marker enzymes assays. It is now clear that loss of cellular cholesterol may also contribute to defective glycoprotein assembly and membrane biogenesis. Such an effect points to the importance of a metabolically active cholesterol pool which the cell synthesizes to make certain that proper assembly is maintained. In view of this, contribution of exogenous cholesterol, supplied in a variety of forms, to such a pool will also be investigated."}
-{"text": "This research proposal constitutes an attempt to develop biological-biophysical data about specific patterns of ribonucleic acid biosynthesis and those enzyme systems involved in their processing, in particular the transfer RNA-methylases, as they relate to the leukemogenic process. It is intended that this information will afford an expanded level of knowledge concerning the biochemical responsiveness of leukemia cells found to be sensitive or resistant to various antineoplastic agents (e.g. vincristine, cystosine arabinoside, cytoxan, methotrexate) administered alone or in combinations. In this connection, information will also be sought as to how such agents can modulate the biosynthesis of particular species of RNA in leukemic cells. The basic techniquesto be employd entail the isolation and purification of mouse ascites tumor cells (e.g. L1210, P383, L5178Y) and certain resistant sublines as well as human peripheral blood leukocytes, cell-culturing where appropriate and variable-interval pulsing with radioactive precursors of ribonucleic acid (uridine, L-methionine). In vitro evaluation of tRNA-methylase activities in various leukemic cells will be conducted using either a homologous or heterologous assay system in the presence of unfractionated tRNA plus partially purified enzyme preparations. Special techniques to be used for characterizing individual RNA species and ribonucleic acid components (nucleoside level) include thermal-detergent extractions, sucrose-gradient centrifugation, polyacrylamide gel electrophoresis and computer analyses of \"RNA-fingerprint\" data."}
-{"text": "This proposal requests funding to assist in upgrading of laboratory animal facilities at The Rockefeller University, with the goal of providing research scientists with pathogen free rodent colonies. In a previously funded 1987 facilities improvement grant from the NIH we upgraded equipment, investigator safety, sanitation, surgery, water and environment components of The Rockefeller University Laboratory Animal Research Center and Field Research Center. These improvements have helped us remain a regional center for training and excellence in laboratory animal science. Matching funds are now being requested for a modification in our basic rodent husbandry care system which will enable our scientists to maintain and work with mice and rats free of common pathogens. We propose, with the assistance of this grant, to expand a limited 1200 sq ft of room with rodent microisolator housing by 2800 sq ft of additional pathogen free experimental animals. our existing limited pathogen free rodent housing was established by Howard Hughes Institute and NIH funding in addition to supplemental Rockefeller University funding. This proposal will enable us to maintain virtually all rodents which originate as pathogen free strains or stocks as pathogen free experimental animals, thus greatly enhancing the biomedical research of this institution. Approximately $45,000,000 in federal research funding is utilized at this university. Nearly two thirds of the laboratories conducting this research require laboratory animals for their studies and utilize the facilities of the Laboratory Animal Research Center."}
-{"text": "Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs, alone, or in combination with other factors, to introduce a point mutation into the alphaB-crystallin gene in one-celled mouse embryos. We found that co-injection of single stranded DNA (ssDNA) along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative non-homologous end joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been reported previously. For this initial study we have been using an extremely time consuming PCR-based restriction fragment length polymorphism (RFLP) assay to detect point mutations introduced into genomic DNA. To improve the detection method, we have developed transgenic mouse lines in which correction of a mutation in a fluorescent protein gives an instant read-out, and many embryos can be screened simultaneously. We engineered a cassette encoding a bicistronic mRNA (mutated red fluorescent protein DsRed / an IRES element / green fluorescent protein). All mouse embryos expressing the transgene should fluoresce green (endogenous control for transgene expression), but only embryos in which the point mutation in DsRed has been corrected should also fluoresce red. We have used several enhancer/promoter elements, which have been used successfully in preimplantation stage mouse embryos or mouse ES cells, to drive expression of the cassette. A successful reporter line would exhibit easily detectable levels of fluorescent protein expression at the earliest stages of embryo development. All promoter/fluorescent protein transgenes were constructed and tested for expression of EGFP and intact DsRed in both transient assays in mouse embryos and in F1 transgenic mouse embryos. Several promoters/enhancers were used in attempts attain early embryonic gene expression. A construct, whose expression is driven by the EF1alpha promoter, was able to generate green fluorescent protein in eight cell stage embryos (48 hr pf) and red fluorescent protein at 72 hr pf. A line of transgenic mice with the EF1alpha promoter / mutated red fluorescent protein / an IRES element / green fluorescent protein construct appears to be extremely promising at this time. Four transgenic mice lines with mutated DsRed protein were established. All four lines show expression of EGFP at 48 hr pf. Sequence analysis of a PCR-amplified region of the DsRed gene, from mouse genomic DNA, confirmed presence of the nonsense mutation in codon 15 of DsRed. RT-PCR analysis shows that three transgenic mouse lines have low copy number of the construct (2 copy) and one has a high copy number (26 copies of the construct). One line of mice (with 2 gene copies) is being bred for further experiments. In addition to our work with ssDNA as a targeting vector, we are also working, in single celled mouse embryos, on another type of gene targeting which we have named Forced Homologous Recombination (FHR). In this case, we use a significantly larger targeting vector, similar to those currently used in ES cell targeting procedures, with regions of homology to a selected gene or chromosomal locus at both 5'and 3'ends. Co-microinjection of the targeting construct with key factors involved in homologous recombination (for example Rad51/Rad54 proteins, which has been shown be able to form a filament-complex with nucleotides in vitro) may increase gene targeting efficiency. We are planning to use a nucleoprotein filament complex instead of the naked DNA vector to further increase alteration efficiency. The presence of long homology arms on both ends should increase specificity of gene modification and also dramatically decrease any random integration of the targeting vector, compared to our ssDNA strategies. Two genes were selected for in vivo testing of this FHR system. The first being the alphaB-crystallin gene. This gene had previously been knocked out, however another neighboring gene, Hspb2, was also knocked out in that mouse line. If successful, the FHR system will enable elimination of only one gene with precise accuracy. The second gene is caspase-6, a knock-out of which was also developed earlier, by eliminating the first exon. However our preliminary data suggests positive identification of caspase-6 by western blot in some tissues, which could possibly arise by alternative splicing or an alternative translation start site. We are working on a construct for FHR study that should eliminate the active center and dimerization domain from caspase-6 with precise accuracy. Generation of these two vectors is presently underway."}
-{"text": "Variation in bitter taste perception may play a key role in diet selection, which has an uncontestable effect on human health. Although bitter compounds are often associated with toxic substances a large number of nutritionally significant food sources contain bitter phytochemicals (e.g., broccoli [1], spinach [2]) many of which can have beneficial value [1, 3] Understanding how bitter taste is perceived and detected is of great importance to understanding diet selection [4] and increasing the acceptability of pediatric medicines [19]. Under normal feeding and drinking conditions taste compounds must inescapably mix with saliva before reaching their receptor targets but very little work has been done examining how salivary proteins modulate taste stimuli and alter bitter taste perception. Seventy percent of all salivary proteins make up a class of proteins referred to as proline-rich proteins (PRPs). PRP production is induced in rats by dietary exposure to tannic acid, a class of plant compounds that animals, including humans, regularly consume. It has been hypothesized that PRPs alter the acceptability of tannic acid by binding to tannins and thereby reduce the perceived intensity of the tannic acid solution [10, 11]. There is evidence to suggest that these proteins may have a similar relationship with other bitters. They are produced in close proximity to bitter taste receptors [13]. Genes for PRPs and bitter taste receptors are interspersed on the same chromosome [5] and lastly, gene linkage studies have implicated a role for PRPs bitter acceptance in humans and mice [15-18]. Specific Aim 1 will establish if long term exposure to bitter compounds can increase the expression of PRPs and establish whether oral exposure is necessary and sufficient to cause induction. Saliva will be analyzed for PRP content before and after exposure to diets adulterated with bitter compounds, including quinine and tannic acid. To isolate the site of exposure necessary for PRP induction rats will also be given tannic acid via either oral or gastric exposure. The ability or inability to induce PRP production is not sufficient to predict interactios between the proteins and the bitter tastants. Therefore, Specific Aim 2 will establish if PRP induction can alter the detection threshold for and superthreshold responsiveness to multiple bitter stimuli. Rats will be used to derive psychophysical detection curves of several bitter tastants in the presence and absence of PRPs. Likewise, responsiveness in the suprathreshold range will be assessed in the presence and absence of PRPs with a series of brief-access tests. Collectively, these experiments will test the hypothesis that PRPs alter taste sensitivity for bittr taste stimuli and ultimately blunt unconditioned taste avoidance responses to these compounds. Together these aims could inform methods for increasing the palatability of healthy phytochemicals and pediatric medicines."}
-{"text": "We propose to investigate the interface between EcoRI endonuclease and its cognate DNA recognition sequence by solution biochemical methods. The research is divided into two complementary efforts which will investigate the enzyme-DNA interface from both sides, i.e., we propose to: 1) Localize the points on the DNA which interact with the enzyme by determining the effects of base substitutions within and immediately surrounding the cannonical hexanucleotide, GAATTC. We will measure the effects of sequence changes on the enzyme kinetics, dissociation constants and the ability of the endonuclease to protect bases from Maxam-Gilbert reagents. 2) Localize points on the endonuclease which interact with the DNA by determining which amino acid residues can be: (A) Crosslinked to synthetic oligonucleotides containing the photo-activatable base analog 5-bromodeoxyuridine at unique points in the recognition sequence. (B) Protected by the DNA from reacting with reagents which modify exposed amino acid residues. We will also seek to determine whether or not the polypeptide chain is comprised of domains, one of which retains most of the recognition specificity. These experiments make extensive use of techniques developed in this laboratory for specifically binding DNA containing the recognition site to the enzyme under conditions which abolish catalysis. The ultimate goal of this research is to elucidate molecular mechanisms of sequence specific DNA-protein interactions."}
-{"text": "Hip fracture and resultant surgical repair entail a significant loss of muscle mass and function. Both injury and surgery, in and of themselves, initiate a cascade of events which lead to the loss of muscle mass. The general premise of this project is that this initial loss of muscle protein during surgery and the accompanying convalescence predispose a generally compromised patient to poor functional or morbidity outcomes. Further, we propose that muscle protein is lost due to a significant alteration in the muscle protein metabolism. The general goal of this project is to investigate interventions designed to ameliorate the negative muscle balance between protein synthesis and protein breakdown. We hypothesize the enhancement of muscle anabolism is required for these patients to recover muscle strength and function. We first propose to restore muscle protein synthesis throughout hospitalization with the administration of an essential am/no acid formula. We further propose that the amelioration of hypercortisolemia after surgery will result in a decreased muscle protein breakdown. The amelioration of hypercortisolemia after surgery will be accomplished with a common antifungal agent, ketoconazole, in order to investigate the resultant affects on muscle protein breakdown. We will measure lean body mass and muscle volume, as well as functional outcomes before surgery and discharge from acute hospitalization to determine the efficancy of these interventions. We will utilize stable isotope methodology to quantify muscle protein metabolism after acute hospitalization. We will also amplify these results by investigating cellular markers of protein breakdown to corroborate our metabolic endpoints. [unreadable] [unreadable] [unreadable]"}
-{"text": "This proposal seeks continued support for the development and continuation of an interdisciplinary Center for Research in Oral Biology. The central goal or purpose of the Center is to assist in the national effort to reduce the toll of oral disease and to promote the general level of oral health. The Center is achieving this goal by developing and supporting meritorious and relevant research projects, by creating an intellectual and physical environment conducive to optimal research productivity, by providing opportunities for significant interaction between investigators with diverse backgrounds, by producing both basic and applied knowledge related to the oral region, and by encouraging the incorporation of relevant research findings into dental education, dental practice, and the surrounding community. The types of projects supported have been the subject of intensive discussion within the Center and by the Scientific Review Committee, and within the committee appointed by the Vice President for Health Affairs to study the future pattern of the Center. The emphasis in the past has been on high quality basic research programs. This emphasis continues, but we are increasing our percent effort directed toward application of basic research findings to problems of patient diagnosis and care and to measures which will lead to improvement of the oral and dental status of the population."}
-{"text": "Receptors for transplantation antigens may be visualized directly on unsensitized cells with the use of an anti-idiotypic antisera. It should be possible to eliminate these cells specifically by treatment of the unsensitized population with sera directed at the specific binding site. Sera have been raised to cells cytotoxic for transplantation alloantigen in strain combinations selected so that only the variable portion of the antigen specific receptor could be recognized. Bona fide anti-idiotypic sera have not been raised. Refinements in the approach under way include 1) use of cytotoxic populations as immunogen with a demonstrably high proportion of receptor-bearing cells, 2) use of parent strains differing only by a point mutation to restrict the heterogeneity of the immune response of one against the other, and 3) use of continuously cultured cytotoxic cell lines to increase the homogeneity of the receptors used as immunogen."}
-{"text": "Multiple sclerosis (MS) is a chronic autoimmune disease characterized by the degradation of CNS myelin. The etiology of the disease is still unknown;both genetic and environmental factors have been implicated. Oligodendrocyte apoptosis may represent an initiating event in MS, with improper removal of apoptotic cell bodies and released myelin fueling the immune response. Low density lipoprotein receptor-related protein-1 (LRP-1) is a cellular receptor, which functions in endocytosis and cell signaling. We have demonstrated that LRP-1 is a key endocytic receptor for myelin in vitro. LRP-1 also inhibits the inflammatory response by controlling the NF?B pathway. We, therefore, hypothesize that LRP-1 could be directly involved in regulation of the development and/or progression of MS. We will test this hypothesis by a combination of in vivo and in vitro experiments. As complete LRP-1 deficiency is lethal, the role of LRP-1 in a mouse EAE model will be determined using mice with conditional LRP-1 deficiency in the myeloid lineage. Our preliminary results suggest that LRP-1 deficiency in this model leads to a significant increase in EAE symptoms. Effector functions of LRP-1- deficient macrophages will be characterized. Finally, we propose to identify LRP-1 ligands in myelin preparations, using a proteomics discovery approach. This research grant proposal should provide the basis for understanding the function of LRP-1 in MS and for potential development of novel therapeutics for MS. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) is a chronic disease characterized by the immune system attack on myelin sheath, which is necessary for proper brain function. This proposal is focused on a cell protein, LRP- 1, which we show is important in the clearance of degraded myelin, and controls of the immune response. We hypothesize that LRP-1 regulates the sequence of events leading to MS development. We aim to identify the mechanisms by which LRP-1 functions in MS, with potential of discovering ways leading towards development of novel treatments for MS patients."}
-{"text": "Inactivation of p53 is the most common genetic alteration detected in human cancers. Underscoring the importance of p53 in preventing tumorigenesis, mice in which p53 has been disrupted invariably develop lymphomas and other cancers. The overall goal of this proposal is to expand our understanding of the p53 tumor suppressor pathway, including upstream effectors and down-stream mediators, and the mechanisms by which it prevents pediatric cancers. Individuals diagnosed with Li-Fraumeni Syndrome (LFS) generally carry a germline p53 mutation and are remarkably predisposed to cancer at an early age. The mutations are highly penetrant and are associated with brain, breast, bone and adrenal cancers. We have recently identified a group of unrelated pediatric patients with adrenocortical carcinoma (ACC) in which 35 or 36 patients have a identical germline p53 mutation (R337H). Analysis of intragenic polymorphisms eliminate a founder effect. Tumors deleted the wild-type allele and express high levels of mutant p53 in the nucleus, indicating that p53 is functionally inactive in vivo. However, these patients are not from cancer-prone LFS families and over-expression of 337H in cell culture-based assays fail to detect a functional defect. Our hypothesis is that 337H is functionally inactive under physiologic conditions and that this mutation contributes to th4e development of ACC through a loss of p53-mediated cell cycle arrest and/or apoptotic activities. To test this hypothesis, we propose to address the following specific questions: 1) Does the 337 H mutation disrupt p53 biological activity under physiological conditions?; 2) Does the 337H mutant have altered biochemical and biophysical properties?; 3) Does the 337H mutation contribute to tumorigenesis? These studies should establish a new class of p53 mutation, a low penetrant allele, that would previously go undetected but nevertheless, significantly contributes to the development of human cancers."}
-{"text": "Schizophrenia is currently thought to be a complex genetic disorder, with altered neuronal development, perhaps very early in life, important in determining vulnerability. Neurogenesis and neurodevelopment occur in the adult olfactory epithelium, and probes of the olfactory system may provide insights into the neuro-biological basis of altered neurodevelopment in this disorder. Indeed, studies of patients with schizophrenia have demonstrated robust behavioral, structural, and functional impairments of the olfactory system. Our work, along with others', has indicated that olfactory brain regions are affected by both neurodegenerative and genetically-mediated neurodevelopmental processes. This project represents the only systematic effort to examine the underpinnings of these chemosensory impairments from a life-span perspective. To date, our efforts have established that: 1)pervasive and stable olfactory deficits exist across the lifespan in schizophrenia;2)no significant medication effects are seen;3)similar psychophysical, structural, and functional deficits exist in first-degree relatives of patients;and 4)underlying facial structures such as nasal and palate volumes are abnormal. Over early fetal life, cerebral morphogenesis proceeds in embryological intimacy with craniofacial morphogenesis. As such, quantitative analysis of craniofacial and cerebral dysmorphology along with a detailed psychophysical assessment of the olfactory system may provide important information concerning the developmental origins of schizophrenia. In this application, we propose an in-depth assessment of the structural and functional abnormalities of the olfactory system from an early neurodevelopmental perspective. We will study 40 schizophrenia patients, 40 otherwise healthy first-degree family members, 40 unrelated healthy controls, and 40 high-risk subjects who present with early symptoms of psychosis. New methods will be utilized to examine the neurodevelopmental contributions to chemosensory dysfunction in patients including nasal/palate volume, structural MRI, and quantitative examination of facial morphology. Predictive utility of these impairments will be probed by examining subjects at increased risk for the development of psychosis. Our working model is that olfactory impairment reflects developmentally disturbed processes of neurogenesis and synapse formation and that these measures may represent specific markers of embryological dysmorphogenesis underlying schizophrenia. Schizophrenia is currently thought to be a complex genetic disorder, with early developmental abnormalities in brain structure and function, being important in determining vulnerability to illness. This study will examine changes in smell abilities in schizophrenia along with detailed measurements of the nasal and oral cavities and mapping of facial and brain structure and topography. We believe that robust olfactory impairment seen in patients reflects a disturbance of an early developmental process and that the analysis of these functions and structures may provide important information concerning the developmental origins of schizophrenia."}
-{"text": "ABSTRACT Chronic neuroinflammation of the olfactory epithelium, as occurs in chronic rhinosinusitis (CRS), results in the loss of the sense of smell, due in part to cytokine-mediated neuronal death and inhibition of olfactory regeneration. Within the scope of the parent grant, the exploration of the effect of chronic neuroinflammation on the olfactory system extends to the axonal projections of the olfactory epithelium to the olfactory bulb (OB). In the brain, including the OB, neuro-immune interactions are increasingly believed to play an important role in the pathogenesis of Alzheimer's disease (AD) and its related dementias. Neuroinflammation is a prominent feature of AD that is highly correlated with disease escalation. The precise etiologic relationship between inflammation and AD histopathology is uncertain, but recent evidence increasingly suggests that systemic inflammatory disease is a risk factor and exacerbator of ADRD. The OB, which is an early site of AD histopathologic change, is connected and in close proximity to the olfactory epithelium. Directly exposed to the outside environment, the delicate olfactory neuroepithelium is subject to microbial exposures and local immune responses. In chronic rhinosinusitis, the olfactory epithelium becomes infiltrated with inflammatory cells producing a variety of pro-inflammatory mediators. A case-control study has linked dementia with a history of chronic rhinosinusitis, suggesting that longstanding nasal inflammation may propagate effects to the brain. The major goal of this project is to use a mouse genetic model of chronic olfactory epithelial inflammation to investigate whether neuroinflammation spreads to the OB over time, either driving deposition of amyloid plaques and neurofibrillary tangles, or exacerbating disease histopathology in mouse AD models. We further plan to investigate the role of the MAP kinase JNK in AD pathophysiology, which is an inflammatory signaling mediator that we have been studying as a therapeutic target for olfactory neuroprotection in CRS. Using genetically-modified mouse strains and pharmacologic agents, we will explore the roles of specific cytokines implicated in ADRD in mediating olfactory inflammation-induced AD-like changes in the OB, and whether these can be modulated by inhibiting JNK activity. The experiments described in this proposal will afford new insights into neuroinflammatory mechanisms that may trigger early stages of ADRD."}
-{"text": "Pediatric cardiac arrest is a common and devastating condition which remains poorly understood. Mortality rates are extremely high and brain injury is the most common cause of death. The majority of research regarding cardiac arrest over the past 50 years has focused on improving rates of return of spontaneous circulation (ROSC), with significant progress leading to increased survival rates. However, without interventions to minimize organ injury, there is an increase in long-term health issues associated with our improved resuscitation practices. This has been termed the post-cardiac arrest syndrome, consisting predominantly of long-term neurological deficits. Indeed, several interventions that have been useful in improving ROSC, have not shown benefit in improving long term outcome. There have been numerous pre- clinical translational studies of cardiac arrest in adult animals demonstrating various pharmacological interventions to improve neuronal survival following global cerebral ischemia. However, to date, there are very few studies in pediatric cardiac arrest, as models are scarce. In the current proposal, we describe the first pediatric cardiac arrest model utilizing mice to study the effects of cardiac arrest on neuronal survival and test new therapies. Epidemiologic studies in adults have suggested that females have better outcomes after CA when compared to males. Numerous experimental studies in adult animal models have recapitulated this clinical data, showing that female animals exhibit significantly less brain injury following cerebral ischemia than males. The sex difference observed in experimental adult animals can be nearly completely explained by the high levels of estrogen in female animals, as removal of endogenous sex steroids (ovariectomy) increases female brain injury to male levels and estrogen replacement returns female injury to intact animal levels. Not surprisingly, we did not observe a gender difference in ischemic injury in pediatric mice, consistent with pre- pubertal state of low estrogen in both male and female animals. Interestingly, when estrogen is exogenously administered we observe a remarkable sex-difference in response to estrogen neuroprotection. We observed that a single intravenous estrogen dose administered at a clinically relevant time point following CA/CPR (30 min) provides protection to the female brain, while having no effect in the male brain. Therefore, the aims of the current proposal are designed to further characterize our novel pediatric cardiac arrest model and begin to elucidate the molecular mechanisms of sexually dimorphic estrogen neuroprotection observed at this developmental stage. Therefore, we will 1) establish the role of estrogen in determining neuronal injury following pediatric cardiac arrest and 2) determine the relative contribution of estrogen receptors alpha and beta (ER? and ER?) in estrogen neuroprotection. 3) Finally, determine the molecular mechanism of estrogen neuroprotection in pediatric cardiac arrest."}