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This application is to request three years of funding for the annual Winter Conference on Brain Research (WCBR). The 2019 meeting will take place Snowmass Village and Conference Center, Snowmass, CO between Tuesday January 29-Saturday February 2. This conference is an ideal forum for exploring new developments in the field of substance use disorders (SUDs). WCBR has a unique format with 90 sessions, including 88 2-hr panels, 2 Pioneer sessions, and 2 short courses in 2018 and 499 scientist attendees. There are daily poster sessions (Sun-Tue) including a special judged poster session that includes the top-ranking poster submissions predominantly presented by young investigators on Fri evening. In 2015 Career Development Workshops for graduate students, postdoctoral fellows, and junior faculty were initiated and maintain high attendance annually. Extended periods of time are set aside for breakout groups, networking and scientific discussions. WCBR is an excellent opportunity for junior investigators and trainees to interact with established investigators in a relaxed atmosphere. The evaluations of recent conferences highlight the strong impact this meeting typically has on emerging as well as established scientists. As the travel funds allocated for emerging scientists have not kept pace with the rising costs of attending small, specialized meetings, we are requesting funding from other sources. We are hoping that the results of our requests enable us to support a keynote speaker as well as a large number of junior investigators to participate in the meeting. The conference includes one keynote speaker and one scientist who holds a Brain Talk Town Meeting open to the entire community. For the 2019 WCBR meeting, Fred (Rusty) Gage Ph.D. Interim President of the Salk Institute and Adler Professor in the Laboratory of Genetics and member of the National Academy of Sciences will speak on Stem Cells and Neuropsychiatric Disease. There is an annual School Outreach Program organized in collaboration with local middle and high school teachers in which up to 10 WCBR scientists visit community schools. WCBR?s program is developed annually based on abstract submissions in August each year. At WCBR 2018, twenty-two panels, one Keynote, and one Pioneer session were specifically on abused drugs and another 19 panels and one short course were on reward-related topics. Thus, a large proportion of the conference is highly relevant to understanding the neurobiological bases of drug addiction as well as neurological and neuropsychiatric disorders consistent with NIDA?s mission. WCBR always provides a unique opportunity to discuss recent advances in the understanding of the pathophysiology and treatment of those conditions. The fact that a large proportion of the participants are junior investigators and a significant number are graduate students and postdocs ensure that this conference has an impact on how future generations of neuroscientists view these issues.
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{
"pile_set_name": "NIH ExPorter"
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ABSTRACT Support is requested for a Keystone Symposia meeting entitled RNA-Based Approaches in Cardiovascular Disease, organized by Drs. Thomas Thum and Roger J. Hajjar. The meeting will be held in Keystone, Colorado from March 26-30, 2017. Cardiac diseases are the leading cause of death worldwide. RNA-based mechanisms and therapeutic approaches are emerging fields in cardiovascular science. This meeting will present and discuss latest developments using both coding RNA and noncoding RNA- (such as microRNAs, long noncoding RNAs and circular RNAs) based approaches to better understand and develop new therapeutic strategies for cardiac diseases. Attendees will benefit from workshops, specific sessions and industrial perspectives covering everything from basic science to clinical translation employing RNA therapeutics in cardiovascular medicine. Opportunities for interdisciplinary interactions will be significantly enhanced by the concurrent meeting on Molecular Mechanisms of Heart Development, which will share a keynote address and two plenary sessions with this meeting. Relevance to NHLBI: Cardiovascular disease (CVD) is a major cause of morbidity and mortality in the United States. Among the NHLBI?s strategic mission is the goal to advance our knowledge of CVD while identifying new potential targets. Novel treatments are being formulated based on a better understanding of the signaling pathways involved in the pathogenesis of cardiovascular diseases. Targeting genes to the heart through microRNA and RNA based deliveries have the potential to alter our approach to patients with cardiovascular diseases. This meeting will bring together scientists from a wide array of avenues. Through this combination of investigators with multidisciplinary backgrounds, diverse scientific perspectives will be brought into focus on cardiovascular diseases.
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{
"pile_set_name": "NIH ExPorter"
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Evidence suggests that for the most part gp4l neutralizing epitopes are cryptic, and this is most likely related to the functional role of the transmembrane protein (TM) in mediating virus entry. It has been shown that gpl2O-CD4 binding causes the HIV envelope to undergo a series of conformational changes, which may be driven by the formation of a series of structural intermediates within gp41, leading ultimately to virus entry. Previous work in the applicant's laboratory has identified two regions within the ectodomain of gp4l which play critical roles in this process and recently a crystal structure has been reported for the structure formed by the specific interaction of these two domains. This work supports the theory that it is the transitory nature of the entry event and the structures associated with it rather than the absence of appropriate structural determinants which accounts for the seeming lack of neutralizing epitopes within gp41. The applicants propose that these structural components, which form and function only during virus entry, and remain unexposed or are not present in the "native" fusion-inactive envelope complex, constitute a novel set of neutralizing epitopes within gp4l. The objectives of this research are the induction and characterization a humoral immune response targeting these "entry relevant" gp4l structures. It is hypothesized that immunization with constructs mimicking those highly conserved, gp41 structures involved in virus entry will elicit the production of broadly neutralizing antibodies targeting these structures.
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{
"pile_set_name": "NIH ExPorter"
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DESCRIPTION: Females produce speech with a higher fundamental frequency (f0, voice pitch) as compared to male-produced speech. This higher f0 results in relative under sampling of the spectral envelope which should, in turn lead to lower intelligibility. However, excerpts from natural speech produced by females is as or more intelligible than male speech. This application proposes a series of complementary speech perception and production experiments designed to uncover gender-specific acoustic attributes that lead to this enhanced intelligibility. In particular, females have been characterized as speaking slower, with a more "breathy" voice, and with greater pitch excursions or "swoopiness." While these descriptions have in the past been considered derogatory, they may, in fact, be signs of adaptive production by females to compensate for the negative effects of a high voice pitch. In order to test this possibility, a large database of speech samples from oral reading will be analyzed for gender differences in breathiness, dynamic f0 range and speaking rate. A large number of acoustic measures theoretically related to breathy phonation will be computed. Using Principal Components Analysis. A composite acoustic measure of breathiness will be developed. Breathiness will then be analyzed by gender and by vowel. These measures can serve as much-needed gender-specific normative data on production. These data may be important for more sensitive diagnoses of vocal pathology, for synthesis of realistic-sounding female speech, and for the creation of gender-specific templates for automatic speech recognition. In addition, any gender differences uncovered by these analyses will form the basis of perception experiments designed to test their consequences on intelligibility. It is predicted that females produce high tense vowels as more breathy than similar lax vowels and that this pattern increases the size of the resultant vowel space. To test these predictions, synthesized vowels will be created to mimic various acoustic aspects of male- and female-produced speech. Listeners will attempt to identify these stimuli when presented in babble noise. If the female pattern of breathiness is adaptive, then the resulting vowels should be easier to identify than vowels that do not vary in breathiness. Similar predictions are made for "swoopiness." If the perception tasks reveal that gender-specific acoustics can enhance intelligibility, then these data may be important for development of signal processing techniques for enhancing communication. This series of perception-production studies will be interpreted within a framework that proposes that speakers faced with individual intelligibility challenges vary their speech production in ways that enhance the auditory cues to phonetic identification, thereby aiding the listener.
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{
"pile_set_name": "NIH ExPorter"
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The overall objectives of our current MBRS program are to: (a) provide research experiences for undergraduate and M.S. level graduate students; (b) provide a vehicle for research for faculty at our institution; (c) provide an institutional model for research; (d) increase the pool of minority students who will enter terminal degree programs with emphasis in biomedical science; (e) provide more diverse dimensions to the research that is being conducted at the university; and (f) expand the scope of biomedical research at the university. These projects contained in this proposal will increase the research opportunities available to both faculty and students and they will enhance the overall research programs in the departments of animal science, biology, chemistry and psychology. The objectives of the subprojects contained in this proposal are (1) to determine the mechanism of apoptosis and glucocorticoid resistance in human leukemic cell lines; (2) to study Protein V as a bacterial Fc receptor; (3) to determine the neuroendocrine and cardiovascular effects of prepubertal and prenatal chronic stress; (4) to synthesize and study potential antimelanotic amino acids; (5) to investigate the inhibition of phospholipase A/2 by lipocortin; (6) to synthesize and determine the antitumor activity of binuclear gold(I) complexes, and (7) to study the behavioral, cardiovascular and neuroendocrine changes in hypertensive vs. hyperactive rats. Students involved in this program will assure a supply of highly trained and motivated candidates for graduate and professional schools.
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{
"pile_set_name": "NIH ExPorter"
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Ions are essential components of biomolecular systems. One third of all proteins contain metal ions as integral parts for structural or functiona purposes. Binding of negatively charged phosphate groups is ubiquitously involved in regulation, signal transduction and various other processes. Whereas is a plethora of experimental structural and thermodynamic information about protein-ion systems, our understanding of the mechanism for specific recognition and protein/ion selectivity remains elusive. Computational and theoretical studies of protein-ion systems using classical models are very challenging due to the lack of accurate and yet computationally tractable classical models for simulating ions in proteins. We propose to systematically investigate the binding of divalent metal ions and phosphate containing ligands to proteins using quantum mechanical calculations and classical molecular dynamics simulations. We will rigorously examine the different types of physical forces in protein-ion interactions using quantum mechanical energy decomposition, and develop a new classical model to accurately describe the physical interactions between ions and protein/water environment. With this new classical model, we will obtain quantitative understanding of the thermodynamic driving forces underlying the specificity and selectivity from molecular dynamics simulations. Given the fundamental importance of protein-ion binding, this research will have a broad impact on advancing our scientific knowledge about ions in biomolecular structure and functions. This research will also lead to computational methods and public software tools that will enable accurate prediction of protein-ion binding and ultimately design of new molecules and proteins targeting specific ions.
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{
"pile_set_name": "NIH ExPorter"
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Muscle and nerve cell functions are largely due to the function of transmembrane ion channels. Mutations in ion channel genes can lead to uncoordinated movement, or to the loss or attenuation of particular behaviors. The role of a particular potassium channel and its regulators in coordinating muscle contraction and in executing a set of ritualized male mating behaviors of the worm, Caenorhabditis elegans, will be studied. In C. elegans, a set of four genes, including sup-9 and unc-93, is known to encode a potassium channel and its putative regulators. Gain-of-function mutations in either of these genes lead to paralysis; loss-of-function alleles cause the animal no harm. Gain-of-function mutations in one gene can be suppressed by loss-of-function alleles in another of these four genes, presumably by eliminating channel function. We have isolated a set of unique sup-9 alleles that suppress the paralysis conferred by a gain-of-function allele of unc-93, but that leave male worms incapable of mating. The SUP-9 channel is found in the membrane of all muscle cell types but also in a small subset of neurons. We intend to fully characterize the mating defect conferred by these alleles and to determine whether the defect is due to aberrant channel activity in muscle cells or in neurons. We also will determine how the role of the SUP-9 channel compares with that of other K+ channels. We recently isolated a worm strain that appears to contain a spontaneous mutation that suppresses only one male-specific aspect of the unc-93 gain-of-function phenotype, indicating either that the SUP-9 channel is regulated differently in different tissues or that unc-93 is involved in more than one signaling pathway. We propose to map the location of this suppressor and undertake its molecular characterization. This research will be an important contribution to our understanding of the role of potassium channel function and regulation and the role of K+ channels in coordinating animal behavior. Lastly, this research will help delineate the connections between motility, functions of particular muscle cells, and organismal behavior.
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{
"pile_set_name": "NIH ExPorter"
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PROJECT SUMMARY/ABSTRACT: Immunotherapy with monoclonal antibodies (mAbs) has been successful in settings such as autoimmunity and cancers, and therefore, treatment options with antibodies especially re-designed molecules based on broadly neutralizing antibodies (bNAbs) should be explored in the context of HIV-1. A caveat is that HIV-1 has the ability to rapidly escape from antibodies by generating mutations in its variable env gene. Therefore, there is an urgent need to gain insight into HIV-1 escape from bNAbs to aid in more effective combination antibody strategies to be used towards HIV-1 therapy, cure and prevention. As bNAbs are already being tested in clinical trials, it is imperative that optimal antibody combinations are evaluated not only for their neutralization capability but also their ?ease of escape? by diverse viruses. The overall objective of this proposal is to measure the ability of genetically diverse strains of HIV to escape from broadly neutralizing antibodies and design an antibody cocktail capable of restricting escape. To accomplish this goal, Dr. Lynch, who is an expert in HIV-1 escape from bNAbs has put together a collaborative team for an interdisciplinary approach using molecular virology (Dr. Lynch), computational methods (Drs. Barton and Fischer) and a humanized-mouse model (Dr. Klein) to study HIV-1 escape. Our central hypothesis is that HIV-1 escape from combination bNAbs will be limited when the mutations required to escape all bNAbs exert the maximum replicative fitness cost across diverse viruses and, therefore, replication cannot easily be restored through compensatory mutations. We will test this hypothesis by (i) defining a library of viable escape pathways for single bNAbs in diverse viruses with in vitro and in vivo approaches, (ii) determining the fitness cost or ?ease? of escape for virus-bNAb pairings bioinformatically, and (iii) identifying the optimal combination of antibodies that maximize fitness costs of resistance in diverse HIV-1 subtypes and testing this combination in vitro and in vivo. Our outcome will be the identification of optimal combination antibody cocktails to limit the ability of diverse HIV-1 viruses to escape from antibody pressure. These findings will inform all clinical trials using bNAbs or bNAb-based molecules, and ultimately, these studies will define a rational pipeline to characterize antibody escape pathways in the future.
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{
"pile_set_name": "NIH ExPorter"
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The goal of this research program is to construct a model of human red/green color-vision defects that is based upon the molecular biology of the color-vision genes. In the April 11, 1986 edition of Science, Nathans and colleagues (including one of the authors of this proposal (Piantanida)), reported (1) the nucleotide sequences of the three genes that encode the human cone photopigments. (2) the existence of multiple genotypes among color-normal males, and (3) the occurrence of hybrid genes (and often one or both normal color- vision genes) in persons with red/green color-vision defects (Nathans et al, 1986 a,b). The proposed research project would continue that investigation of the molecular genetics of red/green color-vision defects, concentrating initially on sequencing the hybrid genes, and eventually producing the first comprehensive DNA-based model of normal and defective human color vision. The hybrid genes, which appear to have arisen by unequal intragenic recombination of homologous regions of the red-and green-cone genes, were identified in the studies cited above by polymorphism of their restriction fragment lengths. However, that method does not provide sufficient resolution to determine the exact point of recombination. Because the hybrid genes appear to be the key to understanding red/green color-vision defects, the proposed research will concentrate on specifying their nucleotide sequences. DNA samples from color-defective males will be digested with restriction enzymes and the restriction fragments analyzed by Southern blotting. A determination of genotype will be made by stoichiometric analysis of the Southern blots, and restriction fragments of interest will be cloned into lambda vectors, identified by plaque hybridization, subcloned into an M13 vector, and sequenced by the dideoxy method. In addition by hybrid genes, the genotypes of some persons with red/green color-vision defects include a variable number of ostensibly normal color-vision genes. More generally, the results of Nathans et al. (1986a) show that color-vision genotypes are not related to the six traditionally-defined defective red/green color- vision phenotypes in a simple manner. The proposed research is aimed at clarifying the relationship between color-vision genotypes and phenotypes. Specifically, it will (1) explore further the number and types of genes found in color-normal and color- defective persons in an attempt to develop a complete genotype catalog; (2) expand the red/green color-vision phenotype catalog based on existing psychophysical data so that subjects can be sorted into more precise phenotypes by pseudoisochromatic plate tests, small- and large-field anomaloscopy, neutral point determination, and other psychophysical tests; and (3) most important, develop a model of defective human red/green color vision based upon a comparison of genotypic and phenotypic features, which will have important implications for models of normal color vision.
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{
"pile_set_name": "NIH ExPorter"
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We propose a demonstration and dissemination project that extends a cardiovascular disease risk-reduction program shown to be effective among patients with Diabetes Mellitus (DM) in an integrated HMO population, to a vulnerable outpatient population receiving DM care in 11 Federally Qualified Health Center (FQHC) clinics in the Portland, OR metropolitan area. Adults with DM and other risk factors who take aspirin, statins, and ACE Inhibitors can reduce their risk of cardiovascular events by as much as 40%; the proposed study will adapt intervention strategies used by Kaiser Permanente to increase the percentage of DM patients on these medications in the study clinics. Intervention strategies derive from the Chronic Care Model and include primary-care-team-based care, active case management, clinical prompts built into the electronic medical record (EMR) common to the community clinics, linked pharmacy datasets with the EMR to monitor patient adherence, and a panel reporting tool that prioritizes clinical follow-up to those most at risk. We will determine the effectiveness of the dissemination of the intervention, and its impact on patient adherence, by measuring changes in DM population prescription rates for the target medications and changes in prescription refill rates, using a pre- post comparison within clinics and a staggered, randomized implementation across clinics. We will also conduct a process evaluation to identify the factors important to dissemination and implementation success. Our overarching goals are to identify and resolve issues in disseminating a successful program from a large, well-organized health system into community clinics, so that the program may be implemented more widely in other community health centers and practice settings with modest EMR infrastructure resources. The proposed study will pave the way for future research on disseminating evidence-based care using safety net clinics' EMR. PUBLIC HEALTH RELEVANCE: Adults with diabetes who take aspirin, statins, and ACE Inhibitors can dramatically reduce their risk of cardiovascular events. Therefore, five years ago Kaiser Permanente (KP) launched an initiative to put as many of its members with diabetes as possible on this drug regimen, using the electronic medical record to identify patients not taking the medications, and to remind clinicians to start these patients on the medications; in addition, primary care case management teams contacted eligible patients. We propose to implement a similar program in eleven of Oregon's community health centers, to assess whether KP's program can be successfully disseminated in those settings, and whether it can impact the percentage of eligible patients who are prescribed and refill the target medications.
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{
"pile_set_name": "NIH ExPorter"
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The importance of hydrophobicity in protein folding is universally recognized. A particularly exciting phenomenon is the sudden large scale de-wetting transition that occurs as two large hydrophobic solutes are brought together. But are biological systems like proteins prone to such drying transitions? We have recently demonstrated that the wild-type Melittin tetramer indeed exhibits a fast de-wetting transition, and that spontaneous drying is sensitive mutation of certain hydrophobic residues. A similar study of the BphC enzyme revealed that the collapse is not induced by a drying transition. In this proposal, we aim to (a) investigate sensitivity to the topology of the hydrophobic groups; (b) identify key sequences associated with drying; (c) design bioinformatics tools to identify protein candidates for drying transitions; (d) investigate whether wild-type proteins are optimized for de-wetting; and (e) design algorithms for speeding up protein folding by reducing the attractive forces between protein and water. Hydrophobic interactions are also expected to be closely tied to enzymatic modulation, which is regulated by the interaction of a small molecule (ligand) and a protein. Initial results indicate that concave binding pockets lead to increased hydrophobicity and, thus, solvated protein-ligand complexes are very sensitive to surface topology. We are currently undertaking a detailed study using all atom molecular dynamics to verify these initial results, which suggest adding terms for hydrophobic enclosure to implicit solvent models. Another major impediment to rational drug design is the lack of realistic force fields. In a detailed QM/MM study, we have recently demonstrated the significance of induced atomic charges when a peptide undergoes conformational changes or moves into different environments. Correcting for this effect was shown to significantly improve the predicted binding affinities of ligands to proteins. We propose to develop a second generation polarizable force field that incorporates induced charges in addition to induced dipoles. This project will integrate two of our existing force fields, which account for fluctuating charges and fluctuating dipoles separately. Perhaps most significantly, it appears that the critical bottleneck in high-resolution protein prediction is the lack of adequate conformational sampling. It is a high priority of this proposal to develop improved sampling methods for biological systems such as proteins in aqueous solution. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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Work on the obtainment, examination, review, and interpretation of data relating to cancer mortality that have been produced historically in successive studies from the General Registrar Office of England and Wales. This shall include analysis of relation of mortality from cancer to occupation and social class, analysis of trends over time, as well as comparison with relevant material from other countries. This analysis shall be performed in collaboration with the Project Officer.
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{
"pile_set_name": "NIH ExPorter"
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Deliberation entails the serial examination and evaluation of outcomes. While there are detailed, mechanistic, and quantitative theories of non-deliberative decision-making, such theories of deliberative decision-making are still lacking. This is due to a lack of key experimental knowledge of the mechanisms of deliberation. Deliberative decision-making entails the sequential consideration of possibilities, requiring three steps in a repeated cycle: (1) prediction of the consequences of one's actions, (2) evaluation of those predicted consequences, and (3) selection of the best action. An important difficulty that has limited our ability to study deliberative decision-making is that the process of deliberation is covert, that is, the transient information being considered is not reflected in immediate behavior. However, new mathematical techniques now allow decoding of represented variables from neural ensembles at very fast timescales, enabling the observation of those transient, covert processes. The goal of this proposal is to track the covert prediction of reward outcomes as alternatives are evaluated. We have preliminary data that structures known to be involved in motivation and evaluation (ventral striatum, orbitofrontal cortex) show a transient activation of reward-related activity at certain deliberative decision-points. Combining newly available multi-structure recording techniques, newly developed tasks, newly improved neural ensemble analysis techniques, and computational modeling, we will examine the relationship between the representations of future possibilities in hippocampus and the covert reappearance of reward-related information in structures known to be involved in motivation and reward and decision-making.
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{
"pile_set_name": "NIH ExPorter"
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It has been estimated that more than one third of patients with neurological disorders with problems affecting the neuromuscular system. Striking progress has been made in understanding many of these diseases, and in the effective application of the new knowledge to treatment. This progress has been made possible by the intimate interaction of basic and clinical scientific approaches. At the same time, the analysis of human neuromuscular diseases has contributed to the understanding of the basic biology of nerves, muscles, and nerve-muscle interactions. The goal of this proposal is to foster the training of both clinical and basic scientists in the areas of neuromuscular biology and disease, so as to encourage even more intense interactions between these approaches. The faculty, laboratories and programs involved are well-established, highly interactive, and productive. The areas of research focus on the neuromuscular system, but entail a wide range of approaches and methodologies. Representative disease-oriented areas include: amyotrophic lateral sclerosis; spinal muscular atrophy; peripheral neuropathies; myasthenia gravis; muscular dystrophies; and autoimmune myopathies. A wide variety of research approaches are used, and basic methods include those of molecular biology, immunology, morphology, pharmacology, cell culture, and electrophysiology. Clinical material and clinical trials serve as lynch pins of the program. The trainees will be both clinicians and basic scientists with doctoral degrees, (many MD/PhDs) whose goals are to perform significant research in the fields of neuromuscular diseases and biology; Support for four postdoctoral fellows is requested is requested. We will stress the recruitment of minority applicants. The training program involves intensive laboratory research, and postdoctoral fellows are encourage to audit relevant courses and to participate in the well-organized programs on seminars and conferences. Special emphasis is placed on the integration of disease-oriented and basic science research approaches. The facilities available are those of well-equipped ongoing productive laboratories, in the rich environment of the John Hopkins Medical Institutions.
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{
"pile_set_name": "NIH ExPorter"
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In recent years, pain research has made revolutionary advances at levels ranging from genes to behavior. However, one basic level of organization that has proved particularly resistant to analysis is the intrinsic circuitry underlying pain transmission and modulation within the first central relay area, the spinal dorsal horn. There is much evidence that changes in intrinsic dorsal horn interneurons contribute to plasticity underlying inflammatory and neuropathic pain, but further understanding has been limited by a dearth of methods for studying this intrinsic circuitry. In recent years a novel technique was developed that has made it possible for the first time to dissect the organization of the synaptic connectivity of closely spaced neural elements. With this technique, laser scanning photostimulation (LSPS), synaptic responses are recorded in a single neuron while focal stimulation produced by laser-induced glutamate uncaging is delivered to multiple sites in the tissue surrounding the recorded neuron. The resulting map reveals the location of local excitatory and inhibitory pre-synaptic neurons that give monosynaptic input to the recorded neuron. LSPS has transformed our knowledge of circuitry in a many areas of the central nervous system, but has not yet been used in the pain system. We now propose to use this technique to study the organization of pain (nociceptive) circuitry in the dorsal horn. The proposed experiments will map the locations of dorsal horn neurons that give rise to local excitatory and inhibitory synaptic input to lamina I projection neurons and lamina II interneurons (islet, central, vertical, and radial cells). This approach is based on the hypothesis that the pattern of intrinsic connectivity in the dorsal horn is spatially organized, and that this spatial organization is a fundamental determinant of dorsal horn function. We further hypothesize that this spatial organization differs for excitatory and inhibitory inputs, and is cell-type specific. The application of this technique to the dorsal horn will advance our understanding of the new concept of a modular organization that has been proposed from recent studies of the dorsal horn, and open up an entire field of investigation into the organization and plasticity of dorsal horn circuitry.
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{
"pile_set_name": "NIH ExPorter"
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We have been investigating a number of biophysical processes associated with nerve excitation and their relationship to the MR signal. Uri Nevo, a former STBB post-doctoral fellow, and now an Assistant Professor at Tel Aviv University, successfully constructed and tested an experimental system in our lab to interrogate organotypic cultured brain slices using diffusion MRI. This work showed promising results relating changes in the measured apparent diffusion coefficient (ADC) map to changes in environmental conditions to which cultured tissues were subjected. One hypothesis that emerged from these studies is that active processes occurring at many different length scales (cell streaming, water flow across membranes, etc.) are responsible for some signal loss in the diffusion weighted MRI signal. This insight prompted the development of a theory to explain how microscopic fluid flows affect the measured diffusion weighted MRI signal and the ADC measured in tissues (i.e., pseudo-diffusion) and an experimental model system, the Rheo-NMR, in which well-characterized flow fields can be produced, which create known amounts of pseudo-diffusion. The importance of these combined studies is that if such microscopic motions, like streaming, water flow across membranes, etc., manifest themselves as an additional signal loss in diffusion weighted MRI experiments, then we can use this information to infer different aspects of cell function and vitality, including excitability. We are now continuing these studies with a recently hired graduate student from the University of Maryland, Ruiliang Bai. We have also been collaborating with Bradley Roth to try to examine different physical mechanisms that could be exploited or used to detect neural currents directly using MRI. One approach we examined previously was whether small displacements caused by Lorentz forces produced in strong magnetic fields (like those within a large clinical MRI scanner) could be employed to measure neural currents in vivo using MRI. Our calculations showed that the induced displacements of nerves caused by Lorentz forces in tissues would be too small to be detectable by MRI using existing technology. In the area of Transcranial Magnetic Stimulation (TMS), Pedro Miranda and his group in Lisbon, in association with STBB, has performed detailed calculations using the finite element method (FEM), to predict the electric field and current density distributions induced in the brain during TMS. Previously, we found that both tissue heterogeneity and anisotropy of the electrical conductivity (i.e., the conductivity tensor field) contribute significantly to distort these induced fields, and even to create excitatory or inhibitory "hot spots" in some regions that were previously not predicted. More recently, we have been developing more realistic FEM models of cortical folds, containing gyri and sulci. We showed that this more complicated cortical anatomy also significantly affects the distribution of induced electric fields within the tissue, and the location and types of nerve cells that could be excited or depressed by such stimuli. These phenomena could have significant clinical consequences both in interpreting or inferring the region or locus of excitation and in determining the source of nerve excitation. We are beginning to marry our macroscopic models of TMS with microscopic models of nerve excitability in the CNS in order to predict the locus of excitation in TMS and even the populations of neurons that are excited or depressed. Recently, we have also been applying these advance FEM models to explain the physical basis for Direct Current Excitation (DCE).
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{
"pile_set_name": "NIH ExPorter"
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The overall long term objectives of the studies proposed in this application are to identify and to understand the ionic mechanisms that underlie the abnormalities in the transmember potentials of the subepicardial ventricular cells that survive in the epicardial border zone of the infarcted heart. It has been suggested that one or more of these electrophysiologic abnormalities may lead to the serious ventricular arrhythmias known to occur after infarction. Therefore, by more clearly defining and understanding the mechanisms for these electrophysiologic changes, we will provide information that may lead to the development of effective therapeutic interventions needed in this clinical setting. We will use the technique of disaggregated single cells to separate these cells from the epicardial border zone of the infarcted myocardium. Then, by using a variety of electrophysiologic techniques we can determine the underlying basis for the electrical abnormalities. For this proposal, we have focused on clarifying the abnormalities of some of the ionic currents known to be integral to normal cell electrophysiology (iNa, iCaL, iCl(Ca), iK). In addition, upon recognition of the dysfunction of a normal ion channel in cells from the infarcted heart, we will proceed in defining the sensitivity of these altered ion channels to certain pharmacologic agents, e.g. Class I agents, adrenergic amines, Class III antiarrhythmic agents, etc. Finally, we will quantitate and compare resting Cai as well as amplitude of Cai transients in cells dispersed from the epicardial border zone with control cells. In particular, we will focus on rates of relaxation of Cai transients during pharmacologic interventions (e.g. beta adrenergic stimulation) and specific pacing protocols. For these experiments, we will also combine whole cell voltage clamp techniques with the measurement of Cai transients using fura-2 fluorescence microscopy. In this way we can test the hypothesis that alterations in normal cardiac function as well as ion channel function result from or result in measured changes in Cai.
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{
"pile_set_name": "NIH ExPorter"
}
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Avoidance of autoimmunity appears to rely on a carefully orchestrated series of activities involving an appropriate interplay between genetics, the environment, and components of both the innate and adaptive (acquired) immune systems. When this process fails, a variety of autoimmune disorders can arise; including type 1 diabetes where insulin producing pancreatic p cells are destroyed. Adaptive CD4'^ T helper (TH) cells orchestrate the nature and duration of immune responses through distinct properties of lineage-specific cell subsets (e.g., THI, TH17, regulatory T cells (Treg), etc.). The overall objective of Project 2 is to characterize the interplay between antigen presenting cells (APC) and TH subsets, identify their mechanistic contribution to the pathogenesis of TID, evaluate the potential of these cells to serve as markers for autoimmune disease activity. In addition, a major emphasis will be given to evaluate the contributions of specific candidate genotypes previously identified in genome wide association studies (GWAS), for their contribution to immune regulation (Project 2 - ILISRAP, TLRS; Project 1 - IFIHI). This project emanates from literature suggesting that patients with TID exhibit a remarkable loss of immunoregulation whose cellular contributors include proinflammatory APC, unstable and functionally defective Treg, and persistent T effector cells (Teff) that are refractory to regulation. Project 2 will test the hypothesis that in TID, CD4^ T cells are functionally defective and that the molecular & cellular basis for this defect resides in interactions between the innate and adaptive immune response (including dendritic cells, natural killer (NK) cells, & iNKT cells) as well as the balance between Teff and Treg subsets. We believe Project 2 finds marked innovation through the integrative approach afforded by the POI mechanism, the use of novel methodologies to isolate the cellular source of defects, the study of genetic loci underrepresented in TID research and quite importantly, our ability to explore lymphoid and pancreatic tissues and cells from the nPOD program.
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{
"pile_set_name": "NIH ExPorter"
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A continued collaboration with the Naval Research Laboratories has enabled a SQUID magnetometer to be used to study inter-ictal spike discharges in epileptic patients. Correlation of the MEG activity and the corresponding EEG signal for these discharges permits signal averaging of spikes associated with the same neurologicl event. These mesurements allow a magnetic field map at the surface of the scalp to be obtained from which a prediction of the source of the epileptic focus can be made. Computer and electronic processing techniques have been developed to analyze these MEG and EEG signals with the aim of producing an efficient method of localizing the sources of the epileptic discharges in a selective manner. Several patients have been scanned using these techniques. To enhance the collection of MEG data associated with a single spike discharge, a seven channel array detector has been specified and will be installed shortly. Magnetic field maps generated on patients going to surgery predicted epileptic source localization consistent with intraoperative electrical recordings. Refinements of the mapping procedures including MEG normalization by EEG spike amplitude and modeling of contributions of non-sphericity of the scalp over the temporal lobe appear to be substantial improvements in the state-of-the-art.
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{
"pile_set_name": "NIH ExPorter"
}
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The specific aims for the project are 1) to develop technical quality of care measures for asthma that are valid, reliable, sensitive and specific and have predictive value related to asthma outcomes, and can be easily and inexpensively applied in the health care setting used, 2) to assess whether the technical quality of care measures for asthma can be applied to inpatient and outpatients in the health care setting used 3) to assess whether the technical qualith of care measures for asthma can be applied accross the fee for service, managed care organization, Medicaid, and Medicare payment systems in the health care setting used, and 4) to develop a model system to identify technical quality of care measures for other chronic medical conditions, involving the settings outlined in specific aims 2 and 3 in the health care setting used.
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{
"pile_set_name": "NIH ExPorter"
}
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Due to their recent demonstration of efficacy in a number of malignancies, monoclonal antibodies (MAb) are becoming increasingly important in cancer therapy. Most efforts to develop new antibody-based molecules are focused on identifying those with the highest possible affinity for the tumor antigen. Using small, single- chain Fv (scFv) molecules that ranged in affinity for the same epitope of HER2 from 1x10-7 M to 1x10-11 M. We showed that high affinity may impair the ability of an antibody to penetrate into a solid tumor, leading to perivascular localization and potential suboptimal therapeutic efficacy. This work validated a "Binding Site Barrier" hypothesis posed by Weinstein that stated that antibodies of very high affinity would be limited in their ability to penetrate solid tumors. A limitation of our earlier work was that scFv are rapidly eliminated from the circulation, thus limiting the ability to study tumor penetration over time. We have recently generated full-length IgG versions of the scFv molecules described above and are in the position to elucidate the role of affinity in tumor targeting and penetration and determine the mechanisms underlying this process. Preliminary data indicate that high affinity also detracts from tumor targeting and penetration of Ig. The hypotheses underlying this proposal are that 1) IgG molecules with very high affinity for tumor antigen will demonstrate a reduced ability to penetrate into solid tumors, 2) a major mechanism behind the restricted penetration of high affinity antibodies into solid tumors is the internalization and degradation by tumor cells and 3) lower affinity IgG molecules may be superior to higher affinity antibodies in mediating anti-tumor effects. We will evaluate the roles of binding affinity, antigen shedding, antigen/MAb internalization and normal tissue antigen expression on the tumor targeting and tumor penetration of anti-HER2 MAbs. We will also determine if changes in affinity, and the attendant impacts on tumor targeting and penetration, influence the anti-tumor efficacy of unconjugated anti-tumor MAbs, thereby providing information that can help guide future rational development of anti-tumor MAbs. This research is directly relevant to public health, as it will guide the development of new antibodies for the treatment of cancer. Learning how the binding strength (affinity) of an antibody for its target on the tumor cell surface affects the ability of the antibody to move into the tumor and kill tumor cells will allow us to create more effective antibody-based treatments for cancer.
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{
"pile_set_name": "NIH ExPorter"
}
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White adipose tissue (WAT) is the main lipid reservoir that becomes dysfunctional in obesity. It is important to understand how breached lipid homeostasis leads to metabolic disease. Efficient long chain fatty acid (FA) and cholesterol trafficking depends on a transmembrane transporter protein CD36. However, the mechanism through which CD36 switches from uptake to mobilization of lipids and how lipids traffic between the endothelium and adipocytes is not understood. We had previously identified a WAT-specific plasmalemmal interaction between prohibitin-1 (PHB) and annexin A2 (ANX2). To study its role on the surface of endothelial cells (EC) and adipocytes, we have been supported by grant R01DK088131 from the NIDDK. We have shown that PHB/ANX2 binding in the context of lipid rafts mediates FA transport from EC to adipocytes, hence promoting adipocyte lipid deposition. As we recently reported, PHB and ANX2 interact with CD36 on the cell membrane within a complex assembled in response to extracellular FA. Our preliminary data suggest that FA also trigger interaction of PHB and CD36 on the surface of tumor cells, in which function of these proteins has been independently linked to cancer aggressiveness. In this competing renewal application, we propose to investigate the assembly of the PHB/ANX2/CD36 complex and its role in lipid import and export in WAT endothelium, adipocytes, and cancer cells. Our underlying hypothesis is that a spike in either extracellular or intracellular FA brings together PHB, ANX2 and CD36, which then cooperate in either FA uptake or FA mobilization, respectively. By using mice and cell culture models in which PHB or CD36 are deleted in endothelial cells or adipocytes, we will test if these proteins are necessary for lipid import into adipocytes (Specific Aim 1) and for lipolysis-induced lipid mobilization from WAT (Specific Aim 2). Our collaborator, Dr. Maria Febbraio, has generated a mouse model for tissue-specific CD36 deletion, and preliminary data demonstrate CD36 importance in EC for FA and cholesterol homeostasis. We will also use the inter-cellular lipid transport assay that we have designed to study lipid exchange between EC and adipocytes and the function of PHB/ANX2/CD36 interaction in lipogenic and lipolytic conditions. By using cell culture systems in which the protein interaction is disrupted either genetically or pharmacologically, we will also test the function of the PHB/ANX2/CD36 complex in lipid transport from WAT to malignant cells and in cancer chemoresistance and progression (Specific Aim 3). Finally, based on the reported requirement of CD36 palmitoylation for its localization to lipid rafts and for activation of lipid transport, we hypothesize that S-acylation is the trigger of the PHB/ANX2/CD36 complex assembly. Specifically, based on our preliminary data, we hypothesize that CD36 S-acylation occurs upon extracellular or intracellular FA concentration increase. We will collaborate with Dr. Askar Akimzhanov, an expert in protein S-acylation, to test if this process enables FA-induced assembly of the PHB/ANX2/CD36 complex to serve as a switch for context-dependent lipid uptake or mobilization.
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{
"pile_set_name": "NIH ExPorter"
}
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Victimization of young, college women is a problem with potentially devastating consequences. Approximately 2 million women are new freshmen each year (US Dept. of Ed., 2001 ). In a recent report, Hingson et al. (2002) estimate that 600,000 (13.3%) college students were assaulted because of drinking by other students over a 1-year period. Humphrey and White (2000) found that 6.4% of women reported being raped during their first year in school. Research findings indicate that 50% of sexual assaults in college involve alcohol (Abbey, 2002). Based on these figures, 3.2% or 64,000 freshmen women experience an alcohol-related rape annually. Clearly, victimization (nonsexual and sexual) is a significant alcohol-related problem on college campuses. The research proposed in this R01 application focuses on the longitudinal relationship between alcohol consumption and victimization among college women. The PRIMARY OBJECTIVES of the proposed investigation are to 1) describe the rates of alcohol consumption and alcohol-related victimization, across four years of college attendance, 2) assess the temporal relationship between alcohol consumption and alcohol-related victimization (sexual and nonsexual, verbal and physical), 3) assess risk factors for experiencing victimization during college, and 4) assess primary (e.g., injury, psychological trauma) and secondary (e.g., academic, psychological) consequences of alcohol-related victimization. Two Iongitudinal research components will be used to achieve these research objectives. Component 1 involves a brief telephone survey, administered annually during the Fall semester, of the drinking patterns, victimization and other alcohol-related problems that occur in a cohort of women entering college for the first time during the Fall semester of 2003. Component 2 of the research involves an 8-week prospective assessment of drinking patterns and victimization experiences administered annually during the Spring semester, to a sub-sample of women randomly selected during Year I from Component 1 participants. Component 2 will use state-of-the-art technology (Interactive Voice Response) to collect daily data on alcohol consumption and any victimization that occurs. Event-based measures will be used to provide detailed data on victimization experiences. This research is innovative in the use of long- and short-term measures, within a longitudinal design, to assess alcohol-related victimization of college women.
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{
"pile_set_name": "NIH ExPorter"
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Previous work has shown that in frog striated muscle fibers there is a mechanical coupling between the sarcolemma and the column of interdigitating arrays of myofilaments. An ultrastructural study will be made of myofibers, at points where special stress or strain of this coupling can be expected, in an attempt to find an anatomical basis for it; points such as highly stretched fiber tips and the attachments of the festoons of the sarcolemma in shortened fibers. The mechanical strength of coupling and its capability for transferring active tension are being tested in single myofiber preparations where differential stretch or shortening of fiber segments results in segmental inactivation of the myofilament column.
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{
"pile_set_name": "NIH ExPorter"
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This research will define the mechanisms by which the immune system initiates and controls inflammation. We will evaluate biochemical processes involved in the chemotactic responses of polymorphonuclear leukocytes (PMNs), monocytes, and macrophages. We will characterize receptors for chemotactic factors on leukocytes and define the nature of the transductional events which follow occupancy of the receptor and lead to directed migration. We will also see if abnormalities of chemotactic factor receptors are associated with inflammatory diseases such as periodontal disease. We previously showed that transmethylation reactions mediated by S-adenosylmethionine are required for chemotaxis and that chemotactic factors alter the methylation of phospholipids. We recently found that the state of microtubule assembly affects cellular phospholipid methylation. Alterations in phospholipids following binding of chemotactic factors may produce biophysical changes in the cell membrane which are necessary for chemotaxis. Using models for studying chemotaxis in vitro, we will determine how chemotactic factors induce changes in the cellular phospholipid composition and what role such changes play in cellular orientation. We recently defined a model for the study of a partially uncoupled receptor involved in chemotaxis. Equine PMNs have a high affinity receptor for N-formylated chemotactic peptides (NFP) but do not orient nor migrate directionally towards the NFP. They do, however, orient and migrate to C5a. Interestingly, Equine PMNs do secrete lysosomal enzymes and produce superoxide in response to the NFP. Using this model we will study the nature of the defect in horse PMNs. With the binding assays we have developed, we will study PMNs from patients with juvenile periodontosis to see if the chemotactic defects in patients are due to abnormalities of chemotactic factor receptors. We will continue studying the effects of endotoxin on the function of macrophages from normally susceptible and resistant mice. We will determine how "priming" of normal mice with endotoxin renders them resistant to the leukopenic and anti-inflammatory effects of endotoxin. In sum, we will better elucidate biochemical mechanisms of inflammation as well as their control and characterize the factors which govern the effectiveness and intensity of inflammatory reactions.
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{
"pile_set_name": "NIH ExPorter"
}
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Although obesity is one of the outstanding health problems in the United States, little is known about the factors which lead to its development at the cellular level. The aims of this research are to delineate some of the enzymatic profiles of adipose tissue during its normal growth and development and during the development of obesity. Data from normal animals will be compared to that from animals with genetic obesity and early overnutrition. The activity of thymidine kinase and its variants, lipoprotein lipase and hormone sensitive lipase will be measured. The nature of some of the differences noted between lean and obese animals will be investigated. The results will be correlated with changes in fat cell number and size. The data will be analyzed with the objective being to understand the cellular mechanisms which lead to obesity and to develop an enzymatic profile which could be used to detect the onset of a hypercelluar or hypertrophic state in biopsy samples from an at-risk human population.
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{
"pile_set_name": "NIH ExPorter"
}
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The purpose of the project is to study the chemical and physical properties and metabolism of proteoglycans in a number of tissue and cell systems. Topics of present interest include: 1) Protein chemistry and immunology of the core protein of proteoglycans from the Swarm rat chondrosarcoma; 2) Biosynthesis of core protein; precursors and processing to mature proteoglycans; 3) Effects of growth factors (insulin-like growth factor 1) on the regulation of proteoglycan metabolism in organ cultures of bovine articular cartilages; 4) keratan sulfate-proteoglycan and dermatan sulfate-proteoglycan in chick, cat and human cornea; 5) analyses of sulfated oligosaccharides on proteoglycans and sialoproteins in bone and cartilage tumor cells.
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{
"pile_set_name": "NIH ExPorter"
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This is the administrative core for the Southeastern STI CRC (SE STI CRC), in which 6 projects and 3 cores are united under the general theme "STI Prevention". Core A supports time and effort for the PI (P Frederick Spariing), the co PI (Marcia Hobbs) and administrative assistant Sharon Graham as well as a part time accounting technician, plus travel for all project Pis to the annual meeting of the combined STI CRCs. Because this is a regional center with Pis from four universities in the mid Atlantic or SE states (UNC-Chapel Hill, USUHS-Bethesda, MCV-Richmond and Emory- Atlanta), we also request travel funds to bring all the Pis together In Chapel Hill three times yearly. Annual reports and overall supervision of the budget are prime responsibilities of Core A. Core A is responsible for oversight of the science in each of the projects as well as Core B, the Microbiology core, and Core C, the Immunology core. Core C is located at Duke University. Core A organizes regular meetings of the project Pis, provides feedback to Pis of the component projects, and generally serves to help keep the science focused and on track. Core A also facilitates interactions with the other STI CRCs including planning and execution of future clinical trials or other research that is undertaken by the group as a whole. If necessary. Core A also works with the project investigators and NIH staff to revise budgets in projects, as dictated by progress in each project, and changing goals of the STI CRC system.
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{
"pile_set_name": "NIH ExPorter"
}
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Heavy habitual consumption of alcohol leads to progressive injury and fibrosis in several tissues, including the liver. However, many heavy drinkers do not develop advanced liver fibrosis (i.e., cirrhosis) despite years of alcohol abuse. More modest consumption of alcohol (<2drinks/day) has actually been associated with certain health benefits, such as protection from cardiovascular disease. The effects of modest alcohol consumption on the liver have been difficult to predict, however, because evidence suggests that this inhibits fibrosis progression in some types of liver injury (e.g., nonalcoholic fatty liver disease - NAFLD) but accelerates the evolution of cirrhosis in others (e.g., chronic hepatitis C - HCV). Research is needed to clarify the mechanisms that account for the diverse hepatic responses to alcohol. This project is grounded in the HYPOTHESIS that alcohol consumption provokes epigenetic events that modify liver repair responses that are specified by the underlying genetic profile. Our Specific Aims are: 1) to determine if a) there are liver gene expression and/or epigenetic signatures for advanced liver fibrosis and b) alcohol consumption influences this fibrogenic profile; 2) to identify gene polymorphisms that associate with susceptibility to alcohol-related alterations in the fibrogenic profile; 3) to determine the effects of SAMe deficiency and SAMe supplementation on epigenetic modifications and the liver gene expression profile for advanced liver fibrosis in individuals with alcohol-induced cirrhosis. We will take advantage of large well-annotated bio-repositories that house frozen liver samples, serum/plasma, DNA, and clinical information from patients with NAFLD, chronic HCV, and alcoholic liver disease (ALD). The largest and most comprehensive tissue collection was obtained from >1000 patients with NAFLD and includes sizeable sub-populations who are either non-drinkers or social drinkers. Therefore, initial work in Aims 1 &2 will focus on characterizing the liver gene expression, epigenetic signature and haplotype profiles of these groups. Parallel liver gene expression and epigenetic analyses will be conducted in non- drinking and modest drinkers with chronic HCV, our next largest cohort. Because liver biopsies are not generally indicated in patients with ALD, analysis will be restricted to cirrhotic ALD samples that were acquired at the time of liver transplantation or during an already approved and funded clinical protocol that is investigating the effects of SAMe on liver fibrosis. In aggregate, these studies will provide novel information about how alcohol induces epigenetic events that modulate hepatic expression of genes that regulate liver fibrosis, and identify gene polymorphisms that influence these responses. Such knowledge will help to guide recommendations about the risk/benefits of alcohol use, particularly in individuals with underlying liver disease. This has important public health implications given the fact that social use of alcohol is virtually ubiquitous. PUBLIC HEALTH RELEVANCE: These studies will provide novel information about how alcohol induces epigenetic events that modulate hepatic expression of genes that regulate liver fibrosis, and identify gene polymorphisms that influence these responses. Such knowledge will help to guide recommendations about the risk/benefits of alcohol use, particularly in individuals with underlying liver disease. This has important public health implications given the fact that social use of alcohol is virtually ubiquitous.
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{
"pile_set_name": "NIH ExPorter"
}
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The long-range objective is to understand enough about the factors that control mammalian cell growth to be able to apply this knowledge to the control of malignant growth. As a result of recent work, a broad picture is now available of the control of growth of mammalian cells. Application of this knowledge requires additional information and experimentation. Detailed information is needed of the specific factors that control the growth of the epithelial cells that are most important in the origin of tumors, in order to design rational procedures to restrict tumor growth. Among other things, it is important to know whether these epithelial cells produce inhibitors of their own growth, such as the growth inhibitors that are produced by kidney epithelial cells. The kidney epithelial cell growth inhibitor is active on certain lung and mammary cells in culture. With CCL 64 mink lung cells, 60% inhibition of [3H]thymidine incorporation is observed at a 0.1-nanogram concentration of the growth inhibitor. Injection of the growth inhibitor in vivo into human mammary carcinomas growing in nude mice inhibits [3H]thymidine incorporation into the tumors.
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{
"pile_set_name": "NIH ExPorter"
}
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In our 1 M GWAS, 4 of the 10 SNPs most significantly associated with body mass index (BMI) mapped near or within the MAP2K3 gene. Fine mapping of this region revealed that specific regions of this locus were duplicated, and a large number of the database SNPs from this region were false SNPs. Validated SNPs that mapped uniquely to the MAP2K3 locus were genotyped in 3500 full-heritage Pima Indians and replication was assessed in a second sample of 3800 American Indians. Two tag SNPs were associated with BMI in the full-heritage Pima sample and the associations replicated in the second sample. The effect of specific variants on BMI in Caucasians was evaluated using genotypic data from the GIANT consortium. One of the tag SNPs was additionally associated with BMI among 123,800 Caucasians, suggesting that variation in MAP2K3 affects BMI in multiple ethnic groups. Our prior 100K GWAS identified a SNP associated with decreased insulin-stimulated glucose disposal. This SNP mapped within an intron of MAP3K5 which encodes the apoptosis signal-regulating kinase 1 (ASK1). ASK1 is required for the TNF-induced activation of JNK and p38 pathways and may play a pivotal role in the development of insulin resistance and type 2 diabetes. Seven tag SNPs were genotyped in the population-based sample of 3500 full-heritage Pima Indians, among which 415 non-diabetic subjects had been metabolically phenotyped. One SNP was modestly associated with diabetes where the risk allele was associated with reduced insulin sensitivity among the non-diabetic subjects. SNPs in this region genotyped in a GWAS in Caucasians were similarly associated with diabetes, where the risk alleles were consistent between the Caucasians and Pima Indians. Most of the associated SNPs mapped with intron 1 or 2 of MAP3K5, suggesting that the functional variant may affect gene expression. In our recent global gene expression study of muscle biopsies from 125 non-diabetic Pima Indians, the MAP3K5 transcript was indeed positively correlated with the rate of insulin-stimulated glucose disposal and the transcript level was also negatively correlated with SNP genotype. Therefore we propose that a functional variant within the proximal promoter or introns 1 or 2 of MAP3K5 reduces expression of this gene which leads to impaired insulin sensitivity and type 2 diabetes. Among the top signals for percentage of body fat in our GWAS were variants that mapped within the lysophosphatidylglycerol acyltransferase 1 (LPGAT1) gene. LPGAT1 belongs to a large family of acyltransferases which are involved in a variety of biological processes including pathways that regulate energy homeostasis and body weight. Variants located within and adjacent to LPGAT1, including 2 novel variants identified by sequencing, were genotyped in the sample of 3500 full-heritage Pima Indians and the second sample of 3800 American Indians. Two tag SNPs were significantly associated with BMI in both samples. Analysis of LPGAT1 cDNA from human preadipocytes identified an additional exon whose sequence could potentially serve as a mitochondrial targeting peptide. In vitro functional analysis of two variants provided suggestive evidence that a 27 bp deletion in the 5-UTR may affect transcriptional or post-transcriptional regulation.
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{
"pile_set_name": "NIH ExPorter"
}
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The Comprehensive Oral Health Research Center of Discovery (COHRCD) at The University of Iowa will focus on craniofacial anomalies. A comprehensive set of major research projects coupled to technology transfer and the development of new research opportunities through pilots will be supported by a core infrastructure representing a wide range of laboratory, clinical and dissemination activities. Our center builds upon a 40-year history of clinical and research studies on craniofacial anomalies at Iowa and continues the tradition established over the past decade of interdisciplinary and inter-institutional research that includes not only a broad-based internal constituency but collaborative projects and interactions with colleagues in the United States and overseas which offer access to novel populations and innovative techniques. The proposed COHRCD builds upon a foundation of seven comprehensive science projects ranging from gene discovery in animal models to phenotype classification and behavioral studies. Pilot projects will explore the interfaces between imaging, clinical medicine and molecular development. Technology transfer will involve collaboration with industry and other institutions so as to develop a better understanding of how materials can be developed and used to improve treatment of craniofacial anomalies. A clinical resources core will support the major research projects as well as investigations into novel techniques for craniofacial imaging. A clinical resources core will support the major research projects as well as investigations into novel techniques for craniofacial imaging and health policy research into health care delivery for patients with craniofacial anomalies. Dissemination activities will be centered in a core that will utilize communications technology to research not only researchers and health professions but also a wide range of the lay public, including patients and school children. Administration and Biostatistics Cores will provide overall support for the Center. The COHRCD at The University of Iowa represents an integrated program that will continue to make use of well-established, high-quality, investigators as well as bringing in new investigators to continue a long-standing tradition of excellences in craniofacial research. The outcomes include the potential for improved diagnosis and treatment of some of the most common developmental anomalies as a result of fundamental research and technology transfer and effective dissemination of these results to other professions, to patients and to the general public.
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{
"pile_set_name": "NIH ExPorter"
}
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The main objective of the interdisciplinary Prevention And Methodology Training (PAMT) program (T32 DA017629) is to produce two types of well-trained scientists: prevention scientists who apply the most appropriate methodology in their research and can do this even when the methodology is advanced and cutting-edge; and methodologists who understand and are committed to prevention, and who work on improving and disseminating methodology for use in prevention research. The pre- and post-doctoral training program involves the Prevention Research Center (PRC) and the Methodology Center (MC) at Penn State, and the Departments of Human Development and Family Studies, Biobehavioral Health, and Communication Arts & Sciences. PAMT has successfully trained 26 productive scientists since its inception: twenty were funded by the T32 and six through matching funds provided by Penn State. The PAMT program seeks five more years of funding for several reasons. First, during its five years, due to the high visibility of both the MC and PRC and the role of Penn State in prevention research, we have attracted a very bright and diverse group of early career scientists who have been very productive during this period: six have been awarded individual NRSAs; 89 articles have been published in prestigious journals; and 122 presentations have been made at national conferences. Second, the PAMT program is positioned to meet a critical set of training needs that have been identified as central to NIDA's portfolio and the nation's needs in prevention research as identified in two new, prominent reports: Preventing Mental, Emotional, and Behavioral Disorders Among Young People: Progress and Possibilities (IOM, 2009); and a review committee report from the NIDA Council on the NIDA prevention research portfolio (NIDA, 2009), which recommended increasing both research and training related to the interface of methodology and prevention science The PAMT program is specifically designed to facilitate this interface; each PAMT fellow works closely with two mentors--one who specializes in prevention and one who specializes in methods--to assure that fellows simultaneously address both applied aspects of prevention and basic issues in methods. Most mentors have active NIH grants, ensuring that the fellows are working on issues related to public health that are both current and meet high scientific rigor. Thus the mentoring and training program established in PAMT propels young scientists to address research questions in substance abuse prevention as a result of engaging in methodology training in the field of prevention research.
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{
"pile_set_name": "NIH ExPorter"
}
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We compared the regional myocardial distribution of the commonly used flow indicators; flow limited iodoantipyrine, partially flow limited rubidium chloride and 9uM microsphere. The analysis of such data, given the appropriate mathematical model, may give answers to a) the correction factors for flow measurements, b) distribution of permeability surface area product in the heart and c) flow heterogeneity in the beating heart, working or non-working. We will try and correlate regional blood flow to regional tissue contents of high energy phosphate compounds (obtained from myocardial biopsy) in the working heart. From this we shall attempt to delineate the determinants of supply and demand in heart muscle. BIBLIOGRAPHIC REFERENCES: Yipintsoi, T.: Single-Passage extraction and permeability estimation of sodium in normal dog lungs. Circ. Res. 39: No. 4 523-531, October, 1976. Bardfeld, P.A., Yipintsoi, T., Koerner, S.K., Crane, R., Hagstrome, J.W.C., and Veith, F.: 131I fibrinogen in the detection of pulmonary allograft rejection. Surgery 80: No. 5, 575-580, November, 1976, in press.
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{
"pile_set_name": "NIH ExPorter"
}
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Leukocyte adhesion must be tightly controlled for leukocytes to patrol the body as non-adherent cells, yet stop and emigrate from the blood into tissues at sites of infection or inflammation. This involves control adhesiveness of both the leukocyte and the endothelial cell to which it adheres. While some of this control consists of regulation of expression of adhesion molecules, more rapid regulation also occurs. On leukocytes, adhesiveness is increased as integrins are rapidly rearranged into clusters. However, rapid (seconds to minutes) rearrangement of adhesion receptors is not a feature of most cell types. Endothelial cells, like leukocytes, regulate their adhesiveness rapidly, so receptor rearrangement may play a role. It was recently demonstrated that ICAM-1 clusters in response to activating signals. Whether this clustering occurs before, during, or after leukocyte binding is not known, and will have consequences for the role these receptor movements in regulation of inflammation. Dimerization of ICAM-1 is known to greatly increase binding to its counter-receptors, but the effect of higher-order clustering has not been tested. Our hypothesis is that, as with integrins on leukocytes, rearrangement of adhesion molecules on endothelial cells regulates leukocyte adhesion. We will focus on ICAM-1 to test this hypothesis by completion of the following specific aims. Aim 1: Determine the time course and mechanism (diffusion vs cytoskeletal transport) of ICAM-1 rearrangement in response to cytokine stimulation and receptor engagement. Aim 2: Determine the effects of ICAM-1 clustering and cytoskeletal linkage on leukocyte adhesion under flow. If our hypothesis proves true, this new role for adhesion molecule rearrangement on endothelial cells would represent a novel level of regulation of inflammation that could be a potential target for therapeutic intervention.
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{
"pile_set_name": "NIH ExPorter"
}
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The project calls for the maintenance of the following CTEP Information Systems: the Main Administrative System, the Phase II System, and the Drug Distribution and Protocol Monitoring System (DDPMS) with further development of linkages between these systems and with outside data bases. This project is a recompetition of the Cancer Therapy Evaluation Program Information System (CTEP-IS) contract that assists in the management of large amounts of clinical information, protocol administration data and drug information. The system will be expanded to include detailed scientific data on all protocols and drugs undergoing Phase I-III evaluation, and will be able to meet additional current and future needs of the CTEP such as maintenance of Investigational New Drug Exceptions and Adverse Drug Reaction files and automated support for the site visit monitoring function within the Investigational Drug Branch.
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{
"pile_set_name": "NIH ExPorter"
}
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This Competitive Renewal will build upon biomarker discoveries from the previous period. In four Aims we will focus upon the overall hypothesis that inflammatory candidate biomarkers identify patients at risk for incident symptomatic knee OA (SKOA), or those patients with established SKOA, who are at increased risk for disease progression. Together with our collaborators on this grant, we propose to validate and replicate our initial discoveries using four OA cohorts: 1) the NIH Osteoarthritis Initiative (OAI, M. Hochberg); 2) a second cycle OAI+expanded (e)NYU (OAI+eNYU) discovery cohort using high quality radiographs; 3) the Johnston County Osteoarthritis Project (JoCo, J. Jordan); and 4) the Genetics of Generalized Osteoarthritis longitudinal study (GOGO Long, V. Kraus). In Aim 1, we will determine whether candidate Genomic biomarkers (peripheral blood leukocyte expression of inflammatory genes) or plasma Protein/Lipid (GPL) biomarkers predict increased risk of disease severity or progression in subjects with symptomatic radiographic knee OA. We will: a) validate discovered GPL biomarker panel from NYU in the OAI; b) construct a panel of GPL biomarkers from OAI+eNYU; c) replicate this panel in GOGO Long and JoCo. In Aim 2 we will determine whether candidate genetic biomarkers (SNPs from GWAS assays that map to inflammatory genes and exonic variants from whole-exome sequencing assay) predict increased risk of disease progression in subjects with radiographic knee OA. We will: a) construct a panel of SNP biomarkers from OAI+eNYU; b) validate this panel in GOGO+JoCo; c) construct a panel of exonic variant biomarkers from eNYU+OAI; d) validate this panel by holdout in eNYU+OAI. In Aim 3 we will perform an integrative analysis, applying a predictive multi-assay analyses approach to identify potential pathogenic interactions among genetic variations, gene expression and plasma protein/lipid expression. We will: a) perform integrative modeling using GPL+SNP biomarkers in OAI+eNYU; b) validate this model in GOGO and JoCo; c) perform integrative modeling using GPL+SNP+exonic variant biomarkers in OAI+eNYU. In Aim 4 we will determine whether genomic, genetic and protein/lipid biomarkers predict increased risk of incident symptomatic knee OA. Relevance: Osteoarthritis is a common disease that affects multiple joints, resulting in joint destruction, loss of function and disability. Base on our preliminary data we expect that this proposal will identify prognostic biomarker test(s) that can predict individuals at risk for disease onset or of progressing rapidly to joint destruction. Validation of such prognostic biomarkers will facilitate the development new disease-modifying OA drugs, and, in the future, could enhance clinical management of the disease, as in decisions regarding administration of drugs.
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{
"pile_set_name": "NIH ExPorter"
}
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This NICHD Ruth L. Kirschstein National Research Service Award application seeks to continue to train pre-doctoral students in reproductive, perinatal, and pediatric epidemiology. This field has experienced slower growth relative to other fields in epidemiology, despite a clear public health need. The long-term objective of this application is to expand the field of reproductive, perinatal, and pediatric epidemiology through increased investigator-initiated research by solidly trained scientists and to ultimately reduce th population burden of reproductive and pediatric adverse outcomes. Didactic and applied training will be provided by two Boston University School of Public Health centers: the Slone Epidemiology Center and the Epidemiology Department. Trainees will: 1) gain a solid understanding of theoretical and applied epidemiology and associated biostatistics, 2) learn the basic biological processes of and major clinical features of pregnancy and/or childhood, 3) contribute to a research project under the guidance of a program mentor, and 4) learn to write bio-medical manuscripts and grant proposals, and 5) demonstrate the ability to conduct independent, original research. These learning components are achieved through coursework, clinical shadowing, conducting research, and participation in national professional conferences; they culminate in completion of three epidemiologic dissertation studies. Major strengths of the program include 1) easy incorporation into an established, accredited epidemiology doctoral program that attracts high level students; 2) established course curricula in relevant topics; 3) a abundance of reproductive, perinatal, and pediatric research resources; and 4) faculty mentors with expertise in epidemiologic methods and applied epidemiology in reproductive, perinatal, and pediatric outcomes and pharmacologics, environmental exposures, infectious disease, social/behavioral factors such as illicit drug use, and racial/ethnic disparities; and 5) faculty mentors with track records in training academic reproductive, pediatric, and perinatal epidemiologists. An emphasis is placed on the responsible conduct of research through workshop attendance and mentoring. The program is overseen by a director who is a recognized reproductive epidemiologist and whose commitment to the field and links to the classroom, research, and mentoring components ensures a continued successful training program. PUBLIC HEALTH RELEVANCE: The Boston University Reproductive, Perinatal, and Pediatric Epidemiology pre- doctoral training program teaches advanced epidemiology, clinical knowledge, and research skills to launch the next generation of independent researchers in this field. The long-term goal is prevention of adverse reproductive, perinatal, and pediatric outcomes.
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{
"pile_set_name": "NIH ExPorter"
}
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In this revised application for a Mentored Patient Oriented Research Career Development Award, the candidate, Smit S. Sinha, will develop expertise in the psychoneuroendocrinology of anxiety, with emphasis on the endogenous opioid system and its interactions with the hypothalamic-pituitary-adrenal axis in panic disorder. The endogenous opioid system is an integral aspect of the human stress response, and exerts widespread regulatory influence on specific neurotransmitter and neurohormonal systems located in key brain regions thought to regulate stress, anxiety and panic states. Of particular relevance to panic disorder is the evidence for opioid regulation of carbon dioxide sensitivity, which is abnormally heightened in panic and is a strong biological marker of the illness. Opioids also regulate attachment and separation behaviors, disruptions of which are strongly implicated in the development and maintenance of the illness. This proposal will also investigate HPA axis function in panic disorder. Disruptions of HPA function, particularly abnormal and increased central corticotropin releasing hormone (CRH) drive, have been implicated in the pathogenesis of major depressive disorder and PTSD. However, whether panic disorder involves central CRH dysregulation is not known. This is despite the prominent role of CRH in anxiogenesis and respiratory regulation. Preliminary data from the candidate and mentors suggest that panic disorder involves significant abnormalities in the opioid system and the CRH system. The candidate's research plan will 1. Assess the central functioning of both the endogenous opioid system and the CRH system with naloxone and metyrapone administration, respectively 2. Relate central opioid activity and CRH activity to CO2 sensitivity 3. Address functional interactions between these systems and 4. Assess the effects of medication treatment on these systems. The career development activities build on Dr. Sinha's background in clinical research and capitalizes on the resources of Columbia University. The overall goals are to acquire skills in the psychoneuroendocrinology of anxiety and stress with application to the pathophysiology of panic disorder. The comprehensive educational plan provides expert mentorship and didactic instruction in the key areas applied in the research plan: 1) neuroendocrinology 2) psychophysiology 3)research design and statistics and 4) clinical trials methodology. [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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The field of atherothrombosis is poised for advances in basic science and novel therapeutics, yet faces considerable challenges to clinical translation due to 1) the very success of current therapies (rendering event rates low and hence clinical trials large and lengthy) and 2) the lack of validated predictors of clinical outcomes to evaluate new therapies short of endpoint trials, and to target cost-effective preventive therapies to asymptomatic individuals nonetheless at high risk, as well as to those with established disease. Bringing basic science discoveries to the bedside with minimum delay will require major advances. This meeting will bring together investigators of various disciplines to confront these issues. Emerging science of genetics, lipid metabolism, metabolic syndrome, stem cell biology, thrombosis will be coupled with discussions of emerging biomarkers and rapidly evolving imaging technologies. This conjunction should spur progress in confronting these challenges and reducing the rapidly advancing ravages of atherothrombosis worldwide. Cardiovascular disease, namely heart attacks, strokes, and gangrene of the limbs, is a rising challenge not only in developed nations but increasingly worldwide. The major contributor to this worldwide epidemic is hardening of the arteries, a frequent cause of blood clots. Advances in basic scientific research afford new opportunities for understanding, predicting, preventing, and treating the complications of hardening of the arteries or atherosclerosis and the formation of blood clots that obstruct critical blood vessels that supply the brain, heart, and limbs. This meeting aims to foster interdisciplinary approaches to speed the practical application of the basic scientific advances being made in the laboratory. This effort will hasten the translation of research discoveries to promote health of the heart and blood vessels at home and abroad.
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{
"pile_set_name": "NIH ExPorter"
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We have shown that trans-rearrangements between (rather than within) antigen receptor genes are formed by site-specific recombination between variable segments from one immune receptor locus and joining segments from another. We have demonstrated that such events occur in the peripheral T-cells of all normal individuals but are 10-100 times more frequent in the peripheral T-cells of patients with ataxia- telangiectasia (AT). These trans-rearrangements (1) affect and alter the repertoire of immune receptor diversity, (2) suggest that an underlying defect in AT may be chromatin -hyperaccessibility+, and (3) provide a possible screening test for people at increased risk for the development of lymphoid-specific chromosomal translocations, and therefore lymphoid malignancy. We have completed a pilot study of individuals involved in the agriculture industry in which we have demonstrated an acquired transient -AT-like+ picture in individuals exposed to a variety of pesticides and herbicides. We have demonstrated a related increase in Hodgkin+s disease patients receiving chemotherapy, an iatrogenic cancer risk increasing influence. We are in the process of analyzing women involved in the agriculture industry or exposed to well water that might be contaminated by pesticides in a particular Canadian providence to determine if this assay correlates with the risk of this population for lymphoid malignancy. We have also studied the effect of a particular agent, 2.4 D on the incidence of these rearrangements in foresters divided into subgroups on the basis of the method of application of this insecticide. While these studies await verification and extension in large scale population-based studies, we have turned to a murine model because it offers more opportunity for performing refined genetics and also for identifying specific instability-inducing agents. We have found that scid mice, if given low dose (100 cGy) total body irradiation within the first 48 hours of life, show a remarkable and profound increase in the number of TCRVG/TCRJB trans-rearrangements when assayed at one to two weeks following the exposure. Underscoring the relevance and predictive value of this assay for trans-rearrangements, all of the mice so irradiated are dead of thymic lymphoma by 20 weeks of age. - biomarkers, risk assessment, - Human Tissues, Fluids, Cells, etc.
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{
"pile_set_name": "NIH ExPorter"
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In brain, the over-activity of stress hormones and endogenous excitatory amino acids (EAA) causes shortening of dendrites in the CA3 region and suppresses neurogenesis in the dentate gyrus (DG). Yet, even these changes, although impairing some functions such as declarative memory, may not constitute "damage" but rather a form of adaptive plasticity that is protective against permanent damage and also reversible and potentially treatable with antidepressants and mood stabilizers. We focus our studies on the DG-CA3 because it is where both neurogenesis and remodeling of dendrites occurs. The DG-CA3 is the critical entry point for information processing by the hippocampus and it is also vulnerable to damage because of its circuitry. Hence it is also a nexus for the remodeling effects of stress that involve glucocorticoids and excitatory amino acids and which cause impairment of hippocampal dependent memory. We believe that the remodeling of dendrites holds the key to whether the cumulative effects of stress may be ultimately irreversible. Our main hypothesis is that dendrite remodeling in DG-CA3 under stress is a reversible, adaptive process involving EAA and glucocorticoids that, like a fuse box, reduces the likelihood of permanent damage to this important and vulnerable system and affords some neuroprotection and the opportunity for reversibility. The DG-CA3 involves collaterals that excite other CA3 neurons and both feed-forward and feed-back effects that are delicately balanced and subserve memory processing; Chronic stress leads to activation of EAA release resulting in a chronic "on" state that drives dendritic remodeling. This drives the reorganization of the microtubules and associated proteins which is dependent on extracellular modulators such as PSA-NCAM expressed in DG-CA3 that facilitates movement of cellular processes and modulates BDNF. We predict that removal of PSA from NCAM will prevent stress-induced remodeling and lead to permanent damage to CA3 pyramidal neurons. Translational studies have shown that hippocampal shrinkage occurs in major depression and bipolar disorder as well as Cushing's Disease, where it is partially reversible, lending hope to the ability to reverse hippocampal atrophy in psychiatric disorders.
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{
"pile_set_name": "NIH ExPorter"
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There is a need to determine in more detail the biological, psychological, and social factors involved in tobacco smoking behavior in persons who are aware of the harmful health consequences of continued tobacco use. The brain is the principal organ of behavior in the body. Hence, a great deal of interest is not focused on brain imaging of nicotine an tobacco smoking using a variety of imaging techniques. One brain mapping procedure is the quantitative electroencephalographic technique, where it is possible to quantify brain changes using colored topographic mapping. Such studies have shown that tobacco smoking produces an EEG pattern consistent with a behavioral wakeup effect. To date, no topographic EEG studies have been reported that compare the effects of pure nicotine via routes of administration such as nasal spray to that of tobacco smoking. In this research we will compare the effects of nicotine nasal spray to those of tobacco smoke using the quantitative electroencephalographic technique with colored topographic mapping. Subjects are recruited bvia advertisements in local newspapers, via community bulletin boards, and other public places. To participate, individuals must be healthy, drug-free male and female cigarette smokers. Following a screening procedure and signing of the consent form subjects will be scheduled for two testing sessions. During one session the subject will receive nicotine nasal spray (preceded by placebo nasal spray), and during the other session the subject will smoke a research nicotine-containing cigarette (preceded by a very low nicotine-containing placebo cigarette). Blood will be drawn from arm veins to measure nicotine concentrations in the blood during both sessions. A total of 60 mls (two tablespoons) of blood will be drawn during each of the two sessions. During smoking or administration of nasal spray, EEG readings will be recorded by computer using scalp electrodes (placed using local adhesive on the scalp). Each study sessions will last approximately two hours.
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{
"pile_set_name": "NIH ExPorter"
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Pancreatic ductal adenocarcinoma (PDAC) is a painful, deadly, and incurable disease for which effective treatments remain to be discovered and tested. The OVERALL OBJECTIVES of this study are to define mechanisms underlying the regulation of PDAC cell growth, by focusing on further characterizing a novel pathway for which individual components can be targeted pharmacologically. Previous studies along with our preliminary data indicate that mitotic aberrations and PDAC cell growth, two highly interrelated processes, rely on signaling from Aurora A (AurkA) to the HP1?-G9a pathway. Mutations or aberrant expression of either AurkA or the HP1? -G9a complex are implicated in the development of neoplasms of different origins, including PDAC. We will test the CENTRAL HYPOTHESIS that a novel AurkA-HP1?-G9a pathway regulates mitotic progression and PDAC cell growth in a manner amenable to pharmacological inhibition using combination therapy. Congruently, our SPECIFIC AIMS are: 1. To characterize the function of the HP1?-G9a complex as a downstream mediator of the effects of AurkA on mitotic progression in PDAC cells; 2. To characterize cellular and molecular mechanisms that contribute to the inhibitory effects of the AurkA-HP1?-G9a pathway on PDAC growth; and 3. To characterize the effects of combined targeting of the AurkA-HP1?-G9a pathway on PDAC progression in orthotopic xenografts and genetically engineered mice models. Thus, our research will span from the level of molecules to cell populations to the whole organism. These studies are feasible for our laboratory, which has developed the appropriate conceptual framework, reagents, trained personnel and established collaborations to execute the proposed research. The design of this proposal is innovative as it seeks to maximize the yield of new mechanistic knowledge and pharmacological interventions, which impact on PDAC tumor growth. Since PDAC is a dismal disease, the discovery and proposed study of this novel AurkA-HP1?-G9a pathway bears significant biomedical relevance.
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{
"pile_set_name": "NIH ExPorter"
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In simplest terms, osteoarthritis can be understood as the wearing away of articular cartilage, a tissue with a limited ability to repair itself. Findings from recent studies suggest that progressive degradation in cartilage after injury is closely related to the elevation and activation of specific enzymes, called metalloproteinases. In addition to possible treatments, joint load such as during physical therapy or exercise can affect matrix biosynthesis and the degradative events depending on the courses of loading. A better understanding of the mechanobiology of injured cartilage and the effects of cyclic loads on tissue degradation is an important step for optimizing the treatments for cartilage degeneration. In this application, we hypothesize that cyclic load modulates tissue degeneration following injury and affects the efficacy of metalloproteinase inhibitors. Taking advantage of an in vitro model system developed recently in our laboratory, we propose to study how daily load (immobilization vs. low and moderate cyclic compression) affects specific degradative events and the treatments of cartilage following defined subfracture injury. Our specific aims are: 1) to determine the effects of daily load on matrix biosynthesis, cytokine level, matrix degradation, cell apoptosis, and biomechanical properties; and 2) to determine the efficacy of matrix metalloproteinase inhibitors (MMPI) on preventing matrix degeneration in the presence of daily load. The outcomes will be tested at the molecular, cellular and structural levels by determining the level of cytokines, elevation/activation of metalloproteinases, matrix (proteoglycan and collagen) loss and degradation, matrix biosynthesis, collagen network integrity (tissue swelling), and tissue biomechanical properties in the cartilage for up to 7 days after injury. The significance is two fold, first to determine the mechanobiology of injured cartilage and second to understand how mechanical load influences therapeutic treatments such as the use of MMPIs. We believe the results of this study will significantly impact the ways to treat cartilage degeneration in traumatic osteoarthritis and other types of arthritis.
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{
"pile_set_name": "NIH ExPorter"
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The initial research in this proposal will define and compare the molecular effect of the juvenile hormone mimics (Altosid TM and Altosar TM) with juvenile hormone on the insect vitellogenic system. Such a study will determine the mechanism of action (i.e., mode of action at the level of translation and/or transcription) for the mimic in insects and will indicate certain guidelines to be followed when other tissues are studied. Once we have defined the effects of Altosid TM and Altosar TM on the insect vitellogenic system, the program will center on the specific molecular events which are regulated by these mimics in crustaceans. This research will include dose-time responses of the molecular event in at least two different crustaceans (Balanus galeatus and Rhithropanopeus harrisii). The resulting data will indicate if the response is physiological or toxic and will indicate the critical levels which can be tolerated in nature. Positive evidence (i.e., mimic stimulation of transcription in crustaceans) will provide an emphasis to the research in Section III, which involved testing these mimics on frog embryos and on a sensitive mammalian cell line (HeLa cells). Such a cell line is of course not typical of the tissue which may come in contact with Altosid TM and/or Altosar TM; but it will allow us to determine if there is an universal effect of the mimic when it comes in direct contact with a susceptible cell.
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{
"pile_set_name": "NIH ExPorter"
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DESCRIPTION: (Applicant's Abstract) Persistent corneal wounds with epithelial defects pose therapeutic challenges to the clinician and can result in ocular morbidity, visual damage, and increase the patient's risk for potential eye loss. Currently, there are no approved clinical treatments that accelerate and/or promote corneal epithelial healing therefore limiting therapeutic options for corneal epithelial defects to providing merely an environment conducive for healing. Thus, an agent that enhances corneal repair without significant untoward effects would be a major therapeutic advance, particularly for corneal alkali burns. Thymosin B4 (TB4), a 43 amino acid protein, accelerates full thickness wound repair in the skin of normal and healing-impaired rats and stimulates keratinocyte migration in vitro (Malinda et al., 1999). In preliminary studies in rats, the applicant showed that TB4 accelerated corneal re-epithelialization, decreased inflammation in vivo and promoted human corneal epithelial cell migration in vitro. In other experiments, the applicant found that TB4 also decreased corneal polymorphonuclear leukocyte (PMN) infiltration and cytokine levels in a murine alkali burn model at 7 days post bum. However, the mechanism(s) by which TB4 exerts these effects is unknown. The goal of the studies proposed herein is to elucidate mechanisms by which TB4 promotes corneal repair and regulates inflammation. This proposal will test the overall hypothesis that TB4 promotes corneal wound repair by its anti-inflammatory effects. The aims of this study are: 1) To test the hypothesis that TB4 decreases corneal PMN infiltration after moderate (non-perforating) and severe (cornea perforates) alkali bum by altering production of cytokines and chemokines; 2) To test the hypothesis that TB4 regulates cytokine and chemokine production in cultured human corneal cells (epithelial and keratocytes); 3) To test the hypothesis that TB4 regulates PNIN production of soluble mediators of inflammation and proteases. Using in vivo and in vitro model systems to analyze the mechanism(s) of TB4 activity, the proposed studies are highly relevant to the possible clinical application of TB4 in the treatment of corneal epithelial wound healing disorders.
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{
"pile_set_name": "NIH ExPorter"
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Lymphoid stem cells (also termed common lymphoid progenitors or CLPs) are clearly defined by their ability to differentiate exclusively into each type of lymphocyte. Our preliminary data suggest that numbers of CLPs in adult mouse bone marrow are significantly reduced in aged mice, suggesting that previously reported defects in lymphocyte development in aged mice are the result of defects in the factors governing the maintenance and/or differentiation of CLPs. We will explore these possibilities with three specific aims. In Aim 1, we will utilize DNA content analysis of young versus old CLPs isolated by cell sorting and in vivo BrdU labeling to determine whether our observation of reduced cellularity of CLPs in aged mice is due to decreases in proliferation among CLPs or decreases in CLP generation versus survival. In Aim 2, we will utilize IL-7 driven stromal cell-free cultures to assess whether decreases in CLP cellularity in aged mice reflects diminished responsiveness to IL-7. In Aim 3, we will establish BM chimeras to determine whether age-associated decreases in frequencies of CLPs reflect cell autonomous versus environmental defects.
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{
"pile_set_name": "NIH ExPorter"
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We propose to undertake a broad multidisciplinary approach to drug abuse problems. Chemical, biochemical, pharmacological, physiological, and behavioral techniques will be used to explore marihuana, the narcotics and narcotic-antagonists, the inhalants, and other classes of abused substances. Specific binding of the cannabinoids; the role of neurotransmitters, Ca ions and cyclic-GMP in the action of the narcotics and narcotic antagonists; and the development of new methods for the study of the behavior effects of inhalants, and abuse potential will be stressed. Considerable emphasis will also be placed on the pharmacology of the cannabinoids, their behavioral interactions, and the role of pharmacokinetics and metabolism in these processes. New narcotics, narcotic antagonists, and cannabinoids will be synthesized in an effort to gain a greater insight into how these drug classes act. Of particular interest will be the study of d-morphine, d-codenine and d-naloxone. We also propose extensive studies on the abuse potential and pharmacokinetics of LAAM and its metabolites.
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{
"pile_set_name": "NIH ExPorter"
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We are investigating the kinetics and chemistry of isoenzymes of yeast alcohol dehydrogenase which have been produced by mutation and selection in our laboratory. It is our hope that these investigations will lead to a better understanding of the function of this and other enzyme molecules, through a determination of which amino acid substitutions will lead to particular kinetic alterations. We also hope to answer questions of general evolutionary interest, such as: in how many ways can an enzyme molecule change as a selective response to a given environmental alteration, and what fraction of the molecule is potentially involved in this change? Our mutant enzymes, some of which have manyfold changes in Km and Vmax, have been produced by selection for resistance to allyl alcohol. The mutants are resistant because of a shift in the NAD-NADH balance in the cell, which in turn shifts the equilibrium between allyl alcohol and its poisonous product acrolein. BIBLIOGRAPHIC REFERENCES: Wills, C., Phelps, J., and R. Ferguson. 1975. Further Evidence for Selective Differences Between Isoalleles In Drosophila. Genetics 79:127-141. Wills, C., and J. Phelps. 1975. A technique for the Isolation of Yeast Alcohol Dehydrogenase Mutants with Altered Substrate Specificity. Arch. Biochem. Biophy. 167 627-637.
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{
"pile_set_name": "NIH ExPorter"
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At present, positive pressure ventilation is usually applied with large tidal volumes and only exceptionally at respiratory rates above 20 per minute. A novel method of jet ventilation uses instead of endotracheal tube a small cannula introduced transtracheally or between the vocal cords to inject jets of oxygen in the trachea. For this method, in our previous work a fluidic logic controlled ventilator was developed which is also capable of operating at high respiratory frequencies. There is no direct proof that the currently-used rates are the optimum for positive pressure ventilation and not just an expression of technological limits of present respirators. Using the jet ventilator, it was demonstrated experimentally and clinically, that high frequency jet ventilation (HFJV) up to the rate of 200 per miinute maintains satisfactory gas exchange at low intratracheal pressure and without circulatory impairment. During weaning from controlled ventilation, HFJV could be superimposed on spontaneous breathing. In the proposed research, high frequency jet ventilation at respiratory rates between 100 and 600 per minute will be studied on dogs to evaluate gas exchange in the lungs and determine the physiologic effects of HFJV on hemodynamics. It will be determined how the efficiency of HFJV will be influenced by changing inspiration/expiration ratio and changing the catheter flow characteristics. HFJV synchronized with various phases of the heart action will also be evaluated. Finally, it will be studied whether HFJV can be applied transtracheally for long-term ventilation as an alternative to the endotracheal intubation. In clinical studies, the objective is: 1. to evaluate the use of HFJV for weaning from respiratory support as an alternative to intermittent mandatory ventilation; 2. to study the use of HFJV during laryngoscopy in anesthetized patients; and 3. to study the application of HFJV through endotracheal tube for controlled ventilation in intubated patients requiring respiratory support.
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{
"pile_set_name": "NIH ExPorter"
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PROJECT SUMMARY Alcohol consumption has been linked to increased risk of colorectal cancer (CRC). However, the underlying mechanisms have not yet been fully defined. Emerging evidence from animal studies suggest that alcohol associated gut dysbiosis and subsequent gut barrier dysfunctions and endotoxemia may be an important and underexplored pathway. However, the impact of long-term alcohol intake on not only the taxonomic makeup but also the functional capacity of the gut microbiome among healthy individuals has not been established, and how these dysbiosis will influence colorectal carcinogenesis remain unknown. Alcohol-associated passage of luminal endotoxin into systemic circulation is hypothesized to activate both adaptive and innate immune systems characterized by a release of antibodies, cytokines, and other inflammatory mediators, which may increase subsequent risk of inflammation related diseases, including obesity, and diabetes, both of which are well-established risk factors for CRC. However, the role of endotoxemia in colorectal carcinogenesis has not been studied. We therefore hypothesize that long-term alcohol intake induce gut dysbiosis, impair the gut barrier function, and followed by endotoxemia and inflammation to increase risk of CRC. We will test this hypothesis leveraging rich data collected a large and well-characterized prospective cohort (the Health Professional Follow-up Study) with a healthy sub-cohort with stool collection and metagenomic and metatranscriptomic profiling (Aim 1a) and 250 nested CRC cases and 250 controls with archived pre- diagnostic blood and ongoing plasma proteomic profiling (Aim 1b and 1c). Specifically, we will investigate whether long-term alcohol intake induce gut dysbiosis through perturbations in microbial composition (e.g. Proteobacteria) and function of tumor-permissive immune signatures and procarcinogenic pathways among healthy individuals (Aim 1a). We will also investigate whether alcohol-associated colorectal carcinogenesis is mediated by gut barrier dysfunction and endotoxemia through first identifying circulating markers of gut wall integrity loss, bacteria translocation, endotoxemia, inflammation associated with alcohol intake (Aim 1b), and then investigate their associations with subsequent risk of CRC (Aim 1c). Our findings will provide novel mechanistic insight into gut microbial and endotoxemia mediated pathogenesis of alcohol-related CRCs. We will also provide clinically translatable data including specific microbial targets, and circulating biomarkers and network that reflect susceptibilities to subsequent risk of alcohol-related CRCs.
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{
"pile_set_name": "NIH ExPorter"
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Studies directed toward the total synthesis of Harringtonine and related alkaloids will be continued. The proposed route involves a convergent synthesis of Cephalotaxine via conjugate - addition reactions of functionalized organometallic reagents to gamma-oxido vinyl sulfones.
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{
"pile_set_name": "NIH ExPorter"
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A population-based case-control study of lung cancer is proposed to determine if employment in wood-related occupations is associated with increased risk of lung cancer, and to identify specific exposures in these jobs which might explain any increased risks found. Specifically, we will: a) test the hypothesis that persons employed in operational jobs in any of the four major segments of the wood industry are at increased risk of lung cancer; b) test the hypothesis that persons exposed to respirable wood dust in particular, as well as several other potential carcinogens present in wood-related occupations, are at increased risk of lung cancer; and c) determine whether excess risk associated with any of the above factors varies by histologic type, or is modified by other known risk factors such as tobacco use, dietary intake of beta-carotene and vitamin C, or family history of lung cancer. Male cases aged 18-74 years who reside in an 11-county area of western Washington where wood-related occupations are particularly common will be identified from the Cancer Surveillance System of the Fred Hutchinson Cancer Research Center (a SEER registry). Controls of similar age from the same geographic area will be selected by random digit dialing. Approximately 769 cases and an equal number of controls will be interviewed over a three-year period. Subjects will be interviewed by telephone using a structured questionnaire and experienced interviewers. A standard mailed self-administered questionnaire will be used to collect limited dietary information. Exposure to wood dust will be assessed on an individual basis by two industrial hygienists, taking into account job title and typical duties, type of manufactured product, and participation in activities known to produce measurable concentrations of respirable dust. For industries in western Washington in which total and respirable dust levels are not well-characterized, random full-shift sampling will also be accomplished during the first year. Approximately 680 samples will be taken at a minimum of eight industrial sites over two seasons. Stratified analysis and logistic regression will be used to control for the effect of major risk factors and to identify any important effect modification by these factors. Polytomous logistic regression will be used along with pairwise logistic regression to investigate differences in risk among specific histologic types of lung cancer. It is estimated that if wood dust is etiologically related to lung cancer, it may account for 15% of male lung cancer cases in parts of western Washington and 3% nationwide. This would represent approximately 3,000 cases per year in the United States.
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{
"pile_set_name": "NIH ExPorter"
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PROJECT SUMMARY In humans, it is clear that reduced function of the circadian clock is associated with increased incidence of neurodegeneration (e.g., Alzheimer?s Disease; AD). While it has been hypothesized that circadian dysfunction and neurodegeneration may be linked, the mechanism for these proposed links remains uncertain. My lab has evidence that the circadian clock components in Drosophila regulate sensitivity to cell death and neurodegeneration pathways after prolonged exposure to light. The doubletime protein kinase (DBT), which phosphorylates key circadian clock proteins to control their daily accumulation, is the common link. In various genotypes with reduced DBT kinase activity, the DRONC caspase, which cleaves various target proteins to initiate cell death pathways, becomes activated and cleaves tau protein, which is associated with enhanced degeneration in a fly eye model for tauopathy. DBT interacts with two other circadian proteins recently discovered by my lab (SPAG and BDBT) to maintain inhibition of DRONC activation. In turn, SPAG may interact with heat shock protein 90 (HSP90) and tau to reduce tauopathies. Furthermore, activation of the DRONC caspase occurs more broadly in cells that do not express the transgenes knocking down DBT activity and is signaled by the circadian neuropeptide PDF. These findings lead us to propose a mechanism in which the circadian component DBT kinase phosphorylates DRONC caspase to inhibit its activation and DRONC- dependent tau cleavage, which can lead to tauopathy. The inhibition of cell death seems to require phosphorylation of DBT. The work proposed will test this mechanism and map out the biological consequences in the fly model. Aim 1. Determine the biochemical pathway of DBT-dependent caspase activation and tau interactions. Specifically, my lab will investigate whether DBT kinase directly phosphorylates the DRONC caspase to inhibit its cleavage, and whether cell cell-death inducing stimuli like UV stimulate autophosphorylation of DBT, dissociation of SPAG cochaperone and BDBT FKBP from DBT and cleavage of DRONC. The capacity of BDBT cochaperone to regulate activation of DRONC kinase will be investigated in novel bdbt CRISPR mutants, and the nature of SPAG cochaperone interactions with HSP90 and tau will be assessed. Aim 2. Determine whether tau cleavage in the fly eye in response to reduced DBT activity leads to typical tauopathy features that contribute to enhanced neurodegeneration, or whether the effect of reduced DBT is mediated independently of tau via cell death (caspase-directed) pathways. We will try to induce eye degeneration by expressing these proteins only in the adult fly instead of throughout eye development in order to more accurately mimic age-dependent neurodegeneration. We will determine the extent to which cell death pathways are induced in the fly eye by DBT kinase reductions and the extent to which various aspects of tauopathy are downstream consequences (e.g., tau phosphorylation, NFT formation, and mitochondrial and cell cycle dysfunction). Also, the capacity of cleaved tau to produce tauopathy downstream of DBT kinase reductions will be tested, and the role of DRONC caspase activation in the neurodegeneration will be assessed by antagonizing DRONC with RNAi or potentiating it with nonphosphorylatable forms of DRONC. Aim 3. Determine the mechanisms for the interaction between DBT-mediated caspase activation and aging, light and the circadian rhythm PDF signaling pathway. We have found that light induces caspases in the optic lobes after 7 hrs of exposure, independent of circadian time, in our flies with reduced DBT activity in the circadian cells of the brain. The effect involves signaling by the circadian PDF neuropeptide and can occur in cells that do not express the transgenes reducing DBT kinase activity. We will determine the importance of circadian and visual photoreceptors for this process, and whether wild type flies produce tauopathy as a consequence of aging. The cells in which the DRONC caspase activation occurs will be determined, and whether the PDF neuropeptide receptor produces an increase in DBT kinase phosphorylation and a decrease in DBT levels in the cells. Finally, it will be determined whether the caspase activation we observe affects a previously observed light-dependent neuronal remodeling process in the optic lobes.
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{
"pile_set_name": "NIH ExPorter"
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These studies are intended primarily to characterize the mechanisms involved in active calcium transport by renal epithelia. Proposed experiments include examination of the membrane processes and second messengers involved in parathyroid hormone (PTH) stimulation of calcium absorption in renal cortical thick ascending limbs of Henle (cTAL), a nephron site of the physiological hypercalcemic action of PTH. In order to accomplish this characterization, electrophysiological techniques combined with digital fluorescence imaging techniques will be applied to single perfused segments of renal tubules. Biochemical and isotopic flux studies will be performed on cultured Madin-Darby canine kidney (MDCK) cells. Fluorescence ratio microscopy imaging will be used to measure cytosolic calcium in single perfused thick limbs. This technology is established in our lab and preliminary feasibility data are included. Other pilot studies described in this application support the use of confluent MDCK cells as a model with which to examine PTH-sensitive calcium transport. We have established that PTH, but not calcitonin, specifically stimulates transepithelial calcium transport in MDCK monolayers grown to confluence on porous filters. Further, challenge with PTH triggers phosphatidyl inositol hydrolysis in these cells. Thus, from a physiological standpoint MDCK cells simulate completely the response to PTH in cortical thick limbs. However, unlike cTALs, MDCK cells can be grown in sufficient number to permit detailed biochemical analysis and characterization of the receptor events involved in mediating the response to PTH. Studies have been designed to test hypotheses related to: 1) regulation of cytosolic calcium in PTH-sensitive, calcium transporting cTALs; 2) PTH-induced second messengers; and, 3) regulation of PTH-dependent calcium transport by prostaglandins, and other factors known to alter distal calcium transport in vivo, including the concentration of calcium in and ionic composition of extracellular fluid. These studies should provide valuable information on the regulation of intracellular calcium in PTH-sensitive distal target cells, characterization of receptors and second messengers involved in active calcium absorption, and insight into down-regulation and other regulatory factors modulating physiological PTH action in target renal cortical thick limbs and in a cell culture model of distal tubule cells.
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{
"pile_set_name": "NIH ExPorter"
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The rapid rise in atmospheric CO2 levels presents a significant long-term threat to human health.(1) Much of the current rise in CO2 levels may be ascribed to the use of non-renewable fuels and, as a result, there has been great interest in studying energy conversion reactions such as H+ reduction and H2 oxidation.(2) Some of the best H2-producing and -consuming catalysts are [FeFe] hydrogenase enzymes which are able to operate near the thermodynamic potential of the reaction at high turnover frequencies.(3-6) Because understanding the chemical basis for their high efficiency may aid in the design of new synthetic catalysts for H2 production and consumption,(7) much attention has been given to the structure of the active site of the enzyme (the H- cluster).(8, 9) The H-cluster has a biologically unprecedented structure with a 2Fe core ligated by three CO and two CN- ligands, each of which are typically toxic in their free states. Although it is known that the CO and CN- ligands derive from tyrosine,(10-13) the mechanistic details of their formation and assembly into the H- cluster are scant. In addition, the Fe centers are bridged by a five-atom dithiolate bridge; the identity of the central atom-currently thought to be N-has been the subject of much debate, and is important because it is thought to serve as a pendant base that can kinetically facilitate H+ migration.(9, 14-20) Owing to the unusual structure of the H-cluster and its central importance in affecting the rate and redox potential of the featured H2 chemistry, the mechanism of its biosynthesis is of high interest. In this proposed work, I aim to elucidate several aspects of the mechanism by which the set of maturase enzymes HydE, HydF, and HydG promote H- cluster formation. I will focus on three questions: what are the geometric and electronic structures of Fe- containing intermediates in the early stages of cluster maturation, what are the molecular precursors that give rise to the dithiolate bridge, and can the central atom be identified directly by EPR spectroscopy? To address these questions I will perform advanced EPR experiments on specifically labeled isotopologs of both intermediates in H-cluster synthesis as well as the mature H-cluster. These isotopic labels will be introduced through the use of labeled substrates (most often tyrosine) thereby allowing for a specific isotopic label to be traced from the molecular precursor through intermediates and finally into the mature H-cluster. For each intermediate, orientation-selective EPR experiments such as HYSCORE and ENDOR will enable determination of the distances and orientations of the labeled nuclei with respect to the electron spin (modeled as a point- dipole), thereby providing detailed geometric and electronic structure information. These EPR experiments will be performed in conjunction with complimentary stopped-flow FTIR and Mossbauer spectroscopies. Overall, this work will address the mechanistic details of tyrosine degredation into CO and CN- by HydG, the structures of Fe-containing intermediates in this process, the identities of the final product(s) o the HydG reaction, and the molecular precursors to the dithiolate bridge.
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{
"pile_set_name": "NIH ExPorter"
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Recent nmr studies which have provided new evidence for the catalysis of a number of reactions of amino and keto acids by vitamin B6 (pyridoxal or pyridoxamine, respectively) are being followed up by studies of reaction pathways by several methods. These techniques include effect of structure of substrate and catalyst on the kinetics and mechanism of reaction, variation of conditions required for optimization of reaction rates, and detection and isolation of intermediates. The relationship between these reactions and the metabolism of amino and keto acids in biological systems will be studied. The reaction types under investigation are: decarboxylation of alpha amino acids; catalysis of alpha-Beta carbon-carbon bond fission, elimination of electronegative substituents from Beta- and gamma- positions of alpha amino acids; mechanisms of racemization and its relationship to the mechanism of transamination; mechanism of formation of and isolation of transamination intermediates in metal chelate systems and metal-free systems; and the mechanism of catalysis of oxidative deamination of alpha amino acids by pyridoxal and transition metal ions. The relationship of the latter reaction to the metal- catalyzed insertion of oxygen to form pyridoxal oxime will be investigated. Information obtained on non-enzymic catalysis of reactions of molecular oxygen with amino acids by pyridoxal and metal ions will be applied to the interpretation of the mechanism of action of Cu(II)-containing amino acid oxidase enzymes. Reaction rates and mechanisms will be studied by following disappearance of reactant and growth and disappearance of intermediates and reaction products with the aid of proton and carbon nmr 13, circular dichroism, electronic spectrophotometry and Raman spectra. Equilibria will be determined by potentiometric and spectrophotometric techniques. Additional information on reaction pathways will be obtained by C14 and O18 labelling of reactants.
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{
"pile_set_name": "NIH ExPorter"
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Tuberculosis is almost a uniquely human condition because, although the bacterium can infect a range of hosts, it only proceeds to transmission in humans and higher primates. Transmission is a consequence of the tissue response to the bacterium that is probably driven by bacterial components released by the intracellular bacilli. During the previous funding period we demonstrated that Mycobacterium releases its peripheral cell wall components into its host cell, and that these lipids are capable of spreading to neighboring, uninfected cells. We isolated and identified the major lipid species and, in a novel granuloma development model, determined that trehalose mycolates were the most bioactive. We have found that, under certain circumstances and in certain tissue sites, trehalose dimycolate (TDM) induces a destructive tissue response that is highly reminiscent of late stage granuloma breakdown in humans. In this current application we propose to characterize the host gene expression profile in late-stage, degenerative human tuberculosis granulomas, and compare and contrast this with the nature and mechanisms behind the murine response to bacterial cell components in our granuloma model to delineate the tissue reaction that leads to transmission. The specific aims of this proposal are as follows: 1. What is the transcriptional profile within the human granuloma during necrosis and transmission? We intend to determine the gene expression profiles in specific regions within late stage human granulomas during caseation and necrosis. This should determine the "major players" in driving tissue destruction leading to transmission. 2. The development of a tissue breakdown model to identify the role(s) of different factors involved in tissue remodeling and granuloma dissolution. Murine tuberculosis granulomas do not progress to caseation the way human granulomas do however we can manipulate the response to bacterial cell wall components to give a much more comparable result. We intend to validate experimentally the factors implicated in specific aim #1 in driving the tissue breakdown. 3. How is TDM recognized by the innate immune system? The recognition of the potent biomodulator TDM is dependent on both the host receptors, and the mechanism of presentation of TDM. We propose to identify the receptor(s) involved and elucidate their cellular and tissue distribution, and the signaling pathways through which they operate.
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{
"pile_set_name": "NIH ExPorter"
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Abstract Neuropathic pain reflects a myriad of changes in the periphery, spinal cord, and subcortical and cortical structures. Interestingly, despite this baffling array of changes, many approved treatments for neuropathic pain recruit, augment, or mimic bulbospinal inhibition including morphine, noradrenaline (NA) re-uptake inhibitors, and clonidine. Although clinical studies suggest a baseline deficit of descending inhibition in patients with neuropathic pain, we believe that activation of this pathway is key to its treatment. GBP (GBP) is commonly used as a safe and effective treatment of neuropathic pain. GBP analgesia depends on an interaction with the 124 subunit of calcium channels, but the circuits activated by GBP for analgesia are not entirely known. We and others recently demonstrated that GBP activates the descending bulbospinal noradrenergic pathway after nerve injury and after surgical incision in rodents. We also demonstrated that orally administered GBP significantly increases NA concentration in cerebrospinal fluid and decreases opioid requirements after surgery in patients with chronic pain. These findings argue that activation of descending inhibition, rather than peripheral or spinal actions, is pivotal for GBP analgesia. The goals of this proposal are to identify the mechanisms by which GBP activates descending inhibition. Since descending inhibitory noradrenergic axons originate from the locus coeruleus (LC) and adjacent nuclei in the brainstem, we focus on the LC as a target of action of GBP. Initially, we will utilize immunocytochemistry and microdialysis methods in LC to examine the local mechanisms by which GBP activates NA neurons, focusing on the roles of GABA and glutamate inputs. Secondly, we will perform behavioral tests and measure NA content in microdialysis samples from spinal dorsal horn to examine the link between actions of GBP to reduce hypersensitivity after nerve injury and to increase spinal NA release, as a consequence of activation of NA neurons in LC. The proposed studies will not only provide critical tests of our hypothesis of this primary site and mechanism of action of GBP for analgesia but also probe mechanisms by which the descending NA system can be recruited to treat neuropathic pain. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Gabapentin is widely used to treat chronic pain, but how it works is not well understood. We recently found that gabapentin activates a natural pain-relief mechanism, and in this proposal, we will study how gabapentin stimulates the body's own pain relieving system to work in chronic pain. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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During the past funding cycle, our research has emphasized the importance of the NAD(P)H oxidase as a source of reactive oxygen species and confirmed that it plays a crucial role in the hypertension caused by angiotensin II. In addition, we have shown that the extracellular superoxide dismutase (ecSOD) seems to play an important compensatory role in conditions associated with increased vascular oxidant stress, for example in angiotensin H-induced hypertension and in two transgenic mouse models tmice overexpressing either p22 phox or nox1). Very recently, we have found that mice lacking ecSOD, ecSOD mice, the hypertension caused by angiotensin II infusion is dramatically enhanced, again supporting a critical role of the ecSOD in modulating oxidant stress and hypertension. In the present studies, we plan to gain further insight into regulation of this enzyme and how it modulates blood pressure. In aim 1, we will examine the hypothesis that H2O2 is responsible for the increase in ecSOD in response to angiotensin II. To accomplish this, we will treat vascular smooth muscle cells in culture with angiotensin II in the presence or absence of PEG-SOD or PEG-catalase. We will also study vascular smooth muscle cells that are deficient in p47 phox from p47 phox-/-mice and cells overexpressing catalase (Tg catvsmc mice). In vivo, we will examine the ability of angiotensin II to increase ecSOD expression in p47 phox-/- mice and Tg catvsmc mice. We anticipate that ecSOD expression will not be increased in p47 phox-/- mice or in Tg catvsmc mice. In aims 2 and 3, we will examine mechanisms responsible for the augmented hypertension caused by angiotensin II in the ecSOD -/- mice. In aim 2, we will examine the effect of angiotensin II on renal sodium excretion in wild-type and ecSOD / mice and examine the effect of a sodium free diet on blood pressure in these animals. In aim 3, we will make use of a mouse that we have recently created that will allow vascular smooth muscle specific deletion of ecSOD and examine the effect of angiotensin II on blood pressure and renal sodium excretion in these mice. The effect of angiotensin II on vascular reactivity, superoxide production and nitric oxide bioavailability will be studied in these mice as well. This aim will allow us to understand the relative importance of vascular vs. non-vascular sources of ecSOD in modulation of blood pressure and vascular tone. Finally, in aim 4, we will study the effect of deletion of ecSOD in mice overexpressing the NAD(P)H oxidase subunit p22 phox. These transgenic mice, which we created during the past funding period, have a modest increase in vascular free radical oxygen production and a 3-fold increase in ecSOD expression. We hypothesize that elimination of ecSOD in these mice will result in a hypertensive phenotype with a marked increase in vascular free radical oxygen production. Overall, these studies should further our knowledge of how vascular oxidant stress is modulated in vivo and in particular provide new information regarding the importance of the ecSOD in this process.
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{
"pile_set_name": "NIH ExPorter"
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Project Summary/Abstract Alzheimers disease (AD) is characterized by the deposition of amyloid B protein (AB), a small peptide derived from B- and y-secretase cleavages of the amyloid precursor protein (APP). We recently demonstrated that the last 37 amino acids (LRP-C37) of low-density lipoprotein receptor-related protein (LRP) without the NPXY motifs to be necessary and sufficient to increase AB production. Since LRP-C37 alone was a potent inducer of AB production, we used this domain as bait in a yeast 2-hybrid screen, resulting in the identification of Ranbinding protein M (RanBP9). Indeed transient transfections of APP and RanBP9-FL or FL-derived RanBP9- N60 robustly increased secretion of AB in varieties of cell lines, indicating that RanBP9 alters APP metabolism. Most importantly, immunoblot quantification of RanBP9 protein levels demonstrated that RanBP9-N60 and RanBP9-FL were elevated more than six and four-folds in the brains of AD patients and APP J20 transgenic mice respectively. We also found that like LRP and two of its key ligands, RanBP9 is genetically associated with late-onset AD. To gain a better insight on the in vivo role of RanBP9 in the pathogenesis of AD, we generated transgenic mice over expressing RanBP9 in the brain as a part of an ongoing NIH R03 grant. By crossing B6C3-Tg85Dbo mice (APdE9) carrying APPswe, PSEN1dE9 mutations with RanBP9 transgenic mice, RanBP9/APdE9 triple transgenic mice were generated, which produced more CHAPSO-soluble AB and c-terminal fragments (CTFs) compared to APdE9 mice as early as 3 months of age, suggesting that RanBP9 increases amyloidogenic processing of APP in vivo. This R01 proposal is an extension of the R03 project. As loss of synapses is a better correlate of the extent of cognitive deficits in Alzheimers patients and since RanBP9 is present in substantial amounts in neurites and is a strong inhibitor of neurite outgrowth, we next want to examine in this proposal, whether RanBP9-induced altered processing of APP also leads to dendritic and spine injury. We have successfully produced RanBP9 transgenic mice as well as heterozygous null mice for the first time. We propose to compare the pattern of dendritic arborization, spine density, presynaptic and postsynaptic protein levels in the hippocampus and frontal cortex followed by tests for learning and memory skills at 2, 5 and 10 months of age in eight groups of mice, i.e., RanBP9-629 single transgenic, APdE9 double transgenic, RanBP9-629/APdE9 triple transgenic, RanBP9-599 single transgenic, RanBP9-599/APdE9 triple transgenic, RanBP9-/- or RanBP9+/-, RanBP9-/- or RanBP9+/-/APdE9 and wild type litter-mate controls. Neuron Studio, software for automated spine density analysis, will be used to analyze dendritic branching points and spine numbers in Lucifer-yellow-stained pyramidal neurons after obtaining images by laser scanning confocal microscope. If RanBP9 is confirmed as a bona fide target in vivo in this study, the triple transgenic mice may prove to be useful as an accelerated model for synaptic and behavioral deficits.
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{
"pile_set_name": "NIH ExPorter"
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To assess the effect of an acute insulin infusion on the metobolic clearance rate of dehydroepiandrosterone in elderly men and women.
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{
"pile_set_name": "NIH ExPorter"
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Abstract Chronic heart failure, the final step of different cardiovascular diseases (CVD), remains the leading cause of cardiovascular death without a cure. To date, the therapeutic approaches by blocking or inhibiting over-activated single pathway such as inflammatory cytokines and oxidative stress which have been firmly established to play a causative role in CVD turn out to be ineffective or even harmful. In this context, the therapeutic concept of traditional herb medicine that aims to maintain systemic homeostasis by orchestrating multiple signaling pathways rather than blocking or inhibiting single over-activated signaling pathway raises a novel research direction. However, this concept has always been criticized as the 'mystical' claim for herb medicines to treat CVD, due to the contradictory findings and the lack of mechanistic analysis of herb medicine's efficacy and safety for the treatment of CVD. Indeed, the molecular mechanism by which herb medicines protect against maladaptive cardiac remodeling and heart failure remains to be established. Utilizing a standardized American ginseng extract from the National Research Council of Canada, Institute for National Measurement Standards (NRCC-INMS) under the guidelines of National Center for Complementary and Alternative Medicine (NCCAM), we have mechanistically explored the cardiac protective actions of ginseng, the root of genus Panax that has been used as a folk medicine for several thousand years in Asian countries. Our findings strongly support a novel working hypothesis that American ginseng suppresses oxidative stress and inflammatory responses, as well as the subsequent maladaptive cardiac remodeling and dysfunction via coordinating autophagic activity and Nrf2 signaling in the heart. To test this hypothesis, we propose three specific aims as follows: Aim 1. To determine role of autophagy and Nrf2 in regulating the American ginseng-mediated suppression of oxidative stress and inflammatory cytokine production in macrophages as well as oxidative stress, hypertrophic growth and cell death in cardiomyocytes; Aim 2. To determine the active components of American ginseng for the coordination of autophagy and Nrf2 pathway thereby blocking the vicious cycle of oxidative stress and inflammation in vitro; Aim 3. To determine the active components of American ginseng for the suppression of maladaptive cardiac hypertrophy and dysfunction via activating both myocardial autophagy and Nrf2 in the heart; These experiments will demonstrate for the first time that American ginseng-activated autophagy is essential for American ginseng-activated Nrf2-mediated cardiac protection. Overall, the outcome of this proposal will not only provide a novel molecular mechanism of American ginseng-mediated cardiac protection, but also establish biomarkers for the sub-fractionating of the active components of ginsengs for cardiac protection.
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{
"pile_set_name": "NIH ExPorter"
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Besides unique patients with immunodeficiency and immunodysregulation disorders lacking known diagnoses, our intake includes patients with combined immunodeficiency, common variable immunodeficiency (CVID), variants of hyper-IgE syndrome or autoimmune lymphoproliferative syndrome (ALPS), Evans syndrome, caspase-8-deficiency state (CEDS), X-linked Magnesium defect with EBV infection and Neoplasia (XMEN), and PASLI (p110 delta activation mutation causing senescent T cells, lymphadenopathy, and immunodeficiency) disease. Our evaluation includes functional screening and gene sequencing, and a subset of patients is also being intensively studied using biochemical analyses, gene expression microarrays, flow cytometric analyses, in vitro functional tests, and other technologies. These experiments have provided leads for sequencing of new candidate genes not previously associated with disease. Additionally, we are using comparative genomic hybridization (CGH) arrays, whole exome sequencing, whole genome sequencing, and other technologies to determine genetic causes of new immunological diseases in an unbiased manner. In FY2015, using these approaches, we identified from our hyperimmunoglobulinemia E cohort a new combined immunodeficiency disease. TRIANGLE disease is characterized by recurrent sinopulmonary infections, persistent cytomegalovirus viremia and problems with other viruses, and severe autoimmunity including cytopenias and systemic lupus erythematosis. Patients also have developmental delay. We found that the disease is caused by autosomal recessive mutations in the tripeptidyl peptidase II (TPP2) gene. Loss of TPPII perturbs intracellular amino acid homeostasis, leading to compensatory lysosomal expansion with lysosomal degradation of a key glycolytic enzyme called hexokinase-2. The impaired glycolysis in turn causes defective immune effector functions. In FY2015, we also participated in several collaborative studies that involved the identification of two new multi-organ autoimmune diseases with lymphoproliferation and immunodeficiency (CTLA4 haploinsufficiency with autoimmune infiltration (CHAI) disease, and STAT3 gain-of-function disease) and one combined immunodeficiency with lymphoproliferation (PASLI disease caused by PIK3R1 loss-of-function mutations). Additionally, we also participated in a collaborative study that elucidated the mechanism of LRBA deficiency as interfering with CTLA4 intracellular trafficking and thus why this disease is responsive to CTLA4-targed therapy. Collectively, these discoveries now provide molecular diagnoses for some forms of complicated CVID. They have provided new insights into regulation of the human immune system, and our elucidating their pathogenic mechanisms has also led to new medical therapies tailored to each molecular diagnosis.
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{
"pile_set_name": "NIH ExPorter"
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The genetics of rheumatoid arthritis suggest that a combination of inherited alleles and environmental factors contribute to the development of arthritis. T cell reactivity is implicated in RA based on the linkage of RA to particular MHC alleles. Our animal model, proteoglycan-induced arthritis (PGIA) has many similarities to RA and so has the potential to provide important information on the mechanism of human disease. Similar to RA, PGIA is critically dependent on auto reactive T cell activation. In PGIA, activation of Th1 cells and the production of IFN3 are critical for development of severe arthritis. In this, the "conventional" model of PGIA, the route of antigen exposure is intraperitoneal. We demonstrate that altering the route of antigen exposure from intraperitoneal to intradermal induces a Th17 instead of a Th1 T cell response. These findings suggest that antigen presentation changes the early events in T cell polarization and that the innate microenvironment at these different tissue sites may provide unique signals that foster T cell differentiation. Very little is known about how autoimmune responses initiated at different tissue sites might affect disease. The overall goal is to understand the rules governing T cell differentiation at distinct tissue sites. Our strategy in aim 1 is to determine the cytokine environment which contributes to Th cell polarization and to determine if the immunopathology of the resulting arthritis is similar (or different) in Th1- versus Th17-mediated PGIA. In aim 2 and aim 3 we will begin to dissect the mechanism by which the route of antigen exposure contributes to T cell polarization. In aim 2 we will locate the lymphoid tissue sites where T cells are initially primed and produce cytokines and attempt to redirect the differentiating T cell population by altering the cytokine environment. In aim 3 we will identify the antigen presenting cells which drive Th1/Th17 mediated PGIA and determine the cytokines and co-stimulatory molecules involved in T cell polarization. If we can begin to understand how these T cell subsets are initiated we may be able to identify different subtypes of RA which could lead to the development of more effective therapeutic strategies for selective blockade of disease. This is an important and exciting area of investigation, which is expected to shed new light on the mechanisms of CD4+T cell mediated disease in RA, and more generally in autoimmune disease. PUBLIC HEALTH RELEVANCE: Public Health Relevance Statement Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune disease characterized by synovial proliferation and infiltrating of leukocytes which leads to destruction of cartilage and bone. Pro-inflammatory effector T helper cells have been divided into three subsets designated as T helper (Th) 1, Th2 and Th17. Th subsets are distinct subsets based on their cytokine secretion pattern, chemokine receptor expression, and clearance of infectious organisms. Models of autoimmune disease can be divided into either Th1-dependent or Th17-dependent. It is presently unclear what causes this heterogeneity in effector T cell differentiation. Based on preliminary results we hypothesize that the context in which the antigen is first encountered in the immune system determines the T cell effector choice. We hypothesize that contact with environmental antigens or infectious agents at different tissue sites predispose T helper cells to differentiation into Th1 or Th17 cells. Different tissue sites have unique cytokine and co-stimulatory microenvironments that are predisposed to the generation of specific Th cytokine phenotypes. The activation of different CD4+ T cell subsets in RA may contribute to disease heterogeneity. This proposal will elucidate the mechanism (s) by which the route of antigen exposure affect/modify fundamental T cell polarization. These studies aim to develop novel insights into the autoimmune disease process that will facilitate the diagnosis and treatment of RA.
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{
"pile_set_name": "NIH ExPorter"
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Pre-exposure prophylaxis (PrEP) refers to daily or intermittent oral administration of antiretroviral drugs designed to protect high-risk HIV-negative individuals from infection. As PrEP is, at present, the most promising biomedical prevention tool, the Centers for Disease Control and Prevention (CDC) is currently reviewing PrEP data and developing detailed public health guidelines for its safe and effective use. Any roll-out of PrEP to MSM in the U.S. will require the development of messages that facilitate understanding of partial efficacy, proper administration, and sustained risk reduction practice. Although the swift development of PrEP-specific interventions is critical, we currently lack basic knowledge about unique cognitive, affective, and behavioral dynamics that may make a significant difference in both efficacy and effectiveness of PrEP as a biomedical prevention strategy. This project applies empirical findings and theoretical models from behavioral economics, social psychology, decision-sciences, health communication, and neuroeconomics to the development of PrEP messaging and communication strategies, in order to: (1) Examine the independent and cumulative impact of four elements of PrEP messaging - modality, frame, specificity, and messenger - on comprehension and acceptability of PrEP as a biomedical prevention strategy;(2) Estimate the impact of each of these factors on individual risk-perception and predicted sexual behavior in the context of PrEP use;(3) Identify individual-level factors, (i.e. demographics, risk tolerance, health numeracy), that might moderate the impact of message factors on comprehension, acceptability, risk perception, or risk compensation;and (4) Provide specific recommendations regarding the optimal message elements and communication strategies for PrEP in order to maximize comprehension and acceptability, while positively impacting risk perception and reducing the likelihood of risk compensation. To accomplish these aims, we will enroll 800 MSM living in the New York City area and randomly assign them to experimental conditions in a 2x2x2x2 factorial design. Data collection will include ACASI measures of outcomes (comprehension, acceptability, risk perception, and risk compensation) as well as potential moderators in four areas: (a) dispositional;(b) cognitive;(c) affective/motivational;and (d) behavioral (sexual risk and substance use). In addition, we will conduct 2 focus groups with each of 5 "special interest groups" (e.g. transgender women, serodiscordant couples) in order to gather detailed data regarding the specific needs, concerns, and issues among populations most likely to be offered PrEP. The proposed project has the potential to exert a sustained and powerful influence not only on the development of PrEP-specific messaging, but also on our understanding of the cognitive and affective processes underlying comprehension of risk-reduction messages, risk perception, and risk compensation. PUBLIC HEALTH RELEVANCE: Pre-exposure prophylaxis (PrEP) is a new biomedical approach to HIV prevention with the potential to become a powerful tool. This study examines specific elements of PrEP messaging and communication hypothesized to have a significant impact on comprehension, acceptability, risk perception, and risk compensation. Study findings have the potential to move the field toward a more interdisciplinary approach to understanding the behavioral mechanisms that underlie acceptability and adherence to biomedical prevention strategies.
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"pile_set_name": "NIH ExPorter"
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This is a proposed K24 award for Dr. Frederick Hecht, Professor of Medicine at the University of California (UCSF), and director of research at the UCSF Osher Center for Integrative Medicine. Dr. Hecht is an internist with postdoctoral training in clinical epidemiology. He is director of the T32-funded UCSF Training in Research in Integrative Medicine (TRIM) fellowship program, and principal investigator of an NCCAM funded Center for Excellence in CAM Research PO1 investigating the metabolic and immunologic effects of meditation. He has mentored more than 25 postdoctoral fellows and junior faculty and published over 130 peer-reviewed articles. With this K24, Dr. Hecht aims to mentor four postdoctoral and two predoctoral fellows in the TRIM program each year, serving as primary mentor for half. He will mentor a regular panel of five to seven junior faculty with K awards. He will develop his research and mentoring capabilities with advanced seminars on research design and biostatistics. He has assembled an outstanding team to mentor the mentor in specific areas and provide additional enrichment for trainees, including Dr. Peter Bacchetti, director of the UCSF Biostatistics Consult service, and Dr. Bernard Lo, former director of the UCSF Program in Medical Ethics. UCSF provides an extremely rich and supportive scientific environment for Dr. Hecht's research and that of his mentees; including the second largest NIH funded CTSA, which is closely integrated with Dr. Hecht's K24 plans. The specific research aims of Dr. Hecht's research program are: Aim 1: Test the health benefits of mind-body interventions. Aim 2: Apply innovative approaches to test the mechanisms of action of integrative medicine therapies. Aim 3: Develop and test new models of integrative medicine approaches to lifestyle interventions. Aim 4: Develop models of integrative medicine research in medically underserved settings. Overall, Dr. Hecht's research program aims to bring integrative medicine approaches to bear on some of our most important chronic health problems, including obesity, diabetes, and hypertension. This K24 application aims to provide mentoring and a research environment to develop an exceptionally strong and diverse group of researchers who will play leading roles in the future of integrative medicine research.
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{
"pile_set_name": "NIH ExPorter"
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This program of research proposes to examine immunological and viral-immunological factors and interactions which may be involved in the pathogenesis of multiple sclerosis (MS). The laboratory models that will be utilized include acute and chronic experimental allergic encephalomyelitis (EAE) in its usual state as well as under suppression with COP I, myelin basic protein and, possibly, galactocerebroside; organotypic cultures of central and peripheral mammalian nerve tissues both as test objects for the presence and action (demyelination) of cellular and circulating immunological factors and effector (macrophage, enzyme) systems and as models of the pathological response; and the myelinated axons of the rabbit eye as the respond to the injection of components of the immunological response as they may act alone or in concert with the rabbit's response. The laboratory procedures which will be used to examine cell and serum factors from animals and man, and the models' responses include electron microscopy, immunohistochemistry, and immunological studies of suppressor T-cells. The possible viral involvement in demyelinative disorders will be examined in a number of studies including mechanisms by which viruses may interact with autoimmune disease, suppressor cell activity particularly in response to measles virus in MS patients, and the mechanism (s) of measles-induced suppression. Experimental allergic neuritis (EAN) and the Guillain-Barre Syndrome (GBS) will also be examined in studies similar to those for EAE and MS. Finally, immunological manipulations of cultured tissues will be employed in analysis of the cellular-membrane interactions in myelinogenesis.
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{
"pile_set_name": "NIH ExPorter"
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The overall goal of this project is to enhance the detection, assessment and family care of Alzheimer's disease in the rural counties of West Virginia through a state-wide educational program. The specific aims include: 1) to provide training in assessing the cognitive status of elderly clients and the adequacy of their family caregiving arrangements by health care professionals in 28 W.V. counties; 2) to provide training for clinical preceptors (medicine, nursing and social work) at eight rural health network sites; 3) to implement a training dissemination methodology which addresses problems raised in the current grant funded project; 4) to compare the education outcomes from the two projects; 5) to evaluate the patient care outcomes from the training of the local health care professionals; 6) to facilitate networking within counties between health care professionals involved with Alzheimer's disease care; and 7) to facilitate networking state-wide between health care professionals involved with Alzheimer's disease care. The methodology includes: 1) training of staff development personnel from home health, mental health, senior service, and adult protective service agencies; 2) training county professionals in 28 counties at 14 local workshops using the staff development trainees as trainers; 3) train clinical preceptors (medicine, nursing, social work) at eight Rural Health Initiative Networks sites; 3) evaluate educational outcomes at all training sites; 4) evaluate patient care outcomes from the training of local health care professionals; 6) create and disseminate 28 county resource directories for older adults; 7) create and disseminate a state- wide data base of services for older adults with dementing illnesses.
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{
"pile_set_name": "NIH ExPorter"
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Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. Mutations in splicing factor Prp8, for example, cause a severe form of human genetic disorder Retinitis Pigmentosa. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy. Splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNAs and over 100 protein factors. Many lines of evidence point to Prp8 as a key spliceosomal protein that interacts intimately with RNA at the catalytic core, potentially helping the formation and stabilization of the catalytic core. Prp8 is one of the largest and most conserved nuclear proteins known, but it does not have obvious sequence homology with any other known protein. Further structural and biochemical analyses would provide valuable insight into Prp8's function in splicing. However, these studies are hindered by difficulties in obtaining large quantities of full-length Prp8. Identifying, expressing, and purifying domains of Prp8 will provide a valuable alternative approach for characterizing Prp8. This proposal uses a unique high throughput approach to identify domains of Prp8 that can be expressed in soluble forms in E. coli and determine structures of these domains. Structures of these domains and comparison with other known structures can provide important information on the function of Prp8 in splicing, directing future mutational/genetic experiments. These soluble domains are also valuable resources for characterizing Prp8's biochemical properties, such as its interaction with RNA, other protein partners, and among different Prp8 domains. Once structural and biochemical characterizations of individual domains are completed, similar characterizations of regions of Prp8 composing multiple domains can be performed. This approach is a critical step toward generating a complete picture of Prp8 that cannot be obtained otherwise, significantly advancing our understanding of the molecular mechanisms of pre-mRNA splicing. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
}
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Escalating societal concern over the risks of exposure to extremely low frequency (ELF) electromagnetic fields has been fueled by epidemiologic studies which correlate increased incidence of leukemia or birth defects with proximity to power distribution systems. Unfortunately, the phenomenologic nature of these studies make it difficult to differentiate the pathogenic potential of electromagnetic fields from interacting factors (e.g., socioeconomic) that might as well explain an accelerated rate of disease. Conflicting reports at the basic science level have polarized the scientific community on this issue, resulting in only a weak acknowledgement of any potential hazards of 60 Hz electromagnetic exposure. Fundamentally, it is not yet clear if ELF, low intensity field exposure can modulate cell behavior, and if so whether specific characteristics of the field (frequency, intensity, duration) will differentially affect the response. Further, it is not known if a cell's interaction with the field is selective; whether fields perturb the activity of some cell types while remaining innocuous to others. To establish the extent to which ELF electromagnetic fields can influence cell behavior, it is essential to conduct a systematic, controlled study in which well defined (in both biological and physical terms) cell types are exposed to uniform fields of known frequency, intensity and duration, and the subsequent cellular response quantified. The objective of this four year study is to isolate those specific aspects of ELF electric fields (frequency, intensity, duration) which modulate cell behavior, and to identify the biophysical and morphologic properties of a cell which will promote its susceptibility to this exposure. Our preliminary results, at both the in vivo and in vitro level, demonstrate that extremely low intensity (less than 100 mu V/cm), frequency specific (10-100 Hz) electric fields can be a potent influence on both tissue adaptation and cell activity. Importantly, these results also suggest that the field:cell interaction depends not only on the characteristics of the field, but also on the size, shape, and surface characteristics of the cell which is being exposed. Ironically, it may well be this morphologic specificity which is responsible for the inconsistent and disparate reports of electric field interactions with biologic systems. We propose that the capacity of an electric field to modulate cell behavior is dependent not only on the frequency and strength of the field, but that the interaction is strongly dependent on the physical properties of the cell which is exposed. These experiments will be performed in an in vitro system in which the intensity of the induced electric field is both uniform and carefully controlled. Representative cell types of specific size, shape and surface properties will be examined. Biochemical and morphologic assays will be used to quantify electric field interactions.
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{
"pile_set_name": "NIH ExPorter"
}
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The proposed study has three central objectives: 1) an evaluation of the impact of the new Massachusetts mental health law on involuntary civil commitment of the mentally ill; 2) the preliminary development and evaluation of a "dangerous to others" assessment instrument; and 3) a survey of legal and psychological concepts of dangerousness and their relationship to social policy objectives. Subjects will be in-patients at the Massachusetts Mental Health Center, the Boston State Hospital, the Gardner State Hospital and Bridgewater State Hospital. Socio-demographic, clinical and behavioral variables will be studied with relation to their influence on decision- making for or against the release of patients. Patients will be interviewed by project staff and will be followed up one year later by direct interview and telephone. Court proceedings for subjects for whom involuntary civil commitment is sought will be observed and data collected relevant to the adequacy of the litigation, the adequacy of the protection of the patient's civil rights and the quality of the operation of the new Massachusetts mental health statute.
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{
"pile_set_name": "NIH ExPorter"
}
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Gene targeting, homologous recombination between a newly added, exogenous DNA sequence and the cognate chromosomal sequence, in mouse embryo-derived stem (ES) cells allows the specific modification of any chosen genetic locus in living mice. First, a cloned copy of the chosen gene is mutated in vitro using standard recombinant DNA technology. The modified gene is then introduced into ES cells where homologous recombination transfers the mutation, created in a test tube, to the genome of the living cell. The ES cells carrying the mutant gene are then used to generate germline chimeras. Finally, intercrosses of heterozygous siblings are used to generate mice homozygous for the mutant gene. We propose to use this technology to genetically separate the functions of a complex multifunctional gene during mouse development. We hope to achieve this goal by identifying and modifying DNA cis elements responsible for mediating the complex expression pattern of the gene during development as well as by developing an inducible switch which will allow turning on or off the gene during different periods of development. In addition, we propose to use gene targeting to develop new approaches for cell lineage and mosaic analysis.
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{
"pile_set_name": "NIH ExPorter"
}
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The ability to elicit Abs with broad neutralization activity against HIV-1 has been a long sought-after but elusive goal of HIV/AIDS vaccine research. The long-term goal of this proposal is to understand how broad-spectrum neutralizing antibodies (BnAbs) are generated. The majority of the rare BnAbs (2F5, 4E10 and b12) isolated from HIV-1-infected individuals have the common characteristics of long IGHV-CDR3s with positively charged amino acids, structural features often seen in autoantibodies. The main hypothesis to be tested in this proposal is that natural induction of such autoreactive antibodies may be curtailed by the checkpoint control mechanisms occurring during B cell ontogeny. Therefore, a system that models a human immune system, is responsive to HIV infection/immunization and has demonstratable defects in B cell checkpoint control processes is an attractive platform to test this hypothesis. The humanized BLT and GTL mouse models are generated by transplantation of 3-irradiated NOD/scid and NOD/scid IL2r?-/- mice, respectively, with fetal liver and thymus tissues in addition to autologous CD34+ HSCs. The humanized mice allow efficient engraftment of multi-lineage human hemato-lymphoid cells, support a productive HIV-1 infection, develop Ab responses upon immunization with recombinant HIV gp140 trimer and demonstrate autoimmune properties. We will test the ability of these mice to generate strain-specific nAb and BnAbs against HIV-1 upon optimized immunization with gp140 Env trimers. Towards these goals, the specific aims of this proposal are: 1) To test several approaches including the addition of human cytokines and/or activated T cells to induce optimal antigen-dependent class Ig switching and a predominant IgG response in the HIVgp140 immunized BLT and GTL models. 2) To interrogate whether neutralizing antibodies are generated as a result of optimized immunization by HIV pseudotyped virus microneutralization assays. 3) To characterize and quantitate the quality of the neutralizing antibody (nAb) response by cloning and expressing anti-HIV Envelope (Env) Abs isolated by FACS sorting of gp140-trimer binding single B cells from B1-like, conventional B (B2) and Ab secreting cell (ASC)/plasma cell subpopulations. Sequence analysis and binding/neutralization studies of Abs derived from gp140-trimer binding B cell clones will provide a detailed molecular signature of BnAbs. Overall, our studies are aimed at developing a new small animal model to understand the long-standing question of how BnAbs are generated and to set the groundwork for vaccine studies that will result in generation of such BnAb responses. PUBLIC HEALTH RELEVANCE: A major reason for the failure of HIV vaccines is the lack of induction of broadly neutralizing anti-viral antibodies in the host. We propose to utilize new humanized mouse models to study the mechanism(s) by which BnAbs are produced and the barriers that must still be breached to elicit these types of antibodies by vaccination. The results of our studies will establish a new small animal HIV vaccine model and set the groundwork for vaccine studies aimed at the generation of potent and broadly neutralizing antibody responses to HIV.
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{
"pile_set_name": "NIH ExPorter"
}
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This project is designed to investigate alternative methods of infecting Macaca nemestrina with HIV-1 to enhance pathogenesis. One method, direct inoculation of bone marrow with subsequent in vivo passage of bone marrow, was concluded early in 1998. This route of infection did not appear to enhance pathogenesis in this species. Another project was the inoculation of neonate macaques (<1 week of age), whose immature immune systems would be less likely to clear or control the infection. After the first group of infants was infected by inoculation via intravenous and intrarectal administration, a serial in vivo passage was instituted to increase the pathogenicity. To date, three pairs of infants have been inoculated as part of this in vivo passage. After infection was confirmed in the first pair, peripheral lymph nodes and blood were used to generate a virus stock for passage. This virus stock was then inoculated into the next pair of infant macaques, by intravenous an d in trarectal inoculation. The lymph node and blood harvest for virus stock was again repeated and passaged into the third pair of infants. To date, the level of viremia in the infants has been increasing with each passage, and is becoming more persistent. At the same time, all the infants undergo a series of developmental and behavioral tests, as do normal infant macaques at the Infant Primate Research Laboratory. The results of these tests will be compared with the normal standards, as developed by the IPRL, to determine subtle motor and learning delays that may be attributed to infection with HIV-1. FUNDING NIH grant RR00166.
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{
"pile_set_name": "NIH ExPorter"
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Various homologs of cannabidiol (CBD) were synthesized where the aromatic side chain was replaced by H and C1 to C9. They were prepared to study the SAR with regard to anticonvulsant activity. The syntheses were accomplished by condensing the appropriate resorcinol with p-mentha-2,8-dien-1-o1 in the presence of less than 0.5% BF3.Et2O/MgSO4 in CH2CL2 and separating the CBD's by either column or high pressure liquid chromatographic techniques.
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{
"pile_set_name": "NIH ExPorter"
}
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PRINCIPAL RESEARCH CORE a) Intervention Development Program- This Program will focus on developing innovative patientcentered interventions for mood disorders. To facilitate dissemination and implementation, we will emphasize the core values of efficiency, portability across a range of clinical settings and flexibility across diverse clinical and sociodemographic groups. During years 1-3, much of the intervention development program's work will focus on Developmental Project #1 described below. Work for this project, however, will emphasize generalizable methods, structures and procedures (e.g. common theoretical models for motivational enhancement and self-management support, common database structures for web-based programs, common logical structure for treatment algorithms, common procedures for training and supervision, common self-management content, common methods for selfmonitoring). Center scientists will contribute to the Intervention Development program as follows: [unreadable] Behavioral Science, Evette Ludman- This component of the Intervention Development Program will build on our expertise in theory-based behavioral intervention design and implementation, treatment material development, treatment fidelity, and quality assurance. During years 1-3, Dr. Ludman will adapt educational and self-management materials for the web-based intervention in Developmental Project 1. Long-term work will focus on generalizable intervention models for use across a range of mood disorders and treatment settings. Continuing previous work 55, she will also collaborate with the Methods Core to develop tools assessing the underlying mechanisms by which self-management interventions change behavior and clinical outcomes. Our self-management interventions are influenced by social cognitive and self-determination theories, and incorporate strategies of cognitive behavioral psychotherapy and motivational enhancement approaches to health behavior change. Key emphases are patient activation and patient-provider collaboration as core components of effective disease management. Goals include increasing motivation and confidence regarding self-management and developing specific self-management skills relevant to mood disorders (e.g., cognitive symptom management, goal setting and problem-solving). The guiding theories emphasize both intervention content and the counseling strategy or communication style. Self-determination theory posits that an "autonomy supportive" (vs. "controlling") style creates healthhcare partnerships that foster autonomous motivation for self-management. Autonomy support is demonstrated by the extent to which providers listen fully, take the patients' perspective, provide meaningful information, and offer choices for treatment and self-care. These specific strategies for supporting autonomy are built into each intervention. Each intervention includes an individually tailored balance on motivational enhancement, education/skills training, self-efficacy enhancement and goal setting/action planning. All work will be coordinated with the Information Technology and Applying Evidence to Practice components.
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{
"pile_set_name": "NIH ExPorter"
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The effect of retrovenous, hypothermic, diastolic myocardial perfusion (RVDP) (myocardial oxygen supply) combined with ventricular unloading (myocardial oxygen demand) will be studied in sheep subject to coronary artery ligation. Percent salvage of injured myocardium at risk and prevention of necrosis as assessed by radiolabelled microspheres, Microfil and Tetrazolium staining techniques is hypothesized to be greater in injured hearts supported by combined retrovenous diastolic perfusion and left ventricular bypass. Utilizing zero delay after coronary ligation, the optimum effect on myocardial salvage and function as measured by pressure-dimension analysis for treatment study groups of retrovenous diastolic perfusion and left ventricular bypass will be determined. Closet chest, catheter retrovenous diastolic perfusion (myocardial O2 supply) and intraaortic balloon pumping (IABP) (O2 demand) will be studied in sheep after four hours of coronary ligation to simulate the clinical situation of myocardial injury. The hypothesis will be tested that closed chest combined RVDP and IABP can be practically applied and salvage significant amounts of ischemic myocardium (combined effect greater than when either RVDP or IABP is utilized separately). Myocardial reperfusion as would occur with myocardial revasularization will also be evaluated in these sheep treated with RVDP and IABP. The verification of significant salvage or prevention of injured myocardium in sheep with closed chest combined RVDP and IABP could provide a new clinical therapy for acute coronary occlusion and treatment prior to emergency myocardial revascularization. Similarly RVDP could be utilized in certain patients undergoing cardiac diagnostic or therapeutic (percutaneous transluminal coronary angioplasty, enzymatic thrombolysis) procedures to ensure myocardial perfusion.
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{
"pile_set_name": "NIH ExPorter"
}
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During this past year we have made major progress on developing and applying several of the essential modeling methodologies for input/output, or nonparametric, models of point-process input neural systems such as the hippocampus. First, we have pursued development and application of adaptive estimation procedures for decomposition studies of the hippocampus. Using this approach, we have been able to successfully estimate linear and nonlinear properties of negative feedback elements (analogous to inhibitory interneurons) given input/output data for only the feedthrough element in the presence and absence of inhibition, i.e., without direct observations from the feedback pathway. Second, we have developed a new means for estimating input/output properties of neural elements using artificial (feedforward) neural networks, which we have demonstrated is particularly powerful for estimating higher order nonlinearities from electrophysiological data. We are particularly excited about the potential for expanding this approach for estimating higher order cross-kernels for multiple-input conditions. Third, for the first time we have applied our nonlinear systems approach at the level of intracellularly recorded synaptic potentials. We have recently modeled the closed- and open-loop AMPA and NMDA components of glutamatergic synaptic input to hippocampal neurons, have derived a representation of the dynamics of the positive feedback to the NMDA receptor-channel, and can accurately predict control EPSPs to a wide range of input patterns. In addition, the model can account for differences between closed- and open-loop NMDA receptor-mediated synaptic responses. These are two of the most widely studied receptor subtypes, as the dynamics of their interaction determine the direction and magnitude of activity-dependent synaptic plasticity in hippocampus, neocortex, and other brain regions. Finally, we have made considerable progress in developing highly efficient circuit designs for analog VLSI implementation of kernel models of hippocampal neurons, a direction that should provide the means for developing massively parallel architectures for simulating global hippocampal system properties.
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{
"pile_set_name": "NIH ExPorter"
}
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Unraveling the molecular mechanisms which promote the development of asthmatic inflammation represents one of the most challenging and important areas that need to be elucidated in order to understand this disease. We hypothesize that interactions between the innate and adaptive immune systems alter airway cells in a manner that promotes the initiation and propagation of the chronic airway inflammation associated with the differing asthmatic phenotypes. The individual projects in this program examine a diverse group of mediators, cytokines, chemokines, and airway cells. Each project will, however, relate to the central theme and share substantial commonality in the molecular mechanisms to be studied. Project 1 will define the molecular events that determine the outcome of differentiation of naive or uncommitted T cells to T helper (Th)1 or Th2 cells, and the relation of this process to the asthma phenotype. Project 2 will investigate the mechanisms underlying the novel observation that galectins stimulate chemotaxis of eosinophils and mononuclear phagocytes, possibly through direct binding to chemokine receptors. Project 3 will examine the regulatory mechanisms of lipopolysaccharide (LPS) on cytokine/chemokine synthesis in alveolar macrophages, highlighting the role of the Toll-like receptor 4 (TLR4) and its downstream signaling pathways. Project 4 will define the in vivo mechanisms by which kinins stimulate expression of chemokines and chemokine receptors in human airway epithelial cells, thereby focusing the recruitment of inflammatory cells to the airway. Three Cores are proposed for this Asthma and Allergic Diseases Research Center (AADRC): an Administrative Core A, a Clinical Core B, and an Animal Core C. A major strength of this Program Project will be the experimental design of sharing samples (bronchial biopsy and bronchoalveolar fluid) from each subject among all four projects, allowing the diverse results to be combined into a comprehensive analysis of the asthmatic phenotype. This AADRC program is designed to leverage diverse expertise, resources, and experimental targets into a focused investigation of the origins of asthmatic inflammation. Taken together these projects may characterize key molecular mechanisms that contribute to the subsequent chronic inflammatory airway milieu that is the hallmark of the asthmatic phenotype. Efforts to elucidate and then modify key events leading to asthmatic inflammation may ultimately be exploited for prevention of asthmatic disease.
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{
"pile_set_name": "NIH ExPorter"
}
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The primary objective of the Immune Analysis Core is to establish standardized assays of cellular and humoral immune responses for the immunotherapeutic approaches being developed and tested in Individual Research Projects 1, 2 and 3 of the RPCI Ovarian Cancer SPORE. It will be also be responsible for enumerating and characterizing myeloid derived suppressor cells (MDSCs) by flow cytometry for Research Projects 4. The Core will routinely provide Ovarian Cancer SPORE investigators with high quality, state-of-the- art flow cytometry, cellular monitoring by ELISPOT, proliferation and cytotoxicity assays, and multiplex cytokine bead array. The specific aims of the Immune Analysis Core are to: 1) Provide cell banking services for immune monitoring assays requiring viable mononuclear cells. 2) Provide state-of-the-art, multi-color, fluorescence activated cell sorting and analytical support for Individual Research Projects, Career Development and Developmental Research Projects. This includes high-speed sorting and multi-color analysis of cellular subsets. 3) Provide state-of-the-art ELISPOT, ELISA, proliferation and cytotoxicity assays for the functional characterization of antigen-specific T cell responses. 4) Provide state-of-the-art patient sample handling and assessment of immune responses, with standardized SOPs, quality assurance, quality control and data management practices to monitor assay performance and validate data quality. In addition to these established immunoassays services, the Immune Analysis Core will be instrumental in developing new methodologies and making them available to the SPORE investigators. It also has a significant educational role, working with all SPORE investigators including PI's, technicians and young investigators on their assay development, proper use of instrumentation and interpretation of their data.
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{
"pile_set_name": "NIH ExPorter"
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Project Abstract The primary objective of this proposal is to develop and commercialize a safe and more effective therapy for the treatment of Crohn's Disease, an inflammatory bowel disease. Crohn's Disease is a chronic inflammatory disorder of the gastrointestinal tract that affects some 500,000 patients in the US. Selexys Pharmaceuticals is developing a fully human anti-PSGL-1 antibody drug directed against P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin on circulating leukocytes that binds selectins and chemokines, for the treatment of Crohn's Disease. PSGL-1 mediates the first step in the recruitment of leukocytes to sites of inflammation by binding to selectins, cell adhesion molecules that are upregulated in Crohn's Disease patients. PSGL-1 also binds chemokines that amplify the inflammatory response through chemotaxis of inflammatory cells to endothelial cells that line the gut. In preclinical studies in animal models of Crohn's Disease, anti-PSGL-1 antibodies have been shown to block recruitment of leukocytes resulting in a profound therapeutic effect. Thus anti-PSGL-1 antibodies represent a novel new potential therapeutic approach in the treatment of Crohn's Disease. In Phase 1 of this Fast Track Proposal, Selexys is proposing to conduct primate safety and toxicity studies in cynomolgus monkey with a human anti-PSGL-1 IgG2 antibody that has been engineered to lack effector function and complement binding. Pharmacokinetic (PK), pharmacodynamic (PD) and immunogenicity bioassays in support of this proposal are currently being developed by Selexys. Manufacture of drug substance to support the proposed preclinical studies and clinical trials is underway at Cytovance Biologics in Oklahoma City. Selexys is proposing to conduct a PK pilot dose ranging study in cynomolgus monkey (Macaca fascicularis) to assess safety and PK of the anti-PSGL-1 antibody. The pilot study will guide dosing plans to conduct an IND enabling 4-week IV repeat dose toxicity study in cynomolgus monkey. In Phase 2 of this application Selexys proposes to conduct a Phase I safety study in healthy volunteers leading to a Phase IIa safety and efficacy trial in Crohn's Disease patients. The primary goal of the proposed study is to characterize the antibody prior to commencing a Phase IIb six months chronic dosing trial. The overarching goal of the program is to develop and commercialize a humanized antibody to PSGL- 1 to treat Crohn's Disease patients. In doing so Selexys will address an inflammatory bowel disease with major unmet medical needs with a therapy that may be safer than current approved biologic therapies and that has the potential to sustain remission and/or ameliorate the devastating effects of this disease.
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{
"pile_set_name": "NIH ExPorter"
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The purpose of this Core is to provide the high degree of technical expertise required to perform and nterpret advanced MR imaging studies, to facilitate the collection of image guided surgical specimens and to relate the quantitative information derived from the imaging data to molecular markers of the biological properties of the tissue. It should be stressed that the many of the procedures required to achieve this objective are non-standard, requiring specialized knowledge, cutting edge equipment and sophisticated experimental or computer analysis in order to extract the information necessary for testing the hypotheses associated with each of the Scientific Projects. The decision to create a single Core that would integrate the maging and tissue analysis reflects the high priority placed upon acquiring image guided samples that can provide a direct link between the parameters extracted from the non-invasive metabolic and physiologic maging data and the ex vivo markers of biological behavior. The first function of the Core is to ensure that the clinical and research MR scanners at UCSF that will be used for the patient studies have the necessary pulse sequences, radiofrequency coils and analysissoftware to routinely acquire reliable and reproducible imaging data. It will also define the most appropriate imaging protocols for each of the Projects and provide radiological expertise for consistent interpretation of the data. A key component of the study is the quantitative analysis of the imaging data. The Core will contribute to this by making available specialized post-processing software packages for registration of MR and CT images, generation of maps of relevant metabolic and physiologic parameters, as well as software tools for visualization and quantitative analysis. To facilitate the interpretation of the data it will maintain a database of parameters extracted from the imaging data that is linked to the relevant clinical endpoints. The Core will be responsible for all aspects of the acquisition and processing of image guided tissue samples. This will include ensuring that they are processed and archived correctly, as well as performing the molecular and morphological analysis with immunohistochemical techniques. It will provide the expertise in pathology for interpretation of the processed tissue sections and extraction of appropriate quantitative parameters or ordinal scores. For correlation with the MR parameters and clinical endpoints the staff in the Core will maintain a tissue inventory and database of associated molecular parameters that can be linked to the clinical and imaging databases. Finally the Core will provide consistent oversight that will ensure the stringent compliance with regulations governing informed consent and patient confidentiality, as well as management of the images and specimens.
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{
"pile_set_name": "NIH ExPorter"
}
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These experiments have been initiated to characterize embryonic stem cells, especially human embryonic stem cells (hESC), for studies of human dopaminergic neuronal function. In spite of the great importance of dopaminergic neurons for drug abuse, in addition to their therapeutic potential via transplantation, there have been enormous obstacles to the direct study of human dopaminergic neurons. Until very recently such neurons have been obtainable only from human fetal material. The advent of human ES cells has made the derivation human dopaminergic neurons in vitro in unlimited quantities for research use a possibility. Our initial experiments were focused on mouse ES cells, and promoter systems which could be employed for transgene expression. It was found that the CMV and especially the EF1 promoter led to long-term stable transgene expression in ES cells. Interestingly, expression via both the EF1 and CMV promoter disappeared as the ES cells differentiated into dopaminergic neurons, using an established system for dopaminergic differentiation by stromal cell-mediated differentiation. In addition, promoters derived from the tyrosine hydroxylase and GAD65 genes are now being investigated. Since the aim of these studies involved primarily the use of human ES cells, and there was very little data in the literature concerning characterization of most of the lines in the NIH stem cell registry, we therefore determined to characterize the general properties of the hESC lines from BresaGen Inc., in terms of pluripotency, differentiation, stable maintenance, and gene expression patterns. Small and large scale oligonucleotide and cDNA arrays were also employed to characterize gene expression patterns in undifferentiated hESC. Experiments were also undertaken to obtain dopaminergic neuronal differentiation from hESC. It was found that co-culture with the stromal PA6 cell line induced dopaminergic differentiation with a defined and reproducible time course. Cells positive for tyrosine hydroxylase were first detected after 10 days of co-culture, with maximal numbers of cells positive for tyrosine hydroxylase being present after 20-23 days of co-culture. Differentiated dopaminergic neurons expressed a number of markers for mature dopaminergic neurons, including transcripts characteristic of neurotransmitter function and response to growth factors. Most colonies in each culture were found to contain dopaminergic cells after differentiation, although a minority of cells within each colony were dopaminergic and other cell types including non-CNS cells (e.g., cells positive for smooth muscle actin) were also present. After transplantation into the brain, some dopaminergic neurons were found to survive, although numbers of non-neuronal cells (e.g., smooth muscle actin positive cells) were much larger than numbers of CNS cells. Therefore, significant improvements will be needed before dopaminergic neurons derived from hESC can be employed for therapeutic transplantation. In addition, a number of experiments were performed to identify the factors which are responsible for stromal cell-mediated differentiation. Factors identified as possible contributors include hepatocyte growth factor and FGF8; however, these factors alone are not sufficient to induce dopaminergic differentiation. We have also identified a variant hESC line, BG01V, with karyotypic abnormalities, which can be grown more easily than the normal BG01 hESC line, but which undergoes dopaminergic differentiation with the same pattern and time course as BG01. This cell line may be very useful for identifying the factors responsible for stromal cell-mediated dopaminergic differentiation. Dopaminergic neurons derived from hESC were found to be sensitive to the dopaminergic neurotoxin MPP+, providing a cellular model through which neuroprotective agents can be tested in human cells. The neurotrophic factor GDNF was found to be protective against MPP+ toxicity, and increased expression of the dopaminergic transcription factor Nurr 1. We have recently also completed a comprehensive characterization of gene expression in neural stem cells and dopaminergic neurons during differentiation from hESC using the Massive Parallel Signature Sequencing (MPSS) technique. For these experiments, differentiating dopaminergic neurons were isolated by fluorescent-activated cell sorting on the basis of polysialyated N-CAM expression on cell surfaces. These sorted cells were found to be capable of complete differentiation, as indicated by neuronal function defined by electrophysiological criteria. Current experiments are focused on developing genetically altered cell lines for specific purposes, such as monitoring the status of differentiation via reporter transgenes, and employing hESC-derived dopaminergic neurons to characterize the effects of drugs of abuse on human dopaminergic neuronal function. In addition, alternative protocols for producing differentiated human neurons from hESC are being developed; including testing effects of differentiation-promotion agents and alternative protocols for cell-mediated differentiation. These alternative differentiation protocols can be used to produce cell types which are useful for studies of drugs of abuse on specific human neuronal phenotypes and also for neuronal transplantation protocols.
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{
"pile_set_name": "NIH ExPorter"
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Asthma and allergic rhinitis, the most common chronic diseases of childhood in the United States, are major public health problems. Characterized by variable airflow obstruction and airway inflammation, childhood asthma is thought to have its origins in fetal and infant development. Environmental exposures influencing oxidative balance during critical time windows may have long-lasting effects on child airway and immune function, epigenetic programming of inflammation, and consequent risk of symptomatic asthma. In Project Viva, increased wheeze risk in the first two years of life was associated with higher fetal life exposures to sources of oxidative stress/inflammation (adiposity, cigarette smoke, traffic pollution, and acetaminophen). Conversely, higher prenatal maternal antioxidant dietary intake reduced early-life wheeze risk. With 12 years of longitudinal data, Project Viva has the best design to assess whether these fetal exposures have long-lasting adverse or, in the case of antioxidants, protective effects against asthma or airway inflammation that persist into adolescence. We hypothesize the following: (1) At age 12, lower fetal life exposure to dietary antioxidants and higher fetal life/early childhood exposure to these sources of oxidative stress/inflammation will (a) increase risk of allergic rhinitis and active asthma; and (b) be relatd to intermediate age 12 phenotypes including differential DNA methylation of nasal cells, fractional exhaled nitric oxide (FeNO), and airflow obstruction. (2) Differential DNA methylation of nasal cells will relate, not only to allergic rhinitis, but also to FeNO and active asthma. Finaly, (3) The relation of fetal life maternal dietary antioxidants and sources of oxidative stress/inflammation with nasal and pulmonary outcomes at age 12 will be captured by the following biomarkers in cord blood (a) Pro- and anti-inflammatory biomarkers in innate- and adiposity-related pathways [soluble TNF-? receptor-II, IL-6, and C-reactive protein; leptin and insulin-like growth factors I and II] and (b) Differential methylation of cord blood in genome-scal scans by the Illumina 450K BeadChip. We will validate the function of top nasal cell methylation marks on gene expression in the same nasal cell specimens. As well as performing external replication, we will externally validate the function of the top methylation marks in cord blood and nasal cells on gene expression in the Asthma BRIDGE project - a biorepository with methylation and gene expression data from blood, pulmonary macrophages and bronchial epithelium. By defining the longitudinal progression of epigenetic, inflammatory, and heterogeneous clinical respiratory responses to modifiable exposures influencing oxidative balance, this study will make a unique contribution to development of strategies for prevention and targeted treatment of asthma and allergic disease.
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{
"pile_set_name": "NIH ExPorter"
}
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Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths among American Indian and Alaska Native (AI/AN) populations. Further, AI/ANs are twice as likely to be diagnosed with advanced rather than localized CRC compared to non-Hispanic Whites, suggesting that lack of access to adequate cancer screening and treatment may play a role. Yet, few studies have tested culturally appropriate interventions to increase CRC screening among AI/ANs. Our research team has developed and tested a novel intervention package to promote CRC screening in an urban and underserved Asian minority population. This intervention, which involved a health educator, a culturally-tailored video, and a pamphlet, was remarkably successful: 70% of the intervention group were screened compared to 28% of the control group. The goal of the proposed study Is to culturally adapt this successful CRC screening intervention with AI/AN patients. We will modify the media for conveying the screening intervention by using digital stories, a new media methodology that capitalizes on the tradition of Native storytelling. The CRC screening intervention will be implemented in partnership with the only 2 urban clinics in Washington State that serve the AI/AN population: the Seattle Indian Health Board and NATIVE Health of Spokane. Our specific aims are to: 1) Estimate the effectiveness of digital stories accompanied by an educational pamphlet to improve CRC screening rates in AI/ANs;2) Using qualitative methods, examine the adoption, implementation, and maintenance of our CRC screening intervention at these 2 urban Indian clinics. Today, over 55% of AI/ANs live in urban areas, yet urban AI/AN people have been called the "invisible minority." Because neither the Indian Health Service nor the National Center for Health Statistics systematically collects information on this group, the needs of AI/ANs living off reservation lands are rarely recognized and they are under-represented in research. To our knowledge, this will be the first CRC screening intervention targeting urban AI/ANs.
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{
"pile_set_name": "NIH ExPorter"
}
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A robust Pilot and Feasibility Program will be an integral portion of the CONSORTIUM FOR HEREDITARY CAUSES OF NEPHROLITHIASIS AND KIDNEY FAILURE. The Rare Diseases Consortium will serve as an active incubator for novel ideas in nephrolithiasis research and patient care. Plans are to fund 2 projects at all times for the duration of this award. For this purpose, $100,000 has been budgeted each year, allowing approximately $50,000 per pilot project. One of the two pilot projects will be funded by money pledged from the Mayo Clinic Rochester research committee. The primary focus will be to support new protocols with high potential for advancing patient care amongst one or more of the patient cohorts in our consortium. Proposals from junior investigators will also be given special consideration for funding, although the program will also be open to established investigators as a means of encouraging them to develop new projects or avenues of research within this field. Projects with a high potential to produce preliminary data making them competitive for additional extramural funding will also be given higher priority. In years 1-2, we will fund a pilot project on developing technology for harvesting viable hepatocytes from livers removed at transplant. This project synchronizes well with aims of Project 1, and will open the door towards novel model systems and potential therapies for PH. The second pilot project will be funded by the Mayo Clinic Research Committee ($50,000 per year for 5 years pledged). These funds expand the overall resources and scope of the Rare Diseases Consortium. All proposals will be selected by the full Rare Diseases Consortium Pilot Project Committee, using a rigorous selection processed. Several possible candidates for future Pilot Projects have been identified. However, we have intentionally elected to not select this additional proposal at the time of submission. Instead, we will issue a new RFA and select the most promising proposal at the time of potential grant funding. In this manner, we will maintain flexibility and not lock the consortium into specific areas, but instead allow the possibility that novel areas can be supported that emerge over the next 12 to 18 months.
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{
"pile_set_name": "NIH ExPorter"
}
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Patient engagement in HIV care encompasses a cascade of events from HIV diagnosis to retention in care. Each step is vital in achieving the goal of virologic suppression and improved health outcomes. Compromising this clinical imperative is the nearly 50% of HIV-infected individuals in the US who are not in continuous HIV care. The HIV epidemic poses a particularly severe problem in Hispanic populations. While Hispanics comprise 16% of the US population, they comprise 20% of new HIV infections. Immigrants make up 42% of new HIV diagnoses in Hispanics. Emerging interventions to improve retention in HIV care in the general HIV-infected population have not been adequately studied in Hispanic immigrants, a growing segment of the population where risk for poor outcomes is a precarious reality. Factors specific both to Hispanic cultures and to immigrant status are essential to highlight for effective intervention development. A better understanding of these factors could lead to improved, tailored approaches to retention in HIV care for Hispanic immigrants, improvement in health outcomes, and a reduction in ongoing HIV transmission. CANDIDATE: My specialized training in clinical infectious disease, public health, and Hispanic studies, coupled with extensive resources at Harvard University and Massachusetts General Hospital (MGH), uniquely position me to accomplish these aims. I have five peer-reviewed research articles directly related to HIV outcomes and the consequences of adherence to care in resource-limited settings, and extensive clinical experience caring for HIV-infected individuals as well as immigrants. RESEARCH: I will conduct formative qualitative research with Hispanic immigrants infected with HIV and their medical providers to understand factors (e.g. individual preferences, structural barriers) and agents (e.g. role of the family) that influence decision-making around retention in primary HIV care. I will give specific attention to generating novel intervention ideas to improve retention in HIV care through these in-depth interviews with patients and the multi-disciplinary providers within an HIV provider team including physicians, nurses, social workers, case managers, and community health workers (Aim 1). I will expand the Andersen Model of Health Services Use to include conceptual models of health disparities in order to evaluate societal, clinic/system- level, and individual determinants of HIV care utilization. Guided by this conceptual model, formative research, and community stakeholder feedback, we will identify the most significant and modifiable factors that determine retention in HIV care for the study population in order to inform the development of an intervention. We will develop and pilot test a culturally-tailored intervention (Aim 2). The intervention will target Hispanic immigrants who are not retained in HIV care, and for whom there are limited culturally relevant interventions. We will intervene at a particularly vulnerable period in the HIV care cascade, retention in care, where care interruption poses substantial consequences for both the individual, due to risk of clinical decline and risk-taking behaviors, and for public health, due threat of resulting HIV transmission. Standardization of the intervention will occur through iterative refinements based on participant and community advisory board feedback. Through a focused randomized pilot trial (Aim 3), we will further refine study procedures and perform preliminary power analyses in preparation for a larger, more definitive test of the intervention directed through submission of an R01 proposal at the end of the award. TRAINING/MENTORING: My long-term career goal is to become an independent investigator with expertise in developing culturally-tailored community-based interventions to improve outcomes in HIV and other chronic diseases for vulnerable populations. I will need rigorous training in methodologies that will enable me to fully capture factors, such as culture and context, and processes, such as acculturation, that influence use of HIV care for Hispanic immigrants. Training in community-engaged methods will facilitate effective development and deployment of novel interventions in the community setting. To complement my prior training in clinical care and HIV outcomes research, I have outlined a plan for intensive training in community-based participatory research, qualitative research, behavioral theory, and intervention development. This training will occur through graduate-level coursework as well as through intensive supervision with my mentors, who are internationally- recognized leaders in areas of research directly related to the proposal. My mentorship team also has a strong foothold in community-based partnerships for research and HIV care delivery in Massachusetts and nationally. Furthermore, this project advances the institutional strategic priorities of the CDC, NIMH and the Office of AIDS Research, notably, to reduce HIV health disparities and to improve medical care for people living with HIV. The successful completion of this research will position me as an expert in retention in care for HIV-infected Hispanic immigrants, and more generally will enable me to address challenges in vulnerable populations burdened with chronic illnesses that require innovative models for disease management.
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{
"pile_set_name": "NIH ExPorter"
}
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This proposal will evaluate the immunoregulation that develops during adoptively transferred or actively induced Experimental Allergic Encephalomyelitis (EAE). The analysis will involve donor contribution to the disease process, variables associated with the obligatory in vitro expansion of a transfer active cell population, and recipient participation in the development of paralytic EAE following cell transfer. The studies will define the activity and point(s) of control expressed by acquired or induced suppressor cells and will test the hypothesis that EAE effector cell activity is not controlled by the usual patterns of immunologic regulation. Planned experiments will also determine the relationship of the restriction imposed on cell interactions, as defined by the Major Histocompatibility Complex (MHC), to the restrictions observed in adoptive transfer of EAE. A similar analysis of MHC regulation in an autoimmune disease will be conducted with various inbred strains of rat as well as irradiation induced chimeras (employing the Brown-Norway (BN), Lewis (L) and LXBN F1 hybrid for chimera production). These latter studies of the chimera response will determine if actively induced disease, compared with adoptively transferred disease, is MHC restricted or if the initial requirements/restrictions for immune cell interaction are distinct from those MHC defined restrictions of target tissue destruction.
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{
"pile_set_name": "NIH ExPorter"
}
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The last months of pregnancy are particularly important for the development of the child's brain, and the consequences of premature birth on its development can be substantial. Prematurely born children are at higher risk of various cognitive impairments and exhibits more behavioral disorders than full-term born children. Thus early detection and management of at risk children are essential. There is growing evidence of significant volumetric abnormalities in subcortical structures of premature neonates, which may be associated to negative long-term neurodevelopmental outcomes. Understanding these abnormalities could help elucidate the underlying pathophysiology and enable early determination of at-risk patients, both of which would inform the design of novel treatment strategies. However, to date there is still a lack of sensitive, reliable, and accessible algorithms capable of characterizing the influence of prematurity on the anatomy of neonatal brain subcortical structures. In addition, few studies have looked directly at the long-term neurodevelopmental implications of these neonatal subcortical structures abnormalities. Predicting long-term neurodevelopmental outcomes early on ? and preferably at neonatal ages ? is likely to have a transformative effect on their outcome. Our preliminary data indicate significant morphological differences in the putamen, ventricles, corpus callosum, and thalamus between preterm and term neonates. We propose to develop biomarkers of prematurity by statistically comparing the morphological and diffusion properties of subcortical structures between preterm and term neonates using brain MRI. These results will further be used in a sparse learning framework to predict long-term neurodevelopmental outcomes of prematurity. Hypotheses: By combining subcortical morphological and diffusion properties, we will be able to: (1) delineate specific correlative relationships between structures regionally and differentially affected by normal maturation and different patterns of white matter injury, and (2) improve the specificity of neuroimaging to predict neurodevelopmental outcomes earlier. Aim 1: Build a new toolbox for neonatal subcortical structures analyses that combine 1) a group lasso-based analysis of significant regions of shape changes, 2) a structural correlation network analysis, 3) a neonatal tractography, and 4) tensor-based analysis on tracts. Aim 2: Ascertain biomarkers of prematurity in neonates with different patterns of abnormalities. Aim 3: Assess the predictive potential of imaging and clinical features on neurodevelopmental outcomes among premature children at 12 and 18 months and 6-8 years of age. Impact: This application will provide the first complete subcortical network analysis in both term and preterm neonates. In the first study of its kind for prematurity, we will use sparse and multi-task learning to determine which of the biomarkers of prematurity at birth are the best predictors of long-term outcome. The expected findings could improve our ability to predict these outcomes and enable the design of early treatments ? before years of pathological brain development and symptoms occur.
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{
"pile_set_name": "NIH ExPorter"
}
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Sepsis represents the systemic response to infection, and the ensuing inflammatory cascade leads to the development of multi-organ failure and high mortality. The acute respiratory distress syndrome (ARDS) represents a process of devastating lung injury that complicates sepsis and contributes to propagation of the host inflammatory response long after the initial infection has been treated. ARDS results in loss of surfactant, alveolar collapse, and refractory hypoxemia. While mechanical ventilation is necessary to support patients with ARDS, ongoing damage to the lung is inevitable with delivery of high airway pressures to injured lung parenchyma. Thus, novel molecular strategies that target amelioration of lung injury through preservation of surfactant expression hold promise for improving overall outcomes from sepsis. It has long been proposed that significant surfactant loss occurs through protein destruction by nitric oxide (NO) and its metabolites. Excessive production of NO during sepsis and ARDS results from upregulation of NOS2 expression in parenchymal and inflammatory cells. We and other investigators reported a novel mechanism by which NOS2- derived NO interferes with surfactant expression and propagates lung dysfunction. In a murine model of endotoxemic lung injury, we demonstrated that NOS2-derived NO downregulates expression of surfactant protein-B in lung epithelial cells. Although systemic NOS2 inhibitors have not improved survival in human sepsis, and exogenous surfactant replacement has not shown benefit in ARDS, we postulate that the ability to modulate localized NOS2 expression in the lung represents an exciting approach toward restoring "renewable" surfactant expression during ARDS. Our overall hypothesis is that NOS2-derived NO in lung epithelial cells plays a critical role in propagation of lung injury during sepsis through modulating expression of surfactant proteins. To test our hypothesis, we propose three Specific Aims. In AIM 1, we will determine the role of NOS2 in a murine model of sepsis-induced lung injury that mirrors human ARDS. Wild type and NOS2-deficient mice will be subjected to cecal-ligation and puncture (CLP) and antibiotic treatment, followed by low tidal volume mechanical ventilation. We will assess NOS2 expression, lung physiology, cellular inflammation, vascular leak, cytokine levels, surfactant function and expression of surfactant proteins. In AIM 2, we will examine the direct effect of NOS2 expression in the lung epithelial cell on surfactant expression and function during acute lung injury. We will selectively modify SPB and/or NOS2 expression within the lung epithelial cell using two transgenic animals and appropriate controls. In AIM 3, we will explore the role of NOS2-derived NO on regulation of human surfactant protein-B expression. We will perform transfection analyses, in vivo binding studies (chromatin immunoprecipitation assays), and S-nitrosylation assays in human and murine transformed cell lines and in primary murine distal lung epithelial cells isolated from wild-type and NOS2-deficient mice. PUBLIC HEALTH RELEVANCE: Sepsis represents the systemic response to infection, and the ensuing inflammatory cascade leads to the development of multi-organ failure and high mortality. The acute respiratory distress syndrome (ARDS) represents a process of devastating lung injury that complicates sepsis and contributes to propagation of the host inflammatory response long after the initial infection has been treated. Achievement of the AIMS of this proposal hold significant promise for gaining novel insights into the pathobiology of sepsis-induced ARDS that can inform the development of new treatment strategies for these devastating syndromes.
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{
"pile_set_name": "NIH ExPorter"
}
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Although Alzheimer's Disease (AD) is characterized classically by dementia associated with a variety of neuropathologic features, it is likely that there is no single pathologic change that alone is the basis for the cognitive and behavioral impairment. In this proposal, we hypothesize that the pathogenetic mechanism for dementia involves quantitative and variable neuronal cell body and synaptic losses occurring concurrently in multiple brain structures to reach a threshold of loss that results in dementia, especially when combined with the aging process. To substantiate multifactorial origins of dementia, we will identify losses that occur in the brains of patients diagnosed as having dementia due to AD by comparing them with the brains of cognitively normal aged subjects. Consecutive series of tissue sections will be prepared form neuronal regions known to be involved in cognitive and behavioral function, including the hippocampal complex, amygdala, portions of the frontal and temporal lobes, and the nucleus basalis of Meynert. As a marker of synaptic terminals, we will validate and extend the use of antibodies to synapsin. Using these immunohistochemical methods and standard histochemical techniques, we will identify losses in the number and/or density of synaptic terminals and neuronal cell bodies. To determine the extent of neural loss necessary and sufficient to produce dementia, we will quantify deficits in AD patients compared to controls using an image analysis system. Starting in year 2, we will collaborate with other MADRC laboratories to expand the data set by quantifying pharmacologic and biochemical losses in AD. We will statistically analyze the accumulated data with a multivariate approach to characterize the cumulative neural losses in multiple loci that result in the dementia of AD.
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{
"pile_set_name": "NIH ExPorter"
}
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Lung cancer is the leading cause of cancer-related death in the United States and is responsible for over 160,000 patient deaths from their lung cancer this year, more than that of breast, colorectal, prostate, colon and other cancers combined. With the existing therapeutic efforts, patients with lung cancer have a 5-year survival rate of less than 15%. There is a tremendous unmet need for treatment of Non-Small-Cell lung cancer, represent 85% of all lung cancer patients. Efforts have recently been directed toward developing novel therapies based on a growing understanding of molecular oncology. Understanding the molecular mechanisms involved in the pathogenesis of lung cancer can provide opportunities to develop innovative therapies for NSCLC. In this proposal, we proposed a novel multi-targeted siRNA therapeutic for NSCLC treatment. We will test this drug candidate in A549 and H460 xenograft tumor models for its antitumor efficacy and safety profiles. We will also use HKP and PTL nanoparticle to further enhance the this siRNA cocktail therapeutics, targeting EGFR, VEGF, Cox-2 or PDGF oncogenic genes. Furthermore, we test this drug in combination with current targeted therapeutics of small molecules and monoclonal antibodies, such as Tarceva, Iressa and Avastin, in the animal models, for an alternative therapeutics to treat NSCLC. PUBLIC HEALTH RELEVANCE: Lung cancer is the leading cause of cancer-related death in the United States and is responsible for over 160,000 patient deaths from their lung cancer this year, more than that of breast, colorectal, prostate, colon and other cancers combined. There is a tremendous unmet need for treatment of Non-Small-Cell lung cancer, represent 85% of all lung cancer patients. In this proposal, we use a novel RNAi technology approach to develop a multi-targeted siRNA therapeutics for NSCLC treatment. The HKP and PTL nanoparticle technologies will greatly enhance systemic delivery of this siRNA cocktail therapeutics, targeting EGFR, VEGF, Cox-2 or PDGF oncogenic genes. Combining this siRNA drug with current targeted therapeutics of small molecule and monoclonal antibodies will provide us a powerful weapon to fight this deadly disease, NSCLC.
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{
"pile_set_name": "NIH ExPorter"
}
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The information required for adequate diagnosis, treatment and monitoring of cancers is so complex that a panel of measurements, used in sum, may provide the best answers. The concept is embodied in SELDI-TOF mass spectrometric (MS) peptide profiling, an emerging technique for serum based cancer detection. Even though SELDI has thus far only produced low complexity spectra, the patterns, when analyzed as groups, have the potential to create learning algorithms with diagnostic accuracies as good as or better than conventional biomarkers. We have developed a system to capture peptides on magnetic reversed-phase beads, followed by MALDI-TOF MS, to yield increasingly complex, yet very reproducible patterns. This has clear advantages, as more displayed peptides provide more opportunity to select unique patterns ('barcodes') for cancer subtypes and stages, and to predict and monitor clinical outcome. Extreme care has also been taken to standardize specimen collection, handling and storage to avoid the introduction of artifact. Pilot projects at MSKCC with a variety of malignancies suggest that peptide patterns thus obtained appear to hold information that may have direct clinical utility. The goals of this project are to (i) automate our prototype serum peptide profiling platform and implement machine learning methods that use the resulting peptide patterns ('barcodes') for sample classification [R21]; and (ii) to test the 'barcode diagnostic' model in a high-throughput setting, using well defined and carefully observed groups of thyroid carcinoma patients [R33]. R21 aim one is to automate serum sample processing and analysis; aim two is to automate all data processing, to examine pattern selection and sample class prediction methods, and to integrate all software platforms; aim three is to develop routine MALDI-TOF/TOF tandem MS sequencing of 'barcode' peptides. R33 aim one is to define reproducibility of serum patterns in patients with thyroid disease; aim two is to determine barcodes that can distinguish patients with thyroid cancer from those with benign thyroid nodules; aim three is to assess if serum peptidome barcodes can identify occult metastasis in a large group of thyroid cancer survivors. [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
}
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Normal activity of salivary glands is controlled by neural stimulation and recent studies on rodents suggest that experimental stimulation with synthetic catecholamines, especially isoproterenol (ISP), leads to alteration of salivary gland structure and function. This study is proposed to determine the role of the Beta-adrenergic activity, elicited by ISP, on the morphological development and biochemical differentiation of the major salivary glands, and therefore, demonstrate a neural control mechanism in their development. ISP treatment of postnatal rats will begin prior to the period in which the salivary glands show the greatest amount of development. The effects of the Beta-adrenergic antagonists propranolol will also be studied after treatments comparable to those of ISP. Morphological development of the parotid, submandibular, and sublingual glands will be determined by electron microscopic studies and biochemical differentiation will be determined by monitoring changes in protein patterns in the glands by acrylamide gel electrophoresis. It is anticipated that excessive Beta-adrenergic stimulation by ISP will induce exaggerated changes in the glands representative of discrete cellular changes which progress more slowly in normal development. Inhibition of normal development by propranolol will strengthen the supposition that Beta-adrenergic stimulation plays an important role in controlling development of the major salivary glands.
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{
"pile_set_name": "NIH ExPorter"
}
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The Sexually Dimorphic Nucleus of the Preoptic Area (SDN-POA) of the rat hypothalamus is a morphological marker of the process of sexual differentiation of the brain. The differentiation of this nucleus (in terms of volume) takes place during the neonatal life of the animal. In addition, this process is influenced by the gonadal steroid environment during the early postnatal period. 30% of the presumptive SND-POA neurons can be specifically identified during development by administering tritiated thymidine on day 18 of gestation to the pregnant female. We have utilized this fact in order to determine the migratory pattern of immature neurons as they form the SDN-POA during the first ten days of postnatal life. In the proposed studies, the ability to specifically label presumptive SDN-POA neurons will allow the SDN-POA to be used as a model system in order to continue to investigate the interaction of the gonadal steroid environment of the perinatal animal with the morphological development of the central nervous system. Thymidine autoradiography, light and electron microscopy, and catecholamine histofluorescence will be used in order to investigate the following questions. 1) What is the morphology of SDN-POA neurons and the surrounding neuropil? 2) Do gonadal steroids influence SDN-POA neuronal morphology during differentiation by affecting neurogenesis, neuronal migration, neuronal death and/or neuronal organelle formation? 3) What is the time course of development of the sexual dimorphism of catecholamine varicosities in the SDN-POA? 4) Can the short-tailed opossum be used as a model animal in order to describe the development of the SDN-POA during the 'prenatal period'? The results of this research program will help explain the mechanisms by which the hormonal environment influences a) neuronal growth and development and b) the sexual characteristics of an individual.
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{
"pile_set_name": "NIH ExPorter"
}
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DESCRIPTION: This proposal is directed at understanding the requirements for proper chromosome segregation during mitosis. Using a clever screen to detect mutations which lead to increases in ploidy, the PI previously isolated a conditional mutation in the IPL1 gene which has significant sequence homology with a number of kinases implicated in chromosome segregation in higher eucaryotes (e.g., human Aur1, Drosophila "aurora"). He has done a great deal of work characterizing this (and related) genes which is described in the Progress Report. This previous work motivates the work proposed in the current grant, and thus will be briefly reviewed. He has demonstrated that the IPL1 is an essential gene., and that it is required in each cell cycle. Mutants missegregate chromosomes, but do not arrest thus suggesting they have a defect in a mitotic checkpoint. In order to find possible substrates for the putative kinase, he employed a genetic technique: he screened for mutations which were lethal in the presence of tsipl1 mutation at permissive temp (they couldn't lose the wild type IPL1 gene on a URA3 plasmid). This careful search generated 14 mutations; it was initially impossible to classify them into complementation groups because they exhibited unlinked noncomplemention. However, he characterized their phenotypes with respect to chromosome segregation, etc., and cloned 4 of them. They turned out to be: TUB3 and TUB1 (the alpha subunit of tubulin), CIN8 (one of 2 yeast kinesins), and BUB1 (one subunit of a checkpoint kinase). All of these can be related to chromosome segregation, and seem reasonable candidates for target proteins. He isolated two high copy suppressors of tsipl1: one is a dominant negative fragment of the gene GLC7(PP1) that encodes the catalytic subunit of protein phosphatase1;the second was GLC8, a regulator subunit of PP1. The data obtained is consistent with the model that PP1 acts as an antagonist of IPL1, and that GLC8 can act to activate or inhibit PP1 depending on its concentration. All the data presented, and it includes both biochemical assays and genetic tests, is consistent with this hypothesis. For example, a mutation of GLC8 or O/E of GLC7(PP1) lead to a missegregation phenotype similar to that express in the ipl1 mutant. He proposes that IPL1 phosphorylates a target(s) necessary for proper chromosome segregation, that PP1 inactivates the target, and suggests 3 cogent models which will test their interaction and which are be tested in the proposal. All the experiments in this proposal are aimed at understanding the role of the Ilp1 kinase (and to some extent, the PP1 phosphatase) in chromosome segregation fidelity. He proposes to determine the location of the Ilp1 kinase; he suggests that it may be located on microtubules. Since the protein is present at relatively low abundance, he presents several alternative approaches, including in vitro assays to determine association with microtubules. He proposes experiments to examine the checkpoint in ipl1 mutants; these include experiments to investigate kinetochore separation done with Andrew Murray. Extensive experiments are proposed to understand the relationship of the Ilp1 kinase, alpha-tubulin, Cin8 kinesin, and Bub1/3 kinase (to determine if they are the targets). These include direct measurements of the phosphorylation state of the putative targets in WT and ipl1 cells. The PI suggests three different in vivo approaches (with appropriate controls), as well as a in vitro assay for target proteins that give positive results above using purified Ilp1 kinase. He has already partially purified the Ilp1 kinase. A third approach makes use of an altered 2hybrid system. Since the interaction with the Ilp1 kinase with a substrate is likely to be transitory, he proposes making a mutation which, in other related kinases, maintains protein stability but eliminates kinase activity. (In the case of Human Cdk2 kinase, the mutant binds its substrate but cannot phosphorylate it and release it.) He will combine the mutant ipl1 with gal4DB, and fuse the potential targets with the galAc domain. The experiments will also be done with IPL1. He suggests that should the conserved mutation act as proposed, it might be a dominant negative mutation; O/E suppressors of the dom neg ipl1 mutation might identify target genes. A series of experiments are proposed to investigate the relationship between PP1 and Ilp1 kinase. He will ask whether Ilp1 kinase is dephosphorylated by PP1, and if so, whether that event affects its activity. He will determine if Ilp1 phosphorylates the regulatory subunit of PP1 (encoded by GLC8). The precedent from mammalian cells suggests specific residues that may be targets, and further suggests that these might interfere with activation of GLC8 by other kinases. These experiments include techniques analogous to the ones above, and include both in vitro and in vivo approaches. The PI already has purified PP1 and has partially purified Ilp1 kinase. He will also ask, if Ilp1 kinase acts on certain target proteins, whether PP1 dephosphorylates them. The final work proposed is planned for during the last 2 years of the grant. The PI suggests that the other sli mutants isolated may identify target or regulators of IPL1. An alternative explanation would be that the sli mutations lead to an overproduction of PP1. He suggests experiments to test all three hypotheses, as well as the cloning and characterization of the wild type sli genes.
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{
"pile_set_name": "NIH ExPorter"
}
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As a working hypothesis a model of protein folding has been developed. The three-dimensional screened precession data of crystals of Nuclease-T (an enzymically active, non-covalent complex of two fragments of staphylococcal nuclease, residues 6-48 and 49-149) bound with deoxythymidine-3'-5'-diphosphate (pdTp) and Ca2 ion have been collected at 2-4 degrees C to beyond 2.7 A (see the report of the previous year). Phases were calculated for the observed data using both the coordinates of the intact enzyme bound with pdTp and Ca2 ion, as reported by M.J. Legg (1977, Ph.D. dissertation, Texas A & M Univ.) and the same model partially refined against the nuclease-T data (by collaboration with G.H. Cohen, LMB, NIAMDD). The nuclease-T molecule with pdTp and Ca2 ion can be fully traced in these maps and is highly similar to the intact nuclease model. Cytochrome c synthetase catalyzing the formation of thioether bonds between heme and yeast apocytochrome c is found in a yeast mitochondrial fraction. The activity of this enzyme markedly increases by addition of NADP(H) or NAD(H) (the reduced form being more effective than the oxidized) and a heat stable postmitochondrial factor. The yeast enzyme can catalyze also the covalent bonding of heme to horse apocytochrome c.
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{
"pile_set_name": "NIH ExPorter"
}
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This phase I/II study evaluated the safety, tolerance, pharmacokinetics and antiviral activity of the anti-HIV antisense oligonucleotide GEM 91 administered by continuous intravenous infusion for up to 14 days. The drug infusion produced transient elevation of hepatic transaminases in a few subjects, transient mild thrombocytopenia in some and sterile superficial thrombophlebitis in one subject. No significant reduction in HIV plasma RNA was seen with this study treatment. The study is now closed and results are being analyzed for publication.
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{
"pile_set_name": "NIH ExPorter"
}
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