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The goal of this project is the bioproduction of the anticancer natural chemical perillyl alcohol and the flavoring ingredient carveol from the write citrus-derived monoterpene limonene. The enzyme used in the conversion is a novel limonene hydroxylase enzyme cloned from a Bacillus steorothermophitus moderate thermophile which utilizes limonene as a sole carbon source. The MSU Mass Spectrometry Facility has been vital to the success of this project.	{ "pile_set_name": "NIH ExPorter" }
1. We have examined the possibility that daily blood pressure rhythms are produced by variations in the production of prostanoids. Comparing circadian blood pressure and heart rate rhythms in wild type and COX2-/- deficient mice by radiotelemetry we found that absence of COX2 did not alter the robust cardiovascular rhythmicity observed in wild type mice. 24 hour mean arterial blood pressure tended to be higher in COX-2-/- than WT mice (127 plus/minus 4 vs. 119 plus/minus 3 mm Hg; p=0.136). Plasma volume was not different between WT and COX-2-/- mice. Mean arterial blood pressure was not significantly altered by either low or high Na diets in WT mice (114 plus/minus 4 mm Hg at low Na, and 114 plus/minus 2 mm Hg at high Na). In COX-2-/- mice, the low Na diet did not significantly alter blood pressure (130 plus/minus 7 mm Hg) whereas a high Na diet elevated blood pressure by about 10 mm Hg (142 plus/minus 5 mm Hg) in the night time. Glomerular filtration rate was lower in COX-2-/- mice regardless of genetic background. Thus, whereas the expression of COX2 modifies the blood pressure response to salt intake, it does not seem to play a major role in the generation of circadian blood pressure or heart rate rhythms.[unreadable] [unreadable] 2. The most widely used method to assess circadian rhythms is to determine the locomotor activity using running wheels and counting of wheel revolutions as measure of activity. We have developed this method as a complementary approach to determinations of blood pressure and heart rate rhythms. Wheel running activity is examined in cages equipped with stainless steel running wheels (Mini-Mitter, Oregon). Since photic input is a strong determinant of rhythmicity the lighting conditions have to be considered. When located in the animal room running activity can be assessed under conditions of a 12h light/12 h dark lighting cycle. In order to assess true endogenous rhythms independent of light cues, experiments have to be performed under conditions of 24 hour darkness. We have set up housing conditions permitting light control independent of the room lighting by using a ventilated box with its own light controls. Running wheel revolutions are quantified by a magnetic reed switch next to the wheel, and data are stored on a PC using Vital View software (Mini-Mitter, Oregon). We are currently collecting data from an 8 day period and analyze these data with Matlab software (The Mathworks, Inc. ME) and the Clocklab Toolbox (Actimetrics, IL). We have used this approach to further examine the locomotor behavior of NKCC1-deficient mice using 3 female WT and 3 female mutant animals. We found that wheel running activity is dramatically depressed in NKCC1-/- mice. Furthermore, depiction of running data in double-plotted actograms indicates that wheel running activity in NKCC1-/- mice showed either absence of significant periodicity or a greatly reduced period strength as indicated by the low Qp values whereas running in WT mice occurred in a robust circadian fashion with a period length of about 24 h as dictated by the light cycle. These data are unexpected since the shaker-waltzer behavior of circling and head bobbing characteristic for inner ear abnormalities creates the impression of hyperactivity in NKCC1-/- mice. We are currently developing a method of tracking mouse movements using a camera and computer-assisted motion analysis.	{ "pile_set_name": "NIH ExPorter" }
The effect of progesterone on gonadotropin secretion is stimulatory or inhibitory depending on the dose of progesterone used and the time of administration in the estrus cycle or to the estrogen-primed castrated rat. Progesterone does not alter gonadotropin secretion in the castrated rat in the absence of estrogens. An animal preparation has been developed in our laboratory using low dose estradiol treatment which induced progesterone sensitivity but does not bring about endogenous LH surges. A single injection of progesterone results in the stimulation or suppression of gonadotropins depending upon the dose of progesterone. Other models using the adult rat and the use of progesterone, 17Alpha-hydroxyprogesterone and their 5Alpha and 20Alpha reduced metabolites will be developed to bring about stimulatory, inhibitory or divergent patterns of FSH and LH release. The effect of these steroids on the secretion of LHRH by the hypothalamus will be evaluated by measuring medial basal hypothalamic LHRH, blood LHRH and serum FSH and LH. Methods for measuring LHRH turnover will be explored and sites of synthesis located by immunocytochemical techniques and quantified by RIA after isolation of specific areas by the punch technique described by Palhowits. The functional correlation between estrogen receptors, progesterone receptors, LHRH binding sites and the pituitary sensitivity to LHRH will be examined during different patterns of FSH and LH release. The effect of progesterone on cytoplasmic and nuclear estrogen receptors, their molecular forms, ability to translocate to the nucleus, nuclear retention, and replenishment will be studied in detail. Similar studies will be done with progesterone receptor in attempts to determine the effect of estrogen as well as progesterone on progesterone receptor dynamics.	{ "pile_set_name": "NIH ExPorter" }
A major impetus exists for the identification of structural brain anomalies in children with developmental disorders of higher cerebral function, to provide potential data on cerebral reorganization, as well as data upon which to base hypotheses regarding brain-behavior correlations in young children. In vivo evidence of structural anomalies may also demonstrate that some developmental abnormalities of higher cortical function reflect deviant brain organization rather than delayed maturation. A net computerized method of brain morphometry based upon high- resolution magnetic resonance imaging (MRI) is utilized in this project to provide an in vivo neuroanatomic evaluation of children prototypic for subtypes of developmental language disorders (DLD) and autistic spectrum disorders (ASD). Three analytic routines are applied to the MRI data for external validation of the behaviorally- and neuropsychologically-defined DLD and ASD: 1). Volumetric computation of whole brain and individual substructures; 2). Recognition of regional abnormalities of proton density as a function of signal intensity heterogeneity; 3). Geometric and shape characterization of brain structures and focal abnormalities. It is hypothesized that each subtype of DLD and ASD is characterized by a morphometrically distinguishable pattern of localized structural brain abnormality when compared to age- and sex-matched children with mental deficiency and control children with headaches. The prototypic abnormalities will be further correlated with the electrophysiologic and genetic characteristics defined by these respective projects.	{ "pile_set_name": "NIH ExPorter" }
SUMMARY: In 2016 a total of 224,390 patients in the US were diagnosed with non-small cell lung cancer (NSCLC) and 16% of these patients (35,902) were diagnosed as early stage (I and II) and eligible for adjuvant cytotoxic chemotherapy (adj chemo). However, more than 50% of these patients may have low risk disease and hence may not receive added benefit from adj chemo, while suffering its side-effects. From an economic standpoint, unnecessary adj chemo for early stage NSCLC results in a loss of over $35,000 for each quality- adjusted life year lost. With increased lung cancer screening, we can expect an increase in diagnosis of early stage NSCLC. Two large completed randomized clinical trials of NSCLC (International Adjuvant Lung Cancer Trial (IALT) and JBR10) involving surgery with and without adj chemo, only found survival benefit in higher stage patients (>=Stage III). Unfortunately there are currently no validated predictive companion diagnostic (CDx) tools to identify (1) which stage II NSCLC are at a lower risk for disease recurrence and hence will not receive additional benefit from adj chemo and (2) which stage 1A, 1B patients are at elevated risk and hence will benefit? Extant genomic assays have only been shown to be prognostic (i.e. they predict mortality or recurrence) in early stage NSCLC, 1?5, but this does not imply they are predictive (i.e. they do not predict treatment response). Recently, our group validated the computerized histologic risk predictor (CHiRP), an approach that relies solely on computer extracted morphologic measurements (e.g. cellular orientation, texture, shape, architecture) from standard H&E tissue slide images to predict early recurrence in early stage NSCLC. CHiRP has been shown to be prognostic with an accuracy>85% in three independent clinical cohorts (N=290); higher compared to what has been previously reported for molecular based prognostic tests. However, to show that CHiRP is predictive, we need access to randomized clinical trial data involving early stage NSCLC patients treated with surgery and surgery+ adj chemo. The only two trials that fit these criteria are IALT and JBR10. Since molecular tests are tissue destructive, validation is more difficult compared to a tissue non-destructive approach like CHiRP; clinical trial groups are often reluctant to share tissue blocks since it is a valuable resource. For this study we have obtained preliminary approval for use of the slide images from IALT and JBR10 to establish CHiRP as a predictive Affordable Precision Medicine (APM) solution. This Academic-Industry partnership will leverage long-standing collaborations between (1) the Madabhushi group at Case Western who bring expertise in computational histomorphometric imaging, (2) the Velcheti group at the Cleveland Clinic (CCF) with clinical expertise in treatment and management of early stage NSCLC, and (3) Inspirata Inc., a cancer diagnostics company which has recently licensed a number of histomorphometry based technologies from the Madabhushi group and who will bring quality management systems and production software standards to help create a pre-commercial CHiRP test.	{ "pile_set_name": "NIH ExPorter" }
The ubiquitin-proteasome system is believed to play a vital role in regulating proteins involved in maintaining genomic integrity. Genetic and biochemical studies in yeast and mammals have demonstrated that several ubiquitin conjugating enzymes and ubiquitin protein ligases including Ube2A, Ube2B, Ube2rE, Ube2N, BRCA1/BARD1, CDH1/APC are essential for the cellular response to DNA damage. However, the physiological substrates for these enzymes remain unknown. We have recently developed a unique signaling-based functional proteomic system that can specifically identify genome-wide ubiquitin-degraded proteins in response to DNA damage. This novel strategy will lead to the identification of substrates for these critical enzymes involved in the surveillance of genomic integrity. In addition, this combinatorial approach will result in the discovery of new regulatory ubiquitin components pivotal to the control and progression of cell cycle, initiation of transcription, DNA repair and apoptosis in response to genotoxic stress. The goal of this proposal is (1) to develop a large-scale in vitro protein expression technology that allows the synthesis of approximately 30,000 full-length proteins and (2) to apply this newly developed technology to identify genome-wide ubiquitin-degraded proteins in response to DNA damage signaling and (3) to characterize the role of identified proteins in maintaining genomic integrity. Taken together, the proposed work will test the hypothesis that ubiquitin-mediated circuitry is essential to ensure genomic stability. This investigation should fill significant gaps in the DNA damage checkpoint signaling pathway, and provide important insights into genomic stability regulated by ubiquitin. Given the importance of proteolytic regulation in human diseases, information gained from the proposed studies will be of direct relevance to biomedicine, including the identification of novel targets for biochemical agents that could interfere with the regulation.	{ "pile_set_name": "NIH ExPorter" }
Epithelial-stromal interactions are thought to play a critical role in maintaining normal corneal homeostasis, and in regulation of corneal wound healing following injury and refractive surgery. For example, following PRK, injured epithelial cells release several cytokines and growth factors that modulate stromal cell death, proliferation, and differentiation. While the importance of the cross-talk between corneal epithelial and stroma cells is clear, a more detailed investigation of the role of specific cytokines in mediating epithelial-stromal interactions requires an in vitro model in which culture conditions can be tightly regulated. Unfortunately, current in vitro models do not adequately mimic the in vivo 3-D geometry and mechanical properties of corneal tissue. Two of the key barriers to successful tissue engineering of an in vitro anterior corneal construct are: (1) the development of a 3-D extracellular matrix which has high mechanical stiffness, yet supports maintenance of the quiescent stromal cell (keratocyte) phenotype, and (2) the development of a corneal epithelial cell line that differentiates and stratifies in vitro. We have recently performed pilot experiments in which cell-seeded 3-D collagen matrices were compressed to achieve high stiffness tissue equivalents. These constructs support differentiation of corneal keratocytes, and have several unique properties which may be ideal for stromal tissue engineering. We have also developed a unique human telomerase-immortalized corneal epithelial cell line (hTCEpi), which expresses key differentiation markers under stratified, air-lifted culture conditions. The purpose of the proposed research is to test whether we can combine these two unique technologies to produce a novel 3-D model that can be used for studying epithelial-stromal interactions during homeostasis and wound healing. To accomplish this, we propose to: (1) determine whether compressed collagen matrices can be used to support the differentiation and stratification of corneal epithelium under serum-free conditions in vitro, and (2) investigate the feasibility of inducing injuries to these constructs in order to simulate anterior corneal wound healing. Accomplishing these aims would establish the overall feasibility of using compressed collagen matrices as a model system for studying epithelial-stromal interactions during homeostasis and wound healing, and thus provide a foundation for more detailed studies in which the specific factors mediating these critical interactions can be investigated. PUBLIC HEALTH RELEVANCE: Epithelial-stromal interactions play a critical role in normal corneal function and in regulation of corneal wound healing following injury and refractive surgery. The goal of the proposed research is to develop a novel, multi-layered 3-dimensional culture model that can be used to investigate these interactions under controlled, in vitro conditions.	{ "pile_set_name": "NIH ExPorter" }
This proposed 5-year development period will be used for research and training activities that will give Dr. Steven Y. Hong the skills to become an independent investigator. Dr. Hong's long-term career goals are: 1) to become an independent researcher with a faculty position at Tufts University School of Medicine conducting HIV clinical and translational research in resource-limited settings; and 2) to explore optimization of care of HIV-infected patients in resource-limited settings with a special emphasis on improving the delivery of antiretroviral therapy (ART). His short-term goal is to develop a sustainable model of tracing ART patients who have become lost from care that can be applied in resource-limited settings to minimize the emergence of HIV drug resistance and optimize patient outcomes. This training will include formal class study, a research project, and research mentorship from experts in international HIV clinical research, ART program optimization, epidemiology, biostatistics, geographic information systems (GIS), and health systems research. The training will focus on: 1) specialized epidemiologic methods; 2) data management and statistical analysis; and 3) GIS technology. The Division of Geographic Medicine and Infectious Diseases at Tufts Medical Center and the Tufts Clinical and Translational Science Institute are well known for their strong commitment to biomedical research and career development of early-stage investigators. Along with the GIS Center at Tufts University, this institution is an ideal environment for the proposed training activities. The candidate has assembled a team of outstanding mentors with varied and relevant expertise to provide guidance in his career development: Dr. Christine Wanke in international HIV clinical research, Dr. Michael Jordan in ART program optimization in resource-limited settings, Dr. Norma Terrin in biostatistics, Patrick Florance in GIS, and Dr. Philip Odonkor in health systems research. The clinical research will take place in Namibia, in collaboration with the University of Namibia Medical School and the Namibia Ministry of Health and Social Services. Loss to follow-up (LTFU) is currently one of the largest impediments to achieving ART treatment success in resource-limited settings where an average of 21% of patients are classified as LTFU in the first six months after starting ART, and up to 25% at 12 months. As such, interventions to reduce attrition from ART programs are of the utmost importance. Defaulter tracing is a common method utilized in these settings to contact and link patients who have interrupted care or treatment back into care and decrease LTFU. Optimizing methods for defaulter tracing may be a critically untapped minimal-resource intervention which could lead to high yield results. Intensifying defaulter tracing to daily tracin with the goal of linking patients back into care within 48 hours of a patient being without medication has the potential to reduce LTFU and significantly improve ART treatment outcomes. This project seeks to determine the efficacy of intensifying the tracing of patients on ART in Namibia who have become lost to care to daily tracing, in order to decrease LTFU rates and improve patient outcomes (population ART adherence, virologic suppression, and mortality). First, predictors of and reasons for LTFU will be identified in a pilot tracing study o 400 patients. Using these data to design and optimize the intervention, a cluster randomized controlled trial will be conducted in Namibia of 8 patient tracing intensification sites compared t 8 standard-of-care sites (3,200 total patients) in regards to the proposed outcomes. The Specific Aims of the project are as follows: (1) To identify predictors of and reasons for LTFU; (2) To determine the efficacy of patient tracing intensification in decreasing rates of LTFU (primary outcome), compared to standard- of-care defaulter tracing; and (3) To determine the efficacy of patient tracing intensification in improving clinical and virologic outcomes (secondary outcomes), compared to standard-of-care defaulter tracing. This research has the potential to identify a minimal-resource strategy that can be applied throughout Namibia and other resource-limited settings to address the challenge of patient retention, thus reducing morbidity and mortality in the millions of people on ART globally. PUBLIC HEALTH RELEVANCE: High rates of loss to follow-up (LTFU) of patients on antiretroviral therapy (ART) in resource-limited settings can lead to the emergence of HIV drug resistance, treatment failure, and death. This project will assess predictors of and reasons for LTFU and determine the efficacy of intensifying the tracing of ART patients in Namibia that have become lost to care, in order to decrease LTFU rates and improve patient outcomes. This research is relevant because it has the potential to identify a minimal-resource strategy that can be applied throughout Namibia and other resource-limited settings to decrease LTFU, thus reducing morbidity and mortality in the large number of people receiving ART in resource-limited settings.	{ "pile_set_name": "NIH ExPorter" }
Little is known about how the size of the primordial follicle pool at birth affects the rates of follicular growth/atresia and the timing of the onset of estrous cyclicity in postnatal life. This application proposes to use several mouse models to test the hypotheses that: the number of primordial follicles at birth is determined by the number of germ cells present in embryonic life, and the initial size of the primordial follicle pool at birth affects the rate of follicular growth/atresia in the neonatal and prepubertal periods so that the number of primordial follicles at the onset of maturity have become normalized (returned to normal levels). The specific aims for this project are to determine whether (1) a genetically-induced excess of primordial follicles at birth is due to a surfeit of germ cells and/or somatic cells during embryonic life; and evaluate whether an above number of primordial follicles at birth increases the rates of follicular growth/atresia and the onset of cyclicity in postnatal life, (2) a genetically-induced deficiency of primordial follicles at birth is due to a deficiency of germ cells and/or somatic cells during embryonic life; and evaluate whether a below normal number of primordial follicles decreases the rates of follicular growth/atresia and the onset of cyclicity in postnatal life, (3) a chemically-induced deficiency of primordial follicles at birth affects the rates of follicular growth/atresia and delays the onset of cyclicity in postnatal life, and (4) the genetic and chemical manipulations affects primordial follicle numbers via common mechanisms. The results of the proposed studies will provide critical information about the embryonic factors that establish the size of the primordial follicle pool. It also will clarify how the size of the primordial follicle endowment affects the rates of follicular growth/atresia in postnatal life and the timing of the onset of puberty.	{ "pile_set_name": "NIH ExPorter" }
Pain associated with knee osteoarthritis (OA) is characterized by (a) high levels of variability among individuals;and (b) modest associations with radiographic measures of tissue damage. Ethnic background is one important factor that contributes to variation in pain reports and pain-related behavior. Recent studies indicate that African-American, compared to white, individuals exhibit lower pain tolerance levels and relatively impaired pain regulatory mechanisms (e.g., blood pressure responses to stressors). There also is evidence that endogenous opioid release contributes to the function of these mechanisms. The overall aim of the proposed study, then, is detemine whether changes in opioid neurotransmission evoked by thermal heat stimulation partially mediate ethnic differences in thermal pain responses among patients with knee OA. Thirty-six men (18 African-American, 18 white) with knee OA will undergo PET bain imaging with the opiodergic radioligand [18F] fluoroethyl-diprenorphine under two conditions: (a) exposure to thermal heat stimulation, tailored to individual pain thresholds, that will produce similar, moderate, levels of thermal pain intensity across patients;and (b) exposure to thermal heat stimulation that will produce perceptions of warmth across patients (sensory control condition). Patients will produce visual analogue scale (VAS) ratings of pain intensity and unpleasantness following stimulation. We expect that African-American, compared to white patients, will (a) produce significantly greater increases in pain unpleasantness ratings;and (b) exhibit lower opioid neurotransmission in medial pain system structures (e.g., amygdala, anterior cingulate cortex) from sensory control to painful thermal heat stimulation, even after controlling for psychosocial variables such as depressive symptom levels. We also expect that stimulation-evoked change in opioid neurotransmission in medial pain system structures will partially mediate the ethnic group difference in change in pain unpleasantness ratings. We anticipate that the proposed research will generate new sttudies that will lead to (a) improved understanding of additional physiologic or psychosocial variables that contribute to ethnic differences in pain responses and endogenous pain regulatory function;(b) development of new or refinement of current pharmacologic or behavioral/psychosocial interventions for pain management;and (c) reductions in ethnic disparities in patients'expectations of and preferences for these interventions.	{ "pile_set_name": "NIH ExPorter" }
Vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty play a central role in cardiovascular diseases, the current leading cause of death in the western countries and the predicted number one killer worldwide by 2020, but the underlying molecular mechanism is poorly understood. Using RNA differential display analysis in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and age- and gender-matched Wistar Kyoto rats (WKY), we have identified a novel gene, named hyperplasia suppressor gene (HSG) which encodes a protein of 757 amino acids and is widely expressed in various rat tissues. We have also cloned human and mouse HSG genes, which share 95.2 and 98.4 % sequence homology, respectively, with rat HSG (rHSG). The expression of rHSG is markedly downregulated in hyperplasic SHR VSMCs relative to normal WKY VSMCs. In addition, rHSG expression is also overtly down-regulated by proliferation-stimulating factors such as platelet-derived growth factor (PDGF)?Cbasic fibroblast growth factor (bFGF), and endothelin-1 (ET-1) in cultured primary WKY VSMCs. Enforced expression of rHSG using adenoviral gene transfer markedly inhibits growth factors-mediated VSMC proliferation by inhibition of ERK1/2 MAPK activation and subsequent cell cycle arrest in G1/Go, and reduces balloon injury-induced neointimal formation by 90% in vivo, thereby preventing balloon injury-associated restenosis. Thus, we have identified and characterized a widely expressed and highly conserved novel gene, HSG, which exhibits an important role in regulating VSMC proliferation, and might provide a potential molecular target for the treatment of vascular proliferative disorders.	{ "pile_set_name": "NIH ExPorter" }
Imaging based dosimetry for individualized internal emitter therapy Summary/Abstract The current standard practice for internal emitter therapy rarely involves pre-treatment planning to optimize the radiation absorbed dose to the tumor while avoiding critical organ (typically bone marrow) toxicity. This is in stark contrast to planning for external beam therapy where precise absorbed dose calculations to tumor and surrounding organs are now mandatory. Such therapy optimization has not been used in therapies such as radioimmunotherapy (RIT) primarily because accurate internal emitter dose estimation is relatively difficult and because clinical studies thus far have failed to show a consistent relationship between radiation absorbed dose and effect. These past studies typically relied on suboptimal methods of dose estimation and/or did not account for biologic factors that are also expected to affect outcome. The long-term goal is clinical implementation of effective individualized treatment planning for radionuclide therapies such as I-131 tositumomab RIT in non- Hodgkin's lymphoma (NHL). The objective in the present application is to develop accurate imaging based methods for tumor and bone marrow dosimetry and to develop predictive models for tumor response and bone marrow toxicity incorporating key dosimetric factors as well as biologic factors, such as differential proliferation, radiosensitivity and sensitivity to the unlabeled antibody, that will be determined by biomarker studies. The central hypothesis is that integrating dosimetry and biology will enable better prediction of RIT outcome than is obtained with the strictly dosimetric measures commonly used. The hypothesis will be tested in refractory NHL patients undergoing I-131 RIT in standard clinical care and in frontline patients on a phase II clinical trial. To accomplish the objective of this application the following specific aims will be pursued: 1) Develop SPECT reconstruction methods that utilize CT-image information (without needing explicit segmentation of target boundaries) to more accurately estimate the 3-D activity distribution in targets, because dose heterogeneity impacts the effect of the therapy;2) Develop imaging based bone marrow dosimetry coupling SPECT/CT with Monte Carlo radiation transport and accounting for variations in marrow composition as determined by quantitative CT;3) Using patient data develop a multivariate regression model for predicting therapy outcome (response, toxicity) based on dosimetric factors and biologic factors from biomarker studies (e.g., Ki-67, p53 and FLT3-L from immunohistochemistry/immunoassay);and 4) Develop a mechanistic model (with and without modification for low dose hyper-radiosensitivity) to determine the equivalent biologic effect for predicting therapy outcome. The proposed work is innovative because unlike past studies focusing purely on dosimetry this work combines dosimetric factors with biologic factors to arrive at the optimal model for individualized treatment planning. The contribution is highly significant because once the methodologies and predictive models for treatment optimization are established, they will be used in the future by physicians for patient selection and to tailor RIT on a patient-by-patient basis to considerably improve the efficacy of the treatment. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it is expected to provide the clinicians with the methodology and predictive models essential for future radioimmunotherapy (RIT) treatment optimization based on accurate pre-therapy dosimetric calculations and results from widely available biomarkers. The efficacy of RIT is likely to improve substantially with such individualized planning, hence the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge and the application of that knowledge to extend healthy life and reduce the burdens of illness and disability.	{ "pile_set_name": "NIH ExPorter" }
The adult central nervous system (CNS) has a very limited intrinsic ability to regenerate after injury. Traumatic brain injuries or neurodegenerative diseases cause neuronal cell leads to long-lasting functional impairments. The profound effect observed from these injuries results from a series of events that occurs immediately following injury and persists for several weeks. These events take place not only at the intrinsic level via intracellular signaling pathways but at the extrinsic level as well. Recent evidence indicates that in order to promote significant regeneration in the damaged CNS, a combinatorial approach addressing both the intrinsic and extrinsic barriers to regeneration is necessary. Our goal is to understand how modification of the post-injury microenvironment will affect nerve regeneration. Specifically, our hypothesis is that transplantation of neural progenitor cells (NPCs) into the retina combined with administration of an epidermal growth factor receptor (EGFR) inhibitor via an engineered construct will enhance nerve regeneration in an optic nerve axotomy model. This hypothesis is based on two observations: (1) NPCs can differentiate into neurons and incorporate into existing neural networks, and (2) administration of EGFR inhibitors to damaged optic nerve and spinal cord result in regeneration of injured neurons. We will use an optic nerve crush injury model to determine the effectiveness of our treatment. At the site of injury, we will implant a polymer construct allowing controlled delivery of the EGFR inhibitor, followed by injection of NPCs into the vitreous of the eye. Our specific aims are to: (1) develop and characterize a drug delivery system for the controlled release of the EGFR inhibitor, (2) develop and characterize a construct for in vivo delivery of the inhibitor at the injury site, and (3) assess the effects of a combinatorial treatment approach on nerve regeneration in vivo in an optic nerve injury model. Combinatorial treatments provide a promising new option for therapy in CNS repair. We believe this approach in conjunction with cell transplantation therapy will provide important information regarding nerve regeneration in the mature >CNS. Accomplishing the specific aims outlined will provide an understanding of nerve regeneration and the glial scar microenvironment in vivo. Relevance: The central nervous system has very limited repair capabilities. This limitation is due to neuronal cell death and a post-injury cellular environment that is not favorable to regrowth of nerves. A combinatorial treatment of transplantation of neural progenitor cells and administration of an epidermal growth factor inhibitor can address both of these factors simultaneously, thus providing a new avenue for therapy.	{ "pile_set_name": "NIH ExPorter" }
In order to establish an in vivo function of Saccharmoyces cerevisiae DNA polymerase II (a probable homologue of mammalian DNA polymerase epsilon) various temperature-sensitive II mutants were isolated by the plasmid shuffling method. It has been shown that the temperature-sensitive mutants (ts9, 18, and 31) have not only temperature-sensitive DNA polymerase II activity, but also exhibit temperature-sensitive chromosomal replication in vivo. Furthermore, 3'-5' exonuclease-deficient DNA polymerase II mutants exhibit high spontaneous mutation frequency, although chromosomal replication is not affected by these mutations. These results are consistent with the notion that DNA polymerase II participates in chromosomal replication in yeast. These results strongly support our current three DNA polymerase model that is able to explain how the three DNA polymerases participate in yeast chromosomal replication. The CDC7 gene is required for the last step before initiation of chromosomal replication in the yeast Saccharomyces cerevisiae. To understand the regulatory mechanism of initiation of chromosomal replication, we have isolated the gene on a multicopy plasmid which suppresses temperature-sensitive cdc7-1. This gene is identical to DBF4 which we have previously isolated by complementation with a cell cycle mutant dbf4. Furthermore, we find that CDC7 on a multicopy plasmid also suppresses temperature-sensitive dbf4-l and dbf4-2. From these results, we concluded that DBF4 gene product directly interacts with CDC7 gene product (a serine-threonine protein kinase). Finally, we have isolated and sequenced three new genes which suppress temperature-sensitive dbf4-1 mutant cells and characterized them extensively.	{ "pile_set_name": "NIH ExPorter" }
Stroke, silent cerebral infarct (SCI), and cognitive impairment are frequent and highly morbid complications of sickle cell disease (SCD) in children. Current approaches to the prevention and treatment of neurological complications of SCD include screening by transcranial Doppler ultrasound (TCD) to identify children with elevated cerebral blood flow velocity who are at increased risk for strokes; these children are then typically treated with chronic transfusions indefinitely. Hydroxyurea (HU) reduces the frequency of painful crisis, acute chest syndrome and transfusion and may have beneficial effects on central nervous system (CNS) complications of SCD. The safety of HU in infants and children has been demonstrated recently in a NIH-sponsored phase III trial; however, the exact indications for the use of HU in children remain unclear, as well as its efficacy in preventing CNS complications of SCD. Our preliminary data suggest that, if the cumulative frequency of abnormal TCD, SCI and stroke could be reduced by 50%, the majority of pediatric hematologists would prescribe HU to all young children with SCD. The long term goal of this project is to perform a primary prevention trial to demonstrate the neuroprotective effect of HU and broaden the indications for HU in children. The goals of this proposal are to: 1) conduct a feasibility trial demonstrating the acceptability of a randomized trial of HU to reduce the CNS complications of SCD; 2) demonstrate that sedation for MRIs can be safely performed in young children with SCD using a standardized protocol; and 3) create the leadership, network of clinical centers and other procedures necessary to conduct a definitive phase III trial demonstrating the efficacy of HU for primary prevention of neurological complications of SCD. The primary endpoint for the feasibility and definitive phase III trials will be the development of abnormal TCD, SCI or stroke. To begin the feasibility trial, we have obtained CTSA support for pilot studies at Johns Hopkins and Washington University; over the next two years, these sites will screen 40 participants 12-48 months of age and randomly assign and follow 20 participants for two years. Two additional centers (Children's Hospital of Philadelphia and the University of Alabama, Birmingham) will begin enrollment during the course of the R34 (20 patients screened and 10 participants randomly assigned per site), to provide a total of 80 participants screened, 40 randomly assigned, and a minimum of 70 participant years of follow-up. Participants must have TCD measurements that are well below the threshold for transfusion and MRIs that are without evidence of SCI. Participants in the pilot studies will continue into the proposed R34 and phase III trials, to complete 3 years on HU or placebo. The information from the feasibility trial is necessary to demonstrate the safety and practicality of a definitive phase III trial. The results of these studies could lead to true primary prevention of CNS complications of SCD, including abnormal TCD, SCI, neurocognitive impairment and stroke. In doing so, this study could also reduce the burden of chronic transfusions and change clinical practice by broadening the indications for HU.	{ "pile_set_name": "NIH ExPorter" }
The role of factors within the primary familial environment is one of the critical problem areas in the understanding of factors contributing to the development of adult psychopathology, particularly the schizophrenic and sociopathic disorders. This research program concentrates on three lines of experimental investigations in collateral but related areas which are aspects of an overall integrated research program. The first is an examination of parent-child interaction in families of non-psychotic disturbed children in a variety of controlled situations designed to identify the idiosyncratic interaction stresses within the family. Their behavior to these stresses is examined with a view toward identifying those components which may contribute to the shaping of particular coping patterns characterizing adult schizophrenics. The cohort of families includes some who are at risk for developing schizophrenia. The second area is the assessment under relatively controlled conditions of factors in the intra-familial relationships which may differentiate groups of disturbed adolescents who vary in types of presenting problems and symptoms. The third phase of this research is concerned with developing methods for early detection of adolescents at risk for adult pathology, and for early intervention in a way which will increase their effectiveness as adults. BIBLIOGRAPHIC REFERENCES: Goldstein, M.J. and Jones, J.E. Adolescent and familial precursors of borderline and schizophrenic conditions. In P. Hartocollis (Ed.), The Menninger Conference on the Borderline Syndrome. New York: Brunner-Mazel, in press, 1976. Goldstein, M.J., Rodnick, E.H., Jones, J.E., McPherson, S.R. and West, K.L. Familial precursors of schizophrenia spectrum disorders. In L. Wynne and R. Cromwell (Eds.), Second Rochester International Conference on Schizophrenia, New York: Wiley-Interscience, in press, 1976.	{ "pile_set_name": "NIH ExPorter" }
Insulin-dependent diabetes mellitus (IDD) is a serious lifelong disease that remains replete with life threatening complications despite the many advances in patient care. We propose to perform a study in newly diagnosed persons with IDD to learn whether immunization to therapeutic insulin can be abrogated through the use of orally administered insulin. We will also gather information about the autoimmunity to pancretic beta cells underlying IDD and beta cell loss attenuated after clinical diagnosis."	{ "pile_set_name": "NIH ExPorter" }
Regulations by direct interaction between Ca2+-calmodulin and ion channels have now been found in many systems including the mammalian photoreceptor and olfactory receptor. We intend to continue to investigate this phenomenon using Paramecium where it was first explicitly demonstrated. We also found that in vivo mutations defective in the C-terminal lobe of calmodulin weaken Ca2+-calmodulin dependent K+ currents, while those in the N-lobe delete their Na+ currents. We will continue to examine whole- cell currents as well as single- channel currents to study this functional bipartition with existing and newly generated Paramecium mutants. We will try to extend this functional bipartition hypothesis to mammals by testing the effects of the two classes of mutant calmodulins on cNMP-dependent channels in olfactory neurons and ryanodine receptor/Ca2+-release channels. For our long-term project of Paramecium behavioral genetics, mutants and revertants will continue to be examined electrophysiologically. We also intend to make Paramecium an efficient system for reverse genetics, i.e. to generate live mutants each lacking a macronuclear gene that is homologous to a mammalian sequence. Genes of neurobiological interests will be tested first.	{ "pile_set_name": "NIH ExPorter" }
Bacterial pathogens must acquire iron to replicate and survive in mammalian hosts. The iron-porphyrin heme, bound to circulating hemoglobin, contains up to 80% of bodily iron. This fact has led to the hypothesis, which is backed by experimental evidence, that heme serves as a source of iron during infection. However, in the anthrax-causing bacteria B. anthracis, deletion of iron-regulated surface determinant (Isd) genes, which code for surface proteins that bind heme, did not reduce B. anthracis virulence in animal models of infection. These results suggest other factors contribute to iron uptake in this deadly pathogen. A hallmark of Isd systems is the presence of a conserved protein module termed the near-iron transporter (NEAT) domain, which mediates the transfer of heme into Gram-positive pathogenic bacteria. In silico analysis of the genome of B. anthracis indicates a non-Isd gene, designated BAS0520, is annotated to encode for a single NEAT-domain protein. The objective of this proposal is to determine if BAS0520 represents the missing link mediating heme uptake during anthrax infection. Specifically, we hypothesize BAS0520 is a surface protein that extracts heme from host hemoglobin, thereby promoting heme transport into the bacterial cell and enhancing iron-dependent replication in mammalian hosts. This hypothesis will be tested with two aims: 1. Determine the mechanistic function of BAS0520. Biochemical approaches will define the molecular and structural factors of the NEAT domain of BAS0520. 2. Determine the role of BAS0520 in iron acquisition and anthrax disease. Growth studies and animal infection models using fully virulent strains will be used to define which mechanisms of iron uptake are important for anthrax disease.	{ "pile_set_name": "NIH ExPorter" }
Acute myocardial infarction is the leading cause of death in most industrialized nations. The estimated annual incidence in the US is 865,000 events, with ST-segment elevation myocardial infarction (STEMI, severe AMI) comprising an estimated 500,000 events per year. Fibrinolytic therapy is widely utilized to restore coronary blood flow due to its widespread availability to the broad cross-section of patients. The current regimen, including tissue plasminogen activator, aspirin, clopidogrel and heparin, still induces inadequate coronary reperfusion in 30-40% of patients and early thrombotic reocclusion in 5-10% patients. Moreover, successful recanalization causes detrimental reperfusion injury that accounts for up to 50% of the final size of a myocardial infarct. Net clinical adverse outcomes remain 10-12% at 30 days after treatment and 16-17% STEMI patients die during 1-year follow-up. Moreover, both morbidity and mortality are dramatically increased in patients experiencing peri-procedure bleeding. Importantly, most of the recurrent MI and major bleeding events occur in the first hours and days after treatment. Consequently, the search for more efficacious and safer acute antithrombotic agents with effective attenuation of reperfusion injury remains the holy grail' of drug development. APT102 is an optimized human apyrase with two amino acid substitution that has significantly higher activity than the wild-type apyrases. This enzyme inhibits platelet activation and limits vascular inflammation by enzymatically hydrolyzing extracellular ADP and ATP. Adenosine is further generated by CD73 which prevents tissue damage during hypoxia and in the setting of cardiac reperfusion injury. In addition, APT102 prevents platelet desensitization and maintains homeostasis. With the Phase I award, we demonstrated that in the r-tPA induced fibrinolysis model in dogs, treatment of APT102 completely prevented re-occlusion, maintained normal blood flow, and profoundly reduced infarct size of hearts by 80% compared with clopidogrel. Meanwhile, APT102 did not prolong bleeding time, as did clopidogrel. The goal of this Phase II SBIR grant application is to rigorously determine whether in the coronary fibrinolysis model in dogs, APT102 promotes improved myocardial perfusion and left ventricular function with minimal bleeding compared with clopidogrel. The long-term goal is to develop APT102 as a highly effective antithrombotic and anti-inflammatory therapy with minimal bleeding risk that will profoundly improve efficacy and safety of acute treatment for AMI and other thrombotic patients.	{ "pile_set_name": "NIH ExPorter" }
Diarrheal is a leading cause of death and illness throughout the world and causes 560 million deaths of children under 5 years old. Rotavirus (RV) causes about 70% of viral diarrheas and even with two vaccines kills 215,000 children yearly. RV diarrhea is mainly due to excess Cl- secretion that drives fluid loss, but the cellular pathways responsible for RV-induced fluid secretion are not well defined, which limits efforts to develop and testing of potential anti-diarrheal drugs that can be used to treat children when protection by vaccines is insufficient. Activation of chloride (Cl-) secretory channels is regulated by intracellular calcium (Ca2+) and cyclic nucleotide (cAMP) signals These signals in turn activate Ca2+-activated Cl- channels (CaCC), such as anoctamin (Ano) family, and cAMP-activated Cl- channels (cAMP-CC), such as the cystic fibrosis transmembrane regulator (CFTR). RV diarrhea is the classic example of Cl- secretion due to elevated Ca2+ because a hallmark of RV infection is increased Ca2+ signaling, which is induced by the RV nonstructural protein 4 (NSP4). Ano channels are the prominent CaCCs in the gut; however, the Cl- channels responsible for RV diarrhea have yet to be identified. Additionally, NSP4 is considered the primary initiator of RV-induced Ca2+ signals; however, the mechanisms by which NSP4-derived signals cause Cl- channel activation and fluid secretion have not been adequately explored. In Aim 1, we will identify the Cl- channels responsible for RV diarrhea using both in vitro and in vivo approaches. Little is known about the expression of different Cl- channels throughout the human intestine, particularly children, we will use human intestinal enteroids (HIEs) to determine gene expression of the key Cl- channels and their regulatory proteins. Next, we will measure the ability of RV to activate individual Cl- channels using a fluorescence quench assay and determine whether those channels contribute to fluid secretion using the enteroid swelling assay (ESA) in the presence of specific Cl- channel blockers. Lastly, we will determine whether inhibitors of Ano1 or CFTR will attenuate diarrhea and if so whether intestinal specific deletion of those channels also reduces diarrhea. In Aim 2, we will determine whether RV NSP4 activates both Ca2+ and cAMP through activation of the sensor stromal interacting molecule 1 (STIM1). STIM1 activation can generate Ca2+ signals through the store-operated Ca2+ entry (SOCE) pathway, as well as cAMP signals through the store-operated cAMP signaling (SOcAMPs) pathway. We will use live cell imaging of intestinal cell lines and HIEs expressing fluorescent Ca2+ and cAMP biosensors to determine whether RV and NSP4 activate both SOCE and SOcAMPs and use the ESA to determine whether the Ca2+/cAMP signals generated by NSP4 cause fluid secretion. Lastly, we will determine whether blockers of SOCE or SOcAMPs will attenuate RV diarrhea in vivo. Our results will generate critical insights into the mechanisms of viral diarrhea and new biosensor HIE model systems that can be applied to future studies enteric virus, such as noroviruses and astroviruses.	{ "pile_set_name": "NIH ExPorter" }
Renal disease leading to chronic renal failure represents a severe public health problem. Diabetes mellitus is the largest single cause of renal disease. The long term objective of the proposed studies is to contribute in the understanding of the pathogenesis of diabetic kidney disease at the molecular level. In diabetes, hyperglycemia affects many metabolic pathways and each of these changes may contribute to the development of diabetic complications. One of the major alterations is the nonenzymatic glucosylation of proteins. This process occurs in two steps: a first, reversible one (formation of Amadori products) and a second, irreversible one (formation of crosslinked products). Because of their long half-life, basement membrane macromolecules are primary targets of this process. In diabetic kidney disease, one of the most prominent alterations is the thickening and increased permeability of the glomerular basement membrane. Therefore, it is imperative to understand the effect of nonenzymatic glucosylation on the glomerular basement membrane and its components. We propose: First, to isolate calf glomerular basement membranes and incubate them in the presence of glucose in order to promote their nonenzymatic glucosylation; these incubations will be performed in the absence or the presence of aminoguanidine, a substance known to inhibit crosslink formation. Second, to examine the extent of glucose incorporation and crosslink formation and the glucosylation-induced structural alterations of the intact glomerular basement membrane, using spectrophotometric and high resolution scanning electron microscopic techniques. Third, to extract three major macromolecular components of the glomerular basement membrane (type IV collagen, laminin, and entactin/nidogen) using denaturing agents, gel permeation and ion-exchange chromatography and to determine possible glucosylation-induced structural alterations using gel electrophoresis, autoradiography and rotary shadowing electron microscopy. Fourth, to examine the ability of the extracted macromolecules to interact with each other and define glucosylation-induced defective interactions using turbidimetry, solid phase binding assays, and rotary shadowing electron microscopy. These studies will broaden our knowledge on the mechanisms underlying structural and functional alterations of the glomerular basement membrane in diabetes and hopefully will lead to proposals aiming at reducing diabetic complications.	{ "pile_set_name": "NIH ExPorter" }
The ability of vascular endothelial cells to adjust their cytoplasmic properties under the stimulus of fluid shear stress is a key factor in the physiological control of the blood-vessel wall interface. Adjustments to fluid stress control exchange and cell traffic across the endothelium, influence humoral regulation, and are one of the main determinants of lesion formation in large arteries. Cytoplasmic actin plays a key role in these processes because it serves to define the cell's shape, mechanical properties, and motility. Actin polymer (F-actin) stability, arrangement, and extent of crosslinking and attachment to the substrate and the lumenal membrane determine the local stiffness of the cytoplasm. There exists today no measurements of the dynamics of actin in endothelial cells or the consequence of these dynamics for the mechanics and biology of endothelial cells under flow conditions commensurate with those experienced in vivo. Aim 1 will determine actin dynamics in individual human umbilical vein endothelial cells (HUVEC) before and after the application of steady fluid shear to their surfaces. Cells are microinjected with caged resorufin-actin monomer (G-actin). After incorporation of the caged-actin into the cytoskeleton, spatial uncaging of its fluorophore will yield the F-/G-actin ratio, F-actin turnover time, and the G-actin diffusion coefficient. In Aim 2, F-actin turnover data will be combined with a detailed 3D picture of F-actin structure in endothelial cells determined using high-resolution electron microscopy with the goal of developing a model to explain the mechanical properties of the cell and how these properties are altered by fluid flow. In Aim 3, the measurements of actin dynamics and internal structure will be extended to disturbed and unsteady flow to create a complete biomechanical picture of normal in vivo conditions. These studies will provide new insight into the basic physiology of endothelial structure and function under flow conditions characteristic of those experienced in vivo. They will provide new data on cell stasis and motility as well as the coupling between arterial flow and the cell, and thereby contribute to our understanding of the pathologies associated with atherosclerosis and intimal hyperplasia in vascular grafts.	{ "pile_set_name": "NIH ExPorter" }
As phosphorylated proteins play a significant role in normal and abnormal cellular function, there is a critical need for the production of pure, analytically characterized phospho-proteins/polypeptides for use in assays designed to capture the phosphorylation signatures of different cancers. Phospho-protein/polypeptide standards can serve as reference controls for quantitative immunoassays, immunogens for generating phospho-specific antibodies, and can National Cancer Institute (NCI) 65 be utilized in kinase inhibitor screens as well as protein-protein interaction studies.	{ "pile_set_name": "NIH ExPorter" }
Our laboratory has a strong interest in pharmacogenetics. We have been active in studying how germline genetic variants can alter pharmacokinetics, response, and toxicity of various anticancer agents, thereby contributing to interindividual variation in clinical outcomes in therapies with an already narrow therapeutic window. We have established a molecular link between these polymorphisms and their phenotype as it relates to drug treatment. Most of our work has been focused on genetic variations in drug metabolism and transporting candidate genes such as ABCB1 (P-glycoprotein, MDR1), ABCG2 (BCRP), SLCO1B3 (OATP1B3, OATP8), CYP3A4, CYP3A5, CYP1B1, CYP2C19, CYP2D6, UGT1A1, UGT1A9 and several others. Drug transporters mediate the movement of endobiotics and xenobiotics across biological membranes in multiple organs and in most tissues. As such, they are involved in physiology, development of disease, drug pharmacokinetics, and ultimately the clinical response to a myriad of medications. Genetic variants in transporters cause population-specific differences in drug transport and are responsible for considerable interindividual variation in physiology and pharmacotherapy. Thus, we are interested in studying how inherited variants in transporters are associated with disease etiology, disease state, and the pharmacological treatment of diseases. We are also interested in non-candidate gene approaches where large numbers of polymorphisms are explored to establish a relationship with clinical outcome, and experiments are conducted to validate potential causative alleles resulting from exploratory scanning. We have worked with Affymetrix to beta-test the DMET chip that contains 1,256 genetic variations in 170 drug disposition genes, and are currently establishing a clinical trial where patients treated at the NCI will be genotyped with the DMET chip to explore potential links between these genes and various treatments of several cancers. We are currently making progress in validating the results from the initial DMET chip experiments. While many of these studies have been conducted in order to explain some of the genetic influence on pharmacokinetic variability, we also have a strong interest in clarifying genetic markers of pharmacodynamics and therapeutic outcome of several major anticancer agents since this field has been rather poorly studied. We have evaluated genetic polymorphisms in the XRCC1 gene and its association with radiation therapy in prostate cancer and found that XRCC1 genetic variants may affect the outcome in patients who received radiotherapy for localized prostate cancer. ERCC1 polymorphism & platinum-base chemotherapy: Genetic polymorphisms in ERCC1 are thought to contribute to altered sensitivity to platinum-based chemotherapy. Although ERCC1 N118N (500 C>T, rs11615) is the most studied polymorphism, the impact of this polymorphism on platinum-based chemotherapy remains unclear. This is the first study in which the functional impact of ERCC1 N118N on gene expression and platinum sensitivity was explored. The aim of this study is to investigate if the reduced codon usage frequency of AAT, which contains the variant allele of the silent mutation, has functional impact on ERCC1 in a well-controlled biological system. Specifically, the ERCC1 cDNA clone with either the C or T allele was introduced into an ERCC1 deficient cell line, UV20, and assayed for the effect of the two alleles on ERCC1 transcription, translation and platinum sensitivity. Both ERCC1 mRNA and protein expression levels increased upon cisplatin treatment, peaking at 4h post-treatment, however there were no differences between the two alleles (p>0.05). These data suggest that N118N itself is not related to the phenotypic differences in ERCC1 expression or function, but rather this polymorphism may be linked to other causative variants or haplotypes.Sorafenib is an inhibitor of UGT1A1 but is metabolized by UGT1A9: Implications of genetic variants on sorafenib exposure and sorafenib-induced hyperbilirubinemia. Several case reports suggest sorafenib exposure and sorafenib-induced hyperbilirubinemia may be related to a (TA)(5/6/7) repeat polymorphism in UGT1A128 (UGT, uridine glucuronosyl transferase). We hypothesized that sorafenib inhibits UGT1A1 and individuals carrying UGT1A128 and/or UGT1A9 variants experience greater sorafenib exposure and greater increase in sorafenib-induced plasma bilirubin concentration. Sorafenib exhibited mixed-mode inhibition of UGT1A1-mediated bilirubin glucuronidation in vitro. The DMET genotyping platform was applied to DNA obtained from six patients, which revealed the ABCC2-24C>T genotype cosegregated with sorafenib AUC phenotype. Sorafenib exposure was related to plasma bilirubin increases in patients carrying 1 or 2 copies of UGT1A128 alleles. UGT1A128 carriers showed two distinct phenotypes that could be explained by ABCC2-24C>T genotype and are more likely to experience plasma bilirubin increases following sorafenib if they had high sorafenib exposure. This pilot study indicates that genotype status of UGT1A1, UGT1A9, and ABCC2 and serum bilirubin concentration increases reflect abnormally high AUC in patients treated with sorafenib.Romidepsin causes a number of cardiac effects, one of which is heart rate changes. We compared three polymorphisms in the cardiac transporter, ABCB1 (MDR1, P-gp), to heart rate changes following romidepsin treatment in 64 patients. The ABCB1 alleles that were previously associated with increased intracardiac ABCB1 expression were also related to the smallest increases in heart rate following infusion (P=0.033). We conclude that ABCB1 polymorphic variants are related to romidepsin-induced heart rate increases.ABCB1 polymorphisms are associated with variability in exposure to docetaxel and 99mTc-sestamibi before and after tariquidar coadministration. The ABCB1 (P-gp) inhibitor, tariquidar, is not associated with increases in docetaxel pharmacokinetics (PK); however, the impact of variants in ABCB1 have not been studied with this combination. We hypothesized that individuals carrying variants associated with decreased ABCB1 expression and efflux capability would have greater changes in docetaxel PK following tariquidar. Preliminary analyses suggest that individuals carrying ABCB1 variants have similar baseline plasma docetaxel AUC when compared to those carrying wild-type alleles; however, the tissue distribution of an ABCB1 model substrate (99mTc-sestamibi) at baseline appears be greater in extrahepatic tissues of those carrying variant ABCB1 diplotypes. Tariquidar coadministration with docetaxel and sestamibi appears to alter plasma pharmacokinetics and tissue distribution of these compounds differently based on ABCB1 genotype. Wildtype ABCB1 carriers experienced an increase in plasma docetaxel exposure, an increase in the volume of distribution of docetaxel, and an increase in the concentration of sestamibi in heart, lung, and tumors as compared to patients carrying variant alleles. Therefore, it appears that ABCB1 alleles heavily influence the biodistribution of docetaxel and sestamibi into the bodily tissues thereby affecting toxicity and efficacy.	{ "pile_set_name": "NIH ExPorter" }
Despite significant recent advances in the biochemistry of contractile proteins and the regulation of muscle contraction very little is known about myosin linked regulation in mammalian striated muscle, particularly cardiac muscle. Our initial studies show that the 18,000 M.W. subunit of cardiac myosin (Lc2) acts as an inhibitor of actin - myosin interaction. This suggests the possibility of a myosin linked control of actin - myosin interaction (contractile event) in mammalian cardiac muscle. This project will study the effects of removal of the cardiac myosin Lc2 on the control of actin-myosin interaction as determined by measurement of actin-activated Mg2 ion ATPase. The studies will be done using pure actin and regulated actin as cofactors. With pure actin as cofactor we shall study the effects of Ca2 ion on the affinity of myosin for actin in the presence and absence of the light chain. We shall study the mechanisms that reverse the (Lc2) inhibition of actin-myosin interaction. Attention will be focused on the effects of binding of phosphorylated and dephosphorylated Lc2 to the light chain deficient myosin in relation to measurements mentioned above. Hybridization of the Lc2 deficient cardial myosin with the homologous light chains from skeletal, smooth muscle and molluscan myosin will be performed and the effects of Ca2 ion and/phosphorylation on the actin-myosin interaction studied. In vivo studies (James Scheuer, Montefiore Hospital) will be done in isolated perfused rat hearts. Effects of various interventions (increased or decreased contractility) on the state of phosphorylation of myosin (Lc2) will be determined. It is expected that these studies will provide unique basic information regarding the role of myosin in regulating the contraction of cardiac muscle.	{ "pile_set_name": "NIH ExPorter" }
How the extracellular environment influences intracellular signaling pathways that regulate cell growth and differentiation has been a major challenge over the past decade. Mutations in genes controlling these processes result in cellular transformation and cancer development. A family of proteins containing LIM domains has been recognized as playing important roles in the control of gene expression, cell fate determination, remodeling of the cytoskeleton, and as potential mediators of communication between the cytosol and the nucleus in response to extracellular signals. LIM domains function as versatile protein modules, capable of acting within diverse cellular contexts and in multiple subcellular compartments. Many have been shown to participate in direct protein-protein interactions. LIM domain-containing proteins have been classified according to sequence homologies among the LIM domains and overall protein structure. Group 3 proteins are cytosolic and contain 3 to 4 tandem LIM domains at the C-terminus in association with distinct N-terminal domains. Some are components of fusion proteins derived from chromosomal translocation present in some cancers. A major challenge is to understand how group 3 LIM proteins couple extracellular stimuli to specific cellular responses. The PI has identified and characterized a novel group 3 LIM protein, Ajuba ("curiosity" in Urdu, an Indian dialect). Ajuba shuttles between the cytoplasm and the nucleus of cells. Deletion of a leucine-rich nuclear export signal in Ajuba results in an accumulation of Ajuba in the cell nucleus. In embryonal carcinoma cells nuclear accumulation of Ajuba results in a cell growth arrest and spontaneous differentiation. Ajuba associates with the cytosolic adapter protein Grb2 leading to a Ras-dependent activation of mitogen-activated protein kinase activity. When expressed in Xenopus oocytes, Ajuba promotes meiotic maturation. Thus Ajuba is a cytosolic protein that transduces signals to the nucleus in response to extracellular stimuli. In this proposal the PI has designed experiments to 1) determine how Ajuba alters MAPK signaling pathways; 2) determine how Ajuba regulates multipotent embryonal carcinoma cell proliferation and differentiation; 3) determine the signals regulating cytosolic-nuclear cytosolic translocation of Ajuba; 4) identify cytosolic and nuclear proteins that interact with Ajuba and determine how these interactions affect Ajuba function in specific subcellular compartments.	{ "pile_set_name": "NIH ExPorter" }
Serotonin (5-HT) is a neurotransmitter that has been implicated in the aetiology of numerous Mental Health disorders, including depression, anxiety, social phobia, schizophrenia, obsessive-compulsive and panic disorders, migraine, and eating disorders. There are 12 5-HT receptor subtypes with similar recognition properties yet widely divergent physiological roles that belong to the same family of G Protein Coupled Receptors (GPCRs), hindering the discovery of subtype-selective drugs. Knowledge of the 3-dimensional structure of these receptors would enable the design of selective drugs. However, solving the structure at atomic resolution of membrane proteins such as GPCRs has been hampered by the difficulty to purify sufficient amounts of homogeneous and functional protein. Here, it is proposed a novel, proprietary expression system combined with a purification method to generate large amounts of high quality purified receptor, which could solve the bottleneck for structural elucidation of these important drug targets. In Phase I, the feasibility of the proposed approach will be tested for all the 12 human 5-HT GPCRs. In Phase II, this approach will be developed resulting in purified homogenous protein for each human 5- HT receptor subtype. This high quality purified material will be a highly valuable product enabling structural elucidation of these receptors. [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
The Administrative Core will be critical in providing overall leadership and central administrative support to the Chemistry Center, including establishing and implementing, in collaboration with center staff, assay providers and the NIH Network Science Officer, a Compound Probe Development Plan (CPDP) for each program. One of the most critical functions of the Administrative Core will be communication with other Centers within the MLPCN, including coordination of studies, transfer of compounds, and efficient communication of data to facilitate iterative compound synthesis and testing. In addition, the Administrative Core will be responsible for organization for all Center meetings, travel arrangements for attendance of MLPCN meetings, preparation of progress reports and noncompetitive renewal applications. Finally, the administrative core will be responsible for recruitment and staffing as well as budgetary management of the Program. The following specific aims summarize the major functions of the administrative core: AIM 1. To establish goals of each probe optimization effort and initiate probe optimization by assigning lead chemist and coordinating communication between the Vanderbilt chemistry center, other MLPCN centers, the MLSMR, and the NIH program administrators. AIM 2. To effectively manage the integration of activities of medicinal chemistry, informatics and pharmacology components within the Vanderbilt Specialized Chemistry Center. AIM 3. To coordinate and manage communication and transfer of reagents, data and novel probes to the scientific community. AIM 4. To recruit and maintain staff for the proposed MLPCN facility to support and advance the MLPCN goals.	{ "pile_set_name": "NIH ExPorter" }
A genetic component to the development of schizophrenia has been established, although markers for a genetic locus have not yet been identified in consistently replicated studies. Molecular genetic screening of large pedigrees, using restriction fragment length polymorphisms (RFLP's), have been successful, however, in finding markers linked to another neuropsychiatric disorder, Huntington's disease, and identifying other promising regions of the genome in Alzheimer's disease and manic-depressive disorder. A linkage to the proximal long arm of chromosome 5 in schizophrenia has also been reported, although it is not confirmed by other independent studies. Over the past 5 years, the present investigators have identified over 200 families in the United States and a catchment area of Northwest London, United Kingdom, with multiple ill members with schizophrenia. Thus far, approximately 100 of these families have had blood samples taken from available relatives and lymphoblastoid cell lines maintained in culture for continual DNA studies. The present proposal is for the extension of the collection of these pedigrees and for follow-up clinical evaluations for the application of molecular genetic techniques to the screening of these cell lines. Only funding for the clinical evaluations and maintenance of the cell collections is being requested in this application.	{ "pile_set_name": "NIH ExPorter" }
The specific aims of this project are: (1) to search for the primary action and site of action of the diabetic alleles (db and db 2J) that cause a disease in mice resembling maturity-onset diabetes mellitus in man; (2) to define the physiology, biochemistry, and histopathology of the disease caused by these two alleles in different genetic backgrounds; and (3) to compare the effects of the diabetes alleles with those caused by unrelated alleles, in different genetic backgrounds; and (3) to compare the effects of the diabetes alleles with those caused by unrelated alleles, such as obese (ob) and the yellow alleles (Ay, Avy, and Aiy) which also cause hyperphagia, obesity, hyperinsulinemia, and mild hyperglycemia.	{ "pile_set_name": "NIH ExPorter" }
We will develop a new method to measure the time courses of activation of biochemical regulatory networks that control changes in synaptic strength which underlie processing and storage of information in neural networks. The proposed method will permit unprecedented time resolution and will enable measurement of the time courses of activation of at least 20, and eventually as many as 50 to 100 enzymes in brain tissue that has been rapidly frozen at intervals as small as one second following an electrical or pharmacological stimulus. The method will be immediately applicable to basic research on, and target development for, mental illnesses and Alzheimer's disease. Upon scale-up, it will be applicable to screening for drugs to treat these diseases. The method will involve substantial adaptation of two existing technologies: "plunge-freezing" and "Selected/ Multiple Reaction Monitoring" (S/MRM) by mass spectrometry. Once developed, both technologies can be scaled up for medium or high throughput screening. The project has three aims. First, we will develop a plunge freeze apparatus to rapidly freeze slices of hippocampal tissue at accurate time intervals following application of a stimulus to the perfused slice. We will accomplish this by making modifications and additions to a plunge-freeze apparatus now commercially available from Leica (Leica EM GP). We will devise an optimal design for a sample chamber to maintain the health of slices during perfusion, and to deliver electrical stimuli to the Schaffer collateral pathway, a major hippocampal axon tract, prior to rapidly freezing the slice by plunging it into a -1900 C liquid propane/ethane bath. We estimate that freezing time to the center of the slice upon plunge will be ~ 200 msecs or less. This freezing time is compatible with a resolution of one second for time intervals following application of a discrete stimulus. Second, we will develop methods to measure the activation of a panel of 20-25 protein kinases or their key substrate proteins located at positions in the regulatory networks that are believed to control synaptic plasticity in excitatory synapses in the hippocampus. Each enzyme or substrate that we will measure is regulated by addition of a phosphate group to key residues in the protein structure. Mass spectrometry will be used to measure changes in the levels of these phosphorylated sites in the frozen slice tissue. Third, once the assays are developed, we will carry out "proof of principle" experiments by combining the technologies developed in Aims 1 and 2 to acquire time courses of activation of each the enzymes in hippocampal slices after delivery of stimuli that alter synaptic plasticity. PUBLIC HEALTH RELEVANCE: This project will develop a new method for measuring activity in biochemical pathways that do not function properly in individuals with mental illnesses or Alzheimer's disease. The method will be used to support research on the causes of mental illnesses and Alzheimer's disease and for efficient screening for new drugs to treat these diseases.	{ "pile_set_name": "NIH ExPorter" }
Pakistan has the 4th highest burden of child mortality in the world, with an estimated 500,000 annual child deaths. Infectious diseases are the biggest killers, causing 60% of all deaths under 5 years of age. WHO estimates that neonatal infections (15%), diarrheal illnesses (17%), and pneumonia (21%) cause over 50% of all child deaths in Pakistan. Although the infectious morbidity and mortality is huge, Pakistan has no formal training programs in infectious diseases. Consequently, there is a tremendous shortage of individuals with the requisite skills to conduct independent research in infectious diseases affecting Pakistani children. The Department of Pediatrics at the Aga Khan University in Karachi, Pakistan has a strong track record in research relevant to improving child survival in developing countries. In collaboration with colleagues at Emory University and the Centers of Disease Control and Prevention in Atlanta, the Department can offer unique training opportunities in infectious diseases to Pakistani trainees. The long-term aim is to prepare a cadre of individuals who will provide the evidence, leadership, and enthusiasm needed to reduce the burden of childhood infections of major public health importance in Pakistan and other developing countries. The Infectious Diseases and Child Health in Pakistan (IDCHIP) Research Training Program will provide opportunities for structured long- and short-term training in Karachi and Atlanta with a particular focus on vaccine translational research and neonatal infections. Vaccine science is a key tool to close the health gap between children in rich and poor countries. Vaccine translational research aims to accelerate rational introduction of new vaccines (e.g. against rotavirus diarrhea and pneumococcal pneumonia) in developing countries by assessment of disease burden, vaccine safety and impact studies (trials and demonstration projects), and economic and policy analyses. Reducing neonatal infection rates is also a high priority for Pakistan with tremendous potential for improving neonatal survival rates. Thus building research skills in these two areas has particular relevance for reducing infectious disease-related child mortality in Pakistan. Non-technical Summary: Pakistan has a very high burden of infectious disease-related child deaths. This training program will equip individuals will research skills necessary to reduce the burden of infections in newborn babies and vaccine-preventable childhood illnesses in Pakistan.	{ "pile_set_name": "NIH ExPorter" }
This proposal is for a Mentored Patient-Oriented Research Career Development Award. The candidate's immediate goal during this award is to gain expertise in the design, implementation and analysis of epidemiologic studies in the field of epilepsy. The long-term career goals are to develop a large-scale epilepsy epidemiology program in an urban population that will serve as a basis for studies on incidence of epilepsy, epilepsy prognosis (including predictors of response to treatment), mortality, morbidity, genetics and economic costs of epilepsy. In order to attain these goals there are two major areas of focus for this award. The first area of focus is a formal program in epidemiology and biostatistics in the Environmental Health Department of the University of Cincinnati College of Medicine. The candidate will take formal coursework in epidemiology and biostatistics with the anticipation that a Masters degree in epidemiology will be obtained. The second area of focus is the development of a research project in epilepsy epidemiology. The research portion of this award will examine health related quality of life (HRQOQ issues in patients with newly diagnosed epilepsy and single seizures. Epilepsy is a chronic neurologic condition that may affect HRQOL by interfering with employment or driving. Patients take a daily dose of medication that may produce adverse effects. Patients with a new diagnosis of epilepsy or a single seizure may have a greater impact on HRQOL because of important changes in lifestyle that need to be made after the diagnosis is made. Prior HRQOL studies have been performed in patients with intractable seizures. The specific aims of the project are to: 1) Assess initial HRQOL measures in a cohort of patients with newly diagnosed single seizures or a new diagnosis of epilepsy using a specific HRQOL in epilepsy inventory (QOLIE-89). 2) Prospectively examine for subsequent changes in HRQOL in this cohort of patients. 3) Examine for potential mechanisms that may be responsible for changes in HRQOL. 4) Determine if seizure recurrence negatively impacts HRQOL. This study will prospectively gather HRQOL data and neurologic histories and examinations in the cohort every four months for a minimum of two years. Mentors for this project are Drs. Joseph Broderick and Michael Privitera. Dr. Broderick has significant experience in developing stroke epidemiology projects and will provide the candidate the necessary guidance and support to develop his own research program. Dr. Privitera has led the epilepsy program at the University of Cincinnati since 1987 and will serve as a resource for clinical epilepsy. After the completion of this award, it is anticipated that the candidate will develop larger scale projects in the area of the epidemiology of epilepsy.	{ "pile_set_name": "NIH ExPorter" }
The specific etiology of a majority of cases of keratitis and uveitis, as well as the exact immunologic mechanism of corneal graft rejection is unknown. Yet these entities frequently cause significant visual loss and blindness. The purpose of the proposed project is to elucidate the effector mechanisms involved in the immunopathology of anterior segment ocular inflammation (including graft rejection) and to test experimentally the efficacy and side effects of new therapeutic approaches. These studies will deal with two major subjects: 1) the role of soluble mediators ("lymphokines") of cellular immunologic reactions in ocular inflammatory disease, and 2) the use of blocking antibody serum (BAS) in the prevetion of immunologic corneal graft rejections. Lymphokines will be produced by guinea pig lymph node cells and purified and characterized. The lymphokines will then be placed in contact with eye tissue both in vivo and in tissue culture. The ability of lymphokines to cause tissue injury in the presence or absence of inflammatory cells will be investigated. An antibody will be made to a purified lymphokine preparation and its ability to block or suppress lymphokine-mediated tissue damage will be evaluated. Guinea pig anti-rabbit lymphocyte serum will be prepared and modified chemically so that it will not bind complement. The characteristics of modified BAS and the mechanisms by which it protects corneal grafts from rejection will be studied by in vivo and in vitro studies.	{ "pile_set_name": "NIH ExPorter" }
The NIH neuroprosthesis program has fostered so much success in the area of cortically controlled neuroprostheses that the FDA has approved multiple human trials to test the safety and efficacy of cortical implants for brain machine interfaces (BMI). One important application of BMI technologies is the direct cortical control of prosthetic limbs. Recent advances in this field have led to the creation of the most capable prosthetic arms yet developed, including the DEKA 'Luke arm' and Johns Hopkins APL 'Modular Prosthetic Limb'. However, a critical gap in this effort is the lack of somatosensory feedback which is needed to support propriception and tactile sensations for the artificial limb. Without these sensations, users will never achieve maximum benefit from these advanced limbs, because without sensory feedback, these devices will remain as numb, extracorporeal 'tools', rather than integrated fully functional limbs. Our goals are twofold: to better understand the nature of sensory feedback and the way in which peripheral sensory activity is conveyed to primary somatosensory cortex (S1), and to develop a somatosensory neural interface (SSNI) that will provide the user with proprioceptive feedback for their neuroprosthesics limb. We have previously proposed that primary afferent microstimulation (PAMS) in the dorsal root ganglia (DRG) can be used to deliver surrogate somatosensory feedback to the central nervous system. We have demonstrated that in cats, PAMS can recruit small populations of afferents from a variety of sensory modalities (Gaunt et al. 2009) and that this stimulation can transmit meaningful activity to S1 (Weber et al. 2011). The success achieved during the development of this animal model generated a number of new questions and hypothesis upon which a series of new experiments are proposed. Specifically, these experiments focus on characterizing the ability of PAMS to 1) transmit sensory information to S1 in anesthetized cats when the PAMS patterns are based on neural activity recorded in the DRG during movement, 2) transmit discriminable sensory information to S1 in anesthetized cats when the PAMS patterns are based on fabricated static and dynamic inputs, and 3) transmit discriminable sensory information to S1 in awake standing cats, useful for modifying postural responses to ground support perturbations. These experiments range from further investigations of the capabilities of PAMS to testing the ability of PAMS to predictably modify motor behaviors. This work will further the development of a SSNI, critical for the future of BMI based prosthetic limbs, as well as address fundamental questions regarding the role of sensory feedback in the control of normal motor behaviors. PUBLIC HEALTH RELEVANCE: Trauma, vascular disease, and diabetes are leading causes of limb amputation and sensory deficits in both civilian and military populations. The number of people living with the loss of a limb is expected to more than double to 3.6 million by 2050. Thus, there is an urgent and rapidly growing need for advanced prosthetic limbs that can restore the motor and sensory functions that are lost after amputation. The goal of this proposal is to develop a technique for providing proprioceptive sensations to users of prosthetic limbs using patterned electrical stimulation of sensory neurons in the dorsal root ganglia (DRG). This approach is similar, in principle, to that of the cochlear implant which uses patterned electrical stimulation of auditory nerves to restore hearing to people with profound deafness. The proposed study will determine if DRG stimulation is effective in: 1) delivering proprioceptive information to the brain and 2) providing feedback that is useful for maintaining balance during postural perturbations.	{ "pile_set_name": "NIH ExPorter" }
[unreadable] Communication between the nucleus and cytoplasm is mediated by large proteinaceous structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). The long-term goal of this project is to elucidate the molecular sequence of events required for translocation through the NPC. We hypothesize that the framework for selective, facilitated NPC translocation is based on physical interactions between shuttling transport factors and a family of NPC proteins that contain regions with multiple FG-type repeats (F, phenylalanine; G, glycine). We also propose that key events for regulation of mRNA export directionality are controlled at the NPC by the action of multiple factors. To analyze the mechanism and regulation of protein and mRNA transport through NPCs, we propose three aims. In aims one and two, we will build on our recent results documenting the first in vivo tests of NPC translocation models. We propose to use S. cerevisiae mutants with minimal repertoires of the FG binding sites for protein and mRNA transport factors. To define the requirements for FG repeat numbers, FG types and critical FG binding sites in NPC substructural locations, the mutants will be assayed for protein import and mRNA export defects. NPC-transport factor association will also be analyzed using microscopy strategies. A biochemical approach will be used in aim two to detect changes in protein-protein interactions during extrusion of the mRNA-protein complex through the NPC. In aim three, we will investigate the mechanism for activation of the DEAD-box helicase Dbp5 by the essential mRNA export factor Gle1 and production of soluble inositol hexakisphosphate (IPS). We will identify the IP6 binding site in Gle1/Dbp5, and test for roles in RNA unwinding and remodeling of RNA- protein complexes. Studies in yeast and human tissue culture cells will also be conducted to test for regulation of Gle1 and IP6 in other aspects of mRNA processing. This proposal represents an area of basic science research that has the potential to provide novel insights into multiple disease processes. Transport factors and NPC proteins are targets for viral inhibition of cell function and mediators of viral RNA export. Inositol signaling defects are associated with disease states including cancer cell growth, inflammation, neurotransmission, and organ development. Knowledge of the NPC translocation mechanism will be key for designing therapeutic strategies to selectively target these pathways. [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
This project will continue to focus on describing and understanding processes involved in the storage and retrieval of words and pictures. The current working model is described by four assumptions. First, both verbal and pictorial symbols are encoded in terms of their sensory and meaning attributes, with these attributes representing qualitatively different types of information. Sensory features consist of visual and associated phonemic characteristics, and meaning features refer to interpretation, to associative and semantic characteristics. Second, access to phonemic information is presumed to be more direct for words than for pictures. Pictures must be meaningfully understood before they can be named. Third, focusing attention on a particular type of feature enhances the importance of that feature to the memory trace. However, such focus does not prevent the activation of other types of features linked to a very familiar stimulus. Fourth, and finally, retrieval is conceptualized as a redintegrative process with amount recalled depending upon the perceived similarity between the retrieval cue and the encoded trace and upon the number of functionally activated alternatives or set size. The primary objective of this project is to evaluate and refine this conceptualization. Empirical emphasis will concentrate on identifying the nature of memory representation and retrieval as stimulus type and conditions are varied.	{ "pile_set_name": "NIH ExPorter" }
The overall objective of this research is to localize and determine the functional organization of brain structures which control the secretion of the anterior pituitary; attention will focus on somatostatin (SRIF) neurons which inhibit the secretion of growth hormone (GH) and thyroid-stimulating hormone (TSH). The specific aims are to: 1) Test the hypothesis that the SRIF neurons which inhibit GH and TSH secretion under nonstress and stress conditions reside in groups that are anatomically and functionally separate; 2) Locate the SRIF neurons essential for GH responses to feedback and other physiological signals, including response to stress; 3) Test the hypothesis that SRIF neurons play a major role in producing the marked species differences that characterize the control of GH secretion. In the first series of studies, in vivo and in vitro approaches will be used to identify and localize the neural structures that inhibit GH and TSH secretion under nonstress and stress conditions, using ablative procedures (electrolytic and ibotenic acid lesions). and to study the effects of such lesions on SRIF release as assessed in vivo by push-pull perfusion and in vitro by perifusion of preoptic- basal hypothalamic (PO-BH) tissue. In situ hybridization and immunocytochemistry will also be used to localize the SRIF neurons involved. The next series of studies will use most of the same methods to locate the SRIF neurons essential for GH/SRIF responses to feedback and other physiological signals. Emphasis will be placed on the organization of neural circuits that inhibit GH secretion in response to stress and the role of corticotropin releasing factor in this regard. A final series of studies will use some of the same in vivo and in vitro approaches to investigate the bases for the remarkable species differences that characterize the control of GH secretion; in addition to rats, these experiments will involve hamsters and a few rhesus macaques. The latter will be used only for in vitro perifusion of PO-BH tissues obtained as they become available from experiments of others. These projects should provide new and useful information about the organization and function of brain structures that control GH secretion and growth.	{ "pile_set_name": "NIH ExPorter" }
Alterations in vascular permeability are a defining feature of diverse processes including atherosclerosis, inflammation, ischemia/reperfusion injury and angiogenesis. Mechanisms which govern increases in vascular permeability are under intense investigation. However, little is known about processes which determine barrier protection or restoration. Platelets and platelet-derived products are essential to maintaining the integrity of the endothelial cell barrier and are intimately involved in vascular homeostasis and pathobiology. We have shown that sphingosine 1-phosphate (Sph 1-P) is a phosphorylated lipid angiogenic factor released from activated platelets which ligates specific endothelial differentiation gene (Edg) receptors to stimulate endothelial cell chemotaxis and angiogenic responses. Sph 1-P accounts for the majority of the strong endothelial cell chemotactic activity of blood serum, and was strikingly effective in enhancing the ability of fibroblast growth factor to induce angiogenesis in the avascular mouse cornea. Importantly, Sph 1-P produced rapid, sustained, and dose-dependent increases in the barrier integrity of human pulmonary artery and lung microvascular endothelial cells. Furthermore, Sph 1-P potently reversed barrier dysfunction elicited by the edemagenic agent, thrombin. The exact mechanisms by which Sph 1-P enhances barrier function are unknown, however, our data strongly implicate an essential role of endothelial cell cytoskeletal dynamics in this response. Sph 1-P-mediated barrier enhancement was dependent upon actin filament rearrangement and Rac GTPase-dependent recruitment to the cortical actin cytoskeleton of known cytoskeletal regulatory proteins such as cortactin, p21-associated kinase (PAK), LIM kinase and cofilin. In this proposal we will examine the molecular basis of Sph 1-P- induced barrier enhancement and have targeted cortical cytoskeletal interactions with cellular adhesive proteins which promote vascular integrity. SA number 1 will characterize the Sph 1-P-mediated rearrangement of the cortical cytoskeleton following shear stress and assess cortactin, PAK, and cofilin involvement. SA number 2 will investigate Sph 1-P-induced alterations in zona adherens (beta/gamma catenin complex) interaction with the actin cytoskeleton via platelet-endothelial cell adhesion molecule (PECAM) and tyrosine phosphorylation. SA number 3 will define the role of activated p125 focal adhesion kinase (FAK) and Rho/Rac GTPases in Sph 1-P-mediated regulation of focal adhesion structure/function. Finally, SA number 4 will define the role of the barrier-protective PKC delta isotype in Sph 1-P-mediated cytoskeletal rearrangement using antisense strategies, PKC over-expression constructs, pharmacologic and myristoylated peptide inhibitors, and immortalized stable PKC over-expressing cell lines. Given the profound physiologic derangements which accompany the vascular leak seen in multiple vascular pathobiologies, Sph 1-P infusion may provide a novel therapeutic intervention for consideration in these devastating disorders.	{ "pile_set_name": "NIH ExPorter" }
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Simian varicella virus (SVV) infection of primates shares clinical, pathological, immunological and virological features of varicella zoster virus (VZV) infection of humans. Latent SVV infection can be established in cynomologous or African green monkeys (AGM) by exposing SVV-seronegative monkeys to an SVV infected cage-mate monkey. This year we conducted portions of three experiments with AGMs. Two groups were latently infected with simian varicella virus (SVV) and treated for possible reactivation utilizing two different treatments. Animals GV . were only irradiated with a single dose (200cGy of X-rays, animals HC were were irradiated with a single dose (200 cGy) of X-irradiation and then treated with tacrolimus (80-300 mg/kg/day). The third group of animals were group housed and one animal from every group acutely infected with SVV. This group passed the acute phase and is in the process of developing latency. In three months, this final group will undergo irradiation, as described above, and be given daily treatment with the immunosuppressive drug, prednisone (1 mg/kg/day), as a possible trigger of virus reactivation. Samples taken are being analyzed for virus load, histopathological changes and evidence of SVV reactivation.	{ "pile_set_name": "NIH ExPorter" }
Hepatitis C virus (HCV) infection is a major cause of morbidity and mortality. Injection drug users have the highest prevalence of HCV infection among all populations in the U.S. at risk. We will study prospectively the natural history of HCV infection among all populations in the U.S. at risk. We will study prospectively the natural history of HCV infection in a cohort of drug users with or at risk for HIV infection. Subjects will be recruited from among participants already enrolled in a large longitudinal study of the natural history of HIV infection in drug users. We will, therefore, be able to take advantage of the large and complex sets of data being collected in that study, and we will be able to link new data that will be collecting on the natural history of HCV infection with those data. Participants will have detailed standardized interviews on demographics, medical history, sexual and drug use behaviors, HIV and immunological testing, and testing for HCV infection, including quantitative HCV RNA levels, including quantitative HCV RNA levels, HCV genotyping, anti-HCV antibodies and, in a subset of participants, HCV sequencing. Persons found to have HCV infection will be referred to the study hepatologist to be evaluated for liver disease and they will be offered standard medical therapy for their HCV infection. Our aims are to determine 1) the effects of HIV infection and its associated immunodeficiency, HCV genotype, and drug use behaviors on HCV viral load and progression of liver disease, 2) the effects of HIV infection and immunodeficiency on response to therapy for HCV infection, 3) the effects of highly active anti-retroviral therapy and control of HIV viral load on the natural history of HCV infection, and 4) whether genetic diversity of HCV is facilitated by immunodeficiency.	{ "pile_set_name": "NIH ExPorter" }
Almost all archaea and half of bacteria contain Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins (CRISPR-Cas) adaptive immune systems that protect them against foreign genetic elements invasion. While diverse, all CRISPR-Cas function through three common steps: (i) adaptation, i.e., acquisition of foreign DNA segments (spacers) into CRISPR arrays; (ii) CRISPR array transcription and transcript processing to produce mature CRISPR RNAs (crRNAs), and (iii) interference, when Cas effector enzymes are guided by crRNAs to matching targets leading to target cleavage and ultimate invader genome destruction. As any immune system, CRISPR-Cas must be capable of self/non-self discrimination to prevent autoimmune death of the host caused by acquisition of spacers from own DNA followed by self-interference. A remarkable mechanism of self/non-self discrimination called ?priming? operates in type I CRISPR-Cas systems: acquisition of spacers from DNA containing partial matches to pre-existing CRISPR array spacers is dramatically stimulated compared to acquisition from DNA devoid of such sequences. While partially matching crRNAs that promote primed adaptation are incapable of efficient interference, all components of the interference machinery are required for primed adaptation. The mechanistic relationship between the interference and adaptation modules of CRISPR-Cas response during primed adaptation is not clear. In this work, protein-nucleic acid complexes and nucleic acid intermediates of CRISPR interference and primed adaptation by the Type I CRISPR- Cas system of Escherichia coli, the best-studied microbe, will be characterized in vivo and in vitro, and host functions that affect both processes will be revealed. In addition to uncovering a functional link between CRISPR interference and primed adaptation, programmable effector complexes with expanded targeting potential will be created as a result of proposed work and highly sensitive quantitative biophysical methods to study effector-target interactions will be developed.	{ "pile_set_name": "NIH ExPorter" }
Operons are an important feature of the C. elegans genome. Their transcripts are polycistronic pre-mRNAs that get processed by 3' end formation and trans-splicing. These two evens occur in close proximity between operon genes. Although 3' end formation is generally accompanied by transcription termination, in operons it is not. We have identified the key sequences and many of the key trans-acting proteins and snRNAs responsible for carrying out these events, and we are determining what roles they play and how. The key sequence required for both the trans-splicing and for preventing transcription termination is the Ur element that occurs ~50 bp downstream of the 3' end cleavage site. We will determine what binds there. We are also studying the roles of the protein components of the snRNP that does this specialized transsplicing event. Functional studies involving mutants and RNAi of these components and biochemical studies in an in vitro splicing extract are proposed. We have discovered that one of these proteins is bound to a novel snRNA, Sm Y, currently the only snRNA whose function is not known. We will study the role of this RNA in trans-splicing and operon pre-mRNA processing. We have identified several proteins likely to play roles in 3' end formation and transcription termination as suppressors of a mutation that inserts a transcription termination site into the first gene of an operon. One of these proteins is an exonuclease, another is an SR protein, and three others all have a domain that indicates they perform their functions by binding directly to RNA polymerase. We will determine what functions these proteins have in 3' end formation. These studies should provide important new insight into how 3' end cleavage can occur without accompanying transcription termination.	{ "pile_set_name": "NIH ExPorter" }
HIV-1 clade C is currently responsible for more than 50% of new HIV infections and is now the most commonly transmitted subtype worldwide. While HIV-1-associated dementia (HAD) continues to be a major neuropathological manifestation of AIDS among clade B-infected individuals in the US and Europe, the incidence of HAD in regions like India and sub-Saharan Africa, where clade C infection is prevalent, appears to be lower. Whether the low apparent prevalence of neuroAIDS is due to underlying differences in HIV-clade pathogenesis or simply an artifact of confounding variables such as data sampling, clinical diagnosis or opportunistic infections is unclear. Potential differences in clade B and C neurovirulence and the mechanism by which HIV-1 infected brain mononuclear phagocytes (MP;perivascular macrophages and microglia) mediate pathogenesis have yet to be investigated. Our recent preliminary data indicates clade B viral strains may produce more neurotoxins, such as glutamate, during HIV-1 infection than clade C viral strains. We have shown previously that HIV-1 clade B infection of MP leads to enhanced glutamate production through the enzyme glutaminase. In this R21 application, we hypothesize that neurotoxicity is mediated through brain inflammation and the dysregulation of glutaminase in HIV-1-infected macrophages. In comparison to HIV clade B, we hypothesize clade C isolates will demonstrate decreased infection efficiency, altered cytokine/chemokine profiles, and decreased glutamate production by infected MP, providing mechanistic insight into differences between clade B and C infection of the brain. We will apply human monocyte-derived macrophage (MDM) infected by a panel of HIV-1 strains (HIV-1 laboratory clade B strains, primary clade B strains and primary clade C strains), to a severe combined immune deficient (SCID) HIV-1 encephalitis (HIVE) mouse model, thus modeling macrophage driven HAD in vivo. This approach will utilize laboratory assays that mimic HIV-1 infection and immune activation of brain MP to investigate the effects of the CNS immune response on production of inflammatory factors and neurotoxins as well as neuronal injury as it occurs during HAD. This application will establish a foundation of work detailing the differences between viral clades and the potential implications for HIV-induced brain inflammation and dementia. Determining the mechanisms by which HIV-1 infected MP and cytokines influence neuronal injury may identify new therapeutic strategies for treating HAD and other neurodegenerative disorders. HIV-1 viral strain clade C is currently responsible for more than 50% of new HIV infections and is now the most commonly transmitted subtype worldwide. While HIV-1-associated dementia continues to be a major neurological complication of AIDS among HIV-1 clade B-infected individuals in the US and Europe, the incidence of HIV-1-associated dementia in regions like India and sub-Saharan Africa, where clade C infection is prevalent, appears to be lower. This application will establish a foundation of work detailing the differences between viral clade B and C and the potential implications for HIV-induced dementia. It is our hope that determining the mechanisms by which HIV-1 influence neuronal injury may identify new therapeutic strategies for treating HIV-1-associated dementia and other neurodegenerative disorders.	{ "pile_set_name": "NIH ExPorter" }
The goal of this proposal is to investigate the mechanisms by which poly ADP-ribosylation (pADPr) of proteins regulates gene expression. The level of protein pADPr reflects the relative activities of the poly(ADP-ribose) polymerase (PARP) enzyme, which utilizes NAD to create pADPr-modified proteins, and the poly(ADP-Ribose) glycohydrolase (PARG) enzyme, which removes pADPr moieties. My studies in Drosophila first revealed vital roles for PARP protein in the establishment of silent chromatin domains as well as in the chromatin loosening and transcriptional activation of a subset of inducible chromosomal loci. Subsequently, increased expression of inactive PARP1 protein has been implicated in the formation of condensed and silent chromatin domains, whereas upon the stimulation of PARP enzymatic activity, chromatin decondenses and becomes transcriptionally active. At present, the main gaps in our understanding of the PARP1-dependent transcriptional regulation are (1) the mechanism of PARP protein targeting to specific chromatin domains, and (2) the mechanism of local PARP activation. In preliminary studies, we have successfully identified novel chromatin-associated PARP1 partners by use of a Tandem Affinity Purification (TAP) strategy together with sucrose gradient purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Candidate interactors arising from this approach were functionally analyzed for influence on PARP1, using standard genetic approaches combined with immunostaining and confocal microscopy. Based on this work, we have identified the variant histone H2Av as a protein that promotes targeting of PARP1 to chromatin. Recent studies of histone H2Av by other groups have suggested this protein triggers DNA repair, apoptosis and heterochromatin formation. Ser137- phosphorylated H2Av has been shown to co-localize with foci of local PARP1 activation in vivo. In this proposal, we will focus on two aspects of PARP1 targeting to chromatin. First, we will characterize the mechanism through which H2Av controls PARP1 protein incorporation to chromatin and regulates chromatin-directed PARP activity. Second, we will evaluate functions of the PARP1 protein domains, defining which specify PARP1 interactions with chromatin. We are will address these questions with the following specific aims: Aim 1. To characterize the mechanism by which H2Av targets PARP1 to chromatin. Aim 2. To determine the role of core histones in the PARP1 protein binding to chromatin. Aim 3. To investigate the roles of automodification and phosphorylation in regulating PARP1 targeting to chromatin in vivo. Relevance to Public Health Statement: Understanding how an organism can utilize PARP protein ability to modulate chromatin and transcription will provide fundamental insight into genetic processes that are essential for growth, development, and pathogenesis. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
Cognitive decline and cognitive impairment are common in older adults and are associated with significant morbidity and mortality. However, few interventions have been identified to prevent or delay cognitive decline. Widespread environmental pollutants may be related to cognitive decline in later years through neurotoxic effects or adverse effects on cardiovascular health and are potentially modifiable. If a causal relationship exists, intervention on such risk factors would have substantial benefits. The proposed work will explore whether exposures to two common environmental pollutants, lead and traffic-related air pollution, are associated with cognitive decline. Evaluation of the relationship between lead and change in cognitive test performance over time will be conducted using a subset of participants in the Nurses'Health Study (NHS), a cohort of community-dwelling older women. Generalized estimating equations will be used to evaluate whether higher levels of lead biomarkers are adversely associated with change in cognitive test performance over time. In addition, menopause-related bone mineral density releases lead previously stored in bone into the systemic circulation and may increase the vulnerability of women to the adverse effects of past lead exposure. To evaluate the whether recent exposures, including menopause-related reintroduction of lead, is associated with greater declines in cognitive function in postmenopausal women, the relationship between blood lead levels and change in cognitive performance over time will be assessed in a subset of women whose blood lead measurements reflect exposure during this critical period. Evaluation of the relationship between traffic-related air pollution and cognitive test performance will be conducted in subset of participants in the Normative Aging Study (NAS), a cohort of older men. Mixed models will be used to evaluate whether higher levels of exposure to black carbon, estimated using a validated spatiotemporal land-use regression model, have an adverse effect on the level or the change in cognitive performance over time. Understanding the role of these exposures on cognitive function may suggest beneficial interventions and help to elucidate the underlying biologic processes of this detrimental condition. PUBLIC HEALTH RELEVANCE: Identification of and intervention on modifiable risk factors for cognitive decline in older adults would be expected to have significant benefits. If exposure to lead or air pollution is related to cognitive decline, interventions to lessen exposure, including increasing bone health or regulation of pollutants would be beneficial. In addition, identification of risk factors for cognitive decline may provide valuable insight into the underlying biological mechanisms for initiation and progression of cognitive decline and the associated disease state of dementia. )	{ "pile_set_name": "NIH ExPorter" }
Angiogenesis in mature tissues is a complex process involving dissolution of the existing matrix, migration and proliferation of endothelial cells, formation and maturation of new vascular structure and finally deposition of new extracellular matrix. Recent from our laboratory as well as others has demonstrated that some of these growth factors including VEGF and FGFs can induce functionally significant angiogenesis when used in the setting of myocardial ischemia. The mechanisms of these effects are, however, poorly understood. In particular, our as well s other studies suggest a central role of nitric oxide (NO) in the mediation of many effects of VEGF including vasomotion, changes in vascular permeability and stimulation of endothelial cell growth. These biological effects of VEGF together, with the capacity of this cytokine to induce an inflammatory response appear to be at the core of its ability to initiate and sustain angiogenesis. In order to study these issues and to address the role of cell type specific of gene expression in angiogenesis, we propose to take advantage of mice lines with knockout of iNOS and eNOS as well as macrophage-deficient mice. The availability of these mutants in combination with established mouse models of angiogenesis and inducible transgenic tissue specific expression of desired genes, provide a powerful combination in the study of the molecular mechanism of angiogenesis. The project will concentrate on the following two Specific Aims: I. Role of NO in mediation of VEGF-induced effects in coronary circulation; II. Cell type specificity of VEGF-dependent NO generation. Taken together these two Aims will provide new information regarding fundamental biological processes involved in regulation of angiogenic response and lead to better understand of the molecular mechanism of VEGF action.	{ "pile_set_name": "NIH ExPorter" }
On the basis of the work done in the first grant period with acyclovir (ACV) and Epstein-Barr Virus (EBV) we have constructed a project extending this work and consisting of two parts: the first describes experiments designed to evaluate and characterize virologically and biologically the effects of four promising new antiviral agents (B10LF-62, BVDU, FIAC and FMAU), all nucleoside analogs and one of them a congener of ACV, on EBV replication both in isolated nuclei and in cell culture systems. The second part deals with the mode of action of the drugs. In this part based on what we have learned from our studies with ACV we will investigate drug metabolism and purify and characterize EBV-specified DNA polymerase, a key enzyme in the drug interactions, with respect to (i) kinetics of inhibition by phosphorylated drug, (ii) the ability of drug-triphosphates to serve as substrate, (iii) effect of incubation on primer-template efficiency, (iv) question of chain termination, (v) identification of incorporated drug moieties in EBV DNA, and (vi) reversibility of drug effects correlated with impact on latent infection. Data obtained from these studies will facilitate our understanding of the molecular mechanisms of action of the drugs and help in the development of selective antiviral agents.	{ "pile_set_name": "NIH ExPorter" }
The major objective of this proposal is to characterize cellular and subcellular mechanisms involved in the neuronal storage and release of serotonin (5-HT). Although found only in relatively small numbers of central and peripheral neurons, this neurotransmitter has major effect on behavior and mood. During the previous project period two specific serotonin (5-HT)) binding proteins (45 kDa and 56 kDa SBP) were purified and characterized. These proteins are found only in that subset of 5-HT-storing cells that is derived from neuroectoderm. It was proposed that 56 kDa SBP may be a precursor of the 45 kDa protein and that 45 kDa SBP may play a role in the storage of 5-HT in synaptic vesicles. These hypotheses will be tested in the proposed research (i) In order to determine if 56 kDa SBP is the physiological precursor of the 45 kDa molecule a determination will be made of (a) the extent of sequence homology between 56 kDa and 45 kDa SBP; (b) whether metabolically labeled 35S-56 kDa SBP is synthesized before 35S-45 kDa SBP and whether or not the 56 kDa protein is converted to the 45 kDa material. Monospecific antibodies to 45 kDa and 56 kDa SBP that were prepared during the prior project period will make these studies possible. This investigation will be facilitated by studying a recently characterized cell line (MTC cells) that produces and stores both 5-HT and SBP. If SBP is really an intravesicular 5-HT storage protein, it would be expected to be synthesized with a signal sequence, translated on polysomes attached to the RER, and cotranslationally segregated in the lumen of the RER. These predictions will be tested by in vitro translation using poly(A)+RNA extracted from the rat brain. The products (and their resistance to proteolysis) of in vitro translation with a rabbit reticulocyte lysate system will be compared with those synthesized on dog pancreas microsomes, in order to determine if cleavage of a signal peptide occurs and if there is vectorial transport of newly synthesized SBP into microsomes. If the initial experiments show that 56 kDa is produced before the 45 kDa SBP the intracellular compartment where the 56 kDa leads to 45 SBP conversion occurs, will be located. This will be done by following the fate, under control and experimental conditions, of pulse labeled SBP as it passes through successive subcellular compartments during a chase period. In additional experiments the ontogeny of serotonergic synapses will be studied. In particular, whether the timing of the appearance of 45 kDa SBP during ontogeny coincides with the development of serotonergic synaptic vesicles will be determined. Finally, the possibilities that 45 kDa SBP is co-released with 5-HT from activated serotonergic cells and is recaptured will be tested.	{ "pile_set_name": "NIH ExPorter" }
In this Competitive Grant Revision to the Fast-Track SBIR: "A Compact X-ray Station for Protein Crystallography (CXS)," we describe proposed enhancements and upgrades to the Compact Light Source (CLS) that will result in a substantial increase in x-ray flux to the CXS. The CXS is a project to design and build a combination of x-ray optics, endstations, and software that use the monochromatic, tunable x-ray beam produced by the CLS. The CXS project, though not yet complete, has already resulted in an almost turnkey scientific instrument - a synchrotron-like x-ray system - for structural biologists at academic and corporate research centers. In order to make a significant reduction in data collection time, especially for small crystals, enhancements to the CLS are needed. In response to the NIH Notice: "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications" - NOT-OD-09-058, we plan to expand the scope of the CXS grant to include several hardware upgrades to the CLS and to fund additional operations time. These efforts will result in significantly more x-ray flux and better reliability of the CLS, which together will greatly improve the impact and performance of the CXS. PUBLIC HEALTH RELEVANCE: The Compact X-ray Station for Protein Crystallography (CXS), along with the Compact Light Source (CLS), provides the technology to bring state-of-the-art macromolecular crystallography to structural biologists in their own local or regional laboratories. This tool will allow a wider community of researchers to conduct a broad range of structure determination experiments that will lead to better understanding of the role of proteins and other macromolecular assemblages involved in disease and therapy.	{ "pile_set_name": "NIH ExPorter" }
This is an AIDS Clinical Trial Group (ACTG) ongoing study designed to evaluate the potential late effects of therapies received in HIV-infected and HIV-uninfected infants, children and adolescents, currently or previously enrolled in pediatric ACTG protocols. The major aim is to longitudinally follow patients who have participated in perinatal and pediatric ACTG clinical trials in order to monitor late consequences of therapy, including long-term survival and quality of life.	{ "pile_set_name": "NIH ExPorter" }
There is increasing evidence that lymphocytes can target and inhibit myeloid leukemia cells both in the allogeneic and autologous settings. The potent graft versus leukemia (GVL) effect associated with allogeneic stem cell transplant (SCT) conveys activity against leukemia and contributes to the decreased relapse risk compared with patients who do not receive an allogeneic graft. While GVL can induce long-term remission, the potentially lethal complication of graft-versus-host disease (GVHD) limits overall effectiveness of this treatment approach. We hypothesize that GVL would be enhanced, and GVHD reduced or eliminated, if the target antigens that drove those responses were identified, and if T cells with GVL antigen specificity could be isolated and adoptively transferred to patients with leukemia in the allogeneic setting, and that this effect could be increased by vaccination with appropriate leukemia antigens. We have identified an HLA-A2-restricted nonomer peptide, PR1, as a target antigen of CTL that inhibits myeloid leukemia progenitors and kills myeloid leukemia bone marrow cells. We have found that PR1 is processed from both proteinase 3 (PRTN3) and neutrophil elastase (ELA2), which are aberrantly expressed in leukemia. After vaccination with PR1, immune responses were observed in the majority of patients, and persistent remission was documented in some patients with AML, CML, and MDS. Immune-based therapies with low toxicity could be useful in combined treatment strategies to induce remission in patients with leukemia in the allogeneic setting to enhance GVL and minimize GVHD. In this proposal, we will (1) determine whether phenotype, function, and T cell receptor (TCR) affinity differ between PR1-CTL elicited after vaccination compared to those derived from healthy donors;(2) determine whether aberrant trafficking of PRTN3 and ELA2 can reverse tolerance and facilitate the expansion of central memory PR1-CTL;and (3) determine whether PR1-CTL expressing high affinity TCR can be transferred to patients with AML after T cell-depleted haplo-identical SCT to boost GVL and diminish GVHD. Lay Description: Stem cell transplantation has been used to successfully treat patients with leukemia, but the complication of graft-versus-host disease and the limited availability of suitable stem cell donors limit our use of this otherwise life-saving treatment to less than 25% of the potential patients who could benefit. To improve the effectiveness of SCT, we have identified some of the leukemia-associated target molecules recognized by the donor lymphocytes that mediate an immune effect against the patient's leukemia. We used one of these antigens, PR1, as a vaccine to boost immunity against leukemia in some patients with refractory disease. We found that PR1 vaccination was associated with little toxicity, and it boosted immunity in the majority of patients and induced remission in some patients, which has lasted up to seven years. In this proposal, we will study the molecular basis for immunity to this target, and we will use a strategy identified during the previous grant period to expand donor lymphocytes against this antigen, that we then transfer to patients with leukemia. If successful, this technique could eventually be used to reduce GVHD while retaining the beneficial effect of long-term remission after SCT.	{ "pile_set_name": "NIH ExPorter" }
E. Justification A key goal of this Core is to provide access to state-of-the-art imaging technologies to researchers at CN and GWU to perform studies on cells and animal models in this area of IDDR. In addition, the Cellular Imaging and Analysis Core assist investigators in their studies on a variety of children's diseases through the expertise of the staff and the affiliated users. The services offered include assistance in 1) preparation of samples and examination of the cells and tissues by light microscopy and EM (see projects* 1-6), 2) quantitative analysis of the data obtained for each project and 3) obtaining cells and tissue samples to carry out genomics/proteomics analysis (see project#6). For these services we make use of dedicated staff in the Core and also through the communal participation of the users with specific expertise (see section E.2, 3). Additionally, we work together with other Cores including, the Genomics and Proteomics and the Biostatistics and Informatics Cores. Use of the recently acquired laser capture microscope (see section E.6.) has enhanced our ability for targeted extraction of diseased cells and tissues from animal or patient materials further enhancing the analysis of these materials with the help of the Genomics and Proteomics Core. Additionally the services provided for establishing neural cell cultures and cell lines have been valuable for subsequent analysis of these purified populations of cells for quantitative analysis of their molecular profiles through genomic and proteomics analysis. In the coming years, we will increase the support our Core provides in carrying out live imaging and quantitative analysis of imaging data. To this end, we have a new Director with extensive and outstanding experience in both these aspects and have hired a new Core Manager who has background and several years of experience in performing quantitative image analysis. In addition, we have started working closely with the Biostatistics and Informatics Core to enable the users to appropriately design and carry out suitable statistical analysis of their data. We are also working in close collaboration with other IDDRCs nationwide - one example of which is the work that Drs. Corbin and Huntsman's have been performing in collaboration with the IDDRC at the Kennedy Krieger Institute at John's Hopkins Medical School. Their work with Dr. Walter Kaufmann (an expert clinician and scientist in the area of Fragile X Syndrome) makes use of the Cellular Imaging Core and has recently resulted in a publication on inhibitory synaptic defects in a mouse model of Fragile X Syndrome (Olmos-Serano et al., 2010).	{ "pile_set_name": "NIH ExPorter" }
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Chemotherapy has long been used to provide tumor control during cancer treatment. However, there are several limitations of chemotherapy. First, free chemotherapeutic agents tend to distribute to the whole body, causing the familiar treatment side effects associated with chemotherapy. Second, a lack of a noninvasive manner to assess chemotherapy drug concentration post treatment has hindered study of how drug distribution/dose affects tumor control. Temperature sensitive liposomes (TSLs) may provide a way to overcome both of these hurdles because they localized drug release at the tumor site and allow noninvasive imaging of the drug concentration distribution. However, in order to have local drug release, and accurate measurement of drug concentration, local hyperthermia (HT) must be applied and the temperature must be accurately measured. The hyperthermia causes the lipid membrane of the liposome to break down, and the increased permeability in concert with the absolute temperature quantitatively influence the signal received from the imaging. Consequently, the project we are proposing will help explain the relationship between tissue temperature and drug release were in previous experiments the temperature from the hyperthermia device was estimated but not measured. The primary goal of our TSL research is to develop a technology that can be moved from the lab/animal domain into the clinic/human domain. Our initial work with the CIVM in Viglianti et al. 2004 and 2006 demonstrated that we could image and quantify drug distribution delivered with TSL. From this work the measured signal was dependent on drug/TSL concentration in addition to temperature. Temperature data from animals that were not imaged allowed us to make assumptions about the animal's temperature that were necessary to convert the measured signal to absolute concentration of drug. Although a first approximation, the previous work allowed Ponce et al. (2007) to demonstrate that the temperature distribution was just as important as total drug delivered in the efficacy of TSL. This was possible by changing the sequence of the applied hyperthermia and the administration of the TSL. In our experiment, the effects of HT are necessary for multiple reasons. Biologically, HT causes the vascular system of the tumor to become more "leaky" and increased the perfusion of the tumor. This effect is taken advantage of in the clinic in order to improve tumor oxygenation, which in turn leads to improved radiation response. To maximize the benefits of this effect, accurate temperature monitoring is essential. Attempts to do this non-invasively are currently on going. The method that is suitable for our purposes utilizes the proton resonant frequency (PRF) shift that occurs with changing temperature. To do this, we will use a spoiled gradient recalled acquisition in steady state (spoiled GRASS) pulse sequence. By using the pre and post heating phase data of a given ROI and the temperature independent phase data from an oil standard, the temperature of the tissue can be derived. The oil standards are used because they allow us to map the field inhomogenity and to correct for phase drift of the sample in the magnet. In our experiment hyperthermia is used for targeting of the liposome contents through TSL release. The rat will be treated with HT while inside the magnet, and the LTSL will be injected after HT. The study that we are proposing overcomes one of the main limitations of previous studies by Viglianti et al. (2004,2006) by actively monitoring the temperature of the animal instead of using steady state heat flow equations to estimate it. By noninvasively monitoring the temperature of the tissue during liposome content release, we can see how the temperature maps of the tissue will help predict the final destination of the chemotherapy drug release. This will potentially enable one to "paint" chemotherapy drug onto the tumor by altering the temperature maps so that the desired end doxorubicin concentration is received. The project that we are proposing uses a rat fibrosarcoma model with liposomes injected before hyperthermia. We will then attempt to relate a non-invasive temperature map obtained from a spoiled gradient recalled echo pulse sequence in a 2T magnet to the T1 based DOX concentration measurements in order to better understand how heating affects in vivo drug concentration and further demonstrate the translation of the imagable liposome system to the clinic.	{ "pile_set_name": "NIH ExPorter" }
The validity of an earlier finding that patients with Huntington's Disease, a genetic disorder, exhibit an increased incidence of a rare allele for plasma cholinesterase is being tested on a sample of 100 patients and 100 matched controls.	{ "pile_set_name": "NIH ExPorter" }
This proposed study of the stress/mental health relationship utilizes a research and development strategy to investigate individual and situational stress interaction in states of mental health. The major study focus of the R & D action is on the role of stress in hypertension and depression in black persons and families. There are three objectives: 1. To conduct the second year activities of a major five-year study on stress, hypertension and depression in Black families; 2. to conduct second year phase of the three-year pilot study of the development of a taxonomy of Black families; and 3. to seek additional funding to conduct a collaborative study on the impact of five community mental health centers in the provision of services to Black communities, and to conduct a study on the development of Black infants of adolescent families. METHODS: The major study will be conducted as a survey, longitudinal (5-year) design with a sample of 400 probands and controls and their families. Variables to be studied include life stress events, stress coping styles, resources and assets, cognitive stress set, personal and familial health history, blood pressure, depression and other affective states. The study design combines case-control and family-cluster panel methods, and makes assessments retrospectively and prospectively. The exploration of a predictive model for the target diseases is proposed. The pilot studies will include simple experimental designs as well as survey, and ecological analysis procedures. For objective 3, the emphasis will be on developing critical MH data systems (i.e., mini-clearinghouse) and providing technical assistance to local and national individuals, institutions and communities via workshops, seminars, conferences and publications. SIGNIFICANCE: The total R & D efforts of the Center will have a significant impact on the local and national black mental health research and service efforts.	{ "pile_set_name": "NIH ExPorter" }
This revised ADAMHA Research Scientist Development Award application proposes studies of the ontogeny of motivation, reward and learning, and their physiological and neural substrates. The experiments exploit a developmental system of independent ingestive behavior in infant rats (described by my lab) and techniques for neural analysis recently adapted to infants. The proposed experiments are divided into two related research programs: (a) on ingestive development and (b) on early reward and learning. (a) Studies of ingestive development will assess sensory and physiological controls of ingestion in neonatal rats and describe how these controls develop, including assessing how such development is influenced by experience. Neural mechanisms underlying the developing ingestive system will be studied with brain transection techniques and deoxyglucose autoradiography. (b) Studies of early reward and learning will make use of ingestive rewards to describe and contrast the properties of basic forms of early learning as well as assess their long term effects. Neural mechanisms of early learning will be investigated using unilateral olfactory training techniques and deoxyglucose autoradiography. These experiments provide an ontogenetic analysis of a motivational system, one that can be followed from birth and manipulated in a controlled fashion, a system in which maturing neural systems can be related to changes in behavioral organization. This motivational system will be used to gain a better understanding of how specific early experiences influences the course of individual development, and determine how forms of early learning are represented in the brain. The award will contribute to my continued professional growth in areas of developmental analysis and expand my skill in neural and physiological analysis of behavioral mechanisms. Training plans are detailed in the proposal.	{ "pile_set_name": "NIH ExPorter" }
The complexity and relationships of various classes of RNA in the mouse brain are being studied by nucleic acid hybridization strategies. A population of nonadenylated messenger RNAs (poly(A)-) equal in complexity to that of polyadenylated (poly(A) plus) mRNA has been discovered. In terms of MRNAs encoded by single copy DNA, there is essentially no sequence overlap between the poly(A) plus and poly(A)-mRNAs. The biological significance of these two classes of mRNA is unknown, but our recent investigations show that a wide variety, perhaps all, of the poly(A) plus mRNAs are encoded discontinuations in the genome (split genes). Other studies indicate that most poly(A) plus mRNAs are derived from much larger percursor nuclear RNAs and that these mRNAs are also regulated, in terms of their presence in the polysomes, post-transcriptionally. Preliminary experiments suggest that these observation of poly(A) plus mRNA may not apply to poly(A)-mRNA. Many brain-specific mRNAs may be nonadenylated.	{ "pile_set_name": "NIH ExPorter" }
ronchiolitis obliterans is an obstructive disease of the small airways of the lung that may develop after either hematopoietic stem cell or lung transplantation. It is associated with considerable morbidity and mortality in long-term transplant survivors and thus represents a major impediment to the success of these therapies Mortality due to bronchiolitis obliterans-associated respiratory failure may occur in more than 55% of patients developing this complication typically within 3 to 5 years following transplantation. The pathogenesis of bronchiolitis obliterans in hematopoietic stem cell and lung transplants appears to share a common final pathway of immune-mediated injury and inflammation to airway epithelium and sub-epithelial structures that results in granulation tissue and fibro-proliferation. Following hematopoietic stem cell transplantation, a chronic inflammatory process develops as a consequence of transplanted T cells targeting allo-antigens expressed on the respiratory epithelium of the recipient. In contrast, the inflammatory injury that develops in the transplanted lung results from alloimmune-dependent and independent mechanisms. Other additive factors to these immune-based inflammatory injuries include viral infections and micro-aspiration. Treatment is based primarily on increased systemic immunosuppression which results in attendant increased risk of infection and morbidity. As a consequence, conventional therapy for bronchiolitis obliterans has not improved long-term outcome of this disorder. Recently, cyclosporine inhalation solution (CIS: made by the APT pharmaceutical company) in solution with propylene glycol has been shown to improve overall survival and chronic rejection-free survival in lung transplant patients. These findings suggest targeted delivery of immunosuppressive therapy to the diseased organ warrants further investigation as this may minimize the morbidity associated with systemic immunosuppression. However, inhaled cyclosporine has not been studied in the treatment of BO following hematopoietic stem cell transplantation. This program represents collaboration between the Hematology branch of the NHLBI, The Critical Care Medicine Department of the NIH Clinical Center, and The Pulmonary Medicine Department at The University of Maryland. Studies are being done in association Aldo Iacono, MD, the Medical Director of the Lung Transplant Program at the University of Maryland. Dr. Iacono pioneered the development of inhaled cyclosporine for lung transplants. Our group was awarded a bench to bedside award to study the safety and efficacy of inhaled cyclosporine for the treatment of BO. Two distinct patient populations will be offered enrollment on this clinical protocol: hematopoietic transplant recipients with BO and lung transplant recipients with BO referred from the University of Maryland. Patients will undergo dose titration of inhaled cyclosporine to a maximum dose of 300mg, three times weekly. Clinical parameters, including pulmonary function tests, will be measured in addition to laboratory markers of the anti-inflammatory response to CIS measured in the blood and from bronchio-alveolar lavages. Adverse events associated with treatment will be recorded. The primary objective is assessing the safety and efficacy of inhaled cyclosporine as a new therapy in hematopoietic transplant patients and lung transplant patients with established BO and to promote a better understanding of the pathogenesis of BO in these two transplant groups. This study is actively accruing patients with preliminary efficacy having already been observed in the first cohort of stem cell transplant patients being treated with this agent.	{ "pile_set_name": "NIH ExPorter" }
The objective of this proposal is to develop a glycoside adjuvant or immune agonist capable of concurrently stimulating both innate and adaptive immunity, and driving the induction of both cellular (Th1) and humoral (Th2) immune responses in a synergistic manner. The results of this proposal may lead to the development of a novel subclass of clinically applicable synthetic saponin of triterpene, gypsogenin, which can serve as immune agonists that are stable at room temperature, non-toxic, well characterized, and capable of promoting both innate and adaptive immune responses. To develop this adjuvant, this proposal seeks to add to the triterpene i) oligomannans that can serve as ligands for innate immunity receptors, e.g. toll-like receptors (TLRs) and lectins, and ii) lipophilic chais, that may be designed to serve as ligands for innate immunity receptors. Additionally, the aldehyde group of triterpene, by forming imines with the amino groups of certain cell surface receptor(s), can also provide co-stimulatory signals requisite for eliciting Th1/Th2 immunity. The triterpene group can facilitate the delivery of exogenous antigens to the cytosol of antigen-presenting cells to be processed for presentation to and activation of cytotoxic T lymphocytes (CTL). The concurrent stimulation of both innate and adaptive immunity receptors in the same cell would synergistically drive a much more effective immune response to the immunogen than would be elicited by adjuvants that target these different receptors individually. In this proposal we seek to test the hypothesis that the inclusion of an adjuvant that simultaneously triggers receptors of innate and adaptive immune immunity within the same cell can synergistically improve immune responses to an immunogen as compared to adjuvants that target these receptors individually. This hypothesis will be tested in the following specific aims - 1) Synthesize two immune agonists, having an aldehyde-carrying triterpene (gypsogenin) linked covalently to a mannan oligosaccharide and a lipophilic chain. 2) Assess the ability of the synthetic saponins to enhance cellular and humoral immune responses to the model antigen recombinant hemagglutinin protein (rHA) in mice. Immune responses will be evaluated for the production of IgG2a and IgG2b, antigen-specific lymphoproliferation, the production of Th1 and Th2 cytokines (IL-2, IL-4, IL-5, IFN-r, and TNF-a), and the induction of CTL. 3) Evaluate saponin's critical micellar concentration, haemolytic effect, stability and acute toxicity.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION (provided by principal investigator): The prevalence of obesity and chronic sleep loss are at record levels among Americans and evidence continues to emerge to support a causal link between the two conditions. Metabolic and cardiovascular abnormalities related to sleep disruption are particularly evident in individuals with obstructive sleep apnea (OSA), a disorder traditionally associated with male gender. While more prevalent in men, OSA is underrecognized in women in part because its clinical and polysomnographic features differ from those of men. Women with polycystic ovary syndrome (PCOS) are particularly susceptible to OSA with at least a 5-fold higher risk for its development compared to obese women without PCOS. The overarching aim of this SCOR application is to therefore establish the basis for the apparent gender difference in prevalence of OSA by focusing on the mechanistic role of sex steroids in the pathogenesis of the disorder as well as its metabolic complications. Four Projects sharing integrated hypotheses, aims, and methods, plus an Administrative Core are proposed. In Project 1 (Van Cauter. principal investigator) and Project 2 (Ehrmann. principal investigator) subjects with and without OSA will have detailed assessments of sleep, metabolic, and cardiovascular function; studies will be conducted in serum and urine for metabolomics and in fat biopsies for adipocyte function. Obese men and women with and without OSA will participate in Project 1: those with OSA will be treated with continuous positive airway pressure (CPAP) and its impact on baseline measures will be assessed. Project 2 will enroll obese women with PCOS, with and without OSA. Those with OSA will receive CPAP or will be randomized to receive depot leuprolide to suppress ovarian steroid output over 12 weeks, reassessed at 6 weeks, and then randomized (double-blind, placebo controlled) to 6 weeks of either micronized estrogen + placebo or micronized progestin + placebo. The independent effects of androgen, estrogen, and progesterone on OSA and metabolic function will be assessed. Project 3 (Mittendorfer, principal investigator) will focus on mechanisms responsible for increased plasma triglyceride (TG) concentration, a finding common to both OSA and PCOS. Studies of VLDL-TG kinetics will be undertaken before and after modulation of plasma glucocorticoid, progesterone, and testosterone concentrations. In Project 4 (Brady. principal investigator) primary human adipocytes will be prepared from fat biopsies obtained in Projects 1 - 2. Insulin sensitivity will be determined by phospho-specific immunoblotting in conjunction with glucose uptake and anti-lipolysis assays. In parallel, adipocytes from these subjects will be cultured for 1-5 days prior to metabolic assays to ascertain if removal of from circulating factors will improve insulin signaling, or if insulin resistance persists in vitro. Finally, the Administrative Core will have oversight of all Project functions; interface with the Metabolomics Laboratory at Duke University (C. Newgard, Lab Director); and coordinate meetings of the External Advisory Committee. [unreadable] [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
This study of hormonal regulation is targeted to define the biochemical mechanisms whereby age-dependent changes perturb physiological control systems and, thus, lead to failure to maintain homeostasis in the aged. Investigations are focused on the biochemical interactions of hormones, which are mediated via cyclic AMP and cyclic GMP. Topics investigated include: 1) hormone receptors in membranes; 2) adenylate cyclase and guanylate cyclase activities; 3) phosphorylation of membrane proteins by cyclic nucleotide-dependent and -independent protein kinases; 4) regulation of protein phosphorylation by Ca2 ion and Ca2 ion-dependent regulatory protein (Calmodulin).	{ "pile_set_name": "NIH ExPorter" }
Polyvalent anti-immunoglobulins, an anti fab, and class-specific anti IgM, anti gamma, anti gamma 2, and anti IgA were purified by affinity chromatography on columns of myeloma proteins covalently coupled to sepharose. Purified anti immunoglobulin was covalently coupled to Sephadex G-200, and lymphoid cells were fractionated on columns thus prepared. Most of the immunoglobulin bearing lymphocytes adhered to the columns and could be eluted with soluble myeloma proteins or normal immunoglobulin. Most of the thymus derived cells passed through the columns. These columns provide a means of specifically separating T- lymphocytes and B-lymphocytes.	{ "pile_set_name": "NIH ExPorter" }
In chronic obstructive pulmonary diseases such as chronic bronchitis, and in cystic fibrosis, the airway epithelium is constantly exposed to neutrophil elastase (NE), an inflammatory protease. The cellular response to NE dictates the balance between epithelial injury and repair. A critical factor in this balance is the activation of the epidermal growth factor receptor (EGFR), a member of the ErbB receptor tyrosine kinase family. Activation of EGFR requires homo- or heterodimerization of the receptor. A major EGFR heterodimerization partner in airway epithelial cells is ErbB2. To date, the only ligand known to activate ErbB2 is MUC4. MUC4 is a major membrane-tethered, respiratory tract mucin with epidermal growth factor (EGF)-like domains. We have made four key observations supporting a critical role for MUC4 in activating the airway epithelial response to NE: (1) NE acts on airway epithelial cells to enhance MUC4 mRNA stability and increase MUC4 protein levels. (2) NE induces tyrosine phosphorylation of EGFR. (3) NE stimulates proliferation of normal human bronchial epithelial cells in serum-free and EGF-free media. 4. MUC4, ErbB2 and EGFR colocalize in injured superficial airway epithelial cells in vivo. These observations support the hypotheses that NE triggers a molecular cascade of events in airway epithelial cells by inducing the production of MUC4, a key regulatory molecule in the cascade, activating ErbB2, and thereby promoting the heterodimerization/activation of EGFR. EGFR activation is a critical prerequisite for epithelial proliferation. The Specific Aims to be tested in this proposal are: (1) To determine whether NE induces MUC4-activation of the ErbB2/EGFR receptor tyrosine kinase cascade, resulting in epithelial cell proliferation. (2) To identify the RNA stability domains and RNA-binding proteins regulating NE-induced expression of MUC4, a key molecule in this signaling cascade. (3) To determine the relevance of these signaling pathways in mediating epithelial proliferation in vivo in chronic bronchitis and cystic fibrosis. Our ultimate goal is to use information from this project to identify new biologic targets for rational therapies to induce normal airway epithelial proliferation and differentiation following injury.	{ "pile_set_name": "NIH ExPorter" }
Malaria remains a major cause of illness and death worldwide. Plasmodium vivax malaria is an especially important cause of morbidity in South America. Two major unresolved issues concerning the pathogenesis of P. vivax malaria are: 1) what molecules from the parasite activate the innate immune system and cause inflammation and, 2) what are the receptors that are ligated by these microbial products. Our studies suggest that Toll-like receptors (TLRs), acting primarily through the Myeloid Differentiation Factor 88 (MyD88) adaptor molecule, are critical for initiation of this early inflammatory response and pathogenesis of malaria. In addition, we have evidence of Nod-Like receptor (NLR) activation, inflammasome formation and processing of caspase- 1 in human patients during malaria. We hypothesize that upon P. vivax infection, innate immune receptors in phagocytes are responsible for the intense cytokinemia as a consequence of hyper-responsiveness of Toll-like receptors (TLRs), and the assembly of inflammasomes. However, the most basic details of malaria-related cytokinemia are not well understood, although our studies in P. falciparum allow us to construct a hypothetical model of disease surrounding the delivery of parasite DNA to innate immune sensors. We further hypothesize that the innate immune response results in priming of the innate immune system which triggers the production of cytokines via phagocytosis of nucleic acids of via formation of immune complexes. We will evaluate (i) the levels of circulating parasite DNA, anti-nucleic acid antibodies, as well as immunocomplexes (ICs); (ii) the immunostimulatory activity of DNA-containing ICs on triggering cytokine production and caspase-1 activation via nucleic acid sensing innate immune receptors expressed by phagocytes; and (iii) as indicative of malaria priming, we will look for the expression of genes related to the TLRs, NLRs and IFN signaling pathway as well as the responsiveness of peripheral blood mononuclear cells (PBMCs) from patients infected with P. vivax. Hence, we hope to define the contribution of ICs, pro-inflammatory priming, and inflammasomes on systemic inflammation observed during acute P. falciparum malaria. We hope to identify immunomarkers of disease, whose expression indicates checkpoints that are critical for the pathogenesis of P. vivax infection. Such knowledge should contribute to new insights on the molecular basis of inflammation during malaria and help to improve clinical management of malaria patients. Finally, this project will be done in collaboration with Dr. Carolina Gallego-Marin from CIDEIM (International Centre of Medical Research and Training) in Cali, Colombia, with the purpose of developing her career as an independent investigator in Colombia. The research will be performed primarily by the LMIC collaborator; initially 50% of the work will be done at CIDEIM and 50% in Massachusetts, and gradually the amount of time Dr. Gallego spends in South America will increase, with the ultimate goal of exporting cutting edge technology and expertise to the scientific community in Colombia.	{ "pile_set_name": "NIH ExPorter" }
Conduct Phase I studies of immunotherapeutic agents in patients with advanced cancer. These studies will be conducted under protocols which have been approved by the project officer. The goal of the protocols will include the evaluation of dose, route, and schedule of administration of immunotherapeutic agents and the effects of these agents on in vivo and in vitro aspects of the immune response.	{ "pile_set_name": "NIH ExPorter" }
Osteoporosis is a major public health problem, especially in women. It is mainly characterized by low bone mineral density (BMD). BMD has high heritability of > 60%. Women have much lower BMD than men. We and others demonstrated that some BMD genes/genomic regions are gender specific. Menopause is a most significant physiology event in female's life and is associated with female-specific rapid bone loss. Bone marrow mesenchymal stem cells (BMMSCs) and peripheral blood monocytes (PBMs), are precursors for osteoblasts (bone formation cells) and osteoclasts (bone resorption cells), respectively. Our hypothesis is that changes in the protein expression profiles in female BMMSCs and PBMs underlie mechanisms of female BMD variation and are associated with menopause. Our major goals here are to identify proteins differentially expressed in BMMSCs and PBMs in women: 1) with high vs. low BMD; 2) before and after menopause, and thus identify proteins (and their genes) associated with female osteoporosis and menopause in BMMSCs and PBMs. The proteins identified significant in females will be examined in male samples. Together with Project 2, we will recruit 80 otherwise healthy females and 80 age-matched otherwise healthy males aged 50-55, including 40 subjects with low and 40 with high BMD (age matched population bottom or top 20% respectively) for each sex, all Caucasian. Each female BMD group includes 20 pre- and 20 age matched post-menopausal women. We will take fresh bone marrow (for which we have had extensive published research experience) and peripheral blood samples from each subject. BMMSCs and PBMs will be isolated and equally divided into two aliquots, one for Project 2 and one for Project 3. In this Project 3, we will extract the total proteins from aliquots of isolated BMMSCs and PBMs. Proteomic profiling experiments and analyses will be performed on Females using MD-nano-LC-MS/MS. Significant differentially expressed proteins identified will be verified by Western blotting with female samples for their importance in females. Significant proteins verified in females will be examined by Western blotting with male samples for sex-specificity. The results obtained in Caucasians will be cross checked for ethnic generality/specificity in Chinese samples. The molecular and cellular functional studies of the identified proteins and their genes will be pursued as exemplified in Project 2 of this SCOR. The major results (particularly those obtained from PBMs) of this study may be used to design customary diagnostic protein antibody chips and/or protein markers for prognosis of female osteoporosis. In particular, the results will be used to provide some functional evidence, when combined with the DMA polymorphism data in Project 1 and DMA microarray data in Project 2 of this SCOR to powerfully and efficiently identify genes and some of their functions for female osteoporosis.	{ "pile_set_name": "NIH ExPorter" }
This study will focus on families with significant history of pre-eclampsia and eclampsia in an effort to find polymorphic DNA markers which cosegregate with the tendency to develop this disease. Once linkage is found, DNA markers will be developed which might be used clinically to test for a genetic susceptibility to pre-eclampsia, and also to begin the search for the "pre-eclampsia gene" and its products.	{ "pile_set_name": "NIH ExPorter" }
Alterations in the organization of the phospholipids in the lymphocyte plasma membrane may affect several lymphocyte functions. This proposal is designed to assess the role of phospholipid packing in the interactions of lymphocytes with reticuloendothelial cells and with the targets of cytolytic T lymphocytes (CTL). Immature and activated lymphocytes isolated from tissues bind merocyanine 540 (MC540), a fluorescent lipophilic probe for loosely-packed lipids, whereas lymphocytes isolated from the peripheral circulation do not. Whether this difference in phospholipid packing is responsible for differences in the interactions of these lymphocytes with other cells will be tested in several ways. Using centrifugation to produce a controlled and reproducible force to distinguish adherent from nonadherent cells, the strength of the interactions of peripheral blood lymphocytes, thymocytes and splenocytes with macrophages and endothelial cells will be measured. Using flow cytometry, thymocytes and splenocytes stained with MC540 will be fractionated according to fluorescence intensity and the adherence properties of the fractions determined. The dependence of cellular interactions on lipid organization will be examined in vivo by following clearance from the circulation of reinfused lymphocytes which differ in phospholipid packing. Two biophysical approaches will be used to investigate the mechanisms by which altered phospholipid organization could affect adherence. Fluorescence recovery after photobleaching will be employed to assess whether increases agglutinability of thymocytes with loosely-packed lipids results from increased mobility of membrane receptors. Fluorescence transfer methods will be used to determine whether the loose packing of lipids in activated lymphocytes is accompanied by local aggregation of a lymphocyte adhesion-related membrane protein, LFA-1. Finally, phospholipid organization in CTL and their targets will be assessed by MC540 staining to evaluate the potential role of this parameter in the resistance of CTL to self-lysis and in the efficiency of killing of target cells.	{ "pile_set_name": "NIH ExPorter" }
The project will develop a portable microanalytical system based on nanoparticle labels/ immunochromatographic electrochemical biosensor (IEB) for on-site rapid detection and quantification of tobacco smoke exposure (TSE)-induced multiple biomarkers in biological matrices. The detection of TSEinduced biomarkers is based on the principle of immunochromatographic separation and nanoparticleenhanced bioelectrochemical detection. To improve the sensitivity of electrochemical detection in the immunochromatographic platform, apoferritin-templated metallic phosphate nanoparticle labels will be employed to amplify detection signals of those biomarkers. The first goal of this Project is to establish nanoparticle-based competitive electrochemical immunoassay for (a) cotinine as a marker of tobacco smoke exposure. The detection system will be tested and evaluated using human plasma and urine samples (Project 1) and mouse plasma, urine, and saliva (Project 2). Secondly, Project 3 will establish nanoparticlebased multiplexed electrochemical immunoassay for sequence specific nitrotyrosine, sequence - independent nitrotyrosine (identified by Project 1 and 2, and Core A and B) and the pleiotropic, non-specific marker, TNF-alpha. Finally, this Project will build a prototype Immunochromatographic/ Electrochemical Biosensor (IEB) device for on-site application. The device will provide for analysis of exposure (cotinine), selected markers of specific (nitrated proteins) and general markers of stress (e.g. TNF-alpha) in a single platform.	{ "pile_set_name": "NIH ExPorter" }
Chronic pain resulting from orofacial disorder can be intractable and often involves neuropathic processes. Analgesic drugs used for treating acute pain are not very effective for chronic pain, partly because most analgesic drugs are designed to target neurons. Compared to neurons, the role of glial cells in chronic pain is less defined. Increasing evidence shows that spinal.glia (e.g., microglia and astrocytes) are markedly activated following nerve injury and contribute to the development of neuropathic pain, one of the best characterized chronic pain conditions/However, it is largely unknown as to how glia interact with neurons in chronic pain conditions. The overall goal of this application is to investigate how injury-inducedneural activity from the periphery results in an activation of spinal microglia, and further, how glial products influence dorsal horn neuron activity. Results from our previous studies have shown that nerve injury induces a drastic activation (phosphorylation) of p38 MAPK (mitogen-activated protein kinase) specifically in spinal microglia, and blockade of this activation suppresses the development of neuropathic pain. Therefore, p38 phosphorylatipn can be used as a pain-related marker for spinal microglial activation. Our preliminary data show that spinal infusion of the chemokine fractalkine strongly activates p38 in the spinal cord, which provides further evidence that fractalkine is involved in mediating neuronal/glial interaction and neuropathic pain. We propose to produce a nerve injury condition (spinal nerve ligation) to mimic the neuropathic aspect of orofacial pain and test our hypotheses via the following 3 specific aims: (1) To establish that fractalkine, liberated by injury-evoked nerve activity, causes p38 activation, in spinal microglia; (2) To define whether p38 activation induces the synthesis of the proinflammatory cytokines IL-1J3 and IL-6 in spinal microglia via an upregulation of the transcription factor NF-KB; and (3) To determine whether IL- ip, IL-6, and fractalkine rapidly enhance synaptic transmission and also induce the expression of the pronociceptive genes NK-1 and COX-2 in spinal laminae I-II neurons. We will combine the methods of behavioral testing, whole cell patch clamp recording in spinal slices, immunofluorescence, in situ hybridization, Western blot, and kinase assay to accomplish these aims. Our studies will reveal new mechanisms for neuronal- glial interactions, which may help develop new and more effective therapies for the management of chronic pain including orofacial pain conditions.	{ "pile_set_name": "NIH ExPorter" }
Two-thirds of the >38 million people with HIV/AIDS reside in Sub-Saharan Africa. Access to antiretroviral therapy (ART) in less wealthy nations is expanding quickly, creating an urgent need to understand those factors influencing adherence, a key determinant of ongoing efficacy of ART, so that the creation and spread of medication-resistant virus is limited. The proposed research team has identified suboptimal adherence among 29% of clinic attendees taking ART for >6 months in Moshi Tanzania, while documenting lack of virologic suppression in 32% and antiretroviral drug resistance in 10%. We propose to follow 750 newly diagnosed HIV+ persons, 500 previously diagnosed HIV+ persons and 750 HIV-negative persons in over 3 years in this region. The newly diagnosed HIV+ and HIV- cohorts will be obtained through consecutive sampling in a Voluntary Counseling and Testing (VCT) site and the on-going HIV+ cohort will be similarly drawn from the Infectious Disease Clinic (IDC) in the tertiary care medical center 5 miles away. This research team has a track record of successful past and ongoing research at both the VCT and the IDC sites. Baseline data we will used to examine associations between psychosocial (including trauma, mental health symptomatology, coping strategies and life events), sociodemographic, and economic factors and health status, adherence to ART, and health promotion and high risk activities. Longitudinal analyses will examine causal relationships between changes in the independent and dependent variables. Results will lead to the creation of more effective prevention and care models for the area and other similar areas. Studying the population of ART naive patients in a resource poor setting provides a unique window of opportunity to gather data designed to increase our understanding of underlying variables associated with maladherence and risk behaviors. Psychosocial determinants are a unique strength of this proposal. The study will result in a minimum of 7 peer reviewed articles and dissemination of findings at annual national (US and Tanzanian) and international AIDS, Public Health and Mental Health conferences, including the annual Tanzania national AIDS conference. A policy related report will be disseminated to national and international leaders.	{ "pile_set_name": "NIH ExPorter" }
Specific Aims: This study will evaluate whether the case management and nursing, nutritional and psychosocial support service component of Washington's 1989 Medicaid expansion for pregnant women has succeeded in averting deleterious and expensive birth outcomes. Specifically, this study will 1) examine the effect of support services and case management on use of prenatal care, birth outcomes and medicaid payments, 2) explore whether the impact of these programs is greater in certain groups of women, such as teens, and 3) determine whether the more support and case management services used, the better the birth outcomes attained. Background and Significance: Despite their lack of financial barriers to care, women insured by Medicaid have higher rates of inadequate prenatal care and poor birth outcomes than privately insured women. Addressing the social service needs of low-income pregnant women has succeeded in improving birth outcomes in several small scale programs, and how is being implemented by a number of state Medicaid programs. This project will help determine whether these large scale, statewide programs can also succeed in reducing adverse birth outcomes. During this time of shrinking public resources, evaluations of the effects of existing Medicaid expansions on birth outcomes and costs will help policy makers decide whether to maintain or expand these programs. Research Design and Methods: A pre- and post-intervention study design will be the primary method used to evaluate the effects of the support service and case management programs on prenatal care use, birth outcomes and Medicaid payments. We will compare the change in the study's outcome measures before and after Washington's implementation of these programs in the Medicaid-AFDC populations of Washington and Colorado, a "control" state without support service or case management programs. The study groups will include singleton births of Washington and Colorado residents insured by the Medicaid-AFDC program during seven baseline and seven impact months. Similar analyses will be performed for subgroups of women such as teens, African-Americans and unmarried women. A secondary, cross-sectional analysis will compare the study's outcome measures among subgroups of women receiving different levels of support and case management services during the post-Medicaid expansion period in Washington State only. We will use Washington's First Steps Database, which has linked birth, fetal death and infant death records with Medicaid eligibility and claims, for the analyses of Washington's births. Co-investigators in Colorado will create a similar linked database for the analyses of Colorado's births. The intervention of interest is the support service and case management component of Washington's Medicaid expansion. The major outcomes include one measure of the use of prenatal care (percent of expected prenatal visits), two measures of birth outcome (birthweight and newborn length of stay), and four payment measures (Medicaid payment per delivery for maternal care, neonatal care, post- neonatal care, and maternal and infant care combined). We will adjust for potential confounding factors, including maternal demographics, prior pregnancy loss, preexisting medical conditions, geographic location, availability of obstetric providers and medicaid enrollment pattern in our analyses.	{ "pile_set_name": "NIH ExPorter" }
Continued investigation at the ultrastructural level on the extent of resin penetration into treated enamel surfaces and the effectiveness of the seal against further acid attack. Examination of specific areas of enamel which show structural variations. A correlated scanning and transmission microscopic approach will be used. The effect of removal of the organic matrix of enamel will be investigated. A correlated microradiographic and transmission electron microscopic study of sections cut through a carious lesion will be initiated. Use of selected area diffraction will be continued to determine if there are any variations in the enamel crystal population.	{ "pile_set_name": "NIH ExPorter" }
Malaria is caused by the protozoan parasite, Plasmodium and inflicts much suffering in the developing world with more than a million deaths recorded each year. In addition, a significant number of cases of malaria are imported into nonmalarious areas by tourists, military personnel, and migrant workers. The prospects for control of malaria by chemotherapy have been seriously impeded by the emergence of drug-resistant parasite strains. This accentuates the need to study the biochemistry of this parasite so that potential target for chemotherapy can be identified. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the de novo synthesis of deoxyribonucleotides by the direct reduction of the corresponding ribonucleotides. The important role of RNR in DNA synthesis and cell division makes this enzyme an excellent target for chemotherapy. Several different classes of inhibitors targeted against RNR for cancer and viral chemotherapy already exist. Critical to the development of a chemotherapeutic agent directed against RNR from the malaria parasite is a detailed understanding of the biochemistry of this complex enzyme. The proposed investigation is an attempt to characterize ribonucleotide reductase from plasmodium falciparum (PF). Specifically we aim to: 1) Purify the native enzyme from PF culture lysates by chromatographic methods and assess its catalytic activity. 2) Test different classes of known mammalian and viral RNR inhibitors for antimalarial activity by measuring the effects on [3H]-hypoxanthine incorporation into nucleic acids. The kinetics of inhibition (Ki) of these inhibitors will be studied with native and/or recombinant enzyme preparation. 3) Overexpress and purify the recombinant RNR subunits in E. coli and/or baculovirus expression systems. Compare the activities of the recombinant and native RNR. 4) Study kinetic properties and the allosteric regulation of the PF RNR. 5) Study the in vivo expression of RNR subunit genes in asexual blood stages upon addition of DNA damaging or modifying agents by Northern blot analysis and nuclear run-on assay.	{ "pile_set_name": "NIH ExPorter" }
Galactomannan of Asperigillus fumigatus was measured in serum of patients with aspergillosis and experimentally infected rabbits. Normal volunteers showed an increase in serum antibody to cryptococcal polysaccharide in response to vaccination with this antigen.	{ "pile_set_name": "NIH ExPorter" }
This is a proposal to establish laser microdissection instrumentation using Laser Microdissection and Pressure Catapulting (LMPC) technology at the University of Kentucky. Laser microdissection technology has become a standard for cutting-edge biomedical research at many institutions and clearly will revolutionize studies of gene and protein expression. Numerous NIH-supported projects at UK focus on gene and protein expression approaches, in many cases with a clear focus on particular cell types, tissue lesions, genetically modified loci, and even subcellular regions. The ability to incorporate computer-automated, highly selective specimen collection will vastly accelerate research progress, refine interpretation, and drive the discovery of novel mechanisms in various physiological and pathological conditions that are the focus of UK's research enterprise. In particular, major user projects at UK emphasize detection of subtle changes in tissue, cell, and subcellular compartments in various cell types, cancerous lesions, and plaques, with aging, Alzheimer's disease, inflammation, traumatic brain injury, oxidative stress, and infection and heavily integrate translational goals with the concept of personalized medicine. The integrated system proposed here (Zeiss PALM MicroBeam) has the power, accuracy, and flexibility to address the diverse needs of the Major Users in this application and will allow researchers to focus on homogenous cell collection derived from extant tissue. Coupled with the molecular expertise at UK, this system will allow our researchers to investigate the critical interplay among different cell types at the transcriptional and proteomic levels during pathological processes, leading to rational development of new drugs and individualized pathological diagnoses. Thus, the proposed system will not only specifically enhance progress in multiple NIH-funded projects at UK, but will facilitate the development of new methods that may well benefit investigators at many other institutions as well. Relevance: In recent years, advances in molecular measurement technology (e.g., proteomics, gene expression arrays) have yielded important insights into disease processes. The equipment proposed here will bring sampling technology (i.e., the ability to select an area of tissue for study at the cellular or even subcellular level) up to the same standard. The combination of advanced molecular analysis and cell-level collection specificity will allow us to investigate the roles of different kinds of cells in diseases such as Alzheimer's, cancer and heart disease, leading to rational development of new drugs and individualized medicine approaches for treatment.	{ "pile_set_name": "NIH ExPorter" }
The objectives of the proposed research are to identify genes controlling replication during meiosis and to determine the role of these genes in the biochemical reactions of replication. Genes controlling replication (in Saccharomyces cerevisiae) will be detected by the isolation of temperature sensitive mutants with lesions blocking pre-meiotic DNA synthesis. The mutants are obtained by screening mutagenized clones for thermosensitive defects in induced meiotic recombination. Based on prior results a significant fraction of recombination deficient isolates, when tested by isotope incorporation, should have specific lesions in pre-meiotic replication. After genetic analysis, to determine dominance and segregation, suitable mutants are organized into a minimum number of functional groups (i.e., genes) primarily by genetic complementation tests. These experiments provide the basic genetic background for subsequent research. Our second objective, a start in identificatton of the biochemical reaction(s) controlled by each gene, begins with a determination of the thermosensitive period, representing the temporal span of a specific gene function. Genes with thermosensitive periods coincident with replication are further analyzed for the effects of the restrictive temperature on the kinetics of DNA synthesis. This provides a preliminary division of the genes into those which affect precurson (deoxynucleotide) formation or polymerization, from those affecting replication initiation.	{ "pile_set_name": "NIH ExPorter" }
This is a revised application for a K23 award to support Dr. Alexander Butwick's proposed research and training focusing on clinically effective and cost-effective strategies for reducing hematologic-related maternal morbidities, specifically postpartum hemorrhage (PPH) and early postpartum anemia (EPA), in women undergoing cesarean delivery (CD). Severe PPH can cause major obstetric morbidity and is a leading cause of maternal death in the US; EPA is an underappreciated adverse outcome, which may lead to long-term deleterious psychological, physical, and cognitive effects among postpartum women. Currently, risk- stratification for severe PPH and EPA and therapeutic approaches for EPA lack consensus and are predominantly non-standardized. For women undergoing CD, innovative approaches are needed for: (1) preoperative risk stratification for PPH and, (2) postoperative screening and cost-effective treatment regimens for EPA. These approaches comprise the central aspects of the proposed research. In Aim 1, Dr. Butwick will perform a nested case-control study, using hospitalization data at Stanford University School of Medicine (SUSM), to determine predictors and develop prediction models for severe PPH in patients undergoing prelabor CD and intrapartum CD. In Aim 2, Dr. Butwick will assess the incidence of and risk factors for EPA in women post-CD using linked hospitalization databases from Kaiser Permanente Medical Care Program - Northern California (KPMCP-NC). In Aim 3, Dr. Butwick will perform a cost-effectiveness analysis to assess the costs, benefits, and harms of oral versus intravenous iron therapy for the treatment of EPA. The candidate is an obstetric anesthesiologist at SUSM with additional training in Clinical Epidemiology, and is therefore uniquely qualified to address these aims. Furthermore, Dr. Butwick is supported by a multidisciplinary and collaborative team with expertise in Clinical Epidemiology, Obstetrics, Perinatology, Health Services Research, and Anesthesiology. The candidate's proposal will benefit from the diverse strength of resources at SUSM (the Department of Health Research and Policy, the Stanford Center for Clinical Informatics) and at Division of Research, KPMCP-NC. To accomplish his research goals and prepare him for a career as an independent investigator, Dr. Butwick has created a multi-disciplinary career development plan incorporating: (1) graduate level didactic training in health outcomes research with mentoring in cost-effectiveness analyses and analytic approaches for medical decision-making; (2) mentoring in advanced epidemiologic techniques, integrating electronic health information and linked hospitalization databases; (3) targeted education in obstetrical and perinatal medicine; and (4) mentoring in leadership in interdisciplinary research in obstetric anesthesiology and perinatology. Successful completion of this research will form the basis for future R01 applications to validate the clinical effectivenes and cost-effectiveness of computerized predictive rules for severe PPH and to investigate individualized therapeutic strategies for EPA and other major peripartum morbidities.	{ "pile_set_name": "NIH ExPorter" }
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Recent findings suggest that the neural substrates of phasic (short-duration onset responses) and sustained (longer-duration responses) startle increases may be partially different. Other findings suggest that the sustained component may be especially relevant clinically. The central goals of this project are to develop better animal models of clinical anxiety (based on sustained fear);to develop a greater understanding of the neural substrates of sustained and phasic fear, how they are similar and how they are different;and to identify potential targets for therapeutic intervention. During the first several months of this project, we completed experiments which compared the predictive validity of these two measures. Thus, several drug treatments that either are or are not clinically effective for anxiety reduction were evaluated for their effects on phasic and sustained startle increases. In general, we found that the effects on sustained startle increases more accurately predicted their clinical effects than their effects on phasic startle potentiation. This suggests that sustained fear paradigms may be better models of at least some types of clinical anxiety and perhaps therefore, more useful for evaluating novel compounds for anxiolytic activity. We have also collected initial data showing that prefrontal cortex lesions exacerbate sustained startle fear while having no effect on phasic fear. These results suggest a key role for the prefrontal cortex in controlling anxiety. Other activities have been devoted to optimizing procedures that will be used in years 2-5 of this project (e.g., lesions of the bed nucleus of the stria terminalis).	{ "pile_set_name": "NIH ExPorter" }
The serotonin transporter (SERT) is an integral membrane glycoprotein that is responsible for the reuptake of serotonin from the synapse. Drugs that inhibit the SERT are effective in several psychiatric disorders including depression. Recent studies have shown that the SERT can be regulated in vitro. Less is understood about its acute or chronic regulation in vivo. Different approaches have been used to address this issue, with inconclusive results. We have used in vivo chronoamperometry, a fast voltammetric technique, to demonstrate that there are areas in brain such as the CA3 region of the hippocampus, where the active clearance of exogenously ejected serotonin is primarily a function of SERT activity. This technique, then, is capable of generating rapid kinetic, quantitative measures of SERT function in vivo. In the proposed experiments, we will use this approach to study if long term administration of two different selective serotonin reuptake inhibitors (SSRIs), paroxetine and fluoxetine, produce a time-dependent subsensitivity of the SERT. The influence of treatment parameters such as route of administration, duration of administration, and drug-free washout period will be evaluated. Based on our preliminary data, we will explore if activation of terminal 5-HT1B autoreceptors alters the kinetics of SERT function in vivo. If so, then we will examine whether such a phenomenon impacts on any apparent regulatory effect of chronic SSRI treatment on SERT activity. In addition to examining the CA3 region, two other brain areas where the active clearance of 5-HT is due exclusively to the SERT will be studied. These experiments provide a new approach to study whether the SERT, a primary target for several types of antidepressants, is capable of being regulated either acutely or chronically in vivo.	{ "pile_set_name": "NIH ExPorter" }
Epidermal growth factor (EGF) was discovered 30 years ago in murine submandibular glands. Only recently have physiologic actions of the EGF system begun to be understood. Funding is requested for a conference, "The Physiologic Roles of the EGF System: EGF, TGF-alpha, and the EGF Receptor". Although there have been several meetings on the EGF system, this one is unique in having its in vivo physiologic roles as the primary topic. Attendance is limited to 60 participants from a variety of disciplines, to insure interaction between seasoned and new investigators. There will be 7 sessions, each with 4 to 7 oral presentations and open discussion. The first 2 present and overview, and update information on the EGF system. The next 4 will spot-light specific organs where data are emerging on how the EGF system functions in normal physiologic mechanisms; these are the Oral Cavity and Digestive Tract, the Nervous System, the Kidney, and the Reproductive Systems. The last covers transgenics and mutants of the EGF system. Data on the EGF system in less fully investigated organs will be explored in a poster session. Current data on the EGF system in specific organ physiology have developed somewhat independently of each other. It is a principal goal of this conference to bring workers from varied backgrounds together to: 1) inform each other of their findings on the in vivo functions of EGF, TGF-alpha, and their receptor; 2) generate a unified view of the physiology of the EGF system; and 3) formulate aims, directions and strategies for future research. An equally important goal is to underscore the impact of salivary research on current developments in biomedical research in general. EGF was first isolated from submandibular glands, and one of its first effects noted was acceleration of incisor eruption. Subsequent demonstration of the wide distribution of the EGF system has long indicated some very basic physiologic roles for it--some of these are now beginning to be understood, and they are the primary topic of the proposed conference.	{ "pile_set_name": "NIH ExPorter" }
The purpose of this K08 AHRQ Patient-Centered Outcomes Research (PCOR) Mentored Clinical Investigator Award is to establish Dr. Anita Misra-Hebert as an independent investigator studying natural experiments in redesigning primary care delivery systems. Her goal is to develop high-level skills in implementation science quantitative and qualitative research methods, with an emphasis on defining contextual factors that may promote success or failure of specific primary care redesign efforts in diverse practice settings. Dr. Misra-Hebert will gain practical expertise in PCOR primary care redesign through both formal and informal educational programs and through close work with her mentors during this project. Dr. Kurt Stange, her primary research mentor, is a recognized expert in the study of contextual factors in primary care practice redesign. Her co-mentors include Dr. David Aron, a recognized expert in implementation science research methods, Dr. Michael Rothberg, an expert in quantitative research methods, specifically related to observational studies with large datasets, and Dr. Adam Perzynski, a sociologist and an expert in qualitative and quantitative research methods. Environment Located in Cleveland, Ohio, Cleveland Clinic is a nonprofit, multispecialty academic medical center that integrates clinical and hospital care with research and education, and one of the largest and most respected hospitals in the country. Cleveland Clinic is structured as a group practice. The physicians on staff are salaried employees and are not in private practice. The practice is structured to complement the group practice model. The Medicine Institute at Cleveland Clinic is leading efforts across the institution to promote value-based care initiatives for patient care. The Medicine Institute Center for Value-Based Care Research specializes in health services and clinical research regarding novel approaches to care that can improve quality, reduce cost, or both. The Center includes 4 primary investigators, nurses, statisticians and research assistants, as well as affiliated health services researchers from across the Cleveland Clinic. Dr. Misra-Hebert will be fully supported in this environment to conduct research to evaluate best practices in primary care redesign efforts. The ability of the Cleveland Clinic and its Medicine Institute to support Dr. Misra-Hebert's research and development is greatly enhanced by the Cleveland Clinical & Translational Science Collaborative, which provides collaborative clinical research support and infrastructure across the institutions represented on her mentoring team. Research Project This goal of this study is to provide evidence of best practices for the delivery of team-based primary care with evaluation of contextual factors in the implementation of primary care redesign efforts. As the complexity of healthcare delivery increases, primary care is moving from a model of care delivery in which individual clinicians provide care to individual patients to team-based care models. To understand whether these new models have actually improved value in care delivery, it will be important to measure outcomes relevant to patients and those specific to team-based care. Team approaches will need to be optimized based on diverse contextual factors. The initial project will focus on the evaluation of contextual factors relevant to a specific team-based redesign effort involving an enhanced role of medical assistants compared to usual care and will include evaluation of patient and provider experience. The specific aims of the proposed research are as follows: Aim 1: To evaluate patient-centered clinical quality of care outcomes in the team-based care-medical assistant model (TBC-MA) compared to usual care. Aim 2: To identify contextual factors (patient, provider, practice) that contribute to the sustained implementation of the TBC-MA model. Aim 3: To assess patient and provider experience in the TBC-MA model compared to usual care. Later work during the career development award will include study of the longitudinal effects of team-based care, and redesign implementation that involves non-clinician care team members in improving care coordination for primary care patients.	{ "pile_set_name": "NIH ExPorter" }
This is a renewal application for a NIDA Independent Scientist Award (K02). The candidate, Amelia J. Eisch, Ph.D., is a tenured Associate Professor in the Department of Psychiatry at UT Southwestern Medical Center. Dr. Eisch is a pioneer in understanding the reciprocal relationship between certain forms of hippocampal plasticity such as adult hippocampal neurogenesis and behaviors relevant to addiction. The first award period of this K02 provided Dr. Eisch with the protected time she needed to accomplish her past goals, which included renewing her NIDA R01 grant on opiates and neurogenesis, securing additional federal funding, advancing her work from correlative to more mechanistic and causative studies, and being promoted to Associate Professor and receiving tenure. This K02 renewal is requested in order to continue this protected time and to allow Dr. Eisch to utilize the momentum she has established in understanding the proposed reciprocal role between new neurons in the adult brain and addiction. The receipt of a K02 renewal award would allow her continued protection from administrative burdens, and thus allow Dr. Eisch to advance: (1) research on the relationship between adult-generated neurons and behaviors relevant to addiction; (2) dissection of the cellular, molecular, and genetic control of adult-generated hippocampal neurons; (3) newly developed collaborations on translational profiling, ultrastructural analysis, and circuit-level impact of new neurons; (4) application of new techniques for these collaborations (BAC-TRAP, optogenetics, EM, electrophysiology); (5) data collection for grant applications: the 10-year renewal of her existing R01 on opiates and neurogenesis, a new NIDA R01 relevant to the molecular control of hippocampal neurogenesis, and a new NIDA R01 relevant to behavioral and circuit-level impact of adult-generated neurons. By providing this protected time, Dr. Eisch can work closely with her collaborators and rapidly advance her new ideas, thus benefitting the fields of addiction research and stem cell biology and neuroscience as a whole. The stability and protected time offered by this K02 award would ultimately support at least two new projects exploring the intriguing relationship between adult-generated hippocampal neurons and behaviors relevant to addiction. As these studies hold great potential to improve our understanding of the complex mechanisms by which drugs of abuse affect brain function, they therefore may open new avenues for treatment of addiction and relapse to drug seeking. As these studies also will also shed much-needed light on how adult-generated neurons influence complex behavior - such as drug/context association, extinction from drug-seeking, and behavioral response to stress - they will be important to future efforts to harness neural stem cells for repair of the injured, even addicted, brain and to our understanding of what new neurons can/will do in the adult brain.	{ "pile_set_name": "NIH ExPorter" }
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The pilot study will assess the dopaminergic effects of branched-chain amino acids (BCAA) in ten 18-40 year old male healthy subjects. The effects on dopamine activity will be measured with plasma catecholamine and prolactin levels, and dopamine D2 receptor availability (assessed with [11C]-raclopride and positron emission tomography following the oral administration of a BCAA mixture).	{ "pile_set_name": "NIH ExPorter" }
This ENCODE consortium project aims to identify and classify chromatin regulatory elements in the human genome in systematic fashion. This goal is being pursued through the application of chromatin methods and ultra high-throughput sequencing to acquire genomewide maps that reflect the distributions of histone modifications and chromatin proteins in a given cell type. By assaying multiple cell types, the project will build an increasingly comprehensive catalog of chromatin regulatory elements and gain insight into their cell typespecificities. Although there are dozens of different modifications and many other structural components in chromatin, only a fraction can be measured with existing antibodies and reagents. The rationale of this supplement request is that an ability to characterize these additional modifications and proteins as they occur across the genome would augment our understanding of the structure and regulation of functional elements elucidated by ENCODE, and reveal additional elements of yet unknown significance. Requested funds would be use to screen dozens of additional chromatin epitopes with the goal of identifying suitable reagents and methods for genomewide mapping. The screens will be pursued through a combination of conventional methods and high-throughput techniques. Reagents identified through this process will then be applied within the existing ENCODE pipeline to characterize genomewide distributions of the novel epitopes and their relationships to known or novel regulatory elements. Chromatin regulation is of wide-ranging importance in human development and disease. The proposed systematic identification and characterization of chromatin regulatory elements in the human genome will offer valuable insight into the structure and function of chromatin, and provide a resource for investigators in chromatin, genomics, cancer and many other fields of research. All data collected in the context of the proposed project will be made available pre-publication to the greater scientific community as soon as reliability has been confirmed. 2.	{ "pile_set_name": "NIH ExPorter" }
This application to the Global Network (GN) for Women's and Children's Research asks for the Global Network Research Unit of Aga Khan and Columbia Universities to be renewed for a third 5 year term. We believe that our track record, especially over the last 5 years, as documented in the full application, justifies an award for the next 5 years. The strengths of this application include a stable and highly productive team devoted to carrying out meaningful research to improve the health of women and children, large and varied populations for study, and an ongoing birth outcomes registry of 20 rural clusters of 500 deliveries per year each with excellent follow up. Pakistan, with a population of 180,000,000 people and some of the highest maternal, fetal and neonatal mortality rates anywhere in the world, provides every type of population that could be considered for GN studies. We have the largest number of rural clusters of any site in the GN with an active birth registry in each, and have also demonstrated access to a number of urban hospitals for deliveries and access to urban children for research purposes as well. To date we have recruited more than 220,000 subjects for GN studies. In the next 5 years, we plan to bring substantial external resources to the GN by building strong relationships between industry, foundations and the GN to do research meaningful to all three institutions. We will actively participate in the deliberations of the GN Steering Committee and continue to mentor more junior investigators at our site and other sites in the GN in research study design, and manuscript and grant preparation. One of our major goals is to improve facility performance in the care of mothers and infants. Our proposed common protocol alms to reduce facility fetal and neonatal mortality using a defined outcome related to facility care, e.g., the sum of intrapartum stillbirths and early (pre- discharg) neonatal deaths. Using a simple cellular telephone-based data collection system, rapid feedback on the defined outcome, and a quality assurance program aimed at reducing intrapartum related deaths; it is our hope to achieve a substantial improvement in facility-based pregnancy outcomes.	{ "pile_set_name": "NIH ExPorter" }
Mountain States Group's current Idaho Partnership for Hispanic Health (IPHH) NCMHD phase one grant has completed significant planning work. IPHH has built a Core Team partnership among five research, community, and health care organizations; established a functioning Hispanic Community Advisory Board; completed a comprehensive community health assessment; determined the project's disease focus and intervention model; and planned out the pilot intervention. The proposed five-year implementation study will continue IPHH's partnership work with the Hispanic community in southwest Idaho. The disease focus -based on community assessment results, community decision-making, and secondary data - will be metabolic syndrome. The intervention - an adaptation of the promotores (community health worker) indigenous health education, outreach and advocacy model -will be implemented to build on Hispanic family and community strengths. The defined community will remain the southwest Idaho Hispanic population. The project has four study aims. 1) As behavior change is the first line of prevention and treatment of metabolic syndrome, the intervention will work at the family level to research the impact of the intervention on changes in diet and physical activity of 680 family members. 2) The intervention will also work at the community level to create change in community norms and practices on diet and exercise, and to build resources that support and reinforce community level behavior change. Baseline and end-of-project data will document these community level impacts. 3) As community involvement is vital to all aspects of the study, the contribution of community participation will also be evaluated through qualitative surveys and interviews with the Core Team and CAB throughout the project. 4) Finally, IPHH will study the local capacity built to sustain the promotores intervention model. The project will document changes in Hispanic health care utilization of local clinic and hospital services, and demonstrate the value of local health facility investment in the promotores program. Project partners include Mountain States Group as applicant organization and partnership convener, University of Washington as research and evaluation partner, Centra de Comunidad y Justicia (Center for Community and Justice) as a community partner, Boise States University Department of Nursing as health care education partner, and Idaho Commission on Hispanic Affairs as dissemination partner. The project directly addresses several Healthy People 2010 objectives, including increase consumption of fruit to at least two daily servings, increase consumption of vegetables to at least three daily servings, and increase moderate physical activity to at least 30 minutes per day. The study also emphasizes the critical role of preventive services in preventing and managing chronic diseases.	{ "pile_set_name": "NIH ExPorter" }
[unreadable] The goal of this proposal is to make a state-of-the-art ultrasound imaging system available for NIH-funded researchers at the University of Wisconsin (UW) Medical School. The Section of Cardiovascular Medicine and the UW Department of Population Health Sciences have a strong history of successful and productive collaborations that have resulted in high-quality research in cardiology and the epidemiology of cardiovascular disease. Acquisition of an ultrasound imaging system will facilitate and significantly enhance the scientific quality of several ongoing NIH-supported research projects described in this grant application. The Siemens Acuson Sequoia C512 is a state-of-the-art, multipurpose instrument for cardiac and vascular ultrasound imaging. All of the funded research studies for which we are seeking a shared ultrasound system will use it as a tool to better understand the physiological consequences of sleep-disordered breathing, hypertension, diabetes mellitus, congestive heart failure, and myocardial infarction. The three "major" users are an ideal group to share instrumentation given their common research interests in sleep-disordered breathing and cardiovascular disease, their need for high-quality ultrasound to assess cardiovascular structure and function, their strong collaborative relationships with the Principal Investigator, and their status as active, R01-funded investigators. All six investigators use several ultrasound-based research techniques including the evaluation of carotid intima-media thickness, brachial artery reactivity, cerebral blood flow, coronary blood flow, and left ventricular systolic and diastolic function. Because diagnostic ultrasound has no known biological effects, serial studies can be performed longitudinally to evaluate various stages of disease processes and their responses to interventions. Since the research equipment needed to perform these studies currently does not exist in the UW Medical School, it must be borrowed or time purchased from an already oversubscribed clinical laboratory, an expensive and inconvenient arrangement. The necessary space, administrative support, supervision, and technical expertise already are in place to assure that the requested equipment will be operable immediately and highly productive at UW. There is significant opportunity for greater collaboration, research productivity, and cost savings if an ultrasound system is made available at the UW Medical School as a shared resource. [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
Our lab is interested in the process of chromosome segregation and how defects in this process can affect the development of a multicellular organism. Over the past few years we have focused on the meiotic divisions that produce haploid gametes. We have been studying a class of temperature-sensitive (ts) embryonic lethal mutants from C. elegans that arrest in metaphase of meiosis I. In wildtype animals, oocytes in prophase of meiosis I are fertilized by sperm. Following fertilization, the oocyte chromosomes undergo two meiotic divisions, discarding the extra chromosomes in the polar bodies. These first meiotic divisions are important as any errors in chromosome segregation at this stage can lead to embryos with an abnormal number of chromosomes, which would likely lead to lethality. In our mutants, the oocyte chromosomes arrest in metaphase of meiosis I and never separate their chromosome homologs and never extrude polar bodies. In order to molecularly identify the genes required for the first meiotic division, we have mapped our mutants and sequenced candidate genes. Five of the six genes have now been identified and they encode subunits of the Anaphase Promoting Complex or Cyclosome (APC/C). This complex serves as an E3 ubiquitin ligase that targets proteins for destruction (by the 26S proteosome) during the metaphase to anaphase transition of the cell cycle. We have named our mutants "mat" for their defects in the metaphase to anaphase transition during meiosis I. These ts mutants also display defects in spermatocyte meiosis; primary spermatocytes arrest in metaphase of meiosis I with a normal meiotic spindle, yet fail to separate chromosome homologs. Thus, these mutants disrupt meiosis in both oocytes and spermatocytes. To address the role of the mat genes in mitosis, we have performed shift-up experiments during embryogenesis and larval development. Temperature shift-up experiments during embryogenesis do not result in embryonic phenotypes, however, somatic defects in the gonad, vulva, and male tail are apparent in adults. This observation suggests that mitotic divisions in the soma are affected by the mat mutants. For many of the alleles, these shift-up experiments also result in sterility, suggesting mitotic defects in germline proliferation. We have constructed double mutants of many of these APC/C mutants and have found that many combinations are synthetic lethal at the permissive temperature. We are currently using some of the double mutants that are still viable at the permissive temperature to re-examine the role of APC/C during the mitotic cell cycles during embryogenesis and larval development. Specifically, we hope to observe more severe mitotic defects during the development of the germline and various tissues. To further understand the role of these mat genes during development, we are characterizing their expression patterns using antibody staining and GFP transgenic lines. We have also completed a genetic suppression screen to identify extragenic regulators or substrates of these APC/C subunits. The majority of our 29 suppressor mutations are dominant. We anticipate finding novel molecules that shed light on how APC/C functions and is regulated in different tissues and at different times during the development of a multicellular organism. We also want to examine the composition of the APC/C in C. elegans. For these studies, we will generate transgenic lines expressing epitope-tagged APC/C subunits and then purify the complex with epitope-specific antibodies. These proteins that we purify will be subjected to mass spectrometry to identify the components of the APC/C. We will then examine whether this complex varies during development. In a separate study, we are examining the function of the C. elegans Myt1 ortholog. Myt1 belongs to the Wee1 family of kinases and is thought to down regulate Cdk1 during the cell cycle. RNAi studies with the Myt1 ortholog, wee-1.3, result in sterility. Mothers injected with dsRNA quickly become sterile; the oocyte chromosomes are no longer paused in diakinesis of meiosis I. We propose that WEE-1.3 normally functions to keep maternal CDK-1 inactive during oogenesis, and that upon fertilization, CDK-1 becomes activated to allow for the meiotic and mitotic divisions of the embryo. We are further characterizing this phenotype and plan to use RNAi screens to identify other components of this pathway.	{ "pile_set_name": "NIH ExPorter" }
For many years this laboratory has been developing and using radiation target analysis, a method for determining the mass of biologically active molecules. The laboratory has developed a variety of experimental techniques to properly utilize the inactivation of biochemically active material by ionizing radiation, specifically that from high-energy electrons. Radiation target theory has been successfully extended to complex biological systems. These techniques and theories have been utilized in studies of enzymes, receptors, transporters, and other structures of wide interest in the biomedical field. Often these radiation studies resulted in discovery of unexpected features or properties of these biologically active materials. The objectives of present studies are to determine the precise nature of damage in macromolecules caused directly by ionizing radiation and to utilize this knowledge in the application and extension of radiation target analysis. Several projects have been brought to fruition during the past year. The enzyme hyaluronan synthase is of great medical importance in human infections by streptococcus. Radiation studies revealed that each enzyme molecule requires fifteen molecules of a specific lipid in order to function. The enzyme ribonucleotide reductase is crucial in AIDS infection. It has a unique structure involving a stable free radical. In isolated subunits, this radical confers extraordinary radiation sensitivity which is not seen in the intact holoenzyme. Another enzyme, phenylalanine hydroxylase, is especially important in humans: a defect in this enzyme is the cause of phenylketonuria in newborns. Radiation studies of this enzyme have been continuing for several years. The enzyme has complex regulation mechanisms; in the purified state, radiation studies revealed the different structures involved in several of these mechanisms. Now the technique has been applied to the enzyme in the crude state, revealing unanticipated differences from those seen previously. It appears that purification has altered the structure of the enzyme complex. The method of radiation target analysis continues to reveal unique molecular properties of biologically active structures. Often these properties were unanticipated from studies using other techniques. This new knowledge has given a greater understanding of the mechanisms of these biological functions. In some cases these properties offer the possibility of external control or modification of these active structures.	{ "pile_set_name": "NIH ExPorter" }
Proposed studies will assess hypotheses related to a fundamental biological problem, how sleep affects brain structure and plasticity. We will examine the processes of adult hippocampal neurogenesis (AN) and the expression of genes and proteins facilitating AN. We showed that sleep deprivation strongly inhibits proliferation of new cells and the percentage of new cells showing mature neuronal properties. We now propose to study effects of sleep fragmentation and REM deprivation on AN and to evaluate a prediction of delayed functional effects of sleep fragmentation. AN is regulated by a cascade of proteins, including phosphorylated cyclic AMP response element binding protein (pCREB) and brain-derived neurotrophic factor (BDNF). Our pilot data shows that 48 hrs sleep deprivation depresses hippocampal expression of BDNF as well as synapsin-1, a vesicular protein induced by BDNF. In neocortex plasticity-related genes are expressed during wake. Brain protein synthesis is increased during sleep. These findings suggest that wake and sleep states play distinct roles in support of brain functions and plasticity. We will assess the following model: The light/sleep phase provides the milieu for gene translation and protein synthesis, including cell proliferation. REM sleep is required for protein docking and stabilization. However, the interactions of wake and sleep and the effects of sleep fragmentation and REM deprivation on proliferation gene expression and protein synthesis in hippocampus have not been studied. We will separately analyze the light phase sleep arid dark phase wake periods and determine effects of sleep fragmentation and REM deprivation. Cell proliferation will be studied by the bromodeoxyuridine (BrdU) method. Gene expression in hippocampus is assessed by RT-PCR. We propose to restore proliferation, during sleep fragmentation and to suppress proliferation during sleep by local changes in BDNF. Our studies utilize a new, well-controlled, non-stressful method of sleep deprivation, the intermittent treadmill. Dependent variables will be correlated with sleep parameters and delta EEG activity during sleep. Much evidence shows that suppression of AN could be critically involved in the neuro- and cognitive pathology associated with aging and with prevalent human diseases, including major depressive disorder, and obstructive sleep apnea. Chronic sleep fragmentation and restriction are common to these conditions.	{ "pile_set_name": "NIH ExPorter" }
India has long experienced a numerical shortage of females, and this sex ratio imbalance is likely to grow with the widespread adoption of sex-selective abortion technologies. Although considerable research has documented levels, trends, and differentials in India's population sex ratio, we know little about the consequences of India's imbalance in the numbers of women and men for critical family, demographic, and health-related behaviors. This project uses data from three different sources-the 2005-06 National Family Health Survey (NFHS-3), the 2004-05 India Human Development Survey (IHDS), and the 1981, 1991, and 2001 Censuses of India-to examine the impact of India's imbalanced population sex ratio on key family formation behaviors (e.g., age at marriage, fertility, likelihood of divorce, social and geographic exogamy, the values of dowries);on sexual behaviors and health outcomes (e.g., age at first sex, engagement in premarital and extramarital sex, number of sexual partners, commercial sex, forced sex, and contraction of sexually-transmitted disease, including HIV/AIDS);on gendered family dynamics (e.g., women's roles in household decision-making, other dimensions of women's empowerment and autonomy, domestic violence);and on women's socioeconomic attainment (e.g., women's literacy, educational attainment, labor force participation, occupational attainment). Multilevel fixed-effect and random-effect regression models will estimate the impact of the local marriage market sex ratio, measured with census data, on these outcomes measured in the NFHS-3 and the IHDS. By capitalizing on the substantial age-graded and geographic variation in adult sex ratios across India, and by applying statistical techniques appropriate for the analysis of multilevel data structures, the proposed analysis holds promise for substantially enhancing our understanding of how India's increasing overabundance of males relative to females will affect multiple dimensions of family life and population health. PUBLIC HEALTH RELEVANCE: India is the world's second most populous country and projected to become the largest by 2050. Growing imbalances in India's sex ratio have the potential to alter substantially multiple dimensions of demographic and family-related behavior, including the sexual behaviors that contribute to the spread of HIV/AIDS. Consequently, learning more about the family and health consequences of India's growing male surplus and female deficit is an important scientific objective.	{ "pile_set_name": "NIH ExPorter" }
Recent developments in peptide chemistry have increased interest in the potential biological significance of these substances. Attribution of a peptide structure to many important hormones, releasing factors, chalones, kinins, specific behavior-inducing substances, and the possibility of the role of peptides in cancer therapy, suggest that these substances may play an important role in the regulation of biological functions. It has already been proven that some of the biologically active peptides are present in urine, which may be a convenient source for their isolation. The general aim of this study is the isolation, purification, identification and determination of the biological, therapeutic, and diagnostic significance of urinary peptides, especially their effect on normal and neoplastic cell growth. The peptides are isolated by ethanol extraction, gel filtration, free-flow high-voltage electrophoresis and thin-layer chromatography. Since only a small fraction of the urinary peptides is expected to have functional significance, a battery of bioassays will be set up to evaluate the activity on various normal and neoplastic cells (DNA, RNA and protein synthesis), animal tumors, intestinal and vascular smooth muscle, cardiac functions and behavior. The peptides proved active will be identified by amino acid analysis and sequence determination, and will be reproduced by synthesis. According to preliminary experiments, 10% of the 119 peptides studied up to now inhibit DNA, RNA, or protein synthesis in cultures of human osteosarcoma cells. Four substances of this group showed no inhibitory action on DNA synthesis in normal cells (human embryonal fibroblasts) and no effect on smooth muscle and heart. Six fractions produced remarkable inhibition of DNA synthesis (from 75% to 97%) in human leukemic cells. The active fractions have been selected for further study.	{ "pile_set_name": "NIH ExPorter" }
Autism Spectrum Disorder (ASD) is a group of developmental disorders which affects approximately 1% of children, a prevalence rate that is increasing annually. The molecular basis of ASD is likely to be complex and heterogeneous, but given the central role of the inhibitory neurotransmitter GABA in cognitive control, development and plasticity and a growing body of evidence linking GABAergic dysfunction to autism and autistic phenotypes, the GABA system is a strong potential area for ASD research. Recently, it has become possible to measure GABA concentration non-invasively in the brain using magnetic resonance spectroscopy (MRS), and in an extremely novel set of experiments we have shown that differences in regional GABA concentration among healthy subjects have measurable behavioral correlates. For example, GABA concentration in sensorimotor cortex correlates with performance at a somatosensory discrimination task (see below); this may have particular relevance for autism, as atypical response to tactile stimuli is a common (and diagnostic) feature of autism and one that can substantially impair behavioral function This proposal arises from a collaboration between Dr Richard Edden (PI) and Dr Stewart Mostofsky (Co-I) to apply the extremely novel pairing of MRS of GABA with somatosensory paradigms to investigate whether there is a measurable GABA deficit in autism, and whether this correlates with autism associated differences in somatosensory processing. The findings from examination of GABA MRS and its association with autistic behavior will provide a foundation for investigating novel treatment therapies, specifically use of medications that increase GABAergic activity. In Aim 1, we will compare measurements of GABA concentration in a group of 20 children with ASD between the age of 8 and 12 years with 20 age-, gender-, and IQ-matched typically developing children (TDC) as the control group. In Aim 2, we will perform psychophysical measures of tactile discrimination to investigate whether there are differences between children with ASD and TDC, and in Aim 3 we will test whether the abnormal tactile processing commonly observed in ASD arises from a GABAergic deficit.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: Primary Congenital Glaucoma (PCG) is an autosomal recessive disorder caused by unknown developmental defect(s) of the trabecular meshwork and anterior chamber angle. Although PCG is the most common form of glaucoma in infancy, nothing secure is known about its specific ambryologic pathogenesis despite studies of various animal models. Furthermore, although a single chromosomal assignment has been proposed there is no method to detect carriers by any clinical, biochemical or molecular techniques. Therapy is primarily surgical with variable success, as more than one surgical intervention might be necessary to control intraocular pressure in a number of these patients. Thus, there is significant morbidity associated with both this condition and the currently available treatment options. The investigators will apply genetic an physical mapping strategies to isolate the gene(s) for PCG. Two complementary linkage approaches to gene mapping will be used: outbred family linkage studies of the "sib pair" method and homozygosity mapping. The latter approach will utilize consanguineous Saudi Arabian pedigrees. Alternative strategies for initial gene localization will include: (I) allele-frequency dependent homozygosity mapping (AHM) and (ii) pooling of genomic DNA samples from affected and unaffected individuals in single large pedigrees. Other traditional mapping approaches to be use for positional cloning include YAC contig building, PFGE mapping, eye cDNA library screening, and exon trapping. Mutation scanning detection methods including heteroduplex analysis, chemical cleavage of mismatched bases, SSCP analysis, DGGE, and direct DNA sequencing will then detect specific mutations. The identification of a PCG gene has potent implications for both ocular and human development and have the potential to improve the quality of life of the affected individuals. Further, it will enable the accurate identification of siblings who may be carriers of the condition, and has the ultimate capacity to modify or prevent the devastating visual consequences of this disorder in future generations.	{ "pile_set_name": "NIH ExPorter" }
Benign glottic lesions are generally believed to result from mechanical trauma to the vocal folds, and affect million of working Americans. However, the extent of trauma and loading conditions that lead to structural damage, inflammation, and ultimately lesions such as nodules, polyps or cysts, is not well defined. In order to better understand the loading conditions and vocal behaviors that can lead to damage, greater understanding of material properties, stress distributions, and the resulting fiber damage is necessary. Vocal fold tissues experience a type of loading that is unique among soft tissues of the body. They undergo vibration and are loaded in the axial and transverse directions in tension, compression and shear, while most soft tissues are loaded either in tension or compression in a single direction. Because of this difference in load bearing, the structure (extracellular matrix fiber arrangement) and material property trends of the vocal folds differs from other softer tissues, making study of the vocal fold tissue properties critical for understanding the mechanism of structural damage leading to lesions. The overall goal of this proposal is to evaluate the material properties of the vocal folds, the stress experienced during phonation under varying hydration, elongation and subglottal pressure conditions, and the resulting structural damage to the tissue. The results will potentially describe the forces of vibration that result in tissue damage- either fatigue damage or fiber rupture. Methods of material property estimation and structural damage evaluation are novel and will contribute to knowledge about vocal fold tissue properties. Knowledge gained from this research is essential for a more complete understanding of clinical management of voice disorders. The approach to this research will involve three specific aims. The first aim will focus on characterizing vocal fod material properties. It will employ acousto-elastography, which is a novel measure of the tissue acoustic properties that is linearly related to strain and nonlinearly related to tissue stress. Th results of these methods will be useful for establishing the effects of dehydration on tissue stiffness and informing computer model parameters. The second aim will focus on finite element modeling of vocal fold vibration and the resulting interstitial fluid dynamics and stress distributions. Elongation, subglottal pressure, hydration and stiffness will be varied and resultin stresses evaluated. The third aim will focus on excised studies and evaluation of tissue damage under varying subglottal pressure, hydration and elongation phonation challenges. The potential long term implications of this work are (1) better characterizing the vocal behaviors that might lead to lesions to inform therapeutic recommendations and (2) a better understanding of the mechanism of lesion formation to inform interventions.	{ "pile_set_name": "NIH ExPorter" }
A central theme in diabetic cardiovascular disease is the dysfunctional accumulation of lipids in critical cell-types (e.g., cardiac myocytes and macrophages) which underlie the intrinsic cardiomyopathy and accelerated atherosclerosis present in the diabetic patient. The unifying hypothesis of the program project grant is that lipid alterations in these cell types are due to fundamental changes in peroxisomal lipid flux mediated by alterations in intracellular phospholipases, PPAR mediated transcriptional regulation of critical regulatory proteins and altered lipid second messenger generation which collectively predispose candidate target cells to lipid accumulation and maladaptive pathophysiologic responses. In Project 1, we will examine the role of a novel peroxisomal calcium-independent phospholipase A2, iPLA2gamma, as a regulator of peroxisomal lipid synthesis and fatty acid beta oxidation in the cardiac myocyte. Additionally, Project 1 will examine the role of iPA2beta as a potential mediator of altered lipid metabolism and electrophysiologic dysfunction in diabetic myocardium. Dr. Kelly's Project, the role of PPARalpha as a primary determinant of the cardiac metabolic and functional phenotype present in the diabetic state will be examined utilizing mice over-expressing PPARalpha in cardiac myocyte specific fashion and a mice null for PPARalpha. Physiologic and biochemical alterations resulting from PPARalpha over-expression and the ventricular hemodynamic and metabolic abnormalities in diabetic myocardium will be compared. In Project 3, the role of altered peroxisomal lipid metabolism and intracellular phospholipase A2, activities in contributing to monocyte migration, lipid second messenger generation and lipid accumulation will be examined. The contribution of these factors in mediating the accelerated vascular response to mechanical to mechanical injury in diabetic rats will be examined. The contribution of these factors in mediating the accelerated vascular response to mechanical injury in diabetic rats will be examined. Dr. Muslig's Project the hypothesis that diabetic cardiomyopathy develops as a result of abnormal stimulation of Gq and Gi mediated signaling pathways leading to alterations in intracellular phospholipase activity, peroxisomal lipid metabolism and lipid second messenger generation will be studied. The contributions of cardiac myocyte phospholipases to G-protein signaling will be examined. Collectively, these studies represent a synergistic, targeted, multi-disciplinary investigation aimed at elucidating the role of altered lipid metabolism and second messenger generation in critical cardiovascular cell types as the primary determinants of the excessive cardiovascular mortality and morbidity in diabetic patients.	{ "pile_set_name": "NIH ExPorter" }
The carcinogen methylazoxymethanol (MAM) acetate induces tumors in rats predominantly of the liver and colon. Since MAM does spontaneously decompose to liberate a reactive alkylating moiety this marked organotropism is surprising and suggests that MAM might be metabolized selectively by those tissues sensitive to its effects. We have investigated the hypothesis of Schoental (Br. J. Cancer 8: 436, 1973) that MAM might be a substrate for alcohol dehydrogenase. Our results indicate that, in fact, NAD ion-dependent dehydrogenase activity using MAM as substrate is detectable in 169,000 xg supernatant fractions from liver, duodenum, cecum and colon, tissues sensitive to the acute and chronic effects of MAM acetate. Jejunum and ileum, relatively resistant to this agent, had no measureable activity. We also found that MAM is substrate for horse liver alcohol dehydrogenase and that pyrazole, an inhibitor of alcohol dehydrogenase, could prevent MAM acetate-induced acute lethality. These results suggest that the NAD ion-dependent dehydrogenase activity observed with MAM might be due to alcohol dehydrogenase. Other dehydrogenases might also be important, however, since NADP ion-dependent enzymatic activity is detectable in tissues other than liver. The importance of these reactions in the induction of tumors is being investigated. Also, the effects of other carcinogens might be influenced by NAD/NADP-dependent dyhydrogenases. In this regard Phillips, et al. (J. Natl. Cancer Inst. 58:629, 1977) reported that pyrazole decreases the metabolism and toxicity of dimethylnitrosamine. We have confirmed these findings and have extended our studies to include additional carcinogens.	{ "pile_set_name": "NIH ExPorter" }

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