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In the previous studies, we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine Ne-(4-amino-2-hydroxybutyl)lysine, and established that hypusine biosynthesis occurs by two sequential enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We demonstrated that hypusine modification is essential for the activity of eIF-5A and for mammalian cell proliferation. We have cloned and characterized the structural and catalytic properties of the two enzymes of the hypusine pathway, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH), with the aim of developing specific inhibitors. In this reporting period, we have carried out the structure/function studies of DOHH to characterize the basis of the strict specificity of the hypusine modification in one protein. We have also conducted the structure/function studies of eIF5A, and identified the key structural elements of eIF5A critical for its biological activity in supporting growth and protein synthesis.[unreadable] [unreadable] Examination of the DOHH sequence alignment reveals that it is a super helical protein containing eight tandem alpha helical hairpins (HEAT motifs). DOHH is a dyad of symmetrical N-terminal and C-terminal halves, each half consisting of four HEAT repeats, connected by a variable insert. Either the N-terminal half or the C-terminal half was inactive alone and in mixtures, an indication that DOHH does not contain two independent active sites, and that the two symmetrical halves form one active site. We have validated the proposed HEAT-repeat structure by CD spectral analysis. DOHH contains four distinctive, strictly conserved His-Glu motifs. Each the His and Glu residues in these motifs is critical for the DOHH enzymatic activity. We have identified six residues of these motifs (His56, His89, Glu90, His207, His 240 and Glu241) as the iron coordination sites and two residues (Glu57 and Glu208) as those anchoring the protein substrate eIF5A(Dhp). [unreadable] The predicted super helical structure of DOHH is entirely different from the beta jellyroll structure (termed the double stranded beta helix (DSBH)) of the majority of other protein hydroxylases, e.g. the Fe(II)-and 2-oxoacid-dependent dioxygenases, prolyl or lysyl hydroxylases, indicating that DOHH has evolved uniquely and exclusively for eIF5A hydroxylation . [unreadable] [unreadable] Structural requirements of eIF5A as a substrate for DOHH: Importance of the deoxyhypusine side chain[unreadable] The basis of the strict specificity of hypusine modification lies on the specificity of molecular interaction between its biosynthetic enzymes and the protein substrate. In an effort to determine the basis of the specificity of DOHH, we first tested the ability of three eIF5A forms, the precursor, the intermediate and the mature form (eIF5A(Lys), eIF5A(Dhp) and eIF5A(Hpu)) to compete with 3H-labeled eIF5A(Dhp) in the DOHH reaction. eIF5A(Lys) did not inhibit DOHH reaction, even when it was added 100 fold excess over the level of 3H-labeled eIF5A(Dhp), suggesting that the eIF5A precursor does not bind to DOHH. The hypusine-containing mature form, eIF5A(Hpu) showed low inhibition. Thus, the 4-aminobutyl side chain of deoxyhypusine residue seems to be critical for binding to DOHH. In order to determine how much of the eIF5A polypeptide backbone is required for the DOHH reaction, we tested eIF5A(Dhp) fragments with stepwise truncation of 10 amino acids from the N-or C-terminal of eIF5A(Dhp) as substrates. Whereas the eIF5A(Dhp) peptide, aa 1-90 was a good substrate for DHS and DOHH, aa 1-80 and aa 30-90 were poor substrates. Thus, truncation of more than 30 amino acids from the N-terminus, and 74 amino acids from the C-terminus caused a drastic reduction of the substrate activity for the DHS as well as for the DOHH, indicating that a large portion of eIF5A(Dhp) N-terminal domain is required for binding and modification by the two enzymes.[unreadable] [unreadable] Identiffication of amino acid residues of DOHH critical for binding of its protein substrate[unreadable] In order to determine the amino acid residues of DOHH involved in the binding of its substrate, eIF5A(Dhp), we generated 36 mutant enzymes with individual alanine substitution of strictly conserved amino acids and examined their activities, iron contents and abilities to bind the substrate protein. The alpha-helical contents of the mutant enzymes estimated by CD (circular dichroism) analyses were comparable to that of the wild type enzyme, suggesting that there is no gross alteration in the folded structures of these mutant enzymes. Of these 36 mutant enzymes, the eight alanine substitution mutant enzymes of the His-Glu motifs were totally inactive. R26A, G63A, R88A, E93A, R175A, R183A, S202A, G214A, Q215A, M237A and G247A exhibited a large reduction in the activity (by 60-95 %). The iron contents were significantly reduced in six mutant enzymes of the His-Glu motifs, H56A, H89A, E90A, H207A, H240A and E241A, but not in others. The GST fusion proteins of the wild type and mutant enzymes were used to pull down eIF5A(Dhp) using GSH sepharose resin. Whereas the wild type enzyme and a number of mutant enzymes effectively pulled down eIF5A(Dhp), the binding of the substrate protein was undetectable with E57A, and E208A and drastically decreased in G63A and G214A, indicating that the activity loss is due to their inability to bind the protein substrate. The importance of the acidic charges of the two glutamic acid residues (Glu57 and Glu208) was confirmed by substitution of these residues with Gln, Asp and Asn. Only the aspartic acid substitution mutant enzymes (E57D and E208D) were able to bind the substrate protein and were partially active. The glutamine or asparagine substitution enzymes (E57Q, E57N, E208Q and E208N) failed to bind and hydroxylate eIF5A(Dhp). Based on these and above (structural requirement of eIF5A(Dhp)) results, we conclude that the amino groups of the deoxyhypusine side chain of eIF5A(Dhp) is anchored by the two carboxyl side chains of Glu57 and Glu208 at the DOHH active site. This critical ionic interaction between the deoxyhypusine side chain of eIF5A(Dhp) and the acidic groups of Glu57, and Glu208 of the enzyme provides the basis of strict specificity of DOHH reaction.[unreadable] [unreadable] Mutational analysis of eIF5A: Identification of amino acid residues critical for the biological activity of eIF5A and the hypusine modification. [unreadable] To investigate the features of eIF5A required for its activity, we generated 49 mutations in the human eIF5A-1, with a single amino acid substitution at each of the highly conserved residues or with N-or C-terminal truncations, and tested the mutant proteins in complementing the growth of a S. cerevisiae eIF5A null strain. Growth supporting activity was abolished in only a few mutant eIF5As, those with substitutions at or near the hypusine modification site (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A) or those with truncation of 21 amino acids from either the N-or C-terminus. For the Lys50 substitution proteins and G52A and K55A, the inactivity is largely due to the lack or impairment of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical importance of Lys47 and Gly49 in eIF5A biological activity, possibly in its interaction with downstream effector(s). These results demomstrate that both N- and C-terminal domains of eIF5A are required for its biological activity and that the hypusine residue and the hypusine containing loop play a critical role in the biological action of eIF5A possibly in its interaction with its downstream effectors.	{ "pile_set_name": "NIH ExPorter" }
Bisphenol A (BPA) is an estrogenic chemical that is widely used in the manufacture of policarbonate plastics and epoxy resins. Because BPA leaches out of plastic food and drink containers, including baby bottles, as well as the BPA-containing dental prostheses and sealants, considerable potential exists for human exposure to this compound. We have demonstrated for the first time that treatment of ovariectomized female rats with BPA dose- dependently inhibits both the hippocampal synaptoplastic and the positive cognitive effects of estrogen administration. These effects of BPA can already be observed at a dose of 40 [unreadable]g/kg that is below the current U.S. Environmental Protection Agency reference daily limit for human exposure. Moreover, several publications have also raised the same worrysome issue that low- dose BPA might compromise normal sexual development and function of the brain. However, the majority of these studies, including our own, investigating BPA effects on the brain have been performed in rats. Hence, the implications of these alarming results to human health are intensively debated. Therefore, we propose to reveal the effect of BPA on estrogen-induced spine synapse formation in the brain of nonhuman primates, which will provide more relevance to human health. There is ample evidence that remodeling of synaptic contacts might mediate the beneficial effects of estrogen on both hippocampus- and prefrontal cortex-dependent executive functioning, particularly working memory. Thus, we hypothesize that BPA exposure may interfere with estrogen-induced synaptogenesis in the hippocampal CA1 and CA3 areas and dentate gyrus, as well as in the prefrontal cortex of nonhuman primates. This hypothesis will be tested using ovariectomized, adult young vervet monkeys that will be treated with vehicle- (controls), estrogen-, BPA- and estrogen+BPA. Furthermore, regarding the receptor target(s) of BPA, we will test whether BPA interferes with the positive effects of estrogen on the hypothalamic expression of oxytocin (O) and the progestin receptor (PR). Neurons producing PR and O contain different estrogen receptors, ERa and ER[unreadable], respectively. Light and electron microscopic unbiased stereological calculations will be used to determine the total number of spine synapses in the hippocampus and prefrontal cortex, and the number of hypothalamic PR- containing and O-producing neurons. Bisphenol A (BPA) is an estrogenic chemical that is widely used in the manufacture of policarbonate plastics and epoxy resins. Because BPA leaches out of plastic food and drink containers, including baby bottles, as well as the BPA-containing dental prostheses and sealants, considerable potential exists for human exposure to this compound. We have demonstrated for the first time that treatment of ovariectomized female rats with 40 [unreadable]g/kg BPA, which is below the the current U.S. Environmental Protection Agency reference daily limit for human exposure, inhibits both the hippocampal synaptoplastic and the positive cognitive effects of estrogen administration. This study, has been performed in rats. Thus, the implications of these alarming results to human health are intensively debated by the chemical industry. Therefore, we propose to reveal the effect of BPA on estrogen-induced spine synapse formation in the brain of non-human primates, which will provide more relevance to human health. [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
The biogenetic-type synthesis of the alkaloids salutaridine, narwedine, dihydrolyfoline and decodine will be carried out, along with syntheses of several phenolic macrolides. Ortho metalation reactions of a variety of ligands with palladium, rhodium, and nickel and their utility in the synthesis of organic natural products will be explored.	{ "pile_set_name": "NIH ExPorter" }
The possible role of motor development on psychological function is once again a topic of great theoretical and practical importance. The revival of this issue is stemming from a different approach to the topic, away from Gesell's interest in the long-term prediction of psychological functions from early motoric assessments,toward an attempt to understand how locomotor experience orchestrates psychological changes. Research to date is quite promising in pointing to a linkage between crawling experience and cognitive, perceptual, social, and emotional changes. However, this work, having been conducted within a quasi-experimental framework, is intrinsically limited in its contributions, despite its heuristic implications for normal and clinical populations and for brain development. Enough research has been conducted with positive findings, however, to warrant the difficult but necessary task of determining whether crawling experience is playing a causal role as an inducer or facilitator of psychological development or whether it is a maturational forecaster - a sign of important psychological changes to come. This proposal describes an experimental approach to addressing the potential role of locomotor experience on the development of sensitivity to peripheral optic flow. Three experiments are proposed in which locomotor experience is manipulated in a powered-mobility-device and developmental changes in sensitivity to peripheral optic flow are measured on the basis of infants postural compensations to side wall movement in a moving room. Experiment 1 focuses on the efficacy of infants~ ability to control the powered-mobility-device and the consequent changes in sensitivity to peripheral optic flow that result from self generated experiences in such a device. Experiment 2 examines the role of active versus passive experiences in the mobility device and consequent changes in sensitivity to peripheral optic flow, whereas Experiment 3 looks at whether differential attentiveness to the environment while locomoting contributes to the aforementioned perceptual changes.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: Technological change is a central feature of health care markets and over the past few decades has revolutionized the treatment of many medical conditions. Although breakthrough scientific advances are an important driver of the pace of medical innovation, market incentives - such as those provided by the patent system - are often a critical determinant of which potential technologies successfully make the transition 'from the lab to the market.' The key contribution of this project is to develop new data (Aims 1 and 3) and new empirical methods (Aims 2 and 4) to investigate how patents shape the development and diffusion of medical innovations. Specifically, we apply these new data and new methods to estimate two relevant parameters: the extent to which patents provide incentives for the development of new technologies, and the extent to which patents on existing technologies hinder subsequent innovation. The more effective patents are in inducing research investments, the stronger the case for longer or broader patents. On the other hand, the larger the costs of patents in terms of hindering subsequent innovation, the weaker is this case. Evaluating these costs and benefits of patents is a key input into optimal policy design. To estimate the first parameter - how the prospect of stronger patent protection affects research investments - we develop a new empirical method (Aim 2) based on the following idea: because pharmaceutical firms file patents prior to starting clinical trials, shorter clinical trials grant firms longer efective patent terms. Hence, if longer patent terms encourage more research investments, we should see higher levels of research investments on treatments for patient groups that require shorter clinical trials. We apply this method to a newly developed data set measuring research investments on treatments for cancer patients (developed in Aim 1). To estimate the second parameter - how patents on existing technologies affect subsequent research investments - we develop a new empirical method (Aim 4). The key idea behind our approach is to take advantage of two facts: first, patent applications are quasi- randomly assigned to patent examiners at the US Patent and Trademark Office; and second, patent examiners differ in their likelihood of awarding a patent to any given application. We apply this method to a newly- developed data set measuring research investments related to the human genome (developed in Aim 3) and quantify the extent to which patents on human genes have hindered subsequent medical innovation. The new empirical methods (Aims 2 and 4) will be applied to two particular classes of medical technologies - cancer treatments and gene-based diagnostics and treatments - but each method can be applied in other contexts. In addition, the new data construction methods (Aims 1 and 3) - text-based matching with clinical trial enrollment lists, and MeSH-ICD matching of research investments to the relevant groups of patients - can also be applied in other contexts.	{ "pile_set_name": "NIH ExPorter" }
This is an R01 grant application for five years of funding to apply novel advanced image analysis techniques and introducing technology that would improve the targeting in CT guided interventions. CT is currently in the United States the most common imaging modality used to guide biopsy and tumor ablation. The majority of liver tumors such as hepatocellular carcinomas are visible on contrast-enhanced CT or MRI obtained prior to the procedure. Yet, these tumors may not be seen or may have poor margin conspicuity on unenhanced CT images acquired during the procedure. This may increase the procedure time, and/or lead to non-diagnostic cytopathologic assessment, requiring repeat biopsy or sub-optimal ablation applicator placement. We aim to enhance the current information available to the interventional radiologist, by registering the high-resolution pre-procedural images with low quality intra-procedural images. Anatomical details (including the tumor margin) visible on the pre-procedural images will be superimposed on the intra- procedural images. The precise position of ablation applicator and biopsy needle would be thus estimated with respect to the real contour of the tumor as appearing on the pre-procedural scan. Non-rigid registration is demonstrated as a technology to achieve alignment of images with good accuracy, even in the presence of organ motion. However, up to date it has not been used for fusing pre- and intra- procedural data during CT guided interventions in a clinical suite, since it requires significant computational infrastructure and often these methods are not sufficient robust. We have developed a new non-rigid registration method based on biomechanical model, validated in a prospective study in the area of image-guide neurosurgery. Recently, this new technique is also validated on retrospective data from RFA patients. Pre-procedural MRI and unenhanced intra-procedural CT images are aligned within 5 minutes, with 2mm accuracy. This proposal aims to demonstrate the feasibility of this technology in an interventional radiology suite during image-guided abdominal interventions, without impact on the medical decision. Our predictions for biopsy and ablation will be compared with the histological findings and ablated area on post- procedural MRI. Both mathematical methods and visual inspection by two independent radiologists of results will be employed in assessing the results. The success will be determined by the accuracy, robustness, execution time of the non-rigid registration algorithms. This proposal will benefit public health by developing and assessing key technologies to enable enhanced navigation during the image guided biopsy and ablation. The capacity to visualize the tumor and tumor margin throughout the procedure will better enable the interventional radiologists to achieve better diagnosis, and coagulation necrosis for the entire tumor mass, without unexpected site effects. PUBLIC HEALTH RELEVANCE: This work will lead to new technologies for precise targeting in CT guided interventions. These technologies will facilitate better diagnosis and treatment of liver neoplasms, by enabling the interventional radiologists to more accurately place (i) the biopsy needle and (ii) the ablation applicator, and so, they can (i) improve the specificity in detecting even small tumors and (ii) achieve complete tumor ablation with less risk of removing healthy liver tissue located adjacent to the tumor.	{ "pile_set_name": "NIH ExPorter" }
The objective of this study is to investigate the role and molecular mechanisms of tissue kallikrein in cardiac protection after myocardial infarction in normal and genetically modified animals. Our recent studies have shown that tissue kallikrein gene or protein delivery protects against organ damage in the heart, kidney and brain through anti- oxidative, anti-apoptotic, anti-inflammatory and angiogenic effects. These results indicate that kallikrein is a pleiotropic agent ideal for combined gene and stem cell therapies for cardiovascular diseases. Therefore, we hypothesize that tissue kallikrein gene delivery or kallikrein modified-mesenchymal stem cell (TK-MSC) implantation provides superior benefits in cardiac repair by inhibiting apoptosis and promoting neovascularization and cardiomyocyte regeneration. We intend to fulfill the following specific aims: 1) determine the effect and signaling mechanisms of tissue kallikrein on angiogenesis, arteriogenesis and ventricular remodeling in rats after myocardial infarction, and on migration, growth and capillary tube formation in endothelial cells;2) determine whether tissue kallikrein directly activates kinin B2 receptors via proteolysis, independent of kinin formation, to prevent cardiomyocyte apoptosis and inflammation and cardiac dysfunction in kininogen- deficient rats after acute myocardial infarction;3) determine the viability and effects of TK-MSCs on cardiac function as well as their paracrine effects on cardiomyocyte apoptosis and inflammation in rats after acute myocardial infarction;4) determine the effects of graft TK-MSCs on cardiac repair and remodeling by promoting neovascularization and cardiac regeneration in rats with post-infarction heart failure. Our long-term goal is to develop a novel therapeutic strategy for myocardial repair and regeneration of damaged myocardium using kallikrein gene and cell-based therapies. This study should generate new and important information to provide the impetus for developing therapeutic regimens in the prevention of heart failure. Project Narrative: Our objective is to investigate the molecular mechanisms of tissue kallikrein in cardiac protection in animal models with acute and chronic myocardial infarction. Our long-term goal is to develop a novel therapeutic strategy for myocardial repair and regeneration of damaged myocardium using kallikrein gene- and cell-based therapies. This study should generate important information to provide the impetus for developing therapeutic regimens in the prevention of cardiac dysfunction and heart failure.	{ "pile_set_name": "NIH ExPorter" }
Environmental or occupational exposure to manganese (Mn) causes a neuropathy resembling idiopathic Parkinson's disease (PD), characterized by motor deficits and damage to dopaminergic (DAergic) nuclei of the basal ganglia. Mitochondria! dysfunction, oxidative damage, and protein aggregation have been implicated in the pathobiology of PD. The complexity of the vertebrate brain has hindered understanding of molecular mechanisms associated with this disorder. The nematode, Caenorhabditis elegans (C. elegans) and mammals share a highly conserved genetic code with all genes responsible for DA biosynthesis, packaging, and reuptake present and functional in the worm. Exposure of C. elegans to Mn results in DAergic neurodegeneration, consistent with observations in mammals. Thus, the C. elegans offers an innovative and powerful platform to evaluate the molecular and functional mechanisms associated with Mn-induced DAergic neurodegeneration and a unique model for evaluating gene-environment interactions. Our 1 overall hypothesis is that knockdown and loss-of-function mutations of the PD-associated genes, dj-1, and pink1, and their related chaperone proteins confer selective vulnerability to DAergic neurons rendering them susceptible to an accelerated neurodegeneration upon exposure to Mn. We will test the following Specific Aims: (1A) Knockdown and loss-of-function mutations in dj-1 and pink1 render C. elegans susceptible to oxidant-induced DAergic neurodegeneration which is amplified by Mn exposure, (1B) Overexpression of DJ-1 and PINK1 protects C. elegans against oxidant-induced DAergic neurodegeneration mediated by 6-OHDA and Mn, (2) Knockdown of heat shock protein 70 (hsp70) and carboxyl terminus of Hsc70-interacting protein (CHIP) orthologues inhibits DJ-1 and PINK1 translocation to the mitochondria rendering C. elegans more susceptible to 6-OHDA and Mn-induced DAergic neurodegeneration. Using RNAi, site-directed mutagenesis and overexpression of PD-associated genes to generate the worm phenotypes, we will assess DAergic neurodegeneration by GFP fluorescence and a-synuclein aggregation assays. Oxidative injury will be assayed by measuring F2-and F3-isoprostanes, ATP and mitochondrial membrane potential. Understanding the relationship between genetic vulnerability and Mn exposure will provide critical insights into the mechanisms of Mn-induced toxicity and the development of novel therapeutic neuroprotective strategies. [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
Extensive laboratory and surgical experience by the Principal Investigator has proven that cartilage regeneration and biological resurfacing of damaged joints is possible using periosteal grafts. However, the major limitation is an age-dependent decrease in the potential of periosteum to produce cartilage in subjects beyond early adulthood. The next step is to document age-related changes in the cellular events in periosteal chondrogenesis and factors regulating patients. The periosteal explant culture model developed by the Principal Investigator makes it now possible to pose and test, in vitro, a logical series of hypotheses to achieve this objective. In Specific Aim 1, it will be determined if the age-dependent decrease in chondrogenic potential of the periosteum correlates with changes in chondrocyte precursor cell frequency, proliferation and/or differentiation. Specific Aim 2 will focus on regulation of proliferation, testing the hypothesis that growth hormone (GH) and insulin-like growth factor (IGF-1) regulate proliferation of chondrocyte precursors in the periosteum. Next, Specific Aim 3 will test the validity of our model, that proliferation and differentiation are sequence-dependent, permitting more accurate timing of the study or control of either of these events. Finally, in Specific Aim 4, it will be determined if the age-dependent chondrogenic potential of the periosteum is related to altered activity of certain growth factors (or their receptors) known to regulate chrondrocytes, IGF-1 and TGF-beta, will be determined. The impact of society and the improvement in the quality of life for so many thousands of patients suffering from, or at risk of developing, arthritis would be profound if arthritis could be prevented in many, and treated in others, with biological resurfacing.	{ "pile_set_name": "NIH ExPorter" }
The overall goal of the project is to validate the feasibility of a microwave tomographic (MWT) imaging technology for assessment of functional and pathological conditions of extremity soft tissue. The successful management of a fractured bone involves an understanding of the two major components (boney and soft tissue elements) of any extremity segment. The diagnosis and evaluation of the boney component is obvious to the treating physician by radiographic studies. The accurate assessment of the soft tissue component of the injured extremity segment remains a major deficiency in management of fractures. Consequently there is an important need to develop an effective and rapid method of non-invasive assessing of extremities soft tissue viability. Previous studies (NHLBI grant No HL65657) demonstrate that microwave tomography is feasible for non-invasive assessment of myocardial viability. Our most recent studies suggests that the technology might be feasible for rapid soft tissue functional imaging of extremities Both the imaging capabilities of this technology for assessment of functional conditions of extremities soft tissues within a very short msec range of acquisition cycle and the use of low, safe levels of non-ionizing radiation with cost efficiency have potentials for significant improvement of public healthcare. In this three year study we plan to (i) develop 2D MWT system;(ii) develop an appropriate image algorithm and (iii) assess feasibility of the technology for extremities soft tissue imaging, including sensitivity and resolution of the technology. Relevance of this project to public health. There is important need to develop an effective and rapid method for assessment of conditions of extremities soft tissue during trauma and recovery. The goal of the project is to validate the feasibility of novel imaging modality (Microwave Tomographic Imaging) for non- invasive (non-destructive) imaging of functional conditions of extremities soft tissue. There is an important clinical need to develop an effective, rapid and cost-effective method of non-invasive assessing or imaging of extremities soft tissue viability. The overall goal of the project is to validate the feasibility of a microwave tomographic (MWT) imaging technology for assessment of functional and pathological conditions of extremity soft tissue. The imaging capabilities of this novel technology and the use of low, safe levels of non-ionizing radiation with cost efficiency have potentials for significant improvement of public healthcare.	{ "pile_set_name": "NIH ExPorter" }
The objective of the MBRS-IMSD Program at the University of California, Irvine (UCI) is to increase the number and academic excellence of underrepresented (UR) undergraduate and graduate students that complete PhD degrees in biomedical sciences and advance to competitive postdoctoral positions. During the past decade, program increased by severalfold the institutional rate at which UR undergraduates enter PhD programs following graduation and the number of UR graduate students completing PhD programs at UCI. Program elements are academic preparation, research training, intense mentoring and professional development. Independent paid research conducted under the direction of faculty mentors serves as a core element to prepare MBRS students for graduate school and research-focused careers. Research training begins in the freshmen year with the development of original projects that are developed by participants. Over 100 faculty with funded research programs serve as preceptors of MBRS trainees. The program offers a series of components to increase the interest, motivation and academic preparedness of undergraduates (freshmen to seniors) to enter PhD programs in biomedical sciences, including a peer tutoring/mentoring program of science classes, a seminar series, journal club, progress report sessions, workshops on scientific communications, and application to graduate school. The graduate component is designed to provide a comprehensive training for UR Ph.D. students to excel in graduate school. The program provides summer research training for incoming UR PhD students to prepare them for the graduate core classes, a workshop to prepare oral exams after the first year, a series of activities for second year students to prepare a dissertation research project and competitive proposals for extramural funding, presentation of papers at national conferences, professional development workshops on the academic postdoctoral positions search and counseling and orientation about graduate studies and career development in a non-departmental setting.	{ "pile_set_name": "NIH ExPorter" }
Mosquitoes Culex pipiens is a major vector species for Japanese encephalitis and filariasis in China, and it is an important vector of West Nile Virus in the USA. Vector control is an important means of mosquito-borne disease prevention and management. Pyrethroid insecticide is currently being promoted worldwide for disease vector control because of their high efficacy, rapid rate of knockdown, and low mammalian toxicity. Due to wide- spread and improper use of insecticides, resistance to pyrethroids is very prevalent in Cx. pipiens mosquito species in China. The long-term goal of this research is to elucidate the genetic and molecular mechanisms of pyrethroid resistance in Cx. pipiens mosquitoes, and to develop more accurate and cost-effective resistance detection methods that are applicable to field samples. During the past three-year granting period, we have established deltamethrin resistant strains of Cx. pipiens, characterized novel genes associated with deltamethrin resistance in Cx. pipiens, developed PCR-based knockdown resistance (kdr) mutation detection methods and conducted quantitative trait loci mapping for pyrethroid resistance. Because the classic genetic mapping study in our previous studies cannot identify genetic transcripts differentially regulated between susceptible and resistant individuals, this renewal application will use high-throughput transcriptome analysis and population genetic approaches to determine the relationship between pyrethroid resistance at organismal level and transcription activity and gene nucleotide polymorphisms at molecular level. Here we focus on insecticide resistance at the organism level because mosquitoes may possess compensatory mechanisms under the selection by insecticides. Studying individual genes in cell lines will not reveal resistance at the organismal level because it ignores gene-gene and gene-environment interactions. The specific aims are: 1) to identify the major candidate genes for resistance to deltamethrin in Cx. pipiens using transcriptome analysis, 2) to determine the function of the candidate genes in pyrethroid resistance; and 3) to establish association between pyrethroid resistance at organismal level and gene polymorphisms and transcription activity at molecular level. We anticipate that this research will significantly enhance our understanding of resistance to pyrethroid insecticides in Cx. pipiens mosquitoes. The knowledge from this application will greatly facilitate the development of new resistance diagnostic methods for this important disease vector in China and the world. PUBLIC HEALTH RELEVANCE: Culex pipiens is a major vector species for Japanese encephalitis and filariasis in China, and it is an important vector of West Nile Virus in the USA. Insecticide- based mosquito control is an important method, but Insecticide resistance represents a major obstacle in the struggle against vector-borne disease. This project will better understand the genetic mechanisms of resistance to pyrethroid insecticide in Cx. pipiens pallens mosquitoes, and will lead to novel diagnosis methods for resistance. Thus, the proposed research will greatly enhance the monitoring and management of insecticide resistance.	{ "pile_set_name": "NIH ExPorter" }
The Eleventh Gordon Research Conference on Fertilization and the Activation of Development will provide a forum for presentation and discussion of new developments and ideas in this exciting, rapidly advancing field. This meeting will be held at the Holderness School, Plymouth, New Hampshire, July 27 - August 1, 1997. This conference has been held every two years since 1974 and is the only Gordon Research Conference concerned with the interaction of the male and the female gametes in the vital process of fertilization and activation of the fertilized egg to begin the process of embryo formation. It provides a venue for the interaction of biochemists, cell biologists, molecular biologists, physiologists, and biophysicists working in the field. This conference has been singularly successful in fostering collaborations between widely disparate experience and seniority and between research laboratories in this country and in Europe, Japan, Australia and Latin America. These collaborations have borne many fruitful results. There will be nine sessions in the conference, following the Gordon Research Conference format: 1) The mechanism of sperm activation; 2) Penetration of egg surface coats and sperm-egg binding; 3) Initiation of egg activation; 4) Egg activation pathways; 5) Plenary Lecture on signal transduction; 6) Membrane fusion mechanisms; 7) The cell cycle and early development; 8) Current topics and contributed papers; 9) Integrins and disintegrins in fertilization. This will be a truly international meeting with speakers and discussion leaders from Europe 97), Mid East (1), Far East (2) and Central America (1) as well as the U.S. (24). Among these 35 speakers are eight women and three representing ethnic minorities.	{ "pile_set_name": "NIH ExPorter" }
The overall objective of this research program is to identify proteins that regulate the activity or subcellular localization of the alpha subunit (G- alpha) of heterotrimeric G proteins. Regulatory proteins may be proteins whose interaction with G-alpha was not previously appreciated, or they may be novel proteins or protein families. The Regulators of G Protein Signaling (RGS proteins) are a recent example; RGS proteins enhance the intrinsic GTPase activity of many G-alpha subunits, thus tending to decrease the activity of the G proteins. Four proteins that interact with G- alpha-s have been identified in the yeast two-hybrid assay. Two of the four proteins are NEFA and the mu subunit of the clathrin-associated protein AP2. The other two are essentially unknown, one being homologous to a predicted protein of unknown function identified in the C. elegans genome project, and the other described only as an anonymous brain mRNA encoding a 1405 amino acid protein. Subsequent to the identification of NEFA in the two-hybrid assay, we confirmed a physical interaction between NEFA and G-alpha-s. The objective of the present application is to determine the functional significance of this interaction, and to initiate experiments to confirm and characterize the interactions between the other three proteins and G-alpha-s. The first specific aim is driven by the hypothesis that the identification of the proteins NEFA, AP2-mu, C16C10.10, and AB002373 in the two-hybrid assay is due to a physical interaction between each of these proteins and G-alpha-s. 1) The physical interaction between G-alpha-s and proteins identified in the initial yeast two-hybrid screen will be confirmed. The second specific aim is based on the hypothesis that the physical interaction between NEFA and G protein alpha subunits is physiologically relevant. 2) The subcellular and regional distribution of NEFA and its cognate mRNA will be characterized, and the possibility of a functional interaction between NEFA and certain subtypes of G-alpha will be evaluated. The third specific aim is to assess the hypothesis that the existence of a physical interaction between a given target protein and G-alpha-s, as determined in aim l, reflects a functional interaction between the protein and some subtype of G-alpha. 3) Functional consequences of physical interactions, confirmed in aim l, between G-alpha and any of the other three target proteins will be determined.	{ "pile_set_name": "NIH ExPorter" }
The purpose of this project is to characterize the extents and time scales of structural reorganization associated with redox state changes in biological redox proteins in disordered media, such as liquids, oriented membrane multilayers and frozen glasses, using x-ray scattering in the small to mid-angle domain (0.005 [unreadable]-1<q<2 [unreadable]-1). Protein dynamics and nuclear reorganization energies are controlling features of biological electron transfer, although the amplitudes and time scales of these motions have not been well characterized. The goal of this project is to correlate structural reorganization, detected by solution x-ray scattering, directly with biological electron transfer activity in three redox protein systems: cytochrome c, rutheniated cytochrome c, and the bacterial photosynthetic reaction center. Initially, proof-of-principle experiments will be carried out using chemically generated, stable oxidized and reduced redox states of proteins.	{ "pile_set_name": "NIH ExPorter" }
An experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease is critical for the study of human AIDS. Simian immunodeficiency virus (SIV) infection of macaques satisfies this criterion and is therefore a relevant model. SIV induces an immunodeficiency syndrome in infected macaques that is remarkably similar in pathogenesis to human AIDS. An important use of this animal model system is the detailed study of pathogenesis and viral determinants of disease since many studies of this type are not feasible in humans. The purpose of this project is to investigate host and viral factors involved in variable disease progression in SIV-infected macaques and the lack of disease in African primates infected with their own strains of SIV. PATHOGENESIS OF SIVsm-INFECTION OF MACAQUES: To investigate the role of host factors in SIV-infection of macaques, we used a well-defined molecularly cloned virus (SIVsmE543-3). Susceptibility to SIV infection of primary PBMC in vitro varies significantly between individual macaques. Interestingly, the susceptibility phenotype correlates with the extent of in vivo viral replication following in vivo inoculation. However, although macaques with low susceptibility phenotype initially maintained low levels of viremia for variable periods of time, eventual progression to AIDS was observed coincident with increasing viremia. These latter data suggest viral and/or immune escape in these animals. We evaluated the immune responses in four macaques that progress rapidly to AIDS in under 6 months. These animals developed transient SIV-specific CTL and antibody responses and failed to develop antibody to either tetanus toxoid or Hepatitis A, consistent with a global immune defect. In collaborative studies with the Martin laboratory, the phenotype of CD4+ T cells was evaluated in SIVsm-infected RP macaques. SIVsmE543-3-infected RP macaques showed profound and early depletion of memory CD4+ T cells explaining the immune deficits in these animals. Virus from RP macaques was molecularly and biologically characterized. Sequence analysis of env genes cloned from three SIVsm-infected animals exhibited common substitutions in the env gene. These substitutions were unusual in that they involved residues that were generally conserved and that were known to affect binding of env to CD4 or coreceptor. Full length infectious molecular clones of various RP viruses were derived from plasma of RP RhH635. This macaque was inoculated with virus isolated from tissues of one of the initial RP study animals. The 635 viruses used CCR5 as a coreceptor and replicate less efficiently in primary macaque PBMC and macrophages than the parental SIVsmE543-3 strain. Pilot studies with three selected viruses were performed in two rhesus macaques per virus. One of these viruses demonstrated an interesting phenotype of low initial viremia but increasing levels over the first 3-4 months of infection associated with depletion of CD4+ T cells. Previous studies in this lab demonstrated that infection of PT macaques with SIVagm9063 results in AIDS. SIVagm9063 was generated by animal passage and thus might not be reflective of the virulence of the parental strain. We assessed the pathogenicity of the SIVagm9063 and two primary SIVagm isolates in PT macaques. Infection of macaques with any of the three isolates resulted in high levels of primary plasma viremia by 1 week after inoculation. Viremia was quickly controlled following infection with SIVagm155; these animals have maintained CD4+ T cell subsets and remain healthy. The plateau levels among SIVagm90 and SIVagm9063-inoculated macaques varied widely from 100 to a million copies/ml of plasma. Three of four animals from each of these groups progressed to AIDS. Set point viremia and the degree of CD4+ T cell loss at 6 months post infection were not significantly different between macaques inoculated with SIVagm90 and SIVagm9063. However these parameters were significantly different in SIVagm155-inoculated macaques (p values < 0.01). Considering all the macaques, the degree of CD4+ T cell loss by 6 months post infection, correlated with the plateau levels of viremia. Thus, similar to SIVsm/mac-infection of macaques and human AIDS, viral load is an excellent prognostic indicator of disease course. SIVlhoest/sun Infection of Macaques. Previous studies in this laboratory showed that the related SIVlhoest and SIVsun strains are capable of inducing AIDS in PT macaques. Long-term follow-up of these animals demonstrated profound CD4+ T cell depletion and eventual progression to AIDS. Survival was prolonged compared to infection of macaques with SIVsm or SIVagm. Although these animals exhibited high levels of primary viremia, progression to AIDS occurred despite moderate to low chronic viremia suggesting other mechanisms than direct virus-induced cell killing in the pathogenesis of this infection. ASYMPTOMATIC INFECTION OF NATURAL HOST SPECIES A second goal of this project is to study the mechanisms underlying the apparent lack of pathogenicity of SIV for their natural host species, with emphasis on SIVagm from vervet monkeys. SIVsm, SIVagm and SIVlhoest are capable of inducing AIDS in macaques but are not virulent for their natural host. These studies have utilized a molecularly cloned, pathogenic SIVagm9063-2 that induces AIDS in pigtailed macaques but results in asymptomatic infection of African green monkeys. Natural and experimental infection of vervets (Chlorocebus pygerythrus) with SIVagm was evaluated by in situ hybridization, plasma viral load assays and limiting dilution coculture to identify the numbers and distribution of infected cells in tissues. Virus was found in lymphoid tissues and the gastrointestinal tract and in the lung macrophages of one animal. Plasma viral load varied widely from undetectable (<1000 copies/ml) to 800,000 copies/ml. We recently expanded these studies by evaluating plasma viremia in a cohort of 12 naturally infected vervets from Tanzania. Plasma viral load varied from 1000 to >100,000 copies/ml in these animals. PCR and sequence analysis of portions of the envelope gene of these novel SIVagm isolates revealed wide genetic diversity, although all isolates clustered phylogenetically with previously characterized SIVagm isolates from vervets, confirming that these source animals were indeed vervet AGM. The kinetics of viremia of one of these viruses in naive vervets was evaluated in a pilot experiment using intravenous inoculation of 1 ml of blood from one naturally-infected donor, AG1. These animals exhibited high levels of primary viremia. The range of set point viremia was similar to that observed in the naturally-infected cohort. These animals have remained asymptomatic for 1 year of follow-up. We have evaluated the viral kinetics of a natural SIVagm isolate in AGM. Two species of AGM were evaluated, vervet monkeys (the species of origin of SIVagm90) and sabaeus monkeys from the Barbados. The virologic outcome in sabaeus and vervet AGM was surprisingly divergent. Inoculation of sabaeus AGM with SIVagm90 resulted in low and variable levels of primary viremia and low setpoints (<100 to 10,000 copies/ml). In contrast, inoculation of vervet AGM resulted in high primary viremia and establishment of moderate plateau levels (10,000 to 100,000). Regardless of the extent of viremia, CD4+ T lymphocytes remained stable throughout infection of AGM with no significant relationship between viral load and CD4+ T cell loss.	{ "pile_set_name": "NIH ExPorter" }
Coronary heart disease in the leading cause of death, disability, and health care costs in the US. Among patients with established coronary heart disease (CHD), a majority of patients continue to have acute cardiac events, many sudden and unexpected, despite identification and treatment of their disease and traditional risk factors. Substantial evidence indicates that psychosocial stress and the sympathetic nervous system contributes to acute cardiac events. Preliminary evidence suggests that complementary medicine practices, such as traditional acupuncture (TA), may beneficially alter outcomes in patients with CHD, although the mechanisms responsible for the observed benefit are unknown. We propose to conduct a randomized, blinded, controlled study of the effects of the complementary medicine practice of traditional acupuncture (TA) compared to sham TA and waiting control group on the primary outcome of: 1) autonomic nervous system imbalance (heart rate variability);and the secondary outcomes of: 2) an inflammatory marker (high-sensitivity C reactive protein);and 3) psychological stress, depression, hostility, anxiety, social support, and quality of life;and the exploratory variable of arterial vasomotion (brachial artery reactivity testing). We hypothesize that the complementary medicine practice of TA can serve as an additive/alternative treatment for the prevention of acute cardiac events in CHD patients. The results of this randomized controlled trial will: a) yield new data regarding the pathophysiologic mechanisms effected by the complementary medicine practice of TA in CHD patients, and b) provide pilot data for a large multi-center study of the effect of TA on acute CHD events, including sudden cardiac death.	{ "pile_set_name": "NIH ExPorter" }
Proposed is a continuation of a productive program of research that has the overall goal of elucidating the mechanism(s) underlying cognitive decline with aging. To achieve this goal, we originally organized and will continue a program of research that includes 4 research projects, 3 essential core facilities and a group of talented investigators. The research program is driven by the now strongly supported hypothesis that oxidative stress in the brain leads to age-related oxidative damage and is a major determinant of the rate of cognitive aging. As such the 4 research projects focus on a systematic assessment of these issues. Project 1 will determine the role of now identified age-related oxidation sensitive proteins and highly significant shifts in the redox state of most brain regions in cognitive decline with age. Project 2 will determine the role of and mechanism by which insult-induced decline in protein phosphates results in persistent activation of a number of kinases, and oxidative stress that leads to AD-neuropathology and cognitive decline. Project 3 will determine the mechanisms underlying the function of immediate early gene products that control intracellular Ca2+ channels and intracellular Ca2+ homeostasis during cognitive aging and oxidative stress in the CNS. Project 4 will assess the signaling mechanism(s) by which the important ovarian steroid, progesterone, enhances LTP and reduces cognitive decline with aging, with a focus on its effects on NMDA receptors. All of these research projects are interactive in their mechanistic focus on the overall hypothesis of the program, the sharing of ideas, tissues, and the use of behavioral assessments following insults or interventions as important functional readouts in animal models shared and employed by all projects. This is achieved through an Administration Core (Core A) that will oversee the program and provide biostatistical support, an Animal Resources and Behavioral and Assessment Core (Core B) that will provide care for and behaviorally characterize all animal models and an Electrophysiology Core (Core C) that will provide assessment of the effects of age, genotypes, insults or interventions on an electrophysiological signature of memory and learning, LTP. This mechanistically driven and statistically-validated, multidisciplinary program of research, that focuses on determining critical molecular and functional mechanisms that underlie cognitive aging, will enhance our understanding of the role of oxidative stress in cognitive aging, and generate potential targets for effective intervention.	{ "pile_set_name": "NIH ExPorter" }
The computational power of the nervous system relies on the ability of individual cells to integrate multiple inputs and generate an output targeted to the appropriate partner(s). These functions would be impossible without the asymmetric localization of proteins within the cell. For example, many of the proteins that are in the axon are specialized for functions like transport and electrical conduction. In contrast, many of the proteins in the dendrites are specialized for receiving information from other cells. Restriction of localization is important for limiting the activity of a particular protein to a specific cellular compartment, but it can also be an important regulator of biochemical reactions. Many proteins behave differently when localized to a multiprotein complex due to the ability of direct binding partners to alter their conformation. This function of localization has important implications for how neurons process information that is relevant to many therapeutic interventions since it means that the behavior or activity of a protein cannot always be predicted from in vitro studies. This proposal has the goal of understanding how CaMKII and two proteins that directly bind to it, dCASK, a scaffold protein, and Eag, a potassium channel, are regulated by localization and cellular context. To achieve an understanding of how cellular context can influence the activity of these proteins we propose two sets of experiments. In Aim #1 we will use a transfected cell system to understand how dCASK and CaMKII interact to change the location, activity and potential substrates of CaMKII in response to calcium influx. We will do a parallel set of experiments in dCASK mutant transgenic flies that express a YFP tagged dCASK transgene that restores normal behavior. We will determine where in the cell dCASK has to reside and what proteins it has to complex with to carry out its behavioral function. Aim #2 addresses how Eag, a voltage-gated potassium channel, is localized and regulated by other proteins that bind to it. We have discovered a novel role for RNA editing in localization that has implications for many other genes that undergo this type of modification. We will also investigate the function of an alternative splice product of the eag gene that does not conduct ions, but instead functions as a scaffold for CaMKII and other signaling proteins. We address these issues by using homologous recombination to create mutations in the eag gene that selectively alter each of these processes and assess the impact on neuronal gene expression, excitability and behavior.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: (Applicant's abstract) The yeast genome sequence is now known, a major technical accomplishment that offer unprecedented opportunities for study of the cell biology of the simplest eukaryote. Knowledge of the interactions of regulatory and structural proteins with DNA in vivo is important for application-of the yeast DNA sequence to its function. This proposal addresses how to accomplish that goal. It builds on emerging applications of DNA methyltransferases to map protein-DNA interactions in living cells by developing methodology that will allow resolution (<l0 base pairs) study of interactions over extended regions of the S. cerevisiae genome. It describes methodology which will allow quantitative assessment of protein-DNA interactions with automated techniques that are appropriate for large regions of the yeast genome and exportable to larger eukaryotes. To illustrate the utility of the approach, we will map the chromatin organization of about half of chromosome III. Comparing portions of the structure with that determined by classical methods. Many other laboratories are highly interested both in chromatin structure and its role in DNA function and in interactions of regulatory proteins with DNA. To facilitate the research of these scientists, we propose a service which will provide chromatin mapping data for other regions of the yeast genome of interest.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: The functioning of the nervous system depends on the correct specification and "wiring up" of many different neurons in the nervous system. Goulding is interested in elucidating the molecular mechanisms that control the generation of specific neuronal cell types in the developing spinal cord. A large body of evidence suggests that gene regulation by transcription factors plays an important role in patterning the spinal cord. However, very little is known about the mechanisms that control the generation of specific cell types within the spinal cord. The homeodomain transcription factors, En1 and Ev1, are expressed in two populations of differentiating interneurons in the spinal cord. The expression patterns of both genes during development strongly suggest both proteins may function as determinants of neuronal cell type. As a first step toward analyzing the function of these transcription factors, Goulding and colleagues generated taulacZ knock-in mice in order to mark and morphologically characterize the cells that express En1 and Evx1. Goulding and colleagues will use these mice to investigate the fate of these two neuronal populations in mice that lack En1 and Evx1 function. Furthermore, the generation of these knock-in mice will provide valuable tools for further investigating the mechanisms that control cell fate specification and axonal guidance by neurons in the spinal cord. Goulding also plans to examine the regulation of En1 and Evx1 expression by genes that are expressed in the precursors of these cells. The longterm aim of these studies is to molecularly define how these two populations of interneurons are generated during embryonic development.	{ "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. Methods: The sample seemed to contain detergents (Triton, Tween and so on...), which may interfere with the sample analysis, so the sample was washed with acetone to make sure to remove all detergents before we analyze the sample by mass spectrometry. The detailed procedures used for your sample are shown below. Acetone precipitation Acetone was added to the dried sample. The sample solution was placed on ice for 15 minutes and then spun at maximum speed in a refrigerated microcentrifuge for 15 minutes to pellet the protein. The supernatant was removed. The sample was washed three times. Release of N-linked glycans The dried sample was dissolved in Trypsin buffer (0.1M Tris-HCl, pH 8.2 containing 0.01M CaCl2). The sample then was denatured by heating for 5 minutes at 100[unreadable]C. After cooling, the sample was digested with the trypsin (37oC, overnight). After tryptic digestion, the sample was heated at 100[unreadable] C for 5 minutes to de-activate the trypsin. After cooling to room temperature, the sample was applied to a C18 sep-pak cartridge. Before elution of glycopeptides and peptides, the sample adsorbed in the C18 sep-pak cartridge was cleaned with 5% acetic acid to remove any possible contaminants (salts, etc.). Peptides and glycopeptides then were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol each into a microcentrifuge tube. The propanol fractions were dried and then combined in one tube. The sample was reconstituted with 50mM sodium phosphate buffer (pH 7.5) and then the N-glycans were released using PNGase F (New England BioLabs). After digestion, the sample was passed through a C18 reversed phase cartridge and the carbohydrate fraction was eluted with 5% acetic acid and dried by lyophilization. Released N-linked oligosaccharides were permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and profiled by mass spectrometry. Mass spectrometry MALDI/TOF-MS analysis was performed in the reflector positive ion mode using [unreadable]-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol: water) as a matrix. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems). NSI-MSn analysis was performed following the method developed at the Complex Carbohydrates Research Center (Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127-42.). Mass analysis was determined by using on a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. Permethylated glycans were dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument at a constant flow rate of 0.4 [unreadable]L/ min. A full FTMS spectrum was collected at 60 000 resolution. The capillary temperature was set at 210oC and MS analysis was performed in the positive ion mode. For total ion mapping, automated MS/MS analysis (at 28 collision energy), m/z range, 500 to 2000 was scanned with ITMS mode in successive 2.8 mass unit windows that overlapped the preceding window by 2 mass units.	{ "pile_set_name": "NIH ExPorter" }
A major increase in the number of cases of coccidioidomycosis (Valley fever), a fungal respiratory disease of humans, has occurred in southwestern United States during the last five years. Our goals are to develop rapid and relatively simple methods for diagnosis of this disease. Current methods are inadequate because they are primarily dependent on patient antibody detection (antibody production may be delayed or attenuated in immunocompromised patients), and because multicomponent antigens currently used for antibody detection are variable between batches and display some cross-reactivity with heterologous sera (false positive reactions). We have proposed the development of three approaches to diagnosis of coccidioidomycosis and have designed experiments to test specificity and sensitivity of the molecular probes to be used. We will employ a recombinant, complement fixation antigen (CF-Ag) in standard immunoassays for specific antibody detection, monoclonal antibody to the CF-Ag and Coccidioides-specific antigen (CS-Ag) for detection of antigenemia, and a PCR method developed in the P.I.'s laboratory for C. immitis DNA detection in patient fluids. Multiple control and positive patient fluids (>400 sera, CSF, urine, BALs, and sputum) submitted by 8 physician-consultants will be used in open and blind studies for testing the efficacy of our diagnostic methods. PROPOSED COMMERCIAL APPLICATION: This proposal outlines methods for detection of (a) pathogen-specific antibody, (b) antigenemia and (c) DNA in patient sera and other body fluid using (1) bacterial-produced recombinant antigen, (2) monoclonal antibodies and (3) a polymerase chain reaction (PCR) primer-pair derived from a pathogen-specific gene, respectively. Each reagent (molecular probe) can be produced in unlimited amounts, can be stored without loss of activity for unlimited time, can be standardized (quality-controlled), and can be incorporated into individual test kits for clinical application using established testing methods in clinical laboratories.	{ "pile_set_name": "NIH ExPorter" }
Approximately one-third of veterans are current smokers, which is a significantly greater proportion than in the general population. Tobacco smoke exposure can promote airway hyperresponsiveness (AHR), a characteristic feature of asthma defined as airway constriction after exposure to a provocational agent. The prevalence of asthma is greater in veterans than in civilians and contributes significantly to healthcare utilization. Tobacco smoke is known to trigger asthma symptoms such as wheezing and cough; both are manifestations of airway hyperresponsiveness (AHR). Understanding the mechanisms behind AHR is important for patients that continue to be exposed to tobacco smoke (directly or secondhand), particularly asthmatics. Identifying a specific causative agent(s) for the adverse effects of smoking can be difficult given the complex nature of tobacco smoke. However, nicotine, a lipophilic chemical that is the major addictive component of tobacco smoke, may play an important role mediating the effects of tobacco smoke in the lung. Over the past several years, several studies have implicated nicotine in altered lung development and lung carcinogenesis. Nicotine is a ligand for endogenous nicotinic acetylcholine receptors (nAChRs). nAChRs are ligand gated channels comprised of five subunits that have neuronal and non-neuronal functions. The most common non neuronal receptor is the (17)5 nAChR. In this proposal, we hypothesize that chronic nicotine exposure promotes AHR by inducing nerve growth factor (NGF) secretion in lung fibroblasts through 17 nAChR dependent signaling. NGF belongs to the neurotrophin family, growth factors essential for development and survival of neurons. Overexpression of NGF has previously been associated with increased airway innervation and increased AHR after allergic stimulation. To address our hypothesis, we plan to utilize cell culture, organ explants, and a murine model of nicotine exposure. In Aim 1, using a cell culture model of primary lung fibroblasts and cultured lung explants from wild type and 17 nAChR deficient mice, we will demonstrate that nicotine acts on nAChRs to increase NGF secretion/production and increase neuronal growth. In Aim 2, we will demonstrate that airway smooth muscle contraction is increased and mediated by nerve growth factor by measuring bronchial ring contractility using isometric tension in an organ bath system. Bronchial rings will be harvested from unexposed mice and mice exposed to chronic nicotine. In Aim 3, we plan to use an in vivo mouse model of chronic nicotine exposure to show that NGF-related signaling is essential for nicotine induced increases in airway hyperresponsiveness by modifying the effects of nicotine with administration of NGF receptor agonists and antagonists. Airway hyperresponsiveness will be measured using a methacholine challenge in anesthetized, mechanically ventilated mice with the flexiVent. As part of this CDA-2 application, the applicant will engage in coursework geared toward improving grant/manuscript writing skills, and improving experimental design and analysis. She will also gain expertise in additional experimental techniques such as siRNA and isometric tension measurements. The applicant will also attend faculty development courses offered by her institution as well as national associations/professional societies that will address leadership, communication, and promotion. She will attend scientific meetings to present her research. These developmental activities will help the applicant achieve her immediate goals of improving her publication productivity and applying new research techniques, as well as her long term goal of becoming an independent investigator and mentor. With successful completion of the proposed project, we will better understand the mechanisms behind airway hyperresponsiveness related to nicotine exposure and help define alternative ways to treat veterans with asthma, particularly those who are exposed to tobacco smoke. PUBLIC HEALTH RELEVANCE: Cigarette smoking is more prevalent in veterans than the general population (35% vs. 21%). Exposure to tobacco smoke is also associated with airway hyperresponsiveness (AHR), a characteristic feature of asthma. Asthma is a common self reported diagnosis in veterans and contributes significantly to healthcare utilization. In asthmatics, cigarette smoke exposure is often a trigger of cough and wheezing, both manifestations of AHR. Given the increased prevalence of smoking and asthma in veterans, it is important to understand the mechanisms behind cigarette smoke associated AHR. Nicotine, the addictive component of cigarettes, may play an important role in AHR through neurogenic mechanisms. This project is relevant to the VA population because it will improve current understanding of the neurogenic pathways involved in nicotine exposure and may uncover alternative therapeutic interventions to improve disease management for asthmatic veterans, including those who continue to be exposed to cigarette smoke.	{ "pile_set_name": "NIH ExPorter" }
We have established conditions for in vitro assembly of stable synaptic complexes of a pair of viral DNA ends with HIV-1 integrase. These nucleoprotein complexes are intermediates in the integration of HIV DNA into a target DNA. Furthermore, the association of integrase with viral DNA in these complexes mimics all the properties of the association of integrase with viral DNA in preintegration complexes (PICs) isolated from virus infected cells. The synaptic complexes contain a tetramer of integrase tightly bounds to a pair of viral DNA ends. Footprinting of the viral DNA ends within the complex reveals that less than 20 base pairs of terminal viral DNA sequence are protected by integrase. within the SSC, a conclusion that is also in agreement with atomic force microscope images of the stable nucleoprotein complexes. 20bp of terminal viral DNA end sequence are efficient substrates for half-site integration in vitro, and are the only protected region observed in footprinting experiments. However, several hundred base pairs of non-specific flanking DNA sequence are required for efficient SSC assembly, stability and concerted integration. We are probing the function of this non-specific DNA sequence in SSC assembly and stability and propose that non-specific interactions between IN and DNA (distinct from the stable association of a tetramer of IN with the viral DNA ends) are involved. Having established methodologies to assemble synaptic complexes in vitro with purified integrase and HIV-1 DNA substrate, we are attempting both low and high-resolution structural studies combined with biochemical approaches to understand the detailed mechanism of DNA integration. The potential role of cellular proteins in SSC assembly is under investigation. One cellular protein that has been implicated in playing an important role in HIV-1 DNA integration is Lens Epithelial Derived Growth Factor (LEDGF). We find that LEDGF does not stimulate assembly of the SSC and in fact inhibits complex assembly. LEDGF must therefore be acquired by the preintegration complex after the two viral DNA ends are engaged by integrase to form the SSC. Our goal is directed towards X-ray crystallographic studies of HIV-1 intasomes. The two major obstacles we need to overcome are the propensity of the intasomes to self-associate and the requirement of several hundred base pairs of non-specific flanking DNA sequence for their assembly. The role of the flanking DNA is not understood. Mixing experiments with long viral DNA ends, and short viral DNAends that do not assemble intasomes alone, show that the short DNA ends are efficiently incorporated into intasomes if the partner is long. The length requirement of several hundred base pairs is similar to the length required for DNA to be easily able to bend back on itself. Our current hypothesis is that is that the flanking DNA transiently associates with the intasome, perhaps occupying the target DNA binding site. Cherepanov and colleagues have recently solved the structure of the prototype foamy virus (PFV) intasome. PFV belongs to the Spumaretrovirus family. Although it shares only 13% identify with HIV-1 integrase and has an extra domain not found in the HIV-1 protein, the arrangement of domains in the HIV-1 intasome is unlikely to be radically different. However, PFV integrase is sufficiently different that structures of the HIV-1 intasome are required to understand the detailed mechanisms of resistance to inhibitors such as Raltegravir. Four of the domains in the in the PFV intasome structure are disordered. The requirement of several hundred base pairs of internal DNA for HIV-1 intasome assembly and stability does not allow us approach structural studies in the way that has been successful with PFV intasomes. Our first approach was to assemble intasomes with long viral DNA substrate and cut off the internal DNA segment after assembly, but we found that the intasomes dissociate after cleavage of this flanking DNA. However, once target DNA is captured and stand transfer has taken place the internal DNA can be cleaved without loss of stability. A major effort is therefore directed at assembling HIV-1 STCs for structural studies. We are using the approach of assembling STCs on branched DNAs corresponding to the product of integration, a strategy we have successfully applied to the PFV system. We have exploited prototype foamy virus (PFV) as a model system explore novel strategies for obtaining high-resolution structures of HIV-1 intasomes. We have shown that PFV integrase can assemble STCs on product DNA, bypassing the requirement for assembly through the normal reaction pathway. High-resolution structural studies of these complexes shows they are identical to those formed on the normal reaction pathway as previously determined by the Cherepanov group. In addition, we observe additional electron density that likely corresponds to the domains that were missing in the earlier structure. Based on this proof of concept with PFV, we are currently applying a similar strategy to the HIV-1 system. Four of the domains in the PFV integrase structures are disordered. We have tested whether these domains are necessary for function of HIV-1 integrase by constructing heterodimers lacking the corresponding domains. We find that intasomes can be assembled when the outer subunits lack the domains that are missing in the PFV intasome structures. These domains are therefore not essential for function. However stable heterodimers of HIV-1 integraase comprising full-length protein in a dimer with the catalytic domain are inactive and do not assemble intasomes. We propose that that monomeric integrase is an obligatory intermediate on the intasome assembly pathway. Stabilization of the catalytic domain dimer interface therefore blocks intasome assembly. We have engineered a hyperactive mutant of HIV-1 integrase by fusing the DNA binding domain of Sulfolobus solfataricus chromosomal protein (Pdb: 1BNZ) to the N-terminus of integrase. This fusion protein has much higher in vitro concerted activity and, unlike the wild type protein, it is active on oligonucleotide viral DNA substrate. This activity with short DNA substrates overcomes one of the major obstacles to structural studies. In addition, the fusion protein has much better solubility properties than the wild type protein. Intasomes containing the fusion protein have been prepared at concentrations suitable for structural studies. Although these intasomes aggregate much less than those assembled with wild type protein, aggregation is still an issue at the concentrations required for crystallization. We are attempting to identify mutations that further improve the solubility properties of assembled intasomes. Lens epithelium-derived growth factor (LEDGF) is a cellular protein that stimulates HIV-1 replication between ten and one hundred fold. It acts as a tethering factor between the preintegration complex and chromatin. The integrase binding domain (IBD) binds to integrase within the preintegration complex and a domain in the N-terminal region binds chromatin. LEDGF also stimulates the concerted integration activity of HIV-1 integrase in vivo with short oligonucleotide DNA substrates. Partial proteolysis studies indicate that most of LEDGF is disordered, making it a poor candidate to aid our structural studies of HIV-1 intasomes. However, by constructing a peptide library spanning LEDGF, we find a short peptide is sufficient to promote efficient concerted DNA integration and also greatly improves the solubility and physical properties of HIV-1 intasomes. A fusion of this peptide with HIV-1 integrase makes HIV-1 integrase behave in a similar manner to PFV integrase, which to date is the only retroviral integrase for which high resolution structural studies have been successful. We are also s	{ "pile_set_name": "NIH ExPorter" }
Repair and regeneration of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiovascular medicine. Following cardiac injury, fibroblasts enter injury zone and through various processes actively impair contractile function. Converting cardiac fibroblasts within scar tissue into functional cardiomyocytes is a therapeutic approach that has great potential to restore the function of an injured heart. We were the first to identify a combination of miRNAs, miR combo (miR-1, miR-133a, miR-208a, and miR-499- 5p) that directly reprogrammed cardiac fibroblasts into cardiomyocytes, whereas others have used transcription factors to the same effect. Importantly, delivery of miR combo using lentivirus or retrovirus led to a modest, but significant, improvement in cardiac function. This application is focused on improving miR combo as a therapeutic by identifying ways to enhance efficiency, cell specificity, and effectiveness. Preliminary studies have identified an Adeno-associated virus (AAV) serotype that specifically targets fibroblasts and, moreover, displays enhanced transduction efficiency in vivo. Furthermore, TLR3 activation, which strongly augments miR combo directed reprogramming in vitro, may be a novel way to increase effectiveness. Finally, several transcriptional inhibitors have been identified as miR combo targets. These transcriptional inhibitors repressed the expression of key cardiac transcription factors, suggesting a mechanism for miR combo directed reprogramming. Three overlapping areas will be investigated in this project. Aim 1 will focus on optimizing the efficiency, effectiveness, and cell specificity of miR combo in vitro. To that end we will test if the self- complementary (sc)AAV 2/1 expressing a polycistronic miR combo results in the fibroblast-specific delivery of miR combo. Pharmacological agents will define the appropriate level and duration of TLR3 activation for optimal enhancement of miR combo mediated reprogramming. Aim 2 will address the same questions in vivo. Speckle tracking echocardiography will determine changes in regional wall motion in vivo. Histology will quantify fibrosis levels and lineage tracing will evaluate reprogramming of fibroblasts into cardiomyocytes. Pharmacological agents will be used to determine if TLR3 activation enhances the functional improvements that follow fibroblast reprogramming by miR combo. Aim 3 will define the mechanism by which miR combo promotes reprogramming. Gain- and loss-of-function approaches will determine the role of the transcriptional repressors in miR combo directed reprogramming. Luciferase assays will be used to delineate the physical interaction between the miRNAs within miR combo and the transcriptional repressors. Decoy inhibitor approaches will define the role of NF?B, the key transcription factor in the TLR3 pathway, in miR combo directed reprogramming. Findings from these studies will provide important new insights into improving the efficiency, effectiveness, and cell specificity of direct reprogramming for cardiac repair and regeneration as a therapy.	{ "pile_set_name": "NIH ExPorter" }
Despite a wealth of evidence on the factors driving the ?supply side? of helping?when people give help and why?there is a comparative dearth on the ?demand side??when people ask for help and why. Violating the standard economic assumption of ?more resources are preferred to less,? evidence suggests that despite large benefits, services ranging from governmental welfare programs to tutoring programs are vastly underused. The proposed project seeks to explain why people fail to ask for help in the context of social services. Building on work in economics, psychology, and sociology, as well as our own pilot evidence, we hypothesize that in addition to standard economic costs of seeking help (such as time and effort expended on searching for services), there are also potent psychological costs (such as feelings of shame, failure, and indebtedness). These psychological costs, we argue, may be a first-order problem, inhibiting those in need from seeking help for their financial hardships, undermining the effectiveness of welfare policies, and slowing individuals? escape from poverty.In collaboration with a Pennsylvania social services agency specializing in housing assistance, we propose two studies to examine the role of psychological factors in help-seeking. The first study will identify the extent to which people delay seeking initial help for their financial hardships and what factors contributed to those delays. While correlational, it will provide evidence for the relative importance of a wide range of economic and psychological factors among a demographically diverse population of low-income adults and families.The second study, a randomized controlled trial, will use low-cost, scalable interventions to test the importance of two psychological factors. The population of interest will be people who initially failed to get help with the partner organization due to capacity constraints and are subsequently referred to an external service provider. We will randomize participants into one of three groups (a control group or one of two treatment groups) and test the extent to which different messages affect people?s willingness to follow through on the external referrals. One intervention will test the importance of people wanting to maintain a positive image of themselves, while the other will test the importance of people wanting to conform to social norms of not taking resources out of a system without also contributing to it.In addition to advancing our theoretical understanding of help-seeking and filling a critical gap in the literature, the results of these studies could have large policy implications. Should our hypotheses be upheld, they would suggest that incorporating the psychological costs of help-seeking into welfare policies may be a powerful tool in the fight against poverty. Not only can it increase people?s willingness to seek out potentially life-saving services, it can simultaneously improve psychological outcomes as well, relieving those in need from the painful dilemma of how to trade off their financial and psychological well-being.	{ "pile_set_name": "NIH ExPorter" }
The long-term objectives of this study are to understand the mechanisms by which three unique streptococcal flavoproteins catalyze reactions normally ascribed to hemeproteins or to heme- dependent processes. Investigation of the catalytic roles of both flavin and non-flavin redox centers will be essential to the overall evaluation of the key structure-function relationships operative in the three enzymes, which are important in streptococcal oxygen metabolism. Recombinant DNA approaches will be applied to the study of the FAD- containing NADH peroxidase: the specific questions to be addressed include the cysteinyl peptide sequences of the proposed redox- active disulfide and the overall relatedness between the peroxidase and the flavoprotein disulfide reductases. In the context of our mechanistic studies, the roles of two distinct complexes of the reduced peroxidase with pyridine nucleotides in both peroxide reduction and in transhydrogenation will be investigated by static titration and rapid-reaction techniques, as will the specific role of cysteine thiol(s) in peroxide reduction. Studies of the unusual FAD-containing NADH oxidase will focus on the identification of the second non-flavin redox center in this enzyme and on the interaction of this center with the flavin in the overall four-electron reduction of oxygen. The possible role of oxygenated flavin intermediates in the catalytic mechanism will also be investigated by stopped-flow methods and will include studies of the enzyme reconstituted with modified FAD analogs. The possible structural and functional relationship of the NADH oxidase and the NADH peroxidase will be examined by protein chemistry and molecular genetic approaches. There are two major questions concerning the streptococcal alpha- glycerophosphate oxidase. The distinction between hydride- and carbanion-mediated substrate dehydrogenations is an important one, and halogenated analogs of the natural substrate will be used to probe this question in more detail. Secondly, the comparison between this flavoprotein and the FAD-containing anaerobic alpha- glycerophosphate dehydrogenase of E. coli should provide valuable information concerning structural and mechanistic features shared by both.	{ "pile_set_name": "NIH ExPorter" }
Sialoglycoconjugates are cell surface components involved in many aspects of cellular response to extracellular changes and stimuli. Of all the biochemical changes associated with neoplasia, modifications of these components and their biosynthesis have been documented. In order to monitor sialoglycoconjugates in the surface membrane of living cells with the fluorescence microscope and biochemical techniques, we plan to introduce a covalently bonded fluorescent label into the sialic acid moiety. This will be achieved by condensation of dansylhydrazine as fluorophor with the C7-sialic acid aldehyde produced by mild periodate oxidation. Preliminary data show the specifity of the procedure for sialic acids. The fluorophor will be made impenetrable to the membrane by a) complexing with cycloheptaamylose, incorporation into b) polystyrene beads or c) lipid micelles or,d) by synthesis of a hydrophilic, negatively charged derivative. The quantitative nature of the labeling will be evaluated by microspectrofluorometry as well as biochemical analysis. The dansylated products of proteolysis of while cells and of their solubilized surface glycoproteins will be compared to determine their relationship. The procedure will be adapted for electron microscopy by tritium labeling, anti-dansyl-antibody techniques and heavy metal staining of a sulfonated dansylderivative. All the techniques developed will be used to study the mobility of sialoglycoconjugates in the cell surface in response to lectin binding. The technique will allow to monitor and separate cell populations on the basis of their surface sialic acid content and might therefore be useful in flow-microfluorometry as a tool for cancer diagnosis and research.	{ "pile_set_name": "NIH ExPorter" }
One of the most prevalent abnormalities in leukemia patients, the Philadelphia chromosome, a translocation of the ABL1 oncogene on chromosome 9 to the BCR gene on chromosome 22, occurs in adult chronic myelogenous leukemia and childhood acute chronic leukemia. In addition to the translocation, the commercial ES-probe used in clinical cytogenetics laboratories sometimes detects deletions of DNA sequences upstream of the ABL1 gene on the derivative 9 chromosome (in 1/10 to 1/3 of patients). These deletions appear to be prognostic for early blast crisis. We hypothesize that these deletions result in hemizygosity of additional genes adjacent to ABLI. These deletion breakpoints can be refined with high- resolution fluorescence in situ studies (scFISH), which uses short single copy (sc) DNA probes designed from the draft genome sequence. Chromosomal preparations of patient specimens diagnosed with Philadelphia chromosomes in clinical cytogenetic laboratories will be analyzed with these probes. ScFISH, which has been developed in our laboratories, provides an unprecedented level of resolution for delineating sequences associated with inherited chromosomal rearrangements by FISH (Rogan, Cazcarro, Knoll, 2001; Knoll, Cazcarro, Rogan, 2000). We have developed and validated single copy DNA probes (quickly and without cloning) for FISH analysis of more than 20 distinct chromosomal regions. We aim (1) to develop scFISH probes for ABL1 oncogene, BCR and their adjacent regions from the draft genome sequence and verify their locations by FISH; (2) to compare results with scFISH probes to those obtained using the commercially available ES-probes for t(9;22) on the same patient specimens; and (3a) to categorize cytogenetically positive t(9;22) into molecular subgroups based on the sizes of deletion centromeric to ABL1 and the breakpoints within the BCR gene, (3b) to determine if there are preferential sites of breakage on chromosome 9 adjacent to ABL1, and to localize these sites. In contrast with scFISH probes, commercial reagents detecting this translocation routinely used in clinical laboratories comprise large multigenic segments. We anticipate that both the commercial and scFISH probes will detect the chromosomal translocation in some patients, however, only the scFISH probes are expected to delineate deletions in sequences immediately adjacent to ABL 1, and to distinguish the major and minor sites of breakage in the BCR gene that correspond to chronic myelogenous leukemia and acute lymphocytic leukemia, respectively. The proposed study will delineate the chromosomal regions that undergo breakage in these types of leukemia and will establish whether there are common deletion breakage intervals on the translocated chromosome 9. Our long range goal is to classify patients based on the sites of translocation and deletion size, and then to determine if the disease complications can be attributed to the loss of specific genes adjacent to ABL1.	{ "pile_set_name": "NIH ExPorter" }
The Flow Cytometry Shared Resource provides technical expertise, trained personnel, and technical services in flow cytometry to meet the needs of Cancer Center members. The specific services of the Flow Cytometry Shared Resource include 1) quantitative measurement of fluorescent receptors to assess the distribution of specific molecules within cell populations 2) sorting to isolate purified populations of cells based on detection of specific probes such as antibodies or green fluorescent protein. This Shared resource provides analyses using expensive equipment within individual investigators could not afford. The well-trained staff can provide timely analyses. Both the efficiency of the facility and the contribution of CCSG funds reduce investigator costs. Use of this shared resource has increased from 120 hours in 1996 to 892 hours in 1998.	{ "pile_set_name": "NIH ExPorter" }
Premature birth is the number one perinatal health problem in the USA. Relatively little attention has been paid to the function of milk-borne hormones and their potential implications in neonatal health care. Transfer of maternal hormones to the fetus in utero by the placenta ceases with birth; thereafter, milk is the only means of humoral signal transfer from the mother to the newborn. Milk contains hormones and other biologically active substances that may aid the adaptation of the newborn to extrauterine life. This role of milk and the mammary gland may be even more important in prematurely born infants, who normally would still be under placental influences. Based on similarities in their nutritive, immunological, and endocrine functions, the hypothesis has been developed that the placenta and the mammary gland are functionally analogous organs. As an initial objective toward testing this hypothesis, a prolactin(PRL-) secreting cell line has been established from a novel animal model: the short-tailed gray opossum (Monodelphis domestica) as a cost-effective source of species-specific hormone. M. domestica is a marsupial species; thus, it represents a branch of the phylogenetic tree that diverged from the eutherian (placental) mammals 100 million years ago. Marsupials do not have a chorioallantoic placenta and their pups are born at a very early stage of fetal development. The "extrauterine fetus" is readily accessible for experimentation and (by virtue of parallelism between ontogenetic and phylogenetic development) may serve as a model for the milk-related maternal modulation of the developing endocrine system of the premature human infant. The specific aims of the proposal are: 1. To purify opPRL from culture medium conditioned with immortalized opossum pituitary cells (chromatographic fractions will be monitored for PRL-like bioactivity in Nb2 lymphoma assay); 2. To develop a species-specific antibody in rabbits and a homologous radioimmunoassay (RIA) for opPRL; 3. To evaluate immunoreactive opPRL in maternal plasma and milk during lactation and in neonatal plasma during ontogeny, and to investigate the source of milk- borne opPRL by demonstrating transfer of radiolabeled PRL into milk in vivo, and PRL synthesis and secretion by cultured opossum mammary epithelial cells in vitro; and 4. To demonstrate the transfer of metabolically labeled, immunoprecipitable opPRL via milk into the circulation of the offspring during ontogeny. The proposed studies will lead to the development of the first species-specific PRL antiserum in M. domestica, thus providing a valuable experimental tool for pituitary, PRL and mammary gland research in the only laboratory-adapted marsupial species. The experiments will elucidate the sources of milk-borne PRL and will verify if milk is a significant exogenous source of PRL for the neonate/extrauterine fetus.	{ "pile_set_name": "NIH ExPorter" }
The project develops models for analysis of multiple event times. Two projects are considered in this proposal. The first project proposes new methods for regression analysis of a class of stochastic process models called modulated renewal processes. They can be used to model multiple event times representing specific episodes in the disease progression of a patient. In this project we propose extensions of accelerated failure time and transformation models. We also consider parameter estimation in these models. The second project considers matched pair competing risk regression models. Models of this kind arise in biomedical and actuarial follow-up studies. In such studies, matching (or blocking) serves as a method to control for effects of prognostic variables on the variability of the outcomes represented by time and failure type. We propose a class of stochastic process models for analysis of matched pair competing risks. Both projects develop also applications to analysis of follow-up data from bone marrow and kidney transplant studies. We consider prediction of patient post-transplant experience based on pre-transplant characteristics and follow-up history.	{ "pile_set_name": "NIH ExPorter" }
Perennial endemic and potentially pandemic influenza virus infections constitute a major global health problem. It is known that the adaptive CD8 T cell response is vital for clearance of acute influenza virus infection, and that antibodies are important for protection against recurrent infections. Relatively little is understood about how the innate phase of immunity impacts the success of these adaptive components. Natural killer (NK) cells are cytotoxic lymphocytes that respond in the earliest phase of viral infections to control viral spread by direct killing of infected cells, and by secretion of potent cytokines (e.g., interferon-gamma, IFN-g) that enhance the priming of a Th1 adaptive immune response. We have found that depletion of NK cells prior to influenza A virus (IAV) infection results in exacerbated disease, and that these mice may be rescued from early demise by reconstitution of NK cell immunity using adoptively transferred lung NK cells. The long-range goal for this project is to elucidate the molecular mechanisms through which NK cells mediate this protection. The experimental design is one that provides new information about the integrated immune response operating during the early innate and subsequent transitional phases of acute IAV infection-from the recruitment of NK cells into the lungs and draining lymph nodes, through the effector phase of NK cell activation, and ultimately to the establishment of a pulmonary CD8 T cell response. We will exploit novel in vivo and in vitro experimental systems including our NK cell depletion-reconstitution in BALB.B6-CT6 (NK1.1+) mice and ex vivo intact lymph node culture to isolate NK cell responses and functions through three Specific Aims: 1) a determination of the mechanisms of: a) homeostatic NK cell homing to the lungs following adoptive transfer, and b) pulmonary NK cell accumulation during acute IAV infection (proliferation, differential apoptosis, and/or recruitment);2) a determination of the mechanism of NK cell-mediated protection against lethal disease during IAV infection-role of interferon-g, perforin, Fas ligand and TRAIL in control of peak viral load and the virus-specific CD8 T cell response;and 3) a determination of the contributions from proliferation, differential apoptosis, and CXCR3- dependent recruitment to NK cell accumulation in the draining lymph node during acute IAV infection and potential regulation by migratory pulmonary DC and/or macrophages. The results from this project will expand our understanding of the integrated immune response to a major respiratory pathogen, and will impact the design of vaccines in order to exploit the beneficial contributions of NK cells. PUBLIC HEALTH RELEVANCE: Influenza virus infections exact an enormous toll on human populations: seasonal endemic infections result in >35,000 deaths annually in the US, while recurrent pandemic infections constitute a major global threat. The results from this project will enhance our understanding of how innate immunity works to control spread of the acute infection and initiate clearance by the adaptive immune system, and how dysregulation in the innate phase may occur (and be effectively treated) in severe influenza infections. Finally, the findings will impact the design of new vaccination strategies in order to exploit the contributions from natural killer cells.	{ "pile_set_name": "NIH ExPorter" }
The overall objective of the proposed research is to gain insight into the mechanisms by which cariogenic bacteria such as Streptococcus mutans can synergistically and antagonistically interact with other plaque bacteria during dental plaque formation. The specific objectives are: (1) to utilize our quantitative in vitro plaque forming assay to analyze factors involved in heterotypic interactions between oral bacteria; (2) to analyze the interaction and association of the S. mutans dextransucrase with lipids in order to evaluate the role of such molecules in glucan production and adherence; and (3) to continue evaluation of the association between dextranase-producing oral bacteria and S. mutans. The long-term goal is to define interactions involved in regulating the microbial content of plaque which could be used to develop caries control measures based on selective inhibition of specific cariogenic bacteria.	{ "pile_set_name": "NIH ExPorter" }
Categorization is considered a high level visual-perceptual function. It allows individuals to make reasonable guesses based on experience about the function and desirability of a stimulus or object. Monkeys, like humans, can classify whether images blending cats and dogs, so-called morphing from cat to dog are more cat-like or dog-like. In our experiments, monkeys were required to identify whether the morph being presented is closer to a cat or a dog in a sequential two-alternative forced choice task. Single unit recordings in regions of inferior temporal cortex--specifically area TE and rhinal cortex (Rh)--have implicated these regions in late-stage visual processes, such as categorization and stimulus-reward association, respectively. We have previously found that TE-lesioned monkeys are only mildly impaired in a perceptually difficult test of visual categorization (employing dog-cat morphs), and that the performance of Rh-lesioned monkeys is indistinguishable from that of controls. To study how this categorization takes place, our monkeys were trained to touch a bar to initiate a trial, and release the bar during one of two intervals; early (during the presence of a red central target) if they identified the stimulus as more cat-like, or late (following the transition of the central target to green) if the stimulus was more dog-like. Rewards were only delivered for correctly identifying a dog. In summary, if cat, release on red to skip the trial and move on to the next, if dog, release on green to get a reward. We measured the percentage of trials and reaction times when the monkey correctly indicated cat. As the morph level changes from pure cat towards dog, the number of times the monkey indicated a cat decreased sigmoidally. The sigmoid was also shifted toward dog, indicating a bias toward choosing dog - when very unsure, guessing dog prevents missing a potential reward, with a penalty of having to wait through the whole trial for nothing if the stimulus should have been identified as cat. As the morph progresses from all cat to equal cat/dog, the monkeys take longer to make a decision, i.e., the reaction times become longer. We model this behavior as a drift diffusion process. A point starts midway between 2 boundaries, and takes a random walk until it hits a boundary, which represents a decision of cat or dog. The drift velocity depends on the morph level presented. The model has an analytical solution. Here we use the experimentally measured frequency with which the monkey identifies a given morph as dog to determine the drift velocity, which is then used to predict the mean reaction time. We find that for morph levels close to a pure cat the experimental mean reaction times are similar to the theoretical ones. When the morph level is close to a 50:50 mixture, the experimental mean reaction times are faster than the theoretical predictions. It appears that for the easy morph levels the monkey reaches the boundary quickly and decides. For more ambiguous morphs the monkey takes more time to accumulate evidence. If reaching a bound takes too long, the monkey stops accumulating evidence and indicates dog - not wanting to miss a potential reward. The question now is whether there is another process apart from drift diffusion, also stochastic, governing how long the monkey will wait to accumulate evidence before simply terminating the trial by guessing dog. Now, we asked whether decreasing the information available by shortening the stimulus duration would affect any of our study groups: controls, or monkeys with either TE or rhinal removals. We trained the monkeys to touch a bar to initiate a trial, and release the bar during one of two intervals; early (during the presence of a red central target) if they identified the stimulus as more cat-like, or late (following the transition of the central target to green) if the stimulus was more dog-like. A correct response resulted in the delivery of a fixed-size liquid reward, an incorrect response led to a punishment time-out. We presented the morphed stimuli for durations of 25, 50, 100, 250 or 500 ms in an interleaved design. The stimuli appeared on a background of black and white noise. When the stimuli were removed, the background immediately reappeared; the reappearance of the visual noise appeared to mask the after-image. The accuracy with which control monkeys categorized the stimuli decreased as the stimulus presentation became shorter. The reaction times of control monkeys were indistinguishable across the different stimulus durations. Bilateral TE and Rh cortex removals had different effects: TE-lesioned monkeys made more incorrect categorization judgments, but made their decisions as quickly as controls. Rh-lesioned monkeys categorized as accurately as controls, but took longer to respond, that is, their reaction times were longer. The mild impairment in the performance of TE-lesioned monkeys is consistent with our previous results, and may reflect compromised perceptual or associative processes. The slower reaction times of the Rh-lesioned monkeys could be related to the decision process in some as yet undefined manner.	{ "pile_set_name": "NIH ExPorter" }
ABSTRACT Type 2 diabetes is an independent risk factor for the development of heart failure. It contributes to adverse cardiac remodeling and it increases heart failure mortality. While cell therapy offers promise for treating heart failure in nondiabetic patients, the efficacy of cell therapy in diabetic hearts remains uncertain. Our preliminary studies show that diabetes impairs cardiac progenitor cell (CPC) growth, survival and differentiation and that CPCs isolated from diabetic hearts fail to promote myocardial recovery after myocardial infarction (MI). We find that CPCs express Glut1 and that their rate of glucose utilization increases in the presence of high extracellular glucose, independently of insulin. Moreover, CPCs isolated from diabetic mice exhibit a marked and sustained increase in aerobic glycolysis, accompanied by an elevated level of 6-phophofructo-2-kinase/fructose-2,6- bisphosphatase 3?a phosphofructokinase 2 isoform known to sustain high rates of glycolysis. Our studies also show that increased glycolytic activity in diabetic CPCs is associated with lower glucose-derived carbon flux through the pentose phosphate and hexosamine biosynthetic pathways, but higher flux through the glycerolipid biosynthetic pathway. These results indicate that high rates of glycolysis in CPCs alter the activity of ancillary pathways of glucose metabolism, which are important for biosynthetic reactions, maintaining redox balance, and for resistance to stress. That such changes in glucose metabolism affect CPC function is indicated by our observation that increasing glycolysis at the PFK step is sufficient to decrease CPC proliferation in vitro. Informed by these observations, we suspect that diabetic dysfunction in CPCs is, at least in part, caused by a dysregulation of glucose metabolism and that correcting this metabolic defect will be a viable approach to optimize cell therapy for the diabetic heart. We propose to test the hypothesis that diabetes leads to an increase in the glycolytic activity in CPCs, which diminishes flux through key ancillary pathways of glucose metabolism. This metabolic dysregulation decreases CPC proliferation, survival, and secretory activity, and impairs the capacity of CPCs to promote myocardial repair. To test this hypothesis, we will: (1) examine how diabetes affects CPC competence and therapy; (2) elucidate the role of glucose metabolism in mediating CPC dysfunction in diabetes; and (3) determine whether rescuing defects in glucose metabolism improves cell therapy in the diabetic heart. Successful completion of the project will provide new understanding of the metabolic pathways that regulate stem/progenitor cell function and how they affect the outcomes of cell therapy for heart failure. These findings would facilitate the optimization of cell therapy protocols and inform ongoing and future cell therapy trials for the treatment of heart failure in both nondiabetic and diabetic patients.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION (Taken from application abstract): Objective: The proposed project includes development and preliminary evaluation of a novel virtual reality dynamic anatomy (VRDA) teaching tool. The first prototype of the tool will enable a user wearing a see-through, head-mounted display (HMD) to visualize real-time, synthetic 3-D bone anatomy of the knee joint superimposed on a human subject in motion. Subsequent projects will focus on including deformable objects and providing a complete demonstration of the leg. The long term goal is a model of the entire body for demonstrating normal anatomy and musculoskeletal disorders. Relevance: The VRDA tool will have relevance in medical and science education, clinical medicine, and in research of innovative medical interventions. VR presentations of 3-D anatomy also have potential in patient education. Specific aims: 1. To develop, implement, and evaluate a synthetic bone model of knee joint motion using real-time tracking data combined with a kinematic model (constraints) for motion; 2. To evaluate the integration of real and computer-generated 3-D objects, with accurate scaling and proper registration, when using see-through HMDS; 3. To evaluate user interaction with the tool and perform preliminary evaluations of the effectiveness of the VRDA tool; and 4. To demonstrate that the prototype can be extended to include deformable objects. Methods: Data from the Visible Human Project will be used. A synthetic bone model of knee joint motion will be developed, implemented, and evaluated. Methods to scale graphical bones according to anatomical landmarks and to achieve dynamic registration of virtual images of bones with their real counterparts will be developed. The VRDA tool will then be evaluated in a set of user studies involving medical experts and students. Finally, incorporation of elastic string-like models or newer state of the art models of ligaments, tendons, and muscles will be demonstrated to show feasibility of extensibility of the prototype to include deformable objects.	{ "pile_set_name": "NIH ExPorter" }
ABSTRACT Colon cancer (CC) is a clinically and molecularly heterogeneous disease. While the TCGA data has implicated numerous molecular aberrations in cancer etiology and mechanisms, a direct link between genomic events and patient outcomes is lacking. While the TNM (tumor, node, metastasis) staging system is widely utilized and provides prognostic information, CCs show considerable stage-independent variability in outcome indicating that more robust classifiers are needed for prognostic stratification. Prognostic information is critical to guide patient management and surveillance after cancer resection and can inform treatment selection. Using only gene expression data, we identified four consensus molecular subtypes (CMS) of CC with distinct prognoses. We hypothesize that inclusion of additional genomic features will enable more granular molecular subtyping by identifying additional molecular patterns. Toward this objective (Aim 1), we will utilize multi-omics data sets generated from two completed phase III adjuvant chemotherapy trials in CC (NCCTG N0147, NSAPB C-08). We will also develop a supervised prognostic model by integrating comprehensive molecular data with clinicopathological variables and outcome data (Aim 2). Our unique resource for supervised learning is the high-quality survival data from the clinical trial cohorts. We hypothesize that integration of genomic alterations within clinically relevant genes and gene expression levels with clinicopathological variables can improve the prediction of recurrence/survival compared to traditional TNM staging alone. We will include in a step-wise fashion in our training models selected genes and miRNA expression, somatic mutations, minor allele frequencies, somatic copy number alterations as well as CMS and clinical features, to optimize predictive performance. Given that immune and stromal infiltrating cells are well recognized as determinants of prognosis in CC, we propose to characterize tumor immune and stromal markers among distinct CC molecular subtypes and determine their contribution to prognosis (Aim 3). Specifically, we will characterize these transcriptomic markers computationally, and determine whether they can refine molecular subtypes and improve prognostic modeling. Our proposal represents the first comprehensive prediction of CC patient survival using features from both genomic and transcriptomic alterations that will be integrated with immune and stromal markers using state-of-the-art supervised learning approaches. The impact of this work is substantial in that it will identify determinants of recurrence at the molecular pathway level or in the tumor microenvironment, which will help prioritize targets for therapeutic intervention. Furthermore, the outcome of this grant is expected to have practice-changing implications that can further advance the field of precision oncology.	{ "pile_set_name": "NIH ExPorter" }
Background: Three decades of research on end-of-life care in the United States indicate that people who are dying often spend their final days in ICU settings receiving poor quality end-of-life care. This failure to receive desired end-of-life care is attributed, in large part, to communication failures among patients, families and clinicians. In order to improve end-of-life care, interventions that result in improved communication are needed. Long-term Objectives: The long term objectives of this proposal are: 1) to demonstrate the efficacy of a facilitator-assisted interdisciplinary communication intervention in the ICU to improve family and patient outcomes; and 2) to demonstrate the feasibility of making this intervention a routine part of clinical practice in the ICU setting. Specific Aims: The study's specific aims are: 1) to evaluate the efficacy of a facilitator-assisted interdisciplinary communication intervention in the ICU to improve family symptoms of depression, anxiety and post-traumatic stress disorder; 2) to evaluate the efficacy of the intervention on the patient's quality of dying and death as evaluated by both families and nurses; and 3) to evaluate the efficacy of the intervention on processes of care and processes of communication. Study Design: A five year, randomized controlled study at 4 hospitals enrolling 480 patients, 240 assigned to the intervention and 240 assigned to the control group. The intervention includes: 1) in-person interviews by the facilitator with the family prior to the family conference in order to discuss the family's concerns, questions, and communication needs; 2) a pre-conference meeting with the facilitator and the patient's clinicians in which the family's concerns, questions, and communication needs are discussed; 3) facilitator participation in the family conference; and 4) facilitator follow up with the family throughout the ICU stay. The efficacy of the intervention will be measured with quantitative measures collected at enrollment and at three months after the patient's death or discharge from the ICU. We will assess the efficacy of the intervention by comparing scores on questionnaires from the families and clinicians of intervention patients with scores on the questionnaires from the families and clinicians of the control patients who will receive usual care. Significance: This intervention, designed to improve communication and decision-making about end-of-life care in the ICU, offers significant potential benefits for improving patient- and family-centered care for several reasons: 1) communication is an integral component of clinician skill that affects all other aspects of end-of-life care; 2) physicians and nurses in practice do not demonstrate adequate skills for communicating about end-of-life care, especially in the ICU or acute care setting; and 3) preliminary evidence suggests that interventions that have improved communication within the ICU team and between the team and the family have the potential to improve patient and family outcomes. PUBLIC HEALTH RELEVANCE: Twenty percent of deaths in the US occur in or shortly after a stay in the intensive care unit (ICU), a setting in which technologically expensive care is delivered. End-of-life decisions are often made by family members in these settings, but families report experiencing poor communication with clinicians, making these decisions difficult. This proposal addresses the need for improving communication in ICU settings with a randomized trial of an interdisciplinary communication intervention in which a facilitator intervenes to assist and support communication efforts by all stakeholders. The facilitated communication intervention is designed to be easily generalizable to other hospitals and ICUs and, if successful, would improve the quality of care patients and their families receive in the ICU.	{ "pile_set_name": "NIH ExPorter" }
Birth defects occur frequently in humans but occurrence in most cases is sporadic without obvious genetic or environmental etiology. The mouse mutation Disorganization (Ds) is an extraordinary example of a single gene Mendelian trait that has sporadic patterns of occurrence because of its exceptional genetic and developmental features. Ds is one of the rare examples of a trait that is inherited in a true-dominant and gain-of-function manner. Its developmental attributes are even more striking. Ds causes an extraordinary variety of birth defects involving most body parts, but remarkably no two mice are affected in the same way. The defects are always asymmetrical but often show mirror-image symmetry in the affected structure. The nature of the various birth defects suggests both that the Disorganization gene plays a fundamental role in pattern formation and lineage determination during embryonic development, and the nature of the mutation leads to unpredictable anomalies in the normal patterns of development. During the previous funding period, we discovered a new insertion of an ETnII transposable element at the Ds locus, and a BAC with this element and three flanking genes that can cause Disorganization-like birth defects in transgenic mice. However, the location of this ETnll insertion between genes suggests that it did not disrupt coding sequences, but instead may cause chimeric transcripts, it may adversely affect the expression of flanking genes, or the insertion may have disrupted a non-coding functional element. To identify the Ds gene, we propose Specific Aim 1: to complete the comparison between the finished sequence in Ds mice with the corresponding sequence in the DA/Hu strain on which the Ds mutation arose; Specific Aim 2: to test expression patterns (chimeric transcripts and mRNA levels) of the three candidate genes during embryonic development in DS/Ei and DA/Hu embryos; and Specific Aim 3: to test for affected mice in BAC transgenics, ETnll knock-in mutants at the Ds locus in wild-type mice, and the functional consequences of ubiquitous over-expression of each of the three flanking genes in wild-type mice. These systematic studies should lead to identification of the Disorganization gene and to insights into the control of fundamental developmental processes and the ways in which sporadic birth defects arise. [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
The overall goal of this collaborative study is to investigate the transmission dynamics of cryptosporidial infections in children in southern India. In developing countries, cryptosporidiosis is a cause, not only of outbreaks, but also of significant endemic disease and morbidity, resulting in diarrheal disease and long-term consequences such as impaired growth and cognitive function. Although cryptosporidiosis is a cause of immediate and prolonged morbidity, the risk factors that affect acquisition of infection in developing countries are not completely understood. In collaborative studies between the Christian Medical College, Vellore, India and the Tufts-New England Medical Center, we propose to use state-of-the-art epidemiological and molecular methodology to investigate the source and spread of infection to children in this area by assessing risks from the external and domestic environment. In this application, our multidisciplinary team will intensively follow two cohorts of children with protected and unprotected drinking water sources in order to quantify the risk of infection from familial, domestic and environmental sources for a period from weaning and for up to two years in a semi-urban slum area of Vellore, South India, where Cryptosporidium is the most common cause of parasitic diarrhea. This study will determine the time to first cryptosporidial infection in children drinking protected and unprotected water, in the context of maternal antibodies, hygiene, water safety and sanitation practices, as well as potentially transmissible infections in family members, household animals, drinking water and sewage. Molecular information on genotyping will be integrated with clinical and epidemiologic information in a spatial database to build integrated models to assess indicators for infection. The underlying hypothesis is that the time to infection with Cryptosporidium spp. in southern India is dependent on environmental risk factors that can be quantified and modeled. This will be the first prospective study of transmission dynamics of cryptosporidiosis using molecular fingerprinting and geospatial technology. This study has important implications for public health because there are no data from longitudinal studies from developing countries using molecular and geospatial methods to asses the role of familial and environmental risk factors in transmission of cryptosporidiosis in children. These data will provide the basis for the logical development of interventions that are appropriate to the developing country communities in which cryptosporidiosis is common.	{ "pile_set_name": "NIH ExPorter" }
The muscles and bones of the thorax must support many critical functions, some of which may at times conflict. For example, the thorax must stiffen to maintain stability during locomotion, whereas it must expand to bring air into the lungs during inspiration. Yet, to maintain continuous locomotion, animals must breathe and locomote at the same time. An additional function that has been overlooked is reproduction. Animals that have large or many simultaneous offspring have much of the abdominal cavity occupied by offspring, which may interfere with both ventilation and locomotion. Breathing is impaired because the lungs have less room to inflate, whereas the torso may not be able to bend to accommodate locomotion, changing the basic dynamics of movement or resulting in loss of stability. Thus, how do animals carry large offspring, move and breathe at the same time? In this study, I will 1) determine the changes in locomotion (forces and kinematics) for gravid vs. non-gravid females over a range of speeds. 2) Determine the activity pattern of axial muscles during locomotion and whether the activity pattern differs when females are gravid. I will use electromyography to determine whether different muscles are recruited, and/or whether there are differences in timing or duration of activation. 3) Determine the change in ventilation (both while stationary and during locomotion) caused by the gravid condition by both air flow and anatomical means. This study will potentially reveal important design constraints in terrestrial vertebrate evolution, and may improve our understanding of fundamental physiological and biomechanical changes during human pregnancy.	{ "pile_set_name": "NIH ExPorter" }
We propose an integrated, multidisciplinary program of basic and clinical research addressing a central theme: The characterization and therapeutic modulation of the genetic, molecular and cellular attributes of an allogeneic hematopoietic stem cell graft (HSCT) and transplanted host which contribute to the early establishment of innate and adaptive immunity and provide the patient with effective resistance to infections, and the patient's underlying malignancy. The Program Project includes 6 research projects and 5 cores. Project 1 is focused on the early development of NK cells, their acquisition of self tolerance and their recognition of allogeneic normal and malignant hematopoietic cells through activating and unengaged inhibitory KIR receptors. Project 2 will examine the functional characteristics and cytokine modulated activation of recently identified subsets of monocytes, specifically focusing on the attributes of specific types of monocytes that contribute to resistance to invasive Aspergillus infection in immuno-ablated hosts early after transplantation and the capacity of cytokines to selectively stimulate monocytes providing resistance. Project 3 will examine the relative capacities of different types of dendritic cells developing early post transplant to: a) stimulate T cell responses specific for minor alloantigens (GVH) or antigens differentially expressed by leukemic cells (GVL), and b) influence the reconstitution of NK cell populations and, (with Project 1), the repertoire of KIR receptor reactivity in histocompatible hosts. Project 4 proposes the development and evaluation of novel, broadly applicable strategies for rapidly generating donor-derived leukemia-reactive WT1-specific CD8 and CD4 T cells for adoptive cell therapy to treat or prevent relapse. The T cells generated by these strategies will be compared for their activity against leukemic and normal cells both in vitro and in immunodeficient mice bearing leukemic xenografts in vivo. Project 5 proposes the development and evaluation of synthetic heteroclytic analog peptides of predicted and newly identified epitopes of WT1 and JAK2 kinase for use as vaccines to induce or augment leukemia reactive T cell responses in leukemic patients and transplant recipients at responsive stages of immune reconstitution post transplant. Project 6 presents a program of clinical trials evaluating strategies for enhancing early recovery of innate and adaptive immunity post transplant, particularly in older, and HLA disparate recipients of T cell depleted grafts who do not require or receive immunosuppressive agents post transplant to prevent acute or chronic GVHD. These include trials of KGF to promote reconstitution of thymopoiesis and trials of adoptive therapy with donor NK cells or T cells specific for WT1 to treat leukemic relapse or CMV-pp65 specific T cells for persistent CMV infection. We will also evaluate double cord blood grafts in patients lacking 8-10/10 allele matched HSCT donors, focusing on prospective analyses to identify genetic and cellular attributes of the grafts and host that determine the sustained engraftment and establishment of normal hematopoiesis by one of the two grafts. 5 Cores provide: A) HLA and NK receptor genotyping of research specimens; B) evaluation of cells generated for adoptive immunotherapy; C) monitoring of reconstitution post transplant; D) Biostatistics;and E) administrative support and oversight. Relevance to Public Health: This integrated program of research and therapeutics should improve in transplants for hematologic malignancies and elucidate genetic and cellular interactions that contribute to the re-establishment of resistance to infection and disease recurrence.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: Recent trials of monoclonal antibody-based therapy have demonstrated clear anti-tumor activity with minimal toxicity in patients with low grade and follicular B-cell malignancies. In a recently completed trial, approximately 50 percent of patients that had refractory disease or relapsed following standard modalities of treatment responded to therapy with unlabeled chimeric anti-CD20. Although the assumption is that most of this anti-tumor activity is related to antibody dependent cellular cytotoxicity, there is little direct evidence for this mechanism. A growing body of data indicates that transmembrane signaling via CD20 plays a key role in the control of B-cell proliferation and differentiation. The applicants hypothesize that transmembrane signaling via CD20 may be important in the clinical response to anti-CD20 monoclonal antibody therapy. The current study is designed to assess whether anti-CD20 mediates transmembrane signaling in malignant lymphocytes obtained from patients scheduled to undergo anti-CD20 antibody therapy, and to determine whether signaling correlates with clinical response. Malignant lymphocytes will be harvested from patients enrolled in a clinical trial of anti-CD20 moAb therapy prior to the administration of mAb. Harvested lymphocytes will be treated in vitro with anti-CD20 or isotype-matched control antibody. Changes in a variety of parameters at different points along the signaling cascade will be assessed to determine whether transmembrane signaling has taken place, and whether it results in downstream changes. Parameters to be measured include tyrosine phosphorylation, intracellular calcium flux, Bcl-2, Bax and Bcl-XL message and protein expression, cell cycle analysis, proliferation, apoptosis and immunophenotype. Any observed changes will be correlated with clinical response to antibody therapy. The detection of a correlation between antibody-induced signaling and clinical response would supply strong evidence that transmembrane signaling is important clinically, and would impact on the design of the next generation of regimens that utilize this new therapeutic approach.	{ "pile_set_name": "NIH ExPorter" }
Many of our skilled behaviors involve not single actions but sequences of movements. Lesion and imaging studies in humans, in addition to neural recording in sub human primates have demonstrated that the medial motor areas in the frontal cortex are particularly important for the generation of motor sequences. What is less clear is the exact relation between neural activity in the different areas and the spatial and temporal features of sequence performance. Our general thesis is that the more rostral motor areas code for global aspects of sequence production such as serial order or complete sequence specification. Whereas neurons in areas with direct access to motor output such as the SMA, caudal cingulate, and motor cortex reflect sequences by changes in their directional properties. (1) To determine the extent to which the directional properties of cells in the motor cortex reflect aspects of the performance of over-learned motor sequences. The hypothesis is that cells in the motor cortex show a dynamic re-organization of directional properties during sequence production. (2) To determine the relation between neural activity in the SMA and pre-SMA and the spatial and temporal aspects of over-learned sequences. The hypothesis is that global aspects of sequence production will be more commonly coded in pre-SMA, while neural activity in SMA will reflect an interaction between the directional properties of the neurons and sequence parameters. (3) To record the neural activity in the cingulate motor areas during the performance of over-learned sequences. The hypothesis is that cingulate motor areas are an integral component of the cortical system for sequence control. The methods used will be those of standard multi-electrode recording in the target cortical areas of highly trained rhesus monkeys with appropriate instrumentation for behavioral control and monitoring. Our experimental approach differs from other attempts to examine the neural correlates of sequences in several important respects: we will use behavioral tasks which exploit the spatial dimension inherent in sequence performance and we place particular emphasis on changes in the spatial properties of the recorded neurons.	{ "pile_set_name": "NIH ExPorter" }
2-Oxoglutarate (2OG) is a precursor for neurotransmitter glutamate, and an important metabolic intermediate for maintaining the capacity of the Krebs cycle in glutamatergic neurons. 2OG enters neuronal cells through a high affinity uptake system localized to glutamatergic neurons. This uptake system is under negative feedback control by glutamate, biphasic modulation by glutamine (another source for neurotransmitter glutamate) and positive modulation by a putative novel protein, transport activating factor (TAF). TAF accelerates 2OG uptake by several hundred percent. We propose to purify, partially sequence, clone, and raise antibodies to TAF from bovine retina. The results of this investigation will characterize a hormone of significance to neural systems in general but especially to the retina, which has a very high metabolic rate as well as a robust glutamatergic neurotransmission system.	{ "pile_set_name": "NIH ExPorter" }
The Social Relations Model is a model of dyadic processes. A person's behavior is assumed to be determined by the person, the person's partner, and the relationship between the two. The model has been applied to the areas of interpersonal attraction, accuracy of interpersonal perception, family relations, intergroup relations, small group process, animal behavior, interpersonal competence, self-disclosure, stereotypes, personality, self-perception, social influence, jury deliberation, and nonverbal communication. The proposal is to make a number of important extensions of the model. The first set of specific aims concern work that should provide important insights into consensus or agreement. Understanding when and why people agree is a fundamental question in the fields of social psychology, personality, medicine (diagnosis), methodology (rater reliability), and anthropology. The field of social cognition has made important insights into the use of stereotypes which need to be incorporated to the model of consensus. Also the effects of individual differences and relationship closeness on consensus need to be modeled. It is important to make the model simpler to apply. By developing a more economical design and establishing the power of various tests, more investigators should be able to use the model. Work accomplished should make the Social Relations Model more useful to researchers in the social and behavioral sciences.	{ "pile_set_name": "NIH ExPorter" }
There are two developmental objectives in this proposed K02 award: 1) to obtain expertise in qualitative and mixed-methods design to advance the principal investigator's (Pi's) research program on informal and formal long-term care, and 2) to gain knowledge and experience in the development and administration of large-scale, collaborative research initiatives based on the innovation of mixed methods designs. To achieve both objectives, a detailed career development plan is proposed that will offer extensive training in qualitative and mixed-methods research approaches via textbook reading, university-based courses, attendance at institutes, and collaboration with a number of senior colleagues. These activities will allow the candidate to integrate methodological viewpoints and will also provide valuable guidance as he transitions to the administration of research initiatives that rely on multiple R01-level projects and effective mentorship of junior researchers. Research activities based on these training aims will offer the candidate extensive experience in the conduct of complex, mixed methods research in long-term care. Using complementary quantitative and qualitative approaches, the proposed pilot study will: ascertain how various programmatic aspects of adult day service (ADS) programs facilitate the development of strong client-family-staff relationships;determine how ADS program environment and client-family-staff relationships operate independently and in concert to influence outcomes across key stakeholders (i.e., clients, families, and ADS staff);and test and refine a conceptual model of ADS utilization. Quantitative pilot data will be collected from a random sample of 20 ADS programs and 200 elderly ADS clients (65 years of age or over) as well as primary family caregivers and ADS staff persons most closely involved in the supervision and care of each client. The qualitative component will involve in-depth ethnographic research at two ADS sites and 20 client-family-staff constellations embedded in the quantitative component. Case study and thematic analyses will facilitate integration of the qualitative findings with those from the quantitative protocol. This study, an innovative analysis of the process of ADS programmatic and social contexts, will help to develop new conceptual models to aid in the evaluation of ADS and other community-based long-term care services. Moreover, the integrated training and research activities will provide a strong foundation for future, large-scale initiatives revolving around the integration of multiple methodologies in the study of process and outcome trajectories of long-term care utilization.	{ "pile_set_name": "NIH ExPorter" }
This research plan is directed towards improving artificial kidney technology, one of the specific areas of interest to the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases. By the middle of 1977 more than 36,000 patients were being regularly treated for uremia by hemodialysis at an annual cost of $650,000,000. Generally, this treatment required the use of large complicated machines at treatment centers. Sorbent regeneration of dialysate is one approach to substantially reducing the cost of this treatment. In addition, such regeneration lends itself better to the production of a more compact dialyser. In fact, an enzyme-based, sorbent system has been developed commercially (i.e., the REDY system). This system, however, still has significant problems related to the NH4+ produced during the enzymatic hydrolysis of urea. The long term objective of this program is to develop an electrochemical urea oxidation reactor that would be part of a safe, compact and inexpensive system for regeneration of hemodialysate. We envision two alternative approaches to the urea reactor as described below. In either case, however, the reactor would be part of a system composed of charcoal for adsorbing all other nitrogenous waste products and ion exchange resins for reducing the levels of phosphate and K+. In one version of the reactor, urea would be oxidized directly at a catalytic metal electrode in the reactor to produce mostly non-toxic oxygen, nitrogen and water which can then be disposed of readily. In the other version, chloride would be oxidized at the reactor anode to produce hypochlorite which would then completely oxidize the urea. Any unreacted hypochlorite ion would be removed by reduction at the reactor cathode or, if necessary, by adsorption on a charcoal bed. Phase 1 of the program would be devoted largely to selecting the reactor configuration that exhibited the best overall performance. In Phase 2, extensive toxicology tests of treated dialysate would be performed. Reactor design would be optimized and scale-up of the reactor to the proper capacity would be carried out. This would be followed by the construction of a full-size prototype regeneration system. This system would first be tested at the laboratory bench, then with animals and finally it would undergo clinical trials with humans at a kidney treatment center.	{ "pile_set_name": "NIH ExPorter" }
Project summary/abstract This proposal is to synthesize and commercialize Fmoc protected glycoamino acids (GAAs) in large scale. Glycoproteins have been reported to exhibit a pivotal role in processes as diverse as fertilization, neuronal development, hormone activities, immune surveillance, and inflammatory responses. GAAs are the brick to build all glycopeptides and glycoproteins. The synthesis of GAAs requires sophisticated protection/deprotection and activation strategies that is time consuming. Glycoscientists have faced many challenges with obtaining pure compounds in sufficient quantities. Due to glycoscience?s reliance on GAAs, there is an urgent need for a production pipeline to provide these essential building blocks. The goal of our ?Large scale preparation of Glycoamino acids (GAAs) for expanded glycopeptides and glycoproteins synthesis? is to meet this need and commercialize a stable, affordable and diverse supply of GAAs to drive advances in glycoscience. The availability and cost of these GAAs are the keys for advancing glycoscience for many fields of biomedical and materials sciences. In this proposed SBIR Phase I, Chemily will develop the latest synthetic strategy and route to produce the 11 GAAs on 100 g scales. Chemily will also set up and use 10 to 30 L reactors to produce some of the high demanding GAAs on kg scale. Chemily should be able to produce some of the GAAs around $50/g. This will open the access of many synthetic glycopeptides and glycoproteins. In summary, this project should make such a dream come true: scientists can have affordable access to glycopeptides as they have these GAAs building blocks. A stable supply of GAAs will facilitate experiments that advance our understanding of cell surface markers, cell recognition, and protein-carbohydrate interactions. In turn, synthetic glycopeptides offer a unique frontier for research in glycobiology and proteomics as well as for drug discovery & development, drug delivery/targeting, diagnostics development, and biotechnological applications.	{ "pile_set_name": "NIH ExPorter" }
The purpose of this request for renewal is to define the impact of exposure to bioactivated environmental contaminants on a special population: children. Lung disease is the leading cause of death in infants under one year of age and a strong relationship exists between human infant exposure to environmental tobacco smoke, increased respiratory infections in childhood and distal airway disease and asthma in adults. Because most tobacco smoke components acquire the toxicity after metabolism by cytochrome P450 monooxygenases, we have asked how exposure to bioactivated lung toxicants affect neonatal lungs. We have found 1) lungs of neonates are susceptible to acute injury at doses below the threshold for injury in adults and 2) once the injury occurs, bronchiolar development and repair are abnormal and incomplete, even when the animals become adults. In this renewal request we propose to 1) define the mechanisms which render differentiating Clara cells in the immature lungs of neonates more susceptible to injury by bioactivated environmental contaminants and 2) identify the events that result in incomplete bronchiolar repair in injured neonates. We will use a comparative approach to discriminate mechanisms which are unique to a particular chemical class or mammalian species from those which apply to neonates in general. We will compare the injury and repair response of two compounds of different chemical classes and different pathways of activation and detoxification (naphthalene and 4-ipomeanol). We will compare neonates and adults of two species with differing levels of susceptibility as adults to these compound (mice and rabbits). Because we have found that differences in the location of the target epithelial population within the airways alters the injury and repair patterns, we have developed novel methodologies to specifically sample the subpopulations, biochemically analyze the metabolism and mRNA biology in their three-dimensional context. This work will further our understanding of the basic molecular molecular mechanisms by which environmental insults are involved in the origination of lung diseases that begin in childhood.	{ "pile_set_name": "NIH ExPorter" }
This is a new application that proposes to study regulation of chemokine receptor expression by cytokines in experimental granulomas, and to evaluate the contributions of these various receptors to the granulomatous response. The PI previously developed a novel murine model of synchronized T cell-mediated granuloma formation using agarose beads coated with either mycobacterial or schistosomal antigens to elicit the formation of lesions that show type 1 or type 2 cytokine responses, respectively. These studies demonstrated that cytokine and chemokine receptor expression differ in the two distinct granulomata. The experiments proposed here will extend previous studies which examined the contribution of selected cytokines to the development of granulomas in vivo. In Aim 1, flow cytometry and RT-PCR will be used to define the profiles of chemokine receptor expression in leukocyte populations during the course of granuloma development. Aim 2 will characterize granuloma formation in chemokine receptor knockout mice. Aim 3 will attempt to determine the roles of selected cytokines in regulating chemokine receptor expression during granuloma formation, using cytokine knockout mice or anti-cytokine antibodies. Lastly, in Aim 4, the PI will attempt to manipulate granuloma formation in vivo by administering chemokines, and truncated chemokines that function as receptor antagonists, via osmotic pumps.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: The primary goals of this program are: 1. to prepare disadvantaged high school students in biomedical/behavioral science and health professions for pursuit of college science education and careers in the health sciences; and 2. to improve pre-college science education by allowing elementary, middle, and high school teachers to increase their knowledge and skills in modern research tools and techniques, and to transfer this new knowledge to their students. To accomplish these goals, participating students and teachers will: 1. complete a research project in one of the basic science or clinical laboratories at the UCHC or the Veterans Administration Hospital (VAH) in Newington, CT; 2. attend weekly seminars on advanced topics in biomedical sciences or clinical research; and 3. present the results of their research at the close of the program in a symposium attended by the students, teachers, research mentors, other Health Center summer program participants, and other invited faculty and administrators. Participating teachers will be expected to develop a plan for academic-year activities which will foster a continued relationship between the teachers, their research mentors, and the Health Center, which will improve the level of pre-college science education and motivation of students for careers in biomedical research and other health professions. Students will participate in an eight-week series of lectures sponsored by the School of Medicine.	{ "pile_set_name": "NIH ExPorter" }
ADMINISTRATIVE CORE ABSTRACT The Administrative Core for our SDSU/UCSD Cancer Center Comprehensive Partnership will provide oversight of the administrative and financial functions and the personnel and other resources to support the Partnership's day-to-day operations. This includes providing leadership, oversight, and infrastructure to support all scientific, administrative, and fiscal activities of the Partnership to ensure effective and synergistic performance. The leadership of the Administrative Core is crucial to achieving the goals of collaboration within the Partnership. The organizational structure is made up of an Executive Committee (EC), an Internal Advisory Committee (IAC), a Program Steering Committee (PSC), the SDSU and UCSD institutional leadership, as well as faculty, students, and community partners participating through the various Cores and Programs. The EC is comprised of the four PIs, two from each institution. Administrative and scientific leadership is conducted in a collaborative, integrative manner across SDSU and UCSD and takes into account the advice of the IAC and PSC. In the next phase of the Partnership, the Administrative Core will continue to oversee the ongoing coordination and provide administrative support to all the Cores and Projects of the Partnership. The Core will also provide support to foster effective interactions and collaborations amongst SDSU and UCSD investigators, staff, students, advisory committee members, and community members. Specifically, the Core will be responsible for; 1) Convening, coordinating and conducting all meetings, as well as providing agendas and minutes; 2) Facilitating communication between all institutional departments, including with the leadership at both institutions; 3) Providing administrative support and monitoring the progress of all Cores; 4) Working with the Planning and Evaluation Core to solicit, review and select pilot and full projects, as well as monitoring their progress; 5) Encouraging and supporting active participation of SDSU and UCSD faculty and students in Partnership activities; 6) Preparing progress reports for internal and external evaluation, renewals, and summaries; 7) Providing fiscal management; and 8) Organizing and disseminating information to the Partnership community. The Partnership uses the multiple PI model for governance and leadership and the Contact PIs represent and reinforce university commitments. The application proposes to recruit two new faculty at UCSD and two at SDSU. Recruitment efforts at UCSD will be targeted specifically to translational scientists in cancer disparities. At SDSU, the faculty recruits will increase the capacity for cancer research in basic sciences. The new positions will be aligned with the future needs and priorities of the Partnership. If the new recruit is an early stage investigator (ESI), the Partnership and leading institutions are committed to providing support to ensure the future success of this individual, through existing institutional support to support ESIs and activities in the Research Education Core.	{ "pile_set_name": "NIH ExPorter" }
The purpose of this study is to evaluate subclinical abnormalities of glucose metabolism in subjects with previous gestational diabetes and further to study the rate of insulin clearance to determine whether differences in insulin clearance might contribute to abnormalities of glucose metabolism in women with gestational diabetes.	{ "pile_set_name": "NIH ExPorter" }
NKT cells are a population of regulatory T cells that is known to be able to potently modulate immune responses. The antigens that control NKT cell activation are lipids and glycolipids presented by CD1d molecules. A remarkable characteristic of NKT cells is that many are autoreactive to self antigens. This allows them to become activated even when there is no foreign antigenic challenge, and may be critical for their endogenous immunoregulatory effects. The auto-antigens recognized by human NKT cells have not been identified, and little is known about how their presentation is regulated. Here we will: i) characterize the endogenous ligands presented by human CD1d molecules, and analyze which ones are antigenic for NKT cells;ii) follow up on our preliminary results, which indicate that an intercellular lipid messenger called lyso- phosphatidylcholine (LPC) that is produced under inflammatory conditions, is a self antigen that can be recognized by many human NKT cells;iii) investigate the cellular and molecular requirements for CD1d-mediated auto-antigen presentation to NKT cells. These studies will provide new information about the lipid characteristics that are important for binding to human CD1d molecules and for recognition by NKT cells, and will explore how NKT cell activation is linked to broader physiological processes of inflammation through recognition of bio-active lipid mediators as self antigens, and will provide critical insights into the mechanisms by which CD1d- mediated auto-antigen presentation is regulated by APCs. PUBLIC HEALTH RELEVANCE: NKT cells are autoreactive regulatory T cells that recognize lipids and glycolipids as antigens presented by CD1d molecules. The molecular identity of the auto-antigens recognized by human NKT cells remains unknown. In this grant we will: i) identify constitutively presented cellular ligands that are recognized by human NKT cells;ii) investigate lipid messengers as putative inducible auto-antigens that may directly link NKT cell activation to inflammatory responses;and iii) analyze cellular and molecular requirements for auto-antigen presentation by CD1d.	{ "pile_set_name": "NIH ExPorter" }
The long term goal of this research is to develop an efficient method to authenticate rodent cell lines. Misidentification as well as inter- and intra-species cross contamination of cell lines is widespread and has a substantial negative impact on biomedical research. The use of short tandem repeat (STR) analysis has been shown to be an effective means of authenticating human cell lines, STR genetic profiles have been established for a large number of human cell lines, and much of this data is publicly accessible in the ATCC STR Profile Database. Equivalent resources do not currently exist for authentication of rodent cell lines. In order to fill this gap, the specific aims of this application are to develop a multiplex microsatellite marker-based test to generate unique genetic profiles for mouse and rat cell lines and to build a database of these profiles to use for rodent cell line authentication. By developing these resources, it will be possible to provide a cost-effective and accurate method for rodent cell line authentication for the research community. PUBLIC HEALTH RELEVANCE: The use of cell lines is a cornerstone of basic biomedical research and the ability to ensure the authenticity of the cell lines is critical to ensuring the integrity and validity of the research results that guide scientific advances affecting public health. Establishing a method for generating unique genetic profiles for rodent cell lines provides a quick, sensitive, and accurate means to verify the authenticity of those cell lines being used in research laboratories.	{ "pile_set_name": "NIH ExPorter" }
The proposed research represents the initial stages of a research program investigating the potential role of parents' cognitions as mediators of their behavior toward their young children. Based on prior work in social cognition, mothers' attributions (i.e., causal explanations) for their young children's behavior will be investigated. Specifically, mothers' beliefs about possible causes for their infants' easy/difficult temperament behavior will be studied. Two studies are proposed that will address the following specific aims: (a) to identify specific causal attributions made by mothers of 18 to 24 month olds for their infants' behaviors contributing to temperamental easiness/difficultness, (b) to investigate mothers' specific attributions in terms of the causal dimensions that may underlie them, and (c) to examine differences in mothers' attributional patterns as a function of several parent and child variables including parity (primiparous vs. multiparous), age and sex of child, and easy vs. difficult behaviors. The purpose of Study 1 is to identify specific attributions that are representative of mothers' causal explanations for their infants' easy/difficult behavior. This will be done when mothers will generate possible causes for a sampling of 12 infant behaviors (6 "easy" behaviors and 6 "difficult" behaviors) that are described in brief statements and that have been identified in previous research as relevant to parents' notions of easiness/difficultness. Specific attributions provided by mothers will be subjected to a content analysis and will be used in Study 2 for determining causal dimensions that may underlie these explanations. The identification of causal dimensions is sought because attribution theorists view such dimensions as critical to the understanding of subsequent cognitions, affect, and behavior. Thus, in Study 2, a second sample of mothers will be requested to provide attribution ratings (the specific rating scales will be derived from Study 1 based on frequently made attributions) for the same 12 stimulus behaviors presented in Study Principal components analysis will be used to investigate causal dimensions that may underlie mothers' specific attributions. Together, these studies will serve as the foundation for the assessment of parental attributions in future studies designed to investigate relations between such attributions and subsequent cognitions, affect, and behavior.	{ "pile_set_name": "NIH ExPorter" }
The proposed study will attempt to identify and characterize the mechanism of action of cholecystokinin (CCK) on gallbladder and intestinal smooth muscle. The action of CCK will be assessed by measuring its effect on smooth muscle contraction and calcium flux. The effects of CCK on smooth muscle will be compared to the effects of other cholecystokinetic agents. By attempting to discern the mechanism by which dibutyryl cyclic GMP specifically inhibits smooth muscle contraction caused by CCK peptides, we hope to gain information how CCK acts at its receptor. Besides studying the initial steps in the contractile process induced by CCK, we plan to study the persistently stimulated state of smooth muscle contraction caused by CCK. That is, the mechanism by which CCK causes persistent stimulation of smooth muscle contraction after the peptide has been removed from the incubation medium. Finally, we plan to compare the biologic activity and chemical properties of the factor(s) in human serum which causes gallbladder contraction and pancreatic amylase release with those of synthetic CCK octapeptide (CCK8) and purified porcine native CCK (CCK33). By performing these studies we hope to resolve the difference between serum CCK levels measured by radioimmunoassay and those by bioassay and determine whether CCK is the major cholecystokinetic agent in human serum (which has recently been disputed). Studies are planned to examine further our preliminary observations that serum CCK may be bound to a serum protein and to determine if the bound state alters the peptide's biologic activity.	{ "pile_set_name": "NIH ExPorter" }
This grant application describes the proposed total syntheses of a number of potent antitumor agents of natural origin, namely lomaiviticins A and B, cytoskyrin A, amphidinolide N and caribenolide I, as well as a number of related molecules for biological screening. The proposed total synthesis of the lomaiviticins would involve the dimerization of an advanced a-halo enone intermediate to generate the C2-symmetric polycyclic framework. Further elaboration of the sterically crowded central core would then be facilitated by the temporary incorporation of a bridging cyclic tether. Two alternative such tethers have been devised, namely a tetrahydrothiophene and a carbocycle bearing a pendant ester group, increasing the flexibility of the overall strategy. Once the required structural motifs are installed, either of these bridges could be oxidatively cleaved to reveal the necessary functionalities for completion of the target molecules. For cytoskyrin A, a biomimetic cascade process to furnish the cage-like framework of the target molecule has been experimentally demonstrated. Involving the acid-catalyzed dimerization of an anthraquinone derivative followed by a bis-enolization/ double intramolecular Michael addition sequence, careful monitoring of this cascade allows for the selective generation of the central core of not only cytoskyrin A, but also that of a number of other structurally related biologically active natural products. This cascade process would be amenable to the production of not only these natural products themselves, but also to that of specifically targeted analogs, which would be screened for antitumor activity. The proposed syntheses of amphidinolide N and caribenolide I feature a unified, highly convergent strategy involving the sequential asymmetric alkylation of a chiral 1,3-dihydroxyacetone equivalent to establish the complete carbon framework of the target molecules, followed by a highly selective macrolactonization to generate the 26-membered ring in each case. The significance of the proposed work will lie specifically in the area of cancer chemotherapy research, and in the development of new synthetic strategies and technologies for general use in the drug discovery and development process. [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
This application for renewal of the The Advanced IViultlmodai Neuroimaging Training Program seeks continued support for integrated training in neuroimaging through linl<ed Institutional Predoctoral Training and Short-term Research Education components. The goal of the program is to prepare a new cadre of scientists who are focused in the areas of basic and clinical neuroscience and possess the necessary physical science l<nowledge, computational skills, and familiarity with team science to optimally position them for major contributions using and developing the tools of neuroimaging. Designed to facilitate interdisciplinary interactions in neuroimaging through project-based joint mentorship, the program rests on collaboration between faculty members in the Department of Psychology at Harvard University, the Program in Neuroscience at Harvard Medical School, the Harvard-MIT Division of Health Sciences and Technology, the Department of Brain and Cognitive Sciences at MIT, and the Athinoula A. Martinos Center for Biomedical Imaging at Massachusetts Genei:al Hospital. Uniting trainees from diverse backgrounds in a common learning community, this multi-level training program includes predoctoral training as well as a short-term educational program that supports a 2-week Multimodal Imaging Short Course and mentored research training opportunities for individuals at other career stages (e.g., postdoctoral fellows, medical students and residents) who require exposure to and additional education in the technology and application of advanced neuroimaging tools. Through the short course, we are able to extend neuroimaging training beyond the context of traditional graduate-level education. Trainees in the pre-doctoral training program are drawn from Harvard and MIT graduate programs, and carry out research projects jointly supervised by the primary mentor and a co-mentor with complementary expertise. Trainees take additional coursework during the appointment term and attend an AMNTP seminar series and other required programmatic activities. The short-term research education program provides important outreach educational opportunities for scientists at various stages of career development. Central to the research education component is paired mentorship with a senior researcher in the trainee's domain, and opportunities for extended stays to engage in hands-on research experiences. By training these groups to understand the bases of neuroimaging methods and apply them effectively to address the fundamental principles of neuroscience, the ultimate goal of this program is to arm Individuals across the career spectrum with the tools and broad-based knowledge of the fundamental neuroscience and the technologies and analytic methods olin vivo neuroimaging to address crucial questions in neuroscience.	{ "pile_set_name": "NIH ExPorter" }
The Genetics and Molecular Biology (GMB) Training Program at Princeton University has as its primary goal the education of carefully selected individuals for the research, teaching, and industrial needs of this country. The GMB program is the major source of training funds for the Molecular Biology Graduate Program, which has a 53-member training faculty who are currently mentoring 147 graduate students, 98 postdoctoral fellows, and 142 undergraduate majors. We receive approximately 350 applications to our program per year and are successful in attracting some of the best students in the country. Our program is multi-disciplinary and highly collaborative. In addition to 36 training faculty from the Department of Molecular Biology, the Graduate Program includes 5 faculty from Chemistry, 5 from Chemical & Biological Engineering, 3 from Ecology & Evolutionary Biology, 2 from Computer Science, and 2 from Physics. Some of the faculty are also members of the Princeton Neuroscience Institute or the Lewis-Sigler Institute for Integrative Genomics. The faculty provides expertise in a wide range of biological systems and offers training in biochemistry, biophysics, cancer, cell biology, computation & modeling, development, evolution, genetics, genomics, microbiology & virology, neuroscience, and structural biology. Research is performed in well-equipped laboratories with support from state-of-the-art core facilities in the department. The training program consists of formal course work, laboratory rotations, a general exam for PhD candidacy, thesis research, and a diverse array of special activities. In addition, trainees gain teaching experience and receive broad training in responsible conduct of research at different stages of the program. In year one, individual planning is emphasized to help each student select courses and lab rotations that fit his or her intellectual interests and to assist students in identifying an appropriate thesis advisor. We have developed a Diversity Program that has been highly successful in identifying and recruiting under-represented students. As part of our diversity initiative, we have developed mentoring and enrichment activities that benefit all students, and we monitor and assess the progress of students' thesis research throughout their tenure in the program. Evaluation of the program involves faculty and students and helps us to identify the need for new courses, policies, and activities that keep the program fresh. The success of our program is best judged by the success of our graduates, more than 95% of whom are actively engaged in science-related careers.	{ "pile_set_name": "NIH ExPorter" }
In vitro studies with hyperphosphorylated tau from either AD brain or generated in vitro show that this tau binds normal MAPs, disrupts microtubules, and self-assembles into PHFs/SFs (Alonso et al., 1994, 1996, 1997, 2001a, 2001b, 2004). These data provides compelling evidence to propose the following: a) in affected neurons, there is a phosphorylation imbalance;b) hyperphosphorylated tau binds MAPs both to disrupt microtubules and to self-assemble into filaments;c) this microtubule disruption interrupts axonal transport, preventing vesicular transport to the synapse;and d) this interruption results in the slow and steady degeneration of the synapse. Establishing the in vivo relevance of these in vitro results and of different tau phosphorylation sites will provide an explanation of the progression of tauopathies and it will identify new therapeutic targets for neurodegenerative diseases. It is proposed that the abnormal hyperphosphorylation of tau leads to neurofibrillary degeneration by cytoskeletal disruption, and that tau mutations associated with dementia promote their phosphorylation in vivo. The effect of tau hyperphosphorylation upon the cytoskeleton and neurodegeneration in cells in an in vivo-like system, will be investigated as follows: 1) What is the function of tau phosphorylation at positions 199, 212, 231, 235, 262, 396 individually or in combination on tau function, particularly for promoting microtubule activity, for modulating its ability to bind other MAPs, and for its ability to self-assembly? To approach an answer to this question tau phosphorylation at these positions will be mimicked by site directed mutagenesis changing Ser/Thr for Glu at these sites individually or in combination;the pseudophosphorylated forms of tau will be expressed in bacteria and purifyed and their ability to self-assembly, promote and inhibit the promotion of microtubule and its binding to normal tau, MAP1A, MAP1B and MAP2 will be studied. These studies will help understand the influence of tau phosphorylation at different phosphorylation sites. 2) What is the effect of expressing pseudophosphorylated tau in Drosophila's neurons? We plan to use Drosophila as a model system to study effects of peudohyperphosphorylated tau on neurofibrillary degeneration at multiple levels. Transgenic flies expressing pseudohyperphosphorylated tau with Thr212, Ser231, and Ser262 mutated individually or in combination by site-directed mutagenesis to Glu will be generated. The expression will be controlled temporally and spatially so that acute effects on specific type of neurons can be examined and to prevent developmental perturbations. The effect of tau hyperphosphorylation will be examined in axonal transport, neurodegeneration, and learning and memory. To establish parameters for modifier screening, eye degeneration and life-span will also be examined. Some of the proposed studies require little training and can be served as projects for high school and undergraduate students. PUBLIC HEALTH RELEVANCE: The information gained from these studies will provide insight into what different phosphorylation sites on tau are doing in the process of neurofibrillary degeneration, to explain the progression of tauopathies and identify new therapeutic targets for neurodegenerative diseases. The generation of pseudophosphorylated transgenic flies will provide a useful model of neurofibrillary degeneration for future studies, for drug screenings and for genetic modifiers screening.	{ "pile_set_name": "NIH ExPorter" }
The secretion of glucocorticoids (GCs) represents the final step in a neuroendocrine cascade beginning within the CNS. Somatic and psychogenic stressors, as well as circadian drive, initiate the cascade by releasing hypothalamic ACTH-secretagogues. A major advance in recent years in the recognition that CRF is only the principal of numerous secretagogues, icnluding vasopressin (AVP), oxytocin (OT) and catecholamines. Superimposed upon this complex regulatory system are the negative feedback actions of GCs, acting at both adenohypophysial and CNS sites. The demonstration of dexamethasone (DEX) resistance and hypercortisolism in depression and Alzheimer's disease (AD) was exciting for biological psychiatry, for these neuroendocrine abnormalities testifed to the biological underpinnings of such disorders. Furthermore, that only some patients had adrenocortical abnormalities gave credence to ideas of heterogeneity to affective disorders, and promised a diagnostic tool for sub-typing depressive individuals; however, some of this optimism has waned. While the endocrine abnormalities of DEX resistance and hypercortisolism still testify to the biological reality of affective disorders and of AD, it is not clear yet precisely what that biological reality is. Using various rat models of glucocorticoid hypersecretion and feedback resistance, this proposal addresses the broad issue: What are the mechanisms by which feedback regulation of the adrenocortical axis can fail at the CNS level? Knowledge concerning the anatomical locus of the defect underlying hypersecretion is slowly accumulating and implicate a CNS site of dysfunction. Two particularly interesting regions in this regard are the hippocampus and hypothalamus. We will focus our studies on the putative role of these structures of GC-mediated feedback inhibition of ACTH-secretagogue release, and attendant dysregulatory syndromes resulting from disruption of these structures. Specifically, we will investigate: (1) which ACTH-secretagogues are hypersecreted following destruction of the hippocampus or fornix, (2) the relationship between hippocampal or hypothalamic corticosteroid receptor occupancy and hypophysiotropic factor release, and (3) the nature of alterations in ACTH-secretagogue profile following up- or down-regulation of hippocampal or hypothalamic corticosteroid receptors.	{ "pile_set_name": "NIH ExPorter" }
The incidence of HIV-associated tuberculosis has been increasing worldwide since the beginning of the AIDS epidemic, and is expected to rise even further in the future, especially in developing countries. The accelerating and amplifying influence of HIV infection is contributing to the increasing incidence of disease caused by multidrug-resistant strains of Mycobacterium tuberculosis. Development of new drugs against tuberculosis is thus important for control of both of the infections. Mycobacterial cell wall is an attractive target for rational drug design against tuberculosis, due to the fact that it forms a protective, almost impermeable barrier, on the surface of mycobacteria. Some of the most effective drugs currently used for the treatment of TB affect components of its backbone - mycolylarabinogalactan-peptidoglycan (mAGP) complex. Our long-term goal is to identify processes and enzymes involved in the mAGP assembly. Possible AG biosynthetic gene cluster has been recently identified in the genome of M. tuberculosis and thus the specific aims of this grant proposal are: 1. Identify the genes involved in mycobacterial galactan biosynthesis within AG biosynthetic cluster and determine their biological functions via cloning, overexpression and subsequent biochemical characterization. 2. Establish the function of the putative ABC transporter within the AG biosynthetic cluster by way of preparation and phenotypic characterization of the mutants/conditional mutants. The approach will help define one of the more complex pathways in microbial biochemistry and reveal reactions that should be exploitable for drug development. The research will be primarily carried out at Comenius University, Faculty of Natural Sciences in Bratislava, Slovakia in collaboration with Katarina Mikusova, as an extension of the NIH grant A1-18357.	{ "pile_set_name": "NIH ExPorter" }
The mission of COEC is to improve the public's knowledge of how to reduce the risk and individual susceptibility to environmental factors influencing human health and disease. To fulfill our mission, we will engage in this work: Develop educational materials that communicate the significance and public health impact of EHSC research to the general public, community educators, and public health professionals. Serve a broader audience by strengthening relationships and establishing new partnerships with external organizations. Utilize web technology to promote EHSC research and educate the public.	{ "pile_set_name": "NIH ExPorter" }
Studies are directed toward understanding the mechanisms by which estrogens are formed in peripheral (non-endocrine) tissues. Specifically, models are being sought in which the increased efficiency of estrogen production associated with obesity may be explained. Other studies are directed towards defining the role that binding of serum estrogens to sex hormone binding globulin (SBG) plays in limiting action. In particular, we are studying the consequence of reduced SBG levels in amplifying estrogen effects on target tissues.	{ "pile_set_name": "NIH ExPorter" }
Death of spinal motor neurons is a hallmark of devastating and currently untreatable neurodegenerative diseases. However, little is currently known about the reasons why motor neurons are particularly sensitive to mutations affecting broadly expressed genes, such as SOD1 in amyotrophic lateral sclerosis or SMN in spinal muscular atrophy. It is assumed that cell intrinsic differences in motor neurons underlie the disease susceptibility, but nature of such modifiers on neuronal survival is currently poorly understood. Here we propose to study a recently generated mutation of a mir-17~92 cluster of micro RNAs. The deletion of this cluster results in a striking loss of limb muscle innervating spinal motor neurons, while other spinal neuronal classes appear unaffected. We propose to: first, study the cellular pathology of mir-17~92 null animals to determine whether any other cells besides motor neurons are dying in mir-17~92 null spinal cords and whether individual motor neuron subtypes exhibit different degree of degeneration. We will examine whether motor neuron cell death is due to cell autonomous function of miRNAs or due to the loss of miRNAs in other cells that might impinge on motor neuron survival in a non-cell autonomous fashion. Finally, we will determine whether miRNAs are required both in progenitors and postmitotic neurons to exert their pro-survival function. Second, we propose to dissect which specific miRNAs from the mir-17~92 cluster are involved in motor neuron death and which genes are deregulated in the absence of the miRNAs. We will test whether previously identified pro-apoptotic targets are involved in motor neuron degeneration. Furthermore, we will perform unbiased expression screen and test the function of selected identified biochemical pathways in motor neuron survival. As a lead into future studies we propose to determine whether overexpression of the miRNA cluster might save motor neurons from naturally occurring programmed cell death. We expect that these studies will define cellular pathologies in the developing spinal cord connected to the loss of mir-17 ~ 92 clusters and identify relevant molecular pathways linking the miRNAs and motor neuron survival. Understanding miRNA controlled cell type specific survival pathways might provide new targets for slowing down or arresting progression of motor neuron loss in motor neuron diseases.	{ "pile_set_name": "NIH ExPorter" }
1. To evaluate promising lead compounds for treating drug dependence through a broad panel of receptor and enzyme targets (Profile) in order to identify potential side effect liabilities and/or characterize the selectivity of the compounds for the desired pharmacological target set (Task 1). 2. To perform binding affinity and/or functional activity assessments of active compounds (Task 2). 3. To evaluate test compounds in a set of toxicological and pharmacokinetic assays in order to judge their potential for development as future therapeutic agents. These compounds and assays will be evaluated under Task 3. 4. Large compound libraries will be screened against a single target. (Task 4).	{ "pile_set_name": "NIH ExPorter" }
The cytochromes P-450 are central to the metabolism of xenobiotics and drugs and to the activation and detoxification of carcinogens. Whether a potentially carcinogenic molecule is metabolized to an active carcinogen or to an excretable form may depend on the activity and substrate specificity of the forms of the P-450s which predominate in the target tissue. As a result of this, susceptibility to cancer induction may well depend on the regulation of expression of the genes for specific cytochromes P-450. We are using recombinant DNA techniques and other molecular biological methodologies to study the structure and the regulation of the genes for the P-450s. We have constructed cDNA clones complementary to methylcholanthrene(MC)-induced P-450 mRNA. We have used these clones to quantitate MC-P-450 mRNA levels in control and MC-induced rat liver and to isolate a complete, native MC-P-450 gene. The structure of this gene is now being analyzed and the regulatory regions of this gene will be identified and characterized both structurally and functionally. This gene will also be used to isolate related P-450 genes.	{ "pile_set_name": "NIH ExPorter" }
The Committee on Nutrition of the American Academy of Pediatrics suggests that a logical goal for nutritional support of the LBW infant is to achieve postnatal growth approximating that of a normal fetus of the same post-conceptional age. To date, attempts to achieve sufficient catch-up growth in the LBW infant to result, at discharge, in the infant's weighing the same and having the same body composition as a fetus of comparable post-conceptional age have failed. Our previous studies suggest that this is because the relatively high protein intake required is not utilized completely unless accompanied by an energy intake that results in excessive fat deposition. We, therefore, propose to evaluate a strategy for enhancing accretion of lean body mass without excessive fat accretion. Specifically, we will test the hypothesis that a high-carbohydrate diet relative to an isocaloric high-fat diet will enhance protein utilization but may exert undesirable effects on cardiorespiratory function. To test this general hypothesis, we will examine systematically the differential effects of high-carbohydrate and high-fat energy intakes on weight gain, nitrogen retention, ratio of tryptophan to concentrations of large neutral amino acids, sleep state, substrate utilization, energy expenditure and cardiorespiratory functions of enterally fed, growing LBW infants (Birth weight, 750-1600 g). All infants will receive a protein intake of 4.0 g/kg.d; concomitant energy intake will be either 120 kcal/kg.d (non-protein energy 65% carbohydrate and 35% fat, 35% carbohydrate and 65% fat or 50% carbohydrate and 50% fat) or 150 kcal/kg.d (non-protein energy 65% carbohydrate and 35% fat or 35% carbohydrate and 65% fat). The question whether fat and carbohydrate are equivalent sources of energy is not only important for the nutritional management of LBW infants but may also be relevant to their neurobehavioral development.	{ "pile_set_name": "NIH ExPorter" }
Urinary incontinence is a major health care problem in the United States and an area of high priority for NIDDK. This DK55387 competitive renewal grant will explore several new developments using muscle derived stem cells (MDSC) as a treatment of stress urinary incontinence. We were extremely productive during the initial ROl grant funding period and we would like to thank NIDDK for their support. All key objectives of the previous grant were successfully completed. This resulted in 7 peer review papers, 3 more manuscripts are in press and 3 are near completion and will be submitted. Our findings were publicized at 16 international meetings and through 24 submitted abstracts. As a result of our work, we won 3 international contests and submitted 3 patents. A NTDDK Ki 2 Physician Scientist fellow, 3 PhDs. 2 Ph.D. candidates and 3 medical students entering urology worked on our project. Among them are 3 women (1 African American) and 2 African American men. [unreadable] [unreadable] What questions have been left unresolved? The experiments during the present grant identified new issues. We have evidence that bladder injection of MDSC rather than myoblasts persist in the bladder up to 6 months. MDSC can differentiate into smooth muscle. Most importantly, MDSC were able to improve the contractility of damaged bladder muscle while myoblasts were not. In the renewal grant, we want to investigate several important issues, such as: 1. Will MDSC injection improve function in a damaged urethral sphincter? 2. What is the potential for MDSC to differentiate into neurons and improve urologic function? 3. If MDSC becomes neurons, what neurons do they become, afferent, sympathetic, and/or parasympathetic? 4. Do MDSC become neurons in normal conditions or under conditions of acute or chronic stress and neuropathy? [unreadable] [unreadable] The Key Aims of this grant include: 1. Evaluation of the long-term safety and persistence of allogenic MDSC versus myoblasts urethral injection, 2. Measure urethral MDSC injection to improve sphincter function by assessing leak point pressure (LPP) and urethral strip contractility, 3. Assessment of MDSC ability to improve peripheral nerve functions and differentiate into neurons, and 4. Isolation, purification, and proliferation of human MDSC that would be suitable for clinical trial. We want to strongly emphasize that our stem cell research is in complete compliance with the federal guideline on embryonic stem cell research. We want to underscore that these stem cells have not been obtained from embryos (animal or human) or cell lines of embryonic stem cells.	{ "pile_set_name": "NIH ExPorter" }
Atherosclerosis, the underlying cause of most morbidity and mortality in the Western world, results from a focal imbalance of the normal equilibria of the artery wall. While metabolic cooperation between vascular cells is essential for the maintenance of normal artery, very little is known about the nature of cellular interactions or how they are disturbed during the recruitment of monocyte-macrophages during atherogenesis. Failure of endothelial-mediated arterial relaxation is a prominent example of communication dysfunction in hypercholesterolemia and atherosclerosis. We will examine the hypothesis that compromised gap junctional (Gj) communication in hypercholesterolemia is responsible for inhibition of vasoregulation in atherogenesis. Direct cell-cell communication occurs via Gj communicating channels through which small metabolites and ions can pass between the cytoplasmic compartments of adjacent cells. Northern blot and riboprobe hybridization analyses have demonstrated that both vascular endothelial and smooth muscle cells in tissue culture express mRNA for the Gj protein connexin 43 (cxn43). In this competing renewal, we will investigate vascular endothelial and smooth muscle cell expression of Gj proteins in situ and in vitro as a function of atherogenic processes. Lesions induced during experimental hypercholesterolemia in rabbit and baboon arteries as well as fully developed atherosclerosis in human endarterectomy tissue will be probed for Gj protein transcription and translation by in situ hybridization and immunocytochemical techniques. The spatial and temporal relationships between expression in resident endothelial and smooth muscle cells, infiltrating plasma cells, and lipid filled foam cells will be studied as a function of hypercholesterolemia. In parallel in vitro studies, the influence of altered lipid environment upon vascular cells will be evaluated in terms of RNA, protein expression and functional communication (dye transfer and electrical coupling). The effects of atherogenesis and associated changes in Gj expression and upon direct electrical coupling of vascular cells will be evaluated . A recent novel finding is that lipid-filled macrophage foam cells in human atherosclerotic lesions express mRNA for cxn43 whereas the monocytes from which they are derived do not. The proposed research will delineate the mechanisms associated with vascular cells and macrophage induction of cxn43 with particular emphasis upon the cytokine environment, the role of intracellular cholesterol accumulation, and the transcriptional, translational and signal transduction mechanisms involved. These studies will address a poorly understood mechanism of vascular cell communication at the tissue, cell and molecular level as a function of atherogenesis.	{ "pile_set_name": "NIH ExPorter" }
It is widely thought that the exposure of chondrogenic factors to bone marrow progenitor cells in cartilage lesions can enhance the synthesis of a repair tissue. This proposal is based on the hypotheses that gene transfer can be used as a means to achieve sustained synthesis of specific proteins within a cartilaginous lesion, and that this can be used to augment the differentiation of mesenchymal stem cells toward chondrogenesis in vivo. We have recently found that a natural clot created from freshly coagulated bone marrow aspirate forms a simple, but surprisingly effective, matrix for delivery of gene transfer vectors and genetically modified cells to osteochondral defects. The matrix formed is completely native to the donor and part of the natural reparative process. From our preliminary results we have found that in culture mesenchymal stem cells within coagulated bone marrow aspirates will undergo differentiation into cartilaginous tissue under the proper cytokine stimulation. Furthermore exogenous transgenes seeded into the clot as vectors or genetically modified MSCs will be expressed for several weeks. The experimental scheme of the proposed study is designed to broaden our knowledge of the role of protein factors in the process of chondrogenesis while laying the foundation for development of gene- and stem cell-based approaches to improve the natural repair of cartilage lesions. For this we will address the following Specific Aims: (1) to determine the appropriate vector for delivery of chondrogenic transgenes to MSCs; (2) to evaluate the ability of candidate transgenes to stimulate MSCs toward chondrogenesis within a pellet culture system; (3) to optimize the delivery of chondrogenic genes to bone marrow clots in vitro; (4) to characterize patterns of transgene expression in vivo following implantation of genetically modified bone marrow clots in osteochondral defects; (5) to evaluate the capacity of genetically modified bone marrow clots to induce chondrogenesis in osteochondral defects in vivo; and (6) to elucidate the biological effects of long-term expression of chondrogenic transgenes within the joint.	{ "pile_set_name": "NIH ExPorter" }
The broad aim of this proposal is to elucidate the molecular details of an unusual type of non-Mendelian inheritance in Paramecium tetraurelia. We will combine molecular biology and genetics to study a mutant cell line (d48) that contains a complete copy of the A type variable surface antigen gene in the micronucleus but not in the macronucleus. Wild type cytoplasm allows the incorporation of these sequences into the macronucleus, but in d48 cytoplasm the chromosome is broken incorrectly and the A gene is deleted. Recent experiments have shown that macronuclear transformation of d48 with a recombinant clone containing the A antigen gene (pSA14SB) leads to the correct processing of the micronuclear A gene sequences during the formation of the next macronucleus (K. Aufderheide, personal communication). By microinjecting specific fragments of pSA14SB into the d48 macronucleus we will define the DNA sequences necessary for the correct processing of the A gene. Further experiments will be performed to determine if RNA products from these sequences are important for their function. Micronuclear DNA from the d48 and wild type cell lines will be isolated and used to compare the organization of the micronuclear sequences surrounding the chromosome breakage sites. Additional non-Mendelian mutants have recently been isolated that affect expression of the B surface antigen gene (J. Preer, personal communication). We will clone the B gene and determine if similar molecular mechanisms are involved in these mutations. We believe this research will advance the understanding of developmentally controlled DNA rearrangements in eukaryotes, and perhaps reveal novel aspects of nucleic acid processing.	{ "pile_set_name": "NIH ExPorter" }
The activity of calcium-activated neutral proteases (CANP) and acid proteases has been studied in the squid nervous system as well as the rat erythrocyte ghost. In the squid, nerve cell bodies and axons contain abundant CANP activity, whereas nerve terminals (i.e., optic lobe synaptosomes) do not. In contrast, the axon contains no detectable acid (lysosomal) protease whereas the cell bodies and nerve terminals contain significant levels of these acid hydrolases. Ethanol appeared to increase acid protease activity in the squid nerve terminals (synaptosomes). CANP acitivty was also studied in erythrocyte ghosts isolated from normal and alcohol treated rats. Ghosts prepared from alcohol dependent-intoxicated animals contained more than twice as much CANP activity as control erythrocyte ghosts. Ghosts from acute-intoxicated, and withdrawing animals were about 50% greater in CANP activity. These studies provide information on the role of calcium-activated proteases in nervous tissue and erythrocyte ghosts, and on the effects of alcohol on these proteases. The studies also suggest that alcohol may alter erythrocyte and nervous system proteins by increasing the activity of calcium activated and acid proteases.	{ "pile_set_name": "NIH ExPorter" }
Multiple treatments that can change the course of type 1 diabetes have been identified over the last several years through the efforts of TrialNet, the Immune Tolerance Network and others. Although these immune therapies can stop disease progression in those with elevated blood sugars, the effect has proven to be transient in most study subjects. The ongoing effort of Type 1 Diabetes TrialNet, its Clinical Centers and affiliates is to continue to develop better therapies to prevent and reverse type 1 diabetes in human subjects. The Barbara Davis Center and its affiliate network has been a central contributor to these efforts from the beginning of DPT-1 through TrialNet and we hope with renewal of our Clinical Center to be able to continue to push towards these aims as well as develop the next generation of physician-scientists who can build on the work accomplished so far. Our aim is not only to conduct excellent clinical research which can answer the main study questions being examined: most often metabolic - development of impaired glucose tolerance, overt diabetes or reduced C-peptide, but also to develop immune biomarkers which can help us better understand the pathogenesis of disease progression and identify secondary markers of responder groups that allow us to better tailor our therapies to achieve successful prevention of T1D.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: (Adapted from the application.) The genes encoding U2 small nuclear RNA (snRNA) exist as a perfect tandem array of 5 to >22 copies of a 5.8 KB repeat unit on human chromosome 17q21. This locus is a fragile site which may be induced by adenovirus 12 infection. Further, the locus has been maintained at its present site for >35 Myr through multiple speciation events in the primate lineage. The Principal Investigator proposes a multi-faceted approach to understanding adenovirus-induced chromosome fragility. Six specific aims include: (1) U2 chromosomal sequences required for fragile site induction will be identified and analyzed; (2) The chromosomal functions required for adeno 12 virus fragility will be identified, including requirements for snRNA transcription, chromatin structure, methylation, topoisomerase binding, and the origin and scheduling of replication; (3) The requirement for a high concentration of transcription units for the existence of a fragile site will be determined; (4) The evolutionary history of this primate locus will be traced. DNA sequencing from a variety of primates, as well as human groups, will be performed to compare and investigate mechanisms of inheritance and linkage; (5) The influence of U2 repeat sequences on the rate, boundaries, and stability of gene amplification will be investigated; and (6) The domains and functions of the adenovirus 12 E1B 55 kDa protein which are required for inducing chromosome fragility will be characterized.	{ "pile_set_name": "NIH ExPorter" }
Evaluation of cochlear prostheses for deafness.	{ "pile_set_name": "NIH ExPorter" }
The link between electrical excitation and contraction of a skeletal muscle cell (e-c coupling) involves signaling between two calcium channels: the dihydropyridine receptor (Oqs-DHPR) which is a voltage-gated Ca 2+ channel in the plasma membrane and ryanodine receptor type 1 (RyR1) which is a Ca 2+ release channel in the sarcoplasmic reticulum (SR). The long-term objective of this proposal is to understand the molecular basis for signaling between RyR1 and alpha1s- DHPR. The basic experimental approach will be to analyze the functional consequences of expressing cDNAs encoding DHPRs or RyRs in cultured muscle cells (myotubes) obtained from animals genetically null for one or more muscle proteins: dysgenic (alpha1s-DHPR null), dyspedic (RyR1 null), double knockout (null for both alpha1s DHPR and RyR1) and homerless (null for homer 1,2 and 3). The proposal has four specific aims. The first aim is to quantify "retrograde" signaling, whereby the RyR regulates entry of extracellular Ca 2+, for chimeric and mutant RyRs and to probe the functional importance of this entry. The second aim is to characterize the role of the accessory proteins homer and calmodulin in excitation-contraction coupling and in retrograde signaling. The third aim is to determine whether fluorescence energy resonance transfer (FRET) occurs between fluorescent protein-tagged alpha1-DHPRs assembled into plasma membranes at junctions with the SR and if it does to determine whether the FRET efficiency is affected by pharmacological manipulations of RyR1. The fourth aim is to explore interactions between DHPRs and RyRs in double knock-out (null for both alpha1s DHPR and RyR1), including a determination of whether skeletal-type e-c coupling can occur after expression in double knock-out myotubes of (alpha1c-DHPR and RyR2, the DHPR and RyR isoforms predominant in cardiac muscle. In addition to providing new insights into a basic muscle function, the proposed experiments will also provide knowledge essential for understanding the inherited human muscle diseases hypokalemic periodic paralysis (caused by mutations of alpha1s-DHPR) and malignant hyperthermia and central core disease (caused by mutations of RyR1).	{ "pile_set_name": "NIH ExPorter" }
Priming is a form of implicit memory, and refers to the fact that the mere processing of an item can facilitate subsequent processing of that same item. Priming effects can occur in the absence of conscious recollection of the prior study episode, and it has, therefore, been suggested that priming and explicit memory are mediated by different memory systems. Evidence from memory disordered patients is critical for this hypothesis, since if it is possible to link the breakdown of explicit memory and priming to damage to different brain regions this would strengthen the independent memory systems hypothesis. Many studies have found normal priming effects in amnesic patients and some investigators have reported impaired priming in patients with Alzheimer's disease. However, many difficulties are associated with evaluating priming effects in memory impaired subjects. The priming measures may be less sensitive than explicit memory measures, and the lack of group differences may simply reflect low measurement sensitivity. The degree of priming obtained in a given task is related to processing efficiency (i.e. baseline performance), with less efficient processing resulting in greater priming. Therefore, if patients have even mild processing deficits this may mask priming impairments. The proposed project consists of a series of experiments designed to manipulate baseline performance level and sensitivity of the priming measures. Normal control subjects and three groups of patients will be investigated; patients with probable Alzheimer's disease, patients with Huntington's disease, and amnesic patients. These patient groups differ in the severity of their explicit memory defects, and in the extent to which lexical/perceptual processing efficiency is compromised. Younger normal subjects will also be included in the study. Therefore, it will be possible to determine if the decline in memory function resulting from amnesia or dementia is qualitatively similar to the decline in function associated with the normal aging process. In addition to the behavioral measures, measures of volume loss in specific brain structures will be obtained from MRI. These brain measures will be related to the behavioral indices, using a multiple regression approach, to determine the specific role of each brain region in perceptual/lexical processing, explicit memory, and strength of priming. Specific hypotheses about the relationship between the behavioral and structural brain measures derived from our previous work will be tested.	{ "pile_set_name": "NIH ExPorter" }
Trichohyalin is a major differentiation product of the inner root sheath cells of hair follicle, the medulla of the hair fiber, and is expressed in the epidermis, and other tissues including the hard palate, and filiform ridges of the tongue. It is thought to function in part as a keratin intermediate filament associated protein in these tissues. Trichohyalin is a substrate for transglutaminases (TGases), which crosslink it into polymers, and for the enzyme peptidylarginine deiminase (PAD) which converts protein-bound arginines to citrullines. We have expressed in bacteria domain 8 (representing about 40%) of human trichohyalin, and used it to study these two postsynthetic modification events. PAD converts 60% of the arginines to citrullines, resulting in the complete loss of its a-helical structure. Trichohyalin is used by all three TGases known to be present in the epidermis, but by calculation of kinetic parameters, TGase 3 enzyme uses it most efficiently. The kcat for the TGase 3 enzyme increases by a factor of about 10 following PAD modification. These data suggest that trichohyalin is first modified by PAD before crosslinking. We have explored the regulation of expression of the human trichohyalin gene. Constructs containing 3 kbp of upstream sequences coupled with a b-galactosidase reporter can confer expression in transgenic mice in the tongue and palate, but not epidermis or hair follicle. The proximal promoter of the gene exists in the first 135 bp above the transcription start site, and consists of an AP1 element which confers keratinocyte specific expression, and contains a calcium responsive element.	{ "pile_set_name": "NIH ExPorter" }
Inner ear hair cell loss and lack of hair cell regeneration are the major cause of permanent hearing loss. In this application, it is proposed to test whether mouse embryonic stem cell-generated hair cell- like cells are functional in the sense that they display mechanotranduction currents, appropriate basolateral currents, as well as the ability to form appropriate synaptic connections. It is further planned to develop an in utero cell-replacement treatment to determine correlation between occurrence of graft-derived hair cells and regionalized restoration of the organ of Corti. Physiologically, we expect that such a restoration may lead to alleviation of hair cell loss and hearing impairment in a mouse model. A second Aim addresses the guidance of embryonic stem cells toward hair cell-like cells. It is proposed to devise a protocol of defined inductive steps, which will enable researchers to efficiently generate progenitor cells from embryonic stem cells that are competent to develop along the otic lineage. A third Aim proposes to identify a non-FGF-based activity that is involved in otic induction, which is released from a region of the chicken embryo that is adjacent to the otic placode. PUBLIC HEALTH RELEVANCE Toward finding a treatment for hearing loss, it is proposed to continue research on converting mouse embryonic stem cells into hair cells. First, it is planned to assess whether stem cell-generated hair cell-like cells display the same functional properties as wild type hair cells, particularly cochlear hair cells. Building on this, it is planned to use embryonic stem cell-derived inner ear progenitor cells to treat a mouse model of hereditary deafness and to test whether hearing loss can be alleviated with a cellular treatment. In a parallel Aim, it is proposed to increase the efficacy by which embryonic stem cells can be primed to become responsive to inner ear inducing signals. This will be done by mimicking the inductive steps that lead to establishment of the inner ear during embryonic development. In the third Aim, it is planned to identify a signaling molecule from chicken embryonic tissue that appears to be conducting such a priming, which makes progenitor or stem cells responsive to FGF-based inner ear inducing activity.	{ "pile_set_name": "NIH ExPorter" }
Human biology depends on the way the human genome is expressed. Protein levels in cells rely on the fate of messenger RNA-how pre-mRNAs are spliced, how and when mRNAs are translated, and finally when mRNAs are degraded. Defects in these steps can lead to diseases ranging from inherited disorders to cancer. By their nature as RNA polymers, pre-mRNAs and mRNAs may contain secondary and tertiary structural elements that serve as regulators of mRNA abundance and protein synthesis. Despite the central importance of mRNA regulation in biology, there has not been a systems-level study of how pre-mRNA and mRNA structure controls mRNA fate in living cells. The Center for RNA Systems Biology will use new methods to establish a fundamental basis for understanding and predicting the control of mRNA fate due to RNA structure embedded in pre-mRNA and mRNA sequences. The Center will combine new in vivo chemical probing methods with control of the physical environment of cells to address the following Specific Aims: 1) Determine the roles of RNA structure in pre-mRNAs in controlling alternative splicing and their Relationship to human genetic variation. 2) Define mRNA structures that control translation initiation and protein synthesis in response to a cell's physical environment. 3) Map RNA structural regulation of miRNA-mediated turnover. Ultimately the goal of the Center is to develop maps of relationships between the placement of RNA structure in pre-mRNA or mRNA sequences and mRNA fate. These maps will provide many new insights into human biology and the mechanisms underlying genotypic variation and human disease.	{ "pile_set_name": "NIH ExPorter" }
The objective of this research is to develop and test the validity and reliability of a modified picture-sort food frequency questionnaire between 2 application populations, African-American and Hispanic adolescents. Nutritional patterns developed during adolescence may contribute to obesity and increase the risk for several important chronic diseases later in life, including cancer. With emerging nutrition interventions developed for youth, there is a need for accurate dietary methods to assess the success of these interventions. Traditionally, food frequency questionnaires (FFQs) have been developed and tested in white adults. Since diet is not uniform and varies significantly among different populations, results of studies among white adults, as well as the FFQs themselves, are not readily generalizable to other populations. Considering the limited studies and generally poor to modest results of FFQ validation studies among African-American and Hispanic adolescents, there is a necessity as well as obligation to develop and evaluate methods in minority youth, who have been typically under-represented and under-served in these studies. The picture-sort is a novel FFQ, where foods and drinks are presented as pictures with descriptions on cards and sorted by subjects into piles corresponding to consumption over a given period of time. It is an engaging approach, which requires minimal literacy on the part of subjects compared with a typical pencil and paper food frequency questionnaire. Ethnic and age variations in diet can be taken into account by adding foods and drinks specific to the population as new cards or incorporating them into existing categories. For this study, we will design application sets of cards specific to each of the adolescent populations and test validity and reliability of the approach separately among each sample, African-American subjects will be assessed in year 1 and Hispanic subjects in year 2. Subjects will be recruited through application high schools in Denver. For validity (N=80 for each population), energy and nutrient values from administration of the picture-sort FFQ will be compared with those from the mean of three 24-hour dietary recalls. For test-retest reliability, we will select subjects who have not taken part in the validity study (N=80 for each population) and compare energy and nutrients for application administrations of the picture-sort FFQ conducted application weeks apart.	{ "pile_set_name": "NIH ExPorter" }
PROJECT SUMMARY/ABSTRACT HIV-associated nephropathy (HIVAN) is a renal disease almost exclusively seen in people of African ancestry. More than 2 million HIV-infected children living in sub-Saharan Africa are at high risk of developing HIVAN, if they do not receiving appropriate anti-retroviral therapy (ART). HIVAN is characterized by the collapse of glomerular capillaries and micro-cystic transformation of renal tubules leading to rapid chronic renal failure or death. These changes are caused by the infection of podocytes and renal tubular epithelial cells (RTEc), yet the mechanism is unclear. Two genetic risk variants in the human APOL1 gene (G1/G2) were identified as major risks factors for developing HIVAN in people of African ancestry. Nonetheless, other endogenous factors are needed as well, since people of African ancestry who do not carrying the ApoL-1 risk variants, or HIVtransgenic (Tg) mice, also develop HIVAN. Our program is in a unique position to identify these factors, since we have been exploring this topic in children for the last 20 years funded by the current R0-1, and have collected a large number of samples and tissues during this time. In addition, during the last cycle of the grant, we have (1) identified a new role for Tumor Necrosis Factor-alpha (TNF-?) facilitating the establishment of a productive infection of podocytes cultured from children with HIVAN; and found that (2) HIV-Tat is preferentially recruited to lipid rafts (LRs) in podocytes cultured from children with HIVAN, enhancing the signaling of heparin binding growth factors (HBGF) and activating the expression of HIV-genes. Based on these data, we hypothesize that trans-membrane TNF-?, modulates the infection and injury of renal epithelial cells by interacting with heparan sulfate proteoglycans, lipid rafts (LRs), and globotriaosylceramide (Gb3). A second corollary of this hypothesis is that the recruitment of HIV-Tat to LRs, in synergy with other HBGF, precipitates the collapse of glomerular capillaries. Finally, we hypothesize that HBGF release in the urine of children with HIVAN can become reliable biomarkers to diagnose HIVAN and to follow the clinical outcome of these patients in combination with other non-invasive tests. To test these hypotheses, in aim 1, we will define how transmembrane TNF-? modulates the infection of podocytes and renal tubular epithelial cells (RTEc) cultured from children with HIVAN by interacting with LRs, and Gb3. In aim 2, we will determine how HIV-1 and HIV-Tat, precipitate the collapse of glomerular capillaries by interacting with the Von Hippel-Lindau protein (pVHL) and HIF-2?-inducible HBGF in cultured podocytes from children with HIVAN and new transgenic mice. In aim 3, we will determine the clinical value of a new panel of urinary biomarkers, a podocyte permeability assay, podocyturia, and the APOL-1 genotype, to diagnose HIVAN in children and young adults, and to follow their clinical outcome. In summary, these studies will generate fundamental new knowledge to improve our understanding of the pathogenesis HIVAN, and identify new urinary biomarkers and other non-invasive tests that will be used to establish the diagnosis of HIVAN in children without doing a renal biopsy.	{ "pile_set_name": "NIH ExPorter" }
Abstract Prospective Functional Characterization of All Possible Missense Variants in HNF1A Loss-of-function mutations in HNF1A are the most common cause of maturity-onset diabetes of the young (MODY). Individuals with HNF1A-MODY benefit from specific pharmacotherapy with low dose sulfonylureas and can avoid insulin injections, but distinguishing these patients from those with polygenic type 2 diabetes (T2D) on clinical grounds is challenging. Gene sequencing of HNF1A in population-sized cohorts demonstrates that ~1:50 individuals with diabetes harbors a missense variant in HNF1A. But almost all of these are novel, of unknown functional consequence, and therefore clinically unactionable. In just the past few years, dozens of novel variants in HNF1A have been identified, overwhelming the capacity of clinical laboratories to functionally characterize them. We hypothesize that the ability to functionally characterize variants at the rate and scale they are now being discovered would enable diagnoses and potentially guide therapy for thousands of HNF1A variant carriers. To address these challenges, we propose a prospective experimental approach: 1) In order to establish the function of any missense variant in HNF1A that may be found in future genome sequencing, we will engineer every possible amino acid substitution and measure HNF1A function in a disease-relevant bioassays. 2) In order to relate HNF1A function to diabetes risk and therapeutic response we will correlate molecular function in vitro with clinical phenotypes of ~2000 HNF1A missense variant carriers in vivo. If successful, our pilot study will allow instant functional classification of any missense variant in HNF1A that may be found in clinical or investigational sequencing. The data generated will lay the foundation for future recruit- by-genotype trials to assess sulfonylurea responsiveness in diabetic patients carrying HNF1A missense variants.	{ "pile_set_name": "NIH ExPorter" }
Interactions of cells with their immediate extracellular matrix are thought to be critical in the control of cellular functions such as adhesion, replication, motility and differentiation. Cell surface and extracellular matrix proteins are thought to mediate these interactions, with fibronectin being one of the most thoroughly studied of these adhesive proteins. Recent work has identified a new protein oligomer, called "hexabrachion", as a major component of cell surface fibronectin preparations. At least one of the activities previously ascribed to cell surface fibronectin (hemagglutination) has been shown to be due entirely to this oligomer. Studies are proposed to delineate the role of the hexabrachion in the interaction of cells with their environment. (1) Adhesion assays will be used to determine whether the hexabrachion serves as one of the cell-substratum attachment molecules. Microtiter wells will be coated with hexabrachion, fibronectin, or both and tested for their ability to support adhesion of different cell types. (2) The effect of the hexabrachion on cell spreading and restoration of normal morphology to transformed cells will also be examined both by adding transformed cells to coated wells and by preincubating the cells in the presence of hexabrachion. (3) The types of cells that produce hexabrachions will be delineated by direct examination of culture media for hexabrachions, by enzyme linked immunoadsorbent assay (ELISA), and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunobloting. The effect of cell culture aging on the production of hexabrachion will also be assessed. (4) Monoclonal and polyclonal antibodies to hexabrachion will be used to attempt to delineate the domains of cell adhesion and cell spreading activities and further define the mechanisms of interaction of the hexabrachion with the cell surface and with the other proteins which comprise the extracellular matrix.	{ "pile_set_name": "NIH ExPorter" }
The delivery of cytokines into the subretinal space may play an important role in the treatment of retinal degenerative diseases such as age related macular degeneration and certain forms of retinitis pigmentosa. These diseases, which constitute the leading cause of adult onset blindness spring from a variety of genetic sources, and have few available treatment options. Transplantation of retinal cells, as well as delivery of genes to restore lost function are currently being investigated both in the laboratory and clinical setting as possible treatments. This proposal combines elements of both procedures with the goal of long-term drug delivery into the subretinal space. Preliminary research has shown that single dose injection of cytokines into the eye show short-term prevention of photoreceptor degeneration in rat disease models. In order to achieve long-term delivery and prevention we propose the use of genetically modified cells which can overexpress the desired cytokines upon external pharmacological signaling. The method of action of these primarily neurotrophic cytokines could be simultaneously in the photoreceptor and the retinal pigmented epithelium (RPE). We propose that these biomolecules cause upregulation in photoreceptor metabolism, maintaining viable pre-dystrophic levels of gene expression. In the RPE upregulation prevents dedifferentiation into a wound-healing phenotype allowing maintenance of the immunoisolating blood-retina barrier. We intend to use the genetically altered transplant model to investigate these two hypotheses, following photoreceptor rescue and cellular metabolism. The long term goal of this project is to provide tissue engineering therapy which can be applicable to retinal dystrophies with differing etiology.	{ "pile_set_name": "NIH ExPorter" }
Flagellated protozoa of the Order Kinetoplastida contain a single mitochondrion termed a kinetoplast. Two distinct populations of double stranded circular DNA, the maxicircles and the minicircles, are localized within the kinetoplast as a single highly catenated network. These organisms are generally parasitic and are responsible for a variety of human and animal diseases including leishmaniasis, African sleeping sickness and Chagas disease. We will determine the biochemical mechanisms involved in the replication of the DNA minicircles of the trypanosomatid Crithidia fasciculata. These studies should provide insight into eukaryotic replication mechanisms in general and may reveal unique features of kinetoplast replication that can be targeted in the development of chemotherapeutic agents. We will isolate and characterize minicircle replication intermediates synthesized in isolated kinetoplasts and will develop a model of the mechanics of the replication and partitioning of minicircles. Replication of minicircles in soluble extracts will be investigated as a means of identifying specific enzymatic steps in the replication process and developing assays for the purification of the individual enzymes required for minicircle duplication. Replication products produced in vitro will be characterized by physical and biochemical methods in order to elucidate the various enzymatic reactions involved in the overall replication mechanisms. Antibodies prepared against specific components of the replication machinery will be used to investigate the requirements for individual components in different stages of replication, to determine their cellular localization and to provide reagents for rapid immunoaffinity purification of the various proteins.	{ "pile_set_name": "NIH ExPorter" }
Advances in noninvasive methods of measuring atherosclerosis and arteriosclerosis have changed the field of CV epidemiology by providing more precise and useful phenotypes for CVD expression. Technology is constantly changing, requiring new protocols to match the capabilities of new equipment. Under the leadership of Dr. Kim Sutton-Tyrrell, this program is designed to provide concentrated training in the quality collection of subclinical atherosclerosis measures and how they can be used to understand the process of atherosclerosis. The training program is organized around four major research strengths: vascular aging, women's health, high risk and international populations and genetics. The unifying theme is the application of subclinical atherosclerosis measures to each research area. Core strengths in nutrition, exercise, biostatistics, multi-center collaborations and psychosocial measures are also integrated into the program. This interconnected set of resources will provide a rich substrate upon which the trainees will develop. Trainees will have a primary mentor and will also work closely with other faculty members as well as a peer mentor who is a more experienced trainee. The training program consists of course work, research field work and professional development, all individually tailored to each trainee. Research field work includes recruitment, data collection, entry and management, study operations and data analysis, providing trainees with experience in the full range of research tasks. Professional development activities include focused work with subclinical measures, completion of an independent research project, a remote training experience, grant writing experience, manuscript preparation, participation in national conferences, skill development workshops and training in the responsible conduct of research. Trainees will also have the opportunity to develop laboratory-based and analytical skills in genetic epidemiology. The program is designed to produce investigators skilled in a multidisciplinary approach to cardiovascular research, who are comfortable with using subclinical measures in cross-discipline collaborations and who have the skills to develop new measurement protocols.	{ "pile_set_name": "NIH ExPorter" }
The pH-dependent vesicle membrane binding property of chromogranin A appears to be of fundamental physiological importance with regard to the potential roles of chromogranin A in secretory vesicle biogenesis. This is particularly true in segregating secretory vesicle membranes from other membranes in the trans-Golgi network, and also in transmitting extravehicular signals, such as inositol 1,4,5-triphosphate (IP3) or inositol 1,3,4,5-tetrakisphosphate, for Ca2+ release or uptake to the inside of vesicles. Several vesicle matrix proteins of the secretory vesicles, including chromogranins A and B, bound to the vesicle membrane at the intravesicular pH of 5.5 and were freed from it when the pH was raised to a near physiological level of 7.5. Further, chromogranin A is shown to interact with several integral membrane proteins of the secretory vesicles at pH 5.5, but not at pH 7.5. One of the chromogranin A interacting membrane proteins had a size of 260 kDa and reacted with the IP3 receptor antibody. This result suggested the existence of the IP3 receptor/Ca2+ channel in the vesicle membrane and also the existence of direct communication between chromogranin A and the IP3 receptor/Ca2+ channel. In addition, the pH-dependent interaction of chromogranin A with integral membrane proteins implies an important role for chromogranin A in the sorting process of the vesicle membrane proteins during vesicle biogenesis in the trans-Golgi network. Further study has shown that one of the intraluminal loop domains of the IP3 receptor/Ca2+ channel interacts with chromogranin A at the intravesicular pH of 5.5, suggesting the importance of the intraluminal loop domain in transmitting Ca2+ mobilization signals to chromogranin A.	{ "pile_set_name": "NIH ExPorter" }
This acquisition is for the maintenance of an international bone marrow transplant registry (IBMTR). The purpose of the IBMTR is to pool data acquired from national and international transplant centers and to analyze this data in a disciplined and thoroughly tested manner. To date, a data base of over 3,000 recipients exists with over 1,000 accruing during the past 18 months. The increased frequency of bone marrow transplants supports the need for the IBMTR's continuing acquisition and analysis of the rapidly expanding data base. The data and results acquired through this acquisition shall potentially improve the prognosis for individuals at risk because of congenital or other cause of immunodeficiencies. These results shall assist in the development and production of monoclonal antibody therapy used to abrogate graft versus host disease and provide a more comprehensive data base upon which to design more efficient procedures for allogeneic bone marrow transplantation.	{ "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 DNA genetic information in our cells is packaged as chromatin. The nucleosome core particle is a fundamental repeating unit of chromatin and comprises about 150 base pairs of DNA spooled around a histone protein core. Recent studies show that chromatin is not simply a repressive structure that occludes DNA, but is instead an active participant in gene regulation that associates with multiple chromatin factors and enzymes. Despite the importance of such interactions, we currently lack a structural understanding of how chromatin factors or enzymes recognize the nucleosome. To address this problem, we have reconstituted complexes containing the nucleosome core particle and several chromatin factors or enzymes, and we have grown single crystals of a chromatin factor/nucleosome core particle complex. We request beam time at APS for this challenging project which features large macromolecular complexes, weakly diffracting crystals and large unit cell dimensions.	{ "pile_set_name": "NIH ExPorter" }
Polarized epithelial cells line every organ and are of utmost importance for the function of the human body. Central to epithelial cell function is the establishment and maintenance of biochemically and functionally distinct membrane domains, the apical membrane facing the lumen of an organ and the basolateral membrane that is in contact with connective tissues. Both membranes are separated by tight junctions. To maintain this architecture, epithelial cells must continuously sort newly synthesized and internalized transmembrane receptors to the correct membrane domains in the biosynthetic and endocytic pathways. Our work focuses on the molecular mechanisms that ensure correct targeting to the basolateral membrane from a central sorting station, the recycling endosomes. Previously we showed that cargos destined for the basolateral membrane are recognized by the epithelial-specific clathrin adaptor complex AP-1B for incorporation into AP-1B vesicles. This grant application proposes to follow up on these findings and to investigate the molecular mechanisms of AP-1B function, membrane recruitment, and regulation of the AP-1B-dependent pathway from recycling endosomes to the basolateral membrane. We will employ genetical, biochemical, and cell biological methods to accomplish our research goals. PUBLIC HEALTH RELEVANCE: The primary function of epithelial cells is to ensure the correct nutrient and waste product exchange between the body and the environment. To fulfill these different functions, the surface of epithelial cells is divided into biochemically and functionally distinct membrane domains. Our long-term goal is to understand how polarized epithelial cells establish and maintain this asymmetry with a focus on processes that lead to correct targeting of transmembrane receptors to the membrane domain that faces connective tissues and neighboring cells.	{ "pile_set_name": "NIH ExPorter" }
One of several approaches to the treatment of cancer is the promotion of differentiation of malignant cells into histologically and clinically benign lesions. Malignant cells in human acute and chronic lymphocytic leukemias and lymphomas of B cell origin most frequently exhibit an arrest of maturation as shown, when studied by immunofluorescene, by the presence of monoclonal surface-associated immunoglobulin, without secretory-intracytoplasmic immunoglobulin, and without the corresponding serum monoclonal immunoglobulin. This study will concentrate on in vitro stimulation of maturation of cells from patients with lymphoid malignancy of B cell origin by exogenous cyclic AMP and cyclic GMP, by substances and drugs which elevate endogenous cyclic nucleotides, and by pokeweed mitogen. The role of Ca ions in the process of maturation of malignant and non-malignant B lymphocytes will be studied by using ionophores. Malignant human lymphoid cells and cryostat sections of solid tissues will be studied for B-, T-, and M- (monocytic) cell receptors. Pulse and prolonged exposure to substances triggering maturation will be done in vitro. Quantitation of cells bearing surface-associated (receptor) immunoglobulins and of cells containing intracytoplasmic (secretory) immunoglobulins will be done by immunofluorescence. Secreted immunoglobulins in tissue culture will be measured by radioserological assay. Normal lymphocytes from volunteer donors and established cell lines of B- and non-B-origin will serve as a control.	{ "pile_set_name": "NIH ExPorter" }
Changes that may occur in the expression of protein and mRNA for TGF-beta isoforms in response to various disease states and tissue treatments are being investigated using immunohistochemistry and Northern blot techniques. For example, we have found that there are changes in expression of TGF-beta protein in the mouse brain during experimental allergic encephalomyelitis. In mouse embryos, changes in the localization of protein occur following retinoic acid treatment, while ovarian steroids alter expression of TGF-betas in the mouse uterus. Following heat shock of rats, there are transient increases in TGF-betas in the heart which may mediate the cardioprotective effects of heat shock. This hypothesis is being investigated by measuring recovery of contractile force following ischemia-reperfusion in an isolated perfused heart system. This is being done in hearts from rats exposed to heat shock, as well as in control animals in the presence or absence of TGF-beta or TGF-beta antibodies. Similar changes in TGF-beta expression occur in cultured cardiac cells following heat shock. TGF-beta also can induce its own expression in these cell types. The actions of TGF-beta in the heart during septic shock are also being investigated. Lipopolysaccharide (LPS) treatment of rats causes destruction of the collagen ultrastructure of the heart as determined by scanning electron microscopy and the ability of TGF-beta to reduce the molecular defect is being examined. LPS treatment of cultured cardiac myocytes induces expression of collagenase mRNA and this induction is abolished by TGF-beta. TGF-beta expression in hearts of dogs treated with tumor necrosis factor-alpha, which reduces cardiac output, is also being examined.	{ "pile_set_name": "NIH ExPorter" }
HL-131: To function as an intact barrier, epithelia must maintain constant cell numbers despite sometimes high rates of turnover. If the rate of cell death exceeds proliferation, epithelial barrier function could become compromised; if it lags behind proliferation, cells could amass into tumors. However, little is known about the molecular mechanisms that keep airway epithelial stem cells largely quiescent during homeostasis or in check after a regenerative response to prevent the tissue overcrowding and dysplasia associated with airway remodeling in chronic lung diseases like COPD and asthma. After airway epithelial injury, surviving epithelial cells spread out to maintain barrier function and secrete Wnt7b to induce fibroblast growth factor 10 (Fgf10) expression and proliferation in airway smooth muscle cells (ASMCs). Fgf10 secreted by the ASMCs then acts on surviving club cells to initiate epithelial repair by breaking quiescence and inducing proliferation. The objective of this project is to identify the key signals that keep this Wnt7b-Fgf10 signaling cascade silent during normal homeostasis or once regeneration is complete, but activate the Wnt7b-Fgf10 signaling cascade when airway epithelial injury is sensed. Recent work in the lung has identified a role for the Hippo tumor suppressor pathway in regulating airway epithelial stem/progenitor cell quiescence during normal homeostasis. In addition, it was shown that the Hippo pathway becomes inactivated in surviving airway epithelial cells when they spread out to maintain barrier function shortly after injury, likely to initiate repair. We therefore hypothesize, that the Hippo pathway keeps airway epithelial/stem progenitor cells quiescent during normal homeostasis by inhibiting activation of the Wnt7b-Fgf10 signaling cascade. We have identified substantial aberrant signaling of the above-described pathways in clinical samples of COPD. We further hypothesize that when surviving airway epithelial cells spread out, signals are transduced by integrins to induce Yap/Taz-mediated Wnt7b expression. Interestingly, the scaffolding protein integrin linked kinase (Ilk) has been proposed to be essential for the proper termination of liver regeneration by positively regulating the Hippo pathway. Based on these findings the overall goal of this proposal is to test the hypothesis that: Ilk in the airway epithelium maintains stem cell quiescence by positively regulating the Hippo tumor suppressor pathway to block activation of the Wnt7b-Fgf10 signaling axis and prevent airway remodeling as observed in chronic lung diseases. In Specific Aim 1, we will test the hypothesis that the Hippo pathway blocks activation of the Wnt7b- Fgf10 signaling axis between airway epithelial progenitors and their niche during normal homeostasis. In Specific Aim 2, we will test the hypothesis that during normal homeostasis Ilk positively regulates the Hippo pathway in airway epithelial cells, to inhibit activation of the Wnt7b-Fgf10 repair pathway. In Specific Aim 3, we will determine whether human airway remodeling in COPD patients is associated with deregulated Ilk-Hippo- Wnt7b-Fgf10 signaling.	{ "pile_set_name": "NIH ExPorter" }
Under NCT00025935, we have already screened over 150 patients with depression and have scanned over 35 participants with depression, other conditions, and healthy volunteers. We have analyzed existing data and have already published papers since the start of my tenure here. On the methodological side: we have just completed a paper measuring the reliability of the reward prediction error signal in a reward task. We have included a new metric, namely intrinsic functional connectivity, and published a paper about its role in depression. We have completed the analysis of a double-blind, placebo controlled pharmaco-imaging study and are about to submit the data for publication. We have completed a meta-analysis of EEG and fMRI study related to reward processing in depression and are about to submit the data for publication.	{ "pile_set_name": "NIH ExPorter" }

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