text
stringlengths 1
32.7k
| meta
dict |
|---|---|
The long-range goal of the proposed research is to understand the molecular mechanisms by which mutations in Myosin Binding Protein-C (MyBP-C) cause hypertrophic cardiomyopathy (HCM), an autosomal dominant disorder that affects 1 in 500 people and is the most common cause of sudden cardiac death in adolescents and young adults. Mutations in cardiac (c) MyBP-C are among the most frequent causes of HCM with >149 distinct mutations described so far. However, despite progress in identifying genetic causes of HCM, the molecular mechanism(s) by which any single cMyBP-C mutation causes disease are still unknown. Reduced amounts of cMyBP-C were recently reported in affected human myocardium, suggesting that loss of functional cMyBP-C from an affected allele (i.e., haploinsufficiency) is a common factor contributing to cardiac dysfunction. However, alternative possibilities that affected proteins impair contractile function or that processing and degradation of improperly folded proteins cause aberrant cell function have not been eliminated. These distinctions are critical for designing effective therapeutic strategies to overcome HCM, yet definitive evidence in support of the different possibilities has not been obtained in part because of the limited availability of human biopsies and in part because engineered mouse models do not fully recapitulate either the human disease phenotype or the proximal cell processes that lead to disease. Experiments proposed here will overcome these limitations by utilizing the only naturally occurring large animal model of HCM that both closely resembles the human disease phenotype and that has a known genetic cause. The mutation is a spontaneous missense mutation in cMyBP-C in domestic Maine Coon cats that results in a proline for alanine substitution at codon 31 (A31P). The mutation results in a single amino acid substitution, but causes an anomalous decrease in total amounts of cMyBP-C protein. Because similar decreases in cMyBP-C have been reported for missense cMyBP-C mutations in human myocardium, the feline A31P model offers a unique opportunity to distinguish between three primary factors proposed as causative in human disease i.e., dominant negative effects of a single amino acid point mutation, gene dosage effects, and cellular protein folding/trafficking defects. The proposed experiments will test the hypothesis that reduced amounts of cMyBP-C in sarcomeres (i.e., haploinsufficiency) of affected cats leads to contractile deficits that ultimately cause cardiac dysfunction. Specific aims of the project are to determine 1) the expression level and subcellular localization of total cMyBP-C protein in cats carrying the A31P mutation, 2) effects of the A31P mutation on myocyte contractile properties, and 3) effects of the A31P mutation on the myocardial ubiquitin-proteasome (UPS) system. Collectively, results from these studies will provide critical insights into molecular mechanisms by which mutations in cMyBP-C cause disease and will provide a significant and lasting impact on HCM research by developing a unique animal model that will be a resource for basic research and that will aid in the design and testing of therapeutic strategies for the treatment of cardiomyopathies linked to cMyBP-C. PUBLIC HEALTH RELEVANCE: The proposed studies will investigate basic pathogenic mechanisms by which the A31P mutation in the cardiac myosin binding protein-C (cMyBP-C) causes inherited hypertrophic cardiomyopathy (HCM) in Maine coon cats, a breed of domestic cat. In humans, HCM affects an estimated 1 in 500 people and is the leading cause of sudden death in adolescent and young adults. Because mutations affecting cMyBP-C are a leading cause of HCM in humans, it is anticipated that the proposed studies in Maine Coon cats will provide insights into human disease and will ultimately contribute to advances in disease diagnosis, prognosis, and treatment of HCM linked to cMyBP-C.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Despite improvements in the past 20 years in glycemic and blood pressure control, and the introduction of 'renoprotective'drugs such as renin-angiotensin system blockers (RASB), the incidence of end-stage renal disease (ESRD) in type 1 diabetes (T1D) is not declining. Novel therapies to complement these interventions are urgently needed. Mounting evidence from prospective studies indicates that moderately elevated serum uric acid is a strong, independent predictor of an increased risk of chronic kidney disease and increased rates of loss of kidney function among T1D persons. To study whether uric acid lowering can reduce glomerular filtration rate (GFR) loss in T1D, we have established a consortium that includes investigators from the Joslin Diabetes Center, the Universities of Minnesota, Colorado, Toronto, and Michigan, and the Steno Diabetes Center. Currently, we are envisioning a four-year, multi-center, double- blind, placebo-controlled, randomized clinical trial evaluating the efficacy of the urate-lowering drug allopurinol, as compared to placebo, in reducing kidney function loss among subjects with T1D. The trial will specifically target T1D patients with microalbuminuria or moderate macroalbuminuria and serum uric acid levels e 5 mg/dl, since these are the patients who are at very high risk of having rapid rates of GFR decline and might most benefit from reductions in uric acid levels. Study subjects will be required to have a GFR e 60 ml/min, consistent with the goal of intervening relatively early in the natural history of kidney disease, when kidney function can still be preserved, rather than at later stages when structural changes are far advanced and most of kidney function is already lost. The primary endpoint of the study will be the GFR (as measured by the iohexol plasma disappearance) at the end of the 4-year intervention. Preliminary calculations suggest that ~200 subjects in each treatment arm would provide us with reasonable power to detect a clinically meaningful and achievable reduction in GFR decline in the allopurinol as compared to the placebo group. With the R03 support, we intend to bring this study concept closer to implementation by accomplishing the following Specific Aims: 1.To finalize the Study Protocol;2.To identify the best site specific mechanisms to query the respective patient databases for the quickest identification of eligible subjects;3.To prepare a Manual of Operation;4.To file IRB applications at all study sites. By accomplishing these aims, we will be optimally positioned to successfully apply for an R34 grant, during which we will establish the clinical trial infrastructue and test it in a vanguard phase of the trial. If we can then demonstrate in the full trial that allopurinol can halt or slow GFR decline in T1D subjects, we will have a simple, safe, and inexpensive intervention to prevent or delay kidney failure in T1D that can be applied at the earliest clinically detectable stages of renal injury. It is difficult to overstate how significantthis discovery would be, both from the perspective of public health and that of individual diabetic patients. PUBLIC HEALTH RELEVANCE: The trial that we propose, if successful, will introduce a new pharmacological intervention to prevent or delay kidney failure in T1D. The reduction in morbidity and mortality resulting from this would have a major impact on the lives of T1D patients as well as on society at large, significantly reducing the human and financial costs associated with this condition.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The speed of advancement in data-rich biomedical sciences is limited in part by the lack of awareness and expertise in big data sets and tools, the absence of approaches that productively intersect disparate domains, and the underrepresentation of diverse groups in the workforce. To address these disparities, we propose CREEDS: Community Research Education and Engagement for Data Science. This title represents our commitment to our overall goal of fostering practical skills for a national, diverse and interdisciplinary community of early career researchers. Our three specific aims are: (1) to provide biomedical researchers with the practical skills and insight needed to harness the power and advance the promise of big data science to accelerate scientific discovery, (2) to develop an online social environment to facilitate the exchange of big data ideas, approaches and techniques between novices and experts, and (3) to enhance the diversity of the biomedical big data workforce through targeted recruitment and retention of disadvantaged and underrepresented student populations. We will personally engage 150 graduate students through an intensive, self-tailored, two-week summer school in NYC that will showcase interesting, and current, collaborative case studies in activities at schools throughout NYC. It wil also include training in the Responsible Conduct of Research. Participants will employ active learning techniques to develop their skills of specific new methods and tools through both individual and group tasks on real-life large data sets. The training will raise the skills of students of varied backgrounds to a sufficient level for additional graduate research and will not require any prior computing experience. Students will also receive experience and materials to help them teach others when they return to their home institutions. Additionally, we will mentor another 30 NYC-based graduate students for team participation in four month long DREAM challenges. The DREAM challenge teams will help develop computational skills on solving difficult, real-life biomedical problems. An online social environment, Kipin Hall, will enable participants to discover job opportunities, find data sources and tools and communicate with peers as well as big data experts. We will leverage our extant relationships with organizations serving underrepresented groups to boost participation and enhance this exciting field. To continually improve our program we will actively seek and respond to feedback from our participants, program faculty and our advisory panel. As a result of this project, we will train ovr 180 people to develop team skills to understand, select and use genomics data tools and approaches. We will reach more individuals by placing the summer school on Coursera. Armed with this knowledge, we plan that the next generation of genomics scientists will be better placed to design, analyze and interpret high-throughput genomics datasets. This is the important need for research education that we aim to address with CREEDS.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Uromodulin (Tamm-Horsfall protein) is the most abundant protein in human urine. It is made exclusively and abundantly in kidney tubules. Its cDNA sequence contains structural motifs often seen in cell adhesion molecules. The abundance, specific location and structural properties of uromodulin have long suggested a role for this protein both in renal physiology (fluid and electrolyte transport and crystal and cytokine binding) and in renal disease (casts of uromodulin are though to participate in the pathogenesis of acute renal failure, especially that associated with rhabdomyolysis and with myeloma). The exact function of uromodulin, however, has remained obscure to date. The objective of studies proposed in this application is to help determine the biological function of uromodulin in health and disease. The specific aim of this application is to produce a uromodulin-deficient strain of mice by the technique of homologous recombination and to study the structure and function of kidneys in the uromodulin-deficient mice for the consequences of uromodulin deficiency.
|
{
"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 nitroxide spin label 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl-methanethiosulfonate (MTSSL), commonly used in site-directed spin labeling of proteins, was studied with molecular dynamics (MD) simulations. After developing force field parameters for the nitroxide moiety and the spin label linker, we simulated MTSSL attached to a polyalanine R-helix in explicit solvent to elucidate the factors affecting its conformational dynamics. Electron spin resonance spectra at 9 and 250 GHz were simulated in the time domain using the MD trajectories and including global rotational diffusion appropriate for the tumbling of T4 Lysozyme in solution. Analysis of the MD simulations reveals the presence of significant hydrophobic interactions of the spin label with the alanine side chains
|
{
"pile_set_name": "NIH ExPorter"
}
|
The chloroplast DNA of the unicellular green alga Euglena gracilis will be dissected into specific fragments with a variety of restriction endonucleases. Fragments generated by the EcoRI, Bam and Sal enzymes will be cloned in a plasmid of Escherichia coli. The location of restriction sites generated by the EcoRI, Bam, Sal and other enzymes will be determined. Genes for ribosomal RNA and specific transfer RNAs will be mapped, and several transfer RNA genes will be sequenced.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Neonatal lipid absorption is in part, dependent on the capacity of the liver and intestine to synthesize, excrete, and conserve bile acid. Preliminary studies performed in this laboratory have suggested that neonatal bile acid turnover is deficient, and that this may be a contributory factor in the development of steatorrhea in formula fed premature infants. Studies have, therefore, been undertaken to characterize the state of preparedness of the bile salt related activities of the liver and intestine just before birth. For this purpose fetal dogs and monkeys will be prepared in utero with vascular, intestinal, and biliary catheters. C14- and H3- labeled bile salts and bile salt precursors will be administered intravenously or into the intestine and the appearance of these materials in fetal plasma, liver, bile and other fetal tissues will be determined. Fetal to maternal placental transfer will also be measured by techniques available in the laboratory. The results have already established that bile salt turnover in the dog fetus closely resembles that in the adult, while the primate fetus' mechanisms are grossly deficient. Primate fetal hepatic excretion is reduced while placental transfer is increased in the primate fetus. It would thus appear that mechanisms for hepatic bile salt turnover, like those for bilirubin, are "maturing" during the perinatal period, and that deficiencies in these mechanisms are reflected in deficiencies of neonatal nutritive function.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The goals of Gene Expression in Inflammatory Myopathies are: (1) to characterize patterns of muscle gene expression through the use of DNA microarrays among adult patients with distinct subtypes of inflammatory myopathies and correlate these patterns with clinical phenotypes, and (2) to formulate hypotheses relevant to the pathogenesis of the inflammatory myopathies. High-density oligonucleotide microarrays will be used to assay gene expression in muscle biopsy specimens from patients with inflammatory myopathies. These patients will be phenotypically well described and known to the clinical investigators of this proposal. Data analysis of gene expression profiles will be linked to clinical data to refine disease classification and understand distinctions among and within the major subtypes of inflammatory myopathy. Analysis of differential gene expression will also provide clues to pathogenesis of these disorders. This work may provide further diagnostic approaches to these disorders and contribute to the understanding of their pathogenesis.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Cell cycle checkpoints are implemented to safeguard our genome and the deregulation of which contributes to the pathogenesis of human cancers. Hence, it is of paramount importance to discover and interrogate novel key constituents of the mammalian DNA damage response network. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the essence of an intact S-phase checkpoint in maintaining genome integrity. Although basic framework of the S-phase checkpoint in multi-cellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome band 11q23 translocation disrupting the MLL gene results in poor prognostic leukemias that carry pathognomonic MLL fusions. MLL is a transcription co-activator that is best known to maintain HOX gene expression. The importance of HOXA gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models indicated that MLL fusion-induced HOXA gene aberration alone is insufficient to initiate MLL leukemia. Therefore, further dysregulation must exit and contribute to the ultimate leukemia phenotype. Our recent studies demonstrated a close relationship between MLL and the regulation of mammalian cell cycle. MLL not only assists in the G1/S and G2/M phase transition during a normal cell division cycle but also executes the S-phase checkpoint upon DNA damage. We found that (1) MLL functions as a key effector of ATR-mediated S-phase checkpoint response, (2) activated ATR phosphorylates and thus stabilizes MLL, (3) upon checkpoint activation MLL accumulates at the late replication origin, methylates histone H3K4, and thus delays DNA replication, (4) MLL deficient cells exhibit defects in the S-phase checkpoint response, and (5) MLL fusions work as dominant negative mutants that compromise the integrity of S-phase checkpoint. Here we will determine the mechanisms by which MLL executes the S phase checkpoint response and examine whether and to what extent an S-phase checkpoint dysfunction contributes to MLL leukemogenesis. Our proposal connects MLL/MLL fusions to the S-phase checkpoint response network, which not only provides novel insights into the mammalian cell cycle checkpoint control but also shed light on the pathogenesis of poor prognostic human leukemias.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Although considerable primary sequence data have been developed for voltage-dependent sodium channels by this laboratory and others, and a number of detailed models have been proposed for channel tertiary structure, experimental data have been reported that relates directly to the three-dimensional conformation of the channel in the membrane. Given the difficulties encountered in obtaining x-ray crystallographic information for membrane proteins such as the sodium channel, it is likely that high-resolution structural data will still be some years in coming. In the meantime, studies relating structure to function require the best possible analysis of channel three-dimensional structure in order to proceed. Because of the indirect nature of most structural approaches short of crystallography, a number of different techniques will have to be brought to bear. Building on our recent biochemical studies of the skeletal muscle sodium channel, and our cloning of the primary sequence for the rat TTX-sensitive and TTX-resistant isoforms of the sodium channel and their human homologs, we propose here to continue a multidisciplinary approach to muscle sodium channel structure, and to begin an analysis of the relationship between this structure and its function. We will probe the organization of the sodium channel in the membrane with studies that focus on the membrane topography of specific segments of the primary sequence, on the organization of the large extramembrane regions in the channel structure, and on the structure of the compactly folded internal repeat domains. Specifically, we will complete our analysis of the location of sites of post-translational modification in the channel primary sequence. We will extend our detailed analysis of the location and kinetics of protease-sensitive regions in the channel structure to include a topographical analysis of these regions, and an analysis of their response to alterations in membrane potential or toxin binding. We will extend our use of monoclonal antibodies to probe the interaction and location of the extramembranous domains. We will use antibodies to interhelical loops or to foreign epitopes inserted into interhelical loops to begin an analysis of the folding pattern within the repeat domains. Finally, in collaboration with Dr. Richard Horn, we will begin an analysis of structure function relationships in the muscle channel that takes advantage of the characteristic differences in toxin binding, single channel conductance, and kinetics between the muscle TTX-sensitive and TTX-resistant channels. This analysis will take specific advantage of the availability of expressible full-length clones for the two rat skeletal muscle sodium channel isoforms. Chimeras will be constructed between the two clones and the partitioning of characteristic properties investigated. Initial leads will be pursued with site-directed mutagenesis. Special attention will be paid to the S5-S6 interhelical loop regions in light of their potential role in toxin binding, and their markedly different treatment in key models of channel structure.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The long-term goals of this project are to 1) understand the role of mitochondria in ischemia-reperfusion injury and cardioprotection ; 2) to understand the role of altered ion homeostasis and altered metabolism in ischemia-reperfusion and cardioprotection and 3) to understand changes in cytosolic and mitochondrial signaling involved in cardioprotection and cell death. It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. It has been proposed that cells die following ischemia and reperfusion because of a rise in mitochondrial calcium which leads to activation of the mitochondrial permeability transition pore (MPT). The increase in mitochondrial calcium is proposed to occur via calcium uptake by the mitochondrial calcium uniporter. The composition of the MPT is unknown, but cyclophilin D has been shown to be a regulator of the MPT. We examined the role of calcium uptake by mitochondrial calcium uniporter (MCU) in regulating cell death and metabolism. In collaboration with Dr. Toren Finkel, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU(-/-) mice have no apparent capacity to rapidly uptake calcium. Whereas basal metabolism seems unaffected, the skeletal muscle of MCU(-/-) mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU(-/-) mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU(-/-) mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. Surprisingly, the lack of PTP opening does not seem to protect MCU(-/-) cells and tissues from cell death. Following global I/R in a Langendorff perfused heart model infarct size was indistinguishable between WT hearts and hearts from MCU-/- mice. To further complicate the picture, cyclosporine A, an inhibitor of the mPTP, reduced infarct size in WT hearts, but not in MCU-/- hearts. We have been testing whether loss of MCU leads to alterations in other cell death pathways. We also examined the response of the mouse to addition of isoproterenol. We also examined the role of succinylation in regulating cardiac function and response to ischemia and reperfusion. Succinylation refers to modification of lysine residues with succinyl groups donated by succinyl-CoA. Sirtuin5 (Sirt5) is a mitochondrial NAD+-dependent deacylase that catalyzes the removal of succinyl groups from proteins. Sirt5 and protein succinylation are conserved across species, suggesting functional importance of the modification. Sirt5 loss impacts liver metabolism but the role of succinylation in the heart has not been explored. We combined affinity enrichment with proteomics and mass spectrometry to analyze total succinylated lysine content of mitochondria isolated from WT and Sirt5-/- mouse hearts. We identified 887 succinylated lysine residues in 184 proteins. 44 peptides (5 proteins) occurred uniquely in WT samples, 289 (46 proteins) in Sirt5-/- samples, and 554 (133 proteins) were common to both groups. The 46 unique proteins in Sirt5-/- heart participate in metabolic processes such as fatty acid β-oxidation (Eci2) and branched chain amino acid catabolism, and include respiratory chain proteins (Ndufa7, 12, 13, Dhsa). We performed label-free analysis of the peptides common to WT and Sirt5-/- hearts. 16 peptides from 9 proteins were significantly increased in Sirt5-/- by at least 30%. The adenine nucleotide transporter 1 showed the highest increase in succinylation in Sirt5-/- (108.4 fold). The data indicates that succinylation is widespread in the heart and enriched in metabolic pathways. We examined whether the loss of Sirt5 would impact ischemia-reperfusion (I/R) injury and we found an increase in infarct size in Sirt5-/- hearts compared to WT littermates (68.5+/-1.1% Sirt5-/- vs 39.6+/- 6.8% WT) following 20 minutes of ischemia and 90 minutes reperfusion. We further demonstrate that the degree of I/R injury in Sirt5-/- heart is restored to WT levels by pretreatment with dimethyl malonate, a competitive inhibitor of succinate dehydrogenase (SDH), implicating alteration in SDH activity as causative of the injury.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The overall objective of the proposed project is to design, develop and construct a bone marrow separation device that will offer significantly superior performance compared with current methods of velocity sedimentation. In Phase I Technical Research Associates (TRA) will apply patented pseudo-standing wave ultrasound technologies to separate bone marrow cells by size. This novel device will eliminate the traditional cell concentration problem referred to as the "streaming limit" associated with velocity sedimentation, using an acoustically inverted separation column. Using safe levels of ultrasound the forces generated by TRA's ultrasonic means are significantly higher than traditional "g" forces and will effect rapid cell separation. The use of ultrasonically generated velocity gradients will also eliminate the need for the tedious and difficult formation of density when integrated into commercial separation systems, will provide more efficient collection of specific bone marrow and other biological cells for clinical treatment. The device will also save time, decrease costs and provide unique performance for those interested in fractionation of bulk quantities of cells (or bone marrow components).
|
{
"pile_set_name": "NIH ExPorter"
}
|
In the same treatise in which he states that his historical findings "compel us to formally advance the principle of functional localization as an irrefutable rule", Korbinian Brodmann clarifies that "One cannot think of - human perception (of sound for example) - taking place in any other way than through an infinitely complex and involved interaction and cooperation of numerous elementary activities, with the simultaneous functioning of just as many cortical zones, and probably of the whole cortex..." (Brodmann, 1909 [1, p. 255]). Modern neuroimaging findings now demonstrate how well Brodmann understood his data and support the notion of brain functions as served by a distributed network. Functional magnetic resonance imaging (fMRI) has emerged as an important non-invasive methodology in 21st century studies of language networks and functional connectivity in the human brain. Although its application in studies of child language development have been somewhat more challenging, during the first five years of this proposal, we addressed these challenges by developing child-friendly approaches for studying language processing in children and made technical advances that assure valid data is obtained and analyzed from this population. We have demonstrated that the cerebral distribution of language function changes with age, exhibiting patterns that are influenced by normal development as well as neurological disorders. This large data set (n>300, age 5- 18 years) provides the necessary power for the development and application of new statistical approaches to identify chronologically-linked regions of activation through Independent Component Analysis and model their functional connections through Linear Structural Equation Modeling techniques. This work will advance the understanding of brain-language relations beyond the static models of adult language to the dynamics represented by the period of language acquisition. This data will also serve as a frame of reference for two new studies. The first extends our original longitudinal study of normal language development to cover a period of 11 years in children 5-18 years of age. The second investigates the neuroplasticity of language function associated with childhood onset epilepsy as, a model of chronic brain injury. These studies and methods hold the promise of revealing the ontogeny of both localization and connectionist models foreshadowed a century ago by Brodmann, in the unique context of the developing brain.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Genomes and subgenomic clones of herpesviruses, human cytomegalovirus (HCMV and human herpesvirus-6 (HHV-6), were studied for oncogenic potential in order to evaluate their role in human cancers. CMV: Multiple regions of CMV strain Towne were studied for transforming functions by introducing DNA into rodent cells and human cells, and assayin the selected cells for tumorigenicity by growth in agarose or in nude mice. In this study, the major immediate early gene (IE1), which is a candidate subunit vaccine was also included. We identified two domains, mtrII and mtrIII, in the CMV genome that could transform cells to tumorigenicity. Th mtrII transformed human cells could produce poorly differentiated carcinoma in mice. Both domains contain several protein coding sequences. Data from the digestion of these domains with restriction enzymes suggested that a 79 amino acid (aa) protein in mtrII and a 270 aa protein in mtrIII might be responsible for tumorigenicity. Further mapping is in progress. We found that the IE1 gene is not involved in transformation. HHV-6: Similar studies were applied to HHV-6 to elucidate its role in huma lymphomas especially in AIDS and bone marrow transplant patients. Genomic DNA of HHV-6 isolates from AIDS patients (GS and Z29) and from chronic fatigue syndrome patients (GD) could transform mouse fibroblasts. Two DNA clones (21 kb and 8.7 kb) of GS isolate could transform human keratinocytes HHV-6 DNA, specific to 8.7 kb region, has been detected in an immunoblastic lymphoma by hybridization. Further studies are in progress to determine th specific genes involved in transformation. Identification and characterization of herpesvirus oncogenes are important to safety evaluatio and to development of CMV and other herpesvirus vaccines as well as to development of herpesvirus-based vectors for gene therapy. Since IE1 of CM is found non-tumorigenic in our assay, it may be safely used as a candidate subunit vaccine. If a tumor antigen is identified, a specific diagnostic reagent can be developed for CMV or HHV-6 related malignant diseases.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Our research objective remains the development of artificial lungs and artificial kidneys based on the membrane separation principle. A second generation of mass transfer devices is now emerging in part because of the availability of new materials, in part because of growing sophistication in the design and prediction of performance of mass transfer devices. In the next years, our program will involve: a) continuing theoretical studies of the determinants of gas transfer in blood oxygenators; b) further development of the microporous, flat sheet type of oxygenator, with special attention to water vapor change, control of microporosity of exchange membranes, interaction between blood and microporous materials, and factors of decay in gas transfer performance; c) further design of the microporous coiled tube oxygenator design, with special attention to the induction of secondary flows to achieve high oxygen transfer coefficients with a minimum resistance to blood flow; d) miniaturization of the microporous coiled tube oxygenator and development of branching and manifolding techniques that will permit the construction of an implantable membrane oxygenator; e) further theoretical and experimental study of some factors leading to platelet agglutination and aggregation in flow systems; f) application of the engineering exchanger theory, as it pertains to blood oxygenators, to the design and performance of the natural lung with a view to define design parameters of importance for implantable, artificial lungs.
|
{
"pile_set_name": "NIH ExPorter"
}
|
In the past decade, laboratories around the world have produced tens of thousands of mutant, transgenic, and wild-type fish lines. Maintaining these valuable genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. One of the major recommendations of the 2007 NCRR workshop 'Achieving High-Throughput Repositories for Biomedical Germplasm Preservation' was for performance of detailed studies to improve sperm cryopreservation for important aquarium fish models. Pursuant to this we propose to establish high-throughput processing of zebrafish sperm for stock centers and laboratories by extending a comprehensive cryopreservation pathway, and by intensifying efficiency and output to enable implementation of germplasm repositories. The Specific Aims are to: 1) broaden the pathway to improve quality and provide multiple access points for samples produced by current approaches, 2) intensify the pathway for automated processing and repository development including use of industrial modeling and decision-making tools, and 3) implement two scalable cryopreservation protocols; the large-scale high throughput method for Resource Center use and, reverse-engineered from it, a small to intermediate scale application for other research laboratories to back up their own lines or submit them to the Resource Center. This will improve quality, reduce variability, and increase efficiency. We propose to improve broodstock conditioning, gamete quality, fertilization and reproduction, and to accommodate the variety of approaches, levels of application, and current and future needs of the research community. This will yield two complementary approaches: one at an industrial scale using automated equipment and pooling of samples to yield the highest level of intensity and efficiency; the other, less intensive but highly reliable, designed or dissemination to other laboratories. The materials generated from this external pathway will feed into the central pathway and will provide an additional form of throughput based on the aggregate production from multiple locations. A high level of standardization will be necessary for both approaches to merge efficiently to deliver high-quality samples into large comprehensive germplasm repositories. Thus, we envision this renewal as a funnel to improve and adapt the wide breadth of technical approaches available for zebrafish cryopreservation, and to focus these activities for repository development that can be eventually coupled with commercially available high-throughput equipment, supplies, and reagents specifically optimized for aquarium fish. This approach is practical and extremely feasible. This renewal proposal builds directly on the success of our established pathway and looks to couple efficiently with future advances.
|
{
"pile_set_name": "NIH ExPorter"
}
|
DESCRIPTION (investigator's abstract): Our research goal is to understand the biological role of terminal complement complexes (C5b-7, C5b-8, C5b-9) in inflammation and immune reaction. We and others have shown that eukaryotic cells are protected from the cytolytic C5b-9 h species-specific restriction of C5b-9 assembly and via elimination of membrane-inserted TCC. Cells exposed to limited C5b-9 are therefore survive, but with cell phenotype alteration. Sublytic C5b-9 acts as a mitogen, and this is shown by induction of proto-oncogenes and increased DNA synthesis with cell growth. The Ras, Raf-1, and ERK1 pathway is activated by TCC. and this is one pathway transducing signaling to nucleus. We have cloned a novel gene RGC-32 (Response Gene to Complement), which is activated by C5b-9 and involved in cell cycle. RGC-32 overexpression increased DNA synthesis. RGC-32 complexes with cdc2 in cells, which is increased by C5b-9. In cell-free systems, the cdc2 kinase activity is markedly increased by RGC-32. In terminally differentiated cells such as oligodendrocytes, C5b-9 increases cell survival without cell growth, through inhibition of apoptosis. Our preliminary data indicated both pro- and anti-apoptotic BCL-2 family proteins are regulated by C5b-9. These novel functions of C5b-9 may be involved in cell phenotype alteration during inflammation and immune reactions. Two specific hypotheses are proposed. Aim 1. RGC-32 is involved in cell cycle activation through regulation of cell cycle kinases. To test this, we will examine (i) DNA synthesis and cell growth affected by overexpression of RGC-32 and by inhibition of endogenous RGC-32, (ii) the binding to and activation of cdc2 by using mutated and deleted recombinant RGC-32 proteins, and (iii) the role of TCC signaling on the activity of RGC-32. Aim 2. C5b-9 increases survival of oligodendrocytes by inhibiting mitochondrial pathway apoptosis. This hypothesis will be explored by identifying the steps of mitochondrial pathway apoptosis affected by C5b-9, and by investigating C5b-9-mediated regulation of the pro- and anti-apoptotic BCL-2 family of proteins.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The Clinical Radiation Therapy Research Center has four sections: Clinical Radiation Oncology, Nuclear Medicine, Radiologic Physics and the Cancer Research Unit. It has a number of overall objectives: one is to provide optimal treatment to all cancer patients, and another is to participate with the Radiation Therapy Oncology Group in various clinical trials to develop new concepts of treatment through basic and clinical research. The Cancer Research Unit, which is the basic research section, has four groups: Cancer Biology, Cancer Immunology, Experimental Oncotherapy and Viral Oncogenesis. Research of all four groups is closely integrated and is primarily directed towards improved cancer treatment through obtaining a better understanding of the biological factors involved in the response of normal and malignant cell populations to treatment modalities. The experimental systems developed over the year provide the opportunity for obtaining quantitative information at the biochemical, cellular, tissue and animal levels. These systems include the gastrointestinal epithelium, hematopoietic & immunological systems, several transplantable tumor cell lines, a viral leukemia, and the PDP assay for growth fraction measurements in experimental and human tumors. Cancer treatment regimens utilizing multi-fraction irradiation, chemotherapeutic agents, immune stimulation and combined modality therapy are being quantitatively evaluated. It is felt that an increased understanding of the cellular and tissue events occurring in both normal and malignant tissues, initially unperturbed and following treatment with currently available modalities, will provide a basis for planning cancer treatment regimens with a rational a priori input. BIBLIOGRAPHIC REFERENCES: Concannon, J.P. Carcinoma of the Cervix. University of Pennsylvania Oncology Course, December 1977. Concannon, J.P. Immunity and Lung Cancer, Indian Congress of Radiology, Chandiagarh, India, January 1977.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Microscopic colitis (MC) is a chronic condition that is a common cause of watery diarrhea, particularly in the elderly. The etiology is unknown but widely considered to be an abnormal immune reaction to luminal antigens in predisposed hosts. Drugs and autoimmunity have also been implicated. Although the incidence is comparable to ulcerative colitis and Crohn's disease, there are currently no funded NIH studies of microscopic colitis. In order to advance our understanding of MC, the proposed study will include rigorously and quantitatively defined cases, adequate controls, and extensive exposure information. The aims of the study are: 1) To quantitatively classify microscopic colitis using image analysis microscopy to determine whether the degree of lymphocytic infiltration correlates with etiology, symptoms and prognosis. 2) To investigate the etiology of microscopic colitis by examining medical and lifestyle risk factors including medications, autoimmunity, diet, and smoking 3) To evaluate the association between the adherent microbial flora and MC to assess whether bacterial dysbiosis is linked to presence of MC. As an exploratory aim we will evaluate whether CYP2C19 polymorphisms are more common in purportedly drug-induced disease since the diverse drugs that have been associated with MC are all substrates for this gene. To conduct the study we will obtain detailed dietary, medical and lifestyle information on study subjects who undergo complete colonoscopy for diarrhea. We will obtain colon biopsies from the right, transverse and left colon to evaluate adherent bacterial organisms. We will draw blood to evaluate CYP2C19 polymorphisms and for future genetic studies. The prospective design corrects important limitations of prior research on MC. The proposed study will use methods for colonoscopy-based studies that we have perfected over two decades. Successful completion of the study aims will improve our understanding of risk factors, set the stage for more scientificall grounded future research, and potentially suggest new interventions for a disease that is currently poorly understood.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Diseases affecting the lung's epithelium are not easily treatable and result in significant morbidity and mortality worldwide. Specialized stem cells with the potential to self-renew or give rise to differentiated, functional progeny have been proposed as a critical component of tissue homeostasis for many organs, including the lung. Different potential approaches for the use of stem cells for lung disease treatment include enhancement of endogenous stem cell differentiation or in vitro directed differentiation of stem cells to lung lineages followed by cell transplantation. Both approaches require that the identity and pathways of differentiation of lung stem or progenitor cells be known and well characterized. Embryonic stem (ES) cells have emerged in the last 10-15 years as a promising platform for the development of cell-based therapies, since they can transit through several defined stages in vitro to recapitulate mammalian development. In addition, induced pluripotent stem (iPS) cells offer an attractive alternative to human ES cells. iPS cells are easy to derive, are not fraught with ethical issues and offer the possibility of patient-specific therapies. Nevertheless, the presumptive lung progenitor cells in development have not yet been identified, and this represents a major hurdle limiting the use of ES/iPS cells for lung disease therapies. The overall objective of this project is to define the genetic program of the multipotent epithelial progenitors of the lung primordium. This represents the first step towards our long-term goal of developing cell-based therapies for diseases affecting the lung epithelium. Since the transcription factor Titf1 is the earliest known marker of lung development, we created a new genetic tool, the Titf1-GFP knock-in mouse. This mouse will be used to characterize the progenitor cells of the lungs and the thyroid, both Titf1-positive and of endodermal origin. Using this tool, we will be able in Aim 1 to isolate cells from lung primordium based on GFP fluorescence, define their genetic program by means of DNA microarrays and study the epigenetics of genes important in lung development. We will proceed in Aim 2 to examine the role of FGF and Wnt signaling in lung specification in vitro using the Titf1-GFP ES and iPS cell lines. FGF and Wnt ligands are known to induce lung fate within definitive endoderm in vivo and we will assess whether they have a similar function in vitro. In Aim 3, we will test the functionality of the in vitro derived lung progenitors using novel and established systems, such as a bioartificial lung and air-liquid interface culture respectively. We envision that the outcome of our studies will be to define the optimal protocol for derivation of lung progenitor cells from ES/iPS cells to be used in novel lung disease therapies.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Dr. Feroz R. Papa is an endocrinologist, strongly committed to an academic career as an independent investigator in (-cell molecular biology. His specific interest is the role of endoplasmic reticulum (ER) stress in development of diabetes mellitus type 2 (DM2). Accordingly, he has initiated a postdoctoral fellowship in the lab of Dr. Peter Walter, in the Dept. of Biochemistry and Biophysics at UCSF, studying the Unfolded Protein Response (UPR), a homeostatic mechanism through which eukaryotic cells adjust to ER stress. ER stress occurs when there is an imbalance between the load of client proteins undergoing maturation in the ER lumen and the capacity of the ER to process this load. Pathophysiological conditions provoking ER stress include anoxia, nutrient deprivation, and polypeptide mutations, which result in protein misfolding. The UPR remodels the environment of the ER to respond to ER stress through two mechanisms: 1) a transcriptional upregulation of genes encoding ER chaperones, enzymatic activities, and structural ER components, and 2) a global translational halt, which reduces the load of ER client proteins. The ER-transmembrane kinase/endoribonuclease Ire1alpha is the sensor for the transcriptional arm of the UPR, and the ER-transmembrane elF-2alpha kinase Perk is the sensor for the translational arm. Dr. Papa is proposing a research program which will address the hypothesis that modulating the UPR, in real time using small molecule effectors, can influence the function of beta-cells, and ultimately their survival. He will address this hypothesis through three specific aims. First, using a chemical-genetic strategy, he will sensitize Ire1alpha by gene targeting of IRE1alpha to a cell-permeable ATP analog, 1-NM PP1, which he has demonstrated is a permissive co-factor for signaling by sensitized Ire1 in yeast. Beta-cell-specific gene targeting would render this tissue unable to signal the transcriptional arm of the UPR in the absence of 1NM-PP1 Second, similar gene targeting of PERK will be conducted, conferring pharmacological conditionality on the translational arm of the UPR. Third, the biological consequences of these manipulations on beta-cells will be studied in these knock-in mutants, and in the genetic background of three other mouse mutants which develop DM2 as a consequence of unchecked ER stress: Akita, hlAPP, and perk-/-. Together this work promises to yield important insights into how professional secretory tissues (including, but not limited to beta-cells) adapt to physiological and pathophysiological stresses. Dr. Papa will conduct this work in the Dept. of Biochemistry and Biophysics at UCSF under the mentorship of Dr. Peter Walter, and in collaboration with Dr. Robert Farese, Jr. in the Gladstone Institute of Cardiovascular Disease, and Dr. Kevan Shokat in the Department of Cellular and Molecular Pharmacology. The UCSF Department of Medicine is fully committed to Dr. Papa's career development, and will make all necessary resources available to facilitate the success of the project.
|
{
"pile_set_name": "NIH ExPorter"
}
|
DESCRIPTION OF THE PROJECT AS A WHOLE (adapted from the application): This is an application for a U01 continuation, requesting 2.5 years of continued funding for the Hebrew Home for the Aged CC for the NIA collaborative studies of SCUs for AD. In 1991, the applicant was awarded grant support from the NIA as one of 10 cooperative studies of special dementia care units and received additional support to act as the CC to all 10 studies. In 1995, 2-years of continuation funding was provided to aggregate and process the common-core data, and to plan the cross-site analyses. The applicant was advised to resubmit the application for the meta analytic component after strengthening the hypotheses, performing pilot analyses and providing more detailed analytic plans. The proposed continuation will undertake analyses of data developed from the 10 collaborating studies, the coordination of both analyses and publications, and the ultimate archiving of the projects data. The application requests funding to permit: (1) the application of psychometric analysis to later waves of data collection for comparison to results of baseline analyses; (2) examination of cross-site and longitudinal outcomes associated with the provision of special dementia care; (3) application of meta analyses; and (4) final preparation of data for submission for archival, public-use.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This proposal outlines a 5 year plan for the training of this candidate for an academic career as a Transfusion Medicine Virologist. She has completed 3 years of formal residency training in Clinical Pathology, and is now working in her sponsor's laboratory. Her research interests center upon HIV-1 molecular pathogenesis, in particular, the regulation of HIV-1 replication in resting and activated T cells. She has a strong background in cellular immunology, having received a Ph.D. in this field under Dr. Ralph M. Steinman in the Department of Cellular Physiology and Immunology at The Rockefeller University. This experience poises her to investigate viral infection in primary leukocytes; however, she lacks the experience in HIV molecular biology necessary to attack questions of regulation independently. An integrative research and training program for the Principal Investigator is being established within the Department of Pathology. The candidate's sponsors are Michael H. Malim and Francisco Gonzalez- Scarano, both recognized authorities on molecular HIV-1 pathogenesis. In addition, an advisory board of prominent scientists will insure the candidate's development. While all steps of the virus life cycle offer opportunities for regulation, it is her intention to examine events that occur between fusion at the plasma membrane and integration of viral DNA into cellular DNA. One reason for pursuing this path is that these aspects of HIV replication have received little attention to date. In addition, there is evidence that infection of T cells is blocked at an early step, since 1 in 100 T cells from infected individuals contain HIV DNA, but only 1 in 10,000 contain integrated HIV DNA. A greater understanding of these critical early steps is likely to identify novel virus-host cell interactions that may serve as future targets for therapeutic consideration. She describes three specific aims: (1) measuring HIV uptake by quiescent T cells; (2) examining the kinetics of viral transport to and through nuclear pore complexes in quiescent and activated T cells; and (3) determining the changing composition of post-entry nucleoprotein complexes in resting and activated T cells. These unprecedented studies promise to provide critical new insights into the HIV disease process. Concurrently, Dr. O'Doherty will acquire the expertise necessary for her to become an independent Transfusion Medicine Virologist.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Prematurity is the leading cause of neonatal morbidity and mortality in the United States. Intrauterine infections are an important, and potentially treatable cause of prematurity, and are associated with increased risk of neonatal white matter lesions of the brain and cerebral palsy. However, the mechanisms by which infection leads to prematurity and/or cerebral palsy remain speculative and treatment strategies untested largely because humans cannot be longitudinally studied following infection. We propose to use chronically instrumented pregnant rhesus monkeys at 120-130 day gestation with experimental intrauterine infection, as previously described (Gravett et al, Am J Obstet and Gynecol; 171:1660-1667,1994) to study the temporal and quantitative relationships among infection, cytokines, prostaglandins, steroid hormones, cytokine antagonists, preterm labor, and neonatal white matter lesions of the brain in order to develop effective interventional strategies. After postoperative stabilization in a tether, we will; (1) inoculate Group B Streptococci (GBS) into the amniotic fluid to establish intrauterine infection and preterm labor. Uterine contractility will be continuously monitored and periodic samples of amniotic fluid and maternal and fetal blood (1-4 cc) will be obtained for assays of eicosanoids, steroid hormones, cytokines, matrix metalloproteinases and for microbial studies; (2) utilize antibiotics with and without potent inhibitors of proinflammatory cytokine production (dexamethasone,IL-10) o prostaglandin production (indomethacin) to ascertain the most effective intervention to down-regulate the cytokine/prostaglandin cascade and associated uterine activity; (3) infuse proinflammatory cytokine IL-1beta into the amniotic cavity through indwelling catheters in the absence of infection. Prior to infusion of IL-1beta in the absence of infection, specific novel proinflammatory cytokine inhibitors (IL-1ra and sTNF-R1 PEG) will be used to identify other potentially useful immunomodulators. Samples of the decidua, fetal membranes, tissues, and brain will be obtained at cesarean section for microbiologic, histopathologic studies, immunohistochemistry for cytokines, localization and quantitation of mRNA for cytokines and PGHS-2. Fetal brain will be examined for increased apoptosis associated with white matter lesions. Leukocytes in amniotic fluid and tracheal aspirates will be assessed by flow cytometry Postpartum, the mother will be treated with appropriate antibiotics to eradicate the GBS from the genital tract and returned to the colony. These studies will clarify the pathophysiology of infection-associated preterm labor and will suggest effective interventional strategies.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Two interrelated models have been presented which describe 1) the regulation of surface growth of Streptococcus faecalis by chromosome replication; and 2) a shape-determining mechanism which directs the assembly of envelope precursors into new cell surface. The first model provides that during a round of chromosome synthesis a signal is produced which results in the formation of a growth zone. This site, once formed enlarges the cell surface until a second signal, produced during the terminal stages of chromosome replication, inhibits enlargement and promotes septation. The second model is concerned with the construction and function of the growth zone once it has been formed. This model suggests that surface growth proceeds by a regulated flow of cell wall precursors into a growth zone essentially along two channels. One channel would assemble a bilayered cross wall (possibly by the addition of precursors to the leading edge of the cross wall), while a second channel of precursors would convert this cross wall into two layers of peripheral wall. Peripheral wall formation would be mediated by precursors being used to expand the surface area and increase the thickness of the cross wall layers as they separate into peripheral wall. Under the influence of the chromosome termination signal, the flow of precursors along this second channel would be progressively reduced in the latter portion of the cycle. This reduction coupled with the continued flow of precursors into cross wall synthesis would result in cell division. In attempting to test many aspects of the above models, methods have been devised which can compare the geometry of the envelope with the macromolecular content and autolytic activity of cells. BIBLIOGRAPHIC REFERENCES: Higgins, M.L. Three-dimensional reconstructions of whole cells of Streptococcus faecalis from thin-sections of cells. J. Bacteriol. 127:1337, (1976). Higgins, M.L. and Shockman, G.D. Study of a cycle of cell wall assembly in Streptococcus faecalis by three-dimensional reconstruction of thin-sections of cells. J. Bacteriol. 127:1346, (1976).
|
{
"pile_set_name": "NIH ExPorter"
}
|
Sialyltransferase has been found to be elevated in the sera of breast cancer patients. Specific cancer associated isoenzymes may be present which could be used as markers for breast cancer. Our initial studies using preparative flat bed isoelectric focusing did not indicate any novel isoenzymes of sialyltransferase in sera from breast cancer patients. Preliminary results have indicated two major serum sialytransferase enzymes with PI's of 4.9 and 5.3 in normals and cancer patients. However, in those studies a significant amount of enzyme activity was not recovered from the plates. Therefore, several new techniques will be attempted to increase recovery of enzyme activity while still maintaining good separation and fine resolution. These proposed studies include use of photoaffinity labeled substrates and new techniques in column isoelectric focusing. These analyses should provide definitive information on the usefullness of serum sialyltransferase isoenzyme assays in breast cancer. Other studies will be performed which will assess the serum sialyltransferase inhibitory activity of several nucleoside-sugar antibiotics. The chemotherapeutic potential of these compounds and other N6-substituted adenosine analogs will also be evaluated in rodents with several transplantable mammary tumors. These studies should lead to the development of better therapeutic strategies in the diagnosis and treatment of breast cancer.
|
{
"pile_set_name": "NIH ExPorter"
}
|
PROJECT SUMMARY Cochlear implants (CIs) promote spoken language development in children with bilateral severe-to-profound sensorineural hearing loss. Yet, many of these children do not obtain age-appropriate language proficiency: children with CIs are typically slower at processing spoken language input and generally have less spoken language experience than chronologically age-matched peers with normal hearing. Delayed language abilities have significant consequences for children's performance in everyday environments, which often require children to process continuous speech rapidly. Rapid speech processing requires instantaneous lexical access, or the ability to retrieve stored representations of words that are contained in spoken input. Instantaneous lexical access may be altered in children with CIs, not only due to their history of auditory and language deprivation, but also due to the inability of CIs to fully restore a high fidelity representation of speech. An inability of children with CIs to process language rapidly may be a source of the variability in language and academic outcomes within this population. To better maximize outcomes in children with CIs, there is a need to understand how lexical access is different in children with CIs. However, a real-time measure that quantifies spoken lexical access moment-by-moment in children with CIs is not established. The objective of the proposed project is to explore how children's hearing ability impacts instantaneous lexical access. Participants will be 92 native English-speaking children who are 6-to-10 years old (n = 36 with bilateral sensorineural hearing loss who use CIs; n = 36 with normal hearing; an additional 20 children with normal hearing will be recruited for stimuli norming). The proposed research will examine two cues helpful in lexical access: phonotactic knowledge (i.e., knowledge of how phonemes are sequenced within a word) and conceptual (i.e., semantic) knowledge. Aim 1 will test children's ability to use phonotactic cues during a classification task based on phonotactic probability. The proposed experiment will test children's ability to discriminate between words that have varying phonotactic probabilities based on the English language (Exp. 1.1). In addition, aim 1 will also test the ability of children to use phonotactic cues to predict word identity in a visual world paradigm (Exp. 1.2). Aim 2 will test children's reliance on conceptual (i.e., semantic) cues on lexical access. Using a modified version of the visual world paradigm from Exp. 1.2, this proposal will test whether semantically related images facilitate lexical access (Exp. 2). All experiments will implement integrated eye-tracking and touch-screen technology, which will allow us to both quantify task accuracy as well as observe the time course of lexical access in children. Findings from this research will establish a foundation of knowledge that will help us further understand how prior hearing and linguistic experience impacts everyday speech comprehension. Future studies will be able to capitalize on this knowledge to assist children with CIs in critical stages of their development.
|
{
"pile_set_name": "NIH ExPorter"
}
|
A goal of these studies is to better understand the molecular mechanisms underlying human nervous system development and function, as well as the pathogenesis of certain inherited brain disorders. Our studies have focused on the structural and active-site properties of proteins found in the nervous system including neurotrophic peptides/proteins, lysosomal hydrolases, and other proteins/peptides, which interact with excitable membranes, receptors and venom toxins. Proteins from human and animal tissues are purified by liquid chromatography (ion-exchange, gel permeation and affinity techniques), high performance liquid chromatography (HPLC) and electrophoretic separation. State-of-the-art micro sequencing analysis (gas-phase, liquid-phase and solid-phase), surface enhanced laser desorption ionization (SELDI) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) are performed to characterize proteins. Both isoelectric-focusing and molecular weight determination of Pico mole amounts of proteins are carried out by capillary-zone electrophoresis. Peptide maps of both normal and mutant proteins are generated using chemical (mild acid, cyanogen bromide) and enzymatic (trypsin, AspN, V8 proteases) cleavage and analyzed by HPLC. Several novel scorpion venom neurotoxins, which interact with synaptosomal structures such as human calcium and potassium channels in excitable membranes, are being studied. We are also investigating the association of amyloid peptides with both high and low-density lipoproteins from cerebrospinal fluid in Alzheimer's disease. Our research emphasis is on studies that define post-translational processes not identifiable by DNA sequence analysis, e.g. carbohydrate attachment, protease cleavage, and phosphorylation that could be responsible for abnormal brain function. By a combination of methods described above, we have identified molecular forms of cystatin C in CSF, which is up regulated in pain and also found in the urine of people with varying degrees of renal dysfunction. We are evaluating several novel approaches for the identification of Bio Markers for psychiatric disorders as well as cancer. We have also discovered a simple rapid method for the recovery of peptides/proteins from tissue/cell samples that has been submitted as an employee invention report for assessment of patentability. Several new long chain potassium channel blocking peptides have been characterized from scorpion venom both directly from the protein and more recently by analysis of their cDNA sequence. Additionally, several novel defensin-like peptides have been characterized at both the peptide and cDNA level.
|
{
"pile_set_name": "NIH ExPorter"
}
|
CORE B: ADMINISTRATIVE CORE The overall goal of the Administrative Core is to provide administrative, fiscal and scientific management of this POl Program Project Grant. Key components of this Core will be to provide overall fiscal and administrative coordination. Additional objectives of this Core include organizing scientific interactions communications, and to coordinate interaction with NINDS.
|
{
"pile_set_name": "NIH ExPorter"
}
|
DESCRIPTION (provided by principal investigator): Proton pump inhibitors (PPIs) are among the most widely used medications. It is becoming increasingly common for patients to take these potent acid suppressants on a long-term and continuous basis for erosive esophagitis, Barrett's esophagus and protection against nonsteroidal anti-inflammatory drug-related gastropathy. PPI therapy leads to elevated serum gastrin levels and may impair the absorption of calcium and food-bound vitamin B12. PPI-induced hypergastrinemia has a direct trophic effect on the parathyroid glands, leading to parathyroid hyperplasia, increased parathyroid hormone secretion and bone loss. Furthermore, both calcium malabsorption and vitamin B12 deficiency are associated with reduced bone mineral density (BMD) and increased osteoporotic fracture risk. Consistent with these data, recent studies revealed a positive association between PPI therapy and the risk of osteoporotic fractures. Peripheral quantitative computed tomography (pQCT) can provide a three-dimensional structural analysis of trabecular and cortical volumetric BMD (vBMD) and dimensions. These data are imperative for a valid assessment of the effect of chronic PPI therapy on bone strength. We hypothesize that PPI therapy leads to decreased cortical and trabecular vBMD, cortical dimensions and bone strength. Our primary aims are: 1). To compare the changes over time in cortical and trabecular vBMD, cortical dimensions and estimates of bone strength between long-term PPI users and non-users;2). Among PPI users, to determine whether average PPI daily dose and extent of PPI adherence are predictors of greater loss in bone vBMD, dimensions and strength;3). To compare the changes over time in PTH levels between long-term PPI users and non-users. The proposed study will longitudinally measure radial and tibial trabecular and cortical vBMD and dimensions in two groups of subjects between 55 to 75 years of age recruited at the University of Pennsylvania Health System:1). PPI-na[unreadable]ve patients who are starting long-term continuous PPI therapy for newly diagnosed erosive esophagitis or Barrett's esophagus;2). PPI-non users frequency matched with the PPI users on race, age, sex and body mass index. The pQCT measurements will be obtained at baseline and then yearly over the next 3 years. Longitudinal regression analyses will be conducted using generalized estimating equations or quasi-least squares modeling to compare the changes in pQCT measures of bone strength between the two groups. This study addresses a novel and important clinical question that has a direct impact on the bone health of the enormous population of elderly PPI users. The results of the current study will help determine the need, the intensity, as well the appropriate choice of modality for monitoring BMD in this population and guide the development of effective strategies to prevent and/or reverse this adverse effect of acid suppressive therapy. PUBLIC HEALTH RELEVANCE: Proton pump inhibitors (PPIs) are among the most widely used medications. These potent stomach acid suppressants may reduce bone density and increase the risk of osteoporotic fractures. This study will assess the effect of PPI therapy on bone strength using a valid radiographic tool called peripheral quantitative computed tomography. This project addresses a novel and important clinical question that has a direct impact on the bone health of the millions of PPI users. The data generated by the current project will help determine the need, the intensity, as well the appropriate choice of modality for monitoring bone mineral density and bone structure among chronic PPI users and guide the development of effective strategies to prevent and/or reverse this adverse effect of acid suppressive therapy.
|
{
"pile_set_name": "NIH ExPorter"
}
|
PURPOSE: To investigate self-regulation of keratinocyte migratory function in the wound bed to understand how electric fields facilitate skin re- epithelization and learn to pharmacologically control this process. BACKGROUND: Closure of skin wounds involves the migration of keratinocytes into the wound bed. Keratinocyte migratory function includes a sequence of events that begins with detachment from neighboring cells, succeeds with locomotion to the wound, and ends with re-attachment to neighboring cells, and cell maturation. Multiple factors that influence this migratory function have been identified, but much less is known about how these influences are coordinated by wound keratinocyte. PRELIMINARY OBSERVATIONS: Human cultured and tissue keratinocytes possess all functional elements of a cholinergic system, the enzymes that synthesize or degrade acetylcholine and both the nicotinic and muscarinic types of acetylcholine receptors, and interference with acetylcholine production or reception affects migratory function of human keratinocytes in vitro. HYPOTHESES: Keratinocyte cholinergic system plays an auto-regulatory role in controlling cell functions essential to migration, and its mediator acetylcholine serves as a "pace maker" in coordinating cellular activities mediating migration. Electric fields exploit this physiologic phenomenon. Supporting these hypotheses are the use of autocrine and paracrine acetylcholine by a variety of cells types as a local hormone of cell motility; chemoattractive properties of acetylcholine; and exclusive migration of cells toward the cathode, which is the direction of movement of highly positively charged acetylcholine molecule in electric fields. EXPERIMENTAL DESIGN: To investigate: (1) physiologic gradient of acetylcholine in epidermis; (2) keratinocyte chemotaxis toward acetylcholine gradient in vitro; (3) cellular cholinergic mechanisms mediating self-regulation of keratinocyte migratory function; and (4) efficacies of cholinergic drugs to promote epidermalization of superficial skin wounds in laboratory animals. METHODOLOGY: The molecular biological, biochemical, pharmacological, and immunological techniques are integrated into an in vitro experimental model of skin re-epithelization that provides for direct observation of morphology and measurement of migration distances of keratinocytes stimulated by cholinergic drugs or DC electric-fields. Optimal doses of the cholinergic drug which is found to best reproduce stimulatory effects of acetylcholine and electric fields on keratinocyte migratory function will be administered to euthymic hairless guinea pigs with inflicted blister, burn or abrasive wounds, and the rate of epidermalization will be assayed by image analysis, evaporimetry, and histology. SIGNIFICANCE: These basic studies will provide new and useful information on mechanisms of pharmacological promotion and suppression of keratinocyte migration with cholinergic compounds, including mechanisms of wound failure under effects of nicotine.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Mammalian hosts, from inbred mouse strains to humans, display a wide range of phenotypic responses to various bacterial infections. One of the pathogens eliciting such differential responses in mice is Listena monocytogenes, a Gram-positive bacterium which is a common source of food poisoning. Previous studies have established that differences in immune response are often genetically controlled. Differences in immune response can be characterized using a variety of readouts, from gene expression changes to disease outcome. Currently, gene expression data is the most comprehensive way to describe a biological system. In addition, gene expression values can be used directly in genetic quantitative trait mapping experiments. Therefore, by monitoring differential transcriptional responses to infection, we can identify and characterize molecules critical for control of infection. To achieve this goal we will perform genome-wide characterization of gene expression profiles in differentially susceptible inbred mouse strains following infection with Listeria monocytogenes. To understand how changes in gene expression affect immune function, we have to differentiate between primary and secondary differences. Primary expression differences are caused by genetic polymorphisms at the gene locus itself, which directly affect the level of the gene transcript. Accordingly, secondary differences are those which reflect upstream functional or expression differences. We will use a novel Recombinant Inbred expression mapping approach to differentiate between primary and secondary changes in gene expression in mouse strains differentially susceptible to Listeria monocytogenes infection. Our pilot study will identify molecules differentially regulated in response to infection. Furthermore, it will allow us to refine our methodology for a future comprehensive study of changes in gene expression in differentially susceptible hosts over the course of the infectious disease. Identification of primary gene expression changes will lead to identification of genetic polymorphisms controlling gene expression. In addition, these primary expression differences will serve as starting points for building a multilayer map of transcriptional responses to infection. We hope to use this map to identify novel therapeutic targets for treatment of infectious diseases.
|
{
"pile_set_name": "NIH ExPorter"
}
|
In patients with lung injury and pulmonary edema, alveolar gas exchange is impaired which results in hypoxemia. We have previously reported that hypoxia impairs the lung's ability to clear edema by inhibiting the alveolar epithelial Na,K-ATPase. The focus of this application is to determine the mechanisms regulating the effects of severe hypoxia: 1.5%, 3% or 5% (pO2~10 to 40 mm Hg) on alveolar epithelial function focusing on the regulation of Na,K-ATPase endocytosis and degradation. In the previous cycle of the grant, we reported that in alveolar epithelial cells (AEC) exposed to hypoxia the plasma membrane Na,K-ATPase was rapidly degraded, while the degradation of the intracellular Na,K-ATPase molecules was kept at steady state. We now have preliminary data to suggest that this apparent discrepancy is related to the "acute versus chronic" effects of hypoxia and cell adaptation via the hypoxia inducible factor a (HIF1a). Thus, we propose to dissect the mechanisms that regulate the effects of acute and prolonged hypoxia. We will determine whether a brief exposure of AEC to hypoxia results in mitochondrial reactive oxygen species mediated phosphorylation of the AMP Kinase leading to activation of protein kinase C zeta (PKCQ and Na,K-ATPase downregulation. We reason that prolonged exposure to hypoxia results in cell adaptation via a HIF1amediated mechanism in which the downregulation of the PKC[unreadable] prevents further endocytosis/degradation of the Na,K-ATPase. We will study the effects of hypoxia on the alveolar epithelium via three interrelated aims: in Specific Aim # 1 we propose to determine whether hypoxia activates AMPK and its role in the regulation of alveolar epithelial Na,K-ATPase and fluid reabsorption;in Specific Aim # 2 we will study whether the HIF1a ubiquitin ligase, von Hippel Lindau protein (pVHL), regulates Na,K-ATPase endocytosis/degradation during hypoxia;and in Specific Aim # 3 we will determine whether HIF1a stabilization leads to PKC[unreadable] ubiquitination and degradation as a mechanism of regulating total cell Na,K-ATPase levels and thus, alveolar epithelial function. Experiments have been conducted for each of the specific aims and the preliminary results support the feasibility and importance of this proposal.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The existence of a dietary requirement for fatty acids of the omega-3 or Alpha-linolenic acid family has been an unresolved issue in human nutrition. Like the omega-6 or linoleic acid family, omega-3 fatty acids cannot be synthesized by animals. The high content of these fatty acids, particularly docosahexaenoic acid (22:6 omega-3 or DHA), in the phospholipids of the retina and cerebral cortex suggests an important role in the function of neuronal and photoreceptor membranes. We have now produced omega-3 fatty acid deficiency in rhesus monkeys by dietary deprivation throughout gestation and infancy. The deficiency state is characterized by both biochemical and functional changes. The plasma and tissues exhibit reduced levels of omega-3 fatty acids from birth to two years of age. Levels of DHA are greatly reduced in brain and retina. Visual defects and abnormal electroretinograms occur in deficient animals. We now propose to continue studies of existing omega-3 fatty acid deficient monkeys to investigate these questions: 1. Does the deficient state produce increasing manifestations of visual loss with increased age? 2. Do juvenile deficient animals show impairments in tests of complex learning? 3. Will the correction of the omega-3 fatty acid deficient diet with DHA or linolenic acid correct both the functional and biochemical abnormalities of deficient monkeys? In addition, we propose to begin breeding a new group of deficient adult females in order to investigate the following questions: 4. How rapidly will infants deficient at birth regain normal levels of DHA after dietary repletion with dietary omega-3 fatty acids (DHA or linolenic acid)? Will visual loss then be avoided? These studies will help determine the nutritional needs of primates for omega-3 fatty acids during different stages of development. This work has implications for the optimum diet of pregnant and lactating women, human infants, and perhaps of older humans. Finally, these studies will provide information about the biochemistry of the developing primate brain.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The major focus of this study is to determine if healthy individuals over the age of 80 yrs can respond to endurance exercise training with cardiovascular (CV) adaptations similar to those seen in younger individuals. A secondary focus is to determine whether such CV adaptations are gender specific. Maximal oxygen uptake (VO2max), which is commonly used to quantify the adaptive response to endurance exercise training, is the primary outcome. Ongoing studies from our Claude Pepper Older Americans Independence Center suggest only minimal increases (0.10 L min-1 or from 17.0 to 18.5 ml kg-1-min-1) in VO2max in frail elderly adults (78+ yrs) in response to 3 mo of endurance exercise. However, it is not possible to determine if the failure of the exercise program to induce a larger increase in VO2max is due to the age of the subjects, their frailty, or the relatively short duration of the training program. Therefore, this study will use an experimental control-group design and a 9-mo endurance exercise program and the primary outcome, VO2max, will be assessed at baseline 3 mo, 6 mo and 9 mo. The specific aims are to determine if endurance exercise training in healthy men and women greater than 80 yrs old invokes physiologically significant increases in VO2max and to: a) determine the contributions of central and peripheral mechanisms responsible for any increase in VO2max by measuring maximal cardiac output, calculating stroke volume and oxygen extraction, and assessing skeletal muscle histochemical and enzymatic adaptations; b) further investigate CV adaptations by determining if endurance training increases left ventricular size (preload), or reduces arterial stiffness or enhances endothelial function (afterload).
|
{
"pile_set_name": "NIH ExPorter"
}
|
The purposes of the proposed studies are to: 1) compare isogravimetric capillary hydrostatic pressures in skin and skeletal muscle of the horse and dog; 2) determine the effect of histamine on capillary pressure in the same tissue; 3) determine the effect of local histamine on microvascular permeability of skin and skeletal muscle in both horse and dog; and 4) determine if catecholamines, time and/or histamine blood concentrations are factors in the permeability effect of histamine on the microvasculature. Studies will be conducted on the horse gracilis muscle and digit and the dog gracilis muscle and forelimb. Both in vivo and in vitro studies will be conducted. All studies will utilize constant pressure perfusion. Uptake and washout of radioactive 131I albumin and 86Rb Cl in maximally dilated vascular beds will be used to determine permeability.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Allergic asthma is a syndrome characterized by eosinophilic airway inflammation associated with airway hyperresponsiveness (AHR) and increased mucus production. CD4+ Th2 cells play a critical role in controlling the inflammation seen in asthma; however, the molecular mechanisms that control Th2 cell entry into the lung and the factors that determine whether these cells stay in the lung or leave are not completely known. In the last funding period, we have made considerable progress in answering these questions, which has led us to the central hypothesis of this renewal proposal: Th2 cell trafficking into the allergic lung is controlled sequentially by several discrete regulatory pathways. Here we propose that Th2 cell accumulation in the allergic lung is controlled by at least four discrete steps that include an Initiation Phase, a Propagation Phase, an Amplification Phase and a Resolution Phase. Based on our published and preliminary data, we hypothesize that Th2 cell recruitment into the asthmatic lung is initiated by the activation of mast cells and the release of leukotriene B4 and chemokines. This is followed by a Propagation Phase that is mediated by activation of Toll-like receptors (TLRs) on innate immune cells in the lung and the release of TLR- inducible chemokines. Once Th2 cells enter the lung, we propose an Amplification Phase that is mediated by IL-4 and IL-13 released from Th2 cells that induces the production of STAT6-inducible chemokines from resident pulmonary cells. We propose that the resolution of allergic inflammation is then mediated in part by the recruitment of Th2 cells out of the lung via the afferent lymphatics into the draining lymph node, mediated by CCR7 Th2 cell expression. We also propose that this egress pathway will contribute to Th2 immunologic memory. In this proposal, we will use a combination of transgenic and knockout mice in several adoptive transfer models of murine asthma as well as the study of human patients with allergic asthma to test these hypotheses. Specifically we propose: (1) To determine the role of mast cell-derived chemoattractants in the initial recruitment of antigen-specific Th2 cells into the allergic lung; (2) To determine if TLR-inducible chemokines contribute to the propagation of Th2 cell recruitment into the allergic lung; (3) To identify the key hematopoietic-derived cell that mediates STAT6-dependent amplification of Th2 cell recruitment into the allergic lung; (4) To determine the contribution of CCR7-mediated T cell egress from the asthmatic lung in the resolution of pulmonary inflammation and the establishment and maintenance of Th2 cell memory; and (5) To identify the mediators that control Th2 cell trafficking into the human lung early and late following Ag challenge. The identification of the relevant chemoattractant receptors that control Th2 cell entry into and exit from the allergic lung will not only provide key insight into the asthma pathogenesis but will also define excellent targets for therapeutic intervention. Project Narrative: Asthma is an allergic disease of the lung that is increasing in prevalence and severity, especially among children, despite current therapies. Asthma is caused by the inappropriate activation of T cells by normally innocuous aerosolized antigens. Once activated, these T cells accumulate in the lung, where they drive the asthmatic response through the release of potent inflammatory mediators. The goal of this grant is to identify the key proteins that control the accumulation of these pathogenic T cells in the lung, which will define excellent new targets for therapeutic intervention.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The Science Core exists to provide reagents and services to the various projects within this Program project grant, and to the Neuropathology and Animal cores. The need for specific reagents and services are often common to multiple projects and cores, as they are for the projects on other Program project grants. The most efficient method of operation for providing reagents and services requiring complex, expensive equipment is therefore via a central core with a highly skilled staff. For this renewal ofthe Program project, the Science core will provide recombinant mouse PrP (with and without isotopic labels) for the generation of synthetic mammalian prions, and anti-PrP antibodies, for Project 1. Recombinant Sup35 will be produced for Project 2, in addition to constructs of Sup35 for development of transgenic mouse lines. The Science core will provide fluorescence activated cell sorting, and prepration of various A-beta aggregates (both synthetic and extracted from natural sources), for Project 3. Anti-PrP antibodies including recombinant Fabs will be supplied to the Neuropathogy core (Core C). The Animal core (Core D) will be supplied with isotopically labeled mouse diet in support of Project 1, and screening of all transgenic lines in suport of Projects 1, 2 and 3. Leadership for the Core will be provided by Dr. Kurt Giles, and Assistant Professor with over 15 years experience in neurodegenerative disease research, and by Dr Michael Silber, a Professor with over 30 years industrial and academic experience, who will act as co-director. Drs. Giles and Silber will be assisted by other scientists and research associates to provide the above functions.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This renewal of "Smoking Prevention Through Mass Media and School Programs: (CA38395) provides for further follow-up of students exposed to the educational interventions and further data analyses. The project was funded through the Smoking, Tobacco, and Cancer Program of the Division of Cancer Prevention and Control in 1984 as part of a group of studies evaluating the educational use of mass media in control of tobacco use. Our major question was to test the ability of mass media interventions to add to the established efficacy of school smoking prevention programs. In the original design, two matched pairs of communities were systematically selected to provide student samples. Within these SMSAs specific school districts and feeder systems were chosen to provide a closer match on demographic characteristics. The inception cohorts of 5458 students in Grades 4,5, and 6 were established in Binghamton, NY and Great FAlls, MT, and in Burlington, VT and Billings, MT in Spring, 1985. Those in the first community pair then received four years of the school program only. Evaluation was accomplished through annual surveys of students which combined self- response with saliva collection procedures to enhance reports of tobacco use. Annual surveys were conducted through Grade 8,9, and 10. Data from these surveys were linked for individual students from year to year. Preliminary results from the five-year linked data sets show that the mass media program appeared to provide an advantage in preventing cigarette smoking, as measured by the Smoking Behavior Index or prevalence measures, especially among girls. This apparent advantage also was reflected in a series of variables which were theoretically linked to this outcome as mediators of program effect. Substance use behaviors not targeted by these interventions showed no similar advantage for the experimental group. For this renewal we will follow-up all students from the inception cohort through school and telephone surveys to collect data on smoking behavior and other indicators of program impact when the main group of this cohort is in Grades 10,11, and 12 during 1991. School surveys, for those who have remained in their original school districts, will be conducted by the team which conducted the earlier surveys. Telephone follow-up, for those who transferred to another school district or dropped out of school, will be conducted by a survey research group from the University of Minnesota. The four studies will accomplish assessment of impact of the interventions two years after their completion on 1) those who were exposed to the complete interventions; 2) those exposed to a part of the interventions because they transferred to another school district; 3) those who dropped out of school; and 4) the complete inception cohort. To accomplish these objectives, we have assembled a multi-disciplinary team of investigators and consultants with expertise in public health, human development, health education, mass media, biostatistics, and survey research.
|
{
"pile_set_name": "NIH ExPorter"
}
|
An organism's ability to detect and respond to environmental stimuli is critical for its survival. It is particularly important for animals to incorporate contextual information, such as internal state or external cues, in order to modify behavioral responses and maximize fitness. This behavioral plasticity has largely been studied in the context of learning or adaptation. However, how environmental cues modulate the valence of innate responses to nave stimuli remains poorly understood. Behavioral plasticity can stem from genetic, molecular, and circuit level changes in neural function, ultimately leading to an organism's ability to thrive in a dynamic environment. The overall goal of my thesis is to investigate the molecular and neuronal mechanisms underlying olfactory plasticity in C. elegans. Preliminarily, I have found that while C. elegans grown under sparse culture conditions avoid high concentrations of the volatile chemical 1-hexanol, animals grown at high population density are instead robustly attracted to this odorant. My results indicate that this plasticity in olfactoy responses is mediated by pheromones, which may serve as a population density cue. I will exploit the experimental amenability of C. elegans to identify the genes, neurons and circuits that underlie the responses to 1-hexanol, and investigate how these responses are modified by contextual cues and experience. To do so, I will utilize a novel quantitative approach and high-resolution data analysis algorithms. Insights from this research will shed light on conserved molecular and neuronal pathways involved in sensory plasticity. Many neurological disorders-from developmental disorders such as autism to neurodegenerative diseases such as Alzheimer's and Parkinson's-stem from underlying deficits in neuroplasticity. Therefore, in addition to understanding the mechanisms of plasticity in a healthy context, this work has important implications for understanding the mechanisms underlying disease states.
|
{
"pile_set_name": "NIH ExPorter"
}
|
We have examined the effect of FoxP3 on class I gene expression both in vivo and in cell culture. In cell culture assays, FoxP3 has a dramatic effect on MHC class I promoter activity. However, the effect is cell type dependent. Thus, FoxP3 enhances promoter activity in Jurkat T cells but inhibits it in Hela epithelial cells. Although only a single canonical FoxP3 binding site occurs within the promoter, multiple response elements have been mapped in both cell types. Intriguingly, the single FoxP3 binding site is located within the IRE of the promoter, such that mutation of the IRE abrogates the FoxP3 response. Furthermore, although FoxP3 is known to interact with RUNX1 and RUNX1 enhances class I promoter activity, the two factors function independently. Most intriguingly and consistent with the cell culture findings, class I expression in Tregs in vivo is elevated relative to the CD4+CD25- T cell subset. The functional significance of this over-expression, if any, is being pursued.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Project Summary Cardiovascular disease is responsible for more than 600,000 deaths each year in the US, and more than half of these are due to coronary heart disease (CHD). Myocardial perfusion imaging (MPI) is one of the most effective tools for assessing CHD, and according to the most recent estimate, this technique accounts for >50% of all nuclear medicine studies in adults, nearly 6 million scans in 2014. At present, most of these studies are carried out using 99mTc-MIBI and 99mTc-tetrofosmin, despite the significant limitations of SPECT (single photon emission computed tomography) for MPI including lower spatial resolution than PET (positron-emission tomography); the lack of routine attenuation corrections, which are of particular importance in an increasingly obese population; and the challenge of measuring myocardial blood flow (MBF), which is critical to accurately evaluating global ischemia and microvessel disease. This investigation, ?A Novel F-18 PET Myocardial Perfusion Radiopharmaceutical based on Rhodamine Dyes,? builds upon a currently funded project of the same title, 1 R01 HL108107-01, that concludes on 1/31/2017. As in the foundational R01, the primary objective of this project is to develop an 18F-labeled radiopharmaceutical for PET imaging of myocardial perfusion. The Specific Aims of the original project have been largely fulfilled; and thus, while our central hypothesis remains the same, i.e., that a PET myocardial perfusion imaging agent can be developed based on an 18F-labeled rhodamine dye, we have gathered sufficient preliminary results to initiate a first-in- human-study in the next project period. We have demonstrated that 18F-labeled rhodamine dyes accumulate impressively in the heart, which confirms their significant potential as PET MPI radiopharmaceuticals. The central hypothesis of this proposal is, therefore, unchanged: A PET radiopharmaceutical for the evaluation of myocardial perfusion can be developed based on an 18F-labeled rhodamine dye. We have tested and validated this basic premise in our laboratory and found that the 18F-labeled diethyleneglycol ester derivative of rhodamine 6G ([18 F]Rho6G) shows very high accumulation in the heart, minimal accumulation in the liver, and rapid blood clearance. While 18 F-Rho6G has shown great promise as an MPI agent, we do not yet know if it will perform optimally in humans. Thus, we will explore additional rhodamine-like compounds as potential PET radiotracers for MPI. Accordingly, the new Specific Aims will first build upon our findings with 18F- Rho6G by a) carrying out a first-in-human study with [18F]Rho6G; b) measuring the extraction fraction of 18F- Rho6G, a critical parameter for a clinically useful MPI radiopharmaceutical; and c) evaluating a number of novel rhodamine derivatives and also assessing alternatives cores in anticipation of the possible need for an alternative to rhodamine dyes for human use. Our successful accomplishment of these Specific Aims will lead to the development of a novel and effective PET radiopharmaceutical for MPI that will significantly improve clinical management of patients with CHD by providing an accurate measure of myocardial perfusion.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Molecular imaging continues to evolve into a discipline in its own right with applications to many areas of biology and clinical medicine. The long-term goal of this first-time competitive renewal application is to develop technologies focused on reporter genes for use with PET, optical, and other emerging modalities for applications to gene therapy, cancer biology, developmental biology, as well as many other areas. The general hypothesis is that reporter gene technologies can be developed and utilized to study fundamental molecular/cellular events in living subjects. Excellent progress has been made in this regard over the last 4 year funding period. We have helped to push the field forward by developing and validating enzyme based (Herpes-simplex Virus Type 1 thymidine kinase (HSVI-tk)) and receptor based (Dopamine Type 2 receptor (D2R)) reporter gene assays. We have applied these reporter genes primarily in the area of imaging gene therapy. The specific aims of the current renewal are to take the developed approaches to the next level by providing tools "systems imaging" by imaging intracellular networks in living subjects. Protein-protein interactions are at the heart of many biological process including normal and diseased tissue. In Aim 1 we refine a recently developed strategy to image protein-protein interactions that occur in the cell nucleus through an inducible yeast two-hybrid (IY2H) strategy. In Aim 2 we refine an alternate approach referred to as an inducible split reporter (ISR) strategy which is capable of measuring protein-protein interactions in many locations within a cell. In Aim 3 we test the imaging strategies developed in Aims 1 and 2 using new approaches being used to target the interaction of heat shock protein 90 (Hsp90) with p23 using geldanamycin analogs in tumor therapy models in living mice. Finally, in Aim 4 we develop transgenic models to further test methods for imaging protein-protein interactions in deep tissues of living mice. In all aims, care is taken to develop generalizable approaches that should be applicable to other studies using multimodality reporter gene approaches. The significance of the proposed work is it will help to develop and validate new technologies focused on imaging intracellular communication networks. This should lead to many applications including methods to image the interaction of drugs designed to inhibit protein-protein interaction in living subjects. More rapid methods to validate and translate pre-clinical models for cancer therapeutics into the clinic will likely result.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This protocol compares diabetes and heart disease risk in African-Americans (AA) and African immigrants. One hundred ninety-two AA and 248 African immigrants have enrolled. The AA cohort is known as TARA for: Triglyceride and Cardiovascular Risk in AA. The African immigrants are known as the: Africans in America cohort. The sample of AA participating appears to be representative of the AA population of the United States because the prevalence of obesity (43%), pre-diabetes (22%) and hypertension (21%) is similar to National Health and Nutrition Examination Survey data. However, there is no national data on diabetic or cardiac health of African immigrants. Therefore we are working to establish basic information. To explore risk for diabetes, we are relying not just on fasting glucose but are also on performing oral glucose tolerance tests and measuring A1C levels. In performing these tests, we discovered that the prevalence of pre-diabetes and hypertension is twice as high in African than AA men. In addition, the rate of undiagnosed diabetes was 7% in African men vs. 0% in AA men. In contrast the rate of hypertension, diabetes and pre-diabetes are similar in African women and AA women. Identifying the reasons for why African immigrant men are less metabolically healthy than AA men has become a major focus of research in this protocol. To improve and then maintain good health in African men, it is essential to understand why pre-diabetes, diabetes and hypertension is occurring in African men even though African men are less obese, more likely to be non-smokers, more likely to be married and have similar educational levels and income as African-American men. We are also working on determining whether A1C which has been recommended by the American Diabetes Association as a diagnostic test for diabetes, can replace the oral glucose tolerance test in people of African descent. A1C is a hemoglobin dependent test and African immigrants have a high prevalence of sickle cell trait (i.e. 10 to 40%) and hemoglobin C trait (i.e. 15% in West Africa). Therefore before widespread use of A1C as a diagnostic test for diabetes is instituted in Africa, validation is necessary. When we evaluated A1C in African immigrants, we found that the sensitivity was only 55%. When the Africans are subdivided by presence or absence of variant hemoglobin, the sensitivity of A1C as a diagnostic test fell below 50%. Therefore, our results suggest that A1C might not be an ideal, single test to use to detect diabetes in Africans. We are in the process of examining alternatives such as fructosamine and glycated albumin as well as combining A1C with fasting glucose. The relationship of body size to cardiovascular and diabetes risk is another area of investigation. In our cohorts, the mean body mass index (BMI) in AA is 30.6 kg/m2 but only 26.4 kg/m2 in African immigrants. BMI is a mathematical method used to correct weight for height. Due to the broad range of BMI in the participants in this cohort, it is possible to evaluate the relationship of body size to insulin resistance, a major factor in the development of diabetes, and heart disease. We have found in AA men a waist circumference (WC) of 102 cm predicts both insulin resistance and obesity. This is in agreement with the National Cholesterol Education Program values for whites. But in African men, insulin resistance occurs at a much lower WC, specifically 92 cm. This difference between AA and African immigrant men, suggests that a single WC of risk does not apply to all African descent populations. The situation may be analogous to Asian populations, as the WC of risk is different in Chinese, Japanese and Asian Indians. In AA women we found that a WC of 98 cm predicted both insulin resistance and obesity and this WC of risk was similar in AA, African immigrant, Black South African and West African women. Therefore among populations of African descent, there may be less variation in women than men. However, as the WC of risk is 88 cm in white women, there is a large difference by race. Guidelines which screen for disease might be more effective if this was better fully appreciated. Elevated TG and low HDL are considered lipid hallmarks of insulin resistance. However while elevated TG is a marker of insulin resistance in whites, we have shown that TG is not a marker of insulin resistance in AA. Results from TARA were so impressive that the hypothesis that TG was not a marker of insulin resistance in African Americans was subsequently tested in NHANES data collected from 1999-2001. In this NHANES data set of whites, AA and Mexican Americans, the fact that TG was not a marker of insulin resistance was confirmed. However, TG was a powerful marker of insulin resistance in whites and Mexican Americans. Altogether this research on race differences in the relationship of TG to insulin resistance again demonstrates that to detect disease at time when intervention can affect outcome, there is a need to develop ethnic-specific guidelines. More recently the TG/HDL ratio at a level of >3.0 has been suggested to be a marker of insulin resistance. This is well established in whites. But we have shown using TARA data that this did not work in AA. Having detected that the TG/HDL ratio was not effective predictor of insulin resistance in AA women, we tested the ratio in white South African women, Black South African women, West African women from Ghana and Nigeria and African-American women. We found that while the ratio effectively predicted insulin resistance in the white women, it did not work in any group of women of African descent. Again demonstrating that findings related to insulin resistance in whites may not be applicable globally and systematic testing is necessary. In recent years, it has been reported that low vitamin D levels may influence bone health as well as enhance risk for cardiometabolic disease. As people of African descent have lower vitamin D levels than whites, the adverse effect of low vitamin D in people of African descent may be magnified. Alternatively vitamin D may be sufficient at lower levels in people of African descent than whites. This race difference issue is important to clarify because vitamin D has a relatively narrow therapeutic window. In summary this protocol is dedicated to undertaking epidemiological research which has the potential to improve cardiometabolic health and prevent through early diagnosis diabetes in people of African descent globally.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The intent of this project is to develop new and to improve existing biophysical methodology for the characterization of biological macromolecules in solution, and to apply these methods collaboratively to the study of proteins and their interactions. Experimental techniques employed are analytical ultracentrifugation, static and dynamic light scattering, isothermal titration calorimetry, differential scanning calorimetry, circular dichroism spectroscopy, and surface plasmon resonance biosensing. For the study of reversible formation of multi-protein complexes in solution, we have developed a novel multi-signal analysis for sedimentation velocity analytical ultracentrifugation. It permits the label-free detection of sedimentation coefficient distributions of currently up to three protein components, and thus measure the stoichiometry of multiple hydrodynamically separated protein complexes. We have also analyzed the influence of binding kinetics on the time-scale of sedimentation. Studies were conducted for the proof of principle, followed by first model applications to different biological systems of interacting proteins. These include the collaborative study of the extended hetero-association of plasminogen activator inhibitor (PAI-1) complexes with vitronectin, which is important in redirecting the localization of vitronectin from the circulation to the extracellular matrix where an adhesive function is manifested. The multi-signal sedimentation velocity method has been used to document the occupation of two PAI-1 distinct binding sites on vitronectin as a requisite for the assembly of higher-order complexes. In the collaborative application to the study of a signal transduction complex after T-cell activation, formation of extended triple protein complexes of adaptor proteins was detected. We have collaboratively applied the previously developed techniques hydrodynamic and thermodynamic sedimentation techniques to the study of PH domain of ArfGAPs, the HIV envelope proteins and their interactions, interactions of NK (natural killer) cell surface receptors, malarial surface proteins, clathrin basket assembly, cryptophycin induced tubulin structures, iron regulatory protein 1 and 2 interactions with IREs, and the glycolytic enzyme gucocerebrosidase and its interactions with alpha synuclein (the causative agent in Parkinson?s disease). We have continued our efforts to collaboratively apply our biophysical methodology to the characterization of the filamentation process of alpha synuclein, which is associated with Parkinsons disease. In the area of optical biosensing, progress was made in the computational approach to determine the functional heterogeneity of surface binding sites. The previously introduced model was extended to account for mass transport limited surface binding. This was applied to antibody-antigen model systems, followed by the analysis of integrin receptors to a variety of ligands relevant to leukocyte interaction with tissue proteins, the study of antibodies to anthrax protective antigen, and the study of MAPK-kinases with anthrax LF protein. We have also developed techniques for studying small molecule binding with surface-immobilized nucleic acid and proteins. A first application was the study of inhibitors of HIV reverse transcriptase and human RNAse H to aid in the screening of compound libraries of potential drug candidate molecules, and to analyze the mechanism of the inhibition. A further methodological development is aimed at increasing the recovery of surface captured molecules for interfacing biosensors with mass spectroscopy.
|
{
"pile_set_name": "NIH ExPorter"
}
|
mRNA decay is a critical determinant of gene expression. Loss of mRNA stability regulation results in a variety of diseases including neurological disorders and various types of cancer. mRNA decay pathways and decay factors are conserved from yeast to humans. Our long term goal is to understand the mechanism of eukaryotic mRNA turnover using the budding yeast S. cerevisiae as the model system. A major mRNA decay pathway in yeast begins with deadenylation of the mRNA followed by decapping and 5' to 3' exonucleolytic degradation of the message. Decapping is a rate limiting step affected by several factors. The highly conserved heptameric Lsm1p-7p complex and its interacting partners, Pat1p and Dhh1p, activate decapping by an unknown mechanism. This complex also protects mRNA 3'-ends from a 3' trimming reaction in vivo. It colocalizes with other decay factors and the mRNAs targeted for decay to the cytoplasmic P-bodies where decapping is thought to occur. In vivo, the Lsm1 p-7p complex specifically binds to deadenylated messenger ribonucleoprotein (mRNP) molecules that are targeted for decay and are distinct from the translating mRNPs - these mRNPs are also bound to other decay factors but not to translation factors. [unreadable] These observations led to the following hypothesis: In a process aided by Pat1p and Dhh1p, the Lsm1p-7p complex directly binds to the mRNA 3'-end after deadenylation (so that the 3'-end is protected from trimming) and triggers an mRNP rearrangement that directs the mRNP from translation to decapping. Using a combination of in vivo and in vitro studies, this proposal will test this model by focusing on the following specific aims: (i) To elucidate the molecular events that constitute decapping in vivo. We will determine the exact basis of the block to decay in our collection of decay-defective Ism 1 mutants by determining how the interactions (with other decay factors and mRNA) and the localization (to the P-bodies) of the Lsm1p-7p complex are affected in these mutants, (ii) To elucidate the determinants of the Lsm1p- 7p complex-mRNA interaction. To this end, we will determine the factors that influence this interaction in vivo and the intrinsic RNA binding characteristics of the purified Lsm1p-7p complex in vitro, (iii) To determine the mechanism(s) by which the Lsm1p-7p complex enhances decapping. In principle, the Lsm1p-7p complex could enhance decapping rates in vivo either by directly stimulating the decapping enzyme or by facilitating the disassembly of the translation factors from the mRNA. We will determine the contribution of these two mechanisms to decapping activation. Together, these studies will provide insight into the mechanism of decapping and reveal the manner in which functions of the various decay factors are coordinated. Owing to the homology of the Lsm1p-7p complex to the other Sm-like protein complexes in biology, these studies should also reveal some principles common to this family of protein complexes engaged in various RNA-related functions. [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Addiction is a disease of aberrant learning. Addictive substances, and the environmental cues that predict those substances, hijack normal reward circuitry in the brain and become overvalued relative to all else, leading to a catastrophic cycle of drug craving, seeking, and intake behaviors at the expense of healthy behaviors. These aberrant stimulus-outcome (SO) associations are manifested in frontolimbic (FL) networks that normally drive a wide range of behaviors vital for healthy function. In the addicted brain, these circuits become maladaptive, leading to compulsive behaviors, incapable of the flexibility the system originally possessed. Currently, two major avenues of treatment for addiction exist: behavioral and neuropsychiatric treatments. Behavioral treatments aim to manage maladaptive SO associations by breaking habitual drug taking behavior, though relapse rates are high. Neuropsychiatric treatments broadly target neurotransmitter systems as a whole without regard to specific SO associations, thereby impacting normal reward processing and learning. In the current grant, we aim to test an alternate approach to the treatment of addiction by the precise targeting and modification of SO associations at the systems level through a brain-machine interface (BMI) framework. We propose that by identifying the underlying neural representations of SO associations in FL networks and modifying them through electrical microstimulation, we can provide a means to unlearn addictive behaviors without unwanted side effects. Modifying SO associations for the treatment of addiction requires understanding how SO associations are formed in FL networks and how these formations can be manipulated through targeted electrical stimulation. To do so, large populations of neurons must be recorded and stimulated during SO learning. Recent advances in recording technology enable such experiments. For example, recent studies have manipulated sensory networks through electrical stimulation to affect learning, thus optimizing the control of BMI prostheses. We plan to adopt the same methods to manipulate value signals to alter SO associative learning in FL networks. Our experiments will focus on two major building blocks. First, we will determine optimal stimulation parameters and FL targets to modify SO associations. We will test this by determining the effects of a wide range of stimulation parameters and locations across the FL circuit on value-based learning and decision making (open loop). Second, we will use a BMI framework to decode value information about a given stimulus from large populations of FL neurons in real time as a control signal to preferentially apply targeted stimulation. This will enable closed-loop bidirectional control over SO associations. We will study how stimulation affects SO associations represented in the FL circuit across days in both open and closed-loop conditions.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Human platelets were extracted with acid-ethanol and platelet-derived TGF-beta was purified from the extract by a two-column procedure using sequential gel filtration in the absenceand then presence of urea. Purified TGF-beta is a protein of 25,000-daltons, and it is comprised of two 12,500-dalton subunits held together by disulfide bonds. The purified factor elicits its biological activity at concentrations less than 4pM. Comparative studies showed that platelets contain 100 times more TGF-beta than do other non-neoplastic tissues. Platelets also contain a peptide growth factor related to EGF. These two new growth factors can interact mechanistically. Incubation of TGF-beta with NRK cells for 6 h results in an increased number of cell surface EGF receptors. IGF-II receptors are not affected. Shorter incubations with TGF-beta show that this peptide can also increase the Kd of the high affinity EGF receptor.
|
{
"pile_set_name": "NIH ExPorter"
}
|
PROJECT SUMMARY Schistosomiasis is a neglected tropical disease (NTD) caused by parasitic worms and is second only to malaria as the most devastating parasitic disease. It is currently estimated that more than 200 million people are infected and 700 million more are considered at risk. Humans become infected when doing activities in freshwater (e.g., Schistosoma-contaminated lakes, rivers) such as washing cars and fishing. The highest incidence of schistosomiasis is in children and young adolescents with persistence of disease in adults who have frequent contact with water. Current approaches for the diagnosis of schistosomiasis require high infection levels for successful detection or are unable to determine if a positive diagnosis represents a past or current infection. Nucleic acid-based techniques to detect schistosome DNA have substantially greater sensitivity than the existing microscopy- or antigen-based methods, higher specificity and can diagnose ongoing infections. Lynntech proposes to develop a user-friendly, low-cost, portable PCR-based human diagnostic (called NTDx-S) to detect causative organisms in human samples in low resource settings. The NTDx-S system will be based on PCR- mediated amplification of specific genes, and a lateral flow (LF)-based detection method to produce a specific diagnostic signal. During this effort, Lynntech will expand upon a previously developed schistosomiasis PCR-LF detection assay and develop PCR reactions aimed at the amplification of species specific loci. To ensure the detection of all potential causes of disease, we will also develop a primer set capable of detecting a pan locus, enabling detection of each schistosome species, including hybrids. Two separate LF strips will be developed that detect the pan locus alongside species specific loci from the three main species responsible for disease. These two strips will be designed to detect schistosomes based on their geographical distribution. Finally, we will develop a simple, low power sample preparation protocol, aimed at isolating schistosome eggs from human urine/stool samples. These samples may be processed in the NTDx-S system as the end user requires based on region of use. Schistosomiasis endemic countries require diagnostics which are economical and simple to operate. Therefore, during the development of the NTDx-S diagnostic, efforts will be focused at each step to limit power requirements and achieve a cost-friendly, simple assay. The development of Lynntech's NTDx-S system will enable non-specialized operators in non-traditional settings to perform sophisticated biological testing.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Injury of the brain is a major cause of death and morbidity after exposure to nerve agents that activate the muscarinic receptor system, which often causes long term disability with unusually high accompanying social and medical costs. Current countermeasures such as midazolam and atropine are of doubtful utility for civilian populations because they must be administered within minutes of an attack to be effective. Therapies that can be administered hours to days after the attack are needed. Prolonged status epilepticus (SE) induced by the muscarinic agonist, pilocarpine, triggers a similar series of molecular and cellular events in the rodent brain. Cyclooxygenase-2 (cox-2) is induced strongly in principal hippocampal neurons after a seizure. Our overarching goal is to develop small molecules that act on specific prostanoid receptors to oppose seizure-induced neurodegeneration, neuroinflammation and functional deficits in adults and children subject to chemical attack by nerve agents. Specific aims 1 and 2 are designed to answer several key questions in order to better understand the basis of these neuropathologies: Which prostaglandin receptors are involved in neuronal injury and neuroinflammation after SE, and in delayed sensorimotor deficits? Which cell type(s) release PGE2 and PGD2? We will test the specific hypotheses that an allosteric EP2 receptor activator is neuroprotective after pilocarpine-induced SE, and a DP2 receptor blocker blunts the neuroinflammatory response. In aims 3 and 4 we identify novel small molecule modulators of selected prostanoid receptors that are brain permeant and can be delivered after the period of acute convulsions to blunt SE-induced delayed neurodegeneration, neuroinflammation and cognitive deficits. Successful completion of these studies is an essential step to translate our research findings into a useful countermeasure to a prominent class of chemical threats, and will prepare the ground for subsequent validation in animals exposed to sarin-type nerve agents themselves, followed by GLP preclinical pharmacology studies, IND submission, and Phase I clinical safety trials. [unreadable] [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Sigma-1 receptor (Sig-1R) functions as a chaperon that interacts with multiple proteins and lipids and is implicated in neurodegenerative and psychiatric diseases. This year in a collaboration with Dr. Bonci's lab we found that vesicular glutamate transporters in dopamine neurons play a critical role in neuroprotection against MPTP-induced Parkinsonism in a rat model. Specifically, genetic deletion of the vesicular glutamate transporter enhances the neurotoxicity of MPTP. Although the sigma-1 receptor was not examined in this study, we feel that the result of this study may have a bearing with the sigma-1 receptor because the sigma-1 receptor has been shown to protect against Parkinsonism. In another study on neuroprotection, we found that Sp1 was highly expressed in astrocytes, implying that Sp1 might be important for the function of astrocytes. Sp1/GFAP-Cre-ERT2 conditional knockout mice were constructed to study the role of Sp1 in astrocytes. Knockout of Sp1 in astrocytes altered astrocytic morphology and decreased GFAP expression in the cortex and hippocampus but did not affect cell viability. Loss of Sp1 in astrocytes decreased the number of neurons in the cortex and hippocampus. Conditioned medium from primary astrocytes with Sp1 knockout disrupted neuronal dendritic outgrowth and synapse formation, resulting in abnormal learning, memory, and motor behavior. Sp1 knockout in astrocytes altered gene expression, including decreasing the expression of Toll-like receptor 2 and Cfb and increasing the expression of C1q and C4Bp, thereby affecting neurite outgrowth and synapse formation, resulting in disordered neuron function. Studying these gene regulations might be beneficial to understanding neuronal development and brain injury prevention. Since the sigma-1 receptor can affect chromatin remodeling via an interaction with the nuclear envelope protein, future investigation on the potential interaction of sigma-1 receptor and Sp1 may advance our understanding on the transcriptional regulation of sigma-1 receptor.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Abstract: The goal of the proposed research is to develop novel radiolabeled ultrasmall core shell nanoparticles (C' dots) decorated with alpha melanocyte stimulating hormone peptide analogs (DOTA-?MSH) that target the melanocortin-1 receptor (MC1-R) overexpressed on melanoma cells for targeted radiotherapy. The DOTA-?MSH-C' dots will be radiolabeled with the beta particle emitter 177Lu and the alpha emitter 225Ac. Upon binding, the radiolabeled DOTA-?MSH-C' dots are internalized into the melanoma cells sequestering the radionuclide within the cell near the nucleus for optimal radiation deposition. This approach is innovative because the nanoplatform transforms the properties of the individual components, such as the melanoma avid ?MSH peptide's MC1-R affinity and the pharmacokinetics of the conjugated radionuclides, to yield an ultrasmall nanoparticle with superior melanoma targeting and in vivo therapeutic properties. The central hypothesis is that C' dot nanoparticle targeted therapy with alpha 225Ac and beta 177Lu emitting radionuclides will result in efficacious melanoma treatment due to optimal melanoma tumor targeting and irradiation coupled with rapid in vivo clearance kinetics. Preliminary data supports the hypothesis and shows that the DOTA- ?MSH-C' dots have superior affinity and in vivo pharmacokinetic properties to the melanoma targeting peptide alone. Moreover, initial clinical trials with C' dots demonstrated they were safe in humans, foreshadowing the translational potential of the DOTA-?MSH-C' dots. Development and optimization of DOTA-?MSH-C' dots will be guided by 3 specific aims. 1) Determine the optimized tunable surface chemistry for melanocortin-1 receptor (MC1-R) targeted silica nanoparticles to achieve favorable cellular binding, uptake, subcellular localization. 2) Evaluate 225Ac- and 177Lu- DOTA-?MSH- C' dot conjugates' in vivo targeting specificities, pharmacokinetics and clearance profiles in B16/F10 syngeneic and M21 human xenografted melanoma tumor bearing mice to determine the lead radiolabeled DOTA-?MSH- C' dot platform. 3) In vivo characterization and safety assessment will be performed with the lead 225Ac and 177Lu labeled DOTA-?MSH conjugates to support an IND application for clinical trials. Therapy studies with the 225Ac and 177Lu labeled DOTA-?MSH-C' dots alone or combined with immunotherapy will be investigated. Malignant melanoma is the most deadly form of skin cancer. It is extraordinarily resistant to chemotherapy, immunotherapy and external beam radiation resulting in low durable response rates. Recently, several new therapeutics were approved for melanoma treatment; however, the responses are usually short-lived (6-12 months) due to drug resistance and activation of alternative signaling pathways. The development of radiolabeled DOTA-?MSH-C' dot nanoparticles is significant since their administration will result in highly localized tumor irradiation with low normal tissue exposure for therapy addressing a critical unmet need for the development of novel efficacious treatments for advanced staged melanoma.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Both AIDS and TB are very large health problems in India, The objective of this project is to develop a plan for the coming ten years to strengthen integrated and multidisciplinary research and training at the All India Institute of Medical Sciences (AIIMS), New Delhi, India in the fields of HIV/AIDS and tuberculosis. The University of California in Los Angeles (UCLA) will be the partner institution in the U.S, in this collaboration. Research, training and capacity building needs are truly multidisciplinary and will encompass clinical medicine, drug and vaccine trials, behavioral and social sciences, operational research and health services research. This will include cost-effective strategies for prevention, diagnosis and management of tuberculosis and HIV/AIDS and associated complications, including opportunistic infections. The project envisages training of several categories of health professionals including clinicians, laboratory scientists, research nurses, social scientists, psychologists, biostatisticians, and health care administrators. It will consolidate partnership with other stakeholders in India including Ministry of Health, Directorates of National Programs for Tuberculosis and AIDS, research organizations such as Indian Council of Medical Research (ICMR) and its institutions (Tuberculosis Research Center, Chennai, National AIDS Research Institute, Pune), Department of Biotechnology of the Ministry of Science and Technology, etc. Leadership in key Departments at AIIMS have been identified and agree to participate. Similar steps have been taken at UCLA. Multiple linkages already exist between faculty at AIIMS and UCLA. Administrative support has been obtained at the highest levels. Preliminary meetings took place in New Delhi, February 2002. Key Faculty from AIIMS will visit UCLA, the FIC and other NIH institutes and attend major relevant research meetings in the U.S. between June 2002 and February 2003. Several UCLA Faculty will visit AIIMS. Planning will proceed from June 2002 with repeated opportunities for input and critique by involved faculty at both institutions. This will complement existing or planned U.S, programs including FIC, CIPRA, ICTTRI and international CFAR activities, Current plans for training at UCLA and courses, workshops and cooperative projects at AIIMS in 2002 and 2003 will continue.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The University of Missouri Electron Microscopy Core facility provides the only electron microscopy services and instrumentation for research on the University of Missouri campus. It is through this Core Facility that the major user group performs research that requires transmission electron microscopy. The transmission electron microscope (TEM) currently in the Core Facility was acquired approximately 20 years ago. We have been informed by the manufacturer that new replacement parts are no longer available for this instrument. The instrument continues to be serviced with used parts, but once the supply of any critical part is exhausted, the instrument could soon become nonfunctional. In addition, the existing microscope does not have some features available on newer instruments that are needed by the user group. The objective of this application is to replace the old instrument with a modern TEM that can be equipped with features that will meet the research needs of the major user group and other NIH-funded investigators over the coming 15 to 20 years. The great majority of TEM use in the Core is for NIH-funded research. Core TEM services are currently used to support research on diabetes, aging, cardiovascular disease, nutrition, neurodegenerative disorders, and a variety of other health problems. The time is near when the TEM presently housed in the Core will no longer be able to be serviced. In the absence of a reliable supply of replacement parts, the risk of losing TEM capability altogether is high. The requested TEM will be housed in the existing Core Facility and will be maintained by experienced Core staff. Core staff will supervise use of the instrument under the direction of a Faculty Coordinator for Electron Microscopy Services in consultation with a user Advisory Committee. Training in instrument use will be provided by Core staff and associated faculty. Staff salaries will continue to be provided from a combination of user fees and University funds. A service contract will be maintained with the manufacturer to provide service for the instrument that cannot be performed by Core staff. Service contract costs will be covered by user fees and an institutional subsidy if necessary. The instrument will be available to users outside of the major user group under essentially the same terms that apply to major user group. Broadening the user base will help make the cost per user of maintaining the instrument as low as possible. [unreadable] [unreadable]
|
{
"pile_set_name": "NIH ExPorter"
}
|
Rhabdoid tumor is a clinically aggressive malignancy that generally presents in the first four years of life. Rhabdoid tumors of the central nervous system (atypical teratoid/rhabdoid tumor;AT/RT), kidney and soft tissues are associated with alterations of the INI1/hSNF5 tumor suppressor gene in chromosome 22q11.2. AT/RT is the only pediatric brain tumor for which the primary genetic etiology has been elucidated, and as many as 35% of children may have predisposing germline deletions or mutations of INI1. INI1 is a member of the SWI/SNF chromatin remodeling complex and functions to repress or activate gene transcription. Understanding the role of INI1 in tumor development specifically addresses the goals of the NIH Brain Tumor Progress Review Group, but has wider implications for a variety of pediatric and adult diseases that may arise as a consequence of mutations in genes involved in chromatin remodeling. A continuing goal of this program is to determine the spectrum of clinicopathologic manifestations of heterogeneous germline and somatic mutations of the INI1 gene. In aim 1, we will perform a comprehensive genomic analysis of the 22q11.2 region, including deletion analysis by FISH and MLPA, as well as direct sequencing. We will determine whether specific deletions or mutations are associated with anatomic site, as well as prognosis. In aim 2, we will define the spectrum of de novo and inherited germline deletions and mutations in patients and their families. Preliminary data suggests that there is a bias in the parent of origin of germline mutations, which will be explored in a larger patient cohort. A genome wide approach, using high density single nucleotide polymorphisms arrays, will be used in aim 3 to interrogate the region of chromosome band 22q11.2 which contains INI1, as well as to identify other chromosomal regions that may be related to rhabdoid tumor development. The characterization of potential candidate genes associated with clinical features and outcome will be explored in aim 4 using a combination of mutation and expression analyses. PUBLIC HEALTH RELEVANCE: Rhabdoid tumors of the brain, kidney and soft tissues are clinically aggressive malignancies that primarily affect children under four years of age. The INI1 gene on chromosome 22 is a key tumor suppressor inactivated in the majority of tumors. Understanding the mechanisms by which INI1 is inactivated will be important for treatment stratification, and ultimately designing biologically based therapeutic strategies for patients.
|
{
"pile_set_name": "NIH ExPorter"
}
|
There is currently a deficiency in understanding the events that effect proper cardiovascular development. The long-term goal is to define these events in molecular detail, providing a basis for understanding cardiovascular development in both normal and pathological states. With this goal in mind, the objective of this application is to determine in mechanistic detail how the transcriptional corepressor known as BCOR represses cardiac-specific transcription and how mutations in BCOR contribute to the molecular pathogenesis of cardiovascular developmental defects. In humans, strong clinical evidence suggests that BCOR plays a key role in early heart formation. Although the BCOR repression complex is thought to be involved in chromatin modification in some cell types, little is known about BCOR's specific role in cardiac cells and the purpose of this function in the overall process of cardiovascular development. Thus, the central hypothesis of this application is that BCOR plays an essential role in cardiac development by recruiting chromatin modifying enzymes to regulate the expression of genes involved in cardiovascular development. To test this hypothesis, two specific aims are proposed. The first aim is to define the components and candidate transcription factor targets of the BCOR repression complex in cardiac precursor cells. To accomplish this, protein purification and mass spectrometry methods will be used to identify the BCOR complex components in a cardiac precursor cell line. Protein interaction assays will then be utilized to test specific known cardiac transcription factors for interaction with BCOR. The second aim is to determine the role of BCOR in murine cardiovascular development. This will involve generating a BCOR null mouse and analyzing the effect of BCOR loss on cardiac development and cardiac- specific gene expression. These experiments are significant because they will increase understanding of the molecular events leading to proper cardiovascular development and, in doing so, will confer an awareness of the processes that are likely to be disrupted in cardiovascular developmental disorders. Relevance to Public Health: This research examines the function of a protein that is involved in regulating cardiac development. Ultimately, the information gained from these studies will lead to a more thorough understanding of normal cardiac development, the pathogenesis of cardiac developmental defects, and potential therapies to prevent those defects.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Repetitive administration of the anthracycline antineoplastic doxorubicin in man or experimental animals produce a dose-related cardiomyopathy that may culminate in severe cardiac dysfunction, overt heart failure, or death. We propose (a) to study the cardiovascular effects of long-term doxorubicin administration in dogs equipped with totally implantable telemetry devices and (b) to use the derived information to test a cardioprotective strategy that is based on the hypothesis that cardiac damage is mediated, at least in part, by the release of vasoactive amines (histamine and catecholamines). The first goal of this investigation is to elucidate the manner in which the heart of the intact conscious animal with normal cardiovascular reflexes responds to long-term doxorubicin administration, and to correlate functional changes accompanying the onset of cardiomyopathy and failure with structural changes in the heart. Specific physiological and pharmacological interventions will be employed to characterize serial changes in sympathetic and parasympathetic control mechanisms and the inotropic responsiveness of the heart. Animals on a long-term dosing regimen of doxorubicin combines with histaminic (diphenhydramine and cimetidine) and adrenergic (phentolamine and propranolol) blockade will then be evaluated using the same experimental interventions, and the success of the cardioprotective strategy will be judged on the basis of deviation from the failure profile. This research is important not only because it will provide clinically useful information regarding an important antineoplastic drug, but also because it will permit us to evaluate a heart failure model that is based on a chemically induced myopathy and to demonstrate the usefulness of totally implantable telemetry instrumentation in conducting cardiovascular studies that require weeks and months to complete.
|
{
"pile_set_name": "NIH ExPorter"
}
|
My colleagues and I have provided two lines of evidence suggesting that defects in Platelet-Derived Growth Factor-A (PDGF-A) function result in lethality or developmental deformities during early vertebrate embryogenesis. First, mRNA for PDGF-A and its receptor are maternally-encoded in Xenopus and mice, and are expressed throughout early development. Second, mice harboring a deletion of the receptor for PDGF-A fail to develop normally past 8.5 days and usually die in utero. I would like to extend these preliminary findings in the experimentally accessible Xenopus embryo. Because the temporal and spatial expression patterns of PDGF and receptor are conserved evolutionarily, these studies should have relevance to human physiology and development. The research has two broad objectives. The first is relatively straightforward and builds on my preliminary studies on the role of PDGF during early embryogenesis. The second is more complicated and relies on my observation that PDGF-A and its receptor are markers for anterior development. Using these markers, I propose to identify novel morphogens that specify antero-posterior pattern formation. The first objective is to determine the function of PDGF-A during development. Antiserum to PDGFA and its receptor will be used immunohistologically to identify those cells producing PDGF and receptor protein. This should extend my preliminary results localizing mRNA for PDGF-A and its receptor to ectoderm and mesoderm, respectively, in the gastrula. Recombinant Xenopus PDGF-A will be evaluated for effects on differentiation of mesodermal cells in culture. Finally, PDGF-deficient embryos will be produced and monitored for developmental abnormalities. To create PDGF-deficient animals, I have already constructed mutant PDGF genes that suppress wild-type activity in a trans-dominant fashion. Embryos will be made deficient in PDGF by the injection of synthetic mRNA encoding mutant PDGF. Abnormalities of these embryos may reveal the in vivo function(s) of the growth factor. Of particular interest will be abnormalities appearing at early gastrula to late neurula stages when PDGF and receptor mRNA levels are highest. The second objective is to use PDGF-A and its receptor as markers to identify factors that control antero-posterior patterns of differentiation. Preliminary results show that mRNA encoding PDGF and its receptor are expressed in an anterior to posterior gradient in the gastrula and neurula. Thus, PDGF-A appears to be expressed in anterior ectoderm, while its receptor is localized in the anterior mesoderm. Candidate agents thought to influence axial polarity will be studied in an attempt to induce (or suppress) the differentiation of cells expressing PDGF-A and its receptor. These factors will be evaluated in conjunction with tissues (such as the organizer) for their effect on expression of PDGF-A and its receptor. These experiments may lead to the identification of morphogens that control antero-posterior polarity in vertebrates.
|
{
"pile_set_name": "NIH ExPorter"
}
|
To determine the vulvar, vaginal, and cervical toxicity and/or irritation and acceptability of SG036 gel with Gynol II.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Esophageal carcinoma is endemic in the Ho Nam Province in China. Characterization of antigens unique to these tumors would be useful for diagnosis of this carcinoma. Glycolipids isolated from esophageal tissues obtained from 11 cancer patients, 10 normal individual controls, and a cultured esophageal carcinoma line were examined using several monoclonal antibodies. Several glycolipids differ in amount between the tumor samples and normal controls. Both neutral and sialylated glycolipids containing 3-fucosyllactosamine sequences differ in expression between the tumor and control tissues and a heptaglycosylceramide reacting with antibodies to this sequence is found only in the tumor tissues. The same glycolipids are detected in the cultured cell line, confirming that the tumor antigen is produced by the tumor and is not due to contamination by infiltrating granulocytes.
|
{
"pile_set_name": "NIH ExPorter"
}
|
DESCRIPTION (applicant's abstract): Neurosecretory neurons have a large capacity for morphological and physiological plasticity as a function of endocrine state during adulthood. Of principal interest in this project is the formation of new synapses in the supraoptic and paraventricular nuclei of the hypothalamo-neurohypophysial system during lactation, when the demand for oxytocin (OT) release is high. This morphological plasticity is accompanied by significant increases in the activity of glutamate-releasing synapses which are selective for OT neurons, These changes include a doubling in miniature excitatory postsynaptic currents and an increase in the probability of glutamate release. A similar up-regulation has been found for GABAergic synapses. Profound changes in gonadal hormone secretion during late pregnancy are thought to program these changes, anticipating the increased activity of OT neurons. Furthermore, local OT release feeds back on both types of synapse through autoreceptors on OT neurons and/or their presynaptic terminals. Central OT release is critical for the morphological plasticity, and for the normal expression of OT neuronal firing during lactation. In this project, the mechanisms and consequence of this increased synaptic activity will be investigated. There are four Specific Aims: Aim 1) A) To test whether glutamate release at AMPA/Kainate receptors on OT neurons is enhanced during lactation by a change in the presynaptic regulation of its own release; B) To determine whether NMDA receptors participate in this plasticity; Aim 2) To test the combined and differential contribution of GABAergic and glutamatergic activity to spike patterning in OT neurons during lactation; Aim 3) To test whether OT's direct and presynaptic effects on OT neurons are state-dependent, and whether OT can alter the firing pattern of OT neurons during lactation; Aim 4) To test the time-dependence of the increase in glutamatergic activity during pregnancy, and whether it is controlled by gonadal steroid hormones. These studies are important for understanding how the central control of OT neurons adapts to the demands of increased hormone secretion during pregnancy and lactation.
|
{
"pile_set_name": "NIH ExPorter"
}
|
My laboratory is currently funded by NIMH and other sources to study the molecular bases of memory in Drosophila, but I have not previously received extramural support for studies on aging. Thus, I am an established investigator moving into aging research. Therapies aimed at ameliorating age-related functional declines would be greatly facilitated by a better understanding of the molecular bases of functional senescence. This project will explore the genetic relationship between control of longevity and functional senescence in Drosophila. Additionally, this project will use powerful genetic tools in Drosophila to explore the molecular underpinnings of functional senescence. Together, these studies will shed much light on age-related changes in nervous system function.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Polycyclic aromatic hydrocarbons (PAH's) are widespread pollutants. Many are metabolized to highly carcinogenic derivatives. The principal enzyme activity involved is cytochrome P1-450 dependent aryl hydrocarbon hydroxylase (AHH). PAH's, in addition to being substrates, also induce P1-450. Induction is mediated by the Ah receptor. This receptor also mediates pathogenesis (including carcinogenesis) of many environmentally important polychlorinated hydrocarbons, but by an unknown mechanism. P1-450 is highly inducible in mouse Hepa-1 cells. Non-inducible mutants were isolated. A few are dominant. The majority are recessive and fall into four complementation groups. Mutants in genes B, C and D are defective in Ah receptor functioning. Dominant mutants synthesize a repressor of P1-450 transcription. The main objectives of the proposed research are to clone, analyze, and utilize the B, C, D and dominant genes. Human or rat genomic DNA-derived transfectants of individual B-, C- and D- mutants have been obtained in which P1-450 inducibility is restored. Phage or cosmid libraries of secondary or tertiary transfectants will be made, and plaques or clones containing all parts of the B, C, or D genes identified by probing with human or rat repetitive sequences. The dominant gene will be isolated using a similar transfection strategy. Each cloned gene or gene fragment will be utilized to determine i) how many closely related genes exist; ii) whether the corresponding mRNA is induced by PAH's in human lymphocytes, and whether the degree of inducibility in different persons is associated with susceptibility to cigarette-induced lung cancer, and iii) to map the human genes. Portions of each gene will be ligated to an E. coli expression vector, and the corresponding fusion protein isolated and used to generate antisera. Mammalian cells expressing high levels of the "natural" proteins corresponding to each gene will be obtained by cotransfecting and coamplifying the genes along with a cloned dihydrofolate reductase gene. The proteins will then be isolated from these cells using the above antisera. These proteins (three of which may be structural components of the Ah receptor) will be tested for their ability to bind TCDD, the cloned P1-450 gene, and each other. Finally the AHH regulatory genes or gene fragments will be used to isolate cDNA's and these, and interesting portions of the genes, will be sequenced. These studies therefore promise to give condiderable insight into the structure, role and function of the Ah receptor and the mechanism of regulation of cytochrome P1-450.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Dr. David Aguilar is a non-invasive cardiologist strongly committed to a career in clinical research. He completed his internal medicine residency and cardiology fellowship at the Brigham and Women's Hospital, Harvard Medical School and is currently an Assistant Professor of Medicine at Baylor College of Medicine (BCM). This Mentored Career Development Award (K01) will develop Dr. Aguilar's career through several mechanisms, including weekly mentoring meetings, the Clinical Scientist Training Program, and didactic coursework at the University of Texas School of Public Health and BCM. As part of the award, Dr. Aguilar will address the importance of aortic and left ventricular (LV) diastolic stiffness in diabetic patients with heart failure (HF) and normal ejection fraction (EF) and will test the effects of a treatment strategy with a glucagon-like peptide-1 (GLP-1) receptor activator (exenatide) on aortic and LV diastolic stiffness. Diabetes is commonly seen in patients with HF and normal EF and is associated with increased morbidity and mortality. The mechanisms contributing to these adverse outcomes in diabetic patients are poorly understood and represent a barrier to improving outcomes. We hypothesize that, in patients with HF and normal EF, LV diastolic stiffness will be greater in diabetic patients than in non-diabetic patients and that this increased LV diastolic stiffness will correlate with increased aortic stiffness. We also hypothesize that this increased LV diastolic stiffness will correlate with serum biomarkers of increased myocardial collagen accumulation. The importance of advanced glycation end products (AGEs) in this process will be examined by also testing the hypothesis that increased aortic stiffness and LV diastolic stiffness are directly correlated with increased serum levels of AGEs. Finally, we hypothesize that treatment of type 2 diabetes with exenatide for 12 weeks in individuals with HF and normal EF will be associated with improvement in aortic and LV diastolic stiffness and in serum biomarkers of abnormal myocardial collagen accumulation. Given the increasing prevalence of diabetes and the burden of disease attributed to both diabetes and HF with normal EF, this work has significant public health implications. Dr. Aguilar will study mechanisms contributing to the adverse outcomes in diabetic individuals with HF and normal EF and will test a diabetes-specific treatment strategy in hopes of improving outcomes in this high-risk group of patients.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The study examines regional cerebral blood flow changes in subjects during worry. We have modified the design of the study, instead of SPECT we are using PET imaging with 015 as a tracer. Using the revised schedule, a total of 6 scans have been completed. Presently, we are analyzing the data to determine the total number of subjects necessary for the study.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The objectives of this proposal are (1) to develop methods for quantitating daily exposure to nicotine, tar and carbon monoxide while smoking cigarettes or consuming smokeless tobacco; (2) to apply these methods to study exposure to toxic substances while smoking different numbers or different brands of cigarettes, and (3) to study other factors which are known to influence the intake and effects of tar and nicotine. Assays for quantitating urinary and, if possible, blood concentrations of polycyclic aromatic hydrocarbons, solanesol (a tobacco-specific terpene alcohol), tobacco-specific nitroso compounds and various combustion gases will be developed. Consumption of nicotine will be measured using pharmacokinetic data obtained after intravenous infusion of nicotine in combination with nicotine blood concentration data obtained over 24 hours while smoking in volunteer subjects on a clinical study center, or using casual blood level of nicotine and cotinine in outpatient studies. Using these methods, the effects of smoking different numbers of cigarettes, different tobacco products, i.e., pipes, cigars, snuff and chewing tobacco, and smoking cigarettes with different yields will be studied. In addition, the influence of alcohol and caffeine on nicotine and tar intake, as well as on the metabolism of nicotine and the cardiovascular effects of cigarette smoking, will be studied. Assays developed in our studies will be useful in assessing exposure to toxic components of tobacco smoke in epidemiologic studies and will provide specific information about potential health hazards of different tobacco products. The alcohol and caffeine studies will further our understanding of the influence of these commonly used drugs on smoking behavior and its consequences. Our studies may lead to more effective interventions to reduce the extent and harmful consequences of cigarette smoking.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The purpose of this contract is to provide support for the clinical and basic research staff of the NICHD Intramural Program. The Contractor will perform assays of steroid and peptide hormones on samples of blood, urine, and saliva from NICHD patients and research animals. These assays may number as many as 100,000 per year. The Contractor will also work with NICHD staff members to provide tight quality control on all assays and will be available to discuss and effectuate alterations in procedure to meet the changing needs of the investigators. This service is essential for the functioning of the two clinical branches and two of the basic research laboratories of the NICHD. The needs of the NICHD for hormone assays simply outstrip the in-house capabilities of the Clinical Center Clinical Chemistry Section.
|
{
"pile_set_name": "NIH ExPorter"
}
|
It has recently been found in this laboratory that ethanol can importantly affect meiotic chromosome behavior so that abnormalities are induced. A major goal of this project is to clarify the nature of the effects of ethanol on meiotic chromosome behavior and their implications with respect to the mechanisms of synapsis, crossing over and regular anaphase I chromosome distribution. Other recent work in this laboratory has provided new evidence of homologous chromosome pairing at the premeiotic mitosis. It is an aim of this project to assess the capacity of advance homologous association to provide the prerequisites for synapsis at meiosis and to establish preconditions for crossing over the sort which might influence the distribution of crossover sites. Approaches combine application of new techniques and laboratory findings to experimental material with an appropriate repertoire of meiotic and morphological chromosome variants. It is reasonable to expect that findings will have general applicability.
|
{
"pile_set_name": "NIH ExPorter"
}
|
MRI-guided therapy procedures have the potential to reduce the invasiveness of surgical approaches. The use of intraoperative MRI can also result in the discovery of novel interventional surgical applications. The long-term objective of this Program Project has been to provide the scientific medical and technical infrastructure for the development of intraoperative MRI. The work proposed involves an integrated, coordinated, multi-disciplinary translational research effort in developing MRI-guided procedures which can be tested in future clinical trials. The proposal addresses critical areas of intraoperative MRI which may have significant impact on the future of this emerging field. These specific areas are: MRI-guided neurosurgery; MRI-guided, ultrasound surgery; image processing technologies and display tools for surgical for surgical guidance; and dynamic, adaptive volumetric image acquisition techniques. Project 1 aims to develop an integrated system of pre- and intra-operative image acquisition, online image processing and intraoperative display which allows the utilization of all the accessible intraoperative and preoperative information for image-guided neurosurgery. The objective of Project 2 is to implement MRI guided and and monitored focused ultrasound surgery used in combination with radiation therapy for the effective treatment of small malignant breast tumors. In Project 3 we continue to develop and implement high performance computer integration of pre-operative MR data with intraoperative images in order to guide surgery intraoperatively . The proposed research in Project 4 is to develop, implement and improve fast 3D dynamic adaptive MRI techniques to meet the specific needs of several interventional MR applications. This Program Project emphasizes the interaction of closely related individual research projects. The individual components are not only well integrated but there is also significant cross-fertilization and active interchange between them. We present a plant for the integrated development of therapeutic techniques which are optimized through MRI-guidance, monitoring and control. We have been providing a rapid transfer of new developments from the laboratory to clinical applications. With this translational research we refine and validate the experimental methods and techniques development and implemented previously.
|
{
"pile_set_name": "NIH ExPorter"
}
|
DESCRIPTION (Applicant's Abstract): The hepatic biosynthesis of bile acids constitutes a major excretory pathway of cholesterol from the body. Bile acid feedback inhibits the transcriptional activity of the rate-determining enzyme in the neutral bile acid biosynthetic pathway, cholesterol 7alpha-hydroxylase (CYP7A1), as well as the first enzyme in the acidic pathway, sterol 27-hydroxylase (CYP27). Moreover, bile acids up-regulate the activity of mdr2, a transporter of phospholipid into bile. The mechanisms by which bile acids coordinate the expression of these genes remains undefined, but may have important implications in human diseases such as cholesterol gallstone disease and hyperlipidemia, in which bile acids play a pathogenetic role. This research proposal's broad objective is to explore the mechanisms by which bile acids regulate and coordinate cholesterol homeostasis in the hepatocyte by examining two specific aims: 1) to identify the bile acid-activated signal transduction pathways in primary rat hepatocytes, define the bile acid structural characteristics and PKC-dependence requirement for activation of these pathways. To determine which bile acid-induced signaling cascade(s) regulate the expression of CYP7A1 and CYP27 and the mdr2 transporter. 2) To determine the role of receptor and non-receptor tyrosine kinases in bile acid-mediated activation of the signaling cascades. The results of the studies outlined in this proposal will greatly improve our understanding of the role of bile acids in regulating cholesterol metabolism in the liver.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The objective of the collaborative clinical research effort is to improve the care of patients with bladder carcinoma through studies of different aspects of clinical management. The studies are carried out according to well-designed protocols. A Central Pathology Laboratory is responsible for the uniform collection, classification and analysis of pathologic and cytologic material. A Statistical Coordinating Center is responsible for data collection, organization and analyses and the Administrative Center is responsible for program coordination and control. At each participating institution, the specific aims are: 1. To assess and characterize all patients with bladder cancer, their neoplasms, and the fields from which the neoplasm arise; 2. To study systemic chemotherapy in patients with metastatic bladder carcinoma; 3. To study systemic chemotherapy as an adjuvant to preoperative radiotherapy and cystectomy in patients with invasive bladder carcinoma; 4. To study combined radiotherapy and chemotherapy in patients with inoperable, invasive bladder carcinoma; and 5. To study intravesical therapy in patients with superficial bladder cancer.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Our goal is to determine whether changes in the formation or processing of oxidative DNA damage are associated with neurodegeneration observed after stroke or in aging and age-associated diseases. Stroke is a leading cause of death, and ROS generated during ischemia may contribute to neuronal death. Although stroke is treatable with timely medical help, only 10% of stroke victims recover completely from a major stroke episode. Thus, it is important not only to identify risk factors for stroke, but to identify factors that influence post-stroke outcomes (i.e., reduce disability or death from stroke). It has been proposed that lower BER capacity could partly explain the increased incidence and adverse effects of stroke in older individuals. Therefore, we are investigating the impact of simulated stroke in mice carrying defects in specific DNA glycosylases or other BER enzymes. We are testing the hypothesis that loss of BER capacity negatively impacts the brains ability to recover from acute oxidative stress experienced during a stroke. Using the Ogg1 knockout mice and a stroke model, we previously demonstrated that Ogg1 KO animals had larger infarct volumes and displayed poor recovery following stroke. Our recent results suggest that the Neil1 KO mice are more sensitive to stroke and generates more ischemia and recovers more slowly than wild type mice. In addition, using behavioral studies, we detected a memory deficiency in the Neil1 KO mice. This work suggests that a BER deficiency may be directly associated with cognitive function and we plan to extend these studies to other DNA repair defective mouse models and on another genetic background. Together, these findings underscore the importance of BER as a disease modifier. Efforts to define other BER proteins whose absence impacts the recovery from stroke are ongoing and continue to support our findings that an individual's BER capacity may be a major determinant in the extent of recovery from an acute stressor like stroke. AD is one of the leading causes of neurodegeneration and there are numerous documented cases of neurodegeneration associated with genetic DNA repair defects. Given that there is compelling evidence that DNA repair capacity alters the ability of mice to recover from an acute oxidative stress and that AD is associated with chronic oxidative stress, we tested the hypothesis that a defect in DNA repair might exacerbate the AD phenotypes in a mouse model of AD (3xTg AD). While the studies are still ongoing, there is evidence to suggest that a defect in DNA repair capacity can modulate memory and learning. Full behavioral, memory and learning experiments are underway in mice to quantify the extent to which a DNA repair deficiency impacts the AD features.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Spectral techniques such as fluorescence, phosphorescence, flash photolysis, and ESR are necessary to elucidate the photophysics and photochemistry of environmental chemicals. Because much of the needed equipment is either not available commercially or does not offer the desired futures, we build or modernize/upgrade most of it ourselves. This includes the interfacing to computers for ease of data acquisition and manipulation. Two old spectrophotofluorometers (steady state and phase modulation) that have been combined into one T-configured unit have being upgraded to measure phosphorescence spectra and photobleaching. The laser flash photolysis set-up has a new more powerful laser (Surelite II) for excitation. The choice of excitation wavelengths has been extended from 400nm to the infrared by using a tunable OPO system that is pumped by the 355nm harmonic from the Surelite laser. A flow system that refreshes anaerobic samples after strong laser excitation has been added to prevent the bleaching of the irradiated area. This new laser flash photolysis set-up has been adapted for EMF studies by incorporating an electromagnet and a new analytical lamp to observe the transient spectra in the presence of EMF. The Surelite laser has also been aligned with the EPR spectrometer to generate radicals directly in the cavity of the EPR spectrometer after multi-photon absorption from laser pulses. Our singlet oxygen spectrometers are being presently used to measure the interaction of singlet molecular oxygen with biological and environmental substrates we investigate. In addition, the steady-state singlet oxygen spectrophotometer is being upgraded to measure singlet oxygen production in non-photochemical reactions. This system has also been modified to permit the direct observation of keratinocytes grown in a monolayer. With the aid of this instrumentation we have been able for the first time to detect singlet oxygen directly in cells. To interpret the singlet oxygen phosphorescence data correctly, we have to establish how singlet oxygen properties may be affected by different environment. We have already measured the influence of polarity, proticity and polarizability in a number of solvents and solvent mixtures. Presently, these investigations are being extended over the heterogeneous (micellar) systems, which more closely relates to biological environments. As new technology becomes available, all of the above systems are continually being modified. These changes frequently also require the building of new interfaces and the development of new software for control. We are presently building a prototype photoconductivity cell to measure electrical photoconductivity in dielectric liquids in conjunction with ESR detection. This cell will be interfaced to the time-resolved laser flash photolysis spectrometer (vide supra) to permit studies of systems that cannot be observed optically. The laser flash photolysis system has been upgraded with a tunable laser system which emulates dye lasers. This new system has been adapted for EMF studies by incorporating an electromagnet.In order to understand the photochemistry and photophysics of environmental chemicals it is necessary to use the techniques of modern chemical analysis including spectroscopic techniques of many kinds. The object of this project is to build, test and interface spectrometers that are needed for photophysical studies.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Different mechanisms could explain the origin and heterogeneity of CSC such as (i) differentiation arrest (stem cells), (ii) dedifferentiation (mature cells) and (iii) transdifferentiation (bone marrow stem cells). It is conceivable that all 3 mechanisms may be corrupted by oncogenic events, resulting in an assortment of CSC and explaining their heterogeneity. Defining and characterizing this heterogeneity is of vital importance for understanding CSC biology, and for effective therapeutic translation. Our most recent results in this project include: (1) Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. Here we addressed the impact of epigenetic alterations and cellular context on functional and transcriptional reprogramming of hepatocellular carcinoma (HCC) cells. Our strategy employed a 3-day treatment of established and primary human HCC-derived cell lines grown as a monolayer at various cell densities with the DNMT1 inhibitor zebularine (ZEB) followed by a 3D culture to identify cells endowed with self-renewal potential. Differences in self-renewal, gene expression, tumorigenicity, and metastatic potential of spheres at generations G1-G5 were examined. Transient ZEB exposure produced differential cell density-dependent responses. In cells grown at low density, ZEB caused a remarkable increase in self-renewal and tumorigenicity associated with long-lasting gene expression changes characterized by a stable overexpression of cancer stem cell-related and key epithelial-mesenchymal transition genes. These effects persisted after restoration of DNMT1 expression. In contrast, at high cell density, ZEB caused a gradual decrease in self-renewal and tumorigenicty, and up-regulation of apoptosis- and differentiation-related genes. A permanent reduction of DNMT1 protein using short hairpin RNA (shRNA)-mediated DNMT1 silencing rendered HCC cells insensitive both to cell density and ZEB effects. Similarly, WRL68 and HepG2 hepatoblastoma cells expressing low DNMT1 basal levels also possessed a high self-renewal, irrespective of cell density or ZEB exposure. Spheres formed by low-density cells treated with ZEB or shDNMT1 displayed a high molecular similarity which was sustained through consecutive generations, confirming the essential role of DNMT1 depletion in the enhancement of cancer stem cell properties. In conclusion, these results identify DNA methylation as a key epigenetic regulatory mechanism determining the pool of cancer stem cells in liver cancer and possibly other solid tumors; (2) Human primary liver cancer is classified into biologically distinct subgroups based on cellular origin. Liver cancer stem cells (CSCs) have been recently described. We investigated the ability of distinct lineages of hepatic cells to become liver CSCs and the phenotypic and genetic heterogeneity of primary liver cancer. We transduced mouse primary hepatic progenitor cells, lineage-committed hepatoblasts, and differentiated adult hepatocytes with transgenes encoding oncogenic H-Ras and SV40LT. The CSC properties of transduced cells and their ability to form tumors were tested by standard in vitro and in vivo assays and transcriptome profiling. Irrespective of origin, all transduced cells acquired markers of CSC/progenitor cells, side populations, and self-renewal capacity in vitro. They also formed a broad spectrum of liver tumors, ranging from cholangiocarcinoma to hepatocellular carcinoma, which resembled human liver tumors, based on genomic and histologic analyses. The tumor cells coexpressed hepatocyte (hepatocyte nuclear factor 4 alpha), progenitor/biliary (keratin 19, epithelial cell adhesion molecule, A6), and mesenchymal (vimentin) markers and showed dysregulation of genes that control the epithelial-mesenchymal transition. Gene expression analyses could distinguish tumors of different cellular origin, indicating the contribution of lineage stage-dependent genetic changes to malignant transformation. Activation of c-Myc and its target genes was required to reprogram adult hepatocytes into CSCs and for tumors to develop. Stable knockdown of c-Myc in transformed adult hepatocytes reduced their CSC properties in vitro and suppressed growth of tumors in immunodeficient mice. From these data we conclude that any cell type in the mouse hepatic lineage can undergo oncogenic reprogramming into a CSC by activating different cell type-specific pathways. Identification of common and cell of origin-specific phenotypic and genetic changes could provide new therapeutic targets for liver cancer; (3) The relative contribution of hepatocyte growth factor (HGF)/MET and epidermal growth factor (EGF)/EGF receptor (EGFR), two key signal transduction systems in the normal and diseased liver, to fate decisions of adult hepatic progenitor cells (HPCs) has not been resolved. Here, we developed a robust culture system that permitted expansion and genetic manipulation of cells capable of multilineage differentiation in vitro and in vivo to examine the individual roles of HGF/MET and EGF/EGFR in HPC self-renewal and binary cell fate decision. By employing loss-of-function and rescue experiments in vitro, we showed that both receptors collaborate to increase the self-renewal of HPCs through activation of the extracellular signal-regulated kinase (ERK) pathway. MET was a strong inducer of hepatocyte differentiation by activating AKT and signal transducer and activator of transcription (STAT3). Conversely, EGFR selectively induced NOTCH1 to promote cholangiocyte specification and branching morphogenesis while concomitantly suppressing hepatocyte commitment. Furthermore, unlike the deleterious effects of MET deletion, the liver-specific conditional loss of Egfr facilitated rather than suppressed progenitor-mediated liver regeneration by switching progenitor cell differentiation toward hepatocyte lineage. These data provide new insight into the mechanisms regulating the stemness properties of adult HPCs and reveal a previously unrecognized link between EGFR and NOTCH1 in directing cholangiocyte differentiation. (4) Activation of c-MYC is an oncogenic hallmark of many cancers including liver cancer,and is associated with a variety of adverse prognostic characteristics. Despite a causative role during malignant transformation and progression in hepatocarcinogenesis, consequences of c- MYC activation for the biology of hepatic cancer stem cells (CSCs) are undefined. Here, distinct levels of c-MYC over-expression were established by using two dose-dependent tetracycline inducible systems in 4 hepatoma cell lines with different p53 mutational status. CSCs were evaluated using side-population approach as well as standard in vitro and in vivo assays. Functional repression of p53 was achieved by lentiviral shRNA transduction. The results show that c-MYC expression levels have a differential impact on liver CSC characteristics. At low levels, c-MYC activation led to increased proliferation and enhanced CSC properties including activation of reprogramming transcription factors and CSC marker expression (e.g. NANOG, OCT4 and EpCAM), expansion of side population and acceleration of tumor growth upon subcutaneous transplantation into immunocompromised mice. However, when exceeding a threshold level, c-MYC induced a pro-apoptotic program and loss of CSC potential both in vitro and in vivo. Mechanistically, c-MYC induced self-renewal capacity of liver cancer cells was exerted in a p53 dependent manner. Low c-MYC activation increased spheroid formation in p53-deficient tumor cells, whereas p53-dependent effects were blunted in the absence of MYC overexpression. Our results confirm the role of c-MYC as a master regulator during hepatocarcinogenesis and establish a new gatekeeper role for p53 in repressing c-MYC induced CSC phenotype in liver cancer cells.
|
{
"pile_set_name": "NIH ExPorter"
}
|
P-glycoprotein (P-gp) is an ATP-dependent efflux transporter that plays a critical role in drug distribution, drug-drug interactions and Drug resistance. Drugs that modulate P-gp activity could have therapeutic utility by increasing delivery of other drugs to the central nervous system, or by improving their pharmacokinetic properties. P-gp is an extremely promiscuous transporter that includes a drug binding site, or sites, within a 12- transmembrane helix domain (TMs), which communicates with nucleotide binding domains (NBDs) that bind and hydrolyze ATP to drive conformational changes in the TM domain. Due to the difficulty in studying P-gp in model membranes no kinetic measurements have been made on any elementary step within the P-gp reaction cycle, which remains poorly defined. Knowledge of the rates of these processes is essential to determine which mechanistic models are most accurate, among several proposed schemes. The goals of this proposal are to exploit P-gp in lipid bilayer nanodiscs, to challenge two competing mechanistic models of P-gp with studies that utilize stopped-flow spectroscopy, surface plasmon resonance and single molecule total internal fluorescence microscopy (TIRFM). For the latter two methods, several surface attachment strategies will be compared to optimize data acquisition. The binding and dissociation rate constants of nucleotides will be determined in the presence of several P-gp substrates and inhibitors. An extension of these studies includes the use of single cysteine P-gp mutants covalently adducted with varying drugs, to determine the effect of drug location on nucleotide binding and dissociation. Similarly, binding and dissociation of fluorescent drugs will be determined with varying nucleotides bound at the NBDs. These studies will provide the first kinetic data for P-gp ligand interactions that will elucidate several mechanistic details that have not been previously clarified. Such studies could lead to conformation-specific inhibitors of P-gp with utility in the modulation of pharmacokinetic properties of existing drugs. PUBLIC HEALTH RELEVANCE: P-glycoprotein is a large transmembrane helical efflux transporter that plays a major role in cancer cell resistance to drugs and the distribution and clearance of most drugs spanning all therapeutic areas. The molecular mechanism of P-gp remains unknown. The long term goal of this proposal is to understand the molecular mechanisms of the efflux transporter P-glycoprotein in order to rationally design drugs that target P-gp and improve the therapeutic profile of other drugs.
|
{
"pile_set_name": "NIH ExPorter"
}
|
PROJECT SUMMARY?OVERALL COMPONENT: The NIH has long recognized the necessity to aggressively leverage emerging methodologies like precision medicine, digital health, biomedical data science and virtual reality & simulation based interventions approaches to ameliorate health disparities in racial and ethnic minorities. However, relatively fewer efforts have attempted to conduct trans- disciplinary aging research integrating biological, social and behavioral sciences utilizing emerging methodologies. The overarching goal of the proposed Stanford Aging and Ethnogeriatrics Transdisciplinary Collaborative Center (SAGE) is to increase the diversity of the aging research workforce by mentoring new leaders in aging research and promoting advances in behavioral and social sciences aging research using emerging methodologies including precision medicine, digital health, biomedical big data science and virtual reality and simulation based interventions. By identifying, mentoring and supporting (through dedicated pilot funds and methodological and recruitment support) junior investigators from underrepresented groups (SAGE Scientists) we propose to create a culturally sensitive and culturally competent research workforce focused on promoting equity in healthcare and mitigating disparities in diverse older populations using emerging methodologies. We have in place outstanding strengths in the three principal scientific elements requisite to form a coordinated, sustainable, and far-reaching `aging research to practice collaboration' integrating behavioral, social and biological sciences using emerging methodologies: 1) Behavioral and social scientists and disparities researchers, spanning psychology, psychiatry, demography, epidemiology, evolutionary biology, health services research & economics, computer science, social media science, virtual reality/simulation interventions and digital health; 2). Extensive experience with trans-disciplinary translational research that integrates laboratory assessment of genetic markers, their relationships to both disease risk and treatment relevance with psychosocial research approaches; and 3) Novel and outstanding analytic and high-performance `big-data' computing capabilities, consolidated in our Department of Biomedical (`Big Data') Data Science. With Stanford's setting in one of the most racially, ethnically and socially diverse aging populations in the US, and leveraging the seven-school campus-wide ?Precision Health? initiative that integrates biomedical data science, engineering, business, design, digital health technology, virtual reality interventions and patient care under the recently endowed Center for Population Health Sciences we feel uniquely situated to make game-changing breakthroughs in aging research using emerging methodologies, and to lead such research efforts nationally.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Neonatal thymectomy, which inhibits maturation of select aspects of the immune response, renders animals more susceptible to the carcinogenic action of oncogenic viruses and select chemical carcinogens. In man, several thymus-related immunologic deficiency diseases have been shown to be characterized by a markedly increased frequency of malignant tumors, as has been chronic immunosuppressive therapy. On the basis of the above and related observations, the recirculating thymic-dependent small lymphocyte (T cell) has been implicated in the pathogenesis of a variety of spontaneous and artifically induced tumors and it has been postulated that this cell type constitutes an integral part of the immune surveillance mechanism. The remarkable radiosensitivity of the small lymphocyte has been well documented, as has been the carcinogenic effect of ionizing radiation. The mechanisms involved in such tumorigenesis are not clear, however, and in particular the relationship between the immunosuppressive effects of exposure to biologically significant amounts of whole body radiation and the neoplastic consequences of such exposure remain poorly defined. The purpose of the proposal is threefold: 1) to investigate the effect of neonatal and adult thymectomy on the prevalence of spontaneous and radiation-related tumors in germfree and conventional mice; 2) to determine if the tumorigenic effects of radiation can be modified via reconstitution with defined populations of lymphocytes post-exposure; suppressor and helper T cells are of particular interest in this regard; 3) to correlate the above with the immune status of the host, particularly with respect to the number of recirculating T cells mobilizable via thoracic duct cannulation. Other methods of evaluation include: temporal evaluation of the immunologic capabilities of thymic-dependent lymphocytes from each experimental group including evaluation of relative numbers of suppressor and helper cells; morphologic evaluation of all deaths to include histologic evaluation of tumors; age-specific death rate for neoplasms.
|
{
"pile_set_name": "NIH ExPorter"
}
|
In a series of anatomical experiments, we have identified two routes by which somatosensory information reaches limbic structures important for somatic memory. One route courses dorsally from the postcentral strip through posterior parietal cortex and has access to the limbic system via cingulate cortex. This pathway may be functionally analogous to the occipitoparietal pathway for spatial vision. A second route courses ventrally from the postcentral strip through SII to the insular cortex and then to the amygdala and indirectly to the hippocampus through rhinal cortex. We now have evidence that this second pathway is analogous to the occipitotemporal pathway for object vision. Our neurobehavioral evidence indicates that bilateral insular lesions cause a severe tactile recognition deficit, consis- tent with the suggestion that insular cortex is a critical link in a parieto- insulo-limbic pathway for tactile recognition, and so occupies a position analogous to that of area TE in the occipito-temporo-limbic pathway for visual recognition. By recording neuronal activity from SII in hemispheres that received lesions of selected portions of the postcentral body representation, we demonstrated a loss of the corresponding representation from SII, indicating a serial flow of information from the postcentral strip to SII. An unexpected finding in this study was that, following a postcentral lesion, the deactivated SII region undergoes major functional reorganization. In another experiment we recorded neuronal activity from the postcentral strip in 4 monkeys 10-12 years after the dorsal roots representing the upper limb were severed. The results indicated that the entire representation of the upper limb had been replaced by an expanded face representation, an expansion an order of magnitude greater than previously thought possible. Both of these experiments, involving perturbations of the central nervous system, resulted in a previously unrecognized degree of cortical plasticity in adult mammals and suggest that the potential for reorganization is greater after central than peripheral nervous system manipulations.
|
{
"pile_set_name": "NIH ExPorter"
}
|
D-type cyclins (cyclin D1, D2 and D3) are components of the core cell cycle machinery. Rearrangements of cyclin D genes and overexpression of cyclin D proteins are seen in a large number of lymphoid malignancies. Cyclins D1 and D3 were implicated to play role in formation of T-cell acute lymphoblastic leukemias (T-ALL). The best-documented function of D-type cyclins is their ability to activate cyclin-dependent kinases CDK4 and CDK6 and to allow cell cycle progression. However, there is growing evidence from several sources, including our own work, that D-cyclins play important cell cycle-independent roles. We hypothesize that aberrantly expressed cyclins D1 and D3 contribute to T-ALL formation via their association with novel partners, and facilitate T-cell transformation through mechanisms and pathways independent of the roles of these proteins in cell cycle progression. We also hypothesize that cyclin D1 contributes to T-ALL formation via different molecular mechanism than cyclin D3, i.e. through interaction with different cellular partners. In the work described this application we will study the exact molecular functions of cyclin D1 and D3 in mouse and in human T-ALL. For mouse studies, we will take advantage of a novel knock-in strain of mice expressing tandemly (FLAG- and HA-) tagged cyclin D1 in place of the wild-type protein, which was recently generated in our laboratory. We already demonstrated that this strain allows us to determine the identity of cyclin D1 protein partners (through immunoaffinity purification - mass spectrometry) and cyclin D1 genomic targets (through ChlP-chip) in essentially any major mouse organ or in tumorigenesis. We will also generate a novel mouse strain expressing tagged cyclin D3. We will use these strains to determine the identity of cyclin D1- and D3-molecular partners and targets in a Notch-driven murine model of T-ALL. We will utilize cell lines and gene-delivery systems available in von Boehmer lab to test contribution of these proteins to tumorigenesis in a mouse T-ALL model. Together with the Look lab, we will extend these analyses to human T-ALL, and we will dissect the molecular functions of cyclins D1 and D3 in the oncogenic transformation of human T-cell lineage. The Specific Aims are as follows: Specific Aim 1;To determine the molecular partners and targets of cyclins D1 and D3 and their contribution to tumorigenesis in a Notch-driven murine model of T-ALL. Specific Aim 2: To determine the molecular function of cyclins D1 and D3 in human T-ALL.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The enhancement of pain following injury occurs acutely within a few minutes, and can last for several days or weeks. In some cases, the enhance pain can last indefinitely, either because the injured cannot be repaired, or because of some unknown mechanism that may not be related to peripheral injury. In these cases, the pain is defined as "chronic pain." It has been proposed that long-term exposure to painful stimuli can cause profound changes in the central nervous system that are very difficult to alter and are the cause of chronic pain. Chronic pain has proven to be extremely debilitating for affected individuals, and has been refractory to most treatments. We hypothesize that one of the CNS locations in which pain transmission is altered by persistent nociceptive inputs is the spinal cord. Just as a number of peripheral events contribute to short-term sensitization of peripheral nociceptors (see Projects 2 and 3), it is likely that long-term spinal cord sensitization of peripheral nociceptors (see Projects 2 and 3), it is likely that long-term spinal cord sensitization requires a combination of factors involving damaged peripheral tissue, afferent traffic from that tissue, and spinal responses to the peripheral injury. We propose that understanding, long-term spinal sensitization requires systematic manipulation and evaluation of combinations of peripheral, afferent and central effects that have been implicated individually as contributors to persistent pain. Once these factors are understood, rational treatments can be developed. The specific aims for the next 5 years are to determine: 1. the contribution of primary afferent firing in our present model of long- term hyperalgesia. 2. whether blockade of afferent activity, axonal transport of peripheral nerve ending destruction a) affects the glial activation and allodynia/hyperalgesia produced by peripheral injection of formalin, and/or b) produces glial activation and allodynia/hyperalgesia without peripheral tissue damage. 3 whether activation of spinal cord glial cells (astrocytes and microglial cells) is involved in short- and long-term hyperalgesia. 4. some of the potential mediators that activate astrocytes and microglia and induce long-term hyperalgesia in our formalin model. Combinations of single afferent recording, behavior, and immunohistochemistry will be used on rats and transgenic mice to accomplish these aims.
|
{
"pile_set_name": "NIH ExPorter"
}
|
ABSTRACT Although the respiratory acclimatization to chronic hypoxia has been well described, specific information as to the nature of the cardiovascular component of the acclimatization response is lacking and a convenient animal model has been unavailable. We propose to expose chronically instrumented sheep to chronic hypoxia in an environmental chamber for 4-5 days to test the hypothesis that neurohumoral adjustments, probably initiated by arterial chemoreflexes, provide a redistribution of systemic flow which sustains a pattern of 02 conservation. We will measure regional blood flow distribution in chronic hypoxia to skeletal muscle, GI tract, kidney (cortex), respiratory muscles, brain, and heart by serial injections of radiolabelled microspheres (5 isotopes) using the reference sample technique in the awake sheep. Subsequently, the role of the chemoreflexes in sustaining the regional circulatory adjustments during chronic hypoxia will be determined in sheep after chronic chemoreceptor denervation. Also, reversible vagal cold block will be used to assess the contribution of vagal cardiopulmonary afferents to the control of interorgan flow distribution in chronic hypoxia. Lastly, the contribution of autonomic efferent activity to the regulation of regional flow will be assessed by administration of selective adrenergic and cholinergic blocking agents. Ventilatory sensitivity to periodic C02 breathing will be used to determine the phase of acclimatization and to analyze cardiovascular responses to C02 hypoxia interactions.
|
{
"pile_set_name": "NIH ExPorter"
}
|
AR PAR-16-137 Project Summary/Abstract The Arkansas Agriculture Department is applying for funding through Funding Opportunity PAR-16-137, Competition AB, to plan, develop, and implement a produce safety program in Arkansas to implement the FDA Produce Safety Rule to minimize the risk of serious adverse health consequences or death from consumption of contaminated produce. The Arkansas Agriculture Department intends to seek the adoption of the FDA Produce Safety Rule in its entirety. In Year 1 of the grant, the Arkansas Agriculture Department will work with all appropriate state and federal agencies and other organizations to: assess existing and needed resources; determine infrastructure, organizational and personnel resources needs; and develop a multi-year strategy for the program. The agency and its partners will identify the type and location of commodities grown in the state and capture all farm inventory data in an electronic system that allows sharing with regulatory partners. Arkansas Agriculture Department staff also will secure all necessary legislative and regulatory authority and begin hiring needed personnel identified in the assessment. Funding in Year 2 of the grant will be used to develop and provide education, outreach, and technical assistance to producers and regulators utilizing FDA sanctioned materials. The Arkansas Agriculture Department will enter into a sub-grant agreement with the University of Arkansas, Division of Agriculture Cooperative Extension Service (Extension) for this work. In Year 2, the Arkansas Agriculture Department will develop and implement an On-Farm Advisory Review program. An inspectional program also will be researched, designed, and implemented in Year 2. In Years 3, 4, and 5 of the grant, the Arkansas Agriculture Department will continue to work of the objectives started in previous years if the requirements of a specific goal have not been met. The inspectional program will continue and expand as new producers enter the industry. The Arkansas Agriculture Department will assess and evaluate all processes and progress toward program goals to identify if changes or improvements are needed. Best practices will be identified and incorporated. Extension will continue all outreach, education, and technical assistance efforts, and action will be taken to ensure all regulatory staff remain fully trained on the Produce Safety Rule requirements.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The goal of the research during the past year has been: 1) to complete work on the site at which barbiturates block excitatory synaptic transmission and 2) to determine the mechanism by which barbiturates enhance the action of GABA. Further studies on the sympathetic ganglion using quantal analysis confirmed previous conclusions that the blockade in ganglionic transmission by pentobarbital is due entirely to a postsynaptic action. Thus the number of quanta released was unaltered by pentobarbital but the size of each quanta was severely depressed. The second project involved determining how barbiturates enhance the action of GABA. For these experiments, recordings with sucrose gap were made from the ventral root of the isolated hemisected frog spinal cord. Dose response curves for GABA, B-alanine and glycine were constructed. It was found that barbiturates shift the GABA dose response curve to the left, often with a slight reduction in the slope of the curve. They had no effect on the B-alanine or glycine responses. These results suggests that bartiturates increase the affinity of the GABA receptor for GABA. Experiments are currently in progress to determine if barbiturates can interact with the sites at which bicuculline and picrotoxin act and, if so, whether this interaction can be related to the apparent change in affinity that barbiturates produce in the GABA receptor.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The apical chloride/base exchanger in kidney proximal tubule and gastrointestinal tract works in parallel with NHE3 and is essential for chloride absorption and fluid homeostasis. However, less is known about the molecular identity and the main functional mode of this exchanger. SLC26A6, also known as PAT1 or CFEX, is a member of a large, highly conserved family of ion transporters (designated as SLC26A) many of which mediate anion exchange. SLC26A6 can mediate CI'/HCO3-, Cl/formate and Cl-/oxalate exchanges, indicating that it is an ideal candidate for apical Cl/base exchange in proximal tubule. Our experiments demonstrate that SLC26A6 mediates CI-/HCO3- exchange, is located on the apical membrane of proximal tubule, small intestine and tubulovesicles of gastric parietal cells, and is upregulated by NH4+, a naturally occurring chemical in kidney proximal tubule and small intestine. We further demonstrate the presence of an apical CI/HCO3- exchange in rat and mouse isolated microperfused proximal tubules and in tubulovesicles of gastric parietal cells. We hypothesize that SLC26A6 is the main apical anion exchanger in kidney proximal tubule and duodenum, functions predominantly as a CI-/HCO3- exchanger under physiologic conditions and is responsible for the bulk of chloride absorption. In the stomach, PAT1 may be essential for neutralizing the luminal acidity of H-K-ATPase-containing vesicles during the resting state. The purpose of the current proposal is to ascertain the role of the apical CI-/IHCO3- exchanger and PAT1 (SLC26A6) in chloride (and fluid) absorption in kidney proximal tubule and gastrointestinal tract. Toward this end, we propose to: 1. Characterize the apical CI/HCO3- exchanger in isolated microperfused proximal tubule and ascertain its role in chloride reabsorption in the presence of solutions mimicking proximal tubule milieu, 2. Ascertain the role of SLC26A6 in apical Cltbase exchange and cr reabsorption in kidney proximal tubules using SLC26A6 (PAT1) null mice, 3. Examine the acute and chronic regulation of SLC26A6 (PAT1) and apical CI-/HCO3- exchanger in kidney proximal tubule, and 4. Determine the role of SLC26A6 in gastric and intestinal function, including CI absorption and protection against acid injury in the duodenum and acid secretion in the stomach, using SLC26A6 null mice. Insight into the functional and molecular properties of PAT1 and apical CI-/HCO3- exchange in kidney proximal tubule and upper gastrointestinal tract in physiologic states and examination of PAT1 deficient mice should significantly enhance our understanding regarding the apical CI-/base exchanger and cr absorption in epithelial tissues. They should further facilitate future investigations into the role of this transporter in pathophysiologic states affecting electrolyte and acid-base homeostasis.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This application responds to RFA-CA-09-001, which requests applications for Centers for Population Health and Health Disparities (CPHHD) that will collaborate with other such Centers to promote transdisciplinary research in health disparities. In this application, the University of Washington (UW) and Black Hills Center for American Indian Health (BHCAIH) seek support for a Center for Native Population Health Disparities (CNPHD). Our aim is to directly contribute to improved cancer health outcomes and quality of life for American Indian/Alaska Native (AI/AN) populations - populations that are experiencing dramatic health inequities. Although strides have been made in improving health and life expectancy in Native communities, much work remains, as eloquently argued in the Institute of Medicine report, Examining the Heath Disparities Research Plan of the National Institutes of Health: Unfinished Business'^ and the Surgeon General's report. Mental Health: Culture, Race, and Ethnicity.^ Our CNPHD will strive to address the gaps identified in these seminal publications. Our research agenda articulates a model for AI/AN health and health disparities that is well-grounded in current theory, consistent with that of the CPHHDs, and applicable to all our projects. The many Native people involved in this Center, as well as our Community Action Board and 2 AI/AN sociologists, will help us critically examine the role of culture in the model. Our investigations reiterate a single leitmotif - the examination of genetic, environmental, biological, psychosocial, and cultural influences on cancer health disparities at multiple levels and across the lifespan. Our projects include children, youth, and adults. The proposed CNPHD will fill a striking research gap, since none of the 8 currently funded CPHHDs examines AI/AN health disparities and only 1 targets rural populations. Our Center will truly represent our research partner communities, with 11 AI/ANs serving in Icey roles, for example as Project and Core Leaders or Co-investigators (see Table 5 at end). Most notably, the Co-Principal Investigator is an enrolled member of the Cheyenne River Sioux Tribe and directs a community-based organization. As a result, all proposed projects have deep and meaningful ties to their respective communities. The unique connection between our investigators and the tribal communities imbues our work with a special dynamic. Investigators are more fully invested in the communities with which they work, and this commitment goes a long way to ensuring reciprocity and respect among ail parties involved in the research. Finally, the CNPHD will truly be transdisciplinary. We bring together scholars from across the full spectrum of biological, social, and behavioral sciences. Of special note, we do not limit team science to traditional academic circles, but bring in ideas and Native expertise from community-based leaders and entities. We envision that this approach will eventually move research on AI/AN health disparities beyond the purely descriptive into meaningful action. The CNPHD will pursue an integrated, multi-faceted research program that capitalizes on several large, well-organized, existing efforts and databases, as well as numerous well-established and mutually respectful relationships, to address issues in the genetics, etiology, epidemiology, primary and secondary prevention, clinical assessment, treatment, and survivorship of cancer. Clinical, basic, and social sciences are linked with a community-based participatory perspective to provide a transdisciplinary research program of the highest scientific and programmatic caliber for AI/AN community members, patients, clinicians, researchers, and health decision-makers. Notably, this application follows the overarching requirements for CPHHDs to 1) include scientists from basic, clinical, and social sciences; 2) focus on an underserved population; 3) incorporate principles of community-based participatory research; 4) include interventions directed towards at least 2 levels; 5) offer career development opportunities; and 6) interact and harmonize with other CPHHDs and NIH program staff to conduct transdisciplinary, multi-site, highly interactive research.
|
{
"pile_set_name": "NIH ExPorter"
}
|
NIDA has had a long-standing mission to determine the impact of chronic stress on substance use, abuse, and addiction. This application addresses this important objective in multiple, innovative ways. In particular, we focus on late adolescents with histories of experiencing child maltreatment, a severe and chronic stressor contributing to high risk for maladaptation, psychopathology, and substance use and abuse across the life course. Although numerous studies have identified a linkage between child maltreatment and substance use in adolescence and adulthood, the developmental mechanisms through which the experience of the chronic stress of child maltreatment results in substance use and abuse have received only minimal attention. Child maltreatment is known to impair both biological and psychological systems, and it is essential to investigate how these developmental consequences of child abuse and neglect contribute to the emergence of substance abuse outcomes. In the present application, we're seeking a renewal of our previous investigation of a diverse cohort of preadolescent maltreated and nonmaltreated children studied during a period when there is minimal substance use normatively. We propose to follow-up this sample of children in late adolescence/emerging adulthood, a period when substance abuse normatively escalates. We will capitalize on the solid developmental foundation acquired in the original study, which obtained an extensive assessment of multilevel influences (socio- emotional, interpersonal, family relational, social contextual, personality, psychopathology, attentional, neurocognitive, and neuroendocrine) in maltreated and nonmaltreated children. The multilevel assessment of diverse domains will be continued in the present application. We also will include genetic sampling in order to investigate the potential of protective vs. risk promoting influences of genetic variants on late adolescent substance abuse outcomes and differential effects dependent on exposure to child maltreatment. We will conduct diverse multilevel assessments with the late adolescents, as well as obtain perspectives on the late adolescent's functioning from caregivers and best friends. Assessing the effects of chronic stress on stress-sensitive systems at earlier, more distal periods of development, as well as the capacity to integrate both distal and proximal adaptations to chronic stress in late adolescence has the potential to greatly expand understanding of the multilevel impact of chronic stress on drug abuse. This research will have important implications for public health. In addition to advancing knowledge regarding the multilevel developmental roots of substance use and abuse, the research will provide important direction for prevention and intervention strategies. Given that multiple pathways from child maltreatment to substance abuse are likely to be identified, insights into different intervention targets will be elucidated. PUBLIC HEALTH RELEVANCE: Child maltreatment poses substantial risk across the life course for maladaptation, psychopathology, and substance use and abuse and thus constitutes a critical public health concern. The proposed multilevel, developmental follow-up investigation of late adolescents with and without a history of child abuse and neglect will expand understanding of the adverse effects associated with the chronic stress of child maltreatment on stress-sensitive biological and psychological systems that are implicated in substance abuse. Through increasing knowledge of multilevel developmental processes contributing to substance abuse, the research will provide important direction for prevention and intervention and is of high public health significance.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Numerous studies have demonstrated that only a small percentage of subjects with ocular hypertension actually develop primary open-angle glaucoma. Efforts to identify specific single factors as prognostic indicators of glaucomatous visual field loss have been unrewarding. In a previous retrospective study using a system of multivariate analysis, it was possible to identify accurately those individuals who developed glaucoma among a group of subjects with ocular hypertension. The present study is designed as a prospective evaluation of this multivariate estimate of risk in a selected population of subjects with ocular hypertension. Using the above system, estimates of the probability of visual field loss will be calculated for each subject at the beginning of the study. All subjects will then be followed for five years, following which the predictive estimates of visual field loss will be evaluated.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Alveolar macrophages (AM) produce two chemotactic factors for neutrophils that may be important in inflammatory processes in the lungs, a low molecular weight lipid and a 10 kDa protein. In past studies, we have identified the low molecular weight lipid as LTB4, but the protein species has not been completely characterized. Identification of this protein and the study of the regulation of its production is important in understanding the mechanisms by which inflammatory cells are recruited to the lungs. Our GOALS in these studies are to isolate and purify the protein chemoattractant; to isolate a specific cDNA in order to identify the amino acid sequence of the entire protein; to determine the relative contribution of this protein to the neutrophil chemotactic activity produced by the AM; and to study the regulation of this protein in vitro and in vivo, using an animal model of localized lung inflammation. We have found that porcine and human AM produce large amounts of this 10 kDa protein when stimulated with E. coli lipopolysaccharide (LPS). We have succeeded in purifying it 200 fold in a series of steps including cation exchange HP. We will purify the protein to homogeneity and determine the N-terminal amino acid sequence. We will raise a specific polyconal antibody to the protein in rabbits, and use this antibody to screen a cDNA library made from LPS stimulated AM in order to isolate a clone containing the specific cDNA to this protein. From this cDNA,, we will predict the amino acid sequence of the protein, which will permit a definitive comparison with other known cytokines. The preparation of the specific antibody and the cDNA probe will provide tools for the study of the expression/secretion of this protein in AM from an animal model of localized inflammation in order to understand when this protein is produced in vivo. We will test several pharmacologic interventions, including pre- and post-treatment with corticosteroids, in order to determine whether these drugs block the expression of the protein in vivo and limit lung inflammatory reactions. These studies will help define the role of an important AM product in the genesis of lung inflammation and they will determine whether steps that limit the production of this protein can be useful in limiting some types of lung inflammation in vivo.
|
{
"pile_set_name": "NIH ExPorter"
}
|
DESCRIPTION: Ten departments distributed throughout the Medical School, Trinity College of Arts and Sciences, and the Nicholas School of the Environment participate in the teaching and training activities of the Integrated Toxicology Program (ITP). Instructional training in toxicology is provided in core courses in environmental and/or mammalian toxicology. Each predoctoral student has to fulfill the requirements of the Graduate School, the department to which they are affiliated, and the core curriculum of the ITP. Requirements include successful completion of 60 units of graduate credit (30 of which must be in residence at Duke and 6 in a minor field), completion of a preliminary examination, and completion of a dissertation with oral defense of the thesis. All predoctoral students in the ITP are required to take core courses which include "Essentials of Pharmacology and Toxicology" and a choice of either "Environmental Toxicology" or "Mammalian Toxicology." They are also required to attend the Toxicology Seminar and Symposium Series and the advanced Focused Topics Toxicology Seminar. Additional training in statistical methods is a new requirement of the program. The present curriculum constitutes a significant diversion from that outlined in the previous submission. Increased emphasis on molecular biology and reduced focus on traditional pathology reflects recent trends in toxicology.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Among adult mammals, brain neurogenesis is highly restricted, both spatially and phylogenetically, and has not previously been found in primates. In contrast, neurogenesis is widespread and robust in the adult songbird forebrain, which continues to generate neurons from mitotic ventricular zone (VZ) precursor cells. We previously established a preparation by which neurogenesis could be studied in cultures of the adult avian forebrain. The percentage of neurons generated in these cultures varied as an inverse function of the serum level, indicating that serum might harbor or induce factors which are anti-mitotic for VZ precursors. On this basis, we proposed that the lack of neuronal production by non-neurogenic adult brain might result not from an absence of suitable precursor cells, but rather from their tonic inhibition by serum-born or locally-derived agents. Consistent with this idea, recent reports have demonstrated that neuronal precursor cells are present in cultures derIved from adult rodent brain. Since brain neurogenesis in mammalian embryogeny is VZ-based, just as it is in the adult songbird, we hypothesized that the adult human VZ might continue to harbor neuroepithelial stem cells; these cells may remain neurogenic in selected groups such as the songbirds, yet become vestigial in mammals. We further postulated that the adult human forebrain might also harbor such cells, which although non-neurogenic in vivo, retain the capacity for neurogenesis in vitro, once removed from local tissue influences. In preliminary studies, we indeed obtained evidence of newly produced neurons in cultures of adult human temporal lobe. We now propose to further study this phenomenon, by examining the mitotic capability, lineage potential of, and regulatory constraints upon, these precursor cells of the adult mammalian forebrain. Although the emphasis of this proposal is upon adult human forebrain, these experiments will include study of both rat and human brain cells: Rat tissue will be used as a more accessible alternative to human brain for experiments requiring many matched samples run in parallel, such as comparisons of the effects of defined growth factors upon VZ cell proliferation and differentiation. In this proposal, we will ask, and hope to answer, the following questions: 1. Can mitotic precursor cells with neuronal potential be identified in explant cultures of the adult human forebrain ventricular zone? 2. At what regulatory level is neurogenesis suppressed in the adult mammalian brain in vivo? What humoral signals are operative in the temporal and spatial restriction of adult neurogenesis? 3. Can typically quiescent glial phenotypes, particularly oligodendrocytes, also be generated anew from resident precursor cells in the adult human brain? 4. Are individual adult human forebrain ventricular zone precursor cells multipotential? Does the cell type generated by the ventricular zone stem cell depend upon its ambient environment? The establishment of adult human neurogenesis in vitro may permit us to induce this process in vivo, whether from endogenous or introduced progenitors. In pursuing this work, we hope to develop an operational rationale by which induced neurogenesis may become a viable option in the repair of structurally-damaged brain.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The levels of mRNA in cells reflect a balance between rates of gene transcription and rates of mRNA turnover. Although attention has focused on the control of transcription in the regulation of gene expression, the rates of mRNA turnover can be just as important in determining final mRNA concentrations. Rather than a single default pathway of mRNA decay, rates of mRNA turnover can differ dramatically among different mRNA species in a given cell type, between the same mRNA species in different cells, and with the same mRNA species and cell type when cells are exposed to distinct environmental stimuli. The mammalian tristetraprolin (TTP) protein can bind to AREs in the mRNAs encoding tumor necrosis factor alpha and granulocyte-macrophage colony stimulating factor and lead to their destabilization, through a process that may involve the removal of the poly(A) tail, or deadenylation. There are three other mammalian members of the TTP protein family. All of these proteins can act in the same way as TTP in cell transfection and cell-free assay systems to promote the deadenylation and instability of mRNA targets that contain AREs. Although recent knockout studies have identified potential roles for these proteins in widely disparate physiological processes, their target mRNAs are as yet unknown. In order to fully understand the molecular nature of how TTP and its family members act to promote mRNA deadenylation and destruction, it would be desirable to conduct studies in a simpler, genetically tractable organism in which only one member of this protein family is expressed. Drosophila melanogaster meets these criteria, expressing only a single member of this protein family (TIS11). The Drosophila Tis11 gene, however, may be differentially spliced to produce two different proteins. Essentially nothing is known about the function of this protein in Drosophila, although its transcript is expressed in the early embryo and in the adult.
|
{
"pile_set_name": "NIH ExPorter"
}
|
This research evaluates the utilization and costs of computed tomography (CT) scanning for a large heterogeneous population. Factors associated with utilization differential across hospitals and small geographic regions are examined. Independent variables regional analysis include CT scanner density, percent Medicare coverage, percent Medi-Caid coverage, hospital beds/1000 population, regional percent medical specialists and other variables. Independent variables for hospital utilization include percent specialized medical staff, hospital case mix, hospital ownership, teaching programs, volume of scans at non-hospital facilities, and year scanner installed. Cost functions for CT scanning are estimated and analyzed. The relationship of CT costs to charges, other ancillary service costs, hospital-based-physician reimbursement, profit motive, and other hospital and system characteristics are analyzed. The data sources for these studies are mandated annual hospital surveys, discharge abstract summaries, financial disclosure reports required by the State of California and charge surveys. Data on non-hospital and mobile scanner costs and utilization are collected by a survey of these units. Case studies will be conducted in four to six hospitals with CT scanners and newly installed nuclear magnetic resonance units (NMR) in order to determine the extent to which NMR may replace CT as an imaging modality of choice. Cost and utilization forecasts for CT and NMR will be produced. The findings of this research project are significant because of the prominence of CT scanning as a prototype high cost medical technology. While there is a considerable body of knowledge on the diffusion of this medical technology the understanding of its utilization is deficient. These sutdies will add to the body of knowledge on utilization of medical technology as it reaches a steady state. Knowledge of this complete cycle and factors associated with utilization and cost are essential to policymakers and regulators seeking to implement cost containment and utilization control strategies. Information on NMR utilization and its relationship to CT will provide new insights into the newest of imaging technology.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The vetibulo-ocular refle (VOR) reduces motion of images on the retina by evoking eye movements in the opposite direction from head movement. Motor learning calibrates the VOR gradually correcting the reflex whenever image motion is persistently associate with heat turns. In addition, motor learning in the VOR in the VOR depends critically on the function of the cerebellum, and the principles uncovered in studies of the VOR may apply generally to many motor systems that are thought to rely on cerebellum- dependent learning to maintain normal sensory-motor function and for recovery of function following brain damage. The investigator proposes to develop a model system that would take advantage of the genetic tools as well the pharmacological and surgical manipulations available in the mouse to study motor learning in the VOR. Gene knockout mice will be used to more directly link cellular pathways to cerebellar physiology and physiology to motor learning in the VOR. This application aims to perform the necessary preliminary description of the basic behavior and physiology of the VOR in normal mice and to begin to use some interesting cerebellar mutants that are available to analyze the neural mechanisms for the induction of learning.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Renal failure resulting from chronic glomerular disease is an important cause of death and disability in the United States. The adult idiopathic nephrotic syndrome is a sub-type of chronic glomerular disease uniquely suited for the study with an aim to identifying causes of glomerular injury and evaluating the effectiveness of therapy. We have begun and propose to continue a multi-center collaborative controlled study of the adult idiopathic nephrotic syndrome with an aim to: 1. Correlate the clinical manifestations, course, and response to therapy of patients with nephrotic syndrome with the specific pathological changes observed in kidney tissue. 2. Ascertain whether measurable statistically significant benefits in mortality and morbidity are conferred within the period of observation by currently proposed therapeutic agents. 3. Establish an organization of cooperating investigators and hospitals, and a framework for the controlled study of renal disease that might ultimately be extended to other investigations.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The objective of this research proposal is the development and evaluation of immunoassays having clinical utility for the "early" detection and diagnosis of pancreatic carcinoma. In the year 2009, an estimated 42,000 new cases of pancreatic carcinoma will be diagnosed in the United States. Pancreatic carcinoma is the tenth most common form of cancer in men and women today, yet it is the fourth leading cause of cancer deaths. The most common symptoms of the disease, jaundice, abdominal pain, and weight loss, together with other presenting factors are nonspecific in nature. Thus, diagnosing pancreatic carcinoma at an early stage of tumor growth is difficult at best, requiring considerable suspicion and extensive diagnostic work-up, up to and including exploratory surgery. Our laboratory has developed and characterized the PAM4 monoclonal antibody (murine, chimeric and humanized versions), providing evidence as to its potential for clinical detection, imaging and therapy of pancreatic carcinoma. Our recent developments employing an in vitro enzyme immunoassay to quantitate PAM4-reactive antigen in the blood of patients, appears quite promising for detection of pancreatic carcinoma and its discrimination from pancreatitis, as well as other cancers and normal individuals. Early detection and diagnosis of pancreatic carcinoma, as well as appropriate staging of the disease, would almost certainly provide a survival advantage. With this in mind, we intend to further develop and assess the ability of the PAM4-based immunoassays, both immunohistochemical detection in tissue specimens and enzyme immunoassay to detect and quantitate the antigen in both serum and pancreatic fluids, to provide "early" detection and diagnosis at a time-point when therapeutic intervention may have better opportunity for successful outcome. The specific aims of this project are: 1. To evaluate the performance profile of the PAM4-Immunoassay for diagnostic accuracy;2. To evaluate the clinical utility of the PAM4-Immunoassay for a-accurate diagnosis of patients presenting with symptoms suggestive of pancreatic cancer;b- early detection of pancreatic carcinoma in asymptomatic individuals at high risk for pancreatic cancer;c- assessment of tumor response or early detection of relapse;3. To evaluate the nature of the antigen to which PAM4 is reactive. PUBLIC HEALTH RELEVANCE: Pancreatic carcinoma is an insidious disease with a particularly high mortality rate. In large measure, this is due to the location of the tumor where it can grow in a silent fashion. Symptoms that might suggest the patient seek medical assistance are usually not evident until an advanced stage of tumor growth. Compounding this challenge is that currently available treatment procedures have not been able to provide a cure for the overwhelming majority of patients. Our goal is to provide an antibody approach for early detection and diagnosis of the disease at a time when curative procedures have a better opportunity for successful outcome. We have developed monoclonal antibody PAM4 that has a high specificity for pancreatic carcinoma as compared to benign pancreatic disease, other types of malignancies, and normal individuals. Thus, the detection of the PAM4-reactive antigen provides a high likelihood for the diagnosis of pancreatic cancer. The aims of the proposed research are to further define the specificity of the antibody as to its ability to provide accurate diagnoses, and to examine the value of the immunoassay for "early" detection of pancreatic carcinoma, prior to the development of symptoms, in a group of patients considered to be at high risk for development of the disease.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Mononuclear non-heme iron active sites are present in a wide range of enzymes involved in a variety of important biological functions requiring dioxygen. These include the lipoxygenases (fatty acid hydroperoxidation), bleomycin (anticancer drug involved in DNA cleavage), intra- and extradiol dioxygenases (degradation of aromatic rings), tetrahydropterin dependent hydroxylases (phenylalanine metabolism), and the alpha-ketoglutarate-dependent enzymes (substrate hydroxylation and ring closure). Both the ferrous and ferric oxidation states are involved in catalysis for different enzymes in this class, and substrate and oxygen bound intermediates have been observed. Much less is known about the active sites in these enzymes relative to heme systems as the non-heme iron centers are less spectroscopically accessible. The general goals of this research program have been to develop new spectroscopic methods for the investigation of non-heme iron active sites and to apply these methods to the study of the above enzymes to obtain molecular level insight into the catalytic mechanisms and to understand the differences in the active site geometric and electronic structure which relate to differences in O2 and substrate reactivity. These studies should also contribute significantly toward elucidating the similarities and differences between non-heme and heme iron sites. Studies thus far have emphasized variable-temperature variable-field magnetic circular dichroism (VTVH MCD) combined with other excited state spectroscopic methods to probe the geometric and electronic structure of non-heme ferrous and ferric sites and to define the unusual electronic structure of the NO complex of non-heme ferrous sites and its relation to possible oxygen intermediates. The specific aims of this proposal are to: l) Complete the development of VTVH MCD as a powerful probe of non-heme ferrous active sites; 2) Develop Fe L- edge X-ray absorption spectroscopy as a new probe of non-heme iron active sites, particularly for ferric centers and oxygen intermediates; 3) Extend studies on lipoxygenases to correlate to the two conflicting crystal structures and to the mammalian enzymes with systematic mutations which influence reactivity, define active site intermediates, and probe the interaction of the active site with inhibitors: 4) Continue studies on bleomycin to understand each of the steps of the catalytic mechanism, determine the effect on the iron site due to the interaction with DNA, and define the relation of bleomycin to heme and other non-heme iron systems; 5) Correlate results on the extradiol dioxygenases with parallel data on the intradiol dioxygenases to determine differences in substrate-iron active site interactions which relate to differences in activation and could influence the position of ring cleavage; 6) Define the interaction of the ferrous site of phenylalanine hydroxylase with the pterin cofactor, probe the key steps of the catalytic mechanism, and determine how mutations which affect the enzyme's reactivity change the active site and its interactions with cofactor and substrate; 7) Understand the interaction of the ferrous site of clavaminate synthase with the alpha-ketoglutarate cofactor, define the interaction of this binary complex with O2 and analogs, and determine the interactions of this site with different substrates which lead to hydroxylation versus ring closure chemistry.
|
{
"pile_set_name": "NIH ExPorter"
}
|
: The individual, family, and societal costs of substance abuse in the United States are staggering. Over a million premature deaths of Americans each year are attributed to smoking and illicit drug use accounts for at least another 12,000 deaths. About 30 percent of the nation?s youth reside with parents addicted to alcohol and drugs (AOD), increasing the risk of future use among young people. Substance abuse is implicated in motor vehicle crashes, crime, and lost productivity at work. In 1995, the economic cost of AOD was $276 billion. Despite the enormity of the problem, there are huge gaps in educating health professionals about substance abuse nd methods to study these phenomena. The goal of the competing continuation application is to train (2) predoctoral and (4) postdoctoral scholars each year for a total of 5 years. Predoctoral trainees are nurse scholars earning Ph.D.?s. Two postdoctoral nurse scientists and two postdoctoral trainees in other health disciplines (psychology, medicine, or social work) will increase the cadre of highly qualified individuals to conduct state-of-the-art substance abuse research. The overall goals and specific aims are congruent with AOD research training priorities set forth by the Institute of Medicine, the National Institutes on Drug Abuse and Alcoholism and Alcohol Abuse, the 2010 goals for Healthy People,Pew Health Professions Commission, and guidelines for training scientists at NIH. The significance of the current and the proposed competing continuation projects is that is promises to increase: a) the number of women researchers studying AOD phenomena which are currently underrepresented in NIDA, NIAAA, NIOSH, and the Institute of Justice, b) the number of women with AOD phenomena being studied. The specific aims of the project are to: (1) identify risk /protective factors and consequences of addictive behavior of varied population; (2) test intervention models including treatment delivery, treatment efficacy, and post-treatment follow-up; (3) acquire skills in statistical procedures for quantitative and qualitative methods; (4) acquire skills in data analytic methods including longitudinal designs and multivariate testing; (5) acquire skills in grant-writing; (6) acquire skills in writing for publication; (7) acquire skills in the recruitment and retention of underrepresented persons for study; (8) articulate a position on ethical conduct of research; and (9) form and maintain collaborations in the conduct of research within and across disciplines. The training environment at the University of Washington is extremely strong. The school of nursing has been ranked the top school in the U.S. consistently since 1984 and is among the top three in the U.S. in faculty-acquired external research funding. The program faculty represent a number of university schools and departments and the majority are currently conducting research funded by NIDA. Follow-up data collected from former trainees every two years shows that most are making satisfactory progress in substance abuse research careers.
|
{
"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 goal of this study is to determine whether or not standard of care treatment using an intralesional steroid (Kenalog-10) in patients with alopecia areata has a suppresive affect on adrenal gland function.
|
{
"pile_set_name": "NIH ExPorter"
}
|
The research currently planned on the histochemistry of the autonomic innervation of the eye has several objectives. First, to improve our understanding of the normal neurohistology of the eye. Here special emphasis is paid to retinal histochemistry and to the relationship between the autonomic innervation to melanin-containing cells of the eye. Second, the same histochemical methods can be used and are currently being used to study the penetration after local or systemic administration of autonomic drugs or of marker dyes. And third, the information gathered from these studies can be used as a basis for special projects in experimental pathology, projects which stress neuro-vascular relationships.
|
{
"pile_set_name": "NIH ExPorter"
}
|
1. Capsular polysaccharides (CPS) of Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of S. oralis although closely related, are functionally distinct. CPS protects pathogenic S. pneumoniae from phogcytic killing whereas RPS of commensal S. oralis mediates interactions with other members of the dental plaque biofilm community. We recently completed comparative structural and molecular studies of ribitol-containing types of RPS (i.e. RPS4Gn and RPS5Gn) and closely related CPS (i.e. CPS10A, CPS10B, CPS10C and CPS10F). The results provide important insights into RPS structure, function and evolution. They also opened an approach for further comparative characterization of related S. pneumoniae serotypes, including pneumoniae CPS39. The structure of CPS39 was determined from sugar composition analysis performed at the Complex Carbohydrate Research Center in Georgia and high resolution NMR spectra. The repeating unit of this polysaccharide resembles those of CPS10A and RPS4Gn but contains arabitol rather than ribitol. From previous studies of CPS10A and RPS4Gn, we were able to assign each linkage in CPS39 to a gene in the cps39 locus. The only ambiguity involved the genes designated wcrC in the cps10A and cps39 loci. Whereas WcrC of CPS serotype 10A links Gal 1-2 to ribitol, the transferase encoded in the cps39 locus appears to link Gal 1-1 to arabitol. Thus, we suspect that the corresponding gene in the cps39 locus is not wcrC, but instead, a distinct gene. The goal of this work is to define the genetic basis of CPS and RPS structure. 2. The presence of Gn- or G-types of RPS on many strains of S. sanguinis, S. gordonii and S. oralis accounts for coaggregations noted between these bacteria and plaque bacteria such as Actinomyces spp. that have RPS-binding adhesins. In addition to Gn- or G-types of RPS, we suspect that other polysaccharides described from strains or S. oralis and S. mitis mediate interbacterial adhesion and thus, represent novel types of RPS. To test this hypothesis, we are preparing specific antibodies against the later polysaccharide for use as probes to identify the corresponding bacteria in naturally occurring oral biofilm communities. Following identification of these bacteria in vivo, we plan to isolate and characterize neighboring bacterial cells for the presence of RPS-binding complementary surface adhesins. These studies constitute a critical test of our hypothesis that a wide range of bacterial surface polysaccharides function as specific recognition molecules for dental plaque biofilm development and that RPS structure influences community composition. 3. Although saliva is a primary source of carbon and nitrogen for growth of different plaque species, little is known about how bacteria in biofilm communities degrade and utilize salivary proteins and glycoproteins for growth. We hypothesize that the close association of different cell types in dental plaque favors cooperative degradation of salivary proteins and glycoproteins by members of the biofilm community. To test this hypothesis, we are developing robust experimental models for studies of bacterial growth and biofilm formation in filter-sterilized whole saliva. In recent studies, the number of A. naeslundii T14V in co-cultures with S. oralis 34 was significantly greater than the number seen in actinomyces monocultures. Moreover, the actinomyces observed in co-cultures were closely associated with streptococci. In other experiments, we compared growth of different streptococci including strains of S. gordonii, S. oralis and S. mutans. In general, strains of S. gordonii and S. oralis grew well in saliva whereas strains of S. mutans grew poorly or not at all. Currently, we are exploring whether growth of S. gordonii and/or S. oralis in saliva promotes associated growth of S. mutans, a possibility that has important implications for initial colonization of cariogenic S. mutans in the absence of sucrose. Interestingly, S. gordonii DL1 and S. oralis Uo5, commensal species that grow well in saliva, appear to have a greater array of cell surface glycoside hydrolases than S. mutans UA159, which grows poorly or not at all in saliva. To assess the contribution of specific glycoside hydrolases for growth of S. gordonii DL1 and S. oralis Uo5 in saliva, we are introducing unmarked, in-frame deletions in genes predicted to encode these proteins. We anticipate that growth studies performed with well-characterized wild type and mutant strains, individually and in co-cultures, will provide new insight into the underlying basis of dental plaque development, opening new approaches for prevention, diagnosis or treatment of plaque-associated oral diseases.
|
{
"pile_set_name": "NIH ExPorter"
}
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.