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Carcinogenesis is thought to originate from a single cell which accumulates a series of well-defined, albeit randomly obtained, genetic mutations, leading to carcinogenic transformation. Despite the inroads into elucidating the carcinogenesis process, there still remain gaps in our understanding of how mutations drive cell transformation and subsequent cancer progression. Although gene mutations clearly contribute to cancer, no single mutated gene or combination of mutated genes occur in all or even the majority of cancers. Moreover, as Vogelstein and Kinzler {Nature Rev Cancer 2004) point out, it is not gene mutations, but primarily chromosome-level aberrations that cells exhibit following transformation to the cancer state. Although it has long been assumed that cancers arise from mutated differentiated cells, mutated stem cells are now also considered candidate cancer precursors due to their self-renewal and differentiation capacity. Although genomic instability is ubiquitous across malignant cancers, its mechanistic underpinnings remain unclear. Inhibition of stability genes, alterations in mitosis genes, aberrant centrosomes, aneuploidy, telomere dysregulations and, for virus-linked cancers, virus-induced fusion of genetically-mutated cells, are all candidate causes of the instability. Given that gene mutations and particularly chromosome aberrations decrease the fitness of a cell, how is it that cancer cell populations are able to sustain and proliferate in the face of the high degree of chromosomal damage per cell? If the answer lies in the existence of cancer stem cells, one needs to ask how this privileged cell compartment is itself exempt from the deleterious effects of chromosomal damage.	{ "pile_set_name": "NIH ExPorter" }
This project tests the hypothesis that growth failure in children with Rett Syndrome can be shown to be nutritionally-mediated, particularly with respect to dietary energy, based on clinical features of the disease, e.g., feeding difficulties and increased energy expenditure due to repetitive motor activities.	{ "pile_set_name": "NIH ExPorter" }
This study proposes to test the feasibility of using severity and quality of care indices for cross site comparisons of outcome for psychiatric emergencies treated in the ED. The study will be conducted at two test hospitals in Rhode Island, one of which has an inpatient psychiatric unit. As part of the study, we propose to define psychiatric emergencies as those patients with a psychiatric complaint and/or who receive psychotropic drugs, counseling, and/or a psychiatric discharge from the ED. An advisory panel of physicians from New England and with expertise in the area of psychiatric emergencies will be convened to 1) finalize the definitions prior to the beginning of data collection and 2) review the preliminary results after data collection is completed. In addition, we propose to test the feasibility of using mortality, follow up compliance, and unscheduled ED visits within 15 days of the initial visit to measure patient outcome. We will identify the incidence of psychiatric emergencies and determine the degree of variance existing in outcomes at each site. Two nurses with experience related to psychiatric emergencies in the ED will be hired half-time to abstract patient records. Data quality will be enhanced by abstracting records within twenty four hours of admission. We expect to collect about 2500 abstracts, 1500 at one hospital and 1000 at the other. The nurse abstactors and the physicians will validate from copies of the emergency department records the reliability and validity of the case selection and abstracting at both sites. The various indices will be correlated with the expert opinion of the physicians to evaluate the sensitivity of the indices. Logistic regression and other multivariate analysis will be used to determine if the independent and dependent variables reflect impact of EMS effectiveness on psychiatric emergencies. It is our intent to use the proposed measures to replicate this study New England wide.	{ "pile_set_name": "NIH ExPorter" }
In investigations of the mechanisms of regulation of the replication of equine infectious anemia virus (EIAV), cDNA libraries prepared from cells infected with EIAV were probed so as to isolate putative Rev-encoding cDNAs. Three structurally distinct variants generated by alternative splicing were identified. Two Rev proteins (18 and 16 kDa) were encoded by two cDNAs, while the third encoded only a 16 kDa species. The two Rev proteins were shown to potentiate the cytoplasmic expression of transcripts containing the env region of EIAV. This marks the first demonstration of EIAV rev gene function. Identification of the EIAV RRE (rev responsive element) is in progress. The lymphoproliferative disease virus (LPDV) of turkeys is acutely oncogenic, but determination of the complete nucleotide sequence of its genome died not reveal the presence of an oncogene. Protein sequence analysis of gag-pol gene products indicates that LPDV, although most closely related to avian oncoviruses, possesses a unique gag protein (p31) of unknown function and generates its protease by a distinct mechanism. A cDNA predicted to encode a transmembrane tyrosine kinase receptor with sequence features characteristic of known fibroblast growth factor (FGF) receptors was isolated from a human mammary epithelial cell and was subsequently shown to represent FGF receptor-4 (FGFR-4). FGFR-4 bound both acidic and basic FGF (Kd - 10-15 and 120 pM) but not keratinocyte growth factor (KGF). To determine whether FGF receptors contribute to the development of human tumors, screened RNAs were prepared from cell lines derived from a variety of solid tumors. High levels of the FGFR-4 transcript were specifically (relative to FGFR-1) and frequently detected in human mammary and kidney carcinomas, but only infrequently in other tumors.	{ "pile_set_name": "NIH ExPorter" }
Our work can be divided into five major categories: neuroanatomy, neuropharmacology, molecular neuroendocrinology, physiology, and behavior. Mapping receptor localizations using the in vitro binding and autoradiography techniques yields important basic information about brain organization and function. We have localized the cannabinoid receptor in brain. The molecular biology group uses in situ hybridization to examine the regulation of gene expression of neuropeptides, monoamine synthesizing enzymes, and adrenal steroid receptors, which have been implicated from clinical studies in the pathophysiology of psychiatric disorders. Behavioral studies are designed to assess a variety of factors associated with anti-anxiety drug withdrawal determinants of drug response, Maudsley rats, taste aversion learning, kindling, stress-related CNS changes, and central administration and monitoring of neuroendocrine factors in primates.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: (Applicant's Abstract) The past several years have seen an alarming increase in the abuse of centrally acting sympathomimetic stimulants and an increase in the incidence of cardiovascular toxicity. Due to the close temporal relationship between drug use and the onset of most clinically significant toxicities, most reports of the cardiovascular effects of these drugs have focused on the acute toxic responses. Very little attention has been paid to the possibility that repeated stimulant use may alter cardiovascular function in such a way as to increase the potential for the development of serious cardiovascular toxicity. Therefore, this proposal was designed to test the hypothesis that the repeated administration of sympathomimetic stimulants produces structural and/or functional changes in the cardiovascular system that increase the potential for the development of serious cardiovascular toxicity. These studies will attempt to determine whether the repeated administration of cocaine, 3,4-methylenedioxymethamphetamine (MDMA) or methamphetamine (METH) alters arterial pressure, heart rate, electrocardiographic activity size and baroreceptor or Bezold-Jarisch reflex function. Histologic and morphometric analysis will also be performed to determine whether repeated stimulant administration produces damage or structural changes in the heart that are visible at the light or electron microscopic level. Two types of dosing schedules will be employed. One will involve administration of cocaine, METH or MDMA on a daily basis for up to 2 months. The other type of schedule will involve large, closely spaced doses given for 4-14 days. The latter types of stimulant dosing schedule is similar to those often used to produce neurotoxicity in monoaminergic systems. The proposed studies represent a unique combination of "state of the art" techniques for cardiovascular recording in conscious animals and histological analysis to examine the changes in cardiac, cardiovascular and cardiovascular reflex structure/function elicited by the repeated administration of sympathomimetic stimulants. Examination of these changes should provide new insight into the potential for repeated stimulant use to produce cardiac and/or cardiovascular toxicity. The mechanisms mediating these changes will provide the basis for future studies.	{ "pile_set_name": "NIH ExPorter" }
The HLA class I genes are the most polymorphic loci in the human genome, resulting in the ability of the system to bind and present a great variety of antigenic peptides to cytotoxic T lymphocytes. HLA-C molecules are expressed at a low level on the cell surface compared with HLA-A and -B. Unlike HLA-A and -B, HLA-C surface expression is not down-regulated by Nef upon HIV infection. We previously demonstrated that HLA-C allotypes are expressed at variable levels on the cell surface in a manner that is allotype-specific, and higher surface expression mediates greater selection pressure on HIV-1 and better viral control overall. We further showed that polymorphism in the miR-148a binding site in the HLA-C 3prime untranslated region (3 prime UTR) region partially explains differential expression of the various HLA-C allotypes, where miR-148a inhibits expression of HLA-C alleles that have an intact binding site but does not affect expression of escape HLA-C alleles that contain a disrupted binding site, which is not recognized by miR-148a. In general, the escape alleles are expressed at a higher level than the inhibited alleles (although there are exceptions). We have now demonstrated that the expression levels of miR-148a, as marked by MIR148A gene variation, correlate positively with HIV viral load among those individuals who carry at least one inhibited HLA-C allele. There is no correlation between the level of miR-148a and HIV control among those individuals who carry two copies of HLA-C escape alleles. Alternatively, the genotype marking higher miR-148a expression levels (which lead to lower HLA-C expression) results in protection against Crohn's disease (CD) among those who carry at least one inhibited HLA-C allele. These data underscore the importance of immune gene interactions in human disease and the independent role of HLA-C expression levels in HIV viral control and risk of CD. Natural progression of HIV-1 infection depends on genetic variation in the human MHC class I locus, and the CD8+ T cell response is thought to be a primary mechanism of this effect. However, polymorphism within the MHC may also alter innate immune activity against HIV-1 by changing interactions of HLA class I molecules with leukocyte immunoglobulin-like receptors (LILR), a group of immunoregulatory receptors mainly expressed on myelomonocytic cells including dendritic cells (DCs). We previously characterized HLA allotype-specific binding capacities of LILRB1 and LILRB2 as well as data from a large cohort of HIV-1-infected individuals to test whether LILR-HLA class I interactions influence viral load in HIV-1 infection. Analyses in persons of European descent, the largest ethnic group examined, showed that the effect of HLA-B alleles on HIV-1 control correlates with the binding strength between corresponding HLA-B allotypes and LILRB2 (p = 10-2). Moreover, overall binding strength of LILRB2 to classical HLA class I allotypes, defined by the HLA-A/B/C genotypes in each patient, positively associated with viral replication in the absence of therapy in patients of both European (p = 10-11-10-9) and African (p = 10-5-10-3) descent. This effect appears to be driven by variations in LILRB2 binding affinities to HLA-B and is independent of individual class I allelic effects that are not related to the LILRB2 function. In vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of DCs, suggesting an impact of LILRB2 on HIV-1 immune control through altered regulation of DCs by LILRB2-HLA engagement. HLA-disease associations have been shown in some cases to link to the peptide-binding characteristics of individual HLA class I molecules. In collaboration with Dr. Malini Raghavan at the University of Michigan, we showed that polymorphisms at the HLA-B locus profoundly influence the assembly characteristics of HLA-B molecules and the stabilities of their peptide-deficient forms. In particular, dependence on the assembly factor tapasin is highly variable, with frequent occurrence of strongly tapasin-dependent or independent allotypes. In vitro refolded forms of tapasin-independent allotypes assemble more readily with peptides compared to tapasin-dependent allotypes, and, during refolding, reduced aggregation of tapasin-independent allotypes is observed. Paradoxically, in HIV-infected individuals, greater tapasin-independent HLA-B assembly confers more rapid progression to death. Consistent with these findings, allotypes such as HLA-B57:01 and HLA-B27:05 that are associated with slow progression to AIDS display low or intermediate tapasin independence. Some studies also suggest protective effects of HLA-Bw4 homozygosity in HIV infections, and low tapasin independence is strongly prevalent within the HLA-Bw4 serotype. Conversely, some allotypes such as HLA-B35:03 and HLA-B35:01 that are associated with more rapid AIDS progression are highly tapasin independent for their assembly. Together, these findings demonstrate significant variations in the assembly of HLA-B molecules and indicate influences of HLA-B-folding patterns upon infectious disease outcomes. Further studies are needed to better understand the influences of HLA-B assembly/stability characteristics upon disease outcomes in other disease contexts and upon global CD8+ T cell responses during infections. A recent genome-wide association study (GWAS) involving patients with hemophilia A who were exposed to but uninfected with HIV-1 did not reveal genetic variants associated with resistance to HIV-1 infection, beyond homozygosity for CCR5-delta32. Variation at the HLA and KIR loci is usually not extensively tested by standard GWAS, because most HLA alleles are not efficiently tagged by any single-nucleotide polymorphism (SNP) present on the genotyping arrays and because of the extreme insertion/deletion polymorphism within the KIR locus. Given the importance of HLA and KIR for both innate and acquired immunity, we tested the same hemophilia cohort on which GWAS was performed, to determine whether variation within these loci may influence HIV acquisition as they do for post-infection events. Controls were randomly drawn from the general population in order to avoid the frailty bias that is inherent to virtually all HIV+ cohorts (ie, the enrichment of alleles associated with better HIV control in cohorts of chronic patients, due to longer survival which confounds association results). Use of a random control population is essential when probing for an effect of HLA on HIV infection, since HLA class I is the only locus genome-wide to consistently show an effect on control of HIV after infection. Analysis of the data indicates that HLA class I and KIR genes do not appear to impact HIV acquisition among hemophiliac patients exposed to contaminated blood products. Host genetic factors could still be involved in the resistance phenotype observed in highly exposed seronegative (HESN) individuals (eg, low-frequency variants poorly tagged by genotyping arrays or structural variants, such as insertion and deletion polymorphisms), and this should be investigated further.	{ "pile_set_name": "NIH ExPorter" }
The pyridine nucleotide NAD is directly involved in the regulation of cell growth. We observe that carcinogenic N-nitroso compounds cause a rapid depression of cellular NAD. This depression appears to be related to NAD-dependent nuclear modification reactions associated with carcinogen-induced damage to DNA and/or repair of DNA damage. A major objective of this proposal is to study in detail the ability of N-nitroso compounds of known carcinogenicity to cause acute depression of NAD in 3T3 cells and in mitogen stimulated human lymphocytes. Several classes of direct-acting and indirect-acting carcinogens will be examined to determine whether acute depression of NAD is a general effect of carcinogens. The extent of DNA damage caused by N-nitroso compounds and the rate of DNA repair of carcinogen induced DNA damage will be studied in normal and NAD depleted 3T3 cells. Sensitive, quantitative methods involving gas chromatography-mass spectrometry and high pressure liquid chromatography for measuring NAD-dependent nuclear modifications as mono and poly adenosine diphosphoribose (ADPR) will be developed. The extent of these NAD-dependent nuclear modifications will be studied in normal cells and in cells exposed to carcinogenic N-nitroso compounds. Carcinogen induced DNA damage and the rate of DNA repair will be studied in the presence of the methylated xanthines caffeine and theophylline which are specific inhibitors of NAD-dependent modifications.	{ "pile_set_name": "NIH ExPorter" }
This application, Novel serotonergic, pro-cognitive antipsychotic therapies, is in response to PA-08-142: Pharmacologic Agents and Drugs for Mental Disorders (SBIR [R43/R44]). Schizophrenia is a devastating brain disorder that affects approximately 0.7% of the global population. The symptoms of schizophrenia can be grouped into three major categories: positive (e.g. hallucinations and delusions), negative (e.g. social isolation and inappropriate emotional response), and cognitive symptoms. The cognitive symptoms of schizophrenia include impaired attention and disruption of working memory, an essential type of short term memory. These fundamental cognitive processes are critical for the performance of day-to-day activities, and their disruption in schizophrenia is a key factor underlying the inability of patients to integrate successfully into society. Indeed, the cognitive deficits associated with schizophrenia are recognized as a core component of the disorder. Currently available antipsychotic therapies are effective at ameliorating the positive symptoms of schizophrenia, but they are not effective at treating the negative or cognitive symptoms of the disease. Moreover, these therapies often cause significant side effects such as motor disturbances, weight gain, and diabetes. For these reasons, schizophrenia represents a major area of unmet medical need which must be addressed by the discovery and development of new antipsychotic therapies that are effective at treating the cognitive symptoms of schizophrenia with reduced potential for side effects. The overall goal of this proposal is to optimize a series of lead compounds we have identified with dual 5-HT2C receptor agonist and 5-HT6 receptor antagonist activities. Compounds with this novel pharmacological profile hold promise for yielding safe and effective treatments for the positive as well as the cognitive symptoms of schizophrenia. Such compounds are likely to have a significantly reduced risk for generating the undesirable side effects that are typically observed with currently available antipsychotic therapies. Successful completion of the specific aims outlined in this proposal will result in the identification of lead compounds with desirable pharmacological and drug-like properties. Furthermore, proof-of-concept for this novel target profile will be achieved by demonstrating antipsychotic and pro-cognitive efficacy in animal models with a selected lead compound. These achievements will provide a solid basis for a Phase II SBIR application to support further research aimed at selecting a clinical development candidate for the treatment of schizophrenia and its cognitive symptoms. A clinical development candidate with this novel profile would comprise a valuable asset within the pharmaceutical industry and would provide the basis of a successful commercial outcome for the research program. The research outlined in this proposal will directly benefit patients and their families and caregivers by leading to the discovery of safer and more effective medicines for the treatment of schizophrenia.	{ "pile_set_name": "NIH ExPorter" }
We have demonstrated that several human states are characterized by hyperactivity or hypoactivity of the central stress system, which explains not only mood changes but also the propensity of patients with such disorders to develop developmental, metabolic, cardiovascular or autoimmune complications. These changes in the central nervous system also influences fertility by affecting various components of the reproductive system. We have previously reported that CLOCK/BMAL1, the self-oscillating transcription factors that generate circadian rhythms both in the central nervous system and periphery, rhythmically repressed GR-induced transcriptional activity, indicating that CLOCK/BMAL1 functions as a reverse phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing the biologic actions of diurnally fluctuating circulating glucocorticoids. We performed one human study and revealed that this negative regulation on GR transcriptional activity by CLOCK was also functional in humans. As an extension of this circadian rhythm project, we screened microRNAs regulated in a circadian fashion in granulosa cells of mouse ovaries. MicroRNAs are short hairpin-like RNAs that demonstrate strong biological actions on reproduction, and specifically, granulosa cells by influencing proliferation and apoptosis, as well as steroidogenesis of these cells. Granulosa cells, on the other hand, are components of ovarian follicles required for their proper development and steroid hormone production. We have found that primary granulosa cells obtained from mouse ovaries showed circadian oscillation of several CLOCK-related genes, such as Per1/2 and Cry1/2. We found that expression of 10% of over 600 known microRNA we screened were in circadian rhythms. We further focused on miR-196a, as it showed the most significant circadian oscillation. We found that its target gene HOX8B, a transcriptional repressor important for the regulation of cell growth and apoptosis, demonstrated diurnal expression through miR-196a. We are now examining importance of our finding in the biologic action of granulosa cells. Aging is an important factor for reducing the chance of successful pregnancy, eventually developing ovarian failure and menopause, but the biological mechanisms underlying this physiologic process have not completely been elucidated as yet. It is also possible that pathologic infertility, such as by malnutrition, stress and exercise, might share part of the mechanisms responsible for aging-dependent ovarian failure. To examine impact of aging on ovarian functions, we again examined microRNA expression in primary granulosa cells obtained from mouse ovaries. We obtained granulosa cells from 2-year old mice and tested expression of 600 microRNAs by employing the cells of young mice (6-9 week old) as controls. In this screening, we found that 37 microRNAs were significantly regulated in aged cells. We focused on miR-503 and -322, as these microRNAs showed the most significant reduction (over 20-fold) in aged granulosa cells and form a gene complex on chromosome X regulated by the shared promoter. We found in the subsequent protein expression profile analysis that several subunits of the ATP synthase, a component of the mitochondrial respiratory chain, were down-regulated by inhibition of these microRNAs, suggesting that miR-503 and -322 are required for mitochondrial activity. Recent publication indicated that these miRNAs down-regulate expression of mitophagy transcripts, such as Nix/Bnip3L, Ulk1, Gabarapl2, Sh3glb1, Atg12, Becn1, and Bcl2l1, thus these microRNAs maintain mitochondrial biogenesis and energy production by suppressing expression of mitophagy-inducing genes. Since reduction of mitochondria is a key feature of aged ovaries with reduced reproductive activity, it is likely that miR-503 and -322 are mediators of mitochondrial dysfunction observed in aged ovaries, and further, underlying factors for increased incidence of infertility in older subjects through modulation of mitochondrial biogenesis/activity. Crosstalk between the gonadotropin-regulating cAMP signaling system and that of the steroid hormone receptors, such as progesterone (PR), estrogen (ER) and glucocorticoid receptor (GR), are important for the regulation of ovarian functions. To elucidate their crosstalk at the transcriptional regulation, we focused on the CREB-regulated transcriptional coactivator 2 (CRTC2), a cofactor known to be specific for CREB (thus cAMP signaling)-mediated transcriptional regulation, and tested its effect on PR- or GR-induced transcriptional activity. We found that CRTC2 functions as a coregulator of GR and PR, stimulating former transcriptional activity, while repressing latter activity. We further found that CRTC2 physically interacts with the ligand-binding domain of PR and GR through its C-terminal portion harboring the transactivation domain in vitro and in vivo. We are now examining biologic importance of this crosstalk in granulosa cells. 11beta-hydroxysteroid dehydrogenase type-2 (11bHSD2) is responsible for inactivation of circulating cortisol by converting it into inactive cortisone. High levels of circulating cortisol provided by mother are toxic to fetal development and cause abnormal metabolic traits in later adult life. Thus, placenta expresses 11bHSD2 to block entrance of maternal cortisol, whereas abnormal pregnancies, such as those complicated with diabetes mellitus, pregnancy-induced hypertension and eclampsia, are associated with reduced expression of this enzyme. To examine roles of 11bHSD2 in placental development and activity, we employed human chorioblastoma BeWo cells as a model system, and found that forced reduction of 11bHSD2 expression attenuated proliferation of these cells and secretion of differentiation markers, such as human chorionic gonadotropin and progesterone, suggesting that 11bHSD2 is necessary for normal development of placenta, in addition to its well known activity of inactivating maternal cortisol.	{ "pile_set_name": "NIH ExPorter" }
In the last fiscal year we determined the etiology of this disorder. It is caused by mutations in the GLI3 zinc finger transcription factor and is thus allelic to the Greig cephalopolysyndactyly syndrome. In this fiscal year we plan to determine mutations in additional families to perform genotype-phenotype correlation, study the intracellular distribution and transcriptional activation properties, and study animal models of the disorder. Our preliminary data show that the truncated mutant GLI3 protein has nuclear tageting by using green fluorescent protein tagged GLI3. In addition, we will study patients with Greig cephalopolysyndactyly syndrome to determine the clinical and molecular overlap of these disorders. We have preliminary data showing that microdeletions may be a common cause of Greig syndrome.	{ "pile_set_name": "NIH ExPorter" }
Two color flow cytometric lymphocyte phenotyping of peripheral cells from Wegener's patients.	{ "pile_set_name": "NIH ExPorter" }
NJ/NY Hazardous Materials Worker Training Center ECWTP Abstract The NJ/NY Hazardous Materials Worker Training Center has a long history of effective training that meets the requirements of OSHA 29CFR1910.120, providing hazardous materials knowledge and skills to over 450,000 workers since 1987. Our Center will train workers about safety issues during clean-up hazardous waste sites, issues related to generation, treatment and storage of hazardous materials, and emergency response. The courses develop competency in workers to critically analyze dangerous situations, and enable them to identify safe work practices. Key principals of adult education are incorporated into our training, and training courses are developed to include peer-learning, hands-on activities, and development critical thinking skills. The Center has a robust evaluation plan, and will continue to expand the types of date collected on impact of training on workplace practice, as well as how training can more effectively influence workplace safety culture. The ECWTP will utilize focused strategies to recruit, train and employ underserved residents living in disadvantaged communities for construction and environmental remediation work. The Center has long-term, effective partnerships in minority and underserved communities that help reinforce occupational health and worker education, and mitigate health disparities at the community level. Our Center includes training in environmental justice, health and safety, and life skills, preparing trainees for a career in the environmental industry. Over the next five years, the Center proposes to train 300 workers in 75 courses in the ECWTP.	{ "pile_set_name": "NIH ExPorter" }
The Neurofilament proteins (NFPs) and the glial fibrillary acidic protein (GFAP) are major cytoskeletal elements in the nervous system. Preliminary studies suggest that each NFP is present in filaments isolated from newborn rat brain and spinal cord and that the NFPs are phosphorylated at this stage of development. In the present proposal these studies will be extended. The appearance of the three NFPs will be determined in the rat CNS and PNS during fetal and neonatal life by both the immunoblot technique and immunohistochemistry utilizing monoclonal antibodies. These results will then be correlated with the appearance of morphologically distinct neurofilaments as identified by electron microscopy. Subsequently, phosphorylation of the NFPs in the newborn and fetal rat CNS and PNS will be examined to determine whether these proteins are phosphorylated immediately after their synthesis or only at a later state of neuroaxonal differentiation. The amount of covalently bound phosphate associated with each NFP in the rat CNS will also be analyzed using a developmental approach. The proposed studies on the appearance and phosphorylation of the NFPs during fetal and early postnatal life may provide insight into possible structural, differential or functional roles of the NFPs during nervous system development. Similarly, the in vivo phosphorylation of GFAP will be investigated, initially in the adult rat CNS. Since it has been demonstrated that GFAP is present in rat brain and spinal cord at 14 and 16 days of gestation, respectively, and in rat sciatic nerve at birth, it will be feasible subsquently to ascertain at what stage of development GFAP is first phosphorylated in the CNS and PNS. These developmental studies are designed to examine the role of GFAP phosphorylation in the differentiation of astrocytes and Schwann cells.	{ "pile_set_name": "NIH ExPorter" }
Therapeutic approaches to hemoglobinopathies such as sickle cell anemia as well as thalassemia include strategies to alter globin gene expression during erythropoiesis. Understanding the developmental switch that determines expression of embryonic, fetal and adult hemoglobins is of particular relevance as in adults with sickle cell anemia or beta-thalassemia, increased fetal hemoglobin levels can be associated with more mild forms of disease. Stimulation by erythropoietin results in the proliferation and differentiation of erythroid progenitors and increased production of transcription factors such as GATA-1 and SCL/Tal-1, further induction of erythropoietin receptor expression, and activation of globin gene expression. The requirement for GATA-1 and SCL/Tal-1 for embryonic hematopoiesis suggests that these factors are also upstream of events leading to differentiation along the erythroid lineage. We use transfection of these transcription factors into erythroid cells to determine their direct effect on erythropoietin receptor expression and globin gene expression. GATA-1 can transactivate the erythropoietin receptor via a GATA-1 binding site in its proximal promoter, suppresses GATA-2 and epsilon globin gene expression in K562 cells with an embryonic erythroid phenotype. In K562 cells, SCL/Tal-1 increases expression of the erythropoietin receptor. The 5 UTR flanking the erythopoietin receptor coding region contains three E-box motifs, potential binding sites for SCL/Tal-1. Reporter gene assays demonstrate that in addition to the GATA-1 binding site in the proximal promoter, these three E-box motifs are important for transcription activation in erythroid cells. Stable clones of erythroleukemia K562 cells containing a SCL/Tal-1 expression vector were isolated and the resultant expression profile assessed incorporating real-time RT-PCR and expression microarray techology. Examination of target genes in the SCL/Tal-1/K562 cells show an increase in expression of GATA-1, beta-globin and carbonic anhydrase I. These data suggest that in addition to activation of the erythropoietin receptor, there was a shift to a mature erythroid phenotype by SCL/Tal-1. As an approach to genetically manipulate globin gene expression, we are using a multiribozyme to target mutant globin transcripts. Ribozymes introduced into K562 cells were able to suppress alpha-globin production. Ribozymes specific for the beta-S globin transcript are now under investigation. Combinations of globin specific ribozymes with vectors targeted to increase gamma-globin production can be of potential use in maintaining alpha/beta-like globin chain balance. - Sickle cell anemia, hemoglobinopathy, globin, ribozyme, therapy, transcription, GATA-1, SCL/Tal-1	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: Angiogenesis is required for proper development of the embryonic circulatory system and is an important step in the progression of many eye diseases, including retinopathy of prematurity (ROP). Therefore, understanding how the normal regulatory systems in the endothelium keep angiogenesis in check has great clinical implications. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an important regulator of angiogenesis. We have shown that multiple isoforms of PECAM-1 are expressed in vascular beds of different tissues in a developmentally regulated fashion. The ability of these isoforms to differentially activate intracellular signaling pathways suggests specific roles for these isoforms during vascular development and angiogenesis. However, the physiological role PECAM-1 and its isoforms play in these processes requires further investigation. The main objective of this proposal is to delineate the physiological role of PECAM-1 and its isoforms in retinal vascular development and angiogenesis, as well as in regulation of retinal EC adhesive and migratory properties, and to elucidate the function of genes whose endothelium expression is differentially regulated by PECAM-1. Specifically, we will demonstrate the role of PECAM-1 in the development of retinal vasculature and neovascularization and determine how these processes are affected in the absence of PECAM-1. We will determine the expression pattern of PECAM-1 isoforms during retinal vascular development and neovascularization, as well as in retinal endothelial cells (EC). We will evaluate the specific roles of PECAM-1 isoforms in the regulation of EC adhesion and migration. To further elucidate PECAM-1's mechanism of action, we will identify and perform functional studies of genes such as endoglin and connective tissue growth factor whose endothelium-specific expression is differentially affected by the lack of PECAM-1. These studies will provide insight into the physiological role of PECAM-1 in retinal vascular development and angiogenesis and in modulation of EC adhesion and migration. This knowledge will be instrumental in the development of new treatment modalities for a variety of eye diseases with a neovascular component.	{ "pile_set_name": "NIH ExPorter" }
This project addresses three separate issues related to the kinetics of HbS polymerization and the formation of domains. The three principle issues are: (1) the effect of components of the red cell on homogenous nucleation; (2) the effect of domain formation on mass distribution of both polymerized and free HbS in the gelled sample and (3) the effects of the subunit tertiary structure on the thermodynamics and kinetics of polymerization. Ferrone lists three specific aims in this project A.)Measurements of the homogenous nucleation rates of HbS in the presence of membrane preparations including inside-out vesicles of Hb A and Hb S cells with and without spectrin. Any preparation in which effects are observed in the initial observations will be subjected to detailed studies. B.) Polymer domain formation will be studied in a series of hemoglobins prepared by site directed mutagenesis. C) Double mutant models for partially liganded hemoglobins (e.g. Hb S/M Hyde Park or partially saturated NOHbS) will be characterized kinetically and thermodynamically in dilute and in concentrated solutions.	{ "pile_set_name": "NIH ExPorter" }
We previously constructed a sequence tagged site (STS)/Bacterial Artificial Chromosome based physical map in mouse t-complex region and sequenced 43 BACs in conjunction with Mouse Genome Consortium. The mouse t-complex on chromosome 17 has long been a focus of interest for developmental biologists. Because many embryonic lethal mutations are localized in the region, corresponding genes of importance during development are inferred to reside there. [unreadable] Based on the physical map the tw5 embryonic lethal locus mutation was refined to a single, 175 Kb BAC. Histological studies have demonstrated that mice homozygotic for tw5 die at the gastrulation stage. The candidate BAC rescues the tw5 lethality, restoring viability. Our collaborator Dr. K. Abe is currently focusing on the search for the gene mutated in the tw5 embryonic lethality.[unreadable] Currently, we have focused our attention on the mouse homolog of human ADAMTS10 gene that maps to the same interval. Several metalloproteases play critical roles in cell-cell signal transduction. ADAMTS-10 gene, a zinc metalloendopeptidase with thrombospondin domains, is a potential candidate for important functions during development based on its structural domains. Recently ADAMTS10 was found to be a candidate gene for Weill-Marchesani syndrome (OMIM#277600). We have found that mouse ADAMTS-10 is alternatively spliced in a tissue-specific manner, and encodes protein isoforms that either include or exclude the thrombospondin domains by truncation of the open reading frame. We have cloned and characterized 3 major isoforms from embryo, kidney and lung and find that the embryo, for example, expresses an isoform that has a truncated translation product. The truncated embryonic and full-length kidney isoforms were fused a to V5-epitope in a Gateway vector system and transfected into 293 cells. We find that both isoforms are stably expressed and localize to cytoplasm as determined by immunocytochemistry. We are currently completing the characterization of the spatial and temporal expression of ADAMTS10 in the embryo.	{ "pile_set_name": "NIH ExPorter" }
This document describes a proposal to investigate: (1) the development of malignant lymphoma in homosexual men with persistent, generalized lymphadenopathy (PGL) and AIDS-like immune dysfunction (acquired immunodeficiency syndrome); (2) factors responsible for the development of this state; and (3) the development of PGL or AIDS or malignant lymphoma in sexual contacts of these patients. Patients will be examined at 6 month intervals, and have serial lymph node biopsies and blood specimens taken. The lymph nodes will be investigated for morphologic expression, and for quantitative and qualitative changes in lymphocyte populations. In addition, DNA content analysis, oncogene expression, chromosomal karyotypes, and staining for various viral antigens (EB, CMV, HTLV) will be performed on the nodes. Serial evaluation of immunologic function will be performed at 6 monthly intervals, including B and T lymphocyte numbers and subsets, Interleukin I and II levels, natural killer and macrophage cytotoxicity. Serologic evidence of infection to a variety of agents (CMV, EB, HTLV, Hepatitis B, toxoplasmosis) will be studied. In addition, a careful epidemiologic evaluation will be performed. All of these parameters will be evaluated serially, in order to note changes which may be associated with the development of lymphoma. Patients will be compared to friend controls who are sexual contacts and with other homosexual controls by means of serial evaluation at six month intervals, with epidemiologic questionnaire, viral serologic and immunologic studies, identical to those performed on the patients, in order to discover those factors responsible for the development of the lymphadenopathy syndrome, or for lymphoma, or for AIDS in the normal homosexual population.	{ "pile_set_name": "NIH ExPorter" }
Program Director/Principal Investigator: Schryvers, Anthony B. ABSTRACT Neisseria gonorrhoeae, the bacterial pathogen that causes gonorrhea, is now considered an ?Urgent Threat? due to the recent emergence of strains with multi-drug resistance to the antibiotics that are frequently used during treatment. Due to the emergence of these `superbug' strains, we are progressing towards the spread of untreatable gonococcal infections. This situation has added to the urgency for developing a vaccine to prevent these infections, as untreated gonococcal infections can lead to pelvic inflammatory disease, ectopic pregnancy, infertility and invasive infections. Development of a gonococcal vaccine has been challenging due to the remarkable ability of the bacterium to vary its surface components and suppress the development of a protective immune response against reinfection in humans, and due to the lack of appropriate animal models available to study infection and immunity of this pathogen. The primary focus of this proposal is to further develop a protein-based vaccine that targets the constitutively-expressed gonococcal transferrin binding protein B (TbpB), which captures iron from human transferrin. It is an ideal target since the transferrin receptor in N. gonorrhoeae is required for survival on the mucosa and we have shown that it is capable of reducing colonization in a mouse model. We have demonstrated that transferrin-binding defective mutants of TbpB induce a more protective immune response than the wild type proteins, and are currently finalizing the antigenic composition of our TbpB-based vaccine. In this project, we will scale-up and optimize production and purification of our antigens, develop an optimal vaccine formulation and perform all the steps required for implementing Phase I trials in humans. Cross-protection will be evaluated in a lower genital tract colonization model and a model of pelvic inflammatory disease using female transgenic mice expressing human CEACAM receptors and human transferrin, which facilitate gonococcal mucosal attachment and growth, respectively. Together with an industrial sponsor, Vaxiron, Inc., we will develop quality control tools and metrics for assessing vaccine antigen formulations, transfer production of our vaccine formulation to a contract manufacturing organization, complete all the quality, stability and toxicology studies required for regulatory approval to proceed to a future Phase I clinical trial after the culmination of this project.	{ "pile_set_name": "NIH ExPorter" }
The objective of this project is the completion of the construction of the University of Mississippi's National Center for Natural Products Research (NCNPR) Phase II research wing. This will entail addition of the top two floors, with completion of a portion of the first floor of this research wing, with a total of about 55,000 gross SF of new research space. Note that funds are already committed for the construction of the basement and substantial completion of the first floor;this is slated to begin in fall 2009, with an anticipated completion date in early 2011. This application requests funds to construct the second and third floors, and partial build-out for completion of the first floor, of the Phase II research wing as envisioned in the original NCNPR planning and in the Phase II facility programming document. This proposed project, if awarded, can be timed so as to be completed in concert with the currently funded component as a single, continuous construction. At the foundation of this NCNPR program are a number of PHS and other federally funded research projects aimed at discovery, design, and synthesis of new drugs and development of new drug delivery systems for unmet therapeutic needs in human health, an important national priority. Early development activities to facilitate commercialization of these technologies will also be a critical function for this program. Such efforts are key components for realization of the full potential economic impact of these natural product-based discoveries. In addition, the NCNPR has a strong and growing research effort to expand the scientific basis for enhancement of the authentication, quality and safety of botanical products used as dietary supplements in the US. Completion of this construction as planned will foster translation of basic research into clinical studies and commercial development as follows: 1) provide laboratories for scaling up extraction and isolation of bulk natural products in quantities that will support advanced development activities;2) provide laboratories for scale-up synthesis and analog development;3) provide Good Laboratory Practice-compliant analytical facilities that will support bioanalytical research, product and regulatory package development;4) provide Good Manufacturing Practice-compliant facilities for production of active pharmaceutical ingredient (API);5) provide laboratories for preformulation, formulation and stability studies to characterize APIs;6) provide laboratories for cellular and molecular mechanism of action and toxicity studies;and 7) provide laboratories for expansion of discovery efforts with greater emphasis on microbial and marine natural products. The requested funding will support construction to include 55,127 gross SF (partial first floor, 7,117;second floor 24,005;third floor, 24,005). Four laboratories of about 860 SF will be included in the first floor section, with eight laboratories each on the second and third floors;laboratory and building/mechanical support areas and office/cubicle/conference spaces are also included on each floor. Total net laboratory, office and support spaces to be constructed with this requested funding on the three floors will be 23,011 SF of new laboratory space, 9,773 SF of new office space, and 15,460 SF in support areas.	{ "pile_set_name": "NIH ExPorter" }
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cryo-electron microscopy provides density maps of biomolecular complexes in their functional states, but only at low resolution, unlike X-ray crystallography, which provides atomic-resolution structures of biomolecules but usually not in a physiological state. Computational methods to combine information from both techniques hold the promise of generating physiologically accurate, high-resolution structures of biomolecular complexes. To combine experimental data from these two sources, the Resource developed a novel method, molecular dynamics flexible fitting (MDFF;http://www.ks.uiuc.edu/Research/mdff) [1,2], to fit atomic structures into cryo-EM density maps. MDFF employs molecular dynamics (MD) to perform the fitting, which allows flexibility while maintaining a realistic conformation. The standard MD force field is modified by incorporating the EM density map as an attractive potential that drives atoms into high-density regions. Furthermore, restraints are applied to preserve secondary structure of the biomolecules. MDFF setup and analysis are performed with the Resource's molecular visualization program, VMD, and MDFF simulations are conducted using the Resource's MD simulation software, NAMD. Since NAMD is highly scalable and supports simulation of large systems, MDFF can be applied to large macromolecular complexes such as the ribosome [3].	{ "pile_set_name": "NIH ExPorter" }
Both data management and biostatistics have become increasingly more important to productive research and the resulting understanding of Alzheimer's disease, Previously considerable effort was expended for the ADRC Clinical and Neuropath Cores in standardizing what data to collect and how to collect and store it;in the future emphasis on statistical analysis will increase. In the next grant period, we will maintain and update existing Clinical and Neuropathology Core ADRC databases and develop data entry and database systems for a "core" set of data collected for the Oldest Old and Down syndrome cohorts to be compatible with current clinical data. We propose to develop an interface for researchers to select variables and to specify criteria for selecting subjects for export to statistical software packages. A web based data dictionary will be supported for all ADRC investigators to view. To improve Neuropath repository management, we will develop a centralized system for autopsy results, molecular lab results, inventory and tracking of frozen/blocked tissue, etc. An inventory system for information on triple and double transgenic mouse brains of different lines at different ages will be created. In order to facilitate ADRC research, we will be proactive about ADRC statistical consulting services including study design with power analyses, statistical analyses, and the review of grant applications for appropriate informatics and statistical analyses. We will also conduct workshops to introduce ADRC researchers to data analysis and teach them to vet their data using statistical software. Finally, we will continue to prepare timely submissions of clinical and neuropath data to the National Alzheimer's Coordinating Center (NACC) and to apply changes to these data as they are mandated.	{ "pile_set_name": "NIH ExPorter" }
Human Usher syndrome, the most frequent cause of deaf blindness, is characterized by congenital deafness, due to loss of sensory hair cells, and progressive retinal degeneration, due to retinitis pigmentosa. Although nine of the genes responsible for Usher syndrome and one genetic modifier gene have been identified to date, we still lack an understanding of the normal functions of these genes or what goes wrong in the disease. This is primarily because the ten known genes encode a surprisingly broad range of different types of proteins, all with multiple isoforms, and we currently lack animal models and tools to study their functions. Due to recent advances in zebrafish genetic technology, it is now possible to isolate mutations in any gene and easily produce transgenic animals with altered gene expression. This project will develop libraries of mutants, transgenics, and antibodies to characterize all the genes known to contribute to Usher syndrome. Together, these libraries will provide the first resource ever generated to dissect the distinct functions of the complete set of known genes that underlie a human disease. PUBLIC HEALTH RELEVANCE (provided by applicant): Usher syndrome, the leading cause of deaf blindness, is a genetic disorder that affects tens of thousands of Americans. Mutations in any one of at least a dozen different genes can cause Usher syndrome, but the processes that lead to loss of hearing and vision are unknown. This project will develop a complete resource of animal models and tools necessary to understand the molecular and cellular biology of Usher syndrome.	{ "pile_set_name": "NIH ExPorter" }
The objectives of this grant request are the upgrading and improving of the laboratory animal facilities at the Southern Illinois University School of Medicine and the provision of needed items of equipment to provide care and maintenance of research animals to better support ongoing biomedical research. The specific aims are to provide additional animal holding space by converting a clothes change/locker room to an animal holding room, to slope the floors in rooms containing runs so that the water will not pool under the runs and to replace cat cages with marginal floor space. The equipment and renovations are required to maintain the health of the animals, to improve efficiency of operations, to provide necessary additional animal holding space and to assist in maintaining AAALAC accreditation.	{ "pile_set_name": "NIH ExPorter" }
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global infectious disease emergency. A major hurdle in combating TB is the fact Mtb is able to persist for long periods of time in host tissues, in a quiescent state. These bacilli are able to reactivate and cause pulmonary TB, when the immune system is compromised. Hence, a complete understanding of TB latency and reactivation is required for the effective control of TB. The research models and tools necessary to perform these studies are now available. Nonhuman Primates (NHPs) are excellent models of TB, especially to study the progression of experimental infection to latency, and to study the pathology and biology of granulomatous lesions - the hallmarks of TB infections. We have established a model of human TB, by exposing NHPs to true Mtb aerosols. While many research groups focus on the bacterial factors of latency and reactivation, we would like to leverage our highly tractable model to identify host signatures and mediators of this process. We show that pro-inflammatory immune signaling pathways, initially induced in NHP TB lesions, are overwhelmingly silenced over the course of next several weeks. This transcriptional reprogramming could be a host response to changes in bacterial replication and physiology. Further, these responses could reflect the efforts of the pathogen to prevent excessive immunopathology during the infection of lungs. The central hypothesis of our proposal is that host granuloma responses can be used to predict latent and reactivation TB. We propose to perform a systematic study of the "transcriptome" and the "miRNAome" of NHP lung lesions. Temporal profiles will be obtained from NHPs infected with a low-dose of Mtb aerosols, accurately modeling long-term latent infection. Profiles will also be obtained from NHPs in which latent TB is reactivated by simian AIDS. These system-wide profiles, in conjugation with the clinical, microbiological and immunological data obtained from infected NHPs will generate statistical learning algorithms and mixed effects computational models of latent and reactivation TB. The relevance of some of the most informative set of genetic predictors available from the data collected will be tested back in both the NHP model, as well as in human patients. The expression profiles of CCL24, CCL25 and CCL27 show negative correlation with all other chemokine ligands and receptors in primate TB granulomas. The expression of these three chemokines is significantly increased in late, rather than early lesions. We hypothesize that these chemokines are important for the long-term maintenance of primate lesions harboring latent Mtb bacilli. The expression of LAG3 was induced more than 40-fold in early primate lesions relative to late ones. LAG3 is a novel marker of Treg cells. We hypothesize that LAG3 is responsible for negatively regulating protective immune responses generated by effector T cells in primate TB lesions. The expression of "latency" specific genes CCL24/25/27 and the "active-TB: specific gene LAG3 will be silenced in NHPs using a novel lipidated-siRNA nanoparticle based approach. The progression of latent disease and its immunological and molecular correlates will then be studied in these animals. Finally, the expression of an immune response to these and other "latency" and "reactivation"- specific profiles will be determined in human patients of latent and active TB, as well as TB/AIDS co- infected patients. These systems-biology studies will likely exponentially enhance our understanding of TB latency and reactivation in a host that mimics both TB and AIDS in the closest possible manner to humans. Eventually, these advances may empower clinicians better to detect and treat latent TB. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global infectious disease emergency. A major hurdle in combating TB is the fact Mtb is able to persist for long periods of time in host tissues, reactivating when the immune system is compromised. Hence, effective and long-term control of TB requires a better understanding of TB latency and reactivation. Nonhuman Primates (NHPs) are excellent models of TB. We have recently established a model of human TB, by exposing NHPs to Mtb aerosols. Pro-inflammatory immune signaling pathways are initially induced in NHP TB lesions, but silence over the course of several weeks. This could be a response to the progression of Mtb to a latent phase of growth within these NHP granulomas. We will employ a systems biology approach to study the host granulomatous response to Mtb latency and reactivation, using our NHP model. Towards this, we have assembled a highly diverse and collaborative team including microbiologists, aerobiologists, bioinformaticians, mathematicians/statisticians, veterinarians, veterinary pathologists, as well as infectious disease and critical respiratory care clinicians/clinician-researchers.	{ "pile_set_name": "NIH ExPorter" }
PROJECT SUMMARY Improving STEM-focused curriculum is a primary objective of the current U.S. administration and is crucial for ensuring that upcoming generations receive the training and skills necessary to compete in the existing global economy. To that end, there is an urgent need for additional effective teaching tools able to reach a generation that requires instant access to information and advanced technology. Of particular interest to this proposal is the development of a highly effective, marketable, and interactive educational video game (iEVG) that focuses on STEM topics and targets 5th and 6th grade students?the age at which interest in STEM subjects is developed or lost. The creation of carefully developed and critically-evaluated educational video games is timely and necessary to ensure the promotion of desirable learning principles for 5th and 6th grade students. The long-term goal of our interdisciplinary team of media arts specialists, computer programmers, scientists, writers, students, teachers, and key members of the community is to produce growing libraries of educational video games with immersive graphics and audio, challenging gameplay, and a well-rounded delivery of STEM-focused educational content related to environmental health. Planning and implementation activities based on our specific aims are as follows: (Aim 1) Create an educational video game and teacher lesson plan with significant commercial potential that increases awareness of the importance of clean water in human health. (Aim 2) Evaluate the game for effectiveness in the delivery of STEM educational goals, including an increased understanding of lead poisoning, watershed dynamics, and clean water. The creation of an evaluated and highly effective STEM-focused (iEVG) that incorporates the Next Generation Science Standards will provide teachers with an innovative classroom tool that is engaging to the students, while improving interest in STEM subjects and increasing STEM knowledge.	{ "pile_set_name": "NIH ExPorter" }
Mentoring in Substance Abuse Treatment Research: This K24 Mid-Career Investigator Award in Patient- Oriented Research will provide an intense phase of career development and mentoring in substance abuse treatment research, specifically in the areas of mechanisms of substance use disorders, development of therapeutic interventions, and clinical trials of specific treatments of substance use disorders. The application addresses two areas of need in clinical research: (1) increased patient-oriented research focused on gender differences in substance use disorders, and (2) the need for more clinical investigators committed to a career in substance abuse patient oriented research. More specifically, over the next five years, this K24 would allow the investigator to accomplish her near term career goals which are closely related to the specific aims of this K24 application: AIM 1: The investigator will focus her research efforts on substance abuse research projects that address gender differences in substance use disorders in terms of risks and disease mechanisms, treatment services use, and new and effective treatments;AIM 2: The investigator will provide more-intensive substance abuse patient-oriented research mentoring to Harvard Medical School adult and child psychiatry residents (PGI-VI), research fellows, and junior faculty of both clinical and nonclinical departments;and AIM 3: The investigator will develop specific expertise in qualitative research methodologies, including assessment of group process outcomes, to help enrich and deepen her research on gender differences in substance abuse treatment outcomes. Accomplishing these aims will increase the knowledge base of substance abuse treatment research through both the investigator's enhanced productivity in patient oriented research in gender differences in substance use disorders, and also her mentoring of junior investigators who will acquire both the fascination and research skills to go on to productive careers in substance abuse treatment research.	{ "pile_set_name": "NIH ExPorter" }
Men and their families facing a prostate cancer diagnosis need information and support as they sort through key decisions and adjustments they must make over the subsequent weeks, months and years. The disease and its treatments have substantial and long-lasting effects on quality of life, and our goal is to reduce those effects and speed the process of regaining control over one's quality of life. CHESS (the Comprehensive Health Enhancement Support System) is a Web-accessed system of resources that has been accepted and used, and has successfully improved quality of life for patients with a variety of serious health conditions. The research proposed here would add depth and needs-assessment-based focus to CHESS, make it a system capable of delivering individually-tailored information and resources, and enhance its usefulness for the partners of patients. In addition, we will create procedures for a human Cancer Information Mentor (based on the CIS Information Specialist role) to have a continuing information- supportive relationship with a patient. This project follows and parallels one for breast cancer patients, allowing both substantial efficiencies in development and operation, and the opportunity to compare the two experiences. 360 prostate cancer patients from three hospitals will be randomly assigned to four conditions in a crossed design: either 1) CHESS or open Internet access, and 2) a personal Cancer Information Mentor or not. Where the patient has a partner (often a spouse) interested in participating, that partner will also be offered a separate codename and password for CHESS/internet access. We expect that both CHESS and a Mentor will produce better quality of life on each of four dimensions than accessing the Internet alone, and that the two together will multiply each other's strengths, producing greater benefits than either alone. Drawing on Self-Determination Theory, these effects are expected to occur due to CHESS and Mentor effects on autonomy, competence, and relatedness, each of which is assessed here with two mediating variables. Additional process analyses will distinguish the selectively-variable nature of CHESS and Mentor content and immediate outcomes such as knowledge of one's condition, information overload, etc.	{ "pile_set_name": "NIH ExPorter" }
Treatment of schizophrenia (SZ) patients with monoaminergic antagonist drugs (typical or atypical neuroleptics) has an antipsychotic effect, but negative symptoms and cognition are not significantly improved. Hence, there is an urgent need to find new molecular targets for the development of pharmacological agents active on cognitive deficits and negative symptoms. In this regard, nicotine receptor agonists are some of the most promising treatment options currently under investigation (Freedman et al, 2008). Postmortem examination of the brain of SZ patients reveals: a) a decrease of high and low affinity nicotinic acetylcholine receptor (nAChR) subtypes in telencephalic GABAergic neurons, which is possibly alleviated by nicotine abuse, and b) a neuropathology of cortical, hippocampal and striatal GABAergic neurons that includes decreased expression of GAD67, the 67 kDa isoform of the glutamic acid decarboxylase enzyme that synthesizes GABA, and decreased expression of other GABAergic proteins including reelin and the NR2A subunit of the NMDA receptor (Guidotti et al, 2005; Lewis et al, 2005; Lisman et al, 2008; Woo et al, 2004; 2008). Evidence suggests that this GABAergic neuropathology of SZ brain is accompanied by the overexpression of DNA-methyltransferases 1 and 3a (DNMT1 and DNMT3a) in GABAergic neurons (Costa et al, 2007; Ruzicka et al, 2007; Veldic et al, 2005; 2007; Zhubi et al, 2009). DNMTs are the enzymes that catalyze the methylation of the cytosine carbon atom in position 5' of CpG dinucleotides of several gene promoters. To model this neuropathology in mice, we will use offspring of mothers subjected to restraint stress during pregnancy. These mice are characterized by increased levels of DNMT1 and 3a and decreased GAD67. Our preliminary experiments in normal mice (Satta et al, 2008) suggest that nicotine, acting at central nAChRs (1422, 17) present in GABAergic neurons, reduces the expression of DNMT1 and elicits GAD67 promoter demethylation, thereby upregulating the expression of GAD67. Hence, the use of synthetic nAChR ligands to selectively downregulate DNMT in GABAergic neurons may be an innovative attempt to control the downregulation of GAD67 and other genes operative in selected populations of telencephalic GABAergic neurons of SZ patients, while leaving the function of DNMT in cells that do not express nAChRs intact. To be investigated is whether selective 1422 nAChR agonists (A-85380, ABT-594), partial agonists (AMOP-H-OH), or 17 nAChR agonists (PN -282987), partial agonists (GTS-21) or positive allosteric modulators (galantamine) are better suited to downregulate DNMT and upregulate GAD67 expression in telencephalic GABAergic neurons. Thus, the proposed research is aimed at studying the action of nAChR ligands on the epigenetic regulation of GABAergic neurons to better understand the pathogenesis and pharmacological treatment of sensory/cognitive components of SZ, bipolar disorder and other diseases with psychiatric or cholinergic components.	{ "pile_set_name": "NIH ExPorter" }
Plasma concentrations of zidovudine (ZDV) and other nucleoside analog drugs have shown little correlation with clinical events. Because they must be metabolized intracellularly to elicit a biological effect, intracellular concentrations of the phosphorylated metabolites of nucleoside analogs might correlate with drug effects. The studies herein propose to use a sensitive and reproducible radioimmunoassay method, suitable for routine monitoring of total phosphorylated ZDV in peripheral blood mononuclear cells (PBMC), to define the pharmacokinetics of ZDV phosphorylation in PBMC from patients receiving ZDV, and examine the relation between intracellular concentrations and certain prognostic indicators of HIV disease. This includes a two armed study with a total of 40 patients. One arm will examine the rate of ZDV phosphorylation in ZDV-naive patients and the length of time required to reach steady-state concentrations while on standard dose ZDV (500 mg/day). The second arm will investigate the dose-response in patients on an escalating dose regimen. A two-year study combined both patient groups, and investigates the effects of long-term ZDV therapy on intracellular pharmacokinetics, including clearance and phosphorylation upon re-introduction of drug. Concurrent measurements of the prognostic indicators will be made. This study may suggest that longer dosing intervals and/or lower doses of ZDV are adequate to maintain intracellular concentrations. Additionally, an HPLC/RIA method will be validated and used to determine pharmacokinetics of ZDV triphosphate. this 10 patient study will be nested into the larger study because of practical limitations of the method, but the same parameters will be investigated. Similarly, a pilot study investigating the pharmacokinetic differences of ZDV phosphorylation in patients on combination ZDV/dideoxycytidine will also be performed in an effort to investigate synergistic efficacy. This study will be nested in site patients enrolled in ACTG 155. Finally, the development of new assays for measurement of intracellular nucleoside analog metabolites is planned. Eventually, modifications of dose to maximize effect might be possible through routine intracellular monitoring of nucleoside analog metabolites.	{ "pile_set_name": "NIH ExPorter" }
The DCCT represents the largest clinical trial to evaluate whether patients with insulin-dependent diabetes mellitus whose blood glucose levels are maintained in the normal/near normal range, will have the same, fewer, or more complications than patients whose levels are in the usual diabetic range. Retinal changes are the major endpoints. Renal, cardiac, and neurological assessments are also made.	{ "pile_set_name": "NIH ExPorter" }
It is proposed to complete development of a new type of noninvasive transducer that acquires systolic, mean and diastolic pressure, heart and respiratory rate and oxygen saturation to be used on premature and neonatal infants and small animals. The anesthetized weanling piglet tail will be used for direct comparisons. Human studies will employ the little finger and indirect wrist (radial artery) comparisons on subjects with very small little fingers. The number of births in the US in 1998 was 3,942,000 and among them the number of low birth weight infants (below 5 lb - 8oz) represents 7.8% of the whole population or 307,476 babies per year, and the number of very low birth-weight infants (below 3.3 Ibs.) is 45,000 per year. In veterinary medicine it is estimated that the market size for this device is around 20,400 units per year. Rats are used extensively in drug screening; this device is ideally suited for use in the rat tail. The outcome of this research will be a single instrument that indicates systolic, mean and diastolic pressure, pulse and respiratory rate and oxygen saturation, all derived from a single, member encompassing transducer. The neonatologist, the premature infant physician and the small-infant pediatrician have no single instrument that provides all of these vital signs.	{ "pile_set_name": "NIH ExPorter" }
The long-term goal of this work is to identify secretory protein biomarkers for lung adenocarcinoma diagnosis and prognosis in animals and in humans. In addition to gas exchange, the lung is an organ for host defense through inflammatory responses. Inflammation is a protective process that facilitates pathogen clearance and repairs tissue injury in the lung. However, exuberant inflammation can cause severe consequences and lead to carcinogenesis. One commonly induced pro-inflammatory gene group during pulmonary inflammation is the interleukin 6 (IL-6) family cytokines. Upon binding to their cell-surface receptors, IL-6 family members trigger activation (phosphorylation) of signal transducer and activator of transcription 3 (Stat3) by Janus-activated kinases (JAKs). To assess the consequences of STAT3 persistent activation in the lung, a doxycycline-controlled CCSP-rtTA/(tetO)7-CMV-Stat3C bitransgenic mouse model was generated recently in our laboratory that over-expresses STAT3C (the constitutively active form of STAT3) in alveolar type II (AT II) epithelial cells. In sequential steps, Stat3C over-expression promoted inflammation and adenocarcinoma in the lung with a high frequency (80%). This supports a concept that persistent inflammation triggered by the Stat3 signaling induces lung adenocarcinoma. In searching for human lung cancer samples, Stat3 up-regulation was highly associated with adenocarcinoma and squamous cell carcinoma. Therefore, Stat3 and its downstream genes can be used as biomarkers for lung cancer diagnosis in animals and humans. By Affymetrix GeneChip microarray analysis, multiple Stat3 downstream genes were identified in CCSP-rtTA/(tetO)7-CMV-Stat3C bitransgenic mice. A set of mostly-changed-genes from this list showed similar changes in human adenocarcinoma, confirming that Stat3 downstream genes are suitable for lung cancer diagnosis and prognosis. Since many Stat3 downstream genes are plasma proteins, we plan to use them for blood testing in both animal lung adenocarcinoma models and in human lung cancer patients. The central hypothesis for this proposal is that secreted protein products of Stat3 downstream genes can be used for diagnosis and prognosis of lung adenocarcinomas in animal models and in humans. Two aims are proposed to test the central hypothesis: 1) Characterization of Stat3 downstream genes as lung cancer biomarkers in animals. Three animal models that developed lung adenocarcinoma in association with inflammation will be used. Although they represent different molecular mechanisms for inducing lung adenocarcinoma, Stat3 gene up-regulation and activation are the common mechanism in all three animal models; 2) Characterization of Stat3 downstream genes as lung cancer biomarkers in humans. Putative secreted biomarkers will be screened in lung tissues and blood samples that are from human adenocarcinoma, squamous cell carcinoma and small cell lung cancer. After these studies, we would like to formulate a panel of protein biomarkers for diagnosis and prognosis in the blood of animal models and in human lung cancer patients.	{ "pile_set_name": "NIH ExPorter" }
In this proposal we plan to study the solution conformation and dynamics of a group of compounds which are structurally related to nucleic acid components by H1, H2 minus(P31) P31, P31 minus H1 C13 and C13 minus(H1) NMR spectroscopy. The two fold objectives of investigating these compounds are: (a) Many of the compounds which are structural analogs of nucleic acid components are powerful chemotherapeutic especially, antileukemic agents. Knowing their three dimensional dynamic solution geometry should enable to provide some chemical and conformational basis for the treatment of disease. (b) A by-product of the above study is that it will lead to an understanding of the structural factors--ie steric, electronic and electrostatic-which precipitate a particular conformation in a given molecule. This information is of fundamental importance in the general understanding of the interaction between various parts of the same molecule as well as intermolecular interactions and should be of help to unravel the solution geometry of the loop region of t-RNA which contains so many unusual basis.	{ "pile_set_name": "NIH ExPorter" }
Human T-cell leukemia viruses (HTLV) are the etiologic agents of specific forms of leukemia/lymphomas in man. The mechanism by which these viruses transform T-cells in vitro and induce malignancies in vivo is not known. Viral replication and cellular transformation is dependent on the two virus-encoded trans-acting regulatory gene products termed Tax and Rex. Tax acts to increase the rate of transcription. The ultimate function of the Rex phosphoprotein is to increase the level of the viral structural and enzymatic proteins (Gag, Pol, and Env) expressed from the incompletely spliced viral mRNAs that contain the cis-acting Rex-response element (RxRE). The precise mechanism of Rex function is still unclear, although the current theory on how Rex achieves its effect is through binding of Rex to the RxRE and facilitating nuclear export of the incompletely spliced viral RNAs containing the RxRE to the cytoplasm. It appears that the phosphorylation state of HTLV-II Rex determines the efficiency of binding of Rex to target RNAs in vitro. Furthermore it has been proposed that the HTLV Rex protein may play a role or control the switch that determines whether virus exists in a latent or productive state. Thus, the phosphorylation state of Rex in the infected cell may provide this regulatory control. This proposal focuses on understanding the genetic and biochemical basis of HTLV-II rex gene function with particular emphasis on the importance of posttranslational modification by phosphorylation. The Specific Aims are: 1) to identify and characterize functional domains within the HTLV-II Rex protein; 2) to characterize HTLV-II Rex phosphorylation in vivo and determine its role on HTLV-II replication and cellular transformation; and 3) to characterize the kinase/phosphatase responsible for regulating Rex function. A clear picture of Rex mechanism of action will be important to understanding the pathogenesis of HTLV in man.	{ "pile_set_name": "NIH ExPorter" }
Morphine remains one of the most frequently prescribed drugs for the treatment of moderate to severe pain, including pain due to cancer or surgery. However, the long-term use of this excellent pain reliever in man is limited by side-effects that include analgesic tolerance and opioid-induced bowel dysfunction, with constipation being the most common and debilitating symptom. The long-term goals of this study are to elucidate the mechanisms that lead to tolerance to many of the effects of opioids, including slowing of gastrointestinal transit but not constipation. The main hypothesis to be tested is that differences in the cellular signaling properties of morphine determine the development of tolerance in the ileum but not the colon. Preliminary data suggest that morphine tolerance in the ileum is associated with an uncoupling of the ? opioid receptor from its downstream signaling proteins. Unlike the ileum, the colon which is the major site for constipation does not develop tolerance to repeated administration of morphine. The major objective of specific aim 1 is to test the hypothesis that down-regulation of ? arrestin2 is associated with morphine tolerance in the ileum. The specific goals are to characterize the concentration and temporal relationship for ? arrestin 2 downregulation and tolerance development. Functional and biochemical studies will be utilized to correlate the effect of chronic morphine in-vitro and in-vivo utilizing ?-arrestin2 knock-out mice. Specific Aim 2 will test the hypothesis that ? arrestin2 acts as a scaffolding protein to regulated downstream signaling including MAP kinase, Src kinase, Akt and protein kinase C. Preliminary findings suggest that unlike morphine induced antinociceptive tolerance, in the ileum downregulation of phospho-ERK correlates with tolerance development suggesting fundamental differences in the mechanism for opioid tolerance in the gastrointestinal tract from CNS. This aim will also examine if downregulation of ? arrestin 2 is mediated via altered ubiquitination. Specific Aim 3 will explore the effect of long-term morphine on isolated enteric neurons from the adult mouse myenteric plexus. In this aim, single enteric neurons from the colon and ileum will be characterized and tested to determine morphine- induced changes in electrical excitability and effects on sodium, calcium and potassium channels in wild-type and ? arrestin2 knock-out mice. The information obtained from these studies will increase our understanding of the mechanisms of opioid tolerance and ultimately physical dependence in the gastrointestinal tract and in the brain. PUBLIC HEALTH RELEVANCE: There is a pressing need for new therapies that act upon the underlying mechanisms of opioid-induced bowel dysfunction. The major public health implication of this research is the use of the information to develop medications to treat chronic pain that are devoid of constipation and to learn mechanisms of tolerance that will help in ultimately understanding physical dependence to opioids in the brain.	{ "pile_set_name": "NIH ExPorter" }
[Mild traumatic brain injury (mTBI) is a major clinical problem in United States Veterans and reported by 315,897 service members since 2000, representing 82.3% of all TBIs reported in military personnel. Although the majority of people with mTBI recover within a few weeks, 25% of military personnel with mTBI have persistent symptoms lasting for at least 3 months post-injury.] However, the factors that contribute to developing persistent symptoms after mTBI are unknown. Understanding the risk factors involved in the persistent sequelae after mTBI could provide clinicians with the ability to identify Veterans at risk and enable early interventions. One potential factor recently identified in a study of mTBI patients is pre-exposure to stressful life experiences. Chronic early life stress is highly prevalent in the United States, and a major cause of early life stress in childhood is neglect. Maltreated children are at risk for developing long-term emotional, cognitive and medical problems that emerge during middle age. One potential mechanism linking early life stress to neurological problems in adulthood is hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. Early life stress results in a heightened and prolonged cortisol response in response to stress encountered in adulthood. Using brief daily maternal separation in rat pups to model childhood neglect, we have found that the combination of early life stress with mTBI in adulthood impaired hippocampal-dependent learning and increased corticosterone levels in response to restraint stress. Based on these preliminary data, the main hypothesis of this proposal is to determine if early life stress prior to mTBI is a risk factor for persistent memory problems after mTBI and if these memory impairments can be improved with cognitive rehabilitation in combination with a glucocorticoid receptor antagonist. To test this hypothesis, the following aims are proposed: 1) To determine if early life stress prior to mTBI results in persistent memory impairments and 2) To determine if early life stress prior to mTBI potentiates the response to stress in adulthood and if a glucocorticoid receptor antagonist in combination with cognitive training improves memory deficits. These studies will address a highly significant clinical question, do predisposing factors contribute to the development of persistent neurological sequela after mTBI? We will determine if an early life stress event prior to mTBI exacerbates memory impairments and test whether implicit cognitive training in combination with a clinically approved drug, mifepristone, can mitigate the effects of early life stress on mTBI outcome during cognitive rehabilitation. [This proposal may improve Veteran health by providing scientific support for screening for early life stress in Veterans who have had a mTBI and use these screening results to indicate whether this combinatorial treatment may be potentially efficacious in Veterans.]	{ "pile_set_name": "NIH ExPorter" }
Investigation has focused in the mechanisms of interaction between corticosterone releasing hormone (CRH) and other regulators in the control of the hypothalamic-pituitary-adrenal axis in normal conditions and during adaptation to stress. Studies during aging in two strains of rats which differ in their responsiveness to stress, showed reduced concentration of pituitary CRH receptors in old rats. Despite the decrease in receptors in both strains, basal and stress stimulated ACTH were comparable to those in young rats. However, stress induced corticosterone responses were more prolonged in old rats. These data suggest that a defect in adrenal responsiveness to ACTH may have an important role in the alterations of the hypothalamic-pituitary adrenal axis during aging. Previous studies showed that sustained stress is accompanied by decreases in pituitary CRH receptors and pituitary desensitization to the primary stress, but a hypersensitivity to a novel stress. The role of CRH and VP on these changes were studied by analysis of plasma ACTH and corticosterone responses to acute stress in rats receiving chronic minipump infusions of CRH alone or in combination with VP. CRH and CRH plus VP infusion caused the expected decrease in CRH receptors, but in contrast with the changes in chronic stress, ACTH responses to acute stress were markedly reduced in rats receiving the infusions. Plasma ACTH responses to acute CRH or CRH plus VP injection, as well as releasable ACTH pools measured in vitro by stimulation with 40 mM KCL were also reduced following chronic CRH treatment. The data indicate that sustained pituitary account for the enhanced ACTH responses to a novel stimulus during chronic stress. Studies in forebrain neuronal cell cultures showed that CRH stimulates cyclic AMP production. Similar to the pituitary, this effect was enhanced by the protein kinase C stimulators VP and phorbol esters, and inhibited by glucocorticoids. The data supports a role for CRH in the regulation of extrahypothalamic neuronal function.	{ "pile_set_name": "NIH ExPorter" }
The synthesis of proteins by ribosomes is a fundamental step in the expression of genetic information by living organisms. A simple, reliable, rapid, and generalizable method for stopping this fundamental process for a specific protein with a drug would be an enormously useful tool in biomedical and translational research. It would benefit basic studies of protein function by allowing conditional expression of proteins of interest. It would provide a means for regulating protein production from gene and cellular therapies in vivo. A method for controlling synthesis of specific proteins would also allow assessment of how inducible synthesis of specific proteins contributes to particular biological responses or to disease, a line of investigation which has not been possible. In the proposed work, we will develop a novel generalizable method for shutting off synthesis of genetically tagged proteins using a small-molecule drug, and use this method to address outstanding questions on the role of new protein synthesis in nervous system adaptation. We will express proteins of interest as fusions to a potent degradation signal that undergoes autocatalytic removal. By default, the proteins will be released from the degradation signal and function normally. Application of a specific non-toxic cell-permeable drug will preserve the degradation signal on subsequently synthesized proteins, leading to their rapid degradation. We will perform experiments to validate the utility of this method in primary cells and animals, to determine the protein degradation pathways on which this method relies, and to extend the strategy to controlling production of secreted proteins. We will further use this method to test a long-standing hypothesis that synthesis of specific synapse-regulating proteins is required for memory consolidation in transgenic mice. If successful, these experiments will be groundbreaking in establishing a completely new method for controlling protein production that is rapid, robust, simple, and generalizable. The proposed research will have broad benefits in biomedical research by providing a generic method for regulating protein expression that allows more rapid kinetics than transcriptional control methods but, like transcriptional regulation, produces functional proteins without a permanent fusion tag. This work may thus facilitate studies on protein functions in general, including genome-wide screens, and provide a means for tightly controlling gene and cellular therapies in vivo. This work will also produce the first experimental tools capable of addressing the importance of specific protein synthesis events in normal physiology and disease. Thus the proposed experiments have the potential to produce a major advance in our ability to control protein function for elucidating biological mechanisms and for controlling biological therapies.	{ "pile_set_name": "NIH ExPorter" }
Project Summary/Abstract Inhibitory circuits formed by GABAergic interneurons (INs) contribute to processing and encoding of cortical information by shaping the spatial and temporal structure of neural activity. Consistent with this critical role of INs in normal brain functions, IN malfunction has been implicated in a wide array of brain disorders such as schizophrenia, autism, and epilepsy. Despite their importance, detailed wiring diagrams of inhibitory local circuits remain largely unknown due to huge diversity of IN types. Furthermore, it is poorly understood what principles govern assembly of inhibitory microcircuits. Filling these knowledge gaps will provide us with wiring and developmental principles of cortical inhibitory circuits, which in turn dramatically facilitate our understanding of how cortical circuits work. One fundamental cortical circuit module contains an excitatory principal neuron (PN) locally innervated by distinct IN subtypes (an IN-PN circuit). In the neocortex, PNs are grouped by areas, layers, and remote projection targets, which represent their functional attributes. It has been shown that distinct classes of PNs display unique homotypic- and heterotypic-connections and convey different neuronal signals. However, little is known about cellular and axonal organization of distinct IN subtypes sending inputs to defined PNs. To address this question, we have developed a novel genetic strategy combining rabies virus (RV)-mediated retrograde monosynaptic labeling and intersectional approaches. The major objective of our proposal is to provide wiring and developmental principles of INs sending inputs to defined PN subtypes at a cell type-specific resolution. Previous studies showed that layer 5 (L5) PNs receive a larger number of inhibitory inputs from parvalbumin (PV)-expressing INs than L 2/3 PNs and this connection feature is controlled by PN identity. Thus, we hypothesize that distinct PN types defined by areas, layers, and long-range projection targets have different organization of input INs, which is shaped at least in part by PN identity. To test this hypothesis, we will dissect the following subjects using an intersectional retrograde monosynaptic tracing, genetic manipulation of PN identity, and mouse genetics. In Aim 1, we will elucidate organization of PV-, somatostatin (SOM)-, or vasoactive intestinal polypeptide (VIP)-expressing INs sending inputs to distinct PN types defined by cortical areas, laminar positions, and remote projection targets. In Aim 2, we will examine developmental processes of IN-PN circuits containing specific PN types innervated by PV-, SOM-, or VIP-INs. In Aim 3, we will generate ectopic PNs by genetic manipulations of transcription factors that control PN identities and examine organization of input IN subtypes. Through these experiments, we will gain wiring and developmental principles of IN-PN circuits in a cell type-specific manner, which will pioneer novel approaches for diagnosis and treatment of brain disorders.	{ "pile_set_name": "NIH ExPorter" }
The Integrated Program in Cellular, Molecular and Biomedical Studies (CMBS) at Columbia University Medical Center is a Ph.D. granting program that combines faculty from all the basic science departments. The CMBS Program is an umbrella program that presents students with a unique opportunity to obtain individualized training in basic cell and molecular biology, microbiology, structural biology, biophysics, genetics, immunology, neurobiology, computational biology, as well as translational biomedical disease-related research. Our hope is to train the next leaders in the field of biomedical research and also to provide training for future leaders in other areas where a biomedical research background will be of great benefit. The CMBS program is an accredited degree-granting program that was first established in 1986 and has been supported by this Training Grant since 1987. The program has a distinguished, well- funded faculty of 135 trainers, whose research expertise represents nearly all the areas of modern cellular and molecular biology, neurobiology and computational biology. There are currently 69 students in this program. Seventy two students have graduated from the CMBS Program in the past five years and have gone on to postdoctoral positions in outstanding laboratories, careers in the pharmaceutical of biotechnology industry, or careers where they use their biomedical training to provide other societal benefits. Students take core courses in molecular genetics, molecular and cell biology as well as statistics during their first year and complete three laboratory rotations. In the secod year, students take their qualifying examination and a course in the Responsible Conduct of Research. The CMBS Program hosts a student research seminar series and a biennial student-faculty retreat. Most students graduated in 5-6 years. The CMBS Program remains the premier cellular and molecular biology graduate program at Columbia University Medical Center and support from this Training Grant is crucial for the continued success of this program. PUBLIC HEALTH RELEVANCE: The goal of this program is to provide students with a unique opportunity to obtain individualized training in basic cell and molecular biology, microbiology, structural biology, biophysics, genetics, immunology, neurobiology, computational biology, as well as translational biomedical disease-related research.	{ "pile_set_name": "NIH ExPorter" }
To advance the work already performed in our laboratory with rats, adrenal medulla tissue was grafted to the denervated putamen of the rhesus monkey in our continuing research on brain tissue transplantation. Graft survival is erratic. In the most successful animal, the behavioral response produced by the graft has lasted one year. An instrument (the brain grafter) that facilitates grafting was developed and a patent has been awarded. Adrenal survival may be enhanced by the addition of nerve growth factor and other trophic factors.	{ "pile_set_name": "NIH ExPorter" }
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Background: The Tulane Resource Allocation Committee (TRAC) membership is composed of core and affiliate scientists who are responsible for the equitable allocation of animal resources. Methods: The ninth year of operation of the TRAC saw continued refinement of operations of the committee, development of policy statements, and better reporting and analysis of allocation data. Analysis of breeding colony demographic, morbidity, and mortality data as well as allocation data assist in colony management decision-making. Results/Discussion: A total of 55 investigator applications were received requesting a total of 492 animals. Approximately 78% of animal allocation has been to affiliate (outside) investigators and 22% to core investigators for the last reporting period. Because of the rapid growth of the research program, 29 investigator requests for 326 animals were initially deferred until housing space and/or animals were available for assignment. At the end of 2010, five investigator requests for 39 animals remain deferred.	{ "pile_set_name": "NIH ExPorter" }
Open angle glaucoma (OAG) is a prevalent disease that results in irreversible loss of vision through the death of retinal ganglion cells (RGCs). Although several risk factors for OAG have been identified, including elevated intraocular pressure (IOP), the exact molecular mechanisms leading to OAG remain poorly understood. It remains to be determined how systemic blood pressure (BP) and gender contribute to OAG. Finally, available therapies delay disease progression but offer incomplete protection, and development of new therapeutic strategies has been hampered by the lack of animal models of OAG. Our preliminary data establish mice deficient in the nitric oxide (NO) receptor soluble guanylate cyclase ?1 (sGC??1 -/-) as a new animal model for OAG, and identify sGC??1 -/- mice as a unique model to study the relation between blood pressure, gender, and OAG. Furthermore, this new animal model of moderately increased IOP and OAG represents an important tool to teststrategies for disease prevention and treatment of elevated IOP and OAG. We hypothesize that sGC??1 -deficiency predisposes RGCs to modest IOP increases in a gender-specific manner. Our hypothesis will be tested in three specific aims. Aim 1 - Determine the role of gender and BP in the development of OAG in sGC??1 -/- mice. Objective A - Determine whether male sGC??1 -/- mice develop OAG. Objective B - Test whether sex-hormones affect the development of OAG in sGC??1 -/- mice. Objective C - Investigate whether BP modulates OAG risk in sGC??1 -/- mice. Aim 2 - Test whether sGC??1 -deficiency predisposes to optic neuropathy associated with elevated IOP. Objective A - Study the effect of increasing IOP (with microbeads) on the retinal nerve fiber layer (RNFL) and the optic nerve (ON) in young WT and sGC??1 -/- mice. Objective B - Assess retinal and ON damage in mice in which sGC??1 is selectively deficient in RGCs. Aim 3 - Investigate the ability of available compounds that enhance cGMP signaling to prevent optic neuropathy in OAG associated with sGC??1 -deficiency. Objective - Serially measure the impact of treating sGC??1 -/- mice with the cGMP-elevating compounds sildenafil (a phosphodiesterase (PDE) 5-antagonist), brain natriuretic peptide (BNP, a peptide activator of membrane bound guanylate cyclase (pGC)), and cinaciguat (an sGC-activator) on AqH outflow, IOP, RNFL thickness, and ON axon count. We anticipate that the studies described in this proposal will further our knowledge of the etiology o OAG by elucidating the role of impaired NO-cGMP signaling, gender, and BP in the development of OAG. We furthermore expect to identify cGMP signaling as a promising therapeutic target in the treatment of OAG. PUBLIC HEALTH RELEVANCE: Our preliminary data identify mice deficient in the ?1 subunit of soluble guanylate cyclase (sGC??1 -/- mice) as a novel and unique model of open angle glaucoma (OAG). This research project aims to i) elucidate the role of impaired NO-cGMP signaling, gender, and ocular perfusion pressure in the development of OAG, ii) further our knowledge of the etiology of OAG, focusing on the susceptibility of retinal ganglion cells to changes in IOP, and iii) identify cGMP signaling as a therapeutic target in the treatment of OAG.	{ "pile_set_name": "NIH ExPorter" }
We seek to understand the psychological and biopsychological aspects of normal and pathological aging in terms of attention and attentional processes. We are also concerned with applying that knowledge to develop strategies for improving attentional and cognitive functioning. This year we report on an investigation into compensatory variations in sustained attention situations which result in disproportional improvements in performance in the elderly. A particularly powerful variation was the increase in exposure time of the stimuli-the elderly improved their performance while the young and middle-aged were unaffected by the increase in exposure time. We have also begun the development of an attention-switching functional test which will be used in our attempts to provide early markers of Alzheimer's Disease.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION (provided by principal investigator): Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly fatal neurodegenerative disease of largely unknown cause. It is clinically characterized by progressive skeletal muscle paralysis and eventual respiratory failure, with a mean survival of about 40 months after symptom onset. No prospective investigations have attempted to determine what factors accelerate or slow disease progression. Recent clinical and epidemiological studies suggest that diverse environmental and lifestyle factors are associated with ALS and that these generate oxidative stress (OS). We hypothesize that these extrinsic factors act throughout the disease course, generating varying levels of OS, and thus influence disease progression. We propose to test this hypothesis in 420 newly diagnosed ALS patients from 11 ALS centers across the US. We specifically aim: 1. To determine whether markers of increased exposure to OS, measured via questionnaire or biomarkers, are associated with the progression of ALS. ALS progression will be determined with a widely used and well-validated ALS functional scale (the ALSFRS-R) every 3 to 6 months for 24 months. At baseline and follow-up, we will obtain measures of OS biomarkers (urinary 15-F2t-isoprostane and 8- oxodeoxyguanosine, plasma paraoxonase I [PON1] levels, and PON1 functional status) and, through structured interviews, measures of current environmental, psychological, dietary, and lifestyle factors associated with OS. 2. To examine the associations between OS biomarkers, an OS index, and survival of patients with ALS. An OS index will be developed based on a sum of the external factors (Goodman et al. 2007). Survival will be followed at the ALS Centers during the grant period and thereafter by using National Death Index data. We will test whether increased and continuing OS as measured by the index and biomarkers affect survival; 3. To determine whether a variety of environmental, lifestyle, and psychological factors are associated with increased levels of OS biomarkers at baseline; 4. To evaluate associations between lipid profile and ALS progression, as measured by ALSFRS-R and survival. Lipids will be analyzed at several time points to determine whether high cholesterol and LDL are associated with longer survival, as recently reported (Dupuis et al 2008); and 5. In exploratory analyses, to determine whether OS markers and exposures are associated with distinct subtypes of ALS, such as bulbar- or spinal-onset ALS, and ALS with or without fronto-temporal dementia (FTD). To our knowledge, this is the first prospective, interdisciplinary, in-depth multicenter epidemiological investigation of OS in ALS. Our project will increase the understanding of the disease mechanisms involved in disease prognosis and may be the first step toward new treatment and prevention approaches, such as multiple anti- oxidant therapy, to target oxidative stress sites in ALS.	{ "pile_set_name": "NIH ExPorter" }
We are studying the structures of three antibodies the F ab of humanized OKT4A, and two metal-binding mutants of the single-chain F v of the catalytic antibody 43C9. Computational models of the antigen binding regions of all three antibodies have been constructed and will be compared to the crystal structures to ascertain the predictive power of the modeling techniques. High resolution structures will be needed to clearly define the mechanisms and properties of antigen binding by these proteins.	{ "pile_set_name": "NIH ExPorter" }
A sustained-release formulation of recombinant hGH (rhGH) would allow a simpli-fied administration regimen and the opportunity to investigate the endocrine and metabolic effects fo continuous of rhGH. ProLease hGH is a formulation of rhGH (somatropin, Genentech, Inc.) intended to provide continuous release of hGH for up to 28 days. Since rhGH is primarily administered to children w/growth disor-ders, there is particular interest in a new hGH product that reduces the need for daily injections.	{ "pile_set_name": "NIH ExPorter" }
There is accumulating evidence that dopamine loss at locations outside of the striatum such as the substantia nigra or the globus pallidus may play a significant role in the development of parkinsonism. Recent studies in animals, primarily rodents) have suggested that dopamine may also act at the level of the subthalamic nucleus (STN). In these animals, a direct dopaminergic projection from the substantia nigra pars compacta to the STN has been demonstrated. Furthermore, it appears that the activity of STN neurons is modulated through actions of dopamine at pre- and post-synaptic dopamine receptors in the STN, and that dopamine depletion in the STN leads to altered firing rates and more intense bursting in this nucleus. The extent and anatomy of the dopaminergic innervation of the STN, the function(s) of dopamine within the STN, and the effects of dopamine loss in the STN in the parkinsonian state have not been explored in primates. With the proposed studies, we will examine the anatomy and function of the dopamine supply to the STN in vivo in primates. With the experiments under aim 1, we will analyze the extent of the dopaminergic innervation and characterize the subcellular localization of dopamine receptors in the STN of normal and MPTP- treated (parkinsonian) monkeys, using a combination of light-microscopic and high resolution electron microscopic immunocytochemical methods. The experiments under aim 2 will study the functional effects of dopaminergic compounds, locally administered in the STN, on the activity of STN neurons in normal and parkinsonian monkeys. We will examine the effects of dopamine receptor agonists and antagonists, injected locally into the STN with a microinjection/recording device. In addition, microdialysis experiments will be carried out to measure the presynaptic effects of dopamine on GABA release in the STN, as well as electrophysiological recordings in the primary targets of STN projections, i.e., the external and internal pallidal segments. Finally, under aim 3, we will study the behavioral effects of dopamine receptor blockade in the STN in normal animals, and activation of dopamine receptors in the STN in parkinsonism, with the expectation that disruption of dopaminergic transmission contributes to parkinsonism, and replacement of dopaminergic function in the parkinsonian state may ameliorate parkinsonian symptoms. Taken together, these studies will provide us with a thorough examination of dopaminergic functions in the primate STN, will help us to understand further the effects of dopamine loss at this extrastriatal site, and may identify the STN as a target for focal dopamine replacement strategies in parkinsonism, such as gene delivery methods or grafting. PUBLIC HEALTH RELEVANCE: The neuronal activity in the subthalamic nucleus is strongly affected by the dopamine depletion in the basal ganglia that occurs in Parkinson's disease, making this nucleus the primary targets of functional neurosurgery to treat parkinsonism. Activity changes in the subthalamic nucleus are usually seen as secondary events, triggered by striatal dopamine loss. However, there is accumulating evidence that dopamine loss within the subthalamic nucleus also contributes to the neuronal activity changes, and to the generation of parkinsonism. We will explore this hypothesis in parkinsonian primates through anatomical, electrophysiologic, biochemical and behavioral experiments. These studies will expand our knowledge of the pathophysiology of parkinsonism, and may identify the subthalamic nucleus as a location at which current dopaminergic therapies work. If significant dopaminergic effects are seen in the subthalamic nucleus, it may emerge as a new target for highly specific local dopamine replacement therapies for parkinsonian patients.	{ "pile_set_name": "NIH ExPorter" }
Currently for lymphoma patients, 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/ computed tomography (CT) is used for interim restaging or therapy monitoring after 1-4 cycles of chemo-/chemoimmunotherapy. An excellent negative predictive value (NPV) of >80% with only a moderate positive predictive value (PPV) of between 25%- 70% has been reported. It has been shown that a substantial portion of the false-positive FDG-PET findings in this setting were caused by early post-therapy inflammatory changes. This hampers the accuracy and reliability of this test and, hence, its acceptance as an early prognostic tool in the clinical practice setting. An important recent advance in the area of molecular imaging of lymphoma is the development of the PET tracer18F-fluorothymidine (FLT) as an in vivo marker of cell proliferation. FLT uptake in lymphomas has been shown to be proportional to the Ki-67 index, a recognized histopathological marker of cell proliferation and since less affected by postherapy inflammatory changes caused by macrophages/monocyte infiltration, it is also a more tumor-specific tracer when compared to FDG. This higher tumor- specificity may be advantageous in early response assessment following chemo-/chemoimmunotherapy, radiation therapy (RT) or combined modality therapy. In this application, we propose to compare the predictive values of interim FLT- PET/CT and FGD-PET/CT in diffuse large B-cell lymphoma (DLBCL) patients after 2 cycles of R-CHOP chemotherapy. Our hypothesis is that FLT-PET/CT will have a substantially higher PPV when compared to FDG-PET/CT (i.e., e 91% vs. d 68%) while providing a similar or only slightly lower NPV and, therefore, prove superior to FDG-PET in this setting. We propose to prospectively study 137 DLBCL patients at 5 institutions. Study patients will be scanned by FLT-PET/CT and FDG-PET/CT 18-24 days after the second cycle of R-CHOP. After completion of six cycles of chemotherapy, conventional staging methods (CSM) will be performed in all patients to assess response and determine appropriate patient management. Patients will be followed for event-free survival (EFS) and overall survival (OS) for at least 24 months post- therapy or until definite determination of persistent disease (by biopsy) or disease progression (both considered an event). Specifically, we intend to: 1. Investigate whether the PPV of FLT-PET/CT is significantly higher (i.e., e 91% vs. d 68%) than that of FDG-PET/CT by following-up patients for at least 24 months posttherapy or until evidence of or disease progression. 2. Investigate whether the EFS of patients with FDG-PET/CT-positive and FLT-PET/CT negative scans is not significantly lower than that of patients with concordantly negative FDG-PET/CT and FLT-PET/CT scans (i.e., <65% vs. 85%) and, therefore confirming that the NPV of FLT-PET/CT is similar to that of FDG-PET/CT. 3. Correlate interim FLT- PET/CT and FDG-PET/CT with the International Prognostic Index (IPI), a well-established predictor of outcome in DLBCL. The demonstration of superiority of FLT-PET/CT over FDG-PET/CT is expected to lead to greater confidence in using interim PET in patient management. This increased reliability could potentially even render repeat biopsy unnecessary for midtherapy treatment decisions. This would represent a significant advance in the use of PET imaging for personalized therapy with an expectedly positive impact on patient management and/or outcome of patients with DLBCL. PUBLIC HEALTH RELEVANCE: Currently for lymphoma patients, 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/ computed tomography (CT) is used for interim restaging or therapy monitoring after 1-4 cycles of chemo- /chemoimmunotherapy. However, its accuracy is suboptimal which hampers its acceptance as an early prognostic tool in the clinical practice setting. In this investigation, we will determine whether the novel tracer 18F-fluorothymidine (FLT) combined with CT (FLT-PET/CT) is superior in this setting. The demonstration of superiority of FLT-PET/CT over FDG-PET/CT is expected to lead to greater confidence in using interim PET in patient management potentially rendering repeat biopsy unnecessary for midtherapy treatment decisions. This would represent a significant advance in the use of PET imaging for personalized therapy with an expectedly positive impact on patient management and/or outcome of patients with lymphoma.	{ "pile_set_name": "NIH ExPorter" }
Cardiovascular and renal complications, including atherosclerosis and lipid abnormalities, diabetic cardiomyopathy, stroke and renal disease, comprise the major morbidity and mortality in diabetes. The complications of diabetes mellitus in mice appear to be largely the same as in humans. The purpose of the Cardiovascular Pathophysiology and Complications Core (CPCC) is to provide rapid, comprehensive, and accurate screening for cardiovascular disease and complications of diabetes in mouse models. The mouse phenotyping tests used by this Core are modeled after and directly translatable to tests used to assess patients with diabetes. Core services include assessment of a) cardiac morphology and function, including morphology, histology and echocardiography;b) vascular regulation, including resting measurement of blood pressure and response to vasomotor perturbations;c) exercise capacity and metabolic function;d) comprehensive renal function;e) microvascular function;and f) circulating markers of cardiovascular disease including electrolytes, indices of renal function, glycated hemoglobin, and serum lipids. The range of phenotyping tests performed by the CPCC allows for thorough investigation of the presence, correlation with, and modification or amelioration of cardiovascular disease, as well as diabetic complications associated with specific genetic manipulations in the mouse.	{ "pile_set_name": "NIH ExPorter" }
Osteoarthritis is characterized by the progressive degeneration of the articular cartilage of the joints, ultimately leading to inflammation, pain, and impairment of mobility. In this proposal, we will address the molecular biology of osteoarthritis by means of manipulation of an in vitro chondrogenic cell culture system, application of gene knock-in technology to generate and then characterize mice that express mutated forms of cartilage types II and IX, and cartilage oligomeric matrix protein (COMP), and identification of candidate genes involved in cartilage development and maturation by means of differential gene expression profiling. The objective of the Morphology/Biomechanics Core (Core Leader: Rocky S. Tuan) is to provide state-of-the-art facilities and technologies for the characterization of the morphological, structural, gene expression profiling, and mechanical property testing of the cells and tissues generated from the Research Projects of the Program Project. The Morphology Component of the Core, will be responsible for routine histology, immunohistochemistry, in situ hybridization, ultrastructure, and confocal laser scanning microscopy, and will be carried out at the Orthopaedic Research Laboratory at Thomas Jefferson University. The Biomechanics Component will be supervised by Dr. Lori Setton in the Mechanical Engineering Department at Duke University, and will be responsible for the application of the osmotic loading technique to determine the mechanical properties of joint articular cartilage in the experimental mouse models that harbor the extracellular matrix gene mutations. Both Components of the core are staffed by experienced investigators with established, published experience in the application of these techniques in cartilage cells and tissues. The Morphology/Biomechanics Core therefore serves a vital function in the end-point analysis of cartilage structure and function, and is essential for the successful completion of the studies proposed in each of the Research Projects.	{ "pile_set_name": "NIH ExPorter" }
LRRK2 and Parkinson's disease cell biology Parkinson's disease is a disorder of movement, cognition and emotion, characterized neuropathologically by neuronal degeneration and deposits of protein aggregates termed Lewy bodies. While most cases are sporadic, rare genetic forms of the disease, caused by mutations in alpha-synuclein, parkin, DJ-1 and PINK1, are helping to elucidate pathogenesis. In previous studies, we have defined the role of alpha- synuclein protein interactions (including interactions with synphilin-1 and parkin) in PD-related cell biology. Mutations in leucine-rich repeat kinase 2 (LRRK2) have recently been found to cause autosomal dominant PD. Our overall hypothesis is that identifications of LRRK2 protein interactions will help elucidate pathogeneses of LRRK2 related PD, and possibly sporadic PD. We have identified interactions between LRRK2 and several other proteins, including parkin, synphilin-1, WSB-1 and CARD7. We have found that that mutant LRRK2 causes direct cellular toxicity, and have initial data that for at least some of the LRRK2 mutations, GTP binding and kinase activity are necessary for toxicity. In Specific Aim 1 we will identify LRRK2 interacting proteins using the yeast two-hybrid system and co-immunopreciptation from transfected cells, and define interaction domains of these proteins. In Specific Aim 2 we will study the LRRK2 interactions in expression studies in cells in culture, and in mouse and human tissue, including postmortem human sporadic and mutant LRRK2 PD tissue. We will study the role of these interactors in LRRK2 cellular toxicity, by using siRNA, and by modifying the interaction domains. In Specific Aim 3 we will determine whether LRRK2 kinase activity is critical for cell toxicity, and determine whether LRRK2 can phosphorylate the interactors-and if so, we will determine whether this has a role in toxicity. These studies will help define the role of LRRK2 and its interacting proteins in cellular pathogenesis related to PD, and potentially identify targets for future therapeutic interventions. [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
Background: Cancer registries traditionally have relied upon hospital data to estimate site-specific cancer incidence in a population. In the last few years, the CDC has helped expand the role of registries to provide key information on patterns of cancer care. As a result, registry data have great potential for research on trends of cancer care in the population as well as patterns of care and disparities in care access and outcomes of vulnerable groups such as minorities and the underserved. With the diagnosis and management of many cancers shifting from the traditional hospital setting to the ambulatory setting, more complete diagnostic, treatment, and follow-up information from physician offices and other non-hospital settings is required. We have found that instances of low data quality are not randomly patterned, and thus can be statistically predicted with known accuracy. On this basis it is feasible to develop targeted audit protocols (TAP) that meet or exceed program thresholds for data quality. Objectives: This 3-year project is designed to improve the completeness, timeliness, quality and use of recent first course of treatment and stage data in the North Carolina registry;and to describe patterns of care in randomly selected samples of 600 white and nonwhite cases of female breast cancer cases stages l-lll (N=1,200) and 600 white and non-white cases with prostate cancer, all stages (N=1,200) contained in the NC registry in the two most recent diagnosis years available This work builds upon the past collaborative work of Wake Forest University and the North Carolina Central Cancer Registry (NC CCR) on patterns of cancer care. In our proposed work, we plan to implement efficient strategies for data quality assessment and improvement, and assessment of patterns of cancer for breast cancer and prostate cancers within the total sample, by race/ethnicity groups, and by markers for poverty. Specifically, in this three year study we would: 1) Test the efficiency and effectiveness of a targeted audit protocol (TAP) for registry data to improve the overall quality of registry data. 2) Assess the quality, completeness of staging and first course of treatment collected by the NC CCRby electronic edits and professional case record re-abstraction. 3) Describe the percentage of reported cases with female breast cancer that receive standard of care in North Carolina. 4) Describe the patterns of care with prostate cancer in North Carolina, considering patient (distance to radiation facility, age, race, disease status such as stage/grade/serum markers, urban/rural zip code) and system, or facility level variables (procedure volume, registry hospital, provider type). 5) Participate in the analysis of aggregate data and follow common protocols of the collaborative PoC group.	{ "pile_set_name": "NIH ExPorter" }
Appropriate breathing requires 1) feedback concerning the level of CO2 from central chemoreceptors, and 2) a tonic 'drive', partly from CO2, and partly from other sources including the rostral ventrolateral medulla (RVLM). Recent work established that 1) central chemoreception is present at many brainstem locations, and 2) the retrotrapezoid nucleus (RTN) is a key RVLM site that provides both chemoreception and a tonic drive to breathe. We ask: Why are there so many central[unreadable] chemoreceptor sites? How do they work? What is the physiological role of the[unreadable] RTN in the control of breathing? We will evaluate chemoreceptor and RTN function during sleep and wakefulness in a chronic unanesthetized rat model using a microdialysis probe to deliver substances to the RTN (or other site). The probe tip is 1 mm in length and 240 mum in diameter, a volume of 45 nl. It allows repeated application of neuroactive substances at the same site in the same animal with continuous measurement of ventilation and oxygen consumption (whole body plethysmograph), arousal state (EEG, nuchal EMG), body temperature and blood pressure (telemetry), and, in some cases, blood gases and pH. Approximately 2/3 of the[unreadable] experiments use this model; 1/3 an anesthetized, ventilated rat with phrenic activity as the measure of respiratory output. For studies of chemoreception physiology, we produce focal tissue acidosis by CO2 microdialysis in both models. For studies of mechanism, we alter neural function by injection/dialysis within the focal region of acidosis in the anesthetized rat. For studies of the RTN, we inhibit neurons reversibly by dialysis with muscimol, a GABA-A receptor agonist, in the chronic model. Central chemoreceptor physiology is significant; CO2 is a key component of the respiratory control system and CO2 retention in disease causes morbidity. Chemoreception and RTN function vary with arousal state, and thus are likely to be important in sleep disordered breathing, and the RTN is hypothesized to be an animal homologue for the arcuate nucleus, described as abnormal in SIDS victims.[unreadable]	{ "pile_set_name": "NIH ExPorter" }
Project Summary/Abstract High blood pressure is often associated with excessive salt and fluid retention from the kidney. The peptide hormone angiotensin II (Ang II) is one of the most important factors in maintaining body salt and fluid and blood pressure homeostasis by regulating salt and fluid reabsorption from proximal tubules of the kidney. Thus increased production and actions of Ang II in proximal tubules can cause salt and fluid retention and consequently increase blood pressure. Ang II exerts powerful effects on proximal tubular sodium and fluid transport by activating cell surface receptors on apical and basolateral membranes of proximal tubule cells. However, we have evidence that a) circulating and paracrine Ang II is taken up by proximal tubule cells in culture or by the kidney via an AT1 (AT1a) receptor-mediated mechanism;b) microinjection of Ang II directly into the cells can induce intracellular calcium responses;and c) intracellular Ang II can induce mRNA expression of the major sodium and hydrogen antiporter, NHE-3, in isolated rat renal cortical nuclei. In this project, we hypothesize that circulating and paracrine Ang II is taken up by proximal tubule cells through AT1a receptor-mediated endocytosis, and that after endocytosis, Ang II acts as an intracellular hormone to increase the expression and activity of NHE-3, increase salt and fluid reabsorption by proximal tubules, and thereby induce hypertension. This hypothesis will be tested in four specific aims. Is Specific Aim I, we will test the hypothesis that in polarized proximal tubule cells, apical membrane AT1a receptors play a dominant role in mediating intracellular uptake of luminal Ang II, whereas basolateral AT1a receptors play a relatively minor role in mediating intracellular uptake of interstitial Ang II in vitro. In the absence of apical AT1a receptors, AT1b receptors or the endocytic receptor megalin are unable to assume the role of AT1a receptors. In Specific Aim II, we will test the hypothesis that in polarized proximal tubule cells, apical membrane AT1a receptor-mediated intracellular uptake of Ang II is primarily regulated by the non-canonical cytoskeleton microtubule (microtubule-associated proteins, MAPs)- and caveolin-1-dependent mechanisms, rather than by the canonical clathrin-coated pits-dependent pathway. In Specific Aim III, we will test the hypothesis that in polarized proximal tubule cells, expression of an intracellular Ang II fusion protein induces the expression and increases the activities of NHE-3 in AP membranes via the activation of PLC/Ca2+/PKC[unreadable]/[unreadable]2 and calcineurin/NF-[unreadable]B signaling pathways. Finally, Specific Aim IV will test the hypothesis that intrarenal adenoviral gene transfer of an intracellular Ang II fusion protein selectively in proximal tubules increases salt and fluid reabsorption, promotes salt and fluid retention, and thereby increases blood pressure by increasing the expression and activity of apical NHE-3 via activation of AT1 (AT1a) receptors. These studies will provide new insights into important roles of intracellular or intracrine Ang II in the regulation of salt and fluid transport in proximal tubules of the kidney and in the pathogenesis of hypertension.	{ "pile_set_name": "NIH ExPorter" }
The functional organization of the primate retina is investigated using techniques which identify single cells or single populations of neurons. With Golgi impregnation, electron microscopy (EM), Golgi-EM, formaldehyde-induced fluorescence (FIF) for catecholamines, and immunohistochemistry, the component elements of the neuronal circuitry are identified. By these studies of the structure, connections, synapses, and neurotransmitters of Macaca retina, the functional role of neurons in the processing of the visual image in the retina can be defined. Dopamine-containing amacrine (DA) cells were localized in whole, flat-mounts of the rhesus monkey retina using FIF. Their distribution parallels that of the rod photoreceptors in a topographic pattern across the retina with peak densities of both DA cells and rods occurring at the same location. This evidence suggests a functional role for dopamine in the rod neuronal circuitry of primates. Bipolar cells, the interneurons which connect photorceptors in the outer plexiform layer (OPL) to amacrine cells and ganglion cells in the inner plexiform layer (IPL) have only been found to contact one to seven cones in the OPL. Using Golgi impregnation, a new bipolar cell type which connects to as many as three times the number of cones as the other bipolars has been identified. These giant bistratified bipolar cells also have an axon that arborizes in two separate strata of the IPL. Aside from the bistratification of this arborization, which has never been described for any bipolar cell type in primates and is rarely reported in mammalian retinas, the highly asymmetric arrangement of this axon terminal suggests it may be involved in a function other than the formation of the concentric receptive field center responses of ganglion cells.	{ "pile_set_name": "NIH ExPorter" }
Nephrolithiasis (NL) affects up to 10% of the population producing significant pain and suffering, as well as great economic costs (up to $5.3 billion per year in the United States). Treatment strategies are imperfect and have not improved substantially over the last 30 years, in part because the key pathogenic steps remain poorly defined. Therefore, we have assembled a multidisciplinary team to define genetic risk factors for NL. A key and unique resource of our application is the Rochester, MN cohort of the Genetic Epidemiology Network of Arteriopathy (GENOA), which has conducted genome-wide linkage and association studies to identify genes influencing blood pressure and end-organ complications of hypertension (HTN). Members of the well-characterized GENOA cohort will be phenotyped for kidney stone risk via 24-hour urine measurements of lithogenic factors including calcium, oxalate, citrate, and uric acid excretion, as well as overall crystallization inhibition (upper limit of metastability, ULM). Extensive pre-existing genotyping data of the GENOA cohort will be analyzed via genome-wide linkage, together with selected NL candidate gene associations (vitamin D receptor, soluble adenylate cyclase, intracellular protease with no lysine WNK4, chloride channel CLCN5, calcium sensing receptor, urinary prothrombin fragment 1, urate anion transporter 1, oxalate-formate exchanger Slc26a6, Tamm-Horsfall Protein, osteopontin, and bikunin). These studies will determine if specific loci or candidate genes associate with corresponding urinary lithogenic factors (e.g., 24-hr excretion of calcium, oxalate, citrate, or uric acid;ULM). Genetic linkage and association analyses will control for diet and other environmental factors, and assess potential gene-environment and gene-gene interactions. These will be the first studies to assess genetic determinants of urinary lithogenic factors other than calcium in a population-based cohort, taking into account the important covariate of diet. Specific Aims are: Aim #1: Use variance component methods for univariate and multivariate quantitative trait linkage analyses to determine if urinary lithogenic measures (24-hr excretion of calcium, oxalate, citrate, and uric acid;crystallization inhibition) map to specific genomic regions in the GENOA cohort, controlling for dietary factors;Aim #2: Use family-based association methods to determine if urinary lithogenic measures (24-hr excretion of calcium, oxalate, citrate, and uric acid;crystallization inhibition) associate with polymorphisms in selected candidate genes in the GENOA cohort, adjusting for dietary factors, and determine if gene-environment and gene-gene interactions are present. Our goal is to establish genetic associations with urinary NL risk factors in a community- based cohort. The vast experience and infrastructure of GENOA investigators and use of pre-existing genotyping data will allow completion of these studies at a fraction of the cost otherwise required. Results should provide new insight into the pathogenesis of NL, and may lead to identification of new potential treatment targets.Kidney stones are common, and it is known that specific changes in the urinary composition are important risk factors, for example higher than normal calcium excretion. However, the genetic factors that cause these urinary changes have not been well defined. Therefore, in this grant we determine if specific genetic loci or candidate genes associate with kidney stone risk factors in a well-studied population for whom vast amounts of genetic data are already available, the Olmsted County GENOA cohort.	{ "pile_set_name": "NIH ExPorter" }
This project would continue the research funded by grant MH41704 on human concepts, their representation in the mind, and their relation to word meaning. The work investigates two main issues. The first issue, category learning, involves the coordination of empirical experience with category members as well as prior expectations and beliefs about what kinds of categories are sensible and likely. Although much research has focused on one or the other of these issues, the present research addresses their interaction. In particular, the research follows up the unexpected finding of the lab's previous work that when knowledge relates some aspects of a concept, those aspects are preferentially learned, but other aspects are not learned less well (than a control group). A series of experiments on concept acquisition explores the possible interactions of empirical and knowledge-based learning to explain this effect. A second series of experiments investigates how prior knowledge influences the construction of properties included in a concept representation. Although we tend to think of an object's properties as given to us by perception, in fact, people notice and encode different aspects of an object depending on their prior categorization history. The proposed experiments manipulate causal knowledge of an entity in order to discover whether such knowledge can influence the unitization of experience, thereby altering the perception of a part. The second main issue is the relation between word meaning and concepts, as revealed through the phenomenon of polysemy -- the fact that many content words have numerous related meanings or senses. A series of studies is proposed that examines how people can understand a word when it is used in a novel sense, which occurs frequently in normal language use. Unlike previous work on this topic, the studies will use a realistic linguistic setting and will obtain online measures of comprehension to test its hypotheses. This research investigates some of our most basic thought processes -- how we perceive, identify, think about, and talk about the objects and events in our world. As these processes are part of the basic building blocks of intelligent behavior, they are critical to understanding both normal and abnormal behavior. A complete understanding of concepts is necessary to understanding the breakdown of thought in brain damage and mental illness.	{ "pile_set_name": "NIH ExPorter" }
The population of cancer patients is growing rapidly due to demographic changes, earlier identification, and increased survival. Cancer treatment is being dehospitalized due to changing patient preferences, high inpatient costs, and the pressure of prospective hospital reimbursement to limit expenditures on high-cost, long-stay patients. Rising prevalence and changes occurring within the health care reimbursement system necessitate that policy makers learn more about the major cancers. The proposed study will build directly upon the Brown University Cancer and Aging Study. Through this study, the health care utilization patterns of a population based cohort of lung, breast, and colorectal cancer patients (N = 1572), identified in Rhode Island, have been documented. Each patient will have been followed for the 2 year period following diagnosis. The proposed study will continue to follow this cohort of patients for 3 additional years yielding a patient database containing health care utilization and cost data for a 5 year post-diagnostic period. Health care utilization data will continue to be collected at 9 of the state's hospitals, all of the state's radiation therapy centers, and most of the state's medical oncology practices. Cost data provide by Blue Cross/Blue Shield and HCFA will be merged with the patient utilization data. Patients participating in the interview phase of the Cancer and Aging Study will continue to be interviewed annually. Interviews will document quality of life and functional status. In addition to examining the medical care epidemiology of patients diagnosed with lung, breast, or colorectal cancer, the proposed study will also determine the influence of health status, family and community resources, and demographic factors on the provision, cost, and location of medical treatments patients receive. The relationship between treatment receipt and health state measures of quality adjusted survival, controlling for age and normal disease-stage specific survival rates will also be examined.	{ "pile_set_name": "NIH ExPorter" }
Project Summary Synthetic Biology has the potential to positively impact human health is many ways. However, dependency on using chemical synthesis methods has limited the ability to rapidly and accurately generate gene-length products for on-demand production of whole genomes. This is particularly useful for combatting sequence diversity in rapidly mutating viruses for vaccine development to prevent world-wide epidemics. Currently, diminishing yields in oligonucleotide synthesis using the phosphoramidite chemical method limit DNA synthesis lengths to ~200 bases, and incur a very high error rate. Therefore, products of phosphoramidite synthesis must be filtered for deletions using expensive and time-consuming gel purification methods. In addition, to generate an entire gene of >1000 bases, shorter strands must be assembled with polymerase and ligation-based methods to achieve the desired full-length product, which can take many days to accomplish. Thus, there is an unmet need for methods capable of kilobase synthesis to rapidly generate artificial genes for whole genome production. Harnessing the power of an enzyme to produce the DNA is an appealing solution. The only known enzyme fully dedicated to template-independent ssDNA synthesis is terminal deoxynucleotidyl transferase (TdT). In uninterrupted homo- polymerization, TdT can produce > 8000 bases at a rate of 16 bases / minute. However, because its 500 million- year old biological function in the immune system is to increase antigen receptor diversity, TdT only will randomly add bases to the 3? end of DNA. This presents a major challenge in using TdT in controlled, stepwise synthesis of a desired DNA sequence. Therefore, it is necessary to engineer a TdT variant that is ideal for stepwise synthesis using blocked nucleotides as substrates. To accomplish this, a method developed for massively parallel CRISPR-Cas9 editing platform to generate thousands of TdT variants in yeast will be applied, followed by screening of TdT activity with a novel high-throughput reporter system. The reporter system will leverage scalable Illumina sequencing to readout incorporation of bases by TdT at a DNA double strand break. Select TdT variants will then be studied for increased base incorporation efficiencies by measuring steady-state kinetics on purified proteins. In parallel, a DNA synthesizer designed for enzymatic synthesis will be built for synthesizing gene-length strands > 1000 bases using these high-performance TdT variants. By exploiting the power of TdT, our platform has the potential to improve the length, speed, error rate, and cost by orders of magnitude over the traditional phosphoramidite method.	{ "pile_set_name": "NIH ExPorter" }
The experiments outlined in this proposal are designed to provide a deeper understanding of the mechanisms by which viruses select and package multipartite genomes into a single particle. During the previous funding period we have focused our attention on the bipartite, positive-strand RNA nodavirus Flock House virus (FHV), which represents a relatively simple system to address this issue. By using a combination of molecular and cell biological approaches we discovered that specific genome packaging relies heavily on defined subcellular location of the viral coat protein and the genomic RNAs. Specifically, our data suggest that the two genomic segments are packaged independently and in different cellular micro-environments. In addition, we have evidence that both ER and mitochondria play a critical role in this process. Our data have been summarized in an updated model for nodaviral RNA packaging and we intend to confirm and further refine this model in the next funding period. We believe that the experiments outlined in this proposal will not only illuminate an important aspect of molecular virology but also reveal new facets of cell biology. In aim 1, we will determine the cellular location of viral RNA2, the message of FHV coat protein, and map its site of translation. In aim 2, we will determine how coat protein trafficks from the ER to mitochondria, the site of viral RNA synthesis, and whether cellular factors are involved in this process. In aim 3, we will identify determinants of specific FHV genome packaging, specifically whether genome segments are packaged co-translationally or co- transcriptionally and how these mechanisms determine formation of reassortants during mixed infections.	{ "pile_set_name": "NIH ExPorter" }
CD8 T cells are essential for clearance of intracellular pathogens. A current goal of vaccinology is to develop methods for inducing protective T cell mediated responses against some of the world's most intractable infectious diseases, including malaria and the human immunodeficiency virus. Effective vaccination requires the generation of long-lived memory cells with protective capabilities. Induction of a productive CD8 T cell response to robust infections represents a best case scenario but such models have yielded valuable insight into the regulation of memory CD8 T cell development. Thus far, vaccines geared toward promoting CD8 T cell immunity have generally not met with success. Therefore, identifying the checkpoints in memory CD8 T cell development along with the elements controlling those checkpoints continues to represent an important research goal. Our studies have focused on the detailed analysis of the factors that control the generation of central and effector memory CD8 T cells in response to bacterial and viral infections. Our results along with those from a number of other groups, have shown that activation of na?ve CD8 T cells in response to infection results in the generation of a heterogeneous population of effector cells with distinct phenotypic and functional properties. Our preliminary and published data supported by this long-standing grant, indicate that a population of early effector cells (EEC) are the sourc of the two major effector subsets: memory-precursor effector cells (MPEC), which generate memory cells, and short-lived effector cells (SLEC) which, at least in a primary response, are a terminal lineage. This proposal is aimed at exploring the central hypothesis that commitment to the memory lineage is designated at the EEC stage and further that EEC are themselves heterogeneous with respect to their developmental potential. This hypothesis will be examined in three specific aims: Aim 1. To determine the developmental potential of EEC in response to the external milieu. Aim 2. To identify the metabolic pathways in early effector cells leading to memory development. Aim 3. To identify the molecular pathways that specify effector subset lineage development.	{ "pile_set_name": "NIH ExPorter" }
This protocol is designed to determine whether there are differences in tolerance and side effects associated with two dosing schedules of AZT when used to treat HIV-infected children.	{ "pile_set_name": "NIH ExPorter" }
We are requesting funds for a research career development program in Pediatric and Medical Oncology at the Fred Hutchinson Cancer Research Center and the University of Washington. The Career Development Program will interface with our Pediatric and Medical Oncology Research Training Programs at the Fred Hutchinson Cancer Research Center (FHCRC) and the Departments of Pediatrics and Medicine at the University of Washington (UW) with the goal of developing clinical investigators. It will support physicians who have completed three years of fellowship training in the pediatric or medical oncology training programs. Prior to entry into the program, fellows will have acquired clinical skills and basic laboratory tools of modern biology. Those most qualified and motivated to enter into investigative endeavors in clinical research will be selected. During their two to three years supported by the career development program, trainees will gain the tools and experience necessary to conduct clinical research, translate their findings to patients, and generate sufficient data to help establish independent research careers. The aim of their projects will be to contribute new and useful information relevant to clinical oncology and to develop the clinical research capabilities of the trainees. Clinical research projects in the areas of bone marrow transplantation, immunology and immunotherapy, transplantation biology, normal and malignant hematopoiesis, human immunogenetics, neuro-oncology, molecular biology, and gene therapy, as well as other programs, will be used for training. This program involves the laboratory and clinical research facilities of the Fred Hutchinson Cancer Research Center and the University of Washington, and the clinical facilities of the Seattle Cancer Care Alliance and the Division of Hematology-Oncology at the Children's Hospital and Regional Medical Center, the primary clinical pediatric service of the University of Washington.	{ "pile_set_name": "NIH ExPorter" }
The specific goal of this application is to identify and characterize the molecular events leading to elastic fiber alterations and calcification in pseudoxanthoma elasticum (PXE) as a pertinent disease model to better understand the dynamics governing the super-assembly and homeostasis of elastic fibers. These are large, complex molecules that provide mechanical properties to elastic tissues and regulate cell fate in many developing tissues. These fibers are essential for the development of life but how they are assembled and maintained is not fully understood. Pseudoxanthoma elasticum (PXE) is a heritable disease characterized by elastic fiber fragmentation and calcification. PXE was long thought to be a prototypic connective tissue disease but the PXE phenotype was unexpectedly linked to a gene (ABCC6) apparently unrelated to elastic fibers. We have obtained preliminary results showing that circulating factors from PXE patient sera altered elastic fibers formation in vitro. This data suggested that PXE is a metabolic disorder with secondary extracellular matrix remodeling and that circulating factors might either participate in or exacerbate the development of the disease phenotype. Hence, we hypothesize that abnormal circulating factor(s) arise in the blood as a secondary consequence of ABCC6 failure to export its substrate(s);these PXE factor(s) then percolate from the circulation into the connective tissue and either preclude the initial deposition of elastic fibers or promote structural alterations that ultimately result in their calcification. To test this hypothesis, we propose to undertake two specific aims corresponding to (i) the molecular characterization of elastic fiber defects induced by the presence of serum from PXE patients in culture of dermal fibroblasts (ii) the identification of the serum factor(s) responsible for the these defects. We expect that our proposed experiments will lead to the identification of factor(s) interfering with elastic fiber and the possible development of therapeutic measures alleviating the symptoms of PXE. We also anticipate from our studies a new level of understanding of elastic fiber formation and aging in vascular tissues. PUBLIC HEALTH RELEVANCE: Elastic fibers are large, complex molecules that are important for life by providing specific mechanical properties to elastic tissues. To understand how elastic fibers are maintained over time, we are exploring the pathologic mechanism underlying pseudoxanthoma elasticum, a heritable disease, characterized by progressive elastic fiber fragmentation and calcification.	{ "pile_set_name": "NIH ExPorter" }
The identification of separate disease 'subtypes' or 'intermediate phenotypes' within the syndrome of schizophrenia would facilitate future research on etiology, identification of salient genes and biological markers, and enable more effective prevention and intervention. One promising schizophrenia 'subtype', the 'deficit syndrome' (DS), is characterized by persistent and primary negative symptoms. The deficit syndrome differs from general negative symptoms in its emphasis on primary negative symptoms, predictive power for outcomes, and specific risk factors. While a substantial literature supports the differentiation of 'deficit' from 'non-deficit' syndrome schizophrenia, there is an important gap in the evidence, which this proposal seeks to address. Methodological concerns and geographic limitations of studies have not allowed researchers to fully answer three important questions concerning this subtype: 1) effects of acute psychosis; 2) potential effects of medication treatment; 3) cross-cultural generalizability. In particular, no studies to date have effectively ruled out both the effects of acute psychosis and prolonged medication treatment in the assessment of the deficit syndrome. This proposal seeks to address this gap via a secondary data analysis of the sole existing representative, non-acute, population-based, sample of 'untreated or minimally-treated' schizophrenia patients obtained from a landmark psychiatric epidemiology study in a non-Western context (China). Utilizing this population-based sample of chronic, untreated psychotic illness--who have had untreated illness for an average of 10.5 years and are thus likely to have distinct clinical features such as greater symptomatology--offers an extraordinary test for construct validity of the deficit syndrome. We propose to utilize a representative sample of 389 patients diagnosed with psychotic disorders obtained from a stratified random sample of 4 provinces in China (a sampling frame of 113 million adults). This sample offers unique advantages over prior studies in its: 1) large size (n=389); 2) representative, population-based sampling with data obtained via a clinician-administered interview; and 3) large numbers of 'untreated/minimally-treated' patients (n= 208), thus affording a unique opportunity to comprehensively test the validity of the 'deficit syndrome' subtype within a non-acute, representatively-sampled 'untreated and minimally-treated group' in China. We first seek to confirm the deficit syndrome construct in a group of psychotic patients 'substantially exposed to medication treatment' in this sample (i.e., havinge1 psychiatric hospitalizations), then to assess the construct validity of deficit syndrome within a unique 'untreated or minimally treated' group of psychotic patients. Our specific aims are: 1) To identify cases of deficit syndrome and assess construct validity of deficit syndrome among 'treated' patients with psychotic disorders in China to determine whether this subtype demonstrates the same pattern of correlations to key demographic and clinical variables already established in Western samples. We control for developmental effects of illness in all analyses. Confirming the construct validity of deficit syndrome among the treated group in China will establish a baseline condition to test Aim #2. 2) To identify cases of DS and assess construct validity of deficit syndrome among the 'untreated or minimally treated' psychotic patient group to determine whether this subtype shows the same pattern of correlations to key demographic and clinical variables as the 'treated' group in China. 3) To explore among the full sample (n= 389) whether the relationship between the two treatment groups and key construct validation variables differ in magnitude by group. To model statistical interaction of 'deficit syndrome categorization' X 'treatment group status', we will examine whether the distribution of our variables used to demonstrate construct validity between DS and non-DS varies by treatment groups This will be the first study to utilize an untreated, non-acute, population-based sample of schizophrenia patients to establish construct validity for deficit syndrome, thereby controlling for acute psychosis, exposure to medication use, and validating deficit syndrome in a representative, non-Western based sample. This study has promise to provide evidence for deficit syndrome as a distinct disease 'subtype' within schizophrenia, thus contributing to future etiological, genetic linkage, and intervention studies in schizophrenia. This R03 (small grant) will be the first of several proposals from the Global Mental Health Program at Columbia that will initiate a novel and productive program of research examining subtypes and course determinants of untreated schizophrenia in non-Western societies to examine schizophrenia's cross-cultural aspects.	{ "pile_set_name": "NIH ExPorter" }
A method was developed to determine rates of incorporation of palmitic acid into different brain regions in the awake rat. The regional cerebral metabolic rate for palmitate, rCMRpalm, was determined. rCMRpalm ranged from 2 x 10-5 mol/g-sec at the internal capsule to 9.3 x 10-5 mol/g-sec at the median eminence, and was proportional to the regional cerebral metabolic rate for glucose. rCMRpalm is a measure of turnover of structural brain lipids in vivo. In Fischer-344 rats at different ages, rCMRpalm declines between 1 and 3 months, when myelination also declines, but is age invariant between 3 and 34 months. The latter findings indicates maintenance of cerebral integrity in the absence of disease. rCMRpalm is independent of cerebral blood flow. It is unaffected by acute visual or auditory deprivation in awake rats, but is reduced by anesthesia induced by pentobarbital.	{ "pile_set_name": "NIH ExPorter" }
The central goal of this proposal is to define how syndecan interactions modulate lung injury responses in systemic inflammatory diseases. Dysregulated lung injury responses to systemic inflammatory diseases, such as sepsis, can lead to acute lung injury (ALI) and are a major cause of morbidity and mortality. However, many patients survive the initial phase of lung injury and do not progress to ALI or to the more severe form of ALI, acute respiratory distress syndrome (ARDS). These data suggest the importance of endogenous protective mechanisms that attenuate or reverse disease progression, but the underlying biology remains to be elucidated. Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans function as coreceptors on the cell surface and also as soluble HSPGs in the extracelular environment because its ectodomain can be shed under inflammatory conditions. Syndecans bind to ligands through its heparan sulfate (HS) chains, a glycosaminoglycan that binds to and regulates several inflammatory mediators implicated in lung injury. However, the precise role of syndecan interactions in inflammatory lung injury has yet to be determined. Syndecan-1 null mice show increased lung injury and mortality when subjected to animal models of systemic inflammatory diseases, such as endotoxic shock, Gram-positive toxic shock, or sepsis. Syndecan-1 shedding is induced in the lungs of wild type mice by the systemic inflammatory challenge, and inhibition of shedding exacerbates lung injury, whereas administration of purified syndecan-1 ectodomain or HS improves disease parameters. Based on these data, this proposal will examine the overall hypothesis that syndecan-1 modulates, in part, the highly complex mechanisms of lung injury and repair in systemic inflammatory diseases in 3 Specific Aims. Aim 1 wil define how syndecan-1 facilitates the resolution of lung inflammation. Aim 2 will determine how syndecan-1 attenuates lung injury and inflammation in sepsis. Aim 3 will establish that temporal syndecan-1 interactions regulate its shedding at the cell surface. These studies should define the key functions of syndecans in lung injury and repair, and provide a mechanistic foundation for the design and development of new therapeutic approaches against inflammatory lung diseases. PUBLIC HEALTH RELEVANCE: Correctly coordinated inflammation protects from infection and helps heal tissues. However, excessive and inappropriate inflammation can damage tissues and lead to serious complications associated with high morbidity and mortality, such as lung injury, dysfunction, and failure. This grant application will investigate how one of our own molecules called syndecan corrects the dysregulated inflammatory response in the lung, with the goal of identifying new molecular targets for the effective therapeutic control of inflammatory lung diseases.	{ "pile_set_name": "NIH ExPorter" }
Parathyroid hormone-related protein (PTHrP) is a newly identified tumor peptide which causes hypercalcemia in patients with the syndrome of humoral hypercalcemia of malignancy. Unlike its relative PTH, PTHrP is produced in many normal tissues and emerging evidence suggests that smooth muscle is a main site of production and activity of PTHrP. Our recent studies show that PTHrP is abundantly expressed in vascular smooth muscle cells and subject to regulation by Ang II and other vasoconstrictor peptides. Since both PTH and PTHrP exhibit vasorelaxant activity, we hypothesize that this protein is produced to counter balance the effects of either humoral or mechanical vasoconstrictor signals. The overall goals of this project, are to: 1) determine the mechanisms which control PTHrP gene expression in vascular smooth muscle; 2) characterize the secreted form(s) of the protein and; 3) study its activity in vascular smooth muscle cells and in intact aortic strips. Using a well characterized rat aortic smooth muscle cell culture model we will study the mechanisms by which Ang II regulates PTHrP gene expression in smooth muscle cells (SMC) and establish the relative importance of transcriptional and post-transcriptional control. Separate studies will apply gene transfer and molecular biological techniques to identify cis-acting elements in the PTHrP promoter and 3' UTR which account for the induction of gene expression. We will also examine regulation of PTHrP expression in rat aortic strips in vitro in response to vasoactive agents. We plan to characterize the secreted forms of PTHrP using region-specific RIAs and determine its glycosylation status. In parallel studies, PTHrP and putative N-and C-terminal fragments will be evaluated for their ability to bind to and activate established intracellular signaling pathways in SMC and to affect selected markers for the differentiated SMC phenotype. We will use specific PTHrP antagonists and the aortic strip technique to directly test the hypothesis of local production of PTHrP. In summary, we believe that our findings will implicate PTHrP as an autocrine regulator of vascular smooth muscle tone and finally explain the long-recognized but under-appreciated effects of PTH-related peptides in this tissue.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: Dr. Monthaporn S Bryant is a physical therapist and an assistant professor in the University of Texas Medical Branch, Division of Rehabilitation Sciences, Galveston and an adjunct assistant professor in the Baylor College of Medicine, Department of Physical Medicine and Rehabilitation in Houston. Her research work has focused on the rehabilitation of persons with Parkinson's disease (PD) at the Michael E. DeBakey VA Medical Center (MEDVAMC) in Houston. Dr. Bryant has experience in rehabilitation intervention for gait, gait analysis and exercise in persons with PD, as well as experience in conducting clinical studies within the VA system. The aim of the proposed research project is to study the effectiveness of multidirectional treadmill training (MDTT) in improving gait and balance in veterans with PD. Despite advances in both medical and surgical treatments, postural and gait instability are still significant problems, and necessitate rehabilitative interventions. The project will target veterans with PD who experience gait difficulty, impaired balance, freezing and/or fall. The first phase of the study will develop dose-response profile of MDTT to be used in the second phase. The second phase will study the effectiveness of MDTT in both clinical and biomechanical quantitative outcomes. Clinical outcomes will include PD symptoms, clinical gait and balance evaluations. Quantitative evaluations will include gait kinematics and computerized posturography. The design will be a randomized, wait-list controlled trial to evaluate the efficacy of MDTT to improve gait and balance. The intervention will consist of walking on a treadmill at a fastest tolerated speed in 4 directions (forward, backward, sideways) while supported in a harness for safety. The training will occur 3 times per week for 8 weeks. Follow-up assessments will be obtained at 4 months after the end of MDTT. Veterans with PD will be recruited from MEDVAMC in Houston. Dr. Eugene C Lai (Neurologists), Dr. William Paloski (Biomechanics), Dr. Kenneth Ottenbacher (Rehabilitation), and Dr. Michele York (Neuropsychologist) are members of a mentoring team for supporting Dr. Bryant's interests and training goals. The specific purposes of this career development proposal are to: 1) understand overall management for PD, and correct use of standardized PD clinical scales; 2) develop a dose-response profile of MDTT to gait and balance; 3) study the changes from MDTT in both clinical and quantitative outcomes; and 4) study the influence of MDTT on cognitive function and other non-motor symptoms of PD. Dr. Bryant will be trained in overall management and assessment of PD, biomechanical measurement (motion analysis), data acquisition and interpretation, non-motor symptom and cognitive function assessment. These tools will be used for the assessment of changes from MDTT. PUBLIC HEALTH RELEVANCE: Relevance to Veterans Health: An estimated 40,000 Veterans with Parkinson's disease (PD) receive care from VA facilities annually; many have difficulties with gait and balance leading to falls and subsequence disability. The VA is committed to improving care and finding a cure for PD as evidenced by creation six Parkinson's Disease Research, Education, and Clinical Centers in the country. The proposal is highly relevant to the VA patient care mission. The proposed research seeks to demonstrate the efficacy of a new rehabilitative intervention to address gait and balance deficits and to better understand the possible mechanism underlying the intervention. Veterans with PD will be recruited to participate in the proposed study and the results will be generalized to other Veterans with PD who receive care from VA facilities. In summary, care and quality of life in Veterans living with PD will be improved.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION (Author's abstract): The goal of the research proposed in this application is to gain an objective clinico-pathological understanding of behavioral disorders seen in dementia of the Alzheimer type, in order to provide a framework for the assessment and treatment of these disturbances. Behavioral disorders are seen in approximately 60 percent of patients with a clinical diagnosis of probable Alzheimer's disease (AD), and are a major cause of disability and institutionalization. These disturbances lack objective clinical criteria, their neuropathological basis is unknown, and effective treatments for them are largely unavailable. Preliminary studies suggest that some behavioral disturbances commonly seen in dementia may be associated with abnormalities of circadian rhythms, and that degenerative changes in the neurological structures thought to be necessary for circadian rhythmicity may occur in AD. These findings may have important implications for diagnosis and treatment. The specific aims of this research are to test whether abnormalities of specific circadian parameters will define specific subtypes of behavioral disorders in AD (pacing and sundowning), and to determine the pathologic alterations responsible for these syndromes. This will be accomplished with a detailed longitudinal clinical evaluation of AD patients being cared for in the same hospital environment, followed by post-mortem brain analysis. The clinical evaluations (75 patients studied once every 6 months for 3 years) will include objective measurement of the circadian cycles of rest-activity and core body temperature. The neuropathological examinations (approximately 15/yr) will focus on the suprachiasmatic nuclei (SCN) and the optic nerve, structures found to be altered in AD patients. Findings in the SCN and optic nerve will be correlated with clinical findings and also with pathologic changes elsewhere in the brain.	{ "pile_set_name": "NIH ExPorter" }
Gram-negative gliding bacteria associated with periodontal diseases will be fractionated and their exopolysaccharide, lipopolysaccharide, and peptidoglycan components purified, chemically characterized, and assessed for endotoxic activity. A separate group of seemingly novel gram-negative bacteria also associated with periodontal tissues will be characterized in terms of morphological and physiological traits as an aid in the identification of them, and representative members of subgroups fractionated and assayed as above.	{ "pile_set_name": "NIH ExPorter" }
Numerous recent studies link development of type 2 diabetes (T2D) to endoplasmic reticulum (ER) stress, a condition that occurs whenever protein-folding requirements overwhelm protein-folding capacity in the secretory pathway. Notably, there is mounting evidence that ER stress contributes to diminished glucose-responsive insulin secretion in ?-cells, to ?-cell apoptosis, and to general peripheral insulin resistance, all hallmarks of T2D. ER stress triggers the unfolded protein response (UPR) pathway, which slows translation and transcriptionally upregulates genes that enhance ER protein-folding capabilities. If homeostasis is not restored through these outputs, the UPR triggers apoptosis instead. We hypothesize that key component of the UPR, act as a toggling switches between homeostatic and apoptotic outputs, ultimately controlling ?-cell fate. Our project goal is to study these switches at the molecular level using interventional approaches. PUBLIC HEALTH RELEVANCE: Type 2 diabetes mellitus (T2D) affects 18 million Americans, with national healthcare and lost productivity costs exceeding $100 billion per year. T2D begins as a state of compensated insulin resistance;frank disease develops when approximately 50% of insulin-producing pancreatic islet ?-cells of affected individuals undergo cell death. A detailed understanding of how 2-cells die is necessary to rationally mount an assault on T2D. We hypothesize that the stress from having to overwork may be responsible for ?-cell death in T2D. In this proposal we are testing this concept using molecular and cellular approaches.	{ "pile_set_name": "NIH ExPorter" }
Most animals have the ability to selectively attend to some stimuli while others are ignored. Mechanisms that endow organisms with this capability are of much general interest since malfunctions in these processes induce debilitating pathologies manifested in a variety of circumstances, e.g., when cognitive function is disrupted by aging, in attention deficit/hyperactivity disorders (AD/HD), and when hallucinations associated with schizophrenia are observed. My laboratory studies processes that regulate afferent transmission in the context of motor control. An advantage of this approach is that we are able to directly assess functional consequences of alterations in the efficacy of sensori-motor transmission (evoked movements can be monitored). Specifically, we study a situation where afferent activation will induce a motor response if it occurs during one phase of a motor program, but it will not evoke a response if it occurs during the antagonistic phase. The general goal of our research is to characterize cellular/molecular processes responsible for this type of phasic control. Proposed work will focus on a form of synaptic plasticity that has recently been identified in several loci in the brain. When this plasticity is present, synaptic transmission is a graded function of the baseline membrane potential (i.e., the membrane potential prior to and during afferent activation and spike initiation). As neurons are depolarized, the amount of transmitter that is released per action potential is increased. Proposed experiments will characterize mechanisms that mediate this interesting, but relatively poorly understood type of plasticity. Additionally, we will test a hypothesis that postulates that it can play an important role in regulating sensori-motor transmission during a motor program. Thus during motor programs sensory neurons in our system are rhythmically depolarized via input from central pattern interneurons. We suggest that under normal conditions this input is needed to up-regulate synaptic transmission to make it functional. Consequently, ongoing activity is not easily disrupted. Under more extreme conditions, very strong peripheral activation may induce functional sensori-motor transmission, but a disruption of ongoing activity is more likely to be beneficial under these circumstances.	{ "pile_set_name": "NIH ExPorter" }
DESCRIPTION: This application was received in response to RFA AI 93-11 entitled "Causes and Consequences of Thymus Involution". The goal of this project is to determine the consequences of thymic involution on T cell development and to investigate procedures to rejuvenate the involuted thymus. Specific Aim 1 will investigate the effects on thymic involution of T cell subpopulations by quantifying the frequency and absolute number of CD4-CD8- double negative and CD4 and CD8 single positive thymocytes in the involuted thymus. In order to determine how the proliferative and differentiative potential of thymocytes from the involuted thymus compares to cells from the neonate, thymocytes will be analyzed in a novel long-term thymocyte culture system developed in this applicant's laboratory. Specific Aim 2 will investigate whether decreased cell numbers in the involuted thymus are due to failure of normal cytokine production by thymic stromal cells or inability of the thymocytes to respond. Particular focus will be placed on evaluating the role of various endocrine hormones on Dymic function in mice of various ages. Finally, Aim 3 will build upon preliminary data indicating that administration of insulin-like growth factor (IGF-1) to mice results in a reversal of thymic involution. Experiments will be performed to investigate if the increase in thymocyte numbers following IGF-1 treatment is selective for one thymus population or affects all subsets comparably. Numbers and functional activity of T cells that appear in secondary lymphoid organs also will be examined. It is hoped that the results obtained will further elucidate a basic biological process and will provide insights into preventing declines in thymopoiesis that result from disease or therapeutically induced stress.	{ "pile_set_name": "NIH ExPorter" }
Our diverse mechanosensory system encompass the salient senses of hearing, balance, touch, and proprioception, as well as less conscious senses like the detection of blood pressure and gut stretch. The mechanosensory cells that mediate these senses are structurally and functionally dissimilar, yet share a central feature. Unlike other sensory signaling modalities, which use second messengers to relay sensory information, mechanosensation occurs by the direct opening of mechanically gated ion channels by applied forces. Despite its importance, we currently do not know the identity of this channel or, with few exceptions, the remainder of the transduction machinery. The long-term goals underlying the proposed research are to define the molecules of the transduction machinery and understand how they work in concert to transduce mechanical stimuli into electrical signals. To understand mechanotransduction, this research takes advantage of the ease and elegance of a genetically tractable model organism, Drosophila. Fruit flies make an ideal organism for research on mechanotransduction for several reasons: renowned molecular-genetic tools, the ability to electrically record from mechanosensory bristles, and surprising similarities between the development and physiology of fly mechanosensory neurons and that of vertebrate hair cells. The scientific approach taken here can be divided into two parts: a molecular-genetic path to identify the genes involved in mechanosensory transduction and an electrophysiological approach to understand mechanosensory responses. Many of the experiments utilize the mechanosensory transduction channel, NompC, as a biochemical and genetic toehold into the transduction machinery. A first step in this proposal is to better define NompC's expression pattern. Antibodies against Nompc and in situ hybridization on mechanosensory organs will determine what cells express NompC and where within those cells it is expressed. NompC does not act alone, the transduction machinery likely encompasses many molecules. To identify molecules that interact with NompC and that are therefore likely comprise the transduction machinery, yeast two-hybrid assays with portions of NompC will be used. A genetic enhancer/supressor screen will be undertaken in a sensitized nompC background to identify new genes that interact with nompC. Other mechanosensory genes will be identified from existing mutants and new temperature-sensitive mutants will be generated. An understanding of the transduction process cannot be complete without accompanying biophysical analyses of the mechanosensory response. Because this requires electrical and mechanical access to the mechanosensory neuron that is not currently available, an isolated mechanosensory neuron preparation will be developed that will allow this access. An in-depth electrophysiological characterization of transduction in these neurons will be undertaken using whole-cell, voltage-clamp recording. Finally to understand the biophysical properties of NompC, such as gating and permeation, electrophysiolgical experiments on heterologous cells expressing the NompC channel will be undertaken. The experiments proposed in this application represent the next step in the assembly of a comprehensive picture of Drosophila mechanotransduction. Because their transduction and developmental pathways are so similar, the information from Drosophila mechanosensory neurons can be used as a paradigm to further understanding of the molecules and transduction in vertebrate hair cells.	{ "pile_set_name": "NIH ExPorter" }
It is the objective of this laboratory to provide plasma lipoproteins and purified apolipoproteins isolated according to carefully standardized procedures for use by the individual projects within the program. This core facility is central to the Program Project and every member utilizes it. This core has three main purposes which are: I. Lipoprotein Production a). Large-scale isolation and purification of lipoproteins and related products from normolipidemic human serum by ultracentrifugation b). Analytical preparation oflipoproteins from experimental samples (including mouse sera, dyslipidemic human sera, and tissue culture media) by FPLC II. Apolipoprotein Production Isolation of human HDL apolipoproteins apo A-I and A-II III. Characterization of Lipoprotein and Apolipoproteins Using the Following Techniques: a). Agarose gel electrophoresis of lipoproteins b). SDS-PAGE ofapolipoproteins c). Immunoblotting of apolipoproteins d). Electron microscopy of native and reconstituted lipid-protein complexes The benefits arising from the centralized service provided by the Core Laboratory include: 1) uniformity of materials and techniques; 2) optimal use of space, supplies and equipment, especially centrifuges and FPLC.	{ "pile_set_name": "NIH ExPorter" }
Alcoholic liver disease is a serious health condition that results in a significant amount of deaths related to liver disease each year in the U.S. A number of pathway alterations are associated with the profound effects of ethanol on the liver including oxidative stress and epigenetic modification of histones; however, the molecular mechanisms linking these processes are unknown. From an initial proteomics-based survey, we have identified a hepatocellular protein target of oxidative modification that was found to be oxidized to a greater extent after acute, high-dose ethanol exposure. Specifically, this protein, phosphohistidine phosphatase 1 (PHPT1), was differentially oxidized via ethanol-induced oxidative stress at an amino acid residue that is important for substrate binding to its active sit. It is hypothesized that this oxidation event will have a significant impact on the phosphohistidine levels of PHPT1 substrates such as ATP-citrate lyase (ACL) which will then influence the level of the ethanol-induced epigenetic modification, histone H3 acetylation, a modification that is potentially mediated through the acetyl-CoA pool generated by ACL. This hypothesis will be tested by 1) characterization of ethanol-induced oxidative modifications in mouse immortalized hepatocytes (AML12 cells) and primary hepatocytes on a global-scale, validation and characterization of PHPT1 oxidative modifications and assessment of hepatocellular proteomic response to alteration in PHPT1 activity and 2) quantification of ethanol-mediated alterations in the phosphohistidine level and cell localization of ACL and correlation of these findings to hepatocellular histone H3 acetylation status as well as other ACL-mediated histone acetylation sites. In Aim 1, mass spectrometry in addition to conventional biochemical tools will be employed to identify and quantify various oxidative modifications of PHPT1 as well as resultant activity changes. The effect of alteration of PHPT1 activity will then be assessed using global-scale protein expression profiling. In Aim 2, novel proteomics and mass spectrometry-based methods will be used to accurately quantify the phosphohistidine level of ACL and other phosphohistidine-containing proteins after ethanol exposure. Localization of ACL and other proteins will be determined through global-scale investigation of nucleocytoplasmic trafficking after ethanol exposure followed by confocal microscopy and biochemical validation. Acetylation of histone H3 that is mediated by ACL-generated acetyl-CoA will be monitored via stable isotope metabolic tracing. The results from this project could reveal a novel link between ethanol-induced oxidative stress, cellular metabolism and subsequent changes in hepatocellular epigenetic modification. Additionally, the results from this study could lead to the identificationof proteins or pathways that can be targeted for epigenetic-based therapeutic intervention in order to treat hepatic pathway alterations that have occurred through excessive alcohol use in humans.	{ "pile_set_name": "NIH ExPorter" }
Principal Investigator/Program Director (Last, first, middle): Miller, Josef, M. RESEARCH &RELATED Other Project Information 1. * Are Human Subjects Involved? l Yes m No 1.a. If YES to Human Subjects Is the IRB review Pending? l Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 00004969 2. * Are Vertebrate Animals Used? m Yes l No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? m Yes m No IACUC Approval Date: Animal Welfare Assurance Number 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or l Yes m No partnership with International Collaborators? 5.b. If yes, identify countries: Spain, Sweden 5.c. Optional Explanation: Please See Other Attachments 6. * Project Summary/Abstract 8691-Abstract.pdf Mime Type: application/pdf 7. * Project Narrative 1485-Narrative.pdf Mime Type: application/pdf 8. Bibliography &References Cited 0013-Bibliography.pdf Mime Type: application/pdf 9. Facilities &Other Resources 9467-FacilitiesResources.pdf Mime Type: application/pdf 10. Equipment 3211-Equipment.pdf Mime Type: application/pdf 11. Other Attachments 9617-Scientific_Environment,_CollaboratMioinmse_PTuyrpeE:dagpep.lpicdaftion/pdf Tracking Number: Other Information Page 6 OMB Number: 4040-0001 Expiration Date: 04/30/2008	{ "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. This career development plan will support the candidate's goals of mastering new imaging methodologies to investigate multiple aspects of a specific brain system in schizophrenia. This systems approach will allow a deeper understanding of the mechanisms associated with declarative memory (DM) impairments that are mediated by the medial temporal lobe (MTL) memory system (hippocampus, parahippocampal region and cerebral cortex). Guidance from accomplished investigators, state-of-the-art imaging facilities, and didactic training provide the infrastructure needed to achieve these goals. The rich institutional environment at UCLA further supports the candidate's long-term plan to develop a productive and independent program of research using imaging methods to better understand the pathophysiological underpinnings of brain disturbances in neuropsychiatric disorders. DM impairments and hippocampal volume reductions are shown particularly vulnerable to disease processes in schizophrenia and to indicate a genetic predisposition towards the illness. However, it is uncertain whether abnormalities of declarative memory (DM) reflect disturbances in regional brain activity and/or in functional connectivity and whether such impairments relate to structural abnormalities within this neural network. To address these questions, the candidate will combine structural, functional and diffusion tensor imaging to map structural and functional abnormalities of the MTL memory system in schizophrenia patients, first-degree relatives of patients and healthy comparison subjects recruited through a larger NIMH study, "Transmission of Vulnerability Factors for Schizophrenia." The research plan includes advanced structural image analysis techniques and functional magnetic resonance imaging (fMRI) and diffusion tensor imaging (DTI) strategies that will be employed to dissociate the structural and functional correlates of MTL disturbances including declarative memory deficits in schizophrenia and to establish the presence of genetic liability effects.	{ "pile_set_name": "NIH ExPorter" }
Described here is a multidisciplinary program which is aimed at elucidating the cellular mechanisms involved in the accumulation and remodeling of muscle connective tissue and the reduced range of movement that occur in aging and/or inactive individuals. The first hypothesis is that the amount and properties of the connective tissue in the muscle belly are influenced by the type of activity that the muscle is subjected to and that stretch is the important factor. The second hypothesis is that connective tissue remodeling is linked with adjustments in sarcomere number so that there is a reduced range of movement as well as an increase in stiffness. The questions asked include: can regular exercise delay the onset of muscle stiffness by altering the distribution and nature of the connective tissue?; also, which type of stretch (active, passive, dynamic) is the main factor in preventing muscle connective tissue accumulation and sarcomere loss? The form and amount of muscle connective tissue will be studied by quantitative transmission, EM, and light microscopic techniques. Changes in endomysial and perimysial connective tissue will be evaluated and the rates of synthesis of collagen and contractile proteins will be measured to shed light on the remodeling process. Changes in the functional length of muscle fibers will be determined by measuring sarcomere number and sarcomere length. The cellular mechanisms involved in the addition and removal of sarcomeres will be studied concurrently with those involved in connective tissue remodeling and these will be related to the physical and physiologic characteristics of the muscles studied. These will include measurements of muscle force, rate of force development, compliance and elasticity. Both the morphologic and physiologic changes will then be related to the functioning (force production, compliance and elasticity) of the aging musculoskeletal system. The data should be of considerable value to those involved in physical therapy, the management of elderly respiratory, orthopedic and neurologic patients as well as elderly patients in general.	{ "pile_set_name": "NIH ExPorter" }
An accelerator-based neutron irradiation facility for neutron capture therapy is proposed which is based on a 2.5 MeV proton beam bombarding a lithium target. The quality of the neutron beam (in terms of neutron energy spectrum and gamma ray contamination) generated by the proposed facility design has been demonstrated previously to be suitable for treating deep-seated tumors. However, to accomplish neutron capture therapy during reasonable treatment periods (approximately 1 hour) requires a beam current of about 30 mA of 2.5 MeV protons. This beam current translates to a heating rate of 75 kW at a heat flux in excess of 1000 watts/cm2. Many of the building blocks for the proposed neutron irradiation facility have already been developed and demonstrated including the radiofrequency quadrapole proton accelerator and the moderator system. A key building block heretofore unavailable is the focus of this proposed Phase I study, viz, the target and associated target cooling system. The scope of the proposed study will focus on the analysis and design leading to the assessment of the technical and economic feasibility of the proposed irradiation facility with particular emphasis on the target and associated cooling system. This program will include preparation of drawings/specifications in preparation for construction of the neutron irradiation facility during Phase II.	{ "pile_set_name": "NIH ExPorter" }
In accordance with our strategic plan and endorsed by our external advisors, the former Mouse Models Core has expanded to offer multiple imaging modalities and thus reorganized into the Small Animal Modeling and Imaging (SAMI) Core to reflect this improved, integrated focus. The primary purpose of the SAMI core is to provide proficient generation and examination of rodent models of cancer for basic and translational research. SAMI continues to provide specialized consultations and services for the derivation, characterization, and preservation of genetically-engineered mice, and a variety of tumor cell lines for xenograft and syngeneic transplant mouse models. Tumor development, progression, metastasis, and response to therapy in transgenic or transplanted mouse models of cancer can be evaluated using modern multimodality imaging including bioluminescent and biofluorescent imaging, ultrasound, magnetic resonance imaging, and beta particle imaging. Within the last funding period, the core has experienced significant growth, including the addition of imaging sen/ices, expert personnel, a new Scientific Director, acquisition of new lab space and capital equipment, and provision of several new services including an MRI, an ultrasound, bioluminescence and biofluorescence, beta-imaging, intravital microscopy, cryopreservation and rederivation, and ES cell culture. The Core requests CCSG Suppori of $17S,286, which is S1% of its operational budget. Over 80% of users were Moffitt members and peer-reviewed. Usage increased in all services. The core derived 66 founders or chimeras for 12 genetically engineered mouse models and cryopreserved or rederived 8 mouse lines. Genomic DNA was prepared from 774 mouse tail biopsies and genotyped via 20 hybridization blots. Twelve luciferase-expressing tumor cell lines were distributed for use in xenograft mouse models. In FY 2010, the Caliper IVIS workstations were utilized for 519 hours by 15 members; 4S4 hours on the IVIS-200 and 85 hours on the IVIS-100. The Visual Sonics Vevo 2100 US system was used for 165 hours by six Pis. The Varian 7T MRI instrument was used for 486 hours by five investigators.	{ "pile_set_name": "NIH ExPorter" }
Novel means first to better characterize and then to treat infection with major respiratory pathogens using existing or newly developed strategies are a primary focus of this important project within the Clinical Research Section. Beginning with the emergence of the pandemic strain of A(H1N1)pdm09 influenza in April 2009, our section undertook clinical research efforts to help better characterize and treat infection with both novel and seasonal subtypes of influenza. An initial treatment trial involving open-label administration of two units of hyperimmune plasma to 98 hospitalized patients with severe influenza suggested that investigational arm participants might have fewer days in the hospital, in the ICU, on mechanical ventilation, and may have improved disposition at Day 7. Based upon these highly suggestive trends, the CRS then launched a follow-up multicenter trial (called IRC005) enrolling patients with severe influenza A infection who were randomized in a double-blind manner to receive either high-titer (hyperimmune) plasma or a low-titer (control) plasma in addition to standard-of-care treatments. 140 subjects out of a desired goal of 150 were randomized in this trial, with the primary endpoint being an assessment of clinical status using an ordinal outcome score as measured at Day 7. The major finding of IRC005 was that the use of high-titer immune plasma did not confer a clinical benefit over non-immune plasma in patients hospitalized with influenza A, and thus this immunotherapy could not be recommended as a useful adjunctive treatment strategy for future patients. As a separate trial launched through the INSIGHT clinical trials network, we also conducted an international multi-center randomized, double-blind, placebo-controlled study of hyperimmune intravenous immunoglobulin (IVIG) versus standard-of-care in hospitalized patients with severe influenza A or B. This clinical outcome trial was preceded by successful completion of a multi-center pilot trial in 31 patients through domestic US sites within the INSIGHT network that showed that administration of IVIG to hospitalized patients or outpatients was safe, significantly boosted HAI titers against the infecting influenza A subtypes, and could be conducted at clinical sites while maintaining the blind. By June, 2018, the trial reached and actually exceeded the accrual goal of the desired number (320) needed to power the endpoint of clinical improvement at Day 7 of illness. Similar to IRC005, however, no treatment benefit was found for IVIG in patients with influenza A. Surprisingly, a significant treatment benefit was found for patients hospitalized with influenza B infection despite the lower antibody titers present in IVIG against influenza B virus. We have also completed two randomized international multicenter trials evaluating 1) the virologic and clinical correlation of triple combination anti-influenza treatment versus monotherapy in at-risk populations (IRC003), and 2) the use of virologic assessments in measuring the effects of oseltamivir versus placebo in mild outpatient disease (IRC004). In IRC003, 80 of 200 (40.0%) participants in the combination arm had detectable viral shedding at Day 3 compared to 97 of 194 (50.0%) (95%C.I. 0.2-19.8%, p=0.046) in the control arm. Despite this effect on viral shedding, however, there was no observed benefit in multiple clinical endpoints. Further investigations are needed to understand the lack of clinical benefit when a difference in virologic outcome is identified. In addition to the ongoing interventional trials mentioned above, we continue to contribute to the management and oversight of two large international observational protocols for outpatients or hospitalized patients with seasonal influenza infection, as well as a third protocol looking at the genomic host response, all administered under the auspices of the INSIGHT network. In the realm of ongoing biodefense-related initiatives, we continue to 1) monitor yearly the clinical and psychological status of a subset of patients previously exposed to anthrax as a result of the October 2001 anthrax attacks, and 2) continue to enroll patients on a protocol designed to permit diagnosis and characterization of patients presenting with unusual, previously undiagnosed infectious or inflammatory conditions. The Special Clinical Studies Unit (SCSU) with the NIH Clinical Center was one of three special high containment patient care units within the US originally called upon to hospitalize and provide care to medically-evacuated HCWs exposed to or infected with Ebola virus in 2014-15. The section continues to provide the medical oversight to the SCSU in the event that patients or staff suffering high-risk exposures to select agents require observation and/or care under conditions of high containment. We have also aligned ourselves closely with the Special Pathogens Research Network of 10 domestic U.S. medical centers capable of caring for Ebola-infected patients in the event of medical evacuations from overseas. The section helped design and orchestrate the only multicenter randomized controlled safety and efficacy study (PREVAIL II) of putative MCMs in the treatment of patients with confirmed Ebola infection during the recent West African crisis. The first investigational countermeasure studied was a triple monoclonal antibody product ZMapp. Although the goal was to enroll a total of 200 patients, the trial was terminated in early January 2016 due to the extinction of new cases of Ebola in the affected countries. By that time a total of 71 evaluable patients had been enrolled at sites in Liberia, Sierra Leone, Guinea, and the United States. While the observed posterior probability that ZMappTM plus oSOC was superior to oSOC alone reached 91.2%, this fell short of the predefined threshold of 97.5% required to establish efficacy. However, these data suggest at least the possibility of a potential beneficial effect of ZMapp had it been possible to complete full accrual. In concert with WRAIR, we successfully completed a phase 1 randomized, double-blind dose-escalating safety and immunogenicity trial of (VSVG-ZEBOV) vaccine in a prime-boost strategy in late 2014 and early 2015. This study established the safety parameters of the experimental vaccine as well as provided comparative immunogenicity data on the three different dosing levels tested. In Fall 2016 NIAID also launched an ongoing pre-exposure vaccination protocol called PREPARE utilizing VSVG-ZEBOV vaccine to immunize HCWs, BSL-4 Laboratory staff, and other at-risk personnel against Ebola virus infection. The protocol features randomization to a homologous booster immunization at month 18 to determine whether the booster further augments antibody levels induced by the primary immunization. Current enrollment as of early August 2019 now exceeds 160 participants. Finally, with the emergence of the 10th outbreak of Ebola virus infection in the Democratic Republic of the Congo (DRC) in August 2018 that has now grown to be the second largest recorded outbreak in history, NIAID collaborated with the Institut National pour la Recherche Biomedicale (INRB), the WHO, and several international partners to design and implement a randomized controlled trial (RCT) of 4 different promising investigational countermeasures against Ebola in that country. As of early August, 2019, the RCT has been open at 4 different Ebola treatment centers and is rapidly approaching its accrual goal of 725 patients. Results of this RCT should help determine which countermeasure(s) provide the best treatment against this highly lethal infection.	{ "pile_set_name": "NIH ExPorter" }
In our experimental conditions, we are[unreadable] determining "colocalization" in the resolution limit of light microscopy. In this PPG, the "colocalization" term[unreadable] will imply that two molecules may share the same microenvironment and/or can be elements of the same set[unreadable] of macromolecular complexes which are resolved by the optical system. Forexample, two proteins forming part[unreadable] of two different molecular complexes may show positive "colocalization" tests due to the lack of the optical resolution[unreadable] to detect each molecular complex. Still, the positive test will indicate that the two proteins are located proximally[unreadable] within the optical resolution. Thus, in "colocalization" determinations it is critical to achieve the maximal x,y,z[unreadable] resolution. In this PPG, we will use developed methodologies in the laboratory to achieve the highest resolution[unreadable] attainable with optical microscopy. We have maximized the optical linearity and the quantity of light recorded by the[unreadable] detectors in conjunction with image restoration analysis to recover the light lost by the optical imperfections of the[unreadable] system. This methodology has allowed us to reach a resolution in the x,y plane of about 100-200 nm (see Figs. 1-3).[unreadable] Definition of "colocalization" and random "colocalization". In a cell labeled with two fluorophores, one red (R)[unreadable] and one green (G), the number of voxels above threshold that are labeled with the red fluorophore is nRand the[unreadable] number labeled with the green fluorophore is nG. The number of colocalized voxels, i.e. those voxels containing[unreadable] intensity signals above threshold from both fluorophores is ncoioc. The percentage of "colocalization" of G with R is[unreadable] given by 100^^- (eq. 1); and the percentage of "colocalization" of R with G is given by 100^^ (eq. 2). In[unreadable] n* nc[unreadable] these measurements we will estimate the probability that the measured "colocalization" could occur by chance by[unreadable] calculating % random "colocalization" from ((area_1x area_2)/total areaA2) x100, where area_1 and area_2 are the[unreadable] areas above threshold (eq. 3).[unreadable] The "colocalization" method is generally based on the user ability to set the intensity threshold, and has the major[unreadable] draw back of the lack of a mathematical model to adjust the threshold of both images that will define the degree of[unreadable] paired pixel overlap. Most importantly the pixel overlap method has the limitation of being a binary test that[unreadable] determines whether the two paired pixels in two images have or not intensities above the intensity threshold and does[unreadable] not consider whether the two stained proteins have a landscape of intensity staining with a high degree of correlation,[unreadable] as it would be expected if they are elements of a common complex. The application of a correlation measurement[unreadable] was recently described by Li et al. (2004)7.[unreadable] In the following sections, I will discuss and apply developed algorithms to quantify the degree of protein-protein[unreadable] association by two methods, the INTENSITY CORRELATION ANALYSIS and the INTENSITY THRESHOLD[unreadable] "COLOCALIZATION"ANALYSIS.[unreadable] Intensity correlation analysis. In the present analysis, we acquire high resolution images to compare the[unreadable] correlation of pixel intensities in equivalent x,y coordinates of paired images from cells double stained for two different[unreadable] proteins. The prediction is that if two proteins are elements of the same macromolecular complex, the intensity[unreadable] staining landscape of the two images should have a x,y pixel to pixel positive correlation. On the contrary, if the two[unreadable] proteins are localized in distinct compartments the result will be a negative correlation. Finally, if the proteins in the[unreadable] two images are labeled in a diffuse non structured pattern (random), the correlation will tend to 0.[unreadable] This method is based on the principle that for any set of values the sum of the differences from the mean equal zero,[unreadable] i.e., ?N(A-a)=0, where a is the mean of the distribution with N values of Al. In the experiment N is the number of[unreadable] pixels, and A is the intensity for each pixel. If we have two set of values in two arrays 1 and 2 with N pixels per array[unreadable] having a random distribution of intensities A-,and 6, for arrays 1 and 2 , the sum of the product of their differences will[unreadable] also tend to zero, thus IN(A-a)(S,-Jb)~0. On the other hand, if the two intensities are positively correlated, the product[unreadable] will tend to be a positive value (^(Ara)(Brb}>G) and if they are negatively correlated the product will tend to a[unreadable] negative value (LN(Ara)(Brb)<0). To perform the analysis, we generated two intensity arrays 1 and 2 of equivalent[unreadable] x,y coordinates of the paired images. The pixel intensity values of the arrays were normalized to their individual[unreadable] maximum and a 3rd array was obtained from (Ara)(Brb). Rows with 0 values with the same x,y coordinates in both[unreadable] PHS 398/2590 (Rev. 05/01) Page 382 Continuation Format Page[unreadable] CONTINUATION PAGE Principal Investigator/Program Director (Last, first, middle): Ping, Peipei (Stefani, Heart Biology Core)[unreadable] images were eliminated. We generated the correlation plot between the first two arrays and between array 3, and[unreadable] arrays 1 and 2. As indicated, positive, negative or 0 values of EN(A-a)(S,-6) would be an indication of positive,[unreadable] negative, and 0 correlation. To evaluate the statistical[unreadable] significance of the data we used the non-parametric[unreadable] Psign test by counting the number of positive N+ and[unreadable] negative N .numbers of the 3rd array (Ara)(Brb). We[unreadable] generated the intensity correlation quotient (ICQ) from[unreadable] N+/(N+-N.)-0.5. Values of ICQ are 0> for positive[unreadable] correlation, <0 for negative correlation and - 0 for[unreadable] random correlation. The correlation coefficient r[unreadable] (COR) between arrays 1 and 2 was calculated from[unreadable] r=2xy/(NSxSy) wher r = correlation coefficient, xy =[unreadable] product of deviation scores, N = sample size, Sx =[unreadable] standard deviation of X (intensities in first image), and[unreadable] Sy = standard deviation of Y (intensities in second[unreadable] image).[unreadable] We initially performed intensity correlation analysis[unreadable] using as an experimental "colocalization" model the[unreadable] same protein tagged by two different antibodies at[unreadable] different sites. Confocal images were at 0.038[unreadable] urn/pixel in the x, y axis, and every 0.1 urn in the z[unreadable] plane. We used as the target protein caveolins (1-3).[unreadable] Caveolin 3 (CAV-3) was immunostained with anti-[unreadable] CAV3 antibody and all the caveolins with a generic[unreadable] anti-caveolin antibody (anti-CAVg). Since in the heart,[unreadable] CAV3 is the most abundant caveolin, anti-CAVg[unreadable] should mainly stain CAV3. As a model for non-[unreadable] "colocalization" (segregated localization), we[unreadable] displaced by 4 pixels the x,y plane of the anti-CAVg[unreadable] labeled image. In Figure 6, panels A,B are single[unreadable] confocal sections of cardiomyocytes immunostained[unreadable] for CAV3 and caveolin generic CAVg, respectively.[unreadable] Panels C and D are the regions marked with squares[unreadable] in (A) and (B) at higher magnification with the[unreadable] corresponding overlay in E. Note the strikingly similar Fig. 6. Intensity correlation analysis using "colocalization" and non[unreadable] pattern of pixel intensity distribution for CAV3 (C) and "colocalization" experimental models. (A,B) Single confocal sections[unreadable] CAVg (D) that generates the yellow signal in E. In of cardiomyocytes immunostained for CAV3 (A) and caveolin generic[unreadable] CAVg (B). (C, D) Regions depicted in (A) and (B) at higher[unreadable] contrast, when the region D was displaced by 4 pixels[unreadable] magnification with their overlay (E). (F) overlay of (C) and (D) but with[unreadable] in the x,y plane, the overlay shows a clear separation[unreadable] the region D displaced 4 pixels in the x,y plane. (E1-E3) Intensity[unreadable] between green and red signals (F). correlation analysis for (C, D). (E1) Correlation plot of CAV-g vs.[unreadable] CAV3. (E2.E3) Plots of CAV-g and CAV3 vs. (A,-a)(Brb). (F1-F3)[unreadable] In the same figure, graphs E1-E3 illustrate the[unreadable] Intensity correlation analysis of F. (D) (CAVg) was displaced by 4[unreadable] intensity correlation analysis for (C) and (D). E1 is the pixels in the x,y plane; the analysis shows a negative correlation. (G1-[unreadable] correlation plot of CAV-g vs. CAV3 pixel intensities. G3) Intensity correlation analysis of two arrays with random numbers.[unreadable] The points are not randomly distributed (compare with[unreadable] G1 for a random plot) with a correlation coefficient of 0.84. E2 and E3 are the plots of CAV-g and CAV3 vs. (Ara)(Br[unreadable] b). As expected for a positive correlation, the majority of the (A,-a)(Brb) have positive values (red dotted line marks[unreadable] the zero value of the (Ara)(Brb) axis). The ICQ was 0.36 with Psign test<0.001. We can conclude from these data[unreadable] analysis that the pixel intensities are highly correlated as expected from the dual labeling of the same protein. Graphs[unreadable] F1-F3 show an equivalent analysis for the pair of images generating the F overlay. As indicated, in this case the[unreadable] region D (CAVg) was displaced by 4 pixels (0.038 um/pixel) in the x,y plane. As result of the displacement, the[unreadable] analysis should give a negative correlation. In fact, the correlation coefficient of the plot in F1 was -0.24 and ICQ -0.15[unreadable] with PSign <0.005. Graphs G1-G3 show the results of a random distribution. In this case, the data arrays were made[unreadable] with random numbers. G1 shows the random distribution of pixel intensity in the correlation and G2, G3 quasi equal[unreadable] number of positive and negative (Ara)(B,-b) values.[unreadable] PHS 398/2590 (Rev. 05/01) Continuation Format Page[unreadable] CONTINUATION PAGE Principal Investigator/Program Director (Last, first, middle): Ping, Peipei (Stefani, Heart Biology Core)[unreadable] Intensity threshold "colocalization". The most difficult task to measure "colocalization" by changing the intensity[unreadable] threshold in both images is to establish criteria of setting the intensity threshold values which define the areas above[unreadable] threshold to be compared. The intensity threshold should eliminate non-specific signals as detector noise, non-[unreadable] specific antibody binding, autofluorescence, and blur[unreadable] from optical imperfections. In "cotocalization" studies.[unreadable] 100 D CAVg OVER CAV3[unreadable] the intensity threshold is typically set in an arbitrary[unreadable] manner bv the user and "colocalization"[unreadable] measurements are questionable. To overcome this[unreadable] caveat, we have developed mathematical criteria to[unreadable] set the threshold levels. Most importantly, in the[unreadable] same set of paired images, curves are constructed to u[unreadable] measure "colocalization" level as function of the 3[unreadable] intensity thresholds selected. We found convenient to[unreadable] plot % "colocalization" level vs. random % 8[unreadable] OCAVgOVERCAV3[unreadable] "colocalization" (calculated from eq. 2 taking into CAV3OVERCAVg[unreadable] account the total area and the two sets of thresholded (4 Pixel shift)[unreadable] areas), using various threshold levels in paired[unreadable] images. In this manner, we can have an estimate of 100.00 10.00 1.00 0.10[unreadable] the level of contamination random "colocalization". in % RANDOM COLOCALIZATION[unreadable] the "colocalization" measurements, (see below). We[unreadable] have calculated intensity threshold values in two Fig. 7. Threshold intensity "colocalization" analysis as function oi[unreadable] different manners: METHOD 1. Varying the intensity threshold intensity. % "colocalization"vs. % random "cotocalization" is[unreadable] threshold as a function of the average intensity. plotted for random "colocalization" (r), CAVg over CAV3 (a),and[unreadable] CAV3 over CAVg (b) (for panels C,D, Fig. 6), and CAVg over CAV3[unreadable] independently in both images, and in a sequential[unreadable] (c), and CAV3 over CAVg (d) (overlapped images, Fig. 6F). The[unreadable] manner, and METHOD 2. Adjusting the threshold[unreadable] CAVg image in panel F was 4 pixel x,y shifted. Note the high CAVg[unreadable] intensities of both images to attain the same area over CAV3 of ca. 85% at 1 % random "colocalization". The 4 units x,y[unreadable] (number of pixels) above threshold in both images. In pixel shift dramatically reduced the "colocalization" level.[unreadable] METHOD 1 the threshold in each image is first Calibration=0.038 urn/pixel.[unreadable] calculated from the average intensity of all the pixels in each image, thereafter the same procedure is applied in the[unreadable] resulting images and so on. In this manner, a set of images are generated where the threshold values are the[unreadable] average intensities of the respective previous images. This method has the advantage that all the pixel intensity[unreadable] values are considered to calculate the threshold values, reducing asymmetries and differences of the total point[unreadable] histogram between both images. In METHOD 2, intensity threshold values are calculated in each image with the[unreadable] constrain that the number of pixels above threshold (thresholded area) is the same in both images and areas in[unreadable] decrementing steps from 100% to ca. 1% of the total area are calculated. In a given area having an identical value[unreadable] for both images, one subset of pixels in the same x, y coordinates will have intensities in both images while a second[unreadable] set of pixels will have intensity values only in one image. The "percent colocalization" is calculated from the ratio of[unreadable] the number of pixels with intensities (>0)in both images with the number of pixels with intensities (>0)in one image.[unreadable] In this algorithm, the colocalization level of Image A over Image and of Image A over A is the same. Minor[unreadable] differences can arise from the computational limitations to define the right threshold to obtain two identical areas in[unreadable] both images. As in METHOD 1,"%colocalization" will be plotted vs."% random colocalization". These two methods[unreadable] are testing pixel overlap in a binary manner at various threshold intensities and thresholded areas. Therefore, this[unreadable] analysis is also taking into consideration the degree of correlation of the landscape of intensity staining in both[unreadable] images.[unreadable] Figure 7 illustrates METHOD 1 by evaluating the "colocalization" level as function of the threshold intensity[unreadable] determined sequentially by the average intensity of the individual images. The graph illustrates the relationship[unreadable] between percent "colocalization" and percent random "colocalization". As previously discussed, a positive[unreadable] "colocalization" has to be much higher than the random "colocalization". Plots (a and b) are the CAVg over CAV3 and[unreadable] CAV3 over CAVg "colocalization" levels, respectively. Plot (r) is the theoretical percent random "colocalization". It is[unreadable] clear that at 1% random "colocalization", the percent "colocalization" of CAVg over CAV3, and CAV3 over CAVg is[unreadable] much higher being 85% and 64%,respectively. Plots (c and d) correspond to panel F of Fig.6, where 4 pixel units[unreadable] (0.038 urn/pixel) were shifted in the x,y plane. As for the intensity correlation analysis, the pixel shift practically[unreadable] eliminated the "colocalization" level between CAVg over CAV3 and vice versa. Identical results were obtained by[unreadable] using METHOD 2 (not shown). Equivalent results are illustrated with the three methods in Fig. 16, showing their[unreadable] application to investigate '"'colocalization'" of cytochrome C-GFP fusion protein expressed in cardiomyocytes via[unreadable] adenovirus and a mitochondria marker (mitotracker TMRE).[unreadable] PHS 398/2590 (Rev.05/01) Continuation Format Page[unreadable] CONTINUATION PAGE Principal Investigator/Program Director (Last, first, middle): Ping, Peipei (Stefan!, Heart Biology Core)[unreadable] Figure 8 shows another example of "colocalization" measurement varying the threshold as a function of the average[unreadable] intensity (METHOD 1) by comparing the "colocalization" level of a transfected labeled protein with a native[unreadable] immunolabeled protein. Confocal images were at 0.054 urn/pixel in the x, y axis, and every 0.1 urn in the z plane.[unreadable] Panels A, B and C are single confocal sections of a cultured neonatal rabbit myocyte transfected with p38-GFP fusion[unreadable] protein (green) and immunostained with anti-TAB1 antibody (red). Regions 1 and 2 are displayed at a higher gain in[unreadable] D,E and H,l, respectively. Note the distribution outline in defined clusters of TAB1 and P38 which has a filamentous[unreadable] pattern in the cytoplasm. Graphs (G) and (K) show the relationship between percent "colocalization" and percent[unreadable] random "colocalization" for regions 1 and 2, respectively.[unreadable] In the graph, each data point was obtained by[unreadable] sequentially setting the threshold as function of the[unreadable] average intensity. The percent "colocalization" and[unreadable] percent random "colocalization"was calculated from eqs.[unreadable] 1-3. The theoretical curve of random "colocalization"was[unreadable] plotted as a reference (labeled RANDOM). With a .RcgioncV c ? Region 1 I \|_\| Region 2 \| Region2[unreadable] minimal threshold value, the area above threshold equals[unreadable] the total area of the region; thus, the values of percent[unreadable] "colocalization" and percent random "colocalization" are[unreadable] 100. As the intensity threshold values are increased as a[unreadable] function of the average intensity, the thresholded areas[unreadable] are reduced resulting in diminished "colocalization". The[unreadable] arrow marks the "colocalization" level when the percent[unreadable] random "colocalization" is 3% (see table at the right[unreadable] bottom). Panels F and J show the corresponding masked XUM1K00DOM1C0OLOC1A.U0ZATO0H.1[unreadable] DCAV3 OVER UVg[unreadable] thresholded areas (yellow shows the overlap). The ? NCX OVER CAVg COLOCALIZATION ANALYSIS (Arrow in G and K)[unreadable] "colocalization" level (40-50%) is much higher than the CYTOPLASM (Region 1) NUCLEUS(Region Z)[unreadable] Total Area = 131 \rn\2 Total Area = 104 (im2[unreadable] expected random "colocalization"for those areas. These P38Area =25 prn2 P38Area =20 ym2[unreadable] TAB1Area =21 pm2 TAB1 Area = 20 Mm2[unreadable] determinations indicate a high level of "colocalization" in P3B Over TAB1 =41% P38 Over TAB1= 50%[unreadable] the nucleus and cytoplasm for TAB1 and P38. Similar <-> 100%.0RAN1D0.O0 M CO1L.0OCALIZ0A.1TION 0.01 TRAanBd1omOvCeorloPc3.8==35.0% RTaAndBo1mOCvoelor cP.3=8=3.502%[unreadable] values were obtained using METHOD 2. Panel L shows Fig. 8. "Colocalization" measurements in neonatal heart cells[unreadable] examples of minimal and maximal "colocalization"in adult transfected with p38GFP (green) and immunolabeled with anti-[unreadable] heart myocytes. Minimal "colocalization" was observed TAB19 antibody (red} (seetexO. _[unreadable] between caveolin generic (CAVg) and Na/Ca2+ exchanger (NCX). Note that the curve for NCX over CAVg follows[unreadable] closely the theoretical curve for random "colocalization". In contrast, maximum "colocalization" was obtained when[unreadable] the same protein (caveolin) was immunostained with monoclonal (CAV3) and polvclonal (CAVg) antibodies. In the[unreadable] latter condition, one should expect 100%"colocalization" if the antibodies would have 100%efficiency to stain the[unreadable] target protein. Evidently, this is not the case, and only a fraction of caveolin can be simultaneously stained by both[unreadable] antibodies.[unreadable] Evaluation of intensity correlation and intensity threshold analyses. The major advancementin our studies[unreadable] is the achievement of high spatial x.v.z resolution bv image restoration in conjunction with the use of[unreadable] analytical user-independent colocalization methods (intensity correlation analysis and intensity threshold[unreadable] colocalization). These methods will provide a strong argument to define whether two proteins are elements or not of[unreadable] the same macromolecular complex, and,in particular for this PPG, to quantify with rigorous algorithms protein-protein[unreadable] "colocalization" and association of proteins with mitochondria. Both "colocalization" methods, intensity correlation[unreadable] analysis and intensity threshold "colocalization" have advantages and drawbacks. The intensity correlation is a[unreadable] powerful statistical method that evaluates the correlation between the landscapes of paired images. One draw back[unreadable] is that not clearly interpretable results are obtained if the images have regions of positive and negative correlation. To[unreadable] circumvent this deficiency the user should evaluate different cytoplasmic and membrane regions. Another drawback[unreadable] of this analysis is that it does not give the "colocalization" level as the overlap factor as in the intensity threshold[unreadable] analysis. On the other hand, the major draw back of the intensity threshold method is the mathematical definition of[unreadable] random "colocalization" due to the lack of a valid estimate of the total area where the protein can be distributed[unreadable] randomly. This is not the case for cytosolic proteins that can randomly distribute in the cytoplasm and the total area is[unreadable] directly measured from the region of interest. In general, proteins are targeted to regions making very difficult to[unreadable] measure the surface of the targeted area. For example, the total area that one has to consider for membrane proteins[unreadable] is difficult to estimate. Nevertheless, the fact that when positive, "colocalization" gives a "co/oca//zaton"/tandom[unreadable] "colocalization" ratio of several orders of magnitude higher which makes it easy to define the "colocalization" level. In[unreadable] fact, if "colocalization" remains significant in the limit of random "colocalization" (-0.1%), it is a strong argument of a[unreadable] PHS 398/2590 (Rev. 05/01} Continuation Format Page[unreadable] CONTINUATION PAGE Principal Investigator/Program Director (Last,first,middle): Ping, Peipei (Stefani, Heart Biology Core)[unreadable] positive finding. Moreover, as control. I have been able to detect lack of "colocalization" of two membrane proteins[unreadable] that do not co-immunoprecipitate such as NCX and caveolin as shown in Fig.8L. In view of these considerations,[unreadable] one of the major advantages of the intensity correlation analysis is that it is not dependent on the estimate of random[unreadable] "colocalization". Thus, by using both types of analysis. I am confident that reliable "colocalization" measurements will[unreadable] be obtained in the limit of the microscope spatial resolution. The implementation of the intensity correlation analysis is[unreadable] very laborious and time consuming. For example, the analysis of 1024x1024 pixel image takes about 6 hrs.At[unreadable] present, I am developing a program to increase the processing speed and to automatically analyze various[unreadable] predetermined regions in the image. Alternatively, I am also developing a program that will determine the degree of[unreadable] correlation from the x and y first derivative of the landscape. This approach may increase the sensitivity and[unreadable] discrimination of the analysis, since it will take also into account the slopes of the valleys in the landscape.[unreadable]	{ "pile_set_name": "NIH ExPorter" }
Project Summary/Abstract Atopic dermatitis (AD) is a highly pruritic chronic episodic inflammatory skin disease that often presents between 3 and 6 months of age but may also present during later childhood or in adulthood. In the US, AD affects roughly 10-20% of all children of all races and ethnic groups. The prevalence of AD is increasing worldwide and it is a major public health burden. The majority of children with AD will also develop asthma and seasonal allergies. From the recent Global Burden of Disease study, AD was among the top 50 most prevalent diseases worldwide and it had the second highest disability rank of all non -malignant skin diseases. Many genes have been associated with the onset of AD. These genes are mostly associated with skin barrier changes (e.g. FLG) or immune dysregulation. Antigen-presenting cells (APC) are known to be involved in the initiation of the skin immune response. APC presentation of antigen to the immune system is guided by human leukocyte antigen (HLA) molecules, which are encoded within the major histocompatibility complex (MHC) located on chromosome 6. Six large population genetic studies of children with AD have been published and three of these studies demonstrated an association between the 6p21.3 cytoband (i.e. the MHC) region and AD. In a preliminary study, we have shown, for the first time, that specific variation of the HLA II DRB1 allele results in amino acid variations at position 9 (pocket 4), position 26 (pocket 4), and position 78 (pocket 4) that were marginally associated with the prevalence of AD and markedly associated with the persistence of AD. Importantly, unlike many other associations between HLA alleles and diseases (e.g., diabetes), the pathogenesis of AD is known to be associated with cutaneous inflammation and HLA II is known to be expressed on APCs that have an effect on the inflammatory cascade. We plan to focus on the HLA region of the genome using new technology to better understand the genetics of AD and the interplay between skin barrier dysfunction and immune dysregulation. This proposal has two goals. The first goal is to define HLA variation associated with the onset and persistence of AD. The second goal is to demonstrate that HLA variation is associated with immune dysregulation in those with AD.	{ "pile_set_name": "NIH ExPorter" }
Methods are being devised and tested to improve the detectability of trace organic compounds using mass spectrometry. The development of new derivatization reagents and methods to impart improved electron capture properties and ionizability for negative ionization mass spectrometric detection has been continued. A variety of derivatives are being tested for quantitative analysis of anandamide (arachidonylethanolamide), an endogenous cannabinoid agonist found in mammalian brain and peripheral tissues, for kynurenine and kynurenic acid, and for the amino acids nitrotyrosine and tyrosine as biomarkers of peroxynitrite from nitric oxide released in tissues. Liquid-chromatography - electrospray mass spectrometry is being investigated for trace detection analyses of lipids related to anandamide and products of the kynurenine pathway metabolism of tryptophan, and for oxidized nucleosides. Liquid chromatography - matrix assisted laser desorption mass spectrometry is being tested as a means of analyzing NAD and NADP as products of tryptophan metabolism. (Formerly LCS)	{ "pile_set_name": "NIH ExPorter" }
Interphotoreceptor retinoid-binding protein (IRBP), the major soluble protein component of the interphotoreceptor matrix (IPM), has access to M[unreadable]ller cells, photoreceptors, and RPE. The mechanism by which IRBP protects retinoids from isomeric and oxidative degradation while targeting their delivery/release between the above cells during the visual cycle is poorly understood. Our long-term goal is to understand at the molecular level how IRBP accomplishes its remarkable functions. The mechanism underlying IRBP's function or role in disease remains unknown because little is understood about its structure-function relationships. Its structure is unusual being composed of tandem homologous "modules" each ~300 residues in length. Although the individual modules have some functional activity, they are not equivalent, and important interactions take place between them. A critical gap is that little is known about the structure of the full-length protein and quaternary association of the "modules". However, obtaining IRBP at the concentrations needed for X-ray crystallography has been problematic as the protein denatures and precipitates when concentrated above 3 mgs/ml. In the current funding period, we purified to homogeneity full-length bovine, xenopus, human and zebrafish IRBPs in stable and functionally active pristine forms, devoid of fusion tags. These protein solutions can now be readily concentrated without denaturation or precipitation. We have optimized conditions for growing diffraction-quality crystals of these full-length IRBPs. Preliminary structure elucidation analysis for Xenopus IRBP suggests that the single module structure may be substantially modified in the full-length functional protein. Analyses of the expression, purification, stability, crystallization, ligand-binding, anti-oxidant activity, and homology-modeling data on these IRBPs have led to our hypothesis that quaternary association of the "modules" contributes to the structural scaffold(s) that bind and protect retinoids from degradation, and that the "modules" contribute unequally in these roles as well as in target retinoid delivery and release at the cell surface. This hypothesis will be evaluated through the following complementary aims. Aim 1: To determine the crystal structures of IRBPs composed of four modules (human, bovine, Xenopus), and two modules (zebrafish). Aim 2: To define binding-interactions of physiologically relevant ligands with IRBP and elucidate the molecular basis of IRBP's protective/anti-oxidant roles. Aim 3: To determine how IRBP efficiently targets retinoid removal/delivery. Aim 4: To determine the structural and functional "hot-spots" in IRBP. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: The mechanism by which IRBP protects retinoids from isomeric and oxidative degradation while targeting their delivery/release between photoreceptors, retinal pigmented epithelium and M[unreadable]ller cells during the visual cycle is poorly understood. A critical gap is that little is known about the structure of the full-length protein and quaternary association of the "modules" that comprise the structure of IRBP. Analyses of the expression, purification, stability, crystallization, ligand-binding, anti-oxidant activity, and homology-modeling data on these IRBPs have led to our hypothesis that quaternary association of the "modules" contributes to the structural scaffold(s) that bind and protect retinoids from degradation, and that the "modules" contribute unequally in these roles as well as in targeting retinoid delivery and release at the cell surface. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]	{ "pile_set_name": "NIH ExPorter" }
The extensive variation at some of the immune response genes is central amongst the host genetic determinants that contribute to the variability in risk of virtually all human diseases. We have studied the genetic effects of the highly polymorphic KIR and HLA loci, as well as other related, less polymorphic loci on several diseases. Our contributions to the general understanding of these effects are summarized here. Variation at the HLA class I locus has a stronger influence on HIV-1 disease outcome than that of any other genetic locus identified to date. HLA class I molecules have two fundamental roles in determining the strength of the immune response against HIV: 1) regulation of the acquired response through presentation of antigenic epitopes to cytotoxic T lymphocytes (CTL), and 2) modulation of the innate response by serving as ligands for the killer cell immunoglobulin-like receptors (KIR) expressed on natural killer (NK) cells. In both regards, HLA-B has been the primary focus relative to HLA-A and C because the strongest genetic and functional associations with HIV disease outcomes have involved this locus. Recently, however, a scan for genetic variants that influence control of viral load indicated that a dimorphism 35kb upstream of the HLA-C gene (-35C/T) had one of the two strongest genome-wide effects on the level of HIV plasma viremia in early, established HIV infection. Notably, the -35C variant that associates with low viral load has also been shown to associate with high HLA-C mRNA levels in a co-dominant manner amongst a group of individuals of European ancestry. These findings suggest that certain HLA-C allotypes may have a primary role in restricting HIV replication through innate and/or acquired immune mechanisms that have been previously overlooked. Using a cohort of 1698 individuals, we showed that the -35 variant associates very strongly with HIV outcomes during the early phase of infection by influencing steady- state viral load, and to a weaker extent with the very late phase of infection by influencing time to death. These temporal data implicate two at least partially distinct mechanisms of HIV restriction associated with the -35 variant. No individual HLA-C allotype appears to be clearly better than others in terms of controlling HIV through antigen presentation to CTL, unlike that which has been observed at the HLA-B locus. However, higher levels of HLA-C on target cell surfaces may generally result in more effective antigen presentation to CTL or enhance NK cell activity, thus boosting the immune system and leading to better viral control by the host. These data strongly implicate high HLA-C expression levels in more effective control of HIV-1, potentially through better antigen presentation to cytotoxic T lymphocytes or recognition and killing of infected cells by natural killer cells. It is well established that HLA class I plays an important role in determining the rate of progression to AIDS after infection with HIV-1. It is less clear, however, how HLA diversity affects HIV transmission. We previously showed that the HLA-Bw4 and Bw6 epitopes of the HLA-B molecules are associated with risk for HIV transmission between heterosexual couples. The presence of HLA-Bw4 in HIV-1-infected men was associated with a decreased risk of male-to-female HIV-1 transmission. More recently, we further investigated the association of individual HLA-B alleles for their respective contributions to the risk of HIV-1 transmission from HIV-infected men to their female partners. We also tested for an association of HLA-B alleles present in the female partners of HIV+ carriers on risk of becoming infected. Among the HIV-infected men, three HLA-B alleles showed a significant association with infectivity. Men positive for HLA-B35Px (B3502, B3503, and B5301), a group of alleles previously associated with rapid AIDS progression, showed an increased risk of transmitting the virus to their female partners, whereas men positive for B27 or B57, alleles which is associated with slow AIDS progression, transmitted the virus to their partners less frequently. On the other hand, HLA polymorphisms among female sex partners did not affect their susceptibility to HIV infection. In summary, class I HLA polymorphism appears to affect HIV-1 infectivity but not susceptibility. The HLA alleles B35Px, B27 and B*57 may influence both HIV-1 transmission and post-infection risk of AIDS through control of HIV-1 RNA levels in semen and blood. NK cells are critical in the early containment of viral infections. Epidemiological and functional studies have shown an important role of NK cells expressing specific killer immunoglobulin-like receptors (KIRs) in the control of HIV-1 infection, but little is known about the mechanisms that determine the expansion of these antiviral NK cell populations during acute HIV-1 infection. One particular activating KIR (KIR3DS1), in combination with its putative ligand, an HLA-B allele with isoleucine at position 80 (HLA-B Bw480I), has been previously shown by us to be associated with slower HIV-1 disease progression. They have also shown that KIR3DS1+ NK cells can effectively suppress HIV-1 replication in HLA-B Bw480I+ target cells in vitro. Furthermore, a subset of inhibitory alleles from the same locus, KIR3DL1, that show high cell surface expression levels have similarly been associated with slower disease progression toward AIDS in the presence of their ligand, HLA-B Bw480I. However, the mechanisms underlying their protective role are not understood. In collaboration with investigators at Harvard University, we assessed clonal NK cell expansions during acute HIV-1 infection by quantitative PCR and flow cytometric analysis. The results indicate that NK cells expressing the activating receptor KIR3DS1 and, to a lesser extent, the inhibitory receptor KIR3DL1 specifically expand in acute HIV-1 infection in the presence of HLA-B Bw480I, the putative HLA class I ligand for KIR3DL1/3DS1. The early accumulation of highly activated NK cells may provide a potent first-line defense allowing for the initial control of acute HIV-1 replication while adaptive immune responses are still developing. These data demonstrate for the first time the impact of the HLA class I ligands on clonal NK cell expansions during an acute human viral infection.	{ "pile_set_name": "NIH ExPorter" }
The objective of the research is to gain an understanding of the effect of level of protein intake on calcium metabolism in man. Efforts will be made to study the physiological mechanisms whereby high protein diets cause hypercalciuria. Four population groups, namely 1) young adult males, 2) older males and 3) pre- and 4) postmenopausal women will be studied. Subjects will be fed carefully controlled diets for varying lengths of time and will be given both low and high protein diets. Calcium, phosphorus and magnesium will be about 500, 800 and 350 mg, respectively, and will be the same at both protein intakes. Parathyroid function will be assessed directly by measuring serum parathyroid hormone levels and indirectly by determining urinary cyclic AMP. Other analyses indicative of calcium metabolism will also be made. Renal function will be examined by measuring glomerular filtration rate, calcium clearance, fractional excretion of calcium and fraction reabsorption.	{ "pile_set_name": "NIH ExPorter" }
Over twenty distinct transforming genes have been identified in the genomes of oncogenic retroviruses. Each of these oncogenes has a homologue in the chromosomal DNA of a vertebrate species. Current evidence indicates that this highly conserved set of genes may play a vital role in cell proliferation and/or differentiation. In addition, inappropriate expression of some of these genes has been implicated in the genesis of cancer. Our hypothesis is that deregulation of oncogene expression may be a possible general mechanism for the induction of neoplasia in humans. Our efforts have been concentrated on the cellular homologue of the ras gene, the oncogene carried by Harvey and Kirsten Sarcoma viruses. In this study we are investigating the role of ras gene expression in the induction of rat and human mammary carcinomas. In a study of more than 200 human breast carcinomas, we have observed elevated expression of c-rasH in 70% of estrogen and progesterone receptor positive tumors and 40% of estrogen and progesterone receptor negative tumors. Whereas, an amplified or rearranged c-rasH gene has not been detected in human mammary carcinomas. Thus, the mechanism by which c-rasH gene expression is deregulated in these tumors remain to be determined. The goal of this project is to elucidate the mechanisms of oncogene involvement in the neoplastic transformation and progression.	{ "pile_set_name": "NIH ExPorter" }
Orthotopic liver transplantation (OLT) is the only treatment available to treat acute liver failure patients but annually the scarcity of transplantable organs results in the deaths of nearly 2000 patients awaiting transplant. Current liver support to extend the viable waiting period of these patients based in fresh porcine hepatocytes in the extracorpeal perfusion systems. As a cost effective alternative to these treatments, MCA proposes to encapsulate a proprietary human cell line for use in cell transplantation therapies. A novel encapsulation procedure will provide the means for a long term treatment correction of liver failure. Successful recent reports of liver cell transplantation therapies support the potential of this minimally invasive treatment as an alternative modality to bring an inexpensive and noninvasive treatment to the high populations of chronic hepatitis patients in developing countries which may or may not have access to the comprehensive medical centers required for OLT. The United States chronic liver failure market targeted by MCA represents approximately $875 million annually and world wide may be as high as $2 billion. PROPOSED COMMERCIAL APPLICATIONS: Whole liver transplantation is the only treatment available to treat acute liver failure patients but scarcity of transplantable organs annually results in the deaths of 2000 patients awaiting transplant. However recent results suggest that hepatocyte transplantation, per se may be sufficient to restore normal liver function under some circumstances. Current liver support to extend the viable waiting period of these patients is limited to the availability or primary human hepatocytes. In order to address this need, MCA proposes to exploit proprietary methodology to generate and provide functional human hepatocytes for transplantation. The United States market targeted by MCA represents approximately $875 million annually and world wide may be as high as $2 billion.	{ "pile_set_name": "NIH ExPorter" }
Breast cancer is the most commonly diagnosed cancer among women in the United States. There is currently a controversy on whether the Epstein-Barr virus (EBV; Human Herpes Virus 4) plays a role in breast carcinogenesis. We propose to test the hypothesis that EBV is commonly found in invasive ductal carcinomas of the breast but not in normal adjacent tissue, and that the presence of EBV is associated with a more aggressive phenotype, a poor prognosis, and epidemiolgic risk factors that are associated with suppression of the cellular immune system including a diet high in animal fat or animal protein, a history of blood transfusion, and cigarette smoking. This study will be conducted in the Iowa Women's Health Study (IWHS), a prospective cohort study of postmenopausal breast cancer risk in over 40,000 older Iowa women who were first enrolled in 1986. We propose to retrieve tumor blocks from incident invasive ductal carcinomas first diagnosed in the IWHS between 1988 and 1990 (N=258) in collaboration with the State Health Registry of Iowa. Based on extensive prior experience, we expected to have 184 useable tumor blocks available for analysis. Diagnostic slides and pathology reports will be reviewed in order to grade the tumors and to select appropriate normal and tumor sections from paraffin blocks. Tumor blocks will be processed in th Mayo Tissue Acquisition and Processing Core. We will attempt to identify the presence of EBV in the tumor and adjacent histologically normal tissue using PCR-based methods to identify EBV, and immunohistochemical methods to identify EBNA-1 expression, allowing us to estimated the prevalence of EBV (any EBV; EBV-1; EBV-2) infection in a relatively large sample of invasive ductal carcinomas. We will also correlate EBV status in tumors with prognostic factors including tumor size, axillary lymph node status, estrogen and progesterone receptor status, and histologic grade. As secondary aims, we will evaluate whether EBV status predicts long-term (great than or equal to 10-year) survival after breast cancer diagnosis (overall survival; death due to breast cacncer), and whether certain epidemiologic risk factors that alter immune function allowing reactivation of EBV infection are more strongly associated with EBV+breast cancers. We have carefully designed this study within the resource constraints of the small grant program in order to clearly address our main hypotheses, and to begin to address our secondary aims, albeit with limited power, in order to generate preliminary data on which to base future studies within this cohort as well as other study populations. This proposal also fits well into the goals of the small grants program in cancer epidemiology since it address an emerging, high risk/high gain hypothesis that requires the types of data we will generate in order to justify a more definitive study.	{ "pile_set_name": "NIH ExPorter" }
Angiotensin (AII) has been shown to stimulate jejunal sodium (Na+) and water transport in vivo. However, the mechanism of action and physiological role of the intestinal effects of the hormone are unclear. Therefore, this study will show that the increase in jejunal Na+ and water absorption following extracellular fluid (ECF) reduction is mediated by the renin angiotensin system, demonstrate that ECF depletion results in increased jejunal Na+ and water absorption which is independent of changes in intracellular fluid (ICF) volume, define the site of action of AII within the sympathetic nervous system (SNS) leading to increased Na+ and water absorption and determine the intestinal cell types that have specific yield-adrenergic binding sites and receptors for AII. Three general protocols will be employed to accomplish these aims. First, jejunal electrolyte and water absorption will be measured from closed intestinal sacs in anesthetized rats following non-hypotensive hemorrhage, water deprivaton and Na+ depletion. All are models of ECF delpetion but either have no effect or decrease and increase the ICF respectively. Plasma aldosterone, AII and ECF volume will be determined in these animals. Jejunal absorption also will be determined following adrenalectomy, nephrectomy and after treatment with spironolactone, captopril and a specific renin inhibitor in ECF-depleted rats. The site of interaction of AII within the SNS will be determined by circulating blood containing AII through the vascularly isolated head or mesenteric circulationof a recipient animal, while simultaneously measuring jejunal absorption from closed sacs in the recipient. To determine the intestinal cell types having yield and AII receptors, both light and electronmicroscopy autoradiography for I25I AII and 3H-prazosin will be performed in combination with flouresence microscopy for catecholamines and immunohistochemistry for DBetaH. The long term objectives of this study are to increase understanding of the normal physiological control of intestinal absorption by hormones and the SNS.	{ "pile_set_name": "NIH ExPorter" }
Project Summary We propose that ASM (acid sphingomylinase) is a critical regulator of RTKs (receptor tyrosine kinases), and that ASM can serve as a novel therapeutic target for various cancers, including gliomablastoma multiforme (GBM). Also as loss of function of ASM may cause the severe neuron degeneration phenotype in the rare familial Niemann Pick Disease, type A, our studies may provide molecular insights into this disease. The plasma membrane is a lipid bilayer composed primarily of phospholipids, as well as sphingomyelins, cholesterol, glycosphingolipids and other less abundant lipid molecules such as ceremides. The composition of the plasma membrane lipids is dynamically regulated and undergoes rapid exchanges with intracellular organelles such as Golgi, endosomes and lysosomes through secretion, exocytosis and endocytosis. For many transmembrane and membrane-associated proteins, including RTKs, specific interaction with various lipid molecules in the plasma membrane is an integral part of regulation to maintain their protein structure and function. However, the mechanisms by which specific lipid molecules regulate the dynamic activities of RTKs and other transmembrane or membrane-associated proteins are not well characterized. In this application, we propose to investigate the roles of ASM (also called SMPD1) in regulating the RTK-mediated cell signaling processes. We have recently found that the levels of ASM (acidic sphinomyelinase) are highly elevated in GBM and our studies reveal that ASM is required for the activation of multiple RTKs. ASM is an enzyme involved in sphingolipid metabolism that hydrolyzes sphingomyelin to produce ceramide. Ceramide, with a biophysical property of self-association, is involved in establishing a lipid microenvironment that promotes protein-protein interactions. Mutations of human ASM gene cause Niemman-Pick disease, type A, an inherited disease that induces massive loss of Purkinje neurons in the cerebellum and patients usually die by 2 or 3 years of ages, but the underlying molecular mechanism of ASM deficiency for the disease remains unresolved. In this application, we will investigate the mechanism by which ASM regulates the RTK signaling pathway, by following specific aims: Specific aim 1: To determine how ASM regulates the activation RTK receptor proteins. Specific aim 2: To investigate the involvement of ASM sphinomyelinase activity in RTK signaling. Specific aim 3: To examine whether ASM regulates RTK signaling at multiple levels. As co-activation of RTKs is critically important in GBM and in a multitude of human disorders and diseases, elucidation of this new regulatory mechanism may provide novel targets for prevention and therapeutic treatment. Our studies should also provide molecular underpinning how loss-of-function of ASM causes neuron degeneration in the human diseases such as Niemman-Pick disease, type A.	{ "pile_set_name": "NIH ExPorter" }
The overall Aim of this Project is to determine the physiologically relevant mechanisms of signal transduction that regulate contractility by pathways not dependent on cytoplasmic Ca concentration. The domain dependent functions of the upstream regulators of Ca?+-sensitization by the RhoA-Rho kinase pathway, the guanine nucleotide exchange factors (GEFs) PDZ.RhoGEF and LARG, will be determined in conjunction with the structural identification and recombinant expression of GEF domains in Project 2. The role of a membrane associated protein (p120 catenin) as a potential RhoA-binding partner guanine nucleotide dissociations inhibitor (GDI) will be determined. The kinetics of myosin phosphatase regulation by Rho-kinase and its dependence on the expression level of the endogenous phosphatase inhibitor, CP1-17 will be quantitated in order to determine their relative physiological relevance. The dependence of myosin phosphatase activity on the specific long and short splice isoforms of the regulatory subunit (MYPT1) expressed in mammalian smooth muscle will be evaluated, based on the hypothesis that expression levels of the ratio of the two isoforms determine localization of the phosphatase and the mechanism of its regulation through differences in their phosphorylation sites and myosin binding properties. A major, novel, objective of this Project is to determine the functions of a protein, LPP, newly discovered to be selectively expressed in smooth muscle, on cellular phenotypes, including morphology, adhesion to substrate, cytoskeletal structure and motility. Localization and stimulus induced translocation of signaling proteins, co-localization of proteins, such as p120 catenin with RhoA or CP1-17 with myosin phosphatase or telokin and telokin and LPP partners identified in two-hybrid screens will be performed in Project 3. p120 catenin, LPP, recombinant RhoA, telokin, RhoA- and other protein-mutants will be supplied by Core B. Abnormalities of the RhoA pathway are thought to be involved in asthma and hypertension.	{ "pile_set_name": "NIH ExPorter" }
The formation of accumulated, abnormally folded proteins is associated with more than twenty different clinical syndromes, each of which is associated with a distinct `misfolding' protein. These misfolded proteins aggregate and form the hallmarks of many neurodegenerative diseases, termed amyloids. Amyloid formation is associated with neurodegenerative disseases such as Alzheimer's, Parkinson's, and Huntington's disease, as well as localized diseases such as Type II Diabetes. Of the amyloid-related diseases, polyglutamine (polyQ) diseases represent a set of heritable neurological disorders, comprising at least nine diseases. In polyQ diseases like Huntington's disease (HD), our knowledge of how and why an expanded tract of polyQ leads to aggregation and amyloid formation is largely hampered by the fact that there is little high-resolution structural and dynamic insight into this process. Furthermore, the ability of a polyQ protein misfolded species to engage in aberrant protein interactions has become increasingly studied. Particularly, the interactions of polyQ proteins with molecular chaperones and their resistance to proteolysis are poorly understood. Challenges posed by polyQ proteins are manifold and, therefore, new biochemical and biophysical approaches developed to tackle such problems would greatly aid our understanding of polyQ diseases. To accomplish this goal, this application seeks to further investigate the aggregative properties of polyQ proteins at high-resolution, namely the N- terminal fragment of the huntingtin protein (implicated in the progression of HD), by using by nuclear magnetic resonance (NMR) spectroscopy. Newly developed NMR methods, such as dark-state exchange saturation transfer (DEST) and lifetime line broadening, offer the capability to achieve atomic resolution on the dynamic exchange occurring in the self-assembly of polyQ proteins, in addition to their interactions with molecular chaperones such as Hsp70 and Hsp40. While this proposal will focus on the development and application of new NMR methodologies to such challenging systems, the study will be extended for investigation using a variety of other biophysical techniques, including electron paramagnetic resonance (EPR), fluorescence spectroscopy, and calorimetry. The outcome of the proposed studies on amyloid formation by polyQ proteins will significantly advance our understanding of HD and other polyQ diseases, in general, by achieving structural and dynamic insights with atomic-level resolution and will aid in the development of therapeutic strategies for such diseases.	{ "pile_set_name": "NIH ExPorter" }
Depression is an illness that is poorly understood and treatments are only effective in a certain percentage of the population. Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed antidepressant medications. SSRIs act indirectly via 5HT receptors, including the 5HT1A receptor that couples to heterotrimeric G proteins. Regulators of G protein signaling (RGS) are a family of intracellular proteins that negatively modulate receptor-mediated G protein signaling. Using mutant mice we have identified a subset of 5HT1A receptors coupled to G?i2 and negatively regulated by RGS proteins that appear to mediate the beneficial actions of SSRIs. This led us to consider that RGS protein modulation of 5HT1A receptor signaling may play an important role in depression and so present a novel target for the treatment of depression. However, there are >20 mammalian RGS proteins and a general inhibitor will likely have a broad range of effects. In this proposal we seek to identify the specific RGS protein that modulates 5HT1A receptors and therefore controls behavioral responses to 5HT1A receptor activation related to mood disorders. The proposed work will use a combination of behavioral, biochemical and molecular biological approaches to provide knowledge of RGS proteins in serotonin signaling in animal models of depression and implicate RGS proteins as drug targets to promote the antidepressant actions of currently available SSRI's without enhancing their unwanted effects. PUBLIC HEALTH RELEVANCE: Depression is a poorly understood illness that affects approximately 10 million Americans. Moreover, treatments for depression are ineffective in a majority of patients. We have recently identified a family of intracellular proteins (RGS proteins) that appear to enhance the beneficial actions of selective serotonin reuptake inhibitors (SSRIs) which are the major drugs used for the treatment of depression. In this proposal we seek to identify the particular RGS protein(s) involved and so identify a novel target for treatment of depressive illness.	{ "pile_set_name": "NIH ExPorter" }

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