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The long-term goals of the proposed research are to develop tissue-engineering materials (TEM) with enhanced biocompatibility, and flexibility to tailor mechanical properties for a wide range of applications. There are two specific aims for this proposal. 1) To develop a biocompatible tissue-engineering scaffold based on visible light-cured polymer that is filled with bioactive glass. 2) To assess the biocompatibility of unfilled and bioactive glass-filled polymer scaffolds. Enhanced biocompatibility will be attained via three basic mechanisms: polymer design, nature of the photo-activation system, and by incorporating bioactive glass (BAG) in the polymer. We will synthesize biodegradable methacrylated monomers using Polyethylene glycol (PEG) and polylactic acid (PLA). By changing the number of PEG repeat units in the PEG-PLA methacrylated monomer, we will systematically control the length of the chain segment between adjacent lactate groups. Consequently, the amount of acidic by-products released per unit volume of the polymer that degrades, and therefore the biocompatibility of the scaffold, will be controlled. This design will indirectly control hydrophilicity and cross-link density of the polymer, and thus the rate of release of acidic products. By adding BAG in the polymer the acidic degradation products will be neutralized as they are released. We will also use a visible light-photoinitiator system along with a polymerizable coinitiator. The latter will be bound within the polymer network, minimizing leachability. Visible light, that is deeper penetrating, will potentially cause a more uniformly cured TEM than currently used UV activation. This will minimize residual monomer in the TEM, and thus enhance biocompatibility. The amount of residual monomers in the TEM cannot be directly measured but can be indirectly determined by measuring degree of conversion (DC) and Knoop hardness (KHN) of the cured TEM. We will measure and compare DC and KHN of the various TEMs. We will also evaluate the effects of polymer design and BAG on acidity by measuring and comparing the pH of solutions surrounding cured samples of TEMs with or without BAG. A photoinitiating system based on visible light that penetrates deep into biological tissues will provide a means of curing scaffolds across intervening structures, e.g., skin and muscles. This will be key to applying less invasive surgery in tissue and organ repair, minimizing damage to surrounding tissues, and reducing recovery time and its associated expenses. PROJECT NARRATIVE: This project seeks to develop improved tissue engineered composite materials with enhanced biocompatibility, and the flexibility to tailor mechanical properties for a wide range of applications. A photo-initiating system based on visible light that penetrates deep into biological tissues will provide a means of curing bioactive glass-reinforced polymer scaffolds across intervening structures, e.g., skin and muscles. This will be crucial to the use of less invasive surgery in tissue and organ repair, minimizing damage to surrounding tissues, and reducing recovery time and its associated expenses.
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{
"pile_set_name": "NIH ExPorter"
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A study of the kinetics and mechanisms of metal ion transfer and other reactions of metal-peptide complexes is proposed. The metal ions include Cu(I), Cu(II), Cu(III), Ni(II), Ni(III), Co(II), Co(III), and Pd(II). The mechanisms include proton transfer, electron transfer and metal ion substitution reactions. The nature of copper and nickel transport in blood and of metal-peptide coordination in proteins will be examined.
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{
"pile_set_name": "NIH ExPorter"
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The central goal is the development of new MRI techniques, or new combinations of established techniques, for distinguishing tumors from normal tissue or inflammation. This is an important clinical diagnostic problem when diagnosing or staging cancer. While conventional MRI can often detect tumors, it is often difficult to distinguish them from imflammation or edema. A secondary goal is relating MR images to tumor vascular patterns and tumor oxygenation. Tumor oxygenation is an important factor in determining the response to radiation therapy. These goals will be approached by combining the information obtained from images of protons, fluorine, and sodium. The proton images will be used for their anatomical detail. The fluorine images will be of fluorinated blood substitutes and will dilineate vascular anatomy and measure the vascular oxygen tension. The sodium images will be of endogenous sodium which in biological systems shows two components of T2 decay. Chemical shift reagents will be used to identify the intra and extracellular components. We will determine whether the short T2 component can be used to distinguish tumors from edema. The work will be done in a variety of animal tumors of known vascularity and radiosensitivity. If successful, the techniques can be transferred to human imaging and should lead to improved tumor diagnosis.
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{
"pile_set_name": "NIH ExPorter"
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Medical ultrasound techniques are widely used for pre-clinical investigations. These non-invasive imaging modalities are crucial for researchers to observe changes in organ/tissue structure, size, and blood flow during disease stages as well as in response to pharmacological interventions. The requested equipment is an ultra high-frequency ultrasound machine (Vevo3100, FUJIFILM, VisualSonics) specifically manufactured to image mice, rats, and rabbits. The Vevo 3100 system will be equipped with a linear phased array ultrasound probe specific for animals in the size range of rabbits that is not currently available on our existing equipment. The Vevo 3100 system also has several upgrades in the ultrasound technology including; 1) improved resolution, 2) reduced artifact, 3) real time image processing, 4) left ventricular tracing, 5) improved workflow for rapid data acquisition, 6) speckle tracking analysis (for strain analysis), 7) an imaging station equipped with injection mount (for echo guided tissue injections), and 8) a high throughput analysis. All these functionalities would make this system a necessary part of our goal to produce high quality data in a time and cost efficient manner. Additionally, the Vevo 3100 system with high- resolution ultrasound has applications in nephrology by providing excellent delineation of renal tissues, the technology to quantify and analyze kidney size, and the ability to measure blood flow throughout the pelvic region. The functionality of this novel Vevo 3100 will allow our cardiovascular research groups (Drs. Roth, Patel, Dillmann, Hammond, Ross, and Mahata) as well as our colleagues in the nephrology department (Drs. Sharma, Singh, and Rajasekaran) the opportunities to expand their experimental capabilities.
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{
"pile_set_name": "NIH ExPorter"
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High concentrations of readily chelatable (mobile) Zn2+ are found in various brain regions, yet the details of zinc signaling pathways in areas such as CNS development, learning and memory formation, and motor and sensory function, remain elusive. These dynamic pools of mobile Zn2+ may be tracked using fluorescent zinc sensors and are the focus of many studies aimed at understanding the function of zinc- enriched neurons; however, few have attempted to elucidate the molecular mechanisms of Zn2+ during neurogenesis or clarify zinc signaling pathways in motor and sensory functions. To address these deficiencies, we propose the development of a category of zinc-selective sensors that combine the advantages of tunable small molecule sensors and genetically encodable tags. We will design, prepare, and characterize reaction-based small molecule-protein hybrid fluorescent zinc sensors for targeting subcellular organelles and cell membranes. Specifically, we will prepare a reaction-based hybrid green fluorescent zinc sensor for targeting the mitochondria, nucleus, cytosol, and endoplasmic reticulum in proliferating and differentiated neural progenitor cells (NPCs) and use it to map mobile Zn2+ pools in NPCs at discrete locales. Hybrid probes will be characterized in vitro using purified proteins and functionalized small molecule sensors, protein tag-expressing genes cloned to include subcellular targeting sequences, and in cellulo imaging carried out to monitor sensor localization and relative endogenous zinc levels. We will also develop a red-emitting reaction-based hybrid probe for quantifying mobile zinc in NPCs. This will be approached through the design, preparation, and characterization of a protein tag fused to a fluorescent protein, followed by in cellulo fluorescence microscopy for calibration and then endogenous Zn2+ quantification. Finally, we will develop a novel cell-surface displayed hybrid fusion protein for probing synaptic zinc because imaging zinc transmission with high spatiotemporal resolution requires novel sensors that can be localized to neuronal cell surfaces. We will employ a transmembrane protein fused to a protein tag that can be coupled with an extracellular zinc sensor for imaging in cell culture, and with collaborators, in brain tissue slices of relevance to auditory function. This study will be of great importance to the fields of bioinorganic chemistry and neuroscience by providing novel sensors that will be utilized to directly probe mobile zinc signaling pathways in various neurobiological platforms and which can then be built upon to address various broader biological questions.
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{
"pile_set_name": "NIH ExPorter"
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For nine years the International Meeting of the Institute of Human Virology (Meeting) has provided a forum for dedicated scientists in the fields of virology, most specifically HIV/AIDS and cancer biology to present new data and exchange ideas and views. The Meeting serves as a unique conference with the following aims: 1. To disseminate the most current research findings within the selected program topics; 2. To critically assess the impact of new findings on future directions for research; 3. To provide a forum for discussion of current and emerging issues; 4. To identify, support, and build the next generation AIDS researcher; 5. To increase participation by women ethnic/racial minorities, and other individuals who have been traditionally under represented in science, yet over represented in the populations impacted most by the disease and to increase participation by individuals from the developing world. The Institute of Human Virology (EHV), with the financial support requested in this application, will accomplish these aims: a) by attracting world renown senior scientists as presenters and participants; b) by carefully organizing each dedicated conference session with theoretically divergent perspectives and ethnically diverse participants; c) by enriching the scientific sessions with an open schedule to foster dialog and interaction between and among investigators; d) by reaching out to and promoting the participation of young investigators in poster sessions and scholarship initiatives, and e) by ensuring that conference results are shared with the broader scientific community through publication of all speaker and poster abstracts. The Meeting is further strengthened by a strictly enforced press policy, one that protects data presented at the Meeting for future peer-reviewed publication. This policy prohibits the members of the press from disclosing any information presented at the Annual Meeting without the permission of the presenter. An estimated 650 scientists and clinicians from around the world are expected to participate in this five-day meeting scheduled for August 29 - September 2, 2005 at the Baltimore Waterfront Marriott. To increase participation by FDA and NIH scientists the meetings in 2006-2009 will be alternate between Bethesda and Baltimore in suitable locations. The Meeting for 2005 will offer a combination of single track and dual track lectures. The incorporation of dual track sessions is a result of feedback from our participants who like some of the more specialized sessions that may not be of interest to the entire group but may not be topics at more general meetings they attend including topics like HTLV. This change also allows for more time to be set aside for oral presentations selected from a call for abstracts. Each conference session opens with a senior scientist's overview presentation on the current base of knowledge in the topic of discussion. This overview provides a basis for evaluation of the data presented by a panel of speakers, each sharing the results of their work. The Annual Meeting Program Committee, responsible for the program content, is encouraged to invite speakers with differing perspectives to stimulate open discussion. Posters are displayed throughout the meeting. One evening is set aside for a reception in the poster area to encourage attendance and interaction. Evening social events are provided to encourage participants to meet with current and potential collaborators, and to talk with presenters. The Annual Meeting Scholarship Initiative provides funds to ensure the participation of promising women, ethnic and racial minorities, and other individuals who have been traditionally underrepresented in science as well as participants from the developing world.
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{
"pile_set_name": "NIH ExPorter"
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My objectives in seeking a K08 career development award are two-fold: 1) to examine the multipotentiality of post-natal human keratocytes and human neural crest stem cells in ovo and;2) to develop my career as an independent investigator in stem cell biology by hands-on research experience, didactics and mentorship. The most common causes of human corneal blindness are visually significant stromal scarring and endothelial cell dysfunction. In the US, it is predicted that with the advent of refractive surgery, the supply of donor corneas suitable for transplantation will be significantly reduced. Because of these challenges, there is significant interest in pursuing the use of cells that have the ability to self-renew, differentiate into multiple cell lineages, and remodel tissues in vivo, in the treatment of corneal disorders. While there have been recent reports of human cornea stem cells that can be induced to express markers consistent with multi-potency in cell culture, little is known about the multi-potentiality of differentiated cornea stromal cells. Our preliminary data indicate that human keratocytes isolated from postnatal corneas have the ability to differentiate into neural crest derivatives in the chick embryonic environment. This is the first evidence, albeit early, that human keratocytes and postnatal (versus embryonic) keratocytes retain the multi-potentiality of the neural crest precursors from which they are derived as partially restricted progenitors. The working hypothesis of this proposal is that human postnatal keratocytes retain the multi-potency of their neural crest precursors and have the ability to differentiate into neural crest derivatives, including other ocular tissues. Further, the chick embryonic microenvironment likely contains the adequate signals required to differentiate human neural crest stem cells into ocular tissues, as well as other neural crest-derived structures. Three specific aims will be addressed;Aim 1;characterize the multipotentiality of human post-natal keratocytes, using the chick embryonic environment as an assay system. Aim 2: examine the effects of age and differentiation status on the multipotentiality of human keratocytes. Aim 3;explore the potential for human neural crest stem cells to form neural crest ocular derivatives in ovo. RELEVANCE (See instructions);The availability of donor corneas often limits the ability to treat corneal scarring, the second most common cause of blindness worldwide. Our goal is to understand the ability of post-natal human keratocytes and neural crest stem cells to differentiate into ocular tissues in an embryonic environment. This will serve as a basis for determining the feasibility of creating specialized cells for use in regenerative medicine.
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{
"pile_set_name": "NIH ExPorter"
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Project Summary In a given year, over 60% of adolescents in the U.S. directly experience interpersonal violence or witness family or community violence. Although exposure to interpersonal violence increases adolescents? risk for numerous psychosocial difficulties, not all adolescents develop problems. Physiological stress regulation is a critical factor underlying the vulnerability of violence-exposed youth. A growing body of evidence links interpersonal violence exposure to aberrant patterns of arousal and regulation in parasympathetic (PNS) and sympathetic (SNS) activity, particularly to social challenges. It is believed that the dynamics of interpersonal violence (betrayal, stigmatization, powerlessness) render youth more likely to perceive interpersonal stressors as threatening and mobilize threat-related ANS stress responses. Unfortunately, research evaluating these claims is limited in important ways, leaving significant knowledge gaps regarding (1) how violence-exposed adolescents regulate autonomic responses in age-normative interactions that pull for interpersonal violence dynamics and (2) whether adolescents? autonomic responses in these contexts are related to psychosocial functioning across domains (e.g., mental health, interpersonal functioning, health risk behavior). The proposed project adds to a recently awarded NIH grant that will validate a developmentally sensitive assessment of these violence dynamics using virtual reality (VR) technology (HD092956). Specifically, this project will add assessments of autonomic regulation (heart rate variability and cardiac pre-ejection period) to the VR assessment to examine associations between adolescents? violence exposure, ANS regulation during the VR assessment, and adolescent adjustment across various domains (mental health, interpersonal functioning, and health risk behavior). We will also explore potential moderators of these associations, including gender and parenting, as well as individual differences in the synchrony of PNS and SNS regulation. Findings will advance knowledge of how adolescents regulate stress during age-normative interpersonal challenges and inform the development of larger prospective studies that can examine longitudinal relations between violence exposure, stress regulation in real world contexts, and adolescent adjustment. Since the VR assessment is highly portable, it can be disseminated broadly to researchers to further advance theory and research on the cascading effects of violence exposure on stress regulation and adjustment. In addition, our paradigm could be incorporated into clinical research on trauma-informed interventions targeting stress regulation and violence dynamics or provide a platform for new interventions for improving stress regulation and enhancing competencies through the rehearsal of triggering or age-salient challenging interactions.
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{
"pile_set_name": "NIH ExPorter"
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Thyrotoxic osteoporosis, which is accompanied by a high fracture risk, is known to arise from pro-resorptive effects of high thyroid hormone. We reported that TSH-signaling deficient mice lacking the TSH receptor (TSHR) display severe osteoporosis, suggesting that low TSH levels also contribute to thyrotoxic bone loss. That haploinsufficient euthyroid TSHR mice had an equally profound phenotype suggested that the effects of TSH were independent of thyroid hormones. Nonetheless, it remains unclear whether TSHR activation by stimulating antibodies in Graves' disease reduces the hyperthyroid bone loss that is due to high thyroid hormones and low TSH. We further showed that TSHR activation inhibits osteoclast formation, function and survival, as well as the production of TNFa. When TNFa is ablated from TSHR-/- osteoclasts the enhanced osteoclastogenesis and osteopenia are both rescued, suggesting that TNFa plays a key role in thyrotoxic bone disease. Recently, we observed that TSH, when injected intermittently as far apart as once every two weeks, prevented and restored ovariectomy-induced bone loss by inhibiting bone resorption and stimulating bone formation. We hypothesize that TSH preserves the skeleton through potent anti-resorptive and anabolic actions, and that a loss of these actions contributes to the bone loss of hyperthyroidism. We will use genetically modified mice and state-of-the-art molecular approaches to understand the role of TSH in hyperthyroid bone loss. We will first attempt to rescue the TSHR-/- phenotype by deleting TNFa or its receptors, p55 or p75, in double mutants, or by transgenically reconstituting TSHRs in TSHR-/- osteoclasts or osteoblasts. Next, we will determine whether stimulating anti-TSHR antibodies given by injection or produced in vivo by adeno-TSHR immunization attenuate hyperthyroid bone loss. Finally, using mice in which TSHRs are restored cell-selectively in osteoclasts or osteoblasts on a TSHR-/- background, we will examine which cell contributes to the prevention and restoration of post-ovariectomy bone loss by TSH. These foundation studies should allow us to consider skeletal protection by TSH in post-menopausal women whose TSH levels are suppressed by thyroxine therapy for non-cancer causes.
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{
"pile_set_name": "NIH ExPorter"
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The proposed project will utilize the comprehensive dataset from the Comparison of Age-related Macular Degeneration (AMD) Treatments Trials (CATT), a multi-center non-inferiority clinical trial, sponsored by the National Eye Institute, to assess the efficacy and safety of ranibizumab (Lucentis) and bevacizumab (Avastin) when treated monthly or PRN (pro re nata). The specific aims of this proposal do not include the major goals of the CATT Study, which were already accomplished. Instead, the proposed project involves additional secondary analysis of the CATT dataset to address the following important new aims: (1) To describe the response pattern of visual acuity over time during the 2 years of treatment, and to determine whether the short-term response of visual acuity (after 1 or 3 injections) predicts the long-term (2 years) visual acuity response. (2) To describe the morphologic changes captured by OCT and fluorescein angiography over time during 2 years of treatment, and to evaluate whether the morphologic responses at 3 months predict the long-term morphologic responses at 2 years. (3) To assess the associations between vision and morphologic changes from anti- VEGF treatment at 2 years. (4) To describe treatment patterns and identify the prognostic factors associated with a low number of treatments in patients treated PRN for 2 years. (5) To assess genotype-phenotype associations with respect to the morphologic features and severity of neovascular AMD at presentation. (6) To evaluate the association of anticoagulation medications with retinal hemorrhage in patients with neovascular AMD. The CATT dataset is a unique resource to further evaluate the effects of Lucentis and Avastin on visual and morphologic changes, to elucidate the association between visual and morphologic responses, and to determine the factors (genetic and systemic drug exposure) that may be associated with features of neovascular AMD at presentation. The analyses of the CATT dataset will provide useful information to the treating ophthalmologists and their patients on what to expect from treatments, and to help monitor and optimize the treatment of the neovascular AMD by personalized medicine. The very large sample size (N=1185), high quality of the dataset, as well as the rich experience and high productivity of the investigators along with the full supports of the CATT Research Group, support the feasibility and likelihood of high yield of the proposed project.
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{
"pile_set_name": "NIH ExPorter"
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We proposed to examine patterns of maternal interactive behavior with infants and young children who are physically/cosmetically handicapped. Our aims are to document the mother's accommodation to her child's behavior (expected or deviant) and to relate divisions of maternal style to the emergence and patterning of competent behaviors in the child. Competency is considered here in a broad ecological framework, covering 3 interrelated aspects of child functioning: social competence, e.g. the ability to get the mother's attention; affective competence, e.g. the ability to maintain emotional equilibrium under stress; and cognitive competence, e.g. a capacity for concentration. Qualities of maternal behavior such as responsivity, contingency of vocalization, or effective limit-setting are related to competency in the normal young child. Investigations of maternal interaction with physically handicapped infants and young children at cognitive risk reveal differences, which presumably have negative consequences for the child's later successful functioning. Ample evidence exists of later cognitive and social impairment in physically/cosmetically handicapped children. No study has followed such children from infancy into the preschool period, documenting changes in mother and child behavior as the child moves into a wider social system. We propose to investigate maternal style, infant and child competency, and the relation between them, in a sample which is sufficiently broad to allow a consideration of the impact of the child's sex, the location, severity and degree of possible remediation of his handicap and the family's social background. Thirty mother-child pairs in each of 2 groups (1. healthy, full-term deliveries; 2. cosmetic/physical handicap: full-term deliveries with conditions such as cleft lip of limb deformation) will be seen at 9, 12, 18, and 24 months. Further, we will continue to see our current homogenous sample of 14 normal and 14 cosmetic/physical handicap mother-child pairs (followed now from 9-24 months) at ages 3 and 4. Competency and maternal style will be examined by videotape analysis of semi-structured naturalistic situations, using the Maternal Style Scale, the Infant Competency Scale, standardized developmental testing, and measures of child coping, attention focus and behavioral symptoms. In this way, we hope to idenfity characteristics of the child, his illness, and of maternal response associated with subsequent healthy or maladaptive functioning.
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{
"pile_set_name": "NIH ExPorter"
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Relationship to Alzheimer's disease and its related dementias This application is a response to NOT-AG-18-039 . This pilot study, using 60 subjects from one clinic, will be the first to test Alzheimer's disease (AD) patients directly on a thorough battery of standard, objective diagnostic tests of the vestibular system (ENG). Subjects will be patients with mild to moderate AD. We will estimate the prevalence of vestibular disorders in AD and compare their performance to that of healthy controls in the parent study who will have been tested on the same test battery. Therefore, we will be able to determine if the prevalence differs from age-matched controls. We will use clinical data to determine if severity of cognitive impairment or reduced hippocampal volume on MRI are related to increased prevalence of vestibular impairment.
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{
"pile_set_name": "NIH ExPorter"
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Bone tissue deficiencies, including poor implant-bone integration, non-union fractures, and bone loss associated with diseases such as osteoporosis, trauma, joint replacements, and tumors, have tremendous socioeconomic impact in terms of disability and related health care costs. Biomaterial-based strategies to enhance implant osseointegration and bone formation will enable the development of biologically active and integrative orthopaedic and dental implant technologies to address these pressing clinical issues. The objective of this project is to engineer biomaterials presenting cell adhesive motifs that specifically bind to integrin receptors involved in bone formation in order to promote implant osseointegration and bone repair. Our central hypothesis is that precise presentation of pro-osteogenic, integrin-specific ligands will direct osteoblastic differentiation, implant osseointegration and bone repair. We have formulated this hypothesis based on our work with two engineered integrin-specific ligands that recapitulate the secondary structure of their native ligands (triple-helical GFOGER peptide from collagen-I for a2b1 integrin, recombinant FNIII7-10 from fibronectin for a5b1 integrin) and promote osteoblastic differentiation, implant osseointegration, and repair of bone defects. Aim 1: Engineer integrin-specific coatings that enhance screw-bone integration in healthy and ovariectomized rats. Aim 2: Engineer poly(ethylene glycol)-based hydrogels presenting integrin-specific ligands as grafting templates and BMP-2 delivery vehicles for the repair of non-healing bone defects. This research is innovative because it focuses on engineering novel biomaterial coatings and hydrogels with specificity for pro-osteogenic integrins to promote bone formation and repair. This work will establish the extent to which presentation of integrin-specific ligands enhances screw-bone integration in both healthy and osteoporotic bone. Also, we will engineer novel hydrogels as grafting materials and BMP-2 delivery vehicles for enhanced repair of non-healing bone defects. Collectively, these studies will establish novel bioactive materials that enhance bone formation and implant integration for improved bone repair in various clinical applications.
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{
"pile_set_name": "NIH ExPorter"
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Project Summary The intestinal epithelium exhibits a remarkable capacity of self-renewal which reflects the activity of multi- potent intestinal stem cells (ISCs) that divide and differentiate to produce the different cell types that comprise the intestinal epithelium. Control of ISC activity is thought to be mediated by factors produced by other cells that reside either in the stromal layer surrounding the intestinal crypt, by cells in the adjoining epithelium, and by the microbiome that resides in the small intestinal lumen. These factors create an environment called the ?stem cell niche?. Our past work and preliminary findings indicate that epithelial WNTs, stromal myofibroblasts and monocytes, and commensal derived factors are all components of the ISC niche. This renewal application seeks support to continue to collaborate in the Intestinal Stem Cell Consortium (ISCC) to extend our previous work defining the role of epithelial, stromal, and luminal intestinal factors that regulate ISC proliferation to acquire knowledge to regenerate and rebuild the human intestine. Gaining a molecular understanding of the role that these cells and factors play in the niche will be essential to consider when building intestine. Our proposed studies within the goals of the ISCC are follows: Goal 1: define essential niche components that contribute to intestinal epithelial homeostasis by (a) determining what microbial communities inhabit the human intestinal crypt and what their role is in regulating the ISC and (b) evaluating how intestinal myofibroblasts and monocytes affect proliferation and lineage differentiation of ISCs. Goal 2: determine niche components that are altered following injury and evaluate whether they play a role in epithelial regeneration by (a) dissecting the molecular signaling pathways in damaged epithelium that stimulate the ISC through WNT3, and (b) determining the specific role of human WNT2B in ISC?induced regeneration. The mission of the ISCC is to characterize the minimal, required niche cells and factors that support ISC in health and disease, using an integrated, multidisciplinary team science approach. Our proposed research complements and synergizes with the other eight projects of the ISCC to achieve the ultimate goal of developing both humoral and tissue therapy for gastrointestinal diseases via modulation of factors that comprise the ISC niche. Combining and sharing our skills and resources will allow rapid advancement of ISC biology and application of new knowledge to repair and build an intestine.
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{
"pile_set_name": "NIH ExPorter"
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The post-translational processing of initiator methionine during protein synthesis is a universal biological process that is conserved from prokaryotes to eukaryotes. Methionine aminopeptidases are the enzymes catalyzing the removal of initiator methionine. One gene is known in prokaryotes while two genes are known in eukaryotes encoding methionine aminopeptidases. The importance of initiator methionine processing is underscored by the fact that deletion of the methionine aminopeptidase genes in either prokaryotes or eukaryotes is lethal. Of the two methionine aminopeptidase genes in eukaryotes, the type 2 enzyme (MetAP2) has been shown to be the direct target for the fumagillin family of angiogenesis inhibitors, including its analog TNP-470. Work in the past several years has provided strong evidence that MetAP2 is a physiologically relevant target for TNP-470. It has also been found that inhibition of endothelial cell proliferation by TNP-470 is mediated by the tumor suppressor gene p53. Thus, TNP-470 is capable of activating p53, which induces the expression of p21that is responsible for the cell cycle blockade of endothelial cells. These studies reveal a unique role of MetAP2 in the progression of the endothelial cell cycle. Recently, a novel anticancer drug entering Phase II clinical trial known as bengamide was found to inhibit both MetAP2 and MetAP1 in vitro and to block cell cycle in both G1 and G2/M phase. It is hypothesized that MetAP1 may play a role in the cell cycle at the G2/M phase. The major objective of the current proposal is to further delineate the physiological functions of MetAP1 and MetAP2 employing yeast as a model system and to identify isoform-specific inhibitors for MetAP1 and MetAP2 by high throughput screens. The functions of the different domains in MetAP1 and MetAP2 will be investigated by creating various yeast mutants expressing different domains of these enzymes and determining the phenotypic changes using DNA microarray. The different known activators of p53 will be systematically examined to identify potential mediators of p53 activation by TNP-470. Molecular probes of bengamide will be prepared to confirm its interaction with MetAP1 and MetAP2. High throughput screens will be conducted to identify specific inhibitors for MetAP1 and MetAP2. The newly identified inhibitors for MetAP1 will be employed to assess the physiological role of MetAP1 in the cell cycle progression in the G2/M phase.
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{
"pile_set_name": "NIH ExPorter"
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The past and current theme of my research is the role viruses and virus-related genes play in the etiology of naturally occurring cancers. Within this framework our major approaches have included the population-level seroepidemiology, the immunobiology, and the virology of the agents involved. Until about 3 years ago most of our attention was directed to the study of the feline leukemia (FeLV) and feline sarcoma (FeSV) viruses in cats, since this represented the only outbred mammalian species where such studies could be conducted at both the population and individual host level. Recently we have devoted an increasing proportion of our laboratory effort to the study of human T-cell leukemia viruses (HTLV) and to hepatitis B virus (HBV). The studies with FeLV led to the first evidence that RNA tumor viruses are horizontally transmitted, the first evidence of retrovirus-induced immunosuppression, the identification of the first mammalian onc gene protein (in this case gag-fes) and the best evidence to date of immunosurveillance in a naturally occurring cancer. Studies with HBV led to evidence for replication in sites other than liver (lymphocytes). Studies with HTLV have let to identification of an HTLV agent as the apparent cause of AIDS, the first identification of the HTLV env gene products, the first demonstration of the pX putative transforming protein(s) of HTLV, and the link between HTLV-like agents and lymphoma development in monkeys. Hemophiliacs were shown to be infected with HTLV, and transfusion-associated AIDS were linked to HTLV exposure, using tests based on our observation that the env gene proteins are the most immunogenic for use as serologic markers. We plan to continue studying all 3 agents, as well as the newly identified primate virus, but with primary emphasis on the HTLV family. Our approaches will include studies on immunoregulation, and the characterization of viral and virus-induced proteins, both as markers for seroepidemiology and as keys to understanding leukemogenesis and lymphoid cell function. We will also study the role of HTLV-type agents in diseases such as aplastic anemia and AIDS. In the feline system our primary emphasis will be on mechanisms of FeLV-induced immunosuppression, and the role of FeLV and various onc genes in the causation of "virus-negative" cat tumors. With HBV we will continue to study the role of lymphoid and myeloid-stage infections and we will develop the monkey HTLV-related lymphoma and AIDS models to parallel our studies of HTLV in people.
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{
"pile_set_name": "NIH ExPorter"
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The vertebrate retina receives, processes and transforms visual information. Light absorbed in photoreceptors is transformed into electrical signa.ls that evoke light modulated release of glutamate onto horizontal and bipolar cells (HCs and BCs). HCs provide lateral feedback regulating transmitter release from photoreceptors according to conditions of ambient illumination, while BCs transfer light signals forward to amacrine cells (ACs) and ganglion cells (GCs). ACs are retinal interneurons with mainly inhibitory and modulatory roles. GCs transform light signals into trains of action potentials that propagate through the optic nerve to brain visual centers. As images pass through retinal circuitry, they are decomposed into component parts, so that at the retinal output specialized sets of GCs extract different components of the image. Some signal highlights, others shadows, movement, direction or color. Signal processing within the retina is achieved by sets of neural circuits. These are composed of patterns of connections among neurons, and specialized neurotransmitter to neurotransmitter receptor interactions. The research program studies the relationships between receptor expression on retinal neurons, the neural circuitry of the retina, and retinal information processing tasks. Zebrafish provides a model of human genetic disease. Through studies of this model, genetic and other perturbations of human vision may be better understood. We examine zebrafish retinal function using acutely dissociated retinal neurons, light responses from in vitro eyecup preparations, and zebrafish retinal slices. Using the voltage probe (oxonol, DiBaC4(5)) to study glutamate responses in dissociated zebrafish retinal HCs we have found both short and long term effects on membrane potential. The short-term effect is classical excitation. The long-term effect is a post excitatory restoration of membrane potential (after-hyperpolarization or AHP). AHP also occurs in OFF-type BCs but not in ON-type BCs. It peaks about 5 min after glutamate application. Glutamate antagonists (CNQX) block AHP. Some glutamate agonists (kainate, AMPA, (S)-5-Fluorowillardiine) evoke the response whereas others (NMDA, D-aspartate, SYM 2081, DL-AP4) do not. This pattern suggests AMPA receptor activation. Some HCs are tonically depolarized. Glutamate evokes only AHP signals in these cells. Resting potentials are restored by nifedipine in tonically depolarized cells, suggesting a sustained, depolarizing action of L-type Ca2+ channels. AHP is not blocked by the L-type Ca2+ channel-blocker nifedipine, however, and it is insensitive to [Cl-]o. AHP is blocked by [Li+]o substitution for [Na+]o and by ouabain. This suggests that Na+ entering through ionotropic AMPA channels stimulates Na+, K+ -ATPase. The electrogenic action of the ATPase restores membrane potential, generating the AHP response. Patterns of ATPase immunoreactivity support localization in the outer plexiform layer (OPL) as cone pedicles, HCs, and BCs are strongly labeled. Persistent stimulation of distal retina by photoreceptor glutamate may induce increased expression and activity of Na+, K+ -ATPase, with a consequent impact on distal glutamate responses. Heightened ATPase activity may be one of several adaptive mechanisms protecting distal retinal neurons against excitotoxicity from tonically released photoreceptor glutamate. Cell structure, a key element in discerning retinal circuits, is beautifully delineated in the zebrafish model. Neurons in retinal slices can be stained in isolation shotgun fashion by spraying DiI coated microcarriers onto the cut surface with a gene gun. This is the Diolisitic technique. Diffusion of the lipophilic tracer along membranes reveals the shapes of individual neurons. In a study of 300 stained zebrafish retinal neurons different subtypes of HCs, BCs and ACs were observed. Based on cell body shape, and the presence or absence of an axon, 3 HC morphologies were identified corresponding to stellate and elongate types earlier seen in cell culture. Based on branching patterns of axons and dendrites within the retinal inner plexiform layer 17 BC morphologies and 7 AC morphologies were discerned. Cells with ON-type (branching in IPL sublamina b), OFF-type (branching in sublamina a) morphological signatures were about equally prevalent. Mixed a/b branching patterns were also observed. In previous studies using Lucifer-dye filled patch electrodes 13 morphological types of bipolar cell were identified. There is substantial agreement on morphological types between the two studies. Whole-cell patch recording and puff pipette techniques have identified glutamate receptor mechanisms on the BC dendrites of many of these types showing a distribution of 3 basic glutamate receptor types: AMPA/kainate (OFF cells), mGluR6 (ON cells) and glutamate-gated chloride currents (Iglu, ON cells). Diolisitic studies, however suggest that in addition to 3 distinct strata within sublamina a of the IPL, and 2 within sublamina b, there is also a third stratum of sublamina b, intermixed with the retinal ganglion cell layer. ARPE-19 cells are a self-immortalized human retinal pigment ethelial cell line that transforms into neural like morphologies with processes and growth cones after treatment with the vitamin A derivative phenretinide. Studies are currently underway to examine expression of neurotransmitter receptors in these cells.
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{
"pile_set_name": "NIH ExPorter"
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Ectopic pregnancy (EP), the implantation of a fertilized egg outside the uterine corpus, is a serious acute medical condition that also confers increased risk of substantial future reproductive morbidity, including subsequent ectopic pregnancy and infertility. The U.S. Centers for Disease Control and Prevention (CDC) documented a nearly 4-fold increase in EP occurrence between 1970 and 1989, from 4.5 to 16/1000 reported pregnancies. The most recent U.S. surveillance data, from 1992, estimate lower rates (11/1000 pregnancies), but the shift toward outpatient management of the condition and the reliance on hospitalization records for national surveillance compromise the accuracy of this estimate. Although sparse, available evidence indicates that EP rates may not be declining; and a number of important risk factors-including tubal sterilization, advanced maternal age and Chlamydia trachomatis and other asymptomatic sexually transmitted infections -have become increasingly common. As EP surveillance in the U.S. based on nationally representative sources has become untenable, it has been suggested that other surveillance strategies should be tried, including use of automated databases available at selected healthcare plans. The current application proposes to provide population-based data on long- term (is-year) trends in EP rates within a large U.S. health maintenance organization via analysis of data from an extensive set of administrative data sources. In addition to overall and age-specific trends over time, analyses will include evaluation of inpatient vs. outpatient management and medical vs. surgical treatment modalities. We will also document trends in potentially important etiologic factors during this same interval, including their occurrence among women with ectopic pregnancy. These analyses will provide data to: i) better assess the current status of ectopic pregnancy as a public health problem in the U.S.; 2) evaluate potential risk factors that may merit more in-depth investigation in future etiologic and prevention studies; and 3) provide case selection and programming approaches for establishing more widespread surveillance and evaluation of this condition by aggregating population-based data from healthcare settings with similar resources. [unreadable] [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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Most top-level searches of scientific literature include querying of structured fields such as author, subject, or affiliation. A free-text search of abstracts or full texts entries would be more flexible allowing queries with any word combination including ranges of names and identifiers. Unfortunately, free text searches usually yield incomplete and often erroneous results since the naming of biologically important molecules (genes, proteins, substrates) is not standardized. Unless a specific query issued to a retrieval service (e.g. PubMed) covers all possible aliases of a given protein or gene the results may be insufficient or simply wrong. The system proposed here translates the problem of looking up literature pertaining to a certain protein to the sequence level. By correlating existing identifiers, names, and synonyms of proteins with their sequences this lookup increases the accuracy and coverage of the results. A particular challenge that our system will uniquely address is the following. Increasingly structural and functional genomics projects bring up proteins for which nothing is known. If someone published some new experimental that will actually name such a protein, this important knowledge will likely be lost to the genomics investigator because PubMed alarms need to be activated by keywords and names. Our system could fill in the gap: users will be able to deposit sequences corresponding to proteins of unknown function/name. If experimental information will be published for the same or a related sequence the original investigator will be notified. PUBLIC HEALTH RELEVANCE: The experimental and computational data appearing daily in publications is critical to the advancement of biological research. However, the sheer quantity and high frequency in which new data is published turns bench scientists into research librarians trying to sift through the flood of information while searching for relevant and reliable data. Furthermore, as biological research is increasingly driven by the study of proteins and genes that mostly lack annotations, or even an identifiers, there is a need to access the literature by using sequence data alone. By automating the process of searching and discovering relevant information as it becomes available, the proposed system promises to save time and increase the coverage of relevant and reliable data retrieved by a given search in an intuitive and "easy to consume" format.
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{
"pile_set_name": "NIH ExPorter"
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Hydroxysteroid dehydrogenases (HSD)s constitute an important class of steroid metabolizing enzymes. The reversible oxidoreductase reactions mediated by these enzymes have dramatic effects on the hormonal activities of corticosteroids, progestins, androgens, and estrogens. By using mechanism-based irreversible inhibitors of HSDs and studying their mechanisms of inactivation, information relevant to the pharmacologic manipulation of this class of enzymes can be obtained. The specific aims of this proposal are: 1) the synthesis of novel mechanism-based irreversible inhibitors of three different HSDs, 2) the enzymatic evaluation of these inhibitors, and 3) the elucidation of the structures of covalent HSD-inhibitor adducts. The proposed mechanism-based inhibitors are acetylenic alcohols whose enzymatic oxidation by a particular HSD will generate a conjugated acetylenic ketone that can be attacked by an enzyme nucleophile to form a covalent adduct and thus inactivate the enzyme. Three different series of acetylenic compounds, one series for each of the three hydroxysteroid dehydrogenases included in the study, will be prepared. Kinetic and mechanistic data needed for the structure/activity correlations will be obtained from measurements of rates of HSD inactivation under a variety of experimental conditions. Structural data will be obtained from carbon-13 nuclear magnetic reasonance (NMR) experiments done with HSDs inactivated by enzyme-generated acetylenic ketones that contain less than 99% carbon-13 enrichment in both carbon atoms of the acetylenic bond. Solution and/or solid state NMR experiments are proposed. Structural conclusions based on carbon-13 chemical shift data from these NMR experiments will be made possible by comparison of the observed chemical shifts with those found in appropriate model adducts.
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{
"pile_set_name": "NIH ExPorter"
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The long-term objective of this research is to understand how the cerebral cortex acquires and stores information, which can lead to neurobiological interventions in maladaptive thought and behavior, and impaired learning in development and aging. Although the cerebral cortex is by far the largest part of the human brain and is a major structure that subserves learning and memory, it has been neglected because of the need for animal models of cortical memory. However, recent studies have established that the auditory cortex develops memory-like changes in the frequency tuning of individual cells during fear learning, termed "receptive field plasticity". This neural plasticity is associative, highly specific, rapidly acquired and retained indefinitely, like major forms of human memory. Cortical control of subcortical fear centers is known but not understood. The specific aims of this project are to determine the nature and extent to which the overall functional organization of the auditory cortex is involved in fear learning and memory. The specific goal of this research project is to provide the first comprehensive investigation of the extent to which learning modifies the spatial representation of behaviorally relevant acoustic frequencies in the primary auditory cortex. Guinea pigs will be trained with pure tones in habituation, sensitization, classical, and instrumental conditioning. Further, the development and long term retention (weeks), and the degree of reversibility of plasticity will be delineated. The neurobiological bases of fear conditioning, which is basic to acquired anxieties, phobias and other behavioral pathologies, requires direct investigation of cellular mechanisms because pharmacological treatments can reduce the elicitation and expression of these emotions but not eliminate their cause.
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{
"pile_set_name": "NIH ExPorter"
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Perhaps the major unsolved problem in diabetes is the pathogenesis of the long-term complications. There is ample evidence that in diabetes a variety of proteins are modified by non-enzymatic glucosylation, in direct proportion to the degree of hyperglycemia. Glucosylated hemoglobin, by far the most thoroughly studied protein, has proven useful as an aid in monitoring the control of diabetic patients. We plan to investigate the structure and function of other modified proteins with emphasis on those that may contribute to diabetic complications. We plan to utilize a combination of borohybride reduction, boronate affinity chromatography, and high performance liquid chromatography to accurately quantify glucosylated amino acids. Thus far, estimates of glucosylated hemoglobin and albumin have been seriously hampered by lack of accurate quantitation. We hope to develop an analytical scheme that detects chemical adducts rather than one that depends on incorporation of tritium label. This approach will be combined with mass spectroscopy in order to identify further products of the Maillard reaction scheme including possible sites of protein-protein cross-linking. Special emphasis will be placed on the following proteins: serum albumin, lens crystallins, and glomerular basement membrane collagen. We plan to establish the relationship between non-enzymatic glucosylation and life span of these proteins. In addition, we hope to gain insight into how glucosylation at specific sites on these proteins might affect function and fate in diabetic tissues.
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{
"pile_set_name": "NIH ExPorter"
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The United States currently has the largest number of older adults at any time in its history, and this number is expected to rapidly increase over the next 20 years. This rapid increase in the aged population highlights the need to better understand changes that accompany aging and which may contribute to mental health and well-being in later life. One important and relatively unexplored aspect of aging relates to the changes that occur with decision making in the social and emotional realms. Making the correct social and emotional decisions during the lifespan is an essential contributor to well-being. One model that describes social and emotional flourishing with aging is Socioemotional Selectivity theory. According to Socioemotional Selectivity theory, there is an increasing focus on positive emotion over the adult lifespan such that older individuals selectively choose social interactions where the likelihood of positive emotional experience is high. In this setting, social contacts are limited to good friends and family, and greater satisfaction and well-being is achieved while the number of social contacts decreases. The underlying neural mechanisms that support this enhanced Socioemotional decision-making have received relatively little attention, but are important as they may protect from social isolation and mental illness. The specific purpose of the current proposal is to examine how the brain correlates of two well defined aspects of Socioemotional decision making - primary appraisal and affective learning about social objects (i.e., people) - are altered with aging. We will focus on two specific brain areas involved in these decision-making processes, the ventromedial prefrontal cortex and the amygdala. We plan to collect and analyze structural and functional magnetic resonance imaging data and self-report measures of social network characteristics and well-being. The overarching hypothesis is that age-related differences in ventromedial prefrontal cortex-amygdala circuitry during Socioemotional decision- making are closely related to Socioemotional selectivity and well-being in aging. Aim 1 will investigate age-related changes in the structure-function relationships between the ventromedial prefrontal cortex and amygdala during different aspects of Socioemotional decision making. Aim 2 will investigate how the ventromedial prefrontal cortex-amygdala circuitry relates to social network characteristics and well-being across the lifespan. The results of these studies will elucidate the neural bases of healthy Socioemotional decision-making and well-being in aging.
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{
"pile_set_name": "NIH ExPorter"
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Most visual and visuomotor functions hinge on our visual system's ability to localize objects in dynamic scenes-to determine the positions of objects as we move or as objects in the world move around us. However, dynamic localization requires that the visual system must be able to both assign and update object positions, and it remains unclear which visual areas play these necessary roles and what mechanisms are responsible. Assigning and updating object locations is especially relevant considering that the eye is rarely stabilized and objects are constantly drifting across the retina as they move. Given the sluggish nature of visual processing, how do we perceive the positions of moving objects with clarity and precision? To understand how the visual system assigns and updates object positions, we must approach the task of localization not as a static process, but as a dynamic one. The goal of our proposed experiments is to characterize the neural pathways and mechanisms that determine perceived position in dynamic situations. Our preliminary results suggest that two mechanisms are required to perceive the positions of objects when motion is present on the retina: trailing edge deblurring and edge-shifting. Without these mechanisms, we would perceive the world as a blurry smear, unable to accurately predict the future locations of objects or interact with them as they move around us. We hypothesize that these two independent mechanisms integrate information between early visual areas (V1) and the motion sensitive region (MT+). Our pilot data for the proposed experiments suggest that (1) MT+ carries precise information about object position, supporting the hypothesis that it serves an integral role in object localization, and (2) feedback as well as feed-forward connections between V1 and MT+ may be necessary to assign and update the positions of objects when retinal motion is present. Using functional magnetic resonance imaging (fMRI), transcranial magnetic stimulation (TMS), and psychophysics, we will characterize and isolate the neural locus of the two mechanisms and establish their causal roles in perceiving object position. Dynamic localization is the default situation faced by the visual system, but many neurological disorders produce specific deficits in dynamic visual and visuomotor localization, including optic ataxia, dyslexia, akinetopsia, autism, and schizophrenia, among others. Until we understand how position is determined in the normal brain for images that are constantly moving across the retina, we lack the necessary insight to develop diagnostic tools, predictive markers, and therapies for these impairments. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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Comparison of the deduced amino acid sequences of three viral myc sequences (from avian acute leukemia viruses MC29, MH2, and OK10) with cellular proto-myc sequences from trout, chicken, mouse, and human cells indicates that there is a single position where each of the viral sequences differs from the cellular sequences. This position and its neighbors are completely conserved in the vertebrate sequences examined. Since this is unlikely to have occurred by chance alone, the mutations in this position may influence the oncogenic potential of the viruses. A simultaneous comparison of the trout, chicken, and human cellular myc sequences indicated that while on the whole these sequences are highly conserved, each sequence contains unique regions that likely were introduced by insertion events. The genome of the AIDS virus, HTLV-III, contains an open reading frame termed sor that is conserved among different isolates of the virus. Antibodies raised against a bacterially-expressed protein containing sor sequences precipitated an Mr 23,000 protein. This protein comigrated with protein precipitated by some sera of HTLV-III-infected humans and, thus, is likely the product of the viral sor region. When radiolabeled HTLV-III-infected T-cells were fractionated and the fractions assayed for sor by radioimmunoprecipitation, the sor protein was determined to be predominantly located in cytoplasm.
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{
"pile_set_name": "NIH ExPorter"
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Stem cell therapy and Ab clearance through neprilysin (NEP) gene delivery have recently been shown to be effective treatments on mouse Alzheimer's Disease (AD) models. It is still unknown whether their effects are additive. We propose here to test this question by the transplantation of an embryonic stem cell (ESC) line that has been modified to express NEP under the control of a doxycycline-inducible promoter. Through this combined strategy of gene and cell therapy, it is expected that the impaired neural circuit may be repaired by the formation of synapses between the engrafted and host neurons. It is also expected that the engrafted cells may secrete neurotrophic factors and achieve local environment enrichment to improve neuronal survival. Moreover, the engrafted cells may be induced to overexpress NEP to degrade A and reduce plaque burden. Therefore, the proposed study may lead to a more effective treatment option for AD. The innovation of the proposed strategy lies not only in the potential of combined benefit from gene and cell therapy, but also in its ability to allow external control of the biochemical activity of engrafted cells. By transplantng the proposed genetically modified cells, we will test whether NEP expression after cell graft can be controlled by an orally administrated drug, and how this affects brain A levels and disease progress. The ability to control the activity of engrafted cells is of paramount importance to future clinical applications as it can help to achieve optimal therapeutic results and, more importantly, avoid possible adverse effects caused by the over-supply of therapeutic cells. Although AD is studied in the current proposal, we envision that the proposed concept and technology can be readily modified to deliver other therapeutic cells and genes for a wide range of neurological disorders.
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{
"pile_set_name": "NIH ExPorter"
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G protein signaling pathways in the striatum mediate a range of critical neuronal processes that control behavior, locomotion, pain perception and underlie drug abuse and addiction. The normal functioning of these pathways is hinged on the tight control of signal duration mediated by the Regulators of G protein signaling (RGS) proteins. Our long term goal is to elucidate the mechanisms governing the function of RGS proteins in neurons as a necessary prerequisite to understanding neurological disease processes and therapeutic means of their treatment. The main focus of the proposed studies is on RGS9-2, a striatum specific regulator that crucially controls signaling efficiency through dopamine and opioid receptor systems which are exploited by dugs of abuse. However, the molecular mechanisms of RGS9-2 function are largely unknown and need to be elucidated for better understanding of the neurochemical basis of addiction. To this end, we have recently discovered that RGS9-2 in the striatum exists in a complex with a novel neuronal protein which we named R7 Binding Protein (R7BP). Preliminary data suggest that R7BP serves as a critical modulator of RGS9-2 function in neurons. This HYPOTHESIS will be tested by addressing the following SPECIFIC AIMS: 1. To determine the mechanisms mediating stability and localization of RGS9-2. Proposed studies will determine the mechanisms by which R7BP circumvents the proteolysis of RGS9-2 and test the hypothesis that R7BP also determines the localization of RGS9-2 in neurons. 2. To understand the role of R7BP in the regulation of RGS9-2 catalytic activity. We will use a combination of in vitro enzymatic approaches and protein-protein interaction assays to determine the kinetic mechanism of R7BP action in the regulation of RGS9-2's ability to stimulate the GTPase activity of G proteins. 3. To further characterize the molecular composition of the G protein inactivating complex in striatal neurons. We will use proteomics approaches to identify additional components of the macromolecular ensemble involving the RGS9-2/R7BP complex. These studies should provide an understanding of the regulation of signaling in the striatum and generate insights into the molecular mechanisms of G protein signal disruption caused by drug abuse and neurological diseases.
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{
"pile_set_name": "NIH ExPorter"
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Abstract A? oligomers are considered the most toxic structural form of Amyloid beta, causing synaptotoxic changes underlying cognitive decline in Alzheimer?s disease. CogRx has developed the world?s first highly brain penetrant drug that selectively displaces oligomers from synaptic receptor sites and clears oligomers from the brain into the cerebrospinal fluid (CSF). This first-in-class drug, CT1812, allosterically modulates a key protein regulator of oligomer receptors (the sigma-2/PGRMC1 protein complex), destabilizing the oligomer binding site, increasing the off-rate of A?Os and allowing rapid clearance into the CSF, however the interactions between the receptor components and their role in downstream signaling pathways are unknown. CT1812 restores synapse number and cognitive performance to normal in AD mouse models. CT1812 has been demonstrated to be safe and well-tolerated in healthy volunteers dosed once daily for 14 days in a placebo- controlled Phase 1a trial, and is currently being evaluated in a follow-on placebo-controlled safety trial in AD patients. The study will provide critical information about the mechanism of action of this innovative differentiated and first in class compound. Successful commercialization of this drug will require partnership with a large pharmaceutical company to support Phase 2 and 3 clinical trials and marketing, and detailed understanding of the molecular mechanism of action of CT1812 has been cited by potential development partners as being a critical requirement for engaging in such a partnership. CogRx has no other support available for elucidating the details of CT1812?s mechanism of action. This Fast-Tract SBIR proposal will provide crucial details about this novel mechanism of action and will enable the application of the proprietary portfolio of sigma-2 ligands at CogRx to other diseases. This information will increase the chance for partnering opportunities necessary for the clinical development of CT1812 for AD patients and will expand the pipeline portfolio of CogRX into new clinical areas involving autophagy pathways.
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{
"pile_set_name": "NIH ExPorter"
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This research plan will study the mechanism of pressure induced interruption of optic nerve axonal transport in animal eyes with acute experimental pressure elevation. More specifically, the effects of pharmacologic reduction in ocular blood flow upon this transport interruption will be investigated. In addition, this effort will test the hypothesis that pressure induced deformation of structurally weaker regions of the nerve head lamina cribrosa causes this transport interruption. Finally, analysis of preferential involvement of optic nerve axons by this phenomenon will be made to test the hypothesis that specific axon types are most susceptible to this damage.
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{
"pile_set_name": "NIH ExPorter"
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Theiler's murine encephalomyelitis virus (TMEV) is an endemic murine pathogen that induces a demyelinating disease (TMEV-IDD) of the central nervous system (CNS) in susceptible mouse strains with accompanying CNS histopathology characterized by mononuclear cell infiltrates. TMEV-induced demyelinating disease is initiated by CD4+ T cell immune responses to viral proteins early in disease and self proteins in later disease. In susceptible strains of mice such as SJL, TMEV establishes a persistent infection in macrophages, induces a CNS infiltration of macrophages, T cells, and B cells, which results in chronic progressive paralysis. The chronic paralytic nature of the TMEV-induced clinical disease course as well as the CNS pathology presents as a very good model for the human CNS demyelinating disease, multiple sclerosis (MS). One of the major goals of this proposal is to define the cellular and molecular mechanisms that regulate the immunopathogenesis of this chronic inflammatory CNS demyelinating disease. Mononuclear cells infiltrate tissue sites during inflammatory processes and a family of chemotactic cytokines called chemokines have been implicated in this gradient-induced cell migration. The central hypothesis of this proposal is that chemokines are expressed in the CNS during TMEV-IDD and regulate immune responses including cell migration patterns that are required for demyelinating disease development and progression. This hypothesis will be tested by examining the following specific aims: 1) determination of the mechanisms of chemokine function in the initiation of TMEV-IDD 2) determination of the mechanisms of chemokine function in the progression of TMEV-IDD; and 3) determination of chemokine receptor expression and function in the development of TMEV-IDD. These studies will help to understand the immunopathogenesis of MS and provide a basis for the development of novel therapies designed at targeting molecules involved in the migration and accumulation of pathogenic lymphocytes and monocytes into the CNS during the induction and progression of inflammatory demyelinating diseases.
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{
"pile_set_name": "NIH ExPorter"
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PROGRAM SUMMARY This proposal seeks to renew a successful UCSF Asthma and Allergic Diseases Cooperative Research Center dedicated to identifying molecular phenotypes (endotypes) of asthma and understanding how these endotypes contribute to disease pathophysiology. The proposal builds on our track record of using cell and molecular biology tools, animal models, and human studies focused on the airway epithelium to dissect asthma mechanisms, relate mechanisms to disease phenotypes, predict responses to existing therapies, and identify new therapeutic targets. Work from our Center demonstrated the central importance of direct effects of the type 2 cytokine IL-13 on airway epithelial cells, identified the type 2 asthma endotype as the dominant feature in a large asthma subgroup, established the ability of asthma endotyping to predict therapeutic responses, and showed how IL-13-induced changes in secretory cells cause mucus plugging in fatal asthma. Recent studies from our group and other laboratories implicate other pathways, notably the interferon (IFN) and ER stress pathways, in some individuals with asthma. Despite the considerable progress made by our Center and many others, there is still an urgent need for a more complete understanding of asthma disease mechanisms and more effective therapies for the many individuals with type 2-high or type 2-low asthma who do not respond well to current treatments. This proposal includes two projects that are highly interrelated and share a focus on the epithelium as both a key participant in asthma pathogenesis and a useful sensor for asthma endotyping. Project 1 will determine mechanisms and consequences of heightened epithelial sensitivity to IL-13, examine the basis of IL-13-induced changes in physical properties of mucus that cause airway obstruction, and dissect the contributions of epithelial ER stress in both type 2- and IFN-high asthma. Project 2 will determine the clinical significance of interferon-driven inflammation and airway epithelial ER stress in asthma, establish whether interferon-driven inflammation and airway epithelial ER stress are resistant to and predict poor response to existing asthma therapies, and determine whether specific inhibition of airway epithelial ER stress with a novel therapeutic, KIRA8, improves AHR, inflammation and mucus production in allergic asthma models. A Clinical Subject and Biospecimen Core will recruit and carefully characterize participants with asthma and healthy controls and provide biospecimens that will be used extensively in both projects. An Administrative Core will coordinate Center activities. Through the proposed studies we expect to gain new insights into the mechanistic bases of asthma endotypes and better understand how to target novel pathways important in specific endotypes.
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{
"pile_set_name": "NIH ExPorter"
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ABSTRACT Noroviruses are now the leading cause of viral gastroenteritis in children. Development of pediatric norovirus vaccines is underway, yet little is currently known about the development of humoral immunity against norovirus in children. We aim to help close this gap by defining how natural infection primes pediatric humoral immunity at the serological and B cell levels. This work generates new immunological tools to understand the development of norovirus immunity, and leverages samples being collected from an existing birth cohort study in Nicaragua (R01AI127845-03, Becker-Dreps/Vinje: Natural history, immunity and transmission patterns of sapovirus in a Nicaraguan birth cohort). First, we will characterize and compare memory B cell repertoires after first and second norovirus infections of the same genogroup in three previously nave children. From these primary and secondary infections, we will generate stable populations of B cells to determine frequencies, specificities, and function of corresponding antibodies. Functional assays will be performed by assessing histo-blood group antigen (HBGA) blockade to a panel of diverse noroviruses. These studies will enable us to determine whether primary exposure to norovirus elicits broadly-active antibodies against a range of norovirus genotypes, or if immunity is genotype-specific, and how the breath of the response changes after secondary infection. Following second infections, we will examine whether the predominant response is to pre-existing norovirus strains or to the newly-infecting strain, to better understand how primary infections shape the immune response to subsequent infections. This will provide insight into the question of whether antigenic seniority (boosting of pre- existing immunity to an ancestral strain) may be occurring, as has been shown in previously-infected adults receiving a norovirus vaccine candidate [Lindesmith, et al. 2015 PLoSMed]. Next, we will clone norovirus-specific pediatric mAbs that are found to have HBGA blocking activity from these natural infections. Of highest utility to vaccine development, we will use these mAb to map epitopes targeted in natural GII.4 infections. We posit that epitopes targeted by natural infection may be important to include in pediatric norovirus vaccines, to best mimic the development of natural immunity to norovirus. Overall, a better understanding of priming to norovirus in nave individuals is now needed to inform the design and schedule of pediatric norovirus vaccines. This project would provide the most in-depth view to date of B cell stimulation and antibody specificity following norovirus infection in nave children.
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{
"pile_set_name": "NIH ExPorter"
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Obsessive-Compulsive Disorder (OCD) is a psychiatric illness that affects approximately 3% of Americans (Narrow et al., 2002), and is associated with substantial impairment (Riggs et al., 1992) and poor quality of life (Olatunji et. al., in press). Cognitive models assert that information-processing biases confer vulnerability to OCD (e.g., Rachman, 1997). For example, biases towards detecting certain types of threats may play a role in the extent to which triggering stimuli enter into consciousness. While this bias may also occur in other anxiety disorders, OCD may be more uniquely characterized by problematic disengagement, wherein the patient cannot disengage from negatively valenced stimuli. Unfortunately, techniques for assessing these information processing characteristics have often been lacking. The emotional attentional blink paradigm, which assesses the extent and duration of attentional capture by emotional stimuli, represents a novel opportunity to empirically test for abnormalities in information processing. Such a research agenda could better inform our understanding of the etiology of OCD and facilitate the specificity of cognitively informed psychological treatments. SPECIFIC AIMS: Cognitive theories propose that information-processing biases confer vulnerability to OCD (Rachman, 1997, 2002; Salkovskis, 1985). We aim to test a disengagement model of attentional bias that may be central to the etiology and maintenance of OCD. DESIGN: This new investigator initiated R03 will compare a sample of 30 patients with OCD, 30 patients with Generalized Anxiety Disorder, and 30 nonclinical controls on the emotional induced attentional blink task. SIGNIFICANCE: The proposed study represents an initial step along a line of research clarifying the role of cognition, emotion, and disengagement in the etiology of OCD. The emotional induced `attentional blink' (a diversion of attention toward an irrelevant but salient stimuli) experimental paradigm could serve as a more reliable method for assessing the cognitive processes that have been implicated in more recent theories of the etiology of OCD. More importantly, if OCD is marked by an emotion induced attentional problems that are specifically reflected in difficulty with disengagement; this could be an important target for treatment and prevention programs and could provide a novel link between biological and cognitive models of OCD. PUBLIC HEALTH RELEVANCE: Information-processing biases have been proposed as risk factors for Anxiety disorders. This project will test a disengagement model of attentional bias that may be unique to obsessive-compulsive disorder in hopes that this research agenda may ultimately inform the development of more efficacious treatments for this debilitating disorder. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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In many autoimmune diseases such as arthritis, and lupus the tissue injury is caused by the interaction of inflammatory cells such as neutrophils with the immune complexes (IC) deposited on tissues. Such a tissue injury also results in glomerulonephritis leading to kidney failure and death in these disease states. Recent studies with gone knock out mice have clearly demonstrated that Fc gamma receptors (FcgammaRs) play a major role in IC mediated autoimmune diseases. Therefore understanding the regulation of function FcgammaRs has important therapeutic implications. In humans, two types of low affinity FcgammaRs for IgG, CD32A and CD16B, are coexpressed on neutrophils. Both bind ligands with overlapping specificity, however, only CD32A is capable delivering signal for phagocytosis. Recent studies from our laboratory have demonstrated that CD32A is functionally inactive in resting neutrophils. However, once neutrophils are activated by fMLP, a bacterial chemoattractant peptide, CD32A is converted to a functionally active state and can bind ligand efficiently. On the contrary, activation of neutrophils with PMA, a neutrophil activating phorbol ester, completely abolished CD32A binding to antibody-coated erythrocytes. Interestingly, the neutrophils expressed on cultured cell lines are constitutively active. These results suggest that the avidity modulation is cell type and activation signal specific and the regulation of ligand binding may be one of the mechanisms by which human neutrophil regulates CD32A function. We hypothesize that the molecular changes that occur during neutrophil activation alter the functional state of CD32A. In this grant we propose to determine the molecular basis for this signal specific and cell type dependent regulation of CD32A functional state. Specifically, we will: 1. determine whether the neutrophil activators alter the CD32A-dependent EA binding by influencing the 2D and 3D affinity of CD32A; 3, determine whether phosphorylation of ITAM motif of CD32A is altered by cell activation and correlates with change in ligand binding using immunoblotting, 2D amino acid analysis, and CD32A cytoplasmic domain mutation studies; 3. analyze whether neutrophil activation alters the cytoskeleton interaction of CD32A, receptor clustering, and change in the lateral mobility of CD32A. Since neutrophil activation occurs in vivo during infectious and autoimmune diseases it can be hypothesized that dysregulation of CD32A functional state occurs in vivo leading to the expression of high avidity CD32A which enables the neutrophils bind IC efficiently resulting in tissue injury in these diseases. The data obtained from the proposed research will be useful in understanding and designing therapies to inhibit IC mediated tissue injury in autoimmune and infectious diseases.
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{
"pile_set_name": "NIH ExPorter"
}
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The MSM RCMI Center of Excellence for Clinical and Translational Research (MSM R-CENTER) is designed to establish an institution-wide organizational framework and research infrastructure for promoting clinical and translational research. Our approach to translational research reaches from bench-to-bedside as well as from bedside-to-curbside in underserved minority communities. The RCMI program's decade long investments in the development of the MSM research infrastructure served as a strong foundation for establishing the MSM R-CENTER five years ago. However, it is also recognized that it is the investment in human capital - our students and faculty - that will define the long-term success of MSM's clinical and translational research enterprise. The major objective of re-engineering our research enterprise is to establish a research environment that will enable MSM to emerge as preeminent in training the next generation of minority clinical/translational investigators who will use discovery science to reduce health disparities. This competing R-CENTER renewal proposal builds upon the foundational infrastructure developed with the careful stewardship and investments of RCMI resources over the past decade. Our approach is to further enhance our research capacity in clinical and translational science by building effective partnerships and seeking synergies that leverage the collective strengths of our talented faculty, trainees, students and collaborators. The R-CENTER's institution-wide organizational framework and key functions are designed to strengthen the transformative process begun during the first funding cycle. Aim I: Transform Our Institution - Prioritize growth in clinical and translational research through focused investments, strategic collaborations with alignment of clinical services, research infrastructure and technology transfer. Aim II: Transform Our Research Environment - Support a clinical and translational research home with an administrative structure that streamlines and monitors access to resources, supports best practices in regulatory compliance and research efficiency. Aim III: Transform the Community of Biomedical Scientists - Foster innovations in team science and prioritize training the next generation of clinical and translational scientists in cutting edge approaches to addressing health disparities. Aim IV: Transform Our Community - Establish a continuous learning healthcare system with bidirectional communications, a community-centered model of clinical and translational research that advances discovery science and translates new knowledge into improvements in the health status of minority communities.
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{
"pile_set_name": "NIH ExPorter"
}
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Traumatic events, particularly during early life, have far-reaching consequences including increasing an individual's vulnerability to depression and anxiety disorders. However, there is a paucity of information concerning the impact of early traumatic experiences on the development of neural circuitry, and its relation to adult vulnerability to neuropsychiatric disorders. Moreover, it is known that there is considerable heterogeneity in response to traumatic stress in relation to later development of neuropsychiatric disorders. In the US, 20-30% of individuals exposed to traumatizing events subsequently exhibit symptoms of posttraumatic stress disorder (PTSD). Nonetheless, the characteristics of neural circuitries associated with either risk or resilience to these disorders re unknown. Understanding these issues is now possible with the advent of a novel approach capable of imaging resting-state functional connectivity (RSFC) in awake animals. This advancement is unique in its noninvasiveness, whole-brain coverage and high sensitivity to neuroplasticity, and thus is ideal for studying the dynamic changes of neural circuitry across brain development and under selective perturbations. By utilizing this approach, we propose to investigate the impact of early trauma on the development of the neural circuits implicated in stress-induced disorders in an animal model. Specifically, with a longitudinal design in which traumatic stress is administered during juvenile, adolescence or adulthood, we will characterize the impact of early trauma on the developmental trajectories of the neural circuits of medial prefrontal cortex (mPFC), amygdala (AMYG) and hippocampus (HP). In addition, we will examine the difference in these circuits in animals exhibiting high vulnerability to developing PTSD-like behaviors. This vulnerability will be evaluated based on cut-off criteria of an established PTSD animal model. Our preliminary data showed that the neural circuits of mPFC, AMYG and HP are still immature during adolescence. We also demonstrated that trauma exposure can induce long-lasting effects on the same neural circuits in adult rats. Intriguingly, vulnerable rats showed much weaker RSFC strength within the mPFC-AMYG circuit compared to resilient rats, implying that RSFC may predict vulnerability to PTSD. Based on these pilot data, we plan to accomplish the research objectives by pursuing three specific aims. In Aim 1, we will characterize the normal developmental trajectories of the neural circuits of mPFC, AMYG and HP. In Aim 2, we will evaluate the impact of early trauma exposure on the developmental trajectories of these neural circuits. In Aim 3 we will assess the neural substrate underlying the vulnerability to PTSD in an animal model. The proposed work is innovative, because it combines novel neuroimaging tools and behavioral measurement to investigate the development of critical neural circuits and their vulnerability to traumatic stress. The impact of this research is highly significant because understanding the role of early trauma in neuroplastic changes in the circuitries subserving mood and anxiety disorders is critical to earlier diagnosis and treatment of these disorders.
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{
"pile_set_name": "NIH ExPorter"
}
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The syndrome of copper deficiency in young pigs includes spontaneous rupture of the heart and of elastic and muscular arteries. It has been the main objective of this project to interpret the cardiovascular abnormalities in terms of the mechanical properties and composition of the connective tissues and of the biogenesis and interrelation of their components. It is intended to apply the knowledge of the pathogenesis of this model of disease toward an understanding of the normal biogenesis and function of connective tissues. The foundation will be laid for the application of the principles established to the exploration of naturally acquired and heritable disorders of connective tissue in man and animals. Specific aims within the overall objective have been to elucidate (1) the biosynthesis of elastin, (2) properties of the elastin monomer, (3) crosslinkage of elastin and collagen, (4) fine structural organization of aortic fibers, and (5) mechanical properties of aortic components in health and disease. The approach is: (a) to maintain copper-deficient pigs as a source of supply of soluble aortic elastin and collagen and of the partially crosslinked fibrous proteins which composed the vessel wall in this disease; (b) to develop in vitro systems for study of the kinetics and intermediates in biosynthesis of these proteins; (c) to characterize the properties and interactions of soluble elastin and collagen and their reactions with other proteins of the intercellular matrix; (d) to establish the crosslink sequence and enzymes responsible for the stabilization of the fibrous proteins; and (e) to correlate the structural organization by electonmicroscopy with the mechanical properties of the diseased vessels.
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{
"pile_set_name": "NIH ExPorter"
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Our long term goal is to gain a molecular understanding of morphogenetic processes in higher organisms by examining a number of events which take place in the development of the limb. In order to meet this goal we will continue to analyze an unique collection of mutations which affect some aspect of limb morphogenesis in the avian embryo. Conceptually we follow the partition of the developmental events into a morphogenetic phase and a phase of cytodifferentiation as first proposed by Zwilling. For studies on the morphogenetic phase we will analyze the events by which the lateral plate mesoderm acquires limb properties through an interaction with somite mesoderm. We will concentrate on chondrogenesis as a measure of cytodifferentiation and we will analyze this developmental event in terms of the coordinate expression of three cartilage specific macromolecules, proteochondroitin sulfate, collagen and a proteochondroitin surfate glyco-protein linker molecule. These three macromolecules will be analyzed as individual entities. They will be further analyzed as they interact with each other (e.g. proteochondroitin sulfate with collagen and proteochondroitin sulfate with glyco-protein linker) in the formation of extracellular matrix.
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{
"pile_set_name": "NIH ExPorter"
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DESCRIPTION (adapted from the application) Support is requested to provide expenses for younger investigators to attend the Banff Conference on Solid Organ Allograft Pathology, to be held June 7-12, 1999. This conference is the fifth in a series that has been held biennially, beginning in 1991. The overall objectives of the Banff conferences are: to provide a venue for focus on the pathology and pathophysiology of solid organ allografts; to foster collaborative studies in the areas of allograft pathology and pathophysiology; and to enable broad representation and collaborations, interdisciplinary interactions, and consensus building. The conference in 1991 led to development of the Banff Working Formulation for classification of renal allograft pathology. In 1995, the Banff working formulation for liver allograft rejection was developed from consensus discussions held at the Third Banff Conference. The most recent conference, in 1997, led to a revision of the Banff classification for kidney allograft pathology, Banff 97, soon to be published in Kidney International. Attendance at the conferences has grown steadily, from 16 in 1991 to 123 in 1997. The conferences are attended by an international group of pathologists and clinicians, most with strong research interests in transplantation. Formats are a mix of presentations and open informal consensus discussions, which have proven to be productive forums for exchange of ideas and development of collaborations. The conferences provide a stimulating environment for young professionals interested in the science and practice of transplantation, and junior faculty have attended the conference in increasing numbers in recent years. The specific goals of the Fifth Banff Conference are: 1) updating of Banff Classifications for renal and liver allograft rejection, as well as classifications for acute rejection of heart, lung, and pancreas allografts; 2) development of a consensus working classification for grading/staging chronic "rejection" in liver allografts; 3) consideration of feasibility of and information to be gained from baseline and protocol allograft biopsies; 4) fostering of collaborative studies of protocol biopsies; 5) stimulation of continued studies for identification of early predictors of late allograft dysfunction; and 6) refinement of the diagnosis of and summary of pathogenic significance of viral infections in solid organ allografts. Topics to be covered at the Fifth Banff Conference include: 1) updates of the schemas for rejection grading for kidney, liver, heart, lung, and pancreas allografts: 2) protocol biopsies, rationale and methods; 3) early predictors of late allograft function; 4) chronic allograft dysfunction; and 5) viral infections in allografts. Funds from this grant would provide travel support, registration, and housing for 16 young investigators to come to the Banff Conference to present their work and participate in the formal sessions and informal consensus discussions that characterize these conferences. The work of young investigators will be highlighted as platform presentations or in interactive poster sessions.
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{
"pile_set_name": "NIH ExPorter"
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Point mutations have been implicated as important etiological factors in a number of human genetic disorders including several of the haemopathies, osteogenesis imperfecta, as well as in the activation of some oncogenes. Little is known, however, about the mechanisms by which such mutations occur in mammalian cells. Determination of mechanistic pathways by generating mutational spectra using DNA sequence analysis has proven extremely powerful in bacterial systems. Similar studies in mammalian cells have been limited due to technical problems associated with the rapid isolation of target gene sequences from mammalian cells. We are in the process of analyzing both spontaneous and induced point mutations in a Chinese hamster ovary (CHO) cell line, AS52, that carries a single copy of the bacterial gpt gene transfected and functionally integrated into the CHO genome. The gpt gene is analogous to the mammalian hgprt gene and mutations at either locus can be isolated by selecting for resistance to the purine analog, 6-thioguanine (6TG). The small size of the gpt gene (456 base pairs) provides for the convenient rescue of mutant gpt sequences from the CHO genome for DNA sequence analyses. Our data suggest that genomic sequences at the site of the gpt integration may influence sequence stability in cloning experiments. We are in the process of performing cloning experiments utilizing alternative E. coli hosts known to stabilize genomic inserts. In addition, we have begun to amplify the gpt gene sequences using the polymerase chain reaction (PCR) technique. This approach bypasses the requirement of cloning each mutant gene and provides enough DNA to allow direct sequence analysis without further subcloning. Initial studies with the PCR technique indicate the entire 456 base pair gpt structural gene can be amplified from 1 g of genomic DNA. Using these amplified DNA sequences we will perform DNA sequence analysis and determine the utility of the PCR technique in generating point mutational spectra from mammalian cells.
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{
"pile_set_name": "NIH ExPorter"
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The overall goal of this program is to elucidate the structural features of antibiotics in the vancomycin class which lead to their ability to complex specifically with certain peptides involved in the biosynthesis of bacterial cell walls. As the first step in the project, the structures of ristocetin A, actinoidins A and B and several other members of the vancomycin group will be determined, looking both at the peptide and the carbohydrate portions. Second, a study will be made of the structural specificities of all the available antibiotics of this group which have defined structures and also certain derivatives of them. These studies will employ Ac2-Lys-D-Ala-D-Ala and related peptides having one or more of the residues replaced by other amino acids. Binding constants will be obtained by UV measurements. Details of binding of the antibiotics with the peptides will be determined by NMR spectroscopy. Finally, after ascertaining the minimum structural features of the antibiotics that are needed to obtain specific peptide binding, new compounds of this type will be prepared and tested for binding and antibiotic activity. In some cases, semisynthetic compounds will be prepared by altering the structures of existing antibiotics, in others they will be prepared de novo taking advantage of a biomimetic synthetic route. By this rational approach to antibiotic design it may be possible to prepare compounds with substantially improved activity.
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{
"pile_set_name": "NIH ExPorter"
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The Flow Cytometry & Cell Sorting Shared Resource is a critical technology for members of the NYU Cancer Institute (NYUCI). Flow Cytometry allows analysis of the light scattering and fluorescence properties of individual cells and the rapid, statistically detailed analysis of 10,000s of cells. The data on individual cells is retained, and subpopulations can be identified in multiple dimensions, showing distinct phenotypical cell types. Cells with particular fluorescence profiles can also be purified and collected for growth or further analysis using the cell sorting. The Flow Cytometry & Cell Sorting Shared Resource offers users who cannot afford and maintain their own Sorters and Analyzers access to this technology, as well as the technical expertise required to design experiments and evaluate experimental results. The facility has a Beckman Coulter MoFlo sorter with 4 lasers and up to 9 colors of Fluorescence, a Becton Dickinson 5-laser 14-color ARIA II cell sorter in a BSL-2 biosafety enclose for primary human cell sorting, an iCyt/SONY Reflection parallel cell sorter, as well as a 5-laser 17-color Becton Dickinson LSRII analyser. The shared resource also has a Becton Dickinson FACScalibur and FACScan analyzer. All cell sorting experiments are performed by highly-trained shared resource staff, and analyzers are run by investigators who have been trained by the shared resource staff. The scientific director also participate in training activities including individual consultations, public seminars and hands on training of users. The facility performed 3,887 hours of analysis and 3,111 hours of sorting during the most recent 12 month period.
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{
"pile_set_name": "NIH ExPorter"
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This proposal outlines a multidisciplinary research effort by four principal investigators to continue development of a series of conformationally and topographically constrained peptide analogues of dynorphin that have high receptor potency and selectivity for the kappa opioid receptor and its subtypes, potent agonist or antagonist in vivo activities, high stability in vivo, prolonged biological effects at kappa opioid receptors, and unique biological activities. This approach combines aspects of computer assisted molecular design, synthetic organic amino acid and peptide chemistry, conformational analysis and molecular mechanics and molecular dynamics calculations, and biochemical, biophysical, pharmacological and physiological studies. The specific aims of this research include: 1) continued developments in the design, synthesis and evaluation of novel conformationally and topographically constrained peptide analogues of dynorphin targeted for kappa opioid receptor types and subtypes; 2) to utilize topographically fixed or biased amino acid residues in conjunction with computer aided drug design methods to develop kappa receptor selective ligands; 3) to comprehensively examine the potency and selectivity of all new ligands for kappa vs mu and delta receptors using radioligand binding assays - highly selective and potent analogues for kappa receptors will be radiolabeled to high specific activity; 4) comprehensive examination of kappa vs. mu vs. delta binding distribution in the brain using autoradiography; 5) evaluation of opioid agonist and antagonist activities in vitro using especially the GPI and MVD assays to establish receptor selectivity and potency; 6) evaluation of in vivo kappa receptor properties of ligands highly selective for kappa receptors and kappa receptor subtypes using such tests as in vivo analgesic assays including the tail flick and hot plate assays, and the acid induced mouse abdominal constriction test; 7) careful evaluation of the enzymatic stability of key compounds and their serum half-lives, so as to develop analogues that are stable with good biodistribution; and 8) to carefully examine the conformational of properties the most potent and selective analogues by 1D and 2D nuclear magnetic resonance spectroscopy, molecular mechanics calculations, and other biophysical methods. The long-term goal of this research is to develop an understanding of the physiological roles of kappa opioid receptor types and subtypes, and to develop specific ligands so that they can be used for the treatment of pain and disease.
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{
"pile_set_name": "NIH ExPorter"
}
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The discovery of presenilins as ubiquitous intramembrane proteases in metazoans exemplifies an emerging trend in biology. The traditional distinction between basic and applied research has become increasingly blurred, as studies initiated with a strictly disease-oriented focus uncover fundamental biological mechanisms. In this renewal application, ten investigators who have collaborated successfully during the last decade on the normal and pathological biology of presenilin (PS) wish to extend their productive interactions into new experiments that will further illuminate the structure-function relationships of PS/ ? -secretase in normal biology and the role of this unusual multi-protein complex and various related gene products in the genesis of Alzheimer's disease. Based on extensive preliminary data in each of our four interrelated projects and supported by three vital cores, we will pursue numerous Specific Aims that include: 1. attempting to determine the structure of PS/ ?-secretase by performing cryo-electron microscopy of the purified, active complex as well as x-ray crystallography of the individual components;2. extending our recent discovery that cholesterol dramatically enhances the catalytic activity of purified ? -secretase and that membrane lipids in general appear to represent the most potent regulators of both A[unreadable] production and Notch cleavage;3. performing extensive genomic, functional genetic and protein analyses searching for novel lateonset AD candidate genes potentially implicated in presenilin-related pathways;4. using advanced microscopy approaches in living cells (bimolecular fluorescence complementation;FLIM) to image and quantify the conformations and interactions of presenilin with APP, with new candidate genes emerging from (2), and with lipids and ? -modulating compounds identified herein;5. identifying common binding partners and common subcellular processing pathways for the (32 subunit of NaJ, APP and APLP2, three proteins that are processed identically by both (3- and y-secretase. 6. performing an unbiased proteomics screen to more fully define the " ? -secretome", i.e., the extent of unknown ?-substrates. These and numerous additional aims and sub-aims will be pursued collaboratively across our 4 projects, with progress in one project modifying experimental plans in another. In short, we are anxious to continue our close interaction to bring greater clarity to exactly how PS and its associated proteins and lipids process a host of substrates within membranes in health and disease. Lay summary: Ten experienced scientists who have collaborated productively for the past decade wish to address the structure and the functions of an unusual protein-cutting enzyme that is required for life in all multi-cellular animals and implicated in the causation of Alzheimer's disease. PRINCIPAL INVESTIGATOR: Dr. Selkoe has been associated with the Harvard Medical School (HMS) since 1972, first as a resident in Neurology and, since 2001, as the Vincent and Stella Coates Professor of Neurologic Diseases at HMS. He is renowned as a pioneer and leader in the field of Alzheimer's Disease research, having made contributions fundamental to several aspects of AD molecular pathogenesis and to the preclinical development of novel treatment strategies for the disease. His work has been recognized by many awards and honors, including LEAD and Merit Awards from NIA, MetLife, Potamkin and Pioneer Awards, and numerous distinguished lectureships. In recent years, his work has centered on mechanisms of amyloid processing, expression, and catabolism. Dr. Selkoe's focus has expanded to include the molecular biology of presenilins, His laboratory is a national and international center for research and training of pre- and post-doctoral students, many of whom have gone on to productive careers in neuroscience research. In this proposal, Dr. Selkoe has brought together much of the top Alzheimer's talent at HMS in collaborative projects. His leadership is evident in the successful evolution of the Center for Neurologic Diseases, of which he is co-director, and his direction and growth of the current program project. He is eminently qualified to lead this program. REVIEW OF INDIVIDUAL COMPONENTS CORE A: ADMINISTRATIVE AND EDUCATIONAL CORE, DR. DENNIS SELKOE DESCRIPTION (provided by applicant): The Administrative and Educational Core will continue to play a key role in the program, integrating the activities of this highly interactive consortium of 4 independent labs and 3 core units (comprised of more than 25 scientific personnel) at the Massachusetts General Hospital and Brigham and Women's Hospital. The 10 co-investigators of the program are strongly committed to enhancing scientific collaboration, communication and education, as we have done during the two preceeding terms of this Program. In this regard, we have found the three specific forums proposed in this core to have been highly effective tools in the previous funding periods. They are therefore included in this renewal of Core A as a means of achieving the important goals of scientific cooperation and exchange that define a NIH Pogram Project Grant. 1) Regularly scheduled Project Leaders'Meetings, attended by all the Project Pi's, their coinvesigators, the Leaders of Core B and C and and selected project scientists, provide ongoing opportunities to examine new developments in each of the 4 projects, and to review one or more of the projects in depth at each meeting, with analysis of progress and problems. 2) Our superb Scientific Advisory Board will continue to consult with the program scientists on an ad hoc basis throughout the year and then convene for an intensive annual on-site review of major new developments in each of the projects. 3) Our successful Seminar Series "Basic and Applied Biology Relevant to Neurodegeneration" will soon enter its 9th year, presenting local and out-of-town speakers, including our own investigators, who inform the Program members and the Boston scientific community at large of new developments in presenilin biology, the pathogenesis of AD and numberous relevant basic research issues. In addition to running the program's vital educational and information-sharing activities, Core A will, as before, manage all the day-to-day fiscal and administrative issues related to our large multi-site program, including regular income/expense reports, inter-institutional personnel and reagent exchanges, joint publications, regular exchange of all publications emanting from the grant, preparation of non-competing renewal applications, and timely and efficient communication with the National Institute on Aging.
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{
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The molecular mechanisms which govern the developmental specificity of human beta-globin gene transcription has been studied in K562 cells, a human eyrthroleukemia line that expresses minimal beta-globin. Protein- binding analysis reveals the 5 region contains three elements bound by trans-acting factors, beta-protein 1 (BP1) and beta-protein 2 (BP2). In vitro mutagenesis of each element in a beta-globin vector containing chloramphenicol acetyltransferase (pCAT) followed by transient transfection into K562 cells increased levels of CAT activity 5.5-fold higher than wt beta-CAT, consistent with their silencing role. Mutagenesis of all three elements, however, resulted in activity significantly lower than wt beta-CAT. BP1 and BP2 motifs have overlapping binding sites with high mobility group proteins (HMG1+2), DNA-bending factors shown here by circular permutation assay to extrinsically bend the beta-globin distal promoter. Theoretically, mutations in all beta-protein binding sites could affect the binding of HMG1+2 sufficiently to impede DNA-protein and/or protein-protein interactions needed to facilitate the low level, constitutive gene expression. Placing two helical turns of DNA between BP1 and BP2 motifs also increased expression 3-fold, indicative of spatial constraints required for optimal silencing. However, insertion of the HMG1+2 DNA- bending motif (also equivalent to two turns) facilitates beta-silencing by re-establishment of BP1-BP2 proximity. Thus a combination of general DNA-bending and specific transcriptional factors appear to be involved in beta-globin silencing in the embryonic/fetal erythroid stage. To further define this relationship an in vivo study using transgenic animals is underway. Preparation of a cosmid containing the uLCRbeta locus with a mutated BP1 silencing motif will permit an in vivo analysis of adult beta-globin gene expression in transgenics during the embryonic/fetal developmental stages.
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{
"pile_set_name": "NIH ExPorter"
}
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The inflammatory response to acute Sindbis virus (SV) infection of the central nervous system (CNS) has been characterized in a murine model. This disease is representative of arboviral encephalitis, and it serves as a model to study immune reactions within the CNS. Cerebrospinal fluid inflammation during SV encephalitis, which reaches a peak early in infection, is composed of T cells and natural killer cells. Parenchymal inflammation is maximal later, and appears more complex in cellular composition. The virus is subsequently cleared from the CNS, and mice with the wild-type infection recover uneventfully. The primary goal of the proposed experiments is to characterize the development of the CNS T cell response during SV encephalitis over the full course of the infection. Specific investigations will focus on detailed phenotypic study of T cells that infiltrate the CNS, as well as characterization of their functional properties. These include cytokine production as well as anti-viral proliferative and cytotoxic responsiveness. Other experiments will investigate the significance of preliminary findings which have shown that gamma delta plus T cells accumulate preferentially within the CNS, especially early in infection. Since the specific molecular interactions between T cells and cerebral capillary endothelial cells likely regulate lymphocyte extravasation into the CNS and the development of perivascular inflammation, further goals of this proposal will be to explore the nature of adhesion events between these two cell populations during acute encephalitis. This will be performed using both an in vitro cell adhesion assay between lymphocytes and tissue sections of brain parenchyma from infected animals, as well as with an in vivo assay of T cell homing into the CNS. Blocking experiments with monoclonal antibodies are planned in order to determine the relevant molecular interactions between T cells and CNS microvascular endothelium both in vitro and in vivo. The in vitro assay may also serve to screen for and identify presently uncharacterized receptor-ligand interactions that mediate binding in this setting. Exploring these features of T cells will be important for understanding not only the pathogenesis of acute viral encephalitis, but also other inflammatory conditions of the CNS which are presumed to be T cell-mediated.
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{
"pile_set_name": "NIH ExPorter"
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The Chemistry Core laboratory will provide routine assays for the individual projects within the Program Project. Assays that will be performed on a routine basis include insulin, non-esterified fatty acids, palmitate, leptin, renin activity, aldosterone, corticosterone, dexamethasone, and norepinephrine. Techniques will include radioimmunoassay and radiozymatic assay, ELISA and HPLC with UV detection. We anticipate that usage of the chemistry core will entail approximately 400 assays per year. The core will provide resources for establishment of additional assays should they be needed by individual investigators.
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{
"pile_set_name": "NIH ExPorter"
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The broad, long-term objective of this project is to increase cultural competency in a wide array of dental providers using a web-based tool (EthnoDent). Washington state's population, in addition to the United States, is becoming increasingly diverse in its' ethnic and racial makeup. Between 1995 and 2025, international migration will bring to-Washington state an additional 394,000 people (Campbell, 1996). The U.S. culture is becoming more heterogeneous with the influx of many minority cultures who have their own beliefs and traditions and may only share one commonality of living in the same country - poorer oral health status (Brookins, 1993; Ahmann, 1994). Low-income and minority populations in the U.S. have benefited less than other groups in spite of advances in knowledge of oral health diseases such as childhood caries (Seow, 1998; Milnes, 1996). As the population's diversity increases, providers will care for multi-cultural patients who may not attribute the same traditions or meanings for health and illness. Underlying traditions and values arising from cultures may govern the group's perceived meaning of health, illness and approach to curing. An inability to communicate cross-culturally may impair health professionals' effectiveness at providing the most appropriate care to patients, potentially affecting patient adherence. The current project proposes to develop a web-based tool (EthnoDent) which will focus on reducing cultural barriers between providers and multi-cultural patients in the area of children's oral health. The specific aims of the proposed study are; 1) To qualitatively describe the Native American/Alaska Native (NA/AN), Asian/Pacific Islander (A/PI), and Latino/Hispanic (L/H) cultures (i.e., traditions, beliefs/values, practices) with respect to children's oral health. 2) To develop a web-based tool (EthnoDent) based on the collected information, which will contain information about the cultures, their languages, beliefs, traditions, and practices with respect to children's oral health. 3) To describe the user profiles of EthnoDent and manage the evolution of system changes based on feedback from these profiles. 4) To determine whether dental providers' (e.g., first-year dental students, dental assistant and dental hygiene students) knowledge and cultural competency is increased with the use of EthnoDent The long-term vision of this project is to reduce disparities in children's oral health through enhancing the providers' attitudes and skills in communicating with multi-cultural families.
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{
"pile_set_name": "NIH ExPorter"
}
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Our review of the literature as well as our own work indicates that the probability of lethal disease occurring in otherwise normal animals when stressed is low; however, when an animal has some predisposition to disease and is then subjected to stress, lethal disease occurs much more consistently. Using this concept, we have predisposed guinea pigs to arrhythmias by using digitalis. When our digitalized animals are then stressed, their cardiac rhythm changes from a normal one to an abnormal potentially lethal one. This then is a good animal model of psychosomatic digitalis-toxicity. This model is an important one because digitalis is the fourth most commonly prescribed drug in the U.S. and has a low therapeutic:toxic ratio. Environmental factors have never been seriously considered as a reason for individual variability in response to digitalis. We are currently improving our model by using a free-ranging rather than a restrained subject and by using IV administration of our cardiac glycoside, ouabain, rather than IP. Our objectives are to use this improved model to learn the relative importance of the autonomic nervous system in the production of the arrhythmias; we will do this by pharmacological and surgical ablations and by neurophysiological recording and stimulation; we also plan to carry these experiments into the brain in an attempt to locate sites responsible for the psychosomatic effect. Another objective is to begin to analyze whether specific emotional states are responsible for the effect or merely a generalized arousal. Finally, we shall explore how manipulation of psychological contingencies can protect the animal from the lethal outcome; this will be done (a) by manipulating controllability over shock and by training the subject to maintain a constant heart rate by operant techniques.
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{
"pile_set_name": "NIH ExPorter"
}
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The University of Kentucky Superfund Basic Research Program will focus on the environmental health impacts of chlorinated organics with a more specific focus on RGBs and TCE. The Program will include five separate biomedical research projects that seek to better understand the biochemical and cellular mechanisms associated with exposure to such chemicals, as well as the associated impacts on such diseases as cancer, diabetes, hypertension, and cardiovascular disease, i.e. diseases common in Kentucky. One of the unique aspects of the research program will be the investigation of the role of nutrition in mitigating the impacts associated with such exposures. In addition, separate projects will examine the use of nanotechnologies and biosensors in detecting such chemicals in the environment and will explore novel techniques for remediation, with a particular focus on groundwater contaminated by TCE. The latter project will have specific potential for application as a result of ongoing remediation studies associated with the Gaseous Diffusion Plant in Paducah, Kentucky. The Research Translation Core will translate Program research to five targeted audiences: Government, Industry, Broad Audiences, Health Professionals, and Academia. Consistent with the guidelines of the RFA, the specific aims of the Research Translation Core are summarized as follows: 1. Communicate important research outcomes to specific targeted audiences to ensure the accurate and timely use of the knowledge and technologies generated by individual research projects, thus contributing to the reduction of environmental contamination and the improvement of public health. Specific target audiences include: a) government, b) academia, industry and health professionals and c) broad audiences (including the general public and impacted communities). 2. Leverage research translation activities and targeted research translation projects through effective partnerships and other communication tools. Specific partnerships include: 1) The Kentucky Water Resources Research Institute, 2) The Kentucky Research Consortium for Energy and Environment, 3) The Kentucky Cooperative Extension Service, 4) UK College of Communications and Information Studies, 5) UK Center for Clinical and Translational Science, and 6) Other SBRP Research Translation Cores, (see attached letters of support) 3. Develop and implement a mechanism for tracking and evaluating the effectiveness of the translated research to confirm the utility and appropriateness of the communication tools selected. These efforts, promoted through eight specific translation projects, will contribute to a reduction in environmental contamination, an increase in target audiences'knowledge about the relationship between Superund chemical exposure and health outcomes, and a subsequent improvement in human health.
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{
"pile_set_name": "NIH ExPorter"
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The time course and intensity of human hepatic regeneration will be studied by measurement of rates of DNA synthesis by isolated hepatocyte nuclei. The relationships among cyclic nucleotides, hormones, electrolyte concentrations, amino acids, and hepatocyte proliferation will be studied in isolated hepatocytes from normal and regenerating rat liver.
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{
"pile_set_name": "NIH ExPorter"
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During the past year our research efforts encompassed two areas; (A) The modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins, and (B) the continued development and applications of vibrational infrared spectroscopic imaging techniques. (A) Infrared spectroscopic measurements demonstrate the critical role of the purple membrane lipid environment of Halobacterium halobium in mediating conformational changes in bacteriorhodopsin (BR), an integral membrane protein. The strategy consists of assessing the flexibility of BR by monitoring the temperature dependence, pressure dependence and retinal-apoprotein coupling of vibrational spectral features characteristic of the conformational reorganizations and dynamical properties of both the transmembrane helices and surrounding lipids. Photocycle activity was correlated to helix mobility in reconstituted membranes, in addition to probing aII to a1 helical conversions. Raman and infrared spectroscopic measurements were performed on membrane bilayer systems comprised of lipid systems containing a saturated and unsaturated chain. The double bond of the saturated chain occupies one of the possible multiple chain positions. The properties, dimensions, and behavior of the bilayer's microheterogeneity, or fluctuating microdomain structures, were elucidated. (B) Our mid-infrared spectroscopic chemical imaging techniques, combining step-scan interferometry with an infrared microscope, were substantially refined and increased in spectroscopic capabilities by incorporating a variety of state-of-the-art infrared sensitive two-dimensional focal plane array detectors. These imaging systems, exhibiting instrumental multiplex/multichannel advantages, operate from 4000-400cm-1 and provide high fidelity, chemically specific images at nearly diffraction limited conditions. Applications consisted of imaging the effects of antineoplastic drugs on rat cerebellar tissue and examining intact brain sections from transgenic mice to elucidate the pathology of lipid distribution induced by the neurodegenerative Niemann-Pick C disease.
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{
"pile_set_name": "NIH ExPorter"
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Jenny J. Lin, MD, is a general internist at the Mount Sinai School of Medicine. The candidate's long-term goal is to become an independent clinician-investigator working at the intersection between cancer survivorship and chronic disease management. More specifically, the candidate is interested in understanding the cognitive and emotional factors that affect cancer survivors' self-management of chronic comorbid diseases and to use this knowledge to develop, test, and disseminate behavioral interventions that can improve cancer survivors' management of comorbid illnesses. The candidate became involved in this area as she transitioned from being an established clinician-educator to a clinician-investigator and started to examine barriers to and facilitators of medication adherence to hormonal therapies in breast cancer survivors. The proposed career development plan combines a closely-mentored cancer control and behavioral sciences project with multidisciplinary, didactic training in health psychology (including behavior change and health beliefs), survey design, and complex data outcomes analysis. Diabetes is one of the most common comorbid illnesses affecting cancer survivors. This chronic disease and its complications are associated with considerable morbidity and mortality in cancer survivors. Additionally, diabetes may also impact cancer risk and prognosis. However, little is known about the impact of a new cancer diagnosis on patient self-management behaviors for cancer survivors with diabetes and of the impact that cancer has on diabetes control. Under the mentorship of cancer researchers, Juan Wisnivesky, MD, DrPH; Nina Bickell, MD, MPH; and Michael Diefenbach, PhD, the candidate will study the short- and longer-term impact of a new diagnosis of early-stage breast or prostate cancer on diabetes management and outcomes. The goals of the research component will be (1) to assess the relationship between new cancer diagnosis and diabetes control; (2) to evaluate the impact of cancer diagnosis on diabetes self-management behaviors, such as medication adherence, glucose self-monitoring, diet control and exercise; and (3) to elucidate the cognitive and emotional factors (such as health beliefs, depression, anxiety and stress) that mediate the association of cancer with poorer diabetes self-management behaviors and control. To achieve these goals, the candidate will prospectively assess diabetes control via hemoglobin A1c levels and monitor adherence to oral diabetes medications in a cohort of 200 diabetic patients with newly diagnosed early-stage breast or prostate cancer and 150 age- and gender-matched control diabetic patients without cancer. Adherence will be measured with electronic monitoring caps and via patient self-report using a standardized survey instrument. Self-management behaviors will be assessed using standardized survey measures, and validated survey instruments will be used to assess symptoms of depression, stress and anxiety, health beliefs and mental models of disease. Patients will be surveyed at enrollment and at 3, 6 and 12 months. We will use multivariate methods and structural equation modeling to test the proposed aims.
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{
"pile_set_name": "NIH ExPorter"
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DESCRIPTION(provided by applicant): Neuronal cell death by apoptosis is often associated with cerebral ischemia. Currently, a number of factors that influence neuronal cell death have been identified. Among them, members of the Bcl-2 family represent a group of proteins that serve as key regulators of apoptosis by promoting either ceR survival as in the case of Bcl-2 and Bcl-XL, or cell death as in the case of Bax. Upregulation of the pro-apoptotic factor Bax has been reported in the affected area of the brain, implicating the participation of this protein in promoting neuronal cell death. In healthy living cells, Bax is predominantly a cytosolic protein and its ability to modulate cell death is associated with its translocation from the cytosol to mitochondria during apoptosis. This pro-apoptotic activity of Bax however, can be blocked by coexpression with Bcl-XL, a pro-survival member of the Bcl-2 family that is essential for neuronal development and is localized mainly to mitochondria. The long-range goal of this project is to define the underlying mechanisms by which Bax and Bcl-XL regulate apoptosis to enable the development of neuroprotective compounds. The objective of this application is to define the molecular basis by which Bcl-XL inhibits Bax activity as a first step towards accomplishing the long-range goal. The central hypothesis for this proposal is that Bcl-XL blocks the pro-apoptotic function of Bax by preventing its redistribution from the cytosol to mitochondria during apoptosis. We have developed a simple method of tracking the movement of Bax by tagging it to the green fluorescent protein. This enables us to determine how the presence of various Bcl-XL mutants affects the intracellular distribution of Bax by confocal microscopy. In addition, we have begun to study how Bcl-XL exerts its effect. With a unique epitopespecific rnonoclonal antibody that we have generated against Bcl-XL, we have purified a major Bcl-X1 associated protein by immunoaffinity chromatography and identified it as ATP synthase subunit. To accomplish the objective of this application, the following specific aims will be pursued: 1) determine the functional domains of BCI-XL involved in inhibiting Bax translocation to mitochondria, 2> determine the specificity and universality of BCI-XL interaction with ATP synthase subunit, 3) determine the sites of interaction between Bcl-XL and ATP synthase subunit, 4) determine the role of ATP synthase subunit in regulating Bax redistribution to mitochondria and cell death, and 5) determine whether ATP synthase subunit can regulate the channel forming properties of BcIXL Upon completion of this proposal, we expect to have gained further understanding of the structural/functional properties of BCI-XL and its binding protein and the underlying mechanism of Bax inhibition by Bcl-X1 during apoptosis.
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{
"pile_set_name": "NIH ExPorter"
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Specific Aims: 1 - to refine a novel set of low cost patent pending interactive home AMD education and monitoring tools that combine tests evaluated favorably in Phase I together with additional novel tools currently in prototype. 2 - To clinically evaluate the effectiveness of these tools in reducing the delay between onset of symptoms and presentation, thus potentially minimizing vision loss. Significance: Macular degeneration is the leading cause of blindness in those over 60. New anti-VEGF treatments introduced in 2005 are effective if given early but patients typically wait for up to 5 months after the onset of symptoms to see a healthcare professional, leading to unnecessary vision loss. This results in incremental costs of care (which most often accrue to Medicare), a higher societal burden and lower patient quality of life. In earlier studies, we have identified critical reasons for patient delay and significant gaps in the current standard of care. Our solutions address the most significant reasons for patient delay in a cost-effective format capable of distribution to millions of patients, that, if successful, will accelerate presentation and minimize vision loss for a significant population of patients. Background: There are almost 10 million people with early AMD in the US, with the number expected to rise to 17.8 million by 2050. 2,3 of this population, 10 to 15% of dry patients will eventually convert to wet AMD.4 The conversion from dry to wet AMD can occur suddenly and has been historically difficult to recognize and respond to by patients. Delays in presentation of up to 5 months are common.8 In a recent study of patients with newly diagnosed wet AMD, the most common patient-cited reasons for delay were lack of confidence in symptoms, and a lack of appreciation of the urgency of the problem. The current gold standard of home monitoring, the Amsler Grid, developed in the 1940's, does not address the top reasons for delay, nor does it address the variety of symptoms that patients experience with advancing disease. New electronic technologies for improving home monitoring AMD have demonstrated sensitivity and specificity, but their cost and learning curve can limit broad-scale distribution. A solution is required that 1) addresses the multiple reasons for delay, 2) is low-cost and amenable to distribution across large populations, 3) includes multiple interactive elements that address patient compliance and individual needs, and 4) fosters appropriate and timely action. Research design and methods: 1) conduct focus group evaluation of current prototype tools. 2) Refine the prototypes based upon patient feedback. 3) Conduct a clinical evaluation of the performance of the novel tools, measuring drop in visual acuity prior to presentation after onset of wet AMD. If successful, tools will enable broad-scale outreach, reduce educational disparities, foster routine eye exams, help accelerate patient presentation, help reduce vision loss, and help lower the socioeconomic burden of AMD. PUBLIC HEALTH RELEVANCE: Age-related macular degeneration (AMD) is the leading cause of blindness in adults over the age of 60.1 New injectable treatments introduced in 2005 are effective if given early but patients typically wait for up to 5 months after the onset of symptoms to see a healthcare professional, leading to unnecessary vision loss. This results in incremental costs of care (which most often accrue to Medicare), a higher societal burden and lower patient quality of life. The current study refines a patent pending novel group of low cost tools and assesses their ability to reduce presentation delay.
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{
"pile_set_name": "NIH ExPorter"
}
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The proposal deals with very early development of peripheral and central taste systems in the rat. Three of the specific aims build on the principal investigator's demonstration that taste papillae will form in vitro from organ cultures of the embryonic rat tongue lacking normal sensory innervation. Specific Aim 1 will determine the potential for development in organ culture as a function of the age of the embryonic tissue. It will attempt to establish embryo age thresholds and cut- offs. Specific Aim 2 determines if in vivo molecular characteristics of tongue are maintained in organ culture. Aim 3 determines the effects of co-culturing the tongue with gustatory and nongustatory sensory ganglia on papilla and taste-bud formation in vitro. Specific aims 4 nd 5 deal with second order taste neuron development and utilize brainstem slices of the NST to explore basic changes in neuron circuitry and function. Specific Aim 4 tests the hypothesis that the adult level of sensitivity of NST neurons to excitatory and inhibitory neurotransmitters is acquired gradually following birth. Aim 5 tests the hypothesis that intrinsic neurotransmitter characteristics of rat NST cells are altered by dietary sodium deprivation. These in vitro systems have not previously been used to study development in taste, a sense that influences food selection and nutrition.
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{
"pile_set_name": "NIH ExPorter"
}
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The purpose of this project is to identify optimal treatment for malignant diseases in children through continuing collaborative studies within the Children's Cancer Study Group. Statistically valid selection of these treatments which are most effective in prolonging survival of children with cancer cannot be achieved in a reasonable period of time within single institutions. Interinstitutional collaborative studies provide a more efficient means of achieving such a goal. These studies utilize standardized protocols variously designed to determine (a) dosage and toxicity of new drugs (Phase I.1, (b) efficacy of select drugs for specified malignancies (Phase II), and (c)-the.best of alternative treatments for a given disease by-means of randomized clinical trials (Phase III). A multidisciplinary diagnostic and therapeutic effort is essential for the current optimal management of the child with cancer. The proposed project will formalize our efforts to develop a coordinated, cooperative approach to management within our own institution that includes among others, chemotherapists, radiation therapists, surgeons, pathologists, immunologists, cytogeneticists, and pharmacologists. To enhance our ability to care for the infants and children of North Carolina with cancer, the joint collaboration with an interinstitutional cooperative group becomes essential. Such a collaboration will promote interdisciplinary functions, enrich teaching and clinical research within our institution, and help to introduce optimal approaches to the diagnosis and treatment of childhood cancer to our referring physicians and others at the community level.
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{
"pile_set_name": "NIH ExPorter"
}
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Assembly and Secretion of Apo B Containing Lipoproteins: The dyslipidemia of insulin resistance is very common and is associated with increased assembly and secretion of atherogenic apolipoprotein B (apoB) containing lipoproteins, particularly the triglyceride (TG)-enriched very low density lipoproteins (VLDL). Recently, hepatic steatosis, or non-alcoholic fatty liver disease (NAFLD), has emerged as an additional component of the phenotype. Pharmacologic approaches that would benefit both plasma lipid levels and hepatic fat are limited. A direct path to reducing plasma levels of atherogenic apoB-containing lipoproteins would be inhibition of apoB or TG synthesis and/or assembly into a VLDL. However, efforts to take this path have been associated with severe steatosis e.g., inhibition of microsomal triglyceride transfer protein (MTP) or modest steatosis e.g., inhibition of apoB synthesis using antisense oligonucleotides (ASO). Our preliminary data suggests that autophagy can play an important role in protecting the liver from steatosis while effectively reducing VLDL secretion. Thus, when we treated mice with MTP ASO, significant reductions in VLDL secretion were associated with severe steatosis. However, treatment with apoB ASO led to similar reductions in VLDL secretion but no increased steatosis. Further investigations revealed that apoB ASO treatment stimulated endoplasmic reticulum (ER) autophagy that was associated with increased fatty acid (FA) oxidation. Using this as a foundation, we will test the following hypothesis that reduced availability of apoB in the ER, without concomitant reductions in ER TG, stimulates autophagy, which protects the liver from steatosis with the following aims: Aim 1a. To characterize the effects of inhibiting apoB synthesis, without altering TG delivery to the ER lumen, on hepatic lipid homeostasis and autophagy using mouse models. Aim 1b. To characterize the molecular basis for ER autophagy in cells with inhibited apoB synthesis. We will then extend our studies beyond the specific model of inhibition of apoB synthesis to address the more common states where hepatic lipid metabolism is perturbed: increased FA delivery to the liver, insulin resistance with increased apoB secretion, and perturbations of ER function associated with ER stress. Here we will test the hypothesis that ER autophagy is a mechanism for maintaining homeostasis in the secretory pathway whenever abnormal VLDL assembly and/or secretion lead to ER stress and the UPR with two aims: Aim 2a. To characterize, in greater detail the mechanism whereby FA-induced ER stress and the UPR stimulate autophagy. Aim 2b. To characterize the effects of overexpression of apoB on ER stress, the UPR, and autophagy. The detailed and broad-based studies of autophagy and apoB proposed herein could provide new approaches to reducing VLDL secretion while avoiding hepatic steatosis.
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{
"pile_set_name": "NIH ExPorter"
}
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Although the prevalence of asthma in the U.S. is increasing, the pathogenic mechanisms of asthma are not well characterized. In particular, mechanisms initiating and maintaining airway mucosal inflammation and airways hyperresponsiveness remain largely undefined. Among the possible mechanisms that may contribute to the pathogenesis of asthma, airway innervation, especially the sensory innervation, may exert important and, as yet, unrealized influence on airway responses. There is mounting evidence that infections or irritant exposure occurring during infancy and early childhood may contribute to asthma in children and adults. Therefore, the primary goal for this project is to characterize responses of airway neurons to infections and irritant exposures during early postnatal periods and to investigate changes in airway neurons that are initiated during early postnatal life but that may contribute later to the occurrence of airway inflammation and hyperresponsiveness. Experiments are designed to test the hypothesis that airway infections and irritants not only evoke an initial release of neurotransmitters from airway nerve terminals, but may also stimulate neurotransmitter synthesis lasting weeks or longer thereby increasing the amount and availability of neurotransmitter released in the airways subsequent to an exposure. The proposed studies will also test the hypothesis that infections and irritant exposures occurring during critical periods of early growth and development selectively sensitize airway neurons so that responses to subsequent exposures will be greater than normally expected. The specific aims for the project are 1) to quantify innervation density and responsiveness of different populations of nerve fibers in airways from fetal, postnatal, young and adult rats in various age groups of normal animals and after infections or exposure to inhaled airway irritants, 2) to examine neural responses in adult rats that were exposed to airway infections or irritants during early postnatal life, and 3) to determine the possible role of nerve growth factor in mediating neuronal responses to airway irritants and infections.
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{
"pile_set_name": "NIH ExPorter"
}
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We have demonstrated that circulating phosphate is a critical determinant of normal growth plate[unreadable] maturation. Hypophosphatemia impairs appptosis of hypertrophic chondrocytes, both in vivo and in[unreadable] vitro, leading to the development of rickets in growing animals. Interestingly, while hypophosphatemic[unreadable] Vitamin D Receptor (VDR) null mice, mice with diet-induced hypercalcemia/ hypophosphatemia and hyp[unreadable] mice all develop progressive expansion of late hypertrophic chondrocytes due to impaired apoptosis of[unreadable] these cells, Npt2a null mice have not been reported to develop rickets. Our preliminary data[unreadable] demonstrate that, while deletion of this renal Na-dependent phosphate transporter leads to the same[unreadable] degree of hypophosphatemia as is observed in the other models, the growth plate phenotype in these[unreadable] mice is transient. An expansion of the late hypertrophic chondrocyte layer, accompanied by impaired[unreadable] apoptosis of these cells is observed at 16 days of age, whereas by 35 days of age, there is resolution of[unreadable] this abnormality.[unreadable] The studies in this proposal will address the hypothesis that a specific transport mechanism for[unreadable] phosphate is present in hypertrophic chondrocytes and mediates apoptosis of these cells. They will[unreadable] examine whether there is an intrinsic difference in phosphate transport in cultures of hypertrophic[unreadable] chondrocytes from hyp mice, Npt2a null mice and wildtype mice. They will also determine whether[unreadable] intrinsic cellular factors modify the susceptibility of chondrocytes, isolated from these animals, to[unreadable] phosphate-mediated apoptosis.[unreadable] Investigations will be performed to address the hypothesis that a difference in circulating hormone[unreadable] levels contributes to the difference in the growth plate phenotype of the Npt2a and hyp mice. Primary[unreadable] chondrocytes, isolated from these mice will be treated with PTH, 1,25-dihydroxyvitamin D and FGF23 to[unreadable] address the hypothesis that these agents modulate phosphate transport and/or susceptibility to[unreadable] phosphate-mediated apoptosis. To address the hypothesis that enhanced 1,25-dihydroxyvitamin D[unreadable] action is responsible for normalization of the growth plate of Npt2a null mice, 1,25-dihydroxyvitamin D[unreadable] action will be blocked by mating them with VDR null mice. Hyp mice will be treated with 1,25-[unreadable] dihydroxyvitamin D to address the hypothesis that impaired 1,25-dihydroxyvitamin D action and /or[unreadable] increases in PTH contribute to the development of rickets in this disorder.[unreadable] These investigations are expected to reconcile the difference in the growth plate phenotype[unreadable] observed in hypophosphatemic mouse models and to identify a role for circulating hormones in[unreadable] modulating the susceptibility of hypertrophic chondrocytes to phosphate-mediated apoptosis.
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{
"pile_set_name": "NIH ExPorter"
}
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With the rapid advances in communication technologies, children are increasingly exposed to the radiofrequency (RF) fields starting from a very early age. As cellular telephones are a new, but increasingly common source of a relatively high RF fields, concerns are increasing that this technology could have negative health effects. Investigation need to be initiated immediately while there are still some populations with no or low exposure. In line with the NIEHS's mission to promote the public's right to a healthy environment, the aim of this proposed research is to understand whether exposure to cellular telephones in childhood can have effects on the central nervous system and whether children might be particularly at risk. The Danish National Birth Cohort (DNBC) represents a unique resource, which will allow us to efficiently evaluate whether exposures [in children] lead to behavioral and developmental effects. The cohort consists of over 100,000 Danish children, who were born between 1996 and 2002. Detailed information on lifestyle factors, dietary habits and environmental exposures has been collected. When these children reached the age of seven, we began to ask about current use of cell phones by children. Our results among 10% of the children suggest that cell phone exposure might be related to behavioral and developmental problems. These findings need to be replicated for the rest of the cohort, [and to evaluate if our findings are limited to "early users", who might be different from the rest of the population. Also, several additional potential confounders have been suggested, and we plan re-analysis adjusting for these factors.] We also propose to investigate other central-nervous system-related outcomes such as seizures, autism, migraines, and sleep disturbances. The proposed novel project is the first large scale study to examine health effects of cell phone use early in life. Given that there are over two billion subscribers worldwide and that children are beginning to use cell phones at earlier ages, studies of children have been determined to be of the highest priority by both national and international organizations, including the World Health Organization and National Academy of Sciences. Children may be at greater risk because: 1) their exposure to the RF might be higher due to differences in the anatomy, tissue conductivity and other factors;2) they might be more susceptible due to the developing organ and tissue systems, particularly of the nervous system;and 3) children born in the last decade are part of a unique population that will have lifelong cell phone exposure. [Our preliminary results have results have received a widespread scientific and media attention. Several issues have been raised post publication.] Here we propose to address these issues and thus to significantly advance our knowledge on health effects of RF field exposure in a potentially vulnerable population and contribute to the risk assessment and policy development of this ubiquitous and rapidly increasing exposure. We believe such a study is urgently needed and at present it can only be done using data sources outside the U.S. PUBLIC HEALTH RELEVANCE: To date potential health effects of cell phone use [in children] have not been adequately examined. As this exposure is extremely prevalent, even very small effects of cell phone exposure could have a large public health impact. [We have reported an important result which needs replication: further information will have immediate policy implications.] If our findings are confirmed reducing exposure in this very vulnerable population is easily achievable.
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{
"pile_set_name": "NIH ExPorter"
}
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The Administrative Core will be responsible for integrating all scientific and infrastructure activities of the Conte Center. Drs. Robert Schwarcz (the Center PI and co-PI of the Core) and William Carpenter (co-PI of the Core), together with the Steering Committee, will have overall responsibility for all administrative functions of the Center. The Steering Committee will include the key scientific leadership for this application: Dr. L. Tonelli (PI, Project 2); Dr. L.E. Hong (PI, Project 3); Dr. R.W. Buchanan (PI, Project 4); Dr. R. McMahon (Biostatistician); and Dr. J. Gold (Neuropsychologist), and will meet monthly to review the progress of each of the projects. They will also review the Biostatistics and Data Management Module, which provides statistical and data base support for the projects. In consultation with NIMH program staff, the Core will establish an External Advisory Committee and consult/meet with the advisors regulariy to review plans. The External Advisory Committee will include experts in relevant translafional neuroscience, clinical research and drug discovery academic and industry labs. The Steering Committee will also be directly involved in: review of proposals for Pilot and Feasibility projects; facilitating the involvement of post- doctoral research fellows and junior faculty in Center activities; involving all Center scientific staff in Center progress through an annual retreat; overseeing training in responsible conduct of research and HIPAA regulations, diversity recruitment; overseeing compliance with UMB and Federal Human Subjects and Vertebrate Animals regulations; implementation of an Optional Summer Research Experience for undergraduates; dissemination of research results and sharing of research data; and coordinating the provision of staff support [funded by the host institution (MPRC) rather than this grant] for administrative functions of the Center; data management and data entry; and IT staff, as well as IT hardware and software resources. RELEVANCE (See instructions): The Administrative Core will be responsible for the close oversight and optimal integration of all planned Conte Center activities. Led by experienced investigators. Core personnel will facilitate investigator interactions and productivity, internal and external educational projects related to Center science, and relations with regulatory groups. ^
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{
"pile_set_name": "NIH ExPorter"
}
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The broad goal of this research is to learn how a single epithelial cell sheet becomes patterned during embryonic development into discrete domains of different cell types. The epidermis of the nematode Caenorhabditis elegans, will be studied as a simple model epithelium. This epithelium becomes subdivided into three major cell subtypes during embryogenesis. Particular emphasis will be placed on the mechanisms by which one of these subtypes, the seam cells, are specified. Because the mutations studied here lead to severe defects in embryonic development, these studies should serve as models for human birth defects. In addition, by examining genes that cause proliferating embryonic cells to select particular paths of differentiation, this work should contribute to our understanding of mechanisms by which normal controls of growth and differentiation are abrogated in malignantly transformed cells, resulting in cancer. The zygotically expressed gene, zen-3, is essential for normal epidermal patterning and differentiation of seam cells during embryogenesis. Patterning and seam differentiation defects in six zen-3 mutants will be studied by immunofluorescence with epidermis-specific antibodies to characterize the range of defects resulting from changes in the activity of zen-3. The development of individual epidermal cells in a representative zen-3 mutant will be followed by cell lineage analysis using a video system that records the position of all cells throughout embryogenesis. To assess the time of action of zen-3 function, the temperature-sensitive (ts) period of a ts zen-3 mutant will be determined. A yeast artificial chromosome that carries zen-3 (+) function will be identified and fused to a duplication carrying a cell-autonomous marker. This fused duplication will be used to define the cells in which zen-3 is required by performing genetic mosaic analysis. To initiate studies of other genes involved in seam specification and epidermal patterning, an existing collection of 55 mutants defective in epidermal development will be screened by immunofluorescence and time-lapse. This screen will be used to narrow in on one or a few genes for future detailed investigations. Finally, laser ablations of early blastomeres will be performed on wild-type embryos to identify cells required for seam cell differentiation. These experiments will ultimately lead to the elucidation of epidermal patterning at molecular resolution.
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{
"pile_set_name": "NIH ExPorter"
}
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Proposed work will continue research in the areas of social, cognitive, and perceptual development. The research in social development will focus on friendship relations and problem solving, on effects of various instructional sets on children's interactions, and on the relation between attachment formation in infancy and subsequent establishment of peer relations. The cognitive development research will be directed toward examination of children's problem solving in naturalistic settings. It will further use analytic techniques developed from story grammars to understanding children's inferences in reading and listening to stories, and viewing pictorial sequences. Additional cognitive development work will attempt to determine the nature of mental representations people construct of their spatial environment. In the area of perceptual development perceptual-motor development will be examined in the context of speech production as an auditory-motor task and in the context of development of eye movement control. Other perceptual research will continue to examine development of sensitivity to pictorial depth information in infants. In a final project on perceptual development, children's perception of melodies and their learning to read musical notation will be investigated.
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{
"pile_set_name": "NIH ExPorter"
}
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The technique of microwave inactivation of tissue will be used to study the in vivo effects of paraquat and diquat on labile metabolites in mouse lung. Mice will be given a range of doses of paraquat or diquat and sacrificed over varying periods of time from 1 hour to several days after i.p administration of these compounds. This dose and time schedule will allow investigation of the early destructive phase and later proliferative phase of paraquat on the following: (1) high energy compounds: ATP, ADP, AMP, creatine phosphate, Pi, (2) cyclic nucleotides: cAMP and cGMP, (3) serotonin and acetylcholine, (4) membrane ATPase, (5) mitochondrial respiration, (6) pyridine nucleotides: NADPH, NADP ion, NAD and NADH and (7) intermediary metabolites: lactate, pyruvate, alpha-ketoglutarate, succinate. Changes in the in vivo levels of these compounds will provide information on the effect of paraquat and diquat on the energy and redox state of the lung. Correlation of these effects of paraquat and diquat may provide an insight into the toxic mechanisms of these compounds.
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{
"pile_set_name": "NIH ExPorter"
}
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Several adverse effects of marijuana (cannabis) use are recognized including those on central nervous system function. Among the most prominent effects of cannabis on neural function are deleterious influences on memory, which may be related to damage to the hippocampus. In the brain, the effects of cannabinoids are transduced by the CB1 receptor (CB1R). CB1Rs are highly expressed during development, couple with Gi/Go, inhibit neurotransmitter release, and hyperpolarize neurons. Whereas a considerable amount of information is known about cannabis and CB1R action in the mature nervous system, relatively little is known about effects on the developing brain. Increasing evidence suggests that suppression of neuronal activity during critical periods of brain growth will trigger neuronal apoptosis. Suggesting that the developing brain is also sensitive to effects of CB1R activation, we observe that acute cannabinoid exposure induces apoptosis in the developing, but not mature hippocampus. Based on these observations, we propose to test the hypotheses that (1) the developing hippocampus is especially sensitive to the effects of CB1R activation. (2) Cannabinoid toxicity during early development is due to inhibition of neuronal activity. To test these hypotheses we will perform in vivo and in vitro studies using contemporary neuroscience methods and CB1R knock-out mice. We anticipate that these studies will result in new insights into the potential effects of cannabinoid action on the developing brain.
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{
"pile_set_name": "NIH ExPorter"
}
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Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) of unknown etiology characterized by chronic gastrointestinal (GI) tract inflammation. The key pathological mechanism in IBD may involve a dysregulated immune response to GI tract antigens or infections. Inflammatory Th1 responses characterized by interferon-gamma and tumor necrosis factor-alpha synthesis correlate with IBD, whereas Th2 responses characterized by interleukin-4 and transforming growth factor beta-1 synthesis correlate with protection from IBD. Susceptibility genes contribute to IBD, but inheriting these genes is not sufficient for disease development. Undefined environmental risk factors play a disease-determining role. We hypothesize that insufficient sunlight for vitamin D biosynthesis is an environmental risk factor for IBD. We propose to use the hormonally active vitamin D metabolite, 1,25-dihydroxy vitamin D3 to inhibit IBD development and to treat established IBD in murine models of IBD as a first step in developing a novel therapeutic strategy for this incurable disease. PROPOSED COMMERCIAL APPLICATION: Incurable IBD afflicts two million individuals and costs about $1.2 billion annually to treat. The steroids, immunosuppressants and surgical treatments for IBD symptoms have undesireable side-effects. An FDA-approved short-term treatment (mouse-human IgG1 monoclonal antibody to tumor necrosis factors- alpha) has projected annual sales of $100 million. The commercial potential of a long-term vitamin D-based therapeutic might be greater than $100-150 million.
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{
"pile_set_name": "NIH ExPorter"
}
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The aim of this interdisciplinary program is to determine the structure-function relationships and mechanisms of proteins in normal cardiovascular signaling and subject to abnormalities that cause disease. Major components (Projects 1, 2 and 3) address the mechanism and structure of small GTP-binding protein RhoA, its effector proteins and its mechanisms of regulation. RhoA regulates vascular smooth muscle contraction, cardiac hypertrophy and formation of stress fibers, and its downstream effector, Rho-kinase, was recently implicated in hypertension. The cellular mechanisms of RhoA and Rho-related proteins will be determined in Project 1, their atomic structures in Project 2, and their subcellular localization in Project 3 with proteins largely produced in Core B. The mechanisms of Ca2+-sensitization and the mechanisms of Ca}+-desensitization will be determined in Projects 1 and 3, and its subcellular localization of signaling proteins (sensitivity and de-sensitivity) in Project 3. The molecular structure of LPP, a newly discovered to be smooth muscle specific protein will be determined with combined X-ray crystallography and NMR spectroscopy (Project 2). The program will complete the development of a powerful structural method, energy filtered scanning transmission electron microscopy (STEM-EELS), that is designed to obtain compositional information at nanometer resolution about the cellular distribution and movement of calcium and other elements. This method will be used to quantitatively map calcium bound to a cardiac intercalated disc and vascular smooth muscle cell membranes, and accumulated in mitochondria and other organelles and will relate these findings to the normal functions and abnormal effects of calcium. The high level of biomedical significance of this is derived from the involvement of the smooth muscle regulatory proteins in asthma and high blood pressure, and from the importance of calcium in the development of ventricular fibrillation, the most common cause of sudden cardiac death.
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{
"pile_set_name": "NIH ExPorter"
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One of the most pervasive problems for stroke survivors is movement deficits. Recent research strongly supports prolonged practice of functionally-relevant activities of the upper limb, even though therapy time is quite limited by the current medical economic system. This grant focuses on new developments in human- robot interactions (haptics) that have revealed prospects in the areas of motor teaching and rehabilitation. Specialized robotic devices combined with computer-displays can tirelessly exert force, augment feedback, and redirect error in order to speed up, enhance, or trigger the motor relearning process. These approaches could extend and greatly enhance the recovery process. The first strategy that often comes to mind for teaching movements is to guide the limb along the desired path. However, a promising alternative approach is to make movements more difficult by deflecting them from the desired path. People develop, through practice, the ability to counteract forces that distort the mechanical world, and if these forces are properly designed and applied, a desired movement pattern occurs when the forces are eventually switched off. We and others have also obtained similar results by distorting the visual world using prisms or virtual reality displays. In these studies, the subject sees something unexpected that is perceived as an error. Our results point to a single unifying theory: Errors induce learning, and judicious error augmentation (through forces or visual distortions) can lead to lasting desired changes. Interestingly, this process appears to bypass conventional learning mechanisms that require intense concentration - results are the same if the subjects have a conversation or listen to music. They often consider it a game. Until now very little of this research has been functionally relevant because the devices' ranges of motion were small, were two dimensional, and were lacking an appropriate visual interface. Three dimensional movements introduce the daunting new challenge of gravitational effects that could reduce (or perhaps heighten) the potential of error augmentation training. Our lab has spent several years developing a large- workspace, three dimensional haptics/graphics system. The aims of this grant are to build on our promising body of evidence and expand our error augmentation training work to a large workspace in three dimensions. Accordingly, the experiments below further refine our understanding of error augmentation (Aim 1), expand our approaches to three dimensions (Aim 2), and then move towards clinical application by testing our approaches on stroke survivors (Aim 3). [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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This project investigates the developmental neurobehavioral effects of analgesics used for pain relief during labor and delivery. A rhesus monkey model has been developed for induction and monitoring of labor, intrapartum administration of analgesics and neurobehavioral evaluations during the first year of life. During the last project year, a study was completed using epidural bupivacaine, the second most common form of obstetric analgesia in this country. Results from the neurobehavioral test battery indicated that bupivacaine did not affect neonatal status or cognitive functions in infancy. Bupivacaine did alter the course of neurobehavioral maturation at two important time periods during brain development. The increase in manipulatory activity that normally occurs at 2 months of age was delayed by bupivacaine. The increase in motor disturbance behaviors that normally occurs at 10 months of age was prolonged and exacerbated by bupivacaine. We also completed experiments in guinea pigs demonstrating that epidural bupivacaine administered to the dam reaches fetal brain and that accumulation in fetal brain depends on the maturity of the fetus. Finally, assays of plasma protein throughout pregnancy in rhesus demonstrated that alpha acid glycoprotein, the main serum binding protein of bupivacaine at low doses, more than doubles during late pregnancy, while albumin, the secondary serum binding protein, decreases by a similar percentage. Together these studies demonstrate that bupivacaine administered epidurally to the mother during labor reaches fetal brain and can have long term effects on behavioral maturation.
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{
"pile_set_name": "NIH ExPorter"
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The proposed research is directed toward the differences that exist in the regulatory as well as structural properties of phosphofructokinase in tissues of diverse metabolic function. Particular focus will be placed on the regulator properties of a new isozyme found in brain and on the comparison of its properties with other isozymes. Further studies of isozyme hybridization under conditions that might exist in vivo will be carried out. The regulatory properties of the principal hybrid, A2B2, will be examined.
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{
"pile_set_name": "NIH ExPorter"
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A simple and direct strategy for the stereocontrolled total synthesis of several proven or potential cytotoxic guaianolides is proposed. The synthetic studies are based on a novel acid catalyzed cyclization which efficiently generates a cis-fused hydroazulene intermediate appropriately functionalized for conversion into the guaianolide family of sesquiterpene lactones. The synthesis exploits the unique reactivity of tropone, a highly functionalized seven membered ring system as the key precursor to the bicyclo [5.3.0] decane species. The principal target molecule zaluzanin C, has demonstrated antileukemic activity and the other target species have structural features such as the Alpha-methylene-Gamma-lactone group, which suggest potential anticancer activity as well. To date, very few guaianolide syntheses have been reported and in view of the large number of these natural products with anticancer potential, further effort must be directed toward developing efficient syntheses of these compounds.
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{
"pile_set_name": "NIH ExPorter"
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Project Summary Chagas disease is a severe inflammatory syndrome caused by infection with a protozoan parasite, Trypanosoma cruzi. Although transmission occurs mostly through an intermediate insect host, the infection is silent and chronic carriers pose a significant risk through the transfer of infected products made from donated blood of these individuals to blood recipients. Up to 500,000 new cases and 66,000 deaths are reported annually in countries in South and Central America where the parasite lives. The combination of immigration and expansion of the range where the parasite host lives is, however, leading to large numbers of infected individuals in countries that until recently did not have Chagas disease. The United States is foremost amongst these. Control and prevention of Chagas disease relies on the accurate detection of infection. Improvements in the currently available diagnostics are required. In our research we have found a novel way to identify pieces of the parasite (certain proteins) that could be used as components of a new diagnostic test. Since they have the ability to tightly bind antibodies from infected individuals and are often selected from a variety of individual diagnostic leads, we stitch them together as ?multiple antibody-binding epitopes? to enhance the diagnostic power. These ?polyproteins? maximize the ability to react with infected serum, enabling the new test. We have used computational searches to identify a number of novel diagnostic candidates in T. cruzi and our preliminary data indicate several that hold promise as important components with a new diagnostic test. The research in this proposal will develop an improved and cost- effective diagnostic test to permit screening for T. cruzi infection / Chagas disease. The proposed research will proceed in two steps: In step 1 we will optimize and tightly define the production parameters of two lead polyproteins. In step 2 we will integrate these diagnostic leads into a format consistent with use in standard reference laboratories/ blood centers as well distribute them to our extensive network of collaborators in a wide variety of T. cruzi-endemic regions for further evaluation. If successful, in the phase II application, prototype tests and adjunct test formats will be produced and extensively evaluated as potential commercial tests.
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{
"pile_set_name": "NIH ExPorter"
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The goal of this senior postdoctoral fellowship application is to educate the applicant in three areas: 1) genomics and 2) proteomics, including both the laboratory techniques and the in silico tools necessary for such studies, and 3) the statistical methodology necessary for comparing the relative strength of selection on different genes introgressing across a hybrid zone. The specific aims of this proposal are: 1. To identify the protein components of mouse seminal fluid using proteomic and genomic approaches. The genomic approach will use existing data on ESTs from mouse prostate to identify candidate genes and the proteomic approach will use high-throughput LC-MS/MS to directly identify components of mouse seminal fluid; 2. To sequence candidate genes for the mouse seminal fluid proteins identified above a) in five different rodent species for analysis by statistical tests of the Neutral Theory to look for site-specific evidence of selection (positive and negative) on these genes and b) in wild-derived M. m. domesticus and M. m. musculus populations to look for fixed or nearly fixed polymorphisms in these genes for use in hybrid zone studies outlined in Aim 4 below; 3. To identify true orthologous genes for seminal fluid proteins by asking whether evolutionary similar genes fall within syntenic regions of mouse, human, and other mammalian genomes; 4. To test for selection on specific seminal fluid protein genes by plotting their introgression across the Mus musculus hybrid zone in Europe: a) to discover candidate genes (i.e., shown to be under selection by Aim 2a) that have a fixed or nearly fixed polymorphism between the two subspecies (Aim 2b above); and b) to compare clines of introgression of seminal fluid protein genes across the hybrid zone for seminal fluid protein genes with clines for genes under relatively low selection (e.g., many nuclear allozymes) and genes under high selection (e.g., X and Y chromosome polymorphisms). [unreadable] [unreadable] Relevance: Seminal fluid proteins show dynamic evolutionary histories, significant positive selection and variable selective pressure between lineages. As our understanding of the roles of seminal fluid components grows, so will our understanding of factors that influence reproductive success in humans and other animals. This proposal will provide a comparative genomics view of mammalian seminal fluid proteins from rodents and primates, which should lead to a better understanding of, and ability to cope with human problems involving infertility. Furthermore, the major source of seminal fluid proteins is the prostate, a common site of male cancer. Disease research may benefit from studies of selection, since positive selection is often associated with human disease genes. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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Our experiments investigate the mechanism of action of excitatory amino acids as synaptic transmitters and neuromodulators in the vertebrate CNS, utilizing cell culture and electrophysiological techniques. Much of our work is on the N- methyl-D-aspartate (NMDA) receptor subtype. Permeation and block of NMDA receptor channels by divalent cations has been investigated using whole cell recording. Ba, Ca, Mn and Sr are all permeant, while Co, Mg, and Ni are voltage dependent blockers. Reversal potential measurements with 0.1 to 50 mM extracellular Ca, and 50 to 150 mM extracellular Na, analysed using an extended constant field equation, show Ca to be approximately 10 times more permeant than Na. However binding of permeant divalent cations is suggested by their block of inward Na current, and alternative models are needed to describe permeation. Low concentrations of zinc and cadmium also block responses to NMDA, however their action is similar at +60 and -60 mV, and thus due to an action at a different site from that for Mg. Zinc acts as a noncompetitive NMDA receptor antagonist, and thus does not interfere with the initial binding of agonist. Fluctuation analysis shows a reduction in open time during Zn and Cd antagonism; possible models under consideration include ultra fast channel block and allosteric modulation to substrates of reduced conductance and lifetime. Excitatory synaptic transmission in hippocampus and spinal cord has been studied under voltage clamp. Epsps are produced by two components of synaptic current: a fast inward current of decay time constant 1-5 ms due to activation of kainate/quisqualate receptors, and a slow component of time constant circa 80 ms due to activation of NMDA receptors. The slow component of the epsp is blocked by selective NMDA receptor antagonists, including low concentrations of zinc, is voltage sensitive in the presence of Mg, and has a Ca-dependent reversal potential. Glycine (1 muM) is a potent modulator of the slow epsp. Conditioned medium from hippocampal glial cell cultures also potentiates the slow epsp and responses to NMDA suggesting that release of modulatory substances from glial cells warrants further investigation. Analytical techniques developed to study the synaptic release process include deconvolution analysis, and a novel nonstationary fluctuation analysis of synaptic currents.
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{
"pile_set_name": "NIH ExPorter"
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The historical approaches to training biomedical scientists have been very successful at creating a talented, creative, community of scientists, but have failed to produce meaningful improvement in the participation of individuals from underrepresented racial and ethnic minority (URM) groups in this community. The thesis of this proposal is that the absence of change is an unintended consequence of the fundamental culture and practices of biomedical research and research training. The culture and practices can be well modeled and interpreted drawing on several well-established social science theories, including: 1) Communities of Practice, which describes how individuals with common interests and goals engage to achieve those goals, especially the processes by which new individuals enter the group, gradually acquire (or fail to acquire) the informal knowledge and practices of the group, and become full participants;2) Social Cognitive Career Theory, through which its variables of self-efficacy, outcome expectations, personal goals, and contextual supports/barriers provide insights into forces guiding individual development and career choices;3) a comparison of the strengths and inherent limitations of mentoring (as practiced in biomedical research training) versus coaching (as practiced in the development of athletic talents). From these theoretical frameworks, an experiment is proposed to test the hypothesis that: a hybrid model employing sophisticated coaches to complement what scientific mentors typically provide can mitigate the unconscious processes that significantly impair professional advancement of young URM scientists. The experiment would be a randomized controlled trial of a coaching-based model for a diverse cohort of 160 PhD students who have a strong desire to pursue academic careers. A community of these individuals would be created from around the U.S. and sustained through an annual professional development sequence, periodic electronic meetings throughout the years, and a mediated social network to sustain professional development and progress toward an academic career. PUBLIC HEALTH RELEVANCE: To continue and enhance the prominence of the U.S. in biomedical research, we must find ways to identify, develop and employ the talents of a broader spectrum of our population. The proposed model represents a substantial shift in thinking and approach, and challenges conventional assumptions and practices, but if successful could finally achieve a breakthrough in the efforts to diversifying the biomedical workforce. It also could be applied to many disciplines and talent pools because it is a generalized model that more systematically approaches development of human talents starting from well-developed social science principles.
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{
"pile_set_name": "NIH ExPorter"
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We are in the gradual process of developing a Picture Archiving and Communications System (PACS) for our Radiology Department. At the present time, we have completed construction of the first component for that system; the component is a viewing station that we term the Arizona Viewing Console or AVC. We have begun characterization of this viewing console and have performed usage studies. As a result of our experiences with this console, we have concluded that certain of its performance specifications should be changed. This research proposal is directed toward implementing those changes and measuring their effects. The proposal is divided into five tasks: (1) modify the Arizona Viewing Console, (2) construct a high performance viewing console, (3) provide an image control software environment, (4) refine our display characterization facilities and (5) evaluate console performance and acceptance by radiologists. The modifications to the AVC are for attaining increased speed of access to the images by expanding the semiconductor memory of the system and to allow more flexible software to be implemented. The modifications are to be tested and then integrated into the design of the high performance console. We will produce a machine independent software environment for handling images n the PACS; one element of this environment will be providing images to the display console. This environment will make possible efficient evaluation of the display console when functioning as a part of the PACS system. It will also strongly reduce the amount of effort which must be invested in implementing changes and adding new functions. Characterizing and evaluating the display as part of a more complicated system is an extension of work in our ongoing research. The characterization consists of physical, psychophysical and clinical evaluation. We will be adding capabilities to a cart we have designed and constructed for in-place measurement of CRT characteristics. The most fundamental important change will be the additional CCD camera-based sensors to the present photomultiplier. In addition to physical measurement techniques, we will apply psychophysical methods to judging the effectiveness and acceptability of the various configurations of the viewing consoles. These system evaluations will be extended to evaluations where we will use a fiber-optic network connection to image acquisition modalities for comparisons to film-based diagnosis.
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{
"pile_set_name": "NIH ExPorter"
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Despite extensive efforts focused on preventing and treating age related cognitive decline, this debilitating condition continues to plague the American population, and is projected to increase dramatically in prevalence unless appropriate interventions are discovered. Due to the irreversible nature of neurodegeneration, prevention, early diagnosis and treatment are imperative. Although oxidative stress is known to be involved early in the etiologies of both normal aging and neurodegenerative disease, lack of a noninvasive assay for brain oxidative status has limited development of antioxidant based interventions. The goal of this proposal is to provide a noninvasive assay for human brain antioxidant concentrations that can be used to: identify influences responsible for compromised antioxidant levels, diagnose reduced antioxidant capacity prior to appearance of symptoms or structural damage, and monitor antioxidant capacity throughout therapeutic intervention. In specific aim 1, a novel noninvasive magnetic resonance spectroscopy (MRS) assay will be used to quantify levels of the two most concentrated non-enzymatic antioxidants in the central nervous system, reduced glutathione (GSH) and vitamin C (Asc) in two homogenous human populations. In specific aim 2, this antioxidant profile will be measured under the respective endogenous and exogenous influences of aging and diet. The research design will start with measuring inter- and intra- individual variation of the antioxidant profile under constant experimental conditions. Next, this noninvasive assay will be used to determine whether previous post mortem findings of a 25% decrease in human brain Asc concentration with increasing age will be replicated in vivo. Finally, antioxidant concentrations will be measured in the elder group both before and after administration of vitamin C supplementation. Double edited H MRS will be used to 1 measure antioxidant profiles from anatomically matched cortical volumes of interest in vivo. Editing is a specialized form of MRS routinely used to resolve one signal of interest from overlapping signals in vivo. Double editing is a novel application that will be used to resolve two signals, i.e. Asc and GSH resonances in the same amount of time previously required to measure one. Seven additional neurochemical concentrations will be co-measured with but resolved from Asc and GSH, and will be evaluated for dependence upon age and diet, as will serum Asc levels. Public health relevance: Oxidative stress is involved in the process that leads to age related cognitive decline, which is expected to afflict an increasing number of Americans unless effective interventions are discovered. The goal of this project is to develop a noninvasive means to measure human brain antioxidant capacity and to explore the extent to which this novel assay can be used to design experiments aimed at discovering new interventions. This work could lead to improved cognitive health of the aging American population. Public health relevance: Oxidative stress is involved in the process that leads to age related cognitive decline, which is expected to afflict an increasing number of Americans unless effective interventions are discovered. The goal of this project is to develop a noninvasive means to measure human brain antioxidant capacity and to explore the extent to which this novel assay can be used to design experiments aimed at discovering new interventions. This work could lead to improved cognitive health of the aging American population. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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Project Summary/Abstract The mitochondrial pathway of apoptosis is the major mechanism whereby cells in vertebrate organisms undergo programmed cell death. It is vital for homeostasis of the immune system, tumor suppression and the regulation of cell number in development. Mitochondrial outer membrane permeabilization (MOMP) and release of intermembrane space proteins such as cytochrome C are central to this process. MOMP is controlled by the Bcl-2 family of proteins. Pro-apoptotic members, Bax and Bak, are activated by another subgroup of the Bcl-2 family, BH3-only proteins, and induce the release of intermembrane space proteins. Anti-apoptotic members of the Bcl-2 family inhibit this process. Based on the finding that BH3-only proteins bind to anti-apoptotic Bcl-2 family members to induce apoptosis, drugs that mimic this effect were developed and are being used in cancer therapy. Better understanding of how Bcl-2 family proteins interact with the MOM and induce permeabilization will allow generation of new or even better drugs. The central hypothesis of this proposal is that there are MOM proteins that co- operate with pro-apoptotic Bcl-2 family proteins to generate lipidic pores in the MOM. The object of this application is to identify these molecules and to determine how they contribute to Bax and Bak activation followed by pore formation. Our Specific Aims are: Aim 1. Identify MOM proteins that co-operate with Bid for induction of MOMP. Unknown MOM proteins assist Bid in Bax mediated-permeabilization of membranes. MOM proteins will be fractionated and reconstituted in proteo-liposomes to identify the active fraction by the dextran release assay. We will investigate whether the identified MOM protein knockdown has the same inhibitory effect as Bid deficiency in mouse embryonic fibroblast (MEF) lines. Aim 2. Determine how Bax activation is regulated prior to MOMP. Using a dextran release kinetics assay, we will determine the time course of Bax conformational change and oligomerization in relation to membrane permeabilization. We will also determine the mechanism of Bcl-xL inhibition by analysis of its effect on release kinetics. We will follow up these experiments by examining cytochrome C release kinetics using mitochondria isolated from cytochrome C-GFP expressing cells in time lapse fluorescent microscopy. Aim 3. Demonstrate lipidic pores induced by Bax and Bak and determine whether Bax localizes to these pores. Our cryo-EM data suggest that Bax generates lipidic pores in the membrane. We will use high-pressure freezing/freeze substitution-EM to demonstrate the existence of these pores in OMVs and mitochondria both in isolation and in cells. This technique will allow us to detect pores with immunogold or nanogold labeled Bax. Mapping the distribution of Bax along the edge of the pores will enable us to determine whether Bax pores are mainly lipidic or proteinaceous.
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{
"pile_set_name": "NIH ExPorter"
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Thyrotropin-releasing hormone (TRH) produced by neurons in the hypothalamic paraventricular nucleus (PVN) has an important role in the regulation of energy homeostasis by establishing circulating levels of thyroid hormone under normal conditions and during special circumstances when metabolic adaptation is necessary such as fasting, infection and chronic illnesses. Mechanisms whereby metabolic signals regulate TRH neurons in the PVN will be explored, facilitated by the use of the Cre/loxP recombinase system in transgenic mice in which Cre-recombinase is expressed selectively in TRH cells. Attention will be given to the importance of direct vs indirect leptin signaling during fasting and in association with diet-induced obesity, the role of AGRP as an inverse agonist at MC-4 receptors, participation of CREB, ERK and AMPK signaling pathways as mediators of leptin's diverse actions on hypophysiotropic TRH neurons, and cannabinoid/glutamate interactions as a novel mechanism involved in fasting-induced suppression the HPT axis. The importance of the hypothalamic dorsomedial nucleus (DMN) as a relay station between the arcuate nucleus and TRH neurons in the PVN will be explored and the hypothesis tested that the DMN integrates signals from leptin- responsive arcuate nucleus neurons and the subparaventricular zone to give rise to the circadian periodicity of the HPT axis. Mechanisms by which endotoxin suppress hypophysiotropic TRH neurons will also be studied, focusing on effects mediated by cAMP-response element modulator (CREM), inducible cAMP early repressor (ICER), corticotropin-releasing hormone (CRH) and the role of increased T3 levels in the mediobasal hypothalamus as a result of endotoxin-induced increased type 2 iodothyronine deiodinase levels in tanycytes. Thyrotropin-releasing hormone (TRH) plays a major role in energy homeostasis by establishing circulating levels of thyroid hormone under normal conditions and during special circumstances such as fasting and critical illness when changes in thyroid status are required for adaptation. The proposed studies will elucidate how this specialized group of neurons in the hypothalamic paraventricular nucleus is regulated by metabolic signals and the neuroanatomical pathways and modulators involved, providing insight into disorders commonly referred to as the nonthyroidal illness syndrome.
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{
"pile_set_name": "NIH ExPorter"
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Behavioral experiments in rhesus monkeys in order to determine the period of maximal susceptibility to the effects of temporary and permanent unilateral and bilateral lid closure on development of visual acuity. Light and electron microscopic studies of the visual pathways of normal and amblyopic monkeys.
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{
"pile_set_name": "NIH ExPorter"
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Traumatic brain injury (TBI) is a leading cause of death and disability in the United States. The CDC reports that each year 1.4 million people in the U.S. sustain a TBI. There is a high incidence of co-occurring alcoholism and other substance abuse among brain injured individuals. Approximately 40% of patients in post-acute rehabilitation facilities have moderate to severe problems with substance abuse and alcohol (Brain Injury Association, 2005). Furthermore, based on surveys, Corrigan (1995) estimates 20% of persons who did not have a history of substance abuse are vulnerable for abuse after TBI. Research has highlighted the links between TBI and substance abuse, and the special needs of these injured individuals in treatment. Substance abuse (SA) treatment professionals rarely are exposed to instruction in this area and need to be able to identify, assess, and more reliably treat the brain-injured individual This proposal will update and expand a curriculum currently used and written for an in-person course to create a new training program for substance abuse counselors. This new course, to be called "The Unknown Dual Diagnosis: Chemical Dependence and Traumatic Brain Injury," will be adapted to a Computer Assisted Instruction (CAI) format. The proposed Web-based CAI course will be carefully designed to resolve problems noted with the face-to-face course. It will be self-directed, allowing individuals to proceed at their own pace. It will be highly interactive, with prompts, questions, and case studies, implemented with audio and video as well as interactive graphics. The course will be tailored for SA treatment providers targeted at both operations and management level staff. With approximately 60,000 substance abuse providers nationally, there is a ready market for this course. This course will be evaluated by experts in TBI and substance abuse, and providers of treatment in the field of substance abuse.
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{
"pile_set_name": "NIH ExPorter"
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Cranberry Proanthocyanidins may be the constituents responsible for the association between consumption of cranberry juice and decreased risks of urinary tract infections because these compounds prevent the adhesion of uropathogenic p-fimbriated E. coli to uroepithelial cells in vitro. Proanthocyanidins are oligomeric flavonoids present in many botanicals and nutritional supplements. However, cranberry proanthocyanidins exhibit structural heterogeneity in the degree of polymerization, nature of interflavan bonds (A and B type), pattern of hydroxylation of flavan units and substitution with anthocyanins. This structural heterogeneity creates difficulty in structure to bioactivity. Biomedical research on the health benefits and risks of increased consumption of cranberry proanthocyanidins is severely limited by lack of information on structure/bioactivity relationships that can be used in standardization of cranberry products. Our core hypothesis is that the bioactivity of cranberry proanthocyanidin oligomers is a function of specific structure. Therefore, given the large diversity of individual oligomers, we would expect large variation in bioactivity. The goals of this proposal are: 1. Characterize structural heterogeneity (i.e. intra-molecular bonds, nature of the substitutions and degree of polymerization) of proanthocyanidin structures in the standardized NIH-NCCAM cranberry products using online and offline liquid chromatographic separations coupled with mass spectrometry, and 2. Relate the structural characteristics of the proanthocyanidin fractions to bioactivity in cell culture and in vitro models of inflammation, bacterial adherence and oxidation. We will carry out 3 types of experiments to characterize and prepare cranberry proanthocyanidins for assays of bioactivity; 1, experiments to optimize online and offline chromatography, 2, chemical degradation studies coupled with mass spectrometry of products to rapidly characterize structure, and 3, fragmentation studies using tandem mass spectrometry. We will then assay proanthocyanidin fractions for bioactivity using 3 methods; 1. Attenuation of LPS induced expression of cycloxygenase 2 in macrophages, 2. In vitro model of adherence of p-fimbriated E. coli to uroepithelial cells, and 3. specific association of proanthocyanidins to low density lipoproteins and inhibition of copper induced oxidation. [unreadable] [unreadable] [unreadable]
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{
"pile_set_name": "NIH ExPorter"
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In the past years of support of this project we developed a model to quantify in vivo for the first time in human subjects the rates of amino acid transmembrane transport of naturally-occurring amino acids into and out of muscle, and the rate of muscle protein breakdown, while at the same time also quantifying muscle protein synthesis. The general goal of this proposal is to extend our previous studies of the response to exercise to investigate the following hypotheses related to the effect of amino acid intake on muscle protein metabolism after exercise: Hypothesis 1. There is a curvilinear dose response of protein synthesis to the ingestion of a single bolus of AA which reaches a plateau, beyond which more AA intake causes no further stimulation of protein synthesis. Hypothesis 2. There is an interactive effect between sucrose and amino acids such that the addition of sucrose to any dose of AA will stimulate muscle protein synthesis to a greater extent than would be expected from the individual responses to that dose of AA or that dose of sucrose given separately. Hypothesis 3. An interactive effect between sucrose and AAs is due to the insulin response to sucrose, rather than to the energy (calories) provided by the sucrose. Hypothesis 4. Addition of sucrose to the AAs will increase the clearance of exogenous amino acids from the blood and intracellular compartment. Consequently, a second dose, taken one hour after the first dose, of sucrose plus AA will have the same stimulatory effect on net muscle protein synthesis as the first dose, whereas the response to a second dose of amino acids alone, taken 1 hour after the first dose, will have a diminished response as compared to the response to the initial dose. All studies will be performed using stable isotope methodology and arterial-venous sampling across the leg and muscle biopsies. The results of this study will also contribute to our understanding of the mechanism whereby nutritional intake affects muscle protein metabolism.
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{
"pile_set_name": "NIH ExPorter"
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Project Summary Mitochondria are important organelles within the cell that participate in a number of cellular processes including the generation of energy through oxidative phosphorylation. Mitochondria possess their own genome that is vital to mitochondrial function including oxidative phosphorylation. Mutations within this genome can cause mitochondrial dysfunction. A link has been established between obesity and mitochondrial dysfunction. It has recently been proposed that mutations to mitochondrial DNA might be one of the mechanisms that lead to whole body metabolic dysfunction. We have preliminary evidence that mitochondrial DNA mutations might induce mitochondrial dysfunction and impair whole body metabolic homeostasis thus ultimately leading to an obesity phenotype. Mitochondrial DNA is replicated and repaired by a single DNA polymerase (mitochondrial DNA polymerase gamma, or PolG). Work utilizing a homozygous whole body knock-in mutation within PolG (PolG-/-) results in a supraphysiological increase in both mitochondrial DNA point mutations and deletions and a progeroid like phenotype within mice. Although the accumulation of mtDNA mutations is associated with mitochondrial dysfunction in these mice, the drastically reduced life span of the mice make them a poor model to study the association of mtDNA mutations and metabolic function. We have collected preliminary data utilizing a heterozygous version of the same mouse model (PolG+/-). Our data show an obese phenotype and a more physiologically relevant level of mitochondrial DNA mutations that might precede mitochondrial dysfunction and further alter whole body metabolic homeostasis. Using the heterozygous PolG+/- mouse model, this proposal will seek to establish a link between mitochondrial DNA mutations, mitochondrial dysfunction, and whole body metabolic homeostasis. The first aim of the proposal will identify if mitochondrial DNA mutation load impacts mitochondrial function and dynamics and whole body metabolic homeostasis. The second aim of the proposal will examine if skeletal muscle overexpression of heat shock protein 72 (HSP72), a proposed positive regulator of mitochondrial function, can rescue the metabolic dysfunction via a reduction in mitochondrial DNA mutation load. To accomplish this, we have already generated both whole body heterozygous PolG mice (PolG+/-) and PolG+/- mice crossed with muscle specific HSP72 overexpression via a transgene (PolG+/- x HSP72mTg). Whole body and tissue specific metabolic homeostasis will be assessed along with factors that impact mitochondrial biogenesis, dynamics, and degradation. The proposed studies will fill a knowledge gap and address the question of a potential link between mitochondrial DNA mutations and metabolic dysfunction.
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{
"pile_set_name": "NIH ExPorter"
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Ritonavir administered alone or in combination with reverse transcriptase inhibitors, demonstrated considerable antiretroviral activity in and were generally well tolerated by individuals with HIV-1 infection. This multiple-dose, open-label, randomized, multicenter trial in HIV-1-infected male and female patients with CD4 cell counts of 100-500 cells per microliter is assessing the efficacy and safety of the combination of ritonavir with saquinavir, a second protease inhibitor whose bioavailability is dramatically increased by co-administration with ritonavir. In addition, the study will determine whether this combination of protease inhibitors can retard or prevent the emergence of drug-resistant viruses. During the first year of study patients were randomized to one of the following four treatment regimens: ritonavir 400 mg BID and saquinavir 400 mg BID; ritonavir 600 mg BID and saquinavir 400 mg BID; ritonavir 400 mg TID and saquinavir 400 mg TID; and ritonavir 600 mg BID and saquinavir 600 mg BID. Subjects not achieving plasma RNA levels below 200 copies/ml were allowed to add up to 2 nucleoside inhibitors of reverse transcriptase to their regimen. Seventeen subjects were enrolled at this site, and 9 remain in the trial. The nine subjects who are still participating in the study have experienced a significant reduction in their viral activity. These nine subjects will be studied for an additional 52 weeks to further assess virologic response with serial viral RNA determinations using the Roche PCR assay.
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{
"pile_set_name": "NIH ExPorter"
}
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A major goal in neuroscience is to understand the formation and development of synapses, the tiny membrane specializations that enable nerve cells to communicate with each other. The sequence of molecular signals leading to synapse formation ("synaptogenesis") is qualitatively well known for the more accessible neuromuscular junction (NMJ) [2]. However, very little is known of the quantities (concentration, duration, onset, etc.) of the various neurochemical signals involved in synaptogenesis. Intriguingly, all but one of the axons innervating a given myotube at birth retract after a period of ~1 week as a result of a synaptic competition process that remains, for lack of quantitative methods, poorly understood. Our overall objective is to uncover some of the rules governing the formation and elimination of synapses at the NMJ using a microfluidic cell culture system developed under a previous R01 (which we seek to renew for the first time). Our approach is based on substituting the presynaptic neuron by an artificial microfluidic device that delivers known doses of various synaptogenic neurochemicals to micrometer-scale areas of the membrane of cultured myotubes. We will focus on the three key factors - agrin, neuregulin, and the neurotransmitter acetylcholine (ACh) - secreted by the nerve tip during synaptogenesis. We will measure muscle cell responses that are specific to ACh receptors (AChRs), such as AChR aggregation/disaggregation, degradation/synthesis, insertion, co-localization with other receptors and cytoskeletal proteins, intracellular signaling pathways, etc. Under previous support, we have developed a microfluidic mimic of the innervation process that allows for focally stimulating >80 single, isolated ("microengineered") myotubes using laminar flow streams (orthogonal to the myotubes). We have found that a) focal application of agrin entices myotubes to recruit new AChRs to the stimulated area;b) when the microtracks are formed with Matrigel, a basal lamina extract, the microengineered myotubes display AChR clusters of complex, in-vivo-like morphologies even before agrin is applied, similarly to what happens in vivo;and c) when agrin is focally applied to those agrin-predating clusters, AChRs are degraded at reduced rates, suggesting that a putative role for agrin in vivo is to help stabilize AChRs. We seek to continue these investigations by studying the dynamics and spatial patterns of various AChR-specific responses upon (competitive, synergistic, or combinatorial) stimulation with agrin, neuregulin, and ACh. PUBLIC HEALTH RELEVANCE: The sequence of molecular signals leading to synapse formation ("synaptogenesis") is qualitatively well known, but a quantitative description is lacking because present experimental setups for the study of synaptic development do not allow for a precise control over the many variables involved in synaptogenesis. We will use a device that mimicks the local secretion of neurochemicals onto muscle cells (thereby the device will be "pretending" to be a neuron). By manipulating the variables involved in the formation of the synapse, we will be able to quantitatively describe and understand the mechanisms responsible for stabilization and competition observed at synaptic sites during development and may provide valuable insights on other neurological processes where synapse formation is critical such as in neurological diseases, memory formation, and learning.
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{
"pile_set_name": "NIH ExPorter"
}
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Objectives: (a) To develop, standardize, and evaluate sensitive and specific assays for antibodies to Bordetella pertussis antigens. (b) To serve as a refer-ence laboratory for pertussis serology by writing reference methods, procuring reference reagents, training visiting personnel, and performing comparative test-ing with outside laboratories. For all assays listed below, protocols have been distributed, visitors were trained, and comparative testing with outside laboratories has been performed. (1) Microagglutination assay: Because the supply of US Reference Antigen for Pertussis Microagglutination Assay, Lot 1 was nearly depleted, production of Lot 2 was initiated and completed. The bacterial cells were lyophilized in vials, testing was performed to demonstrate the equivalence of Lots 1 and 2, and distribution of Lot 2 has begun. (2) CHO-cell neutralization Assay: Stained and dried demonstration plates have been prepared and are available for distribution to collaborators. (3) ELISA Assays: (a) Although Reference Pertussis Antisera (Human), Lots 3 and 4 have been proved to be excellent reference sera for IgG assays, they are suboptimal for IgA assays. Therefore 1500 vials of Reference Pertussis Antiserum (Human) Lot 5 were prepared specifically as a reference for IgA assays. (b) A number of collaborative assays have been conducted with outside laboratories to assess the within laboratory reproducibility and between laboratory comparability of a standardized ELIZA procedure. In addition to the Laboratory of Pertussis, two national control laboratories, one commercial clinical laboratory, two university laboratories and four vaccine manufacturers were included in the study. (c) Protocols and reagents for the standardized ELISA procedure were distributed to more than twenty laboratories. (d) Evaluation of the new ELISA calculation program continues.
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{
"pile_set_name": "NIH ExPorter"
}
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Our goal is to increase understanding of the stromal-epithelial interactions that mediate actions of ovarian steroids in the reproductive tract. Our studies have focused on regulation of two stromally-derived growth factors, hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), and on regulation of specific enzymes called matrix metalloproteinases (MMPs). First, we have developed a nuclease protection assay for detection of monkey HGF transcript, and determined that the endometrium expresses a long (full length) and a shorter (truncated) form of HGF. Second, we tested the effects of exogenous KGF on the reproductive tract of juvenile and adult rhesus monkeys and found that KGF inhibited endometrial epithelial apoptosis, and blocked atrophy of the spiral arteries after P withdrawal. Third, we have immunocytochemically analyzed the distribution of three key MMPs including, matrilysin (Mat), gelatinase-A (Gel-A), stromelysin-1 (Str-1) and the MMP inhibitor TIMP-1 in the macaque endometrium. This analysis revealed that all three MMPs and TIMP-1 were strongly detectable 2 days after P withdrawal whether E2 was present or not. Gel-A and Str-1 were upregulated in the upper functionalis directly within the zone of fragmentation, while matrilysin was expressed in the glands below the fragmenting zone. Therefore, Gel-A and Str-1 may be more directly involved in stromal fragmentation, while matrilysin probably functions in glandular remodeling. Fourth, we defined the critical period for P withdrawal-induced bleeding by replacing P at various timepoints after P withdrawal at the end of the artificial cycle. Replacement of P before but not after 36 h of P withdrawal blocked frank menses. A critical period' of approximately 36 hours of P withdrawal is needed for key factors such as cytokines and MMPs to rise above a functional threshold that inevitably results in menstrual breakdown. P cannot suppress these factors once that threshold is passed.
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{
"pile_set_name": "NIH ExPorter"
}
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This application requests funding in partial support of a 5-day FASEB Summer Research Conference (SRC) to be held in 2012, 2014 and 2016, entitled Biology and Pathobiology of Kruppel-Like Factors (KLFs). Kruppel- like factors are a family of zinc finger-containing transcription factors that represents a rapidly advancing field. Since the identification of the first member of this family, Erythroid Kruppel-Like Factor (EKLF or KLF1) 18 years ago, an additional 21 members have been identified. The study of the KLFs is an emerging area, with rapid increases in the number of NIH grants and publications in the past few years. Studies have indicated that KLFs exert important functions in diverse physiological processes involving the cardiovascular, digestive, respiratory, hematological and immune systems and in pathological states such as cancer, obesity, diabetes, and inflammatory conditions, among others. KLFs are also involved in the reprogramming of somatic cells to inducible pluripotent stem (iPS) cells and in maintaining the pluripotency of embryonic stem cells. These symposia are designed to bring together a group of international leaders in the field of KLF research with the goal of stimulating discussion and fostering collaboration. The potential for therapeutic development in cardiovascular, cancer, diabetes, and inflammatory diseases based on KLF biology is a distinct possibility that may result from the meeting. The SPECIFIC AIMS of the meeting are: 1. To provide a scientific program at the forefront of KLF research that is organized around the themes of physiological functions and organ-based pathophysiology of diseases. 2. To provide a venue for meaningful interchanges among investigators in the KLF field which foster interactions and potential collaborations in the basic, translational and clinicl sciences. 3. To provide a scientific platform with which to encourage active participation and career development of young investigators and trainees. PUBLIC HEALTH RELEVANCE: This application requests partial funding for an international conference on the Biology and Pathobiology of Kruppel-Like Factors (KLFs), a rapidly emerging field. KLF transcription factors are involved in numerous physiological processes in the cardiovascular, hematologic, immune, digestive and other systems. They also control pathophysiological processes including heart disease, inflammation, cancer, obesity, and diabetes. The conference is expected to stimulate scientific discussion and collaboration, and generate new ideas which will advance the field.
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{
"pile_set_name": "NIH ExPorter"
}
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The goals of this two year project are: a) to test hypotheses and explore research questions bearing on illness management strategies and on socio-cultural and psychosocial factors that influence the onset improvement and resolution of an illness perceived by patients seeking treatment in a Mexico City hospital; b) to gain an understanding of the ways in which biomedicine, spawned in America, becomes molded and interpreted in the Mexican social and cultural contexts. The proposed project builds upon the PI's 15 years previous field research of folk therapeutics in Mexico which had examined health seeking behavior and patients' responses to Spiritualist treatment and their attribution for recovery, using multi-method research techniques and analyses, including ethnographic description, participant observation, open ended discussion, interview schedules, standardized health questionnaires and consultation with physicians. The proposed project, employing similar methodologies, will shift the focus from the folk to the professional sectors of Mexico's health care delivery and will thereby allow a comparison of utilization of the different systems. Theoretically the research will contribute to the study of the relation between stress, culture and illness & to the anthropology of biomedicine. Practically, while much is known about folk healing utilization, little is known about Mexican patients' perceptions and expectations of biomedical treatment, or the interaction between the folk and professional medical sectors. The findings will assist health providers to address patients' concerns: thereby improve primary health care delivery in Mexico as well as among Mexican Americans in the United States.
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{
"pile_set_name": "NIH ExPorter"
}
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The long-term objective of the proposed research is to understand how different behavior is generated and controlled by the nervous system. The research will be performed on a new invertebrate model system that comprises fewer neurons than vertebrate brains. Many basic neurobiological questions can therefore be more easily addressed. Unlike many invertebrates, social insects show particularly complex behavioral repertoires that rival those of many vertebrates (social behavior, communication, learning and memory). Many brain compartments perform the same functions as some brain regions of vertebrates or humans do, and their morphological design as well as the physiology of their nerve cells may be strikingly similar. Examples are the olfactory centers of vertebrates and insects, or the similar way of processing visual information by both taxa. The behavior and brain morphology of individuals that are genotypically almost identical (e.g. full sisters) yet differ morphologically and perform strikingly different tasks will be compared to explore the range of neuronal variability possible on the same genetic basis. Such individuals are expected to differ with respect to their sensory thresholds and in the significance they assign to certain stimuli. This will be tested in behavioral tests (stimulus preference, maze performance, learning tests). The brains of differently behaving individuals will be compared morphologically, and special attention will be given to prominent brain structures that control complex decision making and are supposedly involved in inter-individual behavioral differences. Electrical recordings from uniquely identifiable neurons will reveal the physiological basis for the behavioral differences. The proposed research will help to develop new uniquely identifiable neurons to reveal the physiological basis for the behavioral differences. The proposed research will help to develop a new animal model system for investigating learning and memory, the weighing of different stimuli and the control of complex behavior by the brain.
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{
"pile_set_name": "NIH ExPorter"
}
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UT Houston has been a member of both the Neonatal and MFMU Networks since 2001. This renewal application for the Neonatal Network proposes to 1) contribute to the conduct of rigorously designed and executed clinical trials, 2) design and implement at least one major trial of an important management question in the NICU and one in the follow-up clinic, 3) advance the methods in clinical research involving preterm infants. The UT-Houston Network site provides: 1) A large and growing NICU population (9937 births and 1775 ICU admissions in 2009) with racial and ethnic diversity [30-38% Black, 12-31% White, 25- 46% Hispanic (ranges of two practice sites)], 2) High enrollment in Network studies (ranked 2nd and 6th of 16 centers in the two most recently completed large Network trials, 3) Skilled and committed PI and CoPI [Drs. Kennedy and Tyson have served as Pl and Co-PI for the past 19 years, 7 in Dallas and 12 at UT- Houston. Together, they direct the Center for Clinical Research and Evidence-Based Medicine and the Master's Degree Program in Clinical Research at UT-Houston. They are known in the Network for their commitment to rigorous research methods and for helping other investigators with study design. They have played major roles in the design and implementation of numerous completed and ongoing Network randomized trials (Vitamin A, Hypothermia, Phototherapy, Surgery for NEC).], 4) a large pool of talented collaborators to provide multidisciplinary expertise [Dr. Kevin Lilly (Chair of Pediatric Surgery) and Dr. Tyson mentored Dr. Martin Blakely (Pediatric Surgeon, now in Memphis) in developing the ongoing Surgery for NEC Trial. Dr. Nehal Parikh, a K23 awardee and mentee of Drs Kennedy and Tyson, directs the ongoing 6- 24 Hour Hypothermia Trial at our site. Dr. Claudia Pedroza is a statistician with institutional K12 funding to conduct Bayesian analyses of Network studies. This application includes a concept application of a pragmatic management trial to compare the clinical effectiveness, safety, and long-term effects of two commonly practiced but poorly studied management strategies in the NICU - aggressive diuretic and strict fluid restriction vs. restricted diuretic use for the prevention of bronchopulmonary dysplasia.
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{
"pile_set_name": "NIH ExPorter"
}
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The Science & Health Education Partnership of the University of California at San Francisco, in strong collaboration with the San Francisco Unified School District, proposes a National Institutes of Health Science Education Partnership Award Phase I/II Project Pathways: Promoting Access to the Health Sciences through Partnership. Pathways seeks to address an issue of critical local and national need - providing students from backgrounds underrepresented in the sciences with access to high quality science instruction that will promote student success in science and reduce the achievement gap. In this effort we define student success broadly to refer to students' scientific literacy and thinking skills, their understanding of both the process of science as well as scientific content, and their interest in and excitement about science. Pathways has five interrelated specific aims: 1) provide high school students, especially those from groups underrepresented in the sciences, with rigorous science learning experiences and develop their academic science skills, 2) develop strong scientist-teacher partnerships grounded in equitable science teaching strategies and research, 3) build a collaborative community focused on promoting academic success in science for all students, 4) evaluate all Pathways components, and 5) disseminate broadly Pathways materials and successful program elements and strategies. The Pathways approach is unique: it brings teachers and scientists together in an intensive partnership to learn about and test strategies for promoting student success in science; based in the California Science Content Standards, it uses current research discoveries to develop lessons that reflect the dynamic nature of scientific progress; and it uses rigorous evaluation to assess the effectiveness of the Pathways model. This project is relevant to and will benefit public health in two primary ways. First, it seeks to increase high school students' understanding of current biomedical research discoveries and how these discoveries relate both to their own lives and advances in patient care. Second, educators participating in the program will learn strategies to promote the success of underrepresented students in science and reduce the achievement gap - educational attainment correlates strongly with both access to health care and insurance, as well as with overall health.
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{
"pile_set_name": "NIH ExPorter"
}
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The objectives of this proposal are to understand: (1) the relationship between the structure and the many distinctive properties of E. coli initiator tRNA and (2) the molecular mechanisms underlying its specific interactions with components of the translational machinery. An important question is the molecular basis of the highly specific recognition of the tRNA by Met-tRNA transformylase. A combination of structural, biochemical, and genetic approaches will be used to study this. These include (I) NMR spectroscopy to analyze the structure of the tRNA substrate, (ii) crystallography to determine the structure of the protein and the tRNA-protein complex, (iii) investigation of the topology of interaction of the protein with the tRNA using crosslinking experiments, protection experiments and by examining suppressor mutations in the protein that compensate for defects in formylation of mutant tRNAs, and (iv) site-specific mutagenesis to identify amino acids important in the protein for tRNA selection and function. Similar approaches will be used to study interactions of tRNA with other proteins, in particular, in vivo selection and analysis of suppressor mutations in IF2, IF3, or other chromosomal genes. Such work could provide information on interactions between the initiation factors and the initiator tRNA and could lead to identification of new genes involved in translational initiation. Work on identification of intermediates in translation initiation in vivo will continue. Questions of specific interest are: (I) is IF2 a carrier of fMet-tRNA to the ribosome? (ii) Does the 30S ribosome bind first to the initiator tRNA and then to the mRNA or vice versa? (iii) Are the requirements in an initiator tRNA for translational reinitiation the same as for de novo initiation? Finally, the role of the All:U24 base pair unique to eubacterial initiator tRNAs in initiation will be studied along with questions of why introduction of the base pair to an E. coli elongator methionine tRNA prevents accumulation of the tRNA in vivo.
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{
"pile_set_name": "NIH ExPorter"
}
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Cocaine (COC) dependence is a significant public health concern. A widely effective pharmacotherapy has not yet been identified for COC dependence. Innovative strategies are needed to identify an effective pharmacotherapy for COC dependence. Testing medications effective for disorders that share neurobiological substrates with drug dependence, for example, could yield treatments for managing COC dependence. Obesity is also a significant public health concern. Although obesity and COC dependence are typically considered distinct clinical entities, both diseases involve perturbations of central biogenic amine and/or hypothalamic-melanocortin systems. The obesity epidemic has spurred development of medications to promote weight loss. A combination of bupropion (BUP) and naltrexone (NTX) is effective for obesity. The overarching goal of this application is to demonstrate the initial efficacy, safety, and tolerability of BUP-NTX combinations for COC dependence. A mixed-model experiment will be conducted in which separate cohorts of non-treatment-seeking, COC-dependent participants will be randomized to different maintenance doses of BUP (i.e., BUP is a between-subject factor). Participants (N=12) in each BUP cohort will be maintained concurrently on NTX (i.e., NTX is a within-subject factor). The reinforcing effects of intranasal COC will be determined after participants in each BUP cohort are maintained for 4-7 days on each of the NTX doses (i.e., COC is a within-subject factor). COC (0, 25, 50, 100 mg) will be tested with multiple dose combinations of BUP (0, 100, 200, 300 mg/day) and NTX (0, 25, 50 mg/day). The proposed study will also identify the optimal dose combination of BUP and NTX that most effectively attenuates the reinforcing effects of COC. This research will provide critical information regarding the initial efficacy and optimal doses ofa novel drug combination, BUP and NTX, for COC dependence, which will enhance the probability of success when advanced to a clinical trial. Innovations of the proposed research include: 1) testing a combination of marketed drugs that demonstrated modest efficacy when tested as mono-therapies; 2) the use of a sophisticated drug self-administration procedure; 3) providing the impetus for the conduct of a Phase II clinical trial to further demonstrate the efficacy of BUP-NTX combinations for COC dependence; and 4) demonstrating the initial efficacy and optimal doses of a combination of commercially available drugs, as opposed to waiting for novel molecules to be available for testing in humans, thereby impacting clinical research and practice more quickly. In these ways, the proposed project will shift the current clinical research paradigm in pharmacotherapy development and have a significant impact on the treatment of COC dependence. PUBLIC HEALTH RELEVANCE: The research proposed in this application will determine the initial efficacy, safety and tolerability of a novel drug combination, bupropion and naltrexone, as a pharmacotherapy for cocaine dependence. A rigorous, inpatient human laboratory study will be conducted. The proposed study is innovative and important because it will provide the impetus for the conduct of double blind, placebo-controlled trials to further demonstrate the efficacy of bupropion-naltrexone combinations for managing cocaine dependence.
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{
"pile_set_name": "NIH ExPorter"
}
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The purposes of the study are (1) Assess the effectiveness of chronic electrical stimulation of midbrain sites for the relief of chronic pain in humans; (2) Evaluate the efficacy and mechanisms of traditional narcotic analgesia and compare these to chronic electrical stimulation of midbrain sites; (3) Validate experimental models of pain and their potential diagnostic use in chronic pain patients; and (4) Determine and compare the impact of both traditional narcotic and chronic electrical stimulation therapies on the functional, intellectual and emotional well being of these patients. Participants in this study will be (1) chronic pain patients receiving surgically implanted stimulating electrodes for pain control; (2) chronic pain patients maintained on traditional narcotic analgesics who will not receive implanted stimulating electrodes; and (3) healthy normal volunteers. The effects of chronic brain stimulation in surgical patients will be compared to the effects of narcotics previously administered to patients and to effects of narcotic regimes in nonsurgical chronic pain patients. In addition, the effects of narcotics on perceptual and neural mechanisms of experimentally induced pain and on normal psychological functioning will be compared between chronic patients and normal volunteers free of chronic pain.
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{
"pile_set_name": "NIH ExPorter"
}
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Crosslinking by psoralens and other reagents will be used to map aspects of the secondary and tertiary structure of free and ribosome-bound rRNAs. Initial work will focus on the E. coli 16S rRNA. Of particular interest are contacts between distant regions of the polynucleotide chain. Crosslinks between such regions are analyzed by electron microscopy and gel electrophoresis. Improvements in these techniques should greatly speed the analysis of RNA structure and allow direct comparison of the folded structures of rRNAs in bacteria, organelles and eukaryotes. New rapid techniques will be developed to allow the positions of crosslinks to be localized a the level of the nucleic acid sequence. Crosslinking will also be used to analyze for interactions between the 6 RNAs on a functioning ribosome. We will also develop techniques for examining the presece of knotted topologies in RNAs. Chemical modification, exchange reactions and x-ray scattering will be used to examine the structure of ribosome-bound mRNA. Blotting techniques will be developed to allow crosslinking studies of RNAs without purification. These should have broad applicability to studies on RNA splicing, tumor virus RNA structure and the structure of particular mRNA during transcription, processing and translation. The rationale behind all of these projects is the recent realization that isolated large RNAs can form stable interesting compact folded stgructures. Long distance contacts are uniquely informative n establishing and comparing such structures. Simple screens for such contacts could profoundly influence current research in the biophysical chemistry and molecular biology of regulation, tumor viruses and protein synthesis.
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{
"pile_set_name": "NIH ExPorter"
}
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Incarcerated adolescents represent a group at high risk for health consequences associated with risky sexual behaviors. They report greater participation in a number of sexual risk behaviors (including sexual risk in conjunction with drug use), higher rates of sexually transmitted diseases (STDs) and pregnancy than their non-detained adolescent peers. Adolescents, including incarcerated teens, report sexual behavior both with partners with whom there is a relationship (main partners) and partners with whom there may be a friendship or only an acquaintance (non-main partners). Condom use is more common with non-main than main sex partners and substance use in conjunction with sexual risk also appears to vary by partner type. The long-term objective of this research program is to reduce HIV risk behavior among adolescents involved in the criminal justice system who abuse non-injection drugs by developing and establishing the efficacy of an intervention that includes a specific focus on partner-specific decision-making and behavior through presenting relevant information, enhancement of prevention motivation, opportunities to learn and -practice safer sex behavioral skills, and sexual decision-making in conjunction with substance use. Furthermore, we seek to advance knowledge of the mechanisms of action by which interventions reduce HIV risk behavior. In the present application, we propose to develop and pilot test a partner-specific HIV risk reduction intervention for incarcerated adolescents who report substance abuse. In the first phase of the study we will pilot the intervention with 20 incarcerated adolescents and elicit their feedback regarding their experiences and perceptions of the intervention. Following refinement of the intervention, we will conduct a small randomized trial (n=60) to examine the efficacy of the intervention relative to a healthy lifestyles psychoeducation intervention (HLS). We expect that, relative to CBT-SA), participants randomized to the intervention condition will have lower levels of sex-related HIV risk behavior and of substance use in conjunction with sexual risk. We will also examine the potential mechanisms by which the intervention produces a reduction in health risk behaviors. If found to be efficacious, this intervention will help to reduce the acquisition of HIV among substance using incarcerated adolescents.
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{
"pile_set_name": "NIH ExPorter"
}
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SUMMARY HIV affects more women than any other life-threatening infectious agent. It is most often sexually transmitted, where virus must evade the genital mucosal barrier to cause systemic infection. Clinical studies suggest HIV more easily penetrates this defense among women using the injectable progestin depot-medroxyprogesterone acetate (DMPA). Offering biological plausibility for this possibility, our research group showed that DMPA promoted mouse susceptibility to HSV-2 infection by reducing expression of the cell-cell adhesion molecule desmoglein-1 (DSG1) and increasing genital mucosal permeability. DMPA similarly boosted susceptibility of humanized mice to genital HIV infection. Identifying a potential mechanism for these results, we found DMPA treatment of mice lowered vaginal levels of ephrin A3 (EFNA3); an estrogen (E) receptor target gene shown to promote DSG1 expression in epithelial tissue. Providing clinical relevance for our findings, we showed women initiating DMPA use display changes in ectocervical DSG1 expression and mucosal permeability identical to those seen in mice. Likewise, pharmacologically relevant DMPA doses comparably enhanced genital mucosal permeability in rhesus macaques (RMs). These data newly reveal DMPA impairs mucosal barrier protection. However, using a humanized mouse model of HIV infection, we also uncovered that combined treatment with DMPA and intravaginal (ivag) E blocks virus acquisition by enhancing genital mucosal integrity. These results thus identified an unexpected advantage of hormonal contraceptive strategies that combine use of exogenous progestin and E (i.e., they avert loss of barrier protection caused by progestin use alone). As necessary steps in establishing this approach, this proposal will elucidate mechanisms of E-mediated enhancement of genital mucosal barrier function and define capacity of an E-releasing ivag ring (E-IVR) to protect RMs from genital SIV transmission. Expressly, we will use mice to define E-mediated regulation of EFNA3 pathways that induce DSG1 expression and boost genital mucosal barrier function (Aim 1). We will formulate a RM-sized E-IVR to deliver pharmacologically relevant drug doses, and use these rings to compare EFNA3 and DSG1 expression and mucosal barrier function in RM treated with DMPA, DMPA and placebo IVR, or DMPA and E-IVR (Aim 2). Finally, we will compare genital SIV transmission in RMs administered DMPA, DMPA and placebo IVR, or DMPA and E-IVR with repetitive genital challenges with escalating inoculums of the virus (Aim 3). This work will identify mechanisms by which E induces EFNA3 signaling pathways promoting genital epithelial integrity, and demonstrate that E-IVRs block SIV transmission by abrogating DMPA-mediated weakening of genital mucosal barrier function. These studies will thus deliver important new information in a highly relevant clinical model, and justify exploration of similar contraceptive strategies in populations at high risk for HIV acquisition.
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{
"pile_set_name": "NIH ExPorter"
}
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