Protein purification is a vital and expensive step in biomedical research and in the development and manufacturing of therapeutic proteins. Unfortunately, affinity methods, which are at the heart of most protein purifications, often present a bottleneck in the separation process because of slow diffusion of proteins in the pores of chromatographic gels. Protein-absorbing membranes can overcome this challenge because convective flow through membrane pores provides rapid mass transport to binding sites. Such flow can also effectively remove undesired proteins to increase purity. However, membrane absorbers are not widely used for protein isolation because they have low protein-binding capacities. The aim of this work is to modify membranes with functional polymer brushes to increase protein-binding capacities by an order of magnitude and enable rapid, selective protein purification. Additionally, properly designed brushes will be resistant to nonspecific adsorption and provide new methods for purification of "sticky" proteins that are not amenable to column-based purification. This research will involve synthesis and characterization of polymer brush-modified membranes that bind histidine6- and glutathione S-transferrase-tagged proteins with minimal nonspecific adsorption. Preliminary results demonstrated purification of a histidine6-tagged protein in a cell extract, with a purity that greatly exceeds similar resin-based purification. Future work aims at developing brush-modified membranes in polymeric supports with pore sizes that will allow large increases in permeability. This will permit the use of lower pressures and thicker membranes with much higher capacities. Formation of such membranes will require both development of new synthetic methods that are compatible with polymer supports and growth of thicker brushes that rapidly bind more protein. Hence, protein binding and non-specific adsorption will be examined as a function of brush thickness, composition, density, and functionalization, and membrane composition and geometry. With new membranes in hand, a variety of tagged proteins expressed in E. coli will be purified including SNAP-50, human MIP synthase, and SNAPc complex. MIP synthase is vital for inositol biosynthesis, while SNAP proteins are critical in gene expression. Additionally, SNAP-50 provides an example of a sticky protein that cannot be purified with typical affinity gels. PUBLIC HEALTH RELEVANCE: This research will yield rapid, inexpensive methods for isolating remarkably pure proteins. Such techniques will be crucial in production of therapeutic proteins as well as research studies aimed at isolating proteins to understand their structure and health- related function. [unreadable] [unreadable] [unreadable]


Unlike most chronic renal diseases, in which the female gender is a protective factor, this "female advantage" is lost in diabetes as diabetic females develop more renal disease than non-diabetics;we propose that this is due to low levels of plasma estradiol (E2) in diabetic females. The findings suggest that estrogens (including E2 and its metabolites, such as 2-methoxyestradiol (2-ME)) contribute to the pathophysiology of diabetic renal disease. Based on our preliminary data, we hypothesize that: Specific Aim 1: E2 regulates the renal renin-angiotensin system by reducing the expression and activity of the RAS: specifically by 1. tonically downregulating AT1Rs, 2. decreasing Ang II and renin levels, 3. increasing AT2Rs and consequently attenuating the renal functional and structural changes (in particular vascular changes) associated with diabetic nephropathy. Specific Aim 2: E2 and an estrogen metabolite 2-methoxyestradiol (2-ME) attenuate oxidative stress associated with diabetic nephropathy by reducing NADPH oxidase activity and NADPH oxidase-induced O2.- generation in the diabetic kidney. Specific Aim 3: E2 and 2-ME attenuate inflammation associated with diabetic nephropathy by reducing 1. Acute inflammation and markers of the acute phase inflammatory reaction: IL-6, MCP-1 and M-CSF;2. Chronic inflammation: vascular permeability, migration and infiltration of inflammatory cells in target tissues;3. glucose and AT1R-mediated Ang ll-induced NF-kB expression in target cells and resultant 4. NF-kB- induced TGF-b protein expression and subsequent activation of the Smad signaling pathway. Specific Aim 4: E2 and 2-ME attenuate glomerulosclerosis and tubulointerstitial fibrosis associated with diabetic nephropathy by 1. Reducing hyperglycemia/Ang ll-induced cell growth and ECM protein synthesis, namely collagen type I and IV, laminin and fibronectin, 2. Increasing ECM protein degradation, by increasing the activity and expression of matrix metalloproteinases, MMP-2 and MMP-9. The findings from these studies will provide insight into the mechanisms by which estrogens contribute to the pathophysiology of diabetic nephropathy and may stimulate novel ideas for developing gender-specific treatment modalities for diabetic nephropathy.


Helicobacter pylori (Hp) gastric infection is a common and serious infectious disease of humans. Current therapeutic regimens, while successful in 80 percent of the instances of infection, are complicated by expense, duration of treatment, emergence of resistant strains of Hp and lack of patient compliance. An ideal alternative to antimicrobial therapy is prophylactic or therapeutic vaccination for Hp. In this phase I SBIR, we will test the hypothesis that proteolytic digests of Hp incorporated into a novel adjuvant formulation will prevent manifestations of bacterial gastritis and promote gastric bacterial clearance in Hp-infected gnotobiotic piglets, an established animal model of this disease. Piglets will be parenterally immunized with Hp digests prior to and after established gastric infection. Evidence for protection (reduced/absent bacterial cfu, local and systemic immunity, etc) will be sought. Protection will be correlated to patterns of Western blot immunoreactivity of convalescent immune sera and isolated leukocyte in vitro responses to putative bacterial antigen(s). Reactive peptides will be isolated by HPLC, sequenced and identified. From these data, the feasibility of parenteral vaccination for Hp will be determined in this nonrodent model of bacterial gastritis and a phase II SBIR will be prepared if the protection data are positive. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE


This competitive renewal investigates the mechanisms and significance of dopaminergic (DA) and serotonergic (5HT) neurotransmission systems in Tourette's syndrome (TS) using PET neuroreceptor imaging which should provide a more complete understanding of pathophysiology and possible treatments. Our recruitment strategies have focused on completing our original DA and 5HT measure sample size, originally funded for only 45% at Year 1 of this R01. We now request funds to complete this sample size given that we have substantial progress including (1) sustained elevations of dopamine release (DArei) with a new PET scanner which is a reproduction of our published paper, (2) demonstrate reductions in SERT in our TS subject, (3) uniquely carrying out amphetamine induced DArei in TS but also pre synaptic DA transporters (DAT) and receptor density (D2Bmax) in the same individuals, (4) similarly also studying both serotonin transporters (SERT) and 5HT2A receptors in the same subjects. Completion of the sample will determine what the inter-relationships are between these pre, post and intra synaptic measures for dopamine and for serotonin transporters and postsynaptic receptors in TS. In addition to our findings in TS alone, we have found an interesting contrast between TS subjects with and without co-morbid OCD (TS + OCD). In particular we found that TS + OCD had elevated DArei compared to TS - OCD and both elevated compared to controls. We found a significant decline in SERT in midbrain in 9 TS + OCD vs. 2 TS - OCD with trends for elevations for TS + OCD vs. TS - OCD for 5HT2A. In 3 subjects who had both DA and 5HT measures, we found correlations between SERT, DArei and 5HT2A which along with the DA and 5HT group differences were suggestive of the co-morbid effect of OCD in TS. This suggested that lower intrasynaptic 5HT resulted in elevated DArei and 5HT2A and lower SERT in these subjects. Further evaluation in AIM 4 with both DA and 5HT measures will help us directly test the hypothesis about the role of 5HT in modulating our tonic/phasic DA model of TS by additionally incorporating features of OCD. At the end of this renewal, we will have completed our originally funded AIMS and examined new possibilities of 5HT and DA interrelationships and role of co morbid OCD and TS. [unreadable] [unreadable] [unreadable]


With the recent availability of radioimmunoassays for both angiotensin I and angiotensin II, as well as for plasma aldosterone, the problem of screening large numbers of patients with essential hypertension to determine the prevalence of normokalemic primary aldosteronism should be considerably easier. We have proposed that, because of these advances, it may become possible to screen hypertensives on the basis of a single blood specimen upon which determinations for plasma renin activity and aldosterone would be carried out. Since such values are greatly influenced by (1) salt intake, (2) recumbent versus upright posture, and a circadian rhythm, we have been attempting to determine the optimum time and conditions in which a single blood specimen from patients with primary aldosteronism will give the greatest variation from healthy subjects studied under the same conditions. Nineteen patients with subsequently proven primary aldosteronism have now been studied in detail to determine these variables. The results will be applied to unselected patients with essential hypertension. With the use of I131-19- iodocholesterol intravenously, we have developed an adrenal photo-scanning procedure which has detected, preoperatively, aldosterone-producing tumors as small as 1 cm in diameter. With a dexamethasone-modified radiocholesterol scanning procedure, we have been able to distinguish aldosterone-producing tumor from 'idiopathic' aldosteronism (bilateral hyperplasia). Much more work is required before we will know the accuracy with which this distinction can be made. This is important because, to date, a preoperative distinction of these two groups has been virtually impossible. An in-depth study of a case of 'primary reninism' (renin-producing juxtaglomerular cell tumor) has provided new insights into some aspects of the physiology of the renin-angiotensin- aldosterone system in man and provides new directions for further research in our area of interest. We continue to study the relationships between serum electrolyte abnormalities, carbohydrate tolerance, plasma insulin and plasma proinsulin.


DESCRIPTION (provided by candidate): The mammalian olfactory bulb is an ideal system to study general principles of sensory coding. Olfactory bulb circuits convert simple monotonic sensory input from olfactory receptor neurons into complex spatiotemporal patterns of mitral cell activation that are relayed to higher order brain centers. Very little is known about how the olfactory bulb produces this transformation. This application seeks to investigate the intrinsic electrophysiological properties of mitral cells-the principal output neurons of the olfactory bulb-and analyze how these properties pattern mitral cell responses. Using electrophysiological approaches, we will address two key questions: 1) what are the kinetic properties and molecular identity of slowly inactivating K+currents in mitral cells; and 2) how do recurrent and lateral synaptic inhibition interact with intrinsic currents in mitral cells to modulate spike timing and action potential propagation? We expect that the results from these experiments will shed important light on how the olfactory bulb processes sensory information, unveil general principles of how local circuits in the brain interact to perform computational tasks, and aid in the development of paradigms for therapeutic electrical stimulation to treat neurological diseases.


In March 2007, professional societies representing primary care disciplines endorsed the "Joint Principles of the Patient-Centered Medical Home (PCMH)". Consequently, the National Committee for Quality Assurance developed a tool to recognize practices as PCMHs. The tool emphasizes practice structures with a strong emphasis on information technology. It is unclear that these practice structures are associated with the actual provision of primary care characteristics, the theoretical foundation of a PCMH, or patient outcomes. PURPOSE: This career development proposal will 1) examine the correlation between the PCMH recognition tool and a tool measuring the evidence-based features of primary care, and 2) identify the factors underlying each of the tools, and 3) assess the correlation between the PCMH recognition tool and a tool measuring the evidence-based features of primary care with clinical outcomes. This project will lead to an R01 application to develop an improved tool for PCMH recognition, incorporating elements of the original tool correlated with primary care and with improved clinical outcomes. CANDIDATE: The candidate is an assistant professor in a research position jointly appointed in the Department of Family Medicine and the Department of Pediatrics and Human Development at the Michigan State University College of Human Medicine. TRAINING GOALS: The goals of the training are to 1) gain didactic training in health services systems theories and practice and 2) apply the theories to applications of health services research to identify features of the current PCMH recognition tool correlated with evidence-based features of primary care and improved clinical outcomes. RESEARCH METHODS: The research methods include a correlation analysis of total scores of and domains in the NCQA PPC-PCMH and the PCAT tools, a factor analysis of each tool, and correlation of each tool with clinical outcomes at the practice level. OUTCOMES: The outcomes include a greater understanding of the correlation of PCMH recognition tool with primary care attributes and the relationship of each to improved clinical outcomes. BENEFIT: The results will be used to develop policy to appropriately classify practices as PCMHs and to inform reimbursement strategies for PCMHs based on patient outcomes. The work resulting from this study will provide an understanding of the correlation between the current tool intended to recognize clinical practices at patient-centered medical homes (PCMHs) and the evidenced-based features of primary care and patient outcomes. The results will inform policymakers on the utility of the current PCMH recognition tool in appropriately classifying practices as PCMHs and inform payers on the utility of the current tool in identifying practices with better patient outcomes


Toxicity is a major problem in the chemotherapy of cancer. The existence of circadian fluctuation in host toxicity is well established from work in chronopharmacology and chronotoxicology. There are significantly different responses to drugs at different times along the 24-hour day, i.e., hours of changing responsiveness or susceptibility. Can knowledge of rhythmicity in over-all host toxicity and of the rhythmic fluctuation in cell division of normal and tumor tissues be effectively used to treat tumor-bearing hosts with maximal toxicity to the tumor and minimal toxicity to the host's "normal" tissues? Our main objectives have been and still are: (1) To identify susceptibility-resistance rhythms to four diverse chemotherapeutic agents (Adriamycin, actinomycin-D, cyclophosphamide and cytosine arabinoside); and (2) To quantify in three groups of mice the rhythmic fluctuation in 3H-thymidine incorporation into the DNA of bone marrow, thymus, spleen, gut and liver, and to similarly analyze the mitotic index of liver and corneal epithelium. One group will have adenocarcinoma of the breast, another the Lewis lung carcinoma and the third will have L1210 leukemia; each group will be compared with non-tumor-bearing controls. The rhythmicity of DNA will be expolred for all tumors, and the effects that tumors of different ages (for each of the three types) have on the variables under study are being determined. Using the data obtained from objectives 1 and 2 above, we have as objective 3: To use chronotherapy to attack tumors by employing the different agents either singly or in combination. To date we have been able to demonstrate significant advantages of timed treatment by utilizing different protocols designed to give the highest levels of drug when the host is least susceptible and the lowest levels when most susceptible. The advantages demonstrated for mice include an increase in survival time, and in several studies it was shown that chronotherapy could significantly increase cure rate in L1210 leukemia in mice over conventional therapy. The potential for further increasing these advantages is promising.


Actin filaments and microtubules are dynamic cytoskeletal polymers that have important roles in cell physiology and determine cell morphology. The complex cellular morphology of neurons is directly related to the function of neurons in the nervous system. Thus, neurons provide an excellent model system for investigating the dynamics of the cytoskeleton as it relates to cellular functions. Actin filaments are of fundamental importance to the development and maintenance of connectivity patterns between neurons, and the formation and function of synapses. Neurons constantly deliver new cytoskeletal proteins synthesized in the cell body to the distal axon through axonal transport. The process of axonal transport is required for maintenance of a functional nervous system and is impaired in a variety of disease states. Filopodia are slender finger-like cellular projections that are strictly dependent on actin filaments and are required for axon guidance and synapse formation. Understanding the mechanisms responsible for filopodial initiation is thus of direct relevance to the understanding of regeneration and the maintenance of nervous system function. This proposal presents studies aimed at (1) determining the form (e.g., filaments versus monomers) of actin transport in axons, and (2) determining the actin-based mechanism of filopodia initiation. The form in which cytoskeletal proteins are transported is controversial. From in vitro studies using neurons transfected with EGFP-actin, we provide preliminary descriptive evidence suggestive of the transport of actin filaments and experimental evidence in favor of the transport of monomeric actin. A series of studies is detailed to experimentally test the hypothesis that actin filaments are the form of actin transport. Additionally, based on live imaging of EGFP-actin in axons and growth cones we have identified the presence of spontaneously formed actin puncta that serve as precursors to filopodial formation. These observations provide a model system for investigating the dynamic cytoskeletal basis of filopodial formation in living neurons. We present studies aimed at determining the role of extra-cellular signals, signaling pathways, and microtubules in regulating the earliest events in filopodial initiation.


These studies are designed to elucidate how changes in extracellular pH influence contractile activity in porcine coronary and carotid arterial smooth muscle. Changes in extracellular pH will be produced by simulating respiratory acidosis and alkalosis and by simulating metabolic acidosis and alkalosis. The effects of changes in extracellular pH on fast and slow components of contractile events elicited by a variety of stimuli (norepinephrine, serotonin, angiotensin, KCl and electric field stimulation) will be analyzed using kinetic approaches. Changes in intracellular pH, calculated by the DMO technique, will be evaluated as a function of extracellular pH, passive stretch, and active generation of force. Studies at each extracellular pH will be performed at different concentrations of Ca ions in the external medium. Ca ions-sensitive actomyosin will be isolated from the arterial smooth muscle preparations, and the extent to which pH modifies Ca ions-sensitive actomyosin-ATPase activity and superprecipitation of actomyosin will be assessed. Thus the effects of extracellular pH on arterial smooth-muscle will be evaluated at the tissue (mechanical responsiveness), cellular (intracellular pH) and molecular levels (actomyosin interactions). These basic studies, integrated at three levels, will reveal how changes in H ion may modulate contractile activity of arterial smooth muscle. In addition to the intrinsic interest regarding the regulation of arterial smooth muscle, the studies bear directly on important clinical settings associated with acid-base imbalances such as circulatory shock and myocardial infarction. Thus, information gained through these studies could be useful in defining how local myocardial acidosis associated with localized cellular damage will influence the responsiveness of the coronary vasculature to therapeutic interventions.


DESCRIPTION: Occupational asthma (OA) is currently the most frequently reported diagnosis of work-related respiratory disease in developed nations. The prevalence of work-related asthma and work-related wheezing in the U.S. is 3.7% and 11.5% respectively and the proportion of asthma attributed to occupational exposures varies [unreadable] from 28.5% to 36.5%. In the U.S. approximately 3% of the working population reportedly works in the cleaning occupation. Several studies, primarily from the European countries, have found an association between occupational exposures and cleaners. However, the major limitations of these studies were non-inclusion of the work aspect in formulating the definition of asthma as the outcome variable, and poor characterization of exposures. We propose to conduct a focus group study to 1) identify an appropriate [unreadable] population of domestic and industrial cleaners; 2) determine the feasibility of contacting and tracking representative groups of domestic and industrial cleaners, for use in future studies of asthma in this worker group; 3) characterize occupational exposures among domestic and industrial cleaners; 4) use information obtained from the focus groups to develop a Job Exposure Matrix that could be used in future studies of asthma in this worker group; and 5) develop and pilot test an English and Spanish version of a survey instrument for use in future studies of asthma among domestic and industrial cleaners. A total of twelve focus group interview sessions (six sessions involving domestic cleaners and six involving industrial cleaners) are proposed for this study. Approximately 12 people (with the aim of 6-10 people attending), between the ages of 18 and 70 and working as a cleaner for at least one year, will be invited to participate in each session. Each session will be moderated by a bi-lingual moderator, who will then use the open ended semi-structured questionnaire to ask questions regarding occupational task, occupational exposures, and asthma and allergy symptoms. At the end of each session, the research team will meet for debriefing. The focus group interview sessions will be audio taped. The interview session audiotapes will be transcribed and analyzed using the content analysis model using "Ethnograph" qualitative analysis software. The information from the focus group interviews will be used to develop JEM and a draft survey instrument for use among cleaners. [unreadable] [unreadable]


Binding of benzodiazepine recognition sites by various ligands can elicit opposite types of responses, such as proconvulsant/anticonvulsant, or anxiogenic/anxiolytic. In order to answer whether a new class of drug belongs to benzodiazepine (anxiolytic), or beta-carboline (anxiogenic) type of ligand we developed a behavioral animal model that predicts the anxiogenic and anxiolytic potency of a drug. The inhibition of the response by the pure antagonist (partial agonist?) of the benzodiazepine recognition site assumes that proconflict and anticonflict action depends on the binding of the drug to the benzodiazepine recognition site.


Development of endometriosis appears to involve shedding of endometrial cells from the uterine cavity and implantation of the cells on ectopic pelvic loci. It is not known to what extend shedding of endometrial cells occurs in normal women at various stages of the menstural cycle or what factors may be involved in determining the biologic fate of these cells. Methodology used in the past has not allowed quantitative characterization of endometrial cells in the pelvic environment. The purpose of the present proposal is to test various biochemical cell markers for utility in parallel characterization of endometrial epithelial cells and inflammatory macrophages. This will be done by applying both conventional and newly developed marker techiques to endometrial and pelvic fluid aspirates and tissue samples of ectopic pelvic endometrial implants. The cell populations from endometrial aspirates, tissue samples and peritineal fluid will be characterized for a variety of enzyme, antigen and receptor markers using conventional cytochemical analysis and a flow cytofluorometric approach in parallel. The comparative analyiss of endometrial cells and inflammatory cells is important because preliminary studies suggest that pelvic macrophages are different in patients with endometriosis as compared to naormal women. The results of these studies will eventually show: 1. If any characteristic marker pattern distinguishable from normal can be found in aspirates of uterine endometrial cells or pelvic endometriotic implants in women with endometriosis. 2. If free endometrial cells in the peritoneal fluid are more closely related to cells in ectopic peritoneal implants or to cells in uterine endometrium in the same individual. 3. If physiologic cyclic changes exist in endometrial cells in peritoneal fluid in normal women or in patients with endometriosis during the menstrual cycle. 4. If biochemcial indicators of macrophage activation follow a physiologic or rhythmic pattern in each group of patients. 5. If pelvic macrophages from endometriosis patients are capable of inducing changes in adherence, cell growth, biochemical marker expression of normal endometrial cells when cocultured in vivo.


This project will evaluate the translation of the Gold (the Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease) COPD guidelines into primary care practice. This study is based upon the premise that an informed, activated patient will interact with a prepared, proactive team to improve COPD screening, diagnosis and management. Testing a similar hypothesis and using a similar design, the project team has demonstrated a successful translation of the NHLBI ATP III cholesterol guidelines into clinical practice. During phase 1, a needs assessment will evaluate barriers and facilitators to implementation of COPD guidelines into clinical practice through focus groups of primary care patients and providers. Using formative evaluation and feedback from the focus groups, three tools will be developed, refined and pilot tested. These include: a computerized patient activation tool that will be placed in each primary care office's waiting room for patient use; a [web-based], interactive COPD guidelines tool to be used by primary care providers as a decision support tool at the point of care and a COPD patient education toolbox to be used by the practice team. During phase 2, a block, randomized design cluster trial will be performed with one year of intervention within non-academic primary care practices (30 practices) throughout the state of Rhode Island and southeastern MA. The effectiveness of the materials developed in phase I will be tested in phase II regarding physician performance of COPD guideline implementation and the improvement in the clinically relevant outcomes (appropriate screening, diagnosis and management of COPD) compared to usual care. We will also examine the use of a patient activation tool - 'MyLungAge' to prompt patients to talk with their health care provider regarding their lung health and risk for COPD. Products for dissemination from this grant will include the results of the focus groups barriers and facilitators to implementation and adherence to COPD guidelines, a computerized data collection module for quality of care assessment regarding COPD guidelines, a refined computerized patient activation tool, an enhanced [web-based] COPD interactive guideline tool, tailored academic detailing materials, and the results of the randomized clinical trial on the effectiveness of these tools to improve COPD guideline adherence in primary care practice. Public Health Relevance Statement: This project will evaluate the translation of the Gold (the Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease) COPD guidelines into primary care practice. During phase 1, a needs assessment will evaluate barriers and facilitators to implementation of COPD guidelines into clinical practice through focus groups of primary care patients and providers. Using formative evaluation and feedback from the focus groups, three tools will be developed, refined and pilot tested. The effectiveness of the materials developed in phase I will be tested in phase II (an RCT conducted with one year of intervention within non-academic primary care practices) regarding physician performance of COPD guideline implementation and the improvement in the clinically relevant outcomes (appropriate screening, diagnosis and management of COPD) compared to usual care.


The primary goal of this research is to being to common availability the precise, sensitive, nondestructive viscosity and density measurements by J.W. Beams on small (approximately 0.2 microliters) samples of biomacromolecular solutions employing only tiny (less than approximately 10 to the minus 3rd power dynes/sq. cm) stresses. In the coming year improvements will be aimed at allowing these measurements to be made simultaneously, without interference from each other. A new "oscillating buoy" method which has been developed, will be evaluated in addition to the improved rotary machine of Beam's design. Larger numbers of actual experiments will be made, using the recently automated datataking means. Mechanical improvements, improved buoy design, and refinement of the new support and rotation electronics are in latter stages, and will be the primary means of achieving the independence of the two measured variables.


Substance abuse in adolescence is an epidemic which takes a significant toll on society and has deleterious physical, psychological, and social effects on adolescents and their families. As such, a great deal of research in recent years has focused broadly on finding patterns among social and behavioral determinants of substance abuse in adolescence, and specifically on the goal of finding clinically relevant subgroups of adolescents according to substance abuse behaviors. This goal is reflected by a recent NIDA program announcement, PA-15-003, entitled Epidemiology of Drug Abuse; this announcement lists trajectories of drug use and patterns of comorbidity with substance use disorders, two areas frequently explored using subgrouping strategies, as areas of interest. The goal of forming meaningful behavioral subtypes has largely been pursued using mixture models, a broad class of models which seeks to divide the sample into meaningful subgroups, known as latent classes, of subjects. Mixture models have seen extensive use in substance abuse studies in the past decade, with behavioral groupings being linked to imaging data, biomarkers, and genetic information. However, despite the their widespread use, virtually all current applications of mixture models have failed to take into account one critical aspect of behavioral measurement known as differential item functioning (DIF). Given an item intended to measure some underlying construct, DIF occurs when subjects with the same level of that underlying construct differ systematically in their responses to an item measuring that construct due to their gender, race, age, or any other individual characteristic. When DIF occurs and is not accounted for, it may render estimates of the level of the underlying construct biased, and inferences about differences between groups invalid. Though techniques for measuring continuous latent variables such as item response theory (IRT) and confirmatory factor analysis (CFA) have given serious attention to DIF as a fundamental threat to the validity of inferences, there has not yet been systematic study of DIF in the measurement of latent classes in mixture models, and there are no known applications of DIF analyses to mixture model results in the substance abuse literature. The proposed research seeks to rectify this shortcoming by rigorously investigating DIF in mixture models with a focus on behavioral studies of substance abuse. The four aims of the proposed project are to: (1) analytically define the ways that DIF can manifest in mixture models; (2) develop a flexible test of DIF in mixture models; (3) use a computer simulation to test (a) the consequences of un-modeled DIF in mixture models and (b) the efficacy of the test developed in Aim 2; and (4) to use DIF modeling techniques in an empirical study of substance abuse in the transition to adulthood. Through the pursuit of these aims, this project will allow fo mixture models to better consider individual differences, thereby enhancing the generalizability of these results in behavioral substance abuse research.


[unreadable] The FASEB Summer Conference on the Molecular Biology of Lipid Transport and Metabolism will bring [unreadable] together scientists in a variety of related fields with the purpose of defining, describing and analyzing [unreadable] critical scientific problems and recent advances in an intimate and interactive environment. A key feature of this meeting is the opportunity to facilitate the interactions of students and postdoctoral fellows with senior established figures in the field. Such interactions have historically been successful in defining new research agendas and fostering scientific discourse and collaborations. This application requests partial funding for a Conference to be held July 15 to 20, 2006 at the Omni Resort in Tucson, Arizona. This will be the sixth such conference on this theme; the last one was held in the summer of 2003. This conference is unique in its highly multidisciplinary approach to study a key problem of mammalian physiology. Thus, we will bring together a diverse group of senior and junior investigators with expertise in the areas of nutrition, physiology, biochemistry, protein chemistry, cell and molecular biology, and developmental biology, to address recent advances in lipid metabolism and its regulation. The format will consist of seven major oral sessions scheduled over four days (four AM and three PM), an evening Keynote Address and two poster sessions. To date 26 highly noteworthy invited speakers have confirmed their willingness to participate. The seven formal sessions focus on active research areas: 1) Intestinal Sterol Trafficking; 2) Lipid Transport and Bile acid Metabolism; 3) Dietary Lipids in Extra-hepatic Tissues; 4) Fat Metabolism in Model Organisms; 5) Lipid Disorders with Multi-organ effects; 6) Nuclear Receptors with Lipid Ligands; and 7) Cell biology and Genetics of Lipid Metabolism. The ultimate session will be a key note address from Jeffrey Gordon on Intrinsic and extrinsic regulators of intestinal lipid metabolism. All participants will be invited to submit an abstract to one of two poster sessions that will be held during the conference in order to allow all attendees, especially students and junior investigators to present data and participate in discussions. Additional speakers will be chosen from the abstract submissions, and all participants will be encouraged to submit particularly novel late-breaking developments for brief oral presentation in the form of "hot topics". These hot topics sessions will be targeted specifically to accommodate junior scientists. In the past this conference has promoted interactions and scientific discussions among basic and clinical investigators from academia and industry. The limited attendance, state of the art scientific sessions, "hot topics" additions, and novel poster presentations combine to provide an essential forumthat will foster exchange of ideas and further scientific advances in a field where advances in basic research have historically been directly translated into improved therapeutic options for a large fraction of the population. [unreadable] [unreadable] [unreadable] [unreadable]


This research involves an electron microscopic examination and lipid and fatty acid analysis of ground squirrel hearts in the active and hibernating condition and a comparison to the heart of non-hibernating mammals, the white rat and the monkey. There are known physiological differences between the hearts of hibernating and non-hibernating mammals, such as maintenance of a rhythmic heart beat at 1 degrees C in the hibernator and ventricular fibrillation and heart failure in the non-hibernator at 15. degrees C. The electron microscope will be used to continue the critical scrutiny of heart ultrastructure to further corroborate the previous findings relating to the anatomical differences with are sufficient to account for the observed physiological differences. The lipids of the ground squirrel heart in the active and hibernating condition will continue to be analyzed by column and thin layer chromatography to corroborate initial findings of modifications in lipid content during the hibernating state. Additionally, the fatty acids, particularly those bonded to the lipids with are modified during hibernation, will be analyzed to determine shifts in the degree and pattern of unsaturation during the hibernating state. Results from these analyses may further elucidate the factors responsible for the continued automaticity of the ground squirrel heart at near- freezing temperatures. This may in turn further enhance medical knowledge and facilitate the use of hypothermia in various aspects of surgery. Since hibernator hearts do not fibrillate at any temperature from 0 degrees C, to 37 degrees C, this knowledge may eliminate, or reduce the frequency of, the extremely dangerous ventricular fibrillation which occurs in most human hearts at about 25-28 degrees C.


Despite advances in intraaortic balloon pumping and emergency myocardial revascularization, salvage of ischemic myocardium has been limited in man by the lack of effective collateral circulation into an area of acute infarction. We are currently carrying out experimental trials of perfusing ischemic myocardium by retrograde diastolic pulsation of oxygenated blood into the coronary veins draining an area of ischemia via a balloon tipped catheter that can be introduced transvenously. Results to date indicate correction of EKG changes, reversal of dyskinetic areas and improvement in myocardial performance in acutely ischemic myocardium following institution of retroperfusion.


Daniel Mueller will direct the transplantation core. He will be responsible for the training and supervision of the Junior Scientist (to be named) on the project. He will assure the quality of the core?s surgical expertise, and will also manage the financial aspects of the core. This Junior Scientist, to be hired, will have significant expertise in animal husbandry and small animal surgery, and will have a background in a biological discipline such as physiology or immunology. This individual will be trained by the Core leader and his associates to harvest mouse tracheas for implantation under anesthesia, to transplant the tissue under anesthesia (survival surgery), to monitor the transplanted animals during the recovery period following the transplantation, and to sacrifice the animals at the end of the experiment. He/she will then be responsible for the recovery of tracheal graft tissues as well as relevant lymphoid organs (lymph node, spleen) at the end of the experiment, and processing of the tissue per the request of the investigator. This will entail the freezing of tissue blocks in OCT medium and frozen sectioning, as well as the preparation of cellular suspensions from collagenase-treated tracheal tissue. This individual will make his or her expertise available to all scientists on this program project on a pre-arranged schedule, and free of charge. This investigator will also be responsible for purchasing of the relevant expendable supplies and for the maintenance of the small equipment (e.g., tissue forceps) and the cryostat.


[unreadable] [unreadable] About 40% of the women diagnosed with breast cancer will progress to metastatic disease. However, metastases cannot be removed by surgery or radiation, and most metastases are chemoresistant. Therefore, therapies that specifically prevent or eliminate metastases offer great promise in the outcome. Recent studies indicate that enhancement of specific helper or cytotoxic T lymphocyte (CTL) responses to breast tumors through vaccination could potentially lead to the specific elimination of micro-metastases. However, poor vaccine-induced immune and clinical responses underscore the need for improved vaccine strategies. This research proposal is focused on the improvement of vaccine therapy against metastatic breast cancer by combining vaccination with the IL-6 inhibitor curcumin, using the preclinical metastatic mouse breast tumor model 4T1. We previously demonstrated that Mage-b DNA vaccination provides significant protection against breast cancer metastases in this model, albeit not completely. Interleukin (IL)-6 may contribute to decreased vaccine efficacy. It is highly up regulated in the 4T1 primary tumors and metastases, probably via the Nuclear Factor Kappa B (NFkB), and is a potent regulator of dendritic cells (DC) differentiation in vivo. IL-6 activates the expression of transducer and activator of transcription (STAT)3 in immature DC, preventing the DC from maturation and subsequent presentation of antigens to T cells. This may lead to T cell unresponsiveness. Agents that are able to inhibit IL-6 may lead to enhanced vaccine efficacy. Curcumin could be such an agent, since it inhibits IL-6 production. Both Mage-b and IL-6 are frequently detected in human breast cancer, which makes the 4T1 model clinically relevant. The long-term objective of this proposal is to develop a novel non- toxic combination therapy of Mage-b vaccination and curcumin in the 4T1 model, with a focus on its translation into human clinical trials, if successful. The hypothesis is that curcumin improves T cell activation and subsequent immune responses upon Mage-b DNA vaccination, resulting in further reduction of the frequency of breast cancer metastases. The specific aims are as follows:(1) Testing the effect of Mage-b vaccination with and without curcumin on tumors and metastases. For this purpose, mice will be immunized preventively or therapeutically with Mage-b DNA vaccine and challenged with 4T1 tumor cells. As soon as the primary tumor can be felt curcumin will be administered daily. Frequency of metastases, tumor size, and survival times will be determined; (2) Testing the direct effect(s) of curcumin on vaccine- and tumor-induced immune responses, and on proliferation and apoptosis of tumor cells. For this purpose, spleen, lymph nodes, tumors and metastases of treated and control mice will be analyzed for the presence of activated CD4 and CD8 T cells, regulatory T cells and mature DC. In addition, tumors and metastases of treated and control mice will also be analyzed for the expression of NFkB and NFkB-regulated genes that are directly involved in immune responses such as IL-6, TNF1, and STAT3, and for proliferation and apoptosis. PUBLIC HEALTH RELEVANCE: Current treatment of cancer is ineffective against metastases. In this research proposal a novel non-toxic combination therapy of Mage-b vaccination and curcumin will be developed that is effective against breast cancer metastases, using a preclinical mouse model that reflects metastatic breast cancer in humans. [unreadable] [unreadable] [unreadable]


DESCRIPTION (applicant's abstract): The long term goal of this project is to elucidate the mechanisms that regulate functions of the noradrenergic locus coeruleus (LC) system. Our previous results defined afferents to the LC nucleus, and characterized LC dendrites that extend into specific extranuclear zones. However, these findings also generated new questions: (i) what are the inputs to extranuclear LC dendrites, (ii) what circuits are LC afferents a part of, and (iii) what LC output functions are regulated by these afferent circuits? The proposed experiments will answer these difficult but important questions using new anatomical technology and electrophysiology. LC neurons have extensive extranuclear dendrites, and the zones containing these distal dendrites receive numerous inputs that do not innervate the LC nuclear core. However, it has been difficult to identify which of these afferents terminate on LC dendrites vs. other elements in this region. We will retrogradely label afferents that specifically innervate extranuclear LC dendrites using the recently developed transsynaptic tract-tracer, pseudorabies virus (PRV). Dendritic afferents will be confirmed by ultrastructural analyses, and their impact on LC neuronal activity will be determined. In addition to direct afferents to LC neurons, it is important to determine inputs to these direct afferents, and thereby identify circuits that regulate LC function. We will map indirect afferents to the LC in a detailed time-course study of transsynaptic transport of PRV. For prominent indirect afferents, the relays to the LC will be identified and the influence of these afferent circuits on LC activity will be determined. Our preliminary results indicate that the suprachiasmatic nucleus (SCN) is a prominent indirect input to the LC. The SCN is the brain's circadian pacemaker, and controls among other rhythms circadian properties of sleep and waking. As the LC has long been implicated in arousal, we hypothesize that the SCN-LC circuit is a key neural substrate linking circadian and sleep-waking processes. We will test this hypothesis by manipulating SCN activity, recording the effects on EEG arousal, and testing the role of the LC and associated relay nuclei in the effects obtained. This will be the first analysis of a neural link between circadian and arousal processes. These studies will extend our analysis of afferent control of LC function to identify inputs to distal LC dendrites and circuits that regulate LC activity. They will also provide the first demonstration of afferent circuit regulation of an important LC output function, cortical arousal.


Objectives: 1. The total number of primary malignant melanomas studied by our group now approaches 570 cases. Of this group, approximately 250 to 300 cases have complete histological data and clinical follow ups of greater than 5 years. From the investigation of these cases, we hope to determine the parameters for high and low risk categories. These studies include cellular parameters of the primary lesions and a variety of epidemiologic data from the patients. 2. We are attempting to study comparatively the immunologic status of patients in high and low risk categories using percentage of circulating T and B cells, percentage of B cells, T cells and histiocytes in the host cellular response to the tumor, determination of cell mediated cytoxicity to three reference melanoma cell types and determination of host blastogenic response to melanoma antigens. 3. After establishing over the past three years malignant melanoma in guinea pigs, we are in the process now of studying its developmental biology. 4. An ultrastructural study to delineate the different between the fine structure Spitz tumor and malignant melanoma is nearly complete. 5. Additional studies include: Fine Structure of Host Tumor Cells and Host Responding Cells, Fine Structure of Nuclear Tubular Bodies in Human Malignant Melanoma-Paramyxovirus-like Structure, and the Study of Significance of Polyclonism in the Vertical Growth Phase or Intralesional Transformation as Related to the Subsequent Development of Metastases. Bibliographic references: Suffin, S. C., Waisman, J., Clark, W.H.: Congruence of Diagnosis of Malignant Melanoma, Lab. Inv., 32 (3):436, 1975. Leone, P.G., Leone, C., Clark, W. H.: Acquired and Congenital Nevi, Lab. Inv. 32(3):464, 1975.


Diabetes accounts for nearly 22 million physician visits annually and is the third leading cause of repeat outpatient visits in the United States. Depression is also strongly related to increased medical (nonpsychiatric) health care utilization, although its specific effect in diabetes has not been studied. Depression is 304 times more prevalent in diabetes than in the general US population. Depression in diabetes has been associated with several factors (e.g., poor glucose regulation, poor compliance with the diabetes regimen) that could lead to increased health care use. We proved during the last period of NIH support, in a double-blind, placebo- controlled trial, that antidepressants were significantly more effective than placebo in managing depression; 65% of treated patients remitted. We also have shown that in the absence of treatment, diabetic depressions are chronic. We plan to recruit 60 depressed patients with NIDDM from a metropolitan primary care practice, and treat them with antidepressant pharmacotherapy for a period sufficient to detect an effect on health care utilization (1 year). Because of the extended treatment interval, the chronicity of untreated depression in diabetes, and the known response/ nonresponse to pharmacotherapy, it is not ethical, practical, or methodologically necessary to include a depressed, no-treatment control group. A comparison sample of 60 nondepressed NIDDM patients matched for age, sex, other comorbid medical illnesses, treating physician, and diabetes type, severity, and method of treatment is included. Health care utilization will be assessed in both depressed and control patients for the year prior to study enrollment and during the year of treatment. Measurements of psychiatric disease activity, family history of depression, personality characteristics, life stress, diabetes symptoms, glucose regulation, and functional status and well-being, will be taken at specified points during the year of depression treatment. We hypothesize that: 1) depressed diabetic patients will make greater demands for medical health care than the nondepressed matched control sample; 20 medical health care utilization will be greater in depressed diabetic patients whose depression persists compared to patients who become nondepressed during treatment; and 3) utilization in the depression improvement group will be similar to that measured in the nondepressed controls. Failure to recognize and treat active depression in diabetes may fuel higher health care utilization. A component study is described that aims to develop a brief psychometric screening tool with positive and negative predictive values for major depression in diabetes greater than 0.80. Witnessing that improvement in depression is associated with a favorable reduction in health care utilization by NIDDM patients would suggest the potential benefits of depression recognition and treatment to society in general, over and above the recognized patient benefits in mood and quality of life.


The studies outlined in this proposal will focus on cyclin D1, a key cell cycle control protein that is one of the most prevalent oncogenes in human cancers. Studies from this laboratory have identified cyclin D1 as an important mediator of hepatocyte proliferation in the regenerating liver. However, the biochemical and cellular functions of cyclin D1 have not been fully characterized despite extensive study. The purpose of these studies is to characterize a novel effect of cyclin D1 in hepatocytes. Based on preliminary evidence, the primary hypothesis is that cyclin D1 regulates sex steroid hormone metabolism and signaling at the cellular and tissue level, and that these effects play a biologically significant role in the liver. It is also hypothesized that the C-terminus of cyclin D1 plays an important role in modulating sex steroid signaling as well as other biologically relevant effects in the liver. Additional studies will identify novel cyclin D1 binding partners. Identification of novel roles for cyclin D1 in the liver are expected to increase our understanding of an important liver oncogene. [unreadable] [unreadable] [unreadable]


We are examining the three-dimensional (3-D) fine structure of the Golgi complex in rapidly-frozen pancreatic beta cells to provide new data on the sites of formation and sorting for secretory granules, constitutive secretory vesicles, and vesicles destined for the endo-lysosomal pathway. Insulinoma cell lines (HIT-T15 and MIN-6) and isolated rodent islets of Langerhans are being fixed by high-pressure freezing and freeze-substitution. 250-400 nm thick sections of plastic-embedded samples are imaged in a high-voltage electron microscope (HVEM) and serially tilted at 1.5x intervals (q60x) about two orthogonal axes. These digital images are used to generate high resolution (5-7 nm) tomographic reconstructions which can then be analyzed and modeled in 3-D. These reconstructions will be used to examine the relationship between trans-cisternae and the TGN, to resolve novel coat structures on nascent vesicles and granules, and to address hypotheses relating to cisternal progression-maturation and protein trafficking via membranous tubules. Our preliminary data show that small dense-core granules (average diameter 95 nm) form from cis-, medial- and trans-cisternae, not just from the trans-most cisterna or TGN. This suggests that secretory product condenses at many, if not all, levels of the Golgi complex. These dense- core granules appear to be connected to Golgi cisternae and to each other by tubular projections of uniform diameter and density, arranged like "beads on a string". The result is a continuum of developing granules, connected with Golgi cisternae at one end and projecting away from the Golgi stack at the other. C3


The purpose of this project is to understand the molecular mechanisms responsible for replication of picornaviruses in susceptible target cells. This virus family includes numerous human pathogens (poliovirus, coxsackievirus, echovirus, enteroviruses, rhinoviruses, hepatitis A virus). Infection of cells with these viruses leads to major changes in the host cell's structure and metabolic activity. Cellular protein and RNA synthesis are inhibited; the intracellular membrane network becomes rearranged into vesicles that surround and provide a scaffold for viral RNA replication complexes; cellular proteins are subverted into facilitating viral protein and RNA synthesis. The unique combination of viral and cellular proteins together accomplish a highly efficient production of viral RNA, proteins, and particles. We are studying the activities of individual viral gene products, expressed alone or in combination, in cultured human cells, to determine their individual roles in the replication process. Interactions of each viral protein with each other and with cellular proteins are being characterized. The biochemical reactions required for translation of the viral RNA and replication of the viral genome are being defined. Understanding the precise biochemical activities of viral proteins in the replication process will allow the development of new strategies for vaccine development and the design of antiviral drugs.


The Administrative Office, under the direction of the Chairman, Dr. George Lewis, in submission of the annual progress report will state the objectives, goals, and progress of the Gynecologic Oncology Group. Our institution continues to be a major contributor to the group.


This is an ACTG sponsored four center safety, pharmacokinetic, and dose finding trial of AZT for infants one day to 3 months of age who are born to women with HIV infection. Dosing and intravenous and oral AZT pharmacokinetic studies are done on three occasions during the 6 week protocol. Participants are monitored closely for evidence of AZT toxicity and for development and progression of virologic and clinical evidence of HIV infection.


Alcohol misuse is a significant public health problem, not only in the US but around the world. Just in the US, alcohol misuse is estimated to be the second cause of preventable death at a cost of more than $220 billion in a single year. Further, it is also estimated that it reduces the lives of those who died by 30 years. Screening and brief interventions have proved effective in promoting behavior changes that lead to increased awareness and decreased consumption when properly conducted at the primary care setting. However, there remain four main barriers that have curtailed broad adoption of screening and brief interventions by primary care: lack of access to validated tools, lack of training, reluctanc to raise the subject to the patient, and lack of face time. Overcoming these barriers creates a vastly significant clinical, public health, and commercial opportunity. VisionQuest Biomedical and the University of New Mexico have formed a team to develop and test the feasibility of PA?MSTM, a mobile platform for the delivery of screening and brief interventions for alcohol misuse. PA?MSTM technical innovation is that it shifts the delivery paradigm from paper and in-person delivery to software-as-a-service. PA?MSTM is not an electronic form only but a complete set of tools that allow electronic communication between patients, providers, and support groups from screening to brief interventions to follow up care. By enabling on-demand, up-to-date patient-centered screening and brief interventions PA?MSTM clinical innovation is that it extends the reach of primary care and facilitates care coordination and decision making. In Phase I, a prototype PA?MSTM will be developed and tested in a primary care setting on 40 patients who will be followed for two months. Standard metrics of usability, acceptability, and user satisfaction will be assessed in order to prove the feasibility of the prototype and plan for an improved version that will be tested in a multi-center clinical study in Phase II.


Characterization of Platelet Function in Patients with Bleeding Disorders: In FY2015 (the fourth year of this project) we have studied several patients with suspected or documented disorders of platelet function and have identified patients with deficiency of dense granules (storage pool disease), and characterized platelet function in people with congenital or acquired platelet dysfunction. The ability to make a reliable, detailed characterization of platelet function may help investigators who are studying genetics of inherited platelet disorders. One family with abnormal platelet function and other abnormalities has been described and submitted to the journal Blood for consideration to be published (York Platelet Syndrome, T. Markello et al, authors and collaborators). Another patient has been identified who has a novel platelet disorder that may be a variant of Glanzmann's thrombasthenia. He has been referred to the Undiagnosed Diseases Program (NIH/NHGRI) for further analysis and possible genome sequencing to identify the basis of this disorder. Characterization of D-Dimer and Related Coagulation Proteins in HIV-Infected Research Subjects Undergoing IL-2 Therapy: During the fifth year of this project, in support of intramural NIH research protocols, we have measured D-dimer levels and numerous coagulation parameters in research subjects who are infected with HIV or who are normal volunteers. We seek to correlate the D-dimer levels with these various parameters to gain insights into the mechanism for the observed elevations in D-dimer levels in HIV infected patients that correlate with increased morbidity and mortality. Effect of Factor VIII Haplotypes on Inhibitor Development. We have identified a set of 800 DNA samples from patients with severe hemophilia A in an NHLBI repository and have IRB approval to determine their factor VIII protein-coding polymorphisms to determine the effect of factor VIII haplotype on inhibitor development. We expect to complete the DNA sequence determinations in the coming fiscal year, and will see if the effect of protein coding polymorphisms on inhibitor development that was seen in a minority population (African-Americans) can be seen in the population as a whole. We have observed two cases of acquired factor VIII inhibitor in the setting of non-myeloablative stem cell transplantation for sickle cell disease in African-Americans, a report on one of which has now been published.


The long-term goal of this proposal is to provide insight into mechanisms critical for stress-facilitated memory formation, or in other words, the processing of memories under stressful conditions. Two stress- and memory-associated disorders, posttraumatic stress disorder and major depressive disorder, are prime examples of disorders with abnormal stress-facilitated memory processing, and they both have an estimated lifetime prevalence of over 5% among Americans. A key signaling pathway implicated in stress-induced memory processing is the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. [unreadable] Activation of this pathway leads to downstream activation of cAMP response element-binding protein, which results in transcriptional changes needed to form new memories. The activator of the MAPK/ERK pathway that modulates stress-facilitated memory is still not well defined. However, evidence suggests the second messenger, cAMP, may be the upstream initiator as cAMP production can activate this pathway to cause changes in memory. Moreover, calcium-stimulated adenylyl cyclases (AC), AC1 and AC8, couple neuronal activity and intracellular calcium increases to the production of cAMP, thus, implicating calcium-stimulated AC activity in modulating stress-facilitated memory changes. As a paradigm for stress-facilitated memory, we will use a classical conditioning test, which serves as a good model to investigate our hypothesis as both the MAPK/ERK signaling pathway and calcium-stimulated AC activity have been shown to effect learning on this paradigm. Therefore, we aim to examine how this activity may activate the MAPK/ERK pathway to modulate stress-facilitated memory. Through use of a novel transgenic mouse model, which uses a tetracycline-inducible system to replace AC8 expression in AC1 and AC8 double knock-out mice, we are able to assess the time-specific importance of this activity. Moreover, through the use of gene therapy techniques, we can assess the region-specific importance of this activity via lentivirus administration that turns on AC8 expression. Relevance: The research we are proposing will give insight into a mechanism of memory formation under stressful conditions and may reveal a primary role of memory-related symptoms in psychiatric disorders. More specifically, it will look at the time- and region-specific manner in which this stress-facilitated memory mechanism modulates memory formation. This work may eventually lead to the development of therapeutic interventions for people with abnormal memory changes from stress-related disorders as global, non-specific treatments can facilitate a host of unwanted side-effects.[unreadable] [unreadable] [unreadable] PUBLIC HEALTH RELVENCE The research we are proposing will give insight into a mechanism of memory formation under stressful conditions and may reveal a primary role of memory-related symptoms in psychiatric disorders. More specifically, it will look at the time- and region-specific manner in which this stress-facilitated memory mechanism modulates memory formation. This work may eventually lead to the development of therapeutic interventions for people with abnormal memory changes from stress-related disorders as global, non- specific treatments can facilitate a host of unwanted side-effects. [unreadable] [unreadable] [unreadable]


The purpose of the proposed research is to examine developmental changes in children's understanding of attention ("meta-attention"). It is expected that there are changes in concepts concerning the nature of attention and distraction, strategies for attending to stimuli, and variables which facilitate or hinder attention. Meta-attention is important because it is believed to influence attentional behavior, especially the selection and execution of appropriate strategies for problem-solving. Since there is almost no research on meta-attention, the proposed research would fill an important gap. Although the emphasis will be on developing valid nonverbal procedures for assessing meta-attention, a variety of response measures will be used--the child's selection of stimuli to expose, prediction of one's performance, construction of "easy" versus "hard" attentional tasks, selection of pictures or other stimuli from several alternatives, rating pictured situations according to a scale, reaction time, and responses to questions concerning strategies. Although children from age 3 to 14 will be used, the emphasis will be on the grade school years. When possible, the tasks will be adapted for use with preschool children in order to examine the beginnings of meta-attention. The studies will examine meta-attention in several tasks commonly used to measure attention in children--"incidental learning," visual search, dimensional sorting of stimuli, stimulus matching, and same-different judgments.


The influence of parenteral nutrition on bone mineralization and metabolism was evaluated. Four children, partially ad fully dependent on parenteral nutrition were evaluated over 24 hours with dietary assessments, and timed urine and blood collections. Mediators of calcium and bone metabolism were monitored.


EVP 1001 is intended to be a unique, intracellular, cardiac specific magnetic resonance imaging (MRI) agent which specifically enhances both the myocardium and the vasculature. It will satisfy currently-unmet needs for the rapid, non-invasive diagnosis and treatment planning of ischemic heart disease, including simultaneous assessment of both structure and function (stenosis, occlusion, wall motion, myocardial perfusion and delineation of viable versus non-viable myocardium). The preclinical research proposed for Phase 1 will augment the cardiac safety of EVP 1001 and will define the relationship between its pharmacokinetics and its MR enhancement. Does regimens which provide effective cardiac and vascular MR enhancements will be delineated from these data. The unique magnetic properties of EVP 1001 are expected to provide enhancement at low doses compared to current gadolinium agents (3-15 versus 100-200 mumol/kg). Phase 2 will assess the safety and efficacy of EVP 1001 in models of cardiac ischemia, laying the foundation for human trials. When approved, this contrast agent will make optimal use of the powerful diagnostic potential of MRI and its rapid technical evolution, becoming the "one-stop shop" for diagnosis of coronary artery disease and replacing more costly and invasive tests. PROPOSED COMMERCIAL APPLICATIONS: The long-term goal of this research is to register EVP 1001 in the U.S. and elsewhere as a contrast agent for cardiovascular MRI. By combining the noninvasive assessment of both cardiac structure and function, this agent should provide more cost-effective diagnosis and treatment planning than echocardiography, nuclear perfusion/functional imaging, and x-ray angiography. In addition, its use as a vascular enhancement agent may be extended to other regions of the body.


Deposition of amyloid in cerebral blood vessels (cerebral amyloid angiopathy, CAA) is a common condition in the elderly associated with both hemorrhagic and ischemic strokes. CAA is also a potentially important model for understanding the pathogenesis of Alzheimer' s disease, the other major beta-amyloidosis of the central nervous system. At the Alzheimer Research Unit at Massachusetts General Hospital, we have used clinical, neuropathological, and genetic approaches to define the pathogenic steps involved in CAA. These studies have been limited, however, by the static nature of post-mortem brain tissue and the two-dimensional nature of single brain sections. The current proposal seeks to move beyond these limitations by using multi-photon confocal microscopy to observe the development of CAA in transgenic mice over time, and by using multi-photon microscopy and computer-aided image reconstruction to analyze the three-dimensional structure of affected blood vessels. We will use these techniques to examine the processes of 1) amyloid deposition in vessels; 2) breakdown of the amyloid-laden vessel wall; and 3) response to anti-amyloid immunotherapy. We will test whether Abeta initially deposits near vessel branchpoints, whether vessel amyloid affects the deposition of parenchymal amyloid, and whether amyloid deposits preferentially on specific vessels. We will generate a timeline of CAA, from the first deposition of A13 species through formation of amyloid, loss of smooth muscle cells and subsequent vasculopathic changes. We will determine whether there are potential detrimental effects to vessels by anti-Abeta therapies. Studies will be performed on mice doubly transgenic for mutant amyloid precursor protein and presenilin- 1, mice that demonstrate detectable CAA at ages as early as six months. Parallel studies on the three-dimensional structure of amyloid deposition and vessel breakdown will be performed on human tissue taken from collected cases of CAA, including the unique Iowa form of familial CAA. Successful completion of these studies will delineate the pathways involved in CAA initiation, propagation, and vessel damage, as well as determine the feasibility of specific immunotherapy for this currently untreatable disorder.


Endothelial dysfunction is associated with corresponding changes in vascular tone and increases in contractility, a condition that may cause hypertension in susceptible individuals which may have allelic variants in cyp2j-epoxygenases, soluble epoxide hydrolase (sEH), A2A, and A1 adenosine receptors (A2A AR & A1 AR) genes. These allelic variants may have similarities to our transgenic mice which may regulate vascular tone and blood pressure (BP). Our preliminary data have suggested a possible link between adenosine-induced relaxation and opening of KATP channels through A2A AR-cyp2j-PKA-PPAR? pathway. Also, it is possible that a link may exist between adenosine-induced contraction and closing of KATP channels through A1 AR-sEH-cyp4a- PPAR? pathway. A combination of pharmacological tools and transgenic mice would allow us to identify the possible targets as a long term goal to treat population which may have allelic variants leading to hypertension. Therefore, there is a critical need to explore the possible mechanism involving cyp2j2-epoxygenases, sEH/cyp4a, A1 AR/A2A AR, PPAR?/?, PKA/PKC?/? and KATP channels in adenosine-induced vascular responses. Our central hypothesis is that adenosine induces vascular relaxation and decreases in BP through cyp2j-epoxygenases via A2A AR-cyp2j-PKA-PPAR? signaling leading to opening of KATP channels. On the other hand, adenosine induces vascular contraction and increases in BP through sEH via A1 AR-sEH-cyp4a-PPAR? pathway leading to closing of KATP channels. To test this hypothesis, we propose to explore in depth mechanism(s) using A2A AR-/-, A1 AR-/-, cyp2j5-/-, sEH-/-, Tie2-cyp2j2Tr (endothelial-cyp2j2 overexpressed), Tie2-sEHTr (endothelial-sEH overexpressed), wild-type mice, immortal renal endothelial cell line from H-2Kb- tsA58 mouse, mouse aortic endothelial cells (MAEC) and mouse aortic smooth muscle cells (MASMC). Further, we will also explore the possible treatment with sEH inhibitors (AUDA/t-AUCB) in drinking water (or gavage) for A2A AR-/-, cyp2j5-/- and Tie2-sEHTr mice which may have high BP. We will measure BP, and we will use aortas/renal arteries (organ bath/DMT-wire myograph) with treatments (adenosine-receptors agonists & antagonists), cyp-epoxygenases, sEH, adenylyl cyclase, PKC?/?, MAPK and PKA inhibitors, PPAR?/?, EETs and KATP channel (activators & inhibitors). EETs & DHETs will be analyzed (UPLC-MS/MS). Western blot & RT-PCR will be used for proteins & mRNA expression. We propose 3 specific aims to determine: (1) whether the cyp2j-epxygenases or sEH affects BP, adenosine-induced vascular response and EETs/DHETs; (2) whether the presence or absence of A2A AR affects adenosine-induced vascular response through PPARs via cyp2j-epoxygenases, sEH (3) whether the presence /overexpression of cyp2j-epoxygenases or sEH regulate KATP channels through A2A AR-cyp2j-PKA-PPAR?/A1 AR-sEH-cyp4a-PPAR? pathway in adenosine-induced vascular response. Such results can have a positive impact, as the identified components are expected to provide new targets to curb clinical problems linked with dysfunctional endothelium leading to hypertension.


Bridging Interventional Development Gaps (BrIDGs) makes available, on a competitive basis, certain critical resources needed for the development of new therapeutic agents. Investigators do not receive grant funds through this program. Instead, successful applicants receive access to NIH contractors who conduct preclinical studies at no cost to the investigator. In general, synthesis, formulation, pharmacokinetic, and toxicology services in support of investigator-held Investigational New Drug (IND) applications to the Food and Drug Administration are available. Within the fiscal year, three projects were completed. These projects involved therapies for Diabetic Retinopathy, Parkinsons Disease and Radioisotope Contamination. The retinopathy project was discontinued due to milestone failure. An IND was filed for the Parkinsons therapy and the first clinical trial is expected to begin in Fall 2012. NIAID is responsible for supporting additional studies related to radioisotope contamination product. An IND is not expected until late FY13 or FY14. In FY 2012, five new BrIDGs projects were approved for potential therapies for rheumatoid arthritis, metabolic disorder, epilepsy, multiple sclerosis and pancreatic cancer. Other ongoing projects support the development of potential therapies for Fibrodysplasia Ossificans Progressiva, Anemia of Inflammation, Epilepsy, Pancreatic Cancer, Niemann-Pick C Disease, Aromatic L-Amino Acid Decarboxylase Deficiency, Alzheimers Disease, Hyperinsulinism, Chronic Dry Eye, Osteoarthritis, Hypoparathyroidism, and Atherosclerosis. In spring 2012, BrIDGs solicited new applications for the first time under NCATS. 44 applications were received and awards will be made in FY13. Products are being developed for the treatment of Rheumatoid Arthritis, Metabolic Disorder, Multiple Sclerosis, Fibrodysplasia Ossificans Progressiva, Anemia of Inflammation, Epilepsy, Pancreatic Cancer, Niemann-Pick C Disease, Aromatic L-Amino Acid Decarboxylase Deficiency, Alzheimers Disease, Hyperinsulinism, Chronic Dry Eye, Osteoarthritis, Hypoparathyroidism, and Atherosclerosis.


Thermo acoustic tomography (TAT) is a rapidly emerging ultrasound-mediated hybrid imaging modality that promises to have a major impact on diagnostic imaging. TAT combines high ultrasonic resolution and strong microwave contrast in a single hybrid modality. Human brain imaging represents an important imaging application that can benefit tremendously by the development of TAT methods. Existing high-resolution human brain imaging modalities such as X-ray computed tomography (CT) and magnetic resonance imaging (MRI) are expensive and employ bulky and generally non-portable imaging equipment. Moreover, X-ray CT employs ionizing radiation and is unsafe for patients who need long time monitoring of brain diseases or injuries. Alternatively, ultrasonography is an established portable pediatric brain imaging modality, but its image quality degrades severely when employed after the closure of the fontanels and therefore is not effective for imaging adults. The development of TAT brain imaging methods would circumvent these limitations and result a powerful new brain imaging modality that would fill an important void left by the available techniques. The major technical challenge in TAT brain imaging is to compensate for the distortion introduced into the TAT measurement data by the skull. The broad objective of this proposal is to make TAT brain imaging a practical, useful, and highly effective brain imaging modality by developing robust image reconstruction methods that can account for skull-induced signal distortion and other physical factors. By use of information regarding the skull morphology and composition obtained from previously acquired adjunct image data, or alternatively from the TAT measurement data themselves, we will develop and investigate robust imaging models for TAT that account for the effects of skull-induced wave front aberrations and other physical factors related to the measurement process. We will develop reconstruction algorithms for obtaining accurate images from incomplete data sets that correspond to clinically useful measurement configurations. The developed methods will be systematically evaluated in computer-simulation and experimental studies. The first in vivo study of human TAT brain imaging will also be conducted. The specific aims of the project are: (1) To develop imaging methodologies that incorporate the effects of skull-induced phase aberrations; (2) To develop image reconstruction algorithms for practical brain imaging scanning configurations; (3) To validate the reconstruction methods for TAT brain imaging in computer-simulation studies; and (4) To assess image quality in phantom and in-vivo studies.


Phylogenetic analysis is crucial to a wide range of biological and medical research. A new type of data based on gene order and gene content within whole genomes has attracted increasing interest from researchers in the past several years. [unreadable] [unreadable] Specific Aims: The complexity of genome evolution poses many exciting challenges to developers of mathematical models and algorithms. However, the current tools can only be applied to small genomes (such as organelle genomes) evolving via very simple rearrangements events, hence their breadth of usage is limited. We will address these problems by: (1) mathematical modeling and theoretical analysis of complex evolutionary events such as gene duplication and loss; (2) algorithm design and implementation for phylogenetics and gene order reconstruction (3) performance assessment of these new algorithms through extensive testing on simulated and biological datasets; (4) high-performance implementation of the algorithms using algorithm engineering techniques and a flexible approach to parallelization. [unreadable] [unreadable] Contributions and Broader Impact: The broader impacts of the proposed project are several. (1) The development of new theories and algorithms for the efficient reconstruction of phylogenies and inference of ancestral genomes based on complex genome rearrangements will considerably enlarge the scope of research in the field and give rise to interesting new problems in mathematical and computational biology. (2) Efficient and accurate software for phylogenetic analysis and genome comparison, tested on a large variety of real datasets and on an extensive range of simulations, is expected to reveal new evolutionary patterns and to enable the investigation of novel biological questions. (3) A web server hosted by our group (or by our collaborators) will enable biologists to submit their datasets through a user-friendly web interface and get results back within reasonable amount of time, without the burden of installation and learning parallel computation. (4) The project team combines expertise in mathematic modeling, algorithm design, high-performance computing, comparative genomics, and phylogenetics. Students (both undergraduate and graduate) and postdocs on this project will receive valuable interdisciplinary training experience. (5) Both universities have established programs to boost research in computational biology. This project will enable the PIs to establish close interdisciplinary collaborations among departments from both universities and recruit graduate students (especially minorities) to this fast-growing research field. [unreadable] [unreadable] [unreadable] [unreadable]


Stroke prevention may be achieved through lifestyle changes on a variety of issues such as physical activities and medication adherence. It is therefore difficult to overstate the importance of developing and disseminating behavioral intervention programs as a public health measure to prevent strokes. For the same reason, a behavioral intervention program naturally involves multiple components addressing the various issues; and a successful multi-component program is likely a direct result of administering each interventional component in an optimal sequence, based on the intermediate health outcomes, so as to maximize the eventual health outcome such as blood pressure reduction over 12 months. This type of treatment program tailors the intervention sequence according to an individual's own characteristics, and is sometimes called dynamic treatment regime (DTR). This research aims to develop, validate, and disseminate statistical methods to identify optimal DTR through carefully designed randomized community-based studies. We plan to achieve this research goal in four steps. First, we will develop a data analytical technique, called Q-learning, that will enable us to identify an optimal DTR in an unbiased fashion using data from community- based studies. Q-learning is a cutting-edge technique originating from the computer science literature; this research will adapt this innovative idea to clinical applications where data are observed with high level of variability (noise). Second, we will develop statistical designs that facilitate the discovery of optimal DTR through Q-learning while benefiting the trial participants. This will involve novel synthesis of two clinical trial design concepts: sequential multiple assignment randomized trial (SMART) and adaptive randomization (AR). Third, we will validate the proposed theory and methods by using computer simulation and analyzing data from an actual behavioral intervention study. Fourth, we will disseminate the methods by building software with public access and employ the methods in the planning of the next stage of intervention study; this step is intended to close the lag time between novel methods and its clinical applications. Our long-term public health goal is to enhance the capability of developing optimal behavioral intervention curriculums.


The abuse and misuse of opiods is already a significant healthcare problem that is expected to escalate dramatically in the next decade. Traditionally, the treatment of opioid-dependence has been provided in specialty care settings, such as residential therapeutic communities, methadone maintenance clinics, and dedicated inpatient or outpatient treatment programs, yet the availability of these specialty care facilities has not kept pace with demand. As a result, an astounding 80% of opioid-dependent persons currently remain untreated in the U.S. One way policy makers have tried to address this treatment gap is by sanctioning delivery of Buprenorphine therapies by physicians working in office-based environments. Although shown to be generally safe and effective, the potential of this treatment modality remains unfulfilled, as only a small percentage of physicians have taken advantage of this opportunity. The reasons cited for this include concern about possible patient abuse and/or misuse of the drug, the intensive demands associated with providing treatment to a population of addicted patients, and the additional effort and legal liabilities involved in complying with burdensome DEA requirements for prescribing controlled substances. To address this problem, we propose the development and testing of BupreCare, a comprehensive treatment management solution for delivering Medication Assisted Therapy (MAT) to opioid-dependent persons in an office based setting. BupreCare will be centered around three primary components - a specialized drug dispensing device (DDD) pre-loaded with a generic formulation of buprenorphine/naloxone that controls access to medications via limited-use pass codes, a web/phone (IVR) patient portal patients use to obtain these pass codes and report on treatment status, and a web-based administrative portal providers use to setup treatment parameters and monitor patient progress. Phase I development efforts will focus on the development and testing of the BupreCare Induction Kit (BIK), which patients and providers can use to facilitate a safe and convenient induction experience. To this end, a high fidelity looks-like, works-like physical prototype of the BIK will be iteratively refined during usability testing, and then used in a pilot study in which the induction experience of patients using BIK is compared to those receiving treatment as usual (TAU). Outcome measures of interest will include opioid usage at 1-week, as well as treatment cost, opioid cravings, symptom severity, and treatment satisfaction. Patient and provider acceptance will also be measured as part of an acceptance study. Finally, the remaining research and development tasks, commercialization plan, and field trial design proposed for Phase II will also be completed. PUBLIC HEALTH RELEVANCE: The use and abuse of opioids is a significant healthcare problem costing the U.S. upwards of $300 billion annually. However, the vast majority of opiod dependent persons remain untreated for the disorder, even though effective medication therapies, such as Buprenorphine, could be made available to them through their own personal physician. This research will develop and test a low-cost treatment management solution designed to increase the practicality, safety, and effectiveness of medication therapies for opioid dependence when delivered by physicians working in an office-based environment, thereby making the treatment more readily available to larger segments of the patient population.


Retroviral proteases are enzymes that cleave gag and gag-pol precursor polyproteins into functional proteins of mature viral particles, an event necessary for infectivity of virus particles. Therefore, characterization of these proteases and development of specific inhibitors should lead to control of infectivity. Recent evidence suggests that the HIV protease belongs to the aspartic proteinase family and may be a dimmer. The specific aims of this project are: (1) to isolate HIV protease, (2) to characterize its substrate specificity as an aspartic proteinase, (3) to test its interaction with known aspartic proteinase inhibitors, (4) to develop screening methods for tests of candidate de novo inhibitors, and (5) to characterize its structural properties, in order to investigate additional means of inhibiting the protease. The protease will be isolated from infected cell lysates and from an E. coli recombinant expression system using affinity to pepstatin-Sepharose, or to antibody resin, or by conventional means. The amino- and carboxyl-terminal sequences of the isolated protease will be determined to establish the precise boundaries of the protease gene in the viral genome. An assay based on proteolysis of the gag precursor will be developed. Subsequently, substrate specificity toward gag sequences and to aspartic proteinase substrate and inhibitors will be tested in order to determine how related the catalytic mechanism of the protease may be to other proteases. The active site subsite specificity will be mapped by chemically mutating the known gag protein sequence and determining affinities of these analogs to the protease. Physical properties such as the effect of various conditions on enzymatic activity and stability will be tested. These studies will allow us to gain a better understanding of the substrate specificity and inhibitory spectrum of the HIV protease and should allow us to ultimately design even more efficient inhibitors which could possibly be used in vivo to inactivate viral action.


Almost 200,000 new cases of epilepsy are diagnosed every year in the United States. Many of these are caused by an initial precipitating injury (IPI) (e.g status epilepticus, febrile seizures or traumatic brain injury). There is a need to develop interventions that could prevent the occurrence of epilepsy in these patients. The clinical challenge for testing and applying antiepileptogenic therapy is in identifying the subset of those who eventually became epileptic out of approximately 2 million individuals experiencing an IPI each year. The NINDS, in association with the AES, recently published a report identifying the most important research directions that should be undertaken to ultimately find cures for epilepsy. One of the 3 benchmarks considered as a top priority for the near future is the identification of biomarkers for epileptogenesis. At the present time, no biomarkers predictive of the likelihood of developing epilepsy after an IPI are available and this is an important reason why no clinical trials have identified an intervention during the latent period that clearly prevents the occurrence of epilepsy. The main goal of this proposal is to determine whether a new noninvasive electrographic putative biomarker of epileptogenesis in an animal model of chronic epilepsy can be consistently recorded during the latent period, and whether it can be used to reliably identify which animals later develop recurrent spontaneous seizures. The results of the proposed research could be used to facilitate assessment of antiepileptogenic interventions in patients. The putative biomarker we wish to study is an abnormality of the UP-DOWN State (UDS) EEG pattern. The features of the normal UDS pattern consist of an UP-phase and a Down-phase as a slow oscillation with a frequency of less than 1 Hz. The UP-phase is associated with prominent beta-gamma oscillations. The features of the pathological UDS pattern, which are seen only in epileptic animals include: 1- the occurrence of epileptiform events we have termed UPspikes during the UP-phase, and 2 - a prolongation of the UP-phase duration. We hypothesize that this pathological UDS pattern could be a valuable predictor of future seizure occurrence. We also propose to evaluate mechanisms of pathological change in the UDS pattern by analyzing the activity of principal cells and interneurons identified by juxtacellular labeling. We will focus our efforts on the analysis of the UDS electrographic pattern prior to pilocarpine induced status epilepticus and compare it to activity recorded during the latent period before spontaneous seizures occur. We anticipate that the identification of pathological features in the UDS EEG pattern will make them a valuable early diagnostic biomarker of epileptogenesis and predictor of later seizure occurrence. An understanding of the neuronal mechanisms underlying the pathological UDS EEG patterns will provide novel targets for future approaches to the treatment of epilepsy in its earlier stages, and help pave new ways to prevent epilepsy. PUBLIC HEALTH RELEVANCE: At the present time, no biomarkers predictive of the likelihood of developing epilepsy after a traumatic brain injury are available and this is an important reason why no clinical trials have identified an intervention during the latent period that clearly prevents the occurrence of epilepsy. Early diagnosis of progressive epileptogenesis with early intervention is important for more effective treatment options and disease management strategies. The goal of this proposal is to identify cellular mechanisms responsible for generation of UPspikes, which is a new biomarker of epileptogenesis.


PROJECT SUMMARY/ABSTRACT Individual birth cohort studies have identified risk factors for developing childhood asthma, including environmental exposures in early life such as allergens, pollutants, patterns of infection and colonization with viruses and bacteria, and psychosocial stress. Despite such advances, further progress in understanding the root causes of asthma have been hampered by at least two factors. First, procedures and scientific methods are not standardized across cohorts, making it difficult to compare and validate findings. Second, asthma definitions across cohorts vary considerably. In fact, asthma is a syndrome; there are different subtypes of asthma with distinct clinical features (?asthma phenotypes?) and likely different etiologies (?asthma endotypes?). We hypothesize that host factors (genetics, epigenetics) interact with environmental exposures during the prenatal period and early childhood to cause specific endotypes of childhood asthma. We further propose that identification of endotypes and associated molecular biomarkers in early life can provide a new paradigm for asthma prevention. Unfortunately, single cohorts have limited ability to identify asthma endotypes due to small sample size and unique population characteristics. To overcome shortcomings of individual cohorts, investigators leading 12 asthma birth cohorts across the U.S. now propose the establishment of the Children's Respiratory Research and Environment Workgroup (?CREW?) consortium. This consortium proposes to identify asthma endotypes and overcome shortcomings of individual cohorts by: 1) providing a large (nearly 9000 births and long-term follow-up of 6000-7000 children and young adults) and diverse national data set, 2) harmonizing data related to asthma clinical indicators and early life environmental exposures, 3) developing standardized measures for prospective data collection across CREW cohorts and other ECHO studies, and 4) conducting targeted enrollment of additional subjects into existing cohorts. This approach will enable collection of samples that are optimized for a systems approach to understanding how environmental and host factors in early life promote the development of specific asthma endotypes. Collectively, the results of this comprehensive research to identify the root causes of asthma vs. resilience and health will go far beyond what can be accomplished by individual cohorts, and thus provide a foundation for future efforts aimed at personalized prevention of chronic childhood asthma.


Protocadherins are new members of the cadherin superfamily. Despite the great number of protocadherin genes identified in vertebrates, little is known about their biochemical properties, functions or working mechanisms. Xenopus paraxial protocadcadherin (PAPC) was recently shown to play a role in morphogenetic cell movements during early embryonic development. However, the adhesive properties of PAPC still remain ambiguous and poorly understood, and whether its morphogenetic role is realized through its adhesive function or by other mechanisms is unclear. The long-term goals of this proposal are to elucidate the adhesive functions and mechanisms of PAPC and to understand the mechanisms of its morphogenetic activity by identifying the cytoplasmic PAPC-binding proteins. First, the adhesive properties and mechanisms, including adhesive strength, specificity, lateral dimerization and clustering, will be examined and analyzed with the cell aggregation, bead aggregation and cell attachment assays; second, the cellular localization of PAPC will be monitored to assess its role in polarized cell movements; third, the function of PAPC cytoplasmic domain, especially a highly conserved region, on the adhesive and morphogenetic activity of PAPC will be analyzed using deletion mutants; and finally, the proteins that bind PAPC cytoplasmic tail will be identified as candidates for regulation of PAPC adhesive function or PAPC-mediated signaling by means of affinity chromatography and/or the yeast two-hybrid method. The proposed research will shed light on the role of protocadherins in embryonic development and should provide important suggestions to the treatment of birth defects and other developmental diseases.


In this study we will take advantage of the ease of manipulation of yeast for the study of the regulation of mitochondrial biogenesis as influenced by the determinants of lipid synthesis, especially phospholipid synthesis. (A) The purification of CTP phosphatidic acid cytidyl transferase is to be pursued with the hope of preparing specific antibodies to this enzyme, thought to be located mainly in the inner mitochondrial membrane. Such antibodies are to be used to study the synthesis of this enzyme and its regulation. (B) Attempts are being made to isolate lipid mutants, including temperature sensitive mutants for the CTP phosphatidic acid cytidyl transferase to further elucidate the role of lipids in the control of mitochondrial biogenesis. (C) We will continue to probe the mechanism that determines the differential synthesis and assembly of cytochrome oxidase in unsaturated fatty acid auxotrophs of yeast grown in cis or trans fatty acids.