Edward Hawrot - US grants

The nicotinic acetylcholine receptor (AChR) of mammalian muscle is a multimeric integral membrane glycoprotein functioning as a neurotransmitter receptor and a transmembrane ion channel. It is clear that the AChR plays a key role in neuromuscular transmission and it has been extensively studied at the biochemical and electrophysiological level. Additional studies are needed, however, to elucidate the molecular mechanisms involved in the assembly, insertion and regulation of important membrane proteins such as the AChR. Monoclonal antibodies provide the needed specificity and affinity to be extremely useful tools in such studies. Information concerning the regulation of the levels of synthesis of the AChR could be of considerable importance for myasthenia gravis where a deficit of functional AChR is clearly the basis for the clinical manifestations of the disease. The proposed research involves the use of monoclonal antibodies against mammalian AChR to study the in vivo biosynthesis and regulation of the receptor complex and its individual subunits. In this work I plan to use established mouse muscle cell lines which produce AChR. Furthermore, the techniques of somatic cell genetics will be applied in order to obtain mutant variants with altered structure or with defective biosynthesis of the AChR. Such variants would expand our knowledge of the biogenesis of cell surface receptors and other functional membrane proteins and would facilitate and complement studies of the genetic regulation of these membrane proteins.

Nerve Growth Factor (NGF) is required for the survival and development of sensory and sympathetic neurons both in vivo and in culture. The molecular mechanisms of NGF action are presently unknown. It is the objective of this research proposal to develop new tools for the study of high-affinity NGF receptors which would help us understand the underlying mechanisms of NGF action. Discrete, quantifiable, electron-microscopic (EM) markers for occupied NGF receptors are not presently available. One major goal of this proposal is to synthesize biologically active derivatives of NGF that would permit quantiative EM investigations of the surface distribution and mobility of NGF receptors. I intend to synthesize ferritin conjugates of NGF using techniques recently developed for the study of insulin receptors. In addition, I will prepare biotinylated derivatives of NGF, which, with the appropriate secondary visualizing agents, would permit rapid and direct EM visualization of NGF receptors. Studies with biotinyl-NGF conjugates would also provide important new information on the structure-function relationships in NGF action. Furthermore, biotinyl-NGF could be used to bring about rapid, affinity purification of solubilized NGF receptors. Biochemical and immunofluorescent procedures will be used to synthesize and screen various NGF conjugates for biological activity and ability to bind to specific cell-surface receptors on cultured rat sympathetic neurons and on pheochromocytoma-derived, rat PC12 cells. In order to obtain information on human NGF receptors, I plan to study the characteristics of the NGF receptor on human melanoma cells. These studies are directly relevant to an understanding of human familial dysautonomia, where a defect either in NGF and NGF receptor may be involved. Finally, I intend to prepare monoclonal antibodies to NGF receptor in order to obtain additional, highly specific molecular probes of receptor structure and function.

The nicotinic acetylcholine receptor (AChR) is a multimeric, integral membrane glycoprotein that functions as a ligand gated channel at the neuromuscular junction. The biosynthesis, assembly, membrane insertion and localization of the AChR complex provides an attractive system for the study of the mechanisms underlying these important cellular functions. Three complementary approaches will be used to provide additional fundamental information concerning AChR biogenesis, regulation, structure and function. 1) We plan to extend our monoclonal antibody (mAb) studies to focus on the extracellular domains of mouse muscle AChR including the ligand binding site and to identify the characteristics of cross-reacting antigens observed in a variety of electrically excitable tissues, including Drosophila central nervous system (CNS), guinea pig ileum smooth muscle, rat sympathetic ganglia, and the rat pheochromocytoma PC12 cell line. 2) Somatic cell genetic techniques will be applied to the questions of AChR regulation and structure-function relationship. Fluorescence-activated cell sorting in combination with specific immunotoxins and replica-plating will be used to obtain muscle cell variants with altered regulatory, biosynthetic, or structural features involving the AChR. 3) The protein-blotting approach will be used to provide additional structural information on the AChR as well as on possible evolutionarily related structures such as the Alpha-bungarotoxin (BuTX) binding site in the CNS of lower vertebrates. We plan also to utilize this approach to identify the amino acid sequences making up the epitopes responsible for various cross-reactions and to thus map the immunologically identifiable sites with respect to other structural features of the AChR.

Certain snake venoms contain toxins, called alpha-neurotoxins, which cause muscle paralysis by blocking the normal action of the neurotransmitter, acetylcholine, at its site of action on the muscle surface. One of these toxins, called alpha-bungarotoxin, has been particularly useful to biochemists interested in studying the acetylcholine receptor on the muscle membrane. In order to understand the action of these toxins at the muscle receptor and to determine what it is about the alpha-bungarotoxin molecule that targets it for the muscle receptor, synthetic genes can be produced for this protein. With the gene in hand one can develop a procedure for preparing large amounts of genetically- modified toxin. By systematically altering different parts of the toxin protein, one can identify the sites on the toxin which "recognizes" the receptor. The determination of the structural changes in the protein will depend on sophisticated new techniques taking advantage of instrumentation using high magnetic fields and radiofrequency pulses. This type of recognition is also how drugs recognize their target sites. This work will help us understand the chemically-important structures which allow chemicals and biological molecules to interact.

The nicotinic acetylcholine receptor (AChR) is a multimeric, integral membrane glycoprotein that functions as a ligand gated channel at the neuromuscular junction. The biosynthesis, assembly, membrane insertion and localization of the AChR complex provides an attractive system for the study of the mechanisms underlying these important cellular functions. Three complementary approaches will be used to provide additional fundamental information concerning AChR biogenesis, regulation, structure and function. 1) We plan to extend our monoclonal antibody (mAb) studies to focus on the extracellular domains of mouse muscle AChR including the ligand binding site and to identify the characteristics of cross-reacting antigens observed in a variety of electrically excitable tissues, including Drosophila central nervous system (CNS), guinea pig ileum smooth muscle, rat sympathetic ganglia, and the rat pheochromocytoma PC12 cell line. 2) Somatic cell genetic techniques will be applied to the questions of AChR regulation and structure-function relationship. Fluorescence-activated cell sorting in combination with specific immunotoxins and replica-plating will be used to obtain muscle cell variants with altered regulatory, biosynthetic, or structural features involving the AChR. 3) The protein-blotting approach will be used to provide additional structural information on the AChR as well as on possible evolutionarily related structures such as the Alpha-bungarotoxin (BuTX) binding site in the CNS of lower vertebrates. We plan also to utilize this approach to identify the amino acid sequences making up the epitopes responsible for various cross-reactions and to thus map the immunologically identifiable sites with respect to other structural features of the AChR.

The long-term goal of this project is the elucidation of the high-resolution structure of the extracellular domain of the skeletal muscle-type nicotinic acetylcholine receptor (nAChR). Near term, a major focus is placed on elucidating the structure of the ligand binding site with particular emphasis on the binding of the classic, nicotinic, high affinity ligand, a- bungarotoxin (Bgtx). A clear delineation of the contact zone involved in Bgtx recognition would contribute not only towards the elucidation of the molecular basis for receptor-toxin interaction but would also further our understanding of the basic principles that underlie molecular recognition in other important protein-protein interactions. In the first aim we will use multi-dimensional NMR techniques to determine the solution structures of 15N-enriched receptor- derived peptides (an a1 18mer and an a7 19mer) each bound in a stoichiometric complex with a-bungarotoxin. These two peptides, which correspond to the sequences on their respective native receptors forming the major binding determinants for Bgtx, bind Bgtx with nM affinity. Aim 2 will use a site-directed Cysteine substitution approach to map the solvent accessible surface of the nAChR and to identify positions in proximity to the Bgtx binding site. In aim 3, we will study chimeric neuronal/muscle a-subunits expressed in Xenopus oocytes to further delineate the residues contributing to Bgtx binding. New chimeric constructs will be prepared using a4 and a2 subunits to determine whether sensitivity to Bgtx can be conferred with minimal sequence replacements. Recent technological advances in the expression and analysis of recombinant proteins and in NMR-based structure determinations make this an opportune time to pursue these goals. The proposed studies, directed towards the ultimate understanding of how ligand-gated receptors operate at the molecular and mechanistic level, will also be of considerable value in the design of better and more specific neuromuscular blocking agents. Information gained from the proposed studies will also be relevant to the entire family of neuronal nAChRs whose structure and function are less well characterized than those of the muscle receptor. Neuronal nAChRs are involved in cognitive function and also appear to play a role in nicotine dependency and in neurological disorders such as schizophrenia and Alzheimer's disease.

A group of faculty in the Departments of Ecology and Evolutionary Biology (EEB), Molecular and Cellular Biology and Biochemistry (MCB) and Molecular Pharmacology and Biotechnology (MPB) at Brown Uliversity seeks funds for the purchase of an Applied Biosystems, Inc. ABI 377 Automated DNA Sequencer and dedicated thermal cycler for use with the ABI 377. This equipment will be operated as a multi-user facility with four major users (Hawrot, Hendrickson, Rand, Zaret) and eight minor users (Biggins, Cane, Gerbi, Landy, Marshal, Mowry, Wessel, Wharton). The research conducted with this Sequencer ranges from the analysis of mutants of known genes (Biggins, Cane, Hawrot, Landy, Mowry, Wharton, Zaret) to the characterization of new genomic sequences (Cane, Gerbi, Hendrickson, Marshal, Wessel Wharton) to studies of genetic variation and evolution of well-characterized loci (Rand). This piece of equipment will provide a significant increase in research productivity and competitiveness as each of the users currently does sequencing by "hand." At present, there is no automated DNA sequencer at Brown University, nor in the entire state of Rhode Island. For most of the major users, acquiring the DNA sequence of cloned or amplified genes is a major burden to research progress. A substantial proportion of sequencing effort falls on the shoulders of graduate students. Hence, an automated DNA sequencer will have a significant impact on graduate education by allowing students to focus on more intellectually challenging research and direct more effort towards creative experimental design. Brown University will provide 48.8% of the funds for the purchase of the ABI 377, and will pay the salary of the staff member (Charles Setterlund) who will be the dedicated person responsible for operating this equipment.

DESCRIPTION: (adapted from Applicant's Abstract) These studies are intended to construct novel "designer" neurotoxins which can be used for the biochemical and functional characterization of neuronal nicotinic acetylcholine receptors (nAChRs). To elucidate the structure-function relationships of the curaremimetic alpha-neurotoxins, a series of site- directed mutagenesis studies will be performed using a synthetic gene for alpha-bungarotoxin (BGTX) that has been designed to be expressed in E. coli. A strategy of "alanine-scanning" site-directed mutagenesis will be pursued to reveal those residues in BGTX with clear functional importance for its interaction with muscle-type AChR. In order to fully interpret the effects of these mutations, methods to increase levels of expression and/or refolding will be developed to generate quantities of recombinant BGTX sufficient for efficient structural analysis by NMR and/or X-ray crystallography. New information on the molecular basis for neurotoxin selectivity between muscle and neuronal subtypes of nAChRs will be obtained by constructing "chimeric" toxins in which selected regions from alpha-bungarotoxin, a toxin which blocks certain subtypes of neuronal nAChRs, will be inserted into a BGTX background. In addition, point mutations will be introduced into BGTX with the view of developing toxins binding with high affinity to neuronal AChRs. Those studies, together with ongoing NMR-based structural analyses of BGTX/receptor peptide complexes, and the observed highly conserved homolgy among muscle and neuronal alpha-subunits, offer the unique opportunity to re-engineer BGTX and to produce novel toxins selective for one or more of the neuronal AChRs.

nuclear magnetic resonance spectroscopy; biomedical equipment purchase;

A group of four investigators in the Brown University Department of Molecular Pharmacology & Biotechnology (within the Division of Biology and Medicine), the Brown University Department of Chemistry, and the Department of Pharmacognosy and Environmental Health Sciences at the University of Rhode Island, is seeking funds for the purchase of a highfield (500 Mhz) NMR spectrometer for use in frontier biochemical and bio-organic investigations.

Ligand-gated and voltage-regulated ion channels on the surface of excitable cells mediate synaptic transmission and neurosecretion. Not surprisingly, mutations of ion channel genes contribute to a wide variety of pathological disorders that can affect neural differentiation and cause neurodegeneration. This proposal is multi-disciplinary ranging from molecular structure, regulation of ion channel function using biophysical techniques, cell biology, to studying the effects of ion channel mutations on animal behavior. The channels being studied include the multimeric gated nicotinic acetylcholine and glutamate receptors and the voltage-gated calcium channels. These ion channels provide important sites for pharmacological intervention in disease status, such as, addiction, epilepsy, and neuronal death due to trauma or stroke, as well as neurodegenerative disease. Approaches being exploited include, expression and analysis of protein domains by NMR, reconstitution of normal or genetically modified ion channels in cell lines to determine their binding and interaction with neurotoxins, patch-clamp analysis to examine the biophysical properties of channel splice variants, modulation ion channels by scaffolding proteins and protein phosphorylation, slice-recording and analysis of animal behavior. Within this context we will use transgenic methods for targeted ion channel knock-out and knock-in experiments to further reveal the physiological consequences of specific channel mutations and to elucidate the physiological roles of specific channel subunits combinations.

DESCRIPTION: (from the applicant's abstract) The long-term goal of this project is the elucidation of the molecular mechanisms by which the snake-venom derived, polypeptide alpha -neurotoxins block nicotinic acetylcholine receptor (nAChR) function. We will focus on the prototypical long-chain alpha -neurotoxin, alpha -bungarotoxin (Bgtx), the "gold standard" of alpha -neurotoxins. Bgtx has been used extensively in the study of muscle-type nAChRs and a subset of neuronal nAChRs sensitive to Bgtx inhibition (e.g., alpha -7containing nAChRs). The questions that we intend to address are: 1.) Which specific residues and general structural and dynamic features in Bgtx are important for recognition and block of Bgtx-sensitive nAChRs? 2.) Can the receptor specificity of Bgtx be altered through mutagenesis to produce "gain of function" variants capable of interacting in a selective manner with neuronal nAChRs that are normally insensitive to Bgtx? and 3.) How do Bgtx mutations that disrupt or significantly alter toxin activity affect the structure and conformational flexibility of Bgtx? We will pursue a structure-function analysis of Bgtx, utilizing site-directed mutagenesis of recombinant Bgtx produced in Pichia pastoris and double mutant cycle analysis to identify sites responsible for the high-affinity neuromuscular blockade produced by this alpha -neurotoxin. We also intend to identify and characterize "gain of function" mutations of Bgtx which enable the recognition and functional inhibition of neuronal nAChRs that are otherwise insensitive to blockade by native Bgtx. This approach should lead to a new battery of toxins that differentially bind to the various neuronal nAChR subunit combinations. We plan to determine the structure of the recombinant Bgtx by multidimensional NMR, to compare this structure to the NMR solution structure of native Bgtx, and to analyze the backbone dynamics of recombinant Bgtx metabolically labeled with15N. Labeled Bgtx would also allow a characterization of atomic motions within its various functionally important regions. Local confirmational flexibility in Bgtx may be an important factor in the mechanism of Bgtx/receptor recognition and binding and could very well contribute both to the high affinity of Bgtx and to its ability to bind to a large number of nAChRs of varying primary structure. The proposed studies promise to shed light on the fundamental mechanisms by which protein alpha -neurotoxins interact tightly and specifically with their target sites on the cell surface. This information will be useful in providing valuable models for drug design efforts and in developing new pharmacological tools for the study of neuronal nAChRs.

Rhode Island seeks to attain national prominence by building a competitive research platform that is both statewide and sustainable and whose programs will empower and stimulate researchers to take advantage of advanced technologies through the establishment of a center for research excellence in marine life sciences and establishment of core research facilities in genomics and proteomics. The University of Rhode Island (URI) and eight other institutions in the state propose strategic investment in life sciences to support the state's biotechnology and biomanufacturing economy.

Federal, state and local, public, private, and non-profit institutions will support the infrastructure-building program in Rhode Island. The integrative activities in research, education, innovation and communication will serve to:
develop the human capital necessary to support and sustain the growth of competitive research capacity in the life sciences;
broaden the participation of women and underrepresented ethnic and racial minorities in the STEM workforce;
provide researchers with targeted entrepreneurial guidance to encourage research innovation, stimulate technology transfer, and promote commercialization and new business development; and,
develop sustainable communication mechanisms to build and enhance a strong statewide network of the state's and region's scientists, institutions of higher education, and private and public sectors.

The Rhode Island Center for Genomics, the Rhode Island Center for Proteomics, the Rhode Island Center for Marine Life Sciences and the Rhode Island EPSCoR Academy will be enabled through this partnership. Funding supports undergraduate and graduate students (including entrepreneurial fellows), instrumentation, K-12 STEM teacher preparation, and Biosciences/Biotechnology curriculum at community colleges. Expert evaluation of the outputs and impacts of this project as well as dissemination and communication of results is also supported.

Funding is provided through the National Science Foundation's Experimental Program To Stimulate Competitive Research (EPSCoR).
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DESCRIPTION (provided by applicant): The long-term goals of this proposal are to understand the molecular and cellular basis for nicotine's addictive properties and to elucidate the functional role of neuronal nicotinic acetylcholine receptors (nAChRs) in the many centrally mediated behavioral and physiological effects of nicotine. This proposal focuses on alpha3-subunit-containing nAChRs which are found at high density in the medial habenula and which also have been implicated in the ventral tegmental area (VTA), a well recognized component of the reward pathway. The medial habenula is an evolutionarily conserved brain region that has been shown to be very sensitive to the neurotoxic effects of nicotine. Furthermore, it has been suggested that some of the cognitive deficits seen in schizophrenia may be linked to underlying pathology in the medial habenula. The main tool to be utilized in this study is a novel knock-in mouse in which the nicotinic alpha3-subunit has been replaced with one containing five amino acid substitutions that impart sensitivity to pharmacological blockade by alpha-bungarotoxin (Bgtx). This knock-in mouse enables the use of the classic pharmacological antagonist, Bgtx, to probe the functional and behavioral role of alpha3-containing nAChRs in the medial habenula and VTA. First, it will be important to confirm the regional expression of Bgtx-sensitive, alpha3- containing neuronal nAChRs in frozen sections of brains isolated from mice heterozygous for the targeted alpha3/alpha1[5] mutation (+/tm1.1). Autoradiography of radioactive-Bgtx binding sites will be used for this purpose. Following stereotaxic cranial cannulation, Bgtx will be microinjected into the medial habenula and VTA of (+/tm1.1) mice to determine the effect of pharmacological blockade of alphas-containing nAChRs on three nicotine-associated behaviors. These include: 1) the hypolocomotor effect of acute, low-dose systemic nicotine;2) entrained oral preference for nicotine;and 3) nicotine-induced seizures. Relevance: Nicotine is an extremely addictive drug responsible for up to 20% of all preventable mortality in the western world. Nicotine also significantly enhances cognitive performance, and some inherited forms of epilepsy involve nicotinic receptors. In addition, a loss of cholinergic neurons is implicated in Alzheimer's disease, a disorder currently lacking effective treatment. Understanding the molecular interactions of nicotine with its receptors in the central nervous system therefore has significant potential to benefit human health.

[unreadable] DESCRIPTION (provided by applicant): This proposal describes an innovative research approach aimed at ultimately revealing the molecular mechanisms that produce and maintain nicotine dependence. To accomplish this, we propose to develop new and generalizable investigative tools by combining cutting-edge features in emerging transgenic and proteomic technologies, specifically by using novel pharmatope-tagged alpha-3-subunit-containing nicotinic receptors in an existing novel knock-in mouse to harness the powerful and sophisticated capabilities of modern mass spectrometry . This project is a new venture in our lab that seeks to apply the fundamental insights gained from our previous structural and functional work to outstanding problems in the drug addiction area. A detailed understanding of nicotine-induced alterations in intracellular protein-protein interactions (i.e., the interactome) involving the putative regulatory cytoplasmic domain of neuronal nicotinic acetylcholine receptors (nAChRs) promises to provide uniquely valuable insights into how behavioral manifestations of nicotine reinforcement, tolerance and sensitization develop. The core concept is that novel pharmatope-tagged knock-in mice can provide general tools to study regulatory proteins interacting with the unusually large cytoplasmic loop that is a feature common to all nAChR subtypes. Our alpha3-knock-in introduces an alpha-bungarotoxin (Bgtx) binding site into neuronal nAChRs that are normally Bgtx- insensitive. The ability to bind Bgtx will be used to biochemically isolate the receptor under mild conditions, together with associated regulatory signaling complexes. These associated proteins will be identified using tandem mass spectrometry and their phosphorylation sites functionally mapped. To critically test this approach, we will focus on the Bgtx-sensitive alphas subunit expressed in primary cultures of superior cervical ganglionic neurons whose post-synaptic alpha3-containing nAChRs are essential for synaptic transmission. This system will allow us to probe the effects of nicotine exposure on the nAChR interactome profile, and will pave the way for studies of alpha3 signaling complexes in the CNS; in particular, in the ventral tegmental area and medial habenula, regions strongly implicated in nicotine addiction. Public Health Relatedness: Knowledge of the cascade of cellular processes and protein interactions regulated by nicotine will contribute to the development of new health interventions. [unreadable] [unreadable]

EPS-0918284, University of Vermont & State Agricultural College, J. L. Van Houten, linked to EPS-0918033 (University of New Hampshire), EPS-0918078 (University of Delaware), EPS-0918018 (University of Maine), EPS-0918061 (University of Rhode Island)
Collaborative Research: North East Cyberinfrastructure Consortium

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

The North East Cyberinfrastructure Consortium (NECC) unites Maine (ME), New Hampshire (NH), Vermont (VT), Rhode Island (RI), and Delaware (DE) to support cyber-enabled research that requires analyses of large datasets. The project is organized around sharing resources, expertise and facilities in order to make cyber-enabled collaborative research possible in a sparsely populated region and among non-contiguous states.

Intellectual Merit. The consortium has three primary needs to support regional, cyber-enabled research: 1) long-term leases on fiber in specific reaches across the northeast to provide high-speed connectivity with dense-wave division capability; 2) redundant, distributed Data Centers for regional cyber-enabled collaborations; and 3) cyber-knowledgeable personnel to allow researchers to access regional compute, analysis and visualization resources. Much of the physical infrastructure required for the NECC network exists, but there are four key reaches of fiber needed in ME, NH, RI and VT. In ME, two stretches are required to provide a redundant route for national and international connectivity through CANARIE (Canada's advanced network organization) and along the I-95 corridor. A fiber route along the I-89 corridor provides connectivity to Boston for Vermont and New Hampshire to Boston. The researchers have been working with the Northeast Research and Education Network (NEREN) to manage the fiber network once it is in place.

Broader Impacts. The possibility of a fiber network that would provide adequate bandwidth for videoconferencing has led to the NECC regional organization around outreach programs for STEM workforce development and diversity. It is planned to create a new Watershed Project through partnerships among multiple state-based programs for high school and undergraduate students. Students in this project from all the NECC states, NY and Puerto Rico, who otherwise would not even meet, will work together in collaborative watershed research. Following training, teams of high school students and teachers or undergraduates join with state programs to work on watershed science during the summer or through the summer and the academic year. The individual NECC state programs are effective in improving participation in STEM majors and diversity, but with the new fiber network and the ability to communicate over the new cybernetwork, a larger, region-wide effective program, with emphasis on cyber-based communication and research tools, is envisioned. The researchers develop a multi-faceted communication plan that will spread the word about the importance of the cyber-enabled research to the public through innovative television shows, podcasts and educational materials. An Ambassador Program will partner with citizen science groups to inform the public about the importance of a fiber network to education and science and about the potential impact of the cyber-enabled metagenomics study to the economies of the states. The fiber network will have an enormous economic impact on the region. The pilot project on metagenomics of the bacterial communities in blooms in lakes in VT, NH, ME and RI will contribute to the understanding of the origin of these blooms and their toxins that shut down access to recreational and drinking water sources. Lakes in the northeast are extremely important to economies, with estimates of $1.5B in lake-related revenues to NY, VT and Quebec each year from Lake Champlain; $2B annually from lake recreational revenues to Maine; 14,000 jobs and bring in $1.8B in revenues from boating, fishing, swimming, drinking water and property taxes to New Hampshire.

Proposal Number: EPS-1004057

Proposal Title: Infrastructure to Advance Life Sciences in the Ocean State

Institution: University of Rhode Island

The Rhode Island Research Infrastructure Improvement (RII) program focuses on cutting edge research to understand how marine biological organisms are affected by variations in their surroundings due to climate change effects. The goals of the project are to advance Rhode Island's competitiveness in marine life science, foster collaboration among researchers and educators in the state, and build diverse, well-trained workforce teams involving nine institutions of higher education across the state. This project is regionally relevant, nationally significant, takes advantage of unique Rhode Island resources including the Narragansett Bay, and aligns well with the state Science and Technology plan.

Intellectual Merit
A number of studies predict that with ocean warming and acidification, many single and multiple cell organisms will be under stress whereas many pathogenic microbes and parasites will thrive. The resilience of marine organisms to climate change will depend crucially on acclimation and adaptation. The Rhode Island RII program is aimed at understanding, predicting, and mitigating the impacts of environmental stresses on marine organisms and ecosystems. These studies are fundamentally important to the global biosphere (and locally to Narragansett Bay) as nutrient cycles and sustained biological production in the ocean, ultimately determine the availability of seafood. The investigations focus on key individual organisms (e.g., fish, plankton, marine pathogens), food webs, and the spread of infection and disease among marine host populations. The studies will explore how these species are affected by changes in chemical and physical parameters in the ocean such as temperature, acidity, and nutrient availability. Through these activities, the researchers at Rhode Island will be well positioned to make significant intellectual contribution to the assessment of climate change effects on marine organisms and ecosystems.

Broader Impacts
The infrastructure improvements for life sciences research and education and the partnership among public and private institutions of higher education including two- and four-year colleges will assist in building Rhode Island's research capacity. The Rhode Island EPSCoR Academy will coordinate efforts to foster collaboration among diverse institutions, broaden participation to increase diversity, and engage students at all levels in research and educational training. Fellowships for students and programs aimed at increasing the participation of under-represented minority groups are included in this RII project. A unique aspect of this project is the involvement of Rhode Island School of Design (RISD) for interdisciplinary collaboration among scientists and RISD artists and designers. This interaction is expected to enable the development of improved tools and strategies for the visualization and communication of complex scientific information to K-12 students and non-traditional audiences. In addition, collaborations with the University of Rhode Island's Metcalf Institute will enhance science communication by strengthening interaction among scientists, students, and journalists.

Proposal Number: EPS -1005789

Proposal Title: High-Capacity Cyber-Connectivity to the Jewelry District Campuses
in Providence, RI

Institution: Brown University

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

The Rhode Island (RI) Research Infrastructure Improvement Inter-Campus and Intra-Campus Cyber Connectivity project will provide high speed connection and thus facilitate effective collaboration among Rhode Island's institutions of higher education. Brown University's extended campus locations will gain high speed access to the computing facilities in the main campus. Other public universities and 2-and 4-year colleges will be connected to Brown University's research and computing facilities through the Ocean State Higher Education Economic Development and Administrative Network (OSHEAN). Upgrades to OSHEAN will strengthen collaborations of the North East Cyberinfrastructure Consortium comprising of institutions from Vermont, New Hampshire, Maine, Rhode Island, and Delaware and also improve connectivity to New York and Massachusetts. The RI Center for Innovation and Entrepreneurship, which facilitates interaction among researchers and entrepreneurs, will also receive high bandwidth connectivity through the upgrades in this project.

Intellectual Merit
The RI Experimental Program to Stimulate Competitive Research (RI-EPSCoR) focuses on studies of climate change effects on marine organisms, ecosystems, and food webs. These studies are fundamentally important to the global biosphere and locally to Rhode Island's Narragansett Bay. The high bandwidth connection provided by this project will facilitate efficient transmission and sharing of data among researchers across RI to make full use of NextGen DNA sequencing technologies and bioinformatics tools. Integrative metagenomic approaches to monitoring marine ecosystem, understanding the influence of rising ocean temperature and acidity on marine food webs, sustained biological production in the ocean, and identification of disease causing marine organisms will be pursued.

Broader Impacts
The proposed cyberinfrastructure enhancements will increase Rhode Island's research capacity for collaborative work in marine life sciences. The proposed broadband capacity will revitalize the Jewelry District in Providence, RI to a biotechnology based Knowledge District and promote economic growth in the state. The RI-EPSCoR Academy's workforce development and diversity initiatives will be strengthened by the improved cyber connectivity among institutions of higher education in RI. Enhanced connectivity will enable wide area video-conferencing and marine life science research related telecasts to K-12 schools. Workshops to broadly disseminate bioinformatics programs are also included.

DESCRIPTION (provided by applicant): This is an application to expand an existing predoctoral training program in trans-disciplinary molecular pharmacology and physiology at Brown University. The training program is designed to produce graduates capable of establishing independent research in the interdisciplinary fields contributing to modern pharmacological sciences. The training program will be operated within the Graduate School approved Molecular Pharmacology and Physiology Graduate Program, and includes highly experienced training faculty drawn from several departments at Brown University including the Warren Alpert Medical School of Brown University. Funds are requested for 5 years, for 2 predoctoral trainees in year 01 and 4 trainees in years 02- 05. The research productivity of the training faculty is strong, and in its diversity of systems and sophisticated methodologies reflects the type of training that is needed in today's multidisciplinary environment to make fundamentally important contributions to the pharmacological sciences. The three tracks of specialization within the program include Molecular Pharmacology, Structural Pharmacology, and Translational Pharmacology. The training faculty have research strengths in the areas of molecular and cellular signal transduction, structural biology of proteins important in cell signaling, synaptic function and regulation, ion channel biophysics, ion channel and receptor function in human disease and development including cardiovascular disease, and transgenic animal models. The program has a strong identity, and achieves integration and momentum through trans-disciplinary Core courses, a seminar series, interactive laboratory rotations, and highly individualized attention to the development of presentation and writing skills. The program thus offers broad yet Well-integrated training in areas playing an important role in the modern pharmacological sciences. Trainees are recruited from a strong, diverse pool, and there are several proven mechanisms in place to attract and retain students from under-represented groups. The graduates from this training program will have pursued cutting-edge, fundamental pharmacological research, and they will have developed a skill set important for the development of new drugs and therapeutic strategies.

DESCRIPTION (provided by applicant): A consortium of productive biomedical investigators at Brown University and the University of Rhode Island is requesting support from the NCRR Shared Instrumentation Program for purchase of a mass spectrometer (MS) upgrade which would take an existing Thermo LTQ MS and transform it into a Thermo LTQ Orbitrap XL ETD hybrid MS. The resulting LTQ Orbitrap will be a high performance LC-MS and MSn system, combining rapid LTQ ion trap data acquisition with high mass accuracy Orbitrap mass analysis. Significantly, the Orbitrap XL ETD includes two fragmentation regimes, higher-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD), that are complementary to the LTQ's collision induced dissociation (CID) option. These additional, alternative fragmentation capabilities are essential for the success of the diverse projects (phosphoproteomics;posttranslational modification analyses;"middle-down" proteomics) being actively pursued by the participating investigators. The requested new upgrade to the LTQ Orbitrap XL ETD MS is necessary for the collection and analysis of the high quality data required by the consortium's demanding and in some cases highly technically challenging research projects. The distinct benefits of the Orbitrap XL ETD mass spectrometer are its high sensitivity, resolution and mass accuracy, coupled to a fast scan rate. Most of the proposed major users participating in this proposal are pursuing the analysis of highly complex mixtures of proteins derived from cells or tissues. To adequately inventory the protein constituents within a complex proteomic sample, high quality data most be acquired on a time scale consistent with nano-liquid-chromatographic separation and elution. The Orbitrap is ideally positioned to provide the most robust, highest quality analysis of the complex samples described in this application. Once installed, this mass spectrometer will be utilized continuously (i.e., "24/7") through implementation of an in-house developed automated data pipeline which includes automated multidimensional capillary separations of peptides, nanospray LC/ESI-MS, automated data acquisition, and post-processing. Our current in-house technology platform provides for fully automated Sequest searching, quantitation of peptide abundance, statistical validation of database search results, and cross-referencing of observed peptides against an array of publicly available proteomic databases. Protein interactions and dynamics play a critical role in cell signaling, and form an important part of current efforts to explore and exploit the proteome. Systems network biology will be a vital component of our focus on proteins and their interactions in disease and development with the eventual goal of identification of new targets for drug therapy and the development of new approaches to therapeutics. As detailed in this proposal, such studies are presently an integral part of a number of existing and long-running NIH-sponsored research projects and with the recent investments in new faculty lines at Brown University in the Academic Enrichment Plan, the number and variety of proteomic studies will continue to grow over the next several years. It is very clear that the requested instrumentation will have an immediate and long-lasting impact in further propelling the high-quality biomedical research being pursued at Brown University and at the other institutions of higher learning in the state of Rhode Island. PUBLIC HEALTH RELEVANCE: The requested instrumentation upgrade will allow our participating investigators to elucidate the interacting proteins that are involved in the regulation of cell function and physiological response in a number of model systems ranging from asthma to cancer to neuronal signaling. These studies form an important part of current efforts to investigate and capitalize on our expanding knowledge of the proteome. This focus on proteins and their interactions in disease and development is being pursued with the eventual goal of identification of new targets for drug therapy and the development of new approaches to therapeutics. The basic research that will be facilitated by the requested instrumentation will greatly expand our knowledge of the interplay and cross-talk between various signaling pathways both in normal cellular and tissue function and in a variety of human disease states.

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the role of the ?7- nicotinic acetylcholine receptor (nAChR) and its associated proteome (i.e., interactome) in the pathophysiology of Alzheimer's Disease (AD) as a vehicle towards developing more targeted and efficacious treatments. We hypothesize that the regulatory and signaling proteins closely associated in vivo with hippocampal ?7-nAChRs play an important role in normal neuronal functions including those critical to memory, and that the signaling machinery of the ?7 macromolecular complex is adversely affected as AD progresses resulting in changes in the protein interactome of ?7-nAChRs. Using high-throughput proteomic technologies and post mortem tissue, we will focus on the cytoplasmic proteins associated with the ?7-nAChR, which constitutes the second most abundant nicotinic receptor system in the human brain. In Aim 1, we will determine the protein interacting partners of the ?7-nAChR in post mortem hippocampal tissue of aged subjects (70-75 years) with no history of AD. The ?7-nAChR and its interacting proteins will be isolated from the homogenate by ligand affinity pulldown (?-bungarotoxin[Bgtx]-Sepharose beads). 500 ?M methyllycaconitine (MLA) will be added to control homogenates to block selectively the binding of ?7-nAChR to the Bgtx-Sepharose beads. Bound proteins will be eluted with carbamylcholine, fractionated by SDS-PAGE, and tryptic digests prepared. The peptide identities will be determined using state-of-the-art mass spectrometric methods, and then proteins identified in experimental and control samples will be compared to filter out those that bind in a nonspecific fashion. Hippocampal tissue from at least 20 donors will be characterized in order to probe the population variation among interacting proteins associated with ?7-nAChR. These results will serve as the normotypic baseline for studies of pathological tissue. In Aim 2, we will determine how the composition of ?7-nAChR interacting proteins is altered in the post mortem hippocampal tissue of individuals with late stage AD. The proteomic data from AD samples will be compared to the proteomic data from Aim 1 to allow detailed analysis of the effects of AD on the protein interactors of the ?7-nAChR. In Aim 3, we will use label-free quantitative mass spectrometry on data collected in Specific Aims 1 and 2 to determine how the relative levels of proteins that interact with the ?7-nAChR are altered by AD. The mass spectrometry data collected in Aims 1 and 2 will be re-analyzed bioinformatically to quantify changes in ?7-nAChR associated proteins. The mass spectrometry data from a hybrid LTQ-Orbitrap Velos ETD instrument would generate data from which both identifying and quantitative conclusions could be drawn. The data from Aims 1 and 2 would be compared to determine alterations in protein levels caused by the pathophysiological mechanisms of AD. PUBLIC HEALTH RELEVANCE: Using a high-throughput technology and human post mortem tissue, we will study how cellular proteins found in close working association with an important human brain receptor are affected in Alzheimer's disease. We will pursue a proteomic approach to determine whether changes in the composition and function of the proteins found associated with nicotinic alpha7 receptors can be correlated with the disease. The results from our proposed studies should lead to a better understanding of the role of nicotinic alpha7 receptors in Alzheimer's disease. Future detailed study of the proteins to be identified in the proposed work could lead to the development of more highly targeted therapies for this disease.

DESCRIPTION (provided by applicant): The overall objective of this G20 project is to modernize the cage wash system in the primary BioMedical Center animal facility by replacing the 24 year old Basil Steris 4600 Rack Wash system with a new model Basil Steris 4700 Cage and Rack Wash system. By replacing this Rack Washer before it fails completely we aim to uphold the quality and integrity of Brown's AAALAC International certified animal care and use program by continuing, without major interruption or disruption, to maintain and exceed the standards of animal care necessary for effective scientific research. Nearing the end of its useful life, the 4600 Rack Wash system has increasingly been offline for repairs and now requires replacement parts that are obsolete. This proactive replacement of the 4600 will sustain and enhance the research practices and productivity of Brown's animal research programs by providing the physical plant environment and equipment redundancy consistent with, and required for, maintaining animals in a state of well-being through timely cage changing. A modernized Cage and Rack wash system, which also includes replacement of two of the oldest cage wash carts and minor utility renovations, will maintain and improve operational efficiency and staff productivity within the animal care facility by continuous and timely physical plant improvement. It will establish a modernized infrastructure that will facilitate a stable, secure an reliable research environment, providing Brown animal research laboratories with critically necessary cage wash service capacity and redundancy. The Animal Care Program at Brown has an annual operating budget of approximately $2M and the BioMedical Center facility currently supports 29 investigators with ~$9.8M in annual direct funding. This facility improvement will support research and educational activities in multiple academic departments, schools, hospitals and collaborating small companies within our broad and collaborative scientific community and allow continued expansion of these research programs. By supporting Brown's federally funded research and discovery programs, the BioMedical facility improvement is aligned with Brown's mission to advance the understanding of living organisms through studies of the behavioral and biological processes upon which their survival and well-being depends. An award would greatly facilitate human health-related research in Rhode Island and at the regional, national, and global level.