Peter G. Barr-Gillespie - US grants

The P30 Core Center requires administrative assistance beyond that provided by indirect costs. The Administrative Core supports the Core Center PI, Peter G. Gillespie, for management. In addition, the Administrative Core supports the activities of an Administrator and an Accountant, who manage the day-to-day administrative load of the Core Center.

DESCRIPTION (provided by applicant): This proposed project is a supplemental application for renewal of the Mouse Core, a component of the Core Center (P30 DC005983) at the Oregon Health &Science University. This P30 supports twelve principal investigators, funded by the NIDCD and other institutes. The group has a strong focus on and a substantial record of accomplishments in the auditory and vestibular sciences. The Mouse Core supports P30 investigators during their use of mice in NIDCD-funded projects. The increase in powerful molecular and genetic tools for investigation of gene function in the laboratory mouse has greatly increased interest in this animal as a model for auditory and vestibular research. Although the interpretation of experiments exploiting mice with genetic alterations is not always straightforward, the ability to examine the consequences of modification of a single gene has tremendous implications for molecular investigation of the inner ear. The overall goal of the Mouse Core is to enable investigators in the Core Center to carry out experiments with mice, and in particular with genetically modified mice. This ability should enhance present research projects and lead to new experimental approaches, particularly involving collaborations among several investigators. In this revised supplemental application, to provide expertise and services to the Core Center for mouse molecular genetics studies, three specific aims are proposed: 1. To provide mouse husbandry services and training, 2. To provide genotyping services and training, and 3. To provide in vivo electroporation resources. By centralizing these activities into a single facility, we not only allow individual investigators to focus on their individual research projects, but also to create a more cost-effective approach to use of mice in NIDCD funded research programs. PUBLIC HEALTH RELEVANCE: Mouse lines in which specific genes are modified by investigators often offer the best models for human diseases, including deafness. Because the expense and complexity of using mice often prevents investigators from using these powerful models, the Mouse Core will provide expertise that catalyzes introduction of mouse experiments into investigators'repertoires. Core B Mouse Core Co-Directors: Peter Gillespie, Ph.D. and John Brigande, Ph.D.

The Increase in powerful molecular and genetic tools for Investigation of gene function in the laboratory mouse has greatly increased Interest in this animal as a model for auditory and vestibular research. Although the interpretation of experiments exploiting mice with genetic alterations is not always straightforward, the ability to examine the consequences of modification of a single gene has tremendous implications for molecular investigation of the inner ear. The overall goal of the Mouse Core Is to enable Investigators In the Core Center to carry out experiments with mice, and in particular with genetically modified mice. This ability should enhance present research projects and lead to new experimental approaches, particularly involving collaborations among several Investigators. To provide expertise and services to the Core Center for mouse molecular genetics studies, three specific aims are proposed: 1. To provide mouse husbandry services and training, 2. To provide genotyping services and training, and 3. To provide in utero gene transfer resources. Our service approach is to centralize these activities in a single facility. The model allows individual investigators to focus on their specific research projects while enjoying a more cost-effective model for the use of mice in their NIDCD-funded research programs.

DESCRIPTION (provided by applicant): We request funds to purchase an Olympus Fluoview 1000 single-point scanning confocal system, which will replace a 10-year-old Bio-Rad MRC 1024 at the Oregon Hearing Research Center (OHRC). The Imaging Core at OHRC, a microscopy resource for the Oregon Health &Science University (OHSU), was built around this single confocal microscope. Because service contracts are no longer available for this confocal and the instrument is starting to fail, we urgently need to replace it. During its useful lifetime, the existing MRC 1024 has been heavily used by a large number of investigators, resulting in at least 100 publications and several journal cover images;the microscope has contributed to an in- crease in the number and quality of grants awarded, has been used to train at least 100 scientists, graduate students, postdoctoral fellows, and international visiting scientists;the instrument has also been critical in recruiting and retaining faculty. Although this old confocal has become unreliable and unserviceable, many grants depend on data acquired with its help. There is no similar instrument at OHSU that could accommodate the time requirements of the 16 participants to this application. After evaluating the Olympus, Zeiss 510 Meta, and Nikon C1si, we selected Olympus because of use of its ability to fluorescence recovery after photobleaching (FRAP) with excellent time resolution;quality of images obtained during demonstrations;its excellent and intuitive graphics interface (essential for a multi-user envi- ronment);adequate local access to service technicians;recommendations from colleagues;and flexibility of the instrument. We propose to acquire an instrument with 405 nm, 488 nm, 559 nm, and 635 nm laser lines, as well as a Simultaneous Illumination Module (SIM) unit for selective bleaching. The research of the primary users of this instrument aims to understand basic mechanisms involved in audi- tion, notably auditory perception at the molecular level, development/morphogenesis of the auditory system, and the processes involved in auditory dysfunction due to administration of life-saving drugs, and/or aging. Because our mission is to provide training, expertise and instrument access not only to OHRC scientists and auditory neuroscience, but also to the entire region regardless of research discipline, this instrument will be of great utility to a wide range of scientific disciplines. A new, functional, state of the art confocal is a necessity for us to fulfill this mission. PUBLIC HEALTH RELEVANCE: The principal focus of the major users of this instrument is on understanding the structure and function of vertebrate auditory and vestibular systems. High-resolution imaging is essential for identification of the molecular architecture of the auditory mechanosensory apparatus, as well as subsequent downstream structures integral to the perception of sound in mammals and humans. In addition, confocal imaging is critical for determining the distribution of ototoxic drugs in the auditory system to determine new strategies for preventing drug-induced deafness.

DESCRIPTION (provided by applicant): The goal of this proposal is to determine the role of lipids in mechanotransduction by hair cells. We propose three specific aims. In Aim 1, we will determine which lipids are present in hair bundles using mass spectrometry and histochemical localization. In addition, we will measure the turnover of polyphosphatidylinositols and determine the identity, localization, and activities of enzymes that synthesize and degrade them. These experiments are shaped by the hypothesis that hair bundles have an unusual lip d composition and distribution and that these characteristics are important for transduction. In Aim 2, using fluorescence recovery after photobleaching, we will determine dynamics of the bulk hair-bundle membrane as well as the dynamics of individual lipid species. These experiments are motivated by the hypothesis that bundle membranes may have domains of reduced lipid mobility. Finally, in Aim 3 we will determine how lipids regulate transduction and adaptation. These experiments will test the hypotheses that (a) membrane association regulates myosin-1c activity, (b) myosin-1c binding to membranes alters lipid dynamics, (c) the adaptation motor and transduction channel interact in a specialized lipid domain, and (d) that the elasticity measured in the "gating spring" is due to properties of the stereocilia membrane itself. Together, the experiments in this proposal will allow us to determine how lipids regulate transduction and adaptation.

DESCRIPTION (provided by applicant): Transduction of mechanical stimuli - like those of sound and head movements - is the fundamental role of the inner ear and its sensory hair cells. Hair cells transduce deflections of their hair bundles, the sensory organelle, with stunning sensitivity. While well described biophysically, the molecular composition and dynamics of the hair bundle's mechanotransduction apparatus are still mostly mysterious. The goal of our project is to identify key molecules involved in mechanotransduction, then to determine how they interact to form the transduction apparatus. We use a multidisciplinary approach that emphasizes biochemical techniques, including purification of hair bundles and detection of specific proteins by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). In addition, we transfect hair cells with key bundle proteins or their fragments, for example tagged with fluorescent proteins, to allow us to test their roles in transduction or localize them without using antibodies. In the proposed project, we will determine how five key hair-bundle components form the transduction apparatus: (1) the bundle actin- based cytoskeleton;(2) myosin-1c, the adaptation-motor myosin;(3) the tip link, hypothesized to be composed of cadherin 23 and protocadherin 15;(4) the transduction channel, thought to be a member of the transient receptor potential (TRP) family;and (5) the bundle Ca2+ pump, plasma-membrane Ca2+- ATPase isoform 2. These molecules and others we expect to identify together form a transducer that can detect hair-bundle displacements of less than a nanometer, arising from sounds so faint that they are nearly drowned out by air molecules randomly impacting the ear drum and water molecules randomly deflecting bundles. Lay summary. Mechanotransduction by hair cells is a central event in hearing;disruption of transduction leads to profound deafness. Hair cells are the sensory cells of the inner ear;they detect and encode (transduce) sound. The complex of proteins in hair cells that transduce sound are still largely unknown;the goal of this project is to identify and characterize them, as they are essential for hearing.

DESCRIPTION (provided by applicant): Biomedical research at the Oregon Health and Science University (OHSU) has expanded considerable over the past decade and, as part of this growth, the number of laboratories with a primary focus in embryonic development has increased greatly. To support the training efforts of these laboratories, this revised application is submitted to support a basic research training program that will provide predoctoral and postdoctoral students with a rigorous grounding in molecular and cellular aspects of Developmental Biology. The proposed training faculty is comprised of 23 investigators who utilize biochemical, molecular, cellular, and/or genetic approaches to understanding developmental processes in a variety of vertebrate and invertebrate animal model systems including flies, chick, frog, fish, and mouse. Program faculty are drawn from basic science departments and independent research units within the School of Medicine, all housed on the main OHSU campus. All members of the training faculty have active, externally funded basic research programs. The field of Developmental Biology is becoming increasingly intertwined with cell biology and other disciplines, requiring that students receive a broad education in diverse subjects in order to meet future challenges. To accomplish this, all trainees will receive rigorous research training in embryonic development in their preceptors'laboratories, and will participate in journal clubs, seminars, and scientific meetings, including the Northwest Regional Developmental Biology Meeting, the annual trainees symposium, and the OHSU graduate student retreat. Predoctoral trainees will also receive a broad classroom based education in biochemistry, genetics, cell biology, molecular biology, and neurobiology, as well as more in depth training in developmental biology through formal coursework involving both lectures and laboratory exercises. One outstanding feature of the training program at OHSU is the high level of interactions and collaborations that occur among faculty members with diverse research interests and expertise.

Project Summary The long-term goal of this laboratory is reveal the structure of the hair cell's mechanosensitive organelle, the hair bundle, and to determine how structural features of the bundle are responsible for its mechanotransduction function. In this proposal, we focus on the under-appreciated process of actin-core widening, which occurs in stereocilia during development of the bundle. We utilize inner hair cells of the mouse cochlea as our model system; not only do their stereocilia rows show unique diameters and lengths, but all of the tools we deploy in studying bundle function can be deployed with these cells. In Aim 1, we will extend our cryo-electron tomography program to developing inner hair cells, asking specifically when and where peripheral actin filaments are added to the actin core during the widening process. In Aim 2, we will isolate inner hair cells marked with GFP using Fgf8-Gfp;Atoh1-Cre mice, and then subject them to protein mass spectrometry. In conjunction with our collaborators at the Pacific Northwest National Laboratory, we have defined the proteomes of single inner hair cells isolated by fluorescence-activated cell sorting. While we will not analyze single cells in the present project, we will exploit the sensitivity of the new techniques to analyze small pools of cells isolated from a specific region of the cochlea at precise development times. In addition, we will isolate inner hair cell stereocilia at the same time points using pipette aspiration, allowing us to also determine the stereocilia proteome over development. By comparing the whole-cell and stereocilia proteomics data, we will track when each protein enters stereocilia, and mine these data to identify new candidates for complexes that control stereocilia widening. Finally, in Aim 3, we will study three mutant mouse lines (Espn, Capzb, Grxcr1) that have thin stereocilia, using the techniques developed for Aims 1 and 2 to characterize the structural and temporal features of stereocilia. Together, the experiments developed in this project will allow us to determine how the hair cell widens its stereocilia, which is one of the critical steps in development of the hair bundle.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This new project couples proteomics (Gillespie), human genomics (Richard J. Smith, University of Iowa), and mouse molecular genetics (Ulrich M[unreadable]ller, Scripps) in an integrated program for identification and description of molecules essential for hearing and balance. The proteomics work will be done at OHSU/PNNL and is described here. When studying monogenic and complex genetic diseases, standard linkage mapping and/or association approaches to identify disease-relevant genes suffer from low throughput and lack of insight into function. Our approach provides high throughput in a contextual functional framework. We begin by noting that specific organelles are often implicated in disease. Focusing on the auditory system, we will first define the complete proteome (especially the membrane proteome) of the hair bundle, the mechanically sensitive organelle of sensory hair cells, which comprises <<1% of total protein in inner-ear epithelia. This work will be carried out at OHSU/PNNL by the Gillespie lab. Second, to link new genes to auditory disease, we will use next-generation sequencing to interrogate en masse the genes encoding bundle proteins in hundreds of affected families with uncharacterized forms of hearing loss. Sequencing will be carried out at Iowa by the Smith lab. Third, we will generate mouse lines carrying the orthologous human mutations and analyze the functional consequences of these mutations on bundle-protein networks. These mice will be generated by the M[unreadable]ller lab at Scripps, then mutant hair bundles will be examined using proteomic techniques by the Gillespie lab at OHSU/PNNL. This project is iterative, so novel members of disrupted networks will be screened in human families as well. This composite approach provides an understanding of a disease phenotype at the molecular level, knowledge requisite to developing novel approaches to disease treatment.

DESCRIPTION (provided by applicant): The investigators request continued support for the Multidisciplinary Training Program in Neuroscience at Oregon Health & Science University (OHSU). This program, which is about to enter its tenth year, offers broad-based, early stage training for students in OHSU's two Ph.D.-granting neuroscience programs, the Neuroscience Graduate Program and the Behavioral Neuroscience Graduate Program; together these programs enroll about 90 students. The program of training proposed is based on a strong core curriculum that provides a common knowledge base for subsequent training in the classroom and at the bench, together with extensive and varied opportunities for research, beginning as soon as students enter the Program. The training faculty of 65 scientists offers research opportunities that range from fundamental studies of nerve cell function to translational research programs that target nervous system disorders. Through workshops and individual instruction, the Program also provides instruction in scientific communication skills and career development. The spirit of collegiality that has developed at OHSU combined with the diversity of its many research institutes and the close proximity of basic and clinical research facilities provide a unique opportunity for predoctoral students to form cross-disciplinary collaborations during their training, and to gain an appreciation for the growing importance of neuroscience research in the treatment of neural disease. RELEVANCE: This Program will train Ph.D. students in neuroscience, providing them with the knowledge base and technical skills to enable them to conduct original research on the biological bases of nervous system disease.

PROJECT SUMMARY (See instructions): The P30 Core Center requires administrative assistance beyond that provided by indirect costs. The Administrative Core supports the Core Center PI, Peter G. Gillespie, for management. In addifion, the Administrative Core supports the activities of an Administrator and a Financial Analyst, who manage the day-to-day administrative load of the Core Center.

DESCRIPTION (provided by applicant): The Portland Area Communication Research Core Center will support twelve principal investigators in its Research Base, each of whom has one or more qualifying R01 grants funded by the NIDCD and or another institute. Eight other investigators with similar research interests are also members of the Core Center. The twenty investigators are affiliated with a variety of institutes in the greater Portland area: the Oregon Hearing Research Center (OHRC) at the Oregon Health & Science University (OHSU); the Vollum Institute (OHSU); the Department of Otolaryngology; Head & Neck Surgery (OHSU); the Department of Biomedical Engineering (OHSU); the National Center for Rehabilitative Auditory Research (NCRAR) at the Portland Veterans Administration (VA) Medical Center; and the Washington State University (WSU) Vancouver, which is just across the Columbia River from Portland. The group has a strong focus on and a substantial record of accomplishments in the auditory and vestibular sciences, with additional investigators interested in the olfactory system. The goal of the Core Center is to centralize expertise on bioengineering, imaging, and mouse genetics in order to enhance presently funded research projects and to stimulate collaborations among participating investigators. In order to achieve these goals, continued support of three Research Core facilities is proposed. The Bioengineering Core (Alfred Nuttall and Stephen David, Co-Directors) provides computer hardware and software support, support for design and construction of analog and digital circuits, as well as expertise and instrumentation for measurement of hearing acuity in live animals. The Imaging Core (Dennis Trune and Peter Steyger, Co-Directors) centralizes light, confocal, and electron-microscopic imaging through the existing Oregon Hearing Research Center infrastructure. The Mouse Core (Peter Gillespie and John Brigande, Co-Directors) provides expertise on mouse husbandry and genotyping, as well as in utero virus injection and plasmid electroporation to deliver exogenous genes into the inner ear. Support for an Administrative Shell is also requested. Support of the Core Center will facilitate increased efficiency, broader collaborations, and novel approaches in NIDCD-funded research projects.

DESCRIPTION (provided by applicant): This work is designed to understand how hair cells, the sensory cells of the inner ear, use their mechanically- sensitive hair bundle to convert sound to electrical signals. Among other approaches, we identify and quantify proteins in purified bundles using mass spectrometry. Focusing on molecular complexes that contribute to bundle function, our experiments address two fundamental questions: First, how is the hair bundle formed? Second, how does the mechanotransduction complex work at a molecular level? In Aim 1, we will identify protein complexes including radixin, an action-to-membrane crosslinker that is essential for hair-bundle cytoskeleton structure, and SLC9A3R2, a PDZ-domain adaptor protein that binds to radixin. We will also express dominant-negative radixin and SLC9A3R2 constructs to determine how complexes with these proteins control bundle structure. In Aims 2 and 3, we will continue our efforts to identify and characterize the transduction channel itself. We have a pair of strong candidates for the channel, members of the transient receptor potential (TRP) channel family. We propose to locate the channels within stereocilia, determine their interactions with other known members of the transduction complex, and examine mechanotransduction in mice missing one or both of the channels. Ongoing experiments could prove that these TRP channels are not the transduction channel, however, so Aim 3 proposes to improve our ongoing biochemical preparation of the transduction complex, as well as to identify transmembrane proteins in purified stereocilia membranes. Research proposed here will show how mechanotransduction operates in the normal inner ear. As stated in the most recent strategic plan, one of the major goals of the NIDCD is to use genomic, proteomic, informatic, bioinformatic, and expression...approaches...to understand the molecular bases of normal and disordered [hearing and balance]. Understanding how the bundle is assembled and how its transduction machinery normally operates, the focus of the proposed research, is essential for rational design of therapies for hearing loss and balance disorders.

Project Summary The long-term goal of our laboratories is to elucidate the mechanisms that control hair cell development and function, and ascertain the defects in this process that cause deafness. We propose here to identify and study proteins that physically and functionally interact with MYO7A and therefore mediate its function in hair cells. Based on published and preliminary data, we hypothesize that MYO7A has a three-fold function in hair cells, regulating the transport of proteins critical for hair bundle adhesion and actin polymerization and directly controlling mechanotransduction. To test our hypothesis, we will: (i) define the protein complexes that mediate stereocilia adhesion, focusing on those consisting of PCDH15 and GRP98; (ii) determine how MYO7A complexes regulate stereocilia length, focusing initially on the complex with CAPZ that we have defined; (iii) specify how MYO7A and its interacting proteins control mechanotransduction. Our preliminary data show the feasibility of our approach. We have already identified hair bundle proteins that interact with MYO7A and mediate its function. We anticipate that some of the novel interaction partners of MYO7A will be affected in genetic diseases that cause hearing impairment.

Project Summary/Abstract We request funds to purchase a ForteBio Octet Red384 instrument to measure protein-protein interactions. To maximize research productivity, the upgraded instrument will be placed in the Oregon Health & Science University Proteomics Shared Resource. The only instruments that measure rates and affinities of protein-protein interactions available at OHSU provide very low throughput, and new instrument is required to increase research productivity at our institution. The new instrument will be used to study the cause and treatment of several diseases including psychiatric disorders, HIV, hearing loss, and anemia, as well as to study methods for treating those diseases. Purified proteins will be analyzed by binding one partner to cheap and dispensable biosensor probes, then using biolayer interferometry to measure binding of a second protein with high sensitivity. The Octet Red384 can efficiently measure many complete sets of interactions per day, each involving 6-8 protein concentrations and producing both kon and koff as well as KD. The introduction of this instrument will fulfill the growing needs of the researchers at the OHSU to incorporate state-of-the-art tools in protein chemistry into their research. The capability afforded by this instrument will speed the progress of discovery and improve the detection and treatment of human disease.

The 5-year renewal for Grant P51OD011092 requests funding for programs at the ONPRC that support biomedical research. The application reflects a strategic focus on interdisciplinary research to optimize the use and development of nonhuman primate (NHP) models. The aims of this application are to: 1) Conduct state-of-the-art research for which NHPs are uniquely suited for solutions to human health problems 2) Provide a regional and national resource for the conduct of interdisciplinary biomedical research, especially as it relates to NHPs. 3) Pursue the highest standards of humane and responsible animal care. 4) Provide research training and experience for those preparing to enter research and biological teaching careers. To accomplish these aims, support is requested in eight broad areas. 1) Administration provides the administrative and service support required for all aspects of the ONPRC. Units include: the Director's Office; Administrative Services; Business Services; Facilities; Information Systems; Research Library; and Research Safety. 2) Animal Services. Units include: Resources, Facilities, and Operations; Pathology Services; the Tissue Distribution Program; Surgical Services; Behavioral Services; Research, Education and Training; Clinical Medicine; NHP Resources (Obese, Primate Aging, Infectious Disease, Time-mated Breeding, and Japanese macaque). 3) Core Science Services. Units include: Assisted Reproductive Technology, Endocrine Technology, Flow Cytometry, Imaging & Morphology, Molecular & Cellular Biology, Molecular Virology, Magnetic Resonance Imaging, and Primate Genetics. 4) Scientific Components include support for Scientific Divisions (Neuroscience, Pathobiology & Immunology, Reproductive & Developmental Sciences and Diabetes, Obesity & Metabolism) and the Interdisciplinary Research Programs (Biology of Aging, Early Childhood Health & Development, Primate Genetics Research). 5) Pilot Project Program. 6) Improvement & Modernization. 7) Outreach and Community Engagement. 8) NPRC Consortium-Based Activities.

PROJECT SUMMARY The Oregon National Primate Research Center (ONPRC), located on the West Campus of Oregon Health & Science University (OHSU), requests funds to renew grant P51--OD011092 for the next five--year period (May 1, 2019--April 30, 2024). Currently in its 59th year of operation, the ONPRC has served a broad range of local, regional, and national investigators performing biomedical research in nonhuman primate (NHP) models with the goal of improving human health. The goals for the next funding period are reflected in the following Specific Aims: 1) Conduct state--of--the--art research that advances understanding of causes and the development of preventions, treatments, and cures for debilitating human diseases; 2) Provide exceptional NHP expertise and services to researchers at the local, regional, and national levels to advance NIH-- and other federally-- supported research programs; 3) Pursue the highest standards of humane and responsible animal care; and 4) Mentor and train the next generation of translational NHP researchers and educate the public about the importance of biomedical research. To accomplish these aims, support is requested in seven broad areas. These areas are: (1) Administration, which provides the administrative and service support required for all aspects of the ONPRC (Governance, Director's Office, Business Services, Facilities, Improvements and Modernization, Information Systems, Research Library, and Research Safety); (2) Animal Services [Resources & Logistics, Operations, Pathology Services, Surgical Services, Behavioral Services, Education and Training, Clinical Medicine, and NHP Resources (Obese, Aging, Infectious Disease, and Japanese Macaque)] ; (3) Core Science Services [Research Cores (Assisted Reproductive Technologies, Bioinformatics & Biostatistics, Endocrine Technologies, Flow Cytometry, Integrated Pathology, Molecular Technologies, Molecular Virology, Magnetic Resonance Imaging (MRI), and Primate Genetics)] ; (4) Scientific Components, including support for Scientific Divisions (Cardiometabolic Health, Genetics, Neuroscience, Pathobiology & Immunology, and Reproductive & Developmental Sciences); (5) Pilot Research Program; (6) Outreach and Community Engagement; and (7) NPRC Consortium--Based Activities. ONPRC's overarching goal is to develop, study, and share NHP models that can inform the causes of human diseases, leading to better preventions, treatments, and cures. The ONPRC achieves this goal by supporting translational research and expertise in models that are provided locally, regionally, and nationally on a collaborative basis. The host institution, OHSU, recognizes the ONPRC as one of the university's `top five' strengths and has pledged to continue support for programmatic integration and infrastructure development.

Project summary Social housing is known to promote welfare for nonhuman primates (NHPs). Rhesus macaques living in large outdoor corrals experience fewer episodes of diarrhea and lower wounding rates, and have less alopecia compared to those living in other housing types. Further, macaques born in corrals show less stereotypy when housed inside compared to those born in other housing. However, the corrals currently in use at the ONPRC have limitations, including being located relatively far from processing areas (which requires animals to be moved to those areas on trucks), solid walls that prevent animals from viewing their environment, and lack of areas for introducing or re-integrating animals into the group. This supplement will allow us to renovate the design to erect a new, innovative, half acre corral, which will allow us to increase our breeding population by approximately 50-60 infants per year. The plan described in this application expands existing housing with a half-acre corral tied to existing infrastructure (thus reducing the need to transport animals by truck); will provide animals with varied enrichment options (including the ability to visualize their environment through observation windows); and will contain an introduction area to facilitate animal reintegration. Together, the corral redesign proposed in this supplement will increase breeding capacity in support of research, thus enhancing the scientific endeavor while promoting animal welfare.

PROJECT SUMMARY/ABSTRACT Hair cells are the sensory cells of the inner ear that carry out the essential function of mechanotransduction evoked by sound and head movement. It is fundamentally important to characterize global protein expressions and their interactions in hair cells in order to understand the molecular mechanism. However, one of the greatest challenges in protein characterization is the low number of hair cells presented in each inner-ear organ, which urges us to develop sensitive analytical approaches. To this end, our labs develop a microfluidic sample preparation platform, termed as nanoPOTS (nanodroplet processing in one pot for trace samples), for proteomics analysis of low-input biomaterials by downscaling processing volumes to <200 nL. While nanoPOTS is well demonstrated to identify and quantify protein expression from single hair cells, it informs nothing on how proteins interact with each other to implement their functions. The overall objective of this project to develop a sensitive nanoliter droplet-based affinity purification with mass spectrometry (nanoAP-MS) platform to identify protein interacting partners using fewer than 1000 hair cells isolated from utricles or cochleas of the mouse ear. The central hypothesis is that the overall sensitivity of AP-MS assay can be significantly improved by performing affinity purification in nanoliter droplets. Theory suggests this hypothesis should be correct because: 1) Improved protein concentrations by lysing cells in nanoliter volumes will improve protein-bead binding efficiency; 2) Reducing the amounts of affinity beads will reduce non-specific binding, which can otherwise dwarf specific binding; and 3) improved LC-MS will provide sufficient analytical sensitivity to measure low abundance proteins. The central hypothesis will be tested by pursuing two specific aims: 1) To establish a nanoliter droplet-based AP-MS workflow; and 2) To apply this workflow for identification of MYO7A and GIPC3 binding partners in mouse hair cells. We expected the proposed nanoAP-MS platform will increase sensitivity by a factor of 103 or more and allow us to characterize low-abundance protein interaction partners. This research is highly innovative because the nanoAP-MS platform will be the first of its kind to reliably measure protein-protein interactions using a small number of primary cells isolated from physiological environment, including animal models or human biopsies. Statement of Impact: As AP-MS has emerged as powerful technology to discover protein interaction partners and establish protein-protein-interaction networks, the nanoAP-MS technology will enable to examine important protein-protein interactions in small numbers of cells isolated by micropipette, FACS, or laser-capture microdissection, or to examine exceptionally low- abundance interactions like those present in the hair cell's mechanotransduction complex.

ABSTRACT/PROJECT SUMMARY-­ OVERALL    The Oregon National Primate Research Center (ONPRC), located on the West Campus of Oregon Health &  Science University (OHSU), requests funds to renew grant P51-­OD011092 for the next five-­year period (May  1, 2019-­April 30, 2024). Currently in its 59th year of operation, the ONPRC has served a broad range of local,  regional, and national investigators performing biomedical research in nonhuman primate (NHP) models with  the goal of improving human health. The goals for the next funding period are reflected in the following Specific  Aims: 1) Conduct state-­of-­the-­art research that advances understanding of causes and the development of  preventions, treatments, and cures for debilitating human diseases;? 2) Provide exceptional NHP expertise and  services to researchers at the local, regional, and national levels to advance NIH-­ and other federally-­ supported research programs;? 3) Pursue the highest standards of humane and responsible animal care;? and 4)  Mentor and train the next generation of translational NHP researchers and educate the public about the  importance of biomedical research. To accomplish these aims, support is requested in seven broad areas.     These areas are: (1) Administration, which provides the administrative and service support required for all  aspects of the ONPRC (Governance, Director?s Office, Business Services, Facilities, Improvements and  Modernization, Information Systems, Research Library, and Research Safety);? (2) Animal Services [Resources  & Logistics, Operations, Pathology Services, Surgical Services, Behavioral Services, Education and Training,  Clinical Medicine, and NHP Resources (Obese, Aging, Infectious Disease, and Japanese Macaque)];? (3) Core  Science Services [Research Cores (Assisted Reproductive Technologies, Bioinformatics & Biostatistics,  Endocrine Technologies, Flow Cytometry, Integrated Pathology, Molecular Technologies, Molecular Virology,  Magnetic Resonance Imaging (MRI), and Primate Genetics)];? (4) Scientific Components, including support for  Scientific Divisions (Cardiometabolic Health, Genetics, Neuroscience, Pathobiology & Immunology, and  Reproductive & Developmental Sciences);? (5) Pilot Research Program;? (6) Outreach and Community  Engagement;? and (7) NPRC Consortium-­Based Activities.    ONPRC?s overarching goal is to develop, study, and share NHP models that can inform the causes of human  diseases, leading to better preventions, treatments, and cures. The ONPRC achieves this goal by supporting  translational research and expertise in models that are provided locally, regionally, and nationally on a  collaborative basis. The host institution, OHSU, recognizes the ONPRC as one of the university?s ?top five?  strengths and has pledged to continue support for programmatic integration and infrastructure development.   

PROJECT SUMMARY The Oregon National Primate Research Center (ONPRC) is one of seven National Primate Research Centers (NPRCs) established by Congress in the 1960s. The NPRCs are sponsored by the NIH Office of the Director, and serve the nation's biomedical research needs in a unique and essential fashion through cost-effective provision of NHPs and related scientific expertise, specialized facilities, and equipment to federally funded studies of NHPs that comprise a vital translational link between basic research and human applications. The mission of the ONPRC is to improve human health and the quality of life through NHP research programs that advance our knowledge of the causes of human diseases and that develop effective preventions, treatments, and cures. The Oregon National Primate Research Center (ONPRC) P51 Core Grant (P51-OD011092) supports a colony of approximately 4900 breeding and research nonhuman primates (NHPs), including rhesus macaques of Indian origin, Japanese macaques, cynomolgus macaques, Hamadrayas and olive baboons, and a small number of squirrel monkeys. Rhesus and Japanese macaques are bred at the Center. Other species are imported from domestic sources to fulfill project specific research needs. In this supplement proposal to the parent grant, we will test the hypotheses that: 1) When given prior to challenge, inhaled, aerosolized neutralizing monoclonal antibodies can prevent or mitigate SARS-CoV-2 infection in rhesus macaques; and 2) Inhaled neutralizing monoclonals can reduce viremia and prevent pathogenesis when given after challenge. To test these hypotheses, we will test a cocktail of human mAbs with potent and broad SARS-CoV2-neutralizing activity for their effectiveness in pre-exposure protection and/or limitation of viral shedding following aerosolization and administration by inhalation prior to intranasal/intratracheal challenge with SARS-CoV-2. Animals will be followed for virological and immunological outcomes and pathology using standard protocols developed by the Coronavirus Vaccines and Therapy Evaluation Network. At necropsy, tissues will be harvested and processed for virus quantification and histology. For post-exposure studies, macaques will be infected with SARS-CoV-2, and treated one day later with the same cocktail of human SARS-CoV-2 mAbs, comparing aerosol and intravenous delivery at various doses. These proof-of-principle experiments will pave the way for post-exposure aerosol treatments with potent antibodies and nanobodies toward the goal of reducing COVID-19 disease. Our findings will expand the utility of the nonhuman primate model for SARS-CoV-2 and will support the discovery of novel therapeutics for COVID-19, thereby fulfilling the primary purpose of the P51 grant.