Battelle Memorial Institute

A PROTEOMICS RESEARCH CENTER FOR INTEGRATIVE BIOLOGY

PROMOTR: A PROTEOMICS CENTER FOR MOTRPAC

CENTER FOR APPLICATION OF ADVANCED CLINICAL PROTEOMIC TECHNOLOGIES FOR CANCER

A PROTEOMICS RESEARCH RESOURCE FOR INTEGRATIVE BIOLOGY

3D IMAGING & COMPUTER MODEL OF THE RESPIRATORY TRACT

SOLID STATE NMR STRUCTURE/FUNCTION STUDIES OF AMELOGENIN

PROTEOGENOMIC TRANSLATIONAL RESEARCH CENTER FOR CLINICAL PROTEOMIC

CENTER FOR ADVANCED MULTI-OMIC CHARACTERIZATION OF CANCER - PROJECT SUMMARY THE OVERALL OBJECTIVE OF THE PNNL PROTEOME CHARACTERIZATION CENTER (PCC) IS TO COMPREHENSIVELY CHARACTERIZE HUMAN TUMOR SAMPLES PROVIDED BY THE NATIONAL CANCER INSTITUTE (NCI), AND TO INTEGRATE THE MULTI-OMIC MEASUREMENTS TO SUPPORT IMPROVED UNDERSTANDING OF THE MOLECULAR CHANGES THAT CHARACTERIZE CANCER, AND DO SO IN THE CONTEXT OF CLINICAL OUTCOME. PNNL HAS PARTICIPATED IN THE NCI’S CLINICAL PROTEOMIC TUMOR ANALYSIS CONSORTIUM (CPTAC) AS A PCC FOR THE PAST TEN YEARS, WITH RESPONSIBILITY FOR COMPREHENSIVE PROTEOGENOMIC CHARACTERIZATION OF HIGH-GRADE SEROUS OVARIAN, COLON, AND ENDOMETRIAL CANCERS, AND GLIOBLASTOMA. THE PLANNED PNNL PCC WILL BUILD ON THOSE ACHIEVEMENTS TO EXTEND AND ADVANCE THE CPTAC MISSION OF COMPREHENSIVE PROTEOGENOMIC CHARACTERIZATION OF HUMAN CANCERS TO ADDITIONAL CANCER TYPES, MEETING OR EXCEEDING CPTAC KEY EXPECTATIONS OR REQUIREMENTS FOR SAMPLE THROUGHPUT, COVERAGE, SAMPLE SIZE, AND DATA QUALITY. UTILIZING AN ADVANCED ANALYTICAL PLATFORM, PNNL PLANS TO ADD ANALYSIS OF BOTH ACETYLOME AND UBIQUITINOME TO THE PHOSPHOPROTEOME OF PROSPECTIVELY COLLECTED HUMAN TUMORS, TO BETTERS ILLUMINATE KEY BIOCHEMICAL PROCESSES RELATED TO PROTEIN-PROTEIN INTERACTIONS, PROTEIN DEGRADATION, AND SIGNAL TRANSDUCTION. WE WILL ALSO COMPLEMENT THE CORE PROTEOME AND POST-TRANSLATIONAL MODIFICATION (PTM)-OME ANALYSIS WITH GLOBAL METABOLOME AND LIPIDOME ANALYSIS, AS WELL AS SELECTED DATA DRIVEN SPATIAL OR SINGLE-CELL PROTEOMICS ANALYSIS. THIS WILL PROVIDE ADDITIONAL CRITICAL INSIGHTS ON POTENTIAL METABOLIC VULNERABILITIES AND TUMOR HETEROGENEITY AS WELL AS MICROENVIRONMENT CONTRIBUTIONS. THIS MULTI-OMIC ANALYSIS STRATEGY WILL ALSO BE APPLIED TO PRECLINICAL SAMPLES, SUCH AS CELL LINES, ORGANOIDS AND PATIENT-DERIVED XENOGRAFTS. WE WILL ALSO DEVELOP TARGETED MASS SPECTROMETRIC ASSAYS USING INPUT FROM THE CPTAC CONSORTIUM, AND PARTICULARLY THE PROTEOGENOMIC DATA ANALYSIS CENTERS (PGDACS), TO PRIORITIZE TARGETS FOR FURTHER EXPLORING IMPORTANT MECHANISTIC PROTEOMIC CHANGES IN INDEPENDENT COHORT(S). THROUGHOUT THIS WORK OUR MEASUREMENTS WILL BENEFIT FROM FURTHER PERFORMANCE INCREASES (E.G., SENSITIVITY AND THROUGHPUT) BASED ON REFINING, VALIDATING AND IMPLEMENTING DEVELOPMENTS FROM BOTH PNNL AND THE OTHER CPTAC CENTERS. THE PNNL PCC WILL IDENTIFY PROMISING CANCER SIGNATURES AND SIGNALING NETWORKS THROUGH PROTEOMIC AND METABOLOMIC ANALYSIS OF HUMAN BIOSPECIMENS AND RELEVANT PRECLINICAL SAMPLES FOR 2-3 CANCER TYPES SELECTED BY THE CPTAC, USING STATE-OF-THE-ART LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY INSTRUMENTATION, HIGHLY MULTIPLEXED ISOBARIC MASS-TAG LABELING (TMT 16-PLEX), AND INTEGRATED SAMPLE WORKFLOWS, AS WELL AS ADDITIONAL ADVANCED METABOLOMIC, SPATIAL AND SINGLE-CELL PROTEOMIC PLANFORMS, AT A THROUGHPUT OF 300 SAMPLES PER YEAR. WE WILL ALSO EXPLORE MECHANISTICALLY IMPORTANT PROTEOMIC CHANGES IN HUMAN SPECIMENS AND MODEL SYSTEMS USING CUTTING-EDGE TARGETED PROTEOMIC PLATFORMS, ANALYTICALLY VALIDATED AND HIGHLY MULTIPLEXED TARGETED ASSAYS, AND WORKFLOWS MEETING THE CPTAC TIER 2 ASSAY GUIDELINES. TWO HUNDRED HIGHLY SPECIFIC, MULTIPLEXED TARGETED PROTEOMICS ASSAYS WILL BE DEVELOPED AND USED FOR MEASUREMENTS IN 300 SAMPLES EACH YEAR. THE PNNL PCC WILL ACCOMPLISH BOTH UNBIASED AND TARGETED MULTI-OMIC CHARACTERIZATION OF CANCERS IN CONJUNCTION WITH IMPROVING THE DEPTH, THROUGHPUT AND QUALITY OF BOTH UNBIASED AND TARGETED DATA GENERATED BY IMPLEMENTING AND DEPLOYING RELEVANT NEW TECHNOLOGIES, SUCH AS NANOSCALE PTM, METABOLOMIC ANALYSIS, AND SINGLE-CELL PROTEOMICS ANALYSIS. THE PNNL PCC WILL WORK CLOSELY WITH THE OTHER PCCS, PGDACS AND PTRCS IN THE CPTAC NETWORK ON DATA INTEGRATION AND BIOINFORMATICS ANALYSIS, AS WELL AS TRANSLATIONAL APPLICATIONS.

INTEGRATING STRUCTIVE ACTIVITY, BIOKINETICS AND RESPONSE FOR ENP RISK ASSESSMENT

PNNL PROTEOME CHARACTERIZATION CENTER

CONSORTIA FOR HIGH-THROUGHPUT-ENABLED STRUCTURAL BIOLOGY PARTNERSHIPS (U01)

PSP OMICS CENTER OF ACUTE TO CHRONIC PAIN SIGNATURES

APBS: NANOSCALE BIOMOLECULAR ELECTROSTATICS SOFTWARE

MULTI-OMIC 3D TISSUE MAPS FOR A HUMAN BIOMOLECULAR ATLAS

RESEARCH CENTER FOR SPATIOTEMPORAL LUNG IMAGING AND OMICS

PACIFIC NORTHWEST ADVANCED COMPOUND IDENTIFICATION CORE

CENTER FOR LUNG DEVELOPMENT IMAGING AND OMICS

CENTER FOR NOVEL BIOMARKERS OF RESPONSE

NP-MRD: NATURAL PRODUCTS MAGNETIC RESONANCE DATABASE

ROBUST MASS SPECTROMETRIC PROTEIN/PEPTIDE ASSAYS FOR TYPE 1 DIABETES CLINICAL APPLICATIONS - PROJECT SUMMARY/ABSTRACT: TYPE 1 DIABETES (T1D) IS A DEVASTATING DISEASE OFTEN OCCURRING IN CHILDREN AND YOUNG ADULTS RESULTING FROM THE AUTOIMMUNE-MEDIATED LOSS OF PANCREATIC SS-CELLS. IT HAS BEEN CHALLENGING FOR MONITORING THE DISEASE PROGRESSION AND THE EFFICACY OF CLINICAL INTERVENTIONS. THEREFORE, A CRITICAL NEED REMAINS FOR HIGHLY RELIABLE ASSAYS THAT QUANTIFY PROTEINS OR PEPTIDE HORMONES (E.G., INSULIN, GLUCAGON) AND THEIR SPECIFIC ISOFORMS (OR PROTEOFORMS) AS MARKERS OF ENDOCRINE AND EXOCRINE FUNCTION. SUCH ASSAYS WILL PLAY AN IMPORTANT ROLE IN FACILITATING EFFECTIVE MONITORING OF DISEASE PROGRESSION OR EFFICACY OF NOVEL CLINICAL INTERVENTIONS PRIOR TO OR FOLLOWING THE ONSET OF T1D. MOST CURRENT CLINICAL ASSAYS DEPEND ON THE USE OF ANTIBODIES OR OTHER AFFINITY REAGENTS ALMOST EXCLUSIVELY. HOWEVER, THE EXACT SPECIFICITY OF AFFINITY REAGENTS IS OFTEN UNKNOWN OR DIFFICULT TO CHARACTERIZE. TARGETED MASS SPECTROMETRY (MS) PRESENTS A PROMISING ALTERNATIVE TO IMMUNOASSAYS. THEREFORE, THE OVERALL OBJECTIVE OF THIS APPLICATION IS TO DEVELOP RELIABLE, PROTEOFORM-SPECIFIC, AND MULTIPLEX TARGETED MS ASSAYS FOR A LIST OF PROTEIN/PEPTIDE ANALYTES OF SIGNIFICANCE IN T1D. SPECIFICALLY, WE AIM TO DEVELOP MULTIPLEX TARGETED MS ASSAYS FOR THE FOLLOWING PANEL OF TARGETS AS MARKERS OF ENDOCRINE AND EXOCRINE FUNCTION: INSULIN, GLUCAGON, AMYLIN, CHROMOGRANIN, SOMATOSTATIN, PROHORMONE ISOFORMS (E.G., PROINSULIN, PROGLUCAGON), HYBRID INSULIN PEPTIDES, TRYPSINOGEN, GLYCATED CD59, AS WELL AS OTHER MARKERS OF INTEREST TO THE T1D RESEARCH COMMUNITY. TO FACILITATE FULL VALIDATION OF THE ROBUSTNESS AND TRANSFERABILITY OF THE ASSAYS, THE OVERALL OBJECTIVE WILL BE ACCOMPLISHED THROUGH THE COLLABORATIVE EFFORTS OF TWO INDEPENDENT TARGETED MS LABS AND THROUGH A MULTI-LAB ASSAY VALIDATION EFFORT. SPECIFICALLY, AIM 1 WILL BE FOCUSED ON ESTABLISHING OPTIMAL ASSAY CONFIGURATIONS FOR CONFIDENT DETECTION OF ENDOGENOUS ANALYTES IN SERUM SAMPLES FOR SPECIFIC PROTEOFORMS OR PEPTIDES OF INTEREST IN T1D. AIM 2 WILL BE CENTERED ON ASSAY OPTIMIZATION AND FULL ASSAY CHARACTERIZATION IN THE ASPECTS OF REPRODUCIBILITY, STABILITY, SELECTIVITY, LINEARITY, AND LIMIT OF QUANTIFICATION. INTER-LAB ASSAY PROTOCOL TRANSFER AND ASSAY CHARACTERIZATION WILL ALSO BE PURSUED. AIM 3 WILL DEMONSTRATE THE ROBUSTNESS AND UTILITY OF THE ASSAYS THROUGH MULTI-LAB VALIDATION OF THE ASSAYS BY ANALYZING THE SAME COHORT OF CLINICAL SERUM SAMPLES AND BENCHMARKING AGAINST WELL-ESTABLISHED IMMUNOASSAYS FOR SELECTED ANALYTES SUCH AS INSULIN AND C-PEPTIDE. TOGETHER, THE PROJECT WILL ESTABLISH HIGHLY RELIABLE AND EASY-TO-TRANSFER MULTIPLEX TARGETED MS ASSAYS FOR MANY DIFFICULT TO MEASURE T1D MARKERS. THESE ASSAYS ARE EXPECTED TO MAKE A SIGNIFICANT CONTRIBUTION TO THE MONITORING OF PANCREATIC ENDOCRINE/EXOCRINE FUNCTIONS, THE DISEASE PROGRESSION, AS WELL AS THE EFFICACY OF CLINICAL INTERVENTIONS IN T1D RESEARCH.

A SCALABLE MASS SPECTROMETRY PLATFORM FOR PROTEOME MAPPING OF BRAIN TISSUES - ABSTRACT THE BRAIN IS THE MOST COMPLEX ORGAN IN THE MAMMALIAN BODY. BULK ANALYSIS OBSCURES HETEROGENEITY OF CELL TYPES PRESENT EVEN IN THE SMALLEST BRAIN REGIONS. MULTI-OMICS SINGLE-CELL RESOLUTION 3D-CHARACTERIZATION OF BRAIN TISSUE IS CRITICALLY IMPORTANT TO CREATE COMPREHENSIVE BRAIN CELL CENSUSES AND ALTAS. RECENT TECHNOLOGICAL ADVANCES ALLOW FOR SINGLE-CELL TRANSCRIPTOME MAPPING OF MAMMALIAN BRAINS, BUT SINGLE-CELL PROTEOMICS TECHNOLOGIES ARE LAGGING FAR BEHIND TRANSCRIPTOMICS TECHNOLOGIES. THE LACK OF HIGH-RESOLUTION PROTEOME CHARACTERIZATION OF BRAIN TISSUES IN THE BICCN CONSORTIUM REPRESENTS A SIGNIFICANT KNOWLEDGE GAP BETWEEN PROTEIN AND MRNA FOR ACHIEVING A MORE COMPLETE UNDERSTANDING OF HOW DIVERSE BRAIN CELLS ARE ORGANIZED INTO DISTINCT ANATOMICAL AND FUNCTIONAL REGIONS. THE OBJECTIVE OF THIS APPLICATION IS TO ADDRESS THIS GAP BY DEVELOPING A ROBUST SCALABLE MASS SPECTROMETRY (MS) PLATFORM FOR HIGH-RESOLUTION 3D-PROTEOME MAPPING OF BRAIN TISSUES. THE FEASIBILITY IS STRONGLY SUPPORTED BY OUR RECENT PROGRESS IN TECHNOLOGY DEVELOPMENT AND OUR EXPERIENCES IN PROTEOME MAPPING OF MOUSE TISSUES. AIM 1 WILL FOCUS ON THE DEVELOPMENT OF A ROBUST SCALABLE MS PLATFORM THROUGH 1) FURTHER IMPROVING SAMPLE PREPARATION FOR RAPID EFFECTIVE PROCESSING OF SINGLE CELLS AND SMALL TISSUE VOXELS, AND 2) LEVERAGING MULTIPLE DISRUPTIVE TECHNOLOGIES DEVELOPED AT OUR GROUP FOR SIGNIFICANTLY IMPROVING DETECTION SENSITIVITY AND SAMPLE THROUGHPUT. THE NEW PLATFORM IS EXPECTED TO ALLOW FOR RELIABLE QUANTIFICATION OF >3,000 PROTEINS IN SINGLE CELLS AND >6500 PROTEINS IN 100 CELLS WITH ~500 SAMPLES PER DAY. AIM 2 WILL OPTIMIZE AND DEMONSTRATE THE SCALABLE MS PLATFORM FOR 2D-PROTEOME MAPPING OF MOUSE MOP AND HUMAN M1 WHEN COMBINED WITH LASER CAPTURE MICRODISSECTION FOR TISSUE VOXELS. AIM 3 WILL APPLY THE NEW PLATFORM FOR 3D-PROTEOME MAPPING OF MOP/M1 WITHIN THE BICCN CONSORTIUM AND INTEGRATE PROTEOMIC DATA WITH EXISTING TRANSCRIPTOMIC DATA FOR PROTEOGENOMIC ANALYSIS. WE ENVISION THAT THE NEW PLATFORM WILL BECOME A CONVENIENT INDISPENSABLE TOOL FOR HIGH-RESOLUTION 3D-PROTEOME MAPPING OF BRAIN TISSUES IN THE BICCN CONSORTIUM AND EXTEND THE BICCN TOOLBOX. IN TURN, IT COULD MAKE SUBSTANTIAL CONTRIBUTIONS TO IMPROVE OUR UNDERSTANDING OF BRAIN FUNCTION IN HEALTH AND DISEASE.

PATHWAYS AND CRITICAL REGULATORS OF EARLY BETA-CELL DYSFUNCTION IN TYPE 1 DIABETES

MULTIPLEX MASS SPECTROMETRIC PROTEIN ASSAYS FOR PRECISE MONITORING OF THE PATHOPHYSIOLOGY OF OBESITY

NON-INVASIVE BIOMONITORING OF PESTICIDES

DISCOVERY OF PATHWAYS AND MOLECULAR MEDIATORS OF EARLY T1D PATHOGENESIS USING SPATIAL MULTI-OMICS - PROJECT SUMMARY/ABSTRACT TYPE 1 DIABETES (T1D) IS A COMPLEX, DEVASTATING AUTOIMMUNE DISEASE WITH A SLOW PROGRESSION OF PANCREATIC BETA-CELL DYSFUNCTION (E.G., A DECLINE OF FIRST PHASE INSULIN RELEASE OCCURS OVER SEVERAL YEARS) BEFORE THE CLINICAL ONSET OF T1D. UNFORTUNATELY, WE STILL HAVE A LACK OF KNOWLEDGE ON THE MOLECULAR MECHANISMS OF EARLY T1D PATHOGENESIS. THE LONG-TERM GOAL OF THIS PROJECT IS TO GAIN A DETAILED UNDERSTANDING OF IN SITU MOLECULAR MECHANISMS OF EARLY HUMAN T1D DEVELOPMENT THROUGH ADVANCED SPATIAL OMICS PROFILING OF HUMAN ISLET MICROENVIRONMENTS WITHIN CLINICAL TISSUE SPECIMENS AND FUNCTIONAL STUDIES OF NOVEL MEDIATORS. OUR RESEARCH PLAN CENTERS ON THE CRITICAL ROLE OF HUMAN ISLET MICROENVIRONMENTS IN DISEASE PROGRESSION AND THE OBSERVATIONS OF SUBSTANTIAL INTRA-DONOR HETEROGENEITY OF ISLET MICROENVIRONMENTS (IN BOTH PRE-DIABETIC AND RECENT-ONSET SUBJECTS) FOR UNRAVELING PROGRESSION INFORMATION. OUR HYPOTHESIS IS THAT PATHWAYS OR MOLECULAR MEDIATORS OF DISEASE PROGRESSION (OTHERWISE UNATTAINABLE WITHOUT LONGITUDINAL SAMPLES) CAN BE UNCOVERED THROUGH ADVANCED SPATIAL MULTI-OMICS PROFILING OF INDIVIDUAL ISLET MICROENVIRONMENTS IN THE CONTEXT OF INTRA-DONOR HETEROGENEITY FOR EACH PRE-T1D OR NEW-ONSET T1D DONOR. SPATIAL OMICS DATA ON INDIVIDUAL ISLET MICROENVIRONMENTS WILL UNRAVEL “PSEUDO- TIME” PROGRESSION OF ISLET DYSFUNCTION WITHIN EACH DONOR. COMMON PATHWAYS AND MOLECULAR MEDIATORS ACROSS MULTIPLE DONORS WILL BE IDENTIFIED FOR FUNCTIONAL STUDIES. OUR PLAN IS TO PURSUE SPATIAL MULTI-OMICS PROFILING OF ISLET MICROENVIRONMENTS AT TWO DIFFERENT RESOLUTIONS. AIM 1 WILL FOCUS ON CHARACTERIZING INTRA-DONOR HETEROGENEITY ACROSS INDIVIDUAL ISLET MICROENVIRONMENTS AT THE ROI (REGION OF INTEREST) LEVEL WITH EACH ISLET MICROENVIRONMENT AS A ROI. PSEUDO-TIME PROGRESSION AND ITS UNDERLYING PATHWAYS WILL BE UNCOVERED THROUGH CORRELATION ANALYSIS WITH PATHOLOGY SCORES FOR EACH ROI. ONE EXAMPLE OF THE PATHOLOGY SCORES IS THE IMMUNE SIGNATURE (A 20-PROTEIN PANEL DISCOVERED FROM OUR PRELIMINARY STUDIES) WHOSE EXPRESSION LEVELS CAN SERVE AS A PATHOLOGY SCORE FOR EACH ISLET MICROENVIRONMENT. AIM 2 WILL CHARACTERIZE ISLET MICROENVIRONMENTS AT THE SINGLE CELL RESOLUTION WITH THE GOAL TO REVEAL CELLULAR NICHES OF SPECIFIC TYPE OF ISLET DYSFUNCTION (E.G., INSULITIS OR Β-CELL LOSS). AIM 3 WILL EXPLORE THE FUNCTIONAL SIGNIFICANCE OF IDENTIFIED MOLECULAR MEDIATORS THROUGH ASSESSING THEIR ROLES IN CELLULAR COMMUNICATION AND BETA CELL SURVIVAL USING HUMAN ISLET/Β-CELLS AND ACINAR CELL CO-CULTURES. OUR APPROACH IS ENABLED BY MASS SPECTROMETRY-BASED SPATIAL PROTEOMICS, SPATIAL TRANSCRIPTOMICS, AND MASS SPECTROMETRY IMAGING (MSI) FOR LIPIDOMICS AND EXTRACELLULAR MATRIX (ECM)-OMICS. STATEMENT OF IMPACT: WE ANTICIPATE THIS PROJECT WILL ESTABLISH FIRST-OF-ITS-KIND SPATIAL MOLECULAR RESOURCES ON HUMAN ISLET MICROENVIRONMENTS IN EARLY STAGES OF T1D DEVELOPMENT, PROVIDE EVIDENCE OF NOVEL IN SITU MOLECULAR MEDIATORS AND BIOLOGICAL PROCESSES FROM HUMAN PATIENT SAMPLES, AND CONTRIBUTE TOWARDS THE DEVELOPMENT OF NOVEL THERAPEUTIC INTERVENTION STRATEGIES. THESE INNOVATIVE APPROACHES AND DATA RESOURCES WILL ALSO LIKELY ENHANCE THE OVERALL IMPACT OF THE CURRENT RESOURCES FROM THE HUMAN ISLET RESEARCH NETWORK (HIRN) AND ENABLE FURTHER COLLABORATIONS WITH OTHER INVESTIGATORS WITHIN THE T1D COMMUNITY.

REVERSE SENSITIVITY ANALYSIS FOR IDENTIFYING PREDICTIVE PROTEOMICS SIGNATURES OF CANCER

HIV PROTEOMIC CENTER FOR HOST-VIRAL RESPONSE CHARACTERIZATION

SINGLE CELL RESOLUTION OMICS ANALYSIS OF T1D ISLETS

NEXT-GENERATION ULTRA-HIGH RESOLUTION ION MOBILITY SPECTROMETER FOR METABOLOMICS - METABOLISM IS ESSENTIAL TO HUMAN HEALTH AND ITS STUDY VIA METABOLOMICS IS CRUCIAL TO BIOMEDICAL RESEARCH. HOWEVER, CURRENT PLATFORMS FOR MEASURING METABOLITES THAT COMBINE CHROMATOGRAPHY SEPARATIONS WITH MASS SPECTROMETRY HAVE LIMITATIONS. THIS PROJECT AIMS TO DEVELOP A NEW PLATFORM THAT PROVIDES ULTRA-HIGH ION MOBILITY RESOLUTION AND SENSITIVITY TO SMALL CHANGES IN THE METABOLITE STRUCTURE. THE PROJECT WILL ALSO DEVELOP A HIGHLY PRECISE COLLISION CROSS-SECTION CALCULATOR THAT UTILIZES THE NEW PLATFORM'S ULTRA-HIGH-RESOLUTION DATA. THESE DEVELOPMENTS WILL ENABLE HIGHER QUALITY AND MORE COMPREHENSIVE METABOLOMICS MEASUREMENTS BY DISTINGUISHING BETWEEN CHALLENGING ISOMERS SUCH AS ISOTOPOMERS. THE KEY DEVELOPMENTS WILL BE THE IMPLEMENTATION OF GREATLY IMPROVED ION MOBILITY SEPARATIONS AND HIGHLY PRECISE PROPERTY PREDICTIONS. THESE DEVELOPMENTS WILL PROVIDE MUCH GREATER RESOLUTION, HIGHER SPEED, BROADER APPLICABILITY TO METABOLITE CHEMICAL CLASSES, AND IMPROVED SENSITIVITY AND MEASUREMENT DYNAMIC RANGE IN CONJUNCTION WITH MASS SPECTROMETRY. THE NEW PLATFORM DEVELOPMENT EFFORTS WILL INCLUDE THE ESSENTIAL PLATFORM CONTROL AND DATA ACQUISITION CAPABILITIES, RESULTING IN A PLATFORM READY FOR INITIAL METABOLOMICS APPLICATIONS AND DISSEMINATION.

REGULATORY NETWORKS AND BIOMARKERS OF BETA-CELL DYSFUNCTION AND APOPTOSIS

DETECTION OF TOXINS: A COMBINED ELISA MICROARRAY AND ACTIVITY ASSAY PLATFORM

ULTRA-HIGH RESOLUTION GAS PHASE SEPARATION AND ANALYSIS PLATFORM FOR METABOLOMICS

INTEGRATED RISK ASSESSMENT AND MOLECULAR CHARACTERIZATION OF PULMONARY RESPONSE TO E-CIGARETTE EXPOSURE

A PROTEOMICS PLATFORM FOR QUANTITATIVE, ULTRA-HIGH THROUGHPUT, AND ULTRA-SENSITIV

COMPUTATIONAL TOOLS FOR ILLUMINATING THE DARK MATTER OF THE HUMAN VIROME - PROJECT SUMMARY AS GREATER UNDERSTANDING OF THE IMPORTANT ROLE THAT THE HUMAN MICROBIOME PLAYS IN HUMAN HEALTH HAS UNFOLDED IT IS INCREASINGLY RECOGNIZED THAT THERE IS A VAST AND UNCHARTED GROUP OF VIRUSES THAT HAVE DIRECT AND IMPORTANT INFLUENCE ON THE COMPOSI�ON, DYNAMICS, AND FUNC�ON OF THESE MICROBIAL COMMUNI�ES, AND THUS ON THE HEALTH OF THE HUMAN HOST. METAGENOME SEQUENCING EFFORTS AND OTHER HIGH-THROUGHPUT METHODOLOGIES HAVE OPENED THE DOOR TO LARGE- SCALE CATALOGUING AND CHARACTERIZA�ON OF FREE-LIVING VIRUSES AND PROPHAGE, WHICH ARE RESIDENT IN THEIR BACTERIAL HOST GENOMES. HOWEVER, THE DIVERSITY AND LACK OF KNOWLEDGE ABOUT THESE VIRUSES HAMPERS OUR ABILITY TO UNDERSTAND THEIR POTEN�AL IMPACT. ILLUMINA�NG THE REGION OUTSIDE OF OUR CURRENT KNOWLEDGE, SO-CALLED ‘DARK MATER’, IS NECESSARY TO BE ABLE TO UNDERSTAND, MONITOR, CONTROL, AND U�LIZE VIRUS-COMMUNITY INTERAC�ONS TO PROMOTE HUMAN HEALTH. WE HYPOTHESIZE THAT APPLICA�ON OF ESTABLISHED AND NEWLY DEVELOPED PIPELINES USING THESE AND OTHER TOOLS WILL ALLOW VASTLY IMPROVED IDEN�FICA�ON AND CHARACTERIZA�ON OF VIRAL ELEMENTS THAT ARE IMPAC�NG HUMAN MICROBIOME DYNAMICS, AND THAT PROVIDING IMPROVED TOOLS FOR VISUALIZING THESE PREDIC�ONS AND THE RELA�ONSHIPS BETWEEN THEM WILL HAVE A BROAD IMPACT ON THE FIELD. TO ADDRESS THESE GAPS WE WILL DEVELOP ENHANCED METHODS FOR DETEC�ON OF VIRAL SEQUENCES USING GRAPH ASSEMBLIES, BUILD A SEQUENCE- AND STRUCTURE-BASED PIPELINE FOR CHARACTERIZA�ON OF AUXILIARY GENES CARRIED BY VIRUSES, AND PROVIDE NOVEL AND USEFUL WAYS OF VISUALIZING THIS COMPLEX INFORMA�ON TO ALLOW GREATER INSIGHT FROM RESEARCHERS IN THE FIELD.

UNDIAGNOSED DISEASES NETWORK METABOLOMICS CORE SUPPLEMENT

MULTIPLEX SENSOR PLATFORM FOR BIOLOGICAL MONITORING

THE IMPACT OF BRAIN LIPOPROTEIN STRUCTURE AND COMPOSITION ON AMYLOID BETA METABOLISM AND ALZHEIMER'S DISEASE - PROJECT SUMMARY/ABSTRACT ALZHEIMER'S DISEASE (AD) IS A DEBILITATING BRAIN DISORDER CHARACTERIZED BY MEMORY LOSS AND COGNITIVE DECLINE, AFFECTING MILLIONS WORLDWIDE. THE INTRICATE INTERPLAY BETWEEN LIPID METABOLISM AND AD PATHOLOGY IS INCREASINGLY RECOGNIZED, AND BRAIN LIPOPROTEINS (BLPS) ARE THOUGHT TO BE KEY PLAYERS GIVEN THEY SERVE AS THE PRIMARY LIPID TRANSPORT VEHICLES IN THE CENTRAL NERVOUS SYSTEM (CNS). BLPS RESIDE IN THE CEREBROSPINAL FLUID (CSF) THAT BATHES THE BRAIN BUT UNFORTUNATELY, DUE TO THEIR LOW ABUNDANCE AND LIMITED ACCESSIBILITY, A COMPREHENSIVE UNDERSTANDING OF BLPS HAS REMAINED ELUSIVE. WE DEVELOPED A FLUORESCENT LIPOPROTEIN PROFILING (FLP) TECHNOLOGY THAT HURDLES THIS ABUNDANCE PROBLEM TO DEEPLY CHARACTERIZE BLPS IN SMALL VOLUMES OF CSF. USING OUR FLP AND MASS SPECTROMETRY (LC-MS/MS), WE REVEALED THAT BLPS CONSIST OF A SPECTRUM OF DISCRETELY SIZED SUBPOPULATIONS THAT EXHIBIT REMARKABLE PROTEOMIC DIVERSITY. WE POSIT THAT THE DOMINANT ORGANIZING SCAFFOLD ON BLPS, APOLIPOPROTEIN (APO)E, ACTS AS A MASTER REGULATOR OF BLP COMPOSITION BY ADOPTING DISTINCT STRUCTURAL CONFORMATIONS TO FACILITATE DOCKING WITH COMPLEMENTARY PARTNERS THAT IMPART UNIQUE FUNCTIONS. APOE HAS THREE ISOFORMS—E2, E3, AND E4 DIFFERENTIATED BY CYSTEINE-ARGININE MUTATIONS AT RESIDUES 112 AND 158 AND CARRIERS OF APOE4 HAVE INCREASED AMYLOID PLAQUE BURDEN, AND SUBSTANTIALLY INCREASED LIFETIME RISK FOR DEVELOPING AD. WE HYPOTHESIZE THAT CONFORMATIONAL DIFFERENCES BETWEEN APOE ISOFORMS ALTER BLP COMPOSITIONAL SIGNATURES AND THEIR ABILITY TO BIND AND TRAFFIC AMYLOID BETA (AΒ), THE BUILDING BLOCK OF AMYLOID PLAQUES. OUR OBJECTIVES ARE TWO-FOLD: FIRST, TO ASSESS THE IMPACT OF APOE ISOFORMS ON BLP DISTRIBUTION, COMPOSITION, AND ABILITY TO BIND AΒ. TO DO SO, WE WILL PERFORM A COMPARATIVE ANALYSIS OF BLP PATTERNS IN INDIVIDUALS WITH DIFFERENT APOE GENOTYPES AND LEVELS OF COGNITIVE HEALTH USING OUR FLP AND LC-MS/MS, SHEDDING LIGHT ON THE AD-RELEVANT BLP SUBSPECIES. SECOND, WE AIM TO UNRAVEL THE STRUCTURAL DETAILS OF LIPID-BOUND APOE ISOFORMS THAT GOVERN THEIR INTERACTIONS WITH AΒ. USING CRYO-EM AND NOVEL STRUCTURAL PROTEOMICS APPROACHES, WE WILL DERIVE HIGH-RESOLUTION STRUCTURAL MAPS OF AΒ COMPLEXED WITH DIFFERENT APOE ISOFORMS ON RECONSTITUTED HDL AND DETERMINE HOW “ACCESSORY” PROTEINS FOUND ON BLPS MODULATE THEIR INTERACTION WITH AΒ. THESE STUDIES WILL PROVIDE A DETAILED MOLECULAR MAP OF BLP METABOLISM IN THE CNS WHICH CAN BE LEVERAGED FOR THE DEVELOPMENT OF TARGETED THERAPEUTIC APPROACHES TO TREAT AND PREVENT AD.

MULTISCALE MODELING OF CIRCADIAN RHYTHMS

PLATFORM PROVIDING INCREASED THROUGHPUT, SENSITIVITY AND SPECIFICITY FOR METABOLO

A UNIVERSAL MULTIPLEX ASSAY SYSTEM FOR HIGH-THROUGHPUT CLINICAL APPLICATIONS

RAPID ASSAY FOR INTERNALIZED RADIONUCLIDES WITH ADVANCED MATERIALS AND METHODS

PROTEOMICS STUDIES OF TYPE 2 DIABETES USING KNOCKOUT MOUSE MODELS

INNOVATIVE BIOMONITORING DEVICE FOR RAPID DIAGNOSIS OF EXPOSURE TO NERVE AGENTS

KEY EVENTS IN MODULATION OF LUNG INFECTION SUSCEPTIBILITY BY NANOMATERIALS

MASSIVE SINGLE CELL PROTEOMICS FOR CANCER BIOLOGY - PROJECT SUMMARY/ABSTRACT SINGLE-CELL TECHNOLOGIES HAVE BECOME THE CORNERSTONE OF BIOMEDICAL AND CELL BIOLOGY RESEARCH. NEXT- GENERATION SEQUENCING-BASED TECHNOLOGIES HAVE ENABLED LARGE-SCALE CHARACTERIZATION OF TRANSCRIPT EXPRESSIONS IN SINGLE CELLS FROM CLINICAL SPECIMENS AND REVEAL UNEXPECTED CELLULAR HETEROGENEITY RELATED TO PATHOGENESIS. HOWEVER, MANY INTEGRATIVE STUDIES HAVE SHOWN ONLY LOW TO MODERATE CORRELATIONS BETWEEN THE ABUNDANCE OF RNA TRANSCRIPTS AND THEIR CORRESPONDING PROTEINS, THE MAIN DETERMINANTS OF CELL PHENOTYPE. WE HYPOTHESIZE MASS SPECTROMETRY-BASED SINGLE-CELL PROTEOMICS COULD PROVIDE DIRECT INSIGHT ON THE CELLULAR HETEROGENEITY AND INFORM PROTEIN MARKERS RELATED TO DISEASE PROGRESSION AND RESISTANCE TO THERAPY. THE OVERALL OBJECTIVE OF THIS PROJECT IS TO DEVELOP A HIGH THROUGHPUT SINGLE-CELL PROTEOMICS (SCPROTEOMICS) PLATFORM TO ENABLE THE ROUTINE ANALYSIS OF >10,000 SINGLE CELLS AT A DEPTH OF 2000 PROTEINS IN A COST-EFFICIENT WAY. THE DEVELOPED TECHNOLOGY WILL BE DISSEMINATED TO THE RESEARCH COMMUNITY THROUGH CLOSE COLLABORATION WITH A COMMERCIAL PARTNER. WE WILL ALSO APPLY SCPROTEOMICS TO INTERROGATE THE HETEROGENEITY OF BOTH MALIGNANT PLASMA CELL AND IMMUNE CELL POPULATIONS FROM MULTIPLE MYELOMA PATIENTS. WE WILL PURSUE THESE GOALS THROUGH THREE SPECIFIC AIMS: 1) TO ESTABLISH AN ULTRA-HIGH THROUGHPUT SINGLE-CELL PREPARATION METHOD BY COUPLING AN ENHANCED MULTIPLEXING METHOD WITH HIGH-DENSITY NESTED NANOPOTS CHIPS AND MULTI-CHANNEL DROPLET DISPENSING SYSTEM; WE AIM TO PROCESS >2000 CELLS IN A SINGLE MICROCHIP, AND MULTIPLEX-LABEL 36 SINGLE CELLS FOR A SINGLE LC-MS ANALYSIS; 2) TO ADVANCE THE THROUGHPUT, SENSITIVITY, AND QUANTITATION ACCURACY OF LC-MS SYSTEM. A DUAL-COLUMN NANOLC SYSTEM AND A FAIMS-BASED MS ACQUISITION METHOD WILL BE DEVELOPED TO ENABLE THE ANALYSIS OF >860 CELLS PER DAY WITH HIGH QUANTITATION PRECISION; 3) TO APPLY SCPROTEOMICS TO PROFILE ~10,000 PLASMA AND IMMUNE CELLS FROM MM PATIENTS. WE WILL INTEGRATE SCPROTEOMICS WITH EXISTING SCRNA-SEQ DATA TO EXPLORE TUMOR HETEROGENEITY, CHIMERIC ANTIGEN RECEPTOR T-CELLS (CAR-T) MARKERS, AND THE IMMUNE MICROENVIRONMENT IN MULTIPLE MYELOMA. THIS RESEARCH IS HIGHLY INNOVATIVE BECAUSE THE PROPOSED SINGLE-CELL PROTEOMICS PLATFORM WILL BE THE FIRST OF ITS KIND TO ROUTINELY AND RELIABLY CHARACTERIZE > 10,000 SINGLE CELLS AT A THROUGHPUT COMPARABLE TO SINGLE-CELL TRANSCRIPTOMICS. IT IS ALSO THE FIRST SCPROTEOMICS STUDY OF PRIMARY LIQUID TUMOR CELLS ISOLATED FROM THE PATHOLOGICAL ENVIRONMENT, E.G. BONE MARROW OF MM PATIENTS. STATEMENT OF IMPACT: TUMOR HETEROGENEITY HAS INDISPENSABLE IMPLICATIONS IN CANCER EVOLUTION, TUMORAL SPATIAL ORGANIZATION, AND CLINICAL TREATMENT. SINGLE-CELL PROTEOMICS COULD PROVIDE A BASIS TO UNRAVEL THESE COMPLICATED RELATIONSHIPS AND TO CLARIFY THE MECHANISMS OF CANCER PROGRESSION AND SUBCLONE RESISTANCE TO THERAPEUTIC TREATMENTS.

PROHORMONE PROCESSING PRODUCTS AS MECHANISTIC INDICATORS OF ISLET CELL DYSFUNCTION DURING THE NATURAL HISTORY OF TYPE 1 DIABETES - PROJECT SUMMARY/ABSTRACT: TYPE 1 DIABETES (T1D) IS A CHRONIC AUTOIMMUNE DISORDER THAT IS CHARACTERIZED BY THE TARGETED DESTRUCTION OF THE INSULIN-PRODUCING Β-CELLS IN THE PANCREAS. EMERGING EVIDENCE SUGGESTS THAT T1D PATHOGENESIS INVOLVES PROGRESSIVE ISLET CELL DYSFUNCTION, INCLUDING NOT ONLY Β-CELLS, BUT ALSO Α-CELLS AND Δ-CELLS. AS A RESULT, THE DEVELOPMENT OF MINIMALLY INVASIVE BLOOD-BASED MECHANISTIC MARKERS THAT CAN EVALUATE Α-CELL AND Β-CELL (DYS)FUNCTIONS IN VIVO IS CRITICAL FOR UNDERSTANDING THE DISEASE TRAJECTORY AND ASSESSING TREATMENT EFFICACY. THE OVERALL OBJECTIVE HERE IS TO IDENTIFY AND VERIFY THE INTERMEDIATE PRODUCTS OF ISLET PROHORMONE PROCESSING AS NOVEL MARKERS OF ISLET CELL DYSFUNCTION (INCLUDING Α- AND Β-CELLS) CIRCULATING IN THE BLOOD THROUGHOUT THE PROGRESSION OF T1D. THIS GOAL IS BUILT UPON EMERGING DATA ON ALTERED PROHORMONE PROCESSING DURING DIFFERENT STAGES OF T1D AND THE UNIQUE TISSUE-SPECIFIC CHARACTERISTICS OF ISLET PROHORMONES, MAKING THEM IDEAL FOR SERVING AS CELL-TYPE-SPECIFIC MARKERS IN CIRCULATION. WE HYPOTHESIZE THAT INTERMEDIATE PROHORMONE PROCESSING PRODUCTS DISPLAY DISTINCT AND DYNAMIC PATTERNS IN CIRCULATION DURING T1D PROGRESSION AND CAN SERVE AS MECHANISTIC INDICATORS OF ISLET CELL DYSFUNCTION. TO TEST THIS, OUR OVERALL STRATEGY CONSISTS OF FOUR MAIN COMPONENTS: 1) IDENTIFYING PROHORMONE PROCESSING PRODUCTS WITHIN PANCREATIC ISLET TISSUE USING ADVANCED PROTEOMICS; 2) DEVELOPING AN INNOVATIVE, HIGH- THROUGHPUT, MULTIPLEXED MASS SPECTROMETRY-BASED ASSAY FOR ENABLING SIMULTANEOUS MONITORING OF THESE PRODUCTS IN BLOOD CIRCULATION; 3) EXAMINING THE POTENTIAL OF THESE MARKERS AS MECHANISTIC INDICATORS FOR ISLET CELL DYSFUNCTION; 4) EXPLORING THEIR POTENTIAL PREDICTIVE VALUES FOR T1D PROGRESSION. SPECIFICALLY, AIM 1 WILL FOCUS ON THE DISCOVERY OF IN SITU ISLET PROHORMONE PROCESSING PRODUCTS FROM HUMAN ISLET TISSUE SPECIMEN AND INVESTIGATING THE UNDERLYING MECHANISMS OF ALTERED PROHORMONE PROCESSING USING ADVANCED TOP-DOWN AND BOTTOM-UP PROTEOMICS. THE DEVELOPMENT OF A HIGHLY MULTIPLEXED LC-MS ASSAY USING PARALLEL REACTION MONITORING (PRM) WILL ALSO BE AN IMPORTANT PART OF THIS AIM. AIM 2 WILL UTILIZE CROSS-SECTIONAL AND LONGITUDINAL CLINICAL SAMPLES FOLLOWING CLINICAL ONSET FROM VARIOUS TRIALS TO EVALUATE HOW THESE PROHORMONE PRODUCTS CHANGES OVER TIME AND THEIR POTENTIALS AS MARKERS OF ISLET CELL DYSFUNCTION BY APPLYING THE NOVEL MULTIPLEXED LC-MS ASSAY DEVELOPED IN AIM 1. AIM 3 WILL DEFINE THE RELATIONSHIP OF INTERMEDIATE PROHORMONE PRODUCTS WITH CLINICAL T1D PROGRESSION THROUGH LONGITUDINAL SAMPLES COLLECTED DURING THE DIABETES PREVENTION TRIAL (DPT-1). IMPACT STATEMENT: THIS PROJECT IS EXPECTED TO SIGNIFICANTLY DEEPEN OUR UNDERSTANDING OF ISLET CELL DYSFUNCTION IN T1D BY IDENTIFYING AND VERIFYING INTERMEDIATE PROHORMONE PRODUCTS AS NOVEL ISLET-SPECIFIC MECHANISTIC MARKERS. THESE MARKERS OFFER PROMISING CLINICAL APPLICATIONS, INCLUDING MONITORING DISEASE PROGRESSION, ASSESSING TREATMENT EFFICACY, AND OPTIMIZING TRIAL DESIGNS BY IMPROVING PATIENT STRATIFICATION. MOREOVER, THE DEVELOPMENT AND DEPLOYMENT OF A HIGHLY MULTIPLEXED LC-MS ASSAY FOR PROHORMONE PRODUCTS WILL PROVIDE A POWERFUL TOOL FOR FUTURE TRANSLATIONAL RESEARCH BY ENABLING MONITORING OF ISLET CELL FUNCTIONALITY IN INDIVIDUALS DURING THE T1D PROGRESSION.

SYSTEMATIC VALIDATION OF BIOMARKERS PREDICTIVE OF IA AND T1D AND THEIR RELATIONSHIP WITH DISEASE DEVELOPMENT - SUMMARY: TYPE 1 DIABETES (T1D) IS A METABOLIC DISEASE CURRENTLY WITHOUT A CURE THAT RESULTS IN DESTRUCTION OF INSULIN- PRODUCING Β CELLS OF THE PANCREAS VIA AN AUTOIMMUNE RESPONSE. T1D TREATMENT REQUIRES INSULIN ADMINISTRATION AND GREATLY REDUCES LIFE QUALITY AND EXPECTANCY. DEVELOPMENT AND PERSISTENCE OF ISLET AUTOIMMUNITY (IA) CAN BE DETERMINED BY CIRCULATING AUTOANTIBODIES AGAINST ISLET PROTEINS. HOWEVER, NOVEL BIOMARKERS ARE NEEDED TO: 1) BETTER UNDERSTAND THE MECHANISMS THAT TRIGGER AND DRIVE IA; AND 2) PREDICT INDIVIDUAL PROGRESSION THROUGH THE STAGES OF THE DISEASE. TO IDENTIFY NEW BIOMARKERS, WE HAVE RECENTLY COMPLETED A PLASMA PROTEOMICS ANALYSIS FOR THE ENVIRONMENTAL DETERMINANTS OF DIABETES IN THE YOUNG (TEDDY). THIS NESTED CASE-CONTROL STUDY 1 (NCC1) INCLUDED 401 CHILDREN WHO DEVELOPED IA (AT MEDIAN AGE OF 21 MONTHS) AND 94 CHILDREN WHO PROGRESSED TO CLINICAL T1D (AT MEDIAN AGE OF 29 MONTHS), MATCHED AGAINST CONTROLS. WE IDENTIFIED 376 PROTEINS AS CANDIDATE BIOMARKERS; 83 OF THESE PROTEINS WERE VALIDATED AS SIGNIFICANT PREDICTORS OF EITHER IA OR PROGRESSION TO T1D IN LONGITUDINAL ANALYSES OF 6,426 SAMPLES. WE ALSO IDENTIFIED PANELS OF PROTEINS THAT CAN PREDICT, WITH HIGH ACCURACY, DEVELOPMENT OF ISLET AUTOIMMUNITY (AUC=0.918) AND PROGRESSION TO T1D (AUC=0.871) 6 MONTHS PRIOR TO SEROCONVERSION BY MACHINE LEARNING ANALYSIS. LIMITATIONS OF THE TEDDY NCC1 INCLUDED LIMITED FOLLOW-UP AND VERY YOUNG AGE (≤6 YEARS) OF THE PARTICIPANTS. IA AND PROGRESSION TO T1D CAN OCCUR OVER A MUCH BROADER AGE RANGE, AND THE CELLULAR AND SYSTEMIC PROCESSES INVOLVED MAY DIFFER IN YOUNGER VS OLDER CHILDREN. IN THE NEXT CASE CONTROL STUDY 2 (NCC2), TEDDY MONITORED INDIVIDUALS UNTIL 15 YEARS OF AGE, AN ADDITIONAL 501 PARTICIPANTS DEVELOPED IA (AT MEDIAN AGE OF 89 MONTHS) AND AN ADDITIONAL 345 PROGRESSED TO T1D (AT MEDIAN AGE OF 108 MONTHS). HERE WE PROPOSE TO PERFORM A LONGITUDINAL ANALYSIS OF THE NCC2 SAMPLES WITH A HIGHLY MULTIPLEXED, SENSITIVE, AND ROBUST TARGETED PROTEOMICS ANALYSIS TO SIMULTANEOUSLY VALIDATE BIOMARKERS IDENTIFIED IN THE NCC1 AND TO PROVIDE INFORMATION ABOUT THE BIOLOGICAL PROCESSES REGULATED DURING DISEASE DEVELOPMENT. MORE SPECIFICALLY, WE PROPOSE THE FOLLOWING SPECIFIC AIMS: (I) VALIDATE PREDICTIVE BIOMARKERS OF ISLET AUTOIMMUNITY; (II) VALIDATE PREDICTIVE BIOMARKERS OF PROGRESSION TO T1D; AND (III) DETERMINE MECHANISMS UNDERLYING DEVELOPMENT OF IA AND PROGRESSION TO T1D. TO FULFILL THESE AIMS WE WILL PERFORM A LARGE-SCALE TARGETED PROTEOMICS ANALYSIS OF 8 TIME POINTS FROM 48 MONTHS PRE-SEROCONVERSION TO 12 MONTHS POST-SEROCONVERSION OF INDIVIDUALS THAT ONLY DEVELOPED ISLET AUTOIMMUNITY AND INDIVIDUALS THAT DEVELOPED CLINICAL DIABETES, PAIRED WITH CONTROLS, TOTALING 6,400 SAMPLES. THE RESULTS OF THIS PROJECT SHOULD BRING THE FIELD ONE STEP CLOSER TO DEVELOPING CLINICAL BIOMARKERS AND ASSOCIATED ASSAYS THAT CAN PREDICT ALL PHASES OF T1D DEVELOPMENT IN ADDITION TO IDENTIFYING POSSIBLE CAUSES. THIS KNOWLEDGE GAINED WILL LIKELY OPEN OPPORTUNITIES TO DEVELOP PREVENTIVE INTERVENTIONS AGAINST T1D.

HIGHLY SELECTIVE MACROBIOMOLECULAR ISOLATION, SOFT-LANDING AND CHARACTERIZATION USING STRUCTURES FOR LOSSLESS ION MANIPULATIONS - PROJECT SUMMARY/ABSTRACT (30 LINES) THIS PROJECT AIMS TO ADDRESS THE CURRENT GAP IN TECHNOLOGY FOR RAPID AND COMPREHENSIVE (MASS, STRUCTURE) PROFILING OF BIOMACROMOLECULES. WHILE HIGH RESOLUTION ION MOBILITY AND MASS SPECTROMETRY SEPARATIONS TECHNIQUES HAVE BEEN EXTENSIVELY DEVELOPED, THEIR APPLICATION HAS BEEN LARGELY LIMITED TO SMALL MOLECULES (M/Z < 2000). THE ABILITY TO ACCESS STRUCTURE AND MASS INFORMATION IN A HIGH-THROUGHPUT FASHION FOR MACROBIOMOLECULES IS AN ASPIRATIONAL GOAL IN MASS SPECTROMETRY. IN PURSUANCE OF THIS GOAL, WE WILL DEVELOP A HIGH-RESOLUTION ION MOBILITY CHEMICAL ANALYSIS DEVICE BASED ON STRUCTURES FOR LOSSLESS ION MANIPULATIONS, AND IN CONJUNCTION WITH MASS SPECTROMETRY OBTAIN HIGH-THROUGHPUT HIGH-RESOLUTION IMS/MS OF BIOMACROMOLECULES. THE STUDY OF THE SIGNIFICANT HETEROGENEITY IN LARGE BIOMOLECULAR SYSTEMS, I.E., MACROBIOMOLECULES, CRITICAL TO BIOLOGICAL RESEARCH WILL BE ENABLED BY THE DEVELOPMENT OF A NEW HIGH RESOLUTION, HIGH THROUGHPUT ANALYTICAL SYSTEM. THE SLIM PLATFORM, CURRENTLY ENABLED FOR ULTRA-HIGH RESOLUTION ION MOBILITY PROFILING (UP TO THE POINT OF SEPARATING ISOTOPOMERS AND ISOTOPOLOGUES) AND MANIPULATIONS OF SMALL MOLECULAR IONS (<3000 DALTONS) WILL BE OPTIMIZED TO EFFICIENTLY CONFINE, TRANSPORT AND SEPARATE MACROBIOMOLECULES. INITIAL OPTIMIZATION OF THE SLIM WILL BE DONE BY COUPLING WITH A TOF-MS SYSTEM. SLIM-TOF WILL BE USED TO PERFORM HIGH RESOLUTION IMS/MS OF ENSEMBLES OF MACROBIOMOLECULAR SPECIES (WITH AN UPPER MASS LIMIT OF ABOUT 20,000 DA DICTATED BY THE TOF). WE WILL USE SLIM-BASED IMS SEPARATIONS AND OTHER MANIPULATIONS TO REVEAL THE EXTENSIVE STRUCTURAL HETEROGENEITY OF MACROBIOMOLECULES. FURTHER, WE WILL PERFORM SIZE AND MASS SELECTIVE SOFT-LANDING OF MACROBIOMOLECULES USING THE DYNAMIC SWITCHING AND REROUTING (TO DEPOSITION SUBSTRATES) OF A SELECTIVE ION MOBILITY DISTRIBUTION IN THE SLIM. THE SOFT-LANDED SPECIES WILL BE CHARACTERIZED USING CRYO-EM AND COMPLIMENTARY IMAGING TECHNIQUES TO ELUCIDATE THE MORPHOLOGIES OF MACROBIOMOLECULES WITH SELECTED MOBILITIES OR COLLISION-CROSS-SECTIONS. THE COMBINATION OF ION MOBILITY INFORMATION AND IMAGING WILL PROVIDE A DETAILED STRUCTURAL CHARACTERIZATION OF MACROBIOMOLECULES.

SERUM PROTEIN BIOMARKERS FOR PREDICTING TYPE 1 DIABETES

A MULTIMODAL MEASUREMENT PLATFORM FOR UNAMBIGUOUS LIPID IDENTIFICATION - PROJECT SUMMARY FREE FATTY ACIDS (FFAS) AND LIPIDS ARE IMPORTANT BIOLOGICAL COMPOUNDS THAT ARE INVOLVED IN ALMOST EVERY BIOLOGICAL PROCESS. STUDYING THE LIPIDOME CAN PROVIDE INFORMATION ON THE ROLES OF LIPIDS, RANGING FROM NUTRIENT ABSORPTION AND CELL SIGNALING TO CANCER METASTASIS. HOWEVER, IDENTIFYING THE COMPLETE CHEMICAL STRUCTURES OF MANY LIPIDS IS A LONG-STANDING ANALYTICAL CHALLENGE. LIPIDS POSSESS AN ENORMOUS NUMBER OF ISOFORMS (COMPOUNDS WITH THE SAME CHEMICAL FORMULA BUT DIFFERENT ARRANGEMENTS OF ATOMS), BUT CURRENT ANALYTICAL TECHNOLOGY STRUGGLES TO DISTINGUISH MANY OF THEM. THIS MEANS MANY LIPID SIGNATURES REMAIN UNCHARACTERIZED AND THEIR BIOMEDICAL ACTIVITIES REMAIN UNKNOWN. THIS PROJECT WILL ADDRESS THIS SIGNIFICANT GAP IN LIPIDOMICS RESEARCH BY DEVELOPING A NOVEL MULTIMODAL SYSTEM THAT WILL BE USED TO UNAMBIGUOUSLY IDENTIFY FFAS AND LIPIDS IN COMPLEX BIOMEDICALLY- RELEVANT SAMPLES. THIS WILL BE ACCOMPLISHED BY USING A NOVEL HIGH-RESOLUTION ION MOBILITY-MASS SPECTROMETRY (IM-MS) PLATFORM EMPLOYING TWO DIFFERENT TYPES OF FRAGMENTATION. A NOVEL HIGH-RESOLUTION IM MODULE BASED ON 'TRAVELING CUPS' (TERMED CUPPED IMS) WILL PROVIDE NEW STATE-OF-THE-ART IM SEPARATIONS OF FFA AND LIPID ISOMERS, MANY OF WHICH ARE NOTORIOUSLY DIFFICULT TO SEPARATE DUE TO THEIR HIGH STRUCTURAL SIMILARITY. COLLISION CROSS SECTION (CCS) MEASUREMENTS GAINED FROM SUCH HR-IMS SEPARATIONS WILL PROVIDE A KEY PHYSICAL METRIC BY WHICH TO DIFFERENTIATE THESE STRUCTURALLY SIMILAR FFAS AND LIPIDS. A NEW ULTRAVIOLET PHOTODISSOCIATION CAPABILITY WILL BE INSTALLED INTO AN ORBITRAP MASS SPECTROMETER TO PROVIDE INFORMATION ON THE LOCATIONS OF LIPID DOUBLE BONDS ALONG ALKYL CHAINS, WHICH IS OFTEN MISSING IN GOLD-STANDARD MS-BASED WORKFLOWS. THE ORBITRAP-MS WILL ALSO PROVIDE HIGH-RESOLUTION MASS MEASUREMENTS, WHICH CAN BE USED TO DERIVE THE CHEMICAL FORMULAE OF FFAS AND LIPIDS. HAVING CHEMICAL FORMULA INFORMATION IS AN ESSENTIAL FIRST STEP WHEN BEGINNING TO DETERMINE THE STRUCTURE OF AN UNKNOWN LIPID. THE ORBITRAP WILL ALSO PROVIDE COLLISION INDUCED DISSOCIATION CAPABILITIES, WHICH ARE USEFUL FOR IDENTIFYING TO WHICH CLASS OF LIPIDS AN UNKNOWN COMPOUND BELONGS. NEW DATA-DEPENDENT ACQUISITION (DDA) AND DATA-INDEPENDENT ACQUISITION (DIA) METHODS WILL BE DEVELOPED TO ENABLE AUTOMATED ACQUISITION OF MS/MS SPECTRA FROM THE NEW MULTIMODAL SYSTEM, AND INFORMATICS WORKFLOWS WILL BE DEVELOPED TO LEVERAGE DATA PROCESSING SOFTWARE ALREADY CAPABLE OF READING THERMO-FISHER .RAW FILES. A NOVEL UVPD INFORMATICS CAPABILITY WILL BE DEVELOPED TO INTERPRET THE MULTIMODAL DATA FROM THIS NEW SYSTEM. THE DATA WILL BE BENCHMARKED AGAINST GOLD-STANDARD LC-MS/MS LIPIDOMICS WORKFLOWS TO SHOW SIGNIFICANTLY IMPROVED LIPID IDENTIFICATIONS FROM LIPID EXTRACTS OF HELA CELLS, WHICH HAS TREMENDOUS IMPLICATIONS IN DISEASES LIKE BREAST CANCER. THE DISCOVERIES MADE BY THE MULTIMODAL SYSTEM PROPOSED HERE WILL SIGNIFICANTLY ADVANCE THE FIELD OF LIPIDOMICS RESEARCH. FURTHERMORE, THIS RESEARCH WILL SHOW THAT MULTIMODAL SYSTEMS ARE CRITICAL TOOLS THAT WILL ONE DAY BE USED IN GENERAL STRUCTURAL IDENTIFICATION WORKFLOWS WHERE REFERENCE DATA FROM AUTHENTIC COMPOUNDS ARE NOT AVAILABLE.

SLOW-MAS NMR METABOLOMICS

A NOVEL HIGH RESOLUTION MS PLATFORM FOR HIGH-THROUGHPUT SCREENING OF G PROTEIN-COUPLED RECEPTORS - SUMMARY: MASS SPECTROMETRY (MS) HAS PROVEN INVALUABLE IN STUDYING THE MECHANISMS OF CELLULAR SIGNALING AS MS PLATFORMS CAN DIRECTLY PROVIDE AMINO ACID RESIDUE SITE-SPECIFIC PHOSPHORYLATION DATA COMPARED TO TRADITIONAL ANTIBODY-BASED APPROACHES. HOWEVER, LIMITATIONS EXIST IN CURRENT MS APPROACHES IN GENERATING CONFIDENT SITE-SPECIFIC PHOSPHORYLATION QUANTIFICATION. THIS IS PARTICULARLY EVIDENT IN COMPLEX MULTI-PHOSPHORYLATED PROTEIN MOTIFS, WHERE THE DETECTION OF ISOMERIC MULTI-PHOSPHORYLATED PEPTIDES EASILY OVERWHELMS ANY PREDICTION SCORING APPROACH THAT IS SIMPLY BASED UPON THE FRAGMENTATION SPECTRA. THERE ARE MANY BIOLOGICAL EXAMPLES OF HYPERPHOSPHORYLATED REGIONS, WHERE THEY ARE ASSOCIATED WITH RECEPTOR/LIGAND INTERACTIONS, INCLUDING G-PROTEIN COUPLED RECEPTORS (GPCRS), MEMBRANE RECEPTORS THAT ARE THE MOST COMMON TARGETS FOR FDA-APPROVED DRUGS. FOR ACCURATE SITE-SPECIFIC QUANTIFICATION OF PROTEIN HYPERPHOSPHORYLATION WE PROPOSE A TRANSDISCIPLINARY APPROACH USING ULTRAHIGH RESOLUTION ION MOBILITY SEPARATION (IMS) INTEGRATED WITH HIGHLY ACCURATE AND SENSITIVE MS AND MS/MS SPECTRA TO ENABLE THE CONFIDENT CHARACTERIZATION OF HYPERPHOSPHORYLATED GPCR ENSEMBLES WITH GREATLY IMPROVED SENSITIVITY, AND SPEED. WE WILL USE MULTI-LEVEL STRUCTURES FOR LOSSLESS ION MANIPULATIONS (SLIM) TECHNOLOGY (SLIM-ORBITRAP PLATFORM) TO FULLY CHARACTERIZE PHOSPHORYLATION OF GPCR/ANTAGONIST INTERACTIONS UTILIZING CXCR3, WHICH PLAYS A CENTRAL ROLE IN INFLAMMATORY DISEASES THROUGH ITS REGULATION OF T CELL FUNCTION AS AN INITIAL TEST CASE. WE PLAN TO FIRST INTEGRATE ULTRAHIGH RESOLUTION IMS WITH AN ADVANCED ORBITRAP MS PLATFORM FOR UNAMBIGUOUS DECODING OF HYPERPHOSPHORYLATED SITES, EVALUATE THE SLIM-ORBITRAP MS PLATFORM FOR RESOLVING HYPERPHOSPHORYLATED PROTEIN REGIONS, AND FINALLY, PERFORM COMPREHENSIVE SITE-SPECIFIC PHOSPHOPROTEOMICS FOR GPCRS THROUGH SCREENING OF ACTIVATED T CELLS WITH DOSE-RESPONSES OF CHEMOKINE AND SMALL-MOLECULE CXCR3 BIASED AGONISTS.

ENZYME ACTIVITY ENHANCEMENT IN FUNCTIONALIZED NANOPOROUS SUPPORT

TARGETED PROTEOMICS TECHNOLOGY FOR ACCURATE QUANTITATIVE SINGLE-CELL PROTEOMICS - ABSTRACT MULTI-OMICS CHARACTERIZATION OF A BROAD SPECTRUM OF SMALL SUBPOPULATIONS OF CELLS BETWEEN TUMORS AND WITHIN INDIVIDUAL TUMORS AT THE SINGLE-CELL RESOLUTION IS CRUCIAL TO ACHIEVE UNDERSTANDING OF A COMPLETE DISEASE BIOLOGY. FURTHERMORE, BIOLOGICALLY IMPORTANT CLINICAL SPECIMENS ARE AVAILABLE IN LOW QUANTITY (E.G., <10 TUMOR CELLS), REQUIRING ADVANCED SINGLE-CELL TECHNOLOGIES FOR EFFECTIVE ANALYSIS. HOWEVER, SINGLE-CELL PROTEOMICS TECHNOLOGIES ARE LAGGING FAR BEHIND OTHER OMICS TECHNOLOGIES. ANTIBODY-BASED IMMUNOASSAYS ARE USED PRIMARILY FOR TARGETED SINGLE-CELL PROTEOMICS, BUT THEY HAVE INHERENT LIMITATIONS (E.G., LOW MULTIPLEX), AND GENERALLY LACK QUANTITATION ACCURACY. MASS SPECTROMETRY (MS)-BASED TARGETED PROTEOMICS HAS EMERGED AS AN ALTERNATIVE FOR BROAD ACCURATE QUANTIFICATION. HOWEVER, CURRENT SINGLE-CELL MS CAN ONLY ALLOW FOR RELATIVE QUANTIFICATION OF ~870 PROTEINS FROM SINGLE MAMMALIAN CELLS. THERE ARE THREE MAJOR CHALLENGES IN SINGLE-CELL MS FOR ACCURATE QUANTITATIVE SINGLE-CELL PROTEOMICS: 1) INEFFECTIVE PROCESSING OF SINGLE CELLS, 2) INSUFFICIENT MS SENSITIVITY AND LOW SAMPLE THROUGHPUT, AND 3) LACKING WELL-CHARACTERIZED UNIVERSAL INTERNAL STANDARD (UIS). TO ADDRESS THESE CHALLENGES, WE PROPOSE TO DEVELOP A SINGLE-CELL MS SYSTEM FOR RAPID ACCURATE ANALYSIS OF SINGLE-CELL PROTEOME. THE FEASIBILITY IS STRONGLY SUPPORTED BY OUR RECENT PROGRESS IN MANY ASPECTS OF TECHNOLOGY DEVELOPMENT (E.G., INTRODUCING THE `CARRIER' CONCEPT FOR EFFECTIVE PROCESSING OF SMALL NUMBERS OF CELLS INCLUDING SINGLE CELLS, AND DEVELOPING DISRUPTIVE MS TECHNOLOGIES TO IMPROVE MS DETECTION SENSITIVITY AND SPECIFICITY) AS WELL AS OUR EXTENSIVE EXPERIENCES IN HIGH- RESOLUTION LIQUID CHROMATOGRAPHY (LC) SEPARATION FOR SENSITIVE DETECTION AND TARGETED PROTEOMICS ANALYSIS FOR ABSOLUTE QUANTIFICATION OF SIGNALING PATHWAY PROTEINS. THE SINGLE-CELL MS SYSTEM WILL BE DEVELOPED THROUGH 1) ESTABLISHING SUPER-SILAC (STABLE ISOTOPE LABELING WITH AMINO ACIDS IN CELL CULTURE) AS BOTH PROTEOME CARRIER AND UIS, 2) INCORPORATION OF PROTEOME CARRIER SUPER-SILAC (CSILAC) INTO THE SAMPLE PREPARATION WORKFLOW FOR ROBUST PROCESSING OF SINGLE CELLS, AND 3) LEVERAGING CUTTING-EDGING LC AND MS TECHNOLOGIES DEVELOPED AT PNNL WITH INTEGRATION OF ULTRALOW-FLOW LC SEPARATION, HIGH-EFFICIENCY ION SOURCE (THE COMBINATION OF AN EMITTER ARRAY TECHNOLOGY AND SUB-AMBIENT PRESSURE IONIZATION WITH NANOELECTROSPRAY), AND ULTRAFAST HIGH-RESOLUTION ION MOBILITY SEPARATION FOR SIGNIFICANTLY IMPROVING BOTH MS SENSITIVITY AND SAMPLE THROUGHPUT. SUPER-SILAC WILL BE CHARACTERIZED AS UIS FOR ABSOLUTE QUANTIFICATION WITH CRUDE PEPTIDE STANDARDS, WHOSE PURITY WILL BE COST- EFFECTIVELY ACCURATELY DETERMINED USING A COMBINED LANTHANIDE LABELING AND ICP-MS METHOD. WITH 96-WELL PLATE-BASED CSILAC PREPARATION AND WELL-CHARACTERIZED UIS, THE NEW SINGLE-CELL MS SYSTEM IS EXPECTED TO ALLOW FOR RAPID ACCURATE QUANTIFICATION OF A LARGE FRACTION OF HUMAN PROTEOME (~60%) IN SINGLE CELLS WITH ~120 SAMPLES PER DAY. WE ANTICIPATE THAT THE NEW MS SYSTEM WILL EVENTUALLY BECOME A CONVENIENT INDISPENSABLE TOOL NOT ONLY FOR QUANTITATIVE SINGLE-CELL PROTEOMICS BUT ALSO FOR ROUTINE ANALYSIS OF VERY SMALL SAMPLES (E.G., RARE CELLS). IN TURN, IT WILL MAKE SUBSTANTIAL CONTRIBUTIONS TO CURRENT BIOMEDICAL RESEARCH.

ONTOLOGY-DRIVEN GENERATION AND LINKING OF PATHWAY DIAGRAMS IN STROKE PATHOBIOLOGY

SYTEMS ANALYSIS OF NANOPARTICLE BIOCOMPATIBILITY

PROTEOMIC INVESTIGATIONS OF ALCOHOLIC HEPATITIS

VALIDATION AND ADVANCED DEVELOPMENT OF EMERGING TECHNOLOGIES

INTERACTIVE INFORMATICS RESOURCE FOR RESEARCH-DRIVEN CANCER PROTEOMICS

CURRENT RISK ESTIMATES FOR MANNED SPACE FLIGHT ARE BASED PREDOMINANTLY ON HUMAN EPIDEMIOLOGY DATA FOLLOWING LOW LINEAR ENERGY (LET) RADIATION EXPOSUR

HIGH RESOLUTION HIGH THROUGHPUT PROTEOMICS PLATFORM FOR CANCER RESEARCH

MODELING THE IMPACT OF SCIENCE POLICIES ON SCIENTIFIC WORKFORCE GROWTH

MAGNESIUM NMR OF DNA REPAIR PROTEINS

HIGH THROUGHPUT SCREENS OF NOVEL RADIATION SENSITIZERS AND PROTECTORS

A STREAMLINED PLATFORM FOR PHOSPHOPROTEOME MAPPING OF HUMAN TISSUES - ABSTRACT RECENT TECHNOLOGICAL ADVANCES IN GENOMICS, TRANSCRIPTOMICS, AND PROTEOMICS ALLOW FOR RAPID GENERATION OF COMPREHENSIVE 3D-HUMAN TISSUE MAPS FOR BIOMOLECULES DNAS, RNAS, AND PROTEINS AT THE SINGLE-CELL RESOLUTION IN THE HUBMAP CONSORTIUM. HOWEVER, SINGLE-CELL TECHNOLOGIES FOR CHARACTERIZING FUNCTIONAL MODIFICATIONS ARE LAGGING FAR BEHIND BUT EQUALLY IMPORTANT AS THESE EXISTING OMICS TECHNOLOGIES. PROTEIN PHOSPHORYLATION IS ONE OF THE MOST IMPORTANT MODIFICATIONS AND OFTEN USED AS AN INDICATOR OF SIGNALING PATHWAY ACTIVATION (CELL FUNCTIONAL STATE). THE LACK OF HIGH-SPATIAL-RESOLUTION PHOSPHOPROTEOMIC CHARACTERIZATION OF HUMAN TISSUES IN THE HUBMAP CONSORTIUM REPRESENTS A SIGNIFICANT KNOWLEDGE GAP FOR ACHIEVING A MORE COMPLETE UNDERSTANDING OF HOW TISSUE HETEROGENEITY IMPACTS HUMAN HEALTH. THE OBJECTIVE OF THIS TTD APPLICATION IS TO ADDRESS THIS GAP BY DEVELOPING A CONVENIENT STREAMLINED PLATFORM FOR ENABLING AUTOMATED HIGH-RESOLUTION 3D-PHOSPHOPROTEOME MAPPING OF HUMAN TISSUES. THE PROJECT FEASIBILITY IS STRONGLY SUPPORTED BY OUR RECENT PROGRESS IN MANY ASPECTS OF TECHNOLOGY DEVELOPMENT: 1) CARRIER-ASSISTED SAMPLE PREPARATION (CASP) FOR BOTH GLOBAL AND TARGETED PROTEOMICS ANALYSIS OF 1-100 CELLS; 2) A BOOSTING TO AMPLIFY SIGNAL WITH ISOBARIC LABELING (BASIL) STRATEGY FOR HIGH-THROUGHPUT SINGLE- CELL PROTEOMICS; 3) BASIL/TIP-IMAC (IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY) FOR RAPID PHOSPHOPROTEOMIC ANALYSIS OF SMALL NUMBERS OF CELLS; 4) ADVANCED LIQUID CHROMATOGRAPHY (LC) SEPARATION AND DISRUPTIVE MASS SPECTROMETRY (MS) TECHNOLOGIES (E.G., MULTI-EMITTER ARRAY TECHNOLOGY AND SUB-AMBIENT PRESSURE IONIZATION WITH NANOELECTROSPRAY (SPIN) SOURCE) FOR IMPROVING MS DETECTION SENSITIVITY. IN THE UG3 PHASE, AIM 1 WILL FOCUS ON THE DEVELOPMENT OF A STREAMLINED PLATFORM THROUGH 1) IMPROVING PHOSPHO-RECOVERY BY DEVELOPING AN IMPROVED CASP/ONLINE IMAC PLATFORM FOR AUTOMATED PROCESSING AND PHOSPHO-ENRICHMENT, AND 2) LEVERAGING MULTIPLE DISRUPTIVE TECHNOLOGIES DEVELOPED AT OUR GROUP WITH INTEGRATION OF A HIGH-EFFICIENCY MULTI-EMITTER SPIN (MSPIN) SOURCE AND BASIL-BASED SAMPLE MULTIPLEXING FOR SIGNIFICANTLY IMPROVING MS SENSITIVITY BY ~50-FOLD AND SAMPLE THROUGHPUT BY >20-FOLD. THE STREAMLINED PLATFORM WILL ALLOW FOR PRECISE QUANTIFICATION OF ~1,000 PHOSPHOSITES IN SINGLE CELLS AND ~7000 PHOSPHOSITES IN 10 CELLS WITH >1000 SAMPLES PER DAY. AIM 2 WILL DEMONSTRATE THE STREAMLINED PLATFORM FOR ENABLING 2D-PHOSPHOPROTEOME MAPPING OF MOUSE UTERINE TISSUES WHEN COMBINED WITH LASER CAPTURE MICRODISSECTION (LCM) AND STANDARD TUBE-BASED VOXEL COLLECTION. IN THE UH3 PHASE (AIM 3) WE WILL FURTHER OPTIMIZE THE STREAMLINED PLATFORM FOR AUTOMATED ROBUST PHOSPHOPROTEOMIC ANALYSIS OF LCM-DISSECTED HUMAN TISSUE SECTIONS. WE THEN WILL VALIDATE THE STREAMLINED PLATFORM FOR HIGH-RESOLUTION 3D-PHOSPHOPROTEOME MAPPING OF HUMAN BREAST AND UTERINE TISSUES AND OTHER HUMAN TISSUES FROM THE HUBMAP CONSORTIUM. AN EASY- TO-USE VISUALIZATION TOOL WILL BE DEVELOPED TO GENERATE 3D MAPS THAT CAN BE QUICKLY AND EASILY ACCESSIBLE BY THE RESEARCH COMMUNITY. WITH ITS ANTIBODY-FREE FEATURE, THE STREAMLINED PLATFORM CAN BE EQUALLY APPLICABLE TO ANY TYPES OF TISSUES. WE ENVISION THAT THE STREAMLINED PLATFORM WILL BECOME AN INDISPENSABLE TOOL FOR HIGH-RESOLUTION 3D-PHOSPHOPEOTEOME MAPPING OF HUMAN TISSUES IN THE HUBMAP CONSORTIUM AND EXTEND THE HUBMAP TOOLBOX FOR 3D-MAPPING OF FUNCTIONAL MODIFICATIONS. IN TURN, IT WILL MAKE SUBSTANTIAL CONTRIBUTIONS TO IMPROVE OUR UNDERSTANDING OF TISSUE BIOLOGY AND ACCELERATE THE MOVEMENT TOWARD PRECISION MEDICINE.

ADV.PROTEOMICS & METABOLOMICS:TYPE 2 DIABET & PRE-DIABET

SPATIALLY RESOLVED CHARACTERIZATION OF PROTEOFORMS FOR FUNCTIONAL PROTEOMICS - PROJECT SUMMARY/ABSTARCT DIFFERENTIATED CELLS HAVE DISTINCTIVE PATTERNS OF EPIGENETIC MARKS INCLUDING VARIOUS POST-TRANSLATIONAL MODIFICATIONS (PTMS) ON HISTONES THAT MAY WORK IN CONCERT TO CONTROL TRANSCRIPTIONAL PROGRAMS. SINCE EPIGENETIC MARKS ARE OFTEN ALTERED FOLLOWING EXPOSURE TO ENVIRONMENTAL TOXINS AND PLAY MULTIPLE ROLES IN DISEASE PATHOGENESIS, THE ABILITY TO MEASURE HISTONES IN A TISSUE AND CELL CONTEXT IS A MAJOR ANALYTICAL OBJECTIVE AND CHALLENGE. MASS SPECTROMETRY (MS) BASED PROTEOMICS IS A POWERFUL TOOL FOR CHARACTERIZING HISTONE ALTERATIONS IN MULTIPLEXED AND NON-TARGETED FASHION. HOWEVER, CONVENTIONAL BOTTOM-UP (I.E. PEPTIDE-LEVEL) MS CANNOT PROVIDE COMPLETE CHARACTERIZATION OF THE STOICHIOMETRY AND COMBINATIONS OF MULTIPLE PTMS, AND OTHER COMBINATORIAL SOURCES OF VARIATION, THAT COLLECTIVELY MAKE UP ANY SINGLE GENE'S SET OF PROTEOFORMS (I.E. FUNCTIONAL UNITS OF A PROTEOME). TOP-DOWN (I.E. PROTEOFORM-LEVEL) MS ADDRESSES THIS CHALLENGE BY OMITTING THE PROTEOLYSIS AND THUS ALLOWING ACCESS TO THE FUNCTIONAL PROTEOFORMS. HOWEVER, TOP-DOWN MS SUFFERS FROM LOW SENSITIVITY AND DYNAMIC RANGE DUE TO CHALLENGES IN SEPARATION AND DETECTION OF LARGE AND LOW-ABUNDANCE PROTEINS AND LABORIOUS PURIFICATION STEPS REQUIRED TO ACHIVE HIGH PROTEOME COVERAGE. THIS SEVERELY LIMITS OUR ABILITY TO ANALYZE SMALL SAMPLES AND EMPLOY TOP-DOWN MS TO GENERATE PROTEOFORM-AWARE IMAGES OF TISSUES REQUIRED FOR A DEEPER UNDERSTANDING OF HUMAN ORGAN FUNCTIONING IN HEALTH AND DISEASE. WE HAVE RECENTLY DEVELOPED NANODROPLET SAMPLE PREPARATION (NANOPOTS) FOR HIGHLY SENSITIVE BOTTOM-UP PROTEOMICS AND EXTENDED THIS APPROACH TO TISSUE IMAGING WITH 100 ΜM SPATIAL RESOLUTION. HEREIN, WE PROPOSE TO DEVELOP AND DEPLOY NANOPOTS-BASED TOP-DOWN MS TO ENABLE CHARACTERIZATION OF PROTEOFORMS IN TISSUE SECTIONS WITH NEAR SINGLE CELL RESOLUTION. TO INCREASE THE RESOLUTION FROM THOUSANDS OF CELLS TO NEAR SINGLE CELL, WE WILL EMPLOY ADVANCED MS IMAGING (MSI) APPROACHES. MSI DATA WILL BE CROSS-REFERENCED WITH GLOBAL PROTEOMICS DATA OBTAINED VIA MICROSCALE TOP-DOWN MS OF MICRODISSECTED TISSUE REGIONS. THE UG3 PHASE EFFORTS WILL BE FOCUSED ON HISTONES AND KIDNEY AS A DEVELOPMENT PLATFORM AND LEVERAGE A UNIQUE COMBINATION OF MICROSCALE TOP-DOWN LCMS, MSI AND NOVEL IMAGE PROCESSING AND VISUALIZATION TOOLS. IN THE UH3 PHASE, WE WILL CONSTRUCT COMPREHENSIVE PROTEOFORM-SPECIFIC MAPS OF MULTIPLE TISSUE TYPES AND FACILITATE MULTIMODAL MOLECULAR MAPPING OF SPECIFIC FUNCTIONAL UNITS OF THE KIDNEY BY LEVERAGING THE HUBMAP CONSORTIUM ONGOING EFFORTS. SUCCESSFUL COMPLETION OF THIS RESEARCH WILL ALLOW FOR COMPREHENSIVE CHARACTERIZATION OF THE FULL SPECTRUM OF PROTEOFORMS IN TISSUES AND CELLS THUS ADDRESSING AN IMPORTANT AND UNDER- STUDIED AREA OF BIOLOGY AND CRITICAL GAP IN HUBMAP EFFORTS.

HIGH-THROUGHPUT EVALUATION OF BREAST CANCER MARKERS

CATALYTIC CONVERSION OF BIOMASS TO FUELS AND CHEMICALS USING IONIC LIQUIDS

STATISTICAL METHODOLOGY FOR CHARACTERIZATION OF MACROMOLECULAR SIMILARITY

TYING DISTINCT NANOPARTICLE PROPERTIES TO CELLULAR INTERACTIONS, FATE AND RESPONS

DEVELOPMENT OF LEWY BODIES BIOFLUID SIGNATURES BY TARGETED PROTEOMICS

SPATIALLY-RESOLVED PROTEOME MAPPING OF SENESCENT CELLS AND THEIR TISSUE MICROENVIRONMENT AT SINGLE-CELL RESOLUTION - PROJECT SUMMARY/ABSTRACT CELLULAR SENESCENCE IS A PERMANENT STATE OF CELL CYCLE ARREST INDUCED BY MANY DIFFERENT STRESSES. ALTHOUGH SENESCENT CELLS (SNCS) HAVE BEEN DEMONSTRATED WITH BENEFICIAL ROLES IN NORMAL PHYSIOLOGICAL PROCESSES, THEY ARE INCREASINGLY RECOGNIZED AS THE KEY DETERMINANTS OF MANY AGING-RELATED DISEASES, SUCH AS CANCER, OSTEOARTHRITIS, AND TYPE 2 DIABETES. THE SNCS AND SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE (SASP) ARE FOUND TO BE HIGHLY HETEROGENEOUS AND VARY IN DIFFERENT TYPES OF CELLS AND TISSUE REGIONS. CURRENTLY, THERE ARE NO “UNIVERSAL” BIOMARKERS FOR IDENTIFYING THE SNCS IN VIVO. THE FIRST STEP TOWARDS ADVANCING OUR UNDERSTANDING OF CELLULAR SENESCENCE AND DEVELOPING SNC-TARGETING THERAPY APPROACHES IS TO COMPREHENSIVELY CHARACTERIZE CELLULAR SENESCENCE IN VARIOUS HUMAN TISSUES. MASS SPECTROMETRY (MS)-BASED SPATIAL PROTEOMICS CAN PROVIDE DIRECT INSIGHTS INTO CELLULAR HETEROGENEITY AND REVEAL NOVEL PROTEIN MARKERS. HOWEVER, CURRENT SPATIAL PROTEOMICS TECHNOLOGIES ARE LIMITED BY THEIR POOR SPATIAL RESOLUTION AND LOW ANALYSIS THROUGHPUT. THE OVERALL OBJECTIVE OF THIS PROJECT IS TO SIGNIFICANTLY ADVANCE OUR MICROFLUIDICS-BASED SPATIAL PROTEOMICS PLATFORM, TERMED LASER CAPTURE MICRODISSECTION COUPLED WITH NANODROPLET PROCESSING IN ONE-POT FOR TRACE SAMPLES (LCM-NANOPOTS), AND APPLY THIS TECHNOLOGY TO MAP SNCS AND THEIR SASP IN DIFFERENT MOUSE AND HUMAN TISSUES. IN THE UG3 PHASE, WE WILL ESTABLISH A HIGH-THROUGHPUT AND ROBUST SINGLE-CELL ISOLATION SYSTEM AND COUPLE IT WITH NANOPOTS-MS. WE WILL MODIFY THE ZEISS LCM SYSTEM TO ENABLE RELIABLE SINGLE-CELL ISOLATION AND COLLECTION BY DESIGNING AND ASSEMBLING A ROBOT-ADDRESSABLE CAPILLARY SAMPLING PROBE. NEXT, WE WILL DEPLOY THE SPATIAL SINGLE-CELL PROTEOMICS PLATFORM FOR MAPPING OF SNCS AND THEIR SASP IN MOUSE SKIN TISSUE. WE WILL DEVELOP A STREAMLINED WORKFLOW TO IDENTIFY SNCS FROM FASST MOUSE SKINS. IMMUNOHISTOCHEMISTRY OR IMMUNOFLUORESCENCE WILL BE USED TO VALIDATE NOVEL PROTEIN MARKER CANDIDATES. IN THE UH3 PHASE, WE WILL SIGNIFICANTLY ENHANCE PROTEOME COVERAGE AND ANALYSIS THROUGHPUT OF THE NANOPOTS-LC-MS PLATFORM. WE AIM TO ACHIEVE A THROUGHPUT OF >300 SAMPLES/DAY AND A PROTEOME COVERAGE OF >1500 PROTEINS. THE NANOPOTS SAMPLE PREPARATION WILL BE OPTIMIZED FOR FORMALIN-FIXED PARAFFIN- EMBEDDED (FFPE) TISSUES FOR BROAD APPLICATIONS OF SPATIAL PROTEOMICS TECHNOLOGY. NEXT, WE WILL DEMONSTRATE THE IMPROVED PLATFORM IN DIFFERENT TISSUE TYPES, INCLUDING MOUSE LIVER, SKIN DRAINING LYMPH NODES, AS WELL AS HUMAN LIVER, BREAST, AND LUNG TISSUES. WE WILL ESTABLISH COLLABORATIONS WITH DIFFERENT TMCS TO CHARACTERIZE SNCS AND SASP USING SPATIAL PROTEOMICS IN VARIOUS HUMAN TISSUES AND CONTRIBUTE TO MULTIOMICS MAPPING OF THE TISSUES. STATEMENT OF IMPACT: THE CAPABILITY TO GENERATE UNBIASED AND COMPREHENSIVE PROTEOME MAPS AT SINGLE-CELL RESOLUTION WILL ENABLE THE DISCOVERY OF SNC PROTEIN MARKERS ACROSS DIFFERENT CELL TYPES AND ORGANS, AND ADVANCE OUR UNDERSTANDING OF THE IMPACT OF SNCS ON TISSUE MICROENVIRONMENT.

HIGH THROUGHPUT LIPIDOMIC PROFILING WITH HIGH STRUCTURAL SPECIFICITY FOR PHENOTYPIC SCREENING - PROJECT ABSTRACT LIPIDS ARE A DIVERSE CLASS OF BIOMOLECULES THAT PLAY PIVOTAL ROLES IN VARIOUS CELLULAR PROCESSES AND HAVE BEEN IMPLICATED IN THE PATHOGENESIS OF DISEASES SUCH AS DIABETES, ALZHEIMER'S, PARKINSON'S, AND VARIOUS CANCERS. UNDERSTANDING LIPID STRUCTURES AND FUNCTIONS IS CRUCIAL FOR UNRAVELING THEIR ROLES IN COMPLEX BIOLOGICAL SYSTEMS AND DISEASES. UNAMBIGUOUS IDENTIFICATION OF LIPIDS, PARTICULARLY WITH HIGH-LEVEL STRUCTURAL DETAILS AND SEPARATION OF LIPID ISOMERS, HAS BEEN AN ANALYTICAL CHALLENGE. HOWEVER, SUCH KNOWLEDGE IS ESSENTIAL FOR OBTAINING A FULL UNDERSTANDING OF LIPID FUNCTIONS AND DISCOVERING LIPID BIOMARKERS FOR DISEASES. ADDRESSING THE ABSENCE OF RAPID LIPID PROFILING METHODS AND THAT PROVIDE SUFFICIENT STRUCTURAL DETAILS IS CRITICAL FOR PREDICTING, CONTROLLING, AND ENGINEERING LIPID METABOLISM ON A LARGE SCALE FOR BIOMEDICAL AND PHENOTYPIC SCREENING. MY LONG-TERM GOAL IS TO ENABLE THE ELUCIDATION OF THE DETAILED MOLECULAR MECHANISMS OF LIPID METABOLISM THROUGH ADVANCING LIPIDOMIC PROFILING WITH HIGH STRUCTURAL SPECIFICITY AND TO UNRAVEL THE RELATIONSHIP BETWEEN SPECIFIC LIPIDS AND ASSOCIATED FUNCTIONS IN BIOLOGICAL SYSTEMS AND DISEASES. TO ACHIEVE THIS, WE PROPOSE DEVELOPING A NEXT-GENERATION LIPIDOMICS PIPELINE USING MASS SPECTROMETRY-BASED TECHNOLOGIES AND ADVANCED BIOINFORMATICS CAPABILITIES. OUR APPROACH INVOLVES DEVELOPING A MULTI-DIMENSIONAL LIPIDOMIC PLATFORM USING HIGH-RESOLUTION STRUCTURES FOR LOSSLESS ION MANIPULATIONS-BASED ION MOBILITY SPECTROMETRY-MASS SPECTROMETRY, ADVANCED FRAGMENTATION TECHNIQUES, AND GAS-PHASE REACTIONS FOR EFFECTIVE LIPID STRUCTURE ELUCIDATION, INCLUDING RESOLVED ISOMER SEPARATION AND DETERMINING DOUBLE BOND LOCATIONS. THIS INTEGRATIVE PLATFORM WILL BE COUPLED WITH LIQUID CHROMATOGRAPHY AND A NOVEL DATA ACQUISITION APPROACH UTILIZING BOTH DATA-DEPENDENT ACQUISITION AND DATA- INDEPENDENT ACQUISITION FOR A COMPREHENSIVE ANALYSIS OF LIPIDS IN COMPLEX SAMPLES. TO ENSURE ROBUST DATA PROCESSING, LIPID IDENTIFICATION AND STRUCTURE ELUCIDATION, WE WILL DEVELOP ADVANCED BIOINFORMATICS PIPELINES USING ARTIFICIAL INTELLIGENCE APPROACHES. ADDITIONALLY, A HIGH-THROUGHPUT AND QUANTITATIVE WORKFLOW UTILIZING ONLINE SOLID-PHASE EXTRACTION FOR RAPID LIPID SCREENING WILL BE DEVELOPED. THE ROBUSTNESS OF THIS WORKFLOW AND ADVANCED BIOINFORMATICS PIPELINE WILL BE VALIDATED BY FULLY PROFILING THE YEAST LIPIDOME. A COMPREHENSIVE AND STRUCTURALLY DETAILED LIPID ATLAS FOR THE YEAST KNOCKOUT COLLECTION WILL BE CONSTRUCTED AND PROVIDE A FOUNDATION FOR UNDERSTANDING LIPID METABOLISM AND ASSOCIATED FUNCTIONS ANALOGOUS TO HUMAN GENES. WE ANTICIPATE THAT THIS PLATFORM WILL BECOME A FUNDAMENTAL CAPABILITY FOR LIPID PROFILING IN BIOMEDICAL RESEARCH TO UNRAVEL THE INTRICATE RELATIONSHIP BETWEEN LIPIDS AND ASSOCIATED FUNCTIONS IN BIOLOGICAL SYSTEMS AND HUMAN HEALTH. FURTHERMORE, WE EXPECT THIS INNOVATIVE APPROACH TO FACILITATE THE DISCOVERY OF LIPID BIOMARKERS FOR DISEASE DIAGNOSTICS AND DRUG DEVELOPMENT.

VISUAL ANALYTICS SOFTWARE ENVIRONMENT FOR PROTEOMICS DATA INTEGRATION

VALIDATION OF NOVEL PEPTIDE/PROTEIN MARKERS FOR DIAGNOSIS OF TYPE 1 DIABETES

AEROSOL EFFECTS ON COLD SEASON OROGRAPHIC PRECIPITATION AND WATER RESOURCES

SINGLE-CELL (PHOSPHO-)PROTEOMICS PLATFORM FOR FUNCTIONAL ANALYSIS OF TUMOR MICROENVIRONMENT - ABSTRACT TUMOR MICROENVIRONMENT (TME) IS A COMPLEX ECOSYSTEM COMPRISED OF CANCER CELLS, IMMUNE CELLS, STROMAL CELLS, AND OTHER CELL TYPES. THEY INTERACT AND COLLECTIVELY DETERMINE DISEASE PROGRESSION AND RESPONSE TO THERAPY. TO ACHIEVE A COMPLETE PICTURE OF TME, MULTI-OMICS SINGLE-CELL TECHNOLOGIES ARE NEEDED TO CHARACTERIZE DIVERSE INDIVIDUAL CELLS WITHIN TME AT BOTH GENOTYPE AND PHENOTYPE LEVELS. SUCH ANALYSIS HOLDS PROMISE TO MOVE TOWARDS PRECISION MEDICINE. HOWEVER, SINGLE-CELL PROTEOMICS TECHNOLOGIES ARE LAGGING FAR BEHIND OTHER SINGLE-CELL OMICS TECHNOLOGIES. MANY STUDIES HAVE SHOWN THAT MRNA ABUNDANCE IS A POOR SURROGATE FOR PROTEIN ABUNDANCE AND CANNOT PROVIDE ANY INSIGHTS INTO PROTEIN ACTIVITY REGULATED BY PROTEIN PHOSPHORYLATION. THEREFORE, SINGLE-CELL (PHOSPHO-)PROTEOMICS IS CRITICALLY IMPORTANT TO PROVIDE COMPLEMENTARY DATA THAT CANNOT BE ACHIEVED BY OTHER OMICS TECHNOLOGIES. ANTIBODY-BASED IMMUNOASSAYS ARE PRIMARILY USED FOR FUNCTIONAL ANALYSIS OF SINGLE CELLS, BUT THEY ONLY CAN OPEN A SMALL WINDOW INTO THE COMPLEX TME ECOSYSTEM DUE TO THEIR INHERENT LIMITATIONS. MASS SPECTROMETRY (MS)-BASED (PHOSPHO-)PROTEOMICS IS A POWERFUL TOOL FOR GENOME-SCALE PROTEOME PROFILING AND SITE-SPECIFIC QUANTIFICATION OF 1,000S OF PHOSPHORYLATION SITES. HOWEVER, IT DOES NOT MEET THE MERIT FOR SINGLE-CELL (PHOSPHO-)PROTEOMICS ANALYSIS. THERE ARE THREE MAJOR CHALLENGES THAT NEED TO BE ADDRESSED: 1) LOW RECOVERY FOR PHOSPHO-ENRICHMENT, 2) INSUFFICIENT MS SENSITIVITY, 3) LOW SAMPLE THROUGHPUT. WE PROPOSE TO DEVELOP A SINGLE-CELL (PHOSPHO-)PROTEOMICS PLATFORM TO ADDRESS THESE THREE CHALLENGES FOR ROBUST RAPID ANALYSIS OF SINGLE CELLS AT BOTH PROTEOME AND PHOSPHOPROTEOME LEVELS. THE PROJECT FEASIBILITY IS STRONGLY SUPPORTED BY OUR RECENT PROGRESS IN MANY ASPECTS OF TECHNOLOGY DEVELOPMENT. THE NEW PLATFORM WILL BE DEVELOPED THROUGH INTEGRATION OF A HIGH-RECOVERY SAMPLE PREPARATION PLATFORM FOR SINGLE-CELL PROCESSING AND PHOSPHO-ENRICHMENT WITH A HIGH-EFFICIENCY MS PLATFORM TO ENHANCE MS DETECTION SENSITIVITY AND SAMPLE MULTIPLEXING TO IMPROVE SAMPLE THROUGHPUT. WE ENVISION THAT THE SINGLE-CELL MS PLATFORM WILL EVENTUALLY BECOME AN INDISPENSABLE TOOL FOR ROBUST, RAPID, DEEP (PHOSPHO-)PROTEOMIC ANALYSIS OF SINGLE CELLS AND TME. IN TURN, IT WILL MAKE SUBSTANTIAL CONTRIBUTIONS TO IMPROVE OUR UNDERSTANDING OF CANCER BIOLOGY AND ACCELERATE THE MOVEMENT TOWARDS PRECISION MEDICINE.

TOWARDS AN OPERATIONAL FORECAST SYSTEM FOR THE SALISH SEA ??? REFINEMENT, IMPROVEMENT, AND TESTING FOR TRANSITIONING OF THE EXISTING SALISH SEA MODEL TO NATIONAL OCEAN SERVICE (NOS)

MASS SPECTROMETRIC TISSUE IMAGING OF SUGARS, GLYCANS, AND GLYCOCONJUGATES - PROJECT SUMMARY/ABSTRACT THE GLYCOME DESCRIBES THE COMPLETE REPERTOIRE OF CELL CARBOHYDRATES THAT ARE EITHER FREE OR COVALENTLY LINKED TO LIPID OR PROTEIN MOLECULES, HAVING THE SPAN OF BIOLOGICAL ROLES FROM THOSE THAT ARE SUBTLE TO THOSE THAT ARE CRUCIAL FOR AN ORGANISM'S DEVELOPMENT, FUNCTION, OR SURVIVAL. BECAUSE THOSE MOLECULES ARE NOT ENCODED DIRECTLY IN THE GENOME, ONE CANNOT ACCURATELY PREDICT THE PRECISE STRUCTURES OF GLYCANS ELABORATED BY A GIVEN CELL TYPE. FURTHERMORE, SMALL CHANGES IN ENVIRONMENTAL CUES CAN CAUSE DRAMATIC CHANGES IN GLYCANS PRODUCED BY A GIVEN CELL, WHICH CAN OFTEN LEAD TO PATHOLOGY, AND AS SUCH, DEVELOPING CAPABILITIES FOR IDENTIFYING THOSE CHANGES AT THE CELL-SPECIFIC LEVEL IS NECESSARY. HISTORICALLY, NUCLEAR MAGNETIC RESONANCE (NMR) AND MASS SPECTROMETRY (MS) HAVE BEEN USED FOR GLYCOME STRUCTURAL ELUCIDATION, BUT THOSE TECHNIQUES HAVE LIMITED CAPABILITY TO CAPTURE CHEMICAL DIFFERENCES AT THE SPATIAL LEVEL, WHILE LECTINS AND OTHER GLYCAN-LABELING IMAGING APPROACHES PROVIDE ONLY LIMITED INFORMATION ON CARBOHYDRATES STRUCTURES. OVER THE LAST DECADE, MASS SPECTROMETRY IMAGING (MSI) EVOLVED AS A POWERFUL TOOL TO LINK CHEMICAL STRUCTURES WITH TISSUE LOCALIZATION, AND IT HAS BEEN SUCCESSFULLY USED TO REVEAL DIFFERENCES IN METABOLITES, LIPIDS, AND XENOBIOTICS ACROSS NUMEROUS HUMAN TISSUES, CELL TYPES, AND CELL STATES. HOWEVER, MSI STILL HASN’T SHOWN ITS FULL POTENTIAL IN GLYCOME IMAGING DUE TO CARBOHYDRATES' POOR IONIZABILITY, COMPLEX CHEMISTRY, AND AMBIGUOUS MOLECULAR ANNOTATION. I HAVE SUCCESSFULLY SOLVED SOME OF THOSE ISSUES THROUGHOUT MY CAREER, ENABLING A BETTER UNDERSTANDING OF THE GLYCOME-PHENOTYPE RELATIONSHIP. HEREIN, I PROPOSE TO DEVELOP AND ADVANCE MSI TOOLS THAT WILL ENABLE MORE ACCURATE AND COMPREHENSIVE CHEMICAL CHARACTERIZATION OF THE GLYCOME AT TISSUE FUNCTIONAL UNIT AND CELLULAR RESOLUTIONS. I WILL DEVELOP VARIOUS WORKFLOWS, MODIFY AND OPTIMIZE EXISTING MSI APPROACHES, AND, IN COMBINATION WITH AUXILIARY TOOLS, MAP SOLUBLE SUGARS, GLYCOLIPIDS, PROTEOGLYCANS, POLYSACCHARIDES, AND GLYCOPROTEINS FROM THE HUMAN TISSUES. MORE SPECIFICALLY, IN COMBINATION WITH HIGH-RESOLVING MASS SPECTROMETERS, I WILL UTILIZE VARIOUS IONIZATION AND POST-IONIZATION SOURCES, ORTHOGONAL ANALYSES, ON-TISSUE CHEMICAL DERIVATIZATION, IN-TISSUE DIGESTION, AND GLYCAN ENRICHMENT STRATEGIES TO OVERCOME CURRENT LIMITATIONS IN GLYCOME IMAGING. SUCCESSFUL COMPLETION OF THIS RESEARCH WILL CREATE A ROBUST, HIGH- THROUGHPUT CAPABILITY FOR GLYCOME IMAGING THAT WILL ENABLE AN UNDERSTANDING OF HOW, AT THE CELL-SPECIFIC LEVEL, GLYCOME PROCESSING AND GLYCOME REDISTRIBUTION MEDIATE PHYSIOLOGICAL AND PATHOLOGICAL PROCESSES, THUS ADDRESSING A CRITICAL GAP IN THE GLYCOSCIENCES AREA OF THE BIOCHEMISTRY AND BIO-RELATED CHEMISTRY BRANCH OF NIGMS.

MIXTURE MODELING: PESTICIDE DRUG INTERACTIONS

A STREAMLINED PLATFORM FOR PHOSPHOPROTEOME MAPPING OF HUMAN TISSUES

DEVELOPMENT OF A NANOLITER SLOW-MAS NMR METABOLOMICS PROBE

AN ULTRASENSITIVE TARGETED MASS SPECTROMETRY SYSTEM FOR PROTEOMICS ANALYSIS OF SINGLE CELLS

PSEUDOMONAS AERUGINOSA OUTER MEMBRANE PROTEIN MODELING

BIOINFORMATICS FOR PROTEIN MICROARRAYS

A HYBRID IMS-MS PLATFORM FOR ULTRASENSITIVE AND HIGH RESOLUTION GLYCAN ANALYSIS

HUMAN EXPOSURE MODELING FOR PBDE DERIVATIVES

HIGH-RESOLUTION FLEXIBLE MOBILITY ANALYZER FOR POINT-OF-CARE DIAGNOSTICS - PROJECT SUMMARY THIS PROJECT AIMS TO ADDRESS THE CURRENT GAP IN TECHNOLOGY FOR RAPID LIPIDS PROFILING. A POINT-OF-CARE TECHNOLOGY THAT PROVIDES QUICK DETECTION WILL IMPACT HEALTHCARE MANAGEMENT BY LEVERAGING PATIENT’S PROACTIVE PARTICIPATION. IN PURSUANCE OF THIS GOAL, WE WILL DEVELOP AN ION MOBILITY CHEMICAL ANALYSIS TECHNOLOGY, WHICH PROVIDES CHEMICAL INFORMATION WITH MOLECULAR LEVEL SPECIFICITY, FOR DISEASE DIAGNOSTICS AND MONITORING AND IDENTIFICATION OF PATHOGENS. THE PROJECT WILL FOCUS ON A PROOF-OF- CONCEPT TECHNOLOGY FOR A PORTABLE MOLECULAR ANALYZER DEVICE THAT CAN BE UTILIZED TO ANALYZE BREATH, SALIVA, BLOOD PRICKS IN ITS MATURE STAGE FOR MARKERS OF DISEASE. THE NEW DEVICE WILL BE DESIGNED BASED ON EXTENSIVE ION TRAJECTORY SIMULATIONS THAT WILL PROVIDE THE GEOMETRICAL AS WELL AS OPTIMAL OPERATING PARAMETERS. OPTIMAL DESIGNS WILL BE USED TO BUILD PROTOTYPES THAT WILL BE EVALUATED IN TERMS OF SENSITIVITY AND SELECTIVITY FOR DETECTING TARGETED MOLECULES TO DEMONSTRATE A WORKING PROTOTYPE IN CONJUNCTION WITH A MASS SPECTROMETER.

SPATIALLY RESOLVED CHARACTERIZATION OF PROTEOFORMS FOR FUNCTIONAL PROTEOMICS

PAN-OMIC CHARACTERIZATION OF THE MOLECULAR DETERMINANTS OF UTERINE RECEPTIVITY

PROTEIN TARGET IDENTIFICATION OF NITAZENES AND THEIR METABOLITES USING STRUCTURAL AND CHEMOPROTEOMICS - PROJECT SUMMARY NITAZENES ARE BENZIMIDAZOLE OPIOIDS THAT DISPLAY SIMILAR OR HIGHER POTENCY THAN OTHER ULTRAPOTENT SYNTHETIC (UPS) OPIOIDS SUCH AS FENTANYL. ALTHOUGH NITAZENES WERE FIRST SYNTHESIZED AND STUDIED IN THE 1950S, THEY WERE NEVER APPROVED FOR MEDICAL USE, AND THEREFORE WE HAVE LIMITED DATA ON THEIR MOLECULAR PHARMACOLOGY BEYOND INTERACTIONS WITH OPIOID RECEPTORS. GIVEN THAT THE NUMBERS OF NOVEL NITAZENE ANALOGS HAVE BEEN INCREASING ON AN ANNUAL BASIS AND INVOLVEMENT OF NITAZENES IN DRUG OVERDOSES WORLDWIDE, WE NEED A BETTER UNDERSTANDING OF THE MECHANISMS THAT CONTRIBUTE TO THE ADVERSE EFFECTS OF THIS EMERGING UPS OPIOID CLASS. WE PROPOSE TO DESIGN, SYNTHESIZE, AND APPLY NITAZENE CHEMICAL PROBES FOR AFFINITY-BASED PROTEIN PROFILING (AFBPP) TO IDENTIFY NITAZENE-BINDING PROTEINS IN HUMAN BRAIN, HEART, AND LUNG CELLS. IN PARALLEL, WE WILL USE LIMITED PROTEOLYSIS (LIP), A STRUCTURAL PROTEOMICS TECHNIQUE, TO MAP PROTEIN STRUCTURAL CHANGES INDUCED BY NITAZENE EXPOSURES TO FURTHER VALIDATE AND CHARACTERIZE THE BIOLOGICAL CONSEQUENCES OF NITAZENE BINDING ON CELLULAR FUNCTIONS. THE COMBINATION OF THESE TWO PROTEOMICS TECHNIQUES WILL PROVIDE RICH MOLECULAR LEVEL DATA ON BOTH DIRECT PROTEIN-NITAZENE BINDING INTERACTIONS AND THE DOWNSTREAM SIGNALING PATHWAYS THAT ARE PERTURBED BY THESE UPS OPIOIDS. BY IDENTIFYING THE SPECIFIC BIOLOGICAL TARGETS THAT INTERACT WITH NITAZENES IN KEY CELL TYPES, WE WILL GAIN CRITICAL INSIGHTS INTO THE ALTERED SIGNALING CASCADES AND MOLECULAR MECHANISMS RESPONSIBLE FOR NITAZENE- INDUCED TOXICITY, WHICH CAN INFORM FUTURE MEDICAL COUNTERMEASURE STRATEGIES TO TREAT NITAZENE OVERDOSE. THE SPECIFIC AIMS OF THIS PROPOSAL ARE: AIM 1: IDENTIFY NITAZENE-BINDING PROTEINS USING AFFINITY-BASED PROTEIN PROFILING (AFBPP). AIM 2: ELUCIDATE PROTEIN STRUCTURAL AND FUNCTIONAL CHANGES INDUCED BY NITAZENE EXPOSURE

DISCOVERY OF PHOSGENE AND CHLORINE GAS MODES OF ACTION AND THERAPEUTIC TARGETS USING CHEMOPROTEOMIC PROFILING STRATEGIES - PROJECT SUMMARY: PHOSGENE AND CHLORINE ARE TWO TOXIC GASES PRODUCED ON LARGE INDUSTRIAL SCALES THAT POSE OCCUPATIONAL HAZARDS AND CHEMICAL THREATS TO THE PUBLIC AND MILITARY. BOTH TOXICANTS MODIFY PROTEINS IN VARIOUS WAYS AND INDUCE OXIDATIVE STRESS IN THE LUNG WHICH PERTURB CRITICAL FUNCTIONS AND PATHWAYS. HOWEVER, THE PRECISE NATURE AND SITES OF THESE MODIFICATIONS IN THE LUNG PROTEOME HAVE NOT BEEN IDENTIFIED, AND THE POTENTIAL CHEMICAL DIVERSITY OF THESE MODIFICATIONS POSES AN ANALYTICAL CHALLENGE TO UNRAVELING THE MOLECULAR MECHANISMS BY WHICH CHLORINE AND PHOSGENE INDUCE ACUTE LUNG INJURY. THIS LACK OF BIOMOLECULAR CHARACTERIZATION OF PHOSGENE- AND CHLORINE-INDUCED MODIFICATION OF PROTEIN TARGETS HAS CONSEQUENTLY HINDERED OUR ABILITY TO DEVELOP EFFECTIVE COUNTERMEASURES; THERE ARE CURRENTLY NO APPROVED THERAPEUTICS FOR TREATING PHOSGENE OR CHLORINE GAS EXPOSURES. WE PROPOSE TO USE CHEMOPROTEOMIC PROFILING—A SUBSET OF MASS SPECTROMETRY-BASED PROTEOMICS THAT CAN DETECT THE SITES AND TYPES OF DISTINCT CHEMICAL MODIFICATIONS TO PROTEINS—TO COMPREHENSIVELY IDENTIFY SPECIFIC PROTEINS IN THE LUNG THAT HAVE BEEN CHEMICALLY ALTERED BY PHOSGENE AND CHLORINE AND PERFORM PATHWAY ANALYSIS TO IDENTIFY PROTEIN CANDIDATES FOR FUTURE TARGETED COUNTERMEASURE DEVELOPMENT. THE SPECIFIC AIMS OF THIS PROPOSAL ARE: AIM 1: DYNAMIC REDOX PROFILING TO IDENTIFY THE TEMPORAL PROTEIN CYSTEINE OXIDATION EVENTS AND GLOBAL PROTEOME CHANGES FOLLOWING GASEOUS TOXICANT EXPOSURE AIM 2: MAPPING PHOSGENE- AND CHLORINE-INDUCED PROTEIN MODIFICATIONS IN THE LUNG USING ELECTROPHILIC CHEMICAL PROBES AIM 3: DATA INTEGRATION AND NETWORK ANALYSIS TO IDENTIFY SHARED AND UNIQUE MOLECULAR TARGETS OF PHOSGENE AND CHLORINE FOR THERAPEUTIC DEVELOPMENT

DEVELOPMENT OF A HIGH-THROUGHPUT PROTEOMIC, TRANSCRIPTOMIC, AND METABOLOMIC PLATFORM FOR MULTIOMIC PROFILING OF SAME SINGLE-CELLS - PROJECT SUMMARY/ABSTRACT SYSTEMS BIOLOGY SEEKS TO BUILD PREDICTIVE MODELS OF CELL BEHAVIOR BY INTEGRATING INFORMATION ACROSS MULTIPLE MOLECULAR LAYERS. HOWEVER, MANY MODELS RELY ON BULK MEASUREMENTS, WHICH OBSCURE UNDERLYING REGULATORY MECHANISMS AT THE SINGLE-CELL LEVEL. WHILE TRANSCRIPTOMIC ASSAYS ARE INCREASINGLY ROUTINE IN SINGLE-CELL EXPERIMENTS, GLOBAL SINGLE-CELL PROTEOMICS AND METABOLOMICS REMAIN TECHNICALLY LIMITED, PREVENTING COMPLETE MULTIOMIC ANALYSIS OF THE SAME CELL. THE PROPOSED WORK ADDRESSES THIS GAP THROUGH THE DEVELOPMENT OF A HIGH- THROUGHPUT PLATFORM THAT COMBINES NOVEL CHEMISTRY, ADVANCED MASS SPECTROMETRY, AND AUTOMATED NANODROPLET PROCESSING TO SIMULTANEOUSLY PROFILE PROTEINS, TRANSCRIPTS, AND METABOLITES FROM THOUSANDS OF INDIVIDUAL CELLS. CENTRAL TO THIS EFFORT IS THE INTRODUCTION OF MASKED AMINO TAGS, A CHEMICAL INNOVATION THAT DOUBLES THE MULTIPLEXING CAPACITY OF MASS SPECTROMETRY ANALYSES, ENABLING UP TO 70 SINGLE CELLS TO BE ANALYZED IN A SINGLE RUN. THIS CHEMISTRY WILL BE PAIRED WITH NEW HIGH-DENSITY NANODROPLET ARRAYS, AUTOMATED DROPLET SPLITTING FOR SAME-CELL MRNA TRANSCRIPT CAPTURE, AND ION-MOBILITY-ENHANCED LC-MS FOR ISOBARIC-LABELED PROTEOMIC AND METABOLOMIC MEASUREMENTS. PARALLEL INFORMATIC ADVANCES WILL INCLUDE TWO NEW R PACKAGES: METABOR FOR METABOLITE QUANTIFICATION AND COVARR FOR CROSS-MODALITY COVARIATION ANALYSIS, PROVIDING UNIFIED INTEGRATION OF TRANSCRIPT, PROTEIN, AND METABOLITE DATA. THIS PLATFORM WILL BE APPLIED TO HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS AND PANCREATIC ISLET CELLS, WITH A FOCUS ON AMINO ACID SIGNALING IN IMMUNE ACTIVATION AND DIABETES. BY GENERATING DEEP, MULTIOMIC SINGLE-CELL DATASETS, THE PROJECT WILL ADVANCE BOTH FUNDAMENTAL BIOLOGY AND TRANSLATIONAL RESEARCH. THE RESULTING TECHNOLOGY WILL ACCELERATE THE DEVELOPMENT OF PREDICTIVE SINGLE-CELL MODELS, PROVIDE POWERFUL NEW TOOLS FOR SYSTEMS BIOLOGY, WHILE CREATING BROAD OPPORTUNITIES FOR STUDYING CELLULAR HETEROGENEITY IN HEALTH AND DISEASE.

NOVEL CHELATORS FOR HIGHLY EFFICIENT REMOVAL OF TOXIC HEAVY METALS IN HUMANS

NATURAL PRODUCTS MAGNETIC RESONANCE DATABASE (NP-MRD) PHASE II - PROJECT SUMMARY/ABSTRACT SINCE ITS LAUNCH IN 2021, THE NATURAL PRODUCTS MAGNETIC RESONANCE DATABASE (NP-MRD, HTTPS://NP-MRD.ORG) HAS BECOME THE LARGEST OPEN REPOSITORY OF NMR DATA ON NATURAL PRODUCTS (NPS) AS WELL AS THE LARGEST COLLECTION OF GENERAL INFORMATION (NOMENCLATURE, STRUCTURE, BIOLOGY, CHEMISTRY) ABOUT NPS IN THE WORLD. THE NP-MRD CONTAINS SUBSTANTIAL AMOUNTS OF DIFFERENT TYPES OF NMR DATA INCLUDING RAW NMR DATA (FREE INDUCTION DECAYS OR FIDS), PREDICTED NMR DATA AND LITERATURE-DERIVED NMR INFORMATION (CHEMICAL SHIFT ASSIGNMENTS, COUPLING CONSTANTS). THE NMR DATA IN THE NP-MRD INCLUDES 15,000 SETS OF EXPERIMENTAL NMR SPECTRA (FOR 3200 NPS), 411,000 SIMULATED NMR SPECTRA (FOR 21,000 NPS) OBTAINED FROM LITERATURE-DERIVED NMR ASSIGNMENTS, AND 5.1 MILLION PREDICTED NMR SPECTRA (FOR ALL 282,000 NPS). MUCH OF THE RAW NMR DATA IN THE NP-MRD IS THERE DUE TO A CONCERTED EFFORT BY NP-MRD, TOGETHER WITH THE JOURNAL OF NATURAL PRODUCTS (JNP), TO ENCOURAGE RAW DATA DEPOSITION OF NMR DATA USED TO SUPPORT NOVEL STRUCTURE ELUCIDATION. FOR THE SECOND PHASE OF THE NP-MRD (2025- 2030) WE WILL BUILD ON THE MOMENTUM CREATED IN THE FIRST PHASE. SPECIFICALLY, WE WILL OPTIMIZE CURRENT OPERATIONS AND CONTINUE ONGOING EXPANSION OF BOTH THE DATABASE CONTENT AND UTILITY. A MAJOR EMPHASIS WILL BE ON STREAMLINING NP-MRD OPERATIONS SUCH THAT MOST OF ITS OPERATIONS WILL REQUIRE LITTLE HUMAN INTERVENTION. FOR EXAMPLE, RAW DATA DEPOSITION FORMERLY REQUIRED TWO NP-MRD STAFF IN TWO DIFFERENT LOCATIONS TO INTERACT WITH THE DEPOSITED DATA, AND WITH THE DEPOSITOR. TODAY, RAW DATA DEPOSITION GOES AUTOMATICALLY INTO A PARSER, A SET OF QUALITY CHECKS AND STRAIGHT INTO THE NP-MRD REPOSITORY WITH NO HUMAN INTERVENTION UNLESS (RARE) PROBLEMS OCCUR WITH THE DATA. OTHER FUNCTIONS BEING STREAMLINED, INCLUDE CENTRALIZED PROCESSES TO QUERY, ENTER AND ASSESS NP- MRD DATA, BETTER PROCESSES TO ANNOTATE NP STRUCTURES, TOOLS TO MORE EFFICIENTLY GATHER BIOLOGICAL, MEDICINAL, STRUCTURAL, CHEMICAL SHIFT AND ASSIGNMENT DATA FROM THE PUBLISHED LITERATURE, AS WELL AS WORKFLOWS TO CORRECT ENTRIES, REMOVE DUPLICATES AND IMPROVE ACCURACY. IN TERMS OF DATA CONTENT AND UTILITY EXPANSION, MAJOR EFFORTS WILL BE UNDERTAKEN TO INCREASE USER DEPOSITION RATES, SUPPORT THE DEPOSITION, INTEGRATION AND FEDERATION OF MASS SPECTROMETRY (MS) DATA FOR NPS INTO THE NP-MRD, IMPLEMENT FASTER, MORE “INTELLIGENT” NMR, MULTI-SPECTRAL (NMR+MS+CCS+RT) AND MULTI-DATABASE (GNPS, MONA, MASSBANK, HMDB, FOODB) SEARCHES, SUPPORT MORE ACCURATE CHEMICAL SHIFT, J-COUPLING AND OTHER KINDS OF “OBSERVABLES” PREDICTION, AND CREATE NEW TOOLS TO SUPPORT AUTOMATED COMPUTER-AIDED NP STRUCTURE ELUCIDATION (CASE) AND DEREPLICATION. THESE STREAMLINING, OPTIMIZATION AND DATABASE EXPANSION EFFORTS ARE BEING DONE TO ENSURE LONG-TERM SUSTAINABILITY OF THE NP-MRD BY MAKING OPERATIONS CHEAPER, MORE AUTOMATED, MORE USEFUL AND MORE VALUABLE TO THE NP COMMUNITY. OUTREACH TO OTHER NP JOURNALS AND TO THE NP COMMUNITY WILL BE FURTHER EXPANDED, WITH THE EXPECTATION THAT THESE EFFORTS WILL TRIPLE THE DATA DEPOSITION RATE TO THE NP-MRD BY 2030, INCREASE COMMUNITY BUY-IN, INCREASE GRANT OR CO-FUNDING SUPPORT, ATTRACT PAYING SUBSCRIBERS AND ENSURE LONG-TERM SUSTAINABILITY OF THE NP-MRD.

A STATE-OF-ART NMR TECHNIQUE TO INVESTIGATE BIOLOGICALS EFFECTS OF ELECTRONIC NICOTINE DELIVERY SYSTEMS

INVESTIGATION OF RADIATIVE - DYNAMICAL FEEDBACKS IN CIRRUS \NCIRRUS CLOUD-RADIATION INTERACTIONS ARE AN IMPORTANT ASPECT OF THE EARTH\''S ENERGY BALA

ENGINEERING AND PERFORMANCE OF MAGNETIC PARTICLE IMAGING

SPATIAL ANALYSIS OF ALCOHOLIC HEPATITIS LIVER TISSUE

MASS SPECTROMETRY IMAGING: LINKING NEURODEGENERATION WITH ENVIRONMENTAL EXPOSURE

NANOLITER-SCALE AFFINITY PURIFICATION MASS SPECTROMETRY FOR SMALL NUMBERS OF HAIR CELLS - 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.

DEFINING THE FUNCTIONAL DYNAMICS OF THE MICROBIOME

BIOMATERIALS AS DECORPORATION AGENTS FOR RADIONUCLIDES

DEVELOPMENT OF SELECTIVE NANOPOROUS SORBENTS FOR RADIONUCLIDE DECORPORATION

HIGH-THROUGHPUT MULTIDIMENSIONAL BIOSEPARATIONS FOR NEXT-GENERATION PROTEOMICS

MULTIDIMENSIONAL PROTEOMICS PLATFORM

AN INTERACTIVE VOLUMETRIC ATLAS OF THE MOUSE BRAIN

PROTEOMIC ANALYSIS OF OSTEOCYTES

IMPACT OF APOLIPOPROTEIN E ISOFORMS ON BRAIN LIPOPROTEINS - PROJECT SUMMARY/ABSTRACT CEREBROVASCULAR DYSFUNCTION LEADS TO THE DEPOSITION OF AMYLOID PLAQUES AND NEUROFIBRILLARY TANGLES IN THE BRAIN WHICH UNDERLIE THE DEVELOPMENT OF ALZHEIMER’S DISEASE (AD); AN EPIDEMIC THAT AFFECTS MORE THAN FIVE MILLION AMERICANS. BRAIN LIPOPROTEINS (BLPS) ARE LIPID-PROTEIN COMPLEXES THAT ARE ESSENTIAL FOR MAINTAINING BRAIN LIPID HOMEOSTASIS AND PROTECTING AGAINST NEURODEGENERATION. UNFORTUNATELY, DETAILED STUDIES ON BLPS HAVE BEEN SEVERELY LIMITED AS THEIR ABUNDANCE IN CEREBROSPINAL FLUID (CSF) IS 200-FOLD LESS THAN LIPOPROTEIN LEVELS IN PLASMA. IT IS KNOWN THAT BLPS ARE PRODUCED INDEPENDENTLY IN THE CENTRAL NERVOUS SYSTEM AND THEY ARE THOUGHT TO EMULATE COMPOSITIONAL AND FUNCTIONAL FEATURES OF PLASMA HIGH-DENSITY LIPOPROTEINS (HDL). THE DOMINANT ORGANIZING SCAFFOLD ON BLPS IS APOE WHICH HAS THREE ISOFORMS—APOE2, APOE3, AND APOE4 THAT DIFFER BY CYSTEINE- ARGININE EXCHANGES AT RESIDUES 112 AND 158. CARRIERS OF APOE4 ARE AT AN ELEVATED RISK FOR DEVELOPMENT AND INCREASED SEVERITY OF AD. WE HYPOTHESIZE THAT, SIMILAR TO APOA1 ON HDL, APOE ACTS AS A REGULATORY SCAFFOLD ON BLPS AND CONFORMATIONAL DIFFERENCES IN APOE ISOFORMS RESULT IN ALTERED SPECIATION AND PARTICLE FUNCTION THAT UNDERLIES THE APOE ISOFORM-SPECIFIC IMPACT ON DEVELOPMENT OF AD. WE HAVE DEVELOPED A HIGHLY-SENSITIVE LIPOPROTEIN PROFILING TECHNOLOGY UNIQUE TO OUR LABORATORY TO OVERCOME THE BLP ABUNDANCE HURDLE IN CSF. OUR PRELIMINARY WORK SHOWS THAT, LIKE PLASMA HDL, BLPS ARE A WIDELY DIVERSE POPULATION OF UNIQUE PARTICLES WITH DISTINCT PROTEIN COMPLEMENTS. MOREOVER, WE HAVE DEVELOPED A PROTOCOL FOR USING CRYO-ELECTRON MICROSCOPY TO VISUALIZE LIPID-BOUND APOLIPOPROTEINS IN UNPRECEDENTED DETAIL. WE WILL LEVERAGE THESE TECHNOLOGIES TO 1) DETERMINE THE IMPACT OF APOE ISOFORM ON THE COMPOSITION AND DISTRIBUTION OF BLPS AND 2) DETERMINE THE MOLECULAR BASIS OF HOW APOE ISOFORMS STRUCTURALLY REGULATE BLPS. IMPORTANTLY, WE WILL DIRECTLY RELATE DIFFERENCES TO CLINICAL AD ENDPOINTS IN THE SAME INDIVIDUALS. THIS WORK WILL SIGNIFICANTLY ACCELERATE OUR UNDERSTANDING OF BLP COMPOSITION, STRUCTURE, AND FUNCTION AND THE SPECIFIC ROLE OF APOE IN THE DEVELOPMENT OF AD.

HOW MERS-COV REGULATES INNATE IMMUNITY IN PRIMARY HUMAN LUNG CELLS

STUDIES OF ATMOSPHERIC BROWN CARBON CHEMISTRY IN SUPPORT OF THE FIREX CAMPAIGN

MODULATION OF MJO-DIURNAL CYCLE INTERACTION OVER THE MARITIME CONTINENT

COMPETING WATER USES IN THE FACE OF CLIMATE CHANGE

MODELING CELL RECEPTOR SIGNALING PATHWAYS

DEVELOPMENT OF DECISION SUPPORT TOOLS FOR HARMFUL ALGAL BLOOM MONITORING IN A CHANGING CLIMATE USING A COUPLED MODELING APPROACH

EARLY DETECTION OF BREAST CANCER USING POST-TRANSLATIONALLY MODIFIED BIOMARKERS

MULTIDIMENSIONAL PROTEOMICS PLATFORM

STRUCTURE OF THE BRCA1-PALB2-BRCA2 COMPLEX - PROJECT SUMMARY THE BRCA1, PALB2, AND BRCA2 PROTEINS FORM A HETEROCOMPLEX THAT IS INVOLVED IN THE MAINTENANCE OF GENOMIC STABILITY, AND VARIANTS OF THESE PROTEINS ARE IMPLICATED IN MULTIPLE CANCERS. UNFORTUNATELY, THE FULL-LENGTH STRUCTURAL DETAILS FOR BRCA1, PALB2, AND BRCA2 ARE UNSOLVED WHICH OBSCURES A COMPLETE UNDERSTANDING OF THEIR MOLECULAR MECHANISM OF ACTION. OUR LONG-TERM GOAL IS TO UNDERSTAND THE MOLECULAR FEATURES OF THE BRCA1- PALB2-BRCA2 AXIS AND HOW DISEASE RELATED MUTANTS IMPACT THEIR STRUCTURE SUCH THAT BETTER TARGETED TREATMENT APPROACHES CAN BE REALIZED. THE PRIMARY GOAL OF THIS PROPOSAL IS TO PRODUCE FULL-LENGTH WILD TYPE BRCA1, PALB2, AND BRCA2 (ALONG WITH ADDITIONAL PROTEIN PARTNERS BARD1, DSS1 AND RAD51) TO DETERMINE THE STRUCTURE OF STABILIZED HETEROCOMPLEXES. OUR HYPOTHESIS IS THAT THE PAST DIFFICULTY IN SOLVING THE STRUCTURE OF THESE CORE PROTEINS IS DUE TO THE PRESENCE OF INTRINSICALLY DISORDERED DOMAINS THAT FAIL TO ADOPT A STABLE CONFORMATION UNLESS PART OF A LARGER HETEROCOMPLEX. THE RATIONALE AND INNOVATION UNDERLYING THE PROPOSED WORK IS THAT CELL-FREE EXPRESSION ALLOWS SMALL SCALE, VERSATILE AND RAPID OPTIMIZATION OF CONDITIONS TO INCREASE THE CHANCES OF SYNTHESIZING A FULL-LENGTH PROTEIN WITH SIMILAR SOLUBILITY, BIOCHEMICAL AND OTHER CHARACTERISTICS AS THE NATIVE PROTEIN. ADDITIONALLY, CRYOGENIC ELECTRON MICROSCOPY (CRYO-EM) CAN INTERROGATE PROTEINS THAT ADOPT MULTIPLE CONFORMATIONS OR CONTAIN FLEXIBLE OR INTRINSICALLY DISORDERED REGIONS AS BRCA1, PALB2 AND BRCA2 ARE KNOWN TO POSSESS. TWO SPECIFIC AIMS WILL BE PURSUED TO COMPLETE THIS WORK: 1) EXPRESS, PURIFY, AND CHARACTERIZE BRCA1, PALB2, AND BRCA2 COMPLEXES. 2) DETERMINE STRUCTURE OF BRCA1, PALB2, AND BRCA2 COMPLEXES USING SINGLE PARTICLE CRYO-EM. THE SUCCESS OF THE PROJECT WILL HINGE ON RECENT ADVANCEMENTS IN CRYO-EM AND OUR CELL- FREE EXPRESSION PIPELINE. THE PROPOSED RESEARCH IS SIGNIFICANT SINCE IT WILL PROVIDE A FLEXIBLE FRAMEWORK FOR PRODUCTION AND STRUCTURAL CHARACTERIZATION OF BRCA1, PALB2, AND BRCA2 COMPLEXES. THESE AIMS DIRECTLY ALIGN WITH THE FUNDING OPPORTUNITY NIH PAR-23-058 “NCI SMALL GRANTS PROGRAM FOR CANCER RESEARCH FOR YEARS 2023, 2024, AND 2025” AS OUR GOAL IS TO GENERATE AN IMPROVED UNDERSTANDING OF MULTIPLE TUMOR SUPPRESSOR PROTEINS. THIS BASIC RESEARCH PROPOSAL IS BOTH A FEASIBILITY STUDY, AS WELL AS A SMALL SELF-CONTAINED RESEARCH PROJECT THAT CAN BE COMPLETED IN TWO YEARS. MORE IN-DEPTH MOLECULAR PHENOTYPING, AND FUNCTIONAL AND MECHANISTIC INSIGHTS INTO BRCA-RELATED DNA REPAIR PROCESSES ARE BEYOND THE SCOPE OF THE CURRENT PROPOSAL DUE TO BOTH TIMEFRAME AND BUDGET. THAT WORK IN COMBINATION WITH MORE DETAILED STRUCTURAL COMPARISON OF MORE DISEASE RELATED VARIANTS WOULD BE THE FOCUS OF A SEPARATE FOLLOW-ON PROPOSAL. THE EXPECTED OUTCOME OF THIS WORK IS THE SYNTHESIS OF WILD TYPE BRCA1, PALB2, AND BRCA2 ALONG WITH STRUCTURAL CHARACTERIZATION OF THE BRCA1- PALB2-BRCA2 AXIS. IT WILL ALSO LAY THE FOUNDATION FOR A WORKFLOW EXTENDING OUR CELL-FREE EXPRESSION PIPELINE FROM BIOENERGY RESEARCH INTO THE BIOMEDICAL FIELD TO PERMIT ITS APPLICATION TOWARD OTHER CANCER RELATED PROTEINS WHOSE STRUCTURE REMAIN ELUSIVE.

NEXT GENERATION, 'STANDARDS-FREE' METABOLITE IDENTIFICATION PIPELINE

CELL-FREE SYNTHESIS OF INFLUENZA VIRUS-LIKE PARTICLES (VLPS) AS PROTOTYPING PLATFORM FOR VACCINE DEVELOPMENT AND VARIANT CHARACTERIZATION - SUMMARY THE ABSENCE OF PROPHYLACTIC AND LOW-COST INFLUENZA VACCINES ALONG WITH SLOW PRODUCTION RATE MAKES US UNPREPARED FOR THE NEXT PANDEMIC OUTBREAK WORLDWIDE. RECOMBINANT VIRUS-LIKE PARTICLES (VLPS) VACCINES PROVIDE US WITH UNIQUE OPPORTUNITIES TO ADDRESS THESE DIFFICULTIES, BUT THEIR RECOMBINANT SYNTHESIS STRATEGIES ARE LIMITED TO CELL CULTURE PRODUCTION, WHICH HAS ITS CHALLENGES SUCH AS LIMITED THROUGHPUT AND ARCHITECTURAL HETEROGENEITY OF THE OBTAINED PRODUCTS. IN THIS PROJECT, WE AIM TO COMBINE WHEAT GERM CELL-FREE PROTEIN EXPRESSION PLATFORM WITH ACTIVE MICROSOMES OF DIFFERENT ORIGINS (INCLUDING HUMAN) TO SYNTHESIZE GLYCOSYLATED INFLUENZA VIRUS-LIKE PARTICLES (VLPS). THESE PARTICLES WILL FURTHER UNDERGO DETAILED MORPHOLOGICAL AND COMPOSITIONAL EXAMINATION USING ELECTRON MICROSCOPY AND NATIVE MASS SPECTROMETRY TO UNDERSTAND THE ARCHITECTURE OF OBTAINED PRODUCTS AND UNDERSTAND THE MECHANISMS OF THEIR ASSEMBLY AND COMPOSITION VARIABILITY. HIGH- RESOLUTION TOMOGRAPHY WILL BE UTILIZED TO DETERMINE THE BEST VLP CANDIDATES WITH MAXIMAL ANTIGEN OCCUPANCY AND THUS MAXIMAL VACCINE EFFICIENCY. WE HYPOTHESIZE THAT THIS APPROACH WILL BE SUITABLE FOR A STABLE AND STREAMLINED GENERATION OF INFLUENZA VLPS AND WILL OFFER MUCH MORE FLEXIBILITY AND CONTROL OVER THEIR ARCHITECTURAL HETEROGENEITY AND EVENTUALLY SERVE AS A RAPID PROTOTYPING PLATFORM FOR INFLUENZA VACCINE DEVELOPMENT. THE CURRENT PROJECT WILL ALSO INFORM US ABOUT THE UTILITY OF USING SUCH APPROACHES FOR VIROLOGY AND FOR GLYCOPROTEIN SYNTHESIS IN GENERAL.

DEVELOPMENTAL EXPOSURE TO POLYAROMATIC HYDROCARBONS ALTERS FUNCTIONAL CAPACITIES IN METABOLISM AT THE HOST-GUT MICROBIOME INTERFACE CHANGING SUSCEPTIBILITY TO CHRONIC DISEASE ONSET

RESEARCHING UNDERSTUDIED PROTEINS OF CHIARI MALFORMATION - SUMMARY MULTIPLE CHROMODOMAIN-HELICASE-DNA-BINDING (CHD) PROTEINS HAVE BEEN IMPLICATED IN RARE DISEASES. WHILE CHD PROTEINS ARE FUNCTIONALLY KNOWN TO BE DNA HELICASES AND CHROMATIN REMODELING FACTORS THAT CAN REGULATE TRANSCRIPTION, INSIGHT INTO THEIR STRUCTURE AND HOW IT INFLUENCES FUNCTION REMAINS SPARSE. EVEN ALPHAFOLD2 MODELS FOR THESE PROTEINS HAVE LARGE REGIONS OF LOW CONFIDENCE (OVER 60% OF RESIDUES IN CHD8 HAVE PLDDT SCORES LESS THAN 70). OUR LONG-TERM GOAL IS TO UNDERSTAND THE MOLECULAR FEATURES OF THESE PROTEINS AND HOW THEY IMPACT CHIARI MALFORMATION SUCH THAT BETTER DIAGNOSTIC AND TREATMENT APPROACHES CAN BE REALIZED. THE PRIMARY GOAL OF THIS PROPOSAL IS TO PRODUCE FULL-LENGTH WILD TYPE AND DISEASE RELATED VARIANTS OF THREE CHD PROTEINS ON NIH’S LIST OF UNDERSTUDIED PROTEINS RELATED TO RARE DISEASE AND BEGIN THEIR BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION. OUR HYPOTHESIS IS THAT IN VITRO CELL-FREE EXPRESSION CAN OVERCOME THE PRIMARY BOTTLENECK TO PRODUCING THESE PROTEINS TO BEGIN DEEPER MOLECULAR CHARACTERIZATION STUDIES. THE RATIONALE UNDERLYING THE PROPOSED WORK IS THAT CELL-FREE EXPRESSION ALLOWS SMALL SCALE, VERSATILE AND RAPID OPTIMIZATION OF CONDITIONS TO INCREASE THE CHANCES OF SYNTHESIZING A FULL-LENGTH PROTEIN THAT HAS SIMILAR SOLUBILITY, BIOCHEMICAL AND OTHER CHARACTERISTICS AS THE NATIVE PROTEIN. TWO SPECIFIC AIMS WILL BE PURSUED TO COMPLETE THIS WORK: 1) EMPLOY CELL-FREE EXPRESSION TO SYNTHESIZE CHD2, CHD7, AND CHD8 AS FULL-LENGTH WILD-TYPE PROTEINS ALONG WITH 2 DISEASE RELATED VARIANTS OF EACH. 2) BEGIN DEEP PROFILING OF SYNTHESIZED PROTEINS BY EVALUATING PROTEIN-PROTEIN BINDING AND DNA BINDING AND INITIATE STRUCTURAL CHARACTERIZATION. THE SUCCESS OF THE PROJECT WILL HINGE ON RECENT INNOVATIONS IN OUR CELL-FREE EXPRESSION PIPELINE INCLUDING THE ABILITY TO PERFORM BOTH FUNCTIONAL BINDING AND STRUCTURAL STUDIES FROM CELL-FREE EXPRESSION REACTIONS OF ONLY 25-MICROLITER VOLUME AND WITHOUT PURIFICATION. THE PROPOSED RESEARCH IS SIGNIFICANT SINCE IT WILL REVEAL NEW MOLECULAR DETAILS ABOUT 3 UNDERSTUDIED PROTEINS LINKED TO MULTIPLE RARE DISEASES. IT WILL ALSO LAY THE FOUNDATION FOR A WORKFLOW EXTENDING OUR CELL-FREE EXPRESSION PIPELINE FROM BIOENERGY RESEARCH AND INTO THE BIOMEDICAL FIELD TO PERMIT ITS APPLICATION TOWARD OTHER UNDERSTUDIED PROTEINS IN THE FUTURE. THE EXPECTED OUTCOME OF THIS WORK IS THE SYNTHESIS OF WILD TYPE AND MUTANT VARIANTS OF CHD2, CHD7 AND CHD8 ALONG WITH INITIAL BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION. FOLLOW-ON PROJECTS ARE EXPECTED TO PROVIDE MORE IN-DEPTH MOLECULAR PHENOTYPING. THE RESULTS WILL IMMEDIATELY IMPACT THE STUDY OF CHIARI MALFORMATION AND ADVANCE THE UNDERSTANDING OF THE CHD FAMILY OF PROTEINS.

UNCERTAINTIES IN CLOUD FEEDBACKS WITHIN THE CLIMATE SYSTEM ARE LINKED FUNDAMENTALLY TO A DEFICIT OF KNOWLEDGE REGARDINGRELATIONSHIPS BETWEEN ATMOSPH

UTILIZATION OF MOTRPAC DATA TO CHARACTERIZE WHITE ADIPOSE TISSUE MOLECULAR AGING AND ITS POTENTIAL MITIGATION WITH ENDURANCE TRAINING-FROM A SEX-SPECIFIC PERSPECTIVE - ABSTRACT - PACIFIC NORTHWEST NATIONAL LABORATORY – PNNL THE PATHOGENESIS, PRESENTATION AND RISK OF CARDIOMETABOLIC RISK FACTORS AND DISEASES DIFFERS BY SEX. THE SUBCUTANEOUS WHITE ADIPOSE TISSUE (SCWAT) IS A DYNAMIC STORAGE AND ENDOCRINE ORGAN WITH CENTRAL ROLES IN MEDIATING CARDIOMETABOLIC DISEASE RISK THROUGHOUT THE LIFESPAN. AS WE AND OTHERS PREVIOUSLY DEMONSTRATED, THE SCWAT OF ADULT FEMALE RODENTS AND HUMANS DISPLAYS A METABOLICALLY HEALTHY AND CARDIOPROTECTIVE PHENOTYPE RELATIVE TO THAT OF MALES. HOWEVER, DECLINES IN FEMALE SEX HORMONES THAT OCCUR WITH AGE SHIFT SCWAT PHENOTYPES TO MORE CLOSELY RESEMBLE THAT OF MALES AND DRIVE AGE-ASSOCIATED INCREASES IN CARDIOMETABOLIC DISEASE RISK. REGULAR ENDURANCE EXERCISE TRAINING (EET) SUBSTANTIALLY REDUCES CARDIOMETABOLIC DISEASE AND ALL-CAUSE MORTALITY RISK THROUGHOUT THE LIFESPAN, YET THE SYSTEMIC MOLECULAR MECHANISMS OF THE HEALTH BENEFITS OF EET WARRANT ADDITIONAL INVESTIGATION, ESPECIALLY IN TERMS OF SEX AND AGE-ASSOCIATED DIFFERENCES. THE MOLECULAR TRANSDUCERS OF PHYSICAL ACTIVITY CONSORTIUM (MOTRPAC) SEEKS TO DEVELOP A MOLECULAR MAP OF EXERCISE TO BETTER UNDERSTAND THE ROLE OF EXERCISE ON HEALTH AND DISEASE. HERE WE PROPOSE TO UTILIZE BOTH PUBLICLY AND PRIVATELY AVAILABLE DATA FROM MOTRPAC TO UNCOVER MOLECULAR FEATURES OF SCWAT AGING AND THE IMPACT OF EET ON SCWAT MOLECULAR AGING FEATURES IN MALES AND FEMALES. THIS PROPOSAL SEEKS TO ADDRESS THE FOLLOWING: AIM 1) DEFINE SCWAT MOLECULAR AGING INCLUDING THE IMPACT OF SEX ON SCWAT MOLECULAR AGING PHENOTYPES IN THE SEDENTARY STATE; 2) DETERMINE THE IMPACT OF AGE AND SEX ON THE SCWAT RESPONSE TO EET. WE WILL ACCOMPLISH THIS BY FIRST COMPARING DIFFERENCES IN THE SCWAT MOLECULAR LANDSCAPE OF SEDENTARY 6 MO F344 RATS (MOTRPAC PUBLICLY AVAILABLE DATA SET) TO 18 MO F344 RATS (MOTRPAC CURRENTLY PRIVATELY AVAILABLE DATA SET) (AIM 1A). THEN WE WILL EXAMINE CORRELATIONS BETWEEN SCWAT SAMPLE-LEVEL MULTIOMIC FEATURES AND THE AGE OF SEDENTARY HUMAN PARTICIPANTS SEPARATELY BY SEX TO IDENTIFY AGE-ASSOCIATED MOLECULAR FEATURES SHARED BETWEEN HUMAN AND RAT (AIM 1B). WE WILL THEN COMPARE DIFFERENCES IN THE MULTIOMIC RESPONSE TO EET BY COMPARING FEATURE LEVEL AND PATHWAY ENRICHMENTS RESPONSES IN 6 AND 18 MO MALE AND FEMALE RATS (AIM 2). COMPARISON OF SCWAT SEXUAL DIMORPHISM AND THE DYNAMIC RESPONSE TO EXERCISE TRAINING BY SEX AND AGE THROUGH UTILIZATION OF MULTIOMIC DATA SETS GENERATED BY MOTRPAC OFFERS UNPRECEDENTED POTENTIAL TO UNDERSTAND SCWAT MOLECULAR AGING AND ITS ASSOCIATION WITH DISEASE-ASSOCIATE PHENOTYPES—ALL DATA AND ANALYSES WILL BE MADE PUBLICLY AVAILABLE TO FACILITATE DISSEMINATION TO THE SCIENTIFIC COMMUNITY. FINDINGS THUS OFFER THE POTENTIAL TO ELUCIDATE MOLECULAR DRIVERS OF SEXUAL DIMORPHISM IN SCWAT AGING BIOLOGY AND INFORM FUTURE STUDIES TOWARDS A PRECISION MEDICINE APPROACH.

"SUSTAINED GROUND-BASED OBSERVATIONS OF RADIATION BUDGET COMPONENTS AND RELATED PARAMETERS"THE MAGNITUDE AND DIRIBUTION OF SOLAR AND THERMAL IRRADIA

A TROPICAL UPPER-OCEAN STRATIFICATION DATA SET FOR CLIMATE AND HURRICANE RESEARCH.

SUPPORT FOR US HUPO MEETING "FUTURE OF PROTEOMICS"

15.255 APPLIED SCIENCE

APPLIED SCIENCE

STEPHEN GAJEWSKI/BATTELLE MEMORIAL INSTITUTE PACIFIC NORTHWEST NATIONAL LABORATORY GULF OF MEXICO REGIONAL COLLABORATIVE (GOMRC)

OBLIGATION - BIOMASS RESEARCH AND DEVELOPMENT GRANTS

CATALYTIC CONVERSION OF BIOMASS TO FUELS AND CHEMICALS USING IONIC LIQUIDS

PACIFIC NORTHWEST REGIONAL COLLABORATORY (PNWRC) THE PACIFIC NORTHWEST (PNW) IS A MOSAIC OF FEDERAL, STATE, PRIVATE, AND TRIBAL LANDOWNERS WHO HAVE