Institute For Systems Biology

CENTER FOR SYSTEMS BIOLOGY

NANOSYSTEMS BIOLOGY CANCER CENTER

CENTER FOR SYSTEMS ANALYSIS OF THE CANCER REGULOME

SPATIOTEMPORAL TUMOR ANALYTICS FOR GUIDING SEQUENTIAL TARGETED-INHIBITOR: IMMUNOTHERAPY COMBINATIONS (ST-ANALYTICS) - OVERALL PROJECT SUMMARY THE PROPOSED U54 PROGRAM SPATIOTEMPORAL TUMOR ANALYTICS FOR GUIDING SEQUENTIAL TARGETED-INHIBITOR -- IMMUNOTHERAPY COMBINATIONS (ST-ANALYTICS) IS DESIGNED TO DEVELOP THE RECENT CONCEPTUAL ADVANCE THAT TARGETED INHIBITOR + CANCER IMMUNOTHERAPY (IT) COMBINATION TREATMENTS MAY YIELD SIGNIFICANTLY GREATER PATIENT BENEFIT IF THOSE TREATMENTS ARE ADMINISTERED IN SEQUENCE RATHER THAN SIMULTANEOUSLY. ANALYSIS OF RETROSPECTIVE CLINICAL DATA COUPLED WITH IN VIVO THERAPEUTIC MODELING USING SYNGENEIC MODELS OF MURINE MELANOMA STRONGLY SUPPORT THIS CONCEPT. IN FACT, THE PICTURE THAT HAS EMERGED IN MELANOMA IS THAT IMMUNE FACTORS CAN PLAY A STRONG ROLE IN DRIVING RESISTANCE TO MAPK INHIBITOR (MAPKI) THERAPY, AND THAT LEAD-IN IMMUNE CHECKPOINT BLOCKADE (ICB) CAN ‘PRIME’ BOTH THE PRIMARY TUMOR AND DISTAL METASTASES (INCLUDING BRAIN METASTASES) FOR ERADICATION WHEN THE IT IS SUBSEQUENTLY COMBINED WITH MAPKI. THIS OBSERVATION OPENS THE DOORS FOR IMMUNE BASED STRATEGIES, SUCH AS ICB OR ADOPTIVE CELL THERAPY (ACT), AS SEQUENTIAL COMBINATORIAL AGENTS TO PREVENT MAPKI RESISTANCE. HOWEVER, THIS CONCEPT INTRODUCES A NUMBER OF NEW VARIABLES, INCLUDING DOSING, SEQUENCE, AND TIMING. THIS CAN MAKE THE DESIGN AND EXECUTION OF CLINICAL TRIALS THAT CAN YIELD STATISTICALLY SIGNIFICANT OUTCOMES IMPRACTICAL. THIS IS THE SCIENTIFIC AND TRANSLATIONAL PROBLEM WE ADDRESS IN THE PROPOSED ST-ANALYTICS U54. THE ST-ANALYTICS U54 CENTER IS POPULATED BY LEADING SCIENTISTS AT THE ISB, THE UCLA GEFFEN SCHOOL OF MEDICINE, AND YALE, AND IS COMPRISED OF TWO RESEARCH PROJECTS AND TWO RESEARCH CORES, WITH EACH PROJECT INTEGRATING BOTH STATE-OF-THE-ART EXPERIMENTATION AND COMPUTATIONAL WORK. THIS STRUCTURE IS FURTHER DESIGNED TO BRING TOGETHER THE SCIENTIFIC, EXPERIMENTAL, AND COMPUTATIONAL AND ADMINISTRATIVE RESOURCES TO DEVELOP A DATA BASE THAT CAPTURES THE KINETICS OF LEAD-IN MONOTHERAPY TUMOR PRIMING, AND APPLY THAT DATA BASE TO THE DEVELOPMENT OF PREDICTIVE IN SILICO MODELS THAT CAN INFORM THE DESIGN OF SUCH TARGETED INHIBITOR – IMMUNOTHERAPY SEQUENCE COMBINATIONS FOR CLINICAL TRIALS. THIS REQUIRES CLOSE INTEGRATION AND CYCLES OF ITERATION BETWEEN OF STATE-OF-THE-ART EXPERIMENTATION, LEADING EDGE COMPUTATION, AND REALISTIC DISEASE MODELS, CONTINUOUSLY CALIBRATED THROUGH THE ANALYSIS OF HIGHLY RELEVANT, BIOPSIED PATIENT TUMORS. THE RESULTING SCIENCE ALSO PROVIDES EXCITING OPPORTUNITIES FOR HIGH IMPACT STEM OUTREACH. WE PROPOSE TO ACT ON THOSE OPPORTUNITIES BY LEVERAGING A LONG-STANDING SYSTEMS EDUCATION OUTREACH PROGRAM AT ISB THAT ALREADY HAS IMPACTED K-12 STEM EDUCATION IN ALL 50 STATES, AND PLACES AN EMPHASIS ON THOSE COMMUNITIES THAT HAVE BEEN HISTORICALLY UNDER-REPRESENTED IN STEM.

A SYSTEMS ANALYSIS OF DRUG TOLERANCE IN MYCOBACTERIUM TUBERCULOSIS

MAINTENANCE AND DEVELOPMENT OF REPEATMASKER

DEVELOPMENT OF TRANS PROTEOMICS PIPELINE, AN ANALYSIS SUITE FOR MASS SPECTROMETRY

TRANSCRIPTIONAL CONTROL DURING ERYTHROPOIESIS

CLINICAL SAMPLE COLLECTION COVID19 PATIENTS TO ALIGN WITH NIAID NATIONAL STUDY

DFAM: SUSTAINABLE GROWTH, CURATION SUPPORT, AND IMPROVED QUALITY FOR MOBILE ELEMENT ANNOTATION

COMPLETE HUMAN PEPTIDE- AND MRM-ATLAS

ELUCIDATING THE ROLE OF TSR GLYCOSYLATION IN PLASMODIUM PARASITES

STEADY STATES AND CELLULAR TRANSITIONS ASSOCIATED WITH CARCINOGENESIS AND TUMOR PROGRESSION

SYSTEMS BIOLOGY OF INTRATUMORAL HETEROGENEITY IN GLIOBLASTOMA - PROPOSAL SUMMARY (REVISIONS IN ORANGE FONT ) PATIENTS WITH GLIOBLASTOMA (GBM) HAVE A 12-14 MONTH MEDIAN SURVIVAL RATE, ~10% CHANCE OF 5-YEAR SURVIVAL, AND ~90% LIKELIHOOD OF RECURRENCE, EVEN AFTER RECEIVING STANDARD OF CARE (SOC), WHICH INVOLVES TUMOR RESECTION, FRACTIONATED RADIATION THERAPY (XRT), AND CHEMOTHERAPY WITH TEMOZOLOMIDE (TMZ). THERE IS GROWING EVIDENCE THAT THIS POOR PROGNOSIS AND DISMAL THERAPY RESPONSIVENESS EMERGES FROM INTERPLAY OF TUMOR CELL HETEROGENEITY AND NON-GENETIC, TREATMENT-INDUCED SHIFTS OF CELLULAR PHENOTYPIC STATES. NOTABLY, THE SOC HAS BEEN SHOWN TO DRIVE A SHIFT OF TUMOR CELLS FROM A DRUG-SUSCEPTIBLE PRONEURAL (PN) SUBTYPE TO A DRUG-RESISTANT MESENCHYMAL (MES) SUBTYPE. THIS PARTLY EXPLAINS WHY PRIMARY GBM TUMORS OF THE CLASSICAL OR PN SUBTYPE OFTEN RECUR AS THE MORE AGGRESSIVE AND DRUG-RESISTANT MES SUBTYPE. TO COMPLICATE MATTERS FURTHER, EXTRINSIC SIGNALS AND STRESSORS CAN DRIVE DEDIFFERENTIATION OF A HETEROGENEOUS TUMOR CELL POPULATION INTO IMMATURE, GLIOMA STEM-LIKE CELLS (GSCS), WHICH HAVE BEEN IMPLICATED IN TUMOR RECURRENCE. GSCS ARE RESISTANT TO MULTIPLE CYTOTOXIC DRUGS LIKE TMZ, WHICH MOTIVATES THE NEED FOR DISCOVERING NOVEL CYTOTOXIC DRUGS, INCLUDING DRUGS REPURPOSED FROM OTHER INDICATIONS, TO TREAT GBM. NOTABLY, WE HAVE DISCOVERED THAT OFF-LABEL FDA-APPROVED DRUGS ARE EFFECTIVE AGAINST PATIENT-DERIVED GSCS (PD-GSCS) INCREASING MEDIAN SURVIVAL OF PATIENTS BY >3X, BUT CAN ALSO INDUCE TRANSITION OF A SURVIVING SUBPOPULATION FROM A SUSCEPTIBLE PN SUBTYPE TO A MES SUBTYPE – CALLED PN-TO-MES TRANSITION (PMT). HERE, WE PROPOSE TO ELUCIDATE AT SINGLE-CELL RESOLUTION THE MECHANISMS BY WHICH DIVERSE DRUGS INDUCE PMT WITHIN A HETEROGENEOUS POPULATION OF GSCS. WE HYPOTHESIZE THAT EARLY RESPONSE TO DRUG TREATMENTS WILL VARY BY MECHANISMS OF ACTION OF DRUGS AND PATIENT-SPECIFIC CHARACTERISTICS OF PD-GSCS, BUT CYTOTOXIC EVENTS WILL DRIVE THESE RESPONSES ONTO A COMMON PATHWAY THAT CAN BE TARGETED WITH GENETIC AND CHEMICAL INTERVENTIONS TO BLOCK DRUG-INDUCED PMT. WE WILL TEST THIS HYPOTHESIS BY SINGLE-CELL PROFILING OF LONGITUDINAL CHANGES IN TRANSCRIPTION (SCRNA-SEQ), CHROMATIN ACCESSIBILITY (SCATAC-SEQ), AND PHENOTYPES OF UP TO 34 PATIENT-DERIVED GSCS (PD-GSCS) ACROSS 76 FDA APPROVED ANTI-PROLIFERATIVE COMPOUNDS. WE WILL INTEGRATE THE LONGITUDINAL MULTI- OMIC PROFILES TO DISCOVER THE TRANSCRIPTIONAL REGULATORY NETWORK (TRN) THAT MECHANISTICALLY DRIVES DRUG-INDUCED PMT IN EACH PD-GSC. BY COMPARING TRNS ACROSS PD-GSCS AND DRUG TREATMENTS, WE WILL IDENTIFY, PERTURB, AND CHARACTERIZE MECHANISMS OF DRUG-INDUCED PMT IN EACH PD-GSC. USING FDA-APPROVED DRUGS MAPPED TO VALIDATED MECHANISMS, WE WILL PERFORM HIGH THROUGHPUT SCREENS TO EVALUATE THE SENSITIVITY AND SPECIFICITY OF OUR MODEL-DRIVEN APPROACH TO IDENTIFY DRUG COMBINATIONS THAT SYNERGISTICALLY KILL PD-GSCS, WITHOUT INDUCING PMT. OUTCOMES OF THIS PROJECT INCLUDE (I) METHODOLOGY TO ELUCIDATE SINGLE-CELL RESOLUTION TRNS WITHIN SUBPOPULATIONS OF A HETEROGENEOUS TUMOR, (II) INSIGHT INTO MECHANISMS OF DRUG-INDUCED PMT IN PD-GSCS, AND (III) A RATIONAL STRATEGY TO REPURPOSE, AND TAILOR FDA-APPROVED COMBINATION DRUG REGIMENS FOR OFF-LABEL USE IN TREATING GBM. LINES: 30 (0 UNDER/OVER)

CYBERGUT: TOWARDS PERSONALIZED HUMAN-MICROBIOME METABOLIC MODELING FOR PRECISION HEALTH AND NUTRITION - PROJECT SUMMARY THE GUT MICROBIOME AIDS IN THE DIGESTION OF COMPLEX POLYSACCHARIDES, THE ABSORPTION OF VITAMINS, AND THE CONVERSION OF PRIMARY BILE ACIDS, DRUGS, AND OTHER BIOACTIVE COMPOUNDS INTO METABOLITES THAT CAN BE ABSORBED BY THE HOST. THUS, THE METABOLIC ACTIVITY OF COMMENSAL MICROBES IS CLOSELY INTERTWINED WITH HUMAN PHYSIOLOGY AND THE NUTRITIONAL IMPACT OF OUR DIET. HOWEVER, THERE IS LIMITED UNDERSTANDING OF HOW VARIATION IN THE ECOLOGY OF OUR INTESTINAL FLORA MODULATES THE BIOLOGICAL IMPACT OF DIET ON HUMAN HEALTH AND NUTRITION. RECENT WORK HAS SHOWN THAT DIFFERENCES IN THE COMPOSITION OF THE GUT MICROBIOME CAN HELP EXPLAIN PERSON-TO-PERSON HETEROGENEITY IN GLYCEMIC RESPONSES, BLOOD LIPID PROFILES, AND WEIGHT LOSS. IN THIS PROPOSAL, WE PRESENT AN INNOVATIVE PLATFORM FOR PERSONALIZED METABOLIC MODELING OF THE GUT MICROBIOME USING METAGENOMIC AND DIETARY DATA AS CONSTRAINTS. WE PROPOSE THE INTEGRATION OF TISSUE-RESOLVED METABOLIC MODELS OF RELEVANT HOST TISSUES, INCLUDING A CELL-TYPE-SPECIFIC METABOLIC RECONSTRUCTION OF THE GUT EPITHELIUM, INTO OUR EXISTING MICROBIAL COMMUNITY MODEL TO IMPROVE ESTIMATES OF METABOLIC FLUXES BETWEEN THE GUT MICROBIOTA, THE DIET, AND THE HOST. WE WILL CALL THIS HOST-DIET-MICROBIOME METABOLIC MODEL ‘CYBERGUT.’ USING EXISTING MULTI-OMIC DATA FROM A COHORT OF >3,000 ADULTS, WE WILL CONSTRAIN AND VALIDATE CYBERGUT WITH PAIRED MEASUREMENTS OF DIET, HOST BLOOD METABOLOMES, AND GUT MICROBIOMES. IN ADDITION, WE WILL GENERATE CROSS-SECTIONAL TRAINING AND VALIDATION DATA CONSISTING OF PAIRED BLOOD AND FECAL METABOLOMES, FECAL MICROBIOMES, AND DETAILED 3-DAY DIETARY RECALL DATA FROM A NEW COHORT OF 100 HEALTHY PARTICIPANTS. USING THESE DATA, WE WILL REFINE AND TEST TWO NOVEL AND INDEPENDENT DIET-INFERENCE ALGORITHMS, WHICH LEVERAGE STOOL METAGENOMES AND STOOL UNTARGETED METABOLOMES, RESPECTIVELY. FURTHERMORE, USING SAMPLES TAKEN FROM A SUBSET OF THIS NEW COHORT (N=40), WE WILL PERFORM EX VIVO STOOL CULTURING EXPERIMENTS, DESIGNED TO DIRECTLY QUANTIFY METABOLIC FLUXES AND BACTERIAL GROWTH RATES IN VITRO. THESE FLUXOMIC DATA WILL BE USED TO DIRECTLY TEST IN SILICO CYBERGUT FLUX PREDICTIONS IN RESPONSE TO A DIVERSE PANEL OF DIETARY AND HOST METABOLITE INTERVENTIONS. IN ADDITION TO CONTRIBUTING TO THE REFINEMENT AND TESTING OF OUR CYBERGUT MODEL, THE PAIRED DIET, MICROBIOME, AND METABOLOMIC DATA, INCLUDING REPLICATE FLUXOMIC ASSAYS, GENERATED IN THIS PROPOSAL WILL BE AN INVALUABLE RESOURCE TO THE PRECISION NUTRITION AND HUMAN MICROBIOME RESEARCH COMMUNITY. IN SUMMARY, WE WILL BUILD, REFINE, AND TEST A NOVEL PLATFORM FOR TRACKING DIETARY INTAKE AND PREDICTING PERSONALIZED NUTRITIONAL RESPONSES TO DIET, WHICH HAS THE POTENTIAL TO FUNDAMENTALLY ALTER HOW WE DESIGN AND TEST DIETARY INTERVENTIONS.

DEFINING THE IMMUNOGENIC LANDSCAPE OF EARLY MOSQUITO-STAGE P. FALCIPARUM TO ACCELERATE MALARIA TRANSMISSION-BLOCKING VACCINE DISCOVERY - PROJECT SUMMARY THE DISEASE MALARIA IS CAUSED BY VECTOR-BORNE PARASITES OF THE GENUS PLASMODIUM. WITH HUNDREDS OF MILLIONS OF INFECTIONS AND OVER HALF A MILLION DEATHS ANNUALLY, MALARIA REMAINS ONE OF THE FOREMOST GLOBAL HEALTH CHALLENGES. THE TWO WHO-RECOMMENDED MALARIA VACCINES HAVE ONLY MODERATE EFFICACY AGAINST SEVERE DISEASE AND DO NOT PROVIDE STERILIZING PROTECTION, NEITHER DO THEY PREVENT TRANSMISSION OF THE PARASITE TO THE MOSQUITO VECTOR THAT CARRIES IT BETWEEN HUMAN HOSTS. THE MAJORITY OF VACCINE DEVELOPMENT EFFORTS TO DATE HAVE FOCUSED ON MEANS TO PREVENT INFECTION OF HUMANS. HOWEVER, A COMPREHENSIVE MALARIA ERADICATION EFFORT ALSO REQUIRES BLOCKING TRANSMISSION WITH VECTOR-TARGETED INTERVENTIONS. COMPARED WITH VACCINES THAT PREVENT INFECTION BY TARGETING PRE- ERYTHROCYTIC AND ASEXUAL BLOOD STAGES OF THE PARASITE, VERY FEW CANDIDATE ANTIGENS HAVE BEEN IDENTIFIED FOR TRANSMISSION-BLOCKING VACCINES (TBVS) THAT TARGET THE TRANSMITTED FORMS OF THE PARASITE PRESENT IN THE MOSQUITO VECTOR STAGES. THIS DISPARITY ARISES IN PART BECAUSE THE COMPREHENSIVE CHARACTERIZATION OF PROTEIN EXPRESSION THAT INFORMS VACCINES AGAINST PRE-ERYTHROCYTIC AND ASEXUAL BLOOD STAGES HAS NOT BEEN PERFORMED FOR THE STAGES THAT DEVELOP IN THE MOSQUITO MIDGUT, I.E., GAMETES, ZYGOTES, AND OOKINETES. THE FEW TBV CANDIDATES THAT HAVE ENTERED CLINICAL TRIALS, INCLUDING PFS25, PFS48/45, AND PFS230, ARE SURFACE-EXPOSED ON THESE EARLY MOSQUITO-STAGE PARASITES. IMPORTANTLY, IT HAS BEEN DEMONSTRATED THAT ANTIBODIES AGAINST THESE PROTEINS CAN BE GENERATED IN THE VERTEBRATE HOST, CARRIED TO THE MOSQUITO VECTOR IN THE BLOOD MEAL, AND SUBSEQUENTLY INTERFERE WITH FERTILIZATION AND/OR SUCCESSFUL INFECTION OF THE MOSQUITO. WE HYPOTHESIZE THAT EARLY MOSQUITO-STAGE PARASITES EXHIBIT MANY MORE UNDISCOVERED SURFACE-EXPOSED PROTEINS, AND THAT THESE PROTEINS CAN BE TARGETED BY ANTIBODIES TO PREVENT TRANSMISSION OF THE MALARIA PARASITE TO THE MOSQUITO VECTOR. WE WILL USE MASS SPECTROMETRY-BASED PROTEOMICS TO PROVIDE THE FIRST COMPREHENSIVE CATALOG OF SURFACE-EXPOSED PROTEINS IN P. FALCIPARUM GAMETES, ZYGOTES, AND OOKINETES. IN PARALLEL, WE WILL IMMUNIZE ANIMALS WITH EARLY MOSQUITO-STAGE PARASITES AND IDENTIFY PARASITE PROTEINS THAT ELICIT AN IMMUNE RESPONSE. FROM THE SUBSET OF IMMUNOGENIC SURFACE-EXPOSED PARASITE PROTEINS, WE WILL IDENTIFY THE MOST PROMISING TBV CANDIDATES AND EVALUATE THEM AS ANTIGENS FOR TRANSMISSION-BLOCKING VACCINES BY QUANTIFYING THE ABILITY OF ANTIBODIES AGAINST THESE ANTIGENS TO PREVENT P. FALCIPARUM FROM INFECTING MOSQUITOES.

PROTOTYPE SYSTEM FOR AML DIGITAL TWINS - PROJECT ABSTRACT CONVENTIONAL CHEMOTHERAPY HAS REACHED ITS LIMITS IN TREATING ACUTE MYELOID LEUKEMIA (AML), THE MOST COMMON LEUKEMIA IN ADULTS. MANY PATIENTS WHO RECEIVE INTENSIVE CHEMOTHERAPY FOLLOWED BY ALLOGENEIC HEMATOPOIETIC STEM-CELL TRANSPLANT EVENTUALLY RELAPSE. THERAPEUTIC RESULTS REMAIN PARTICULARLY DISMAL FOR RELAPSED OR REFRACTORY AND OLDER PATIENTS, WHO ARE OFTEN UNFIT FOR INTENSIVE THERAPIES. SEVERAL NEW THERAPY OPTIONS WITH THE POTENTIAL TO IMPROVE TREATMENT OUTCOMES HAVE EMERGED, BUT THEY SUFFER FROM HETEROGENEITY IN RESPONSES AND A LACK OF METHODS FOR IDENTIFYING PATIENTS WHO MIGHT BENEFIT FROM SUCH THERAPIES. NEW DETAILED SIMULATIONS CALLED DIGITAL TWINS ARE STARTING TO BE IMPLEMENTED IN MANY SECTORS INCLUDING ENGINEERING, MANUFACTURING, AND MEDICINE. DIGITAL TWINS ARE A SIMULATION OF A REAL-WORLD SYSTEM OR OBJECT THAT ALLOWS FOR EXPERIMENTATION WITHOUT REAL-WORLD CONSEQUENCES. A PATIENT ORIENTED DIGITAL TWIN WOULD SIMULATE PHYSIOLOGICAL RESPONSE OR DISEASE AND ALLOW FOR OUTCOME PREDICTIONS FOLLOWING TREATMENT. WE PROPOSE TO BUILD A PROTOTYPE OF THE ‘AML DIGITAL TWIN’ SYSTEM (AML-DT), AN INTERACTIVE SYSTEM TO BE USED BY DOCTORS WITH THEIR PATIENTS IN WHICH THE PATIENT’S CLINICAL AND MOLECULAR DATA, COLLECTED FROM BONE MARROW ASPIRATES AND PERIPHERAL BLOOD, WILL BE USED TO INSTANTIATE A DIGITAL TWIN MODEL OF THE PATIENT’S DISEASE. THE DIGITAL TWIN WILL ENABLE THE DOCTOR AND PATIENT TO EXPLORE PERSONALIZED MODEL-BASED PREDICTIONS OF DRUG RESPONSE, USING MEASURABLE OUTCOMES IN LIGHT OF BACKGROUND KNOWLEDGE GENERATED FROM PUBLICLY AVAILABLE MOLECULAR DATA FROM AML PATIENTS. IMPORTANTLY, THE AML-DT SYSTEM WILL CONTINUOUSLY IMPROVE BY LEARNING FROM THE EXPERIENCES OF PATIENTS AND THEIR DIGITAL TWIN MODELS. THE WORK, CONDUCTED JOINTLY WITH CLINICIAN-SCIENTISTS AND COMPUTATIONAL RESEARCHERS AT THE INSTITUTE FOR SYSTEMS BIOLOGY, THE UNIVERSITY OF HELSINKI, AND TAMPERE UNIVERSITY WILL CENTER ON PATIENT-SPECIFIC DRUG SENSITIVITY PREDICTION OF VENETOCLAX IN COMBINATION WITH AZACITIDINE BY INTEGRATING WITH AN ONGOING CLINICAL TRIAL, VENEX, THAT INCLUDES EX-VIVO PATIENT SELECTION AND MOLECULAR DATA GENERATION FROM BONE MARROW ASPIRATES FROM AML PATIENTS IN FINLAND. A PROTOTYPE OF THE AML-DT SYSTEM WILL BE DEPLOYED AT THE MIDPOINT OF THE 5-YEAR PROJECT PERIOD, COINCIDING WITH THE COMPLETION OF THE CLINICAL TRIAL. FOLLOWING ASSESSMENT OF THE PROTOTYPE, FURTHER DEVELOPMENTS TO THE AML-DT WILL BE CARRIED OUT TO ACCOMMODATE OTHER AML THERAPIES AND RECOMMENDATIONS MADE BY PATIENT ADVOCATES AND CLINICIANS.

NANO AND BIOMOLECULAR ENGINEERED TECHNOLOGIES FOR NEOANTIGEN-SPECIFIC T CELL CAPTURE AND CHARACTERIZATION - PROJECT SUMMARY MUCH OF CANCER IMMUNOTHERAPY IS FOCUSED ON ENGINEERING OR ACTIVATING TUMOR-ANTIGEN SPECIFIC CD8+ T CELLS AND, TO A LESSER EXTENT, CD4+ T CELLS. IN PARTICULAR, NEOANTIGEN-SPECIFIC T CELLS ARE ATTRACTIVE BECAUSE THEY CAN KILL CANCER CELLS WITH HIGH SPECIFICITY. 1 A GENERAL APPROACH STARTS WITH IDENTIFYING T CELLS THAT RECOGNIZE NEOANTIGENS BROADLY EXPRESSED WITHIN THE TUMOR, ISOLATING THE T CELLS AND DETERMINING THEIR T CELL RECEPTOR (TCR) SEQUENCES. THESE TCRS CAN THEN BE TRANSFECTED INTO PATIENT T CELLS, PERHAPS WITH ADDITIONAL GENETIC ENGINEERING2 TO PROMOTE MORE DURABLE ANTI-TUMOR EFFECTS, AND EXPANDED INTO AN INFUSION PRODUCT FOR PATIENT TREATMENT. 3 IN FACT, THIS APPROACH HAS RECENTLY ENTERED THE CLINIC, WITH ONE TRIAL (NCT03970382) DRAWING FROM INVENTIONS FROM AN NCI- FUNDED CCNE U54 GRANT LED BY THE PI OF THIS PROPOSAL. 4,5 HOWEVER, THERE ARE STILL A NUMBER OF FUNDAMENTAL AND TECHNOLOGICAL CHALLENGES ASSOCIATED WITH ADVANCING NEOANTIGEN-SPECIFIC TCR-ENGINEERED THERAPIES. FIRST, THE DISCOVERY OF NEOANTIGEN-SPECIFIC TCRS RELIES ON GUIDANCE FROM ALGORITHMS, SUCH AS NET MHCPAN 4.1, TO PREDICT ANTIGEN/MHC PRESENTATION (BASED UPON BINDING AND OTHER CONSIDERATIONS), AND MANY NEOANTIGENS ARISING FROM TRUNCAL MUTATIONS, SUCH AS MUTANT KRAS OR MUTANT TP53, ARE PREDICTED AS UNLIKELY, YET HAVE BEEN REPORTED AS CLINICALLY EFFECTIVE TARGETS. 6,7,8 SECOND, NEOANTIGEN-SPECIFIC CD4+ T CELLS AND THEIR CLASS II RESTRICTED NEOANTIGENS, WHILE IDENTIFIED AS IMPORTANT FOR IMMUNOTHERAPY-INDUCED ANTI-TUMOR RESPONSES, 9,10 REMAIN A LARGELY UNTAPPED THERAPEUTIC RESOURCE, WITH PREDICTION ALGORITHMS 11,12 FOR CLASS II ANTIGEN/MHC BINDING LESS DEVELOPED. A THIRD CHALLENGE IS THAT ANALYSIS OF A PATIENT BLOOD FOR NEOANTIGEN-SPECIFIC T CELLS TYPICALLY REQUIRES UPWARDS OF 20M PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS), AND SO ISN'T PARTICULARLY EFFICIENT. HERE WE PROPOSE 3 SPECIFIC AIMS DESIGNED TO ADDRESS THESE OUTSTANDING ISSUES. AT THE HEART OF THE TECHNOLOGY SOLUTIONS ARE COMBINATIONS OF ENGINEERED NANOPARTICLES (NPS) AND BIOMOLECULAR ENGINEERED CONSTRUCTS DESIGNED FOR EFFICIENT AND SELECTIVE CAPTURE, ANALYSIS, AND VALIDATION OF TRUNCAL NEOANTIGEN-SPECIFIC CD4+ AND CD8+ T CELL POPULATIONS. SIGNIFICANT PRELIMINARY DATA IS PRESENTED FOR ALL 3 AIMS, SOME OF WHICH USES COVID-19 PATIENT DATA GENERATED BY OUR WORK DURING THE CURRENT PANDEMIC. 13,14 THE RESULT OF THIS WORK WILL BE A POWERFUL TOOLSET DESIGNED FOR A MINIMALLY-BIASED SEARCH FOR CD4+ AND CD8+ T CELL POPULATIONS AGAINST TRUNCAL NEOANTIGENS (INDEPENDENT OF PATIENT HLA HAPLOTYPE), A TOOLSET DESIGNED FOR THE RAPID VALIDATION AND CHARACTERIZATION OF THOSE NEOANTIGEN-SPECIFIC T CELL CLONOTYPES, AND A PUBLIC DATA BASE OF CLASS I AND CLASS II TRUNCAL NEOANTIGENS AND T CELL RECEPTOR GENES SPECIFIC TO THOSE NEOANTIGENS.

HOST INNATE IMMUNE MECHANISMS CONTROL TEMPORAL EXPRESSION OF FLAGELLIN BY PATHOGENIC SALMONELLA - PROJECT SUMMARY INFLAMMASOMES ARE MULTIPROTEIN COMPLEXES THAT SENSE MICROBIAL INFECTIONS AND RESPOND BY INDUCING A CASPASE- 1-MEDIATED FORM OF INFLAMMATORY CELL DEATH CALLED PYROPTOSIS. INFLAMMASOMES HAVE BEEN IMPLICATED IN THE DETECTION AND CLEARANCE OF A VARIETY OF BACTERIAL PATHOGENS, BUT LITTLE IS KNOWN ABOUT WHETHER THERE IS ACTIVE CROSS-TALK BETWEEN THE HOST SENSING MECHANISM AND THE EXPRESSION OF STIMULATORY LIGANDS BY THE PATHOGEN. WE HAVE FOUND THAT INFLAMMASOME ACTIVATION REGULATES EXPRESSION OF THE NLRC4 LIGAND, FLAGELLIN, BY SALMONELLA. A HOST LIPID STIMULUS RELEASED UPON NLRC4-MEDIATED MACROPHAGE PYROPTOSIS INCREASES EXPRESSION OF FLAGELLIN BY EXTRACELLULAR BACTERIA THAT ENHANCES PYROPTOSIS UPON INTERNALIZATION, ESTABLISHING A POSITIVE FEEDBACK LOOP THAT POTENTIATES SALMONELLA DETECTION AND CLEARANCE. AS INFECTION PROGRESSES, A NATURAL TYPE I INTERFERON-DEPENDENT HOST NEGATIVE FEEDBACK RESPONSE SHUTS DOWN EXPRESSION OF NLRC4 AND THE LYSOPHOSPHOLIPID BIOSYNTHETIC ENZYME IPLA2 TO SUB-BASELINE LEVELS, SWITCHING SALMONELLA TO A FLAGELLIN-LOW PHENOTYPE INSIDE MACROPHAGES. BASED ON THESE FINDINGS WE HYPOTHESIZE THAT SALMONELLA HAS EVOLVED TO CO-OPT NLRC4 ACTIVATION AND LIPID PRODUCTION TO INITIALLY ENHANCE PRODUCTION OF EXTRACELLULAR FLAGELLIN THAT PROMOTES SYSTEMIC SPREAD OF THE PATHOGEN AT THE RISK OF NLRC4-MEDIATED CLEARANCE, AND LATER ON TAKE ADVANTAGE OF DECREASED NLRC4 AND LIPID PRODUCTION (A HOST STRATEGY LIKELY AIMED AT LIMITING EXCESSIVE NLRC4-MEDIATED IMMUNOPATHOLOGY) TO DOWNREGULATE FLAGELLIN INTRACELLULARLY WITHIN MACROPHAGES. IN THIS PROPOSAL WE WILL INVESTIGATE THE HOST CELL-INTRINSIC INNATE REGULATORY CIRCUIT INVOLVING TYPE I IFN SIGNALING, NLRC4 AND IPLA2 ACTIVITY THAT REGULATES THE TEMPORAL SWITCH OF SALMONELLA FROM A FLAGELLIN-HIGH TO A FLAGELLIN-LOW PHENOTYPE INSIDE MACROPHAGES. WE WILL CONDUCT THESE INVESTIGATIONS IN THE FOLLOWING SPECIFIC AIMS: 1) TO IDENTIFY HOW MACROPHAGE PYROPTOSIS PROMOTES EARLY INCREASE IN FLAGELLIN PRODUCTION BY EXTRACELLULAR SALMONELLA; 2) TO DISSECT THE NATURAL TYPE I INTERFERON-DEPENDENT HOST NEGATIVE FEEDBACK RESPONSE THAT ESTABLISHES A NLRC4-LOW AND IPLA2-LOW INTRACELLULAR ENVIRONMENT PROMPTING SALMONELLA TO SWITCH TO A FLAGELLIN-LOW PHENOTYPE INSIDE MACROPHAGES. OUR INVESTIGATIONS WILL IDENTIFY THE TEMPORAL AND BIPHASIC REGULATION OF A PATHOGEN-DERIVED INFLAMMASOME LIGAND BY THE VERY PROCESS OF INFLAMMASOME ACTIVATION AS A NOVEL MODE OF HOST-PATHOGEN CROSS-TALK AND REVEAL A HOST MECHANISM THAT SALMONELLA TAKES ADVANTAGE OF FOR FLAGELLIN DOWNREGULATION AND IMMUNE ESCAPE WITHIN MACROPHAGES. THESE STUDIES WILL ALSO HAVE BROADER IMPLICATIONS FOR UNDERSTANDING HOW HOST INNATE IMMUNITY CONTRIBUTES TO MODULATION OF MICROBIAL EFFECTORS IMPACTING THE DEVELOPMENT AND RESOLUTION OF INFECTIONS.

TAS::89 0222::TAS; NEW; TITLE: ENABLING A SYSTEMS BIOLOGY KNOWLEDGEBASE WITH GAGGLE AND FIREGOOSE; PI: NITIN S. BALIGA

INSTABILITY OF CANCER CELL STATES IN TUMOR PROGRESSION (ICCS) - PROJECT SUMMARY ICCS2020-A1 THIS MULTI-PI PROJECT CONDUCTS EXPERIMENTS TO STUDY CELL STATE INSTABILITY IN TUMOR CELLS, MOTIVATED BY THE THEORY OF “CRITICAL TRANSITIONS” (CT). CTS ARE ABRUPT SHIFTS OF BEHAVIOR OF A COMPLEX NON-LINEAR SYSTEM AND ARE PRECEDED BY SYSTEM STATE DESTABILIZATION. A CANCER CELL POPULATION REPRESENTS A STATISTICAL ENSEMBLE OF CELLS, EACH OF WHICH IS A NONLINEAR STOCHASTIC DYNAMICAL SYSTEM. THE LATTER IS EMBODIED BY THE GENE REGULATORY NETWORK (GRN) AND CELLS ARE NORMALLY IN STABLE ATTRACTOR STATES. WE HYPOTHESIZE THAT CANCER CELLS IN SMALL LESIONS CAN BE POISED BETWEEN EITHER STAYING DORMANT OR EXITING DORMANCY (“ESCAPE”) AND THAT THIS BINARY DECISION IS A CT. THIS IMPLIES THAT TO BE IN SUCH A POISED STATE, THE CELL STATE HAS TO BE DESTABILIZED. THUS, DETECTING CELL STATE INSTABILITY, MANIFEST IN THE CELL TRANSCRIPTOMES, CAN DISCERN IF A SMALL TUMOR IS SAFELY IN A STABLE STATE OR POISED IN THE ABOVE SENSE. MANY AN OBSERVATION SUGGESTS THAT CELL DENSITY OF THE DORMANT TUMOR MAY BE A “BIFURCATION PARAMETER” THAT DRIVES GRN DYNAMICS, VIA INSTABILITY TOWARD THE CT, AT WHICH A CANCER CELL POPULATION CAN JUMP TO THE STATE OF STEADY GROWTH. SPECIFIC AIMS. THE PROPOSED STUDY IS EXPERIMENTAL BUT GROUNDED IN THEORY: CELL STATE INSTABILITY IS MANIFEST IN AN INCREASE OF THE QUANTITY IC THAT WE DERIVED FROM THEORY AND REQUIRES SINGLE-CELL (SC) TRANSCRIPTOMES IN A POPU- LATION TO COMPUTE (=DYNAMICS OF A STATISTICAL ENSEMBLE OF GRNS). AIM 1 (IN VITRO) USES LARGE ENSEMBLES OF MICRO- CULTURES (=CANCER CELL POPULATIONS) TO QUANTITATIVELY SHOW DESTABILIZATION AND BIFURCATIONS OF GROWTH BEHAVIORS. AIM 2 (IN VIVO) REEVALUATES OLD MOUSE TUMOR MODELS IN A NEW SCHEME THAT EXPOSES THE BINARY DECISION (DOR- MANCY VS. “TUMOR-TAKE”) TO TEST THE HYPOTHESIS THAT CLINICAL DORMANCY ESCAPE IS PRECEDED BY CELL STATE INSTABILITY. APPROACH: IN AIM 1, USING MASSIVELY-PARALLEL MICRO-CULTURES, BULK RNASEQ AND SCRNASEQ, WE EXAMINE HITH- ERTO UNDISTINGUISHED GROWTH MODES OF CANCER CELLS AND MEASURE BISTABILITY AS A FUNCTION OF CELL DENSITY (DORMANCY VS. “TAKE-OFF”). IN AIM 2 WE EXAMINE OUR INTRIGUING OBSERVATIONS IN MANY MOUSE MODELS: UNDER SPECIFIC CONDI- TIONS, IDENTIFIED BY TITRATING INOCULUM CELL NUMBERS IN CREATING DORMANT TUMORS, SOME MICE EXHIBIT STABLE DOR- MANCY AND OTHERS A ROBUST TUMOR-TAKE DESPITE SAME INITIAL CONDITIONS. THIS FINDING SUGGESTS A POISED STATE AND DEFINES A BISTABLE REGIME. TUMOR MODELS USING CELLS STUDIED IN AIM 1 WILL BE EVALUATED IN OUR SCHEME TO EXPOSE BISTABLE BEHAVIORS AND IC COMPUTED FROM SCRNASEQ DATA. WE ANTICIPATE THAT TUMORS IN UNSTABLE DORMANCY POISED TO TAKE-OFF DISPLAY HIGHER CELL STATE INSTABILITY (HIGHER IC) THAN THE STABLY DORMANT TUMORS. BUT SC-TRANSCRIPTOMES WILL ALSO REVEAL THE GENES THAT DRIVE THE CT AND HOW THEY ARE LINKED TO THE RISK OF IMPENDING DORMANCY ESCAPE. SIGNIFICANCE: WHILE THIS FIRST-IN-ITS-CLASS STUDY ANALYZES ABSTRACT PRINCIPLES RATHER THAN SPECIFIC MOLECULES, ITS POTENTIAL IMPACT IS TANGIBLE: IT PREDICTS THE FATE TRAJECTORY OF INDOLENT TUMORS IN A NEW WAY, COMPLEMENTING CURRENT QUEST FOR MOLECULAR SIGNATURES TO CLASSIFY TUMORS BY PROGNOSTIC GROUPS, BY DETECTING IN SINGLE-CELL RESOLUTION CELL POPULATION DATA SIGNS OF DESTABILIZATION THAT HERALD AN APPROACH TO THE CT OR “TIPPING POINT” OF DORMANCY ESCAPE. THIS WORK ALSO RAISES AWARENESS OF NON-LINEAR BEHAVIORS FOR THE DESIGN OF MORE RELEVANT ANIMAL TUMOR MODELS.

(PQB-4) SINGLE CELL ANALYSIS STRATEGY FOR MONITORING DRUG RESPONSES OF TUMORS

NLRP3 INFLAMMASOME ACTIVATION AND ITS CROSSTALK WITH RLR SIGNALING AT THE MITOCHONDRIA - PROJECT SUMMARY NLRP3 (NOD-LIKE RECEPTOR PROTEIN 3) IS AN INTRACELLULAR SENSOR THAT DETECTS A VARIETY OF STIMULI INCLUDING INFECTION AND METABOLIC DYSFUNCTION RESULTING IN ASSEMBLY OF A MACROMOLECULAR SIGNALING COMPLEX CALLED THE INFLAMMASOME WHICH PROMOTES CASPASE-1 DEPENDENT PROCESSING OF THE CYTOKINES IL-1B AND IL-18 TO THEIR BIOACTIVE FORMS; THESE CYTOKINES IN TURN UNDERLIE THE PATHOPHYSIOLOGY OF MANY AUTOINFLAMMATORY, AUTOIMMUNE, METABOLIC AND INFECTIOUS DISEASES. NLRP3 IS ACTIVATED BY A WIDE-RANGE OF MOLECULES INCLUDING MONOSODIUM URATE CRYSTALS, VIRAL RNA AND EVEN BACTERIAL PORE-FORMING TOXINS, BUT LITTLE IS KNOWN ABOUT THE MECHANISMS BY WHICH NLRP3 SENSES AND ELICITS A RESPONSE TO THESE CHEMICALLY AND STRUCTURALLY DISSIMILAR STIMULI. WE AND OTHERS HAVE PREVIOUSLY SHOWN THAT NLRP3 ASSOCIATES WITH MITOCHONDRIA UPON ACTIVATION. WHETHER THIS LINK SIMPLY POINTS TO A REQUIREMENT FOR A MEMBRANE PLATFORM TO ACHIEVE EFFICIENT SOLID-PHASE ASSEMBLY OF INFLAMMASOME SIGNALING COMPONENTS OR AN ACTIVE MODULATION OF NLRP3 ACTIVATION BY MITOCHONDRIAL COMPONENTS OR EVEN MITOCHONDRIAL PROTEINS REMAINS AMBIGUOUS. IT IS ALSO UNCLEAR IF AND HOW INTERACTION OF NLRP3 WITH MITOCHONDRIA INFLUENCES MITOCHONDRIAL FUNCTIONS. WE HAVE PREVIOUSLY SHOWN THAT NLRP3 MITOCHONDRIAL RECRUITMENT AND THE ENSUING INFLAMMASOME RESPONSE IS DEPENDENT ON INTERACTION OF A SHORT N- TERMINAL SEQUENCE IN NLRP3 WITH THE OUTER MITOCHONDRIAL ADAPTER PROTEIN MAVS. HOWEVER, MAVS SPECIFICALLY AUGMENTS INFLAMMASOME ASSEMBLY IN RESPONSE TO NON-CRYSTALLINE, BUT NOT CRYSTALLINE NLRP3 ACTIVATORS SUGGESTING AN INTERESTING AND COMPLEX MECHANISM BY WHICH MITOCHONDRIA REGULATE NLRP3 INFLAMMASOME ACTIVATION. IN THIS STUDY, WE PROPOSE THAT ADDITIONAL MITOCHONDRIAL PROTEINS CONTROL NLRP3 INFLAMMASOME ACTIVATION IN RESPONSE TO DIFFERENT ACTIVATORS, CONCEIVABLY IN A STIMULUS-SPECIFIC MANNER. IN RESPONSE TO DETECTION OF RNA VIRUSES MAVS IS ENGAGED NOT ONLY BY NLRP3 BUT ALSO BY THE RIG-I- LIKE RECEPTORS (RLRS) TO PRODUCE TYPE 1 INTERFERONS, SUGGESTING AN UNEXPECTED CROSSTALK BETWEEN THE NLRP3 AND RLR SIGNALING PATHWAYS. IN THIS PROPOSAL, WE WILL ELUCIDATE THE MECHANISMS BY WHICH MITOCHONDRIA/MAVS REGULATE NLRP3 ACTIVATION AND EXAMINE ITS CROSSTALK WITH THE RLR PATHWAY. THIS WILL BE ACCOMPLISHED BY: 1) MASS SPECTROMETRY TO IDENTIFY ADDITIONAL MITOCHONDRIAL PROTEINS THAT ASSOCIATE WITH INFLAMMASOME COMPLEXES IN RESPONSE TO CRYSTALS AND NON-CRYSTALLINE NLRP3 ACTIVATORS, FOLLOWED BY ASSAYS TO EVALUATE THEIR EFFECT ON INFLAMMASOME ACTIVATION AND MITOCHONDRIAL FUNCTIONS, 2) SOFT X-RAY TOMOGRAPHY AND CORRELATIVE FLUORESCENCE MICROSCOPY TO ASSESS A REQUIREMENT FOR SUBCELLULAR MEMBRANE PLATFORMS IN INFLAMMASOME ASSEMBLY, AND 3) MODELS OF VIRAL INFECTION TO IDENTIFY THE PHYSIOLOGICAL CONSEQUENCES OF MAVS-DEPENDENT CROSSTALK BETWEEN THE NLRP3 AND RLR PATHWAYS. THESE INVESTIGATIONS WILL YIELD IMPORTANT MECHANISTIC INSIGHTS INTO HOW MITOCHONDRIA AND MAVS REGULATE NLRP3 INFLAMMASOME ACTIVATION, WHICH MAY BE OF RELEVANCE FOR MODULATION OF NLRP3 ACTIVITY NOT ONLY IN VIRAL INFECTIONS BUT ALSO IN A VARIETY OF METABOLIC DISEASES.

THE EXPEDIT-ISOTOPOMERIC CROSSLINKING MASS SPECTROMETRY (EXPEDIT-ICLMS) TECHNOLOGY FOR MAPPING GLOBAL AND DYNAMIC PROTEIN-PROTEIN INTERACTION NETWORKS

EXTENDING BIOTAPESTRY, A TOOL FOR MODELING GENE REGULATORY NETWORKS

DESIGN AND IMPLEMENTATION OF EFFECTIVE SOLUTIONS FOR ARCHIVING AND PROCESSING SYSTEMS BIOLOGY DATA: RESEARCH INTEGRATED WITH AN ONGOING HIGH SCHOOL

AN LXR PROTEIN INTERACTION NETWORK CONTROLLING MACROPHAGE LIPID TRANSPORTER EXPRESSION IN RESPONSE TO INFLAMMATORY-LIPID CROSSTALK

OCEAN ACIDIFICATION: A SYSTEMS BIOLOGY APPROACH TO CHARACTERIZE DIATOM RESPONSE TO OCEAN ACIDIFICATION AND CLIMATE CHANGE

THEORY AND MEASUREMENT OF CELL POPULATION DYNAMICS WITH CELL-CELL INTERACTION (TMCC)

DYNAMICS OF NON-EQUALIBRIUM CELL STATE TRANSITIONS IN CELL POPULATIONS

LAMARCK REDUX: TRANSGENERATIONAL GENETIC EFFECTS ON PHENOTYPES AND DISEASE

SINGLE-CELL TRANSCRIPTOMICS OF COMPLEX BACTERIAL COMMUNITIES - PROJECT SUMMARY GENE EXPRESSION IN BACTERIA IS HETEROGENEOUS EVEN WITHIN GENETICALLY IDENTICAL CELLS DUE TO THE STOCHASTIC ACTIVATION OF MANY GENE REGULATORY PROGRAMS. THE RESULTING PHENOTYPIC DIVERSITY OFTEN PLAYS AN IMPORTANT FUNCTIONAL ROLE FOR BACTERIAL COMMUNITIES, FOR EXAMPLE, FACILITATING HORIZONTAL GENE TRANSFER. THIS FUNDAMENTALLY SINGLE-CELL BEHAVIOR CANNOT BE RESOLVED WITH POPULATION LEVEL MEASUREMENTS AND, SO FAR, HAS BEEN STUDIED IN PURE CULTURES OF GENETICALLY TRACTABLE ORGANISMS USING LOW-THROUGHPUT REPORTER-BASED ASSAYS. HOWEVER, THE MAJORITY OF BACTERIA IN NATURE RESIDE IN COMPLEX MICROBIAL COMMUNITIES SPATIALLY ORGANIZED INTO BIOFILMS AND COMPOSED OF MULTIPLE INTERACTING MEMBERS. WITHIN SUCH COMMUNITIES, A MULTITUDE OF BEHAVIORS EMERGE FROM THE DYNAMIC INTERPLAY OF NOISY GENE EXPRESSION STATES AND RESPONSES TO THE HETEROGENEOUS MICROENVIRONMENT. ABSENCE OF APPROACHES FOR MEASURING PHENOTYPIC STATES WITHIN COMPLEX POLYMICROBIAL COMMUNITIES SIMULTANEOUSLY AT SYSTEMS SCALE AND WITH SINGLE-CELL RESOLUTION RESULTS IN A LACK OF MECHANISTIC UNDERSTANDING OF BACTERIAL ECOLOGY AND IS THEREFORE A CRITICAL BARRIER FOR THE FIELDS OF MICROBIOLOGY AND MICROBIOME STUDIES. DURING MY POSTDOC, I DEVELOPED A HIGH-THROUGHPUT BACTERIAL SINGLE-CELL TRANSCRIPTOMICS TECHNOLOGY, MICROSPLIT (MICROBIAL SPLIT-POOL LIGATION TRANSCRIPTOMICS), THAT ALLOWS TO MEASURE GENE EXPRESSION STATES IN TENS OF THOUSANDS OF INDIVIDUAL CELLS USING ONLY COMMON LABORATORY EQUIPMENT. IN MY LAB, I AIM TO FURTHER EXTEND MICROSPLIT FOR SINGLE-CELL TRANSCRIPTOMICS OF BIOFILMS, AS WELL AS OF COMPLEX BACTERIAL CONSORTIA. SPECIFICALLY, IN MY FIRST PROJECT WE WILL CREATE A SINGLE-CELL GENE EXPRESSION MAP OF SINGLE- AND DUAL-SPECIES BIOFILMS OF PSEUDOMONAS AERUGINOSA AND STAPHYLOCOCCUS AUREUS BY A COMBINATION OF SPATIAL SINGLE-CELL RNA SEQUENCING AND TIME-LAPSE IMAGING. WE WILL CHARACTERIZE WHERE AND HOW THE SPECIALIZED PHENOTYPIC SUBPOPULATIONS EMERGE AT DIFFERENT STAGES OF BIOFILM DEVELOPMENT AND HOW THEY CHANGE IN RESPONSE TO COMPETING SPECIES. IN THE SECOND PROJECT, WE WILL INTERROGATE THE FUNCTIONAL ROLE OF THE INTERMITTENT AND HETEROGENEOUS ACTIVATION OF DIVERSE METABOLIC AND STRESS RESPONSE PATHWAYS WHICH WE HAVE OBSERVED EVEN IN ISOGENIC CELLS AND IN ABSENCE OF EXTERNAL CUES. SPECIFICALLY, WE WILL TEST THE HYPOTHESIS THAT HETEROGENEOUS SAMPLING OF SUCH STATES MAY PROMOTE INTER-SPECIES INTERACTIONS WITH BACTERIAL PARTNERS EVOLVED TO COEXIST IN THE SAME ENVIRONMENT. IN THIS PROJECT, I AIM TO UNCOVER THE PHENOTYPIC SUBPOPULATIONS ARISING IN KEY SPECIES FROM HUMAN GUT MICROBIOTA GROWN EITHER SOLO OR IN PAIR-WISE COMBINATIONS WITH OTHER CO-OCCURRING SPECIES. WITH THESE DATA, WE WILL USE GENE REGULATORY NETWORK MODELING TO PREDICT THE HIGHER-ORDER INTERACTIONS BETWEEN GUT MICROBIOTA SPECIES AND ENGINEER HIGHER COMPLEXITY CONSORTIA WITH PREDICTABLE BEHAVIORAL TRAITS. THE RESULTS WILL PAVE THE WAY TOWARD BUILDING SYSTEMS- LEVEL UNDERSTANDING OF THE PHENOTYPIC STRUCTURE AND THE EMERGENT PROPERTIES OF A HIGHER COMPLEXITY NATURAL MICROBIOTA. OVERALL, THE DEVELOPED APPROACHES WILL BECOME WIDELY APPLICABLE TOOLS FOR MICROBIOLOGICAL RESEARCH AND THE ACQUIRED DATA WILL PROVIDE A FOUNDATION FOR HIGH-RESOLUTION FUNCTIONAL ANALYSES OF MICROBIOTA AND BIOFILMS.

HOW DOES SPATIAL DIFFERENTIATION OF ECOTYPES ENHANCE MICROBIAL MUTUALISM? -THIS PROJECT WILL INVESTIGATE HOW COOPERATION AMONG MICROBIAL SPECIES BECOME MORE STABLE AND PRODUCTIVE OVER TIME. MICROBES ARE MAJOR DRIVERS OF ECOSYSTEM FUNCTION, RECYCLING CARBON, NITROGEN, AND MINERALS INCLUDING RARE EARTH METALS. THIS PROJECT WILL INVESTIGATE A CENTRAL BUT POORLY UNDERSTOOD MECHANISM THROUGH WHICH MEMBERS OF THE SAME SPECIES SPECIALIZE TO PERFORM SPECIFIC TASKS, DIVIDE LABOR, AND IMPROVE PRODUCTIVITY OF THE ENTIRE COMMUNITY. DECODING THE RULES THAT GOVERN THIS SPECIALIZATION WILL TRANSFORM THE ABILITY TO RATIONALLY ENGINEER MICROBIAL COMMUNITIES FOR BIOTECHNOLOGY, MATERIALS SCIENCE, AND ENERGY PRODUCTION. SPECIFICALLY, OUTCOMES OF THIS RESEARCH WILL PROVIDE A BLUEPRINT FOR ENGINEERING STABLE, PRODUCTIVE SYNTHETIC COMMUNITIES THAT CAN BE DEPLOYED ACROSS THE BIOECONOMY?FROM THE PRODUCTION OF RENEWABLE BIOGAS AND AGRICULTURAL BIO-STIMULANTS TO INDUSTRIAL WASTE UPCYCLING AND CRITICAL MINERAL RECOVERY. NOTABLY, THE PROJECT WILL TRAIN SIX HIGH SCHOOL STUDENT INTERNS, THREE TEACHERS, AND 300 STUDENT AMBASSADORS DRAWN FROM ACROSS ALL 50 STATES THROUGH ON-SITE AND ON-LINE INTERNSHIPS AND THE ESTABLISHED SYSTEMS THINKERS IN STEM AMBASSADORSHIP (STISA). PARTICIPANTS WILL DEVELOP A NEXT GENERATION SCIENCE STANDARDS (NGSS)-ALIGNED CURRICULUM MODULE THAT CONNECTS MICROBIAL ECOLOGY, SYSTEMS THINKING, AND COMPUTATIONAL MODELING TO REAL-WORLD CHALLENGES. THE CROSS-DISCIPLINARY TRAINING OF STUDENTS IN SYSTEMS BIOLOGY, ARTIFICIAL INTELLIGENCE (AI), AND BIOTECHNOLOGY, WILL HELP GENERATE A WORKFORCE REQUIRED FOR TACKLING THE MOST PRESSING CHALLENGES OF THE FUTURE. USING A MODEL SYNTHETIC COMMUNITY (SYNCOM) OF A SULFATE-REDUCING BACTERIUM (DESULFOVIBRIO VULGARIS, DV) AND A METHANOGENIC ARCHAEON (METHANOCOCCUS MARIPALUDIS, MM), RESEARCHERS WILL TEST THE HYPOTHESIS THAT MICROBIAL MUTUALISM IMPROVES THROUGH THE INTERPLAY OF SPATIALLY AND PHYSIOLOGICALLY DIFFERENTIATED ECOTYPES WITHIN THE SAME HABITAT. PRIOR WORK DEMONSTRATED THAT FEW MUTATIONS SELECTED OVER A SHORT EVOLUTIONARY TIMESCALE (<1,000 GENERATIONS) GENERATED ECOTYPES THAT SEGREGATED ACROSS PARTICLE-ATTACHED AND FREE-FLOATING (PLANKTONIC) COMMUNITIES. GENOME-SCALE MODELING REVEALED METABOLIC SPECIALIZATION ACROSS ECOTYPES, BOOSTING METHANE PRODUCTIVITY OF THE ENTIRE COMMUNITY. THE PROJECT PURSUES THREE RESEARCH AIMS. IN AIM 1, LONG-READ GENOME SEQUENCING OF INDIVIDUAL SEDIMENT PARTICLES AND PLANKTONIC BIOMASS WILL BE USED TO RESOLVE THE BIOGEOGRAPHY OF ECOTYPES WITHIN REPLICATE BIOREACTORS. AIM 2 DEVELOPS SYNPRIME?A NOVEL FIVE-COMPARTMENT IN SILICO MODEL INTEGRATING GENE REGULATORY NETWORKS WITH METABOLIC FLUX BALANCE ANALYSIS?TO UNCOVER HOW TRANSCRIPTIONAL REPROGRAMMING DRIVES THE PHYSIOLOGICAL INTERPLAY BETWEEN SPATIALLY SEGREGATED ECOTYPES. IN AIM 3, A LIBRARY OF GENETICALLY CHARACTERIZED ECOTYPES WILL BE ASSEMBLED INTO NEW SYNCOMS THROUGH HIGH-THROUGHPUT PAIRWISE SCREENING AND HYPOTHESIS-DRIVEN COMBINATORIAL ASSEMBLY, ENABLING BOTTOM-UP TESTING OF PARTNER SELECTION PRINCIPLES AND DIRECT VALIDATION OF SYNPRIME PREDICTIONS. TOGETHER, THESE AIMS WILL REVEAL PRINCIPLES OF ECOTYPE SPECIALIZATION AND COMMUNITY ASSEMBLY, PRODUCING A GENERALIZABLE SYSTEMS BIOLOGY FRAMEWORK TO RATIONALLY DESIGN NOVEL SYNCOMS FOR BIOTECHNOLOGY APPLICATIONS. IN ADDITION TO TRAINING A GRADUATE STUDENT AND A POSTDOCTORAL RESEARCHER, THIS PROJECT WILL ENGAGE SIX HIGH SCHOOL STUDENT INTERNS AND THREE TEACHERS IN PROJECT-RELATED SCIENCE ACTIVITIES. THE TEAM WILL DEVELOP A NEXT GENERATION SCIENCE STANDARDS-ALIGNED HIGH SCHOOL CURRICULUM MODULE ON HOW PROCESS PARTITIONING BY MICROBIAL COMMUNITIES DRIVES NUTRIENT CYCLES ACROSS ECOSYSTEMS (E.G., SOIL, OCEANS, AND ROOT). FINALLY, THE CURRICULUM MODULE WILL BE DISSEMINATED TO SCHOOLS ACROSS THE US AND >100 COUNTRIES, WHILE MEASURING KEY PERFORMANCE INDICATORS, INCLUDING NUMBERS OF STUDENTS AND TEACHERS IMPACTED, STUDENT LEARNING, AND STEM IDENTITY. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.

A SYSTEMS BIOLOGY FRAMEWORK TO UNCOVER RULES GOVERNING ROBUSTNESS OF A MICROBIAL COMMUNITY

MRI: DEVELOPMENT OF ION-MOBILITY ENHANCED ELECTRON-TRANSFER DISSOCIATION FOR ELUCIDATING BIOLOGICAL NETWORKS USING A SYSTEMS BIOLOGY APPROACH

ABI INNOVATION: A FRAMEWORK TO PREDICTABLY MANIPULATE A MICROBIAL GENE REGULATORY PROGRAM

A MICROFLUIDIC PLATFORM FOR MULTIPLEXED ELISA-BASED PROTEIN MEASUREMENTS FROM SINGLE CELL SAMPLES (ARRA)

MODULAR INTERPLAY OF TRANSCRIPTION AND TRANSLATION

MOLECULAR ASSEMBLIES, GENES AND GENOMICS INTEGRATED EFFICIENTLY (MAGGIE): COMPONENT 3: SYSTEMS APPROACH IN A MULTI-ORGANISM STRATEGY TO UNDERSTAND BI

LIQUID BIOPSY-BASED TOOLKITS FOR NEOANTIGEN AND COGNATE TCR DISCOVERY FOR CANCER IMMUNOTHERAPY - PROJECT SUMMARY/ABSTRACT NEOANTIGENS DERIVED FROM TUMOR-SPECIFIC MUTATIONS ARE A MAJOR DRIVING FORCE BEHIND THE CURRENT CANCER IMMUNOTHERAPIES. THE INTERACTIONS BETWEEN NEOANTIGENS AND ANTIGEN-SPECIFIC T CELLS ENABLE T CELL RECOGNITION AND TUMOR KILLING. RESOLVING NEOANTIGENS AND ANTIGEN-REACTIVE T CELLS NORMALLY REQUIRES FRESH (OR SNAP FROZEN) TUMOR MATERIALS FROM WHICH THE TUMOR TISSUE CAN BE SEQUENCED AND TUMOR-INFILTRATING LYMPHOCYTES CAN BE ISOLATED AND EXPANDED. UNFORTUNATELY, THE AVAILABILITY OF FRESH (OR SNAP FROZEN) TUMOR TISSUE BIOPSIES IS A SIGNIFICANT LIMITING FACTOR FOR PERSONALIZED IMMUNOTHERAPY. SEQUENCING QUALITY OF PARAFFIN-EMBEDDED PATHOLOGICAL SPECIMEN IS OFTEN SUBOPTIMAL FOR NEOANTIGEN DISCOVERY. SEQUENCING DATA OF A SMALL BIOPSY FROM A SINGLE TUMOR LESION MAY NOT BE REPRESENTATIVE OF THE TUMOR'S FULL CLONAL SPECTRUM. MORE IMPORTANTLY, THE MOLECULAR PROFILE OF TUMORS EVOLVES DYNAMICALLY OVER TIME. BUT REPEATED BIOPSY SAMPLING IS SELDOM FEASIBLE FOR PATIENTS WITH MANY CANCERS THAT REQUIRE INVASIVE BIOPSY PROCEDURES. CIRCULATING TUMOR CELLS (CTCS) AND MATCH BLOOD LYMPHOCYTES ARE GOOD SURROGATES FOR NEOANTIGEN AND COGNATE TCR DISCOVERY. THEY ENABLE CLINICIANS TO REPEATEDLY AND NON-INVASIVELY INTERROGATE THE MUTATIONAL LANDSCAPE AND DYNAMIC EVOLUTION OF THE ANTI-TUMOR IMMUNITY VIA SERIALLY COLLECTED BLOOD SAMPLES. A MAJOR TECHNICAL CHALLENGE HERE IS TO DEVELOP HIGH-THROUGHPUT, LOW SAMPLE VOLUME, MINIMALLY INVASIVE TOOLS TO RESOLVE THE NEOANTIGEN AND COGNATE TCR DYNAMICS AND TO MATCH THE TCR GENES WITH SPECIFIC NEOANTIGENS. WE PROPOSE TO DEVELOP A TOOLKIT FOR LIQUID BIOPSY-BASED NEOANTIGEN AND COGNATE TCR DISCOVERY THROUGH A STRATEGIC INTEGRATION OF A NOVEL ON-CHIP IMAGE CYTOMETRY PLATFORM AND A HIGHLY MODULAR NANOPARTICLE-BARCODED SINGLE-MOLECULE PEPTIDE-MHC TETRAMER CELL SORTING ASSAY. WE WILL PERFORM ADVANCED DEVELOPMENT AND RIGOROUS VALIDATION OF THE TOOLKIT IN THIS PROPOSED WORK. WE WILL INVESTIGATE THE DYNAMIC EVOLUTION OF NEOANTIGEN AND TCR REPERTOIRES DURING IMMUNE CHECKPOINT BLOCKADE USING SERIALLY COLLECTED BLOOD SAMPLES FROM MELANOMA PATIENTS. UPON SUCCESSFUL COMPLETION, IT WILL DELIVER A SIMPLE AND NONINVASIVE APPROACH FOR LIQUID BIOPSY-BASED NEOANTIGEN AND COGNATE TCR DISCOVERY FOR PATIENTS WHOSE SURGICAL BIOPSIES ARE NOT AVAILABLE OR NOT ATTAINABLE, WITH TRANSFORMATIVE POTENTIALS ON PERSONALIZED NEOANTIGEN VACCINES AND T-CELL BASED IMMUNOTHERAPIES. THE NEOANTIGEN AND COGNATE TCR DYNAMICS WILL PROVIDE CRITICAL INSIGHTS INTO THE UNDERSTANDING OF IMMUNOTHERAPY RESPONSE AND RESISTANCE.

CAPACITY: BBSRC-NSF/BIO: GLOBALLY HARMONIZED RE-ANALYSIS OF DATA INDEPENDENT ACQUISITION (DIA) PROTEOMICS DATASETS ENABLES THE CREATION OF NEW RESOURCES (DIA-EXCHANGE) -THE RESEARCH COMMUNITY HAS GENERATED MILLIONS OF DATASETS THAT WERE USED TO ANSWER SCIENTIFIC QUESTIONS OF INTEREST, AND DEPOSITED THOSE DATASETS INTO PUBLIC DATA REPOSITORIES AS PART OF THE MANDATED DATA SHARING POLICIES OF FUNDING AGENCIES WORLDWIDE. ESPECIALLY FOR DATASETS GENERATED TO MEASURE THE PROTEIN CONTENT OF BIOLOGICAL SAMPLES, SUBSTANTIAL ADDITIONAL INFORMATION CAN BE EXTRACTED FROM THESE PUBLIC DATASETS USING NEWER TECHNIQUES, NEWER VERSIONS OF SOFTWARE, AND NEWER REFERENCE INFORMATION DURING RE-ANALYSIS. THIS PROJECT WILL DEVELOP A SOFTWARE INFRASTRUCTURE FOR GLOBALLY HARMONIZED RE-ANALYSIS OF AN EMERGING TYPE OF PROTEOMICS DATASET TO EXTRACT NEW INFORMATION FROM OLDER DATA. THE INFRASTRUCTURE WILL IMPROVE OUR ABILITY TO TURN PROTEOMICS DATA INTO KNOWLEDGE ABOUT GLOBAL RELATIVE PROTEIN ABUNDANCES AND ABOUT INTERACTIONS BETWEEN PROTEINS INFERRED FROM HOW PROTEIN ABUNDANCES ARE CORRELATED. THE PROJECT WILL ALSO PROVIDE OPPORTUNITIES FOR STUDENTS TO LEARN SKILLS IN SCIENTIFIC DATA ANALYSIS. THIS PROJECT IS AN INTERNATIONAL COLLABORATION WITH THE PROTEOMICS TEAM AT EMBL-EBI (HINXTON, UK) AND THE INSTITUTE OF SYSTEMS, MOLECULAR AND INTEGRATIVE BIOLOGY (ISMIB) AT THE UNIVERSITY OF LIVERPOOL (UOL; UK). THIS PROJECT WILL MAKE THE DATASETS GENERATED BY THE METHOD KNOWN AS DATA-INDEPENDENT ACQUISITION (DIA) MASS SPECTROMETRY PROTEOMICS MORE FINDABLE, ACCESSIBLE, INTEROPERABLE, AND REUSABLE (FAIR). THIS WILL BE ACCOMPLISHED BY DEVELOPING AN INDEXING SYSTEM FOR THE LIBRARIES OF MASS SPECTRA THAT ARE USED TO ANALYZE THE DATA FROM DIA EXPERIMENTS. THE PROJECT WILL FURTHER DEVELOP BENCHMARKS AND GUIDELINES TO ADVANCE THE FIELD, AND THEN DEVELOP DATA ANALYSIS PIPELINES TO PROCESS THOUSANDS OF PUBLIC EXPERIMENTS AT SCALE AND MAKE THE RESULTS AVAILABLE TO THE RESEARCH COMMUNITY. THE RESULTING PROTEIN ABUNDANCE MAPS ACROSS THOUSANDS OF EXPERIMENTS WILL ENABLE THE DEVELOPMENT OF PROTEIN INTERACTION MAPS VIA INFERENCE FROM PROTEIN CO-EXPRESSION PATTERNS EXTRACTED FROM THESE DATASETS, SOMETHING THAT CAN ONLY BE ACCOMPLISHED BY THOUSANDS OF DATASETS ANALYZED IN UNISON. STUDENTS WILL BE GIVEN OPPORTUNITIES TO PARTICIPATE IN THE WORK IN ORDER TO BUILD THEIR SKILLS, AND ALL SOFTWARE AND DATA PRODUCTS WILL BE MADE PUBLICLY AVAILABLE TO FURTHER ADVANCE THE FIELD. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.

INTERPLAY OF TRANSCRIPTIONAL, TRANSLATIONAL REGULATORY MECHANISMS AND KINETICS OF AN ENVIRONMENTAL RESPONSE

PHYSIOLOGIC STATE MODULATION BY CONDITIONAL TRANSLATIONAL COMPLEXES

ADVANCING DATA AND METADATA STANDARDS FOR PROTEOMICS MASS SPECTRA

DEVELOPMENT OF A PREDICTIVE FRAILTY CLOCK & LONGITUDINAL INVESTIGATION OF ITS EPIGENETIC DETERMINANTS - PROJECT SUMMARY/ABSTRACT LONGEVITY STUDIES IN MICE ARE EXPENSIVE AND TIME-CONSUMING, AND THERE ARE CURRENTLY NO MEASURES THAT CAN PREDICT MORTALITY IN A MOUSE AT AN EARLIER TIME-POINT. ADDITIONALLY, THERE ARE VERY FEW MEASURES OF THE OVERALL HEALTH OF MICE THAT CAN BE ASSESSED LONGITUDINALLY. IN HUMANS, FRAILTY CAN PREDICT MORTALITY WITH GREATER POWER THAN THE DNA METHYLATION CLOCK. IN MY EARLY POSTDOCTORAL WORK I HAVE VALIDATED A MOUSE FRAILTY INDEX, THAT INCREASES WITH AGE, IS ASSOCIATED WITH MORTALITY AND AGE-RELATED PATHOLOGIES, AND IS SENSITIVE TO INTERVENTIONS. IN THE SINCLAIR LAB I HAVE USED MACHINE LEARNING MODELLING OF THIS MOUSE FRAILTY INDEX TO MAKE THE ANALYSIS OF FRAILTY IN DEATH (AFRAID) CLOCK THAT CAN PREDICT THE LIFESPAN OF MALE C57BL/6 MICE AGED 21 MONTHS OR OLDER WITH ACCURACY OF APPROXIMATELY 1.7 MONTHS. WE HYPOTHESIS THAT FRAILTY CLOCKS THAT INCLUDE A RANGE OF MEASURES INCLUDING PHYSIOLOGICAL AND MOLECULAR MEASURES (BLOOD-BASED ANALYSIS OF FRAILTY IN DEATH, BAFRAID) WILL ACCURATELY PREDICT LIFESPAN IN YOUNGER, FEMALE MICE AND DIFFERENT STRAINS. TO TEST THIS HYPOTHESIS I WILL USE PREVIOUSLY COLLECTED LONGITUDINAL HEALTH AND MOLECULAR DATA FROM C57BL/6 MICE, AS WELL AS COLLECT LIFELONG HEALTH DATA, PLUS BLOOD AND STOOL SAMPLES, FROM UM-HET3 MICE. I WILL USE REGRESSION MODELLING OF ALL MEASURED OUTCOMES TO DEVELOP OPTIMIZED ‘BAFRAID CLOCKS’ THAT PREDICT TIME TO DEATH IN MICE OF BOTH STRAINS, AND BOTH SEXES. FOR AIM 2, I WILL TEST THE HYPOTHESIS THAT EPIGENETIC DYSREGULATION UNDERLIES THE DEVELOPMENT OF FRAILTY IN MICE. I WILL USE LONGITUDINAL ASSESSMENTS, AND CUTTING-EDGE EPIGENETIC TOOLS (INCLUDING TIMESEQ FOR DNA METHYLATION AND CUT&TAG FOR HISTONE MODIFICATIONS) TO INVESTIGATE THE RELATIONSHIP BETWEEN EPIGENETIC CHANGES AND THE BAFRAID CLOCKS. COMPLETION OF THIS PROJECT WILL PROVIDE IMPORTANT TOOLS FOR THE FIELD, AND ALLOW US TO UNDERSTAND WHETHER EPIGENETIC CHANGES PRECEDE THE DEVELOPMENT OF FRAILTY IN MICE.

DEVELOPMENT OF ADVANCED TECHNOLOGIES FOR COMPLETE GENOMIC AND PROTEOMIC CHARACTERIZATION OF QUANTIZED HUMAN TUMOR CELLS

COLLABORATIVE RESEARCH: IMAGINE: QUANTIFYING DIATOM RESILIENCE IN AN ACIDIFIED OCEAN

A LONGITUDINAL SYSTEMS-LEVEL ANALYSIS OF THE HUMAN IMMUNE RESPONSE DURING LYME DISEASE.

RARECYTEFINDER: A BENCH-TO-BITS TOOLKIT FOR LABEL-FREE, WHOLE-SPECTRUM ANALYSIS OF RARE DISSEMINATED TUMOR CELLS IN LIQUID AND TISSUE BIOPSIES - PROJECT SUMMARY/ABSTRACT DISSEMINATED TUMOR CELLS (DTCS), ENCOMPASSING CIRCULATING TUMOR CELLS (CTCS) IN BLOOD AND OTHER BODY FLUIDS, AND ISOLATED TUMOR CELLS (ITCS) IN PERITUMORAL TISSUES AND LYMPH NODES (LNS), ARE VITAL IN THE METASTATIC PROCESS OF CANCER. DESPITE THEIR RARITY, DTCS, AS METASTATIC PRECURSORS, PLAY A CRITICAL ROLE IN METASTATIC COLONIZATION IN DISTANT ORGANS AND ARE ESSENTIAL IN CANCER STAGING, PROGNOSIS, AND CLINICAL MANAGEMENT. IN THE FIELD OF LIQUID BIOPSIES, CTCS, CTDNAS, AND TUMOR-DERIVED EXOMES HAVE EMERGED AS SIGNIFICANT MATERIALS FOR CANCER DIAGNOSIS AND MONITORING THERAPEUTIC RESPONSES. CTCS, IN PARTICULAR, ARE UNIQUE FOR ENCAPSULATING COMPREHENSIVE MOLECULAR INFORMATION, THUS SERVING AS KEY PROXIES FOR PRIMARY TUMORS AND OFFERING INSIGHTS INTO TUMOR METASTASIS MECHANISMS. HOWEVER, THE STUDY OF CTCS AND ITCS FACES CHALLENGES, ESPECIALLY IN DETECTION AND ANALYSIS, DUE TO THEIR EXTREME RARITY AND THE ABSENCE OF A GENERIC, ACCURATE METHOD FOR THEIR UNBIASED DETECTION. TRADITIONAL METHODS, OFTEN RELYING ON EPITHELIAL MARKERS OR MORPHOLOGICAL CHARACTERISTICS, SUFFER FROM LOW SENSITIVITY AND HIGH FALSE-POSITIVE RATES. THIS PROBLEM IS COMPOUNDED IN ITCS EMBEDDED IN THE COMPLEX CELLULAR MATRIX OF PERITUMORAL TISSUES AND LNS, MAKING THEIR IDENTIFICATION AND RETRIEVAL FOR MOLECULAR PROFILING CHALLENGING. THEREFORE, AN EFFECTIVE, UNBIASED APPROACH FOR IDENTIFYING AND ANALYZING THESE RARE DTCS ACROSS VARIOUS SOLID TUMOR TYPES IS URGENTLY NEEDED. TO ADDRESS THIS, WE PROPOSE RARECYTEFINDER, A BENCH-TO-BITS, LABEL-FREE METHOD FOR THE UNBIASED IDENTIFICATION AND IN-DEPTH MOLECULAR ANALYSIS OF DTCS IN DIVERSE LIQUID AND TISSUE BIOPSY SPECIMENS. LEVERAGING GENOME-WIDE COPY NUMBER ALTERATIONS AS A ROBUST MARKER, RARECYTEFINDER AIMS TO OVERCOME THE CURRENT LIMITATIONS OF DTC STUDY, SUCH AS LOW SENSITIVITY AND HIGH FALSE-POSITIVE RATES, AND TO PROVIDE ACCURATE IDENTIFICATION AND COMPREHENSIVE MOLECULAR INSIGHTS INTO DTCS. THE METHOD WILL ALSO ENABLE PRECISE TRACING OF DTCS’ TISSUE-OF-ORIGIN AND TUMOR TYPE (TOTT), IDENTIFYING THEIR TARGETABLE VULNERABILITIES AND PREDICTING DRUG RESPONSES. OUR PRELIMINARY DATA HAVE ALREADY DEMONSTRATED RARECYTEFINDER'S POTENTIAL, AND WE NOW AIM TO DEVELOP IT INTO A FULLY FUNCTIONAL PROTOTYPE THROUGH THREE SYNERGISTIC AIMS: 1) ADVANCED DEVELOPMENT AND VALIDATION FOR CTC ANALYSIS IN BLOOD SAMPLES FROM VARIOUS CANCERS, INCLUDING SYSTEMATIC VALIDATION WITH CLINICAL BIOSPECIMENS AND IMPROVING TOTT TRACING; 2) DEVELOPMENT AND VALIDATION OF A SIGNATURE/DRUG TARGET MAPPER (SDTM) MODULE, EMPLOYING BIOINFORMATIC AND MACHINE LEARNING MODELS TO RESOLVE TRANSCRIPTOME SIGNATURES, DRUG SENSITIVITIES, AND TARGETABLE VULNERABILITIES OF DTCS; 3) EXTENSION OF RARECYTEFINDER TO INCLUDE RARE ITCS IN PERITUMORAL TISSUES AND LNS TO ENHANCE UNDERSTANDING OF ITCS IN TUMOR PROGRESSION AND METASTASIS.

ABI INNOVATION: AN APPROACH TO CONSTRUCT A SYSTEMS-SCALE PREDICTIVE MODEL OF A GENE REGULATORY NETWORK COMPLETE WITH MECHANISMS AT SINGLE NUCLEOTID

INTERROGATION OF SYSTEMS LEVEL MECHANISMS CONTROLLING DNA REPAIR PROCESSES

BIOMARKERS FOR PULMONARY INJURY FOLLOWING DEPLOYMENT

CIBR: PTMEXCHANGE: GLOBALLY HARMONIZED RE-ANALYSIS AND SHARING OF DATA ON POST-TRANSLATIONAL MODIFICATIONS

INTERROGATION OF BACTERIAL PATHOGEN CANONICAL AND NON-CANONICAL PHOSPHORYLATION EVENTS - ABSTRACT OVER THE PAST THREE DECADES, SIGNIFICANT PROGRESS IN CANONICAL PHOSPHOPROTEOMIC ANALYSIS HAS BEEN DRIVEN BY IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC), WHICH ENABLES ENRICHMENT OF SERINE-, THREONINE-, AND TYROSINE-PHOSPHORYLATED PEPTIDES FOLLOWING ENZYMATIC DIGESTION OF CELLULAR SAMPLES. WHILE EFFECTIVE, IMAC METHODS RELY ON HARSH BUFFER CONDITIONS AND OFTEN SUFFER FROM LIMITED SPECIFICITY AND REPRODUCIBILITY. IN CONTRAST, NON-CANONICAL PHOSPHORYLATION OCCURRING ON RESIDUES SUCH AS HISTIDINE, ASPARTATE, GLUTAMATE, CYSTEINE, LYSINE, AND ARGININE REMAIN POORLY UNDERSTOOD DUE TO THE LACK OF ROBUST ENRICHMENT STRATEGIES. THESE UNDEREXPLORED POST-TRANSLATIONAL MODIFICATIONS (PTMS) HAVE BEEN INCREASINGLY IMPLICATED IN CRITICAL CELLULAR PROCESSES, INCLUDING SIGNAL TRANSDUCTION, ENERGY METABOLISM, AND ADAPTIVE RESPONSES. DESPITE THEIR BIOLOGICAL IMPORTANCE, BOTH CANONICAL AND NON-CANONICAL PHOSPHORYLATION EVENTS LACK EFFICIENT, SELECTIVE TOOLS FOR ENRICHMENT TO ALLOW COMPREHENSIVE DETECTION, REPRESENTING A MAJOR BOTTLENECK TO ADVANCE THE FIELD. TO ADDRESS THESE LIMITATIONS, WE HAVE RECENTLY DEVELOPED A NEW CLASS OF AFFINITY REAGENTS, PHOSPHOPEPTIDE “SUPERBINDERS”, THAT ARE BASED ON ENGINEERED SH2 DOMAINS AND ARE CAPABLE OF SELECTIVELY ENRICHING CANONICAL PHOSPHOTYROSINE PEPTIDES UNDER MILD PH CONDITIONS. THESE REAGENTS OVERCOME KEY LIMITATIONS OF TRADITIONAL IMAC METHODS, OFFERING IMPROVED RECOVERY, SPECIFICITY, AND COMPATIBILITY WITH DOWNSTREAM MASS SPECTROMETRY WORKFLOWS. IN PARALLEL, WE HAVE ALSO DEVELOPED A HIGH-AFFINITY PHOSPHOHISTIDINE-SPECIFIC SUPERBINDER, DERIVED FROM A SHORT-CHAIN FAB FRAGMENT, TO ENABLE THE ENRICHMENT AND DETECTION OF HISTIDINE-PHOSPHORYLATED PEPTIDES, REPRESENTING A NOVEL AND TECHNICALLY INNOVATIVE APPROACH TO INTERROGATING NON-CANONICAL PHOSPHORYLATION. THIS APPLICATION SEEKS TO ADVANCE THESE SUPERBINDERS AS NEXT-GENERATION TOOLS FOR COMPREHENSIVE PHOSPHOPROTEOMIC ANALYSIS. SPECIFICALLY, WE WILL OPTIMIZE THE TECHNICAL APPLICATION OF THESE PROTEIN-BASED REAGENTS, BENCHMARK THEIR PERFORMANCE, EXPAND THEIR APPLICATION TO DIVERSE BIOLOGICAL SYSTEMS, AND ULTIMATELY DEVELOP PROTOCOLS FOR THEIR BROAD ADOPTION. AS PROOF OF PRINCIPLE, WE WILL APPLY THESE TOOLS TO MAMMALIAN AND BACTERIAL CELLS TO INVESTIGATE PHOSPHO-SIGNALING NETWORKS. USING ESCHERICHIA COLI (E. COLI) AND MYCOBACTERIUM TUBERCULOSIS (MTB), WE WILL DEFINE THE DYNAMIC REMODELING OF CANONICAL AND NON-CANONICAL PTMS IN RESPONSE TO ENVIRONMENTAL STRESS AND TARGETED PHARMACOLOGICAL PERTURBATION, RESPECTIVELY. IN MTB, WE WILL FURTHER INTERROGATE PHOSPHORYLATION EVENTS ASSOCIATED WITH RESPONSE TO NOVEL ANTIBACTERIAL COMPOUNDS TO ELUCIDATE MECHANISMS OF ACTION. OUR APPROACH REPRESENTS SUBSTANTIAL TECHNICAL INNOVATIONS IN PHOSPHOPROTEOMICS, WITH BROAD IMPLICATIONS FOR ADVANCING PTM BIOLOGY, UNCOVERING NEW REGULATORY PATHWAYS, AND ACCELERATING THERAPEUTIC DISCOVERY.

BILATERAL BBSRC-NSF/BIO: IDENTIFYING MECHANISMS FOR ENVIRONMENTAL ADAPTATION IN BACTERIA

LTQ-ORBITRAP XL FOR PROTEIN IDENTIFICATION AND BIOMARKER DISCOVERY

COLLABORATIVE RESEARCH: A SYSTEMS BIOLOGY APPROACH OF DIATOM RESPONSE TO OCEAN ACIDIFICATION AND CLIMATE CHANGE

FURTHER DEVELOPMENT OF THE FEAST SOFTWARE, AND ITS USE FOR NOVEL GENE PREDICTIONS

MRI-CONSORTIUM: ACQUISITION OF AN ADVANCED MASS SPECTROMETER

IDENTIFYING NETWORK PERTUBATION USING SECRETED PROTEIN PROFILES IN GLIOBLASTOMA

PROVIDING PEPTIDE ATLAS BASED SERVICES THROUGH THE CAGRID INFRASTRUCTURE

RESEARCH ON THE EFFECTIVENESS OF THE OBSERVING FOR EVIDENCE OF LEARNING PROFESSIONAL DEVELOPMENT MODEL FOR IMPROVING GRADES 6-8 SCIENCE INSTRUCTION

BRAIN-REGION AND CELL-TYPE SPECIFIC TRANSCRIPTS FOR INFORMATIVE DIAGNOSTICS

UNRAVELLING THE SEX-FRAILTY PARADOX: IDENTIFICATION OF SEX-SPECIFIC DETERMINANTS OF FRAILTY - PROJECT SUMMARY/ABSTRACT FRAILTY IS A CLINICALLY SIGNIFICANT INDICATOR OF BIOLOGICAL AGING AND A STRONG PREDICTOR OF ADVERSE OUTCOMES, INCLUDING MORTALITY. NOTABLY, WOMEN TEND TO EXHIBIT HIGHER FRAILTY SCORES THAN MEN AT COMPARABLE AGES, YET EXPERIENCE LOWER MORTALITY—A PHENOMENON TERMED THE SEX-FRAILTY PARADOX. THE BIOLOGICAL AND SOCIO-ENVIRONMENTAL UNDERPINNINGS OF THIS PARADOX REMAIN POORLY UNDERSTOOD. THIS PROJECT ADDRESSES THE GAP BY PURSUING THREE INTEGRATED AIMS: (1) TO BENCHMARK THE PERFORMANCE AND POTENTIAL SEX BIAS OF COMMONLY USED FRAILTY ASSESSMENT TOOLS ACROSS MULTIPLE LARGE-SCALE, GLOBAL HUMAN DATASETS; (2) TO IDENTIFY SEX-SPECIFIC, MODIFIABLE MOLECULAR AND ENVIRONMENTAL DRIVERS OF FRAILTY USING AI-POWERED ANALYSIS OF MULTI-OMIC AND EXPOSOME DATA; AND (3) TO TEST CAUSAL BIOLOGICAL MECHANISMS OF SEX DIFFERENCES IN FRAILTY USING GENETICALLY DIVERSE UM-HET3 MICE AND FOUR CORE GENOTYPE (4CG) MOUSE MODELS. AS AN EARLY CAREER RESEARCHER PI KANE WILL BE SUPPORTED BY A TEAM OF EXPERTS IN SEX DIFFERENCES, LARGE-SCALE CLINICAL DATA ANALYSIS AND SYSTEMS BIOLOGY. COMPLETION OF THIS PROJECT WILL RESULT IN BETTER UNDERSTANDING OF THE SEX-FRAILTY PARADOX AND ITS UNDERLYING MECHANISMS, AND INFORM THE DEVELOPMENT OF PRECISION TOOLS AND TARGETED INTERVENTIONS FOR THE DETECTION, PREVENTION AND TREATMENT OF FRAILTY FOR ALL OLDER ADULTS.

ELUCIDATING PHOSPHORYLATION SIGNALING NETWORKS IN INFECTIOUS PLASMODIUM PARASITES

ACQUISITION OF FUSION LUMOS ORBITRAP MASS SPECTROMETER

AN APPROACH USING THE CHANGES OF CIRCULATING RNA SPECTRUM AND MICROBIOME SIGNATURES TO DETECT AND ASSESS THE HEALTH IMPACT OF CHEMICAL WARFARE AGENT

FAIMS-SELECTED REACTION MONITORING TO QUANTITATIVE PROTEIN NETWORKS IN DISEASE

PROSTATE SPECIFIC OR ENRICHED GENES AS COMPOSITE BIOMARKERS FOR PROSTATE CANCER

A PROTEOMIC APPROACH FOR EARLY DIAGNOSIS OF DIABETES

ENHANCING INSIGHT INTO PLACENTAL DYSFUNCTION IN COMMON OBSTETRIC DISORDERS USING PLACENTAL MULTIOMICS - PROJECT SUMMARY ADVERSE OBSTETRICS DISORDERS, INCLUDING PREECLAMPSIA, PRETERM BIRTH, AND FETAL GROWTH RESTRICTION, ARE COMMON WORLDWIDE. HOWEVER, THEY ARE DIFFICULT TO STUDY GIVEN MULTIFACTORIAL ETIOLOGIES AND CO-OCCURRENCE OF DISORDERS. PLACENTAL DYSFUNCTION HAS A ROLE IN THESE DISEASES BUT THE UNDERLYING MOLECULAR MECHANISMS ARE NOT WELL UNDERSTOOD. INTEGRATED MULTIOMIC ANALYSIS CAN ADDRESS THIS GAP BY IDENTIFYING UNDERLYING MOLECULAR MECHANISMS ASSOCIATED WITH INDIVIDUAL DISORDERS AND COMBINATIONS OF DISORDERS. THE GOAL OF THIS STUDY IS TO ADVANCE MECHANISTIC UNDERSTANDING OF THE MOLECULAR NETWORKS ASSOCIATED WITH OBSTETRIC DISORDERS, TO IDENTIFY AND PRIORITIZE NEW DIRECTIONS FOR FUTURE RESEARCH FOR IMPROVING MATERNAL-FETAL HEALTH. WE PROPOSE TO FIND PATTERNS AT THE INDIVIDUAL, NETWORK, AND SYSTEM LEVELS THAT SHED LIGHT ON MECHANISMS OF DISEASE ASSOCIATED WITH NORMAL PLACENTAL PHYSIOLOGY AND PLACENTAL DYSFUNCTION. THE SADOVSKY LAB HAS PROVIDED DE-IDENTIFIED METABOLOMIC, PROTEOMIC, AND TRANSCRIPTOMIC PLACENTAL DATA PAIRED WITH HISTOPATHOLOGY REPORTS AND CLINICAL DATA INCLUDING DEMOGRAPHICS, ROUTINE CLINICAL LABS, MATERNAL COMORBIDITIES, AND DELIVERY RECORDS. THESE DATA COME FROM 333 PLACENTAS FROM PEOPLE WITH SINGLETON PREGNANCIES, INCLUDING UNCOMPLICATED TERM PREGNANCY, FETAL GROWTH RESTRICTION, PREECLAMPSIA, FETAL GROWTH RESTRICTION WITH A HYPERTENSIVE DISORDER, AND SPONTANEOUS PRETERM BIRTH. THE ANALYSES IN THIS PROPOSAL ARE COMPLEMENTARY TO, AND DISTINCT FROM, THOSE IN A SEPARATE K99. IN AIM 1, WE WILL DEFINE PLACENTAL PHYSIOLOGICAL ASSOCIATION NETWORKS TO DETERMINE PLACENTAL REGULATION. WE HAVE PRELIMINARY DATA FOR PAIRWISE ASSOCIATIONS BETWEEN ANALYTES AND PLACENTAL HISTOPATHOLOGICAL FEATURES USING GENERALIZED LINEAR MODELS. WE WILL CONDUCT COMMUNITY STRUCTURE ANALYSIS ON THESE ASSOCIATIONS TO IDENTIFY SUBNETWORKS OF PLACENTAL REGULATION AND EVALUATE DIFFERENCES IN NETWORK IDENTITY AND STRUCTURE. FOR EACH OBSTETRIC DISORDER, WE WILL ALSO EVALUATE THE CONTRIBUTION OF TOP INTERCONNECTED COMMUNITY NETWORKS TO REGULATION OF PLACENTAL PHYSIOLOGY BY EVALUATING MODEL ROBUSTNESS AND MULTICOLLINEARITY. THIS WILL PROVIDE DOMAIN-AGNOSTIC DETECTION OF PATTERNS OF SYSTEMS-LEVEL PLACENTAL REGULATION. IN AIM 2, WE WILL DETERMINE MOLECULAR NETWORK DIFFERENCES AMONG OBSTETRIC DISORDERS. WE HAVE EVALUATED PAIRWISE DIFFERENCES BETWEEN INDIVIDUAL ANALYTES BETWEEN PHENOTYPES, AND WE WILL BUILD ON THIS WORK BY EVALUATING PLACENTAL NETWORK DIFFERENCES BETWEEN DIFFERENT OBSTETRIC OUTCOMES IN TWO WAYS: DETERMINING THE NETWORK STRUCTURE DIFFERENCES BETWEEN PHENOTYPES, AND BUILDING AND EVALUATING CLASSIFICATION MODELS FOR OBSTETRIC DISORDERS. WE WILL THEN PERFORM CASE-STUDY OUTLIER ANALYSIS TO PROVIDE POTENTIAL MOLECULAR INSIGHT INTO PLACENTAL DYSFUNCTION ON THE INDIVIDUAL LEVEL. THIS WILL PROVIDE MOLECULAR NETWORK UNDERSTANDING OF PLACENTAL REGULATION AND HOW IT’S DISRUPTED IN OBSTETRIC DISORDERS AT THE POPULATION AND INDIVIDUAL LEVEL.

TEMPORAL AND SPATIAL EFFECTS ON EXPRESSION AND FUNCTION

DOES POST-TRANSCRIPTIONAL CONTROL OF NLRP3 INFLAMMASOME ACTIVITY IMPACT DEVELOPMENT OF TYPE 1 DIABETES? - THE HUMAN GENOME CONTAINS A LARGE NUMBER OF CIS- AND TRANS-ACTING REGULATORY ELEMENTS THAT MODULATE GENE ACTIVITY AT DIFFERENT POINTS IN THE PROGRESSION FROM GENE TRANSCRIPTION TO TRANSLATION. ALTERATIONS IN NON-CODING OR REGULATORY REGIONS OF THE GENOME ARE LINKED TO AUTOIMMUNE TYPE 1 DIABETES (T1D) IN GENOME-WIDE ASSOCIATION STUDIES (GWAS), SUGGESTING THAT CHANGES IN GENE OR GENE PRODUCT EXPRESSION MIGHT PLAY AN IMPORTANT ROLE IN FUNCTIONAL IMMUNE DYSREGULATION. WE HAVE IDENTIFIED MULTIPLE POST-TRANSCRIPTIONAL REGULATORY ELEMENTS IN THE 3-UTR (UNTRANSLATED REGION) OF NLRP3, A CYTOSOLIC INNATE IMMUNE SENSOR OF METABOLIC DYSFUNCTION, THAT IMPACT NLRP3 MRNA STABILITY AND ARE ASSOCIATED WITH T1D RISK. NLRP3 ACTIVATION RESULTS IN ASSEMBLY OF A LARGE OLIGOMERIC SIGNALING COMPLEX CALLED THE INFLAMMASOME THAT PROMOTES A FORM OF INFLAMMATORY CELL DEATH CALLED PYROPTOSIS AND PRODUCTION OF BIOACTIVE IL-1SS, A PRO-INFLAMMATORY CYTOKINE IMPLICATED IN THE PATHOPHYSIOLOGY OF T1D. IN THE NOD (NON-OBESE DIABETIC) MOUSE MODEL, NLRP3 DEFICIENCY PROTECTS FROM DEVELOPMENT OF T1D THROUGH IMPAIRED PRODUCTION OF IL-1SS AND REDUCED MIGRATION OF PATHOGENIC T CELLS TO THE PANCREATIC ISLETS, SUGGESTING A CRITICAL ROLE FOR THE NLRP3 INFLAMMASOME IN T1D PATHOGENESIS. WHILE THIS SET OF LINKED EVENTS IS WELL DESCRIBED, LITTLE IS KNOWN ABOUT THE POST-TRANSCRIPTIONAL MECHANISMS THAT CONTROL THE EXPRESSION NLRP3 AND SET THE THRESHOLD FOR INFLAMMASOME ACTIVATION AND THE PATHOLOGICAL EVENTS ASSOCIATED WITH DEVELOPMENT OF T1D. OUR GENETIC DATA DOCUMENT THAT A SINGLE NUCLEOTIDE POLYMORPHISM IN THE 3-UTR OF THE HUMAN NLRP3 GENE THAT FUNCTIONALLY INCREASES NLRP3 MRNA IS SIGNIFICANTLY ASSOCIATED WITH T1D RISK SUGGESTING THAT AN INCREASE IN NLRP3 MAY PLAY A ROLE IN DISEASE DEVELOPMENT. WE HAVE FOUND THAT THE NLRP3 GENE ALSO HAS TWO POLYADENYLATION SITES, WHICH EITHER ENCODE FOR A STABLE SHORT 3-UTR OR A LESS STABLE LONGER 3-UTR. THE LONGER 3-UTR HARBORS SEVERAL CIS ACTING REGULATORY MOTIFS, SUCH AS AU-RICH ELEMENTS, MIRNA BINDING SITES AND GAMMA INTERFERON INHIBITOR OF TRANSLATION ELEMENTS, WHICH ARE KNOWN TO SUPPRESS PROTEIN EXPRESSION. BASED ON THESE DATA WE HYPOTHESIZE THAT PREFERENTIAL USAGE OF THE SHORT 3'-UTR AS OPPOSED TO THE LONG 3'-UTR INCREASES THE GENE DOSAGE OF NLRP3 THEREBY SENSITIZING CELLS TO NLRP3 ACTIVATORS AND LOWERING THE THRESHOLD FOR NLRP3 INFLAMMASOME ACTIVATION IN T1D. IN THIS GRANT WE WILL GENERATE MICE CARRYING THE SHORT VS. LONG 3'-UTR VARIANTS OF NLRP3 ON THE NOD BACKGROUND WITH THE GOAL OF DISSECTING THE IMPACT OF GENE DOSAGE OF NLRP3 VIA ALTERNATE 3-UTR USAGE ON THE DEVELOPMENT AND PROGRESSION OF T1D IN VIVO. OUR STUDY WILL GENERATE FOUNDATIONAL MOUSE MODELS TO STUDY THE ROLE OF POST-TRANSCRIPTIONAL REGULATION IN DEVELOPMENT OF T1D AND WILL HAVE BROADER IMPLICATIONS FOR UNDERSTANDING HOW EXPRESSION CHANGES CAUSED BY GENETIC VARIATION IMPACT THE DEVELOPMENT OF COMPLEX AUTOIMMUNE DISEASES LIKE T1D.

CAN A MODEST CHANGE IN REGULATION OF NOD1 EXPRESSION HAVE A MAJOR IMPACT ON INFLAMMATION AND GASTRIC CANCER?

A NON-CANONICAL ROLE OF CASPASE-1 IN REGULATING BACTERIAL ANTIMICROBIAL RESISTANCE - SUMMARY ANTIMICROBIAL RESISTANCE (AMR) IS A PROMINENT HEALTHCARE THREAT WITH AN ESTIMATED 4.95 MILLION CASES ASSOCIATED WITH BACTERIAL AMR IN 2019, AND POSES A PARTICULARLY DIFFICULT CHALLENGE WITH REGARD TO INTRACELLULAR PATHOGENS THAT HAVE EVOLVED TO HIJACK HOST DEFENSES FOR THEIR OWN BENEFIT. A DEEP VIEW OF HOST-PATHOGEN INTERACTIONS INCLUDING A GREATER UNDERSTANDING OF HOW PATHOGENS ADAPT AND CHANGE IN RESPONSE TO HOST CUES IS CRITICAL TO GAIN INSIGHTS INTO THE FACTORS RESPONSIBLE FOR AMR. WHILE THE RESPONSE OF HOSTS TO BACTERIAL INFECTION HAS BEEN EXTENSIVELY STUDIED, THE CONVERSE I.E. HOW PATHOGEN GENE EXPRESSION AND IN TURN PATHOGEN PHYSIOLOGY IS MODULATED IN RESPONSE TO HOST CUES HAS GATHERED LITTLE ATTENTION. LACK OF ROBUST TECHNOLOGIES TO SEQUENCE MINUTE AMOUNTS OF BACTERIAL RNA FROM INFECTED CELLS HAS BEEN ONE OF THE LIMITING FACTORS. USING A RECENTLY DEVELOPED TECHNOLOGY - PATH-SEQ, WE RELIABLY SEQUENCED MACROPHAGE-RESIDENT SALMONELLA TO DISCOVER A ROLE FOR HOST CASPASE-1 IN DAMPENING AMR OF INTRACELLULAR SALMONELLA. THIS WAS MEDIATED THROUGH INHIBITION OF THE BACTERIAL TWO-COMPONENT SIGNAL TRANSDUCTION SYSTEM – PHOPQ, A MAJOR CONTRIBUTOR RESPONSIBLE FOR SALMONELLA'S ABILITY TO RESIST HOST CATIONIC ANTIMICROBIAL PEPTIDES (CAMPS) AND THE DRUG POLYMYXIN B, WHICH IS A LAST RESORT ANTIBIOTIC AGAINST GRAM- NEGATIVE PATHOGENS. INTERESTINGLY, CASPASE-1 WHICH IS CONVENTIONALLY RECOGNIZED AS A PROTEASE IMPORTANT FOR INFLAMMASOME ACTIVATION AND PYROPTOSIS, DAMPENS CAMP RESISTANCE OF SALMONELLA IN A MANNER INDEPENDENT OF ITS CATALYTIC/PROTEASE ACTIVITY. IN THIS PROPOSAL WE WILL THOROUGHLY INVESTIGATE THE MECHANISM BY WHICH HOST CASPASE-1 INHIBITS PHOPQ ACTIVATION AND CAMP RESISTANCE OF INTRACELLULAR SALMONELLA INDEPENDENT OF ITS ACTIVITY. IN AIM 1 WE WILL CONDUCT A MOLECULAR DISSECTION OF THE BACTERIAL PATHWAY AND THE PATHOGEN EFFECTORS DOWNSTREAM OF PHOPQ ACTIVATION THAT ARE TARGETED BY CASPASE-1. IN AIM 2 WE WILL INVESTIGATE HOW HOST CASPASE-1 INHIBITS CAMP RESISTANCE OF INTRACELLULAR SALMONELLA, EITHER BY DIRECTLY ACTING ON THE BACTERIUM OR INDIRECTLY BY REGULATING HOST PROCESSES THAT IN TURN CONTROL PHOPQ ACTIVATION AND CAMP RESISTANCE. TARGETING THIS NON-CANONICAL, PYROPTOSIS INDEPENDENT ARM OF CASPASE-1 MAY BE PARTICULARLY USEFUL IN CURBING AMR OF PATHOGENS SUCH AS SALMONELLA THAT EVADE INFLAMMASOME ACTIVATION AND REPLICATE INTRACELLULARLY. OUR FINDINGS WILL REVEAL A NOVEL ACTIVITY-INDEPENDENT ROLE FOR CASPASE-1 IN CONTROLLING BACTERIAL SIGNALING AND AMR, AND BECAUSE PHOPQ ACTIVATION DICTATES MULTIPLE ASPECTS OF PATHOGEN PHYSIOLOGY SUCH AS INTRACELLULAR REPLICATION AND VIRULENCE, WILL ALSO FORM A FRAMEWORK FOR EXPLORING THE IMPACT OF HOST CASPASE-1 ON PROCESSES BEYOND AMR IN INTRACELLULAR GRAM-NEGATIVE PATHOGENS.

PROTEOGENOMIC RESOURCE FOR THE DEVELOPMENT OF NOVEL MARKERS FOR LYME DISEASE

NOVEL PEPTIDE BASED BIOMARKERS FOR LYME DISEASE DIAGNOSTICS

STRATEGIES FOR DISCERNING CHEMOTHERAPY RESPONSE AND RESISTANCE IN OVARIAN CANCER - PROJECT SUMMARY/ABSTRACT OVARIAN CANCER IS THE FIFTH LEADING CAUSE OF CANCER DEATH AMONG FEMALES. MOST CASES OF OVARIAN CANCER ARE HIGH- GRADE SEROUS OVARIAN CANCER (HGSOC), WHICH ACCOUNTS FOR MOST OVARIAN CANCER MORTALITY. IN SPITE OF THE INCREASING KNOWLEDGE OF THIS TUMOR TYPE, THE USE OF “OLD” DNA-DAMAGING CHEMOTHERAPEUTIC DRUGS REMAINS THE FIRST OPTION FOR OVARIAN CANCER PATIENTS. THE STANDARD TREATMENT OF HGSOC INVOLVES DE-BULKING SURGERY FOLLOWED BY PLATINUM-BASED CHEMOTHERAPY WITH THE AIM TO ELIMINATE ALL THE REMAINING MICRO-METASTASES. UNFORTUNATELY, A SIGNIFICANT NUMBER OF HGSOC PATIENTS DO NOT RESPOND OR ONLY PARTIALLY RESPOND TO THESE TREATMENTS. A PATIENT WHO DOES NOT RESPOND TO PLATINUM CHEMOTHERAPIES HAS TO GO THROUGH THE SAME TREATMENT AND EXPERIENCE THE TOXICITY WITHOUT MEANINGFUL BENEFIT. DESPITE THE RAPID ADVANCES IN CANCER GENOMICS AND PRECISION MEDICINE IN THE LAST DECADE, WHAT REMAINS SURPRISING IS THAT NO ROBUST MOLECULAR BIOMARKER THAT CAN FORETELL PATIENT RESPONSE TO PLATINUM AGENTS HAS BEEN IDENTIFIED IN OVARIAN CANCER, POINTING TO THE NEED FOR THE INCORPORATION OF FUNCTIONAL METRICS AND EARLY-STAGE MOLECULAR CHANGES INTO THE PREDICTIVE FRAMEWORK. WE HYPOTHESIZE THAT EARLY-STAGE MOLECULAR AND FUNCTIONAL ALTERATIONS UPON DRUG EXPOSURE THAT UNDERLIE HOW TUMOR CELLS ORCHESTRATE A RESPONSE TO THE DRUG TREATMENT ARE DICTATING LONGER-TERM THERAPY RESPONSES AND THUS CRITICAL TO THE DEVELOPMENT OF PREDICTIVE MODELS. WE FURTHER HYPOTHESIZE THAT THE DRUG SUSCEPTIBILITY AND BIOENERGETIC DEPENDENCY OF THE PATIENT TUMORS ARE IMPORTANT FUNCTIONAL READOUTS FOR SUCH A PREDICTION. WE PROPOSE TO INTEGRATE EARLY-STAGE TEMPORAL GENOME, TRANSCRIPTOME, METABOLIC PHENOTYPES, DRUG SUSCEPTIBILITY, AND HISTOLOGY INFORMATION INTO MACHINE LEARNING MODELS TO DISCERN A SET OF METRICS MOST PREDICTIVE FOR PATIENT-SPECIFIC RESPONSES TO PLATINUM CHEMOTHERAPY IN HGSOC. THIS IS ENABLED BY A STRATEGIC INTEGRATION OF A FRESHLY PREPARED, PRECISION-CUT TUMOR SLICE CULTURE MODEL, AN INNOVATIVE SINGLE-CELL ON-CHIP METABOLIC CYTOMETRY ASSAY, AND A NOVEL IN SITU SPATIAL METABOLIC PROFILING ASSAY ON LIVE TISSUES. UPON SUCCESSFUL COMPLETION, THIS STUDY COULD DELIVER A PREDICTIVE DIAGNOSTIC ASSAY FOR IDENTIFYING HGSOC PATIENTS WITH A HIGHER RISK OF RESISTANCE TO PLATINUM CHEMOTHERAPY, AS WELL AS PATIENTS WHO MAY BENEFIT FROM SUCH TREATMENTS, PRIOR TO THE ONSET OF THERAPY. THESE RESULTS WILL PROVIDE INNOVATIVE MOLECULAR AND FUNCTIONAL INFORMATION COMPLEMENTARY TO TUMOR GENETICS AND OTHER CLINICAL FACTORS FOR MORE INFORMATIVE CLINICAL DECISION-MAKING AND HELP RE-DIRECT PLATINUM-REFRACTORY PATIENTS TO OTHER THERAPEUTIC APPROACHES OR CLINICAL TRIALS OF NOVEL THERAPIES BEFORE THE TREATMENT.

PRODIGE: A HIGH-THROUGHPUT TOOL FOR JOINT PROFILING OF PROTEIN-DNA INTERACTIONS AND GENE EXPRESSION IN SINGLE CELLS - PROJECT SUMMARY/ABSTRACT PROTEIN-DNA INTERACTIONS CONTROL ACCESS TO GENES IN CHROMATINS AND REGULATE THE SPATIAL- AND TEMPORAL-SPECIFIC GENE EXPRESSION. THESE INTERACTIONS ARE CRUCIAL FOR CELL DIFFERENTIATION AND THE MAINTENANCE OF CELLULAR DIVERSITY. DYSREGULATED PATTERNS OF PROTEIN-DNA INTERACTIONS ARE OFTEN ASSOCIATED WITH DISEASE STATES. APPLICATIONS OF NEXT- GENERATION EPIGENOMIC SEQUENCING TECHNIQUES HAVE ENABLED DECONVOLUTION OF PROTEIN-DNA INTERACTIONS AT SINGLE-CELL LEVEL. HOWEVER, IT REMAINS CHALLENGING TO DEFINE HOW CELL-TO-CELL HETEROGENEITY IN PROTEIN-DNA INTERACTIONS IMPACTS GENE EXPRESSION VARIABILITY. CURRENTLY AVAILABLE METHODS FOR JOINT PROFILING PROTEIN-DNA INTERACTIONS AND GENE EXPRESSION FROM SINGLE CELLS REQUIRE LABORIOUS GENETIC MANIPULATION, SUFFER RELATIVELY LOW THROUGHPUT, AND ARE IMPOSSIBLE TO SPECIFICALLY MAP PROTEINS WITH POST-TRANSLATIONAL MODIFICATIONS, SUCH AS HISTONE MARKS. TO ADDRESS THESE UNMET NEEDS, WE PROPOSE TO DEVELOP PRODIGE – A HIGH-THROUGHPUT TOOL FOR JOINT PROFILING OF PROTEIN-DNA INTERACTIONS AND GENE EXPRESSION IN THE SAME SINGLE CELLS WITHOUT THE NEED OF GENETIC MANIPULATIONS. IT IS ENABLED BY A STRATEGIC INTEGRATION OF REVERSIBLE CELL FIXATION, ANTIBODY-GUIDED CHROMATIN TAGMENTATION, NOVEL DEFORMABLE AND DEGRADABLE BARCODED HYDROGEL BEADS, DROPLET MICROFLUIDICS, AND NEXT- GENERATION SEQUENCING TECHNOLOGY. IN AIM 1, WE WILL DEVELOP TECHNICAL COMPONENTS AND ANALYTICAL PIPELINES OF PRODIGE. WE WILL PREPARE PROTEIN A-TN5 TRANSPOSOME FOR ANTIBODY-GUIDED TN5 TAGMENTATION, FABRICATE MICROFLUIDIC DEVICES FOR SINGLE-CELL PRODIGE ASSAY, AND SYNTHESIZE A BARCODED LIBRARY OF DEFORMABLE AND DEGRADABLE BARCODED HYDROGEL BEADS FOR SINGLE-CELL BARCODING. IN AIM 2, WE WILL OPTIMIZE THE METHOD IN BULK AND SINGLE-CELL LEVELS, AND ASSESS THE PERFORMANCE OF PRODIGE (JOINT ANALYSIS) IN COMPARISON TO OTHER STAND-ALONE COUNTERPARTS. IN AIM 3, WE WILL BENCHMARK PRODIGE TO ESTABLISHED TOOLS FOR JOINT SINGLE-CELL PROFILING OF PROTEIN- DNA INTERACTIONS AND TRANSCRIPTOME. SUCCESSFUL IMPLEMENTATION OF THE PROPOSED PROGRAM WILL DELIVER A HIGH- THROUGHPUT TOOL TO QUANTIFY HOW CELL-TO-CELL HETEROGENEITY IN PROTEIN-DNA BINDING INFLUENCES GENE EXPRESSION VARIABILITY, DECIPHER CELL-TYPE-SPECIFIC PATTERNS OF UPSTREAM PROTEIN-DNA INTERACTIONS AND THEIR TRANSCRIPTIONAL OUTPUTS, AND IDENTIFY PROTEIN-MEDIATED MECHANISMS THAT REGULATE CELL-TYPE-ASSOCIATED TRANSCRIPTIONAL PROGRAMS IN HETEROGENEOUS CELL POPULATIONS AND UNDER DIFFERENT DEVELOPMENTAL AND/OR DISEASE CONDITIONS.

RCN-UBE: NETWORKING NEXT GEN BIOLOGISTS WITH COMMUNITY COLLEGE EDUCATORS -THIS RCN-UBE PROJECT SEEKS TO NETWORK COMMUNITY COLLEGE EDUCATORS AND BIOLOGISTS USING BIG DATASETS (NEXT GEN BIOLOGISTS) TO PROMOTE DATA LITERACY IN COMMUNITY COLLEGE STUDENTS. NEXT GEN BIOLOGY HAS LED TO A HUGE INCREASE IN MANY TYPES OF BIOLOGICAL DATA MAKING IT IMPORTANT THAT ALL BIOLOGY STUDENTS HAVE ACCESS TO EDUCATIONAL EXPERIENCES THAT BUILD DATA LITERACY. HOWEVER, DUE TO THE RAPIDLY EVOLVING NATURE OF SKILLS NEEDED TO WORK WITH BIG DATA, MANY STEM EDUCATORS LACK CONTEMPORARY DATA LITERACY SKILLS. EVEN FEWER HAVE TRAINING ON HOW TO USE DATA IN THE CLASSROOM IN A WAY THAT IS ACCESSIBLE TO STUDENTS AND BUILDS DATA LITERACY SKILLS. THIS PROJECT AIMS TO IMPROVE UNDERGRADUATE STEM TEACHING AND LEARNING BY INCREASING FACULTY CAPABILITY WITH NEXT GEN BIOLOGY DATA ANALYSIS. TO DO THIS THE NETWORK PARTICIPANTS WILL CREATE RESOURCES TO ENGAGE DIVERSE UNDERGRADUATES IN CUTTING EDGE METHODS IN INTRODUCTORY COURSES. THE NETWORK WILL ALSO PROMOTE EQUITY AND DIVERSITY BY ENHANCING PARTICIPANT AWARENESS OF ISSUES THAT IMPACT STUDENT ACCESS TO BIOLOGICAL DATA LITERACY SKILLS AND QUANTITATIVE BIOLOGY RESEARCH METHODS. THIS PROJECT WILL BRING TOGETHER NEXT GEN BIOLOGISTS FROM NON-DEGREE GRANTING INSTITUTES AND COMMUNITY COLLEGE FACULTY TO ENGAGE IN A SERIES OF PROFESSIONAL LEARNING EXPERIENCES DESIGNED TO SUPPORT ALL PARTICIPANTS TO BUILD KNOWLEDGE ABOUT STUDENTS? DEVELOPMENT OF BIOLOGICAL DATA LITERACY AND QUANTITATIVE RESEARCH SKILLS. TO PROMOTE PARTICIPANTS? AWARENESS AND AGENCY, DISCUSSIONS FOCUSED ON DIVERSITY, EQUITY, AND INCLUSION WILL BE INTEGRAL TO EACH PROFESSIONAL LEARNING EVENT. FOLLOWING THE SERIES OF PROFESSIONAL LEARNING EVENTS, MEMBERS OF THE NETWORK WILL CO-CREATE CURRICULAR RESOURCES THAT EQUITABLY ENGAGE STUDENTS IN ANALYZING AND INTERPRETING LARGE DATASETS FOUNDED IN AUTHENTIC RESEARCH CONTEXTS. THE PROJECT WILL GENERATE ROBUST INSTRUCTIONAL STRATEGIES AND CURRICULAR RESOURCES FOUNDED IN CONTEMPORARY QUANTITATIVE BIOLOGY RESEARCH, A SERIES OF GUIDES FOR FACILITATING PROFESSIONAL DEVELOPMENT WORKSHOPS INVOLVING PROFESSIONALS FROM EDUCATION AND SCIENTIFIC RESEARCH FIELDS, AND AN EXPANDED NETWORK OF UNDERGRADUATE BIOLOGY FACULTY AND SCIENTISTS AT RESEARCH INSTITUTES ENGAGING STUDENTS IN QUANTITATIVE BIOLOGY SKILLS. THIS PROJECT IS BEING JOINTLY FUNDED BY THE DIRECTORATE FOR BIOLOGICAL SCIENCES, DIVISION OF BIOLOGICAL INFRASTRUCTURE, AND THE DIRECTORATE FOR EDUCATION AND HUMAN RESOURCES, DIVISION OF UNDERGRADUATE EDUCATION AS PART OF THEIR EFFORTS TO ADDRESS THE CHALLENGES POSED IN VISION AND CHANGE IN UNDERGRADUATE BIOLOGY EDUCATION: A CALL TO ACTION (HTTP://VISIONANDCHANGE/FINALREPORT/). THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.

TUMOR SLICE CULTURE: A NEW AVATAR IN PERSONALIZED ONCOLOGY

EPIGENETIC CONTROL OF ASTHMATIC INFLAMMATION BY ATF3

GENETIC REGULATORY NETWORKS: COMP. & EXT. INVESTIGATIONS

REU SITE: RESEARCH OPPORTUNITIES IN SYSTEMS BIOLOGY -THIS REU SITE AWARD TO THE INSTITUTE FOR SYSTEMS BIOLOGY (ISB), LOCATED IN SEATTLE, WA, WILL SUPPORT THE TRAINING OF 10 STUDENTS FOR 10 WEEKS DURING THE SUMMERS OF 2022-2024. IT IS ANTICIPATED THAT AT LEAST 30 STUDENTS, PRIMARILY FROM SCHOOLS WITH LIMITED RESEARCH OPPORTUNITIES OR FROM AN UNDER-REPRESENTED GROUP, WILL BE TRAINED IN THE PROGRAM. STUDENTS WILL LEARN HOW RESEARCH IS CONDUCTED, AND MANY WILL PRESENT THE RESULTS OF THEIR WORK AT SCIENTIFIC CONFERENCES. ASSESSMENT OF THE PROGRAM WILL BE DONE USING AN ONLINE INSTRUMENT. STUDENTS WILL BE TRACKED AFTER THE PROGRAM IN ORDER TO DETERMINE THEIR CAREER PATHS. THE PROGRAM AIMS TO SUPPORT UNDERGRADUATE STUDENTS TO DEVELOP INTEREST, BUILD CONFIDENCE, AND GENERATE THE KNOWLEDGE TO PURSUE CAREERS IN SCIENTIFIC RESEARCH, SPECIFICALLY SYSTEMS BIOLOGY. ALL STUDENTS WILL PARTICIPATE IN A RESEARCH PROJECT DEVELOPED WITH THE GUIDANCE OF A MENTOR; BE INCLUDED IN ALL EVENTS AT ISB, INCLUDING LAB MEETINGS AND RETREATS; PARTICIPATE IN ONGOING PROFESSIONAL DEVELOPMENT COURSE THAT INCLUDES DEEPENING RESEARCH SKILLS, LEARNING SYSTEMS BIOLOGY SPECIFIC SKILLS, AND EXPLORATION OF THE RESPONSIBLE CONDUCT OF RESEARCH; AND COMPLETE A POSTER WITH THE SUPPORT OF THEIR MENTOR AND OTHER ISB STAFF TO BE SHARED AT AN ISB-WIDE MINI-SYMPOSIUM. POTENTIAL PROJECTS INCLUDE A VARIETY OF WET LAB FOCUSED, COMPUTATIONALLY FOCUSED, AND MIXED WET AND COMPUTATIONAL FOCUSED RESEARCH. POTENTIAL RESEARCH PROJECTS INCLUDE: QUANTIFYING DIATOM RESILIENCE IN CHANGING OCEANS, USING MICROBIAL COMMUNITIES TO CHARACTERIZE DENITRIFICATION, IN SILICO PATHOGEN INVASION ASSAYS, DEVELOPING NEW TECHNOLOGIES FOR ANALYZING EXTRACELLULAR VESICLES, AND NETWORK ANALYSIS OF A STOCHASTICALLY ACTIVATED BACTERIAL METABOLIC PATHWAYS. STUDENTS WITH A DEMONSTRATED INTEREST IN FIELDS SUCH AS SYSTEMS BIOLOGY, COMPUTER SCIENCE, INFORMATION SCIENCE, APPLIED MATHEMATICS, AND/OR ENGINEERING ARE ALL GOOD FITS FOR THE TYPES OF RESEARCH PROJECTS AVAILABLE. STUDENTS ARE SELECTED BASED ON THEIR INTERESTS AND THE STEPS THEY?VE TAKEN TO PURSUE THOSE INTERESTS, INCLUDING COURSES TAKEN, SKILLS DEVELOPED, AND OTHER ACTIVITIES. MORE INFORMATION ABOUT THE PROGRAM IS AVAILABLE BY VISITING HTTPS://ISBSCIENCE.ORG/ABOUT/CAREERS/INTERNSHIPS/SUMMER-UNDERGRADUATE-RESEARCH-EXPERIENCES-PROGRAM/ OR CONTACTING DR. JEN EKLUND AT JEN.EKLUND@ISBSCIENCE.ORG. THIS AWARD IS SUPPORTED BY THE DEPARTMENT OF DEFENSE ASSURE PROGRAM IN PARTNERSHIP WITH THE NSF REU PROGRAM. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.

MAPPING THE DYNAMIC ARCHITECTURE OF THE HUMAN MEDIATOR COMPLEX

EAGER: SHARED PRINCIPLES OF ADAPTIVE LEARNING - ANTICIPATORY BEHAVIOR IN HALOBACTETRIUM SALINARUM

EAGER: DIATOM PROGRAMMED CELL DEATH AT SINGLE-CELL RESOLUTION

MODEL-GUIDED SYSTEMS RE-ENGINEERING OF CHLAMYDOMONAS REINHARDTII

ENHANCING PERSONALIZED INSIGHTS INTO COMMON OBSTETRIC DISORDERS USING LONGITUDINAL DEEP-PHENOTYPING DATA - PROJECT SUMMARY OBSTETRIC DISORDERS ARE COMMON GLOBALLY AND A MAJOR DRIVER FOR DEATHS OF CHILDREN UNDER FIVE AS WELL AS OTHER LIFELONG HEALTH ISSUES. DESPITE THIS WE HAVE A LIMITED UNDERSTANDING OF THE MECHANISMS DRIVING THESE DISORDERS HIGHLIGHTING AN UNMET RESEARCH GAP. HERE, WE COLLABORATE WITH DR. YOEL SADOVSKY TO COMPILE A DEEP-PHENOTYPING PREGNANCY DATASET THAT EVALUATES WOMEN’S HEALTH THROUGHOUT PREGNANCY PROVIDING LONGITUDINAL BLOOD AND URINE MULTIOMICS DATA PAIRED WITH CLINICAL, SURVEY, BEHAVIORAL, AND ENVIRONMENTAL DATA COLLECTED FROM 200 PEOPLE (100 PEOPLE WITH ADVERSE OUTCOMES) PROVIDING A COMPREHENSIVE VIEW OF PREGNANCY. WE HYPOTHESIZE THAT A DATA-DRIVEN SYSTEMS BIOLOGY APPROACH WILL DEFINE NORMAL PLACENTAL AND PREGNANCY SYSTEMS BIOLOGY AND FACILITATE INVESTIGATION OF DISEASE MECHANISMS IN COMMON OBSTETRIC DISORDERS INCLUDING PRETERM BIRTH, FETAL GROWTH RESTRICTION AND PREECLAMPSIA. FIRST, WE WILL EVALUATE MOLECULAR NETWORK DIFFERENCES IN COMMON OBSTETRIC DISORDERS USING PLACENTAL MULTIOMICS (METABOLOMICS, PROTEOMICS, AND TRANSCRIPTOMICS) DATA PAIRED WITH CLINICAL AND PLACENTAL HISTOPATHOLOGY DATA COLLECTED FROM 342 PEOPLE (213 WITH COMMON OBSTETRIC DISORDERS). WE WILL BUILD INTER-OMIC PLACENTAL NETWORKS ACROSS DATATYPES AND OUTCOMES INCREASING OUR UNDERSTANDING OF PLACENTAL BIOLOGY. WE WILL ALSO DETERMINE DIFFERENCES IN MOLECULAR NETWORK STRUCTURES AND KEY TRANSCRIPTION FACTORS ASSOCIATED WITH DISTINCT OBSTETRIC DISORDERS. IN ADDITION, WE WILL USE THE DEEP-PHENOTYPING PREGNANCY DATA TO EVALUATE MOLECULAR NETWORK DYNAMICS AND DEFINE MAJOR TRANSITION STATES OF PREGNANCY. WE WILL ALSO USE IT TO IDENTIFY DISRUPTIONS TO MOLECULAR NETWORKS ASSOCIATED WITH COMMON OBSTETRIC DISORDERS. WE WILL ALSO DEVELOP A NEW APPROACH TO IDENTIFY ANALYTE OUTLIERS IN INDIVIDUALS AT THE EARLIEST TIME POINT OF DEVIATION FROM A HEALTHY PREGNANCY TRAJECTORY, PROTOTYPING A PRECISION MEDICINE APPROACH IN THE CONTEXT OF PREGNANCY. FINALLY, WE ARE PARTNERING WITH GOOGLE DATA COMMONS TO BUILD AN OPEN-SOURCE PERINATAL-SPECIFIC KNOWLEDGE GRAPH TO DISTRIBUTE THE DATA FROM THIS PROPOSAL TO THE BROADER PERINATAL RESEARCH COMMUNITY. ALTOGETHER THIS WILL GENERATE AND PRIORITIZE HYPOTHESES OF THE MOLECULAR MECHANISMS OF COMMON OBSTETRIC DISORDERS, WHICH WILL BE USED TO DEVELOP FUTURE CLINICAL INTERVENTIONS TO PROMOTE MATERNAL-FETAL HEALTH. FINALLY, THIS WORK WILL PROVIDE ME WITH THE BACKGROUND NEEDED TO ESTABLISH AN INDEPENDENT LINE OF RESEARCH.

CAREER: SYSTEMS BIOLOGY AND ENGINEERING OF CLOSTRIDIUM BEIJERINCKII FOR ENHANCE BUTANOL PRODUCTION

THE ROLE OF CORTICOTROPHIN RELEASING HORMONE ON PLACENTAL TRANSCRIPTIONAL NETWORKS AND BIRTH TIMING

A SYSTEMS APPROACH TO UNDERSTANDING OVERALL DRUG RESPONSE IN A HETEROGENEOUS TUMOR-CELL POPULATION

COLLABORATIVE RESEARCH: BRAIN METABOLIC PLASTICITY AND AGRESSION

RAPID: HOST MOLECULAR RESPONSES AFTER EBOLA VIRUS INFECTION

MODELING THE DYNAMICS AND COMPOSITION OF T-CELL RECEPTOR REPERTOIRES IN POST-ACUTE SEQUELAE OF COVID-19 (PASC) - PROJECT SUMMARY POST-ACUTE SEQUELAE OF COVID-19 (PASC), COLLOQUIALLY KNOWN AS LONG COVID, HAS BEEN REPORTED IN 31%-69% OF COVID-19 PATIENTS. THE PREVALENCE OF PASC AMONG PATIENTS, EVEN THOSE WITH MILD INFECTION, AND THOSE WITH PRIOR VACCINATION, UNDERSCORES THE IMPORTANCE OF PRINCIPLED APPROACHES FOR UNCOVERING AND ADDRESSING THE CAUSE OF THESE ISSUES. WE PROPOSE TO CONDUCT A DETAILED ANALYSIS OF THE IMMUNE REPERTOIRE DYNAMICS OF PASC PATIENTS AND COMPARE THEM TO BOTH COVID-19 PATIENTS WITHOUT PASC, AND HEALTHY INDIVIDUALS. THESE WILL BE SECONDARY ANALYSES, CONDUCTED ON EXISTING DATA FROM A PREVIOUS STUDY WITH THE INSTITUTE OF SYSTEMS BIOLOGY AND SWEDISH HEALTH SERVICES, WHICH INCLUDES LONGITUDINAL DEEP IMMUNOPHENOTYPING WITH SINGLE CELL AND PLASMA MULTIOMICS, REPERTOIRE SEQUENCING, ELECTRONIC HEALTH RECORDS, VIREMIA MEASUREMENTS, AND ANTIBODY TITERS FOR 209 COVID-19 PATIENTS. IN AIM 1, WE WILL CONDUCT INFERENCE, ANALYSIS, AND COMPARISON OF T-CELL RECEPTOR REPERTOIRE DYNAMICS IN PASC. USING INTERPRETABLE STATISTICAL, BIOPHYSICAL, AND MACHINE LEARNING APPROACHES, WE WILL CONDUCT A DETAILED ANALYSIS OF T-CELL REPERTOIRES AIMED AT FINDING PASC SPECIFIC CLONOTYPES AND THEIR CORRESPONDING RECEPTOR FEATURES. THIS INVOLVES BUILDING COHORT SPECIFIC MODELS OF THYMIC SELECTION, EXAMINING HOW THESE MODELS DIFFER BETWEEN COHORTS AND OVER TIME, INFERRING THE DYNAMICS OF REPERTOIRE SIZE, SHARING AND DIVERSITY, AND UNCOVERING THE RECEPTOR FEATURES WHICH DRIVE THESE DIFFERENCES. IN AIM 2, WE WILL DEVELOP NEW METHODS FOR THE INTEGRATION OF T-CELL REPERTOIRE AND SINGLE CELL DYNAMICS. THE EXISTING BREADTH OF MULTIOMICS DATA ALLOWS US TO EXPLORE NEW METHODOLOGIES FOR INTEGRATING DIFFERENT MODALITIES OF LONGITUDINAL DATA. FOR T-CELL REPERTOIRES IN PARTICULAR, WE PLAN TO EXTEND EXISTING MODELS OF THYMIC SELECTION TO INCLUDE GENE EXPRESSION OF RELEVANT T-CELL SPECIFIC GENES AND TO STUDY HOW THE EXPANSION AND CONTRACTION OF CLONOTYPES AFFECTS THE DYNAMICS OF T-CELLS IN GENE EXPRESSION SPACE. BY USING INTERPRETABLE BIOPHYSICAL AND MACHINE LEARNING METHODS, WE CAN CONSTRUCT GENERATIVE MODELS OF TCRS INCLUDING GENE EXPRESSION VALUES AND STUDY HOW THESE DISTRIBUTIONS CHANGE IN TIME. RESULTS HAVE STRONG POTENTIAL TO ACCELERATE OUR UNDERSTANDING OF THE ETIOLOGY OF IMMUNE-BASED PASC RESPONSES, WHICH IS ESSENTIAL FOR PRIORITIZING POTENTIAL THERAPEUTIC TARGETS FOR PREVENTION AND TREATMENT. FURTHER, RESULTS WILL ADVANCE METHODS FOR FUTURE RESEARCH ACROSS A WIDE RANGE OF INFECTIOUS DISEASES AND IMMUNE-MEDIATED MEDICAL CONDITIONS.

COLLABORATIVE RESEARCH: TRACING THE FATE OF ALGAL CARBON EXPORT IN THE ROSS SEA (TRACERS)

SIMULTANEOUS DETECTION OF PROTEINS AND WHOLE TRANSCRIPTOMES IN SINGLE CELLS

PROTEOMIC AND LIPIDOMIC PROFILING OF TUMOR-DERIVED EXOSOMES FOR CANCER PREVENTION

POST-TRANSCRIPTIONAL REGULATION IN ENVIRONMENTAL RESPONSE

MAPPING THE ANDROGEN RECEPTOR SIGNALING AXIS

SINGLE CELL IMAGE ANALYSIS FOR SYSTEMS BIOLOGY

MULTISCALE KINETIC ANALYSIS OF CU/ZN EFFLUX IN HALOBACTERIUM SP. NRC-1

RCN-UBE INCUBATOR: NETWORKING SYSTEMS BIOLOGISTS WITH COMMUNITY COLLEGE EDUCATOR

BD SPOKES: PLANNING: WEST: COLLABORATIVE: INCREASING COLLABORATIONS IN PROTEOGENOMICS APPLICATIONS OF GENETIC DATA

PROTOTYPING A SELF-LEARNING DIGITAL TWIN PLATFORM FOR PERSONALIZED TREATMENT IN MELANOMA PATIENTS

MICROBIAL ECOLOGY OF COEXISTING ECOTYPES: ARE ALL PROCHLOROCOCCUS EQUAL?

2012 SYSTEMS BIOINFORMATICS WORKSHOP, AUGUST 27-29, 2012

7TH ANNUAL SYSTEMS BIOLOGY SYMPOSIUM: SYSTEM BIOLOGY AND ENGINEERING

MOLECULAR ASSEMBLIES, GENES AND GENOMICS INTEGRATED EFFICIENTLY (MAGGIE) : COMPONENT 3: SYSTEMS APPROACH IN A MULTI-ORGANISM STRATEGY..........

MANIPULATION OF SMALL QUANTITIES OF DNA WITH INDUCED-DIPOLE TRAPS KING