Washington University

NIH Research Projects · FY 2025 · 2012-04

PROJECT ABSTRACT The overall goal of this proposal is to decipher a novel Ca2+-dependent signaling pathway that protects the genome in human cells in the presence of DNA replication stress. Replication stress, which can cause DNA damage and chromosomal instability, is frequently induced by both environmental agents and endogenous factors (e.g., reactive oxygen species, aldehyde, and oncogene activation). In order to maintain genome stability cells must protect the replication fork structure upon replication stress to avoid fork collapse and DNA damage. Defects in fork protection can result in cell death or transformation, which can give rise to cancer, premature ageing and other diseases. The fork protection mechanisms can also be exploited to sensitize cancer cells to replication stress-inducing agents during cancer treatment. Despite its critical importance, exactly how replication forks are protected after replication stress remain an outstanding question. In our effort to address this fundamental question, we recently discovered a novel Ca2+-dependent signaling pathway that protects stressed replication fork structure. In addition, we have established multiple components in the pathway, including CaMKK2, AMPK and Exo1, that act downstream of intracellular Ca2+, which is elevated after replication stress. Disruption of the pathway causes excessive fork degradation, chromosomal aberrations and compromised cell viability. Building on this exciting finding and our extensive preliminary results, in this proposal we describe a series of studies to further delineate this novel fork protection pathway, focusing on the key players and molecular mechanisms that mediate Ca2+ induction and pathway activation. In Aim 1, we will define a key ion channel responsible for Ca2+ induction in the replication stress response. Our preliminary results suggest that TRPV2 is a major ion channel for Ca2+ induction upon replication stress. We will characterize the molecular functions of TRPV2 in this fork protection pathway and its regulation by a novel interacting protein in the replication stress response. Aim 2 seeks to define the direct signal for Ca2+ induction after replication stress. Our preliminary results suggest that cytosolic self-DNA generated after replication stress has a previously unrecognized role in triggering Ca2+ induction for fork protection. We plan to explore the sources of cytosolic self-DNA in cells after replication stress. In addition, we will determine whether other cytosolic DNA-inducing genetic conditions also activate the Ca2+-dependent signaling cascade. In Aim 3, we will elucidate the molecular mechanisms of TRPV2 activation for Ca2+ induction after replication stress, focusing on the role of cytosolic DNA sensing and signal transduction. These studies will further establish a novel fork protection signaling pathway and provide critical insights into the interplay between replication stress, cytosolic DNA sensing and Ca2+ signaling. This project will not only expand our understanding of the fundamental mechanisms of genome maintenance, but also lay a conceptual foundation for the development of more effective cancer therapeutics targeting the DNA replication stress response.

NIH Research Projects · FY 2025 · 2012-03

Abstract: This proposal seeks the renewal of the Opportunities in Genomics Research (OGR) Program. Since 2007, the OGR Program has been run out of The McDonnell Genome Institute at Washington University in Saint Louis and funded through the NHGRI's Diversity Action Plan. The mission of the OGR Program is to increase the representation of students from underrepresented groups in genome science or genome science-related PhD and MD/PhD programs. We seek to accomplish our mission through the effective execution and evolution of summer undergraduate and academic year post-baccalaureate research programs for students from racial and ethnic backgrounds underrepresented in the sciences, students from disadvantaged backgrounds, and students with disabilities. Both OGR programs provide trainees with cutting-edge research experiences in genome science or related fields and 11 other educational activities that seek to train them to think critically and to write and speak effectively about their research. Workshops and classes are tailored to train students in the core concepts of genome science, bioinformatics, and scientific presentation. Professional development activities focus on helping students prepare for and excel in graduate school interviews and graduate school itself. Over its three funding cycles, student outcomes for the OGR program reveal great momentum. PhD matriculation of students in our summer program rose from 29% in the first cycle to 50% or above the last two cycles. More impressively, PhD matriculation of students in our post-baccalaureate program rose from 44% in the first cycle to 75% in the second cycle and 90% in the current cycle. We expect the implementation of new and modified activities to solidify and extend these gains. Specifically, new activities will promote integration among OGR trainees and trainees in the NHGRI T32 PhD Genomic Sciences program, enhance training in bioinformatics, provide trainees with greater exposure to the diversity of research careers available to PhD scientists, and initiate community outreach events to under-served area high schools and their students. Based on the past success and current plans of our program, we seek support for five more years of funding to continue our mission of enhancing diversity in PhD programs nation-wide. We propose to expand our post- baccalaureate program to five trainees per year, and with university support, to maintain the size of our summer undergraduate program at eight students per summer. We believe our programs will continue to help realize the great scientific and intellectual potential inherent within the diverse population of the United States, much of which currently lies latent due to the underrepresentation of many sectors of our nation's population within scientific research in general and genome science in particular.

NIH Research Projects · FY 2025 · 2012-02

ABSTRACT Economic choice behavior is specifically disrupted in neurological and mental disorders such as frontotemporal dementia, major depression, and drug addiction. To shed light on these diseases and to pave the way for treatments, it is critical to understand the neural underpinnings of this behavior. The past 20 years witnessed considerable progress. Economic choices are thought to involve two mental stages – subjective values are assigned to the available options and a decision is made by comparing values. Clinical data, lesion studies, functional imaging, and neurophysiology link these mental operations to the orbitofrontal cortex (OFC). In particular, previous work in my lab examined neuronal activity in the OFC of monkeys choosing between different juice flavors. We identified different groups of neurons encoding individual offer values, the binary choice outcome, and the chosen value. These variables capture both the input (offer value) and the output (chosen juice, chosen value) of the choice process, suggesting that the cell groups identified in OFC constitute the building blocks of a decision circuit. A series of empirical and computational results support this proposal. Importantly, current notions come primarily from experiments where subjects made simple binary choices, similar to that between steak and salmon a person might make when dining in a restaurant. However, real life decisions are often more complex. For example, in many circumstances, options are defined not by a single good (e.g., steak vs. salmon) but rather by a good bundle (e.g., steak and potatoes vs. salmon and rice). Furthermore, most restaurants offer many dishes – not just two. That is, choices often involve more than two options. Last but not least, choices depend on the internal state of the subject, which can vary. Thus, the same person dining in the same restaurant might choose different dishes on different nights. The overarching goal of this proposal is to assess how the decision circuit adapts or reconfigures itself to generate choices in different behavioral conditions. Neuronal recordings will focus on OFC. In Aim 1, we will examine choices between bundles. Each bundle will be constituted of 2 juice types (A+B vs. C+D design) that, if chosen, will be delivered sequentially. In Aim 2, we will examine choices between three options varying on three dimensions (juice type, quantity, probability). We will alternate binary and trinary choices and examine differences in neuronal activity. In Aim 3, we will assess how changes in the internal state of the subject shape decisions. We will examine choices under risk, where the internal state is defined by 3 parameters. Theoretical considerations suggest that changes in the relative value of the juices reflect changes in circuit connectivity. To test this and other predictions, we will record from large populations of neurons simultaneously, and we will use network inference analysis to estimate the effective connectivity of the circuit. Independent of the outcome of the experiments, fulfilling these Aims will significantly advance our understanding of the neuronal mechanisms underlying economic decisions. Our historic record and robust preliminary results indicate high likelihood of success.

NIH Research Projects · FY 2025 · 2012-02

PROJECT SUMMARY/ABSTRACT People seek out upper limb rehabilitation services to improve performance of activities in daily life, with performance defined by the World Health Organization ICF model as what someone actually does in the unstructured, free-living environment. Wearable, motion sensors now allow for direct measurement of upper limb performance in daily life, but performance is rarely measured or considered in research and care. Data from our 2nd cycle make it clear that improvements in impairments and/or functional capacity assessed in the structured environment of the clinic or laboratory most often do not translate to improvements in performance of activities in daily life. These data point to the clear need to move assessment of performance from the specialized research realm into more universal use in upper limb research studies and clinical care. A major barrier to more wide-spread use of sensors for routine upper limb performance assessment is lack of clinical validation. An elegant solution to the many clinical validation challenges is to move from single variables in individual populations to multivariate categories of upper limb performance in daily life that span patient populations. Our central hypothesis is that, despite the immense variability in biological conditions and their resulting upper limb impairments and functional capacity profiles, upper limb performance in daily life can be organized into a relatively small number of categories. Defined, validated categories could be used across populations to push upper limb rehabilitation research forward and personalize clinical decision-making. We now propose to expand and validate preliminary categories from stroke into a variety of conditions that send people to upper limb rehabilitation services and result in upper limb disability. Wearable sensors will be used to assess upper limb performance in a heterogeneous, longitudinal, observational cohort (N=340) of persons with (stroke, multiple sclerosis, UL fracture, adhesive capsulitis, breast cancer) and without UL disability (controls), and a retrospective, pediatric cohort (n ≥ 400) to address three aims. Aim 1 will identify and validate categories of upper limb performance in daily life that can be applied across conditions that cause upper limb disability in adults. Aim 2 will evaluate responsiveness to change across categories. And Aim 3 will explore how categories identified in adults map to upper limb performance in childhood. Expected outcomes at the end of the proposed cycle are validated categories of upper limb performance that are responsive to change and span patient populations. These categories (and the software algorithms to derive them) will immediately be available for intervention studies to measure outcomes that are most relevant to patients. With anticipated advances in hardware and software over the next few years, future work will implement validated categories readily into clinical rehabilitation care.

NIH Research Projects · FY 2025 · 2011-09

- Dissemination and Implementation in Diabetes Research Core The primary goal of the Dissemination and Implementation in Diabetes Research Core (DIDR) is to assist Washington University Center for Diabetes Translation Research (WU-CDTR) investigators in filling the gap between discovery of new knowledge generated by their diabetes research and the application of this science to ultimately inform and benefit patient and population health. The DIDR Core has been critical in expanding the cadre of investigators conducting translational research in diabetes and building novel partnerships. Over the past five years, the Core has contributed to the evolving research methods that support high quality dissemination and implementation (D&I) studies including innovative research designs, novel D&I strategies, specialized measures assessing current research and practice related to D&I in diabetes interventions, and methods to understand context and tap preferences. Additionally, D&I methods support research conducted within clinical and community settings that represent real-world practice, which necessitates specialized methods to fit research within the normal operating procedures of practitioners and organizations. The DIDR Core actively supports WU-CDTR investigators by providing expertise and resources in D&I science to advance health. The Core also supports clinical and academic partnerships and those that extend to community contexts and non-healthcare sector organizations with missions that directly address determinants of health and reach populations that can benefit from effective interventions and approaches for diabetes prevention and management. Current DIDR Core services build on this experience in implementing DIDR activities and supporting the large number of investigators pursuing D&I research. Based on this successful history, promising trajectory, and systematic core evaluation, we expect continued growth of DIDR services to support D&I research of WU-CDTR investigators.

NIH Research Projects · FY 2025 · 2011-09

Resident MΦs naturally reside freely floating in peritoneal fluid. To counteract the problem that phagocytosis of cargo is inefficient in fluid, peritoneal MΦs produce Factor V to raise its local levels and facilitate formation of interstitial clots upon induction of inflammation. The clots in turn bring MΦs and microbes out of the fluid phase and into a 3-D environment together. This clotting reaction operates with induced MΦ adhesion onto mesothelial membranes to collectively account for the classical "MΦ disappearance reaction" (MDR). MDR in the peritoneum may also require inflammasome activation, which may then lead to cell death that accounts for an extended period of MΦ disappearance. Indeed, when the stimulus inciting the MDR is robust, Factor V+ resident MΦs disappear for several weeks, likely leaving the peritoneal cavity susceptible to future infectious and noninfectious threats for rather long durations. We will study possible links between death and coagulation and use lineage tracing models to determine how resident MΦs repopulate. Furthermore, we will investigate the idea that the peritoneal cavity is left susceptible to immunological challenges during the long duration of MΦ loss following MDR. A prolonged period of MΦ loss following a robust MDR may leave the body cavity vulnerable to infection. It may also promote progression of other diseases associated with altered immunity, like cancers. In particular, ovarian cancer is associated with the peritoneal cavity, where it often metastasizes and expands. Human ovarian cancers are considered one of the most procoagulant tumors. If human counterparts to the Factor V+ MΦs exist, which we will study herein, such cells might limit tumor expansion clinically, as observed in mice. Overall, we will test the hypothesis that Factor V+ resident peritoneal macrophages protect the peritoneal cavity from microbial pathogens but also against ovarian tumors and that the procoagulant activity of these macrophages, if linked to MDR by a microbial or inflammatory trigger, may set up a state of enhanced susceptibility to tumor growth due to loss of tumor-restricting MΦs.

NIH Research Projects · FY 2025 · 2011-08

PROJECT SUMMARY / ABSTRACT The Transplant and Cellular Therapy Program at Washington University has been affiliated with the BMT CTN since its inception, initially as a part of the Case Western Consortium, and as a Core Clinical Center since 2011. During that time, our institution has made significant contributions to the BMT CTN through clinical trials accrual and trial design input. The specific purpose of applying for this Notice of Funding Opportunity (NOFO) is to continue participation as a BMT CTN Core Clinical Center. The long term goal of the proposed clinical trial is to apply novel concepts in immunology and cellular therapies to improve outcomes and further expand clinical application of adoptive transgenic chimeric antigen receptor (CAR) T-cell therapies. While the use of CAR T-cells have emerged as a groundbreaking advancement in the treatment of many malignant diseases, and has shown a remarkable promise in a variety of non-malignant disorders, cytokine-release syndrome (CRS) remains a major cause of morbidity and a significant barrier to a successful outcome. CRS is a hyper-immune reaction driven by excessive inflammatory cytokine activation and proliferation, with interleukin-6 (IL-6) thought to be one of the most functionally important cytokines during CRS. Although there are agents used for the treatment of established CRS, including IL-6 antagonist tocilizumab, there are no FDA-approved agents for CRS prophylaxis. Duvelisib is an oral, dual PI3K-δ,γ inhibitor that is FDA- approved for the treatment of adults with several hematologic malignancies, with acceptable safety profile. Using a protein kinase inhibitor library researchers have identified PI3K-δ,γ inhibitors, including duvelisib, as most potent inhibitors of IL-6 secretion, without attenuating CAR T-cell function. We and others have performed extensive in vitro and in vivo experiments investigating the effect of duvelisib on CRS cytokines, leading to a phase I dose escalation and dose expansion study of duvelisib for CRS prophylaxis in patients with non-Hodgkin lymphomas undergoing CAR T-cell treatments, open for enrollment at Washington University. Preliminary results from that study show that duvelisib is safe and tolerable, and delays the onset and reduces the severity of CRS. Based on these data, we propose a phase III, multi-site, randomized, placebo-controlled study with the primary objective to assess the efficacy of duvelisib in preventing grade ≥2 CRS in patients with large B-cell lymphoma and mantle cell lymphoma undergoing therapies with axicabtagene ciloleucel, lisocabtagene maraleucel or brexucabtagene autoleucel (Aim 1). The expected outcome of this trial is to provide definitive data on the effectiveness of duvelisib for CRS prophylaxis within the study population, without negatively affecting the efficacy of CAR T-cells. In addition, variety of correlative studies will provide further insight into the biologic impact of PI3K inhibition, paving the way to further research (Aim 2). We hypothesize that PI3K inhibition with duvelisib will provide effective prophylaxis against clinically significant (grade ≥2) CRS.

NIH Research Projects · FY 2026 · 2011-07

Cardiac myocyte protein quality control is critical for maintenance of the contractile apparatus and cardiac function. Mutations in chaperone proteins that affect protein quality control induce proteotoxicity, sarcomere dysfunction and cardiac myocyte cell death. One such point mutation in the CRYAB gene in humans, results in an arginine to glycine change at position 120 in the heat-shock protein beta-5 (R120G HSPB5) protein, and provokes autosomal dominant cardiomyopathy resulting in heart failure, need for cardiac transplantation and premature mortality. Strategies to prevent proteotoxicity have focused on stimulating the autophagy-lysosome pathway to remove protein aggregates; and need to be refined for maximizing therapeutic benefit. Our studies during the current grant-funding period have uncovered an essential role for TRAF2, an innate immunity protein, in mediating basal mitophagy in cardiac myocytes. Our preliminary data show that loss of mitophagy with TRAF2 ablation in cardiac myocytes induces marked protein aggregation in cardiac myocytes, despite a lack of effect on general macro-autophagy. In a mouse model of cardiac myocyte specific human HSPB5 R120G mutant protein expression, we have uncovered evidence for mitochondrial accumulation of HSPB5 protein, along with polyubiquitinated proteins and p62 (an adaptor protein essential for aggregate formation). This is accompanied with markedly increased TRAF2 expression in the mitochondria and reduced mitochondrial protein content, pointing to a role for mitophagy in handling protein aggregate pathology. Given recent observations in yeast and mammalian cell lines indicating that mitochondria may facilitate uptake and degradation of cytosolic protein aggregates, we hypothesize that mitophagy facilitates removal of cytosolic protein aggregates of HSPB5 R120G mutant protein in cardiac myocytes to prevent cardiac myocyte death and cardiomyopathy. In this renewal application, we have generated reagents and developed collaborations to test this hypothesis via the following aims. In aim 1, we will examine if mitophagy is required for removal of protein aggregates in mice. Studies will also be performed in human induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) with CRISPR targeted ablation of TRAF2, and in those with CRISPR-knock-in of R120G mutation. In aim 2, we will examine if TRAF2-induced mitophagy is sufficient to facilitate removal of protein aggregates in mice and in human iPSC-CMs. Aim 3 will focus on mechanistic studies to understand the steps involved in mitochondrial uptake of HSPB5 R120G mutant protein uptake in mitochondria. Studies in mice will focus on impairing p62-mediated protein aggregation to examine the role of p62 in mitochondrial protein aggregate uptake. Experiments in yeast and mouse model systems will examine if hsp104, a yeast disaggregase protein shown to be functional in mammalian cells, is required and sufficient for mitochondrial protein aggregate uptake, respectively. These studies will determine the role of mitophagy in cytosolic protein quality control, and efficacy of therapeutically harnessing this pathway to treat proteotoxic cardiomyopathy.

NIH Research Projects · FY 2025 · 2011-05

Washington University School of Medicine and the affiliated Barnes-Jewish Hospital have a 40-year tradition of excellence in musculoskeletal research, patient care, education and training. In 2011, the Skeletal Disorders Training Program (SDTP) was established under the leadership of Dr. Roberto Civitelli, with the purpose of training the next generation of skeletal investigators, and it was renewed in 2015. This SDTP provides the educational and mentoring infrastructure of the WU Musculoskeletal Research Center, a partnership between the Departments of Medicine (Division of Bone and Mineral Diseases) and Orthopaedic Surgery, supported by a NIAMS-funded P30 award, thus integrating the complementary aims of this T32 and the P30 into a unified structure. The SDTP offers 3 pre- and 3 post-doctoral positions and has so far enrolled 27 trainees (12 graduate students and 15 post-doctoral fellows). Of these, 5 have progressed to faculty appointments, 5 to permanent positions in industry; one has been awarded a K99/R00 grant; 5 have received an F-type award. Together, they have published 104 articles directly linked to this program and received numerous awards and recognitions. This SDTP offers research training in 5 thematic areas, reflecting the focus and common interests of the participating faculty: 1) Musculoskeletal Biomechanics; 2) Skeletal Development and Regeneration; 3) The Skeleton and Other Systems; 4) Skeletal Immunology; 5) Tumor-Skeleton Interactions. Mentors selected for this SDTP are drawn from 9 academic Departments and Programs at WU. The program is based on 4 training domains: 1) mentored research training; 2) curriculum coursework; 3) enrichment activities; 4) career development. The training program builds on the graduate programs administered by the Division of Biology and Biomedical Sciences and the Department of Biomedical Engineering and leverages the Institute for Translational and Clinical Sciences and other institutional resources. Specific innovations proposed include,1) development of non-academic research training opportunities; 2) expansion of training in rigor and reproducibility; 3) addition of a mentor education plan; 4) enhanced participation of previous trainees and creation of an Alumni Network; 5) new efforts in increasing participation of URM to the program; 6) increased emphasis on career development training. These new SDTP trained researchers will make the future discoveries in the genetic and molecular bases of skeletal disorders, and eventually improve the treatment of osteoporosis, inflammatory osteolysis, osteoarthritis, bone metastasis, and other skeletal disorders that afflict a large proportion of the elderly population.

NIH Research Projects · FY 2025 · 2011-01

Project Summary/Abstract A majority of heritable disease-causing variation resides in the non-coding portions of the human genome. A leading hypothesis is that most of this variation exerts its effects on cell-type-specific cis- regulatory sequences (CRSs). Interpreting such variation will therefore require quantitative models of the ‘regulatory grammar’ that controls the cell-type-specific activities of CRSs. We define the regulatory grammar of a cell type to be the independent and interacting contributions of transcription factor binding sites (TFBSs) to cis-regulatory activity. Models of regulatory grammar must also include the dependencies of those contributions on the number, orientation, spacing, and affinity of TFBSs. Detailed models of regulatory grammars are still in their infancy, partly because we lack systematic training data for how CRSs behave across diverse cell types in vivo. We propose to address this gap by systematically measuring the activities of CRSs across cell types within intact mammalian tissues. To collect this data, we will introduce a single-cell massively parallel reporter gene assay (scMPRA) that measures the cell-type-specific activities of CRSs in vivo. We will model the resulting data using a formal thermodynamic model in which each TF-DNA or TF-TF interaction is represented by its free energy (ΔG) of interaction. By comparing the magnitudes of the resulting ΔG values, we will quantify the independent and interacting contributions of specific TFBSs, thus deriving quantitative regulatory grammars that capture the differences between cell types within the mammalian retina (Aim 1) and the mammalian brain (Aim 2). By validating our models on sequence variants of endogenous CRSs, we hope to make progress towards a framework for accurately predicting the effects of non-coding genetic variation on the function of CRSs.

NIH Research Projects · FY 2025 · 2010-09

Project Summary/Abstract This PRIDE renewal application from Washington University proposes to build upon our current SIPID/PRIDE CVD-CGE program which has been successful with continuous funding for the past 16 years. The overarching goal of our program is to enhance the diversity of the biomedical research workforce by providing creative and highly-relevant training activities in focus areas related to cardiovascular disease (CVD). We will accomplish this goal by providing mentoring and research training to junior faculty who come from backgrounds traditionally underrepresented (URM) in the biomedical research workforce. Our program (13 cohorts, 76 mentees over 16 years) has been highly successful: at the 10-year follow-up, our mentees had: a) received 171 grants (2.3 grants/mentee); published 1,573 manuscripts (21 publications/mentee); and c) received academic promotions with 48% having achieved rank of Associate Professor/Full Professor. Through the proposed activities listed below, we plan to recruit 8 mentees each of the first 4 years, to be mentored and trained to develop independent research careers in areas relevant to the mission of NHLBI with primary focus on CVD. We are adding two new focus areas based on feedback from current and former trainees, faculty and mentors. Highlights of this renewal application include: 1) Continue the two current cornerstone focus areas: “CVD epidemiology and comorbidities”, adding “Global Health” under this focus area, and “Genetic Epidemiology”; 2) Add two new focus areas: “Implementation Science” and “Data Science/Bioinformatics”, both of which are of strategic importance to NHLBI and NIH; 3) Enhance our comprehensive mentoring and writing program, with a proven track record for the past 16 years, to provide rigor and depth to scientific writing; 4) Form a new “External Mentoring Pool & Recruiting Faculty Committee” composed entirely of our SIPID/PRIDE Alumni (former mentees) that brings additional depth and mentoring capacity “by URMs for URMs”. For this renewal, we will continue to focus on the development of competitive “Small Research Project (SRP)” grant applications during SI-1, while SI-2 will focus on new NIH proposals. Based on mentee feedback, SI-1 and SI-2 has been revamped into an 8-week program that includes “in person”, “hybrid” and “virtual” segments. This new format allows for more time at home, yet maintains the academic rigor of previous SIs. A 3-day in-person overlap between new and returning cohorts will facilitate networking and peer-mentoring. The year-long mentoring program includes monthly mentoring meetings, a 3-day mid-year meeting, and a 4-day annual meeting in Washington, DC. Finally, we will follow mentees for two years with bi-annual updates of their individual development plans, while also focusing on grants and manuscripts writing and academic advancement, with follow up evaluations for up to 10 years. Primary program goals will continue to be focused on mentee success securing independent grant support, scientific publications and academic promotions.

NIH Research Projects · FY 2025 · 2010-07

Project Summary/Abstract The long-term objective of this project is to develop a safe and broadly protective vaccine for enterotoxigenic E. coli (ETEC), a common cause of diarrhea worldwide. This highly collaborative project will rely on the combined resources, and innovation of investigators with complementary expertise in molecular microbiology and microbial pathogenesis, vaccinology, immunology, and advanced state-of-the-art structural biology to investigate novel targets for ETEC vaccine development. Specifically, the project will: (1) examine whether two key surface proteins of ETEC can provoke immune recall (memory) responses that are thought to be involved in sustained protection against infection (2) develop monoclonal antibodies from circulating immune cells obtained from blood samples following natural infections (3) map where these antibodies bind on the two surface proteins that we plan to use in vaccines (4) determine the overall structure of the proteins that we plan to use in vaccines (5) test the ability of these monoclonal antibodies to neutralize protein function and the ability of the bacteria to deliver toxins (6) vaccinate mice with these proteins to test protection against infection (7) test the activity of antibodies from mice and map where antibodies from mice bind the vaccine proteins relative to the human antibodies. The project will answer fundamental questions about the nature of the two proteins that are relevant not only to ETEC but to other important pathogens that secrete similar proteins. Most importantly the project is expected to provide critical information that can be translated into efficient design of a multi-featured vaccine for ETEC that provides broad-based long-term protection.

NIH Research Projects · FY 2026 · 2010-05

Aging is associated with cognitive decline, which can manifest pathologically as age-related dementia. Aging is also the major risk factor for the development of Alzheimer’s disease. There is currently no effective treatment for age-associated dementia and no therapies for Alzheimer’s disease. Challenges associated with brain disorders include a lack in understanding of the underlying mechanisms and poor accessibility of the brain due to the blood-brain barrier. Over the last decade, we have been studying meningeal spaces and their relevance to brain function. Our long-standing interest in the meninges and its role in brain function has led us to characterize meningeal lymphatic vessels and to observe many intriguing changes in meningeal immunity with age, and accordingly to raise the questions: are meningeal immune and lymphatic alterations relevant to age- related cognitive decline? If so, could meningeal lymphatic vasculature or meningeal immunity be therapeutically targeted to alleviate age-related dementia and/or Alzheimer’s disease? Here, we are proposing to test our overall hypothesis, that age-related impairment in meningeal lymphatic function results in impaired meningeal blood vasculature (presumably through build-up of waste products), collectively leading to abnormal meningeal immunity, which subsequently results in age-associated impairment of cognitive function. We propose that improving meningeal lymphatic function (or directly targeting the immune system and/or its soluble mediators) may be a plausible therapeutic approach for age-associated cognitive decline. Three specific aims were designed to elucidate the role of meningeal vasculature and immunity in age-associated cognitive decline, dementia, and mouse models of Alzheimer’s disease using state-of-the-art imaging, surgical and pharmacological techniques in mice, as well as a validation of select findings in human dura specimens from fresh autopsies. Specific aim #1 will establish whether dysfunction of meningeal lymphatics may exacerbate the aging phenotype of meningeal blood vasculature; specific aim #2 will define the changes occurring in meningeal immune cells upon vascular dysfunction; specific aim #3 is a therapeutic aim wherein several approaches to boost healthy meningeal immunity—and thus improve cognitive function—will be tested. Understanding how meningeal vasculature and immunity are changing with age and in diseases, such as Alzheimer’s disease, has broad implications in numerous neurological conditions associated with aging. Our findings, therefore, have significant potential to uncover the etiology of and identify novel therapeutic targets for age-related dementia and Alzheimer’s disease. !

NIH Research Projects · FY 2025 · 2010-05

PROJECT SUMMARY Borderline Personality Disorder (BPD) is a prominent contributor to disability, burden, and increased mortality. Although impairments in self (RDoC Systems for Social Processes: Perception and understanding of self) and interpersonal (RDoC Systems for Social Processes: Affiliation) functioning originate in early childhood, little is known about the developmental psychopathology of these core features in BPD, as opposed to a related psychiatric disorder that often precedes and co-develops with BPD: Major Depressive Disorder (MDD). The current proposal will establish developmental trajectories of interpersonal and self dysfunction from early childhood into young adulthood, examining interactions with environmental factors and associations with aberrant neural circuitry, to predict onset of BPD in early adulthood. In this renewal, we leverage 17 years of previously collected longitudinal data (R01 MH090786) from 348 young children enriched for emotional dysregulation. Now young adults (19-25 years), 36% exhibit BPD above diagnostic threshold. This sample offers an unparalleled opportunity to understand specific developmental precursors for BPD onset. Rich phenotyping, including over a decade of clinical interviews, narratives, questionnaires, observations, and repeated MRI and EEG assessments make this the ideal and highly cost-effective dataset to investigate impairments in self and interpersonal functioning that lead to BPD versus MDD. New data collection in young adulthood includes multi-method assessments of interpersonal and self functioning alongside psychiatric diagnostic interviews. Our motivating hypothesis is that in the context of emotion dysregulation, peer acceptance and aggression (interpersonal dysfunction) from preschool through middle childhood, interacts with self-functioning (unstable, incoherent self-worth and self- concept) in adolescence to uniquely predict BPD onset in adulthood versus MDD. We will examine: (1) how specific aspects of these constructs prospectively relate to adult BPD, as opposed to continuation of MDD; (2) during which developmental periods these constructs provide the most predictive utility, including the moderating effect of specific environmental factors; and (3) assess the predictive and mechanistic role of neural correlates of these constructs in forecasting BPD versus MDD. Findings will inform the optimal timing and content-focus (i.e., specific neural/behavioral self and interpersonal targets) of novel early-intervention for preventing BPD during the earliest developmental periods. This longitudinal research will be able to identify risk factors for the persistence or worsening of interpersonal and self dysfunction and BPD onset, offering the best starting point toward developing a prevention strategy for BPD.

NIH Research Projects · FY 2026 · 2010-04

ABSTRACT Nearly half of U.S adults have hypertension (HTN), a leading cause of the cardiovascular disease (CVD). HTN that develops earlier in life contributes to the early development of end-organ damage, thereby increasing the risk of cardiovascular mortality compared to later-onset HTN. Multiple studies provide evidence for the hypothesis that environmental factors in utero program patterns of fetal and infant growth that result in increased susceptibility to HTN later in life. Vitamin D (VD) deficiency is highly prevalent during pregnancy and has been linked to an increased risk of HTN during childhood. We previously found that macrophage-specific deletion of the VD receptor during early embryogenesis induced HTN by two mechanisms: a) renal-dependent by stimulating the secretion of miR106b to drive JG cell renin production and b) renal-independent by increased macrophage renin production and secretion. Additional studies in rodent models support the role of maternal VD deficiency in developing HTN and chronic inflammation via epigenetic mechanisms. In this proposal, we present preliminary data indicating for the first time that HTN is transplantable by immune cells. Hematopoietic stem cells (HSCs) from fetuses exposed to VD deficiency in utero can permanently transfer HTN to VD-sufficient adult mice. Vitamin D deficiency epigenetically suppresses Jarid2 expression and activates the Mef2/PGC1α pathway in HSCs, which persists in recipient bone marrow, resulting in macrophage renin and miR-106b secretion, both of which represent novel mechanisms by which immune cells contribute to the development of HTN. In humans, we found that this immune cells program causing HTN is preventable in children monocytes from the VDAART trial by antenatal 4400 IU/day of VD supplementation. Importantly, children from VD-supplemented mothers have decreased brachial-systolic blood pressure (BP). Thus, we hypothesize that VD supplementation early in pregnancy prevents epigenetic suppression of Jarid2 expression to avoid the BP-increasing effects of renin and miR106b secretion from myeloid cells. To evaluate this epigenetic immune program in vivo, we will utilize HSC transplantation models to determine in Aim1 if 1) Jarid2 deletion is sufficient to induce the HTN phenotype observed in the setting of in utero VD deficiency, 2) miR106b or renin deletion attenuates the HTN phenotype associated with in utero VD deficiency and 3) correction of VD deficiency early in pregnancy prevents the development of HTN in offspring. In Aim2, we will assess the role of maternal VD supplementation on VDAART children’s BP and monocyte Jarid2/PGC1α/Renin/miR106b pathway activation and determine if plasma miR106b and monocyte RAS activation are early markers of HTN in children at ages 9-11 from a randomized controlled trial (VDAART) of antenatal treatment with VD 4400 IU/d vs. placebo. This proposal will provide evidence for maternal early VD screening and treatment to decrease the incidence of childhood HTN and aim to establish miR106b as a specific biomarker identifying children at risk for CVD.

NIH Research Projects · FY 2025 · 2009-09

ABSTRACT Mammals use pheromones to find mates, regulate reproduction, and organize social behavior. The identification of new pheromones, purified from natural sources, has repeatedly triggered new discoveries about the genes, cells, and circuits essential for social behavior. Recently, my lab identified two novel pheromones present in male mouse urine. These pheromones play an essential role in at least one behavior, the acceleration of puberty in juvenile females. However, several features of these new pheromones, and the responses of the neurons that detect them, suggest an even wider role. Structural clues present in these new compounds hint at a previously-unsuspected mechanism for their detection involving multiple sensory systems, receptor genes, circuit mechanisms, and metabolic pathways. We propose to (1) determine whether a multisensory complex involving both volatile and nonvolatile cues coordinate the discovery, detection, and recognition of these vital cues, (2) identify the receptor genes involved in their detection and consequent behaviors, and (3) test whether stereotyped circuit features contribute to the production and plasticity of suitable behavioral responses. We believe that the new opportunities afforded by these novel pheromones will provide new tools to probe fundamental mechanisms regulating mammalian physiology and behavior.

NIH Research Projects · FY 2025 · 2009-09

Abstract In response to PAR-20-080, NIMH Mentoring Networks for Mental Health Research Education, this application seeks five years of support to respond to training demand, continue scientific leadership to the field, and empower a new generation of leaders in implementation science for mental health services research. This network will extend in creative new ways the Implementation Research Institute (IRI, NIMH R25 funding 2009- 2015, 2016-2019), which has successfully trained 87 new mental health implementation researchers. Implementation science is critical for mental health where most Americans with mental disorder receive sub- optimal care due in large part to challenges in the implementation, sustainability, and scale-up of evidence- treatments. Training demand far outweighs supply but only a handful of programs train implementation researchers and only the IRI in mental health. Therefore we seek a five-year renewal of IRI to pursue three aims: (1) to continue a vibrant mentoring network of Faculty and Fellows who will advance the science and lead the workforce in mental health implementation research; (2) to accelerate training benefits and enhance IRI sustainment by elevating new mentoring leadership in implementation science, and (3) to evaluate training outcomes. We will achieve aim 1 by (a) conducting an innovative, continually renewed annual summer institute; (b) fostering networked collaboration and mentoring from faculty to fellow and peer to peer among IRI fellows; (c) supporting fellows’ travel to externally funded implementation research sites where they will learn from NIH Principal Investigators, thereby expanding their networks; and (d) equipping IRI fellows, via the Translational Science Benefit Model (TSBM), to map pathways for the public health impact of the MHSR implementation science in response to aim four of NIMH’s strategic plan. We will achieve aim 2 by using evidence-based mentoring and leadership development strategies to train an Associate Director designee and new core faculty designees, pairing them with seasoned core faculty and advancing them to mentoring leadership positions in implementation science. We will achieve aim 3 by using evaluation methods that include TSBM case studies, bibliometrics, social network analysis, and competency assessment to evaluate outcomes that include mentoring effectiveness, collaboration, translational benefits, and implementation science to scholarly products.

NIH Research Projects · FY 2026 · 2009-09

Abstract The success of lung transplantation is limited by ischemia reperfusion injury-mediated primary graft dysfunction and allograft rejection, two processes that we have shown are immunologically linked. Current strategies to reduce graft rejection and improve survival are mostly based on targeting adaptive immune cell populations in the recipient. These approaches are only modestly effective and carry high risks of life-threatening infections and development of malignancies. We have shown that neutrophilic graft infiltration during ischemia reperfusion injury is a major driver major driver of alloreactivity. Therefore, a complementary approach to prevent rejection is to target innate immune pathways that are activated during ischemia and reperfusion. The ability to dampen the initial immune response following lung transplantation represents a promising avenue to increase allograft tolerance and improve clinical outcomes. Our recent work has identified that necroptosis, a non-apoptotic form of inflammatory cell death mediates the early inflammatory response after reperfusion of pulmonary grafts. Recent work has suggested that the removal of apoptotic neutrophils by macrophages, a process referred to as efferocytosis, induces synthesis of specialized pro-resolving mediators pointing to mechanistic targets to prevent ischemia reperfusion injury. Also, we have generated new data that selectively inducing metabolic stress in neutrophil apoptosis triggers innate immune responses that limit ischemia reperfusion injury. We have additionally reported that treatment with Resolvin D1, an endogenous pro-resolving lipid mediator, inhibits neutrophil recruitment, downregulates inflammatory pathways and reduces ischemia reperfusion injury. In this proposal, we will use state-of-the-art techniques, including intravital microscopy, single cell RNA sequencing, oxidative lipidomics, multi-omics imaging mass spectrometry and novel mouse strains to perform studies that will 1) define mechanisms and impact of cell death (Aim 1), 2) define mechanisms that contribute to the endogenous production of specialized proresolving lipid mediators after lung transplantation (Aim 2) and 3) define mechanisms of action and immunological impact of specialized pro-resolving mediators (Aim 3) in driving innate inflammatory and alloimmune responses after lung transplantation. Our studies will lay the foundation for novel therapy that will improve outcomes after pulmonary transplantation.

NIH Research Projects · FY 2025 · 2009-09

Project Summary This proposal aims to continue the development of XNAT. XNAT is an imaging informatics platform designed to facilitate common management and productivity tasks for imaging and associated data. We will develop the next generation of XNAT technology to support the ongoing evolution of imaging research. Development will focus on modernizing and expanding the current system. In Aim 1, we will implement platform components, in including a new archive file management system and database interface, a streamlined image annotation interface, a module to support integrations with cloud applications, and a set of software development kits (SDKs) to support development of tools that interact with XNAT data and services. In Aim 2, we will implement two innovative new capabilities that build on the services developed in Aim 1. The XNAT Workspaces module will link Jupyter Notebooks with XNAT, creating a computing environment that enables users to mount specified data sets in their Notebooks, share and clone Notebooks, save Notebooks to version control systems, and publish them as static dashboard. XNAT Workspaces will be linked to XNAT Container Service and high-performance computing and cloud computing resources to enable scalable computation. The XNAT Machine Learning (ML) Studio will provide an integrated application for data science teams to build and annotate data sets, train and validate ML models, and publish their models as deployable applications. ML Studio will implement quality control, logging, and provenance tracking to help users avoid the pitfalls that often plague machine learning efforts. For both Aim 1 and 2, all capabilities will be developed and evaluated in the context of real world scientific programs that are actively using the XNAT platform. In Aim 3, we will provide extensive support to the XNAT community, including developer workshops and hackathons, online documentation, discussion forums, and the XNAT Academy training platform. These activities will be targeted at both XNAT users and developers. Together these aims represent a major advance in imaging informatics that is unique in the field and will greatly benefit an extraordinarily broad research portfolio.

NIH Research Projects · FY 2025 · 2009-08

Project Summary: This grant will develop speckle contrast optical tomography (SCOT) for imaging of cerebral blood flow dynamics in humans. Real-time maps of cerebral blood flow (CBF) at the bedside are a long sought-after assay for neuro-critical care. Most critically, regional CBF measures can indicate which brain regions may be becoming ischemic and are at danger for hypoxic-ischemic injury. Current clinical methods for imaging CBF include positron emission tomography (PET) and arterial spin labeling with Magnetic Resonance Imaging (ASL- MRI). Both modalities provide only snapshots of CBF, though, thus they miss dynamic events, which are important in many clinical scenarios including acute stroke, traumatic brain injury, and preterm birth. Non-invasive optical techniques are an attractive approach for imaging brain blood dynamics at the bedside. The most widely used non-invasive optical neuroimaging technique uses near infrared spectroscopy (NIRS) to measure fluctuations in hemoglobin concentrations. Over the last decade high-density diffuse optical tomography (HD-DOT) systems have made significant advances in resolution and brain specificity making it an increasingly viable surrogate for functional MRI. However, fNIRS and HD-DOT do not measure flow directly. Separately, there have been considerable advances in laser speckle methods for measuring CBF. The most established deep tissue speckle method, diffuse correlation spectroscopy (DCS), has been developed for in vivo tomographic imaging in rat models of focal ischemia and for monitoring brain perfusion in humans. However, in humans DCS has been limited to few spatial measurements, which precludes tomographic imaging, and by instrumentation that permits only very low signal-to-noise ratio (SNR) measurements. This proposal will address these limitations and develop speckle contrast optical tomography (SCOT), a new method for transcranial optical imaging of relative CBF in humans. SCOT allows for the use of lower speed detectors that could readily be implemented in parallel in modern scientific CMOS cameras. Aim 1 will develop SCOT instrumentation with high-density imaging arrays. Aim 2 will develop anatomically derived head models for reconstructing SCOT images. Aim 3 will validate SCOT-based measures in patients with carotid artery occlusions based on comparison to PET and ASL-MRI. Aim 4 will evaluate the longitudinal feasibility of using SCOT for imaging acute stroke. These studies will represent the culmination of several advances in functional neuroimaging made possible by the research teams’ unique combination of clinical and technical expertise across the domains of neurology, functional neuroimaging and biomedical optics.

NIH Research Projects · FY 2026 · 2009-03

PROJECT SUMMARY/ABSTRACT Ischemia/reperfusion (I/R) injury in the liver occurs during liver resection and transplantation. Steatotic livers are highly susceptible to I/R injury than lean livers. No therapeutic strategies are currently available to attenuate I/R injury in the patients with steatotic livers. mitoNEET (mNT) is an evolutionarily conserved mitochondrial outer membrane protein containing a unique 2Fe-2S cluster that is tetrahedrally coordinated by 3 Cysteine and 1 Histidine. This small mitochondrial protein regulates redox, bioenergetics, iron homeostasis and autophagy, all of which are indispensable in liver survival after I/R. It is unknown how hepatic mNT is affected by steatosis and I/R. In this application, we will test the hypothesis that the accumulation of nonfunctional mNT sensitizes steatotic livers to I/R injury. Here we have the three independent but complementary Aims. In Aim 1, we will use mouse hepatocytes to investigate molecular mechanisms behind heightened I/R injury in steatotic livers. In Aim 2, we will extend in vitro findings from isolated mouse hepatocytes to an in vivo model of mouse hepatic I/R. In Aim 3, using discarded human livers, we will investigate molecular mechanisms and test the efficacy and safety of potential therapeutic strategies.

NIH Research Projects · FY 2026 · 2009-01

The overarching goal of this R01 is to explore the hypothesis that a prion-like mechanism of proteopathic spread occurs in inclusion body myopathy (IBM). IBM patient muscle has two distinct pathologies, 1) protein inclusions containing aggregated proteins such as TDP-43 and desmin and 2) rimmed vacuoles consisting of autophagosomes, lysosomes and endosomes. How these two pathologies are pathomechanistically connected is unclear. We suggest that aggregated proteins such as TDP-43 and desmin enter the endolysosomal system damaging the membrane leading to a failure in lysosomal degradation and signaling pathways resulting in muscle degeneration. Our aims will test 1) confirm that muscle derived TDP-43 proteopathic seeds can induce TDP-43 aggregation in recipient myofibers. 2) Demonstrate that proteopathic seeds can induce lysosomal membrane permeabilization resulting in autophagic and lysosomal dysfunction 3) Evaluate myofiber to myofiber propagation of proteopathic aggregates. Upon completion of these aims, we will have defined a unique mechanism of IBM pathogenesis and identified a novel point of therapeutic intervention by blocking the spread of protein aggregates.

NIH Research Projects · FY 2026 · 2008-09

In order to accelerate observational clinical studies and trials and to identify appropriate patients for treatments, highly accurate and accessible diagnostic tests for Alzheimer's disease (AD) are needed. Clinical assessments alone are insufficient for accurate diagnoses, and the current gold standard tests for AD (CSF and PET) are costly, limited to specialized medical centers, and perceived to be invasive. In our ongoing prospective Study to Evaluate Amyloid in Blood and Imaging Related to Dementia (SEABIRD), we discovered that decreased blood plasma Aβ42/40 ratio is an accurate biomarker of amyloid plaques and confirmed this in a community-dwelling population, also demonstrating that blood tests are feasible to screen this population. We have discovered and reported the most accurate Aβ42/40 and phosphorylated-tau blood biomarkers (%ptau217, %ptau205, and %ptau181) and discovered novel forms of neurofilament light (NfL) that promise to quantify the major domains of AD pathophysiology. With demonstration that removal of amyloid plaques leads to disease-modifying treatment clinical benefits, access to antiamyloid therapies will depend on accurate diagnosis, and blood biomarkers will be critical tools for doctors and patients. However, there are multiple questions that need to be addressed to inform about the biology, use, and indications of these rapidly developing and utilized bloodbased biomarkers. These questions include: `What are the diagnostic and prognostic properties of blood Aβ, tau, and neurodegeneration (A/T/N) biomarkers to measure amyloid and tau pathology, clinical cognitive decline, and diagnosis in a real-world clinical patient population?', and `Which demographic, clinical, or genetic factors influence the relationship of blood A/T/N biomarkers to the gold standards of clinical cognitive decline, dementia, and PET tests of pathology?' Our proposed study, called SUNBIRD (Study to Understand Novel Biomarkers in Researching Dementia), will enroll 1000 new participants from primary and specialty care clinics and follow the existing ~1000 SEABIRD participants. We will incorporate high performance bloodbased biomarkers tracking amyloid, tau and neurodegeneration and assess their relationship with tau PET, amyloid PET, and other clinical AD tests. We will follow these cohorts longitudinally using blood samples, EHR data, and in-person and remote cognitive testing. We aim to understand how blood-based biomarkers can identify those with clinical symptoms due to AD pathology, determine if blood-based biomarkers can be utilized in lieu of PET or CSF measures, and also diagnose and stage patients to enable treatment and prevention in the clinic. The proposed work builds on the prior pioneering approach that discovered and validated blood amyloid biomarkers, discovered novel tau phosphorylation sites. These studies will lay the groundwork for rapid deployment to accelerate enrollment of AD trials, clinical diagnosis, and early and accurate diagnosis for the whole population.

NIH Research Projects · FY 2025 · 2008-09

Project Summary (DIAN Supplement) This application is for bridge funding until a resubmission can be reviewed and funded maintaining fundamental Core operations of the Dominantly Inherited Alzheimer Network (DIAN). The eight Cores of DIAN are: Administration, Clinical, Biostatistics, Neuropathology, Biomarker, Genetics, Imaging, and Cognition. The renewal application’s first year was budgeted for $13.1 million (total), and this supplement application reduces the year’s funding by $4.2 million to $8.9 million (total) to maintain the essential operations of the study which without would halt the availability of longitudinal data and resources to the scientific community or even dissolve the entirety of the program. Bridge funding would allow for 16 sites supported through DIAN to maintain study visits for its current active population of over 300 participants and provide the Coordinating Center the ability to continue managing the study; support performances sites; ensure validation and integrity to data collection, collect, manage, and handle data/tissue; and disseminate findings and study resources to the research and medical communities. To reduce funding support, this supplement halts all scientific project activities, limits travel costs to Coordinating Center Core members, delays home health nurse visits for non-U.S. sites in collecting assessments and specimens, and removes in- person family conference meeting support to the DIAN Expanded Registry (DIAN EXR). Additionally, the ongoing DIAN-Trials Unit (DIAN-TU) Amyloid Removal Trial (ART), which has found the first clinical signs of prevention of Alzheimer's disease in the clinic is dependent on this DIAN observational study continuing. If gap funding is not provided, this 10 year seminal trial will be forced to stop short of demonstrating how to prevent Alzheimer's disease. Since its inception, the DIAN has led major scientific advances in the understanding of Alzheimer’s disease (AD) stages, cerebrospinal fluid and plasma biomarkers, mechanistic links to therapeutic targets, and enabled ground-breaking prevention and interventional trials. DIAN has helped define the sequence, timing, and magnitude of longitudinal AD biomarker changes decades before symptoms begin. This work directly led to the development and implementation of primary and secondary prevention trials for autosomal dominant AD (ADAD) and the validation of the amyloid-tau-neurodegeneration (ATN) criteria. In this current funding cycle, the DIAN updated the longitudinal progression from biochemical Aβ imbalances, through sequential tau phosphorylations, to the emergence of tau pathology with symptom onset. This tau staging signature of AD can now be tracked with specific biomarkers and was confirmed in “sporadic” late onset AD (LOAD).

NIH Research Projects · FY 2026 · 2007-09

Project Summary Flaviviruses are enveloped, single-stranded RNA viruses that cause epidemics of debilitating human disease on a global scale. The four serotypes of dengue virus (DENV) cause up to ~100 million infections per year including life-threatening shock syndrome. Zika virus (ZIKV) is linked to microcephaly and congenital malformations. Tick- borne flaviviruses (TBFV) are emergent threats that cause encephalitis, long-term neurological sequelae, and death. No specific therapy is available for any flavivirus. Flaviviruses adopt ensembles of structures in which certain antibody epitopes are exposed or hidden. Elucidating how antibodies recognize different structural ensembles of flavivirus virions can enhance our understanding of their functional properties including neutralizing activity. In this competitive renewal application, our long-standing collaborative team (Diamond, Kuhn, Fremont, Crowe, and Pierson) proposes to continue studying structural mechanisms of antibody recognition and neutralization of flaviviruses. We will define the functional properties of protective antibodies that neutralize DENV and ZIKV in a conformationally-restricted manner. We will use these antibodies to probe the spectrum of structures sampled by flaviviruses and how this influences epitope exposure. We also will perform new campaigns to identify neutralizing antibodies against ZIKV and the distantly-related tick-borne flaviviruses that recognize quaternary epitopes. We will use this information to engineer ZIKV subviral particles (prM-E; SVPs) that optimally enable binding of neutralizing antibodies including those quaternary epitopes spanning dimer rafts. Genetically modified ZIKV SVPs will be tested for improved immunogenicity and protection in mice. Overall, our experiments will provide new insight into the dynamic states of flaviviruses and the nature of accessible neutralizing antibody epitopes. These studies may facilitate the generation of novel sculpted antigens and immunogens with improved capacity to detect and elicit specific protective antibody responses against DENV, ZIKV, and likely other emerging flaviviruses.