Washington University

NIH Research Projects · FY 2026 · 2015-05

PROJECT SUMMARY / ABSTRACT Lung transplantation, the only available therapy for many patients who suffer from end-stage pulmonary failure, continues to be plagued by disappointing long-term survival rates. Our research suggests that the premature demise of many transplanted lungs is in large part due to the use of immunosuppression that is not tailored towards the unique immunological characteristics of this organ. Our work will identify cellular and molecular pathways that can be targeted to prevent rejection and promote tolerance after lung transplantation. To accomplish this goal, we will utilize clinically relevant mouse models of lung transplantation, relevant conditional knockout strains and intravital imaging platforms to examine how local and peripheral immune pathways protect lung grafts from destruction by recipient immune cells. Our three projects are entitled “Local and peripheral mechanisms of T cell-mediated immune regulation after lung transplantation” (Project 1), “Early and late inflammatory events controlling lung allograft homeostasis” (Project 2) and “Targeting and imaging of inflammatory circuits in CLAD” (Project 3). The program project will be supported by a Microsurgery Core and an Administrative Core. Our proposed experiments will yield new information that will result in the development of lung-specific therapies that will improve outcomes for pulmonary transplant recipients.

NIH Research Projects · FY 2025 · 2015-04

PROJECT SUMMARY The overall goal of this proposal is to develop high-throughput mass spectrometry technologies applied to the study of post-transcriptional RNA modifications (PTrMs) and associations with RNA binding proteins (RBPs). Efficient RNA processing and protein translation requires interactions with numerous RNA binding proteins, and can be regulated by chemical RNA modifications. PTrMs have been implicated in such diverse processes as RNA splicing, nuclear export, stability, and translation. The mechanisms by which PTrMs control RNA fate has opened up a new field dubbed “Epitranscriptomics”. Although there are antibody approaches to detect some modifications, there is a need for orthogonal approaches for unbiased identification and analysis of PTrMs. Since small DNA viruses that replicate in the nucleus have both to employ cellular machinery to transcribe and translate their gene products, and also develop ways to counteract host defenses, these viruses harness and manipulate cellular RNA processing pathways. Virus infections thus provide elegant biological models to decipher how RNA transcription and its chemical modifications can be regulated and exploited to direct the host cell machinery towards production of viral progeny. Based on preliminary data generated by our collaborative team, our objectives in this proposal are to employ Adenovirus as a model system to study PTrMs on non-coding and messenger viral RNAs and how they are exploited to counter host defenses and promote efficient viral RNA processing and progeny production. Adenoviruses are large non-eveloped viruses and include over 50 distinct strains which elicit a wide range of effects in humans, from respiratory infections to life-threatening organ problems in people with weakened immune systems. Adenoviruses hijack the host cell machinery to express viral genes, and this is achieved by overtaking RNA-mediated processes. Our preliminary data show how Adenovirus infection exploits the m6A modification on RNA to promote splicing and also alters RNA-protein interactions. Here we will develop improved mass spectrometry (MS) technologies to quantitatively and comprehensively detect RNA modifications and RNA-protein interactions over a detailed time-course of infection. Our MS technology will be paired with cellular and genetic assays to determine how the modifications are utilized by Adenovirus to promote growth and counter host defenses in ways that culminate in human disease. These high-throughput unbiased MS-based technologies and approaches will be broadly applicable to enable new biology in virus-host interactions and epitranscriptomics.

NIH Research Projects · FY 2025 · 2015-04

PROJECT ABSTRACT / SUMMARY Hepatic stellate cells (HSC) are fibroblasts of the liver that are normally localized in the perisinusoidal space of the lobule. In response to liver injury, secreted factors from hepatocytes, macrophages, and other cells trigger a program of activation in HSC. Activated HSC proliferate and migrate to sites of injury and begin secreting components of the extracellular matrix including collagens and other extracellular proteins that make up fibrotic lesions. While HSC activation is important for maintaining liver structural homeostasis and injury repair, the chronic, unrestrained activation of HSC can lead to cirrhosis and liver failure. Prior research conducted in vitro has suggested that high rates of metabolic flux are required to support proliferation, migration, and production and secretion of extracellular matrix proteins by HSC. Glucose uptake and utilization are robustly increased in HSC by activating stimuli. Work conducted during the prior period of support demonstrated that inhibition of the mitochondrial pyruvate carrier (MPC), which prevented mitochondrial metabolism of pyruvate, an end product of glycolysis, diminished HSC activation in vitro and in vivo. Glutamine is another important metabolic substrate in HSC and serves not only as a TCA cycle input through glutaminolysis, but also as a precursor for proline and other amino acids that are enriched in collagen. Our preliminary data demonstrate that inhibition of either the MPC or glutaminolysis is sufficient to attenuate stellate cell activation. Since inhibitors of the MPC and glutaminolysis are in clinical development, these findings suggest a viable way to target HSC activation by metabolic modulation. However, a mechanistic understanding of how modifying mitochondrial metabolism inhibits HSC activation is still lacking. The goal of this application is to understand the metabolic and molecular mechanisms by which inhibiting mitochondrial pyruvate and glutamine metabolism suppress HSC activation. We have hypothesized that the hypoxia inducible factor 1α (HIF1α) transcription factor is regulated by mitochondrial metabolism and plays a key role in HSC activation in metabolic dysfunction-associated steatotic liver disease (MASLD). The proposed work will dissect the mechanisms by which mitochondrial metabolism regulates HIF1α stability and examine whether HIF1α inactivation in HSC abrogates the development of fibrosis in mouse models of MASLD. In addition, we will examine how modulating mitochondrial metabolism influences the interactions between HSC and other parenchymal and non-parenchymal cells of the liver and verify some of our findings in human subjects with MASLD. These studies will markedly advance our understanding of HSC metabolism in vivo, provide a wealth of new information for the field, and potentially identify metabolic vulnerabilities as novel therapeutic targets.

NIH Research Projects · FY 2025 · 2015-04

This application would renew support for the BP-ENDURE program in St. Louis to train undergraduate students in the neurosciences. The objective of the grant is to provide rigorous and critical training in neuroscience to a cohort of students from three partner institutions: Washington University, the University of Missouri-St. Louis and Harris-Stowe State University. By supporting 8 new students per year over two years of research, education, and networking experiences, this proposal will establish a Pipeline to graduate school. The Pipeline emphasizes sustained training in oral and written science communication, discovery science and outreach experience. We seek to be a Program that responds to changes in the research environment by helping our students to pursue important and innovative problems and concepts, to adopt new techniques and to communicate effectively with their peers and the general public. The training will create a community of young neuroscientists through three interactive and immersive courses, full-time summer and part-time academic year research in a neuroscience lab matched to the student’s interests and background, and presentations at conferences. The Pipeline provides intensive workshops for mentors. The curriculum and research environments will remain broad and deep, combining expertise in molecular, cellular and systems-level approaches to the study of neural function and dysfunction. Major new initiatives aimed at accomplishing these goals include: 1) creation of a new course at our Partner Institutions to develop the network of future researchers, 2) the introduction of ENDURE-ing Synapses, a course to bolster neuroscience fundamentals, literature reading and presentation skills and a sense of community among the students, 3) mentoring experiences for undergraduates in the Society for Neuroscience Brain Bee as part of their training in science communication, and 4) refinement of a near peer-mentoring program that has graduate students working with undergraduates and undergraduates working with high school students. These initiatives will ensure our students remain at the forefront of developments in neuroscience research, teaching and outreach.

NIH Research Projects · FY 2024 · 2015-02

PROJECT SUMMARY/ABSTRACT This is a renewal application for a high successful program designed to attract and train both predoctoral and postdoctoral investigators in basic investigation of childhood diseases related to cardiovascular and pulmonary biology. The need for a training program in the cellular and molecular basis of cardiovascular and pulmonary disease, especially of physician scientists interested in childhood diseases, is based upon clear evidence of a declining national pool of such individuals at both the faculty and trainee levels. Currently, few pediatric cardiology or pulmonary fellowship programs in American medical centers provide adequate training in this area of research, either at the bench or in structured clinical investigation. Our program is designed to address this need from two important perspectives: 1) To allow pediatric and other clinical fellows with an interest in basic research to develop research competence and career training; 2) To attract and train talented basic scientists at the pre- and post-doctoral levels to study mechanisms related to cardiorespiratory disease in childhood. Our program has been shaped by feedback from reviewers of the previous program, by advice from our faculty and program advisors, by past trainees, and by the inclusion of new faculty and core programs at Washington University that allow us to expand the training experience of our trainees. In addition to continuing our strong training in developmental/cell biology and the molecular basis of disease, the proposed program will take advantage of institutional strengths in genetics, stem cell biology, genomics, and bioinformatics—all of which are components of the foundation of modern research. In addition, we have a newly incorporated focus on infection of the cardiorespiratory system, a leading cause of pediatric morbidity and mortality worldwide. We have maintained a training curriculum for 4 postdoctoral and 2 predoctoral fellows with an uninterrupted 2-3 year block of full-time investigation. This focus is enhanced by a multidisciplinary core of didactic seminars, journal clubs and formal coursework that are designed for each trainee through consultation with the mentor/program administrators to provide individualized education in key aspects of cardiopulmonary development and disease. Our objectives are: 1) to provide extensive mentoring to the trainee for career development and for developing independent research plans that focus on pediatric diseases; 2) to promote training in basic cell and molecular biology, genomics, and bioinformatics together with clinical translational research; and 3) to guide our trainees’ development so that, after the completion of training, successful competition for independent funding is likely.

NIH Research Projects · FY 2025 · 2014-09

Prevention and control programs, guided by a rigorous evidence base, can accelerate progress toward reducing cancer incidence and mortality in the United States. The number of individuals affected by cancer continues to grow as the U.S. population ages. Reducing the impact of cancer requires complex, cross-disciplinary, and rigorous team science approaches. Accordingly, we need to grow a cancer prevention and control research workforce who are adept at team science. Our ongoing Cancer Prevention and Control T32 aims to train postdoctoral scholars and expand pipeline of prevention and control investigators. Housed in the Division of Public Health Sciences in the Washington University School of Medicine, and Siteman Cancer Center, we intentionally create a community of trainees from a range of public health disciplines, including behavioral sciences and epidemiology. Program Mentors have robust cancer research programs, and represent multiple departments. Our training program includes structured elements such as individual development plans, mentored research, and training in cancer prevention/control. Trainees customize other didactic training to meet individual needs and goals. Our innovative approach includes a cross-disciplinary cancer control journal club and career development seminars. Over the course of our first two cycles of funding, we demonstrated success in recruiting, retaining, and training successful researchers. Of 18 completed trainees, 15 remain in research related positions, 2 are in further training, and 1 is delivering clinical cancer care. Several trainees now have K awards (1 K99-R00, 3 K01s) and other early career or pilot awards. They are well on their way to R01 funding. Our process includes evaluation and adaptation of the program. With this renewal, we will enhance training in rigor and reproducibility, team science, and create pathways to early leadership for our trainees. We formalized mentor training requirements. We will continue to leverage institutional resources, ongoing NIH-funded research, and a close collaboration with the NCI-designated Siteman Cancer Center to sustain and improve our training and impact. Washington University School of Medicine and Siteman Cancer Center offer a rich environment for trainees, and our cross-disciplinary training in cancer prevention and control is a unique resource. This renewal (Years 11-15) allows us to continue to train PhD and MD scientists in rigorous cancer research and train the next generation of prevention and control researchers.

NIH Research Projects · FY 2026 · 2014-09

PROJECT SUMMARY/ABSTRACT Background: Some groups are particularly susceptible to Heart, Lung, Blood, and Sleep (HLBS) disorders which persist over time, even in the face of notable improvements in morbidity and mortality rates overall. The NIH is committed to recruiting and retaining a workforce with the potential to contribute new ideas and innovative solutions to help reduce these disorders across various groups. Objectives: The current program consists of 9 active Summer Institute (SI) research education training programs with the general goal of providing research experiences, skills development and mentoring for early career biomedical researchers. The CC will facilitate the coordination of education and evaluation activities among the SI programs by facilitating: 1) the coordination of program-wide activities (e.g., organization, outreach and recruitment, and candidate screening); 2) the education and support through implementing the small research projects (SRP) and distributions of resources, tools and opportunities; and 3) the development and implementation of an evaluation plan that integrates data from previous cohorts. The evaluation protocol will assess key outcomes, collect and track outcomes across time, and benchmark these outcomes against a comparison group of untrained faculties matched to the program participants. Significance and Innovation: Since this is a competing renewal, the infrastructure and organization already are operational, although we are prepared to make timely and efficient changes as needed. There is continuity, with the contact PI having served in this capacity since the beginning of the project (over 17 years). Finally, knowing whether the training has been successful depends on having relevant comparisons. Our application specifically addresses this issue by adding an MPI who is trained in bioinformatics methods and has access to unique electronic training education records for discovery and recruiting an appropriate comparison sample. Methodology: This project is built around our web-based infrastructure that allows Public and Secure access to program information, including our on-line data entry system, which will be upgraded and further automated during the funding cycle. Summary: Our team has unique expertise and experience to continue as the CC for the program and evaluate those indicators of success outlined in this proposal. Further, we are uniquely positioned to assess the impact of this program by benchmarking our results against a matched comparison group and look forward to tracking the career development of these bright and motivated investigators as they lead us into the future.

NIH Research Projects · FY 2025 · 2014-09

PROJECT SUMMARY Up to 50% of patients with locally advanced cervical cancer treated with the current standard of care will fail this treatment, and there is currently no cure for recurrent or metastatic disease. As a result of our recently completed studies of the connection between cervical cancer metabolism and radiation resistance, we have found that cervical cancer is highly dependent upon glutamine. Recent data has also demonstrated that targeting glutamine metabolism not only limits tumor cell growth, but also improves anti-tumor immunity by reprogramming macrophages in the tumor microenvironment (TME) towards a more pro-inflammatory phenotype. Given that radiation therapy (RT) has also been shown to promote anti-tumor immunity, these findings suggest that the combination of targeting glutamine metabolism and RT may synergize to enhance anti-tumor immunity and achieve long term tumor control. The purpose of this renewal R01 application is to perform preclinical mechanistic studies and a corresponding investigator-initiated clinical trial to support targeting glutamine metabolism with radiation therapy as a novel therapeutic strategy for radiation-resistant cervical cancers. Our working hypothesis is that inhibition of glutamine metabolism enhances radiation sensitivity through synergistic metabolic effects on tumor cells and immune cells within the TME. In Specific Aim 1, we will test whether the combination of the glutaminase inhibitor, CB-839, and chemoradiation improves anti-tumor immune responses using paired human tumor specimens collected in the context of an investigator initiated Phase I/II clinical trial. In Specific Aim 2, using 2D and 3D co-culture systems, we will determine whether the cytotoxic effects of inhibition of glutamine metabolism are mediated primarily through metabolic effects on tumor cells versus the combined effects on tumor cells and macrophages. In Specific Aim 3, using a patient derived xenograft (PDX) library and a novel genetically engineered mouse model (GEMM), we will determine whether the radiation modifying properties of CB-839 are dependent upon metabolic editing of the tumor microenvironment, and test new therapy combinations that will support future clinical trials. This work will generate a mechanistic rationale and test predictive biomarkers for the inhibition of glutamine metabolism and RT, and in so doing complete the first-in-human trial of CB-839 + chemoradiation in cervical cancer. This clinical trial is unique in that it includes an investigational new drug, CB-839, administered with both conventionally fractionated external beam RT as well as high dose rate hypofractionated brachytherapy. This design will provide valuable data in humans regarding the effects of RT dose and fractionation on chemoradiation and CB-839 associated changes in the TME.

NIH Research Projects · FY 2025 · 2014-09

Project Summary/Abstract Programmed axon degeneration (AxD), a.k.a. Wallerian degeneration, is a genetically encoded cellular program akin to apoptosis by which neurons effect the orderly demolition of diseased or damaged axons. Our prior work demonstrated that the chief executioner of AxD, SARM1, is an NAD+ hydrolase allosterically regulated by NAD+ and its precursor NMN. SARM1 activation causes depletion of NAD+ that leads to local metabolic catastrophe and axon dissolution. SARM1-dependent AxD was primarily elucidated in models of acute injury that trigger all-or-none rapid axon loss, but we and others have also identified diverse conditions that provoke chronic SARM1 activation below the threshold necessary to provoke rapid AxD. Such subliminal SARM1 activity likely contributes to common neurodegenerative diseases including ALS, CMT2A and diabetic neuropathy. Our evidence indicates that this activity involves upstream and downstream molecular mechanisms that may not be engaged during canonical axotomy-induced Wallerian degeneration, mechanisms that impair axon resilience and contribute to compromised axon integrity and function in progressive neurodegenerative disorders. To enable the study of this subliminal SARM1 activity, we developed means to both manipulate and monitor the AxD pathway via titrating SARM1 activation and quantitatively measuring cADPR, a SARM1 activity biomarker. Here we use these tools to investigate the regulation of SARM1 by post-translational modification and the mechanisms by which DNA damage induces SARM1-dependent AxD. Our preliminary data show that SARM1 binds to and is ubiquitinated by Parkin in response to mitochondrial damage. We propose experiments to determine the unexplored impact of K63 regulatory ubiquitination on SARM1 activity. Such regulatory events are likely to impact chronic SARM1 activation and progressive disease. We will use kinase inhibitor panels to identify novel SARM1 regulators and employ a fluorescent SARM1 activity sensor and imaging-based screening technologies we recently developed to identify molecular components regulating SARM1 function by performing a CRISPR screen in models of subliminal SARM1 activation using gRNAs targeting all druggable genes including most kinases. We also find that disrupted cellular DNA repair activates SARM1-dependent AxD. DNA damage is associated with peripheral neuropathy in many neurodegenerative diseases including ALS and rare genetic disorders, as well as after chemotherapy. We will define the mechanisms of SARM1 activation due to DNA damage and how SARM1 promotes DNA damage-induced degeneration using human motor neuron models of ALS. Finally, our previous work led directly to development of therapeutic SARM1 inhibitors that are now in clinical development. Here we propose to develop therapeutic SARM1 activators as axon-specific neurolytics to treat localized chronic pain. In total, these studies will define mechanisms that regulate subliminal SARM1 activation relevant to chronic disease, identify alternative therapeutic targets for neurodegenerative disorders, and directly generate novel non-opioid medicines to treat pain.

NIH Research Projects · FY 2025 · 2014-08

PROJECT SUMMARY This application proposes the renewal of a post-doctoral training program in pediatric infectious diseases and immunology (PIDI-TP), based in the Divisions of Pediatric Infectious Diseases (ID) and Rheumatology & Immunology at Washington University School of Medicine (WashU) and St. Louis Children’s Hospital. The long- term objective is to train academic physician-scientists to carry out impactful research in pediatric ID pathogenesis and host response. WashU offers an outstanding training environment for physician-scientists, with nationally recognized hospitals anchoring one of the nation’s premier biomedical research facilities. Many WashU faculty, both within and outside the Department of Pediatrics, are field-leading investigators in infectious diseases and human immunology. PIDI-TP will also take advantage of extensive WashU facilities for genome sequencing and microbial genomics, as well as 100 other Research Cores. Our 45 PIDI-TP mentors, drawn from the Departments of Pediatrics, Medicine, Molecular Microbiology, and Pathology & Immunology, represent highly successful investigators with externally funded research programs and experience in mentoring physician-scientists. As before, the PIDI-TP Program Director (PD) will be David Hunstad, MD, the Arnold W. Strauss Endowed Professor for Mentoring, Professor of Pediatrics and Molecular Microbiology, and Chief of the Division of Pediatric ID. The co-PD will be Megan Cooper, MD, PhD, the Anthony R. French Professor of Pediatrics, Professor of Pathology & Immunology, and Chief of the Division of Pediatric Rheumatology & Immunology. Trainee selection and program activities and outcomes will be overseen by an Executive Committee consisting of the two PDs and two other tenured investigators at WashU who are heavily involved in physician-scientist training, and advised by external faculty who are nationally recognized experts in pediatric ID and/or immunology and associated training programs. Each trainee will be advised by an individualized Scholarship Oversight Committee (SOC) comprised of established investigators with complementary expertise. The program will continue to support two postdoctoral (MD or MD/PhD) trainees per year, to be recruited primarily from our pediatric fellowship programs in Infectious Diseases, Rheumatology, and Allergy/Immunology, but also from Neonatology, Pulmonary Medicine, Nephrology, and Critical Care, drawing in particular on MD/PhD trainees in our robust Physician-Scientist Training Program. The mentored research experience, educational and career development activities for trainees are organized in three arenas: Pathogens, Host, and Omics. A core curriculum for all participants will provide training in study design, biostatistics, human subjects research, animal use and care, scientific writing, grant and manuscript preparation, genomics, and the responsible conduct of research. Career development will be monitored by research mentors, the PDs, the Executive Committee, and the SOCs. Program evaluation will rely on short-term and long-term metrics as well as input from external reviewers and from current and past trainees.

NIH Research Projects · FY 2025 · 2014-07

Abstract This application seeks to renew the funding for the T32 training program at Washington University for physician and doctoral scientists pursuing a career in anesthesiology-related research. The practice of anesthesiology is complex and far reaching. The research interests of anesthesiology span this broad clinical realm and have the potential to impact medicine as a whole. Developments in basic and clinical science have made the fundamental unsolved problems in anesthesiology research scientifically tractable. Despite the potential of its research programs for broad scientific and therapeutic impact, anesthesiology lags behind other specialties in training physicians and basic scientists in research. Our program is directed at meeting this need. The overarching goal is to train a diverse group of anesthesiology scientists with appropriate scientific skills to address the research priorities in anesthesiology-related science. To accomplish this, we focus on: (i) recruiting and training a diverse group of talented early stage anesthesiology scholars; (ii) identifying training opportunities in high priority and high yield scientific areas; (iii) providing expert, accountable and dedicated research mentorship; (iv) programmatically and systematically enhancing research training; (v) assessing trainee’s personalized goals and progress; (vi) providing state-of-the art curricula and facilities as well as networking and educational opportunities; and (vii) providing ongoing mentoring to T32 alumni to facilitate their transition to careers as independent investigators. The Washington University Anesthesiology Department is among the highest funded and most research productive anesthesiology departments in the United States. T32 trainees in the Anesthesiology Department are typically selected from a cohort of research residents, which is one of the largest nationally, based on the vibrant and highly effective Academic Scholars Advancement Program and the Scholars track. Graduates from our T32 have published high impact research, obtained competitive training grants, and continued in academic faculty appointments. Two program directors (PDs), an associate program director (APD) and an executive advisory committee (EAC) administer the training program. Mentors for the program have been carefully chosen based on the quality and relevance of their science and demonstrated mentorship experience and success. Trainee-mentor pairing is facilitated and approved by the PDs, APD and EAC. Trainees and mentors are formally evaluated twice annually for progress and satisfaction. Emphasis during these evaluations is placed on progress towards development of an independent research program, acquisition of career skills and attaining milestones such as manuscript and grant submissions. The PDs, APD, EAC and the trainees evaluate the training program annually, and make ongoing recommendations for improvements. Our program also plays a leadership role in the Early Stage Anesthesiology Scholars (eSAS) initiative and in collaborative ventures with other national T32 programs. Therefore, our T32 trainees also develop key networking and leadership abilities.

NIH Research Projects · FY 2024 · 2014-07

ABSTRACT Dry eyes and contact lens wear often cause foreign body sensation. This abnormal mechanosensation is associated with “Lid Wiper Epitheliopathy” (LWE), a condition induced by the mechanical damage to the marginal palpebral conjunctiva (also known as “lid wiper”). However, the underlying neurosensory mechanism remains elusive. We recently found that a population of primary sensory neurons defined by the expression of MrgprD selectively innervates the lid wiper and is sensitive to shear force. This proposal aims to determine whether MrgprD-expressing sensory fibers mediate ocular mechanosensation and regulate lacrimation. In Aim 1, we will characterize the innervations and mechanosensitivity of MrgprD-expressing sensory fibers in the lid wiper. Using genetic labeling and axonal tracing approaches, we will perform detailed anatomical analysis of organization and terminal ultrastructure of MrgprD-expressing sensory fibers in the lid wiper. In addition, we will test whether MrgprD-expressing sensory fibers in the lid wiper can be activated by shear force by conducting ex vivo calcium imaging. These studies will shed light on the role of MrgprD-expressing sensory fibers in ocular mechanosensation. In Aim 2, we will further determine whether MrgprD-expressing sensory fibers sense shear force during eye movements. We will determine whether genetic ablation or pharmacological silencing of MrgprD-expressing neurons alleviates the ocular mechanosensation induced by enhanced shear force. This study will provide insight on the neurosensory mechanism of the lid wiper mechanosensation. In Aim 3, we will determine whether the lid wiper mechanosensation regulates lacrimation to maintain the lubrication of the ocular surface. Specifically, we will examine whether ablation of MrgprD-expressing sensory fibers affects basal lacrimation and mechanically-induced lacrimation. Furthermore, we will test whether chemogenetic activation of MrgprD-expressing sensory fibers in the lid wiper promotes lacrimation. Finally, we will determine whether pharmacological activation of MrgprD-expressing sensory fibers is a potential therapeutic strategy for promoting lacrimation under dry eye conditions. These studies will reveal the neural basis of ocular mechanosensation associated with physiological tear evaporations and pathological dryness of the ocular surface, which will have a significant impact in our understanding of ocular mechanosensation as a protective mechanism and its clinical implication in dry eye treatments.

NIH Research Projects · FY 2026 · 2014-05

Project Summary The vitamin K cycle supports blood coagulation, bone mineralization, and vascular calcium homeostasis. The activity of vitamin-K-dependent proteins (e.g., coagulation factors) is regulated by the γ-carboxylase, GGCX. Epoxidation of vitamin K hydroquinone (KH2) drives catalysis of GGCX and is regenerated by vitamin K epoxide reductase (VKOR) with the assistance of a VKORL paralog and FSP1. VKOR is targeted by warfarin, a widely used oral anticoagulant whose overdose often leads to severe bleeding. Due to poor mechanistic understandings, modulation of the vitamin K cycle to improve anticoagulation therapy has not been explored. In addition, FSP1 regulates ferroptosis by producing KH2, which also affords potent protection against lipid peroxidation. However, the roles of vitamin K reductases in coagulation and ferroptosis, which are linked to numerous cardiovascular disorders, are unclear. Further, the mechanisms of the tightly regulated GGCX catalysis remain elusive. Thus, there is a need to establish a deeper understanding of the entire vitamin K cycle and the biology underlying blood coagulation so new therapeutic strategies can be developed for thromboembolic and cardiovascular diseases. Our long-term goal is to elucidate the physiological process of the entire vitamin K cycle and the underlying mechanisms. The objective of this renewal application is to modulate the redox state of VKOR to regulate anticoagulation, elucidate the relative contributions of VKOR, VKORL and FSP1 to support coagulation and control ferroptosis, and understand the structural mechanisms of GGCX catalysis. Our hypotheses are: (1) shifting VKOR towards a reduced state enhances vitamin K antidoting by increasing VKOR activity and facilitating warfarin release; (2) VKOR/VKORL are primarily responsible for K antidoting and reducing lipid peroxidation in the ER; and (3) GGCX binding of substrates induces conformational changes to tightly regulate the sequential reactions. Our preliminary data obtained 11 crystal structures of VKOR and a VKORL paralog with substrates and antagonists at different redox states. We also discovered that VKOR at reduced state is highly active but poorly inhibited by warfarin, and that K competition at partially oxidized state releases warfarin. We showed that VKOR/VKORL better support carboxylation and K antidoting relative to FSP1. We identified VKOR partner proteins and showed that reduced glutathione maintains VKOR activity. We obtained the first cryo-EM structures of human and conus GGCX that suggests keto-enol tautomerization as an elegant solution that couples epoxidation and carboxylation across the membrane interface. Armed with our expertise in the vitamin K cycle and membrane proteins, we will test our hypotheses with three specific aims: (1) Identify new anticoagulation strategies by employing VKOR redox states; (2) elucidate the interplay of the vitamin K cycle in γ-carboxylation and ferroptosis; and (3) understand the structural mechanisms of γ-carboxylation. The proposed studies are expected to significantly advance our knowledge of the entire vitamin K cycle and lead to innovative strategies that will improve treatment for thromboembolic and cardiovascular diseases.

NIH Research Projects · FY 2024 · 2014-05

Abstract In the next year, approximately 22,000 Americans will develop glioblastoma (GBM) and nearly the same number will die from it. Further, we can reliably predict that of the 22,000 new cases, 8,500 will be in females while the remaining 13,500 cases will be in males. Moreover, while the median survival for female GBM patients next year is expected to be between 17 and 22 months, for males it will be closer to 16 months. The molecular bases for these consistent and significant sex differences in incidence and survival are unexplained. In the absence of an explanation, it is impossible to fully know what the implications of sex differences are for modeling GBM in the laboratory and for treating GBM in the clinic. Identifying targetable mechanisms underlying sex differences in survival are the focus of this project, and our goal is to improve outcomes for all GBM patients. Building on our published and preliminary studies supported during the prior funding period of this RO1, we now hypothesize that sex differences in cellular senescence contribute to the sex disparity in glioblastoma (GBM) incidence and survival. As radiation or chemotherapy induced senescence is a mechanism of stopping tumor growth, we will focus on the mechanisms that endow female cells with greater ability to undergo senescence than their male counterparts in response to DNA damage. While cellular senescence has been extensively studied in normal and pathological states, studies in cancer have focused almost exclusively on fibroblast and bone marrow stromal cell senescence in breast and prostate cancer models. There has been little to no investigation of the role that cellular senescence plays in brain tumor promotion or treatment response, or any focus on sex differences in cellular senescence. We have two Specific Aims in which we will build on our prior success and utilize the extensively validated model systems for studying sex differences in GBM that we developed. We will apply innovative genomic technologies to define the contributions of sex differences in p21 and Rb functions to the induction of senescence, and will determine whether Brd4 and sex-specific epigenetics are required for sex differences in astrocyte and GBM cell senescence and the senescence-associated secretory phenotype.

NIH Research Projects · FY 2025 · 2013-09

The Washington University SPORE in Leukemia is a highly dynamic translational cancer research program that focuses specifically on leukemias and myelodysplastic syndromes (MDS). We have assembled an outstanding group of investigators with complementary expertise in basic and clinical leukemia research. In this SPORE, we leverage expertise in cancer genomics, immunology, and hematopoiesis to develop innovative translational research in leukemia. Our long-term goal is to develop novel biomarkers and treatments for leukemias and myelodysplastic syndromes and to develop and promote innovative translational leukemia research. To achieve these goals, the following specific aims are proposed. Aim 1. We will exploit institutional expertise in cancer genomics, immunology, and hematopoiesis to develop novel biomarkers and treatments for leukemias and myelodysplastic syndromes. Basic research at WUSM has led to the development of the following four translational research projects, three of which feature innovative investigator-initiated therapeutic trials for leukemias or MDS. Project 1. Chimeric antigen receptor T-cell therapy for T cell malignancies Project 2. Memory-like NK cell therapy for AML relapsed after allogeneic transplant Project 3. Targeted therapy for splicing factor-mutant myeloid malignancies Project 4. Targeting ATR in TP53-mutated MDS/AML Aim 2. We will enhance the infrastructure that supports translational leukemia research. This SPORE will support the following Shared Research Resources: 1) Core A. Biospecimen Processing; 2) Core B. Biostatistics and Bioinformatics; and 3) Core C. Administration. Aim 3. We will recruit and train new investigators in translational research. This SPORE will support a Career Enhancement Program (CEP) to recruit and mentor new investigators in translational leukemia research. The SPORE also will support a Developmental Research Program (DRP) to support innovative translational concepts. Aim 4. We will facilitate inter-SPORE collaboration. Two of the SPORE projects include multi-institutional clinical trials, including participation by peer Leukemia SPORE institutions. We have established a CEP educational exchange and grant review programs with peer Leukemia SPORE institutions. We will continue to organize and participate in joint meetings of Leukemia SPOREs at MD Anderson, Harvard, and Memorial Sloan Kettering Cancer Center.

NIH Research Projects · FY 2025 · 2013-08

PROJECT SUMMARY/ABSTRACT For children with hearing loss (HL), speech perception assessments are fundamental to guiding all aspects of habilitation including device candidacy, validation, and therapy goals. Good speech perception requires not only identifying individual vowel and consonant sounds (segmental) but also perceiving prosodic aspects of speech such as intonation, stress and rhythm (suprasegmental). The role of segmental perception for development of spoken language and reading is firmly established for children with typical hearing (TH). The complementary role of suprasegmental perception for spoken language acquisition and, more recently, for reading development has also been established for children with TH. Even though suprasegmental speech perception contributes essential information to listeners with TH, it has been largely neglected in studies of speech perception for individuals with HL. Given the known significant role that suprasegmental perception has for critically important spoken language and reading skills of children with TH, and the unknown suprasegmental perception abilities of children with mild to profound HL, we seek to understand the manners in which both segmental and suprasegmental perception develop in such children. For children with two HAs and with a continuum of HL, we seek to determine the hearing threshold level (dB HL) above which better results are more likely to be obtained through either bilateral or bimodal cochlear implantation. The motivation for extending studies to children with greater levels of residual hearing is due to changing clinical protocols that now include children with thresholds as low as 60-70 dB HL for CI candidacy, and which stress early receipt of bilateral CIs. A logical question, and one that is not addressed explicitly in these recommendations, is: should children with more residual hearing stay with 2 HAs, continue with 1 HA and receive 1 CI (bimodal), or receive 2 CIs? Specific Aim 1A: For children with mild to profound HL who use two devices, we will determine the contributions of acoustic experience (audibility and duration) to the development of suprasegmental and segmental perception. We predict that children with greater acoustic experience will develop suprasegmental skills that approach their TH peers. Specific Aim 1B: For children with mild to profound HL who use two devices, we will determine the contributions of auditory capacity (select suprathreshold abilities) to the development of suprasegmental and segmental perception. Specific Aim 2: We will assess the risk-benefit associated with CI- and/or HA-use by establishing audibility ranges for which children with 2 HAs perform better on suprasegmental and segmental perception than children with 2 CIs or bimodal devices. Specific Aim 3: We will quantify the relative contributions of suprasegmental and segmental perception to these same children's development of spoken language and reading skills.

NIH Research Projects · FY 2025 · 2013-07

Developmental neuroscience and psychopathology have great potential to inform the understanding of the causal pathways and mechanisms of mental illness. This T32, which is focused on this domain, is co-led by Drs. Luby and Barch (with complimentary expertise in developmental psychopathology and neuroscience), and has been very successful in the first two cycles of funding, enjoying a highly competitive national applicant pool and successfully launching the young scientists completing the program into research careers and external funding. We seek to renew this training grant, adding new expertise in a wider range of neuroimaging measures, as well as new faculty mentors with expertise in the effects of genetics, the gut microbiome, and sleep/circadian rhythms on neurodevelopment and risk for psychopathology. From a public health perspective, an infusion of new research scientists in the area of developmental neuroscience and psychopathology continues to be a high priority. The proposed multi-disciplinary training approach is guided by a conceptual model that recognizes that the risk, onset, and course of psychiatric disorders arises through a complex interplay of brain developmental processes influenced by environmental, psychosocial, cognitive, affective, genetic, and biological factors that interact beginning in utero and continue throughout development. Numerous investigators at Washington University in St. Louis (WUSTL) have a rich track record of experience in many aspects of child neuroimaging and related methods, including a focus on structural and functional magnetic resonance approaches in very early childhood (including neonates), functional near-infrared spectroscopy (fNIRS), evoked response potentials (ERP) and high density diffuse optical tomography (HD- DOT). Further, WUSTL has an international reputation in psychiatric genetics, gut microbiome work, and sleep/circadian rhythms, with many researchers who can bring their expertise to bear on understanding the neurobiology of developmental psychopathology. The program mentors have a rich body of available databases derived from longitudinal studies, several of which began in early childhood and in utero. Mentors also provide a unique multidisciplinary training environment in which to pursue this exciting domain focused on development, given the established collaborations between child researchers in the WUSTL School of Medicine clinical and basic departments, and the state of the program in neuroscience and neuroimaging at WUSTL that has been at the forefront of developmental cognitive and affective neuroscience. Interactions between researchers in basic and clinical developmental neuroscience offer an ongoing opportunity to help train the next generation of young scientists who can pursue questions about the developmental etiology of psychopathology from the perspective of core psychological and neural mechanisms of human behavior that span traditional boundaries of psychopathology, an approach central to the NIMH Research Domain Criteria Initiative (RDOC).

NIH Research Projects · FY 2025 · 2013-06

ABSTRACT Heart disease is the most common cause of death in industrialized nations. The presence of underlying diabetes is the greatest risk factor for the progression of heart disease. During the current grant interval, we have discovered previously unknown lipid metabolic pathways and signaling molecules which lead to the generation of eicosanoid-lysophospholipids. Remarkably, the vast majority of eicosanoids in myocardium were found to be esterified to the glycerol backbone of lysophospholipids. In addition, induction of Type I diabetes in wild-type mice or ischemic damage in isolated wild-type mouse hearts resulted in dramatic increases in pro-inflammatory eicosanoid-lysophospholipids. This new class of phospholipids serve as inflammatory mediators by inducing the release of TNFa from monocytes or macrophages. Importantly, genetic ablation of iPLA2g (PNPLA8) substantially decreased the levels of eicosanoid-lysophospholipids in myocardium in the diabetic state, during myocardial ischemia and synergistically decreased their synthesis in diabetic myocardium rendered ischemic. Accordingly, we propose that iPLA2g plays a central role in the pathophysiologic development of diabetic heart disease and promotes the lethal sequelae of diabetic cardiomyopathy. In Specific Aim 1, we will utilize stable isotope labeling of isolated perfused mouse hearts from genetically engineered cardiac myocyte-specific conditional iPLA2g knockout mice we have generated. These studies will investigate the roles of iPLA2g in the metabolic flux of: 1) non-esterified and esterified eicosanoids; 2) eicosanoid-lysophospholipids; and 3) other salient oxidized phospholipids. Stable isotope pulse-chase experiments followed by penetrating bioinformatic analyses will determine rates of metabolic flux through these newly discovered pathways. Translationally, we will explore the impact of Type 2 diabetes on myocardial ischemic damage and the potential salvage of ischemic myocardium in cardiac myocyte-specific iPLA2g KO mice we engineered. Endpoints of analysis include infarct size, hemodynamic performance, and post-translational modifications of iPLA2g. In Specific Aim 2, we will utilize cardiac myocyte-specific iPLA2b KO mice we have generated to explore the role of iPLA2b in promoting myocardial ischemic damage and arrhythmias in WT vs. iPLA2b KO mice in the context of Type II diabetes. Next, we will determine the ability of iPLA2b to catalyze acyltransferase or transacylase mediated re-esterification of eicosanoid-lysophospholipids to generate oxidized phospholipids which have been implicated in damage associated molecular patterns. In Specific Aim 3, the mechanisms through which a high fat diet induces eicosanoid-lysolipid synthesis accompanied by inflammation and mitochondrial dysfunction will be studied. The roles of lysophospholipases in modulating eicosanoid-lysophospholipid levels and activation mechanisms for iPLA2g will be examined. Collectively, the proposed studies will establish the significance of iPLA2g and iPLA2b in mediating the newly identified pathways of eicosanoid-lysophospholipid synthesis and metabolism and determine their impact on diabetic cardiomyopathy and acute ischemic damage in diabetic hearts.

NIH Research Projects · FY 2025 · 2013-04

PROJECT SUMMARY/ABSTRACT Nucleic acids (NAs) are the major information carrying molecules of life. The ability to use computation to model the structure, dynamic and interactions of DNA and RNA is a key adjunct to experimental study of these biomolecules. For example, pseudouridine-containing mRNA vaccines against Covid-19 are a critical tool in battling the pandemic. DNAs, mRNAs, and miRNAs are targets for a number of antibacterial and antiviral drugs. Design of small molecule drugs binding to nucleic acids in the treatment of cancers and neurodegenerative diseases is one of the hottest topics of current pharmaceutical research. Under this project, we will investigate several key aspects of nucleic acids, and develop the polarizable multipole AMOEBA+ model for simulation of NAs and their interactions. This new force field will be further enhanced via coupling of a machine learning-based potential for local valence features with classical long-range nonbonded interactions. The resulting AMOEBA+NN force field promises chemical accuracy in the calculation of binding for NA systems. Parameters for the AMOEBA+ and AMOEBA+NN potentials will be generated using the new, automated Poltype2 package. Implementation of the force fields into the existing GPU-capable Tinker9 molecular dynamics software will enable state-of-the-art simulation and binding free energy estimation. The applicability of molecular simulation to design of therapeutics is limited by efficiency and accuracy of the calculations. The objective of this proposal is to enable routine, accurate computation of the thermodynamics of binding of small-molecule ligands to NAs. Toward that end, several current systems of biological relevance will be investigated, including binding of metal ions to G-quadruplex structures, fluorogenic ligands with RNA aptamers, novel antibiotic drugs with the FMN riboswitch, and conformational dynamics of the P5abc domain of the Tetrahymena group I ribozyme. The structures and functions of NAs are highly dependent upon their ionic environment. The interplay between RNA local structural dynamics and global/tertiary folding is an intriguing question being addressed experimentally. The ability to simulate these complex energetic effects in the design of novel small molecule drugs and synthetically modified oligonucleotides will be an important growth area for future medical advances. Development of the accurate and transferable next-generation AMOEBA+ and AMOEBA+NN force fields will open new paths toward understand and prediction of the behavior of natural and designed nucleic acid molecules.

NIH Research Projects · FY 2026 · 2013-03

PROJECT SUMMARY/ABSTRACT Mitochondria are centers of metabolism whose activities need to be calibrated to meet changing cellular needs. General dysfunction of these organelles is implicated in many common human disorders, including metabolic syndrome, type 2 diabetes, obesity, non-alcoholic fatty liver disease, heart failure, various cancers and neurodegenerative diseases, and general metabolic inflexibility, most often through unclear means. Defining the pathogenic mitochondrial alterations that contribute to these metabolic disorders and devising new therapeutic strategies to rectify them represent principal challenges in mitochondrial medicine. A potential contributor to this dysfunction is aberrant intra-mitochondrial protein phosphorylation—a process recognized as critical for pyruvate dehydrogenase regulation for more than 50 years, but relatively unexplored otherwise. Recent efforts from our laboratory and others have now revealed that mitochondrial proteins are replete with dynamic phosphorylation that changes reproducibly between healthy and diseased states, and that phosphorylation can alter the activities of proteins involved in core metabolic pathways. Furthermore, we have connected select protein dephosphorylation events to the poorly characterized matrix protein phosphatase PPTC7 and discovered that PPTC7 disruption in mice causes profound metabolic defects and neonatal death. Given these emerging findings, the premise of this project is that reversible phosphorylation may be widely important in calibrating mitochondrial metabolism, and that its mismanagement could contribute to the pathophysiology of mitochondria- related disorders. Rigorous new efforts to reveal how phosphorylation affects mitochondrial protein function and to define the phosphatases that target each site may ultimately enable a new therapeutic strategy focused on manipulation of the mitochondrial phosphorylation network. To this end, we have now extended our phosphoproteomic analyses to 10 mitochondrial phosphatase knockdown lines using a CRISPRi system. This rich dataset forges many unique connections between phosphatases and phosphoproteins and forms the foundation for the further work proposed here. Based on these findings, we propose 1) to establish the functional impact of phosphorylation on putative PPTC7 and HDHD5 substrates involved in core mitochondrial catabolic pathways, including branched chain amino acid and fatty acid catabolism, and 2) to define the role of PGAM5 in regulating mitochondrial cristae architecture by managing an extensive set of phosphorylation events on subunits of the mitochondrial contact site and cristae organizing system (MICOS). Altogether, through a comprehensive approach that combines mammalian physiology, omics-level analyses, and rigorous biochemistry, we aim to make definitive connections between mitochondrial phosphatases and their substrates, establish a broad framework for understanding the role of this post-translation modification in calibrating mitochondrial activities, and ultimately pave the way for a new therapeutic strategy to rectify mitochondrial dysfunction.

NIH Research Projects · FY 2026 · 2013-03

PROJECT SUMMARY/ABSTRACT This renewal application (RFA-HD-21-017) is for support of the Child Health Research Center (CHRC) at Washington University School of Medicine. Pediatric physician-scientists play a crucial role in advancing knowledge that improves child health. To meet the ongoing national need to replenish the pediatric physician-scientist pipeline at the junior faculty level, our program supports a mentored career development pathway for 3 Scholars per year for 2-3 years by leveraging a wealth of biomedical resources across the Washington University School of Medicine campus. As we have done for the past 26 years, the long-term objective of our Center is to develop Scholars who focus their research efforts on pediatric disease-oriented biology by applying recent advances in the basic sciences, including developmental biology, cell biology, immunology, genetics/genomics, and systems biology. The specific aims of this proposal include: (1) providing protected, mentored research experiences with well-established investigators encompassing a wide range of disciplines across Washington University School of Medicine and within the Department of Pediatrics; (2) obligatory educational programs in laboratory management, scientific rigor, statistics, grantsmanship and responsible conduct of research; (3) personalized coursework based on each Scholar’s area of investigation (e.g., bioinformatics, statistics, advanced imaging); (4) ongoing feedback to the Scholars, mentors and CHRC leadership; and (5) promoting the development of physician-scholars with a deep commitment to improving child health through scientific innovation and discovery.  The program has a stellar track record, by far exceeding national benchmarks (”K12 to K08/23” conversion rate of 73% and an outstanding “K to R conversion” rate of 60%; national average <38%), and will ultimately close the knowledge gap between basic scientists and pediatric clinicians. Gary A. Silverman, MD, PhD, will continue to serve as Program Director, and David Hunstad, MD, will continue to serve as Training Director. The CHRC and its Scholars will utilize dozens of institutional state-of-the-art core facilities that provide, for example, genomic and metagenomic sequencing, transcriptomics, metabolomics, mass spectrometry, bioinformatics, cryo-EM and other advanced imaging, CRISPR genome editing and animal model development, to facilitate the study of pediatric disease states. The long-term goals of the CHRC are being realized as its Scholars contribute to our understanding of child health and disease for decades to come, while evolving into the next generation of scientific leaders, role models, and mentors for subsequent generations of pediatric physician-scientists.

NIH Research Projects · FY 2024 · 2012-09

PROJECT ABSTRACT This competing renewal to Suubi+Adherence study (R01HD074949) will examine the longitudinal HIV antiretroviral therapy (ART) adherence and related outcomes, and the potential mechanisms of protective health behaviors among youth living with HIV (YLHIV) who participated in an evidence-based family economic empowerment (FEE) intervention in Uganda and are now transitioning into young adulthood. Sub-Saharan Africa (SSA) has the highest HIV prevalence rate in the world. In Uganda, a poor SSA country hardest hit with HIV, the prevalence of viral load suppression among adolescents and young adults (15 to 24 years old) is 44.9% for females and 32.5% for males. For six years (2012-2018), the Suubi+Adherence study prospectively followed 702 YLHIV (aged 10 to 16 years at enrollment) randomized to two study arms across 39 clinics in Uganda: 1) control arm consisting of bolstered standard of care (BSOC) and 2) intervention arm consisting of BSOC and a FEE intervention. Study findings pointed to superior short-term viral suppression and positive child health and mental health functioning among the intervention arm. This renewal (Suubi+Adherence-Round 2) proposes to examine whether the results are maintained through young adulthood, an incredibly vulnerable transition period, particularly in areas of adherence to HIV treatment, cognitive development, mental health, sexual risk taking behaviors and alcohol/drug misuse. The renewal (2020-2025) will build on the Suubi+Adherence study to conduct one of the largest and longest running studies of YLHIV in SSA during a developmental period marked by profound biological and psychological maturation, and development transitional milestones. In this next phase, we will add a qualitative component to examine participants' experiences with the FEE program as well as long-term effects. Innovatively, we will also assess the impact of the FEE on cognitive functioning. The study specific aims are: Aim 1. To examine the long-term impact of the Suubi+Adherence intervention on: HIV viral suppression (primary outcome) and to explore the long-term impact of the intervention on key HIV treatment adherence outcomes for YLHIV, including participants' ability to access and refill prescribed medication, adhere to prescribed daily medication routines, and engage in HIV care; Aim 2. To elucidate the long-term effects of the Suubi+Adherence intervention on potential mechanisms of change, including: a) economic stability, sexual risk- taking behavior, adherence self-efficacy; b) cognitive functioning; c) mental health functioning; and d) young adult transitions and social support; Aim 3. To qualitatively examine– prospectively and retrospectively: a) multi- level factors affecting participants' maintenance of intervention benefits since Suubi+Adherence intervention initiation (prospectively); and b) participants' experiences with the intervention (retrospectively), including multi- level factors that may have influenced their engagement with the program and their decision-making in regard to ART adherence; Aim 4. To examine the long-term cost-effectiveness of the Suubi+Adherence intervention. The study is aligned with the National Institutes of Health Office of AIDS Research priorities.

NIH Research Projects · FY 2025 · 2012-09

The body's largest collection of immune cells underlies the single layer epithelium lining the gastrointestinal (GI) tract and monitors the luminal contents, which includes trillions of microbes, their products, and substances from the diet. The basal tone of the healthy gut immune system is tolerogenic, despite being exposed to trillions of microbes and their products. While this strong tolerogenic capacity is beneficial to the host to avoid inflammatory responses to innocuous dietary and commensal antigens in the healthy state, the inability to dampen this tolerogenic capacity could be detrimental in the setting of enteric infection and inappropriately dampening this tolerogenic capacity could underlie the pathogenesis of intestinal inflammatory diseases. We propose that the gut has a capacity to turn off tolerogenic responses and generate inflammatory responses. While great progress has been made in elucidating the role of specific immune cell subsets, cytokines, and other factors promoting tolerance or immunity, how the gut immune system switches from tolerogenic responses in the steady-state to protective immunity when needed remains a significant gap in our understanding. Completion of the studies outlined in this proposal will fill this void in our understanding by identifying how inhibiting a major pathway delivering luminal substances activates cellular and humoral immune responses at the mucosa. In prior cycles of this award we have identified how goblet cell associated antigen passages (GAPs) are formed, the stimulus inducing GAPs in the steady-state, acetylcholine (ACh), the stimuli and receptors regulating GAP formation, including the luminal microbiota, cytokines, and epidermal growth factor receptor (EGFR) ligands, and the properties of GAPs in various regions of the GI tract. Further we have identified roles for GAPs, when physiologically present, in supporting tolerance to luminal substances including dietary and commensal microbial antigens. Moreover, we have now assembled genetic and pharmacologic models for the manipulation of GAPs and are poised to dissect the role of GAP inhibition in promoting protective/inflammatory immunity in the absence of enteric infection or overt changes in the gut microbiota. Based upon our prior studies and preliminary observations we hypothesize that when GAPs form in the steady state, they act to imprint the immune system to promote tolerance and when small intestine (SI) GAPs are inhibited they participate in a cascade of events promoting protective immunity. To explore this hypothesis we propose to (Aim 1) define the LP-APCs phenotypes, the origins of Th17 and TFH cells that expand, and the durability of the response that occurs when SI GAPs are inhibited, (Aim 2) define the drivers and specificities of the B cell responses arising when SI GAPs are inhibited and (Aim 3) determine if SI GAP inhibition improves outcomes and is required for appropriate responses during enteric infection.

NIH Research Projects · FY 2025 · 2012-07

Project Summary: Revolutionary advances in imaging drive discovery in the biological sciences and medicine. These advances in imaging sciences depend on innovations in technology throughout the physical and biological sciences. In recent decades, a number of significant breakthroughs have underscored the fundamental importance of this interdependent relationship between technology and biomedical science. One important discovery that culminated in the 2008 Nobel Prize was the work on fluorescent proteins in the laboratories of Shimomura, Chalfie, Inoue, Tsien and many others. “Live cell microscopy would have remained in the hands of a small number of skilled cell biologists had not these groups devised a simple way to use molecular biology to tag vital proteins in living cells. Gene products can be localized not only structurally, as with antibody technology, but also dynamically within living cells. With this technology, it is now possible to place virtually any biomolecule in the context of structure and dynamics; that is, within space and time.” From the current perspective, where are the frontiers of imaging sciences in the 21st century, and what are its biggest challenges? Challenges in basic and translational sciences, often define the imaging science challenges, as illustrated in the following examples. (1) Optogenetic and Chemogenetic techniques have revolutionized the way brain circuitry is probed, and now enable dissection of complex neuromodulatory circuits related to reward, aversion and anxiety. Novel Sonogenetic methods are also just now emerging. Yet these technologies for “writing” information into the brain require advances in imaging to “read” the responses. (2) Computational imaging has experienced a revolution over the last 5 years with the incredible growth in machine learning and deep learning techniques. However, as these methods are translated into clinical applications the challenges in optimizing and validating implementation loom large. (3) Tremendous progress is anticipated in the area of cell therapy and tissue engineering. Yet, these advanced therapeutics are asking for advancements in imaging for personalized therapeutic optimization. (4) Major advances in multimodal imaging technologies have recently been made, most prominently the development of PET/MR hybrid systems. New hybrid imaging systems that combine PET and optical or photoacoustic imaging, for example, advance only through solving imaging science challenges. A new educational and creative paradigm is required to prepare future imaging scientists for solving these problems. Interdisciplinary teams of engineers, physicists, computer scientists, mathematicians, chemists, biologists, and physicians working together at the interfaces between biology, technology, and medicine are needed to collaborate in the development of new imaging technologies and strategies. With these needs and goals in mind, Washington University created the Imaging Sciences Pathway (ISP).

NIH Research Projects · FY 2026 · 2012-04

Washington University Paul Calabresi K12 Career Development Award for Clinical Oncology Project Summary / Abstract The goal of the Washington University (WU) Paul Calabresi K12 Career Development Award (K12 program) for Clinical Oncology is to provide didactic coursework, mentored research practicums and specialized career development programs for clinician scientists possessing an MD, DO, PharmD, nurses with PhDs, or equivalent degree. We propose the renewal of this K12 to continue training clinicians from diverse disciplines, including medical oncology, surgical oncology, gynecological oncology, radiation oncology and pediatric oncology to promote their career development as cancer researchers and to design and administer hypothesis-driven pilot/Phase I, Phase II, and Phase III cancer therapeutic clinical trials. We are requesting five slots per year to support eligible scholars for two to three-year appointments. To enhance our highly successful K12 program, we propose the following: Aim 1: Expand the workforce trained to design and execute rigorous oncologic clinical research. We propose to fund five K12 scholar positions per year at WU over the next five years to train eligible applicants from diverse disciplines focused on patient-oriented cancer research. Aim 2: Optimize existing curricula, career development, short-term training, and mentored, hands-on research experiences pertinent to clinical trials and patient oriented research. We expanded upon our opportunities to offer training in four tracks: Cancer Genomics, Translational Medicine, Patient Centered Outcomes Research, and Cancer Immunology and Cellular Therapy. Aim 3: Continue to develop and enhance robust evaluation and tracking of K12 scholars, faculty, mentors, curriculum, and training opportunities. We will monitor progress, demonstrate outcomes, and use short- and long-term results to improve clinical oncology research training. Successful completion of these aims will result in increased numbers of diverse, well-trained investigators who lead hypothesis driven cancer clinical trials. By partnering with stakeholders early and throughout the translational enterprise, they will disseminate and implement their research findings in real world practice to advance rapid human health and health care improvements.