Washington University

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT Current tobacco treatment is limited by physicians infrequently prescribing cessation medication (<20%), patients infrequently using cessation medication (~33%), and limited medication effectiveness (<30%). A multilevel intervention to personalize tobacco treatment, based on substantial data linking genetically informed markers to treatment response, has the potential to address these critical gaps by addressing multiple targets in the cascade of treatment success. First, precision treatment may increase treatment effectiveness by matching the smoker with the medication that maximizes efficacy and safety. Second, it may increase physicians’ likelihood of prescribing because they expect precision treatment to be more effective than standard treatment. Third, precision medicine may increase patients’ motivation to initiate and adhere to cessation medication, as evidence suggests that smokers express desire for gene-guided treatment and increased motivation to use it adherently. Emerging evidence including a recent Cochrane review and our research suggests that patients’ ability to quit smoking and respond to cessation medication are associated with multiple biomarkers involved in nicotine metabolism (i.e., nicotine metabolite ratio, NMR) and nicotinic receptor function (i.e., genotype of CHRNA5 variant rs16969968). Evaluating precision treatment based on multiple markers in a multilevel intervention using behavior change theories will advance the field of tobacco treatment. Our preliminary work demonstrated: 1) promise of using both genetic and metabolic markers in guiding treatment, 2) high interest in gene-guided treatment and its potential for enhancing medication use among smokers, 3) higher motivation for smoking cessation with use of genetic risk communication feedback tools, and 4) successful use of health informatics to implement a multilevel tobacco intervention in clinic settings. The proposed study aims to test a multilevel intervention precision treatment intervention using both genetic and metabolic markers, directed at physicians and patients, to increase the uptake and effectiveness of tobacco treatment in primary care. We propose a 2-arm cluster randomized controlled trial of 40 physicians and 800 patients who smoke randomized to usual care (UC) vs. precision treatment (PT) based on NMR and genetics in primary care. We hypothesize that PT will enhance cessation success via increased physician prescription of medication, patient adherence, and treatment effectiveness. In Aims 1 and 2, we will test the impact of PT on physician prescribing, patient use of medication, and patient smoking abstinence. In Aim 3, we will evaluate mechanisms of behavior change and implementation outcomes. The study reflects a significant and innovative paradigm shift from a traditional treatment model to precision treatment that includes both metabolic and genetic markers to motivate and guide tobacco treatment for both physicians and patients, integrated within primary care.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Electroconvulsive therapy (ECT) is an effective intervention for treating patients with psychiatric disorders. Specifically, it is an established treatment for patients with Treatment-Resistant Depression (TRD) with a lack of responsiveness to at least two pharmacologic antidepressants. Despite an efficacy of up to 70%, little is known about the circuit-level mechanisms through which ECT alleviates psychiatric symptoms or exerts cognitive impairments. ECT induces generalized seizures and causes neuroplastic changes within functional networks. A better understanding of ECT is urgently needed given that it is a repetitive procedure performed under general anesthesia and its benefits are limited by cognitive impairment following treatment. Electroencephalography (EEG) enables capturing the temporal dynamics of brain activity in different states of arousal and recent developments in high-density EEG technology allow for elucidation of large-scale functional brain networks. Recording EEG during sleep provides insights about sleep structure. In this study, “Disruptions of Brain networks and Sleep by Electroconvulsive Therapy,” we will investigate the impact of ECT on information transfer efficiency in functional networks in relation to sleep slow waves, an EEG marker of synaptic plasticity. The novelty of this proposal stems from our focus on the longitudinal assessment of EEG markers during sleep, wakefulness, and ECT-induced seizures over the course of therapy. We will also acquire depression severity outcomes and memory measures longitudinally. Wireless wearable devices will address previous barriers to the longitudinal study of sleep microstructure in the outpatient ECT settings. Graph-based network analyses of high-density EEG signals allow characterization of information transfer over functional networks. These information measures are investigated locally in specific subnetworks and globally over large-scale functional networks. Longitudinal assessments of EEG throughout the course of ECT alongside clinical and cognitive outcomes will provide a unique opportunity to improve our understanding of the circuit mechanisms underlying the development of cognitive impairments and antidepressant effects incurred during ECT. Furthermore, our findings may shed light on other non-invasive interventions targeting sleep slow wave activity in patients with psychiatric illnesses. Overall, elucidating the impact of ECT on information processing in different states of arousal may have promising clinical implications through identification of potential new biomarkers and therapeutic targets leading to the improvement of public health for TRD patients.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Currently no consensus exists in clinical guidelines for management of patients with asymptomatic carotid artery stenosis (ACAS). Some guidelines recommend carotid endarterectomy (CEA) surgery for patients with ACAS of ≥ 60% diameter. However, it is argued that 95% of all surgical interventions for ACAS in the United States may be unnecessary, generating needless healthcare costs of >$2 billion annually. Our goal in this proposal is to study plaque biology in ACAS patients through PET imaging at the molecular level to help identify individuals who are at 'higher-risk’ for ischemic stroke from plaque rupture and may benefit from carotid surgical intervention. We propose a 2-center patient outcomes study that expands upon data and observations from our earlier single center first-in-human study at Washington University using a nanoparticle PET radiotracer that targets the natriuretic peptide receptor C (NPRC) to determine if it can be used to risk stratify patients with ‘higher-risk’ ACAS. We and others have shown that NPRC is expressed at higher levels in complex plaques with features of vulnerability in patients with ACAS. In our most recent NIH R01-funded proof-of-concept study in a cohort of 42 patients with ACAS, we have shown linear correlation between 64Cu-CANF-Comb PET radiotracer uptake and features of high-risk plaque and correlative 64Cu-CANF-Comb PET uptake to the presence of NPRC in CEA specimens of patients who underwent surgery. We propose the following Specific Aims: Aim 1. To determine the ability of 64Cu-CANF-Comb PET to risk stratify ACAS patients treated with optimal medical therapy (OMT) alone with respect to patient outcomes. In this observational study, 80 patients with ACAS ≥ 60% will undergo 64Cu-CANF-Comb PET/MRI. Patients will be maintained on either OMT alone or receive OMT and CEA as determined by their treating vascular surgeon prior to imaging. OMT will consist of antiplatelet, statin, and hypertension and diabetes management when applicable. All patients will be evaluated with phone interviews every 3 months for a minimum of 18 months to assess for ipsilateral ischemic cerebrovascular event. PET signal will be assessed as a marker of risk for event or progression to CEA in comparison to anatomic features of vulnerable plaque on MRI, with the goal of determining a PET signal threshold which suggests higher risk ACAS. Aim 2: To further understand the role of NPRC in the evolution of carotid atherosclerosis. A. Patients treated with OMT alone will undergo repeat PET/MRI at 18 months, or earlier if they develop symptoms. PET/MRI changes over the 18-month interval will be used to further understand the biology of carotid plaque evolution after treatment with OMT. B. In patients who initially undergo CEA, PET signal will be compared to ex vivo plaque vulnerability and NPRC cellular distribution to facilitate understanding of gene expression using immunohistochemistry (IHC) and cell origin through single cell RNA/CITE-seq transcriptomics. Results will provide information on the potential of a new imaging approach, 64Cu-CANF-Comb PET, to risk stratify patients with ACAS and reveal mechanistic information about the role of NPRC in plaque vulnerability and inflammation.

NIH Research Projects · FY 2024 · 2022-08

This proposed project focuses on quantifying the spatial and temporal diversity in cortical oxygen metabolism and neurovascular coupling, and informing next generation of biophysical models of the Blood Oxygen Level Dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) measurements by integrating new technological and conceptual approaches. Broad and long term objectives of this application are (1) to bridge understanding of brain in action at multiple scales in spatial (subcapillary to whole- brain) and temporal (µs to months) domains, (2) to contribute discovering diversity of hemodynamic responses across cortical tissue layers, functional areas, and microvascular branches, and (3) to support the modelling efforts of BOLD signal by to quantifying hemodynamic responses in cortex of healthy-aging awake mice. Realizing these objectives would contribute to the joined efforts towards improving human health through accomplishing BRAIN Initiative goals of understanding the brain in action, discovering diversity, mapping/linking activity across spatial and temporal scales, and understanding the biophysical basis of fMRI signal. The specific aims of the proposed project can be summarized as: (1) to develop faster, deeper, longitudinal measurements of the cerebral blood flow (CBF), partial pressure of oxygen (pO2), and cerebral metabolic rate of oxygen (CMRO2), (2) to quantify the diversity of CMRO2 and CBF responses to functional activation across different cortical functional areas, cortical layers, and microvasculature types in healthy-aging mouse, and (3) to quantify the effects of standard fMRI calibrations (e.g. hypercapnia, hyperoxia, and caffeine) on the CMRO2 and CBF at rest and during functional activation. The research design and methods that will be used in this project will include (1) developing faster and deeper chronic imaging of absolute oxygen concentration to assess relation between cortical pO2, CMRO2, and blood flow changes during brain activation across the cortical areas and layers, and (2) realistic measurements of the oxygenation, blood flow, and metabolism responses to functional activation at the microvascular scales in healthy-aging mice and in response to calibration procedures that are commonly used in BOLD fMRI imaging. The research environment of Dr. Sencan (the Athinoula A. Martinos Center for Biomedical Imaging at Massachusetts General Hospital and Radiology Department in Harvard Medical School) is equipped with state-of-the-art technologies required to execute the proposed project and is rich with opportunities for training in neuroscience, career development, and interdisciplinary collaborations. She has a team of mentors, collaborators and advisors with diverse and strong expertise, who are committed to support Dr. Sencan’s research, and her career development. All these factors will facilitate the successful execution of the proposed project, the completion of Dr. Sencan’s training in neuroimaging, and her transition to an independent tenure-track faculty position.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA) are significant contributors to age-related dementia and a major economic burden. Current strategies to alleviate AD and CAA pathology and associated cognitive decline are largely ineffective, necessitating the need for additional therapeutic avenues to be explored, particularly with a multi-disciplinary team able to achieve a holistic understanding of vascular, neuronal, and immune events that underlie disease progression. One promising strategy is the augmentation of clearance pathways – including microglia/macrophage phagocytosis of Ab and meningeal lymphatic drainage of cerebral spinal fluid (CSF) - that facilitate the removal of toxic, misfolded protein aggregates that represent pathological hallmarks of AD brains. While the vast majority of prior research has focused on parenchymal Ab pathology and microglial functions, many patients also display CAA, contributing to vascular dysfunction, and implicating a role for parenchymal border macrophages (PBMs; perivascular and leptomeningeal, collectively). Thus, we will explore the complex interactions between the meningeal lymphatic system, CSF flow, PBMs, and parenchymal microglia in AD and CAA. The overall hypothesis of this PPG is that neuroimmune events, particularly aspects of the innate immune system and meningeal lymphatics, underlie AD and CAA pathology. We further hypothesize that devising new therapeutic approaches, harnessing the functions of microglia, PBMs, and the meningeal lymphatics may represent novel targets to alleviate AD-related cognitive decline. In particular, we will explore how dysfunction in cholesterol metabolism, apoE, and downstream TREM2 signaling contribute to homeostatic functions or promote pathological roles of microglia, PBMs, and the meningeal lymphatics, precipitating Ab pathology. Working as a highly collaborative multidisciplinary team, we will utilize newly developed innovative tools to explore these hypotheses, including intra-vital imaging approaches, in-vivo microanalysis, new transgenic mouse lines and unique surgical approaches. The specific projects and cores are as follows: Project 1: Kipnis, PI: Parenchymal border macrophages in AD and CAA. Project 2: Holtzman, PI: CAA: Role of ApoE, innate immunity, and meningeal lymphatics. Project 3: Randolph, PI: Interplay between meningeal lymphatics, high-density lipoproteins and border macrophages in CAA and AD. Project 4: Colonna, PI: The protein tyrosine kinase Syk drives innate immune responses against AD. Core A: Administration (Kipnis, PI); Core B: Imaging and surgery core (Randolph, PI).

NIH Research Projects · FY 2025 · 2022-07

Human respiratory syncytial virus (RSV) is responsible for a major fraction of severe acute respiratory tract infections, including pediatric, elderly, and immunocompromised individuals, and thus has a global impact on human health. Despite this, there are limited prophylactic and specific treatment options available for RSV infections. RSV nonstructural proteins NS1 and NS2, which are unique to RSV among the pneumoviruses, play multiple major roles that are thought to enhance viral infection and to prevent protection from subsequent RSV reinfection. However, many questions remain that are related to how RSV virally encoded proteins shape the host response. Our unpublished preliminary studies show that NS1 partitions to the nucleus in physiologically relevant cell culture models, binds chromatin at promoters and enhancers of immune response genes, and impacts host gene transcription. In addition, we observe that nuclear NS1 interacts with viral proteins NS2 and matrix (M), an indication of further modulation of host factors in the nucleus by RSV viral proteins. Functional correlates of these observations are poorly understood. Based on these novel findings and to fill this knowledge gap, we propose to determine the molecular mechanisms of NS1 as a major modulator of the epigenome and host responses during RSV infection. Nuclear NS1 may also skew antiviral responses while enhancing immune evasion, thereby promoting viral pathogenesis. Our team, with complementary expertise in biochemistry/structural biology, epigenetics, transcriptional regulation, and pulmonology will define the impact of RSV NS1 on host epigenetic transcriptional control, define and characterize the molecular interactions that contribute to nucleocytoplasmic transport of NS1, and determine the impact of RSV NS2 and M protein interactions on NS1 nuclear functions. To obtain mechanistic insights and to assess the impact of these observations during viral infections, we will use an approach that integrates biochemical, structural, genetic, cellular, and virological studies in relevant cell culture models, including primary human lung derived cells. By completing these synergistic Aims, we expect to provide insights into key contributors to disease and define novel targets for antiviral and vaccine development.

NIH Research Projects · FY 2025 · 2022-07

Our prior work showed that Medicaid expansion under the ACA is associated with early detection and improved survival in patients with non-small cell lung cancer, the most common type of lung cancer. However, Medicaid enrollees with lung cancer still have much worse survival compared with their privately insured counterparts, which is driven predominantly by their higher risks of being diagnosed at more advanced stages and under-utilizing stage-appropriate cancer treatment. This suggests that having healthcare coverage is essential, but insufficient, to ensure access to lung cancer care and outcomes. Healthcare access has been conceptualized as a result of the interactions between factors across the individual, provider and healthcare system, neighborhood, and policy levels. However, prior studies addressing factors influencing lung cancer care and outcomes in low-income patients focused on patient characteristics. We and other groups demonstrated that geographic access to cancer care is associated with treatment in patients with breast or colorectal cancer. Medicaid enrollment pre- vs. peri-cancer diagnosis is associated with early-stage cancer diagnoses. Much less is known about the influences of neighborhood contexts, Medicaid enrollment continuation, and Medicaid coverage of lung cancer screening on lung cancer care in Medicaid patients. Tobacco cessation treatment is an integral and essential part of lung cancer treatment. No study has examined the utilization of tobacco treatment and its associated factors in low-income lung cancer patients, an oncology population with high prevalence of nicotine dependence. We hypothesize that neighborhood contexts, Medicaid policies, and hospitals play critical roles in lung cancer care and survival in Medicaid patients. Employing a robust multilevel modelling approach to account for clustering within providers and neighborhoods, we will assess the impacts of neighborhoods, policies, and hospitals on early detection and oncologic treatment for lung cancer, utilization of tobacco treatment, and lung cancer survival in Medicaid patients. To this end, we will develop integrated datasets for Medicaid enrollees diagnosed with lung cancer, which includes nationally representative cancer data, Missouri Medicaid claims and enrollment (also Medicare claims for dual eligible enrollees), Missouri Cancer Registry data, Annual Hospital Surveys, neighborhood contextual measures that we will develop. To our knowledge, this will be the first population-based study to comprehensively assess the contributions of factors at the neighborhood, health policy, and hospital levels to the lung cancer care continuum in low-income patients. The results will help inform interventions by suggesting potentially modifiable factors that could be targeted to improve cancer care in the low-income population. Addressing non-financial barriers to cancer care in Medicaid enrollees could maximize the likelihood that substantial investments in Medicaid programs will translate into true gains in cancer care access and outcomes for low-income patients.

NIH Research Projects · FY 2025 · 2022-07

Abstract/Project Summary: Dr. Umeshkumar Athiraman MD, is a neuroscientist and neuroanesthesiologist with the long-term goal to be an independent investigator focused on understanding the underlying mechanisms of anesthetic conditioning- induced neurovascular protection, development of anesthetic conditioning-based therapeutics for aneurysmal subarachnoid hemorrhage (SAH), and later application of these insights to other forms of brain injury. Dr. Athiraman is a member of Dr. Zipfel’s lab in the Department of Neurosurgery at Washington University in Saint Louis. The lab, department, and the university provide an exceptional training environment. Dr. Athiraman will receive training in the Zipfel lab in SAH animal models, immunohistochemistry, molecular biology techniques and assessment of short and long-term neurobehavioral outcomes after SAH. He will also receive training in optical imaging for functional connectivity assessment in the lab of collaborator, Dr. Adam Bauer. Additional support and mentorship will be provided by the applicant’s host department of anesthesiology. SAH is a severe type of hemorrhagic stroke with extremely high morbidity and mortality. Apart from the initial hemorrhage severity, secondary brain injury due to delayed cerebral ischemia (DCI) plays a significant role in patient outcomes after SAH. While many strategies to combat DCI have been developed in preclinical studies and tested in late phase clinical trials, none have proven efficacious for improving long-term functional outcome. The causes of these failures are likely multitude, but include use of therapies targeting only one element of what has proven to be multifactorial brain injury process. The proposed project examines the impact of a therapy known to have powerful, multifaceted protective effects on DCI after SAH called as – conditioning (anesthetic). Preliminary data shows that isoflurane conditioning provides robust protection against SAH-induced DCI and that this protection is likely mediated via inhibition of two critical molecules – NF-kB and iNOS. The planned experiments will rigorously test the following hypothesis through targeted genetic and pharmacological interventions: 1) Inhibition of NF-kB underlies the DCI protection afforded by isoflurane conditioning; 2) Inhibition of iNOS (a key downstream target of NF-kB) underlies the DCI protection afforded by isoflurane conditioning; and 3) Drugs that mimic the molecular effects of isoflurane conditioning (NF-kB inhibitor, PDTC-pyrrolidine dithiocarbamate; and iNOS inhibitor, 1400W) provide long-term protection against neurobehavioral and functional connectivity deficits after SAH. The results of these experiments will fundamentally establish NF-kB/iNOS pathway inhibition as the key inducer of isoflurane conditioning-induced DCI protection in SAH and identify NF-kB/iNOS inhibition as a promising new therapeutic strategy for SAH. The proposed plan will provide Dr. Athiraman with the training, mentorship and experience to transition to independence in a timely manner and obtain R01 funding.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Although coronavirus disease 2019 (COVID-19) is primarily defined as a respiratory illness, up to 50% of patients experience clinical gastrointestinal symptoms, including nausea, abdominal pain, and diarrhea. We recently found that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, infects and replicates in human small bowel intestinal epithelial cells (IECs). However, the molecular mechanisms by which SARS-CoV-2 drives gastrointestinal pathology and how that impacts the mucosal immune responses and systemic diseases remain unclear. In this proposal, our overall objectives are to better define the cellular signaling of SARS-CoV-2 interactions with IECs, enteric immune responses, and microbiome in the context of health or inflamed intestines (i.e., inflammatory bowel disease (IBD)). Our preliminary data suggest that compared to normal COVID-19 patients, those with IBD who have higher expression of cathepsin L in IECs prior to infection, supported higher viral loads. Our central hypothesis is that SARS-CoV-2 induces a distinctive intestinal pathology and mucosal immune response that is shaped by host factors (IBD), luminal factors (bile salts and microbiome), and therapeutics. Specifically, using healthy and IBD-derived organoids, COVID-19 patient fecal samples, SARS-CoV-2 variants, and a highly tractable and innovative intraluminal injection mouse model, we aim to (1) define the host factors and cellular mechanisms involved in SARS-CoV-2 infection of normal and IBD epithelium, (2) determine the impact of intestinal SARS-CoV-2 infection on intestinal immune response and colitis, and (3) define the environmental factors that influence intestinal infection with SARS-CoV-2. With extensive and collaborative expertise in intestinal biology, virology, and mucosal immunology (including IBD), we expect to address mechanistically interesting and clinically important questions regarding SARS-CoV- 2 enteric infection, immune responses, and intestinal pathology. We anticipate that the knowledge derived from this study will further our understanding of SARS-CoV-2 interactions with the epithelial and immune cells to explain COVID-19 GI symptoms with or without IBD. We also expect the new information gained from this project will expand our understanding of acute and chronic gastrointestinal pathology and symptoms of COVID-19, provide novel strategies to mitigate COVID-19 associated GI diseases, and create a foundational knowledge and tool set for deeper investigations into COVID-19 and potentially pathogenic and emerging coronaviruses of the future.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Cerebral edema is a major contributor to neurological deterioration and the leading cause of in-hospital death after stroke. This pathologic water accumulation results in an increase in brain volume that can be measured after most hemispheric strokes. This brain swelling not only raises the risk of cerebral herniation but also impairs stroke recovery as much as infarct growth does. However, the key biologic factors and molecular mechanisms that mediate formation of cerebral edema remain poorly defined. This knowledge gap has hindered development of targeted interventions to mitigate the consequences of edema in conditions as diverse as brain trauma, tumors, and hemorrhagic as well as ischemic strokes. There is significant variability between patients, with some exhibiting malignant edema and others with none to mild swelling despite similar stroke sizes and severities. The central objective of this proposal is to integrate imaging with genetics to identify key biologic pathways and mediators implicated in cerebral edema. We will acquire serial CT scans from 3,506 patients in an NINDS-funded stroke genetics study (GENISIS) and 1,000 being enrolled in an ERA-NET NEURON-funded study (iBioStroke). We will apply automated analysis pipelines to obtain quantitative multi-dimensional measurements of edema severity. Our primary biomarker is the displacement of cerebrospinal fluid (ΔCSF) that serves as a surrogate for the volume of swelling that has developed after stroke. However, we will also measure hemispheric CSF ratio and lesional water uptake as additional edema phenotypes. We will model edema formation (in relation to time from stroke onset) to evaluate the degree to which biologic factors, such as age, sex, glucose, blood pressure, and renal function, influence edema formation. Our central hypothesis is that inter-patient variability in edema formation can be linked to both targetable clinical factors such as hyperglycemia and informative genetic differences. Our preliminary data has suggested that ΔCSF has a significant heritable component. Specific Aim 1 seeks to quantify the relationship of key clinical factors, such as hyperglycemia and blood pressure, to edema formation. We will leverage genomic data to further dissect which factors are causative in edema formation, using Mendelian randomization. We will also quantify the impact of edema and hemorrhagic transformation on stroke recovery. Specific Aim 2 will identify genes and pathways associated with cerebral edema after stroke. It will employ genome-wide association (GWAS) approaches with multiple edema phenotypes in this large cohort. We will further prioritize genes and pathways using functional annotation tools. Specific Aim 3 will dissect shared versus edema-specific injury mechanisms by analyzing edema in relation to traits such as hemorrhagic transformation, white matter injury and small-vessel disease. It will leverage large existing datasets to boost the power of gene discovery from Aim 2. Once complete, this work will provide the first comprehensive picture of the genetic architecture of cerebral edema after stroke and provide unbiased, novel insights into molecular targets that can inform drug discovery.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Approximately 10% of pregnant women give birth preterm In the United States and worldwide, which not only results in a high rate of fetal mortality but also puts the children at a lifelong risk of negative health consequences such as cerebral palsy, mental retardation, and visual and hearing impairments. Despite years of research, the mechanisms of initiation and propagation of uterine contractions resulting in preterm labor and birth remain unknown. In large part, this is because of our limited ability to monitor the human uterine contractions with sufficient spatial and temporal resolutions. This leads to a lack of critical knowledge of the pathologic factors that alter the normal uterine maturation, initiate preterm labor, and result in preterm birth. In order to address this unmet clinical and research need, our team has recently developed a novel high-resolution and noninvasive electromyometrial imaging (EMMI) system, which uses up to 256 unipolar electrodes to measure uterine electrograms from the patient's abdomen surface and then combines the patient-specific body-uterus geometry obtained by magnetic resonance imaging (MRI) to generate accurate and robust three-dimensional maps of uterine electrical activity during contractions. Because such a powerful experimental tool could permit closer and more precise study of birth-related risks and improve maternal and child outcomes, we believe there could be a significant clinical impact for us to develop a low-cost, wireless, and wearable version in order to make this imaging technology more accessible for outpatient or in-home monitoring settings. We propose to develop and validate the functionality of a unique wearable EMMI system with printed disposable electrodes, wireless power delivery, and telemetry for continuously monitoring of the uterine contraction activities in ambulatory patients. The proposed research activity will involve developing of ultrathin soft sensor patches with printed stretchable electrodes for recording high quality electrograms from the patient’s abdomen and generating accurate and robust 3D maps of the uterine surface; investigating and designing a novel self-capacitance based wireless power transfer instrumentation for wirelessly powering all the sensing and telemetry circuits at each recording site in a fully distributed high-density imaging system; validating the wireless and wearable EMMI system in human subjects and benchmarking its performance against “gold standard” wired EMMI system. Upon successful completion of this study, the entirely new wearable, wireless, and batteryless imaging system developed in the project will facilitate EMMI's clinical translations, allow it to be used outside the delivery room for outpatient setting or in-home monitoring applications, and ultimately enable us to leverage the electrical mapping data for evaluating uterine electrical maturation and contraction patterns during pregnancy and labor and use the results to better understand and treat preterm birth.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY My career goal is to better understand systemic diseases in rheumatology by examining the mechanisms of ocular inflammation and determining which mechanisms are specific to the eye and which are shared with other organs, particularly the joint. This five-year proposal for a Mentored Clinical Scientist Research Career Development Award will help me mature into a productive, independent academic investigator in the field of ocular rheumatology. Advances in this area should complement the body of work in the field to allow for a more comprehensive understanding of systemic autoimmune disorders. Axial spondyloarthritis is strongly associated with HLA-B27 and has frequent extra-articular manifestations such as anterior uveitis, an inflammatory ocular disease that can lead to cataract, glaucoma, and permanent vision loss. While discrimination between pathogenic and bystander immune cells in rheumatologic diseases has been challenging, the intrinsic immune privilege of the eye increases the enrichment for pathogenic cell types. Therefore, studying ocular immune cells and the corresponding populations in the peripheral blood provides a novel opportunity to understand both organ-specific and systemic mechanisms of disease pathogenesis. Using single-cell RNA sequencing (scRNAseq) in three individuals, we have preliminarily identified two subsets of CD8 T cells that collectively accounts for ~50% of all CD8 T cells within the eye during HLA-B27+ anterior uveitis. While both types of CD8 T cells share a transcriptional program, such as expression of the surface receptor CD161, the antigen-specificity is distinct for each population. The first population is a conventional polyclonal CD8 T cell population that recognizes viral antigens, among others, but is able to express IL-17 and therefore have been termed Tc17 cells. In contrast, the second population is an unconventional T cell population that predominantly uses a single T cell receptor gene segment that specifically recognizes metabolites of riboflavin synthesis and are called mucosal-associated invariant T (MAIT) cells. Given the difference in antigen-specificity, we hypothesize that these two CD8 T cell types serve redundant roles in an antigen- independent mechanism of inflammation. We propose to establish whether MAIT or Tc17 cells play a central role in HLA-B27+ anterior uveitis by using scRNAseq and multidimensional flow cytometry to assess (1) whether one or both CD161+ CD8 T cell subtypes are conserved in HLA-B27+ anterior uveitis, (2) what mechanisms of activate these cells within the eye, and (3) whether one or both of these subsets are dysregulated in the circulation prior to entry into the eye. Through successful completion of this research, undergoing the didactic training, and receiving guidance from a multidisciplinary group of experts, I plan to launch my career as an independent translational researcher investigating the ocular manifestations of rheumatologic diseases.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract The goal of this project is to understand how CD53 regulates B cell trafficking, and to determine the CD53- partner network underlying this process. CD53 is a member of the tetraspanin family of transmembrane proteins that organize multi-protein networks on the cell surface to regulate a wide variety of cellular processes such as proliferation, homing and survival. Loss of CD53 is associated with impaired immune system function. CD53 is highly expressed on both normal and malignant B cells, however its role in these cells is not clear. We previously reported that CD53 is required for normal B cell development in the bone marrow. In preliminary data, we now find that CD53 is essential to both normal and malignant B cell trafficking, with the loss of CD53 causing significant impairment in B cell adhesion, migration, bone marrow homing and antibody production. To understand the underlying molecular mechanisms, we have determined the crystal structure of CD53; in the entire tetraspanin family, this the first structure captured in an active conformation. We reveal how conformational changes influence CD53 partner interactions by mass spectrometry (MS) based footprinting. We have used proximity labeling to identify several candidate CD53 partners, whose functions link chemokine signaling to cell motility. These data support our hypothesis that CD53 coordinates a complex of adhesion, signaling and cell motility proteins that facilitate B cell trafficking. Both normal and malignant B cells rely on accurate trafficking to their niches in the bone marrow and secondary lymphoid organs to optimize their maturation and function. Thus, an understanding of the interactions that guide B cell trafficking are important not only for optimizing normal B cell function, but will also reveal potential therapeutic targets of malignant B cells. Using a combination of in vitro and vivo adhesion and migration studies in combination with proximity labeling, quantitative MS, live-cell MS- based footprinting, biochemical and electron microscopic analyses, this dual-PI proposal presents a 5-year plan to: 1) elucidate the CD53-partner interaction network regulating B cell trafficking, and 2) determine the functional consequences of disrupting the CD53-partner interactions. Armed with our newly developed tools, we will reveal a novel network of protein interactions coordinating cell signaling and motility to regulate B cell adhesion, migration, chemokine signaling and niche localization. We will uncover how this CD53-mediated network responds to chemokine signaling during B cell migration and how cross-cell interactions are established during B cell homing. Thus, the proposed studies will significantly advance our understanding of how B cell trafficking is coordinated and regulated. Given the conserved structure and functional redundancy of tetraspanin family members, this will lead to our long-term goal of elucidating tetraspanin/partner relationships that apply to other roles of CD53 in immune system function and malignancy as well as the functions of other tetraspanin family members.

NIH Research Projects · FY 2025 · 2022-07

Project Summary / Abstract Obesity-related metabolic disorders, including insulin resistance, type 2 diabetes, and non-alcoholic fatty liver disease, are thought to be caused by low-grade non-infectious inflammation as a result of lipid-mediated immune cell activation, particularly macrophages. However, it has become clear that focusing on macrophage inflammatory signaling is overly simplistic and fails to explain the complex relationship between increased immune cell recruitment to metabolic tissues and disease pathogenesis. Phenotyping of macrophages in adipose tissue and liver demonstrates induction of a lysosomal lipid metabolism program in diet-induced obesity, suggesting that macrophage recruitment to lipid-overloaded tissues might be compensatory in nature. If so, then enhancing a cell-intrinsic program of lipid hydrolysis and metabolism in macrophages should have beneficial metabolic effects. In preliminary work, we show that overexpression of macrophage TFEB, a master transcriptional regulator of lysosomal and fatty acid oxidation genes, enhances the ability of macrophages to metabolize lipids in vitro and ameliorates diet-induced metabolic dysfunction in vivo, presumably because of reduced fatty acid release from adipose tissue. In this proposal, we will investigate the consequences and mechanisms by which activating macrophage lysosomal lipid metabolism in distinct macrophage subsets impacts obesity-induced metabolic dysfunction and fatty liver disease. In Aim-1, we will explore the role of TFEB in adipose tissue macrophages (ATM) in regulating adipose tissue and systemic metabolic function in obesity. This includes assessing TFEB’s effects on the metabolic handling of lipid-rich exosomes and dying adipocytes found in crown-like structures. Our findings in mice will also be confirmed in a human population where we will determine if increased lysosomal lipid metabolism in ATM associates with metabolically normal or metabolically abnormal obesity. In Aim-2, we will utilize unique genetic models to determine the impact of TFEB in liver resident Kupffer cells vs. recruited monocyte-derived macrophages on diet-induced steatosis and fibrosis. Taken together, this proposal will test the hypothesis that induction of a macrophage lysosomal lipid degradation- mitochondrial fatty acid oxidation gene network via TFEB will enhance macrophage lipid handling and could be leveraged to treat obesity-associated insulin resistance and fatty liver disease.

NIH Research Projects · FY 2025 · 2022-07

Abstract Advanced studies in neuroscience increasingly require sophisticated mathematical and computational skills in addition to a strong knowledge base in neurophysiology. Likewise, novel treatments of neurological diseases often require problem solvers (i.e. engineers) with both advanced computational skills as well as a solid foundation in systems neuroscience. The fundamental premise of this training program is that early engagement of computationally strong students, principally engineers, in understanding fundamental mechanisms of neuroscience will accelerate development of technology and solutions for the diagnosis and treatment of stroke, Parkinson’s disease, amyotrophic lateral sclerosis, traumatic brain injury, pain, epilepsy, paralysis, and other neurodegenerative disorders. With long-established research experience in these disorders existing at Washington University in St. Louis, we propose a training program that will develop common skill sets in engineering doctoral students towards solving difficult problems in neural engineering. Graduates of this novel training program will have the knowledge and expertise to pursue either an academic research career and/or a private sector research career in neurotechnology. We request support for four predoctoral students in the proposed five-year program. Cohorts will include five total predoctoral positions, each of two years in duration.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Atrophy and fibrosis of skeletal muscle after neuromuscular trauma is a significant impediment to the restoration of function after severe neuromuscular trauma. Despite this, dynamic assessment tools for muscle wasting and dysfunction are limited, leaving a critical gap in the orthopedic surgeon’s ability to assess the degree of neurogenic muscle injury and its ultimate prognosis. This gap stems in part from an incomplete understanding of the role of increased expression of transcriptional factors and proteases related to atrophy, and inability to dynamically assess them clinically. Calpain is one of these proteases central to the myofibril destruction of neurogenic atrophy, and therefore has potential to serve as a marker of muscle atrophy. However, translation of this relationship into a diagnostic tool is limited by a lack of techniques for real time assessment of calpain activity. The proposed work seeks to explore the potential for use of optical probes to identify muscle atrophy by examining the relationship between nerve injury and muscle contractility and calpain activity. Aim 1a will determine if calpain expression and activity will increase proportionally with nerve injury and muscle dysfunction. Equal numbers of male and female mice will be subjected to a varying degree of unilateral sciatic nerve crush injury. At a subsequent surgery, at staged intervals, functional recovery will be assessed with walking track analysis and grip strength testing. Hindlimb muscles will undergo ex-vivo contractility testing, as well as histomorphometric analysis and relevant transcriptional factors will be assayed. Calpain activity will be quantified with ELISA kits and with use of a pre-clinical imaging system to detect near-infrared fluorescence (NIR) within the hindlimb muscles after administration of an injectable calpain sensitive probe. In Aim 1b, a unilateral sciatic nerve transection and repair will be performed in the mice, and the same series of functional tests, transcriptional assays and NIR imaging with the optical probe will be undertaken. Similarly, Aim 1c will utilize the same methodologic assessments, at the same time intervals, after removal of a segment of sciatic nerve. The increasing degree of nerve injuries and proposed assessments will help to delineate the canonical pathways of muscle atrophy after nerve injury, and the proposed optical probe will provide a powerful new diagnostic tool. As an orthopedic surgeon with a practice devoted to the care of mangled limbs, I understand the clinical impact of such injuries, but need protected time and resources to develop the skills to study these at a molecular level. In addition to the investigations described above, I will participate in graduate coursework to improve my understanding of molecular biology, as well as optical and biological imaging. I will regularly participate in scholarly activities such as journal clubs and grant seminars through the Musculoskeletal Research Center to enhance my grant writing abilities and improve my understanding of experimental methodologies. In addition to my primary and secondary mentor, I have assembled a mentoring committee to give feedback on results and assist with experimental design. This constellation of planned activities, along with the proposed research methods above will provide me the requisite training and experience to develop as a clinician scientist with an interest in optical imaging of skeletal muscle atrophy.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT The olfactory system encodes and analyzes odorants. Chemicals bind to members of large families of receptor proteins tuned to different chemical features. While we have a comprehensive inventory of the receptor genes that underlie olfactory detection, we are far from having a similarly comprehensive understanding of the chemical features detected by olfactory systems. This proposal leverages four developments—the ability to record the output of an entire olfactory system (the vomeronasal system), a large and structurally-rigid class of odorants (sulfated, carboxylated, and glucuronidated steroids), a new tool to identify the receptor gene(s) that have particular ligand detection profiles, and a new tool for ectopically expressing a chosen receptor gene and studying its function—to develop the first system-wide understanding of how an olfactory system represents the chemical world. The overarching goal is to develop insights analogous to (and with similar predictive power to) the role of cone photoreceptor tuning curves in our understanding of color vision. The specific aims of the proposal are (1) to reveal new principles of combinatorial coding through a system-wide analysis of the vomeronasal system’s coverage, redundancy, and specificity via a large ligand screen and quantitative analysis of the structural features required for activating each receptor; and (2) to reveal relationships between receptor sequence and odorant structure by deorphanizing a large subfamily of vomeronasal receptor genes. Preliminary data suggest that structural rigidity of steroid metabolites contributes greatly to the tractability of these aims, supporting quantitative and predictive analysis on a system-wide scale.

NIH Research Projects · FY 2025 · 2022-07

(must be no longer than 30 lines of text). This R25 Short-Term Research Education Program is titled “Washington University (WU) summeR undergrADuate and health professIons reseArch program iN Cardiovascular diseasE & hematology (RADIANCE)”. The program leverages our extensive experience with two existing programs: 1) the NHLBI- funded “PRograms for IndiviDuals Engaged in Health-Related Research (PRIDE)”, which train junior faculty nationwide, and 2) the WU Institute for Public Health Summer Research Program (IPH-SRP) – Public and Global Health track, which has trained undergraduate and health professional students in health research over the past eight years. Our success with these summer research programs, and our established infrastructure, positions us to seamlessly expand and integrate this new initiative. Cardiovascular disease (CVD) and Hematologic Disorders are leading causes of morbidity and mortality in the United States and globally. We aim to recruit, train, mentor, and develop research skills in 12 undergraduate and health professional students each year. The program objectives are as follows: 1) Recruitment and Engagement: To attract students with interests in research careers, using a multidisciplinary approach to advance research on CVD and Hematologic Disorders; 2) Training and Mentoring: To provide rigorous didactics, structured mentoring, and hands-on research experiences that build knowledge, attitudes, and skills in biomedical, behavioral, and clinical research; 3) Exposure and Development: To foster trainee interests in research, academic, and professional development in CVD and Hematologic Disorders, and to provide research experiences that inform future career and training opportunities; 4) Capstone Experience: Under guidance from mentors and program leadership, to prepare a “capstone experience” summary presentation at the end of the program that synthesizes the trainee’s learning and research experience. The overarching goal of RADIANCE is to increase the biomedical research workforce through targeted educational activities focused on research experiences and mentoring. This proposal aligns with NHLBI's scientific priorities by: 1) delivering comprehensive CVD/Hematologic Disorders teaching and research training; 2) providing a robust scientific research foundation for trainees; and 3) ensuring program access and participation for undergraduate and health care professional students. NHLBI-funded WU Program and Mentoring Faculty will provide trainees with excellent mentorship and experience. We are confident that this initiative will contribute significantly to enhancing the development of the research workforce in areas critical to the NHLBI mission.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Neurotechnologies used to treat brain disorders and diseases can drastically change brain function and behavior, monitor brain activity, and collect and transmit personal health data. Industry-academia (IA) partnerships play a critical role in bringing neurotechnologies to market for public benefit. However, there are significant ethical issues that emerge from these partnerships, especially given the unique capacities of neurotechnologies. If these ethical challenges are not handled appropriately, scientific integrity can be undermined and the objectivity in the design, development, and commercialization of neurotechnologies can be jeopardized. This project addresses a gap in our current understanding of ethical complexities in industry-academia (IA) partnerships within the neurotechnology enterprise by engaging relevant neurotechnology stakeholders (e.g., industry, researchers, ethicists, university officials, patients) to identify promising and practical solutions for addressing these ethical complexities. This project builds logically across three aims that culminate in the development, dissemination, and evaluation of a toolkit that includes stakeholder-informed guidance for ethically and responsibly navigating IA neurotechnology partnerships. Aim 1 will use in-depth interviews with neurotechnology stakeholders to identify risk factors and ethical challenges for IA partnerships, including in the design, conduct, reporting, and application of neurotechnology research. Aim 2 will involve a Delphi consensus panel of neurotechnology stakeholders to establish best practices and solutions for mitigating risk and addressing ethical issues that emerge from IA partnerships. Aim 3 will include developing, disseminating, and evaluating a toolkit that provides guidance and resources for neurotechnology stakeholders to help them ethically and responsibly navigate neurotechnology IA partnerships. We will recommend adoption of specific solutions for each stakeholder group. This is the first project to systematically and simultaneously engage diverse neurotechnology stakeholders to develop practical and actionable guidance for ethical and responsible IA neurotechnology partnerships. We will develop the first publicly accessible stakeholder-informed toolkit that can be adapted to current and forthcoming neurotechnologies resulting from IA partnerships. Taken together, this project will have considerable impact on the conduct of neurotechnology research by identifying practical solutions for balancing scientific values with fiduciary goals.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Heightened performance monitoring and overcontrol (HPM/OC) is a transdiagnostic phenotype comprised of perfectionism, extreme concern for errors, cognitive inflexibility and excessive need for control. There is increasing evidence that the HPM/OC phenotype is identifiable in early childhood and underlies obsessive- compulsive disorder (OCD), social anxiety disorder (SAD) and anorexia nervosa (AN). These psychiatric disorders are severe, chronic, and treatment-resistant disorders with high rates of comorbidity. However, there is surprisingly little research examining the neurobiological and behavioral mechanisms that make up the HPM/OC phenotype in early school-age childhood in relation to the development of transdiagnostic psychiatric symptoms. Moreover, it is unknown how the HPM/OC phenotype interacts with rapid developmental progression during this age or is influenced by social-contextual features, such as social evaluation, peer rejection or parenting styles. Determining answers to these fundamental questions could provide insight into the emergence of the HPM/OC phenotype and provide novel treatment targets for early intervention. The goal of the current proposal is to improve understanding of the HPM/OC phenotype in early childhood in relation to early markers of OCD and SAD by assessing the developmental progression of cognitive facets of performance monitoring and reactive versus proactive cognitive control and the influence of social-contextual features on psychiatric outcomes. We posit that cognitive processes that make up the HPM/OC phenotype, including heightened performance monitoring and more reactive versus proactive cognitive control, interact with various social contexts to differentiate early school-age children with impairing HPM/OC. The early school- age period is when HPM/OC is first evident and is a time of rapid cognitive and social development, making it a pivotal time to understand the developmental psychopathology of this presentation. We will employ cutting- edge and cost-effective electroencephalogram (EEG) event-related potential (ERP) and time-frequency (TF) analyses to examine multiple Research Domain Criteria (RDoC) constructs across three repeated yearly assessments in a sample of 300 community children oversampled for elevated dimensional HPM/OC, ages spanning 4 - 9 years. This fine-grained evaluation will provide an opportunity to characterize 1) how HPM/OC impacts normative neurodevelopmental trajectories of RDoC constructs during an age of high neural plasticity, 2) the developmental progression of cognitive facets of the HPM/OC phenotype in relation to transdiagnostic psychiatric impairment and 3) the impact of social-contextual features that may influence these relationships. This knowledge could have far-reaching effects on our understanding of the early neurodevelopment of the HPM/OC phenotype prior to disorder onset, which could inform early identification of high-risk children and targeted early interventions that could lessen the severity, course and impairment of multiple psychiatric disorders across the lifespan.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Ascending thoracic aortic aneurysm (ATAA) is a major cardiovascular health problem characterized by a dilated aorta that may eventually dissect or rupture. ATAA presents a serious challenge in that the surgery is difficult and dangerous, so aneurysm repair criteria must balance the risk of a dissection and/or rupture with the risk of surgery. Current surgical guidelines are based on ATAA diameter or growth rate, but up to 60% of patients with an ATAA experience a dissection before surgical criteria are reached, hence there is a clear need for additional biomarkers of aneurysm failure. Possible biomarkers fall into broad categories including genetic, microstructural, geometrical, and biofluids, but it is challenging to obtain enough human data to calculate and correlate these biomarkers with critical outcomes such as failure. It is likely that a single biomarker is not sufficient, but composite biomarkers that are not intuitively obvious may be necessary for significant predictions of patient outcomes. In this proposal we will use a combination of models: 1) a mouse model of ATAA associated with Marfan Syndrome, 2) a multiscale, multiphysics model of ATAA growth and remodeling, and 3) virtual patient models derived from real patient imaging data, to determine composite biomarkers that may predict ATAA growth, progression, and failure. Our first Specific Aim is to use a genetic mouse model of ATAA associated with Marfan Syndrome to characterize aneurysm progression and failure in previously unachieved detail, quantifying aortic shape, tissue composition, tissue mechanical properties, and hemodynamics over time. This level of detail is not possible in human patients and is necessary to validate and test hypotheses on the growth and remodeling rules in our multiscale, multiphysics model in Specific Aim 2 and to provide an initial set of biomarkers to evaluate for our virtual patients in Specific Aim 3. Our second Specific Aim is to develop a novel multiscale, multiphysics computational model of ATAA growth and remodeling to produce results that will be compared to the mouse data in Specific Aim 1 and used to predict remodeling progression in real and virtual human patients in Specific Aim 3. In our third Specific Aim, we will use available human ATAA scans from Marfan Syndrome patients to generate a statistical shape model basis for the ATAA geometry, and we will use that basis to generate virtual patients, whose TAA course throughout progression and failure will be created by the model in Specific Aim 2, with parameters determined from published literature and our mouse data in Specific Aim 1. Both real and virtual patient data will then be used to train a machine learning tool to relate the composite biomarkers to the remodeling outcomes and predict failure risk. This plan synthesizes multiple recent advances and supplements them with new ideas to produce a computer system capable of making useful failure predictions for ATAA.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Neurodevelopmental disorders (NDDs) affect >1% of the population and are comprised of multiple phenotypes including autism, intellectual disability, and other developmental delays. Over the last ten years, there has been considerable progress in understand the contribution of rare protein- coding variants in NDDs. However, the role of rare noncoding variation has been less clear due to the limited number of individuals with whole-genome sequencing (WGS) data previously available. We were one of the first to identify aggregate enrichment for promoters and enhancers in the first 516 families assessed by WGS; signals that have now been seen by multiple additional groups. Currently, we have analyzed ~2,700 families with autism and identified enrichment in a top enhancer. In Aim 1 of proposed studies we will expand this work to analyze 10,719 families with NDDs to identify specific noncoding regions using statistical tests to identify enrichment based on sequence context (fitDNM), enriched clustering of variants (GRUMP), and a transmission-disequilibrium based test, respectively. We will perform massively parallel reporter assays (MPRA) to follow up specific noncoding variants in the enriched elements and combine this with deep phylogenetic assessment using existing and new reference genomes (ACES). Our recent work suggests that dosage sensitivity is an important characteristic to consider when testing specific promoters and enhancers for enrichment in NDDs. In Aim 2, we will build a copy number map from 92,081 individuals, without NDDs, from the Centers for Common Disease Genomics dataset. We will specifically assess 1,327,879 noncoding regulatory regions for dosage-sensitivity in the human genome. As in Aim 1, we will follow up identified elements using MPRA and our ACES phylogenetic workup. The primary goal of proposed studies is to improve our understanding of noncoding variants in NDDs. We will do this by 1) identifying noncoding regulatory regions with enrichment of variation in NDDs and 2) developing a dosage sensitivity map of noncoding regulatory regions to inform studies of NDDs. This unique proposal, with its mirrored aims, focuses on noncoding variation as opposed to the majority NDD focus on protein- coding regions and it tests dosage in a large high-quality WGS dataset. This work will be integral to the genetic understanding of NDDs and the consequences of noncoding variation. It will also provide key insights into their role in the overall genetic architecture of NDDs.

NIH Research Projects · FY 2025 · 2022-07

The gram-negative outer membrane (OM) is asymmetric, with phospholipids on the inner leaflet and lipopolysaccharide (LPS) on the outer leaflet. A defining part of the gram-negative cell envelope, LPS is a major signal for infection in animals and is a target of last resort antibiotics, such as colistin. Normally essential, clinical use of colistin has selected for Acinetobacter baumannii strains that do not produce LPS. Although LPS has been implicated in diverse functions, its precise role in gram-negative biology is unclear. Recent work in E. coli suggests LPS is capable of contributing as much rigidity to the cell envelope as the cell wall, a function that requires polyionic interactions between negative moieties on LPS and divalent cations. Analysis of conditional E. coli mutants defective in LPS synthesis and transport revealed formation of filaments and cell chains, suggesting an additional role for LPS in cell division and separation. The essentiality of LPS in either process is unclear. To evaluate the essential role(s) of LPS in gram-negative biology, I will leverage a set of E. coli mutants to conditionally knockdown LPS synthesis and transport, manipulate LPS charge (and thereby its interactions with divalent cations to provide rigidity), and produce a minimal LPS structure. Using this collection in Aim 1, I will systematically characterize the effect(s) of LPS defects on cell growth and morphology to understand the contribution of LPS to both phenomena. In Aim 2, I will test my hypothesis that providing cell envelope rigidity is a primary, essential function of LPS. Aim 2.1 will evaluate the ability of hyperosmotic conditions (which reduce the force exerted by turgor pressure on the cell envelope) to compensate for LPS defects. Because the force of turgor pressure is spread between the cell wall and the OM, Aim 2.2 will test whether increasing rigidity of the cell envelope via cell wall crosslinking can compensate for LPS defects. In Aim 3, I will identify the steps in cell division and separation impacted by defects in LPS synthesis and transport, respectively. This effort will illuminate the mechanistic basis of filamentation and chaining in LPS mutants. Examination of A. baumannii LPS deletion mutants identified a correlation between division and cell survival. I am thus particularly interested in testing whether enhancing division using complementary genetic strategies promotes growth of LPS deficient E. coli. If enhancing cell envelope rigidity is an essential role of LPS, additional septa may offset lethality associated with LPS defects through a positive impact on the structural integrity of the cell as a whole. A major signaling molecule for the immune system and target for last resort antibiotics, a better understanding of the essential role of LPS in gram-negative biology will provide insights into mitigation and treatment of gram- negative pathogens. Through this F31 fellowship, I will develop expertise in microscopy, protein biochemistry, and single cell analysis. Further, it will grant me the time and support necessary to hone my skills as a scientific mentor and communicator to prepare me for a career as a professor and independent investigator.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY/ABSTRACT Gastrointestinal parasitic infection is a widespread global health problem and can be a cause of significant morbidity. Nevertheless, there is limited information on how T cells respond to these parasites. In some cases, despite a robust T cell effector response to the parasite, tolerance to food antigens is maintained or enhanced. This has led to the notion that chronic parasitic infection could explain in part the hygiene hypothesis, which states that elimination of colonization or reduction of recurrent infections due to sanitation and pest control increases the risk of allergic and autoimmune diseases. We aim to understand how intestinal parasites induce effector T cell responses to parasitic antigens, and yet facilitate tolerance to food antigens present in the intestine. We will study two parasites that induce opposite T cell effector responses in mice. We will use a newly identified strain of Cryptosporidium tyzzeri, a commensal protozoan that induces a Th1 response, and H. polygyrus, a helminth that induces a Th2 response. The rationale for using parasites which elicit disparate T helper responses is to uncover potentially convergent mechanisms that may underly prevention of immunopathology during anti-parasitic responses and maintenance of tolerance to innocuous food antigens. In each infection, we will use TCR repertoire analysis to identify parasite-specific TCRs expressed by CD4+ T cells. This approach will allow us, for the first time, to precisely track the fate of such parasite-reactive T cells during these infections as they differentiate from naïve T cells to effectors or regulatory subsets. Given the central role of the transcription factor Bhlhe40 in regulating effector function and IL-10 production, we will also test its role in parasite-specific T cell fates and the development of a Tr1 phenotype which may provide immune tolerance to these parasites. In Aim 1 we will characterize the anti-parasite T cell response to H. polygyrus. In Aim 2, we will evaluate the mechanisms that regulate immune tolerance to Cryptosporidium. In Aim 3, we will determine how infection with these parasites impacts on the tolerogenic T cell response to food antigens and on the induction of food allergy. Overall, these studies will provide novel and important insights into how parasitic infections drive effector T cell responses to parasite antigens while simultaneously promoting tolerogenic or pathogenic responses to food antigens.

NIH Research Projects · FY 2026 · 2022-06

Sexual minority adolescents (SMA; ages 13-17) experience higher rates of intimate partner violence (IPV) than their heterosexual peers, including being more likely to experience psychological and physical abuse, and being four times more likely to experience rape. However, this emerging body of literature has a number of significant limitations. First (Aim 1), studies have largely failed to look beyond victimization of IPV and have not measured IPV perpetration among SMA. Second (Aim 1), studies of IPV among SMA have approached SMA as a homogenous group and have not identified within-group differences in SMAs’ experience of IPV, including by race, ethnicity, sex, or urbanicity. Research has been inconclusive in these regards, making hypotheses difficult, although differences may inform health interventions. Third (Aim 2), identified associations between the experience of IPV and negative health outcomes (i.e. mental health symptomology, substance use, homelessness) are largely cross-sectional: the temporal relationships between IPV and behavioral health for SMA across adolescence remains unclear. Fourth (Aim 3), almost nothing is known of the unique etiological factors that influence IPV experiences for SMA. While cross-sectional studies have identified relationships between sexuality-based minority stress and sexual assault, the prospective literature is nearly nonexistent. Finally (Aim 3), even less is known about how SMA-specific protective factors (such as access to SMA-friendly services) may attenuate IPV trajectories. In general, the only longitudinal studies of IPV among SMA to date are limited by (a) the inclusion of violence only in the context of dating; (b) a failure to assess perpetration or bidirectional IPV; (c) a reliance on single-city urban samples largely composed of adults (18+); and (d) the inclusion of no, or limited, measures of minority stress and resilience. We propose to address these limitations, and to explain the prospective relationships between IPV experiences (including victimization and perpetration), risk and resilience factors, and other behavioral health outcomes among SMA (ages 13–17 at baseline). We rely on methods refined in our prior work (1R01MD012252) to recruit a national sample of SMA (N = 1,500) through a hybrid social media and respondent-driven sampling strategy. We will follow participants for 36 months. Proposed by established PI (Goldbach) and Co-Is (Rhoades, Schrager) in this area, our efforts are centered on identifying targets for future interventions to reduce the significant burden of IPV carried by this population.