Johns Hopkins University

NIH Research Projects · FY 2026 · 2023-02

Summary More than 70 million people worldwide are infected with hepatitis C virus (HCV), and development of a vaccine for HCV is essential for disease eradication. Although direct-acting antivirals are highly effective for treatment, the majority of countries surveyed are not on track to reach the WHO goal of eliminating HCV as a public health problem by 2030, with most countries seeing rising incidence of HCV. Fortunately, there is strong evidence that vaccine-induction of high titers of broadly neutralizing antibodies (bNAbs; antibodies capable of neutralizing diverse HCV variants) could provide protection against human HCV infection. However, we are unable to stimulate bNAbs against HCV with a vaccine because we have not defined the full spectrum of anti-HCV antibodies that are critical for neutralizing breadth or characterized the HCV envelope glycoprotein (E1 and E2) genetic and structural features that favor bNAb selection and maturation. The overarching goal of this proposal is to isolate a large and representative set of E1E2-specific bNAbs, bNAb unmutated ancestors, and bNAb intermediates from Elite Neutralizers (EN), individuals with broadly neutralizing plasma and spontaneous clearance of HCV infection. In Aim 1, we will isolate monoclonal antibodies (mAbs) from E1E2-specific B cells isolated from EN and from controls with chronic, persistent infection (CP). We will infer unmutated germline bNAb ancestors and use B cell receptor-sequencing of longitudinal E1E2-specific B cells to identify bNAb genetic intermediates. We will compare neutralizing breadth, potency, epitopes, and genetic features for EN vs. CP mAbs. In Aim 2, we will use X-ray crystallography or cryo-EM techniques to compare structures of EN bNAbs or CP mAbs in complex with soluble E2 or E1E2 heterodimers. By comparing mAb-E1E2 interactions among CP mAbs, bNAb unmutated ancestors, bNAb intermediates, and mature bNAbs, we will define structural and genetic features of E1E2 necessary for the development of neutralizing breadth. Together, these studies will identify a large, representative set of bNAbs associated with spontaneous clearance of HCV, defining key epitope residues and structural features in E1E2 that could be stabilized to optimize vaccine antigens. These studies will inform structure-based design efforts to improve E1E2-based vaccine candidates, which is an urgent challenge with global public health implications.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY Primary Dysautonomia (PD), distinct from the entity called familial dysautonomia, is a multifactorial condition that runs in families in which the autonomic nervous system (ANS) does not function correctly leading to a range of disabling disease symptoms. Despite the known fact that PD exhibits Mendelian inheritance patterns, underlying genetic origins have not been identified. Treatments are solely based on alleviating symptomatology and there is no rationale for effectuating a cure. To help fill this knowledge gap and start identifying contributing factors to PD, we closely followed sixty-nine families with a dominant Mendelian inheritance pattern of multiple shared symptoms including (i) chronic orthostatic intolerance, (ii) chronic fatigue, (iii) primary focal hyperhidrosis, (iv) chronic itch, and (v) generalized anxiety. We initiated whole-exome sequencing (WES) of the probands in these families with resulting data suggesting a causal relationship between PD and an autosomal dominant inheritance pattern of mutations in genes encoding voltage-gated sodium (NaV) channels. This outcome is strengthened by the fact that we have treated family members with NaV channel modulators which resolved many of their complaints. Based on preliminary results, we hypothesize that what appears to pathophysiologically link diffuse autonomic symptoms, is a disease model in which NaV channel mutations can transform the ANS into an oversensitive nervous system that over time develops incapacitating and persistent disease. To start investigating the involvement of NaV channel mutations in PD, we devised a strategy to help elucidate the link between our targets and anomalous activation of the sympathetic nervous system in patients. Our approach will culminate in the creation of a validated animal model with autonomic dysfunction in which human therapeutics can be tested. Successful completion will provide new insights into genetic causes of PD, a vital step towards understanding the multifactorial nature of this disorder.

NIH Research Projects · FY 2025 · 2023-02

Project Summary Although we spend a third of our lives sleeping, the function of this evolutionarily conserved behavior remains elusive. Most studies investigating the function of sleep have focused on sleep in adults. However, young animals spend an even greater proportion of their lives asleep, and their sleep is deeper and has distinct electrophysiological features. Sleep in young animals is thought to aid brain development by promoting plasticity. These plastic processes occur during critical periods in which neural circuits are shaped by experience. Yet, the molecular, cellular and circuit mechanisms by which sleep functions in critical period plasticity remains elusive. This proposal aims to address this question by using a simple neural circuit in a genetically tractable model organism. In Aim 1, I will define sleep substages using behavioral features and statistical modeling, as well as imaging methods, and then determine if critical period plasticity requires a specific form of sleep. In Aim 2, I will investigate the neurophysiological processes by which sleep promotes critical period plasticity using patch clamp electrophysiology and investigate the genes that are important for this process using single cell RNA sequencing. In Aim 3, I will perform a large-scale RNAi screen using a behavioral assay to find novel genes that are involved in sleep-dependent critical period plasticity. In addition, I will investigate whether sleep-related neural activity is generated by astrocytes to aid critical period plasticity. Overall, these aims will delineate the function of specific sleep states in young animals and identify cellular and molecular mechanisms underlying sleep-dependent critical period plasticity. Because sleep disturbances in early life are predictors of neurocognitive disorders such as autism and attention deficit disorders, this work may have implications for the treatment of neurodevelopmental conditions. To achieve these aims, I have brought together a mentoring team which includes experts in computational modeling, transcriptional profiling, and developmental neural plasticity. In addition, the proposed career development and research plan will capitalize on the exceptional environment, facilities, and resources that Johns Hopkins University provides. My overarching career goal is to obtain a tenure track faculty position and establish my own research program as an independent scientist, and the proposed work and training will form a strong foundation for achieving this goal.

NIH Research Projects · FY 2026 · 2023-02

Modified Project Summary An estimated 41% of adults in the US and one in five of children are estimated to have obesity; over 37 million Americans have diabetes and 96 million adults have prediabetes. The prevalence of these conditions continue to grow, highlighting the need for policies addressing their root causes. The federal government’s new, $50 million Community Choice Demonstration seeks to test strategies that help low-income households move to low poverty neighborhoods. The Demonstration offers an exciting, time-sensitive chance to study the impact of this new targeted housing mobility program on obesity and diabetes risk, and, more broadly, provides a window into the mechanisms through which neighborhoods impact obesity and diabetes risk for low-income populations. Starting in the Fall of 2022, approximately 16,000 households will take part in a randomized controlled trial: one group will receive comprehensive mobility services such as security deposit assistance and landlord outreach to facilitate moving to a lower-poverty neighborhood and a control group will not receive mobility services. This proposed project, the Mobility Opportunity Vouchers to Eliminate Diabetes and obesity (MOVED) Study, will recruit 900 households enrolled in the Demonstration from three sites (Pittsburgh, PA; Cleveland, OH; Nashville, TN). We will conduct baseline and 2-year follow-up in-person surveys to investigate the extent to which the receipt of comprehensive mobility services is associated with changes in measured BMI and HbA1c among adult and changes in BMI z-score among children after 2 years compared to the control group (Aim 1). Aim 2 investigates outcomes along the causal pathway, focusing on potential behavioral, psychosocial, and contextual factors that may differ between the intervention and control groups. Data for this aim includes validated questionnaires, geographically-derived data, and, among a subset of participants, accelerometry data on physical activity and sleep. Aim 3 uses in-depth qualitative interviews to delve deeper into potential mechanisms through which moving to a lower-poverty neighborhood influences obesity and diabetes risk. The proposed research answers recent national calls for innovative research on the impact of housing mobility and neighborhoods on health and offers a time-sensitive chance to provide foundational, policy-relevant knowledge designed to reduce the impact of obesity and diabetes. Modified

NIH Research Projects · FY 2026 · 2023-02

Lena Mathews, MD, MHS is an Assistant Professor in the Division of Cardiology at Johns Hopkins University. Dr. Mathews is applying for a Mentored Patient-Oriented Research Career Development Award in order to obtain the skills, knowledge and research experience to provide the foundation for a career as an independent investigator using implementation science to address cardiovascular health disparities. Dr. Mathews’s career development plan includes interdisciplinary mentorship from Drs. Kunihiro Matsushita, Chiadi E. Ndumele, and Kristin Riekert; didactics and directed learning; and mentored clinical research adapting and implementing a theory-based multi-component intervention to increase cardiac rehabilitation (CR) use among individuals with low socioeconomic status. The specific aims of the research agenda are to: 1) Quantitatively evaluate barriers to CR referral and enrollment in a hospitalized population by SES, accounting for race, sex, and other sociodemographic characteristics; 2) Qualitatively elucidate perceived barriers to CR utilization in a clinical population with low socioeconomic status; 3) Adapt and test the feasibility and acceptability of a navigator intervention, delivered by community health workers and refined by engagement of community stakeholders, to increase CR utilization among individuals with low socioeconomic status. She seeks to build upon her research fellowship training in epidemiology and biostatistics, and her clinical background as a Cardiologist and the Director of Cardiac Rehabilitation at Johns Hopkins, by acquiring additional skills and expertise required to achieve her career goals. This Mentored Patient-Oriented Research Career Development (K23) Award will provide her with the opportunity to: 1) develop skills in advanced epidemiology and health utilization research using electronic health records 2) develop expertise in qualitative methods, to inform intervention design and evaluation 3) develop skills in behavior change theory to develop clinical interventions that engage patients with low socioeconomic status; 4) acquire skills in implementation science methods including community based participatory research; 5) engage in additional career development activities to enable her transition to independence as a clinician-investigator. Developing these skills during the award period will support Dr. Mathews’ long-term goal of becoming an independent clinical investigator, leading efforts to improve the delivery of CVD prevention therapies to medically underserved populations with the ultimate goal of reducing CVD disparities.

NIH Research Projects · FY 2026 · 2023-02

Project Summary/Abstract Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is one of the deadliest pathogens on the planet, and for decades, TB has been the leading cause of death due to infectious disease. The cell envelope of Mtb forms a notoriously tough barrier around the cell, protecting the bacterium from harsh agents in the environment such as antibiotics and host immune responses. At the same time, Mtb imports nutrients from the host cell, such as cholesterol, across the cell envelope. Transport of lipids, metabolites and nutrients across the cell envelope, between the inner and outer membranes is critical for building and maintaining the cell envelope itself, and for import of key factors required for bacterial growth. Therefore, the transport systems that facilitate this trafficking are critical for allowing Mtb to survive and thrive in its intracellular niche, most typically macrophages in the lungs. The MCE (Mammalian Cell Entry) family of proteins are transport systems that have been implicated as virulence factors in Mtb, and are an expanded protein family in mycobacteria compared with other double-membraned bacteria. In Mtb, several lines of evidence suggest that MCE systems are important for importing nutrients such as cholesterol and fatty acids. Recent work on E. coli MCE systems has shown that these are multi-protein complexes anchored in the inner membrane of double-membraned bacteria, and may play an important role in the maintenance of outer membrane integrity, raising the possibility that this may also be a role that MCE proteins play in Mtb. The structure and mechanisms of the highly complex MCE systems in Mtb remain unknown, and studying these is critical for understanding how MCE systems work, what their substrates are, and how they may be linked to virulence. This proposal is focused on biochemical and structural characterization of MCE transport systems from Mtb and the non-pathogenic model, Mycobacterium smegmatis. Using single particle cryo-EM, mass spectrometry, biochemical and functional assays, we aim to study the structure and function of endogenous Mtb MCE systems, decipher which protein subunits come together to form complexes and define protein-protein interactions that are important for the systems to assemble and function. The results of this work will provide important insights into the structure and function of the large, multi-protein MCE complexes in the Mtb cell envelope, and how they influence replication and virulence in the host.

NIH Research Projects · FY 2026 · 2023-02

Cryptococcus neoformans (CN) is an important human fungal pathogen responsible for thousands of deaths each year, primarily in immunosuppressed individuals. CN makes melanin, a pigment that performs a variety of functions in the plant and animal kingdoms. In fungi, melanin reinforces cell walls, shields against ultraviolet radiation and toxic metals, harnesses high-energy electromagnetic radiation, and contributes to virulence. In addition to contributing to virulence, melanization reduces the susceptibility of fungal cells to antifungal agents and can contribute to the difficulty in treating fungal infections, which are often chronic and notoriously difficult to eradicate. Despite its importance, little is known about the structure of melanin because it is insoluble and amorphous, making it difficult to analyze. This research program takes a multidisciplinary approach to studying the problem of cryptococcal melanization, combining biochemical, cell biology, and spectroscopic (solid state NMR) techniques to uncover the mechanisms of melanization and its impact on the host-microbe interaction. The current application proposes to elucidate the vesicular pathway used to export melanin to the exterior of the cell, investigate the role of lipids in melanin synthesis, investigate how melanin affects the interactions between CN and macrophages, and identify small molecule inhibitors of melanogenesis. Agents that target melanin are potentially valuable because they could be applied against a broad array of pathogenic fungi. Four related but independent aims are proposed: Aim 1. To establish the mechanism for CN cell wall melanization; Aim 2. To determine how neutral lipids in lipid droplets influence melanin synthesis and deposition; Aim 3. To establish the mechanism for how melanization subverts the CN-macrophage interaction; and Aim 4. To identify CN melanin inhibitors and establish their mechanisms of action.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY/ABSTRACT Ollier disease (OD) and Maffucci syndrome (MS) are untreatable, poorly characterized, newly recognized cancer susceptibility syndromes. Their genetic bases and pathways responsible for the formation and progression of benign and malignant tumors are not known. Our long-term goal is to identify pharmacological approaches to treat bone deformities and malignant transformation in patients with OD and MS and to prevent/treat related non- syndromic forms of cancers associated with these conditions. Our central hypothesis in this application is that OD and MS are distinct under-characterized cancer susceptibility syndromes caused by variants in multiple genes that disrupt the HIF-1 pathway. The rationale for our project is that phenotypic characterization and identification of the genetic causes of OD and MS have the potential to offer a strong scientific framework whereby new pharmacological strategies to cancer therapy in these patients and patients with the non-syndromic forms of the same cancers, such as chondrosarcomas and gliomas, can be developed. The central hypothesis will be tested by pursuing three specific aims: 1) Comprehensively define the phenotypic features of patients with OD and MS; 2) Discover the causative genes and variants of OD and MS in previously uncharacterized cases; 3) Determine the effect of causative variants of OD or MS in the HIF-1 pathway. We will pursue these aims using an innovative combination of genomic and functional techniques applied to a unique set of deeply phenotyped patients. The proposed research is significant because it will: 1) Define the natural history of novel cancer susceptibility syndromes; 2) Establish a unique OD and MS germline and tumor sample collection for research use; 3) Identify the genetic bases of untreatable cancers, and; 4) Determine the role of the HIF-1 pathway in OD and MS. Our expected outcomes are to define the responsible variants and genes that cause benign and malignant tumor formation in OD and MS and to fully elucidate the phenotypic features and natural history of these disorders. Our results will have an important positive impact providing new opportunities for the development of novel pharmacological therapies to treat patients with these diseases as well as those with related non-syndromic forms of cancers such as of chondrosarcomas and gliomas.

NIH Research Projects · FY 2026 · 2023-02

Project summary: The olfactory epithelium is situated in the nasal passages at the interface with the environment – a location that makes it vulnerable to damage by both infectious and non-microbial threats. While the innate immune defenses of the respiratory epithelium are increasingly well understood, the immune mechanisms protecting the delicate olfactory neuroepithelium have not been fully elucidated. Inflammation of the olfactory epithelium can result in the loss of the sense of smell, which is a debilitating health problem in the United States significantly impacting the quality of life of affected individuals. The current COVID-19 pandemic has highlighted how viral infection and the local immune response of the olfactory epithelium can impair sense of smell function, although the mechanism is unknown. We hypothesize that the lining cells of the olfactory epithelium, called sustentacular cells, play a critical role protecting the underlying neurons by maintaining a strong physical barrier and providing a supporting framework. Once damaged, olfactory tissue has a remarkable and unique neuroregenerative capacity, allowing rapid repair by creation of new neurons. The signals that drive and regulate regeneration by olfactory progenitor cells are unclear. Our preliminary studies in mice reveal that regulated inflammation is important to initiating normal repair after olfactory injury. We also have found that olfactory stem cells deep in the mucosa are capable of communicating with immune cells to mediate inflammation. In this way, we propose that olfactory stem cells provide innate immune protection to the epithelium. We hypothesize that injury to the surface barrier exposes olfactory stem cells to stimuli that drive inflammation and replacement of apical cells. The overall goal of this proposal is to explore neuroepithelial-immune interactions in the olfactory epithelium. In aim 1, we will investigate the innate immune activity of olfactory stem cells and demonstrate whether inflammatory cells and their chemical signals modulate basal cell function. In aim 2, we will explore the role of stem cells in modulating the immune response and in fighting infection. Finally, in aim 3, we will study the immune response of sustentacular cells and olfactory stem cells to inflammatory signals related to two common causes of loss of the sense of smell: nasal polyps and infection with SARS-CoV-2. These studies will significantly advance current knowledge about the olfactory system and create an opportunity to develop innovative therapies for important health conditions impacting the sense of smell.

NIH Research Projects · FY 2026 · 2023-02

Pulmonary arterial hypertension (PAH) is a highly morbid disease that commonly complicates patients with the autoimmune disease systemic sclerosis (SSc) and is a leading cause of death in this population. Right ventricular (RV) adaptation to progressive increases in afterload is the main determinant of outcome in SSc and despite guideline-recommended early detection algorithms designed to identify PAH and therapeutic advances in the treatment of PAH, SSc patients are often diagnosed late, and mortality remains exceedingly high. Although several strategies are available, existing screening algorithms have low predictive accuracy. Thus, an unmet need to better delineate high-risk phenotypes in SSc and improve identification of subgroups at greatest risk of PAH earlier in the disease course when therapeutic interventions may affect prognosis. This research aims to fill this gap by providing noninvasive and objective prognostic quantitative imaging markers and trajectory-based subtype analysis using sophisticated biostatistical and machine-learning (ML) techniques, enabling early identification of subpopulations at risk for adverse, long-term outcomes. Utilizing novel echocardiographic techniques, we recently identified early changes in RV contractility and contractile reserve in SSc prior to the development of overt PAH. Drawing from these findings, in Aim 1, we will specifically determine whether the addition of echo-derived parameters of RV contractility to standard screening can identify distinct clinical phenotypes and high-risk trajectories in the development of PAH, while characterizing the nature and interaction of these trajectories across each of the variables. Our methodology will combine ML with Bayesian multivariate linear mixed modeling to improve characterization and phenotyping of similar subgroups and how trajectories present unique risk for adverse events. Aim 2 focuses on the biologic validation of echo-derived techniques with simultaneous direct chamber-level measures of RV contractile reserve and RV-arterial coupling to determine whether RVLSS is a noninvasive surrogate for RV contractility and RV contractile reserve. Our synergistic and complementary aims will be used to derive and validate a robust early detection strategy in Aim 3, improving upon existing screening methods, enabling the early prediction of PAH in SSc. We are uniquely positioned to study these critical questions given our access to one of the largest and finely phenotyped SSc cohorts in the world, our strong track record of excellence in the noninvasive and invasive assessment of RV function in SSc, and our expertise in the complex multifaceted methodology necessary to complete this project. Early identification and characterization of RV maladaptation to emerging pulmonary vascular disease would be transformative in the clinical management of SSc, a population with exceedingly high morbidity and mortality from cardiopulmonary disease. If successful, our findings may also be applicable to other cohorts who are at-risk for the development of PAH to allow for earlier detection and earlier intervention.

NIH Research Projects · FY 2026 · 2023-02

The objective of this proposal is to create a new Clinical Center (Hub) organized by the Johns Hopkins Medicine Department of Emergency Medicine (JHM DEM) for the SIREN research program. The JHM Hub- and-Spoke network is comprised of nine fully committed Spoke institutions with the capacity to recruit from more than 25 unique clinical sites across five states and Washington, DC. Annual ED census of the network exceeds 1.6 million. Emergency medical services (EMS) systems associated with Hub and spokes have committed to the SIREN program through direct participation in research conducted with the JHM Hub-and-Spoke network. The JHM DEM will leverage extensive clinical research experience and a robust institutional research environment to make immediate and meaningful contributions to the national SIREN program and to improve outcomes for patients with neurologic, cardiac, respiratory, hematologic, and traumatic conditions. The JHM DEM has been continuously engaged in clinical research since 1985 and is a consistent national leader in annual publications and grants, total research funding, NIH funding, and total faculty with funded research. To date, the JHM DEM has enrolled tens of thousands of patients in prospective emergency care research. The JHM Hub-and-Spoke network will achieve success for SIREN through the following Specific Aims: Aim 1: Create a Hub network administrative structure that guarantees efficient and effective recruitment for and execution of SIREN clinical trials. An innovative Hub-of-Hubs network design will allow for rapid scalability to meet individual SIREN trial needs. Tools to facilitate effective and efficient recruitment, protocol execution, data entry, and quality assurance will be disseminated across the JHM SIREN network. Aim 2: Conduct SIREN Clinical Trials That Advance Scientifically Valid, Measurable Health Outcomes in Emergency Care Populations. The JHM SIREN Hub will focus on conducting clinical trials that address clearly defined emergency care conditions using robust scientific methods. The Hub will prioritize standardized outcome measures across all spokes, protocol fidelity, and transparent reporting to guarantee that results are generalizable, reproducible, and relevant to improving emergency care outcomes for patients across the nation. Aim 3. Train future leaders in emergency care clinical trials research. Our Hub network will serve as a training environment for junior emergency care investigators (residents, fellows, junior faculty, nurses, EMS providers). Junior investigators will gain mentored experience in clinical trials research through workshops and didactics developed by the Hub, execution of SIREN trials, and participation in technical working groups including those focused on optimizing trial implementation and development of new trial proposals. These aims will be executed through a tripartite Hub organizational structure that includes (1) an Administrative Core, (2) a Governance Core, and (3) Technical Working Groups.

NIH Research Projects · FY 2026 · 2023-01

Summary This proposal aims to understand the effects of HIV on the humoral immune response to hepatitis B by focusing on HBV-specific neutralizing antibodies (NAbs), B cell repertoires, and monoclonal antibodies that develop after acute hepatitis B infection or after treatment-induced control of infection. This proposal will study 185 persons (74 HIV+) in the MACS-WIHS Combined Cohort Study who had acute hepatitis B while in follow- up and whose outcome of either spontaneous control (n=163) or chronic infection (n=22) was previously determined. We will also study an additional 21 participants with functional cure while on HBV treatment (treatment-induced control). The first Aim focuses on plasma NAb responses during and 30 months after spontaneous control of acute HBV infection in persons with and without HIV infection. We hypothesize that in persons with HIV (PWH), the NAb responses will be weaker and less durable than in persons without HIV infection. Decreased durability of NAb responses could contribute to the increased risk of HBV reactivation with HIV infection. We will also assess plasma NAbs in participants with treatment-induced control of infection. We hypothesize that these participants will demonstrate high NAb titers similar to those with spontaneous control of acute infection. In the second Aim, we will isolate HBV surface antigen (HBsAg)-specific B cells from specimens obtained during acute infection or before and after treatment-induced control. We will compare the molecular features of HBsAg-specific B cell receptors between those with spontaneous control of acute infection, those who develop chronic hepatitis B, and those with treatment-induced control of infection. We will also determine the effects of HIV on these molecular features. In the third Aim, we will clone monoclonal antibodies (mAbs) from these HBsAg-specific B cells and determine the mAbs’ functional characteristics. We expect that the mAbs from persons with spontaneous control of acute infection will bind with higher affinity and neutralize more potently than those who develop chronic infection, and that post-control mAbs from treatment- induced controllers will be more potent than pre-control mAbs. We also expect that HIV will decrease the affinity and potency of anti-HBs mAbs. Innovative aspects of this project include: 1) The unique cohort with a large number of incident HBV infections with either spontaneous control or viral persistence in people living with and without HIV infection, as well as rare individuals with treatment-induced control of HBV, 2) The ability to detect NAb responses in plasma from human subjects, and 3) The ability to isolate HBsAg-specific B cells for ex vivo study. To date, such studies have not been possible, explaining why there is little information on NAb responses in HBV-infected humans. Results from this proposal will contribute to our understanding of the effects of HIV on the immune response to hepatitis B and could facilitate development of a functional cure for hepatitis B, the leading cause of cirrhosis and hepatocellular carcinoma worldwide.

NIH Research Projects · FY 2026 · 2023-01

Project Summary The over-arching goal of this proposal is to establish the predictive multi-scale mathematical model to decipher the mechanism of durotaxis. Durotaxis is the preference of cells migrating toward a stiffer extracellular matrix (ECM) and has important roles in many biological processes, ranging from embryo development to tumor metastasis. Focal adhesion (FA) is the functional unit of durotaxis; it an integrin-based multi-protein transmembrane linkage, through which cell exerts actin cytoskeleton-based traction force to tug the ECM and sense the stiffness. Despite the high relevance to biomedical applications, it is not well understood how FA mediates mechanosensing of ECM stiffness and drives durotaxis, largely because predictive mathematical models lag behind the descriptive experimental finding in the field. At single-FA level, while previous models explain molecular-clutch behaviors in FA mechanosensing, they cannot explain how and why FA- localized protein activities adapt to environments by distinctive spatial-temporal patterns (akin to footprints) that are demonstrated to be essential for durotaxis. The full underlying mechanisms of the FA-localized “footprint” and its exact roles in durotaxis are thus unknown. Further, durotaxis must coordinate movements of cell body and protrusion/retraction of cell edge. While the FA-mediated tractions drive the cell body, how the FA-localized mechanosensing events coordinate with the cell edge dynamics is unknown. Last, at a single-cell level, there exist many FAs at different developmental stages at any time. It is not understood how the cell integrates the mechanosensing activities of individual FAs to drive durotaxis. A predictive model that meaningfully engages with experiments is desirable and likely holds the key to decipher durotaxis. Toward this goal, we have been and will uniquely integrate mathematical modeling in iterative dialogues with experimental testing. The central hypothesis is: FA-localized spatial-temporal dynamics of the traction force generation and transmission defines FA-mediated mechanosensing and durotaxis. The basis of this proposal is our previous findings. We built the first mathematical model that captures the essence of entire FA maturation process. That is, FA evolves from a nascent complex, the centripetally growing FA that couples the retrograde flux of branching actin network, to the mature FA that transmits the stress fiber (SF)-mediated contractions onto ECM. This model uniquely links the FA-localized fine features of protein activities – emerging from FA maturation process – to FA mechanosensing events. The model predicted and was experimentally confirmed that a negative feedback between the elongation and contractility of the FA-engaging SF underlies the FA-localized traction oscillation and mechanosensing of ECM stiffness. Ushered by these findings, our specific aims are to determine: 1) how FA force-transmission and SF elongation cross-talk in FA mechanosensing; 2) how FA mechanosensing affects cell edge protrusion/retraction, and 3) how cell integrates mechanosensation of individual FAs to drive durotaxis. If successful, the proposed research would provide a quantitative platform interpret data and guide durotaxis experimental designs, which has the multi-scale resolutions ranging from FA-localized dynamics, cell edge protrusion/retraction, to cell movement at whole-cell level.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Atrial fibrillation (AF) is the most prevalent sustained cardiac arrhythmia, leading to morbidity and mortality in 1-2% of the population and contributing significantly to global health care costs. For patients in whom AF can- not be treated by drugs, the recommended therapy is catheter-based ablation to isolate arrhythmia triggers and eliminate the substrate for arrhythmia perpetuation. The success rate of catheter ablation in rhythm controlled AF patients is 50-75%, and is worse in patients with persistent AF. The mechanisms by which baseline and post-ablation atrial remodeling, including atrial distension, functional impairment, and fibrosis, contribute to AF recurrence following catheter ablation, are not well understood and the underling factors have not been charac- terized. Understanding atrial remodeling in drug-refractory AF patients and discovering new personalized strategies for successful AF ablation and prevention of AF recurrence is a quest of paramount clinical significance. There is an urgent need to develop new approaches to ablation that account mechanistically for the remodeling of the atrial substrate post-procedure, and thereby improve the efficacy of the therapy and eliminate repeat procedures. The overall objective of this application is to use novel combination of imaging, artificial intelligence (AI), electroanatomical mapping, and mechanistic computational modeling to understand the causes for AF recurrence in drug-refractory AF patients and to develop a new paradigm for personalized ablation that eliminates repeat procedures. Leveraging our advancements in the acquisition of high-quality atrial im- ages, our expertise in AI and particularly deep learning, and our ability to efficiently generate personalized com- putational atrial models, we propose to characterize baseline atrial remodeling in shape, structure and function as well as its progression post-procedure. Using the obtained insights, we will develop a comprehensive abla- tion strategy where AF ablation targets will be determined by reinforcement learning based on the mechanistic knowledge acquired in the proposed studies. The project will culminate in a pilot prospective patient study that will test the new ablation strategy. Successful execution of the project will pave the way for a paradigm shift in the clinical procedure of AF ablation and in the quest to eliminate repeat procedures in drug-refractory AF patients, resulting in a dramatic improvement in the efficacy of the therapy. Importantly, completion of this project will be major leap forward in the integration of imaging, AI, and computational modeling in the diagnosis and treatment of heart rhythm disorders.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Virginia Shalkey Hahn, MD is an Assistant Professor of Medicine and heart failure cardiologist in the Division of Cardiology at Johns Hopkins University. Her long-term goal is to become an independent investigator harnessing bioinformatics to answer clinically relevant questions in the fields of heart failure and cardiometabolic disease. She focuses on the intersection of cardiometabolic disease and heart failure due to the rising prevalence of obesity and its prominent role in Heart Failure with Preserved Ejection Fraction (HFpEF). HFpEF is a common disease with limited effective therapies due to its heterogeneity and limited analysis of human tissue. Her prior work used myocardial transcriptomics to create HFpEF subgroups that had distinct clinical profiles, one of which was composed of women with normal natriuretic peptides, a group that was excluded from most all prior and even recent positive clinical trials for HFpEF. The Specific Aims of the current proposal are to 1) determine differences in myocardial cell composition and cell cluster-specific transcriptomics in HFpEF using single nuclei RNA sequencing, 2) define the myocardial metabolic profiles of HFpEF using multi-omic analysis of endomyocardial biopsy tissue, and 3) measure differences in metabolite uptake/secretion by the heart in HFpEF using coronary sinus sampling. The significance of the proposal stems from the use of human tissue with strong clinical phenotyping, and its likelihood to spawn precision therapies for HFpEF that target unique abnormalities in distinct subgroups, particularly subsets of female patients who are traditionally excluded from clinical trials. Dr. Hahn has assembled a world-class mentoring team well- poised to complete the research and training aims of the proposal. The primary mentor, Dr. David A. Kass, is an internationally renowned cardiovascular scientist with >36 years of experience training several generations of physician-scientists. He has extensive experience serving as primary mentor for NIH career development awards. Co-mentors Dr. Patrick Ellinor and Dr. Kavita Sharma have expertise in cardiovascular bioinformatics, translational research, and HFpEF. Collaborators add expertise in proteomics, metabolism, and bioinformatics. The work is performed in the fertile academic environment at the Johns Hopkins University School of Medicine, where the Division of Cardiology demonstrates a clear commitment and investment in Dr. Hahn's research career. The career development plan focuses on development of a formal bioinformatics skillset through hands-on analysis and a Master of Science degree in Bioinformatics. These skills will distinguish Dr. Hahn from her mentors as an independent investigator with a unique niche in bioinformatics and heart failure. Dr. Hahn and her mentoring team are well poised to address the most pressing knowledge gaps in HFpEF and make major breakthroughs toward better care for our patients.

NIH Research Projects · FY 2025 · 2023-01

PROPOSAL SUMMARY Maternal survivors of IPV and their children often face ongoing abuse and harassment, safety concerns, lack of financial resources, and legal constraints around custody following separation from an abusive partner that may impact children’s health, school engagement, and flourishing and impede their children’s access to health care. The purpose of this exploratory mixed-methods study is to develop knowledge of post-separation abuse and examine how post-separation abuse predicts pediatric health and flourishing outcomes. The specific aims are: 1) Understand post-separation experiences of maternal IPV survivors including post-separation abuse, their children’s health and flourishing, and the structural context of family court, 2) Examine if the prevalence of children’s special health care needs, unmet health needs, school engagement, and flourishing differs for children exposed to post-separation abuse as compared to US children overall, 3a) Test construct validity and reliability of new items developed from the qualitative phase as measures of post-separation abuse, 3b) Determine if post-separation abuse predicts children’s health and flourishing outcomes. The study will begin with a qualitative arm using iterative thematic inquiry to explore maternal survivors experiences of post- separation abuse and their children’s health (n=30). The qualitative arm will also identify new items of post- separation abuse not captured by existing measures of coercive control (WEB) and the risk of lethality (DA) to create a new scale (Maternal Child Experiences of Post-Separation Abuse, or “MCEPSA” Scale) that will be tested in the exploratory quantitative arm. In the second quantitative phase, cross-sectional surveys will be administered to mothers (n=150) to assess their children’s (age 6-17) unmet health needs, special health care needs, school engagement, and flourishing utilizing items from the NSCH, and then compare prevalence rates of children in our recruited sample to national prevalence data from the NSCH using test of proportions. In addition, we will conduct preliminary psychometric testing of the new items from the qualitative arm. Further, the study will use logistic regression to examine which measure (WEB, DA, MCEPSA) of post separation abuse best predicts children’s unmet health needs and special health care needs and to determine if the new items in the MCEPSA are significant above and beyond the WEB and DA in predicting children’s special health care needs and unmet health needs. Finally, the results of the quantitative and qualitative phases will be integrated, and the quantitative data will be presented along with themes and quotes from the qualitative data. This mixed-methods study will document health disparities and provide foundational knowledge for nursing, policy makers, public health professionals, and judicial decision-makers to understand how post-separation abuse and the structural context including family court processes combine in complex ways to affect children’s health and flourishing. The study will identify potential areas of intervention in order to develop differential systems responses to promote positive health for children exposed to post-separation abuse.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Residual subpopulations of antibiotic-susceptible bacteria can remain within host tissues following antibiotic treatment. These surviving bacteria are called persister cells, which are transiently tolerant to high levels of antibiotic, and can cause serious relapsing infection after treatment. Critically, current treatment strategies do not target persisters. To fully eradicate all bacterial cells, treatments are prolonged, increasing patient and clinical costs. Prolonged antibiotic exposure can promote antibiotic resistance, further emphasizing the need to improve treatment efficacy. Improved treatment strategies would simultaneously target all members of the bacterial population, including persisters. However, persisters have been primarily studied in culture, and relevant persister cell-specific drug targets within host tissues are largely undefined. Bacteria behave very differently in host tissues, where nutrient limitation and antimicrobial host defenses activate strong stress response pathways in bacterial pathogens. We predict persisters utilize distinct, potentially novel, survival strategies within the host environment. To study bacterial antibiotic persistence within host tissues, we established a mouse model of doxycycline treatment of Yersinia pseudotuberculosis splenic deep tissue infection. Doxycycline is an effective treatment for human Yersinia infection, but requires 7 days continuous treatment, which has been incorporated into our mouse model. Prior to antibiotic treatment, Y. pseudotuberculosis replicate to form clusters of extracellular bacteria that directly interface with a layer of neutrophils that are, in turn, enveloped by a layer of monocytes. In the initial 4h of doxycycline treatment, we observe a significant decrease in viable bacterial numbers, which correlates with a wave of neutrophil infiltration into the spleen. However, a residual bacterial subpopulation (~10%) remain in the spleen throughout the 7-day treatment. Bacterial cells resume growth and cause lethality when antibiotic concentrations wane, defining these cells as persisters. We hypothesize that interactions with neutrophils and monocytes predispose persisters to survive antibiotic treatment, and prolonged antibiotic exposure promotes additional transcriptional and genetic changes within persister cells. Utilizing our fluorescent reporter system to detect viable, doxycycline-exposed bacteria within the mouse spleen, we will: 1) identify the transcriptional, proteomic, and genetic changes specific to surviving bacteria within antibiotic-treated mice, 2) determine whether specific bacterial targets are critical for antibiotic persistence in the host, and 3) determine if monocyte or neutrophil interactions promote antibiotic persistence. We hypothesize activated neutrophils initially reduce the bacterial burden, and we will determine if evasion of neutrophil-mediated killing promotes persister cell survival. Identifying persister cell survival strategies within host tissues will provide critical information to advance the field and enable the development of more efficacious therapeutic strategies against bacterial infections.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY/ABSTRACT We evaluate the influence of patient social factors and surgeon attitudes on communication and outcomes for children with obstructive sleep-disordered breathing (OSDB). OSDB occurs in up to 20% of children and impacts physical health, behavior, and learning. The primary treatment is tonsillectomy, with or without adenoidectomy, which is the most common major surgical procedure performed in U.S. children. Some groups of children have increased prevalence of OSDB, poorer sleep outcomes, and more complications of surgery. Clinician implicit attitudes may be an important contributor to these differences, as evolving evidence shows that communication impacts patient relationships, care delivery, and outcomes. We have previously shown that surgeons inconsistently offer alternatives to surgery and rarely elicit family preferences when speaking to parents, and that they are less likely to explore emotions of some families unexplained by clinical scenario. These findings, coupled with known differences in OSDB health outcomes, highlight the critical problem that surgeon implicit attitudes may influence communication, decision-making, and outcomes for children with OSDB. Our long-term goal is to improve resource use, healthcare quality, and outcomes for children with OSDB. The overall objective of this application is to develop a comprehensive profile of the complex social and interpersonal dynamics that may affect treatment decisions and cause differences in child health outcomes. To do so, we will establish across three institutions a repository of audio-recorded encounters between a large, heterogeneous cohort of surgical clinicians and parents of children undergoing OSDB consultations. We will evaluate the influence of clinician implicit attitudes and patient social factors on surgeon parent-communication, parent engagement, and quality and use of OSDB care. We will quantitatively code communication behaviors (patient-centeredness, emotional responsiveness, and shared decision-making) of parents and clinicians occurring during child OSDB consultations and test for differences across clinician implicit attitudes, by patient social factors. We will also examine differences in parent trust in clinician, decision regret, and clinical outcomes, and assess whether communication behaviors mediate observed differences in these parent-reported and clinical OSDB outcomes. We will then interview a subset of parents and clinicians to understand the potential influence of patient social factors and clinician attitudes on communication and learn patient-centered solutions to improve communication and parent engagement. Using a stimulated recall qualitative approach, participants listen to audiotapes of their own clinical encounters and directly comment on salient elements of communication that occurred. Findings from this research will directly inform development of interventions to mitigate surgeon attitudes, promote patient-centered communication and engagement, reduce surgical overuse, and improve outcomes for children with OSDB.

NIH Research Projects · FY 2026 · 2023-01

PROJECT ABSTRACT Clonal hematopoiesis is acquired with aging, and in some cases, precedes the onset of both myelodysplastic and myeloproliferative disorders. However, the genetic drivers that underlie the evolution of clonal hematopoiesis and the subsequent disease risk remain poorly understood. This proposal builds on generated evidence showing that telomere length, a genetic and clinically available biomarker, predisposes to clonal hematopoiesis in Mendelian syndromes. We have also found evidence for distinct clonal hematopoiesis mutations that appear to differentially predispose to hypoplastic and myeloproliferative phenotypes. In this application, we will examine the role of telomere length in driving clonal evolution with aging in well- characterized cohorts including a Baltimore-based cohort of community-based women with a large African American subset. Additionally, we will also examine the onset and prevalence of a novel clonal hematopoiesis mutation in the telomerase reverse transcriptase gene that appears to be protective against myeloid malignancies. This K08 grant application is supported by outstanding mentors and in a strong translational environment at Johns Hopkins University School of Medicine with a detailed mentorship and training plan that focuses on genetic epidemiology and computational biology skills. The knowledge has the potential to impact current paradigms related to hematopoietic aging, myeloid clonal disease risk as well as novel biomarkers of specific disease risk and progression.

NIH Research Projects · FY 2026 · 2023-01

Abstract Until and unless we better understand and prevent spillovers of bat-borne viruses into intermediate hosts and humans, we will be severely limited in our ability to stop pandemics. Henipaviruses are bat-borne, RNA viruses that spillover through this route. Nearly half of all reported human henipavirus infections have been associated with contact with sick domesticated animals, though our understanding of drivers of henipavirus spillovers and specific transmission pathways remains severely limited. Building on preliminary data about bat, domesticated animal and human infections, this multidisciplinary study integrates epidemiology, ecology, and anthropology to identify spillover pathways for henipaviruses into domesticated animals in Bangladesh and the risk they pose to human health. Our first aim is to identify drivers of henipavirus spillovers into domesticated animals in Faridpur, Bangladesh. We will use multiplex pan-henipavirus assays to identify infections in Pteropus medius bats and domesticated animals living nearby this roost through cross-sectional and prospective studies. Combined with intensive studies of bat-domesticated animal interactions and weather data, we will build statistical models to identify the relative contribution of each of these factors. Our second aim is to describe which henipaviruses are being transmitted from bats to domesticated animals. Serologic studies of animals in Bangladesh show that they are frequently infected with non-Nipah henipaviruses. Through surveillance for and sampling of sick domesticated animals, we will describe the specific viruses that spillover from bats in Faridpur. Our third aim is to determine the risk of henipavirus transmission from domesticated animals to humans. We hypothesize that undetected henipavirus spillovers in humans are occurring through contact with sick domesticated animals and will conduct cross-sectional and prospective serosurveys of humans who have close contact with sick animals in Faridpur. We remain ignorant about henipavirus spillovers through intermediate hosts – including the specific viruses spilling over, the frequency and distribution of spillovers, and the pathways of transmission – at our own peril. The knowledge gained from this study will be immediately applicable to human and animal health programs in Bangladesh and other countries where henipaviruses circulate in bats. By learning about which henipaviruses infect humans, and how they are infected, we can advise public health surveillance programs on how to optimize detection and epidemiologic investigation of cases across Bangladesh. Our investigations about spillovers in Faridpur can also be scaled- up to other areas of Bangladesh and countries where henipaviruses circulate in bats so that we can truly begin to appreciate the scale of henipavirus spillovers in the global landscape.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Dr. Chanee Fabius is an Assistant Professor in the Department of Health Policy and Management at the Johns Hopkins Bloomberg School of Public Health. Her career goal is to become an independent investigator conducting high-impact embedded pragmatic clinical trials (ePCTs) directed at testing interventions, policies and programs in real world settings to better support the home care workforce and improve care quality and quality of life among those receiving services, including persons living with Alzheimer’s Disease and related dementias (ADRD) and their family caregivers. This award will provide her with the protected time and learning experiences to obtain the knowledge, skills, and capability to develop ePCTs and translate findings into routine home care delivery. Dr. Fabius’s career development award proposes five years of training in mixed methods, human factors approaches in home care research, and applied experiences developing and conducting ePCTs. Dr. Fabius has assembled a stellar mentorship team and advisory panel with expertise in the areas of her proposed training goals. As a result of disease progression, high care needs, and limited financial resources, persons living with ADRD often receive Medicaid Home and Community-Based Services, delivered by home care aides proving hands on assistance with daily activities. Family caregivers and home care aides interact to provide care for older adults, but their relationship is often fraught due to gaps in information, misalignment of role expectations, and lack of shared understanding of each other’s roles in home care. The proposed K01 will: (1) leverage nationally representative data to determine how role sharing between family caregivers and home care aides differs among Medicare-Medicaid enrolled home care recipients with and without ADRD, and is associated with older adults’ participation restrictions; (2) conduct a qualitative study to develop and refine a home care role and preference guide to improve information sharing and clarify role expectations between family caregivers and home care aides caring for older Medicaid HCBS recipients living with ADRD; (3) assess the feasibility and acceptability of delivering a home care role and preference guide to family caregivers of older adults living with ADRD and home care aides. The intervention will be piloted in up to five agencies within 10 triads of older adults living with ADRD, family caregivers, and home care aides. The proposed work is aligned with recent policy and research recommendations calling for interventions that support quality of life for older adults living with ADRD, family caregivers, and home care aides. Findings will support the development and submission of an R01 grant proposal to implement and test an ePCT in Medicaid HCBS to assess the effects of the role and preference guide and quality of life outcomes for older adults, family caregivers, and home care aides.

NIH Research Projects · FY 2026 · 2023-01

Project Summary Understanding how proteins fold is a central quest in biology. Studied for over 50 years, investigations of soluble protein folding have proven invaluable for dissecting the molecular basis of a multitude of diseases. By comparison, folding studies of membrane proteins lag far behind. The knowledge gained from soluble protein studies cannot simply be transferred to inferences about because their solvents are different. The balance of forces encoding a MP embedded in a lipid bilayer must be distinct from that of soluble proteins in water. Our research efforts contribute to filling this key gap in the understanding the physical chemistry of membrane proteins. We will experimentally determine of energetic forces stabilizing membrane proteins along the steeply changing polarity gradient of the phospholipid bilayer interface, quantify backbone hydrogen bond strengths, and expand our repertoire of membrane protein folding models to include those with an a-helical secondary structure. These efforts will be complemented by molecular simulations and other solution biophysics interrogations as needed. Our results have broad ranging impact in the field at large through contributions to information databases used in training computational algorithms and by their incorporation in physically realistic mechanisms for protein folding catalysis by cellular machines.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Spinocerebellar ataxia type 12 (SCA12) is an autosomal dominant neurodegenerative disease, discovered and characterized by our group, notable clinically for tremor, gait abnormalities, and neuropsychiatric syndromes, and neuropathologically for atrophy in cerebral cortex and cerebellum. SCA12 is caused by a CAG/CTG expansion mutation in PPP2R2B, a gene encoding brain-specific regulatory units of protein phosphatase 2A (PP2A). Normal alleles carry 4 to 31 triplets, whereas disease alleles carry 43 to 78 triplets. SCA12 is the second most common ataxia in North India, with cases of SCA12 detected in the United States, Italy, and China. There is no treatment. Our preliminary data indicates that PPP2R2B contains at least 16 exons with multiple alternative splice variants, resulting in at least 8 protein isoforms (termed B1-B8) with different N terminal domains, among which Bβ1 and Bβ2 are by far the most abundant and best characterized. The N-terminal domains contribute to the localization of the PP2A holoenzyme, and hence their relative abundance may be critical in establishing patterns of protein phosphorylation. The repeat is located in the 5’ region of exon 7 in a predicted promoter region, but is also contained within the transcripts of alternative splice variants. Based on extensive preliminary data, we hypothesize that the CAG repeat expansion in SCA12 alters PPP2R2B expression and splicing, leading to changes in PP2A targeting that contribute to SCA12 pathogenesis by alteration of phosphoproteome. Secondarily, we hypothesize that missplicing induced by repeat expansion generates a transcript encoding a toxic polyserine tract that contributes to SCA12 pathogenesis. Our aims are each designed to explore a specific aspect of SCA12 pathogenesis, and simultaneously to explore more general principles of neurodegeneration related to repeat expansion and the function of PP2A, using genetically engineered mice and human iPSCs. In Aim 1, we will determine the effect of the SCA12 mutation on PPP2R2B expression. In Aim 2, we will determine the effect of abnormal PPP2R2B expression on PP2A substrate targeting and the phosphoproteome. In Aim 3, we will determine how changes in PP2A substrate targets and aberrantly expressed polyserine contribute to neurotoxicity. If our hypothesis is correct, SCA12 pathogenesis derives from repeat-expansion-induced changes in PPP2R2B expression that 1) alters PP2A substrates targets and hence the phosphoproteome, and 2) generates toxic polyserine tracts. Establishing these pathways will establish multiple potential targets for therapeutic intervention in SCA12. At the same time, our experiments will more generally establish the potential effect of repeat expansion on promoter and splicing function, and the role of PP2A dysregulation in neurodegeneration, of particular interest given evidence of PP2A dysregulation in other neurodegenerative disorders, such as Alzheimer’s disease.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Schizophrenia (Scz) is a common and debilitating neurodevelopmental disorder. However, the genetic architecture of Scz is highly complex, impeding research progress. Recently, the Scz Exome Meta-Analysis (SCHEMA) Consortium has identified genes with ultra-rare coding variants that each confer substantial risk for Scz. We hypothesize that the presence of these rare variants in human induced pluripotent stem cells (hiPSCs) will lead to neuronal phenotypes relevant to Scz. The PI, Dr. Pan Li, has extensive experience with gene editing and iPSCs, including development of an improved method for seamless introduction and removal of mutations into hiPSCs. She has previously focused on neurodegenerative disorders, but with this application proposes to change to study of Scz. This transition is facilitated by the location of her lab in the Division of Neurobiology, which has a long track record of research into major mental illnesses and of highly collaborative research using iPSCs (eg the HD iPSC Consortium). In Specific Aim 1, isogenic hiPSC lines with protein truncating variants associated with Scz will be genetically engineered by CRISPR-assisted homologous recombination to generate panels of iPSC lines, with each isogenic line bearing a different N-terminal protein truncating point mutation. Knockdown and overexpression rescue experiments will determine whether there is loss of protein expression and thus loss of gene function. In Specific Aim 2, the effect of mutations on gene and protein expression will be studied using RNA seq and quantitative proteomics. In Specific Aim 3, the effects of mutations on synaptic and electrophysiological phenotypes will be delineated. We hypothesize that understanding the cellular phenotypes and altered signaling pathways evoked by the SCHEMA mutations will clarify the cellular pathogenesis of subtypes of schizophrenia, and provide potential therapeutic targets.

NIH Research Projects · FY 2025 · 2023-01

Project Summary Development of an organism requires both stereotyped and stochastic patterning. Stereotyped patterning robustly generates nearly identical structures across individuals. In contrast, stochastic cell fate specification produces randomized patterns that are unique to each individual. Stochastic fate decisions are required for the development of many sensory organs, including visual and olfactory systems. Despite their importance, how the molecular mechanisms controlling stereotyped and random patterns intersect within the same tissue has not been addressed. This project aims to determine how gene regulatory mechanisms are tuned to generate highly regular patterns and stochastic patterns in the same tissue using the Drosophila eye as a model. The Drosophila eye is composed of ~800 ommatidia in a near perfect array. Each ommatidium comprises eight photoreceptors (R1-8) which develop in a predictable fashion. As photoreceptors are differentiating during larval eye development, a wave of morphogenesis driven by Hedgehog (Hh) signaling drives the highly reproducible structure of the eye. Underlying the uniform morphology of the fly eye is a random pattern of photoreceptor subtypes. Two R7 photoreceptor subtypes are defined by expression of light-detecting Rhodopsin proteins. Random patterning of these two R7 subtypes is controlled by stochastic ON/OFF expression of the transcription factor, Spineless (Ss). SsON R7s express Rhodopsin 4 (Rh4), whereas SsOFF R7s express Rhodopsin 3 (Rh3). ss is regulated by an interplay of transcription and chromatin regulation during larval eye development. I found that Hh signaling plays a second role in eye development to regulate stochastic patterning. hh mutants display a reduction in the percentage of SsON R7s. Cubitus Interruptus (Ci), an effector of Hh signaling, binds at an eye specific enhancer in ss. This site overlaps with a binding site for Klumpfuss (Klu), a repressor of ss, suggesting competitive binding and regulation. I hypothesize that Hh signaling is finely tuned to drive stereotyped eye patterning and induce ss transcription in precursors to generate stochastic R7 subtype patterning. I will test this hypothesis by 1) Determining how the Hh pathway regulates stochastic ss expression, 2) Describing how antagonism between Ci and Klu regulates ss expression, and 3) Determining how Hh signaling and chromatin regulation are integrated at the ss locus. Together, these experiments will provide the first analysis of coordination between stochastic and stereotyped gene expression within a single tissue and will enhance our mechanistic understanding of stochastic cell fate specification.