Washington University

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract This research proposal is intended to facilitate training toward the applicant's long-term career goal of becoming an independent investigator in gene regulation in cancer. This research training plan will encompass predoctoral and postdoctoral training necessary to gain advanced technical skills in cancer biology research and to develop skills in scientific communication, grantsmanship, and mentorship. The research objective in the F99 phase is to determine the role of APOBEC3A-mediated mutagenesis in altering transcriptional regulation in cancer. APOBEC3A (A3A) is an endogenous mutagen that catalyzes the deamination of cytosine (C) to uracil (U) in single-stranded DNA and RNA. The genomic mutation patterns generated by A3A are the second most common mutation patterns in cancer, thus understanding the impact of A3A-mediated mutagenesis is essential. A3A expression leads to widespread changes to gene expression, however most differentially expressed genes do not contain A3A-induced mutations. This project will investigate the hypothesis that A3A activity alters pre- transcriptional regulation through DNA mutagenesis and post-transcriptional regulation through RNA editing. Preliminary data show that A3A activity alters chromatin accessibility in a subset of differentially expressed genes, yet these chromatin accessibility changes also lack A3A-induced mutations. DNA methylation is an important epigenetic mark that regulates gene expression in part by coordinating chromatin accessibility. Aim 1a will define the effects of A3A activity on genome-wide methylation patterns and determine if these changes are specific to A3A-induced damage or if other base-damaging agents elicit the same changes. Integrating these data with gene expression and chromatin accessibility data will establish how A3A-induced methylation changes alter chromatin function. To determine the mechanisms by which A3A generates changes to methylation, chromatin accessibility, and gene expression, we will use CRISPR-Cas9 base editing to target A3A activity to a specific locus and evaluate the effects in the presence and absence of functional DNA repair. Aim 1b will investigate how A3A induces changes to the transcriptome through RNA editing. Preliminary data show that hundreds of transcripts have altered stability during A3A expression. A3A RNA editing generates missense mutations in the transcripts of three RNA binding proteins (RBP) important for RNA stability. Aim 1b will determine the effects of these missense edits on RBP function to define how A3A-mediated RNA editing impacts stability across the transcriptome. The K00 research will continue investigation of transcriptional regulation in cancer by focusing on how three-dimensional organization is altered in cancer cells. These studies will integrate higher- order chromatin sequencing and microscopy with advanced bioinformatics. Together, the F99 and K00 training plans will support the applicant's development of advanced technical skills in mechanistic cancer research and the professional training required to become a leading investigator in transcriptional regulation in cancer.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract Cardiomyopathies are major causes of heart failure and sudden death, and they represent a common indicator for heart transplantation. Current therapies have focused on “one-size-fits-all” approaches that improve outcomes in some patients, but not others. Therefore, there is an outstanding need to develop new treatment strategies that improve outcomes for patients. It has been hypothesized that patient outcomes could be improved by taking a precision medicine approach that considers genotype for treating cardiomyopathies; however, it is not clear whether such an approach would be beneficial, and if so, how to best implement this approach. This approach remains unrealized, in part, due to fundamental challenges connecting genotype, phenotype, and treatment options. Here, we will take the critical first steps towards investigating the feasibility of a mechanism-based precision medicine approach by focusing on cardiomyopathy mutations in troponin. Specifically, we will use an innovative combination of biochemical, biophysical, cell biological, and tissue engineering techniques to begin to build a mechanistic understanding of the connections between genotype, molecular and cellular dysfunction, and small molecules that potentially reverse molecular dysfunction for key troponin cardiomyopathy mutations. We have engineered model systems of patient-specific mutations that span from molecules to tissues that enable us to 1) understand the fundamental molecular biophysical mechanisms driving disease pathogenesis for key troponin mutations and 2) probe how molecular dysfunction affects cellular and tissue function in human cells. We will leverage these models to answer the following questions for several key troponin mutations: 1) Is it possible to bin patient mutations into subgroups with common molecular dysfunction? 2) Is it possible to use knowledge of molecular mechanism to identify compounds that reverse the molecular dysfunction of patient-specific mutations? 3) Does reversal of the underlying molecular dysfunction improve cellular and tissue function? The results of these studies will set the stage for the development of new approaches for treating cardiomyopathies.

NIH Research Projects · FY 2025 · 2025-08

SUMMARY Traumatic peripheral nerve injuries (PNI) lead to functional loss and incomplete recovery of peripheral nerve function while also impacting the corresponding brain regions that receive, process, and send out signals to and from the affected nerves. A prolonged duration of denervation due to PNI have adverse implications for both the nerve's prognosis and the associated brain regions. Over time, the absence of normal nerve signaling can cause corresponding brain regions to adapt to the loss, making reintegration more challenging after nerve recovery. This long-term disconnection between the nerve and brain can lead to motor and sensory skill impairments that require retraining that takes many years and might often be unsuccessful. To date, a significant amount of work has been focused on improving accuracy and speed of axonal regeneration, but little has been done to better understand the implications of simultaneous cortical remapping on nerve regeneration. Since the compensatory and regenerative processes associated with damaged PNs and their effect on brain plasticity are poorly characterized there is a critical need to develop novel techniques that can monitor both processes with high spatial resolution. To address this need, we have developed an optical imaging method capable of simultaneous imaging of the sciatic nerve and cerebral cortex structure and function in individual mice longitudinally using an implanted cortical and hindlimb windows. In combination with Thy1-GCaMP6 mice these windows enable visualizing a variety of calcium related processes in live animal both in the cortex and peripheral nerves over the entire nerve regeneration process. The goal of this project is to characterize the synchrony between PN post- injury activity and brain plasticity. To achieve this goal in Aim 1 we will test whether the acute injury evokes immediate response from the sciatic nerve to the corresponding cortical regions. In Aim 2 we will characterize longitudinal response of cortical activation and connectivity after sciatic injury and their relationship to nerve regrowth. Using Arc knockout models, we will investigate how cortical plasticity reflects the sciatic nerve recovery in the injury and repair models. In Aim 3 we will establish the effect of therapeutic electrical and optogenetic stimulation on axonal regrowth, and cortical activation. In this aim we will test whether electrical stimulation on the nerve could modulate functional connectivity and whether cortical stimulation could affect nerve recovery. Overall, this research will bridge the gap in our understanding of how the central and peripheral nervous systems interact during the recovery from PNI. By developing a deeper understanding and providing new treatment approaches, we can significantly improve outcomes for individuals suffering from traumatic PNI.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Gastrointestinal (GI) cancer peritoneal metastasis is one of the most lethal, clinically challenging, and treatment- resistant manifestations. The peritoneal cavity, a potential space lined by the serous membrane known as the peritoneum, is divided into the parietal peritoneum covering the abdominal walls and diaphragm, and the visceral peritoneum enveloping the abdominal organs. Studies on ovarian cancer, a non-GI cancer that frequently develops peritoneal metastasis, have shown that omental macrophages, part of the visceral peritoneum, can promote metastasis in mouse models. However, the relevance of these findings to human GI cancer peritoneal metastasis remains largely unexplored. Additionally, the peritoneal fluid within the cavity harbors a diverse array of immune cells, including both myeloid cells and lymphocytes. My recent study characterized the immune cell landscape in the normal human peritoneal cavity. Contrary to our knowledge from mouse models, where GATA6+ large peritoneal macrophages are predominant, these cells are rare in humans. Instead, the human peritoneal cavity is enriched with LYVE1+CD206+ macrophages, known for their potential immunoinhibitory functions. Moreover, normal human peritoneal cavities contain abundant dendritic cells (DCs), especially the CD1C+ DC2s. Despite these findings, the roles and impacts of these peritoneal myeloid cells on GI cancer progression, T-cell responses, and the effectiveness of immunotherapy are still not well understood. Therefore, this research proposal focuses on colorectal cancer (CRC), the most prevalent and lethal GI cancer in the United States, and aims to elucidate the unique roles of myeloid cells within the peritoneal cavity and peritoneum during CRC peritoneal metastasis. Informed by our previous findings that mouse and human tissues may have differing immune cell compositions, this study will commence by profiling immune responses in patients, ensuring that the findings are translational and clinically relevant. The initial efforts will determine the immune cell phenotypical changes within the peritoneal fluid and peritoneum during CRC peritoneal dissemination to identify the peritoneal metastasis-specific immune responses. Given the established role of LYVE1+ macrophages and monocytes in tumor progression and their prevalence in the human peritoneal cavity, mouse models will be employed in parallel to further investigate their functions during CRC peritoneal metastasis. Building on my PhD and postdoctoral training, this study aims to further track the trafficking of peritoneal myeloid cells and their interactions with T cells to clarify their roles in anti-tumor T cell responses and immunotherapy, setting the stage for my career as an independent researcher. The ultimate goal of this proposal is to develop a deep understanding of the immunological interplay within the peritoneal cavity during GI cancer metastasis, potentially revolutionizing our approach to immunotherapy by targeting these dynamics more effectively.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Sensorineural hearing loss is caused by the death of hair cells in the organ of Corti, and once lost, cochlear hair cells in humans and other mammals do not regenerate. In contrast, non-mammalian vertebrates can functionally recover from deafening injury by mobilizing supporting cells to divide and differentiate to replace lost hair cells. Over the last 10 years, the consensus from many studies is that supporting cells in the embryonic and neonatal organ of Corti retain a limited capacity to divide and differentiate into hair cells under certain conditions, but that this ability declines precipitously prior to the onset of hearing. One facet of such an age-dependent decline in regenerative potential is the function of the transcription factor Atoh1. Ectopic expression of Atoh1 in embryonic or neonatal cochlear tissue can transform supporting cells or adjacent non-sensory cells into hair cells - but this ability appears to be severely diminished after the onset of hearing in mice. We have shown that additional hair cell transcription factors, such as Gfi1 and Pou4f3, can enhance the ability of Atoh1 to generate hair cell-like cells in older animals, but it is not clear how closely these resemble bona fide hair cells, whether reprogramming is feasible in ears that have been chronically deafened, and whether additional interventions can improve the modest regeneration we observe. This proposal seeks to evaluate the feasibility of transcription factor reprogramming in the mature, deafened cochlea. We will also apply these cochlear regenerative strategies to the mature utricle, which we and others have shown to have a greater capacity for regeneration than the cochlea, and also a better response to transcription factor reprogramming.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The mammalian inner, middle and outer ears have different embryonic origins, yet the development of each component of the auditory apparatus must be precisely synchronized in space and time. Understanding the mechanisms that regulate and co-ordinate the development of these structures is of central importance in understanding the basis of the many birth defects that affect hearing. We have identified a Forkhead transcription factor, Foxi3, that is expressed at very early stages in the embryonic head. Foxi3 mouse mutants made in our lab lack all components of the inner, middle and external ears. Our work suggests that one of the first steps in ear induction– the formation of the otic placode – does not occur in Foxi3 mutants. Moreover, the mesenchyme of the first and second branchial arches that generate the middle ear ossicles and the external ear begins to form in Foxi3 mutants, but rapidly succumbs to massive cell death. Moreover, we have recently identified human patients with Foxi3 variants that have a variety of defects in their hearing apparatus To our knowledge, Foxi3 is the only mammalian gene that causes a complete developmental failure of the entire inner, middle and outer ears when mutated by itself. We are therefore extremely interested to understand how Foxi3 orchestrates development of the auditory apparatus at both the cellular and molecular levels. Our data suggests that Foxi3 may act as a “pioneer” transcription factor – its main function in addition to initiating transcription is to epigenetically organize genomic loci containing ear-specific genes in a transcriptionally competent state. Our first two aims will determine the function and mechanism of Foxi3 during development of the inner ear using knockout mice, chick embryo manipulations and state-of-the-art ES cell models, deep sequencing and bioinformatic analysis. Our final aim focuses on the function the Foxi3 gene in the development of the middle and external ear – here we will both study the effects of loss of Foxi3 in mice, but also create mouse mutants that recapitulate the genetic variants seen in some of our human patients.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Preterm birth is the most common cause of cerebral palsy (CP) in the US and, consequently, the most common cause of childhood dystonia, a disabling condition affecting 2 of every 1000 Americans. Despite its high prevalence, dystonia in CP remains difficult to treat because 1) it is hard to predict, preventing early treatment when it is most effective; 2) its neuropathology after preterm birth is unclear; and 3) few treatment targets exist. This proposal addresses these gaps by investigating a novel cortical cause of dystonia after preterm birth. We and others have shown that striatal injury, namely chronic excitation of striatal cholinergic interneurons (ChINs), causes dystonia. Yet, anticholinergic dystonia treatments are variably effective in CP, necessitating a search for other treatment targets. We have shown that cortical injury, more than striatal injury, best predicts dystonia in children born preterm. Cortical pathology is also seen in adults with non-CP dystonias who demonstrate abnormal sensorimotor cortex inhibition, suggesting dysfunction of cortical GABAergic interneurons. The largest class of these interneurons are parvalbumin-positive (PVINs). Our preliminary data show that chemogenetic inhibition of sensorimotor cortex PVINs in mice causes dystonia and that mice born preterm have reduced cortical parvalbumin immunoreactivity and fewer PVINs. Sensorimotor PVINs may cause dystonia via targeted modulation of striatal ChIN activity. Sensorimotor PVINs inhibit glutamatergic cortical output neurons and striatal ChINs receive glutamatergic cortical input. Yet, it is unknown if sensorimotor PVINs modulate striatal ChIN activity via these excitatory corticostriatal neurons. These data support our central hypothesis: sensorimotor cortex PVIN inhibition causes dystonia after preterm birth via striatal ChIN excitation. We will leverage two of our recent scientific advances to test our innovative hypothesis: 1) our novel mouse model of preterm birth that demonstrates dystonia by postnatal day 42, and 2) quantitative dystonia measures in mice that we developed and clinically validated. In Aim 1, we determine whether cortical dysfunction precedes, and potentially predicts, dystonia onset in mice born preterm by longitudinally assessing sensorimotor PVIN number and cortical oscillatory activity using electroencephalography, and dystonic behavior using our innovative clinically-validated dystonia measures. In Aim 2, we determine whether inhibition of sensorimotor PVINs excites striatal ChINs using fiber photometry and chemogenetic inhibition of sensorimotor PVINs and corticostriatal neurons. In Aim 3, we determine whether chemogenetic sensorimotor PVIN excitation can reduce dystonia in mice born preterm and whether this chemogenetic treatment prior to dystonia onset at postnatal day 42 is more effective than treatment after dystonia onset. These studies will establish the critical role of sensorimotor PVINs in dystonia prediction (Aim 1), pathophysiology (Aim 2), and treatment (Aim 3) and provide the necessary foundation for future translational studies testing cortically-targeted treatments for dystonia following preterm birth.

NIH Research Projects · FY 2025 · 2025-08

Project summary Lymphatic filariasis (LF) is a mosquito-borne infection caused by filarial nematodes, leading to acute fever attacks and long-term disability due to hydrocele, lymphedema, and elephantiasis. The Global Program to Eliminate Lymphatic Filariasis (GPELF) is the largest public health intervention initiative based on mass drug administration (MDA), aiming to interrupt the parasite lifecycle by treating the at-risk population with drugs that clear transmissible first stage larvae (microfilariae, mf) in the human host. Despite years of MDA, LF transmission persists or has resurged in some countries that were deemed to have reached elimination targets, threatening the World Health Organization’s elimination goals. To mitigate this risk, understanding the various causes of infection resurgence is essential, and closing this knowledge gap is our focus. Current measures of infection, which rely on counting mf in blood (since adult filariae are not directly accessible) or detection of circulating filarial antigen, cannot differentiate between recrudescence and reinfection, or determine the origin of transmission. We recently demonstrated that a comprehensive full-genome analysis of individual mf from Wuchereria bancrofti infected individuals enables (a) estimation of the number of reproductively active females, (b) genetically tracking these maternal lineages through treatment, and (c) differentiation between recrudescence and reinfection. This prior advancement facilitates determination of parasite population genetics among hosts and geographies. In this proposal, we will test our overarching hypothesis that by reconstructing sibling relationships in longitudinal mf samples and performing genomic characterization in geospatial mf samples it is possible to monitor the number of reproductively active adult females, differentiate new and established maternal families, and understand the population dynamics in elimination contexts. Analyzing longitudinal samples provides insights into temporal changes in the parasite population (Aim 1), while analyzing geospatial samples identifies transmission sources (Aim 2). Together, these analyses will elucidate the causes of persistent or resurgent infections and enable the generation of evidence- based empirical parameter estimates for transmission modeling (Aim 3) to determine the likelihood and pace of resurgence, supporting identification of the optimal surveillance strategies. Collectively, the results of our research will link newly generated population genetic information to transmission dynamics modelling to predict the risk that parasite migration and/or resurgence (or poor treatment response) will impede W. bancrofti elimination.

NIH Research Projects · FY 2025 · 2025-08

Abstract Bats harbor the unique ability to host a wide array of emerging viruses, such as Ebola virus, Nipah virus, Hendra virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). These RNA viruses are highly pathogenic and often lethal to humans and animals. Intriguingly, bats develop no/minimal signs of diseases in both natural and experimental infections. Significant progress has been made to suggest the altered immunological networks and dampened inflammatory signaling in bats. However, the direct viral sensing mechanisms in bats and the unique immunological features that distinguish bats from other mammals remain poorly studied. Inflammasomes are multi-protein signaling platforms that form in epithelial cells and myeloid cells upon stimulation by pathogen and damage signals. Their primary function is to active the inflammatory caspases such as caspase-1. Canonical inflammasome sensors consist mainly of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing (NLR) family proteins. Among these NLR proteins, NLRP6 is a unique pattern recognition receptor that is predominantly expressed in intestinal and liver system. The inflammasome function of NLRP6 has been reported to directly detect the RNA viruses (rotavirus and mouse hepatitis virus) that infect the gastrointestinal (GI) tract. On the other hand, the excessive activation of NLRP6 inflammasome may exacerbate the tissue damage and cause the autoinflammatory diseases. In bats, the GI tract represents one major organ for viral infection, while infections rarely cause symptoms. The long-term goal of our project is to understand the specific inflammasome sensing mechanisms in detecting RNA viruses in the intestinal epithelium of bats and gain the insights of how bats protect themselves from the pathogenesis of inflammation-induced intestinal barrier dysfunction. In this application, we propose to pursue the following specific aims: 1) Determine the cryo- EM structures of bat NLRP6 monomer, elucidate the biochemical foundation of bat NLRP6- dsRNA interaction, determine the cryo-EM structures of bat NLRP6 with viral dsRNA and compare the structural mechanisms of dsRNA sensing and inflammasome signaling among bat, mouse and human NLRP6; 2) Elucidate the RNA virus-induced bat NLRP6 inflammasome signaling in reconstituted intestinal epithelial cells (IECs), analyze the bat inflammasome signaling in Eonycteris spelaea (Es) in response to bat-borne RNA viruses, study the genetic role of bat NLRP6 in regulating inflammasome signaling in bat primary IECs/bat intestinal organoids. The proposed studies will guide the development of therapeutics to target GI inflammatory disorders in human based on the molecular details of bat NLRP6 inflammasome.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Chromatin integrates environmental and intrinsic cellular cues to orchestrate nucleosome modifications, therefore regulating basic cellular functions that are essential for cell fate and identity in normal development. Mutations in enzymes that deposit or remove nucleosome modifications often dysregulate chromatin structure and result in pathological gene expression programs in many human developmental disorders. As the basic unit of the nucleosome, alterations in histone genes themselves have only been recently identified in children with developmental disorders and their mechanistic and functional roles remain largely unknown. Given the increasing number of histone germline mutations and lack of understanding of their impact, it is imperative to establish animal models to elucidate their physiological functions and delineate underlying mechanisms. My goal is to uncover the function of histone mutations during development by integrating biochemical and genomic assays, along with employing animal models through the followings aims: (1) Investigate the mechanism of how histone H4 mutants are recruited to heterochromatin, (2) Determine how histone H4 mutations regulate chromatin accessibility and neural differentiation, and (3) Identify the function of histone mutations during development. The central hypothesis guiding this proposal is that histone mutations alter heterochromatin silencing, impact gene expression, and promote neural differentiation, which altogether contribute to brain defects. This research will provide new insights into molecular mechanisms underlying histone germline mutations and the epigenetic causes of developmental disorders. During the mentored period, I will gain training in the following key skillsets: acquiring expertise in mouse models, expanding my knowledge of mouse brain development and in vivo brain models of developmental disorders, deepening training in grant writing and mentoring, as well as scientific career development. With acquisition of these valuable skills, the well-established biochemical and genomics approaches in the Allis laboratory, the great training in neurogenesis and mammalian brain development from my co-mentor Dr. Hatten, and strong support and expertise from my outstanding collaborators, I will be in a unique position to apply diverse approaches to study histone germline mutations in developmental disorders. Importantly, I will receive additional mentoring from my Scientific Advisory Committee, along with fantastic mentorship from Dr. Allis and Dr. Hatten to facilitate my transition to independence. Together, this training and support from the K99/R00 award will fulfill my career goal of becoming an independent investigator in the field of chromatin and developmental biology.

NIH Research Projects · FY 2025 · 2025-08

Abstract Despite the enhancement of antiretroviral therapy (ART) that allows people living with HIV (PLWH) to have an undetectable viral load, HIV-1 persists in a small pool of latently infected, resting memory CD4+ T cells. A major challenge to the immune control and clearance of HIV-1 infection is the rapid within-host viral evolution, which allows selection of viral variants that escape from T cell and antibody recognition. Thus, it is impossible to clear HIV infection without targeting “immutable” components of the virus. Unlike the adaptive immune system that recognizes cognate epitopes or their secondary structures, the CARD8 inflammasome senses the essential enzymatic activity of the HIV-1 protease, which is immutable for the virus. In HIV-1-infected cells, the viral protease is expressed as a subunit of the viral Gag-Pol polyprotein and remains functionally inactive prior to viral budding. Some non-nucleoside reverse transcriptase inhibitors (NNRTIs) can promote intracellular Gag-pol dimerization and subsequent premature intracellular activation of the viral protease. NNRTI treatment triggers CARD8 inflammasome activation, which leads to pyroptosis of HIV-infected CD4+ T cells and macrophages. Thus, targeting the CARD8 inflammasome can be a potent and broadly effective strategy for HIV eradication. Recently, we found that HIV-1 Gag/matrix binds to CARD8 and prevents its activation by the viral protease. This interaction reduces NNRTI efficacy in clearing HIV-1-infected cells. Our goal is to develop a complete and atomically detailed mechanism of CARD8 sequestration by HIV-1 Gag/matrix. In this project, we will combine structural, biochemical, and functional approaches to elucidate the molecular details of the interplay between HIV Gag/matrix and the CARD8 inflammasome. The specific aims in this proposal include 1) functional characterization of CARD8 interaction with HIV- 1 Gag and MA, and comparison of CARD8 inhibition by matrix from different HIV-1 subtypes; and 2) structural and biochemical elucidation of HIV-1 Gag- and MA-mediated CARD8 sequestration. Our study can elucidate the molecular mechanisms of HIV and CARD8 interaction and provide critical insights to CARD8-based drug development.

NSF Awards · FY 2025 · 2025-08

This award supports research on nonlinear dynamical systems that can model a wide range of engineered and natural processes in the real world, thereby promoting the progress of science, and advancing prosperity and welfare. Compared to linear systems, nonlinear dynamics are notoriously more challenging to analyze and control. The objective of this project is to leverage the more comprehensive theory of linear systems to address outstanding challenges in nonlinear dynamics. The project will achieve this through the concept of super-linearization of a nonlinear dynamical system. The application domains where such linearizations can be utilized include fluid dynamics, epidemiology, biology, neuroscience, chemical processes, plasma dynamics, finance, logistics, robotics, and power grids. In addition, the project has developed a robust outreach plan, which comprises organizing tutorials to introduce a larger part of the control community to super-linearization, and research opportunities to undergraduate students interested in dynamics and control. This research aims to establish the groundwork for a unified theory of super-linearization, advancing the field forward. In essence, super-linearization of a nonlinear dynamical system involves transforming it into a linear system operating in a higher-dimensional state space, where its trajectories align with those of the original system after projection. This project comprises three research thrusts to address specific questions related to the existence, computation, and implementation of super-linearization. These thrusts are interconnected, yet none relies on the success of others to proceed, thereby mitigating the inherent risks associated with this research endeavor. More precisely, the first thrust employs algebraic methods to study super-linearizations, particularly focusing on the case of polynomial vector fields. The second thrust explores geometric aspects, to explore the properties of the space of super-linearizable vector fields, such as whether it is locally an infinite-dimensional manifold. The final thrust combines algebraic and graphical invariant theory to obtain practical insights into super-linearization and its implications to other relevant fields, including optimal control theory. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2025-08

Preterm birth, defined as delivery before 37 weeks of gestation, affects ~10% of pregnancies globally and remains the leading cause of neonatal morbidity and mortality. It often leads to long-term complications such as cerebral palsy, developmental delays, and sensory impairments. Despite extensive research, the underlying mechanisms of preterm labor are not well understood, partly due to limitations in current monitoring tools. Clinical devices like tocodynamometry and intrauterine pressure catheters offer limited spatial and temporal resolution and are either invasive or insufficient for early detection of abnormal uterine contractions. This project aims to develop a wearable, AI-integrated, multimodal uterine monitoring system that combines electrical and mechanical signal analysis to offer a more comprehensive understanding of uterine activity during pregnancy and labor. Building on recent advances in electromyometrial imaging (EMMI), the system introduces several key innovations. First, a soft, stretchable, polymer sponge-based sensor capable of capturing both uterine electrical activity and mechanical contractions will be developed, enabling better insight into excitation-contraction coupling in the myometrium. Second, a wireless, motion- artifact-tolerant data acquisition platform will be designed, incorporating embedded machine learning (ML) algorithms for enhanced signal quality and real-time monitoring, even in ambulatory settings. The system will be validated on human subjects against gold-standard clinical tools in labor and delivery settings to assess accuracy, reliability, and usability. Additionally, AI-based predictive analytics will be developed for labor risk stratification and early detection of adverse outcomes, such as preterm birth and fetal distress. By integrating advanced biosensing materials, wireless technology, and AI-driven data interpretation, this project will transform the monitoring of uterine activity, enabling long-term, at-home pregnancy surveillance for high-risk patients. The proposed system has the potential to improve early detection and management of preterm labor, reducing fetal mortality and improving maternal-fetal health outcomes. The long-term impact of this work extends beyond preterm birth prediction; it will advance our fundamental understanding of uterine physiology, facilitate personalized obstetric care, and provide new tools for remote prenatal monitoring. RELEVANCE (See instructions): This project aims to develop a wearable, artificial intelligence (AI)-integrated system for noninvasive monitoring of uterine contractions, combining spatiotemporal mapping of electrical and mechanical signals to improve early detection of labor risks, including preterm birth. By enabling long-term, in-home maternal monitoring, this technology has the potential to enhance prenatal care, reduce fetal mortality, and improve pregnancy outcomes.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The mammalian centrosome-cilium complex is involved in cell division, signaling, and motility. Defects in the structure or function of the complex are linked to a variety of human disease states, including cancer, microcephaly, and a group of inherited disorders known as ciliopathies that affect many tissues and organs. The centrosome is composed of two centrioles surrounded by pericentriolar material and is the major microtubule organizing center of animal cells. One of the centrioles within the centrosome templates the growth of a cilium, an antenna-like organelle involved in cell signaling and motility. While the field has catalogued many of the hundreds of proteins in the complex, how they work together to create functional centrosomes and cilia is unclear. We propose that specialized, compound (triplet and doublet) microtubules within the complex form dedicated scaffolds for the protein-protein interactions that define the organelle. Triplet microtubules in centrioles and doublet microtubules in cilia are conserved in almost all species with these organelles and are only found in centrioles and cilia. Little is known about how these microtubules are formed and regulated to create functional organelles. Delta-tubulin and epsilon-tubulin, two little-studied members of the tubulin superfamily, are key proteins involved in forming compound microtubules at the centrosome-cilium complex. Null mutations in either delta- or epsilon-tubulin in mammalian cells results in the formation of aberrant, unstable centrioles with singlet microtubules that fail to recruit many centrosome proteins. The molecular mechanisms by which these tubulins act on centrioles are unknown. Here, using a combination of high-resolution expansion microscopy, CRISPR/Cas9 gene editing, biochemistry, and proteomics, we will address outstanding questions about the structure, formation, and function of the compound microtubules. We will: 1) determine the molecular mechanisms by which delta-tubulin and epsilon- tubulin form or stabilize the compound microtubules, 2) define how the compound microtubules elongate, and 3) determine how the compound microtubules scaffold the centrosome-cilium complex. These studies will provide mechanistic insight into the assembly of these microtubule structures, improve our understanding of the centrosome-cilium complex, and provide insight into its dysregulation in disease.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Clostridioides difficile (Cd) infection (CDI) is a leading cause of healthcare-associated infections in the U.S., affecting ~450,000 people and costing ~$5.4B annually. Despite substantial advances in investigating Cd pathogenesis and transmission over the past 20 years, CDI remains a significant public health burden. Recent studies suggest multi-strain Cd infection is more common than previously recognized and may cause worse outcomes versus single strain infection. Another recent development is recognition of cryptic clade Cd strains that can cause CDI but may not be detected with current culture methods or diagnostic assays. A major impediment to studying multi-strain and cryptic clade Cd colonization and CDI is a lack of validated culture methods to optimize recovery of more multiple Cd strains or cryptic clade Cd strains. Typically, a single approach is used to isolate Cd from stool. However, growth of Cd in culture varies by strain, and is impacted by spore shock method (i.e. ethanol or heat shock), spore germinant (i.e. primary bile salt or lysozyme), and nutrients and selective antibiotics incorporated into the media. Another challenge to detecting multi-strain colonization and CDI is the need to type multiple colonies to detect the presence of more than one strain, which can significantly increase time and costs. In addition, the probability of detecting multiple strains will be dependent on the number of strains typed. We hypothesize that a combination of Cd culture conditions employed on the same stool specimen will enhance recovery of multiple and cryptic clade strains of Cd, and that metagenomic approaches can be used to detect multiple strains from culture and eliminate the need to type multiple colonies. The rationale for this proposal stems from the need for validated methods to detect the presence of multiple and cryptic clade strains prior to widespread study of multi-strain and cryptic clade strain Cd colonization and infection. Our central motivation is that whether multi-strain or cryptic clade strain Cd colonization and infection cause worse outcomes needs to be confirmed, as this will have a major impact in our understanding and approaches to Cd pathogenesis, diagnosis, treatment and prevention. We propose to develop methods to optimize detection of multi-strain and cryptic clade strain Cd colonization and CDI with the following aims: 1.1) Identify the combination of growth conditions and selective pressures to optimize recovery of multiple and cryptic clade strains of C. difficile from stool specimens that could be broadly employed to study C. difficile infection epidemiology, diagnosis, treatment, outcomes, and prevention., and 1.2) Identify the presence and relative abundance of multiple strains of C. difficile through culture-enriched metagenomic sequencing. Our proposal is significant because validated methods to detect multi-strain and cryptic clade strain Cd colonization and CDI is a necessary first step to study the prevalence and outcomes of multi-strain and cryptic clade strain Cd colonization and CDI. Our proposal is innovative in that it will change the current paradigm of Cd isolation and characterization from stool. The proposed work is impactful in its goal to develop methods that can be rapidly utilized by multiple investigators.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Our goal in this new WRHR is to leverage the significant resources, mentorship, and track record of successful physician-scientists within WashU and the Department of OB/GYN to train and promote the development of future leaders in interdisciplinary research in women's reproductive health. The WashU WRHR program will have three core content themes – 1) Pregnancy and pregnancy complications, 2) Gynecologic cancer, and 3) Benign gynecology and reproductive health – and four methodologic tracks: 1) Clinical trials and epidemiology, 2) Implementation science, 3) Basic and translational science, and 4) Engineering and data analytics. This program, designed with input from successful OB/GYN physician-scientists who launched their careers at WashU, will allow the WRHR Scholars to immerse themselves in reproductive sciences and use the skills and knowledge of diverse scientific disciplines to conduct rigorous, high-impact research aimed at improving women's reproductive health. We will accomplish our goal by pursuing the following four objectives: 1) Provide robust interdisciplinary research experiences in women’s reproductive health. Each Scholar, with the guidance of Mentors, the Program Director (PD), and the Research Director (RD), will design a research project centered in one of our three core content themes. The Scholars' research projects will be the foundation for subsequent grant proposals as independent investigators directing research teams. 2) Provide effective mentoring to promote Scholars’ retention and productivity. Each Scholar will have an interdisciplinary mentor team that includes a primary mentor and at least two secondary extra-disciplinary and external mentors. The mentors, PD, and RD will guide the Scholars to understand research and the research landscape, develop critical assessment skills, ask creative questions, and identify and overcome challenges. The PD and RD will regularly monitor each Scholar’s progress, provide feedback to overcome obstacles to success, and work to retain them in the pursuit of women’s reproductive health research. 3) Provide formal didactic training and professional development opportunities. We will help each Scholar choose the most relevant discipline-specific courses, seminars, and symposia within one of our four methodologic tracks. Additionally, all Scholars will participate in core professional development programs and peer networking to master essential research execution, management, and leadership skills. And 4) Rigorously and comprehensively evaluate and improve our program. The Advisory Committee will annually use milestone tracking to evaluate each Scholar and the program as a whole. Additionally, WashU-WRHR leaders will participate in the WashU Council of NIH-funded Training programs to learn and share best practices, exchange ideas, and work to continually enhance our program.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Brain function is dependent on the precise wiring of distinct neuron types into functional neural circuits. Neurons communicate with each other through synapses, which are carefully monitored and supported by specialized non-neuronal cells called glia. Astrocytes are a prominent, peri-synaptic glial cell population that regulates synapse development, synapse stability, and neuronal signaling. As neuronal signaling is an energetically demanding process, one critical function of astrocytes is to support the metabolic needs of neighboring neurons. In the healthy nervous system, astrocytes are known to shuttle lactate directly to neurons to facilitate neuronal respiration and ATP generation. Recent data suggests that under pathological conditions, astrocytes can also transfer entire mitochondria to damaged or diseased neurons to boost neuronal metabolism and restore neuronal function. Whether astrocytes donate mitochondria to neurons under homeostatic conditions is not clear. Furthermore, the mechanisms used by neurons to stimulate intercellular transport of astrocytic mitochondria are poorly defined. Mitochondrial dysfunction is a hallmark of normal aging that is accelerated in neurodegenerative disease; thus, understanding the cellular and molecular mechanisms used by astrocytes to support neuronal metabolism is a key knowledge gap that impedes our ability to rejuvenate the brain. To explore astrocyte-neuron metabolic coupling, we leverage Drosophila as a system where we have precise genetic access to neurons and associated astrocytes, where we have sophisticated genetic tools for optogenetic and thermogenetic manipulation of neuronal activity, and where we have a wealth of transgenic tools for visualizing mitochondrial location and function. We found that stimulating motor neuron activity is sufficient to recruit astrocyte mitochondria towards neuronal synapses and can induce astrocyte-to-neuron intercellular transport of mitochondria. Moreover, we found that astrocyte-specific knockdown of the mitochondrial adaptor milton completely blocked entry of astrocyte mitochondria into the synapse-rich neuropil, resulting in reduced motor neuron activity and defective locomotor behavior. In this proposal, we continue to leverage this model system to understand (Aim 1) what are the cellular mechanisms that position astrocyte mitochondria near neuronal synapses and (Aim 2) facilitate astrocyte-to-neuron mitochondria transport. Finally, we aim to identify the neuronal-activity induced cues that trigger intercellular transport of astrocyte mitochondria (Aim 3). Ultimately, we hope that a better understanding of the cellular and molecular mechanisms that couple astrocyte and neuronal metabolism will enhance our ability to alleviate circuit dysfunction in neurodegenerative disease.

NIH Research Projects · FY 2026 · 2025-08

Project Summary Preclinical studies that have shown significant analgesic efficacy of targeting peripheral cannabinoid type 1 receptors (CB1Rs) in diverse pain models contrast with disappointing results from clinical studies where the analgesic efficacy of targeting CB1Rs, including using peripherally selective compounds, has been inconsistent. This discrepancy likely results from a number of addressable issues that we will resolve in this proposal. Perhaps cannabinoid therapeutics tested in humans to date do not sufficiently activate CB1R in the periphery. Inadequate peripheral restriction has hampered prior clinical trials of purported “peripherally restricted” cannabinoids. Partitioning of these compounds into the brain leads to dose limiting psychoactivity. We recently developed VIP36, a novel CB1R agonist that shows robust analgesic actions and dramatically improved peripheral restriction. The improved peripheral selectivity of VIP36 will allow maximal activation of peripheral CB1R without psychoactivity associated with CB1R activation in the brain. Perhaps the way we have done preclinical testing in animals is not predictive of efficacy in humans. In Aim 1 of this application, we test the efficacy of VIP36 in mouse models of post-traumatic headache, post- operative pain, and a new model of nerve injury-induced pain. These models have improved face validity for human clinical pain. We also use behavioral endpoints that assess affective-motivational aspects of pain, which will enhance preclinical validation and provide greater predictive power for utility in humans. Perhaps there are differences in CB1R expression or function between rodents and humans. In Aim 2, we will test whether CB1R is expressed in similar cell populations in dorsal root ganglia (DRG) and trigeminal ganglia in mice and humans. We will also test whether CB1R activation has similar functional effects in mouse and human DRG neurons using electrophysiology. Perhaps prior cannabis exposure blunts analgesic efficacy of CB1R ligands in humans. Frequent cannabis use leads to downregulation of CB1R in human brain. This could lead to reduced efficacy of cannabinoid analgesics. Given the widespread recreational and medical use of cannabis, it is conceivable that CB1R downregulation or tolerance in participants of clinical trials could confound results. In Aim 3, we test whether a history of frequent THC exposure leads to cross-tolerance to analgesic effects of THC and VIP36. We also ask whether frequent THC exposure leads to reduced CB1R expression in dorsal root ganglia (DRG) and trigeminal ganglia in mouse models and in DRG recovered from human organ donors with a history of daily cannabis use. Collectively, our approach is designed to evaluate the potential analgesic efficacy of peripherally restricted CB1R agonists and obtain data that will either support or refute the notion that this is likely to translate effectively to humans. The proposed studies will also significantly advance our understanding of human cannabinoid receptor biology.

NIH Research Projects · FY 2026 · 2025-08

PROJECT SUMMARY/ABSTRACT Despite advances in sickle cell disease (SCD) treatment, patients continue to experience stroke, endure lifelong cognitive disability, and have a reduced life expectancy of 43 years. While previous imaging studies have focused on anemia and hypoxia as risk factors for stroke, they have not adequately explored other potential disease mechanisms in SCD, such as systemic thromboinflammation and blood-brain barrier (BBB) disruption, which may contribute to increased stroke risk. Although thromboinflammation-targeted approaches have shown promise in mitigating BBB disruption and reducing brain injury in animal models of SCD, their effectiveness in humans remains unexplored. To bridge this gap, this K23 proposes a prospective cohort study to investigate the central hypothesis that BBB dysfunction, associated with specific thromboinflammatory pathways, predicts silent infarct progression and cognitive decline in patients with SCD. The study has three main aims. In Aim 1, using both a well-validated MR sequence and a novel MR sequence, it will assess BBB permeability in patients with SCD compared to healthy controls, stratified in relation to existing infarct burden. In Aim 2, the study will investigate specific thromboinflammatory pathways associated with BBB dysfunction by examining unique gene expressions associated with elevated BBB permeability. Finally, in Aim 3, during a 30-month follow-up period, the study will determine whether baseline BBB permeability can predict the progression of ischemic brain injury and cognitive decline (executive function). This K23 proposal will be implemented with the support of an interdisciplinary team comprising the PI, as well as, mentors and consultants who are experts in relevant fields including: SCD, cerebral small vessel disease, advanced MRI methods, platelets and thromboinflammatory disorders, cognitive function in SCD, and biostatistics. The candidate, an adult cerebrovascular neurologist, has a long term goal to become an independent physician-scientist and to integrate advanced neuroimaging with blood-based markers of thromboinflammation to best achieve a comprehensive understanding of cerebral microvasculopathy and its cognitive consequences. The career development plan outlines three key areas: (1) expertise in advanced neuroimaging acquisition, data processing, and application; (2) foundation in systemic thromboinflammation; and (3) foundation in cognitive testing to translate MRI findings into clinically relevant outcomes. Successful completion of the proposed research and career development activities will fill knowledge gaps for the developing PI and provide compelling evidence on the role of BBB integrity and thromboinflammation in ischemic injury and cognitive decline in SCD. Furthermore, it will guide the development of an independent R01 proposal aimed at investigating BBB integrity as a novel neuroprotective strategy for treatment of cerebral small vessel diseases.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Half of US adults age 65 or older report doctor-diagnosed arthritis, with osteoarthritis being the most common type. While osteoarthritis is strongly associated with aging, the mechanisms through which the aging process influences osteoarthritis risk and progression are still being explored. Circadian rhythm disruptions could be a key component. Laboratory studies indicate that circadian rhythms are important factors for maintaining homeostasis of the joint tissues. And genetic and environmental disruption of the circadian clock can lead to osteoarthritis -like cartilage degeneration in rodent models. Circadian clock disruptions also increase risk of obesity, a key osteoarthritis risk factor. But little is known about the relationship between circadian clock disruption and osteoarthritis risk in human populations. Shift work, particularly night shift work, is an extreme, but common example of such a disruption in the real-world. To better understand the links between shift work, circadian clock disruption, and osteoarthritis, our goal is to evaluate the effect of duration of time in night shift work and age at night shift work on osteoarthritis risk (Aim 1) and examine the influence of chronotype on these associations (Aim 2). We will use the UK Biobank, a prospective cohort of half a million people in the United Kingdom with data on genotype, shift work, and other relevant factors for osteoarthritis risk such as age, sex, heavy manual labor, socioeconomic status, and body mass index. Frequency of night shift work is reported by all working UK Biobank participants. Through the National Health Service, the full cohort is linked to hospital data and half the cohort is linked to primary care data, providing reliable means to ascertain osteoarthritis diagnoses and joint arthroplasty. Our team has extensive experience with UK Biobank, with particular expertise examining occupational and genetic risk factors for musculoskeletal disease. Aim 1 will focus on a 100,000-person subset of UK Biobank that reported lifetime job histories, including history of shift work. Associations of years duration of night shift work and night shift work done at different ages will be estimated with osteoarthritis endpoints. For Aim 2, associations of shift work with osteoarthritis endpoints will be estimated in analyses stratified by chronotype, considering both self-reported chronotype and genetic chronotype. Evaluation of the effect of duration of night shift work, age at night shift work, and the modification of effects by chronotype will be a critical step forward in determining if these associations are indicative of the effects of circadian rhythm disruption on osteoarthritis and identifying populations that may be most vulnerable. Circadian clock disruption may be a novel risk factor for osteoarthritis that influences cartilage degeneration both through effects on obesity, and obesity-independent pathways. These disruptions could be a crucial piece of the relationship between aging and osteoarthritis. Importantly, as lifestyle changes and chronotherapy can reduce circadian clock disruptions, these findings can point to new potential ways to prevent osteoarthritis development and progression.

NIH Research Projects · FY 2025 · 2025-07

Career Goal: 1. To further define the role of visual morbidity and autism spectrum disorder phenotype. 2. To understand the role of visual development in ASD morbidity guided by fcMRI biomarkers in both sighted and children with visual comorbidities. Research Project: Autism spectrum disorder (ASD), which affects 2% of individuals in the population, is defined by deficits in social communication and restrictive and repetitive behaviors. ASD is characterized by etiologic heterogeneity with contributions from multiple genes, environmental factors, and co-occurring neurological and mental health comorbidities. ASD is 30 times more common in children with congenital blindness, and decreased visual acuity among a population with comorbid social deficits can be particularly burdensome. 20-44% of children with ASD have visually significant refractive error. Yet, 31% of ASD children (vs. 4% typically developing children) are unable to wear glasses even though their vision is secondarily degraded to the point of legal blindness. Refractive surgery can improve visual acuity in this population. We recently published the results of a pilot study of 24 children pre- and 1-month post- refractive surgery. In addition to gains in visual function, we showed that Social Responsiveness-2 (SRS-2) social awareness scores improved by a median of 15 points post-surgery. The objective of the present study is to quantify improvements in visual acuity (Teller acuity testing), adaptive behavior (Vineland), and social responsiveness (SRS-2) post refractive surgery. We hypothesize that in addition to gains in visual function, SRS-2 and Vineland-3 scores will improve in ASD children post-refractive surgery. If these benefits are validated, pediatric refractive surgery could easily be disseminated, with guidelines informed by this study (and subsequent R01), as thousands of centers throughout the world already routinely perform refractive surgery on adults. Furthermore, the proposed study will tangibly impact the basic science of ASD – regarding the role of decreased visual acuity as a comorbid, contributing, or actively reinforcing factor – by clarifying the visual system and cognitive neuroscientific underpinnings of treatment effects on vision and behavior in this population versus other ASD populations. Career Development: This K23 award will provide me with the necessary training in the areas of behavioral phenotyping of ASD children, as well as the concepts and methods of functional neuroimaging. Training will occur at Washington University School of Medicine, which is a highly collaborative scientific environment and a leading institution for ASD, pediatric ophthalmology, and neuroimaging.

NIH Research Projects · FY 2025 · 2025-07

PROJECT ABSTRACT The overall goal of this proposal is to establish and elucidate novel functions of the ion channel TRPV2 in controlling the cGAS/STING-mediated innate immune response. Serving as the first line of defense against microbial pathogens, the cGAS/STING-mediated innate immune system also senses and responds to self-DNA in the cytosol derived from the nuclear and mitochondrial genomes. Upon the binding of cGAS to cytosolic DNA, a series of signaling events occur to transduce the immune signal through multiple factors (cGAMP, STING, TBK1, IRF3, NF-B) and organelles (ER, Golgi, nucleus) to induce the expression of type I interferons and inflammatory cytokines. This innate immune response promotes pathogen restriction, and directly influences tissue maintenance, tumorigenesis, and cancer treatment outcome. However, despite intensive studies, our understanding of the mechanisms and regulation of innate immune signaling remains limited. In particular, precisely how STING is regulated on the ER and how it integrates various signals before translocating to Golgi to induce a balanced immune response remain outstanding questions. Our recent discovery of a physical and functional interaction between STING and the ion channel TRPV2 on the ER in a Ca2+-dependent genome protection pathway provides an exciting new avenue to address these fundamental questions. In this pathway, cytosolic DNA induced by replication stress triggers cGAS activation and cGAMP production. The binding of cGAMP to STING causes its dissociation from TRPV2 on the ER, leading to TRPV2 derepression and Ca2+ release. The resulting elevation of cytoplasmic Ca2+ then activates CaMKK2 and downstream kinase AMPK to protect the genome from EXO1-mediated aberrant replication fork processing. Interestingly, our unpublished results strongly suggest this TRPV2/Ca2+-dependent pathway also directly regulates STING activation and innate immune signaling. Building on our findings, in this grant application we describe a series of experiments to delineate the multifaceted functions of TRPV2 in the innate immune response. In Aim 1, we will elucidate the mechanisms by which TRPV2-mediated Ca2+ release activates the STING-dependent innate immune pathway. In addition, we will determine whether the loss of Trpv2 can rescue the phenotypes of a Trex1-/- mouse model for the autoimmune disorder Aicardi-Goutières syndrome (AGS). In Aim 2, we will define the role of the physical interaction of TRPV2 in STING regulation and the functional relationship between TRPV2 and another ER protein STIM1 in this regulation. These studies will significantly advance our understanding of the innate immune response system and facilitate the development of new therapeutic strategies for autoimmune diseases, chronic inflammation and cancer.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY/ABSTRACT This project has two overarching goals: (1) to investigate the role of dendritic cell (DC) lysosome activity in immune checkpoint inhibitor (ICI)-associated myocarditis, and (2) to serve as a platform for further research and career training of the PI, Dr. Kenji Rowel Lim, to transition to independence. Although tremendously successful, ICIs in cancer therapy induce several immune-related adverse effects. ICI myocarditis is a highly fatal disease of inflammation-induced cardiac injury, with its incidence expected to rise due to improved diagnosis and in- creased ICI use in the clinic. Effective treatments are unavailable and urgently needed. Research has tradition- ally focused on how unrestrained autoreactive T cell activation from loss of immune checkpoint inhibition fuels ICI myocarditis. Preliminary work by the PI suggests that DCs, which take up and present antigens to T cells for activation, are also affected under these conditions, highlighting a previously unexplored layer of complexity in this disease. In particular, the PI found that DC lysosomes had reduced activity in the absence of immune check- point inhibition. Based on these data, it is hypothesized that reduced DC lysosomal activity in ICI myocarditis leads to reduced degradation of internalized antigens, better preserving epitopes, enhancing presentation, and increasing T cell activation. This hypothesis will be tested by characterizing differences in lysosomal activity between wild-type DCs and DCs deficient in the immune checkpoint receptor PD-1 (PD-1 KO), and establishing a connection between PD-1 signaling and lysosomal activity (Aim 1); determining if reducing lysosomal activity in DCs provokes myocarditis in the setting of cardiac injury (Aim 2); and determining if enhancing lysosomal activity in PD-1 KO DCs prevents ICI myocarditis (Aim 3). This project will advance understanding of ICI myo- carditis biology and uncover potential therapies for this disease. The PI has extensive research experience in molecular biology, genetics, and cardiac immunology, from previous Ph.D. training in Canada and postdoctoral training with Drs. Abhinav Diwan and Douglas Mann. The PI seeks to acquire additional training via a mentored approach to complete the proposed work and transition to independence. In their career development plan, the PI aims to: (1) acquire further scientific training in immunology and lysosomal biology to enable the pursuit of innovative cardiac immunology research, (2) acquire training in leadership, mentoring, lab management, and grant writing to become an effective principal investigator, and (3) actively prepare to secure a faculty position at a top-tier research institution. In addition to their primary mentor (Dr. Diwan) and co-mentor (Dr. Mann), the PI will be mentored by a team of leading experts in cardiac immunology (Dr. Sumanth Prabhu, Dr. Pilar Alcaide), and lysosomal biology (Dr. Marco Sardiello), who will also advise on career development. With the exceptionally supportive research environment at Washington University in St. Louis, this training experience will enable the PI’s successful transition to becoming an independent investigator, advancing knowledge on how the immune system shapes the heart, and using this understanding to develop novel, effective therapies for cardiac disease.

NIH Research Projects · FY 2025 · 2025-07

Summary Niemann-Pick C1-related protein 1 (PfNCR1) is a cholesterol (CHL) transporter that is of great interest as an antimalarial drug target. PfNCR1 resides in regions of the parasite plasma membrane that are in contact with the parasitophorous vacuolar membrane that surrounds the malaria parasite inside its host erythrocyte. It is believed that this transporter defends the CHL-poor plasma membrane from CHL accumulation by pumping CHL out. We have recently obtained a cryo-EM structure of PfNCR1, alone and in complex with a small molecule inhibitor. The structures reveal a CHL tunnel that appears to be blocked by inhibitor. Other known inhibitors are predicted by molecular modeling to block CHL movement through the tunnel. We have identified two new PfNCR1 inhibitors that are predicted to bind away from the tunnel region and are putative allosteric inhibitors. We propose to characterize the mechanism of inhibition of PfNCR1 by these interesting new inhibitors, using structural, physiological, genetic and cell biological approaches. We recently described a region of contact between the parasite plasma membrane and parasitophorous vacuolar membrane in a study of PfNCR1 localization. We proposed that the contact sites could be where hydrophobic solutes are transported, in contrast to the regions where there is a substantial vacuolar space between the two membranes, wherein the protein export machinery resides. Little is known about the region of apposition. Our goal is to take advantage of the PfNCR1 localization to explore the components of this zone. Preliminary proximity biotinylation experiments have shown a number of transporters in proximity to PfNCR1. We propose to study these associations by protein biochemistry and structural studies. The goal is to develop a better understanding of components and interactions in this membrane contact zone, to inform us about function of the region. We anticipate that the proposed studies will give us new insights into an important cellular transporter, PfNCR1, its inhibition, and its function in the unexplored region of apposition between the two membranes that surround the malaria parasite. The proposed work is a terrific collaboration between the cell biology/biochemistry group of Dr. Daniel Goldberg and the structure/physiology group of Dr. Ed Yu. This collaboration is already underway and functioning beautifully. This grant aims to keep this work moving forward.

NIH Research Projects · FY 2025 · 2025-07

Dementia is typically diagnosed in late life, however, the disease process begins decades earlier; mid-life experiences such as work are important modifiable predictors of Alzheimer's Disease and related disorders (ADRD). Work is central to the lives of American adults, but the relationship between work, unemployment, and ADRD across the lifecourse has been understudied. This proposal advances the current literature on how occupations influence dementia risk in the United States in two ways: [1] people work from approximately ages 18 – 65, however little research evaluates age when someone has a particular job, job duration, or changes in work experiences across working years; and [2] occupational classification systems used by population-based datasets have changed to reflect the transition from a manufacturing to information and service based economy (with different physical, environmental, and cognitive demands), however physical, environmental, and cognitive characteristics of work have not been systematically applied to these shifting occupational classification systems. In this proposal, we will evaluate lifecourse work trajectories and ADRD risk through novel applications of sequence analysis, and construct a longitudinal database of physical, environmental, and cognitive demands of work to catalyze research on lifecourse work trajectories and ADRD risk. We will leverage the strengths of two large, longitudinal U.S. cohorts to evaluate the relationships between lifecourse work trajectories and ADRD risk: The National Longitudinal Survey of Youth, 1979 cohort (NLSY), and the Health and Retirement Study (HRS). Our research team has previously published using sequence analysis, and previously used both datasets, demonstrating the feasibility of our proposed project. Work is a modifiable social risk factor that spans decades; a better understanding of work trajectories and features will help identify employment interventions to slow cognitive decline and reduce ADRD disparities.