Washington University

NIH Research Projects · FY 2025 · 2022-06

ABSTRACT The initial adaptive immune response to tumors and many viruses relies on the priming of CD8 T cells to gen- erate cytolytic effector T cells that can specifically target tumors or virally infected cells. The priming of CD8 T cells to these agents is carried out in vivo by a particular type of antigen presenting cell that is a component of the myeloid system and a member of the family of dendritic cells. Classical dendritic cells (cDCs) comprise several closely related lineages that are clearly distinct from other myeloid cells such as macrophages, mono- cytes or granulocytes. Primarily, cDCs serve to activate T cells against infections in the central lymphoid tis- sues, rather than carrying out direct effector functions at sites of infections as the other myeloid lineages do. The cDCs are themselves comprised of at least two major branches, now called cDC1 and cDC2. The cDC1 is a lineage that specializes in the uptake and processing of cell-associated antigens, such as from tumors of virally infected cells and the expression of peptide epitopes on its cell surface in conjunction with MHC-I mole- cules. This form of antigen:MHC-I complex is able to activate CD8 T cells, and not CD4 T cells. This process is called cross-presentation. The cDC2 is not capable of carrying out cross-presentation to viruses or tumors in vivo. The cDC1 has many genetic and molecular differences from cDC2; cDC1 require a distinct set of tran- scription factors for their development that are not required for cDC2. This includes dependence on the tran- scription factors Nfil3, Id2, Irf8 and Batf3. Our recent work showed that the genetic hierarchy among these fac- tors has Nfil3 as the first and initiating factor, acting to indirectly induce Id2 and Batf3 via the suppression of the repressor Zeb2. However, it is still unknown how Nfil3 is induced to initiate this process, and how Nfil3 works to suppress Zeb2 expression. It has recently become important to understand these details because of the clini- cal interest to apply Flt3L administration as a therapeutic in expanding the in vivo population of cDC1. It has been known for some time that Fl3L can expand dendritic cells in general and expand cDC1 in particular. But we have uncovered a surprising and worrisome fact; Flt3L administration will expand cDC1-like cells even in Nfil3-deficient mice, which completely lack cDC1 beforehand. The expansion of cDC1 in Nfil3-deficent mice produced by Flt3L is of the same magnitude as the expansion in WT mice. Thus, Flt3L is inducing cDC1 by a different genetic route than normal cDC1 development. There has been no test of whether such cDC1 cells function normally and will boost an immune response. This application will systematically address this issue by Aim 1) defining the normal process by which Nfil3 is induced, Aim 2) define the mechanism by which NFIL3 drives cDC1 development, and Aim 3) determine whether Flt3-induced cDC1 function normally and determine the mechanism by which Flt3L bypasses the normal requirement for Nfil3 in cDC1 development.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY An immense need for selective and antigen-specific immunotherapy without global immunosuppression exists for autoimmune diseases such as multiple sclerosis (MS) and a closely related condition known as myelin oligodendrocyte glycoprotein (MOG) antibody disease (MOGAD). This has prompted exploration of chimeric antigen receptor (CAR) T cell utilization to specifically eliminate autoreactive cells. We have created a unique version of CAR T cells in which peptide MHCII (pMHCII) was fused with signaling domains in order to recognize specific T cell receptors (TCRs). In preliminary studies we demonstrate that these pMHCII-CAR T cells specifically recognize a cognate TCR in vitro and can selectively kill antigen-specific CD4 T cells in vivo. Efficient depletion of high affinity MOG-specific CD4 T cells was associated with prevention of MOG-induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Modifications in pMHCII-CAR construction led to greater efficiency in eliminating lower-affinity MOG-reactive T cells which was associated with resolution of ongoing EAE. These data suggest an “activation energy” model of autoimmunity analogous to that of a chemical reaction, in which higher affinity self-reactive T cells are needed to provide the activation energy to initiate autoimmunity, but lower affinity T cells are capable of sustaining the autoimmune “reaction.” To address the hypothesis that CAR T cell technology can be used to eliminate auto-antigen-specific T cells and abrogate central nervous system (CNS) autoimmunity in mice and humans without global immunosuppression, we have formulated three specific aims. In Aim 1 we will improve the efficiency of low affinity T cell deletion in vivo. In Aim 2, we will test whether we can successfully target autoreactive CD4 T cells specific to all T cell epitopes of a protein in mice, as we predict that such an approach would be useful for treatment of human disease where the T cell autoantigen is identified by an autoantibody. Finally, in Aim 3, we will directly test whether MOGAD patients show an increased frequency of MOG-specific T cells using pMHCII-CARs for antigen discovery. In sum, our proposed studies will explore the “activation energy” model of autoimmunity and establish an optimal CAR T cell approach to eliminate low affinity autoreactive TCR specificities for the treatment of ongoing autoimmune disease. Finally, we will begin to translate these murine observations to human pMHCII-CAR T cells and assess their potential utility in a relevant human autoimmune CNS disease.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY/ABSTARCT Lung cancer is a leading cause of cancer-related death globally. Nearly a third of patients with non-small cell lung cancer (NSCLC) present with potentially resectable early-stage NSCLC. Despite complete resection, approximately 50% of patients with stage II and III NSCLC recur and die from metastatic NSCLC. There are no reliable biomarkers to predict poor outcomes in early-stage NSCLC. Molecularly targeted therapies and immune checkpoint blockade targeting programmed death-1 (PD-1) or programmed death ligand-1 (PD-L1) have significantly improved the outcomes of patients with metastatic NSCLC, and these agents are now undergoing clinical trials in early-stage lung cancer following standard therapy. The National Cancer Institute (NCI) has launched an ambitious multicenter study, the Adjuvant Lung Cancer Enrichment Marker Identification and Sequencing (ALCHEMIST), to screen nearly 8000 patients with completely resected NSCLC to identify those with activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) and rearrangements in anaplastic lymphoma kinase (ALK) to investigate the role of erlotinib and crizotinib, respectively. Those with tumors lacking EGFR mutation or ALK rearrangement were offered participation in a randomized trial comparing nivolumab, an inhibitor of PD-1 to observation. The ALCHEMIST Genomics Working Group is planning to study the tumor whole genomes, exomes, and transcriptomes from nearly 2000 patients who did not participate in the intervention trials (ALCHEMIST Screening Study) and all those enrolled in the three ALCHEMIST therapeutic trials. This suite of trials with data generated using genomic analyses provides a unique opportunity to explore the role of the cancer proteome in predicting outcomes in patients with resected NSCLC. We propose a Proteogenomic Translational Research Center (PTRC) to study the proteogenomic alterations in resected early-stage NSCLC co-led by the Washington University School of Medicine (WUSM) and the Broad Institute along with investigators affiliated with the NCI-funded National Clinical Trials Network (NCTN) supporting the ALCHEMIST suite of clinical trials. Our overarching objective is to apply mass spectrometry-based global and targeted proteomic analyses to patient-derived resected tumor material to improve upon the predictive biomarkers using somatic cancer genome and transcriptome and clinical characteristics. These discoveries will be translated into targeted assays to predict recurrence following therapy. Since the ALCHEMIST Crizotinib study is still ongoing and has enrolled relatively fewer patients compared to other studies, we will not include those samples in this proposal. The three aims of this project are to develop prognostic assessment tools to predict relapse in patients with resected NSCLC treated with standard platinum doublet chemotherapy (aim 1), standard platinum doublet chemotherapy and nivolumab (aim 2), and standard platinum doublet chemotherapy and erlotinib (aim 3) using proteogenomics.

NIH Research Projects · FY 2025 · 2022-06

Project Summary/Abstract Active motility by apicomplexan parasites controls host cell invasion and egress, steps that are critical to the intracellular cycle of infection. These processes are governed by calcium-regulated secretion of adhesive proteins from micronemes, together with the concerted action of an actin-myosin motor that lies beneath the parasite plasma membrane. Prior studies in Toxoplasma gondii have established the importance of calcium- dependent protein kinases and protein kinase G (PKG) in controlling microneme secretion, motility, egress, and invasion. This cascade is tightly regulated and recent studies have also shown that the c1 isoform of protein kinase A (PKAc1) dampens calcium signaling to shut down microneme secretion and impair motility after invasion is complete. The balance between PKG vs. PKAc1 activities governs the decision to activate motility and promote egress vs. to remain intracellular. Although the role of PKAc1 in blocking premature egress is clear, neither its mechanism of regulation nor the targets that it phosphorylates in order to dampen calcium signaling and block egress have been elucidated. PKA activity is governed by production of cyclic AMP (cAMP) by adenylate cyclases (ACs) and its consumption by phosphodiesterases (PDEs). In preliminary studies, we have identified candidate ACs that control cAMP levels to regulate PKA. In the proposed studies, we will explore their functions to define how they temporally and spatially regulate PKA activity. We have also used a protein degradation approach to create conditional knockdowns of PKA isoforms and verify that PKAc1 controls egress in type II parasites. We will use the tightly regulated conditional knockdown approach to investigate downstream targets of PKAc1 that mediate the egress block using a combination of phosphoproteomic studies to identify substrates and downstream validation studies to test their roles in regulating egress. We will validate the role of specific phosphorylation sites in PKAc1 targets in suppressing calcium signaling, motility, and egress. Collectively, the proposed studies will elucidate the molecular pathway by which PKAc1 down regulates calcium levels, microneme secretion, and motility to prevent egress, thus counterbalancing the activating effects of PKG. Elucidating the molecular regulation of the PKG/PKA kinases in T. gondii will foster future studies to design inhibitors that block these pathways, thus providing new avenues for development of therapeutics.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY /ABSTRACT Anterior segment dysgenesis (ASD) refers to a spectrum of disorders that affect the front of the eye, including the corneal, iris/ciliary body, and lens. Among the characterized ASD genes, the majority encode transcription factors. However, the upstream signaling events that control these transcription factors are unclear. My long- term goal is to establish the gene regulatory networks that underlies the development of the anterior segment of the eye, in order to identify molecular targets for disease prevention and treatment. The objective of this proposal is to study signaling networks and transcriptional programs in lens development. In particular, I have identified mechanistic target of rapamycin complex 1 (mTORC1) signaling as a novel regulator in lens vesicle formation. And I propose to investigate both the upstream regulators and the downstream targets of mTORC1, using a combination of conditional knockout mouse and primary lens epithelium cell models. My central hypothesis is that Fibroblast Growth Factor (FGF) signaling activates mTORC1 in the lens epithelium (Aim 1), and this mTORC1 signaling controls cell adherence during lens vesicle formation through regulating Wnt signaling (Aim 2). In Aim 1, I will elucidate the mechanism underlying how FGF signaling activates mTORC1 in the lens epithelium. In Aim 2, I will investigate how mTORC1 regulates Wnt signaling and the functional significance of mTORC1-Wnt axis in the formation of adherens junctions in the lens epithelium. In addition, I propose to identify mTORC1 mediated transcriptional programs during lens vesicle formation by Single-cell RNA-sequencing. This proposal is innovative because it identifies a novel regulatory pathway in lens vesicle development. It also links signaling pathways and transcriptional factors to elucidate the mechanism of lens vesicle development and disease pathogenesis. Results from this study are significant in that they are expected to impact the understanding of normal and defective development of the lens vesicle, and have the potential to reveal novel molecular targets for preventive purpose of disease caused by congenital lens defects. The mentored phase of this project will be conducted under the guidance of Drs. Xin Zhang and Carol Mason (Columbia University Irving Medical Center), who are experts in eye development and signaling pathways. Additional advice will be obtained from my scientific advisor Dr. Melinda Duncan and Dr. Peter Sims on aspect of lens development and Single cell RNA-sequencing, respectively. Resources for career development along with the research environment at Columbia University are ideal for my transition into an independent investigator studying signaling mechanisms of eye development and disease.

NIH Research Projects · FY 2025 · 2022-06

Treatment-resistant depression (TRD), defined as repeated failure to respond to treatment, is the most virulent form of major depressive disorder (MDD), and claims a disproportionate fraction of morbidity and mortality. The overwhelming costs of depression have motivated a decades-long search for meaningful biomarkers. However, despite substantial effort, and the promise of functional brain imaging as a non-invasive tool for whole-brain measurements, the identification of biomarkers of depression for ready clinical use has remained elusive. The major impediments to progress include substantial phenotypic heterogeneity in typical MDD cohorts and unreliable and imprecise imaging measures. During his graduate and residency years, the principal investigator of this proposal developed novel methods for precise functional neuroimaging, known as `precision functional mapping' (PFM), which have been previously applied to study brain organization in healthy individuals. The present K99/R00 Pathway to Independence award proposal is designed to apply these new methods to address these previous limitations and identify trait and state biomarkers of pathology in TRD. In order to achieve these goals, a comprehensive career development agenda has been structured to train the PI in the evaluation and management of this severely ill population. The PI will also learn how to implement ecologically valid tools for precise behavioral and psychiatric assessments. These training aims will be pursued with the guidance and mentorship of Drs. Charles Conway, Deanna Barch, Eric Lenze, Nico Dosenbach, and Abraham Snyder, world experts in psychiatric assessment, dimensions of psychopathology, human subjects study design, ecological momentary assessment (EMA), cognitive neuroscience, and functional neuroimaging methods. During the training K phase, the PI will acquire a proof-of-principle dataset of extended and repeated resting fMRI data on well-characterized TRD patients, treatment-responsive MDD patients, and healthy controls, in order to optimize the quantitative reproducibility of single-subject functional network maps derived from resting state functional connectivity (RSFC; Aim 1). In the R00 phase of the award, a large cohort (n=90) of TRD, treatment-responsive MDD and healthy controls will be evaluated to identify brain imaging trait biomarkers of treatment-resistant depression (Aim 2). To separate state from trait biomarkers, dense time courses of imaging data, as well as EMA, will be acquired on TRD patients while they undergo electroconvulsive therapy (ECT) treatment (Aim 3). Together, these studies are likely to reveal underlying network features of disease and will determine whether RSFC can be reliably used as a biomarker for TRD. The tools and approach laid out in this proposal will naturally extend to evaluations of other treatment modalities and neuropsychiatric disorders. With the skills and expertise gained through this training and research program, the PI will be well-positioned to pursue an independent career as a physician scientist.

NIH Research Projects · FY 2026 · 2022-06

ABSTRACT Retinal ganglion cells (RGCs) are the sole connection between the eye and the brain. They are particularly susceptible to degeneration, and their damage and death leads to vision loss in conditions like glaucoma, diabetic retinopathy, optic nerve glioma, and optic neuritis. Most treatments for these diseases are not focused on specifically rescuing RGCs, but on relieving apparent drivers of disease progression. For example, current glaucoma treatments focus on reducing elevated intraocular pressure (IOP), but are not effective in the majority of patients. Further, many glaucoma patients also have RGC degeneration without IOP elevations. Thus, new treatments to preserve RGCs in degenerative diseases represent an important unmet clinical need. Although RGC cell death leads to vision loss, RGC death in degenerative conditions is incomplete even in severely affected patients and robust animal models. Understanding how some RGCs natively persist in degenerative conditions can inform the development of new treatment strategies. To identify native coping strategies, we will directly observe cellular traits of individual RGCs prior to and during the course of degeneration, focusing on cellular homeostasis. We have established longitudinal, in vivo, 2-photon imaging of genetically encoded biosensors in RGCs to directly observe energetic and Ca2+ homeostasis at single RGC resolution repeatedly over a protracted period of time. This approach allows for measurements that would normally require either end point sample collection, pooling of RGCs from multiple retinae, or both; limitations that obscure population heterogeneity and individual cell dynamics. We will characterize baseline heterogeneity of energetic and Ca2+ homeostasis, along with dynamics following axon injury and directly relate these measurements with RGC survival or death. Mechanisms of homeostasis are highly relevant to a range of degenerative diseases but have yet to be thoroughly investigated in models of RGC degeneration. Our preliminary data indicate that mouse RGCs that natively survive optic nerve crush have salient features of energetic and Ca2+ homeostasis that can be distinguished from the RGC population as a whole prior to induction of degeneration. These results strongly suggest that homeostatic set-points influence RGC survival outcomes in a severe degeneration model. Further, we will conduct experiments to preserve RGCs in optic nerve crush models by manipulating these pathways to mimic the properties of resilient RGCs using both gene overexpression or repression interventions. Doing so we can validate which of our observations are correlative or causative. The goals of our proposal are thus to: more thoroughly define the homeostatic fingerprint of well surviving RGCs; determine how axotomy induced degeneration impinges on homeostasis of well-surviving versus poorly-surviving RGCs; and translate this information into interventions that preserve RGCs that would otherwise degenerate. Taken together our experiments will identify and validate new approaches towards protection of RGCs.

NIH Research Projects · FY 2025 · 2022-06

Project Summary Ventricular tachycardia (VT) is a dangerous arrhythmia that leads to sudden cardiac arrest if left untreated. VT most often involves regions of the heart that are structurally and/or electrically heterogeneous which provide a substrate for reentry. Currently available antiarrhythmic and catheter ablation therapies are limited in both safety and efficacy. In patients with VT that is refractory to conventional therapy, stereotactic body radiation therapy (RT) has emerged as a promising new treatment. An initial clinical trial showed that a single fraction of 25 Gy ionizing radiation to the heart was associated with greater than 99.9% reduction of VT burden, and this VT reduction persisted for at least 12 months. Importantly, studies at several independent academic hospitals have now demonstrated the efficacy of RT for the treatment of ventricular tachycardia. Despite these promising results, the precise mechanisms by which high-dose radiation reduces VT is unknown. It has been hypothesized that 25 Gray radiation to arrhythmogenic regions of the heart causes late-stage fibrosis thereby preventing re-entry, analogous to scar created by thermal catheter ablation. However, histologic data from explanted hearts of SBRT- treated patients suggests that fibrosis alone cannot account for the magnitude of the observed clinical effect (unpublished). Instead, our preliminary data suggest that radiation to the heart causes functional changes in the electrical substrate that may prevent reentry and reduce VT. We hypothesize that ionizing radiation to the heart leads to changes in cardiac gene expression and electrophysiology. The proposed studies will characterize key molecular and cell-signaling mechanisms by which ionizing radiation influences cardiac conduction. The following specific aims will (1) determine the cellular mechanisms by which ionizing radiation influences cardiac electrophysiology, (2) determine the minimal dose response in a porcine model, and (3) translate biological insights from animal models into humans through analysis of serum-derived biomarkers from RT-treated patients. Defining the acute effects of irradiation on the electrical substrate is expected to facilitate clinical implementation of this promising new anti-arrhythmic therapy and advance the field of cardiac radiation biology.

NIH Research Projects · FY 2025 · 2022-06

Abstract Transcriptomic studies in clinical and biomedical research have mainly focused on changes in linear transcripts to provide knowledge of the genes and co-expression networks implicated in the disease. However, very little is known about the role of circular RNAs (circRNAs) in Alzheimer's disease (AD). CircRNAs are a novel category of non-coding RNAs derived from the back-splicing and covalent joining of pre-mRNA exons and introns. We recently performed a transcriptome-wide analysis of circRNA differential expression in the brain cortex from more than 621 brain samples from two independent and large cohorts of late-onset sporadic AD cases and neuropathology-free individuals. We identified specific circRNAs, including circHOMER1, associated with AD risk and neuropathological traits. This project will use brain tissue to identify additional brain circRNAs implicated in AD from a cohort that is four-fold larger than the cohort in our previous study. We also plan to investigate differentially expressed blood circRNAs in AD cases compared with controls to determine their biomarker utility for creating new prediction models. We will establish a framework for in vitro and in vivo functional characterization of the role of circRNAs in AD. As proof of principle, we will start by defining the role of circHOMER1 in AD-related gene expression and related cellular phenotypes. We have found that circHOMER1 is highly expressed in induced pluripotent stem cell (iPSC)-derived neurons. We will use CRISPR/Cas9 to knock down circHOMER1 as well as use circHOMER1 overexpression (OE) in iPSC-derived neurons from isogenic controls and AD patient-derived neuronal cultures from pathogenic mutation carriers of APP, PSEN1, PSEN2, and MAPT genes. To determine whether circHomer1 accelerate/increase AD-related pathology in vivo, we will generate circHomer1-knockout transgenic mice and cross them with 5XFAD and MAPT-P301S mice. We will also use AAV2/9-mediated circHomer1 OE in the cortex and hippocampus of 5XFAD and MAPT-P301S mice. We recently found that seven-month-old 5XFAD mice display significant reductions of circHomer1 expression, similar to the postmortem brains of AD patients. We will restore circHomer1 levels in the hippocampus and cortex of 5XFAD and P301S mice using AAV2/9 vectors. This proposal will be the first to systematically analyze the role of brain and blood circRNAs in AD and to perform in vitro and in vivo functional studies to characterize the role of circRNAs in AD.

NIH Research Projects · FY 2026 · 2022-06

Alzheimer Disease (AD), one of the major health problem in US and worldwide, is a neurodegenerative disorder that is characterized clinically by progressive dementia caused by pathological changes in brain tissue preceding clinical symptoms by 15-20 years. Diagnostic methods are urgently needed for screening populations for early (preclinical) signs of AD pathology when drug intervention could be most efficient, and providing means for monitoring therapeutic efficacy in clinical drug trials. Recently proposed by National Institute of Aging and Alzheimer Association A/T/N (amyloid/ tau/ neurodegeneration) approach classifies stages of AD by means of AD-related tissue pathology. While brain amyloid plaques and tau neurofibrillary tangles can now be measured in vivo using PET tracers, the neurodegeneration is mostly measured in vivo as tissue atrophy by MRI-based morphological studies. However, histopathological studies demonstrated that the neuronal loss in AD significantly exceeds loss of tissue volume. The objective of this project is to introduce a new, potentially widely available, in vivo MRI-based neuroimaging biomarker that would detect loss of neurons at the very earlier AD stages when this loss is not recognized by volumetric measurements (pre-atrophic neurodegeneration). Our innovative approach relies on MRI-based quantitative Gradient Recalled Echo (qGRE) technique developed in our lab. Preliminary data demonstrate that the qGRE identifies two types of tissues in the hippocampus of people with preclinical and mild AD: one type – tissue with markedly lower neuronal content (that we term Dark Matter as it appears dark on qGRE images), and another type – tissue with a relatively preserved concentration of neurons (that we term Viable Tissue). Based on this approach, we plan to achieve the following Specific Aims: Aim 1 will establish pre-atrophic neurodegeneration as a new imaging biomarker of neuronal loss that precedes tissue atrophy and can detect loss of neurons in early, preclinical, AD stages. Aim 2 will establish pre-atrophic neurodegeneration as a biomarker identifying losses of brain functional connectivity and cognitive performance in early AD. Aim 3 will integrate qGRE-based biomarker of pre-atrophic neuronal loss with plasma Aβ42/Aβ40 measurement that would significantly improve upon individual qGRE and plasma Aβ42/Aβ40 tests with regard to sensitivity and specificity for an early detection of AD pathology. Aim 4 will explore an association between brain topographies of AD-related genes and pre-atrophic neurodegeneration at earlier stages of AD. In Summary, successful completion of the aims of this proposal could significantly improve the current imaging paradigm for monitoring individual patients over time, and for use as a more sensitive measure of the neurodegenerative aspects of AD pathology as compared with current measurements of tissue atrophy.

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY Background: The 2,800 local public health departments (LHDs) in the United States have been increasingly responsible for implementing evidence-based programs and policies (EBPPs) to prevent and control cancer in their local communities, which has immense potential to impact population-level cancer burden. Our team has developed effective strategies to support LHDs and their staff to implement EBPPs, including guided facilitation and training, but the reach and sustainability of these strategies is currently limited. A promising strategy is for LHDs to engage in an academic-public health department (AHD) partnership, in which LHD practitioners and academics collaborate to improve public health practice and education through joint research projects and education opportunities for students. However, research on how AHD partnerships should be structured to improve implementation of cancer-related EBPPs is sparse. Goal: This proposal seeks to understand how to leverage AHD partnerships to facilitate implementation of EBPPs to prevent and control cancer. Methods: Aim 1: We will survey an existing, nationwide network of AHD partnerships to identify 4 high- and 4 low-performing partnerships based on their implementation of cancer-related EBPPs. We will use qualitative interviews and document reviews to refine our existing set of strategies, which can improve the use of EBPPs (e.g., facilitation needed, defining a tailored AHD partnership “package”), based on the structures, processes, and contextual influences among successful partnerships. Aim 2: Building on Aim 1, we will test the effectiveness of these refined strategies designed to improve the adoption of EBPPs for cancer prevention and control by strengthening AHD partnerships. We will conduct a group-randomized study (total N=28 AHD partnerships) to evaluate the effect of strategies to improve the adoption of cancer control and prevention EBPPs by supporting AHD partnerships. A mixed-methods approach will be used to evaluate changes in AHD partnerships and understand how contextual factors may have impacted the AHD partnership’s ability to support EBPP implementation. We will translate and disseminate findings from Phases 1 and 2 to LHD practitioners and academic partners to support cancer prevention and control in LHDs. Innovations and impact: The proposed study is innovative and impactful because it will be first study to focus on local-level collaborations that leverage expertise of LHDs and academics to improve public health practice. Also, its application of bridging factors, a component of the Exploration, Preparation, Implementation, and Sustainment (EPIS) framework receiving a greater focus recently, will contribute to the application of emerging components of theoretical frameworks in dissemination and implementation research. Last, this study examines new models for how public practice and academic public health can work together to meet common goals sustainably, i.e., without ongoing support from outside researchers.

NIH Research Projects · FY 2026 · 2022-05

Project Abstract The goals of this project are two-fold: 1) to evaluate the role of lysosomal dysfunction in lipotoxic cardiomyopathy and 2) to provide mentorship and training to allow the PI’s transition to independence. The prevalence of diabetes and obesity are increasing in the population, and both conditions are independent risk factors for the development of heart failure. Cardiac lipotoxicity has been identified as a potential pathway through which diabetes causes cardiomyopathy, but the understanding of how lipid overload disrupts cardiac function remains limited. Preliminary data acquired by the PI demonstrate that lipid overload-induced cardiac dysfunction is rescued by intermittent fasting. Intermittent fasting does not reduce cardiac triglyceride levels, but protein aggregate pathology is attenuated. Also, the levels of specific cardiac ceramide species are altered, suggesting that the effect of intermittent fasting is medicated by changing the level of individual lipid species, rather than overall reduction in lipids. Based on this data, this project hypothesizes that myocardial lipid overload impairs lysosome function via ceramide accumulation to induce cardiomyopathy and that stimulation of lysosomal biogenesis program by TFEB activation is sufficient to rescue this outcome. This hypothesis will be tested by assessing autophagic flux and lysosomal function in mice models of cardiac lipid overload and in cultured cardiomyocytes under lipid stress (Aim 1), examining cardiac lysosomal ceramide accumulation under lipid stress and the requirement for ceramide synthesis in cardiac lipotoxicity (Aim 2), and evaluating the role of TFEB in stimulating lysosomal biogenesis to improve lipotoxic cardiomyopathy (Aim 3). Completion of these aims will address knowledge gaps in the field of cardiac lipotoxicity and also provide training to facilitate the career development of the PI. The PI has previously obtained PhD training in molecular and cellular biology and has undertaken postdoctoral training in the lab of Dr. Abhinav Diwan, following completion of clinical training in medicine and cardiology. The PI seeks additional scientific training in a mentored setting to address the knowledge gaps described. Specifically, the proposed career development plan will provide additional training in 4 areas: 1) autophagy and lysosomal biology, 2) lipid biology and lipidomics, with a focus on sphingolipid biology, 3) echocardiographic analysis of animal models of cardiac disease, and 4) training in grant writing, presentation skills, and leadership. This plan will be guided by mentorship and research support from Dr. Abhinav Diwan and Dr. Douglas Mann, experts in lysosomal biology and myocardial biology, respectively, as well as by additional mentorship from the advisory committee with renowned expertise in lysosomal biology (Dr. Stuart Kornfeld), lipid biology (Dr. Brian Finck and Dr. Clay Semenkovich), and sphingolipid biology and lipidomics (Dr. Ashley Cowart). This training and mentorship in a highly supportive institutional training environment will enable the PI to develop into an independent cardiovascular investigator and physician-scientist focused on studying the role of lysosomes in cardiovascular development and in the myocardial response to injury.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT Multidrug resistant (MDR) infections caused by the bacterial pathogen Acinetobacter baumannii are increasing at alarming rates. Currently, over 60 % of global A. baumannii clinical isolates are MDR, leading the Centers for Disease Control and Prevention and the World Health Organization to categorize it as a top priority for the research and development of new antimicrobial therapies. In addition to accumulating resistance mechanisms, A. baumannii strains develop tolerance to antibiotics, which can frequently lead to poor therapeutic outcomes even with antibiotic susceptible strains. However, the mechanisms used by A. baumannii to adapt to and tolerate hostile conditions remain largely unknown. We found that A. baumannii employs a novel stress response pathway in which phenylacetic acid (PAA), a metabolite derived from phenylalanine catabolism, acts as a signaling molecule. We established that, in the presence of sub-inhibitory concentrations of different antibiotics, such as trimethoprim/sulfamethoxazole, A. baumannii dramatically increases the transcription of the paa operon which encodes enzymes required to degrade PAA. Conversely, other conditions, like hydrogen peroxide treatment, lead to a repression of the paa operon. The regulation of the paa operon triggers a physiological adaptive response that includes the modulation of pili biosynthesis and biofilm formation. Importantly, we found that artificial augmentation of PAA levels, through the addition of commercially available PAA-derivatives or mutations in PAA degradative genes, disrupts this response Furthermore, mutating initial steps of PAA degradation leads to increased sensitivity to antibiotics and oxidative stress in multiple strains. Here we propose to use our expertise in A. baumannii genetics and pathogenesis to investigate the PAA-mediated stress response in Acinetobacter and determine its importance in virulence. We will determine the breadth of PAA signaling using reporter assays, and we will explore PAA-mediated changes in cell physiology by profiling gene expression under different stress conditions. Further, we will characterize the PAA-dependent mechanisms of cell signaling under stress by measuring cellular levels of PAA and determining the role of important regulatory proteins in this cascade. Finally, we will test the virulence of strains unable to regulate PAA levels in the catheter-associated urinary tract infection and lung infection murine models. Our work will establish the role of PAA as a key regulatory molecule in A. baumannii, determine the biological processes regulated by PAA, and uncover the mechanisms by which PAA triggers adaptations to promote survival under stress. Understanding the fundamental aspects of the PAA stress response will provide a foundation to future clinical studies.

NIH Research Projects · FY 2025 · 2022-05

Project Summary Faces often convey a wealth of information and processing the human face is at the focal point of most social interactions. When we see a person's face, we can easily recognize their unique identity and general features such as race, gender, and age. The gestalt of facial processing enables us to make judgments about a person's mood or other aspects such as their level of trustworthiness. Yet, this simple perceptual task is difficult for individuals with autism spectrum disorder (ASD), a population that spends limited amounts of time engaged in face-to-face eye contact or social interactions in general. Although there is a large body of literature on face perception and many studies have documented abnormal face processing in people with ASD, most existing studies focus on the recognition of faces and emotional expressions or on perception of a particular social attribute (e.g., trustworthiness). It remains largely unclear how the brain represents and evaluates faces in general, and whether/how this mechanism differs in ASD. The study of face processing in ASD is very important because it will not only help us understand the social deficits of this disorder but also provide a unique opportunity to study the factors related to the functional specialization of normal face processing. In this project, we propose to conduct one of the very first studies to investigate neural face representation in individuals with ASD and delineate those brain regions involved in coding facial features. Importantly, by using concurrent functional magnetic resonance imaging (fMRI) and eye tracking and taking advantage of recent advances in deep neural networks (DNNs), we are able to extract association-based features from any face and synthesize new faces for validating model predictions. The primary objectives of this research are two-fold: (1) to establish a general neural representation of faces by constructing and validating neural face models, and (2) to compare neural representations of faces between people with ASD and controls. The collaboration between cognitive neuroscience and computer science in this project provides a unique opportunity to better understand how individuals with ASD perceive human faces, specifically what brain mechanisms are involved in representing faces in general. Obtaining this level of understanding of the neural computational underpinnings of face representation will be unique to our understanding of face processing in controls without ASD as well as those with ASD. In turn, this research may provide insights into the developmental trajectory of this pervasive deficit in autism and potential targets for intervention.

NIH Research Projects · FY 2025 · 2022-05

This administrative supplement application in response to NOT-OD-24-179, will leverage our NHLBI-funded UH3 grant: “Enhancing Cardiovascular (CV) Health in Mothers and Children Through Home Visiting (ENRICH)” to study sleep health during pregnancy in low socioeconomic status (SES) and/or Underrepresented Minorities (URM) women, as defined by NIH. Sleep health consists of multiple domains: sleep regularity, satisfaction, alertness, timing, efficiency, duration and lack of sleep disorders, (i.e., sleep-disordered breathing [SDB] and insomnia). Sleep health worsens over the course of pregnancy. Disparities in sleep health are present in non-pregnant low SES/URM populations, however few studies have characterized sleep health using robust measures in low SES/URM pregnant women. Another important gap that this proposal aims to address is characterizing the extent to which social determinants of health (SDOH) are related to sleep health in low SES/URM pregnant women. Neighborhood environment, sociocultural environment, and health care access are SDOH defined by Healthy People 2030, and domains of influence over the life course defined by the National Institute on Minority Health and Health Disparities (NIMHD) Research Framework. These SDOH are related to sleep health in non-pregnant low SES/URM populations, but the relationships have not been evaluated in pregnancy, a unique time to study sleep due to physical, hormonal, and emotional changes. Finally, the extent to which CV health is related to sleep health in low SES/URM pregnant women will also be investigated in this proposal. We propose a sub-study of the ENRICH trial at the Washington University Clinical Center, to characterize sleep health in 50 low SES/URM pregnant women using an in-home sleep testing device and actigraphy, allowing for collection of objective measures of sleep without requiring the participants to leave their home. Additionally, we will leverage data that is being collected as part of the parent ENRICH trial to study associations of sleep health with SDOH and CV health. Geographic Information Systems tools will be used to expand SDOH measures collected in the parent ENRICH trial to examine the association between community level SDOH and sleep health. This highly significant study will advance our understanding of sleep health and its relationship to SDOH and CV health in low SES/URM pregnant women. Data from this study will serve as the foundation for future work that will result in sleep interventions and updated guidelines for screening and treatment of sleep in pregnancy.

NIH Research Projects · FY 2026 · 2022-05

Accumulated evidence in human breast cancer and mouse models of breast cancer have shown that tumor cells invade collectively through the basement membrane (BM) and continue as collective groups to traverse the collagen-rich ECM to access lymphatic and vascular vessels. Rather than single cells, in the circulation clusters of heterogeneous circulating tumor cells (CTCs), that also contain tumor-associated stromal cells such as cancer associated fibroblasts (CAFs), account for >90% of metastases. To move collectively requires coordinated cell–cell and cell–matrix interactions. Hallmarks of collective cell migration include: 1) Cells remain physically and functionally connected such that the integrity of cell–cell junctions are preserved during movement. 2) A subgroup of cells typically defines the leading edge, and thus, the direction of collective migration. These are known as “leader “cells and differ in function from “follower” cells. 3) Collective movement also involves intimate interaction with accessory stromal cells that release polarity-inducing and pro-migratory factors as well as contribute to path finding by physically remodeling the surrounding ECM. Several hypotheses have been proposed to explain cancer leader cell development during collective migration. Yet how these leader cells develop, arrive and define the front edge, then lead directed collective migration, and whether this phenomenon is necessary and sufficient to effect directed collective migration are largely unknown. We have developed novel microfluidic devices in which to study the collective migration of primary breast tumor organoids in response to multiple environmental signals In the present proposal we propose to use primary breast tumor organoids with their inherent cellular heterogeneity to determine how leader cells develop and function, in response to multiple environmental signals, so as to direct collective migration. To do so we propose two specific aims. Specific Aim 1. To determine how K14 leader cells within primary breast tumor organoids polarize to the leading edge and then function to direct collective migration. Specific Aim 2: To understand chemo-mechanical feedback between CAF-based ECM remodeling and leader-based invasion.

NIH Research Projects · FY 2025 · 2022-05

Program Director/Principal Investigator (Last, First, Middle): GUAN, JIANJUN Project Summary Diabetic patients with critical limb ischemia (CLI) have significantly high rates of limb amputation and mortality. CLI is featured by extremely low blood perfusion and degenerated skeletal muscle. Accordingly, regeneration of vasculature and skeletal muscles will salvage the limbs. Yet the poor endothelial and skeletal muscle cell survival, and inferior cell functions under the hyperglycemia and ischemic conditions of diabetic CLI impair the limb repair. Currently, there is no effective treatment available although growth factor therapy represents a promising strategy. However, growth factor therapy has relatively low therapeutic efficacy in regenerating both vasculature and skeletal muscles, as multiple growth factors are simultaneously needed for vascularization and myogenesis, and these cannot be readily delivered by current approaches. In this project, we propose to use a novel TRIM72 protein with both pro-angiogenic and pro-myogenic properties to regenerate vasculature and skeletal muscles in diabetic CLI. The TRIM72 will be engineered to have longer retention time (slower diffusion rate) in ischemic tissue, thus exhibiting longer therapeutic effect. To deliver the engineered TRIM72 (ETRIM72), it will be encapsulated into ischemic limb-targeting nanoparticles, followed by delivering via clinically attractive IV injection. The nanoparticles will then predominantly accumulate in the ischemic limbs and gradually release ETRIM72. The released protein will promote vascularization and myogenesis by (1) improving the survival of endothelial cell and skeletal muscle cell through cell membrane repair, and activation of cell survival kinase; and (2) stimulating endothelial cell and skeletal muscle cell migration and morphogenesis under the hyperglycemia and ischemic conditions of diabetic CLI. In our preliminary studies, we have developed ETRIM72 by genetically fusing TRIM72 with peptide CSTSMLKAC that targets ischemic environment of ischemic limbs. This first version of ETRIM72 was able to retain in the ischemic limbs significantly longer than TRIM72. After IV injection of ischemic limb-targeting, ETRIM72-releasing nanoparticles, the released ETRIM72 significantly promoted regeneration of both vasculature and skeletal muscles in diabetic ischemic limbs. The function of TRIM72 in promoting vascularization and myogenesis under hyperglycemia and ischemic conditions has not been reported before. Based on our preliminary studies, we hypothesize that controlled release of ETRIM72 will simultaneously increase endothelial and skeletal muscle cell survival, migration and morphogenesis under hyperglycemia and ischemic conditions, leading to accelerated regeneration of both vasculature and skeletal muscles in diabetic ischemic limbs. Aim #1 will test the hypothesis that optimal ETRIM72 release profiles will significantly promote survival, migration and morphogenesis of endothelial cells and myoblasts under high glucose and ischemic conditions. Aim #2 will test efficacy of the ETRIM72-releasing nanoparticles using diabetic murine limb ischemia model. This project is innovative because it engineers a novel proangiogenic and promyogenic protein to simultaneously regenerate vasculature and skeletal muscles in diabetic ischemic limbs. The longer tissue retention time of the engineered protein, together with localized and controlled release are expected to significantly improve therapeutic efficacy. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page

NIH Research Projects · FY 2026 · 2022-05

ABSTRACT White matter hyperintensities (WMH) are ubiquitous in the aging brain. Prevailing theories implicate arteriosclerosis and endothelial dysfunction followed by ischemic and hypoxic injury as a cause, but factors that resist or modify these mechanisms are unknown. Animal studies show that glycolysis is a principal metabolic feature of normal white matter and protective against mitochondrial failure. However, white matter glycolysis has been understudied in in vivo human studies and particularly in the context of WMH. Our laboratory has developed methods, now on a state-of-the-art PET scanner, to quantitatively measure brain glycolysis using multiple radiotracers, including 15O radiotracers to measure cerebral oxygen consumption (CMRO2), cerebral blood flow (CBF) and oxygen extraction fraction (OEF), and 18FDG to measure total glucose use (CMRglc), from which we can derive the rate of aerobic glycolysis (AG). Here we propose to apply these methods to provide gold-standard quantitative estimates of normal human white matter metabolism, and to specifically investigate white matter glycolysis in the context of WMH. In our first two aims, we will compare the topography of metabolic PET measurements to MRI measurements of white matter microstructure and WMH, both in healthy adults without WMH and in adults with WMH. In our third aim, we will analyze longitudinal MRI imaging data in a cohort of adults who have already undergone metabolic PET in our prior and ongoing studies on an older scanner, to test the hypothesis that relative differences in white matter glycolysis will predict subsequent WMH development and progression. Moreover, we will explore potential relationships between neurodegenerative pathology and WMH, which we hypothesize occurs due to effects of the aforementioned pathology on white matter metabolism, thereby reducing its resilience to WMH.

NIH Research Projects · FY 2026 · 2022-05

Abstract RNA serves as the key intermediate for carrying genetic information and translating functional proteins, and numerous types of non-coding RNAs have also been recently discovered that play critical roles in cellular processes. While RNAs are directly transcribed from DNA, their sequence, function, and translation efficiency are all actively modulated in the cell though post-transcriptional localization and editing. These processes are essential for development and cellular function and are dysregulated in many diseases. Despite the importance of post-transcriptional localization and modification, significant gaps remain in our understanding of the timing, mechanisms, and regulation of these events. Several methods have been reported that enable researchers to label and image specific RNAs in living cells or pull-down and sequence edited transcripts from cell lysates. However, significant technological limitations still exist, and addressing these gaps holds promise for the development of new therapeutics and diagnostics. We have previously developed and implemented new methods for covalent labeling and imaging of specific RNAs in cells as well as pull-down and enrichment of A- to-I edited transcripts from cellular RNA. The future directions for our program include expanding this chemical biology toolbox to provide researchers with new and improved technologies for probing these important cellular processes. Additionally, recent studies have offered groundbreaking evidence for the hypothesis that asymmetric localization of RNA serves as a mechanism for regulating editing. Thus, a significant focus of the proposed research is to combine our imaging and editing-specific pull-down methods to advance understanding of the interplay between these two processes. Together, this research aims to serve the scientific community by providing insight into the regulation of RNA localization and modification as well as developing and disseminating robust and well-validated technologies for studying and modulating biological systems.

NIH Research Projects · FY 2025 · 2022-05

Alzheimer’s disease (AD) is a global public health crisis with unknown triggers and no disease modifying therapies. Effective treatments likely must be initiated in the early phases of biological disease, well before brain reserves of the neural substrates of cognition are depleted leading to overt clinical symptoms. These ‘preclinical’ periods and their triggering events, therefore, are highly significant areas of study. Traumatic brain injury (TBI), the leading cause of death and disability in younger individuals (under age 45) worldwide, is also the best-established epigenetic risk factor for AD. Once thought to be a monophasic injury, TBI is now known to initiate a chronic neuroinflammatory and neurodegenerative process that leads through unknown pathological mediators to dementing illnesses including AD, ADRDs, and chronic traumatic encephalopathy (CTE). Synapse loss is a common, early finding in AD, and the strongest pathological correlate of AD-induced dementia—even stronger than amyloid plaques or tau neurofibrillary tangles. Synaptic injury is also implicated in TBI in humans and in animal models. Synapses are challenging to study due to their extremely small size and admixture with the extraordinarily complex subcellular milieu of mammalian neuropil. We developed an innovative, widely accessible super-resolution imaging and image analysis platform called SEQUIN (Synaptic Evaluation and QUantification by Imaging Nanostructure) to enable routine monitoring of synaptic health in animal models and in humans. Our preliminary data demonstrate that delayed cortical synapse loss occurs after diffuse, closed head, mild TBI in a mouse model, suggesting that synaptic neurodegeneration may lead to neurological disability following TBI and sensitize the brain to subsequent AD-related synapse loss, hastening the onset of dementia. We will characterize synaptic neurodegeneration resulting from mild TBI over the lifespan, and determine its ability to predict neuropsychological and behavioral outcomes. We will then assess complement activation—a component of the innate immune system that drives synapse loss in AD and is maladaptively activated after mild TBI— as a mechanism of synaptic neurodegeneration. We will determine whether targeting the complement pathway can improve synaptic health and improve behavioral outcomes using genetic and clinically-translatable pharmacological interventions. Finally, we will assess the impact of mild TBI on synaptic neurodegeneration related to amyloidosis and tauopathy, classic AD-related neuropathological and biochemical processes. We will determine whether complement inhibition can prevent TBI-induced potentiation of neurodegeneration in mouse models of these processes. These studies are expected to reveal intervenable links between early brain injury and long-term neurodegeneration relevant to the individuals at greater risk of AD and related brain disorders due to an earlier TBI. They will also further establish innovative synaptic imaging tools (SEQUIN) that will empower routine synaptic analysis in this and related fields.

NIH Research Projects · FY 2025 · 2022-05

Abstract The endoplasmic reticulum (ER) is best known for its role as the locus of protein folding, calcium storage, and lipid metabolism. The organelle also integrates numerous other molecular pathways and contributes to cellular calcium homeostasis, reduction-oxidation regulation, and cell death. Given the many vital and complex functions of the ER, it is little wonder that its failure can trigger a range of diseases. It has been shown that dysregulation of ER homeostasis may underlie β cell dysfunction and death in type 1 and type 2 diabetes, as well as in monogenic forms of diabetes, including Wolfram syndrome, Wolcott-Rallison syndrome, microcephaly, epilepsy, and diabetes syndrome (MEDS), and mutant insulin gene-induced diabetes caused by pathogenic variants in the WFS1 and CISD2, EIF2AK3, IER3IP1, and INS genes respectively. To further understand the contribution of ER dysfunction to β cell death and design novel treatments targeting ER for diabetes, we need to establish functional studies of gene variants affecting ER homeostasis, design treatments targeting common molecular pathways altered in ER stressed β cells, and identify other ER genes involved in β cell dysfunction and death. In this proposal, we will characterize WFS1 and CISD2, EIF2AK3, IER3IP1, and INS variants using functional assays and bioinformatics and test novel treatments targeting the common molecular pathways altered in β cells expressing pathogenic variants of WFS1 and CISD2, EIF2AK3, IER3IP1, and INS genes. Successful completion of this study will lead to the establishment of precision medicine for hereditary ER diabetes.

NIH Research Projects · FY 2026 · 2022-04

PROJECT ABSTRACT Children in Sub-Saharan Africa (SSA) are burdened by significant unmet mental health needs. A recent systematic review estimated that 1 in 7 children in SSA struggle with a serious mental health issue. The World Health Organization estimates prevalence rates may be even higher (20%). Across SSA, high rates of poverty, HIV/AIDS, food insecurity, stigma and an inadequate health safety net system exacerbate serious childhood behavioral health (CBH) needs and impede an effective response. Disruptive behavioral disorders (DBDs) are particularly concerning as they persist through adolescence and adulthood. DBDs are also highly related to poor physical health and interpersonal challenges in adulthood. Hence, addressing the context-specific social influences on CBH is critical given that children in SSA comprise more than half of the total regional population. If children’s needs are to be met in SSA, then: 1) implementing interventions designed and tested in SSA, and which mobilize resources within existing child-focused institutions is critical; 2) combined interventions that simultaneously target SSA-specific influences on CBH and can be delivered in collaboration with child/family- serving community settings are necessary; and 3) group, community and population approaches to CBH are needed to drive scalable solutions. Guided by Social Action, Asset, and Family Systems theories, the proposed study will examine the mechanisms by which EE and FS interventions targeting social, familial and context- specific drivers affect the mental health of 900 Ugandan children in mid-upper primary school (10 to 14 years). The study uses an experimental, longitudinal design across 30 cluster randomized primary schools to compare single and combination intervention options; influences of EE and FS on economic, perceptual and functioning mediators; and context-specific moderators. The three study conditions are: 1) EE only, 2) MFG-based FS only, 3) combined EE+MFG-based FS. The interventions will be provided for 12 months; and assessments will occur at baseline, 12, 24 and 36 months. The specific aims are: Aim 1: Examine the impact of EE only, MFG-based FS only, and combined EE+MFG-based FS on children’s DBD symptoms and behavioral functioning; Aim 2: Test the influence of EE only, MFG-based FS only, and combined EE+MFG-based FS on family financial stability (e.g., food and housing stability, material assets, savings), parenting and protective family processes (e.g., family organization, caregiver/child interaction, cohesion, support) and perceptions related to help seeking (e.g., stigma) on CBH and functioning; and assess whether these change mechanism mediate intervention effects on DBD symptoms and behavioral functioning, and explore moderation by context-specific moderators of intervention effects; and Aim 3: Qualitatively examine participants’ experiences with each intervention arm. The investigative team will leverage their long-term partnerships in SSA to maximize the public health impact of the research findings and to shorten the time gap from new knowledge to system and population-level improvement.

NIH Research Projects · FY 2026 · 2022-04

Project Summary The COVID-19 pandemic, caused by the virus SARS-CoV-2, represents an acute and ongoing threat to human life. A detailed molecular understanding of the viral life cycle is necessary to illuminate clinically accessible processes that can be targeted for therapeutic intervention. The Nucleocapsid (N) protein is a 420-residue multidomain protein with both folded and disordered regions that underlies genome packaging, an essential step in the virion lifecycle. N protein mediates cytosolic genome packaging by binding to and compacting genomic RNA in a process apparently conserved across the coronaviridae family. Our ability to disrupt genome packaging is limited by the absence of a molecular understanding of these processes. To address this knowledge gap, our proposal is focused on the molecular biophysics that underlies how N protein drives genome compaction. N protein is highly multivalent; it can simultaneously bind to both itself and RNA via a number of distinct interaction sites. Multivalency is encoded across both folded domains and intrinsically disordered regions. While there has been substantial work on the folded domains in other coronaviruses, the molecular biophysics of the disordered regions has been largely ignored. We hypothesize N protein multivalency underlies the molecular basis of RNA compaction, and that the three disordered regions play key roles in determining multivalency, binding affinity, and RNA binding specificity. Through the combination of single-molecule fluorescence and force spectroscopy, ensemble methods, and all-atom simulation, we will dissect the molecular details that underlie these interactions. We also present a novel approach to small-molecule screening that leverages the formation of phase separated protein:RNA liquid droplets as a readout for genome compaction. Our work will offer high-resolution structural insight into the physical basis for two critical steps in the viral life cycle, as well as reveal small molecules that can attenuate genome compaction. More generally, by focusing on fundamental biophysical phenomena that empirically explain behavior from other distant coronaviruses, we believe that our conclusions will be broadly transferable to existing coronaviruses that represent major public health threats (e.g., SARS, MERS) but also to future novel zoonotic coronaviruses.

NIH Research Projects · FY 2026 · 2022-04

The goal of this mentored career development award is to facilitate the candidate’s transition to independence as a physician-scientist studying the molecular and neuronal mechanisms of stroke recovery. The candidate is an MD/PhD neurologist with a background in synaptic physiology and cortical network research. The award will help the candidate gain research experience in the mechanisms of network recovery after ischemic stroke and will facilitate his transition to an investigator with an independent laboratory. The award will also help position the candidate to achieve his long term goal of becoming a successful and productive physician-scientist, a leader in academic neurology, and a mediator of translational research which improves the lives of patients suffering from acute brain injury. The environment in which the proposed research will be conducted is outstanding. The candidate’s co-mentor, Dr. Jin-Moo Lee, is an internationally recognized scientist and neurologist with a proven track record of excellence in training junior faculty. The candidate’s career development plan also includes structured mentorship from multiple physician-scientists at all stages of seniority and exposure to a rich and supportive faculty, ensuring the candidate has role models along the full spectrum of the career trajectory. Didactic learning, presentation at scientific meetings, and rigorous training in the responsible conduct of research will ensure balanced development. The proposed research will examine the role of axonal sprouting in restoration of both local cortical circuits and secondary reconnection to global brain networks. Recovery after focal cortical stroke is associated with remapping of the function of the infarcted region to adjacent, non-infarcted cortex. Recovery is also associated with restoration of disrupted functionally connected networks. While both phenomena (local circuit remapping and restoration of functional connectivity) are strongly associated with stroke recovery, the underlying structural substrate is unknown. The goal of this project is to test the hypothesis that axonal sprouting mediates cortical remapping via reconnection of local circuits. A secondary hypothesis is that further axonal sprouting originating from the remapped cortex mediates restoration of whole-brain functional connectivity by reintegrating the disconnected circuit into global networks. This project further hypothesizes that the degree of recovery of these two processes (local remapping and restoration of functionally connected networks) correlates to the degree of behavioral stroke recovery. Clarifying the underlying mechanisms driving network repair after stroke will provide crucial insights into recovery after ischemic brain injury and will be the basis for future studies attempting to harness these mechanisms to improve outcomes for stroke survivors. This career development award is an ideal mechanism to provide the candidate with valuable research training to complement his clinical focus on caring for patients with acute brain injury and will help him develop a skill set for translating basic science discoveries into effective therapies for patients suffering from stroke.

NIH Research Projects · FY 2025 · 2022-04

Project Summary Suicidality in children is a pressing and understudied public health concern. Rates of suicide in youth have tripled in recent years, yet little is known about the early emergence or development of suicidal ideation and behaviors (SI/SB). Suicidal ideation (e.g., wishing to be dead, expressing desire to kill oneself) and behaviors (e.g., choking oneself) have been identified as early as the preschool period in the context of early-onset depression. However, if and how suicidality presents in non-depressed young children is unknown. Importantly, early suicidality remains stable into school-age and confers risk for later psychopathology, suggesting lasting impact and continuity of this early manifestation. Consistent with the NIMH Strategic Objective 2, to “chart mental illness trajectories to determine when, where, and how to intervene,” the overarching aim of this K01 application is to understand the developmental contexts in which suicidality emerges in order to identify at-risk youth and to inform preventative intervention efforts. The proposed study will address a number of questions central to understanding the development of suicidality in early childhood including how SI/SB is expressed at this early age, the normative development of the understanding of suicide, if children with SI/SB exhibit lack of optimism and/or pessimism, and how children with SI/SB process peer acceptance and rejection. A variety of measures, including child interview and narrative approaches, behavioral tasks, and event related potentials (ERPs) will be administered to three groups of 4- to 7-year-olds from diverse racial and socioeconomic backgrounds: 1) children with a history of SI/SB, 2) children with or at risk for psychopathology but no history of SI/SB, and 3) low-risk healthy children. The inclusion of children with or at risk for psychopathology with no history of SI/SB will address the specificity of risk factors for suicidality relative to depressive symptoms and other forms of psychopathology. This approach has the potential to identify transdiagnostic risk-factors of suicidality in young children. The inclusion of low-risk healthy children will inform our understanding of typical and atypical trajectories of suicide understanding and provide guidance regarding how and when to address expressions of suicidality in childhood as a clinical concern. This will be the first study of children this young with SI/SB to be targeted for a study designed to investigate the developmental antecedents of risk and resilience for SI/SB. Suicide research and prevention is a high priority research area for NIMH and the described research and training activities will enable the candidate to develop an independent research program that addresses the rising rates of childhood suicidality. Specifically, the execution of the proposed project will provide the candidate with training and expertise in suicide research and prevention, child psychopathology, and ERP techniques. A rich training environment and a multidisciplinary team of mentors in each of these areas is detailed. Data from this project will be used in a planned R01 to more deeply investigate racial and/or sociocultural differences in risk factors and developmental trajectories of early- onset suicidality.