Ohio State University

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT. While cannabis, nicotine, and alcohol remain the most widely endorsed substances during adolescence, mechanistic pathways by which the social determinants of health influence substance use outcomes among youth are unclear. Structural racism and discrimination results in poorer quality environments in many neighborhoods within the United States, with a greater disproportionate impact for marginalized populations. Neighborhood-level structural racism (NSR) is the totality of ways by which structural racism disproportionately disadvantages neighborhoods in areas of socioeconomic conditions, environmental health, and educational opportunities. NSR features, such as neighborhood inequities in access to healthy food, green space, housing, and poverty rates, comprise important social determinants of health that negatively affect neurocognitive skills as well as increase the risk for more negative consequences of substance use. This project will use a concurrent mixed method approach to investigate the impact of neighborhood-level structural racism on youth substance use and determine the extent this relationship is mediated by neurocognitive markers of decision-making and distress tolerance. We will use both qualitative and quantitative methods throughout the project to study NSR influences on SU in middle school youth (ages 12-14 years) in the R61 phase and in high school youth (14-17 years) in the R33 phase. We will leverage the availability of the Adolescent Brain Cognitive Development ® (ABCD) Study dataset (N = 11,880) to concurrently conduct quantitative analyses to test the neurocognitive pathways on the influence of NSR. Community engagement research approaches will be implemented throughout both the R61 and R33 phase and include oversight from a Community Advisory Board and a Youth Advisory Board. During the R61 phase, interviews with middle school Latino/a/x and Black youth (N = 30) will obtain youth perceived neighborhood- level risk factors for substance use. The quantitative studies will then investigate whether NSR impact youth cannabis, nicotine, and alcohol through neurocognitive pathways of decision-making and distress tolerance. Qualitative and quantitative findings from the R61 phase will be triangulated to inform the precision of the NSR for high school youth during the R33 phase. In addition, the R33 phase will use photovoice with high school youth (N=30), a photographic technique to identify and represent neighborhood features youth perceive contribute to engagement with substance use. Cohort effects will be tested by comparing findings from the high school cohort (R61) with the middle school cohort (R61). Findings from the project will be disseminated to community stakeholders and policy makers. In addition, findings may inform community-level interventions on the neighborhood features that pose risk to adverse substance use trajectories, as well as at the individual- level for neurocognitive interventions for substance use prevention.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Proteins often fold into three-dimensional shapes and operate as cellular machines with well-defined reaction mechanics – hence the common biochemical expression “structure is function.” However, this rule only applies to the slowest evolving portions of the human genome. Proteins that evolve more rapidly are typically less ordered, both enabling their accelerated evolution and expanding the biochemical landscape on which natural selection may act. Currently there exist few tools and conceptual frameworks to understand the sequence-function relationship of quickly evolving dynamical proteins. Across the tree of life, reproductive proteins evolve at extraordinary rates – typically faster than immune genes – and the Wilburn lab studies the biophysics and molecular evolution of species-specific fertilization in animals. The continuous coevolution of interacting sperm and egg proteins has selected for biochemical properties such as intrinsic disorder, weak binding affinities, etc. that complicate their study. High-field NMR spectroscopy is unique among structural methods in its ability to study such heterogenous protein systems, and I have pioneered NMR studies of fertilization proteins in a classic model of fertilization research (marine abalone). Over the next 5 years, we will interrogate the sequence-to-function relationships of gamete recognition proteins important for species-specific fertilization by pairing high throughput mutagenesis screens with targeted biophysical analyses to better understand the complex interplay of molecular dynamics with protein evolvability and interaction kinetics. NMR methods will be expanded to the study of mammalian fertilization proteins. To facilitate our own work and empower other researchers, deep learning-based analytical tools in evolutionary genomics, mass spectrometry proteomics, and NMR dynamics will be developed. The proposed research will provide an evolutionary framework to better understand the breadth of protein biochemistry encoded by the human genome, and includes diverse training opportunities for undergraduate, graduate students, and postdocs

NIH Research Projects · FY 2025 · 2023-09

This proposal concerns age restricted in-person location policies (ARLPs), a novel tobacco control strategy that could have a critical impact on reducing youth tobacco use. Under an ARLP, tobacco sales would only be allowed in tobacco shops or other adult-only retailers. As the majority of tobacco retailers are convenience stores and discount stores, ARLPs could drastically reduce tobacco retailer density, and thereby reduce adolescent and young adult (“youth”) tobacco access and marketing exposure. Moreover, as convenience and discount stores are often disproportionately prevalent in neighborhoods with higher tobacco retailer density, ARLPs may reduce differences in tobacco retailer density. Evidence is badly needed to support the introduction of ARLPs to the United States. Data are needed from models of the potential reduction in tobacco retailer density, the potential reduction in youths’ “focal retailers” (i.e., the retailers that youth frequently encounter or use the most for tobacco purchases), the potential impact on youth behavior, and the potential compensatory behaviors by youth (e.g., obtaining tobacco through alternative sources). Woven into these questions is the critical concern of impact across communities: Will an ARLP reduce current differences in retailer density and youth tobacco use across all communities? The goal of this project is to model the potential impact of ARLPs on youth tobacco use, both overall and for different priority populations (defined by residence in low-income, racial/ethnic minority, or rural neighborhoods). Aim 1 will use spatial statistical methods to model the potential impact of an ARLP on tobacco retailer density. Across four states (California, Connecticut, North Carolina, and Ohio), investigators will geocode the locations of all current tobacco retailers to model an ARLP’s impact on the retailer landscape. Pairing this environmental data with behavioral data from a cohort of young people living in those states, Aim 2 will use geographic ecological momentary assessment (EMA) to (a) identify young people’s most focal retailers and (b) model the extent to which exposure, access, and use stemming from these focal retailers would be impacted by an ARLP. Investigators will enroll 480 youth (120 per state; ages 16-25; 80% tobacco users; 50% priority population) to collect this real-world, close-to-real-time geographic EMA data. Analyses will model whether an ARLP would have prevented purchases and marketing exposures to a significant extent—particularly among the priority population youth. Finally, Aim 3 will use survey and qualitative methods to determine potential compensatory behaviors by youth. Subgroups of tobacco users will participate in focus groups and all tobacco users from the cohort will be asked to complete an online survey. Analyses will assess whether obtaining tobacco from alternative sources will be harder for priority (vs. non-priority) youth. An external advisory board of key partners will inform all stages of the project, particularly dissemination. Successful completion of this project will provide much-needed information to support state and local ARLPs. In the long-term, such efforts will contribute to reducing and eventually eliminating tobacco-related health differences by promoting health for all populations.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT The 5-year overall survival rate for acute myeloid leukemia (AML) is less than 35%, making it the most fatal leukemia in adults. Natural killer (NK) cells are a type of innate lymphoid cell (ILC) capable of recognizing and killing malignant cells. We have previously demonstrated that NK cells are developmentally and functionally impaired in AML. These impairments correlate to worse overall survival and clinical outcomes, highlighting the importance of understanding the mechanisms by which AML alters NK cell development and function. To better understand these defects, we assessed the transcriptional and epigenetic landscape of NK cells in AML and discovered that mature NK cells from AML patients are globally hypomethylated when compared to normal controls and have abnormal activation of activating protein-1 (AP-1). AP-1, which is a set of transcription factor (TF) dimers primarily composed of Jun and Fos family proteins, has been shown to regulate ILC development and homeostasis, chromatin accessibility, and immune cell exhaustion. The activity of AP-1 is regulated by upstream mitogen activated protein kinases p38, JNK and ERK through transcription activation and post- translational modifications. In this proposal, we will determine how the MEK/ERK signaling branch of the AP-1 signaling pathway contributes to NK cell defects in AML and whether development and function can be restored through AP-1 pathway inhibition. We hypothesize that AP-1 signaling is a critical regulator of NK cell development and function that becomes dysregulated in AML. These studies will first describe the mechanism(s) by which the AP-1 pathway influences NK cell development in the setting of AML (Aim 1). We will use pharmacologic agents as well as CRISPRcas9 editing to understand how aberrant AP-1 signaling skews human NK cell development. We will also determine the functional consequences of aberrant AP-1 activation in mature NK cells (Aim 2). Finally, we will assess the therapeutic efficacy of AP-1 pathway regulation in multiple in vivo models of AML (Aim 3). Results from these studies will further our mechanistic understanding of NK cell development in AML to better inform therapeutic strategies.

NIH Research Projects · FY 2025 · 2023-09

Abstract Idiopathic Pulmonary Fibrosis (IPF) is a chronic, irreversible, aging-associated, and ultimately fatal lung disease. The median survival of pulmonary fibrosis patients is only 4-5 years. There is no treatment to reverse fibrosis and cure IPF. Development of anti-fibrotic therapeutics is an unmet need in the treatment of IPF. The relentless progression of IPF is due in part to the failure of fibrosis resolution. The development of therapies for IPF relies on the comprehensive understanding of fibrosis resolution pathways. Accumulating evidence shows that activation of BMPs signaling induces myofibroblast de-differentiation and fibrosis resolution. A key receptor, BMP receptor II (BMPRII), in BMPs signaling has been shown to be reduced in fibrotic lungs; thus, restoration of BMPRII in fibrotic lungs is a potential therapy to treat IPF. However, molecular regulation of BMPRII stability has not been well studied. In our preliminary data, we discovered that (i) BMPRII is degraded in the lysosome system in response to TGF-β1 and lipid peroxidation inducers, which play critical roles in the development of lung fibrosis; (ii) Nedd4L stabilizes BMPRII; (iii) downregulation of Nedd4L reduced BMP4 signaling and the effects were rescued by overexpression of BMPRII; (iv) overexpression of Nedd4L promoted de-differentiation of lung myofibroblasts. Based on these novel observations, we hypothesized that Nedd4L promotes BMPRII stability and facilitates BMPs/BMPRII-mediated myofibroblast de-differentiation and pulmonary fibrosis resolution. We propose three Specific Aims to evaluate our hypothesis. First, we will determine the regulatory mechanisms by which Nedd4L stabilizes BMPRII. We will identify the ubiquitination and Nedd4L docking sites on BMPRII and determine the effect of Nedd4L-mediated K63-linked ubiquitination on BMPRII internalization and stability. Next, we will determine if Nedd4L suppression is essential for TGF-β1- and lipid peroxidation-induced BMPRII degradation. Finally, we will determine if Nedd4L facilitates lung fibrosis resolution through stabilization of BMPRII and promotion of BMPs-mediated myofibroblast de-differentiation and inactivation. We will determine if elevated BMPRII stability by Nedd4L facilitates myofibroblast de-differentiation in ex-vivo cultured lung myofibroblasts. An inducible fibroblast specific Nedd4L depletion mouse will be used in both resolving and non- resolving lung fibrosis models. The proposed studies will address key knowledge gaps regarding molecular regulation of BMPRII stability and anti-fibrotic effects of BMPs/BMPRII. Success of the proposed studies will suggest that targeting Nedd4L/BMPRII to rescue BMPRII expression in fibrotic lungs may lead to new opportunities to halt pro-fibrotic progression and promote fibrosis resolution.

NIH Research Projects · FY 2026 · 2023-09

ABSTRACT Potential long-term effects of tackle football and other contact sports have created significant public health concerns about the risk of childhood participation. Considering the retrospective and cross-sectional nature of prior work, there are critical knowledge gaps regarding the causality of youth tackle football participation and subsequent clinical outcomes that preclude decision-making at the patient and policy level. Further, modifiable risk factors (e.g., neuromuscular control) may influence exposure to and thus, outcomes from football-related neurotrauma, but prevention remains understudied. Here we overcome some of the previous barriers to progress in this field by acquiring a “true baseline” for athletes before they begin their first season of tackle football and following them longitudinally across the first year of participation. We received funding from Ohio State’s Chronic Brain Injury Program and successfully collected pilot data on 50 youth tackle football players (ages 8-12 years); these data form the basis of our current proposal. Aim 1 will identify risk factors for higher exposure to football- related neurotrauma. We will measure football-related neurotrauma exposure in first-year youth tackle football players during all practices and games using instrumented mouthguards. We will use handheld dynamometry to measure neck strength and will measure neuromuscular control through visual, vestibular, and reaction time testing. Aim 2 will determine the effects of football-related neurotrauma exposure on neurodevelopment in children with no previous tackle football exposure. We will enroll children who are registered for, but have not yet begun participating in youth tackle football, as well as children registered for non-contact sports and children not playing any sports. Aim 2a will determine short-term changes in cognitive function and brain structure and function caused by football-related neurotrauma exposure from pre-season to post-season. Aim 2b will determine longer-term changes across the first year of participation by examining players during the off-season just prior to the start of the next season. Aim 2c will determine what factors (e.g., football-related neurotrauma exposure, exposure to adverse childhood events, pre-existing neurodevelopmental disorders) contribute to differences from typical development. We hypothesize that youth tackle football exposes children to repetitive neurotrauma during a period of rapid neurodevelopment, resulting in observable cognitive impairments and structural and functional changes in the brain, most likely in attentional control networks. Our proposal combines a state-of-the-art approach to determining the effects of football-related neurotrauma on neurodevelopment, a productive collaboration with youth sports programs in Ohio, community engagement and outreach, and a multidisciplinary team of experts. The societal and clinical impact of these findings cannot be overstated. In quantifying football-related neurotrauma exposure (Aim 1) and its effects on neurodevelopment (Aim 2) across the first year of youth tackle football participation, our expected outcomes will inform community stakeholders and policy-makers in their appraisal of the cost and benefit of children participating in youth tackle football.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Interferons (IFN) straddle the balance between protective responses that maintain cellular integrity and the induction of deleterious inflammatory responses that cause irreversible tissue damage. The detection of viral infection or the accumulation of cellular stress triggers the synthesis of both type I (IFN/) and type III IFNs (IFNλ), which activate an overlapping JAK/STAT pathway to induce the expression of effector genes known as interferon stimulated genes (ISGs). While almost all nucleated cells can respond to IFN/ stimulation, the expression of the IFN receptor (IFNLR1) is restricted to a few cell types, primarily the epithelium. Studies on the evolutionary requirement for overlapping IFN functions, revealed context-specific functions for IFNs: IFN/ drive antiviral and proinflammatory responses that can promote tissue damage when unabated, and IFNλ confers antiviral protection with minimal inflammatory induction and reinforces the integrity of the barrier. My previous work has defined how divergent activation of canonical transcription factors (TF), interferon regulatory factors (IRF) and Signal transducer and activator of transcription (STAT), specifies maladaptive IFN/ functions. IFNλ responses also become maladaptive and hinder barrier repair, yet understanding of how parallel signaling events and cell identity disrupt the balance of the IFNλ pathophysiological functions is lacking. My future work will utilize a multidisciplinary approach to fill this knowledge gap. We will 1) determine how non-canonical signal integration, such as NF-kB and MAPK, defines the IFNλ-dependent barrier functions, 2) define how cell identity controls cytokine sensitivity, and 3) leverage genetics to identify novel cell-type specific, non-canonical TF and chromatin modifiers that feed into the IFNλ response. These studies will enhance our basic understanding of the shared and unique functions of IFN, identify cellular susceptibilities to inflammatory damage, and guide the development of therapeutic interventions that dampen excessive inflammatory responses while preserving the primordial antiviral functions of IFNs.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Millions of children receive general anesthetics (GAs) for surgical procedures. Emerging evidence from human epidemiologic and animal studies suggest that short acting general anesthetic drugs can cause acute brain injury, leading to long-term cognitive defects and behavioral problems. In 2016, the US Food and Drug Administration issued a warning about the potential neurotoxic effects of GA exposure in children under age three. As GA use is necessary for many surgeries, avoidance is often impossible. Thus, understanding how anesthetics induce neurotoxicity is of critical importance in public health, especially so that effective neuroprotective strategies can be developed. One promising area of investigation is mitochondria -- as neurons have high energy requirements, they are especially vulnerable to injury and death from dysfunctional mitochondria. However, despite the extensive research of anesthetic-induced developmental neurotoxicity (AIDN) done during the last decades, mechanisms by which mitochondrial impairment leads to neuronal signaling deregulation and cell death remain unclear. Causative relationship between mitochondrial injury and anesthetic-induced long-term behavioral abnormalities has not been explored. To address the aforementioned gap, in our preliminary studies we investigated and found that anesthetics were toxic to mitochondria in developing mouse and human brain cells. Our data also suggest the regulative function of dysregulated non- coding RNAs in anesthetic-induced impaired mitochondrial function. Thus, the overarching goal of this program is to continue to fill the gap of mitochondrial knowledge in anesthetic nontoxicity by investigating the functions and novel regulatory molecular mechanisms of mitochondria in AIDN as well as developing neuroprotective approaches targeting mitochondria. Extending upon our lab's recent research and preliminary findings, our proposed program will focus on the following three independent research areas: 1) Determine functions and brain cell type-specific mechanisms of mitochondrial signaling in anesthetic-induced cognitive dysfunction and abnormal behaviors. 2) Delineate novel posttranscriptional regulation mechanisms by which mitochondrial signaling and functions are regulated in AIDN. 3) Investigate neuroprotective effect of small molecules in AIDN. We will conduct these investigations using both transgenic mouse models and similar human induced pluripotent stem cell models obtained via CRISPR-Cas9 gene editing. Furthermore, this program will use innovative, cutting-edge experimental neuroscience tools, unbiased multi-omic approaches (e.g., gene gain- and loss-of function, multiphoton real time imaging, single-cell RNA sequencing, and high-throughput analysis of neuronal activities). The proposed studies will facilitate a better understanding of GA-driven mitochondrial dysfunction, which may lead to effective therapeutics for preventing AIDN in young children.

NIH Research Projects · FY 2026 · 2023-09

Project Summary/Abstract The coordinated effort of the immune system in host defense is dependent upon the proper release and interpretation of different cytokine signals. Several examples of monogenic diseases targeting the JAK-STAT pathway reveal there is still a critical need to understand basic principles of cytokine signaling and cytokine output. For example, patients with STAT1 gain-of-function (GOF) mutations exhibit a type 1/IFN-gamma bias that antagonizes a type 3/IL-17 immune response important for controlling fungal infection, yet patients also, paradoxically, exhibit chronic and sometimes lethal viral infections. Using a novel conditional knock in STAT1- GOF mouse model, we demonstrate that STAT1-GOF mice exhibit an impaired NK and CD8 T cell effector response and develop a cytokine storm with viral infection. This is due to an impaired tissue-specific and microbiome-dependent deficit in IFN-gamma production by liver NK cells, ILC1s, and iNKT cells early during infection. Continuing with these studies, the goal of this application is to understand how perturbations in cytokine signaling have downstream and long-term effects in immune response. This proposal tests the hypothesis that prior exposure to cytokine stimuli have long-term effects on gene expression and immune cell function. The approach is divided into three specific aims: first, to determine the epigenetic mechanisms underlying how a STAT1-GOF mutation alters transcriptional output; second, elucidate the mechanism underlying the tissue-specific reduction of IFN-gamma in STAT1-GOF mice with viral infection; and three, to determine the consequences of a cytokine storm on CD8 T cell memory. During the mentored phase of the K99, the candidate will be trained in various genomic sequencing technologies and bioinformatic analysis to investigate AIM 1. Alongside research training, the candidate will participate in career development courses and workshops about grant writing, laboratory management, and teaching to fulfill her goal to attain a tenure- track independent research position. The training phase will be in Dr. John O’Shea’s laboratory in NIAMS at the NIH, supported by excellent resources, expertise in the laboratory, and vibrant NIH research community. These studies and career development plan will launch the R00 phase of independent research to investigate AIM 2 and AIM 3. The broader implication of this work is it will uncover basic cytokine signaling mechanisms broadly applicable to the field of immunology and identify potential therapeutic options for patients with altered set points.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Colorectal cancer (CRC) is the third most common cancer among both men and women and the second leading cause of cancer deaths in the United States (US). CRC screening is now recommended for average- risk adults ages 45-75, yet many adults in the US are not within screening guidelines. One recommended CRC screening approach is annual completion of a fecal immunochemical test (FIT). FIT can be completed at home and sent/returned via mail, making mailed FIT programs a promising way to reach people and increase CRC screening. Such programs may be especially important for geographic regions with lower access to healthcare and lack of public transportation, such as the Appalachian region of the US. Mailed FIT programs have been shown to increase CRC screening, but typically fewer than half of participants in these programs who were sent a FIT returned a completed test. This indicates that mailed FIT programs can be greatly improved upon by addressing common barriers to FIT return. A key next step in this field of research is to identify strategies that can address these barriers and subsequently increase FIT return in mailed programs. Our study team will build on our past research and take this key next step. In doing so, we will partner with health centers in Appalachian counties that are located in a CRC “hotspot” within the US (i.e., clusters of counties with high CRC rates). The proposed study will involve a mailed FIT program (called “FIT Starts at 45”) that includes 500 participants from Appalachia who are underscreened and ages 45-74. We will determine the efficacy and cost-effectiveness of two potential strategies for increasing FIT return: FIT kit materials (enhanced FIT kit that includes a video brochure vs. standard FIT kit) and patient navigation (PN) delivery mode (text message PN vs. telephone PN). In a 2x2 between-subjects factorial randomized trial, each participant will be randomized to one of the following study groups: standard FIT kit plus telephone PN; standard FIT kit plus text message PN; enhanced FIT kit plus telephone PN; or enhanced FIT kit plus text message PN. The proposed study will be one of the most innovative and comprehensive mailed FIT programs to date in the US, with each study aim making a significant research contribution. Aim 1 will determine the efficacy of FIT kit materials and PN delivery mode on increasing FIT return. FIT return will be defined as return of a completed FIT by participants within six weeks of FIT distribution. Aim 2 will determine if efficacy differs across key characteristics of participants and their communities (i.e., moderation). Aim 3 will compare the cost- effectiveness of FIT kit materials and PN delivery mode on increasing FIT return. Results will be impactful by providing an evidence base that can help optimize the design, implementation, scalability, and sustainability of future mailed FIT programs, including our own efforts to disseminate the FIT Starts at 45 program.

NIH Research Projects · FY 2024 · 2023-08

1 Project Summary 2 There is growing evidence that canine coronaviruses (CCoVs) can infect humans and be associated with 3 clinical (mostly acute respiratory) illness in children and adults. Human infections with CCoVs with recombinant 4 canine-feline-porcine spike proteins (hCFPL-CoVs) have been confirmed in several countries including Haiti 5 (2017), Malaysia (2018), the USA (2014) and Thailand (2003). The high sequence identity (SI, 99.4%) observed 6 between hCFPL-CoVs from geographically distant Malaysia (CCoV-HuPn-2018) and Haiti (HuCCoV-Z19) 7 suggests that they may be capable of human-to-human transmission or represents temporal clustering. CCoVs 8 have not been recognized previously as human pathogens, and the threat they pose to public health is unknown 9 and may be underappreciated. While complete genome sequencing demonstrated hCFPL-CoVs are canine- 10 feline recombinant alphacoronaviruses, it failed to identify potential ancestral strains likely due to scarcity of 11 CCoV genomic data (only 17 complete genome sequences are available in the GenBank). To address that, we 12 will conduct complete genomic sequencing of up to 200 new CCoVs (from Dr. Decaro) and additional hCFPL- 13 CoVs (if identified in Dr. Gray’s ongoing study). 14 A unique 36 nt (12-aa) deletion identified in the N protein in the SR-rich domain (ΔSR-N) of CCoV-HuPn- 15 2018 may be associated with a recent zoonotic transmission of CCoV-HuPn-2018 and certain biological 16 functions acquired or lost by the virus. While such deletions in the SR-rich region of the N-protein were not 17 previously found in CCoVs, presence of a similar deletion was demonstrated in severe acute respiratory 18 syndrome coronavirus (SARS-CoV) strains early following their emergence into human population but not in 19 SARS-CoV-like bat strains. This deletion was shown to be associated with the altered cellular localization of the 20 N-protein and increased pathogenicity of the SARS-CoV strains bearing them. Because SARS-CoV N-protein 21 plays an important role in inhibition of type I interferon (IFN) production, deletions in it may alter innate immune 22 responses against SARS-CoV as well as other CoVs including CCoV-HuPn-2018. Using reverse genetics, we 23 will evaluate the biological function of this mutation. We propose the following Specific Aims to study the 24 genomics, evolution and human emergence mechanisms of hCFPL-CoVs. Aim 1. Conduct genome-wide 25 analysis of historic and current CCoV strains a) to determine the evolutionary relationship between hCFPL-CoVs 26 and their potentially ancestral strains and b) to identify genetic features associated with CCoV-HuPn-2018 27 infectivity or pathogenicity to human host. Aim 2. To generate and use CCoV-HuPn-2018 infectious clone to 28 investigate the effect of the identified N- deletion (ΔSR-N) on CCoV-HuPn-2018 cellular localization, replication 29 dynamics and the host transcriptome response. These studies will identify the genomic features associated with 30 hCFPL-CoV/CCoV infectivity to humans and generate essential fundamental knowledge regarding the common 31 mechanisms regulating zoonotic transmission of CoVs.

NIH Research Projects · FY 2025 · 2023-08

The Ohio State University (OSU) Clinical and Translational Sciences Institute (CSTI) was formed in 2007, with a vision to advance today’s discoveries to improve health for all. We employed a comprehensive approach to address the full spectrum of T1-T4 science via the education and career development of a highly trained workforce integrated with a robust system of CTR resources. We provided leadership in the CTSA Consortium through sharing of tools and methods to advance CTR; adopted innovations from other hubs; and fully engaged in CTSA Consortium activities (e.g., multisite trials). We also supported community engaged research addressing the most pressing health issues in our communities. Yet, work remains. Thus, we build upon unique strengths at OSU/NCH and in Ohio’s communities. We have leveraged our strategic investment in data sciences, a robust environment of resources, and a vibrant CTR community to address CTS gaps and barriers. We will now address five CTS roadblocks to improve the quality, efficiency, and rigor of CTR: (1) need for improved efficiencies and effectiveness to advance CTR and ensure results are disseminated and implemented into healthcare; 2) education and training innovations do not reach the full CTR workforce, resulting in a declining CTS workforce, lacking the knowledge and skills to advance CTR; 3) limited authentic participation by a wide range of stakeholders across the research life cycle; 4) growth of complex datasets necessitates integration of clinical, environmental, and research data, with need for democratization of data accessibility; and 5) need for improved understanding of the contributions of neighborhoods, built environment, education, and financial resources to health outcomes. To address these roadblocks we will pursue five aims: Aim 1: Develop innovations in methods, approaches, and tools to address pressing roadblocks facing CTR. Aim 2: Support training and career development of the full CTS workforce. Aim 3: Engage voices from across academic and scientific disciplines, patients, communities, and industry to conduct CTR and CTS across the full lifecycle of the scientific process. Aim 4: Deploy an accessible, responsive, and integrated system of research resources. Aim 5: Democratize informatics resources by lowering the barriers of entry for data access and computing resources. With a commitment to metric-driven decision-making, we will evaluate the impact of the CTSI by applying the RE-AIM framework to inform strategic pivots over the next 7 years. This proposal reaffirms our commitment to advance CTS and expands our engagement with stakeholders to increase rigorous, impactful, and relevant CTR. We will develop, share, and adopt innovations through focused CTS to enhance CTR for the communities we serve and those we engage. These resources, skilled workforce, and institutional strengths provide an agile foundation to facilitate rapid responses to emerging public health issues and will ensure our laser focus on advancing today’s discoveries to improve health for all.

NIH Research Projects · FY 2025 · 2023-08

The goal of this new Ohio State University (OSU) Center for Clinical and Translational Science (CCTS) predoctoral training program is to leverage our large, collaborative, and multidisciplinary research environment to increase the reach of CTS education and training across the OSU campus and to recruit and develop an outstanding cohort of trainees to become the next generation of clinical and translational scientist leaders. This new program, built upon the foundation of 14 years of experience with the OSU CCTS TL1 program, aims to provide trainees with knowledge, skills and abilities that will expand the scope of their current and future research and prepare them to confidently communicate with various stakeholders and lead multidisciplinary research teams to drive biomedical and clinical innovations. We aim to foster a learning environment grounded in mentorship excellence through mentorship training, monitoring and constructive feedback. Post candidacy trainees are selected from a wide range of graduate programs in nine colleges including Health Sciences Colleges (Medicine, Optometry, Public Health, Pharmacy, Nursing, Dentistry, Veterinary Medicine), Engineering, Arts & Sciences, and three interdisciplinary graduate programs (Biochemistry, Molecular, Cellular & Developmental Biology, Neuroscience). Trainees enroll in 2 required courses that cover core concepts and include team based projects in CTS that emphasize research design and methodologies that are rigorous, ethical, and intentional to achieve health for all. Trainees are required to fulfill requirements of a Graduate Interdisciplinary Specialization in Biomedical Clinical Translational Science (GISBCTS) selecting from coursework in 4 tracks (Research Methods; Statistics, Data Analysis and Bioinformatics; Community and Communications, and Leadership/Team Science). Leadership and team science skill development and resiliency training at this pre-doctoral stage is expected to favorably impact the career trajectories of our trainees, increase retention of CTS investigators from all backgrounds and ultimately enhance the breadth and impact of CTS for individuals and communities. Trainees attend Lunch & Learn monthly seminars on a variety of research and career development topics facilitated by faculty and staff from across colleges. Trainees select CCTS “Tools of the Trade” in-depth workshops that focus on research topics like grant writing, community engagement, clinical trials management, and technology commercialization. Collaboration with the CCTS workforce development team, community engagement team and the K12 program permits cross-fertilization to enhance training across a spectrum of CTS researchers, expansion of trainee networks, opportunities for peer mentoring and coordinated program evaluation of learners at various career stages. The CCTS T32 Career Development Dinner Series includes informal discussion with alumni and invited guests to highlight the wide variety of research career paths and expand networking opportunities. Trainees are supported for two years of training. Six training slots per year are requested.

NIH Research Projects · FY 2026 · 2023-08

Project Summary The nuclear package that comprises the eukaryotic genome not only stores genetic information but also mediates cell-type-specific gene expression. The hierarchical genome organization is tightly regulated to precisely control cell functions. Interphase chromosomes occupy distinct nuclear spaces, a conserved genome architecture known as chromosome territories. Technological advances over the last two decades have revealed many new aspects of the three-dimensional architecture of the genome. However, understanding the mechanisms that localize and mobilize chromosomal loci and territories in the nucleus requires high-resolution studies in real time under physiological conditions. In the past five years, we have developed CRISPR-based high-resolution live-cell imaging techniques using multiple colors to localize and track up to seven genomic loci simultaneously. Recently, we have replaced fluorescent proteins with small cell-permeable RNA-interacting molecules that improve brightness and reduce the size of tags by >100-fold. Our preliminary data revealed surprising dynamic and structural aspects of the chromatin: (1) homologous and non-homologous chromosomal loci moved at different speeds and in different directions; (2) large-scale chromosomal domains continuously rearranged in minutes in non-stressed conditions, termed chromosome morphological dynamics; (3) chromosome conformations were temperature-sensitive; and (4) transformed and non-transformed cells had distinct chromosome conformations. In mouse embryonic stem cells, the mobility of promoters and enhancers correlates with transcriptional activity for specific genes; however, how chromatin mobility correlates with transcriptional activity is poorly understood and controversial. Building upon our preliminary results, we propose to investigate four key concepts: (i) how chromosomal DNA is organized in individual chromosome territories, (ii) what factors drive chromosome morphological dynamics, (iii) how active genes are positioned relative to non- transcribed DNA regions to craft the landscape of the genome, and (iv) how chromatin movements correlate with transcriptional activities in the nucleus. We will perturb transcription, temperature, and microtubule polymerization to identify factors that govern chromosome dynamics. Integration of non-invasive imaging approaches with biophysical models and RNA-seq data will provide new information on the mechanistic and functional foundations of real-time chromatin dynamics and gene positioning at the single chromosome level in the nucleus.

NIH Research Projects · FY 2025 · 2023-08

Prompt closure of wounds is critical to prevention of infection and sepsis in patients suffering massive burn injuries. The most common challenge associated with treating these patients is the lack of available donor skin. Although new technologies are emerging to treat deep partial thickness burns, only one is commercially available for the treatment of full-thickness burns (cultured epithelial autografts, CEAs). CEAs are a life-saving treatment option; however, they are extremely fragile, prone to damage and require > 3 weeks to manufacture. As early wound closure reduces the risk of infection, fluid loss, mortality and scarring, strategies to quickly and permanently close full-thickness wounds are needed to increase survival and improve outcomes. The current obstacles to rapid in situ regeneration of full-thickness wounds using cell sprays include the lack of viable dermis, low engraftment efficiency and variable survivability of spray-on cells. Our team recently developed allogeneic dermal substitutes with laser ablative dermal papillae that significantly enhanced keratinocyte proliferation. This regenerative platform, consisting of freshly isolated autologous cell sprays and an off-the-shelf, allogeneic dermal template with laser micropatterned dermal papillae and growth factor loaded rapid release nanoparticles, is proposed to facilitate rapid, permanent wound closure via enhanced adhesion and survival of spray-on skin cells and rapid angiogenesis. In Aim 1, spray-on skin cell engraftment and survivability will be examined as a function of the form of the laser micropatterned dermal papillae (width, length, angle). Aim 2 seeks to further enhance spray-on skin cell survivability and epidermal regeneration via enhanced angiogenesis. The role of vascular endothelial growth factor (VEGF)/ platelet-derived growth factor (PDGF)-releasing, high surface area to volume polydopamine (PDA) nanoparticles on the rate and extent of angiogenesis and downstream epidermal regeneration will be assessed in a mouse model followed by a highly translational porcine model. In Aim 3, gradient collagen-silk scaffolds will be fabricated to reduce contraction of the wounds while providing a physical and chemical environment that promotes epidermal regeneration. Finally, the efficacy of the fully optimized laser micropatterned dermal template (dermal papillae form, VEGF/PDGF-PDA loading and concentration and scaffold mechanics) will be examined in a porcine burn model compared to standard autografting and spray-on skin cells alone. The proposed studies leverage the expertise in regenerative medicine, vascularization and large animal models to develop a novel, immediate use technology that can dramatically transform treatment for patients suffering from massive burn injuries and improve outcomes and quality of life.

NIH Research Projects · FY 2024 · 2023-08

HISTONE MODIFICATIONS GUIDING HIV INTEGRATION PROJECT SUMMARY / ABSTRACT Human immunodeficiency virus (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS) with ~36.7 million people currently infected worldwide. Integration of the viral genome establishes an irreversible insertion of the proviral sequence into the host chromatin. The integrated genome ensures effective HIV gene expression and ultimately virus production or the establishment of latency where the provirus remains dormant for an extended time. Retroviral integration is mediated by the viral integrase (IN) protein that is bound to the direct-repeated ends of the viral DNA genome, along with additional cellular and viral co-factors that direct integration to host chromatin sites. The overarching goal of this proposal is to understand how viral integration is targeted to chromatin by histone post translational modifications (PTMs), and how cellular factors that tightly control the localization of these PTMs influence target site choice. LEDGF/p75 is a key cellular transcription co-activator that binds to HIV-1 IN via a C-terminal integrase binding domain (IBD). The N-terminus of LEDGF/p75 contains a PWWP domain that is expected to recognize histone methylated lysine PTMs. Bimodal tethering of the HIV-1 integration complex (intasome) to a transcription-related histone PTM has been proposed to account for the observed ~76% of chromosomal integrations that occur in actively transcribed genes. LEDGF/p75 purportedly targets HIV-1 to nucleosomes containing trimethylation of histone H3 lysine 36 residues (H3K36me3). However, H3K36me3 is most often found at the 3’ ends of transcribed genes, whereas HIV-1 integrates more frequently toward the 5’ ends of genes. Bioinformatic correlations of HIV-1 integration sites with histone PTMs is limited by the data available in the Encyclopedia of DNA Elements (ENCODE) database. Notably absent from ENCODE are genomic maps of the H3K36me2 PTM in any cell type. We performed a ChIP-Seq analysis of H3K36me2 and determined that it was commonly found near the 5’ transcription start site of genes. Moreover, we found that HIV-1 integration sites correlate better with the location of H3K36me2 than H3K36me3. We propose to examine the role of H3K36me2 and H3K36me3 with two Specific Aims. Aim 1 will expand the current understanding of H3K36 methylation and its connection to HIV-1 integration efficiency and site selection in vivo. Aim 2 will probe the influence of H3K36me2 and H3K36me3 on HIV-1 integration in vitro. Our approaches will include technologically advanced mass spectrometry, integration site mapping, and single molecule fluorescence microscopy. The results of these studies will clearly determine the role of H3K36me2 and H3K36me3 during HIV-1 integration.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Recent studies by my group have highlighted the clinical relevance of platelet-derived growth factor-B (PDGFB) secreted by malignant breast epithelial cells and its receptor, platelet-derived growth factor receptor beta (PDGFRβ), expressed on cells of the mesenchymal lineage (e.g., fibroblasts, pericytes, astrocytes) in the promotion of breast cancer metastasis to the brain (BCBM). Our studies revealed: (1) PDGFB ligand promotes primary and intracranial breast tumor growth, (2) mesenchymal-specific PDGFRβ hyperactivity (Fsp1- cre;PdgfrbD849V/+) promotes BCBM in mice, (3) PDGFB ligand expression in the primary breast tumor is prognostic of brain metastases in human breast cancer patients, and (4) inhibition with a small molecule selective PDGFR inhibitor (crenolanib) reduces intracranial tumor growth in mouse models of BCBM. This published work discovered PDGFB-to-PDGFRβ signaling as a clinically relevant signaling node for predicting, and potentially treating, BCBM. Our continued investigation into how breast cancer-derived PDGFB mechanistically modulates the brain TME has further revealed previously unknown immuno-modulatory and vascular effects. The current application will directly test the overarching hypothesis that breast cancer cells expressing PDGFB completely transform the brain metastatic niche, both by creating a pro-tumorigenic immune microenvironment and by invoking vascular changes. In Aim 1, the interaction between breast cancer cells with/without PDGFB and the brain immune microenvironment will be evaluated in vitro (microglial co- cultures and conditioned media experiments) and in vivo (intracardiac injections followed by brain histopathological evaluation and high-plex spectral cytometry). This aim will also evaluate if PDGFB causes changes in the microglial phagocytic response as well as shifts in anti-tumorigenic/pro-tumorigenic myeloid cell phenotypes. These studies will be the first to directly test whether breast cancer-derived PDGFB drives immune evasion in the brain metastatic microenvironment. In Aim 2, the interaction between breast cancer cells with/without PDGFB and the brain microvasculature will be evaluated in vitro (BBB spheroids, microfluidics) and in vivo (intracardiac injections followed by brain histopathological evaluation). This aim will also evaluate a putative mechanism by which PDGFB functions in an autocrine manner to upregulate the pro- angiogenic factor Angiopoietin-1, and whether these vascular changes can be ablated with small molecule inhibitors of PDGFRβ and/or Tie2 (angiopoietin receptor). These studies will be the first to directly test the hypothesis that breast cancer-derived PDGFB alters the brain microvasculature indirectly through an autocrine PDGFB-PDGFRβ-Ang1 axis. Upon completion of the proposed study, we will have determined potentially targetable mechanism(s) by which PDGFB pre-conditions the brain microenvironment allowing for metastasis to this site.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Myocardial infarction (MI) is a major cause of death and disability worldwide, affecting ~800,000 Americans annually. Optimal healing of the damaged tissue requires the delicate balance, both spatially and temporally, of inflammatory and reparative mechanisms to create the fibrotic scar. Cardiac fibroblasts (CFs) are the main contributor to fibrotic remodeling. Following ischemic injury, CFs transition into an activated phenotype that is characterized by increased proliferation, migration to the infarct region, and secretion of fibrotic proteins and paracrine signals. At the same time, dysregulation of the CF response to injury can promote pathological fibrosis, increased risk for arrhythmia, and cardiac dysfunction. While there has been many studies exploring the diverse signaling cascades and stressors that cause CF activation, how these stressors regulate the CF phenotype and paracrine signal generation, both spatially and temporally, remain elusive. Recent work identified stress-induced loss of the cytoskeletal protein, βIV-spectrin, to be an important step in CF activation and fibrosis3. Further, loss of βIV-spectrin was found to depend on Ca2+/ calmodulin-dependent protein kinase II (CaMKII). A broader role has been identified for βIV-spectrin/CaMKII in regulating CF gene expression through an interaction with signal transducer and activation of transcription 3 (STAT3)3,4, a signaling molecule and transcription factor that promotes profibrotic mechanisms. Specifically, CaMKII is activated and promotes loss of βIV-spectrin and redistribution of STAT3 to the nucleus that lead to changes in gene expression. Together, this leads to the hypothesis that the βIV-spectrin/STAT3 complex acts as a signaling node that is necessary for regulating cardiac fibroblast activation, recruitment, and scar formation post MI. To evaluate this hypothesis, Aim 1 will identify the role of the βIV-spectrin/STAT3 complex in CF activation and long-range communication. CFs will be subjected to both biomechanical stretch and neurohormonal stimuli, correlating to MI pathophysiology, to evaluate the effects on CF activation and exosome secretion. To understand how remote CFs migrate to the infarct area, long-range communication signals from spectrin-deficient CFs will be characterized and cultured with fresh CFs to see if they lead to activation. Additionally, this project will offer mechanistic insight into the spatiotemporal regulatory role of spectrin-based proteins in modulating exosome secretion following chronic stress. Lastly, Aim 2 will subject spectrin-preserved and spectrin-deficient mice to MI and evaluate the effects on scar formation and maturation. These studies will offer insight into how specific stress combinations tune the process of fibrotic remodeling following MI, and how these regulatory proteins can affect the overall outcome of MI patients.

NIH Research Projects · FY 2024 · 2023-08

Project Summary In the United States, 47% of adults 30 years or older are affected by periodontal disease, and this rate increases with age to 70% in adults 65 years or older. The periodontal complex, consisting of alveolar bone, cementum, gingiva, and the periodontal ligament (PDL), works to ensure proper tooth attachment and nourishment, distribute occlusal forces, maintain alveolar bone height, and protect the periodontium from invading microbes. Periodontal disease leads to the destruction of one or more of these tissues, which ultimately results in partial or total edentulism as well as reduced function of the masticatory complex, self- esteem, and interpersonal relationships. Most therapies are unpredictable as well as unsuccessful at repairing all three lost or damaged tissues. Bone sialoprotein (Ibsp gene; BSP protein) is a multifunctional, extracellular matrix protein found in mineralized tissues of the skeleton and dentition, including alveolar bone and cementum. Total knockout mice (Ibsp-/-) display reduced acellular cementum, hypomineralized alveolar bone, PDL detachment, severe alveolar bone resorption, tooth loss, and periodontal destruction. To date, nearly all studies on BSP focus on its roles in cranial and postcranial development. However, its dual functions in osteoblasts and osteoclasts also suggest an important role in the coupled process of bone remodeling; Ibsp-/- mice show dramatic defects in alveolar bone socket healing following molar extraction. While the importance of BSP in the periodontal complex is evident, its molecular functions remain unclear. We propose BSP is a key molecule in periodontal development, homeostasis, and repair. The outlined experiments will test our central hypothesis that BSP modulates periodontal development and repair through its functions in key cells for periodontal function: cementoblasts, osteoblasts, and osteoclasts. The overall objectives of this proposal are: 1.) define the origin of BSP and cementoblast lineage using conditional ablation of Ibsp from ectomesenchymal vs. epithelial cell populations; 2.) elucidate the role(s) of BSP in osteoblast and osteoclast function(s) in alveolar bone healing by conditionally ablating Ibsp from osteoblasts and osteoclasts; and 3.) determine the role of BSP in postmenopausal osteoporotic changes to bone metabolism using an ovariectomy (OVX) rodent model of postmenopausal osteoporosis. The knowledge gained from this proposal holds promise for the development of novel and reparative therapies of the periodontal complex. Successful completion of the proposed research will provide key insights into the function(s) of BSP in periodontal biology as well as diseases characterized by excessive osteoblast-osteoclast decoupling.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract PRMT5 is an arginine methyltransferase with key roles in cancer. The gene has pleiotropic functions ranging from gene regulation and development to modulation of DNA double strand break (DSBs) repair. In S-phase and mitosis, DSBs are repaired primarily by homologous recombination (HR) a process in which missing genetic information is copied from another undamaged chromosomal region. In humans, error-free HR is mediated by BRCA1, BRCA2 and RAD51. RAD52, an accessory gene, facilitates an error-prone HR sub-pathway that can produce intrachromosomal deletions (ICDs). PRMT5 appears to modulate chromatin remodeling at the DSB through the error-free BRCA1/2-RAD51 pathway. In fission yeast (S. pombe), we identified physical and genetic interactions between PRMT5 and RAD52. Deletion of PRMT5 increases the frequency of ICDs while deletion of RAD52 decreases ICDs suggesting that they have opposite functions. In cancer cells PRMT5 mutations increases the size of ICDs. Using artificial intelligence algorithms, we discovered multiple likely pathogenic mutations including three driver mutations in the active site of PRMT5 that are likely to destabilize the function of the enzyme. We hypothesize that PRMT5 inhibits ICDs by biasing repair of DNA double strand breaks toward conservative pathways. We developed innovative in vivo assays to probe HR repair mechanisms that produce ICDs. The repair mechanisms and factors involved are conserved from yeast to humans making the S. pombe model system highly tractable. A homology analysis reveals that all identified human mutated residues are present in yeast. In Aim1 we will place PRMT5 in the DNA damage repair epistatic pathway using mutational analysis and sensitivity to various DNA damage drugs. Additionally, we will employ in vivo repair assays to understand the HR pathways by which ICDs are produced in the absence of the PRMT5 function. In Aim 2 we will analyze PRMT5 mutations identified in cancer cells using the Catalogue of Somatic Mutations in Cancer (COSMIC). We will employ modeling techniques and enzymatic assays to test how these mutations affect the enzymatic function of PRMT5 and DSB repair. Most of these mutations are conserved in yeast making this analysis tractable. The findings will be further validated in human cells.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract The proposed 5-year study uses a causally interpretable research design to examine the effects of the Sit Together and Read (STAR) early-literacy intervention on the short- and long-term literacy skills of young children with developmental language disorder (DLD). It also examines the use of caregiver-directed behavior- change strategies as a means to support caregivers’ implementation of the intended treatment strength. STAR is a fully manualized intervention that significantly improves the early-literacy skills and longer-term reading outcomes of children at-risk for future reading difficulty. The proposed study is instrumental in assessing longitudinal impacts for children with DLD and identifying ways to enhance caregiver implementation of the key intervention ingredients. The study features four planned variations with each involving 80 caregiver-child dyads (total N = 320, 80 per condition). Two planned variations allow examination of the main effects of STAR for children with DLD, with 80 caregivers assigned to implement STAR for a 15-week period as compared to an untreated control. The outcomes of interest for these variations are children’s short-term print-knowledge development, literacy trajectories to two-years post-intervention, and reading-difficulty status. Two additional planned variations allow examination of effects of caregiver-directed behavior-change strategies (rewards and encouragement) on STAR implementation as compared to caregivers who do not receive any change strategies. The outcomes of interest for these variations are caregivers’ implementation of the intended treatment strength as well as mediated effects on children’s literacy outcomes. Child outcomes are based on direct assessments of literacy skills captured at six time-points, from study baseline to two-years’ post-intervention; assessors are blind to condition. Caregiver implementation is captured via logged sessions on a bespoke app as well as analyses of audiotaped sessions submitted via the app. Directional hypotheses are threefold. First, we hypothesize that children whose caregivers implement STAR will show significantly greater print-knowledge gains over the 15-week intervention period, and accelerated literacy trajectories through first grade. We speculate that STAR exposure will also significantly reduce the rate of reading difficulty among children. Second, we hypothesize that children whose caregiver implement STAR and receive one of two behavior-change strategies will show significantly greater intensity and dosage of implementation relative to those not receiving these strategies. Third, we hypothesize significantly greater print-knowledge gains and literacy trajectories for children of caregivers receiving behavior-change strategies, and that these effects are partially or fully mediated by intervention intensity and/or dose.

NIH Research Projects · FY 2025 · 2023-08

Compared to Whites, African American/Blacks (AA/B) have a substantially higher (40-50%) colorectal cancer (CRC) mortality rate that is a function of both higher incidence and lower survival rates. Our long-term goal is to understand the differences in immune response that influence this health disparity in CRC mortality. Emerging studies suggest alterations in T cell presence and function in AA/B contribute to CRC disparities, and AA/B CRC patients with low immune infiltrate have particularly poor outcomes. We have found that AA/B CRC patients have disproportionally reduced tumoral MHC class I expression compared with White patients. As MHC class I is critical for presentation of tumor antigens to CD8+ T cells, these results suggest a critical immune mediated mechanism that drives the differences in survival times between AA/B and White patients. The objectives of this project are to understand the mechanisms relevant to T cell alterations within the tumors of AA/B versus White patients and to develop improved biomarkers and therapies to reduce CRC health disparities. Our central hypothesis is that reduced tumoral MHC class I expression drives T cell alterations to enhance tumoral immune escape in CRC from AA/B patients compared with White patients. To test this hypothesis, we have developed robust multispectral imaging capability for studying the relationship between MHC class I expression and CD8+ T cell frequency, localization, phenotype, and function. In parallel, we have developed an autologous humanized CRC TIL-PDX mouse model, created with matched patient-derived tumor and tumor infiltrating T cells (TILs) to investigate CRC disparities. This completely unique approach will be undertaken by a multi-disciplinary team, which includes a surgeon-scientist with expertise in CRC oncology, a cancer immunologist, and a cancer health disparities basic researcher. In Aim 1, we will define CD8+ T cell biology in the context of tumoral MHC class I loss in AA/B versus White CRC patients. We will perform both multispectral imaging of archival tumor samples and phenotypical/ functional studies of fresh samples to define essential differences between AA/B and White tumors. In Aim 2, we will evaluate whether TIL-PDX mice generated from AA/B versus White CRC tumors exhibit differential T cell biology and anti-tumor immunity in the context of tumoral MHC class I expression. We expect that the use of the CRC TIL-PDX mouse model will recapitulates a patients’ tumor immunity in a manner not previously possible to determine the differences in immune mediated processes. In Aim 3, we will assess the ability of IL-15 as a therapy to overcome AA/B tumoral MHC class I loss using a syngeneic CRC tumor-bearing mice. The positive impact of this work will be an improved understanding of the differences in immune-mediated mechanisms to understand health disparities associated with CRC patient outcomes and to develop more effective therapeutic approaches for CRC patients.

NIH Research Projects · FY 2024 · 2023-08

Time-to-event is a ubiquitous outcome measure in clinical diagnosis and assessment of therapeutic effects in many disease areas including stroke (time-to-stroke), respiratory (time-to-first medication for worsening asthma) and sleep diseases (time-to-insomnia-related mortality). The hazard rate is commonly seen in survival analysis as it has the convenient interpretation of instantaneous risk. The hazard ratio (HR) is routinely used as an effect measure when comparing between two treatment groups, largely due to the popular Cox proportional hazards (PH) model. However, the HR is vulnerable to selection bias and not collapsible, which make it a questionable marginal causal effect measure. Restricted mean survival time (RMST) is an alternative measure, defined as the area under the survival curve up to a fixed time point. RMST difference is a more adequate causal effect measure than the HR because (i) it is a collapsible measure, thus avoids discrepancy between marginal and conditional effects; (ii) it does not depend on the PH assumption; (iii) it is essentially a mean difference with simpler interpretation. RMST has become a popular metric of treatment effects in randomized trials recently. However, the development of RMST methodology for observational survival data is lacking. The goal of this proposal is to develop a comprehensive matching-based RMST difference estimation strategy to infer causal effects in observational survival data, and apply such tools to evaluate causal effects of direct oral anticoagulants (DOAC) vs. warfarin on the risk of cardiovascular events in a secondary data analysis. We plan to develop propensity score matching-based RMST estimation methodology and corresponding sensitivity analysis, which do not rely on strong outcome modeling assumptions. The matching method will use an optimal algorithm to create matched sets to mimic a block randomized design and an asymptotically valid post-matching inferential procedure will be developed by accounting for the correlation introduced in matching. Built upon the matched data, the sensitivity analysis will address how much association an unmeasured confounder would need to have with both the exposure and the outcome, to explain away the observed effect. In the secondary data analysis, we will apply our methods to examine the hypothesis that using DOAC has lower risk of composite cardiovascular events including stroke, venous thromboembolism, myocardial infarction, and death, using electronic medical record (EMR) data. We will also explore subgroup causal effects related to gender and race to examine potential health disparity issues. Our proposed work will not only result in novel and valid research methodology for estimating causal effects in observational survival data, but also advance the understanding of how different anticoagulant drugs would impact patient outcomes using a large secondary database. Our general-purpose methodology will be widely applicable to study survival data in heart, lung, blood and sleep disease treatment, and disparity research. This will also enable clinical researchers to rigorously identify causal evidence using increasingly available real-world data.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Recently, synthetic cooling agents, WS-3 (N-ethyl-p-menthane-3-carboxamide) and WS-23 (2-isopropyl-N,2,3 trimethylbutyramide), that solely impart a cooling sensation without a mint flavor, have been detected in e- cigarettes (ECs) and are marketed with “ice” as part of the flavor name (e.g., “watermelon ice”). Unlike menthol, synthetic cooling agents can be added at very high concentrations (>5% by weight) to e-liquids allowing users to get extreme cooling/anesthetic properties during vaping, without menthol’s unappealing properties (e.g., eye- watering, harshness). Synthetic cooling agents have the potential to increase indirect and direct harm to EC users. The proposed study will be the first to systematically investigate the effects of synthetic cooling agents on the appeal, puffing behavior, and toxicity of vaping. We will use a single-blind randomized crossover trial with young adult vapers (N = 120, aged 21-29 years), precisely manipulated e-liquids, a well-characterized commercial EC device, validated psycho-behavioral measures, and a novel puff-playback method to estimate human exposures to HPHCs and other toxicants from vaping e-liquids with and without synthetic cooling agents (i.e., WS-3 and WS-23). Specifically, we aim to determine the impact of e-liquids with (mango ice, tobacco ice) and without (mango, tobacco) the presence of synthetic cooling agents on abuse liability (appeal, sensory effects, demand), EC puffing behavior (i.e., EC topography), and toxicant exposure (harmful and potential harmful constituents). Additionally, topography data collected for all participants for each of the conditions vaped will be averaged to produce four human-derived puffing regimens. Machine vaping will be conducted for each of the four e-liquids using the associated human-derived puffing regimens to determine the range of HPHCs and other toxicants in mainstream EC vapor. Findings would immediately inform the evidence base needed to inform FDA rulemaking surrounding the use of synthetic cooling agent additives in EC products.

NIH Research Projects · FY 2025 · 2023-08

Sudden cardiac death due to stress-induced ventricular tachyarrhythmias remains the major cause of mortality in the world. Mitochondrial metabolic output in ventricular myocytes (VMs) is tightly linked to intracellular Ca2+ cycling in a process called excitation-contraction-bioenergetics (ECB) coupling. Disturbances in this process contribute to arrhythmias not only in acquired conditions such as heart failure (HF), hypertrophy or aging, but in heritable arrhythmia syndromes as well. During catecholaminergic surge, i.e. stress and maximum workload, an increase in intracellular Ca2+ transient amplitude results in increased matrix [Ca2+] influx and accelerated ATP production to meet increased metabolic demand. However, it comes with the risk of increased generation of reactive oxygen species (ROS) by the electron transport chain (ETC). This can overcome antioxidant defenses and adversely affect Ca2+ handling machinery and the ryanodine receptor (RyR2), promoting Ca2+ dependent arrhythmia. Therefore, the main objective of current proposal is the identification of new approaches to maintain mitochondrial ROS and Ca2+homeostasis to improve cardiac function and reduce arrhythmic risk in diseased hearts. In the course of preliminary studies, we discovered that a reduction in the formation of quaternary supercomplexes from the elements of ETC plays a key role in accelerated mito-ROS production in VMs from hypertrophic rat hearts and hearts from genetic rat model of catecholaminergic polymorphic ventricular tachycardia (CPVT). Pilot studies revealed that changes in expression levels of two proteins can underlie less compact ETC organization, namely (1) COX7RP, a key regulator of supercomplex formation, and (2) structural protein OPA1 which controls mitochondria cristae diameter. Importantly, we discovered that expression levels of both these proteins are higher in healthy females vs males, suggesting fundamental differences in ECB coupling between sexes. Accordingly, two specific aims are proposed. Aim 1: To determine the role and mechanisms of RyR2 hyperactivity-mediated changes in mitochondria function. We created a unique gain-of RyR2 function rat model of CPVT to test the hypothesis that RyR2 hyperactivity contributes to activation of Ca2+-dependent protease calpain residing in mitochondria intermembrane space leading to OPA1 proteolysis. Aim 2: To determine the mechanisms and physiological significance of COX7RP-dependent mitochondrial dysfunction in cardiac arrhythmias linked to RyR2 hyperactivity. We hypothesize that COX7RP downregulation is the key contributor to the deficient mitochondria electron transport and increased mito-ROS emission in cardiac hypertrophy and failure exacerbating pro-arrhythmic Ca2+i mishandling.