Ohio State University

NIH Research Projects · FY 2025 · 2021-09

Abstract This research focuses on filling gaps in knowledge about the precise molecular pathways that underpin retinal inflammation and impact crosstalk from ischemic retinal diseases, including diabetic retinopathy, vascular diseases, retinopathy of prematurity, and sickle cell retinopathy. Current treatments are often inadequate to prevent vision loss, and adding selective targeting of additional inflammatory mediators may offer new vision-saving therapies. We have identified that (1) the pro- inflammatory cytokine macrophage migration inhibitor factor (MIF) is a druggable target for preventing retinal gliosis and photoreceptor loss in retinal detachment. (2) MIF is up-regulated in the N-methyl-D- aspartic acid (NMDA) damage model which simulates ischemia-mediated retinal excitotoxicity; pharmacologic and genetic inhibition of MIF increases neuronal survival in this model. (3) Clinically we identified a genetic association of MIF promoter polymorphisms with epiretinal membrane formation. Müller glia/astrocytes (MG) are the predominant components of ERM suggesting that MIF could play an important role in the pathological function of retinal glia. MIF inhibitors are in clinical evaluation for a variety of systemic diseases. While inhibition of MIF’s pro-inflammatory effects may indeed underlie the enhanced neuronal survival from MIF d-DT inhibitors, our recent findings strongly suggest that alternative mechanisms also exist. MIF is highly expressed in the Müller glia/astrocytes and it has been hypothesized to be a glial growth factor. Our preliminary data show that conditional inhibition of MIF in the MG enhances the survival of retinal neurons during damage and affects the MG JAK/STAT pathway. Herein, Specific Aim 1 will test the hypothesis that MIF inhibition promotes neuronal survival in retinal damage by activating the gp130/JAK/STAT signaling pathway of Müller glia/astrocytes. In chick and murine NMDA models, we will use pharmacologic and genetic approaches to assess the impact on MG signaling pathways and neuronal survival induced by MIF inhibition. Specific Aim 2 will test the hypothesis that conditional deletion of Müller glia/astrocyte MIF up-regulates the gp130/JAK/STAT pathway and enhances the survival of retinal neurons. In Specific Aim 3 we will develop a single cell RNA-seq database of damaged and undamaged retina treated with MIF inhibitors and/or MG-specific genetic deletion of MIF. We will comprehensively evaluate the transcriptional changes at single-cell resolution in the glia and retinal neurons that result from inhibition of MIF. This research will define the important functional relationships between MIF and signaling pathways on specific cells during retinal damage. The fundamental knowledge gained from understanding the transcriptome ‘switch’ will set the stage for future studies targeting key molecular pathways that are druggable with minimal side effects, but able to prevent and recover visual loss from retinal damage.

NIH Research Projects · FY 2025 · 2021-09

Project Summary The germline is dedicated to produce gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules—well-conserved membraneless and RNA-rich organelles. The composition and function of germ granules remain elusive owing to their dynamic nature and their exclusive expression in the germline. This project addresses three fundamental questions. How are germ granules assembled and maintained? How are the epigenetic regulators, such as small RNAs, amplified in germ granules and passed on to offspring? What is impact of granules on gene regulatory networks and transgenerational inheritance? This proposal uses C. elegans germ granules as a model system to define their proteome and RNAome. The goal is to identify protein and RNA components that are required for germ granule formation, and characterize their role in germline maintenance and embryogenesis. Furthermore, the genetic and biochemical approaches afforded by C. elegans will be used to determine the mechanisms that drive the production of small RNAs within germ granules. By characterizing their constituents, this research program will advance our understanding of mechanisms of germ granule formation and their function in fertility.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Our proposal investigates a mechanism underlying innate immune dysfunction in acute myeloid leukemia (AML), the leading cause of leukemia-related deaths in the U.S. The innate immune system naturally defends against malignancy, however AML evades immunosurveillance to drive disease progression. Natural killer (NK) cells are the primarily innate lymphoid cell (ILC) responsible for anti-tumor immune surveillance, and reduced NK cell function both in de novo AML and in the post-transplant setting is correlated with poor outcomes. We have recently discovered that AML patients carry a fundamental defect in NK cell development leading to specific depletion of a sub-population of NK cells with critical roles in coordinating innate and adaptive immune responses, as well as mature NK cell development and function. We have shown that NK cells develop from a common innate lymphoid cell precursor (ILCP), which generates a series of NK developmental intermediates (NKDIs) leading to mature, cytotoxic NK cells. ILCPs also give rise to the other members of the ILC family, a diverse group of non-cytotoxic, cytokine-producing “helper” ILCs that are known to be pro-tumorigenic. Our preliminary studies show that AML disrupts the NK lineage, shifting production towards helper ILCs. As these populations all stem from the ILCP, this suggests AML is acting on ILCPs to alter lineage fate specification. Lineage specification occurs through carefully controlled activities of transcription factors that modify the epigenomic landscape generating stable cell type-specific gene expression patterns. Our preliminary studies have uncovered an aberrant, helper ILC-like DNA methylation signature in NKDIs isolated from AML patients and following leukemic cell co-culture. One key transcription factor is the aryl hydrocarbon receptor (AHR), which we have found shifts the helper ILC/NK ratio in the presence of AHR ligands ectopic produced by AML cells. We propose a strategy where the combination of AHR inhibition and hypomethylating agents (HMAs) guides development to restore NK cell differentiation from the ILCP. In this proposal, we will determine how AML drives this fate decision and promotes the generation of helper ILCs by performing detailed epigenetic and functional analyses of ILCPs isolated from normal donors and AML patients, including investigation in an immunocompetent murine AML model. We will investigate functional and epigenetic poising of lineage fate including the role of AHR. Secondly, we will determine the relationship of the NK cell defect in AML patients to epigenetic programming and disease progression, and directly test the impact of HMAs on ILCP and NKDI development. We will also determine the preclinical efficacy of combining both HMA and a novel AHR inhibitor to restore normal NK cell epigenetic programming and enhance NK cell generation to improve outcomes in preclinical models of AML. Maintaining functionally mature NK cells and supporting immunosurveillance is critical to long-term disease control, these studies aim to gain a novel understanding of how AML evades innate immunity and investigate strategies to restore anti-tumor immune surveillance patients.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Tumor suppressor p53 is the quintessential guardian of the genome whose function is inhibited in greater than 50% of all human cancers. Though mutation and deletion of p53 are major contributors to p53 inactivation, overexpression of the negative regulators MDM2 and MDM4 (MDMX) are also known to inactivate p53, thus leading to the cancer phenotype. Our lab has shown that specific types of cell stress initiate the generation of an alternatively spliced isoform of MDM2. The predominant MDM2 alternative isoform, MDM2-ALT1 also known as MDM2-B, functions to primarily activate the p53 pathway by inhibiting MDM2 and MDM4 in a dominant negative fashion. Paradoxically, this isoform is upregulated in several human cancers, such as pediatric high-grade gliomas, astrocytomas, rhabdomyosarcomas (RMS), and liposarcomas, as well as adult cancers such as lymphomas and those of the breast. Thus, MDM2-ALT1 plays opposing roles in cancer progression dependent upon the context of its expression. In the proposed research, we will study the underpinnings of the control of the p53 pathway by MDM2-ALT1 to better understand 1) the specific mechanism by which that MDM2-ALT1 is generated in cancer and 2) the ability of the resultant isoforms to be targeted using splice-switching oligonucleotides. We hypothesize that the expression of oncogenic MDM2- ALT1 is modulated by alterations in protein and RNA nuclear factors during the progression to tumorigenesis and can be targeted to induce splicing changes. We will use assays that identify and measure splice regulation in conjunction with gene editing approaches to identify RNA sequences and their respective nuclear factor- binding partners necessary for regulation of MDM2 splicing. Furthermore, we will use novel genetically engineered mouse models as well as established mouse xenograft assays and novel splice switching oligonucleotides (SSOs) to modulate MDM2 isoform levels. Our work will broaden our knowledge of combinatorial regulation of RNA processing in response to stress and in cancer and interrogate the utility of MDM2 isoforms modulation for rational control of the p53 pathway.

NIH Research Projects · FY 2025 · 2021-08

The incidence of tick-borne diseases has risen dramatically in the past two decades, and continues to rise. Human monocytic ehrlichiosis caused by Ehrlichia chaffeensis (Ech) is one of the most prevalent, life- threatening, emerging tick-borne zoonoses in the US. Ech is an obligatory intracellular bacterium of the order Rickettsiales. Therapy of choice is the broad-spectrum antibiotic doxycycline, which is effective only if initiated early. Currently there is no FDA-approved vaccine for Ech. Our long-term goal is to develop an evidence- based vaccine approach to effectively protect humans by targeting multiple critical steps of the rickettsial infection cycle. Toward this goal, we identified four Ech surface-exposed proteins that have known functions required for Ech survival, and that also lack homology to human proteins, OMP-1/P28, Entry triggering protein of Ehrlichia (EtpE), and VirB2. OMP-1/P28s are immunodominant surface-exposed outer membrane proteins that have porin activity essential for bacterial nutrient acquisition. P28 and OMP-1B are predominantly expressed in mammals and ticks, respectively. EtpE is an invasin that uses its C-terminus (EtpE-C) to bind the host cell receptor to trigger Ech entry. We have shown that the type IV secretion system (T4SS) is essential for Ech survival within the host cell. VirB2 is a T4SS pilus protein that is part of the T4SS machinery. Immunization of mice with recombinant P28, EtpE, or VirB2 proteins generated Ech-specific antibody responses that prevented Ech infection. These data support our premise that these proteins serve as rational vaccine candidates for targeting non-overlapping processes in Ech infection of mammalian host cells. DNA vaccines offer a number of potential advantages over traditional vaccines, including the stimulation of both humoral and T-cell-mediated responses, improved vaccine stability, the absence of any infectious agent, and the relative ease of packaging multi-components and large-scale manufacture. We showed the feasibility of an Ech DNA vaccine in dogs by safely immunizing dogs with the DNA vaccines by percutaneous needle-free jet injection and demonstrating humoral and cell-mediated immune responses to the DNA vaccines. Our hypothesis is immunization with plasmid DNA vaccine encoding P28, OMP-1B, EtpE and VirB2 singly or in combination prevents Ech transmission from ticks to mammals. To test this hypothesis, our Specific Aims are: 1. To construct DNA vaccines encoding P28, OMP-1B, EtpE-C, and VirB2, determine the development of humoral and cell-mediated immune responses in immunized mice, and evaluate protection of immunized mice from infection with Ech cultured in tick cells. 2. To test if immunization of dogs with P28, OMP-1B, EtpE-C and VirB2 can prevent Ech transmission from infected ticks. The immediate outcomes of the proposed studies will be to provide proof-of-principle for a DNA vaccine approach to the Ech vaccine candidates for blocking of Ech transmission from ticks to dogs. The long-term outcome will be development of an anti-infective vaccine against HME in humans that does not cause adverse effects.

NIH Research Projects · FY 2024 · 2021-08

ABSTRACT Ibrutinib, a 1st generation nonselective Bruton’s tyrosine kinase inhibitor (BTKi), has dramatically improved sur- vival for chronic lymphocytic leukemia (CLL) patients, a disease affecting nearly 250,000 U.S. adults. However, up to 38% of CLL patients treated with ibrutinib develop atrial fibrillation (AF). The development of AF on ibrutinib is challenging as drug-drug interactions preclude many standard approaches to treatment, and the risk of bleed- ing when ibrutinib and anticoagulants are combined is markedly increased. Thus, there is need to better under- stand the mechanisms involved in the development of ibrutinib-associated AF, and ultimately identify preventive strategies. Recent animal studies suggest that ibrutinib-associated AF involves pathways through an increase in left atrial volume (LAV) and increased left atrial (LA) fibrosis. There are no clinical data characterizing the effect of ibrutinib on LAV or fibrosis; thus, in Aim 1, we test the effect of ibrutinib on LAV (primary outcome) and LA fibrosis by performing serial cardiac magnetic resonance imaging (CMR) in 50 patients pre- and at 6 months after stating ibrutinib. Additionally, in Aim 1, we will measure blood pressure using ambulatory blood pressure monitoring (ABPM). As background, >70% of ibrutinib-treated patients developed hypertension, and we hypoth- esize that ibrutinib-associated hypertension may be a key factor in the increase in LAV and fibrosis. Finally, in Aim 1, we will measure biomarkers of inflammation, fibrosis, and myocardial damage in relation to LAV. These results will be compared to 50 age-, gender-, and cardiovascular disease-risk matched controls with early CLL where standard of care is observation only. In Aim 2, we will compare the effects of the effects of ibrutinib, a first generation BTKi, with acalabrutinib, a second generation BTKi. In animal work, acalabrutinib was associated with a lower LAV and decreased LA fibrosis as compared to ibrutinib. We will do this by comparing the change in LAV and fibrosis among patients those on ibrutinib from Aim 1 and an additional matched-cohort (n=50) treated with acalabrutinib. Also, in Aim 2, we will compare the increase in blood pressure between these two therapies as conversely, based on our retrospective data, there were higher rates of hypertension with acalabrutinib than reported in cancer trials. Finally, in a 3rd exploratory aim, we will measure and compare AF incidence in our two cohorts (ibrutinib and acalabrutinib). The data in Aim 3 will provide preliminary data for subsequent studies com- paring AF development as a primary outcome between ibrutinib-treated patients and those treated with second generation BTKi’s. The current proposal brings together a multidisciplinary team to expand upon our preliminary data to test the link between ibrutinib and LA remodeling (volume, fibrosis), via CMR imaging techniques, while assessing the relationship between LA remodeling and the systolic blood pressure increase (via ABPM), and AF events. Upon completion, we expect to gain important insights into the association between BTKi use and the mechanism involved in the development of AF in CLL patients. These results will also ultimately inform subse- quent studies testing the most effective strategy for AF control in CLL patients receiving long-term BTKi therapy.

NIH Research Projects · FY 2024 · 2021-08

PROJECT SUMMARY The primary goal of this study is to construct predictive models (classifiers) of pulmonary sarcoidosis and progressive (P) vs. non-progressive (NP) disease that will ultimately serve to improve outcomes of pulmonary sarcoidosis. We have assembled a unique investigative team with expertise in proteomics, immunology, genomics, sarcoidosis clinical care, as well as bioinformatics and statistics. Sarcoidosis is a diagnostically challenging immune-mediated systemic disease. It results in significant morbidity and mortality, primarily due to progressive pulmonary disease, although the factors that drive pulmonary disease and P vs. NP disease are unknown. The strategies to treat pulmonary sarcoidosis, including the triggers to initiate treatment, are non- specific; treatment usually relies on suppressing the immune system with corticosteroids and is associated with considerable side-effects. Transcriptional changes in the lung and blood have defined a signature of P disease in cross-sectional studies. Since proteins are the main effectors of cellular function and their alterations result in disruption of biologic systems and disease development, they are a logical source of biomarkers. Our preliminary data from bronchoalveolar lavage fluid and cells demonstrate significant proteome wide alterations in pulmonary sarcoidosis vs controls and P vs NP disease. We hypothesize that effective markers of disease and those distinguishing progressive from non-progressive disease will reflect biological processes active in disease and progression. Secondarily, by characterizing cellular proteins, global phosphorylation events and cell-specific RNA expression, we will define known proteins/gene/pathways such as the PI3K/Akt/mTOR and other serine-threonine kinase signaling mechanisms as well as novel pathogenic proteins/genes, such as endocytic and aryl hydrocarbon receptor signaling, which will have implications for mechanism and therapy. We will use high-resolution mass spectrometry (MS), advanced bioinformatics and computational tools in well- phenotyped sarcoidosis patients. In Aim 1, we will determine a disease-specific classifier for diagnosing sarcoidosis using a Discovery Cohort of sarcoidosis cases and diseased and healthy controls (already recruited) for the development and Validation Cohort (recruited for this study) of sarcoidosis cases and controls to verify and optimize the classifier performance. In Aim 2, we will identify a protein classifier of P vs NP disease using the same approach as in Aim 2. In Aim 3 we will use a novel single-cell RNA-sequencing approach, CITE-seq to identify transcription from specific cells, and integrate it with protein changes, including examination of global phosphorylation events to identify kinase signaling and discover cell-specific cellular proteins/genes associated with disease and progression in a subset of our Validation Cohort. At the end of this study, we will have defined diagnostic biomarkers of disease and progression that can be translated easily to the clinic. We will also gain insights into the sarcoidosis pulmonary proteins and transcripts that may serve as potential therapeutic targets and provide potential mechanistic information with future study.

NIH Research Projects · FY 2025 · 2021-08

Project Summary A hallmark of eukaryotic cells is their ability to migrate, divide, adhere and respond to environmental cues. Nonmuscle myosin-2 (NM2) motors play an essential role in many aspects of these fundamental cellular processes by forming short bipolar filaments that interact with actin filaments. NM2 motors are binary switches that alter between inactive and active states depending on the cellular context. The precise control of NM2 motor activity is critical for its cellular function as master regulator of the actin cytoskeleton. Aberrant regulation due to mutations in NM2 paralogs contribute to a whole host of diseases including blood and neurological disorders, heart diseases, deafness, nephritis, and cancers. NM2-specific therapies are thus needed, yet the lack of basic knowledge about the structure and regulation of NM2 paralogs is a bottleneck to their development. We aim to develop a detailed structural and mechanistic understanding of how force generation by NM2 motors drive various cellular functions. Using innovative and interdisciplinary techniques including the state-of-the-art cryo- electron microscopy, X-ray crystallography, steady-state kinetics, in vitro motility assays and high-resolution fluorescence microscopy, we will systematically dissect the mechanisms of activation and regulation of NM2. To achieve this, in Aim 1, we will determine the major structural states in the ATPase cycle of NM2 motors to explain enzyme function. In Aim 2, we will determine a high-resolution cryo-EM structure of the inactive state of full- length NM2 to explain its molecular architecture. In Aim 3, we will study the consequences of abolishing the ability to form an inactive state on the dynamics of NM2 filaments in cells. Collectively, our studies will provide a deeper understanding of the structure, function and regulation of NM2. Importantly, this knowledge will advance our understanding of emergent NM2 functions in cells and thus, lay the foundation for future development of NM2-specific therapeutics.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY The gut microbiome changes as humans age and affects many aspects of human health including response to cancer treatments. Increased abundance of the microbe Akkermansia muciniphila has been shown to increase response to immune checkpoint blockade (ICB), a class of anti-cancer agents enable the immune system to identify and attack cancer cells. Rational manipulation of the microbiome is a promising approach to improve cancer and other health outcomes, but as with many studies and trials, the effect of age is understudied. Older adults remain poorly represented in clinical trials for ICB and interventions directed at the microbiome to promote response to ICB. This is problematic because age has been shown to affect the microbiome. Therefore, an intervention related to the microbiome will likely have to be tailored to older adults. In addition, diet-based microbiome interventions are likely to enter clinical practice, but linking food intake to changes in particular microbes has proven challenging. My overall goal is to be an academic researcher studying the role of the microbiome in cancer care for older adults, and how to use diet-based interventions to modify the microbiomes of older adults to promote health. To this end, the objectives of this application are (1) identify the microbiomes of older adults who respond to ICB, (2) modify the microbiome through diet-based interventions, and (3) verify the causal role of the microbiome using preclinical models. To meet these objectives, this study will utilize data from ongoing trials for which I am leading the microbiome collection, data generation and analysis efforts. First, these data will help to define the microbiomes of older lung cancer patients who respond to ICB (SA1). Next, we will test a black raspberry dietary intervention for its ability to promote a pro-ICB response microbiome in older adults (SA2). Finally, pre- and post-intervention human microbiomes will be transferred into mice to assess the effect on response to ICBs (SA3). These aims will support a method for predicting which older adults will respond to treatment, suggest a therapeutic strategy to increase healthspan, and improve our understanding of how the microbiome interacts with the immune system in older adults. This award will provide me with the time to gather knowledge and experience in fields outside my current training, especially clinical geriatrics and the cancer microbiome. My mentorship team includes experts in these fields as well as in nutrition, oncology, and computational modeling. With the dedicated support of these experts and targeted didactics, I believe this award would accelerate my transition to an independent investigator.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT There is a sex-based disparity associated with substance abuse disorders, which is evidenced by preclinical and clinical studies. Females are generally more vulnerable to the initiation, escalation and withdrawal effects of substance abuse behavior than males. Although these differences have largely been attributed to hormonal differences, evidence for non-hormonal factors that regulate addiction has been demonstrated by a number of studies. However, the mechanisms underlying sex chromosome influences on substance abuse behavior represent a huge gap in our knowledge base on the epigenetics of substance use disorders. We propose a novel hypothesis that escape from X-chromosome inactivation (XCI) in females contributes to sex associated differences in addiction behavior. We will apply cutting edge technology and uniquely novel approaches and tools we developed recently to comprehensively investigate the impact of XCI escape on sex associated disparities in addiction. XCI is an epigenetic mechanism that occurs in mammalian females and serves to equalize gene expression between the sexes. Females have two X chromosomes (XX), and during XCI, one X chromosome is randomly chosen to be transcriptionally silenced. However, it is known that a number of X linked genes escape XCI and display bi allelic gene expression. The objective of this proposal is to determine the contribution of XCI escape on sex-associated differences in substance abuse disorder. First, we will use novel cutting edge mouse models to characterize cellular mono-allelic (XCI) or bi-allelic (XCI escape) gene expression of specific X-linked genes associated with addiction to opioids and psychostimulants: monoamine oxidase A (Maoa) and GABAA receptor A3 (Gabra3). I pioneered an innovative approach using a gene specific dual bi- cistronic reporter mouse as a tool to enable the visualization of allelic usage of these addiction associated genes in vivo in a model of addiction. Next, we will determine the molecular landscape of XCI in brain tissue and specific neuronal cells during chronic exposure to opioids and psychostimulants, using a highly innovative single cell RNA sequencing technology. To accomplish these goals, I have assembled a talented, multidisciplinary team of research collaborators in addiction, neuroscience, genetic mouse modelling, bioinformatics and biostatistics. This innovative approach to the study and analysis of gene specific XCI escape as an epigenetic mechanism in the context of substance abuse has the potential to open up a new area of research on the epigenetics of addiction. Further, these genetically modified mice can be used to study XCI escape as an epigenetic mechanism in other neurologic disorders. As an early stage investigator, these studies will also advance my long term objective of becoming a future leader in the epigenetics of substance use disorders.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY / ABSTRACT Estrogenic endocrine disrupting compounds (EDCs) are ubiquitous in pesticides and other industrial products where they remain active in the environment for extended periods. Daughters of women who were exposed prenatally to the estrogenic EDC diethystilbestrol (DES) and dichlorodiphenyltrichloroethane (DDT) exhibit an increased risk of breast cancers. Despite a link between EDC exposure and cancer risk the detailed mechanism(s) that ultimately drive tumorigenesis remains largely unknown. This lack of understanding limits the ability to accurately determine the individual and population risk of estrogenic EDC exposures. The goal of this proposal is to determine the mechanism(s) that links EDC exposure to cancer and to provide biological markers capable of evaluating EDC exposure risk. We have identified a number of EDC-driven reprogramming events within the mammary gland stroma. These events include increased collagen deposition that results in increased mammary gland stiffness and decreased permeability of the extracellular matrix (ECM). Similar stromal tissue changes have been shown to increase cancer susceptibility in animal models and appear to provide a biological connection to EDC- driven tumorigenesis. It is our overarching hypothesis that estrogenic EDCs alter the homeostatic signaling within the mammary gland leading to ECM changes that ultimately drive breast cancer. We propose to test this hypothesis using a well-defined mouse model system with the following Specific Aims: 1) Characterize the estrogenic EDC-induced mechanism(s) that contribute to breast stromal molecular and tissue alterations. 2) Evaluate the contribution of estrogenic EDC-induced stromal alterations to breast cancer risk. These studies will answer several key questions in the field including how the estrogenic activity of EDCs impact stromal alterations, how stromal alterations alter tissue homeostatic signaling during mammary gland development and determine the epigenetic reprogramming events within stromal fibroblasts that propagate an EDC exposure from the womb through adulthood. We expect that this analysis will provide a strong foundation for understanding how environmental EDCs drive tumorigenesis as well as provide potential biomarkers and therapeutic targets for EDC exposure.

NIH Research Projects · FY 2024 · 2021-08

PROJECT SUMMARY Polytobacco use, defined as concurrent use of more than one tobacco product including electronic nicotine delivery systems (ENDS), is rising and high in lesbian, gay, bisexual, and transgender (LGBT) young adults (YA). Between 22-40% of LGBT YA (vs 12-21% of non-LGBT YA) report past 30-day polytobacco use, and LGBT YA are less likely to perceive tobacco use as harmful. Low risk perceptions may reinforce tobacco use and widen existing disparities. The Food and Drug Administration (FDA) Center for Tobacco Products (CTP) is mandated educate the public about tobacco product risks, yet no evidence describes how to effectively frame anti- polytobacco risk communications. The proposed training objectives are for the applicant to develop advanced skills in health communication science; bio-behavioral methods, including psychophysiological measurement; and randomized controlled trials. These skills will be used to determine effective communication of polytobacco use risks to at-risk LGBT YA. This proposal directly supports the FDA’s mandate to educate the public by addressing the research priority area of Communications. While studies indicate that antitobacco communications can successfully increase public knowledge about tobacco use risks, there are limitations to the extant literature, as follows: (1) While strategies for effective risk communication are well-established, less is known about how to frame behavioral choices (e.g., total tobacco cessation vs switching to ENDS) to increase tobacco risk perceptions and intentions to quit in polytobacco users. (2) Antitobacco campaigns often leverage cultural targeting, a broadly supported but costly communications strategy, to increase at-risk population engagement. No studies have experimentally tested the effectiveness of LGBT culturally targeted vs non- targeted anti-tobacco messages. (3) Mobile multimedia messaging has been used to disseminate smoking cessation communications and may be feasible for distributing anti-polytobacco messages to LGBT YA, but this has not been investigated. Using formative and summative evaluation, the applicant will address these gaps with three Specific Aims: (1) Identify absolute and relative risk anti-polytobacco messages that effectively communicate polytobacco risks to YA; (2) Determine the effects of cultural targeting on LGBT YA polytobacco users’ attention to anti-polytobacco messages and perceived effectiveness; (3b) Assess the feasibility of delivering MMS anti-polytobacco messages developed in Aims 1 and 2 to LGBT YA via texting; and (3b) Estimate effect sizes of exposure to anti-polytobacco messages on risk perceptions and tobacco use over time. These aims support the National Cancer Institute’s (NCI) tobacco control research priority to reduce tobacco disparities by determining effective antitobacco message framing and cultural targeting to increase polytobacco risk perceptions and reduce tobacco use in an at-risk population, LGBT YA. Findings will provide public health officials, NCI, and the FDA CTP critical information about messages and digital media that may be leveraged in national health communications to reduce poly-tobacco use in at-risk populations.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract Mitral valve regurgitation (MR) is a growing public health concern, and with an aging population, its prevalence is expected to rise steeply. For MR diagnosis and severity assessment, echocardiographic techniques have long been the standard of care. Assessment based on such techniques, however, has limitations, both in terms of technical challenges and treatment recommendations. As a result, optimal management of MR, especially determining the timing of surgery, remains complex and stands to benefit from tools that provide quantitative and comprehensive characterization of MR. The overall goal of this project is to develop and validate a stress cardiovascular MRI protocol that can lead to a more definitive treatment plan for MR patients. Cardiovascular MRI (CMR) is a well-established imaging technique that provides the most comprehensive evaluation of the cardiovascular system. The reproducibility of CMR-based flow quantification has been shown to be superior to that of echocardiography. Despite these advantages, the additive clinical value of CMR for MR patients has not been established. More recently, evidence has emerged that CMR-based assessment has better predictive power for clinical outcomes for MR patients and thus could play a central role in determining management plans for such patients. Existing CMR techniques, however, have significant limitations, precluding their use in routine clinical care. For example, the flow quantification using traditional 2D phase- contrast MRI (PC-MRI) is sensitive to the placement of the imaging plane, cannot measure the transvalvular flow directly, requires breath-holding, and is susceptible to irregular cardiac rhythm. Recently, 4D flow imaging, due to its volumetric coverage and three-directional encoding, has gained significant interest, but acquisition for 4D flow imaging using existing protocols can be prohibitively long, especially for whole-heart coverage. Also, existing 4D flow imaging protocols only perform imaging under resting conditions, which cannot fully characterize functional impairment that is only unmasked under stress testing. In this work, we will develop and validate a comprehensive CMR protocol that (i) provides ferumoxytol- enhanced 4D flow imaging with whole-heart coverage, (ii) requires minimal planning from the MRI technologist, (iii) is performed in clinically feasible acquisition time, (iv) does not require breath-holds or regular cardiac rhythm, (v) does not require navigator gating, (vi) allows imaging during exercise stress, exposing functional impairment, and (vii) additionally provides cardiac function quantification to explain and interpret stress-induced functional impairment observed in MR patients. In Aims 1 and 2, we will develop and optimize the protocol. In Aims 3 and 4, we will validate the accuracy and reproducibility of the protocol in 55 healthy subjects and 55 patients diagnosed with MR. We hypothesize that the developed protocol leads to a more reliable assessment of MR than possible with TTE alone and set the stage for larger clinical studies where the power of CMR parameters to predict clinical outcomes is demonstrated.

NIH Research Projects · FY 2024 · 2021-08

Abstract: Oral health is vital for overall health and quality of life, as exemplified by the importance of teeth in mastication, speech, and esthetics, and by recent connections made between oral health and diabetes, heart disease, preterm birth, and Alzheimer's disease. Periodontal disease, the breakdown of the connective tissues around the teeth, is one of the most prevalent diseases on earth, affecting 47% of adults and 70% of adults over the age of 65. The periodontal complex is a unique joint composed of two hard tissues, cementum and alveolar bone, and an intervening and unmineralized periodontal ligament (PDL). Periodontal disease leads to destruction of periodontal tissues and tooth loss if left untreated. Therapeutic approaches to regenerate or repair periodontal tissues are unpredictable at present, in part because of gaps in knowledge regarding molecules guiding dental and periodontal development. Our goal is to more successfully promote periodontal tissue repair, regeneration, and return to function. Factors directing cementum and alveolar bone mineralization are key for proper periodontal development and function, and likely play important roles in tissue repair. Bone sialoprotein (gene: Ibsp; protein: BSP) is an extracellular matrix protein highly expressed during cementum and alveolar bone formation. BSP has several putative biological roles based on its highly conserved functional domains involved in collagen binding (hydrophobic N-terminal domain), hydroxyapatite nucleation (polyglutamic acid sequences), and RGD-integrin cell signaling (C-terminal motif). BSP was demonstrated to be important in skeletal development, as genetic ablation in Ibsp knockout (Ibsp-/-) mice resulted in a skeletal phenotype marked by mildly delayed long bone mineralization and reduced trabecular bone remodeling. However, ablation of BSP causes even more dramatic effects in dentoalveolar tissues, where Ibsp-/- mice exhibited lack of cementum, severely hypomineralized alveolar bone, disrupted dental attachment, periodontal breakdown, and tooth loss. We hypothesize that BSP directs osteoblast function and mineralization activities and plays an important role in periodontal and alveolar bone repair. We will test this hypothesis in the following three aims: Aim 1: Define the binding location of BSP on type I collagen to define spatial mechanisms by which BSP may contribute to ECM mineralization. Aim 2: Elucidate the mechanistic roles of the RGD integrin-binding domain and the collagen- binding domain using newly generated cementoblast cell lines and mutant mice with a knock-in mutation inactivating the RGD motif. Aim 3: Investigate the efficacy of BSP to enhance alveolar bone healing using exogenous native rat BSP (nBSP) to investigate its use as a therapeutic in promoting alveolar bone repair. Importantly, insights gained will aid not only in regeneration of alveolar bone surrounding teeth or necessary for dental implant placement, but will also be potentially applicable towards healing critical size bone defects and fractures, and ameliorating or reversing systemic bone disorders such as osteoporosis.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT Germline mutation in the tumor suppressor gene BAP1 is associated with the hereditary tumor predisposition syndrome, BAP1-TPDS (OMIM 614327), that we and others identified in 2011. The syndrome is associated with predisposition to mainly four cancers: uveal melanoma, mesothelioma, cutaneous melanoma, and renal cell carcinoma in addition to a preneoplastic melanocytic skin lesions (BAP1-Inactivated Melanocytic Tumors). Other cancers have been also reported in patients with germline BAP1 mutation but it is not clear whether they are part of the BAP1-TPDS. Since its characterization, more than 200 distinct families have been reported with an increasing number of pathogenic/likely pathogenic variants being deposited in ClinVar. Our our analysis of variants in the Exome Aggregation Consortium (ExAC) database suggests that BAP1-TPDS is underreported in cancer patients. BAP1 is a deubiquitinating hydrolase that has four known functions: (i) cell cycle regulation and cell growth, (ii) DNA damage repair, (iii) chromatin remodelling and regulation of gene expression, and (iv) regulation of apoptosis. Which of these complex functional roles are responsible for its tumor suppressor function is unknown, and needs to be determined to enable identification of the best experimental model system(s) to predict the clinical significance of the variants of uncertain significance. Our goal is to characterize the clinical phenotypes associated with different germline variants of BAP1 in order to dissect its complex functions. We will address the following critical barriers: 1) the limited number of reported germline variants in BAP1 with known clinical phenotype; 2) the need for experimental model system(s) to assess the clinical impact of different coding variants in BAP1; and 3) the need to assess the contribution of non-coding variants in germline inactivation of BAP1. Specific Aim1: To expand the understanding of the clinical phenotypes of BAP1-TPDS and correlate with variants in the gene. Specific Aim2: Establish experimental model systems for evaluation of BAP1 germline missense variants of uncertain significance. Specific Aim3: To assess the contribution of non-coding variants in germline inactivation of BAP1. Scientific and Translational Impact: The outcomes of these studies have the potential to provide clinicians with crucial resources needed to address a major barrier for proper counseling and management of patients and families with germline mutations in BAP1. The results will also provide basic scientists with important resources for further studies of various tumor suppressor functions of BAP1, as well as crucial resources for the NCI ClinGen and ClinVar projects.

NIH Research Projects · FY 2024 · 2021-08

Abstract During pathophysiological conditions characterized by extensive hemolysis (e.g. acquired and genetic hemolytic diseases), free heme and cell-free hemoglobin (Hb) are released into the blood stream and elicit a variety of adverse effects, namely: vasoconstriction, hypertension, and end organ damage. Thus treatment of hemolytic conditions would benefit from scavengers of free heme and cell-free Hb such as hemopexin (Hpx) and haptoglobin (Hp), respectively. A possible functional alternative to Hpx is apohemoglobin (apoHb). ApoHb is derived by removing heme from Hb, and its vacant heme-binding pockets have a high affinity for heme. Hence, apoHb could serve as a novel in vivo heme scavenger instead of Hpx. However, major potential issues with the use of apoHb as an in vivo heme scavenger are its low thermal stability at physiological temperature, and short circulatory half-life (similar to Hb, 5 min). Fortuitously, previous studies have shown that, similar to Hb, apoHb can bind to Hp forming a highly stable complex. The apoHb-Hp complex retains its ability to bind heme, and is more stable at physiological temperature compared to free apoHb. In addition to being able to bind free heme, the apoHb-Hp complex can scavenge free Hb by exchanging Hp bound apoHb αβ dimers for Hb αβ dimers. Therefore, we hypothesize that the apoHb-Hp complex will have the dual ability to bind and detoxify free heme and Hb that are produced during states of hemolysis. The resulting Hb-Hp complex is much less toxic than free heme or cell-free Hb and is readily cleared from circulation via CD163 mediated monocyte/macrophage uptake. To test this hypothesis, we propose the following specific aims. Specific Aim 1: Biophysical and biochemical characterization of the apoHb-Hp complex and its ability to bind heme and Hb in vitro. Specific Aim 2: In vivo determination of heme and Hb transfer and binding to apoHb-Hp. Specific Aim 3: Systematic pre-clinical evaluation of apoHb-Hp to prevent and/or halt the progression of intravascular hemolysis (Hb/heme)-induced pulmonary cardiovascular disease. Specific Aim 4: Microvascular evaluation of apoHb-Hp to prevent vaso-occlusion and reduce vascular inflammation.

NIH Research Projects · FY 2025 · 2021-08

SUMMARY It is our long-term goal to understand computations that underlie sensori-motor transformations in the context of thermoregulatory behaviors. Generating appropriate behaviors in response to sensory stimuli is critical for the survival of any animal. Larval zebrafish will be used for these studies as it is the only vertebrate model which allows comprehensive identification and manipulation of thermoregulatory circuits. Importantly, larval zebrafish is an ectotherm animal and therefore exclusively relies on thermal gradient navigation for thermoregulation. This means that the underlying sensori-motor transformations are robust since accurate thermoregulation is critical for survival. The accessibility of the zebrafish nervous system to optical recording of neural activity enabled us to map thermoregulatory circuits from sensory input to behavioral output for the first time in any animal. This research identified two critical classes of hindbrain neurons which encode the rate of heating and the rate of cooling in the environment. Notably, these heating and cooling responses are computed de-novo in the hindbrain from sensory trigeminal inputs. The aim of this proposal is to uncover the biophysical mechanism of these computations and their role in behavior generation to generate a multiscale model of sensori-motor transformations. The proposed experiments are guided by testable hypotheses about hindbrain computation that are based on our previous circuit modeling efforts. Specifically, the research will investigate the (1) cellular mechanisms of computing heating and cooling responses, (2) how the circuit anatomy supports this computation and (3) how the responses of Heating and Cooling neurons influence turning during thermoregulatory behavior. To this end experiments will combine (1) patch electrophysiology in functionally identified neurons, (2) single cell labeling through electroporations and (3) cell type specific ablations followed by behavioral recordings. This research will fill a critical gap in our understanding of sensori-motor transformations: How computations at different scales, from cellular properties to circuits, interact to generate adaptive behaviors in response to sensory stimuli. The understanding of conserved and divergent principles of sensori-motor transformations across animals and sensory modalities furthermore promises insight into what goes awry in neurological disease states where sensory processing goes awry.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT: Adult skeletal muscle has the ability to repair and regenerate following exercise, trauma or disease-induced damage despite being comprised of multinucleated muscle fibers whose nuclei cannot divide. This property is primarily attributable to adult myogenic precursor cells (satellite cells). When activated in response to local muscle damage, satellite cells proliferate extensively, either self-renew to reconstitute the reserve muscle progenitor pool or differentiate into new skeletal muscle fibers by fusing with each other or into the existing muscle fiber. Because satellite cells display lineage-specific differentiation (muscle cell) and self-renewal, two characteristics of stem cells, they are considered the primary resident adult stem cells of skeletal muscle. While intensive research efforts have advanced our understanding of satellite cell biology since their discovery in 1961, the regulatory mechanism(s) controlling satellite cell number remain unknown. Here we provide evidence implicating FGF6 signaling, which can be modulated by the Hippo pathway mediator TEAD1 in skeletal muscle fibers, in the regulation of adult mouse satellite cell number. We previously investigated a mouse model with transgenic TEAD1 overexpression in the muscle fiber and discovered a remarkable up to 6-fold increase in the number of satellite cells without any changes in overall muscle size. We further determined that paracrine signal(s) from the TEAD1-expressing myofiber signal for the satellite cell pool expansion in this mouse model. Applying transcriptomics to this mouse model, we have identified FGF signaling, i.e. FGF2 and FGF6, as a physiologically relevant pathway regulating satellite cell pool size. Indeed, our preliminary analysis of skeletal muscle from Fgf6 mutant mice reveals a significant reduction in the number of satellite cells. This reduction is further exacerbated in mice, in which the two FGF receptors predominantly expressed by satellite cells are inactivated specifically in the myogenic lineage. Our goal is to determine the role of FGF signaling from the myofiber to the satellite cell in achieving a particular pool size of adult muscle progenitor cells for effective repair of muscle tissue throughout life, and how myofiber-specific TEAD1 is regulating paracrine signaling from the myofiber to contribute to regulate this process. Specific Aims: 1) Determine the role of FGF6 and FGF2 in perinatal SC scaling and adult muscle regeneration, 2) Determine the role of Fgfr1 and Fgfr4 in the SC perinatally and in adulthood, 3) Determine how TEAD-mediated transcriptional regulation within the myofiber governs SC pool scaling. We expect new fundamental findings into how the size of the satellite cell population in muscle is specified during development and adaptively maintained during adult life. Insight into how the number of regenerative cells (stem cells) in muscle is controlled provides an entry into the development of new cell-based therapies against muscle wasting diseases, sport/combat injury, and age-related sarcopenia.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Arterial thrombosis resulting in acute ischemic stroke (AIS) is the leading cause of combined morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only drug approved to treat AIS. Unfortunately, only ~5-10% of patients who present with AIS actually receive rtPA. Risks of rtPA treatment include a significant increase in symptomatic intracranial hemorrhage (ICH), which occurs in up to 6.4% of patients who receive the drug. Moreover, rtPA only achieves 10% recanalization in patients who present with large vessel occlusion (LVO) stroke. These clots, which are commonly platelet-rich, are notoriously resistant to rtPA. A critical need exists to develop thrombolytic agents that: 1. target critical proteins involved in stroke clot architecture, 2. recanalize arterial occlusions, and 3. have a safety profile superior to rtPA. Von Willebrand Factor (VWF) is an optimal target for AIS treatment. VWF binds to glycoprotein Ib (GPIb) of the platelet receptor complex GPIb-IX-V as well as to GPIIb-IIIa, resulting in platelet activation and aggregation. VWF also self-associates, extending into the vessel lumen as a scaffold for platelet and red blood cell adhesion. These processes result in arterial thrombosis as seen in AIS patients. Our preliminary data of cerebral thrombi from stroke patients show that the majority of clots have a platelet shell rich with VWF encapsulating the thrombus core, providing an explanation for the poor arterial recanalization rate associated with rtPA. Aptamers are oligonucleotide-based drugs that inhibit their target proteins with high affinity and specificity. We have isolated and optimized an RNA aptamer that binds to and inhibits VWF (DTRI-031). We have also designed a second oligonucleotide (DTRI-025) that fully reverses DTRI-031 activity within minutes. Our data in small and large animal models of thrombosis demonstrates that DTRI-031 both prevents thrombus formation and lyses fully formed arterial occlusions better than rtPA. The overall goals of this proposal are to 1) correlate elevated plasma VWF to clot VWF in AIS patients and 2) demonstrate VWF inhibition by DTRI-031 can translate into an effective treatment for patients who present with AIS. We will test the hypotheses that 1) VWF is an optimal target for AIS treatment. Our preliminary data shows that LVO AIS patients have significantly elevated plasma VWF. Our preliminary data has replicated these findings in a murine model of stroke. 2) DTRI-031 effectively lyses clots in vitro in blood samples from AIS patients and in vivo in a murine model of stroke. Moreover, DTRI-025 rapidly reverses DTRI-031 activity in AIS in vitro and in vivo. 3) DTRI-031 treatment improves outcomes in a murine model of AIS by increasing recanalization, decreasing infarct volume, and improving functional recovery. Our preliminary data reveals that DTRI-031-treated mice have reduced post-stroke infarct volumes compared to control. Finally, DTRI-031 has an improved safety profile by decreasing ICH, cerebral edema formation and blood-brain-barrier breakdown after AIS compared to rtPA.

NIH Research Projects · FY 2025 · 2021-07

Spinal Muscular Atrophy is a devastating neuromuscular disease caused by insufficient amounts of SMN protein. SMA is caused by loss or mutation of the SMN1 gene and retention of the SMN2 gene. The SMN2 gene is a modifier of phenotype where milder SMA cases having more copies of SMN2. Rarely SMA patients have a missense mutation in the SMN1 gene. We can use these mutations and the protein domains they disrupt to study the function of the SMN protein. We have shown that SMA missense mutations are not functional by themselves but can function in the presence of some full-length wild-type SMN protein. Furthermore, we have shown that N and C-terminal SMN missense mutations can complement each other and rescue snRNP assembly in the complete absence of full-length wild-type SMN protein in mice. We have developed cell line that conditionally removes functional SMN to allow us test SMN missense mutations in culture. We have also used this cell line to test for suppressors of the SMNE134K mutation. We have identified a suppressor in the SmF protein that fully restores snRNP assembly lost due to the SMN E134K mutation. We now have a system to screen for suppressors of SMN missense mutations. In this proposal we will test the SmF suppressor we have found in two different SMN E134K mouse models and determine if this mutation rescues the SMA phenotype and survival of the SMA mouse. Thus, we can study the separate functions of SMN in snRNP assembly from the function of SMN in the axon. We will screen for additional suppressors using other SMN patient derived mutations to test other functional domains of SMN. We will investigate the role of SMN in the axon independent of Sm assembly by introducing HuD and truncated forms of SMN into the SMA mice via scAAV9. We have shown in cells that Smn exon2B is not required for cell survival. We will test scAAV9-Smn∆2 in SMA mice to confirm this finding and rescue the SMA phenotype. Finally we will test the role of profilin in axonal function in the SMA mouse using the SMNS230L mutation. Using genetic mutations we can dissect the functions of SMN in splicing and in the axon to resolve the underlying mechanism by which reduced SMN protein causes SMA.

NIH Research Projects · FY 2025 · 2021-07

7. Project Summary / Abstract The long term objectives of this program are to 1) increase the number of engineers who understand that they have a direct effect on the safety of workers who work with the equipment and work systems engineers design, and 2) increase the number of engineers who choose to go into applied or research positions in the area of occupational safety and health. Traineeships in Occupational Safety and Ergonomics are available in the Department of Integrated Systems Engineering (ISE) at The Ohio State University. These provide educational opportunities to engineering students at the master's level who are interested in pursuing industrial, consulting, or academic careers in occupational safety and ergonomics or related areas. Plans of study typically require 4-5 semesters to complete. Students take courses in occupational biomechanics and ergonomics, cognitive systems engineering, occupational health, occupational safety, human error and systems failure or resilience engineering, and experimental design. Students are trained in responsible conduct of research practices and have opportunities to get involved in cutting edge research that addresses a number of original NORA Priority Research Areas, including Low Back Disorders, Musculoskeletal Disorders of the Upper Extremities, Traumatic Injuries, Emerging Technologies, Organization of Work, Special Populations at Risk, Exposure Assessment Methods, and/or Intervention Effectiveness Research. Research projects, seminars, internships, a safety practicum, and other opportunities expose students to several sectors in the current NORA Sector-Based Approach such as Healthcare, Manufacturing, Public Safety, Service workers, and Warehousing. Students learn from OSU faculty, experienced OHS practitioners, and workers. While students learn about safety and ergonomics fundamentals, they also learn about emerging trends and concepts, such as resilience as an approach to safety & engineering and wellness approaches to occupational safety and health. Laboratory facilities and equipment the students work with are state-of-the-art, including OSU's Spine Research Institute, and the OSU Libraries system is second-to-none. OSU's College of Engineering and the Department of Integrated Systems Engineering continue to refine and improve strategies for recruiting and retaining top-notch graduate students from groups that are underrepresented in engineering, and our training program has participated in these strategies when recruiting participants for the program. Advisory Board members bring experience from labor, industry, research, government, and education, and provide the program with important, relevant perspectives, as well as providing direct support to students through internships, safety practicum sponsorships, guest lectures in classes, and seminars. The training program provides Ohio, a state with more than 921,000 employers and 5.2 million workers, and the nation with engineers with training to identify and address occupational safety and ergonomics hazards in a wide variety of work settings. Program graduates become valuable employees because of the breadth of their training (including in research methods, occupational safety, occupational health, and ergonomics/human factors/cognitive systems engineering), diverse sector exposure (service sector, manufacturing, warehousing, healthcare, and others), and instructor exposure (academics and experienced professionals), or students may opt to build upon that strong foundation of knowledge with further education and training at the doctoral level.

NIH Research Projects · FY 2025 · 2021-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The Cellular Molecular and Biochemical Sciences Program (CMBP) at Ohio State University (OSU) draws faculty and trainees from four related molecular life sciences graduate programs: Microbiology, Molecular Cellular and Developmental Biology, Molecular Genetics, and the Ohio State Biochemistry Program. The goal of the CMBP is to create opportunities for student training not available through other programs on campus by providing: (1) a broader range of rotation choices, (2) interdisciplinary monthly meeting seminars and symposia, (3) coursework that includes an emphasis on the responsible conduct of research, scientific writing, rigor and reproducibility, and training in quantitative skills, (4) career-advancing components including professional development workshops specifically developed for this program, internship opportunities, and Individual Development Plans, and (5) faculty mentor training to ensure a holistic and supportive advisory environment conducive to trainee growth and development. CMBP is a rigorous program designed to attract top students to OSU, and the breadth and depth of the training provided will position CMBP graduates to make significant contributions to biomedical research in academia, government, and industry. In the past few years, sustained and significant institutional support has facilitated interdisciplinary research and graduate training at OSU. The unique combination of opportunities offered through the CMBP and strong matching institutional support has already increased recruitment and retention of the very best graduate students. The resources requested in this proposal would allow us to build on initiatives developed over the last 10 years and continue to further develop a graduate training experience that spans a wide range of topics and activities in the cellular, molecular and biochemical sciences. Specifically, we are requesting funds to appoint eight students annually as CMBP fellows with funding initially for a year, renewable for a second year based upon adequate progress. Trainees will remain members of the CMBP after completion of their funding, thereby assuring their continued mentoring, training and development as they move forward to careers in the biomedical workforce.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Advanced NMR, computational, and hybrid methods for metabolomics The emergence of metabolomics promises to revolutionize the understanding of biological systems from a metabolite perspective with important implications for the diagnosis and treatment of disease. Despite recent progress in the profiling and quantitation of complex metabolomics mixtures, such as urine, serum, and cancer tissue, by NMR and mass spectrometry, important methodological challenges remain. The proposed research program aims at addressing some of these urgent challenges by developing new approaches to integrate NMR with cheminformatics and mass spectrometry for the de novo characterization of unknown metabolites and their chemical and structural motifs, by developing and maintaining advanced webservers and metabolomics databases (COLMAR) for the reliable, efficient, and user-friendly analysis of highly complex metabolomics spectroscopic data, by developing automated tools for the spectral deconvolution and quantitation of complex metabolomics mixtures with synergies for protein NMR applications, and by developing new hybrid approaches to bridge metabolomics with other Omics fields. The proposed research will promote the dissemination of metabolomics as a powerful, versatile, and manageable tool to the biomedical scientific community with synergistic benefits also for protein science. These activities are expected to lead to a better understanding of a broad range of biological questions from a metabolite perspective for the benefit of human health.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT During brain development, neurons must properly differentiate into distinct subtypes to assemble healthy circuits. Thus, disruption of this process can impact neural architecture and wiring, and contribute to disorders such as autism, schizophrenia, and epilepsy. The hippocampus is a brain structure crucial for learning and memory, and its function is compromised in these disorders. Excitatory pyramidal cells in area CA1 provide a major output of hippocampal computations to other brain regions. These cells can be parsed based on their physical position within CA1 as “deep” or “superficial.” Deep and superficial hippocampal pyramidal cells are distinct classes of neurons that exhibit differential molecular signatures, electrophysiological properties, sources of afferent input, and circuit connectivity with local inhibitory interneurons. Determining the mechanisms underlying their differentiation is crucial for understanding hippocampal development and function in both health and disease. Superficial pyramidal cells in CA1 preferentially express the transcriptional regulator Satb2, which controls gene expression by modifying chromatin structure. In humans, mutations of Satb2 cause developmental delay, intellectual disability, epilepsy, and autistic behaviors. Our preliminary data show that knocking out Satb2 during early development in mice disrupts the differentiation of superficial pyramidal cells in CA1. Furthermore, there are non-cell-autonomous changes to the migration and survival of distinct subtypes of interneurons in mutant mice relative to controls. In the present proposal, three specific aims will test the hypothesis that early expression of Satb2 is necessary for hippocampal pyramidal cell differentiation and circuit development in CA1, while later expression is necessary to promote experience- dependent synaptic plasticity. These experiments will use molecular genetic tools in mice to conditionally knock out Satb2 from pyramidal cells during both early and late developmental stages. Aim 1 will use electrophysiology and electrical stimulation to study the strength and plasticity of different sources of afferent input to deep and superficial CA1 pyramidal cells in acute slices. This aim will test the hypothesis that early Satb2 expression is necessary to establish differences in afferent input strength, while later expression is necessary for activity-driven synaptic plasticity of these inputs. Aim 2 will use paired whole-cell recordings between pyramidal cells (deep and superficial) and identified subtypes of interneurons to map circuits and study details of their synaptic physiology. This aim will test the hypothesis that early Satb2 expression is necessary to establish circuit motifs between local inhibitory interneurons and superficial pyramidal cells, while later expression is necessary to recruit new inhibitory synapses in response to environmental enrichment. Aim 3 will use single-cell RNA-seq and ATAC-seq to determine how Satb2 knockout alters gene expression and chromatin accessibility in CA1 at multiple developmental timepoints. This aim will provide molecular insight into how Satb2 controls gene expression in CA1 through development, and how its function may change over time.

NIH Research Projects · FY 2025 · 2021-07

PROJECT ABSTRACT In order to advance science and discover cures for the myriad of human diseases, a workforce comprised of diverse and academically strong physician-scientists trained in hypothesis-driven research, rigorous and data- intensive methods, and evidence-based clinical practice is crucial. This cohort of trainees must have specialized and focused training that integrates rigorous scientific education with state-of-the-art medical training. The overall goal of The Ohio State University (OSU) Medical Scientist Training Program (MSTP) is to provide MD-PhD students with the rigor, depth, and breadth of both scientific and medical education in a way that melds both sets of training to prepare them to be the future leaders of the biomedical workforce. The expectation is that graduates of our program will become faculty members at academic, public, or private research enterprises and spend most of their time conducting research relevant to the improvement of human health. Our program provides the programmatic structure, curricular content, scientific and clinical guidance, mentoring, facilities, and stimulating environment to catalyze the integration of research and clinical training essential for a student to become a successful physician-scientist. We focus on imparting an understanding of the best practices in clinical skills and research that lead to high clinical acumen, outstanding science that is both rigorous and reproducible, and the responsible and safe conduct of research and clinical practice. OSU MSTP students will receive a unique blend of competency-based training from the College of Medicine, the Graduate School, and specific graduate programs that will synergize to increase the trainees’ ability to translate between the scientific and clinical worlds. As part of our program, MSTP trainees will receive instruction in technical skills, grant writing, and written and oral communication that will position them for success in the rapidly advancing academic research field. In addition to technical training, students in our program receive extensive mentorship throughout their journey from a cohort of faculty that have received training and embrace this important role. In prior funding periods, students have earned their degrees in the Biomedical Sciences Graduate Program (BGSP), Neuroscience Graduate Studies Program (NGSP), or Biomedical Engineering (BME). We now have students receiving PhD training in Public Health (PH) and anticipate future PhDs across the University in carefully vetted affiliate programs. Because of our curricular integration and careful oversight by MSTP Leadership, the large majority of our students complete their studies in 8 years. Although our program has doubled in size in the past 10 years, continued growth of our applicant pool and our faculty makes continued expansion appropriate, and we plan to increase our class from 10 students per year to 12. In accordance with NIGMS guidelines, we also request an increase in funding to support 25% of the students in the program. Our goal for this MSTP is to continue to recruit and train the best and brightest students, developing future leaders who will enable the Ohio State University Wexner Medical Center to continue towards its stated mission: To improve people’s lives through innovation in research, education and patient care.