Pennsylvania State Univ Hershey Med Ctr

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY Tsetse flies (Glossina spp.; Diptera: Glossinidae) are the vectors of Trypanosoma spp., parasitic protozoa and causative agents of human sleeping sickness and Animal African trypanosomiasis. Natural populations of tsetse flies can be infected with strains of the endosymbiotic bacterium Sodalis glossinidius (Sodalis). In tsetse flies, Sodalis undergoes both maternal and paternal transmission by invading tsetse cells and colonizing a wide range of tissues, including those inhabited by trypanosomes. Notably, Sodalis reaches high cell densities, replicating intra- and extracellularly in tsetse tissues, while neither triggering a tsetse immune response nor impairing the fitness of the fly. These observations indicate that Sodalis has the ability to invade, survive, replicate and egress from host cells. Whereas Sodalis invasion of insect cells depends on structural components of a protein secretion apparatus, to date, no secreted proteins have been identified. Moreover, the identity of genes and pathways required for intracellular survival, replication and host cell escape remains unknown. This poor understanding of Sodalis physiology has resulted from the lack of robust techniques to genetically modify this bacterium, hence hindering the development of Sodalis-based paratransgenesis of tsetse flies. We have recently developed two methods for the efficient delivery of exogenous DNA into Sodalis cells. Using one of these methods, we have now (1) implemented a powerful technique that combines CRISPR-Cas9 and bacteriophage recombineering to introduce precise modifications into the Sodalis genome, (2) expanded the Sodalis genetic code to enable incorporation of a noncanonical amino acid for protein tagging, and (3) introduced an efficient transposon mutagenesis procedure suitable for high-throughput insertion sequencing. Using these tools, this proposal seeks to establish foundational knowledge regarding molecular interactions between Sodalis and insect cells. Specifically, we seek to (1) define the repertoire of Sodalis proteins secreted inside insect cells, identifying which of these proteins promotes bacterium internalization, and (2) define the set of bacterial genes and pathways that are required for intracellular survival and replication, for escape from within insect cells, and for maintaining a persistent infection in the tsetse fly host.

NIH Research Projects · FY 2025 · 2023-11

Organ formation and tissue patterning during human development rely on collective and oriented cell behaviors. Planar cell polarity (PCP), a signaling pathway conserved from flies to humans, governs this coordination between neighboring cells. PCP is well represented by the ordered alignment of body hairs across the mammalian skin along the anterior-posterior body axis. Deficiencies in PCP protein function result in severe developmental defects including cardiomyopathies, ciliopathies and neural tube defects such as spina bifida. How PCP pathway disruption results in developmental disorders remains poorly understood. Importantly, PCP disruption resulting in developmental defects and embryonic lethality in mice also results in a failure to properly pattern the embryonic epidermis, thus the mouse skin is a suitable model system with hair follicle polarity being a tractable read-out of PCP function. A defining feature of PCP is the asymmetric localization of core PCP proteins Frizzled 6 (Fz6) and Van Gogh like protein 2 (Vangl2) at cell-cell borders. Intercellular interactions of atypical cadherin Celsr1 is required for the Fz6 and Vangl2 localization at cell borders. Yet, we do not understand how Celsr1 adhesion organizes asymmetric PCP junctions or how this is regulated during development. The need to understand how Celsr1 functions as a driver of Fz6-Vangl2 asymmetry at junctions is underscored by the recent identification of novel, predicted pathogenic, Celsr1 mutations in patients with neural tube and congenital heart defects. The overall goal of this proposal is to better understand how these disease-associated Celsr1 mutations, particularly those that map to the Celsr1 domains are responsible for cell adhesion, contribute to human developmental disorders. Using the murine epidermis as model system for PCP function, along with cell and molecular biology, biochemical, advanced optical imaging and in vivo genetic approaches, I will test the hypothesis that disease-associated Celsr1 mutations perturb Celsr1 cell adhesion and alter the establishment of PCP asymmetry during development. This hypothesis will be interrogated in the following specific aims: 1) Determine how disease-associated Celsr1 mutations impact Celsr1 adhesion and signaling, and 2) Determine how disease-associated Celsr1 mutations impact PCP establishment. The proposed aims will reveal pathomechanisms of developmental disorders that target Celsr1 function while also providing key insight into how Celsr1 regulates PCP establishment. Fellowship support to complete the proposed research and crafted training plan, along with guidance from my mentoring team and the environment at the Penn State College of Medicine, will be instrumental in my career development toward my goal of leading an independent and collegial research team.

NIH Research Projects · FY 2025 · 2023-09

Project Summary / Abstract: Overall The overall objective of the Penn State TCORS is to provide a science base for FDA to take regulatory actions to reduce the harm from tobacco products. Our center will accomplish this through a collaboration of experienced and new tobacco regulatory scientists focusing on state-of-the-art basic and translational research on the toxicity and addiction of diverse tobacco products. This includes developing new research methods that have the potential to be used in the tobacco regulatory field beyond the scope of the Penn State TCORS research program. While toxicity is a very broad scientific field, our focus will be on the characterization of oxidants in tobacco smoke and in aerosols, and their biological effects in persons who use tobacco products. We will examine several tobacco products including e-cigarettes (EC), little cigars and nicotine pouches. Overall, these studies are a logical extension of our previous work on oxidants produced by combustible and non-combustible tobacco products and the impact of product design characteristics on their production. In addition, they stem from our previous research on the feasibility of a reduced nicotine standard for cigarettes to minimally-addicting or nonaddicting levels. These studies indicated that some smokers will continue to use lowered nicotine products and that additional strategies are needed to reduce tobacco harm including providing safe alternative sources of nicotine to addicted smokers and determining which tobacco product characteristics are associated with greater harm. Thus, the overall integrative theme of our center is to better understand how product design characteristics of diverse and alternative tobacco products affect their toxicity and addictiveness. Our proposed Center will address several CTP scientific domains including Toxicity, Addiction and Product Composition and Design using a highly interdisciplinary and integrative approach. In Project 1, we will conduct translational studies in both laboratory animal models and EC users to address the hypothesis that inhaled EC oxidants and their potential for harm are influenced by product design features and the biological effects of these oxidants can be measured through biomarkers of both exposure and harm. In Project 2, we will investigate the potential toxicity related to oxidant production in little cigar smokers and design features that may affect human exposure using epidemiologic data and clinical and laboratory studies. In Project 3, we will focus on the nicotine pouch as an alternative tobacco product by conducting a randomized controlled clinical trial to test that hypothesis that biomarkers of harm, including those related to oxidative stress, are significantly reduced in smokers who are provided high nicotine pouches. The PSTCORS will also continue its strong support of career development in tobacco regulatory science through an integrated and multidisciplinary series of educational programs and research support through the Career Development Core. Finally, in support of all Center activities will be a mature and experienced system of cores: Administrative Core, Biostatistics and Data Management Core and Biomarker and Analytical Chemistry Core.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT One of the most common, and untreated, health problems among older adults is mobility disability, observed in nearly one in five (17.9%) of older adults. Mobility disability is typically the first disability to develop and increases future risk for additional disabilities and death. Though the most effective treatments for mobility disability are forms of exercise, especially resistance training (RT), they are rarely used, with fewer than 20% of older adults meeting RT guidelines and even fewer among those with mobility disability. The core problem of disseminating the benefits of RT to older adults is adherence. One potential opportunity to enhance adherence to RT, which has not been tested, is to make RT programs shorter. Studies show that most of the benefits of RT accrue with the first few sets per week, consistent with the law of diminishing utility. While traditional RT programs for older adults, like those offered by Silver Sneakers, are typically 45 minutes three times weekly, fewer than 5% of older adults with free access to these programs participate. Our qualitative work shows that older adults often feel that 45 minute sessions are too challenging (e.g., “I don’t think I have the strength to do it for 45 minutes”) and 75% of older adults we surveyed preferred a 5 minute RT to a 45 minute RT option, assuming they were equally effective. In 2020, we set out to design a brief, home-based RT program that would lead to both high levels of adherence and functional improvement. We called the program FAST (Functional Activity Strength Training) and, to overcome its brevity, FAST was augmented with several standard behavior change techniques (e.g., feedback, reminders, self-monitoring) and a novel form of goal- setting, rarely used in RT studies, for the number of additional repetitions participants should be able to do during the study. In FAST-1, 24 healthy older adults were prescribed 30 seconds of squats and push-ups each day and given no personal supervision. Over 6 months, they performed the exercises on 73% of days and showed large increases in squat and push-up performance (Cohen’s d > 1.0). In FAST-2, we randomly assigned 97 older adults with mobility disability, and those assigned to 30 seconds each of chair stands and steps onto a stepper each day completed exercises on 81% of days (5.7 days per week) and improved their 5 time sit-to-stand test (-2.8 seconds, Cohen’s d=0.53), 30 second chair stand test (+4.2 repetitions, d=1.1) and One Leg Stand test (+3.7 seconds, d=0.40), versus controls. In this multicenter study, we will randomly assign 360 older adults with an SPPB score < 8 to 6 months of daily FAST or to a delayed control group. We will test the impact of FAST on physical performance, walking ability, falls and functional limitations. We hypothesize that FAST will improve these measures more than controls, and that those with greater adherence and perceived effort will improve the most. By rigorously testing FAST, we hope to change the paradigm of RT prescription from “More is better” to “What will people do that works?” and, if FAST proves superior, a future study will test whether FAST leads more older adults to do RT and, thereby, improve the public’s health.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT One of the most common, and untreated, health problems among older adults is mobility disability, observed in nearly one in five (17.9%) of older adults. Mobility disability is typically the first disability to develop and increases future risk for additional disabilities and death. Though the most effective treatments for mobility disability are forms of exercise, especially resistance training (RT), they are rarely used, with fewer than 20% of older adults meeting RT guidelines and even fewer among those with mobility disability. The core problem of disseminating the benefits of RT to older adults is adherence. One potential opportunity to enhance adherence to RT, which has not been tested, is to make RT programs shorter. Studies show that most of the benefits of RT accrue with the first few sets per week, consistent with the law of diminishing utility. While traditional RT programs for older adults, like those offered by Silver Sneakers, are typically 45 minutes three times weekly, fewer than 5% of older adults with free access to these programs participate. Our qualitative work shows that older adults often feel that 45 minute sessions are too challenging (e.g., “I don’t think I have the strength to do it for 45 minutes”) and 75% of older adults we surveyed preferred a 5 minute RT to a 45 minute RT option, assuming they were equally effective. In 2020, we set out to design a brief, home-based RT program that would lead to both high levels of adherence and functional improvement. We called the program FAST (Functional Activity Strength Training) and, to overcome its brevity, FAST was augmented with several standard behavior change techniques (e.g., feedback, reminders, self-monitoring) and a novel form of goal- setting, rarely used in RT studies, for the number of additional repetitions participants should be able to do during the study. In FAST-1, 24 healthy older adults were prescribed 30 seconds of squats and push-ups each day and given no personal supervision. Over 6 months, they performed the exercises on 73% of days and showed large increases in squat and push-up performance (Cohen’s d > 1.0). In FAST-2, we randomly assigned 97 older adults with mobility disability, and those assigned to 30 seconds each of chair stands and steps onto a stepper each day completed exercises on 81% of days (5.7 days per week) and improved their 5 time sit-to-stand test (-2.8 seconds, Cohen’s d=0.53), 30 second chair stand test (+4.2 repetitions, d=1.1) and One Leg Stand test (+3.7 seconds, d=0.40), versus controls. In this multicenter study, we will randomly assign 360 older adults with an SPPB score < 8 to 6 months of daily FAST or to a delayed control group. We will test the impact of FAST on physical performance, walking ability, falls and functional limitations. We hypothesize that FAST will improve these measures more than controls, and that those with greater adherence and perceived effort will improve the most. By rigorously testing FAST, we hope to change the paradigm of RT prescription from “More is better” to “What will people do that works?” and, if FAST proves superior, a future study will test whether FAST leads more older adults to do RT and, thereby, improve the public’s health.

NIH Research Projects · FY 2025 · 2023-09

Project summary: This K08 proposal describes a 5-year research and training plan that will facilitate the transition of Chintan Gandhi, MD to an independent researcher in the field of lung innate immunity and host defense. Dr. Gandhi is establishing himself as a basic and translational researcher focusing on innate immune responses to respiratory infections with a focus on the respiratory syncytial virus (RSV). Although RSV is the leading cause of mortality due to viral respiratory illnesses in children worldwide, there are no virus-specific treatments or vaccines currently available. This is partly due to an incomplete understanding of the interaction between the virus and the immature host immune system. Age at initial infection and male sex are independent risk factors for RSV severity. Surfactant protein A (SP-A), an innate immune protein, regulates phagocytic and inflammatory functions of alveolar macrophages (AMs) through the surfactant protein-A-receptor 210 (SP- R210). SP-A genetic variants and low levels of SP-A are associated with RSV severity. Dr. Gandhi reported associations of young age and the 1A0 variant of SFTPA2 (SFTPA2-1A0) with RSV severity in children. The focus of this proposal is to investigate the underlying mechanisms of those associations using a humanized transgenic neonatal mouse model of RSV carrying the SFTPA2-1A0 variant. The central hypothesis is that SP- A genetic variants and male sex converge to dysregulate the SP-A/SP-R210 pathway in neonatal AMs leading to delayed RSV clearance and excessive inflammation. The central hypothesis will be tested via the following Specific Aims: 1) determine how SFTPA2-1A0 regulates in vivo AM differential responses to RSV in male and female pups, and 2) elucidate mechanisms of the SFTPA2-1A0 mediated dysfunction of SP-A/SP-R210 pathway in RSV clearance and define in vivo therapeutic effects of exogenous SP-A in neonatal RSV infection. These studies will yield important information about the SP-A/SP-R210 signaling as a novel pathway in RSV severity and will also determine if SP-A, its receptor, SP-R210, or the modulation of the SP-A/SP-R210 pathway may be future therapeutic targets for RSV infection. During the award period, Dr. Gandhi will continue to develop his expertise in immunology, molecular biology, and genetics. In addition, Dr. Gandhi will develop skills in flow cytometry and microscopic approaches to study viral dynamics. A multidisciplinary mentorship team has been assembled to ensure the success of this project, and includes expertise in virus biology, pathogenesis, and development of novel antiviral agents (Dr. Lukacher); lung cell purification, culture protocols, and advanced life imaging techniques (Dr. Chroneos); and mechanisms of sex differences in neonatal pulmonary diseases (Dr. Lingappan). The mentorship team will guide Dr. Gandhi in meeting his training objectives through direct research experience, formal didactics, and participation in career development opportunities. The research described in this proposal is innovative and will be a substantive addition to the knowledge gap and will help Dr. Gandhi to become an independent R01-funded investigator.

NIH Research Projects · FY 2025 · 2023-09

Abstract. Consuming a nutrient-dense diet, rich in fruits and vegetables (F&Vs) is at the cornerstone of type 2 diabetes (T2DM) treatment. Yet, among Hispanic adults, only 16% meet the recommended minimum F&V intake recommendations and they have higher rates of T2DM than non-Hispanic whites (16.6% versus 13.3%, respectively). F&V Prescription (F&V Rx) programs are embedded in clinical settings, target medically high-risk patients such as those with T2DM, and provide them with vouchers to purchase F&V at local retailers. Preliminary F&V Rx studies in patients with T2DM have found F&V intake increases of 0.2-1.6 servings per day and hemoglobin A1c (A1c) reductions from 0.35-0.71% [3.8 -7.8 mmol/mol]. However, none of these studies included diabetes self-management education and support (DSME/S) which can lead to A1c decreases from 0.24% to 1.0% [2.6-10.9 mmol/mol]. Our team conducted a pilot F&V Rx study where we incentivized DSME/S attendance by providing a F&V Rx at each session. Although DSME/S uptake is typically very low after referral (around 5-7%), our program had a 73% first session attendance rate. Further, at 7 months post- program, we found a clinically- and statistically-significant reduction in A1c of 1.3% [14.2 mmol/mol] (p=.001). Although the receipt of the F&V Rx vouchers was contingent upon DSME/S attendance in our study, other T2DM-focused F&V Rx studies did not have DSME/S attendance requirements and they also saw A1c reductions. Thus, understanding the effect of a F&V Rx on DSME/S uptake and retention with and without attendance requirements is a key design issue that must be evaluated. This proposal is in response to PAS 20- 160 for short-term, pilot randomized controlled trials (RCTs) to acquire preliminary data regarding trial design. We propose a 3-Arm, 16-week, pilot RCT (n=120) for low-income adults (aged 18+) with T2DM (A1c >7% [53 mmol/mol]). The control group (n=40) will receive usual care (UC). The two intervention groups (n=40 each) will receive UC plus a F&V Rx that is either independent of DSME/S attendance (F&V Rx alone) or dependent on DSME/S attendance (F&V Rx + DSME/S). First, we will systematically explore the impact of a F&V Rx on uptake and retention in DSME/S. Next, we will explore changes in A1c, and diet-related outcomes. Finally, with implementation in mind, we will use Proctor’s implementation taxonomy, and the cultural adaptation framework from Castro et al., to assess program appropriateness, acceptability, and sustainability with both participants and clinical care providers. Our study team and our Community Advisory Board (CAB) have the research, clinical expertise, and established retail network necessary to conduct a F&V Rx trial in a low-income, Hispanic community. The findings of this trial will inform the design of a future, fully powered RCT and address gaps in our knowledge related to how F&V Rx programs impact T2DM-related outcomes. If effective, F&V Rx programs have the potential to improve diabetes self-management and reduce the clinical burden of poor glycemic control, particularly in low-income, Hispanic communities.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT The human X chromosome has long been hypothesized to play a significant role in the etiology of sex-biased diseases and traits, particularly autoimmune diseases like lupus. Despite its importance, the X chromosome is largely understudied in genetic association and functional genomics studies. Such an omission is largely due to its unique biology. The hemizygosity of XY males necessitates XX females to achieve dosage compensation of X-linked genes by the means of X-chromosome inactivation (XCI). Thus, in females most genes are expressed only from the active X (Xa) and remain silent on the inactive X (Xi). However, up to 10% of genes consistently escape XCI in healthy females and are transcribed from both Xa and Xi. And 15-30% of genes variably escape XCI in a subset of females or tissues. We hypothesize such inter- and intra- individual heterogeneity is genetically influenced and disease relevant. However, the genetic architecture of X-linked genes and XCI escape remains poorly understood. We recently showed, for the first time, that XCI escape has significant heritability and variable XCI escape genes have a significantly increased enrichment of heritability in female-biased traits when compared to sex-balanced traits. Although promising, the annotations for XCI states were inferred only from lymphoblast cell lines, which could differ across human tissue/cell types. Recent works have shown disease heritability is enriched in regions surrounding genes specific to disease relevant tissues. Thus, matching each trait to relevant tissue/cell type specific XCI states in heritability analysis could pinpoint the disease and trait relevant tissue/cell types in which escape from XCI plays a significant role (Aim 1). To do so, we first propose an empirical bayes method to infer XCI escape states in an individual sample invariant of XCI mosaicism or presence of transcribed heterozygous SNPs in population scale bulk RNA-seq data. Unlike previous efforts, our method maximizes the samples and X-linked genes assayed to construct the most comprehensive XCI escape landscape across human tissue/cell types to date. Such a complete map will enable robust heritability estimation. Next, to understand the genetic influence on variable XCI escape, we propose a two-step method that accurately models XCI mosaicism and genetic regulation of Xa/Xi by jointly modeling male and female samples to detect associations with Xa and Xi expression levels (Xa-/Xi- QTL) (Aim 2). Our method offers substantial advancement and improves power to detect Xa-/Xi- QTLs compared to other approaches that 1) assume genetic regulation on Xa and Xi are largely similar or 2) attribute total expression of X-linked genes to expression from Xa and Xi. We will apply our approach across tissue/cell types and integrate the identified Xa-/Xi- QTL with existence genome wide association studies to identify tissue/cell type specific Xa and Xi gene and trait associations. We will apply our methods to some of the largest datasets for a variety of traits including lupus, diabetes, and addiction. Overall, our proposed methods will allow comprehensive assessment of the role the X chromosome and XCI escape plays in the etiology of diseases and traits, particularly those that are sex-biased like autoimmune diseases.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Transcription factors (TFs) compose a subset of proteins that regulate the expression of a wide range of genes in cells. Instructing many tissue- and cell-type specific gene expression programs in the body, transcriptional regulation is one of the major mechanisms of cell differentiation and polarization induced by TFs. Understanding the dynamics of transcriptional regulation is crucial since it is a critical component of cell, tissue, organ and system development and its dysregulation can lead to many complex diseases. Transcriptional regulation is best modeled via directed networks in which the edges originating from transcription factors to their downstream targets represent regulatory relationships. However, building, optimizing and analyzing transcriptional regulatory networks (TRNs) is highly challenging due to inherent complexity of such networks. Moreover, the dynamic wiring in these networks evolves over time during cell and tissue differentiation and presents a continuous trajectory, instead of discrete states. Current approaches for understanding this dynamic system are mainly based on gene expression and are underpowered to accurately model such networks because alterations in gene regulation often take place via changes in chromatin architecture. Further, existing methods either disregard or oversimplify the heterogeneous nature of network states in cell populations, thereby leading to a loss of resolution. In this proposal, we hypothesize that continuous cell differentiation trajectories are driven by evolutions in the transcriptional network wiring, which are induced by alterations in the chromatin architecture. Our overarching goal in this research program is to elucidate the continuous evolution of regulatory wirings associated with developmental stages or disease conditions using cell-specific TRNs that are constructed from single-cell epigenomic data. To reach this goal, we will build TRNs using motif analysis coupled with multiple single-cell epigenomic sequencing data, including chromatin accessibility (ATAC-Seq), DNA methylation (BS-Seq), histone modification (ChIP-Seq) and three-dimensional chromatin interaction (Hi-C) at single-cell resolution. We will use these networks to uncover the regulatory changes associated with cell differentiation and discover the key transcriptional regulators that drive the cells along developmental trajectories or across the disease states. We will also detect transcriptional regulatory modules within these networks to discover pathways associated with cell differentiation. Finally, we will apply our approach to multiple domains and test our hypothesis using biological models. Altogether these studies will establish a system of dynamic network models for unraveling epigenetic regulation at a high resolution. This integrated set of models will not only facilitate an accurate understanding of epigenetic regulation in development but will also be a powerful asset for discovering targets for therapeutic interventions for a wide range of complex diseases associated with transcriptional dysregulation.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY During human development, cells interact with one another to drive collective and oriented cell behaviors that control organ formation and tissue patterning. This coordination between neighboring cells is governed by planar cell polarity (PCP), a signaling pathway conserved from flies to humans. An excellent example and functional read-out of PCP, or collective polarization, is the ordered alignment of body hairs across the mammalian skin along the anterior-posterior body axis. Genetic disruption of PCP components leads to severe developmental disorders including cardiomyopathies, ciliopathies, and neural tube defects such as spina bifida. We lack a detailed understanding for how targeting of the PCP pathway leads to developmental disorders. Importantly, PCP disruption in mice that results in developmental defects and embryonic lethality also results in a failure to properly pattern the embryonic epidermis, thus making the moue skin a suitable model system to study the conserved biology of PCP. A hallmark feature of PCP is the asymmetric localization of core PCP proteins at cell borders within a junctional complex organized via intercellular interactions of cadherin family member Celsr1. Our long-term goal is to understand how Celsr1 adhesive interactions organize asymmetric cell junctions to coordinate tissue polarity and how this molecular assembly is perturbed in human disease. The need to understand how Celsr1 adhesion coordinates PCP asymmetry is underscored by the recent identification of novel, predicted pathogenic, Celsr1 mutations in patients with neural tube and congenital heart defects. Previously, our work revealed a role for cadherin-mediated dimerization, or lateral clustering, in the organization of asymmetric PCP complexes. We hypothesize that Celsr1 cis-dimerization regulates trafficking of PCP complexes during PCP establishment and that disease-associated Celsr1 mutations differentially impair Celsr1 adhesion and dimerization interactions to disrupt PCP during development. Using the mammalian skin as a conduit for PCP function, along with molecular biology, protein biochemistry, advanced imaging and in vivo genetic approaches, our research program will uncover the pathomechanisms of human disease-associated Celsr1 mutations and reveal how Clesr1 dimerization regulates PCP establishment and maintenance. These studies will provide novel insight into the mechanisms that regulate PCP and those that are perturbed in human developmental disorders.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract Desmosomes are adhesive intercellular junctions that play critical roles in epidermal homeostasis by mediating robust cell-cell adhesion and by modulating signaling pathways that regulate epidermal differentiation. The importance of desmosomes is highlighted by numerous autoimmune and inherited skin diseases that compromise desmosome function and cause epidermal fragility. These diseases include pemphigus vulgaris (PV), a severe autoimmune epidermal blistering disease caused by autoantibodies (IgG) directed against the desmosomal cadherin desmoglein-3 (Dsg3). The precise mechanism by which PV IgG disrupt desmosome adhesion and trigger signaling pathways associated with the loss of adhesion are not well understood. This limitation has hindered the development of new therapies that can spare or eliminate the need for immunosuppressive treatments which can pose risks for patient safety. In the current proposal, we outline a series of cutting edge imaging approaches that will resolve with previously unachievable detail precisely how PV IgG disrupt adhesion. Aim 1 studies will determine how pemphigus IgG disrupt desmosome dynamics, architecture, and function using cryo-EM, live cell imaging, and molecular dynamics simulations. In Aim 2, we focus on how keratinocytes respond to pemphigus IgG and the role of Ca2+ signaling and endoplasmic reticulum stress pathways in the endocytosis of desmosomal proteins and in the inflammatory responses associated with desmosome disruption in both cultured cells and patient tissues. The outcome of these studies will produce fundamentally new conceptual models for desmosome regulation and will form a foundation for the treatment of skin diseases associated with loss of desmosome adhesion.

NIH Research Projects · FY 2026 · 2023-08

Improving communication between healthcare professionals and family members is especially critical in high-stakes ICU environments where the need for shared decision-making demands that clinicians rapidly establish rapport and therapeutic alliance with family members of non-capacitated patients. Many well-designed trials testing ICU communication interventions have had negative or minimally impactful results, perhaps in part because we have only a rudimentary understanding of how sociodemographic factors, which are defined here as educational attainment, economic stability, and neighborhood environment (as measured by the area deprivation index) impact communication in the ICU. The goals of this R01 are to conduct a mediation analysis that will: 1) examine how sociodemographic factors impact on communication between family members and ICU clinicians; 2) identify mechanisms of action related to how communication quality affects relationships with healthcare professionals and therapeutic alliance; and 3) determine how these factors contribute to or mediate outcomes for family members of ICU patients. This longitudinal, observational study will collect data from 320 family members from 5 ICUs in 3 states. Our primary health outcome is the provision of family-centered care (as measured by the Patient Perceptions of Patient Centeredness questionnaire- Family Version). Secondary health outcomes include family members’ psychological stress (anxiety, depression, symptoms of post-traumatic stress disorder) and measures of patient ICU utilization (e.g., ICU LOS, ventilator days). Mediating factors to be examined include communication quality and therapeutic alliance. Moderating factors include family members’ intrinsic traits (e.g., personality traits and decision-making style). We hypothesize that: 1) certain sociodemographic factors predict poor outcomes and result in less attention to family-centered care and worse ICU patient utilization outcomes; and 2) higher communication quality will improve the therapeutic relationship thus resulting in in improved attention to family-centered care and improved ICU patient utilization outcomes. Using our results, we will adapt a prominent conceptual model of communication to address the high-stakes communication needs of family members of ICU patients. Completing this work will advance the field by providing data to allow new understanding of how sociodemographic and other factors (e.g., communication quality) relate to provision of patient- and family-centered care. The knowledge gained will inform new content and concrete communication strategies for future ICU interventions aiming to facilitate high-quality communication and improve therapeutic alliances in pursuit of achieving patient- and family centered ICU care.

NIH Research Projects · FY 2025 · 2023-08

Machine Learning Prediction of 1-Year Mortality and Recurrence after Ischemic Stroke Using Enriched EHR data PROJECT SUMMARY / ABSTRACT Stroke is the leading cause of death and disability worldwide. It has been estimated that the 1-year risk of death and recurrence after a stroke is around 15% and 10%, respectively. Furthermore, a recent report from the Global Burden of Diseases (GBD) has shown a substantial increase in the annual number of strokes and secondary deaths, especially in low-income groups. Recurrent strokes, with an increasing trend, have a higher rate of death and disability. Thus, it is imperative to identify at-risk patients for recurrence and death for proper and timely evaluation, resource allocation, and targeted prevention. The investigators’ recently published review indicates that ─the multiple clinical scores developed for predicting stroke recurrence have only limited clinical utility. Similarly, current stroke prognostic models vary widely in quality; prediction models of post-stroke mortality are limited by their validation cohort size, breadth of clinical variables, and overall usefulness. The investigators have recently developed machine learning-based models of post-stroke all-cause mortality and recurrence using electronic health records (EHR) data. Despite promising results, our current pilot predictive models are limited to a single health system and may have inadequate generalizability due to implicit bias. This proposal seeks to expand and improve predictive models through the creative use of vetted EHR data for ischemic stroke patients from three large and different health systems (Penn State Health, Geisinger, and Johns Hopkins), caring for more than eight million people in rural and urban areas. This project will further explore the predictive value of social determinants of health (SDoH) when added to the clinical data. The investigators propose an integrative approach to design parameter-optimized and interpretable models, leveraging enriched EHR to identify the risk of ischemic stroke recurrence and all-cause mortality. Aim 1: Standardize EHR-based data across health care centers to identify clusters of ischemic stroke patients with common traits. Aim 2: Develop optimal interpretable ensemble models to predict 1-year mortality and recurrence after an ischemic stroke. Aim 3: Validate, prospectively and externally, ensemble models for 1-year mortality and stroke recurrence. This proposal includes model development with internal, external, and temporal validation and lays the foundation for an impact study to provide evidence of clinical utility. The investigators envision that this study will lead to EHR-based screening tools that can flag high-risk stroke patients for more targeted secondary prevention.

NIH Research Projects · FY 2025 · 2023-08

Perturbation in the replication-stress response (RSR) and DNA damage response (DDR) causes genomic-instability. Replication protein A (RPA) is a single-strand DNA (ssDNA) binding protein with key roles in the RSR and DDR. Genomic-instability occurs in Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency and cancer susceptibility disorder, yet the molecular underpinnings of unstable genome in WAS cells remain uncharacterized. WASp, the protein deficient in this disorder, functions both in the cytoplasm and nucleus. In the nucleus, WASp functions to prevent the accumulation of harmful R loops (RNA-DNA hybrids + ssDNA) and in the pre-repair step of escorting broken DNA ends to the repair sites by the homology-directed repair (HDR) pathway. Accordingly, WAS patient lymphocytes are poorly-equipped to both prevent and resolve DNA damage, which proposes WAS as a “genotoxin-sensitive” immune dysregulation disorder. Our foundational studies have uncovered an essential role of WASp in the RSR by influencing RPA functions under hydroxyurea-induced replication stress, in both immune and nonimmune cells. Therefore, the overall objective of this proposal is to explicate the molecular details of how WASp safeguards normal replication and which proteins and pathways WASp associates with to enable this function during replicative stress. We will test the hypothesis that WASp is required to both prevent and manage replication stress and DNA damage. Specifically, we will define WASp role in mechanisms that process a blocked replication fork (in Aim 1) and address how WASp role specifically on modifying the actin state (G-actin vs. F-actin) influence RSR (in Aim 2). Achieving these aims will propose WASp as a novel RSR factor, and human WAS as a disease of dysfunctional RSR, which may provide new mechanisms for oncogenesis in WAS.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Gastrointestinal (GI) dysfunction after spinal cord injury (SCI) is a highly prevalent, but significantly understudied comorbidity that negatively impacts quality of life for individuals with SCI. Increasing evidence suggests impaired vagal activity is a primary cause of upper GI dysfunction after injury. Under normal conditions, gastric reflexes are coordinated by the vagus nerve. The vagus nerve contains both afferent fibers that convey sensory information to central structures and efferent fibers that carry motor output needed for gastric contractions. Previous work in our lab has demonstrated that following injury, vagal afferents are significantly less responsive to chemical stimuli, including the gut peptide ghrelin. Ghrelin is an orexigenic hormone that normally serves to decrease vagal afferent activity and increase gastric motility by binding to the growth hormone secretagogue receptor (GHSR1a), a G protein-coupled receptor, expressed along the vagal afferents. The cellular mechanisms underlying ghrelin’s ability to modulate vagal afferent activity in both healthy and disease states have yet to be fully elucidated. This proposal will utilize an animal model of SCI combined with molecular and imaging techniques, in vitro patch-clamp electrophysiology, and in vivo nerve recordings to identify mechanisms underlying the loss of vagal sensitivity post-SCI. The proposed experiments will investigate the central hypothesis that GHSR1a-mediated inhibition of calcium currents is dysregulated in gastric-projecting nodose ganglia neurons after SCI. Based upon our preliminary observations, we will test the hypothesis with two specific aims. Aim 1 will determine the precise mechanism underlying GHSR1a modulation of voltage-gated Ca2+ channels (CaV2.2 or N-type) in gastric-projecting vagal afferent neurons of naïve rats using whole-cell patch- clamp electrophysiology. Aim 2 will utilize immunohistochemistry, single-cell quantitative reverse transcription polymerase chain reaction (qRT-PCR), electrophysiological techniques, and in vivo nerve recordings to identify whether the GHSR1a-mediated effects of ghrelin on N-type Ca2+ channel currents and gastric vagal afferent excitability are dysregulated following SCI. This proposal will provide critical information regarding how changes to GPCR-mediated inhibition of CaV channels impairs gastric vagal afferent activity following injury. In addition, the proposed work will benefit future studies investigating the use of ghrelin mimetics to treat gastric dysmotility associated with a broad range of conditions including SCI, diabetes mellitus, and obesity.

NIH Research Projects · FY 2024 · 2023-08

Abstract In the past decade, opioid use disorders have significantly increased in pregnant women and women of childbearing age. In parallel, we have seen a drastic rise in neonatal opioid withdrawal syndrome (NOWS). Despite having well-defined, short-term phenotypic symptoms for NOWS, molecular effects and long-term consequences are incomprehensible. This is of particular concern given that opioid exposure alone, as well as withdrawal, can induce inflammation in the brain, and recent work has shown an increase in pro-inflammatory cytokines and chemokines, which may be promoted by reactive glia as a result of in utero opioid exposure. Moreover, cognitive functioning and memory deficits have been observed in preclinical models of in utero opioid exposure, and intellectual impairments and increased attention disorders have been observed in clinical populations previously diagnosed with NOWS. While much of the research has focused on inflammation and in utero opioid exposure in brain regions associated with reward and cognition, less is known about the brain regions associated with withdrawal symptoms in NOWS, and how this may relate to cognitive function later in life. Understanding the pro-inflammatory effects of in utero opioid exposure and withdrawal may allow for novel targets for the treatment of NOWS, and better understanding of long-term consequences. Therefore, by using an established rat model of NOWS that has previously shown an upregulation of inflammatory markers due to in utero opioid exposure, we will investigate inflammation in a withdrawal-associated brain region, the locus coeruleus, and determine effects on behavioral indices of withdrawal and subsequent cognitive function. More specifically, in a rat model of NOWS, we will utilize immunohistochemistry, qPCR, and multiplex assays to evaluate inflammatory markers including reactive glial cells, toll-like receptor 4 expression, and levels of pro- inflammatory cytokines and associated neuropeptides, and their respective associations with quantified behavioral withdrawal symptoms. In addition, we will test the specific role of reactive glia on physical withdrawal symptoms in the rat neonate by systemically administering the glia-inhibiting agent, minocycline. We will assess the resultant cognitive function at PN21 and PN60, when rats are considered preadolescent and early adulthood, respectively. We will test spatial learning and memory in heroin- or saline-exposed rat pups who experienced precipitated withdrawal at PN10. Additional experiments will also include the use of neonatal, systemic minocycline administration to mitigate cognitive deficits previously observed in preclinical and clinical populations of NOWS. Together, these experiments will allow us to better understand the effects of inflammation on physical withdrawal symptoms, as well as the relationship between physical withdrawal symptoms and subsequent cognitive function, as a result of in utero opioid exposure and precipitated withdrawal, thereby establishing novel targets for the treatment of NOWS and laying the foundation for future studies evaluating its long-term consequences.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Obesity is a global epidemic that greatly increases the risk for developing hypertension and cardiovascular disease. The molecular mechanisms connecting hypertension with obesity are poorly understood, however, and optimal treatment strategies are unclear as some antihypertensive drugs elicit adverse metabolic side effects. This illustrates the critical need to identify new therapeutic targets with a positive metabolic profile for treatment of obesity hypertension. We propose that angiotensin (Ang)-(1-7), a protective hormone of the renin- angiotensin system, provides this ideal target. Ang-(1-7) binds mas receptors (masR) to lower blood pressure and improve metabolic function in obese and hypertensive rodents; but the mechanisms involved are unknown. Our preliminary data show that Ang-(1-7) depressor effects require activation of masR within the arcuate nucleus of the hypothalamus (ARC). More specifically, we show that Ang-(1-7) masR are highly localized to proopiomelanocortin (POMC)-containing neurons that release the inhibitory neurotransmitter GABA. Activation of GABAergic POMC neurons by Ang-(1-7) could lower blood pressure by inhibiting downstream melanocortin-4 receptor (MC4R) signaling in the hypothalamic paraventricular nucleus (PVN), but this has not been explored. This proposal will test the central hypothesis that Ang-(1-7) stimulates ARC POMC neurons to enhance GABAergic neurotransmission onto PVN neurons to lower blood pressure. Aim 1 will determine if POMC masR are required for Ang-(1-7) to inhibit PVN neuronal activity and lower blood pressure. To test this, we will employ a novel transgenic mouse model to determine if deletion of masR from POMC neurons prevents the ability of Ang-(1-7) to enhance GABAergic activity in the PVN and lower blood pressure under normal conditions and in the context of high fat diet-induced obesity hypertension. Aim 2 will determine if high fat diet decreases, and Ang-(1-7) treatment restores, GABAergic transmission onto MC4R-expressing PVN neurons. To test this, we will employ MC4R-GFP mice to determine if: high fat diet decreases inhibitory neurotransmission in MC4R-expressing PVN neurons, Ang-(1-7) treatment reverses these effects, and changes in inhibitory neurotransmission in MC4R-PVN neurons correlate with blood pressure. The findings from this proposal will provide new insight into the neural mechanisms by which Ang-(1-7) lowers blood pressure as well as the potential for therapeutic targeting of Ang-(1-7) in obesity hypertension. Importantly, this proposal will logically build upon the PI’s background in molecular neuroscience and allow her to develop a new skillset in integrated molecular and whole animal physiological and pharmacological methods to assess neural mechanisms engaged by Ang-(1-7) for cardiovascular regulation. The PI will receive strong mentorship and a research framework to establish an independent and novel area of research to help meet her long-term career goal of becoming an independent investigator at an academic research institution where she can conduct collaborative and clinically relevant cardiovascular neuroscience research.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Opioid use disorder (OUD) is defined in the DSM-V as a “problematic pattern of opioid use leading to problems or distress”. More than 36,000 people overdosed on synthetic opioids including fentanyl in 2019 and, with the COVID-19 pandemic, there has been a 30% increase in overdose deaths.1,2 Current treatments for OUD include therapy and medication-assisted treatments (MATs) such as methadone, buprenorphine, and naltrexone. Naltrexone has low compliance rates and there is stigma associated with the use of methadone and buprenorphine, as they are opioids used to treat OUD. Access to these drugs, then, is limited, and relapse rates remain high.3-6,46 Relapse often is precipitated by withdrawal and withdrawal, we hypothesize, is a need state.7 Thus, as the need for fluid is sated by water, for example, the need for drug (i.e., withdrawal) is sated by drug. In accordance, we further hypothesized that glucagon-like peptide-1 (GLP-1), a satiety drug, could be utilized to reduce drug seeking and taking in rodent models of OUD. In support, GLP-1 targeted treatments are effective in reducing responding for many substances of abuse in rodent models.8,55-56,72 Additionally, we found that GLP-1 receptor agonists (GLP-1RAs) can reduce heroin self-administration, cue-induced heroin seeking, and drug-induced reinstatement of heroin seeking.9,24 GLP-1RAs also reduce oxycodone taking and seeking.15 Finally, our preliminary data suggest that a GLP-1RA can reduce cue- and drug-induced seeking of not only heroin, but fentanyl as well (Urbanik et al., in preparation). This finding is consistent with a recent report.13 With fentanyl contributing to the majority of opioid overdoses1, it is critical that we understand where in the brain fentanyl is acting and how treatment with a GLP-1RA might mitigate these effects. Here we will use rodent models, light sheet microscopy, qRT-PCR, and pharmacology to address these questions. For our rodent model, we will utilize an extended access drug self-administration paradigm.22 We predict that: (1) As with other drugs of abuse tested,16,22-23 half of the rats tested will be high drug takers and these rats will have higher cue-induced seeking and greater inhibition of the GLP-1 ‘satiety’ pathway and greater activation of reward substrates compared to low fentanyl taker/seekers. (2) Fentanyl intake, fentanyl seeking, brain activation patterns, and gene expression will be attenuated by treatment with the GLP-1RA, liraglutide. (3) The protective effects of GLP-1RA treatment on behavior and brain will be blocked by the administration of the GLP-1R antagonist, exendin-9 (Ex-9), directly into the lateral hypothalamus. These hypotheses will be tested across three specific aims. If our hypotheses are supported, we will have identified the most vulnerable of fentanyl taking and seeking rats, rescued these subjects from fentanyl seeking via treatment with a GLP-1RA, verified that fentanyl, particularly in the most vulnerable, inhibits the GLP-1 ‘satiety’ pathway and activates reward/seeking substrates, and implicated a key role for GLP-1 receptors in the lateral hypothalamus – a structure critically involved in homeostasis and motivated behavior.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract Sepsis is a global epidemic with a high patient morbidity and mortality, and it accounts for staggering healthcare costs both within the US and worldwide. Clinicians caring for septic patients are unable to distinguish between septic patients who will rapidly recover and those patients who will develop a prolonged disease course marked by immune dysfunction, infectious complications, chronic critical illness (CCI) and death. This affects their ability to weigh the clinical risks versus benefits of immune-adjuvant therapy, in cases where sepsis is characterized by marked immune paralysis. The long-term research goals are (1) to develop novel, rapid and personalized tools with which to predict the prognostic trajectory of septic patients based on underlying immune phenotype, and (2) to integrate this information into the clinical care of septic patients. The subject of this proposal encompasses the first of these two goals, with objectives aligning with the NIGMS Sepsis Research priorities (NOT-GM-19-054). Specifically, we will continue recruitment into an ongoing, prospective, observational, clinical research trial enrolling patients suffering from acute sepsis at a tertiary care, academic medical center. We will utilize a combination of immune-based assays and computational approaches to (i) Validate the prognostic value of rapid, microfluidic cytokine analysis, following ex vivo stimulation of whole blood. Hypothesis: functional immune phenotyping can identify septic patients with subclinical immune paralysis who are prone to secondary infections. Approach: Quantitative analyses comparing cytokine responses to immune adjuvants before and after ex vivo stimulation of whole blood. Microfluidic cytokine analysis provides rapid and precise measurements and that are compatible with a clinical, point of care test. (ii) Compare the CCI syndrome following medical and surgical sepsis. Hypothesis: Iatrogenic tissue damage and inflammation caused by surgery alters the immune response and pathophysiology of CCI in surgical patients, as compared with medical patients. We will use a combination of transcriptomic and metabolomic data to confirm current theories of CCI pathophysiology in surgical patients, and then explore how it differs in patients having medical sepsis. (iii) Develop high fidelity, in vitro models of human myeloid-derived suppressor cells (MDSCs) with which to perform preclinical testing of potential therapies for sepsis-induced CCI. Hypothesis: human MDSCs (immature myeloid cells that expand during chronic infection and suppress immune responses) can be generated through differentiation from peripheral blood mononuclear cells isolated from whole blood. These cells can be used as a novel preclinical model with which to test potential therapies for CCI that are extrapolated from the treatment of cancer. The PI of this grant application is a clinician- scientist and the current recipient of a K08 award from the NIGMS which studies sepsis. He is ready to progress to scientific independence via the R-35 grant mechanism for early-stage investigators.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Insomnia is a prevalent health problem associated with adverse cardiovascular, metabolic, and mental health outcomes. Previously proposed subtypes, based on traditional clinical measures, have poor reliability and validity and have not proven useful for guiding insomnia treatment decisions. Based on a large base of preliminary data from various domains and several investigative groups, we have identified a particular phenotype, insomnia with short sleep duration (ISS), that is associated with increased risk for adverse health outcomes, greater physiological hyperarousal as indicated by hypothalamic-pituitary-adrenal (HPA) axis activation, and worse response to Cognitive-Behavioral Treatment for Insomnia (CBT-I). The proposed study represents the next logical extension of our previous observations: To determine the efficacy of CBT-I in individuals with ISS vs. Insomnia with normal sleep duration (INS) among adults with elevated blood pressure (BP), and to examine the efficacy of trazodone among non-remitters to CBT-I. CBT-I is recommended as first-line treatment for insomnia, and trazodone is a widely-prescribed but grossly understudied medication for insomnia. In addition, our pilot data demonstrate differential efficacy of CBT-I and trazodone in ISS and INS: trazodone, but not CBT-I, increases objective total sleep time (TST), and lowers BP and evening cortisol in ISS. We will conduct a 4-site cohort study followed by a placebo-controlled RCT in 600 adults (≥18y) with insomnia. The cohort study will examine the efficacy of CBT-l among individuals with ISS vs. INS phenotypes (n=300 each), defined by polysomnographic (PSG) TST. The subsequent RCT will compare the efficacy of trazodone vs. placebo among CBT-I non-remitters. Investigators at the 4 study sites (Hershey, Denver, Pittsburg, and Quebec) have a long history of collaboration and successful completion of NIH-funded mechanistic and clinical trial studies. Our primary outcome will be the insomnia remission at 8 weeks, defined as Insomnia Severity Index (ISI) <8; ISI is the gold-standard self-report measure of insomnia symptoms. Secondary outcomes will include ISI (continuous), objective (i.e., PSG and actigraphy) measures of sleep efficiency (in the CBT-I cohort study) and TST, HBP, and evening cortisol (in the trazodone-placebo RCT). In exploratory analyses, we will test whether changes in evening cortisol mediate the effect of trazodone on objective TST and HBP. Outcomes will be assessed at 8 weeks and 6 months following the end of treatment to evaluate the durability of treatment effects. Demonstrating a differential efficacy of CBT-I as a function of insomnia phenotype would aid the goals of precision medicine, which directs therapy on the basis of clinical phenotypes and physiology as well as genetics. Although CBT-I is recommended as first-line treatment for all adults with insomnia, finding a worse response in the ISS phenotype will lead to reconsidering this current guideline in lieu of matching patients’ phenotype to treatment. Demonstrating the efficacy of trazodone among CBT-I non-remitters will fill an obvious and important knowledge gap in insomnia treatment l as it pertains to its current wide-spread off-label use.

NIH Research Projects · FY 2026 · 2023-07

Project Summary Opioid use disorder is a life-long burden for many individuals, imposing high personal, financial, and health costs. Even after prolonged abstinence, many individuals in recovery will go on to relapse, including those that received medication-assisted treatments. Repeated opioid exposure usurps normal reward circuit function by producing long-lasting molecular changes that alter physiology and support continued drug use. These cellular adaptations have been implicated in sustained relapse vulnerability, but we lack a clear understanding of what drives their persistence. There is a further lack of information on the precise molecular adaptations underlying altered circuit function, and in which specific circuits they act to promote relapse. Understanding this “who, what, when, and where,” will be key to identifying new therapeutic targets. Here, we will answer these questions using a multi- level approach that allows us to sequence, manipulate, and record from neurons in specific circuits in the context of opioid self-administration and relapse. Our preliminary data show that both genetically-distinct and genetically-identical neuron subtypes in the ventral tegmental area (VTA) undergo differential molecular adaptations after fentanyl self-administration, which we hypothesize arises from activity-dependent transcriptional changes in specific circuits. We further hypothesize the transcriptional changes are sustained by methylation and demethylation at the gene promoters. We will first record calcium activity in VTA neurons that project to either the nucleus accumbens (NAc) or amygdala (AMY)— projections known to be important for drug intake and relapse, respectively. Then, in the same neurons from the same animals, we will identify which gene networks are transcriptionally changed after self-administration and persist until relapse testing. Next, we will identify the DNA methylation marks driving sustained differential expression, with an emphasis on genes important for synaptic plasticity. Next, we will use CRISPR/dCas9 fusion constructs to manipulate methylation states at our identified loci in specific circuits. This proposal will allow us to define the specific VTA circuits that support opioid intake and relapse, which gene networks support activity of these circuits, and how DNA methylation cements the transcriptional landscape to alter behavior. This award will allow research into neural mechanisms of opioid use disorder with unprecedented resolution, and has the potential to transform how we approach studying the genetics of substance use disorders. Together, this critical information will help inform new treatment strategies to prevent relapse.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Penn State MSTP Training Program: Recruiting, Retaining, and Training Physician-Scientists Penn State College of Medicine Co-Program Directors: Robert Levenson, PhD and Leslie Parent, MD; Overview: This T32 proposal is a new application for MSTP funding for the Penn State MD/PhD program. Led by experienced researchers, physician-scientists, and educators, the MSTP Program will train physician- scientists to conduct innovative, rigorous biomedical research. Physician-scientists, by virtue of their combined research and clinical training, play a unique role in generating new discoveries and are essential for translating these discoveries into interventions that improve health. The number of physician-scientists entering the workforce, however, has decreased in recent years. The proposed MSTP is needed to contribute knowledgeable and skilled physician-scientists who can expand the biomedical research workforce. Measurable Objectives & Outcomes: The primary outcome will be the proportion of students who graduate to careers with a research focus. Secondary measures include students’ research output (peer-reviewed abstracts and publications; submitted and awarded grants), trainee retention, and diversity-specific metrics. Process-level measures will include repeated assessments of mentorship and training quality/impact, clinical and research skills through surveys of students and mentors, and post-graduate surveys. This MSTP provides an intellectually rich and nurturing environment with committed, diverse, experienced, collaborative faculty, leveraging the PD’s prior successful T32 leadership, educational, and research expertise. Project Accomplishment: This five-year proposal will support the training of 44 NIH-funded MSTP students with planned one year appointments, primarily during the preclinical curriculum years. The MSTP will ensure the training of diverse students for the conduct of rigorous, high-quality research through an integrated curriculum, including coursework, seminars, workshops and individually mentored research. Training Plan: The MSTP is guided by three curricular tenets: (1) basic and translational research; (2) experiential learning; and (3) active mentorship from training faculty for career development. The MSTP synergizes with institutional partners, including the Clinical and Translational Science Institute, Woodward Center for Excellence in Health Sciences Education, Institute for Personalized Medicine, Neuroscience Institute, and Cancer Institute. The MSTP also partners with robust graduate programs: Biomedical Sciences, Neuroscience; Bioinformatics and Genomics; Molecular and Cellular Integrative Biosciences; Engineering Sciences and Mechanics; and Anthropology. By integrating clinical and research training over the entire course of the program, we aim to provide trainees with the skills to transition seamlessly between medical and graduate school, preparing them to enter the biomedical workforce as rigorously trained physician-scientists.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Inflammatory immune responses dictate cardiac healing post-myocardial infarction (MI) and are temporally regulated to initiate wound-healing and scar formation. Recent studies have shown that, in addition to immediately after myocardial infarction (MI), circulating, splenic and cardiac inflammatory cells (monocytes, macrophages, and T-lymphocytes) are augmented even at 8-weeks post-MI (chronic HF), and promote pathological left ventricular (LV) remodeling. Moreover, we and others have shown that depletion of specific cell types, including monocytes/macrophages or T-cells during chronic HF blunts LV remodeling and prevents progressive decline in cardiac function, suggesting a critical role of immune cells in this disease. These findings underscore the necessity for identifying specific molecular mechanisms that can be targeted to restrain pathological immune activation for therapeutic immune-modulation. Recent studies have shown that CD4+ T-cells are obligatory for efficient healing, neovascularization, and to check excessive fibrosis during MI. However, global knockout mouse models (such as CD4-/-) fail to consider spatio-temporal alterations that we see during progression from acute-MI to chronic HF. Indeed, our previous studies showed that CD4+ T-cells undergo a phenotypic shift specifically during chronic HF, promote LV remodeling in an antigen-dependent manner, and their depletion from 4 to 8 weeks post-MI blunts progressive cardiac dysfunction. Nonetheless, the molecular signatures that mediate this transition from being protective during MI to pathological during chronic HF are not known. Using RNA sequencing on cardiac CD4+ T-cells flow-sorted from the failing hearts of male mice, we showed that, as opposed to MI, T-cells activated during HF possess upregulated estrogen receptor (ER) α signaling. Since ERα effects are opposed by ERβ activation, we identified a novel ERβ agonistic drug (OSU-ERb-012). Our preliminary data show that OSU-ERb-012 is highly efficacious at inhibiting anti-CD3/CD28 (T-cell receptor) mediated T-cell proliferation and expression of pro- inflammatory cytokines, ex-vivo and in-vivo. Moreover, at 10 mg/kg dose OSU-ERb-012 significantly blunted LV remodeling and HF progression when administered from 4 to 8 weeks post-MI suggesting its therapeutic efficacy. Thus, we hypothesize that OSU-ERb-012 selectively inhibits antigenically activated pathological CD4+ T-cells at clinically translatable doses, ameliorate LV remodeling and progressive cardiac dysfunction during chronic HF, and that ERβ agonists could be used as selective immuno-modulatory drugs. We will test this hypothesis by i) delineating dose-dependent effects of OSU-ERb-012 in ameliorating LV remodeling, ii) testing whether the protective effects of OSU-ERb-012 are mediated through T-cell specific ERβ agonism, and iii) identify effects of OSU-ERb-012 on cardiomyocyte function and cardiac physiology.

NIH Research Projects · FY 2026 · 2023-06

ABSTRACT Human complex traits are jointly influenced by genetic and environmental risk factors, whose exact contributions are often subject to extensive debate. Detailed environmental risk factors are not often available, which makes it hard to jointly assess the genetic and environmental contributions. Yet, the emergence of large- scale national biobanks as well international genetic studies offers a great opportunity to make up for this knowledge gap. In particular, as study participants come from diverse locations, geospatial information of the study participants can be used as a proxy for environmental exposure. Models that incorporate geospatial information of study participants will lead to improved power for association analysis and more accurate heritability estimates. In this application, we propose to develop a Spatial MIxed Linear Effect model (SMILE) for improved association analysis and heritability estimation and Spatial Meta-Analysis Regression Test (SMART) for more powerful meta-analyses of genetic association studies. We will apply them to UK Biobank, MarketScan insurance billing database, TOPMed sequence data, and various large consortia studies on smoking/drinking addictions, lipids levels, and diabetes. To achieve the proposed research aims, we assembled a strong research team with complementary expertise from statistical genetics, addiction genetics, lung function genetics, biomedical informatics, and environmental epidemiology. Methods and tools developed from this study will open up new avenues for analyzing national biobanks such as UK Biobank and All of Us cohorts, and global consortium studies. The results from this study will help elucidate the genetic architecture of complex traits with significant

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY Hepatitis B virus (HBV) infection is a global public health concern. Despite effective vaccines to prevent this disease, current approved treatment rarely leads to a complete cure. There is an unmet medical need for developing new therapeutics for HBV infection that can lead to a sustained response. Targeting HBV capsid assembly process has become an emerging strategy for developing new antiviral treatment for HBV. However, many efforts have been made by using different capsid protein (Cp) constructs expressed in Escherichia coli to mimic or reconstitute native-like viral particles. Yet, these structures cannot correctly represent the native HBV conformations due to the lacks of nucleic acid binding domain of the Cp, viral genome, and viral enzymes, all of which are required for viral replication. Furthermore, the lack of post translational modifications of the Cp also hampers the interpretation between observed Cp structures to the biomedical data obtained from the mammalian cell culture system or experimental animals. To date there is no available high-resolution native HBV structures, which is a major gap in knowledge of the HBV field. In this proposal, we aim to use cryo-electron microscope (cryo-EM) to directly characterize the structures of native HBV capsids and virions from human cells. In Aim 1, we will determine the high-resolution structures of purified intracellular HBV capsids with different types of viral genome. We will address the key questions concerning the structural dynamics of HBV capsids during genome maturation. We will also determine the structure of the HBV reverse transcriptase (RT) and its location during reverse transcription to help understand its mode of action (whether the RT is static or moves). In Aim 2, we will investigate the high-resolution structures of secreted HBV virions. This aim will address the questions concerning how HBV capsids interact with the viral envelop proteins. Finally, experimental findings from these two Aims will be integrated to elucidate capsid dynamics during HBV replication and illuminate the molecular determinant(s) of HBV envelopment. This proposal is expected to solve 4 types of intracellular HBV capsid structures (empty, RNA-filled, single- stranded DNA-filled, and mature partially double-stranded DNA-filled capsids) and 3 secreted enveloped HBV virion structures (empty, mature, and prematurely secreted virions) using cryo-EM to define the conformational changes of the capsid during viral replication, particularly in the context of different viral genome forms and interactions between the capsid and surface proteins. The methodology exploits appropriate mutations of Cp and RT to ensure obtaining homogenous particles of the various types as described above, which can be further computationally classified to minimize cross-contamination. Understanding the native HBV structures will provide valuable new information for HBV biology and guide the design of novel antiviral drugs in the future. The project is anticipated to impact fields ranging from HBV, molecular virology, antiviral drug development, and macromolecular structure and function.