Pennsylvania State Univ Hershey Med Ctr

NIH Research Projects · FY 2025 · 2021-02

Project Summary Gliomas are major primary brain tumors, of which glioblastomas (GBM) are the most common and aggressive forms. The poor outcome of traditional treatment for these tumors demands targeted therapies based on identified mechanisms that drive tumor development. Molecular pathology has classified GBM into subtypes, among which the mesenchymal (MES) group is the most malignant. It is still unclear how GBM MES differentiation is achieved. Recent anatomically based transcriptome studies found that tumor cells associated with the necrotic region have higher expression of the MES signature genes, suggesting that the necrotic tumor microenvironment may contribute to MES differentiation and could be exploited as a therapeutic target. The goal of this project is to mechanistically and functionally study GBM necrosis, and identify vulnerabilities of GBM MES progression for therapeutics. We have established the follow premise for the proposed studies. First, we have developed novel pathologically relevant GBM mouse models showing MES differentiation and extensive necrosis. Second, we identified ferroptosis as a novel mechanism for GBM necrosis. Third, in both patient GBM samples and mouse models, we found that the necrotic tumor areas are infiltrated by neutrophils. Our studies suggested that these tumor-associated neutrophils (TANs) are necessary and sufficient to induce tumor cell ferroptosis. Furthermore, we found that ferroptosis and TANs are associated with the hypoxic tumor microenvironment. We hypothesize that GBM necrosis occurs through neutrophil-triggered ferroptosis, and this process is orchestrated by the hypoxic tumor microenvironment. We further hypothesize that ferroptosis could promote tumor progression and be targeted for therapeutic purposes. We propose the following three specific aims: 1) to determine the mechanism of tumor cell ferroptosis induced by TANs; 2) to determine the role of hypoxic tumor microenvironment in tumor cell ferroptosis; 3) to demonstrate the role of ferroptosis in GBM progression and evaluate therapeutic effects of ferroptosis blockade. We will employ a panel of established human GBM cell lines, newly isolated human GBM cells, and mouse GBM models. GBM necrosis is a diagnostic hallmark, predicts tumor aggressiveness, and has deleterious effects on treatments. The nature and mechanism of cell death associated with this necrosis remain obscure. In addition, whether tumor necrosis blockade could benefit therapies is still unknown. By establishing the GBM models faithfully recapitulating the extent of necrosis observed in GBM patients and identification of ferroptosis as the underlying mechanism of tumor necrosis, this proposal will reveal vulnerabilities of GBM MES progression, which could be a novel avenue for GBM therapeutics.

NIH Research Projects · FY 2025 · 2020-09

Modified Abstract Section Many Black, Hispanic, and rural patients are more likely to receive low quality end-of-life medical care than the general population– in fact, they are 3 times more likely than the general population to die after a lengthy intensive care unit stay. Advance care planning (ACP)– the process of discussing one’s wishes with loved ones and clinicians, and then documenting them in an advance directive (AD)– can help reduce costly/burdensome treatments that are unlikely to reduce suffering or improve quality of life. Though ~60% of Americans engage in ACP, <25% of Black, Hispanic and rural White populations have done so– in large part due to reluctance to discuss death and dying. This study harnesses communities’ existing social networks to deploy two community-based ACP interventions and study their mechanisms of action. By identifying which interventions increase engagement in ACP (and why), this project will help improve quality of end-of-life care, reduce unnecessary suffering, and end-of-life healthcare costs which conserves public health resources. Over the past 5 years, our team has studied an inexpensive and readily scalable serious game called Hello that prompts discussion of sensitive end-of-life issues. Across multiple studies, participants report that playing Hello is enjoyable, eye-opening, and motivating —98% of participants subsequently performed at least 1 ACP behavior. Recently, we developed a nationwide community-based delivery model for ‘Hello’ and confirmed the game’s acceptability by engaging 53 Black, Hispanic and rural White communities in ACP (n=1,165). We now propose a 3-armed, cluster, randomized controlled trial (RCT) in similar populations to compare the efficacy of ‘Hello’ (Group 1) with a nationally promoted structured workbook ‘The Conversation Project Starter Kit,’ (Group 2), and a non-ACP game called ‘Table Topics,’ (placebo/attention control; Group 3). We will randomize 75 Black, Hispanic and rural White communities across the US (20 participants/site; n=1,500). The primary outcome is completion of an AD; secondary outcomes include performance of other ACP behaviors. This study will provide key scientific advancements by: 1) providing efficacy data on two widely used and easily scalable but not yet evidence-based interventions; 2) advancing the science of interventional design by examining the interventions’ potential mechanisms of action (i.e. quality of communication, and role of social environment); and 3) assessing how and why our community-based delivery model engages communities in ACP. Should the RCT have negative findings, we still will have gained a robust understanding of the social environment’s role in population health research. If successful, this project will provide an evidence-based model for engaging Black, Hispanic and rural White communities in ACP, along with a robust understanding of how to design and deliver community-based initiatives relevant for other population health-based research.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY / ABSTRACT Type 1 diabetes mellitus (T1D) may develop after, or as a consequence of, one or more acute pancreatitis episodes. Elucidation of early biomarkers, clinical signs, biomarkers of disease progression, immune and genetic risk factors, most susceptible subgroups, precipitating mechanisms, etiology of acute pancreatitis, and associated pathophysiology of the T1D can help inform best practices in treatment for reducing incidence of T1D onset. In 2020, the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) formed the Type 1 Diabetes in Acute Pancreatitis Consortium (T1DAPC), consisting of 10 Clinical Centers (CCs), along with three satellite centers, and one Data Coordinating Center (DCC). The T1DAPC has developed an observational prospective cohort study, titled “Diabetes RElated to Acute Pancreatitis and its Mechanisms (DREAM).” The DREAM study is enrolling adults with a recent episode of acute pancreatitis (AP) to characterize and understand diabetes mellitus (DM) that develops following AP. The DREAM study will enroll 1200 individuals with the anticipation that up to 800 participants will enter longitudinal follow-up. As of November 2024, the DREAM study has enrolled 806 participants, 459 of whom have entered the longitudinal follow-up phase. The DREAM study has three specific aims: (1) Determine the cumulative incidence and clinical characteristics associated with the development of DM after one or more episodes of AP; (2) Comprehensively characterize beta cell function and endocrine alterations after AP and their relationship with the development of DM after AP; (3) Determine the immunologic mechanisms of DM after AP, including the contribution of β-cell autoimmunity. The NIDDK has issued RFA-DK-25-017 and RFA-DK-25-018 for the T1DAPC Principal Investigators to continue the DREAM study during the period 2025-2030. In response to RFA-DK-25-018, the Departments of Public Health Sciences (DPHS) and Medicine at the Penn State College of Medicine propose to continue serving as the DCC for the T1DAPC. The nine specific aims are as follows: (1) provide overall logistical support for the activities of the T1DAPC Steering Committee (SC) and its subcommittees and working groups; (2) support all the administrative, regulatory, managerial, logistic, analytic and financial functions for accrual and longitudinal observation of T1DAPC patients; (3) provide training and technical assistance to the CC in the course of their implementation of protocols; (4) collect, curate, manage and store all study data and provide it as needed to consortium members to support publications; (5) manage subawards for patient costs, biospecimen analysis and other tasks as needed; (6) develop, support and manage the T1DAPC biobank to process, safeguard and distribute the study biospecimens; (7) design and support new studies for the conduct of studies approved by the T1DAPC SC; (8) prepare reports and presentations for the T1DAPC’s DSMB and to collate replies to their questions; (9) prepare and transfer all study data and biospecimens to the NIDDK Central Repository at the close of the study.

NIH Research Projects · FY 2024 · 2020-08

ABSTRACT Despite the availability of the human papillomavirus (HPV) vaccine that can prevent over 34,800 HPV-related cancers in the US every year, only 51% of girls and boys were up-to-date by 2018. Rural populations are the most impacted by HPV-related cancers. Best practices like the Announcement Approach training and systems communication have proven effective in increasing HPV vaccination, but rural providers struggle to access and implement such best practices. These data prompt the question: “How can academic centers support HPV vaccination in rural primary care practices?” Although never tested for HPV vaccination, the ECHO (Extension for Community Healthcare Outcomes) Model is a promising implementation strategy (practice facilitation) that allows “experts” at academic centers to connect with primary care providers to discuss best practices in care and complex cases managed within local practices. The objective of this R01 is to test two ECHO-delivered HPV vaccination communication interventions in rural primary care clinics. The first will provide Announcement Approach training (HPV ECHO); the second will provide this approach plus systems strategies to communicate with parents who initially decline vaccination (HPV ECHO+). The rationale for the project is that ECHO is a robust, highly-accessible platform to deliver best practices to rural providers and address the context-specific communication needs of parents. Our long-term goal is to improve HPV vaccination rates in rural clinics and reduce the health inequity rural populations experience in cancer outcomes. Aim 1 is to evaluate the impact of HPV ECHO and HPV ECHO+ on HPV vaccination among adolescents. We will conduct a 3-arm cluster randomized trial with 36 primary care clinics in rural Pennsylvania. Clinics will be randomized to: HPV ECHO, HPV ECHO+, or control. Our primary outcome will be change in HPV vaccine initiation (≥1 doses) among adolescents, ages 11-14, at 12-month follow-up. Aim 2 is to evaluate the impact of HPV ECHO and HPV ECHO+ on implementation outcomes. Guided by implementation science frameworks, we will conduct a mixed-methods evaluation to compare interventions on acceptability, adoption, cost, penetration, and sustainability. Aim 3 is to evaluate the impact of interventions’ vaccine information on secondary acceptance of HPV vaccination at the clinic level. We will also follow a subset of 200 vaccine-declining parents for up to 12 months to assess exposure to and impact of vaccine information from study arms versus naturally-occurring sources (e.g., social media) on secondary acceptance. Our expected outcome is to demonstrate the effectiveness of a highly efficient and scalable implementation strategy, ECHO, to support HPV vaccination in rural clinics. This study is innovative in leveraging existing infrastructure at academic centers to deliver best practices for HPV vaccination where they are needed most and in developing a greater understanding of the influences on decision making among vaccine-declining parents. We expect the project to have a significant impact on HPV vaccine uptake as we address the communication needs of both rural providers and parents.

NIH Research Projects · FY 2025 · 2020-06

Abstract Cigarette smoke is a major source of reactive oxidants, including free radicals and aldehydes, which are playing critical roles in the development and progression of most tobacco-caused diseases including lung cancer. With the rapidly growing popularity of electronic cigarettes (EC), there is growing concern about potential harm associated with their use. Using state-of-the-art high resolution analytical methods to detect and measure oxidants, we have demonstrated that EC aerosols contain significant levels of highly reactive free radicals and aldehydes. While these oxidants were detected in all types of EC tested, their levels varied substantially by EC product design, flavor additives, and usage behaviors. Based on these studies and the known importance of oxidative stress/damage and inflammation in lung carcinogenesis, our proposal focuses on the potential impact of EC-derived free radicals and aldehydes in mechanisms involved in lung cancer development. Specifically, we hypothesize that exposure to oxidants from EC use will lead to oxidative stress/damage and inflammation in the lung resulting in increased susceptibility to cancer. We will utilize a translational approach to test this hypothesis by first, in the laboratory, identifying the chemical identity and potential for harm for the major free radicals produced by EC (Aim 1), secondly conducting controlled exposure studies in a relevant mouse model on the impact of EC-aerosols on specific lung cancer-related endpoints and lung tumor development (Aim 2), and thirdly, conduct secondary analyses of samples generated from an NIDA-funded EC clinical trial to examine the impact of long-term switching from conventional cigarettes to ECs on relevant oxidative stress/damage and inflammatory biomarkers (Aim 3). In Aim 1, we utilize advanced electron paramagnetic resonance (EPR) spectroscopy techniques and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodologies to identify major radical species in representative EC devices (including the NIDA developed standardized EC, SREC). Aim 2 will consist of multiple short-term exposure studies in the A/J mouse, both in naïve animals and those pre-exposed to cigarette smoke, and will also test the specific impact of flavorants, radicals and aldehydes on systemic and tissue specific biomarkers of oxidative stress/damage, inflammation and lung-cancer related pathways. Additionally, we will determine and compare the impact of EC aerosol and cigarette smoke exposure on lung tumorigenesis in an NNK-induced A/J mouse model. In Aim 3, the impact of switching from conventional cigarettes to EC on biomarkers of oxidative stress/damage and inflammation in healthy adult smokers will be determined. Our research approach is innovative based upon its novel focus on EC-derived free radicals, use of innovative methods and biomarkers, and its integrative translational design. With the completion of these studies, we hope to provide much needed information regarding the potential lung-cancer related harm associated with free radical and oxidant exposure from tobacco products which can be used for the development of regulatory policies aimed at EC products/usage.

NIH Research Projects · FY 2025 · 2020-04

The cerebellum is no longer just a motor structure. Human imaging studies have pointed to links between the cerebellum and cognition, language, and affect. Preclinical work has also indicated that the cerebellum has many nonmotor functions ranging from aggression to sleep. Expression of autism related proteins within cerebellar principle neurons (Purkinje cells) is sufficient to recapitulate many hallmarks of the condition in mice. These Purkinje cells send their projections to the deep cerebellar nuclei (DCN) and vestibular nuclei (VN), often considered the only cerebellar output nuclei. However, the known output pathways that connect the cerebellum with the forebrain seem insufficient to explain the diversity of behaviors now associated with it. We found that there is an additional, underappreciated output pathway through the parabrachial nucleus that receives direct Purkinje cell input and projects to the forebrain. Unlike the conventional cerebellar outputs, this pathway has significant projections to the amygdala, basal forebrain, prefrontal cortex and others. The aim of this project is to characterize cerebellar inputs to the parabrachial and identify these novel cerebellar output targets. We will focus on the projection to the amygdala. There is a rich behavioral literature describing the cerebellum as a core component in fear extinction, though there is no known neural substrate underlying this. In humans, trauma to the cerebellum is a strong predictor of post- traumatic stress disorder. We will test whether this unconventional cerebellar output can potentially mediate this nonmotor behavior.

NIH Research Projects · FY 2025 · 2020-01

PROJECT SUMMARY There are nearly three million mild traumatic brain injuries (mTBIs) in the U.S. each year, and most occur in patients less than 21 years of age. Clinical assessment of mTBI relies on symptom surveys that cannot accurately predict the duration of symptoms or objectively identify brain recovery. A biologic test would allow physicians to provide individualized recommendations for school and athletics participation, prescribe timely pharmacologic treatments, or initiate early psychosocial services in patients at risk for persistent post- concussion symptoms (PPCS). Non-coding ribonucleic acids (ncRNAs), such as microRNAs, are epi- transcriptional molecules that are altered in patients with mTBI. They can be measured in peripheral biofluids such as serum, or even saliva. Our previous research demonstrates that ncRNA changes in cerebrospinal fluid are reflected in saliva, and that saliva ncRNA levels can predict PPCS. Validation of these findings in a large, independent cohort could yield a biologic measure of PPCS risk (Aim 1), and guide individualized clinical management decisions (Aim 2). This scientific premise forms the basis for our proposed multi-center study. We will enroll 750 adolescents (ages 13-18 years) with mTBI, defined by the World Health Organization and Berlin Consensus Criteria. We will measure levels of saliva ncRNAs enriched in neuronal and glial exosomes at acute (<48 hours), sub-acute (7 days), and chronic (30 days) post-injury time points. PPCS will be defined by persistence of ≥ 3 symptoms on day 30 (compared with pre-injury state, determined by the Post-Concussion Symptom Inventory; PCSI). In 250 participants (training set), we will use a LASSO technique to refine a multivariate model, that employs acute and sub-acute ncRNA levels, along with clinical, social, and psychologic factors, to predict PPCS (while controlling for biologic covariates). Accuracy of the model will be externally validated in the remaining 500 participants (test set). Sensitivity and specificity will be compared to the validated “5P” clinical prediction tool. We will also examine the relationships between concussive symptom phenotypes and ncRNA levels with a factor analysis and hierarchical clustering. In Aim 2, we will use LASSO in a training set (n=250) to refine a second multivariate model, that uses acute and chronic ncRNA levels, along with clinical, social, and psychologic factors to identify concussion recovery. Recovery will be defined by self-report of “no difference from pre-injury” on the PCSI. Accuracy of the model will be externally validated in the test set, and compared to the accuracy of reaction time performance across acute and chronic time points. Our multi-disciplinary team includes experts in pediatrics, neurology, molecular biology, psychology, and emergency medicine with a published track record of collaboration and the expertise necessary for this proposal’s success. The study will yield an objective measure of PPCS risk, concussion phenotype, and clinical recovery. When paired with medical, social, and psychologic assessments, this technology will allow researchers to study mTBI therapies in biologically-defined patient subsets and personalize concussion care.

NIH Research Projects · FY 2025 · 2019-09

Project Summary. According to the Centers for Disease Control and Prevention, there were 70,237 drug overdose deaths reported in the United States in 2017, more than 130 per day, with 67.8% involving opioids [1]. While medications are available to treat the disease (e.g., methadone, suboxone, and extended release naltrexone), relapse rates remain alarmingly high [2-4]. Clearly, new approaches are needed. To this end, we recognize that addiction involves not only hijacking of the reward pathway, but also of the need pathway [5]. As such, we posited that heroin seeking and taking should be reduced by peripheral stimulation of the glucagon- like peptide-1 receptor (GLP-1R) ‘satiety’ pathway. In support, activation of the GLP-1R pathway has been shown to inhibit not only ingestion of palatable sweets, water when thirsty, and salt when sodium deprived, but also responding for alcohol, nicotine, and cocaine in rats and mice [6-12]. Here, we show for the first time that pretreatment with a GLP-1R agonist also reduces heroin taking, seeking, and drug-induced reinstatement in rats. The objective of this application is to test whether treatment with a GLP-1R agonist can reduce relapse in humans with an opioid use disorder (OUD). One advantage of using GLP-1R agonists is that various formulations already are approved for treatment of obesity and type 2 diabetes [13, 14]. UG3 Phase Aim G1 will conduct a randomized, double blind, placebo-controlled pilot study to determine whether once daily treatment with the shorter acting GLP-1R agonist, liraglutide, can safely and effectively reduce craving and brain responses to drug cues among patients in residential treatment for an OUD. UG3 Phase Aim G2 will use well established animal models to test the efficacy and safety of a more risky, longer-acting, but more efficacious, GLP-1R agonist, semaglutide, on heroin seeking and cue/drug/stress-induced reinstatement. Milestones: (1) Demonstrate safety and efficacy liraglutide at approved doses to reduce craving and brain responses to drug cues among patients in residential treatment for OUD who also are receiving counseling only (CO) or counseling+buprenorphine/naltrexone (BUP/NA); (2) Verify that semaglutide is safe and effective in reducing cue/drug/stress-induced heroin seeking in an animal model. If these milestones are met, UH3 Phase Aim H1 will conduct a two-arm, pseudo-randomized, placebo controlled multi-site clinical trial in outpatients with an OUD to test whether treatment with semaglutide vs. placebo will reduce relapse out to 180 days in patients treated with CO and counseling+BUP/NA. UH3 Phase Aim H2 will use animal models to further probe the efficacy and usefulness of semaglutide to prevent initiation of heroin self-administration, to reduce ongoing heroin self-administration, or to serve as a non-opioid “bridge to care”, for example. If our hypotheses are supported, we will show that treatment with GLP-1R agonists can safely and effectively reduce opioid craving, seeking, and relapse in rats and humans, providing a second indication for full multi-site, Phase III clinical trials, and we will lay the preclinical and clinical groundwork for approval from the FDA.

NIH Research Projects · FY 2023 · 2019-09

ABSTRACT The development and progression of scoliosis in children and adults with myelomeningocele is complex and dependent on multiple neurological and musculoskeletal factors. Root neurosurgical contributors to scoliosis in this population may include hydrocephalus/shunt malfunction, Chiari malformation and syringomyelia, and tethered cord. The PI and team propose to analyze the NSBPR to identify these neurosurgical contributions to the need for scoliosis correction in the myelomeningocele population. The investigators will study the frequency of scoliosis correction as well as the variability among clinics, controlling for age, lesion level, and ambulation status (factors that influence scoliosis frequency and/or progression); analyze correlations with other neurological, urological and orthopedic factors among those undergoing scoliosis correction; identify the frequency of scoliosis correction subsequent to TCR for those presenting with scoliosis as the sole, or as one of other clinical indications for TCR; and compare the frequency of neurological/urological deterioration following scoliosis repair with, and without prior TCR to study whether TCR is necessary prior to scoliosis correction. The Penn State Spina Bifida Clinic (PSSBC) is a multidisciplinary clinic of the Penn State Health Hershey Medical Center (PSHMC), a tertiary/quaternary care University based medical center, and the Penn State Children’s Hospital (PSCH), a free standing Children’s Hospital on the University campus that functions as a regional pediatric referral center for the children of central Pennsylvania. The PSSBC has been in continuous operation for 40 years and currently cares for 563 adult and pediatric patients. The PSSBC has been an active participant in the Spina Bifida Clinic Registry Demonstration Project since 2011, and has enrolled a total of 481 patients (including 188 children and 293 adults) representing 85% of the clinic population. The PSSBC brings a unique research perspective for the spina bifida clinic registry project because 1) the clinic is one of only a few in the United States with a large, active, and growing adult spina bifida clinic; 2) it is one of a few clinics in the United States to serve primarily a rural population and could serve as a model to study the role of a spina bifida clinic in a widespread, decentralized, and mostly rural environment; and 3) the clinic is unusual in caring for a sizable number of individuals from the Amish, Mennonite and Brethren in Christ communities.

NIH Research Projects · FY 2026 · 2019-08

Project Summary Accurate DNA replication is essential for genomic stability and cellular homeostasis, and protects against cellular transformation. Obstacles to DNA replication block the progression of DNA polymerases, arresting replication forks. Unless efficiently restarted, stalled replication forks can collapse, resulting in generation of DNA breaks, promoting genomic instability and carcinogenesis. To avoid fork breakage, cells are equipped with mechanisms to stabilize and restart the fork, thus promoting genomic stability. One important fork restart mechanism involves rapid re-initiation of DNA synthesis downstream of the lesion, through repriming catalyzed by the PRIMPOL enzyme, to restore timely DNA replication. This leaves behind a single stranded (ssDNA) gap in the nascent strand. Accumulation of ssDNA gaps has recently emerged as an important intermediate in DNA damage- induced cell death, thus potentially determining the cellular hypersensitivity to genotoxic agents (including cisplatin), particularly in DNA repair-deficient cells. While by themselves ssDNA gaps are not considered cytotoxic, their accumulation has been associated with formation of cytotoxic double strand DNA breaks (DSBs). DSBs can also cause genomic rearrangements, representing a main driver a chromosomal instability. Understanding the basic mechanisms of ssDNA gap repair is crucial for improving human health. However, how ssDNA gaps are processed into cytotoxic structures represents a major knowledge gap. We have recently uncovered three new processes involved in ssDNA gap homeostasis, which represent the focus of this application. Aim 1 will investigate the regulation of PRIMPOL-mediated fork restart by CAF1. We hypothesize that CAF1 promotes PRIMPOL recruitment to arrested forks to initiate fork repriming. We will measure ssDNA gap formation using specific cell-based assays and test the impact of CAF1 on PRIMPOL recruitment to stressed forks using biochemical and imaging approaches. Aim 2 will investigate the suppression of ssDNA gaps by PARP10. We hypothesize that PARP10, by activating PCNA ubiquitination-mediated TLS, promotes ssDNA gap filling in response to genotoxic exposures. We will test if PARP10 enhances the PCNA ubiquitination-dependent recruitment of TLS polymerases for gap filling using cell-based localization and functional assays, and investigate the functional synergy of PARP10 with the BRCA pathway in chemoresistance. Aim 3 will investigate the nucleolytic processing of ssDNA gaps into cytotoxic structures. We hypothesize that processing of ssDNA gaps into DSBs drives genomic instability and cellular toxicity in certain genetic backgrounds such BRCA deficiency. We will measure the mechanisms and regulation of nuclease engagement to nascent strand gaps in cell-based imaging assays, and how this functionally impacts genome stability and cell death by measuring chromosomal integrity and cellular viability. By investigating three novel ssDNA gap processing mechanisms, using state-of- the-art functional, imaging and genetic approaches, our proposal aims to paint a detailed picture of ssDNA gap homeostasis, with significant implications on our understanding of genome stability and DNA damage sensitivity.

NIH Research Projects · FY 2024 · 2019-01

Project Summary/Abstract Histone deacetylases (HDACs) are a group of epigenetic enzymes that are important in regulating gene transcription and other cellular processes. HDAC1 is implicated in various developmental program, including hematopoiesis. We and others have showed the GATA-1 can interact with HDAC1 containing NuRD corepressor complex in FOG-1 dependent manner for GATA-1 mediated gene activation and repression. We have also established that GATA-1 can interact with HDAC1 through a FOG-1 independent manner. This interaction is essential for GATA-1 deacetylation and erythrocyte differentiation in vitro, and erythrocyte and megakaryocyte differentiation in mice. The repression function through this interaction can be two folds, it may through constitutive acetylation of GATA-1 and it may also through loss of HDAC1 interacting proteins. We have performed proteomic analysis and found that GATA-1 interacts with BCL11A through HDAC1 dependent manner. Therefore, we hypothesize that HDAC1 regulates GATA-1 activity through deacetylation and interacting with key regulators that mediates erythroid differentiation and globin gene expression. In this proposal, we will first investigate the role of GATA-1 acetylation in GATA-1 mediated gene transcription program and global GATA-1 recruitment and chromatin accessibility. We have identified key interacting proteins with acetyl-mimicking and non-acetyl-mimicking proteins through LC-MS/MS. We will perform in-depth studies on differential recruitment of other GATA-1 interacting proteins that potentially alter GATA-1 binding landscape and target gene expression in vitro and in a mouse model. We will also study the importance of HDAC1 mediated GATA-1 and BCL11A interaction in fetal globin gene expression and erythroid differentiation. Preliminary Cut and run study showed that the BCL11A KD or mutation reduces GATA-1 recruitment at fetal globin locus but not at adult globin locus, suggesting negative role of GATA-1 in fetal globin gene expression. This claim is further supported by significantly upregulation of fetal globin gene expression in Mx1 cre GATA- 1cKO mice. We therefore will study the mechanism of how GATA-1 negatively regulates fetal globin gene expression. We will study how BCL11A regulates GATA-1 recruitment and activity in fetal globin gene expression and how GATA-1 collaborate with BCL11A for -globin gene repression. The role of GATA- 1/BCL11A interaction in globin gene promoter/enhancer interaction and global genome organization will also be study to have a comprehensive view of the role of GATA-1 in regulating erythrocyte function through interacting with BCL11A. Thus, this study will lead to novel in depth understanding of GATA-1 function in erythropoiesis and beyond.

NIH Research Projects · FY 2024 · 2018-09

PROJECT SUMMARY ABSTRACT Polycystic ovary syndrome (PCOS) is the most common endocrinopathy among women. It is a heterogeneous syndrome with reproductive dysfunction of chronic anovulation and hyperandrogenism, as well as metabolic dysfunction: insulin resistance, glucose intolerance and metabolic syndrome. One such medication that may improve both aspects of PCOS is inositol and it is available throughout the world as a dietary supplement. More specifically we will study a combination of isomers, d-chiro and myo-inositol. The combination is thought to improve insulin (and gonadotropin) signaling by restoring an imbalance in inositolglycans which are second messengers in cell surface signaling. This project is the first adequately powered and designed double-blind randomized clinical trial to test prospectively the effects of inositol supplementation in a double blind study (the INSUPP PCOS study). We are conducting a four-armed study of inositol: Inositol at 1 gm, 2 gm, 3 gm bid vs. placebo bid over a 3 month period in 108 women with PCOS. The purpose of this supplement is for us to complete the total enrollment count of 108 women. We want to successfully complete this project in order to reach our study goals and specific aims, which remain the same. Identify the effect of inositol supplementation on serum testosterone levels during the study. Hypothesis 1 (Primary Outcome): Women with PCOS who receive inositol supplementation will have a significantly greater reduction in serum total testosterone than women on placebo, as well as our secondary markers of hyperandrogenism: SHBG and the free androgen index.

NIH Research Projects · FY 2026 · 2018-08

Project Summary/Abstract This project aims to address a significant knowledge gap regarding the mechanism by which VPS37A deficiency provides a survival advantage for cancer cells, particularly in the context of accumulated intracellular Death- Inducing Signaling Complexes (iDISC) and impaired autophagic flux. The VPS37A gene, located on 8p22, is frequently lost in major solid cancers. Our recent findings have highlighted VPS37A as a crucial regulator of phagophore closure, a pivotal step in the formation of double membrane autophagosomes during autophagy. Notably, the depletion of VPS37A inhibits phagophore closure, leading to the upregulation of the NF-κB signaling pathway. This upregulation is dependent on the LC3-conjugation machinery and the autophagy adaptor p62. Moreover, the inhibition of NF-κB activation through the blockade of IKK or TAK1 induces iDISC-mediated apoptosis in 8p/VPS37A-deleted cancer cells. Additionally, our analysis of the Cancer Dependency Map portal revealed that TAK1, along with its cofactor TAB2, demonstrates a strong functional connection with VPS37A in cancer cell survival. TAK1 and TAB2 are known to localize on autophagosomal membranes. Based on these compelling observations, we propose that the TAK1/TAB2/NF-κB axis associated with the phagophore serves as a gatekeeper, suppressing iDISC activation and promoting cancer cell survival. We are in an ideal position to test this hypothesis in the following Specific Aims: 1) define the phagophore-associated regulators of the TAK1/NF-κB pathway; 2) define the regulators of iDISC activation upon phagophore closure inhibition; 3) explore the roles of phagophore-mediated TAK1/NF-kB signaling in tumor development and progression. Successful implementation of this research will provide valuable insights into the interplay between the TAK1/NF-κB signaling and iDISC/CASP8 cascade on phagophores to control cell death and survival and pave the way for future development of new strategies to treat cancers, especially those with 8p/VPS37A deletion.

NIH Research Projects · FY 2025 · 2018-07

Project Summary/Abstract The goal of this project is to address a fundamental gap in knowledge on how phagophores are closed to form double membrane autophagosomes. During macroautophagy (hereafter autophagy), crescent-shaped phagophores elongate around cytoplasmic material and seal to generate double-membrane autophagosomes that fuse with lysosomes for cargo degradation. As the phagophore rim narrows, the membranes must undergo fission to separate the inner and outer membranes in a process that bears resemblance to endosomal sorting complexes required for transport (ESCRT)-mediated membrane scission. Using our elegant HaloTag-LC3 autophagosome completion assay, we provided the first experimental evidence for the ESCRT machinery in mammalian phagophore closure and identified the ESCRT-I subunit VPS37A as critical factor for the recruitment of downstream ESCRTs to the phagophore. Notably, we found that the N-terminal putative ubiquitin E2 variant (PUEV) domain of VPS37A is uniquely required for autophagosome closure but is dispensable for other ESCRT-mediated membrane abscission processes, including endosome receptor sorting and cytokinesis. Compartment-specific targeting factors initiate the sequential recruitment of the four ESCRT complexes (ESCRT-I, -II, -III and VPS4) to the membrane scission site. While the phagophore-specific targeting factors for the VPS37A-containing ESCRT-I complex are unknown, our preliminary study has revealed that VPS37A PUEV interacts with highly curved membranes containing anionic lipids and lipid packing defects, which are all features of the phagophore rim. We hypothesize that the PUEV selectively interacts with highly curved phagophore membranes to target ESCRT-I to the phagophore. Furthermore, our preliminary work has revealed that ESCRT recruitment to phagophores requires protein ubiquitylation and the LC3/GABARAP conjugation machinery, leading us to believe that the stabilization of membrane-associated ESCRT-I requires additional interactions with phagophore-associated ubiquitylated cargo. We are in an ideal position to test our hypotheses in the following Specific Aims: (1) to determine how VPS37A targets ESCRT-I to phagophores and directs the assembly of downstream ESCRTs during autophagosome biogenesis; (2) to identify phagophore-specific targeting factors for ESCRT-I during autophagy. As autophagy is involved in numerous physiological and pathological processes, these studies will have far-reaching implications for human health and disease.

NIH Research Projects · FY 2026 · 2017-07

Although largely asymptomatic, human cytomegalovirus (HCMV) can cause severe and even fatal disease in a subset of susceptible individuals. While great progress has been made in understanding essential stages of HCMV replication, a detailed description of many of these processes is lacking. Of particular interest in this proposal is the maturation of HCMV virions, namely tegument acquisition and cytoplasmic envelopment. To provide a molecular description of these events, it is important to identify the factors involved, both viral and cellular. This proposal will focus on two viral proteins, UL88 and UL71. We have previously published a role for UL88 in packaging a subset of tegument proteins into the virion tegument layer and the absence of UL88 decreases viral fitness. Previous work has identified UL71 as an envelopment factor that potentially mediates membrane scission, as viruses lacking UL71 are trapped at various stages of budding. The experiments in this proposal will seek to elucidate the molecular details of how UL88 and UL71 drive tegument acquisition and envelopment, respectively. This includes a detailed analysis of functional regions on each protein as well as an investigation into additional factors that potentially contribute to each process. We will investigate the role of EEA1+ endosomes in tegument acquisition and for the membrane scission factor DNM1 in cytoplasmic envelopment. This proposal will utilize a novel fluorescence-based envelopment to identify additional cellular proteins that participate in envelopment. Taken together, these studies will further our understanding of the molecular events that drive the late stages of HCMV maturation and identify novel ways in which HCMV assembly can be targeted as a potential intervention.

NIH Research Projects · FY 2025 · 2016-09

The epidemic of child abuse in the U.S. (>670,000 confirmed annually) causes massive harm to children and the adults they become. Surprisingly, <1% of these substantiated cases of child abuse are identified and reported by early childhood professionals (ECPs) –despite the fact that ECPs take care of >10 million American children, and that infants and toddlers account for >75% of all deaths related to child abuse. Results from our parent randomized controlled trial show that the online iLookOut for Child Abuse (iLookOut) Core training significantly improves ECPs' knowledge and attitudes about child abuse/reporting (effect size=1.09 and 0.66, respectively). Preliminary data from this trial also suggest that (compared to standard training) iLookOut improves ECPs' actual reporting of suspected abuse: i) reports are more likely to be screened-in for investigation by child protection services (86% vs 70%, p<.001), and ii) these screened-in reports are more likely (56% vs 34%, p=.001) to be “high yield” (ie, child abuse is identified and/or social services are recommended). But we know that for gains to be sustained, they must be reinforced …and reinforced again. This is because newly acquired knowledge and habits decay over time unless they are kept in use. Built into the design of our parent study was an exploratory effort to deliver basic, ad hoc follow-up messaging to maintain awareness and promote knowledge retention after completing iLookOut's Core training. But it was beyond the scope of the parent study to fully develop this follow-up intervention, study its impact, or establish its optimal timing. Through several small grants, we built a research-quality platform to deliver brief (5-10 minute) gamified micro- learning activities that reinforce and augment what is taught in iLookOut's Core training, and provide practice opportunities to apply what has been learned. We have piloted this micro-learning in the parent study and shown that ECPs will complete these activities on their smart phones in return for 3 hours of (no cost) professional development credit. This, along with strong evidence for the efficacy of iLookOut's Core training, now positions us to systematically examine how interactive, gamified micro-learning promotes knowledge retention and fosters behavior change (regarding child abuse and its reporting), and to establish that its implementation is feasible. Just as rigorous research is needed to determine the optimal timing to give booster doses of vaccines to bolster a child's immunity, it is likewise essential to understand the optimal timing for administering iLookOut's micro- learning so as to boost ECPs' waning knowledge/preparedness to protect children from abuse. Using validated measures, self-report, and data from state child protection services, this study will 1) measure the rate of knowledge decay following iLookOut's Core training, 2) examine how well micro-learning activities (delivered at variable time points) promote knowledge retention and behavior change regarding child abuse and its reporting, and 3) establish whether iLookOut's micro-learning (as a follow-up to Core training) offers an implementation strategy that is feasible (ie, well-accepted, appropriate to the task, and sustainable).

NIH Research Projects · FY 2025 · 2016-09

Advances in the efficiency, quality and impact of clinical and translational science (CTS) will be achieved by researchers who are trained in rigorous research methodologies and equipped to navigate a pathway from basic discovery to improved health. The overall goal of the Penn State Clinical and Translational Science lnstitute's (CTSI) Institutional Career Development Program is to accelerate scholar transition to research independence. Our priorities include building the expertise of our scholars in translational science, utilizing innovative strategies to engage and guide scholars to enhance research productivity, extending engagement in rural health research, and refining our data-driven continuous programmatic improvement. We will recruit KL2 scholars with outstanding potential to become catalysts for innovation in biomedical research translation. In this application we request support for 4 KL2 scholars and have an institutional commitment for an additional 4 scholars. All grant-funded and institutionally funded scholars will be selected through a University-wide RFA, have access to the same career development opportunities, and same expectations for progress toward development of scientific independence. The selected scholars will be enrolled in an intensive 3-year program designed to prepare the next generation of leaders in CTS. Our Aims are to: 1. Recruit and train talented scholars from multidisciplinary backgrounds in collaborative, interdisciplinary CTS career development. 2. Implement an intensive mentoring/coaching strategy for current and recent scholars to "turn the curve" on scholar transition to independence. 3. Leverage the strengths of our CTSl's rural environment, partnerships and technologies to develop a CTS research practicum in rural health. 4. Refine processes for collection, analysis and utilization of evaluative data guided by the logic model to foster continuous quality improvement of the career development program. We recognize multiple factors contribute to the scholars' transition to independence, including their initiative, persistence, resilience, supportive mentorship, interactions with collaborators, protected research time, and the institutional research infrastructure and culture. Timing for milestones on the path toward independence also varies depending on each individual's prior training. Our programmatic offerings are coordinated with the Translational Endeavors Core, the Community Engaged Research Core (CERC) and the TL 1 program, and include advanced degrees, core seminars, experiential opportunities, individual courses, learning modules, and flexibility for individualized learning. Intended outcomes are competent translational researchers with skills to accelerate discovery by bridging gaps and innovation along the translational research continuum.

NIH Research Projects · FY 2025 · 2016-09

The Penn State Clinical and Translational Science Institute (CTSI) was formed in 2007 to accelerate the impact of biomedical research on health. Penn State has almost $1 billion in research expenditures, and its College of Medicine (CoM) and Penn State Health system serve about 2.0 million people in largely rural regions of central PA. Penn State is the state's land-grant university, a research institution with a strong culture of interdisciplinary team science and a health system committed to the people and communities in our area. We have developed a growing outreach network and partnerships to better understand the communities' needs, address barriers to prevention and treatment, and test novel approaches to health concerns across the lifespan and translational spectrum. As a learning organization, we practice continuous evaluation and data-driven decision-making to achieve continuous quality improvement. Our vision is to be the cornerstone of CTS throughout Penn State and a leader in advancing rural CTS. A key component of our vision is that interdisciplinary teamwork and broad engagement of stakeholders is critical to success. Our specific aims are: Aim 1. Catalyze CTS by engaging researchers, providers and community partners. We will engage researchers and establish unique partnerships across and beyond Penn State. We will capitalize on vast sources of health-relevant data to inform novel approaches to health concerns and social and environmental implications, particularly as they relate to our rural population. We will integrate rural community engagement and collaboration through community activities, consultation services, workforce training, and pilot projects. Aim 2. Promote CTS through innovation and investigator-centered research infrastructure that accelerates protocol development and study completion while ensuring scientific rigor and reproducibility. CTSI provides expansive research support and services to conduct CTS research and clinical trials that meet the needs of the communities we serve. We will pursue the establishment of an academic Clinical Informatics Research Division at the CoM and promote integration with the sophisticated informatics capabilities across Penn State. Aim 3. Effectively share resources and expertise through collaboration with other CTSA Hubs. We will increase our interactions with other Hubs, focusing on Hub collaborations germane to rural communities. Aim 4. Educate a new generation of health professionals and CTS scholars, and empower them to conduct funded, innovative and interdisciplinary research. The Institute will provide training opportunities to a talented, interdisciplinary, and expanding CTS workforce, including scholars and professionals across Penn State and the CTSA consortium.

NIH Research Projects · FY 2025 · 2016-09

Translation of scientific discoveries into effective clinical interventions and best practices to improve public health is a complex process transcending traditional disciplines. The goal of the Penn State Clinical and Translational Science Institute (CTSI) TL1 Training Core is to provide early career scientists with the interdisciplinary training needed to conduct collaborative translational research aimed at transforming knowledge to benefit human health. Our program provides opportunities for predoctoral trainees to gain a foundation in clinical and translational science (CTS) through competency-based, didactic, experiential, mentored training opportunities coupled with career development activities. Drawing students from both the College of Medicine (CoM) and University Park (UP) campuses, the TL1 program is a forum for communication, collaboration, and cross-fertilization of ideas among students from a variety of backgrounds and disciplines. In the past funding cycle (2016- 2019), we trained 27 students in our year-long program (22 PhD, 3 MD/PhD, 1 MD/MPH, 1 MD) and 40 students in our summer program (19 PhD, 21 MD). In this renewal application, we request support for 6 year-long trainees and 16 short-term trainees per year. Trainees in the 1-year program pursue studies leading to a certificate in Translational Science, a MS in Clinical Research, or a unique dual-title PhD in CTS. Training is customized via individual development plans and enhanced by externships and experiential clinical research, development of leadership, communication, and time management skills, and activities focused on team science, entrepreneurship, regulatory science, and community engagement. A short-term training is offered in summer to a mixed cohort of medical and graduate students who are introduced to the fundamental aspects of CTS in a highly interactive format while conducting mentored research projects. We now seek to enhance the TL1 program with aims to: 1) Expand and enhance Penn State's community of predoctoral trainees engaged in CTS by increasing cross-disciplinary training of students by working with the directors of existing undergraduate pipeline programs; 2) Provide customized, competency-based didactic and experiential training through externships and training in collaborative team science, coupled with career development activities, structured mentor/mentee training, and networking activities across CTSA Hubs with shared interests in rural health; and 3) Increase the quality and effectiveness of our CTS training using a data-driven decision-making approach involving assessment of the trainees, their mentors, and program outcomes.

NIH Research Projects · FY 2026 · 2016-02

Project Summary DNA double stranded breaks (DSBs) interfere with cellular viability, but also initiate chromosomal translocations resulting in genomic instability and promoting carcinogenesis. BRCA1 and BRCA2 proteins are essential for homologous recombination (HR)-mediated repair of DSBs. Understanding the mechanisms of the BRCA pathway has broad implications for human health. When replication forks encounter damaged DNA, they arrest and unless properly processed, they collapse leading to DNA breaks and genomic instability. To avoid their collapse, stalled forks can be reversed by annealing the two nascent strands to each other, in a process catalyzed by DNA translocases such as ZRANB3. The BRCA proteins load RAD51 on reversed forks to protect the DNA ends against degradation by the nuclease MRE11. The ability to protect forks against degradation corelates with DNA damage sensitivity. Thus, replication fork protection is essential for DNA repair and genomic stability. However, how protection of stalled replication forks against nucleolytic degradation is achieved represents a major knowledge gap. The PARP family at least 17 members, with various and lesser understood functions than the founding member PARP1. PARP14 has been associated with multiple cellular processes, but mechanistic details are generally sparse. We previously showed that PARP14 loss reduces HR efficiency and sensitizes cells to radiation. Recently, we have identified a novel role of PARP14 in promoting replication fork degradation, genomic instability and DNA damage sensitivity, which is the focus on this application. For this application, our goal is to understand how PARP14 promotes fork degradation, resulting in DNA damage sensitivity of BRCA-deficient cells. Our overall hypothesis is that PARP14 interferes with the RAD51-MRE11 mechanism of control of DNA resection at reversed replication forks to trigger nascent strand degradation, thus enhancing DNA damage sensitivity in BRCA-deficient cells. Aim 1 is to reveal the impact of PARP14 on RAD51-mediated protection of stalled replication forks. We hypothesize that PARP14 interferes with BRCA-independent stabilization of RAD51 on reversed forks, to enhance their degradation. Aim 2 is to uncover how PARP14 engages MRE11 for nucleolytic degradation of damaged forks. We hypothesize that PARP14 binds to stalled replication forks in BRCA-deficient cells and recruits MRE11 to initiate nucleolytic degradation of nascent DNA at these structures. Aim 3 is to elucidate the role of KU in fork protection against nucleolytic resection by EXO1 and MRE11. We hypothesize that KU binding to reversed forks protects them against EXO1-mediated degradation, but enables nascent strand resection by the MRE11-PARP14 complex. Since DNA damaging agents promote genomic instability by inducing nascent strand degradation, potentially underlying their carcinogenesis, successful accomplishment of these Specific Aims would reveal a new mechanism of genome stability and tumor suppression, centered on PARP14. It may also reveal PARP14 as a biomarker for the tumor response to radiation and genotoxic chemotherapy, in the context of the BRCA status.

NIH Research Projects · FY 2025 · 1999-02

Hepatitis B virus (HBV) is a major cause of chronic viral hepatitis that increases dramatically the risk of liver cancer and other end-stage liver diseases such as cirrhosis. The major obstacle to curing chronic HBV infection is the persistence of the viral nuclear episome, the covalently closed circular (CCC) DNA, which is derived from the relaxed circular (RC) DNA contained within the viral nucleocapsid. Moreover, the inability of mouse hepatocytes to support CCC DNA formation remains a major hurdle in the development of an immuno-competent mouse model for HBV infection. This MERIT Award Extension application builds on, and extends, our current work on the disassembly of viral nucleocapsids (uncoating), which releases the RC DNA into the host cell nucleus, a prerequisite step for nuclear CCC DNA formation but also exposing RC DNA for potential degradation or triggering of host cell DNA sensing and immune responses. The main goal of the Extension is to better understand viral and host control of HBV CCC DNA formation and infection. Four Specific Aims are proposed. Aim 1 will elucidate the deficiencies in mouse hepatocytes for HBV infection. Aim 2 will develop cell-free systems for nucleocapsid uncoating and CCC DNA formation. Aim 3 will define the late HBV entry events controlled by the viral capsid and host factors. Aim 4 will assess the role of DNA sensing in HBV infection. RELEVANCE (See instructions): The hepatitis B virus (HBV) is a global cause of chronic liver diseases, including liver cirrhosis and cancer. We propose to elucidate the mechanisms of, and viral and host factors involved in, the viral trafficking events during infection and the disassembly of HBV nucleocapsid, which are essential but poorly understood steps in viral replication and contribute to the host tropism of HBV. These studies will bring novel insights into HBV infection and nucleocapsid disassembly and facilitate ongoing efforts to develop novel antiviral agents targeted at these processes as well as mouse models of HBV infection.