Pennsylvania State Univ Hershey Med Ctr

NIH Research Projects · FY 2025 · 2022-07

Abstract: Self-renewal is an indispensable property that allows stem cells to regenerate and maintain the homeostasis of functionally diverse cell populations. Dergulation of self-renewal in hematopoietic stem cell (HSC) directly links to a number of hematopoietic degenerative disorders including bone marrow failure and various blood diseases, whereas its aberrant activation is a defining and indispensable feature of leukemia stem cells (LSCs) that sustain the malignant phenotypes. We and others have shown that self-renewal of acute myeloid leukemia (AML) stem cells heavily rely on the key component of canonical Wnt signaling pathway, β-catenin and the homeobox protein, HOXA9 that are largely dispensable for adult HSCs. Although these findings reveal a contrasting functional requirement and a potential therapeutic opportunity for AML, development of small molecule inhibitors against oncogenic transcription factors has so far met with very little success largely due to our lack of understanding about the molecular regulations of these proteins in mediating hematopoietic self-renewal, which has significantly hindered progress in developing effective therapeutic strategies to treat the resultant diseases. Intriguingly, we have recently revealed coregulation of both canonical Wnt pathway and posterior HOXA loci including HOXA9 by the long non-coding RNA (lncRNA), HOTTIP. Strikingly, we also report a novel crosstalk between β-catenin and HOXA9 that can override their individual requirement in both mouse and human AML of HSC origins. In search for crucial mediators for β-catenin and Hoxa9 functions, we further identified that protein arginine methyltransferase 1 (PRMT1), which also implicates in DNA damage and repair (DDR), can functionally replace HOXA9 or β-catenin in AML stem cell originated from HSCs. PRMT1 together with DDR complex are unbiasedly isolated along with CTCF/cohesin complex, hematopoietic transcription factors (TFs) and nucleosome remodeling factors as HOTTIP interacting partners by ChIRP-MS in AML cells. These findings not only discover a novel HOTTIP/β-catenin-HOXA9/PRMT1 axis critical for mediating hematopoietic self-renewal, but also lead to central hypothesis that HOTTIP/β-catenin-HOXA9/PRMT1 axis coordinates hematopoietic self-renewal and characterization of the functions of individual components and their crosstalk along the axis regulates hematopoietic specific transcription networks and DDR pathways to modulate hematopoietic self-renewal in the disease setting. In this proposal, we will 1 decipher cooperative action of HOTTIP and hematopoietic TFs in regulating HOXA9 and β-catenin axis; 2) dissect the molecular functions and regulation of β-catenin- HOXA9/Prmt1 axis during normal and malignant hematopoiesis. Success completion of proposed studies not only will establish the molecular principles, but also facilitate the design of specific therapeutics in modulating self-renewal activities in normal and malignant stem cells, which can be potentially translated into patient benefits.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY/ABSTRACT Our goal is to identify the mechanisms responsible for cardiovascular disability in peripheral artery disease (PAD) and to then examine therapies that will reduce the impact of these pathophysiologic processes. We have demonstrated that the exercise pressor reflex (EPR) during leg exercise is exaggerated in PAD patients. The mechanisms for the exaggerated EPR in PAD patients have not been examined thoroughly. Utilizing both human and animal studies, we propose to examine the roles of blood flow restriction (BFR) and ischemia-reperfusion (IR) stress in inducing the exaggerated EPR. We anticipate that a blockade of acid sensing ion channels (ASICs) with amiloride will reduce the exaggerated EPR and enhance the walking tolerance in PAD patients. Aim 1: Determine the role of BFR in inducing the exaggerated EPR in PAD. We hypothesize that BFR leads to a greater H+/lower pH in the interstitium of exercising muscles and thereby accentuates the EPR via stimulating ASICs. We propose to employ BFR in healthy subjects to simulate the BFR in PAD. We speculate that BFR will augment the EPR in the placebo trial and amiloride will reduce the EPR and increase exercise time/load under BFR condition. We also speculate that amiloride will play the same beneficial role in PAD patients. In animal studies, we speculate that BFR by femoral artery occlusion will increase interstitial H+/decrease pH thereby exaggerating the EPR via ASIC subtype 3 (ASIC3) and prolonged occlusion will upregulate ASIC3 expression in muscle afferent nerves of PAD. Aim 2: Determine the role of IR in inducing the exaggerated EPR in PAD. We hypothesize that IR contributes to the exaggerated EPR in PAD and amiloride reduces the exaggerated EPR induced by IR stress via blocking ASICs. Healthy subjects will perform plantar flexion exercise under free flow conditions and after 20 min ischemia followed by 20 min reperfusion. We speculate that IR stress will accentuate the EPR. PAD patients before and after leg revascularization will also perform plantar flexion exercise. We speculate that amiloride will improve the EPR and increase exercise time/load in subjects after IR stress and in PAD patients with revascularization. In animal studies, we will examine the EPR in IR rats at different time courses and speculate that in IR rats satisfied reperfusion will alleviate the EPR and the pressor response induced by activation of afferent nerves’ ASIC3. Aim 3: Determine the effects of ASIC on exercise ability in PAD and fundamental mechanisms. We speculate that amiloride will decrease the pressor response to walking and increase the claudication onset time and walking distance/time in PAD patients. In animal studies, we speculate that exaggerated EPR induced by the IR will be attenuated in ASIC3 knockout rats. We will compare the protein levels of ASIC3 and its current response in muscle afferent neurons between IR rats at different time courses and their counterparts serving as controls. We speculate that ASIC3 expression and its current response in muscle afferent neurons will be amplified during the initiating IR stage and the effects of IR will be reduced by ASIC3 knockout or with sufficient time of reperfusion.

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY/ABSTRACT As part of NHLBI’s ENRICH program, this research will test an implementation-ready intervention designed to improve cardiovascular health (CVH) among high risk mothers and young children during pregnancy and the first two years after delivery. Home visitation (HV) models meeting federal criteria of evidence of effectiveness will be leveraged to compare existing HV programs against those with an additional CVH intervention in a cluster-randomized, multicenter trial. Our multidisciplinary team has pioneered behavioral and lifestyle interventions for women during preconception and pregnancy and for infants during the first years after birth including those using HV for the primary prevention of obesity. We also have extensive research experience linking and integrating community- based healthcare programs with health system electronic medical records (EMRs). For this proposal, we will collaborate with two Nurse-Family Partnership agencies in Northern Appalachia, a medically underserved region with high rates of poverty and poor CVH, including high rates of obesity, heart disease, and tobacco use. We will enroll ≥500 of the 3000 pregnant women required for the UH3 phase multicenter trial. During the UG3 phase, we will pilot our proposed intervention with our community partners and work collaboratively with other ENRICH sites and NHLBI to develop the UH3 phase common protocol. Our proposed intervention incorporates tenets of Social Cognitive Theory and the Framework for Understanding Poverty using a skill-building curriculum that includes role modeling, goal-setting, and skill practice opportunities in the home. Five intervention pillars (Lifestyle Behaviors, Self-Regulation, Responsive Parenting, Home Environment, EMR Integration) are designed to increase maternal knowledge and self-efficacy in their ability to establish healthy lifestyle habits and maternal parenting practices to improve CVH. Consistent with a Hybrid Trial Type 1, the study design aims to understand the context for implementing the intervention. The composite primary outcome for mothers will be a modified version of the American Heart Association’s Life’s Simple 7 one year after childbirth; outcomes will also be assessed 2 years postpartum. The childhood primary outcome will be a composite of seven early life risk factors for poor CVH across with the final assessment at age 2 years. For these outcomes, the moderating effects of social determinants of health and psychosocial indices on outcomes will be explored. In addition, the context for implementation will be evaluated and factors influencing intervention efficacy will be identified to determine in HV, what works best, for whom, in which contexts, why and how. Lastly, we aim to lead network efforts to link and integrate HV summaries into EMRs to allow for data sharing with healthcare providers to enhance intervention effectiveness so that the UH3 protocol can evaluate whether EMR integration with HV programs improves CVH composite indicators as well as health equity.

NIH Research Projects · FY 2026 · 2022-05

Project Summary/Abstract The objectives of this project are to more thoroughly understand the relationship between neonatal abstinence syndrome (NAS) and longitudinal academic performance. NAS is a withdrawal condition due to in utero drug exposure, most commonly opioids. The syndrome affects more than 32,000 newborns annually in the U.S., and its incidence continues to rise. Researchers report poorer development in toddler and preschool years, higher rates of inattention and behavioral problems, and worse and deteriorating school performance in children with a history of NAS, indicating the effects of NAS may last well beyond the newborn period. However, these existing studies do not sufficiently account for the complex interaction of biologic, health, and socioenvironmental influences on childhood development. Thus, the relationship between NAS and long-term neurodevelopment and academic achievement remains largely unknown. As school achievement is directly associated with adult productivity and negatively correlated with participation in crime, a better understanding of NAS and academic performance is urgently needed to optimize outcomes across a lifetime. This proposal aims to assess the independent relationship between NAS, NAS severity, NAS treatment, in utero drug exposure and longitudinal academic performance after controlling for relevant biologic, health, and socioenvironmental variables; to explore the moderator effects of early community and school resource support on NAS; and to learn how families’ school experiences may explain childhood academic performance. The former aims will be accomplished using an inclusive, uniquely-integrated South Carolinian data warehouse. With this data system, a child with a diagnosis of NAS can be linked with his/her mother, and the dyad can be followed so that a broad range of childhood outcomes can be examined in the context of relevant, influential factors. In addition, through qualitative interviews with parents and guardians of children with a history of NAS followed at a Pennsylvania academic medical center, the project will 1) explore if and how caregivers’ experiences regarding early community and school resource support are related to childhood academic performance and 2) analyze themes that emerge around barriers and facilitators of academic achievement in children with NAS. Under this career development award, the applicant receives training in methodology of data-driven, public health studies and patient-oriented, clinical research design; epidemiology, mixed methodologies, community health, and applying public health principles to clinical practice; and moving research to policy. Successful completion of the project will not only provide data to serve as the foundation for future studies evaluating NAS outcomes and treatment, but, in addition to her training and mentorship plan, will prepare the applicant to become an independent physician-scientist able to conduct clinical trials aimed at improving outcomes of children affected by NAS.

NIH Research Projects · FY 2026 · 2022-05

ABSTRACT JC polyomavirus (JCPyV), a ubiquitous human pathogen, causes several devastating brain diseases in immune compromised individuals. The most notable of these JCPyV-associated CNS diseases is the frequently fatal demyelinating brain disease progressive multifocal leukoencephalopathy (PML). PML, an AIDS-defining lesion in the pre-cART epoch, has emerged as a life-threatening complication in patients receiving immunomodulatory agents for autoimmune and inflammatory disorders and treatment for certain hematological malignancies. Among the rapidly expanding list of PML-associated biologics, natalizumab (Tysabri®) has the highest incidence and is an ominous sequela for multiple sclerosis (MS) patients who otherwise benefit from dramatic reductions in relapses using this immunomodulatory agent. Drug withdrawal, the only therapeutic option for PML, is often complicated by a high-mortality cerebral inflammatory reaction. No anti-JCPyV agents are available. Polyomaviruses are species-specific. Lack of a tractable animal model of polyomavirus-induced CNS disease is an acknowledged bottleneck to elucidating PML pathogenesis, the immunological mechanisms that control JCPyV, in vivo evaluation of agents that inhibit polyomavirus replication in tissue culture, and uncovering early events that presage irreversible JCPyV-associated neuropathology. Using mouse polyomavirus (MuPyV), we developed a natural virus-host model of polyomavirus-associated CNS disease. In this R35 application, we plan to leverage our three recent key findings: (1) Mapping JCPyV-PML VP1 capsid protein mutations to MuPyV’s VP1 confers escape from virus- neutralizing antibodies (nAb) while preserving CNS tropism; (2) IL-21 produced by high-affinity anti-MuPyV CD4 T cells in the brain is required for formation and maintenance of MuPyV-specific brain resident-memory CD8 T cells (bTRM); and (3) STAT1-dependent innate immunity limits infection of the ventricular ependyma, a critical barrier to infection of the brain parenchyma. These findings lay the foundation for three key questions to be addressed here: (1) Is the ependyma the staging ground for polyomavirus invasion of the brain parenchyma?; (2) Does the integrity of the CD8 bTRM response to persistent infection depend on subset heterogeneity?; and (3) Does T cell deficiency open the door for outgrowth of nAb-escape virus variants? The proposed studies will make use of cutting edge advances in next-generation sequencing to uncover rare VP1 mutations in vivo, custom cryo EM image reconstruction approaches to define endogenous VP1 nAb epitopes and nAb escape mechanisms, and high-resolution 3D imaging of intact mouse brains to visualize virus CNS entry and spread. Findings from these studies will answer fundamental questions about innate and adaptive immune control of polyomavirus CNS infection and conditions underlying dissemination of virus from the periphery into the brain before development of irreversible neuropathology.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY Polyamines are a class of small organic polycations indispensable for many basic molecular and cellular processes including translation, electrical signaling, cell proliferation, and autophagy. Infectious and hyperproliferative diseases as well as many autoimmune, cardiovascular and neurodegenerative disorders are deeply connected to perturbations in polyamine abundance. Polyamine transport plays a major role in cellular polyamine homeostasis in both healthy and abnormal cells. Understanding the molecular basis of polyamine uptake and secretion has enormous potential to improve human health. However, despite decades of work, this subject continues to mystify. A critical barrier to deeper knowledge in polyamine transport is the complete absence of atomic structures of any polyamine transporter. The goal of this project is to elucidate the fundamental principles underlying polyamine transport and its regulation using a combination of structural and functional approaches. ATP13A2 is a lysosomal P-type ATP-driven pump tasked with the import of spermine and spermidine from the lysosome lumen to the cytosol. Mutations that cripple ATP13A2 function causes a spectrum of neurodegenerative diseases including Kufor-Rakeb syndrome, early-onset Parkinson’s disease, hereditary spastic paraplegia, neuronal ceroid lipofuscinosis and amyotrophic lateral sclerosis. ATP13A2 is, thus, a potential drug target. We have made significant inroads in our preliminary studies to determine high-resolution three-dimensional structures of human ATP13A2. In combination with functional analysis, these structures revealed ATP13A2’s luminal gating and polyamine selectivity mechanisms. Building on these preliminary results, we will leverage complementary electron cryo-microscopy, biophysical, biochemical, analytical chemical and mutagenesis strategies to further subject ATP13A2 to detailed mechanistic scrutiny. Specifically, we aim to investigate: 1) how lipids regulate ATP13A2 activity; 2) whether and how ATP13A2 pumps other cations into the lysosome; and 3) how ATP13A2 shuttles polyamines through the lipid bilayer. By addressing these questions, this research project will provide new insights into the basic operating and regulatory mechanisms of ATP13A2, which will broadly advance our understanding of polyamine transport and lysosome physiology. Structural and mechanistic discoveries from the proposed work may inform future rational design of therapeutics targeting ATP13A2.

NIH Research Projects · FY 2026 · 2022-03

The goals of this research plan are to uncover the molecular mechanism by which mitochondrial Ca2+ signaling promotes pancreatic ductal adenocarcinoma (PDAC) cell migration, invasion and metastasis, and to devise novel strategies to exploit potential therapeutic vulnerability in metastatic PDAC based on our mechanistic studies. The Mitochondrial Calcium Uniporter (MCU) is the only Ca2+ channel on the mitochondrial inner membrane responsible for mitochondrial Ca2+ uptake. Under certain pathological conditions, MCU-mediated mitochondrial Ca2+ overload leads to cell death. Paradoxically, MCU levels are significantly increased during the progression of several types of cancer. In this proposal we use PDAC as a model to study the molecular mechanism by which MCU controls cancer metastasis and progression. Our data indicated that MCU overexpression in PDAC promotes PDAC cell migration, invasion and metastasis through a MCU-Nrf2 signaling circuit. Through non- biased RNA sequencing and interrogation of the TCGA transcriptomic datasets, we identified xCT (SLC7A11, the functional subunit of the cystine / glutamate antiporter system) as a potentially druggable target in MCU- mediated anti-oxidant response and PDAC metastasis. Intriguingly MCU overexpressing PDAC cells are addicted to xCT-mediated cystine uptake. When PDAC cells were deprived of cystine or treated with xCT inhibitors, MCU promotes ferroptosis, a form of lipid ROS-mediated, iron-dependent regulated cell death. In Aim1 we will use genetically engineered mouse model to investigate the role MCU-Nrf2 signaling in PDAC metastasis and progression. We will determine the mechanism by which MCU activates Nrf2 in Aim 2 and define cystine addiction as a therapeutic vulnerability in MCU overexpressing PDAC in Aim 3. The success of this proposal will provide important mechanistic insight for mitochondrial calcium signaling in PDAC metastasis, and will likely provide a novel avenue to prevent metastatic recurrence in PDAC.

NIH Research Projects · FY 2025 · 2022-02

Establishing the mechanism of benefit and dose of exercise required to improve liver histology in patients with nonalcoholic steatohepatitis PROJECT SUMMARY— In this career development program, I will study how exercise improves nonalcoholic steatohepatitis (NASH) and answer three highly significant questions about exercise’s (1) mechanism of benefit, (2) dose, and (3) impact on liver histology. This program builds on my clinical experience in NASH through mentored training in: (1) mechanistic study (Dr. Scot Kimball), (2) exercise (Drs. Kathryn Schmitz, Christopher Sciamanna), and (3) NASH trials (Dr. Rohit Loomba). The central hypothesis of this proposal is that exercise training will activate AMP-activated protein kinase (AMPK) by depleting liver glycogen, leading to liver fat reduction and intermediate histologic endpoint improvement in patients with NASH. Because glycogen is the main substrate used during exercise to generate ATP, I hypothesize exercise will lead to glycogen dissociation from AMPK and subsequent AMPK activation. The central hypothesis is based on a 16-wk proof of concept study in 18 adult patients with NASH in which I found only those who completed a 750 Metabolic Equivalents of Task (MET)-min/wk dose of exercise achieved the minimal clinically important difference in magnetic resonance imaging proton density fat fraction (MRI-PDFF) measured liver fat, that surrogates for histologic response. I observed this in parallel with indirect changes in the AMPK pathway, suggesting AMPK was activated by exercise. While I did not study exercise dose >750 MET-min/wk, it is plausible that higher doses may be even more effective because glycogen depletion requires sustained moderate-vigorous intensity exercise. Given these pilot data, I will conduct a 16-wk clinical trial and randomize adults with NASH to different exercise doses (750 or 1,000 MET-min/wk) or standard clinical care. To ensure adherence, each exercise session will be completed under direct supervision, either in-person or remotely with telehealth, followed by immediate calculation of exercise dose as intensity (METs) x frequency (one session) x time (min). In Aim 1, I will study the mechanism of exercise’s benefit with MR-spectroscopy measured change in liver glycogen and ATP following a single session of sustained moderate-vigorous exercise. In Aim 2, I will discern which dose is most effective in reducing MRI-PDFF after 16-wks of exercise training. In Aim 3, I will measure important intermediate histologic endpoints (NASH activity score) and mechanisms (AMPK activation, AMPK targets). Co- localization of glycogen binding to AMPK in liver tissue will be used to confirm indirect MR-spectroscopy evidence from Aim 1. When I am successful, I will have provided rigorous evidence to decipher the dose required and the underlying mechanisms explaining how exercise training leads to improvement in intermediate histologic endpoints, including NASH activity. This research will inform future trial design by generating data for sample size estimates necessary to study exercise’s impact on long-term histologic outcomes, including liver fibrosis. I am committed to a career as a physician scientist and have constructed my training plan to achieve scientific independence and make substantial contributions to advancing the study of NASH and public health.

NIH Research Projects · FY 2026 · 2022-01

Cigarettes are used by over 34 million U.S. adults and cause more than 480,000 deaths annually due to smoking and smoke exposure. Despite smoking at similar rates and consuming less cigarettes per day, African Americans are more likely to die from several tobacco-caused cancers compared to Whites. Quitting smoking reduces the risk of premature death and adds years to life expectancy; however, a disparity exists in annual quit rates between African Americans and Whites (4.9% vs. 7.1%). This disparity is due to several social determinants. Thus, it is critical to investigate scalable, evidence-based strategies to increase smoking cessation among African Americans. African Americans are twice as likely to use quitlines compared to Whites. However, little is known about the impact of mHealth interventions among African Americans using quitlines. In preliminary studies conducted by Penn State investigators, we found that automated text messaging was feasible for monitoring smoking status and providing smoking cessation support. However, these studies were not designed to assess the impact of interventions among African Americans. A recent study compared engagement and abstinence rates between Black and White smokers in a national texting cessation program and found that Blacks were just as likely as Whites to enroll and remain in the program; yet Blacks were less likely to respond to abstinence assessments and report cessation. Few studies have focused on the behaviors and perceptions of quitline texting services among African Americans. The current project proposes to assess data from the Pennsylvania quitline to inform a tailored mHealth smoking cessation intervention. The overall goal of this Mentored Career Development Award (K01) is to build on the candidate’s advanced postdoctoral training by developing her expertise to investigate and alleviate disparities in tobacco-related disease for African Americans through the use of technology interventions. Career development objectives are to develop expertise in: 1) mHealth applications for smoking cessation, 2) qualitative data analysis, and 3) clinical research skills in ecological momentary assessment. The central hypothesis is that social determinants will explain differences in smoking abstinence between African Americans and White smokers using technology as a cessation aid. The specific aims for this study will be to 1) conduct a secondary analysis of the PA quitline texting program data and provide an overview of service engagement and smoking rates during enrollment and at 6-month follow-up; 2) to conduct qualitative interviews with African Americans who enrolled in the PA quitline texting program to better understand the factors that serve as barriers to engagement and abstinence; 3) To conduct a pilot EMA study to inform a tailored mHealth smoking cessation intervention for African Americans. The research environment is an academic medical center with the facilities, technology, resources, and advanced equipment to support the research and training proposed in this award.

NIH Research Projects · FY 2026 · 2021-12

Abstract: Acute myeloid leukemia (AML) is a heterogeneous disorder of hematopoietic stem and progenitor cells (HSPCs) associated with sequential acquisition of driver gene mutations. These mutations often lead to altered genome organization and transcriptional programs that perturb HSC self-renewal and differentiation. Recently, we discovered that HOTTIP, a posterior HOXA-associated long non-coding RNA (lncRNA), remodels CTCF- defined topologically associated domains (TADs). This remodeling regulates the homeotic gene-associated leukemic transcription program and facilitates AML leukemogenesis, driven by MLL rearrangement (MLLr+) or NMP1 mutation (NPM1C+). One of the top HOTTIP-regulated transcription motifs in AML is CTCF-binding sites (CBSs), suggesting a novel function of HOTTIP in regulating CTCF-mediated genome organization and AML pathogenesis. Indeed, combined RNA-seq, ChIRP-seq, and CTCF ChIP-seq analyses revealed that HOTTIP co-occupied with CTCF in a subset of the AML genome, including HOXA and WNT/β-catenin target gene loci, for their activation. However, it remains unknown whether and how HOTTIP lncRNA directly regulates CTCF- directed genome organization to promote leukemic transcription networks and leukemogenesis. Our preliminary data showed that HOTTIP is capable of directly interacting with key TAD boundary CBSs in the HOXA and WNT target loci via formation of an R-loop structure. We hypothesize that HOTTIP activation mediates aberrant R- loop formation in CBSs to stratify CTCF chromatin boundary for reprograming AML TADs and leukemic transcription networks, leading to HSPC perturbation and leukemogenesis. To test this hypothesis, we will focus on the impact of the altered CTCF TAD boundary and R-loop formation upon HOTTIP activation on AML genome regulation and gene transcription output. In this proposal we will test the importance of the HOTTIP activation- mediated aberrant R-loop formation in modulating the CTCF boundary activity and transcriptional regulation in AML. Specifically, we will identify and characterize critical HOTTIP-regulated CTCF chromatin boundaries in the AML genome. We will then define the novel role of R-loops in HOTTIP-mediated CTCF chromatin boundary definition and genome organization. Finally, we will assess the impact of the HOTTIP-mediated aberrant R-loop formation at specific TAD boundaries on leukemogenesis and HSPC regulation.

NIH Research Projects · FY 2026 · 2021-12

Project Summary/Abstract Approximately 9 million patients with diabetes (DM) are hospitalized annually and over 30% of inpatients without DM experience high glucose (HG) due to their acute illness. HG increases the risk of infectious and non- infectious complications and death, hospital length of stay (LOS), utilization of hospital resources and overall healthcare costs. While glucose control reduces these risks, controlling HG in the hospital is difficult due to multiple barriers such as recognizing and proactively treating glucose abnormalities, and adequately ordering insulin to treat HG in the hospital. Clinical decision support (CDS) is a system that uses computerized person- specific data in the electronic medical record (EMR) proven to improve hospital care. Among the various modalities, alert-CDS is shown to improve care delivery, providers’ proactivity, and glucose control specifically in intensive care settings of academic institutions. However, alert-CDS has not yet been studied outside of intensive care units (ICU), or in community hospitals where most patients receive care. Furthermore, its impact on patients’ outcomes has not been tested in any setting. The proposed project uses an innovative alert-CDS tool we developed and validated which automatically identifies dysglycemia and inadequacies in insulin administration in the hospital. It alerts clinicians with recommendations to support decision making without superseding their clinical judgement. In our pilot study, we found that this alert-CDS tool reduced recurrent high glucose levels and shortened LOS. Based on this promising preliminary data, in this project we propose to study the impact of our CDS tool on clinical, economic and providers’ performance outcomes among non-intensive care patients both in an academic and a community hospital. We propose to make this resource available intermittently in the EMR every 3 months during 36 months, thus allowing us to compare 18 months of intervention and 18 months of standard care. Based on our pilot study, we expect that a sample size of 12,560 subjects will give us an 80% power of detecting 0.34 days (~ 8 hours) difference in length of stay, the primary endpoint of our study. We propose the following aims: Aim 1) To determine the impact of the alert-CDS over conventional care on the clinical outcomes of non-ICU patients in an academic and a community hospital. Aim 2) To determine the impact of the alert-CDS over conventional care on the economic outcomes of non-ICU patients in an academic and a community hospital. Aim 3) To determine the impact of alert-CDS for inpatient glycemic control on providers’ perspectives, competencies and practice performance between an academic and a community hospital. We hypothesize that the tool will increase providers’ knowledge about dysglycemia allowing them to make better decisions about insulin administration. The anticipated success of our study builds upon a well-established multidisciplinary team of investigators strongly supported by leadership stakeholders in both hospitals. Our proposed study has the potential of establishing a new paradigm in the management of dysglycemia in hospitalized patients with a major positive impact on clinical and economic outcomes.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Diabetes promotes cellular concentrations of the stress response protein Regulated in Development and DNA damage 1 (REDD1) in the retina, which contributes to the development of retinal disease and impaired visual function. Indeed, intravitreal administration of a siRNA targeting the REDD1 mRNA has demonstrated some limited therapeutic benefit in improving visual function of patients with diabetic macular edema. However, the approach was abandoned over a decade ago due to its failure to outperform vascular endothelial growth factor (VEGF) blockade, despite the partially effective results of this current standard of care. During the prior funding period, we discovered that the REDD1 protein acts as a critical intracellular redox sensor. Specifically, formation of a redox-sensitive disulfide bond acts allosterically to prevent the normally rapid degradation of REDD1 protein in the context of diabetes. This discovery suggests that REDD1 mRNA knockdown may be a poor strategy for reducing REDD1 protein abundance and activity in the context of diabetes. This renewal application is designed to identify new evidence-based therapeutic strategies for preventing the retinal pathology that is caused by REDD1. The rationale is that inhibiting the specific molecular events that lead to increased REDD1 protein abundance or those that are responsible for its deleterious effects on vision may provide new interventions early in the preclinical and non-proliferative stages of diabetic retinopathy (DR). The central hypothesis is that diabetes activates the REDD1 redox sensor to promote glycogen synthase kinase 3 (GSK3E)-dependent gliosis, neurodegeneration, vascular permeability, and impaired visual function. Aim 1 will explore inhibition of REDD1 allostery as a therapeutic target for DR. The studies will evaluate diabetes-induced retinal defects in a new point mutation knockin mouse that expresses a REDD1 variant that continues to be degraded even after redox- modification. To complement this genetic approach, we will also use cutting-edge artificial intelligence (AI) and molecular binding assays to explore small molecule inhibition of REDD1 allostery as a clinically translatable therapeutic for retinal disease. Aim 2 will examine a role for REDD1-dependent GSK3E signaling in the failed adaptive response of retinal Müller glia to diabetes. The proposed studies will determine if Müller glial GSK3E signaling enhances cytosolic calcium influx by promoting the expression of a stress-induced cation channel, leading to downregulation of synaptic glutamate uptake and consequently retinal neurodegeneration. A new Müller glia-specific MITO-Tag mouse will also be used to explore a role for GSK3E signaling in mitochondrial permeability, mitochondrial DNA leak, and activation of STING (stimulator of interferon genes). Finally, the proposed studies will use ribosome profiling of translationally active mRNAs isolated from the retina of diabetic mice to characterize the reprogramming of Müller glia toward reactive gliosis. This project is expected to have a powerful impact on the field, because it addresses a clinical need for therapeutics that provide interventions in the early stages of retinal disease by targeting specific molecular events that cause loss of retinal homeostasis.

NIH Research Projects · FY 2025 · 2021-09

Project Summary The purpose of this program is to educate upper-division biomedical engineering undergraduate students in the area of biomedical device design and development. The development pathway typically includes discovery and ideation, invention and prototyping, pre-clinical and clinical testing, regulatory decision making, and commercialization. We expect the training will lead to well-rounded biomedical engineers, who can recognize specific needs in a biomedical problem, and develop a proper procedure to design and achieve a solution. The specific aims of this proposed work are to enhance problem recognition by clinical observations and effective peer and multi-disciplinary communications; to improve students’ ability to propose and validate solutions for identified problems; and to integrate multidisciplinary training in research, development, prototyping, pre-clinical testing, regulatory decisions, and social responsibility into one complete program. To accomplish the aims, we provide a linked training experience to enhance student engagement and maintain project continuity. The training includes six phases over the span of a year: (1) clinical workshopping in which the students interview clinicians, discover problems, ideate on solutions, and define their year-long projects; (2) a numerical simulation course in which simulation is integral to the design process; (3) summer clinical immersion in which the students attend clinical conferences, view surgeries, and receive more in depth training on clinical aspects of biomedical engineering; (4) capstone design course in which students work in larger multidisciplinary teams on detailed design, prototyping, and testing; (5) regulatory affairs course including an FDA workshop and mock FDA submission; and (6) commercialization training. A diverse group of ten students per year participates in the full program including clinical immersion, but these students will work in larger multidisciplinary teams during the class projects and capstone design course, thus broadening impact of the program. Besides technical skills, we also consider development of leadership, teamwork and self-direction skills. By the end of the training, we expect participants will be able to apply knowledge learned to a device development process in a self-directed manner. We also expect that the training program will provide a broader impact to the department and institution, and hopefully to future biomedical engineering undergraduate education.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Immune activation is a hallmark of myocardial infarction (MI) and dictate infarct healing and left ventricular (LV) remodeling. It is established, both in preclinical and clinical studies, that immediately after infarction immune cells from the spleen and the bone-marrow (BM) egress into the systemic circulation and traffic to the ischemic hearts. This suggests that the damage signals from the injured hearts are communicated to the lymphoid and the hematopoietic tissues to initiate cardiac-specific immune responses. However, the mechanisms by which these signals are transferred to the immune-rich niches such as the spleen are not known. Recent studies have shown that exosomes, membranous vesicles of 30-100 nm size, are potent intercellular communication vehicles that can shuttle mRNA, miRNA, and proteins to the distant tissues for physiological and pathological immune activation. It is also known that circulating exosomal levels increase during myocyte damage and contain sarcomeric, cytosolic and mitochondrial proteins in their cargo. Despite this understanding, it is not known if exosomes also serve as the antigenic vehicles to carry damage- associated protein signals from the ischemic myocardium to the spleen for subsequent immune activation. Indeed, our preliminary results clearly show that intravenous administration of exosomes released by the ischemic hearts (as compared to sham) into the naïve mice induce i) systolic dysfunction (increased end- diastolic and end-systolic volumes and decreased ejection fraction), ii) gene expression of damage associated signals (S100A8, S100A9 and eotaxin) in the left-ventricles, iii) splenic remodeling, and iv) infiltration of innate and adaptive immune cells in the myocardium. Moreover, MI exosomes expressed tumor necrosis factor receptor-1 (TNFR1; and not TNFR2) on their membranes, a classical pro-inflammatory signaling molecule that have been shown to correlate with HF severity and cardiac dysfunction clinically. Importantly, intravenous injection of exosomes isolated from 1d MI TNFR1-/- mice failed to induce cardiac dysfunction in naïve mice suggesting a potent, and previously unknown, role of exosomal TNFR1 in mediating immune activation post-MI and cardiac dysfunction. Therefore, this led us to hypothesize that exosomes carry cardiac antigens to mediate splenic immune cell activation during MI, are critical for immune cell mediated pathological LV remodeling, and these effects are dependent upon the exosomal TNFR1 expression. Importantly, these are key cellular targets for immunomodulation. We will test this hypothesis by i) determining the pathophysiological role of the spleen in mediating exosomal processing post-MI, ii) defining the source and role of vesicular TNFR1 in exosome mediated cardiac dysfunction, and iii) determining the role of exosomal TNFR1 in mediating immunogenic signaling.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract This K08 Mentored Clinical Scientist Research Career Development Award application details a 5-year training program to advance Dr. Cygan’s career goal of developing an independent research program in hemostasis focused on bleeding tendency in female Hemophilia A carriers. The scientific proposal seeks to investigate bleeding tendency in Hemophilia A (HA) carriers, which remains largely underrecognized and poorly understood. HA is due to mutations within the F8 gene encoding coagulation Factor VIII (FVIII), and is commonly thought to affect only men, while female HA carriers are thought to be only rarely symptomatic. There are an estimated four female HA carriers for every one HA male, and increasing evidence indicates that decreased FVIII levels and bleeding symptoms in HA carriers are not rare. Many have significant bleeding events despite Factor VIII activity levels considered adequate in males. Current understanding of bleeding tendency in this area is lacking, and research investigating female HA carriers would greatly inform their classification and management. The proposal seeks to develop a risk prediction model for bleeding tendency in HA carriers, using parameters other than standard coagulation testing. In Aim 1, a multivariable risk prediction model will be developed and validated. In Aim 2, a cell culture system to model the HA carrier state will be developed and the role of X-chromosome inactivation (XCI) in altering basal and stimulated levels of FVIII and resulting thrombin generation will be evaluated. In Aim 3, the role that natural anticoagulants alter hemostasis and explain differences in bleeding phenotypes between HA carriers independent of XCI will be investigated. During the award period, Dr. Cygan will continue to develop his expertise in benign hematology, and further expand his abilities in experimental design and implementation. A multidisciplinary mentorship team has been assembled to ensure the success of this project and includes expertise in X chromosome biology, Factor VIII expression, clinical hematology and biostatistics. The mentorship team includes Dr. Laura Carrel, Dr. Valder R. Arruda and Dr. M. Elaine Eyster; they will guide Dr. Cygan in meeting his training objectives through direct research experience, formal didactics, participation in institutional seminars and junior career development opportunities. Dr. Lan Kong, a biostatistician, will contribute to Dr. Cygan’s research and career plan as a collaborator and statistical advisor.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT Olfactory impairment may signal prodromal Alzheimer’s disease (AD). We currently do not have an established model that can be tested, in vivo, relating AD neurodegeneration to specific functional deficits in olfaction and memory. This significant knowledge gap impedes the development of functional imaging markers for the evaluation and diagnosis of AD. We have developed several olfactory fMRI paradigms that can probe the dysfunctions in brain regions where early stage AD neurodegeneration occurs. Our preliminary data suggest an AD neurodegeneration-to-function model. We hypothesize that progressive neurodegeneration in MCI disrupts the connectivity of the olfactory network (ON) to the default mode network (DMN) via the Hippocampus, leading to early deficits in olfaction followed by memory impairment. Our research is designed to test this hypothesized model using functional connectivity (FC; synchrony among brain regions) in resting state fMRI and effective connectivity (EC; directed interactions between brain regions) during olfactory task fMRI; neurodegeneration will be evaluated by volumetric MRI (vMRI). Aim 1: Determine age-related changes in the ON-DMN network in cognitively normal subjects. Aim 2: Determine changes in the ON-DMN network in mild cognitively impaired (MCI) subjects. Aim 3: Explore the relationships between progressive changes in the ON-DMN network, and cognitive decline in MCI subjects The over-arching goal of the proposed research will be to rigorously test an AD neurodegeneration model that answers two fundamental questions: a) How are odor-identification and odor-discrimination deficits in AD related to memory impairment and neurodegeneration? and b) Do olfactory deficits and progressive disruptions to ON- DMN connectivity signal the development of AD dementia? Research outcomes can provide numerous avenues for improving olfactory testing as an AD marker. Olfactory tests, which target specific brain areas and processes that are affected earliest in AD, could hold additional promise for early disease detection and prevention in other neurodegerative disease, such as Parkinson’s disease.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ABSTRACT Agonists for dopamine D1–like receptors (D1R) consistently have been shown to enhance cognitive functions (e.g., working memory) in normal, lesioned, and aged murine and primate species, including humans. The D1R also plays important roles in the regulation of synaptic plasticity and cognitive process that are damaged during age-related cognitive decline such as in Alzheimer’s disease (AD). Receptor functional selectivity/biased signaling is a useful approach for discovery of drugs with ability to activate differentially signaling pathways mediated by a single receptor. Our recent work suggested D1 functional selectivity has critical influence in modulation of working memory related behavior and neurophysiology in the prefrontal cortex of young adult rats. These and other data suggested functionally selective D1 ligands could be promising candidates for age-related cognitive decline. Here we propose a discovery project to understand how signaling bias affects the cognitive effects of D1 ligands, and to target novel functionally selective D1 agonists that may become potential IND candidates for the pharmacological treatment of age-related cognitive decline. This will be accomplished by the following three iterative but also independent specific aims, using recent advances in three field, in vitro pharmacology, in vivo behavioral and physiological neuroscience, and in silico ligand-target mechanistic studies. Aim 1 will examine behavioral and neurophysiological changes manifested by differential activation of D1 signaling pathways in aged versus young rats (24-month elderly and 5-month young adult Fisher, TgF344 transgenic AD rats). Pre-selected D1 ligands will be tested by a working memory related delayed alteration response task in the T-maze primarily, for their effects on behavior and neurophysiology. Aim 2 will use computational methods for ligand-target simulation to develop mechanism and accelerate drug discovery. Aim 3 will complete a thoroughly pharmacological screen to elucidate optimal D1 signaling profiles for age-related cognitive decline. This will involve characterization of receptor binding properties and functional assays, and off- target analysis, in not only heterologous expression systems but also brain tissue. The successful completion of proposed specific aims will provide heuristic information on the potential advantages of functionally selective D1 agonists for age-related cognitive decline. Recent clinical studies have shown, contrary to earlier views, that the D1R is a druggable target. Thus, this project will provide a rational foundation for selection of novel candidates for future IND-enabling studies and clinical trials.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract The cerebral ischemia/reperfusion (I/R) injury is a major challenge for the treatment of patients with acute ischemic stroke by intravenous (IV) thrombolysis and endovascular therapy. Currently, there is no effective intervention available to treat/prevent cerebral I/R injury. Emerging evidence suggests that thrombotic and inflammatory responses (thrombo-inflammation) and aberrant endoplasmic reticulum (ER) stress in the brain elicited by cerebral I/R contributes importantly to secondary brain injury and neurologic deterioration. In this proposal we wish to develop a novel miRNA-based therapeutic strategy to simultaneously target these pathological processes of cerebral I/R injury through distinct molecular mechanisms. Recently, decreased expression of miR-30c has been implicated in many pathological conditions in both patients and animal models. In preliminary studies, we show that miR-30c is highly expressed in blood platelets, cerebral microvessels, and cortical/hippocampal neurons in normal mice but its levels decline with age. miR-30c levels in both blood and brain are markedly decreased after ischemic stroke and elevating miR-30c by single IV injection of synthetic miR-30c mimic significantly protects against cerebral I/R injury. We further show that the increased expressions of the direct target genes of miR-30c (including PAI-1 in both blood and brain, elF2α and caspase-3 in the brain) induced by cerebral I/R injury were significantly decreased by IV miR-30c mimic treatment. Based on these exciting new findings from young adult mice, we propose the innovative hypothesis that miR-30c functions as a critical regulator of thrombo-inflammation and ER stress in the ischemic brain elicited by cerebral I/R injury, and thus targeting miR-30c represents a novel therapeutic approach for combating cerebral I/R injury. Following updated stroke Therapy Academic Industry Roundtable (STAIR) pre-clinical guidelines, we will test this hypothesis in aged male and female mice. Specifically, we will determine the efficacy and safety of IV miR-30c mimic as novel stroke therapeutics (Aim1). Using complementary approaches, i.e. the “gain-of-function” (miR- 30c mimic) and “loss-of-function” (anti-sense Morpholino oligos that are designed to specifically compete with miR-30c for binding sites of 3’UTR of each individual target gene and thus acts as a “target protector”), we will identify PAI-1 as a key molecular target of miR-30c in regulation of post-stroke thrombo-inflammation (Aim 2), and identify elF2α and caspase 3 as key molecular targets of miR-30c in regulation of post-stroke neuronal ER stress and neuronal cell death (Aim 3). The long-term goal of these studies is to evaluate if targeting miR-30c is a viable option for stroke therapy in both male and female older populations at high risk for stroke.

NIH Research Projects · FY 2025 · 2021-06

ABSTRACT Innate immunity provides the first line of host defense in response to invading pathogens. Pattern recognition receptors (PRRs) sense pathogen-associated molecular patterns (PAMPs) in viruses and other pathogens. RIG-I/MDA-5 are DExD/H box RNA helicases in the RIG-I-like receptor (RLR) pathway that detect double stranded RNA viral genomes and activate the mitochondrial adaptor protein MAVS. The DNA sensor cGAS detects double stranded DNA and activates STING, a transmembrane endoplasmic reticulum adaptor. Both MAVS and STING activate canonical (IKKa and IKKb), and noncanonical IKK kinases (TBK1 and IKKi) to activate transcription factors NF-kB and IRF3 respectively. Together, IRF3 and NF-kB regulate the expression of type I interferon (IFN) and inflammatory genes that coordinate the innate response and initiate the adaptive immune response against pathogens. The NLRP3 inflammasome also plays a critical role in inflammatory responses by triggering caspase-1-mediated pro-IL-1b cleavage to yield the biologically active form of IL- 1b that drives inflammation and adaptive immunity. NLRP3 also induces a highly lytic form of inflammatory cell death termed pyroptosis via cleavage of gasdermin-D to form plasma membrane pores. The RLR, cGAS- STING and NLRP3 inflammasome pathways are potent inducers of inflammation that must be tightly regulated to avert overexuberant inflammation and tissue damage. TAX1BP1 was first identified as an anti-apoptotic protein that interacts with the zinc finger deubiquitinase A20/TNFAIP3. Our previous work has established that TAX1BP1 restricts cytokine-induced NF-kB activation as well as RLR-induced type I IFN production and apoptosis. TAX1BP1 functions as a selective autophagy receptor by recruiting ubiquitinated cargo to developing autophagosomes via two LC3 interaction regions (LIRs). However, it remains unclear how TAX1BP1 autophagy function is regulated and if TAX1BP1 inhibits other innate immune signaling pathways. In preliminary studies we provide experimental evidence that TAX1BP1 is phosphorylated by both noncanonical and canonical IKK kinases which controls both basal and virus-induced TAX1BP1 autophagic degradation respectively. Using TAX1BP1-deficient macrophages we have demonstrated that TAX1BP1 is a novel inhibitor of both cGAS-STING and NLRP3 pathways. Furthermore, MAVS protein aggregates accumulate in TAX1BP1- deficient cells suggesting a potential aggrephagy function in the regulation of innate immune signaling. The central hypothesis driving the proposed investigations is that TAX1BP1 inhibits RLR, cGAS-STING and NLRP3 pathways by autophagy-mediated clearance of signaling protein aggregates. We will test this hypothesis experimentally with the following Specific Aims: (1) determine the role of TAX1BP1 phosphorylation in its autophagy function, (2) determine the mechanisms of TAX1BP1 inhibition of the cGAS-STING pathway, and (3) determine the mechanisms of TAX1BP1 inhibition of the NLRP3 pathway. Completion of the proposed studies will provide new insights into innate immune regulation and immune homeostasis.

NIH Research Projects · FY 2025 · 2021-05

Abstract Mitochondrial and synaptic dysfunction are early pathological features and a driving force of Alzheimer's disease (AD) pathology. Aβ is found to accumulate abnormally in the brains of AD individuals and in an AD mouse models leading to mitochondrial Ca2+ overload and activation of mitochondrial permeability transition pore (mPTP). Prolonged opening of mPTP triggers outer mitochondrial membrane rupture, release of cytochrome c and activation of downstream cell death pathways. The mPTP has been at the center of extensive scientific research for the last several decades but it still remains as one of the most mysterious phenomena in biology today due to its controversial molecular composition and the lack of structural information of its pore. We have recently demonstrated the novel role of ATP synthase c-subunit ring in forming the channel of mPTP. Nevertheless, the gating mechanism of the ATP synthase c-subunit leak channel and conformational changes initiating its opening are yet to be discovered. In this proposal, we will use single-particle cryo-electron microscopy (cryo-EM) to identify the high- resolution structure and the open conformation of ATP synthase leak channel in the presence of channel modulators. We will also perform in situ structural analysis of ATP synthase in its functional environment within the Aβ-exposed primary hippocampal neurons and in mitochondria isolated from the mouse models of AD by using cryo-electron tomography (cryo-ET). In this project we will also investigate the direct role of ATP synthase leak channel as a novel cell death pathway in AD pathogenesis; we will test whether the pharmacological inhibition of this channel will rescue neurons from Aβ-induced cell death. Successful completion of this proposal will reveal the molecular mechanism(s) of mitochondrial permeability transition, the atomic structure of ATP synthase leak channel, and will aid in the development of new treatments for AD, targeting ATP synthase.

NIH Research Projects · FY 2024 · 2021-05

Project Summary/Abstract The objective of this project is to develop an implantable blood pump system as a total heart replacement. The use of left ventricular assist devices (LVADs) for destination therapy for end stage heart failure is expanding. However, right ventricular failure occurs in 10-40% of LVAD patients. We are developing a system using separate centrifugal left and right pumps with a common controller. The right pump is based on a right heart replacement pump developed for failing Fontan patients. It has unique double inlet ports to connect to the superior and inferior vena cavae, allowing the pump to be placed in the position of the right atrium. The left pump is a single inlet pump of similar design. Compared to currently available biventricular assist systems and total artificial hearts, our approach will: 1) allow unobstructed fit in the anatomy of smaller patients, 2) achieve reliable operation for at least 10 years, 3) automatically adjust left and right pump output to respond to the varied physiologic needs of the patient 4) balance left and right pump outputs to safely control left atrial pressure over a wide range of systemic and pulmonary vascular resistances and central venous pressures, 5) provide pulsatile flow, 6) reduce the incidence of thromboembolism and pump thrombosis, 7) preserve high molecular weight von Willebrand factor to reduce the incidence of bleeding, and 8) provide a development path to a completely implantable, wireless system. Specific Aim 1- Build a biventricular replacement device to replace the failing heart, with the right heart placed in the bicaval position and a left pump anastomosed to the left atrium remnant. Integration of pressure sensors in both pumps will measure inlet pressures (equivalent to right atrial and left atrial pressures), which will be used as inputs to the automatic control system. Specific Aim 2- Develop an automatic control system capable of physiologic control of both pumps. The controller will increase right pump output in response to increased venous return, which mimics the normal Frank-Starling cardiac output response, and control left pump output to maintain left atrial pressure within a physiologic range. The controller will also modulate pump speeds to produce pulsatile flow. Specific Aim 3- Test and optimize the TAH in chronic animal studies to: A) Demonstrate automatic Starling-like cardiac output control and automatic balance control of the pumps over a wide range of physiologic conditions including exercise, and B) examine the biocompatibility of the device by assessing thrombus formation, thromboembolism, von Willebrand factor degradation, and intestinal angiodysplasia. We will study both pulsatile and non-pulsatile modes.

NIH Research Projects · FY 2025 · 2021-05

Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with distinct histological and behavioral hallmarks but its cell-type basis is not clear. Recent discoveries now provide compelling rationale that constituent cell types bear a non-uniform risk profile as a function of disease and age. Human post- mortem study showed inhibitory interneurons (IN) and excitatory pyramidal neurons (IN) undergo differential alterations in gene expression in AD and mouse brain imaging study observed progressive cytological dedifferentiation of synapse architecture due to aging. These results dovetail with our discovery that the differential expression of six gene categories, synergistically involved in synaptic communication specifies neuron-type identity and their diverse morpho-physiological properties. Our central hypothesis is that the distinct transcriptomic identity that bestows unique cell-type properties also confers an inherent risk and resilience to AD risk factors, such as age, sex and genetic makeup. We posit that an altered cell-type specific transcriptome underlies the disparate response of inhibitory and excitatory neuron subtypes in AD and the dedifferentiation of synapses in aging. Through cross-disciplinary systems biology approaches we will identify the vulnerable cell-types in AD, establish cell-type specific AD signatures and further investigate biological relationship between healthy aging and AD pathology. Aim-1 will test the hypothesis that diverse brain cell subclasses confers differential risk through distinct transcriptomic response to progressive stages of AD and aging. A subclass specific longitudinal approach will allow us to prioritize for cells that are fundamental to the genesis of AD. We will determine age-dependent vulnerability in 3xTg-AD mouse by single- cell RNA-Seq (scRNA-Seq) of eight genetically labeled subclass in three major AD associated brain areas, brain-stem, entorhinal cortex, hippocampus and frontal cortex at 3, 6 and 15 months. Aim-2 will test the hypothesis that excitatory PNs and inhibitory INs have differential anatomical, morphological and cell survival trajectories in AD and aging. Synapses in neural circuits control behavior and their progressive dedifferentiation have been implicated in aging and dementia. As the molecular composition of synapses are distinct for each neuron-type they will be differentially impacted by AD and age. We will perform whole brain light-sheet microscopy on PN and IN in 3xTg-AD mouse across three time points 3, 6 and 15 months to capture the subclass specific dynamics of progressive pathological changes in AD. Aim-3 will test the hypothesis that cell specific glycosylation codes confers vulnerability to proteinopathy in AD and aging. Plaques and neurofibrillary tangles are AD hallmarks, however cell specific vulnerability is unknown. Differential expression of heparin sulfate (HS) enzymes create HS diversity akin to a “glycosylation code” that predisposes some cells to form aggregates while protecting others. In 3xTg-AD multiplexed RNA in-situ will measure HS biosynthetic enzyme transcripts, as a function of healthy aging and in pathology.

NIH Research Projects · FY 2025 · 2021-05

Abstract HIV-1/AIDS is a devastating immunodeficiency disease that has resulted in over 35 million deaths across the globe. There is a compelling ongoing need to develop novel treatment strategies to combat the continuing emergence of drug-resistant viral strains. A drug target that has not yet been successfully brought to the clinic is the interaction of the Gag protein with its RNA genome to form the viral ribonucleoprotein complex, which initiates the process of virion assembly. A deeper understanding of the biochemical and biophysical interactions that drive viral ribonucleoprotein complex formation is needed to advance further therapeutic development. Based on recent findings that viral ribonucleoprotein complexes undergo liquid-liquid phase separation to form biomolecular condensates, this proposal explores the molecular underpinnings of Gag-viral RNA interactions. We have assembled an accomplished interdisciplinary team of scientists to work at the crossroads of retrovirology, RNA biology, biophysics, and pharmacology to shed light on our understanding of viral and cellular biomolecular condensates in HIV-1 infection. Our preliminary results suggesting that HIV-1 ribonucleoprotein complexes form in the nucleus inspired this provocative proposal to investigate the interplay of virus replication machinery with nuclear bodies that form biomolecular condensates. In the R21 phase, we will use innovative methods involving biophysics, genetics, state-of-the-art live cell imaging, and targeted pharmacological interventions to examine whether HIV-1 ribonucleoprotein complexes assemble into biomolecular condensates in the nucleus. In the R33 phase, we will extend these studies to probe mechanistic questions focusing on whether nuclear BMCs play critical roles in regulating HIV-1 transcription, latency reactivation, and genomic RNA packaging. Due to the high incidence of substance use disorder in people with HIV-1/AIDS, we will also investigate whether drugs of abuse influence HIV-1 nuclear biomolecular condensates. Elucidating the genetic determinants of HIV-1 condensate formation could lead to the identification of novel drug targets to treat HIV/AIDS.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Obesity is a global epidemic that is associated with excessive central sympathetic outflow to cardiovascular end organs to elevate blood pressure and predispose to hypertension. Accumulating evidence from our laboratory suggests that deficiency of angiotensin-(1-7), a protective hormone of the renin-angiotensin system, provides an important link connecting obesity with sympathetic overactivation and hypertension. Our published observations, combined with preliminary data, support this concept by showing that high fat diet-induced obese mice exhibit circulating angiotensin-(1-7) deficiency, and restoration of this hormone attenuates cardiovascular sympathetic overactivity and hypertension in this model. Our preliminary data expand on these phenotypic findings by providing evidence that angiotensin-(1-7) depressor effects require activation of neural circuits originating in the arcuate nucleus of the hypothalamus (ARC). We show that both systemic and intra-ARC angiotensin-(1-7) lowers blood pressure in mice, with effects prevented by deletion of angiotensin-(1-7) mas receptors in the ARC. We further show that blood pressure lowering effects of angiotensin-(1-7) require activation of specific subpopulations of ARC neurons that are likely proopiomelanocortin (POMC)-expressing, as well as cyclic AMP second messenger systems. We propose that angiotensin-(1-7) selectively activates POMC neurons that release the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). In support of this, we show that: mas receptors are highly localized to GABAergic POMC neurons; and angiotensin-(1-7) increases GABA synthesis enzymes in the ARC without altering POMC gene expression. Based on these data, this proposal will test the central hypothesis that angiotensin-(1-7) activates mas receptors on GABAergic POMC neurons in the ARC to reduce cardiovascular sympathetic outflow and lower blood pressure. Aim 1 will determine if angiotensin-(1-7) selectively increases the excitability of GABAergic ARC POMC neurons using transgenic mouse reporter lines combined with whole cell patch clamp electrophysiology methods. Aim 2 will determine if angiotensin-(1-7) requires mas receptors in ARC POMC neurons to lower blood pressure via GABA release mechanisms using a novel mas receptor conditional knockout mouse model we developed and chemogenetic and pharmacological approaches. Aim 3 will determine if angiotensin-(1-7) decreases sympathetic nerve traffic to cardiovascular organs using sophisticated in vivo isolated nerve recording approaches. These studies will be conducted in male and female mice under control and high fat diet conditions, to determine the impact of sex and obesity on angiotensin-(1-7) activation of this neural circuit. Overall, this proposal will span the cellular to whole animal levels to provide new insight into angiotensin-(1-7) effects on neural circuits controlling sympathetic outflow and blood pressure, and related cellular and neurotransmitter mechanisms. Importantly, these studies have more long-term potential to determine if targeting angiotensin-(1-7) represents a novel approach for the treatment of obesity-related hypertension.

NIH Research Projects · FY 2025 · 2021-02

Project Summary Identification of molecular pathways that are preferentially employed and relied upon by cancer cells compared to normal cells is key to designing novel personalized cancer therapies. PARP10 is a poorly characterized member of the PARP family. We previously showed that PARP10 promotes translesion synthesis (TLS)- mediated bypass of DNA lesions during DNA replication, thereby alleviating replication stress. More recently, we also showed that PARP10 is a novel oncogene. We found that the PARP10 gene is amplified and/or overexpressed in a large number of tumors including breast and ovarian, with very few observed occurrences of downregulation or loss. We found that the PARP10 gene is amplified and/or overexpressed in a large proportion of human tumors including breast and ovarian, with almost no occurrences of downregulation or loss. Moreover, we found that PARP10 overexpression in, non-transformed human epithelial RPE1 cells results in enhanced proliferation, resistance to replication stress, and increased xenograft tumor formation in immunocompromised mice. The opposing phenotypes were found upon knockout of PARP10 in cancer HeLa cells. These findings suggest that PARP10 is a putative oncogene and its expression promotes tumor formation and growth. Mutagenic TLS has been previously proposed to promote transformation by both allowing hyper-proliferation and inducing genomic instability. Thus, we hypothesize that PARP10 expression suppresses replication stress through TLS-mediated bypass of replication arresting structures, thereby allowing hyper-proliferation of cancer cells. We propose here to directly test this, in three specific aims which address the hypothesis at three different levels: Aim 1 will investigate the mechanism employed by PARP10 to modulate PCNA-dependent TLS at the molecular level, using biochemical and cellular localization and interaction assays. Aim 2 will functionally test the impact of this mechanism of cellular processes including genomic stability and DNA replication. Aim 3 will employ a mouse genetic model to unambiguously investigate if Parp10 expression induces tumor formation or promotes tumor growth. Using state-of-the-art cellular, molecular and genomic tools (including: CRISPR/Cas9-mediated genome editing; molecular DNA fiber combing to measure fork stability; next generation sequencing approaches to measure mutagenesis and mutation burden) we will investigate here the molecular mechanisms underlying this novel oncogenic function of PARP10. This may eventually result in validation of a new target for cancer therapy.