University Of Florida

NIH Research Projects · FY 2025 · 2022-09

Hundreds of millions of patients are exposed to general anesthetics (GAs) each year, making the heritable effects of GAs a public health issue of paramount importance. The need to investigate the heritable effects of GAs and develop preventative therapies is also indicated by an unprecedented rise, particularly in industrialized countries, of neurodevelopmental disorders. The origin of most of these disorders is unknown and many are more common in males. To form the basis for clinical studies on this topic, the proposed preclinical project will test the following hypotheses regarding the mechanisms, therapeutic tools, and biomarkers pertaining to heritable effects of GAs: 1) GA-induced secretion of the steroid stress hormone corticosterone (CORT) is essential in the initiation of epigenetic changes in parental germ cells (F0 generation) and, by extension, abnormalities in offspring (F1 generation); 2) both GABAergic GA-induced impairment of the K+-2Cl- (KCC2) Cl- exporter, resulting in impairment of inhibitory GABAAR signaling, and an increase in CORT secretion are required for GA-induced F0 neurobehavioral defects; and 3) F1 males are more vulnerable because F0 GAs act via modification of male- specific F1 brain masculinization. Aim 1: Determine the roles of KCC2 and CORT in the initiation of intergenerational effects of sevoflurane (SEVO). Four clinically used GAs with partially overlapping mechanisms of action, SEVO, propofol, ketamine and etomidate, will be used as pharmacological tools to determine the roles of KCC2 and CORT. The F0 rats will be exposed to GAs on postnatal day (P) 56, P58, and P60 and mated on P85 to generate offspring. The F0 and F1 rats will be evaluated in the elevated plus maze, prepulse inhibition of startle*, Morris water maze, fear-potentiated startle, and forced swimming test and by assessing resting and stress-induced hypothalamic-pituitary-adrenal (HPA) axis activity*. Genome-wide DNA methylation in F0 and F1 germ cells and hippocampi and RNA-seq in F0 and F1 hippocampi and peripheral blood monocytes* will be used to gain insight into epigenomic and transcriptomic mechanisms (* = will be evaluated as potential biomarkers for future human studies). Aim 2: Determine whether shorter parental SEVO exposure that is not sufficient to induce F0 neurobehavioral effects can induce F1 effects, and determine the roles of Cl- transporters and glucocorticoid receptors (GRs). Hypotheses: Shorter F0 SEVO exposure is sufficient to upregulate the F0 HPA axis and reprogram the germline, inducing F1 defects. KCC2 enhancement or Na+-K+-Cl- (NKCC1) and GR inhibition have therapeutic effects. Aim 3: Determine the mechanisms of male-biased intergenerational effects of parental exposure to SEVO. Hypotheses: F1 males are more vulnerable because F0 SEVO affects testosterone (T)- regulated brain masculinization. Because T acts through 17β-estradiol (E2) at estrogen receptor α (ERα) and via T-activated androgen receptors (ARs), we hypothesize that F1 ERα-/- (but not ERβ-/-) males and/or F1 wild type males treated with the AR antagonist flutamide at birth will be less affected. KCC2 enhancement or NKCC1 and GR inhibition will alleviate the F0 SEVO-induced changes in F1 male mechanisms of brain masculinization.

NIH Research Projects · FY 2025 · 2022-09

The majority of persons with HIV (PWH) in the US consume alcohol, despite that alcohol use is associated with lower levels of HIV care engagement and HIV viral suppression. Over the past decade, our research team has established the Florida Cohort to support research and training with a mission to maximize HIV viral suppression and improve health outcomes in PWH. Key features of the Florida Cohort include a focus on alcohol-related issues, academic and community-based partnerships across the state, enrollment of individuals with varied backgrounds and risk factors, and linkage to statewide HIV surveillance data. Meanwhile, several agencies in Florida are implementing new interventions to help achieve HIV viral suppression, including the PositiveLinks (PL) app. The overarching goals of the current grant proposal are to advance understanding of the mechanisms influencing adherence to contemporary HIV therapeutic regimens, and to incorporate alcohol-related interventions into emerging strategies to achieve and maintain HIV viral suppression. We propose to enroll and survey 1200 new Florida Cohort participants, with targeted enrollment for persons with heavy alcohol use. The survey data will allow us to identify multilevel factors contributing to ART adherence and viral suppression among alcohol-using PWH. From the overall cohort, 80 alcohol-using PWH will complete “enhanced monitoring (EM)” for one month. The EM group will wear a wrist alcohol biosensor, report alcohol and other risk factors (e.g., real-time anxiety, depression) and ART adherence through an ecological momentary assessment (EMA) app, and provide two dried blood spots (DBS) viral load tests. The EM data will help disentangle the temporal relationships between alcohol, other risk factors, and ART adherence in real-time. Survey and EM data together with input from the EM participants will be used to inform the development of several aspects of the biosensor-based intervention. Our interdisciplinary team will also meet regularly with a new Community/Provider Advisory Board (CAB), who will provide input related to the ongoing Florida Cohort (e.g., enrollment, survey design) and help develop a plan to integrate the alcohol biosensor-based intervention into the PL app. The specific aims include: 1) Systematically investigate multilevel factors based on the social-ecological model and WHO model that influence ART adherence and subsequently HIV viral suppression in alcohol-using vs. non-using PWH. 2) In 80 alcohol-using PWH, use alcohol biosensors and EMA data to identify alcohol consumption patterns and risk factors most strongly associated with ART non-adherence and poor HIV viral suppression. 3) Develop an intervention that incorporates an alcohol biosensor into an existing mHealth ART adherence intervention, with input from alcohol-using PWH and a Community/Provider Advisory Board. The rich information we collect in this project will establish a solid foundation for our next step: a hybrid implementation/effectiveness trial of an integrated alcohol intervention across a large network of clinic-/community-based HIV settings in Florida.

NIH Research Projects · FY 2025 · 2022-09

Abstract Malaria is a major public health problem for many regions of the world, affecting mainly pregnant women and young children. Plasmodium falciparum infection during pregnancy results in significant fetal compromise and contributes to hundreds of thousands of infant deaths each year. These outcomes are associated with a number of malaria-induced placental pathologies, including infiltration of maternal inflammatory cells into the placenta in response to the infection. This, in turn, is linked to significant damage to the villous placenta, including necrotic death of syncytiotrophoblast. Complete understanding of the critical cellular and molecular mechanisms that drive placental damage and dysfunction in placental malaria, however, remains elusive. Evidence suggests that neutrophils and monocytes accumulate in inflammatory placental malaria, and neutrophils in particular are also implicated in severe malaria in non-pregnant patients. However, few details of the roles of these cells in placental malaria pathogenesis are available. Motivated by exciting preliminary data that show profound lipid peroxidation in placental malaria, we hypothesize that activated innate immune cells, through oxidative mechanisms, directly contribute to syncytiotrophoblast stress and death in placental malaria, thereby precipitating poor birth outcomes via placental dysfunction. The objective of this application is to address this hypothesis using placenta tissue from a malaria endemic population, villous explants and a mouse model that recapitulates key aspects of placental malaria. The study objectives will be achieved through three Specific Aims. First, preserved placental tissues from Kenyan women exposed to malaria will be assessed by spatial transcriptomics to establish the extent to which trophoblast oxidative stress and necroptosis are coincident in natural infection. Second, the impact of trophoblast exposure to activated neutrophils and monocytes will be characterized in an in vitro simulation of placental malaria. Oxidative damage, necroptosis and other cell death mechanisms, and syncytiotrophoblast destruction will be monitored in villous explants exposed to neutrophils and monocytes undergoing respiratory burst. Third the role of neutrophil and monocyte oxidative burst, and the relative role of pro-oxidant malarial hemozoin, in driving placental oxidative stress and damage will be investigated in an outbred mouse model for placental malaria. Successful completion of this research will expand understanding of the mechanistic basis of malaria-induced placental damage and fetal compromise, leading to identification of new targets for adjunctive therapies in malaria during pregnancy. By advancing fundamental knowledge of mediators of syncytiotrophoblast compromise and death, this work will have implications for other pregnancy conditions associated with maternal monocyte and neutrophil activity, placental oxidative damage, and cell death-related placental dysfunction.

NIH Research Projects · FY 2025 · 2022-09

Title: De-Implementing Fall Prevention Alarms in Hospitals Inpatient falls result in significant physical and economic burdens to patients (increased injury and mortality rates and decreased quality of life) as well as to medical organizations (increased lengths of stay, medical care costs, and litigation). The Centers for Medicare & Medicaid Services (CMS) considers falls with injury a “never event”— an error in medical care that indicates a real problem in the safety and credibility of a health care institution. Hospitals are no longer reimbursed for extra costs incurred in the diagnosis and management of inpatient fall- related injuries. Thus, because patient falls are common, costly and interpreted as poor care quality, hospitals are highly incentivized to prevent them. Alarm systems are designed to reduce falls by alerting staff when patients attempt to leave a bed or chair without assistance. There is now strong evidence from our group and others that alarms are ineffective as a fall prevention maneuver in hospitals. Despite this, our group has recently shown that more than one-third of hospital patients are undergoing fall prevention alarm monitoring. In nursing homes, CMS regulates the use of fall prevention alarms as it does physical restraints. Instructions to nursing home surveyors state these devices should be used only when medically necessary and continuously reevaluated. Guided by the Choosing Wisely De-implementation Framework, this project will generate a generalizable approach using coaching and tailored de-implementation strategies to reduce use of fall prevention alarms in hospitals. We will conduct a hybrid II implementation study in 30 medical or medical-surgical units from US non-federal hospitals participating in the National Database of Nursing Quality Indicators. Findings from this study could also support future trials aimed at de-implementing low-quality alarm use in other care settings with known high fall rates (e.g., stroke care, cancer care). Evaluation of high versus low intensity coaching addresses an urgent need to evaluate use of tailored strategies and to establish effective thresholds for coaching within health service settings that have varying resources to support de- implementation efforts

NIH Research Projects · FY 2022 · 2022-09

SUMMARY Identification of new targets and mechanisms underlying neuropathic pain is critical to developing new target- specific medications for better neuropathic pain management. The misuse of and addiction to opioids—including prescription pain relievers, heroin, and synthetic opioids such as fentanyl—is a serious national crisis that affects public health as well as social and economic welfare. The current opioid crisis requires novel approaches to chronic pain management. Our proposal leverages a unique finding, originating from the laboratory of Dr. Rajesh Khanna (originally at the University of Arizona and now at the University of Florida), that peripheral nerve injury- induced upregulation of an axonal guidance phosphoprotein collapsin response mediator protein 2 (CRMP2) and the N-type voltage-gated calcium (CaV2.2) as well as the NaV1.7 voltage-gated sodium channel, correlates with the development of neuropathic pain. Leveraging a pocket on the surface of CRMP2, amenable for in silico screening, the PI’s laboratory performed a virtual screen of nearly 0.3 million compounds (diverse small molecules and natural products). Several of the top 21 ‘hit’ compounds from this screen have been validated in in vitro and in vivo experiments, providing experimental proof of our in-silico predictions. Predicted physico- chemical properties of the hit series fall within ranges of lead- or ‘drug-like’ molecules. We have assembled a diverse multidisciplinary team to test the hypothesis that inhibiting CRMP2 phosphorylation associated with sodium and calcium channel activities to decrease nociceptor activity culminates in reduced pain. Our Specific Aims, guided by quantitative goals, are: (1) to profile CRMP2 phosphorylation antagonists for their ability to: (i) bind CRMP2 and (ii) block its phosphorylation by Cdk5 and (iii) inhibit calcium and sodium currents in sensory neurons using whole-cell electrophysiology with a smaller subset being tested in male/female human DRGs; (2) profile CRMP2 phosphorylation antagonists for ADME pharmacokinetic properties and off-target effects on GPCRs, kinases, ion channels and alternative known pain targets, including opioid receptors; (3) characterize the best CRMP2 phosphorylation antagonists for in vivo efficacy in rodents using a phenotypic screen and spared nerve injury (SNI) model of neuropathic pain and explore the potential of phosphorylated CRMP2 as a marker of target engagement; (4) validate optimized CRMP2 phosphorylation antagonists in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) and assess potential reward and/or aversion. At the end of our study, we expect to have at least one lead series for optimization with the goal of developing a selective and efficacious CRMP2 phosphorylation inhibitor for neuropathic pain with minimal side effects or addiction potential.

NIH Research Projects · FY 2025 · 2022-09

The nuclear receptor (NR) superfamily of ligand regulated transcription factors has proven to be a rich source of targets for the development of therapeutics for a wide range of human diseases. Endogenous small molecule regulation of these allosteric proteins control processes central to most aspects of mammalian physiology. Our lab has focused on synthetic ligand development and structure-function analysis of the NR1F subfamily of NRs known as the retinoic acid receptor-related orphan receptors or the RORs. This subfamily contains three genes that are involved in but not limited to regulation of glucose and lipid metabolism, bone growth, and immune functions. In this proposal we seek to expand our understanding of ligand-dependent regulation of NR1F3 (RORy; gene name RORC), in the context of the intact full-length receptor. There are two isoforms of RORy, RORy1 and RORy2, that differ in only their N-terminal sequence. RORy1 is broadly expressed, and in the liver it plays an important role in circadian rhythms and glucose and lipid metabolism. The expression of RORy2 or RORyt, is T cell specific and has been shown to be the key lineage-defining transcription factor to initiate the differentiation program of TH17 cells making RORyt an essential regulator for TH17 and T,17 differentiation. Importantly, these cells that have demonstrated anti-tumor efficacy and RORyt controls gene programs that enhance immunity and decrease immune suppression. We have reported sterols and oxygenated sterols as high affinity endogenous ligands and others have confirmed these findings and provided key evidence that they are indeed physiological RORy ligands. Although in certain experimental paradigms RORy can recruit coactivators without addition of exogenous ligand, suggesting the receptor may be constitutively active. Recent evidence clearly demonstrates that RORy is dependent on ligand binding for activation. While extensive structural studies on isolated domains of the receptor have provided important insight into high affinity ligand binding for both agonists and antagonists, there is a lack of information on how the modular domains of RORy act together in the context of the intact full-length receptor. Given the importance of RORy as a therapeutic target, it is surprising that we have an incomplete understanding of how small molecules modulate its activity. The mechanism for "turning off' RORy activity appears straightforward; however. we have an incomplete understanding on how the receptor is "turned on." We hypothesize that ligand-dependent structural perturbations manipulate the localization and PTM status of the receptor influencing its coregulator and DNA interactions to modulate of the RORy transcriptome. To provide the groundwork to test this hypothesis, we propose to develop and validate an integrated structural model of intact full-length RORy/DNA complex to expand our understanding of ligand-dependent regulation of RORy. Illuminating RORy activation mechanisms will help develop better tools to study its pharmacology and may lead to new therapeutic strategies by designing functionally selective ligands.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Diffuse large B-cell lymphoma (DLBCL), the commonest type of non-Hodgkin lymphoma (NHL), is highly aggressive and despite antiretrovirals continues to be a leading cause of cancer-related death in persons living with HIV. Notably, up to 90% of HIV-DLBCL are positive for the cancer-causing Epstein-Barr virus (EBV). Thus, understanding how EBV contributes to cancer is essential to discovering new therapeutic approaches. Cancer cells require DNA repair but how EBV engages and reshapes cellular DNA repair is an underexplored area. Our studies on EBV-cancer cells and EBV-transformed human B cells (lymphoblastoid cell lines), the latter an important model of EBV-driven lymphomas in immunosuppressed hosts, converge on STAT3. An oncoprotein, STAT3 is frequently activated in cancer. Several studies have also shown that EBV+ HIV-DLBCL frequently exhibit activating mutations in the Janus kinase (JAK)-STAT3 pathway. We have found that EBV activates STAT3 to circumvent the S phase checkpoint barrier, thereby ensuring cell proliferation but in the process, loses homologous recombination (HR) that repairs DNA double strand breaks (DSB). As a result, EBV- transformed and cancer cells become dependent on other forms of DNA repair, in particular, the error-prone microhomology-mediated end-joining (MMEJ) type of repair. This creates a therapeutic vulnerability to synthetic lethal agents that would otherwise be non-toxic to cells with intact HR. PARP [poly (ADP-ribose) polymerase] inhibitors are among such synthetic lethal agents that target MMEJ. Indeed, we find that EBV-transformed and cancer cells are highly susceptible to MMEJ inhibitors that target PARP and the MMEJ-specific DNA polymerase, POLθ. Supporting this dependence on MMEJ, EBV-transformed cells exhibit genome-wide scars of MMEJ repair, and, EBV+ HIV-DLBCL display higher abundance of STAT3 and POLQ transcripts compared to EBV- tumors; POLQ encodes POLθ. Further, by multiomic analyses of several hundred cancer cell lines, we have identified a STAT3-related gene expression signature that points to a mechanistic link between STAT3 and reliance on MMEJ repair while predicting susceptibility to synthetic lethal therapies. We now propose to investigate how EBV uses the JAK-STAT3 pathway to reshape DNA repair and render EBV+ HIV-DLBCL vulnerable to synthetic lethal therapeutic targeting. Using cell lines, xenografts, and patient-derived EBV+ & EBV- HIV-DLBCL from the NCI AIDS and Cancer Specimen Resource (ACSR), we investigate the link between JAK-STAT3 pathway and DSB repair in EBV+ HIV-DLBCL (Aim 1) and synthetic-lethally exploit JAK- STAT3-dependent DNA repair deficiency to kill EBV+ HIV-DLBCL (Aim 2). These studies specifically address PAR-21-348 by identifying mechanisms and generating new paradigms to reveal how EBV contributes to NHL. In the long-term, these mechanistic insights will uncover novel vulnerabilities and enable the prediction of responses to synthetic lethal therapies to improve outcomes for EBV+ DLBCL in persons living with HIV.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY I am a clinical psychologist interested in understanding the role of environmental exposures in neuro-endocrine development. The goal of this proposal is to receive training to acquire the skills needed to continue my academic career by examining the role of child fluoride exposure in sleep patterns and melatonin production. In this proposal, I plan to train with an expert mentoring team with transdisciplinary expertise covering exposure biology, sleep disorders, toxicology, epidemiology and biostatistics. Specifically, I will train in 1) exposure science with Dr. Manish Arora 2) clinical sleep research with Dr. Michael Thorpy, and 3) toxicology with Dr. Robert Wright. I will also acquire expertise in epidemiology and the epidemiological assessment of pediatric sleep with Drs. Rosalind Wright, Emily Oken and Kristie Ross, as well as biostatistics with Dr. Chris Gennings. The proposed formal coursework and training with my mentors, advisors and collaborators will enable me to acquire the knowledge and skills necessary to become an independent transdisciplinary researcher. Further, it will enable me to achieve my long-term career goal of becoming an environmental health scientist investigating the role of environmental toxicants in sleep and neuro-endocrine development. I propose to leverage biospecimens and collected measures, including urine, saliva, Actigraphy data, and self- reported daytime fatigue from an existent prospective birth cohort, the Programming Research in Obesity, Growth Environment and Social Stress study. I also aim to conduct an in-depth clinical study examining associations of urine fluoride with gold standard measures of sleep among adolescents. This work will address the following aims: 1.) Determine whether childhood urinary fluoride concentrations (CUF) at 4-5 and 6-7 years predict sleep and wake time, sleep duration and sleep efficiency (measured via accelerometry) and daytime fatigue (measured via a validated self-report questionnaire) at later ages; 2.) Examine whether salivary melatonin rhythms at 6-7 years mediate the association between CUF and sleep patterns/daytime fatigue among children as assessed in Aim 1; 3.) Determine whether urinary fluoride levels are associated with physiological sleep parameters and symptoms of sleep apnea among adolescents assessed at the Sleep- Wake Disorders Center at Montefiore Medical Center. The proposed research represents the first study to examine whether early childhood fluoride exposure predicts changes in childhood sleep patterns, daytime fatigue or melatonin rhythms. As such, it will advance the field of pediatric sleep research by providing valuable information regarding a modifiable potential risk factor for pediatric sleep disturbances. It will also help to inform titration of the appropriate dose of fluoride to maximize dental health efficacy and minimize risk. I will conduct this study in a cost-effective manner by levering available resources from a previously funded cohort. Lastly, I will translate the research and training in this proposal to position myself as an independent investigator with a tenure track faculty position.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY The objective of this proposal is to evaluate the comparative effectiveness of promising multi-component interventions for implementing evidence-based tobacco treatment in Lebanon’s national system of safety net primary healthcare centers, which serves more than 50% of the population. This objective is directly responsive to NCI Notice of Special Interest (NOT-CA-20-025): Dissemination and Implementation Science for Cancer Prevention and Control in Low Resource Environments. The tobacco use burden in Lebanon is exceptionally high: 35% of adults are current cigarette smokers, 39% are waterpipe smokers, and 5% are dual smokers. The WHO endorses three interventions as the standard of practice for population-level tobacco dependence treatment: smoking cessation advice integrated into primary care settings; easily-accessible phone-based counseling; and low-cost nicotine replacement therapy (NRT). However, the evidence for these interventions comes primarily from high-income countries and they have rarely been evaluated in low- and middle-income countries (LMICs). Further, recommended interventions are not integrated as a routine part of primary care in Lebanon, as is the case in other low-resource settings. Addressing this evidence-to-practice gap requires research on multilevel interventions and contextual factors for implementing integrated, scalable, and sustainable cessation treatment within low-resource settings. We will conduct a group-randomized trial comparing three arms: 1) Ask about tobacco use, advise to quit, assist with brief counseling (AAA) as standard care; 2) Ask, advise, connect to phone-based counseling (AAC); and 3) AAC+NRT. Our central hypothesis is that connecting patients to phone-based counseling with NRT is the most effective alternative. Our hybrid design is informed by the Exploration, Preparation, Implementation, Sustainment (EPIS) framework which emphasizes key steps and multilevel factors to optimize implementation success. We will pursue the following specific aims: 1) adapt and tailor an existing smoking cessation program to deliver phone-based counseling to smokers in Lebanon; 2) test the effectiveness and cost-effectiveness of a referral-based program that delivers smoking cessation services to primary care patients; and 3) identify the multilevel determinants of implementation and sustainability using mixed methods. The proposed project addresses the evidence-to- practice gap in the provision of tobacco dependence treatment within low-resource settings by developing and testing contextually-tailored multilevel interventions and optimizing implementation success and sustainability. This research is significant for its potential to guide the large-scale adoption of cost-effective strategies for implementing tobacco dependence treatment in low-resource settings thereby reducing tobacco-related morbidity and mortality.

NIH Research Projects · FY 2025 · 2022-09

A central question in behavioral biology is how environmental and genetic factors shape behavioral traits, allowing for substantial individuality. Homeostatic behaviors like feeding and food preferences are prime examples of genetic predispositions interacting with the food environment to produce diverse feeding habits. However, these programs are further modified by host-associated microbes, suggesting the overall systems controlling behavioral phenotype is far more complex than first anticipated. The critical questions that remain to be addressed are: to what extent do host-microbe interactions contribute to individual differences in behavior? Specifically, how does gene regulation of behavior operate in the context of host-microbe symbiosis? Implicit in this question is the hypothesis that perturbation of host-microbe symbiosis results in aberrant behavioral outcomes in certain individuals or populations, a topic fundamental to human health yet unsolved. The goal of my research program is to define how behaviors are shaped by host-microbe interactions. My lab studies foraging and food choice behaviors in the fly Drosophila melanogaster, using a gnotobiotic system I developed that permits precise configuration of the fly microbiome. I hypothesize that feeding behavior and food preferences are the product of host genetics-microbiome interactions, which determine, in part, an individual’s long-term health. Our recent data show that foraging and diet selection preferences vary by the microbiome, and altering the microbiome triggers transcriptional changes in specific cell clusters within the fly brain, involving genes predicted to affect behavior. In this five-year plan, I will leverage fly genetics and state- of-the-art genomics and metabolomics tools to identify the gene regulatory networks and mechanisms underlying microbial modulation of host behavior. Expected outcomes include comprehensive gut endocrine signaling and inter-organ communication maps at single-cell transcriptome scale and metabolite-gene interactions networks. The causal effects of microbiome-responsive genes, neuropeptides, and metabolites on host feeding behavior and food preferences will be revealed. Since we know little about how the interplay of host genetics and microbiome contribute to complex behavior, my research has the potential to advance fundamental knowledge in behavioral genetics but also to identify genes or processes that may function similarly in Drosophila and mammals as targets to treat behavioral disorders, such as eating disorders and obesity.

NIH Research Projects · FY 2025 · 2022-09

The stress hormone, cortisol, coordinates the body’s chronic metabolism, but also coordinates acute responses to stress by regulating stem cell differentiation in different tissues, restraining inflammatory and immune responses from being hyperactive, and coordinates acute metabolic responses to nutrient composition and feeding or fasting. Cortisol is also known as a glucocorticoid because it controls glucose metabolism. Synthetic glucocorticoids (GCs) are wide used for reducing pain and inflammation across many diseases, but can cause insulin resistance, muscle atrophy, and osteoporosis. We have developed an approach to studying GCs that enables us to understand how the glucocorticoid receptor coordinates these activities by binding to specific genes and changing the proteins made by those genes to control tissue-selective activity. This occurs as the GCs bind to the receptor and change its shape, enabling it to interact with different enzymes that control transcription. Previous efforts to understand these processes have been hampered by the fact that most of the existing GCs that we study are very similar. We instead make GCs that have a full range of desirable and undesirable activities, which gives us the statistical variance to understand them, a technique we call ligand class analysis, as we identify different classes of GC ligands that interact with different transcriptional regulatory enzymes to control expression of specific genes. Another major barrier has been the very wide expertise needed to understand the links between the GC chemical structure, receptor structure, the interacting coregulators, the regulated genes, and the tissue selective activities. We have assembled the required expertise and approaches to overcome these barriers by studying GC action on skeletal muscle, stem cell differentiation into bone, inflammation, and immune responses to colitis. We will use a chemical systems biology approach with novel GCs along with an integrative structural biology platform to define the signaling code for the glucocorticoid receptor in these aims: Aim 1 Biology: Identify which GC effects are correlated and which can be separated using physiologically relevant biology for the study of skeletal muscle, T cell differentiation, inflammation, and colitis, and osteoblast mineralization and effects on bone. Aim 2 Chemistry: Development of GCs to perturb GR structure and function in novel ways. We will enlarge three classes of compounds based on specific structural hypotheses to enlarge the chemical- structural space and test a novel steroidal scaffold to access new chemical space. Chemistry will focus on creating a diversity of effects on bone versus skeletal muscle while maintaining anti-inflammatory effects of the compounds. Aim 3 Structure: identify the structural underpinnings of selective GC signaling. We will apply machine learning and regression approaches to our structural and biological data to build and test models defining the mechanisms drive tissue-selectivity and identify which processes are globally coordinated to control differentiation and stress responses at an organismal level. Understanding the structural and molecular mechanisms of selective modulation will lead to breakthrough understandings of allostery and the development of improved GCs for medicine.

NIH Research Projects · FY 2025 · 2022-09

Project Summary: One of dura mater’s most important functionalities is to keep Cerebral Spinal Fluid (CSF) inside of the cranial cavity. To avoid life-threatening complications there exists a need for effective and reliable dural graft replacements post-surgery. A broad selection of dura grafts exists; however, no consensus has been reached on which graft is best suited to avoid dura mater ruptures and adverse immunological responses. Naturally derived and synthetic grafts are most used as dura replacements in the clinic. Native ECM constructs, when properly treated and handled, have the potential to preserve the overall architecture of the tissue matrix. Decellularized constructs have been shown to provide structural support, adhesion sites for cell attachment, and growth factors for cell proliferation. However, the commercially available naturally derived matrices undergo extensive processing, including lyophilization, which may be cause for clearing of relevant ECM proteins needed for sustained tissue health. The research question we propose to answer in this study is Do acellular xenogeneic dural grafts elicit a less deleterious immune response than naturally derived commercial grafts and maintain structural integrity over the long term for in vivo studies? We will examine the rigor of graft sealing strength, risk of infection and/or inflammation, and wound healing/tissue incorporation in vitro and in vivo within a rat model. Our central hypothesis is that native xenogeneic ECM grafts with multiple ECM proteins will perform better than the naturally derived (commercial) ECM dural grafts due to their improved mechanical and immunological responses assayed in vitro and in vivo. This application focuses on two, multi- objective specific aims. In Aim 1 we will perform in vitro studies focused on the mechanical response, structural integrity, immunological response, and scar formation associated with four grafts. We will examine two naturally derived, commercial dural grafts, Biodesign®, and Lyoplant®, and two tissue-based grafts directly from the source—porcine dura, and cadaveric human dura. In Aim 2 we will investigate, in vivo, the border integrity of all grafts by quantifying leakage of CSF. Additionally, we will assess scar formation and immune infiltration in our xenogeneic vs. naturally derived commercially available grafts. The knowledge gained from this work will provide empirical knowledge on the role of tissue-derived xenogeneic grafts for dural replacement. Increased surgeon confidence will enable appropriate graft selection for patients, reduced operative morbidity and mortality following cranial procedures.

NIH Research Projects · FY 2025 · 2022-09

PROJECT ABSTRACT Dystonia is a debilitating movement disorder characterized by involuntary muscle contractions with abnormal and repetitive movements, postures, or both. The current treatments for dystonia, consisting of oral medications, botulinum toxin injections, and deep brain stimulation surgery, fail to improve symptoms in more than a third of patients despite optimal management. The improvements are often unpredictable. Recent studies found exercise therapy primarily involving muscle stretching, muscle relaxation, and range of motion exercises, when added to optimal medical management, further improved symptoms of focal cervical dystonia. But the clinical outcomes were noted to be variable and modest. These studies did not investigate the pathophysiological underpinnings that could explain the variability of treatment outcomes. They did not include progressive resistance training that has the potential to induce central brain changes. The main goal of this proposal is to investigate the brain adaptation effects for progressive resistance exercise-focused cervical and shoulder training (PERFECT) in patients with focal cervical dystonia, which is the most common form of dystonia. We will use functional MRI (fMRI) and transcranial magnetic stimulation (TMS) techniques for understanding the pathophysiological underpinnings. We will randomize patients into PERFECT plus standard-of-care (SOC) group and SOC alone group. Participants in the PERFECT + SOC group will perform personal-trainer-guided exercises twice a week for six months. Participants in the SOC group will continue receiving standard pharmacological therapies at stable doses, and they will not exercise. We will use fMRI and TMS techniques to examine brain effects. We hypothesize that the PERFECT program in focal cervical dystonia will improve the functioning of dystonia circuitries involving the sensorimotor cortex. In Aim 1, we will determine the brain adaptation changes in response to PERFECT at six months compared to baseline with fMRI. We will record seed-based functional connectivity and bold oxygen level-dependent signal in the sensorimotor network. In Aim 2, we will determine the brain adaptation changes in response to PERFECT at six months compared to baseline with TMS. We will use a TMS-based paired associative stimulation protocol to measure sensorimotor plasticity and compare. We will monitor the TMS-based resting motor threshold; motor evoked potential, intracortical inhibition, and intracortical facilitation as additional measures. In Aim 3, we will determine whether the brain adaptation response relates to clinical and functional change. We will assess with blinded dystonia ratings, cognition and mood measures, muscle strength, physical functioning capacity. The outcomes of this proposal will (1) test whether a 6-month long resistance exercise is viable, (2) identify physiological brain markers that are responsive to effects of resistance exercise and are specific to dystonia circuitries, (3) provide essential data to for planning a large scale clinical trial.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT: The long-term objectives of the proposed program are to (i) discover type V CRISPR/Cas systems in exotic microorganisms with unique features, (ii) elucidate a deeper understanding of the rules and mechanisms of CRISPR/Cas and apply it for engineering and improving its activity, and (iii) apply them for gene editing and diagnostic applications for a range of diseases. Although the type II CRISPR/Cas9 is the most studied genome editing tool, the type V CRISPR/Cas12 systems are the most diverse with a wide range of functionally distinct single-effector Cas12a-k nucleases that are emerging as next-generation tools for both genome editing and nucleic acid detection. The central hypothesis is that (i) since the type V systems are most diverse and relatively newer, only a handful (<5%) of these systems have been properly studied, while a vast majority of these systems are understudied and poorly characterized and therefore, a systematic study of these systems will enable novel tools for genome engineering, chromatin imaging, base editing, and diagnostics. (ii) A deeper understanding of the sequence-structure-activity relationship by engineering crRNA and Cas will enable the development of improved tools for metagenomic analysis, combinatorial enzymology, and multiplexing strategies for genome editing and diagnostic applications. While the type V CRISPR/Cas share challenges of poor delivery, low gene correction efficiency, and high off-target cleavage associated with other CRISPR-based genome editing tools, they possess both orthogonal and overlapping challenges for diagnostic applications, including a) low catalytic efficiency or poor sensitivity, b) high tolerance of mismatches or low specificity, c) poor stability for deployment, and d) lack of control, desirable for multiplexing. In the first program, novel orthologs of type V CRISPR/Cas systems will be discovered by metagenomic mining of exotic microorganisms that can thrive at extreme conditions followed by expression and purification of Cas enzymes and crRNAs, identification of protospacer adjacent motif requirement, and testing of enzymatic activity in a high-throughput fashion. In the second program, crRNAs and Cas proteins will be modified with various strategies to improve target specificity and activity. Modified crRNAs and Cas would allow elucidation of mechanisms of CRISPR/Cas systems that could further allow improved detection of target DNA or RNA. Finally, integrating novel and engineered CRISPR/Cas with model systems would enable the development of multiplexed technologies that will have broader impacts in the detection and treatment of a wide range of diseases. The PI's lab has already made significant contributions in all three proposed programs with several key collaborations and publications and is poised to run a successful research and training program. The expected outcomes of the support from the Maximizing Investigators' Research Award (MIRA) for Early Stage Investigators include the establishment of an integrative research program to discover, understand, and engineer unique CRISPR/Cas systems and addressing of major problems in their applications for diagnosing and treating infectious diseases, cancers, and genetic disorders.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Deuterium magnetic resonance imaging, DMI, has demonstrated ability to identify changes in brain metabolism associated with cancer. However, downstream products of glucose metabolism, glutamate/glutamine (glx) and lactate, have intrinsically low signal to noise ratios that make detection challenging. This work posits that imaging of HDO following metabolism of [2H7]glucose will produce a more sensitive means of detecting glycolysis and glucose oxidation in the Krebs cycle. Towards this end we will establish optimal dosing levels of substrate as well as the impact of insulin sensitivity on cerebral metabolism of the glucose substrate. The high signal to noise of the HDO peak allows simple gradient echo methods to be used for detection, facilitating higher spatial resolution in the images. After injection of [2H7]glucose in vivo, a ~15 minute window exists where HDO appearance matches the generation of deuterated glx almost exactly, indicating the HDO can serve as a surrogate of oxidative flux in the brain. To take full advantage of this window, we will develop compressed sensing methods for accelerating acquisition of the 2H images. HDO is freely diffusible, and at long times we believe excess HDO appearance in the brain is related to vascular HDO generated by peripheral metabolism. We will use diffusion weighted imaging to test the hypothesis that suppression of the vascular HDO signal will render the remaining HDO component a faithful reporter of cerebral glucose metabolism. We will compare our new methods to 8F-deoxyglucose - positron emission tomography (FDG-PET) to determine if the two techniques provide complementary information about metabolism in animal models of brain cancer. The overall goal in this proposal is to establish HDO imaging as a robust marker of cerebral glucose metabolism. Relevance Metabolic imaging of the brain is of general interest across all of neuroscience, from basic biochemistry, to cognition science, to the study of pathophysiologies, and in the clinical diagnoses of multiple brain related diseases. The primary method currently used for metabolic brain imaging is FDG-PET, which cannot be used for longitudinal studies or in the pediatric population due to guidelines for total radiation exposure. Development of a magnetic resonance based method, which is safe for repeated use, would significantly enhance our ability to study brain function across all of neuroscience. The basic research described in this proposal will improve the robustness of a new method for detecting brain metabolism based on the detection of HDO following metabolism of a perdeuterated glucose tracer. Glucose is the primary substrate used for energy production in the brain, and is therefore the most appropriate substrate for development in this context.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Stigma adversely affects mental and physical health. Societal devaluation or mistreatment from others can lead to internalization of stigma (i.e., self-stigma) among individuals with marginalized identities or traits. Internalized stigma is robustly associated with impaired mental health and health-related quality of life (HRQOL), often to a stronger degree than are interpersonal experiences of stigma (e.g., discrimination). Experts have suggested that peer support or psychological counseling may help to reduce internalized stigma and its associated health impacts, although very few studies have investigated such interventions. Health-related stigma is an umbrella term that encompasses negative judgment, blame, avoidance, rejection, disparagement, discrimination, or dehumanization of individuals due to a health condition or disease. Such health conditions may be visible (such as obesity, skin diseases, or cancers that result in disfigurement), or concealable (such as HIV, diabetes, or chronic pain). Patients who internalize health-related stigma report avoidance of health care settings and disengagement from disease management behaviors, in addition to showing signs of physiological stress. Thus, health-related stigma compounds disease burden and threatens HRQOL beyond the direct effects of the health conditions themselves. Prior research on health-related stigma has been siloed, such that studies investigate one type of stigma, its effects, and possible solutions in isolation from stigma due to other health conditions. Consistent evidence of stigma’s harms on mental and physical health across health conditions, and shared theoretical foundations for intervention development, indicate the need for a unifying approach. An intervention that reduces internalized stigma among individuals with varying health conditions would have broad impact and would shift the paradigm of how stigma is currently addressed. The current project seeks to determine the effects of a novel, transdiagnostic, group-based counseling intervention designed to help patients cope with and to reduce the internalization of health-related stigma. An intervention and a self-report measure that were previously developed to address only one form of health- related stigma will be adapted to generalize to patients of other health conditions. After piloting, a randomized controlled trial will be conducted to test the effects of the group-based counseling intervention on internalized stigma, mental health, and HRQOL. The counseling condition (which also includes peer support) will be compared to a general peer support group and a waitlist control group. Participants will be 195 men and women who report high levels of internalized stigma due to their health condition. All study procedures will be conducted online, including assessments and treatment. Participants in the counseling and peer support groups will receive 12 weekly group sessions, followed by 2 every-other-week and 2 monthly sessions, for a total of 26 weeks. Findings from this study will provide new insights into the most effective and efficient method for reducing internalized stigma, and improving HRQOL, in patients with a wide array of health conditions.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Preserving movement-related independence is a clinical and public health priority, as well as a major goal of the National Institute on Aging and NIH’s strategic plan for research on aging. Our work has demonstrated that low- functioning (LF) older adults have a more rapid functional decline than those who are high-functioning (HF). The biological mechanisms that lead to accelerated functional decline in LF older adults remain poorly understood, and few therapies are available to prevent its progression. As diverse as the etiologies of physical disability are, a growing body of evidence strongly implicates the mitochondria (Mt) as playing a key role in the initial onset and progression of functional decline in many individuals. Mitochondrial dysfunction has been directly linked to accelerate telomere attrition, genome instability, epigenetic alterations, stem cell exhaustion, cellular senescence, impaired proteostasis, and deregulated nutrient signaling, all key hallmarks of aging. What’s more, perturbations in cellular and mitochondrial iron transport and handling may contribute to increased mutations and deletions, which lead to a reduction in mtDNA copy numbers and thereby negatively affect mitochondrial (Mt) function. Cellular iron import and export are critical for optimal cellular function. Iron levels are modulated by the hormone hepcidin via binding and subsequent degradation of the iron export protein ferroportin (Fn). Iron acquisition (import) occurs through transferrin receptor and is highly responsive to intracellular iron levels. We also have documented increased skeletal muscle Mt iron stores which increased oxidative stress and the susceptibility of Mt permeability transition pore (PTP) opening (a measure of Mt resiliency) with age. Based on our pilot data our central hypothesis is that greater circulating hepcidin levels and muscle iron deregulation (↓Ferroportin → ↑ Cellular and Mt Iron Levels → ↓TfR-1) in LF older adults will lead to Mt dysfunction (↑Sensitivity to PTP, ↑Deletions/Damage, ↓Mitochondrial Respiration) and accelerated progression of functional decline in LF compared to HF older adults. To test our central hypothesis, we will conduct a prospective longitudinal study in which we will follow HF and LF research participants (70 to 80 years) for 3 years and obtain plasma and skeletal muscle biochemical measures of iron regulation and mitochondrial dysfunction at baseline and throughout the follow-up period. The Short Physical Performance Battery (SPPB) will be used to classify LF (SPPB ≤ 9) and HF (SPPB > 10) participants. We will annually assess the participants’ physical function through established measures (SPPB, 6-minute walk, and muscle strength). We will assess changes in health behaviors, including activity levels, dietary intake, and sleep at annual follow-up visits. For the proposed study, we will examine cross-sectional and longitudinal associations of dysfunctional iron regulation with levels of Mt and physical function. If our hypotheses are correct, results will be used to design clinical trials testing pharmacologic therapies that target hepcidin and muscle abnormalities found to be associated with functional decline in LF older adults.

NIH Research Projects · FY 2025 · 2022-09

Project Summary The goal of this project is to understand how gene expression during development shapes the delicate and massively parallel cell biological processes that promote wiring of functional networks within the cerebral cortex. This is an important area of basic research because neural dynamics within cortical networks are the direct correlates of thought and behavior. These cognitive processes emerge as neural circuits form through expression of genes over the course of development. Moreover, cognitive impairment, which defines neurodevelopmental disorders (NDDs), is thought to arise, at least in part, from impaired neural circuit connectivity within the developing cortex. A revelation over the past decade is that NDDs can be caused by de novo genetic loss-of-function SNVs within a single gene. Thus, in-depth study of natural functions of these genes can reveal the neurobiological principles underlying the typically developing cortex and as well as principles that contribute to abnormal cortical development associated with NDDs. In this project, we will explore the hypothesis that expression of NDD-associated genes in the typically developing cortex promotes the assembly of cortical circuits through cell-autonomous regulation of intrinsic membrane excitability. This hypothesis is significant because it is known that neural activity shapes the assembly of developing cortical circuits. However, it remains unknown how genes function at the cellular level to promote activity-dependent in vivo development of cortical circuit motifs known to promote cognitive function and behavioral adaptations. Aim 1 will explore the causal relationships between genetic control of intrinsic membrane excitability (IME), activity- dependent dendritic morphogenesis, and developmental assembly of cortical circuits. Aim 2 will explore causal links between genetic control of IME, neuronal ensemble structure/function, and behavioral adaptations. We will do this by regulating genetic control of IME in developing cortical neurons and then observing the effect of this on cortical ensembles and behavioral adaptions. This research design is important because the brain functions across multiple temporal and spatial scales – indeed, this project attempts to link gene function across the major levels of brain function – gene>neuron>synapse>circuit>ensemble>behavior. The overall impact of this proposed research is that it has the potential to reveal how gene expression shapes the activity- dependent assembly of neural circuits that promote cognitive functions required for behavioral adaptations. Because we focus on natural functions of an NDD gene, these basic insights are also directly relatable to the etiology of cortical wiring impairments associated with childhood brain disorders.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Immunotherapy has shown profound benefit for adult patients but has yet to be fully unlocked for pediatric solid tumors such as osteosarcoma (OSA) where a significant percentage of children/adolescents succumb due to the presence of lung metastasis. OSA, like many poorly tumor immunogenic tumors, is defined by a lack of tumor specific targets and a regulatory tumor microenvironment (TME). Unleashing immunotherapy against poorly immunogenic cancers requires new technologies that activate the TME, while concomitantly engaging both innate and adaptive arms of the immune system to generate sustained cellular immunity. We have developed a novel (FDA approved) RNA-nanoparticle (RNA-NP) vaccine that simultaneously penetrates/reprograms the TME while inducing an OSA specific T cell response. This vaccine utilizes a novel engineering design that layers tumor derived mRNA into a lipid-nanoparticle (NP) “onion-like” package. We have shown that systemic administration of RNA-NPs safely mimics viremia, activating the quiescent immune system in only a few hours for induction of potent anti-tumor efficacy in several poorly immunogenic murine tumors resistant to immune checkpoint inhibitors. These RNA-NPs activate dendritic cells (DCs) that supplant regulatory intratumoral myeloid populations inducing antigen-recall response with long-term survivor benefits in murine metastatic pulmonary OSA models. We have established safety of RNA-NPs in acute/chronic murine toxicity studies, and launched a large animal canine OSA trial which demonstrated that RNA-NP administration is feasible, safe and immunologically active. While RNA-NPs mediate substantial anti-tumor activity, some animals suffer tumor outgrowth that warrant exploration of resistance mechanisms in our non-survivors. We have shown that RNA-NPs can be enriched for tumor specific antigens or configured with siRNAs to target pertinent regulatory axes (i.e. PD-L1), which can be studied in our murine/canine OSA models. The scientific premise for this work is that osteosarcoma is encased by a regulatory myeloid microenvironment that actively subverts adaptive immunity. We hypothesize that myeloid reprogramming of metastatic OSA will lead to safe eradication of disease. Our SPECIFIC AIMS are: 1. Establish mechanisms of OSA treatment resistance that can be overcome with adaptable RNA-NPs. 2. Identify correlates for vaccine response and escape in a comparative oncology canine OSA model. 3. Conduct a multi-institutional phase I/II study evaluating the safety and activity of the most promising RNA-NP formulation in recurrent OSA patients. Successful completion of this study will lead to a novel OSA therapy and a mechanistic understanding of its therapeutic effects that will be co-opted as biologic response correlates in a human clinical trial. .

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Significance: By examination of cancer patient genomic datasets, through the cBioPortal, we identified a new class of recurrent mutations in genes encoding histones H2A, H2B, H3 and H4. This supports an underlying premise, supported by rigorous publications from many groups including ours, that mutations of gene regulatory machinery, including histones are oncogenic drivers of human cancer. Many of these mutations affect residues of the histone folds important for the interactions among histones required for integrity of the histone octamer. Others may affect histone interactions with the chromatin modeling machinery. Histone fold mutations are found in 4% of lung (commonest tumor in US) and colorectal tumors, 6% of head and neck and 12% of bladder cancer. Histone mutations are subclonal. Tumors with histone mutations were significantly more likely to harbor mutations of oncogenic signaling molecules and tumor suppressive epigenetic regulators, suggesting that histone mutations augment the effect of other oncogenic lesions. Preliminary data: We characterized a recurrent mutation that changed amino acid 76 of histone H2B from glutamic acid to lysine (H2BE76K). This mutation disrupts the interaction of the H2B/H2A dimer with the H3/H4 tetramer, preventing formation of the histone octamer. Our Cancer Discovery paper showed that H2BE76K inhibits formation of nucleosomes in vitro and in yeast. Expression of H2BE76K in human cells disrupts chromatin structure, activates gene expression, promotes cell growth, and cooperates with PI3KCA to transform breast epithelial cells. We hypothesize that mutations that disrupt histone structure create millions of dysfunctional nucleosomes. These may deregulate gene expression and drive cancer development by creating new sites of “open” chromatin with loss of nucleosome-mediated gene repression, as well as by altered interactions with regulators of chromatin and gene expression. Approach: We will use cell and mouse models and epigenome profiling to characterize the oncogenic mechanisms of histone mutations, particularly in the transformation of lung cells, through three specific aims: 1) Elucidate mechanisms by which histone fold mutations alter cell growth, gene expression and chromatin structure. 2) Determine how histone fold mutations cooperate with co-occurring oncogenes and tumor suppressive chromatin regulators in cell and animal models. 3) Characterize the effects of histone mutations on tumor heterogeneity and response to therapy. Novelty: This proposal explores a new class of cancer-driving mutations to uncover novel mechanisms of oncogenesis by chromatin disruption. We will use the latest versions of gene editing to engineer histone mutations into cells, analyze tumor cells with advanced technologies for genome wide analysis of chromatin states and create new animal models. Impact: We will focus on lung cancer, where we estimate up to 10,000 patients/year will have tumors with histone mutations. The long-term objective of these studies is to uncover new mechanisms of oncogenesis that contribute to tumor progression, heterogeneity, and therapy resistance, with the aim of finding new targets and pathways for intervention.

NIH Research Projects · FY 2024 · 2022-08

COPD SUBTYPING AND EARLY PREDICTION USING INTEGRATIVE PROBABILISTIC GRAPHICAL MODELS ABSTRACT One of the main obstacles in developing efficient personalized therapeutic and disease management strategies is that most common diseases are typically defined based on symptoms and clinical measurements, although they are believed to be syndromes, consisting of multiple subtypes with variable etiology. Identifying disease subtypes has thus become very important, but so far it has been met with limited success for most diseases. In asthma, a notable exception, it was the clinical characterization that led to successful subtyping; and this is now incorporated in treatment guidelines. Unsupervised machine learning approaches of single data modalities (e.g., omics, radiographic images) have not produced actionable subtypes due to instability across cohorts. Developing data integrative approaches for multi-scale data, which are becoming available for a number of diseases, is expected to lead to robust subtyping and provide mechanistic insights of disease onset and progression. This proposal focuses on developing new computational methods, based on probabilistic graphical models (PGMs), to address this unmet need; and apply them to investigate three problems of clinical importance in chronic obstructive pulmonary disease (COPD), which is the fourth leading cause of mortality in USA. Our underlying hypothesis is that PGMs can integrate and analyze under the same probabilistic framework heterogeneous biomedical data (omics, chest CT scan, clinical) and identify disease subtypes and their main determinants. The objectives of our proposal is to build a comprehensive computational framework for disease subclassification, identify stable COPD subtypes at the baseline and longitudinally, and build interpretable models of the disease The deliverables of this project are: (1) new integrative computational approaches for clinical subtyping from multi-scale data; (2) new predictors of COPD progression and severity; (3) new discoveries of longitudinally stable COPD subtypes; (4) new predictors of future development of COPD; (5) new omics datasets that will be invaluable to future research in the area (baseline and longitudinal). To ensure the success of the project we follow a team science approach. This multi-PI proposal builds on the ongoing efforts of our group in the area of graphical models and their applications in biomedicine; and the ongoing collaboration of the three PIs that have complementary strengths: Prof. Benos (systems medicine and machine learning), Dr. Hersh (COPD genetics and genomics) and Dr. Sciurba (clinical aspects of COPD). It is powered by the access of the investigators to three major COPD cohorts (COPDGene®, SCCOR, ECLIPSE) that contain multiple parallel deep phenotyping and omics data from thousands of patients and controls. Although in this project we focus on COPD, our methods are generally applicable to any disease, therefore our project will have a positive impact beyond the above deliverables. We believe that due to their robust nature and interpretability, PGMs will soon become the norm for multi-scale biomedical data integration and modeling, when genetic and genomic data collection will become routine prognostic and diagnostic tools in clinical practice.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY CRISPR-based technologies have transformed science by enabling targeted DNA modification. DNA editing technologies have enriched our mechanistic understanding of DNA repair, and new insights in repair are frequently used to improve methods for gene editing. However, despite the remarkable achievements in targeted DNA modifications, RNA repair is understudied, and RNA editing tools are limited. The long-term goal of this proposal is to understand the molecular mechanisms that govern RNA repair and to develop disruptive new RNA editing tools for applications in science and medicine. The research proposed here integrates synergistic efforts to develop CRISPR-based tools for dissecting viral mechanisms of host cell take over and identify viral RNA repair mechanisms that support acute respiratory syndrome coronavirus (SARS-CoV-2) replication. In Aim 1, I will repurpose RNA-targeting CRISPR systems for deleting, inserting, and substituting sequences in viral RNAs. I have designed CRISPR-Cas systems to delete regions of ORF7a in the SARS- CoV-2 genome and recreate naturally occurring mutations. Based on my previous work, I anticipate that targeted deletions in ORF7a limit viral suppression of the host interferon response and lead to a replication defect. Further, tools developed in this application will be used to test emerging viral variants for new phenotypes. Aim 2 investigates the antagonistic activities of host antiviral nucleases and host RNA ligases in the replication and evolution of SARS-CoV-2. Aim 3 develops a CRISPR-based RNA capture system to enrich sequence-specific RNAs from a complex mixture. I will use CRISPR-based enrichment to map RNA modifications in SARS-CoV-2 viral RNAs and identify molecular signatures associated with interspecies transitions. Successful completion of the K99 phase of this application will require training in the Biosafety Level 3 facility, bioinformatics training to identify and annotate CRISPR systems, as well as new competencies in RNA biochemistry. Collectively, the research and training objectives outlined here establish a solid scientific foundation that will facilitate my transition to independence.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract The candidate currently serves as an Assistant Professor of Engineering Management and Systems Engineering (EMSE) with a joint appointment in Biological Sciences at Missouri University of Science and Technology (Missouri S&T), a member institution of the University of Missouri (UM) System. Before joining Missouri S&T, the candidate obtained an MS degree in Biomedical Informatics (BMI) and completed a National Library of Medicine (NLM) Postdoctoral Fellowship in BMI at Department of Biomedical Informatics (DBMI) at University of Pittsburgh (Pitt). The candidate’s long-time research goal is to become an independent researcher with an extramurally supported research program concentrating on inferring the activation states of signaling pathways from multi-omics data and utilizing it in precision medicine for cardiovascular diseases. In this K01 application, the candidate has assembled a strong mentoring committee from both Pitt and UM System. The training, mentorship, and research opportunities provided by this K01 award will significantly strengthen her expertise in multi-omics analytics, causal inference, deep learning, and more importantly will help build her expertise in complex cardiovascular diseases and their risk factors. This K01 award is critical in transitioning the candidate into an independent investigator in multi-omics analytics for precision medicine in cardiovascular disease. In this proposal, the candidate proposes to pursue the following aims: develop and evaluate an instance-specific causal inference (ICI) framework to identify causative genomic variants for blood pressure regulation (Aim 1); harmonize a large mixed-ethnic cohort from The Trans-Omics for Precision Medicine program and apply ICI and GWAS to better understand the role of genomic variants in racial disparity in hypertension prevalence(Aim 2); apply and evaluate both population-based and instance-specific predictive machine learning models for hypertension prediction by integrating genomics and other omics data (Aim 3). If successful, this project will develop and evaluate a novel, instance-specific method for discovering individualized genomic variants of hypertension, for better understanding the genomic basis of racial differences in hypertension, and for more accurately and timely predicting the development of hypertension for intervention and prevention. Moreover, the developed methods will be applicable to other cardiovascular diseases and risk factor as well.

NIH Research Projects · FY 2026 · 2022-08

Project Summary The mucosal epithelial barrier becomes disrupted in many conditions such as surgical procedures, trauma, inflammatory bowel diseases, ischemia, infections, radiation, and chemotherapy. Epithelial cell regeneration is essential for mucosal wound closure and healing. Our overall goal is to understand the mechanisms that lead to effective mucosal wound closure. This proposal builds on our recent discovery of an intestinal mucosal wound repair mechanism involving CD74 signaling. CD74 is the receptor for the MIF cytokine. The proposal aims to address significant questions that remain unanswered in the CD74 signaling pathway and wound repair, and are worth exploring. Our research is motivated by three key questions based on our published and preliminary data: What is the mechanism that drives epithelial CD74 receptor expression (Aim 1)? Can we safely and effectively stimulate CD74 (Aim 2)? What are the functional consequences of human genetic variation on CD74 activity (Aim 3)? Knowledge gained from these studies will significantly advance our mechanistic understanding of CD74 biology and mucosal wound repair, and may lead to new therapeutic strategies for promoting mucosal wound healing.

NIH Research Projects · FY 2024 · 2022-08

ABSTRACT. Haitians comprise one of the fastest growing subgroups of immigrants in the US. These immigrants experience high levels of psychological distress (i.e., depression, anxiety, fatigue, and pain), contributing to progressive functional impairment, disability, economic burden, and poor long-term health outcomes. Knowledge of factors contributing to psychological distress early in the post-migration period and longitudinally will help inform type and timing of interventions to reduce the disabling effects of psychological distress and improve quality of life for this underserved population. Yet no studies have examined psychological distress and its underlying biobehavioral, psychosocial, and cultural characteristics in Haitian immigrants within the first few months of residence in the US or over time. While multiple biological processes may be associated with psychological distress, rising evidence suggests that gut microbiome (GM) diversity and composition play an important role via the bidirectional microbiome-gut-brain axis. After migration, changes in GM composition and diversity embody changes in the social determinants of health ([SDoH]; e.g., stress, sociodemographic factors, lifestyle behaviors, dietary patterns, acculturation, environmental and sociocultural conditions) that also contribute to risk for psychological distress. With more time in the US, the GM of recent immigrants becomes more Westernized, with reductions of microbial phylogenetic diversity and native GM species as well as genera-level shifts in microbiota abundance. The SDoH that drive these GM changes are modifiable through culturally responsive interventions. The GM thus has the potential to serve as both an early indicator of risk for psychological distress and a tool to mitigate its effects. The overall goal for this longitudinal pilot cohort study is to investigate associations between psychological distress, GM composition/diversity, and post-migration SDoH in recent Haitian immigrants to the US. Specific aims are to 1) Characterize the GM in 60 recent Haitian immigrants by analyzing self-collected stool samples at T1 (< 6 months in the US) and T2 (6 months after T1) and describe changes in composition/diversity over time and 2) Examine longitudinal associations between post-migration SDoH, GM composition/diversity, and psychological distress among recent Haitian immigrants. I hypothesize that, in recent Haitian immigrants, a) migration to the US is associated with changes in GM composition and diversity over time, and b) changes in GM composition and diversity are associated with changes in post-migration SDoH and psychological distress. The PI has assembled an interdisciplinary mentoring team of senior scientists with expertise in symptom science, GM, biocultural approaches, immigrant health disparities, genomics, and bioinformatics. This project will provide the foundation for the PI to build an independent translational research program focused on developing culturally responsive targeted interventions that mitigate psychological distress, improve long-term health outcomes, reduce health disparities, and increase health equity in ethnic-minority immigrant populations.