University Of Pennsylvania

NIH Research Projects · FY 2024 · 2021-08

PROJECT SUMMARY Background. The risk of stillbirth, preterm delivery, low birthweight and other adverse pregnancy outcomes is extremely high in Sub-Saharan African countries including Botswana, especially among HIV-positive women. Daily iron and folic acid supplementation during pregnancy could reduce adverse pregnancy outcomes by reducing maternal anemia. However, the effectiveness of supplementation in subgroups defined by HIV status, anemia, gestational age, and other clinical, demographic and geographic factors remains unknown and compliance with supplementation guidelines is variable (<50% in Botswana). Cost-effective interventions to provide supplementation to pregnant women in Botswana and other resource-limited settings should be designed to address key barriers to successful supplementation and to reach subgroups that would benefit most from intervention. This K01 provides a unique opportunity to address this gap in the literature. Candidate Overview. My long-term career objective is to become an independent investigator who specializes in interventions to improve maternal and child health in resource-limited settings. My background is in applications of epidemiologic methods to evaluate effects of HIV treatment strategies in adults and infants. A K01 award will provide the necessary additional training and experience needed to become an expert in the design, implementation, and evaluation of interventions to improve pregnancy outcomes in HIV-positive women and underserved populations. Career Development and Training Plan. My education program includes structured course work, professional conferences, directed readings, and a research project with specific aims timed to coincide with these activities. My mentorship team includes international experts who specialize in areas related to my training goals: Dr. Scott Braithwaite (decision science), Dr. Roger Shapiro (HIV and maternal and child health in Botswana), Dr. Donna Shelley (intervention development and implementation), and Dr. Carolyn Berry (qualitative methods). Environment. NYU School of Medicine provides an exceptional environment for me to conduct the proposed study, obtain additional training and mentorship, and successfully transition to an independent investigator. Research Strategy. Using the data and infrastructure from an ongoing birth outcomes surveillance study in Botswana consisting of data from over 130,000 births, my study will: 1) estimate the effect of iron and folic acid supplementation during pregnancy on adverse pregnancy outcomes by HIV status, anemia, gestational age, and other key subgroups to identify populations that would benefit most from intervention; 2) assess barriers to supplementation during and prior to pregnancy; 3) optimize a hypothetical intervention to provide supplementation using computer simulation; and 4) develop and test the feasibility of an intervention to provide supplementation to pregnant women in Botswana.

NIH Research Projects · FY 2025 · 2021-08

Abstract Protein homeostasis is crucial to maintain healthy cells and is predominantly controlled by the ubiquitin proteasome system (UPS) whereby proteins are tagged with ubiquitin, via a cascade of 3 enzymes, resulting in recognition by the proteasome and subsequent degradation. While some proteins are constitutively recognized and degraded by this system, others are marked as substrates for the UPS by post-translational modifications such as phosphorylation. Recently, acetylation of non-histone proteins has emerged as an important mechanism of regulation for the ubiquitin-proteasome system, particularly at the level of E3 ligase substrate recognition. Leveraging our expertise of the ubiquitin proteasome and protein-protein interactions we propose to elucidate the molecular mechanisms and biological pathways resulting in acetylation driven modulation of protein homeostasis (Project 1). Additionally, building on our previous work with proteolysis targeting chimera, we will develop heterobifunctional approaches to modulate protein acetylation states as a novel mechanism to control protein homeostasis for both the study of this fundamental biological regulation and as a potential therapeutic approach (Project 2). In Project 1, we will identify and characterize proteins with stability regulated at the level of post-translational acetylation. Using proteomics experiments paired with RNA-Seq we will generate a database of proteins with intracellular levels directly controlled by p300 driven acetylation, not altered at the level of transcription. Furthermore, we will characterize the molecular recognition of acetyl degron substrates by the relevant E3 ligases using biophysical, biochemical and structural approaches, revealing unique insights into this mechanism of protein homeostasis. In Project 2, we will develop heterobifunctional compounds which recruit an acetyltransferase or deacetylase to a neo-substrate. Building on the concept of chemically induced post- translational modifications, exemplified by proteolysis targeting chimera, we will identify the (de)acetylation machinery most amenable to this approach via chemical biology approaches before designing and synthesising compounds to edit acetylation in native systems.Together these projects provide insights into basic biological processes regulating protein stability and a novel chemical biology approach to modify them.

NIH Research Projects · FY 2025 · 2021-07

Project Abstract Vertebrates repair skin injury through two fundamental biological processes: scar formation and tissue regeneration. Human skin generally heals with scar formation, which may cause severe emotional distress and physical disability. One hundred million new scars appear annually in the US, and although many products are marketed for scar prevention, their results are modest. Consequently, the elucidation of mechanisms underlying scar formation and tissue regeneration may result in new insights with far reaching implications in the development of therapeutics that promotes skin regeneration after wounding. Ear hole closure and wound- induced hair neogenesis (WIHN) are two instances where adult mammals can regenerate full-thickness skin wounds without scar formation, including hair follicles and sebaceous glands. Using both models, we demonstrated that topical pharmacologic activation of transient receptor protein A1 (TRPA1), a receptor expressed on skin sensory nerves promotes regeneration. This improved healing depends on dermal dendritic cells activating γδ Τ cells through interleukin 23. Strikingly, local activation of TRPA1 promotes tissue regeneration in distant injured areas, suggesting that a circulating factor may be induced with an accompanying paracrine mechanism. Our results reveal a new cutaneous neuroimmune-regeneration pathway, and a fundamental advance for the field would be a more comprehensive molecular understanding of signaling pathways involving multiple cell types that promotes mammalian tissue regeneration. In Aim 1, we use a combination of optogenetics, mouse models, and single-cell genomics to investigate whether TRPA1 on skin sensory nerves is necessary and sufficient to promote tissue regeneration in our two wounding models and to identify the molecular mechanisms of how TRPA1 expressing neurons locally activate immune cells. In Aim 2, we use mouse models to elucidate how γδ T cells and their effector cytokines promote systemic scarless tissue regeneration in our two wounding models. Together, our aims define mechanisms of cellular cross talk between nerves, immune cells, and skin, and successful completion will contribute to our overall goal of developing novel therapies to promote scarless skin regeneration in humans.

NIH Research Projects · FY 2025 · 2021-07

Establishment of tendon hierarchical structure is critical to mechanical function. This tightly controlled process requires coordinated cell-cell and cell-matrix communication. During embryogenesis, tendon progenitors organize into linear arrays and establish cell-cell communication prior to assembling ECM, suggesting that cells dictate ECM organization. Cells also clonally expand within linear arrays, suggesting that the ECM also dictates cell organization. Collagen XII is known to regulate collagen fibril assembly by forming bridges between fibrils, and our recent data show that collagen XII-deficient tendons exhibit reduced fibril packing and loss of distinct fiber domains. Interestingly, we also found that these tendons have disordered tenocyte arrangement and gap junction organization, indicating a novel role for collagen XII in cell organization, cell communication, and establishing an organized tenocyte network. However, the extent to which disrupted tendon hierarchical structure due to collagen XII deficiency is driven by disordered cellular arrangement and communication or by the deposition of disorganized ECM remains unelucidated. Therefore, our overarching goal is to establish the temporal roles of collagen XII in regulating tendon cell organization, hierarchical structure, and mechanical function during tendon development and healing. Our global hypothesis is that, in addition to ECM fibril assembly, collagen XII regulates cellular arrangement and communication prior to ECM deposition during development and healing, which is pivotal to establishing normal tendon structure-function. We will use novel tissue-targeted and inducible Col12a1 knockout mouse models to specifically target tendons during development and healing. These mouse models will be used in conjunction with an innovative multiscale approach to assess tissue level mechanics, cell organization and communication, fiber alignment, and fibril size/organization. Aim 1 will define the temporal roles of collagen XII in regulating cell arrangement and ECM assembly during tendon growth and development. Targeted knockdown of Col12a1 will be induced throughout tendon development (Scx-Cre driver; Aim 1a) or following establishment of cell organization (Scx- CreERT2 driver; Aim 1b). Temporal studies will also be conducted using 3D cell-gel constructs to evaluate tissue formation without confounding variables found in vivo. Aim 2 will define the temporal roles of collagen XII in regulating cell arrangement and ECM assembly during tendon healing. Using the SMA-CreERT2 driver, Col12a1 knockdown will be targeted to peritenon-derived progenitors, the primary contributors to healing tendon following injury. In Aim 2a, SMA-expressing cells will be targeted during the proliferative phase, while in Aim 2b, SMA-expressing cells will be targeted at the end of the proliferative phase to isolate contributions to ECM assembly. We will utilize sophisticated and rigorous measures of hierarchical structure/function to define the interplay between cell and ECM assembly in tendon formation through establishment of temporal roles for collagen XII in these processes. These innovative studies will provide guidance for future therapies.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Deciphering how natural genetic variations influence complex phenotypic traits is a central goal in human genetics and evolutionary biology. Over the past decades, empirical studies in model systems have conceptually advanced the genetic understanding of natural variations in physiological and morphological traits. In contrast, how natural behavioral variations arise from genetic changes altering neural functions remains a fundamental mystery. Although numerous correlations between genetic variations and behavioral variations have been identified across a wide range of species including human, the identity and functional nature of casual genetic changes are largely out of reach. The evolution of courtship song in closely related Drosophila species provides a rare opportunity to establish such causal links, by offering extraordinary behavioral diversity and unparalleled genetic, neuronal and behavioral tractability. The overarching goal of this proposal is to leverage this powerful system and employ our innovative platforms to identify causal genetic and molecular changes that mediate the evolution of the nervous system and behaviors. Our research will proceed in two independent but complementary directions. First, by combining a targeted genetic mapping approach and automated behavioral quantification with unprecedented throughput, we will perform a gene-resolution dissection of defined trait-associated genomic regions to delineate the causal genes and mutations underlying a species-specific courtship song trait. Second, building on our knowledge of relevant neurons in courtship song evolution and our expertise in genetically labeling these neurons across species, we will exploit possible neural sites of adaptive changes as an entry and use single-cell transcriptomes to probe gene expression changes, especially cis-regulatory changes, that are responsible for species-specific neural functions and behaviors. Importantly, for each direction, we will examine the phenotypic effects of candidate genetic and molecular changes using integrative approaches such as definitive genetic tests and neuron-specific gene silencing. The two directions are synergistic in connecting the dots between evolutionary changes at different levels of biological organization. This study will lead to a rich understanding of how evolution operates on the genes and the nervous system to yield adaptive behavioral traits, which will allow us to derive principal mechanisms generalizable across species, and in turn, inform the etiological conditions of behavioral disorders and mental disease in human.

NIH Research Projects · FY 2025 · 2021-07

During B cell lineage commitment, a dynamic shift of genes between transcriptionally restricted and transcriptionally permissive compartments at the pre-pro-B to pro-B cell transition results in activation of the B lineage program and repression of alternative lineage programs. While B lineage commitment is generally believed to be driven by lineage-specific transcription factors, we have made the surprising discovery that conditional knock-out of the ubiquitous transcription factor YY1 results in loss of B lineage commitment, allowing subsequent development into the T cell lineage both in vitro and in vivo. Pioneer transcription factors such as Ebf1 promote transcription of B lineages genes and repress expression of alternative lineage genes to initiate B lineage commitment, but stable commitment requires changes in chromatin structures at the pro-B cell stage. As YY1 is a key factor controlling lineage-specific gene regulatory long-range chromatin interactions (LRCIs), we hypothesize that YY1 knock-out in pro-B cells results in loss of chromatin LRCIs that stably maintain B lineage-specific gene expression. Consistent with this, we found reduction of B lineage transcripts after YY1 knock-out. YY1 can also mediate Polycomb Group (PcG) repression, and we found that YY1 knock-out resulted in increased expression of alternative lineage genes, suggesting that YY1 loss abrogates repressive chromatin structures needed to prevent expression of these genes. Thus, we hypothesize that YY1 knock-out in pro-B cells results in lost chromatin structures that stably maintain lineage-specific gene expression, as well as loss of repressive chromatin structures needed to prevent alternative lineage gene expression, thus leading to lost B lineage commitment. To test this, we will determine chromatin folding patterns, nuclear localization of key genes, chromatin accessibility, and epigenetic structures in wild-type and YY1-null pro-B cells to define the genomic structures regulated by YY1 during B lineage commitment. To determine if analogous effects of YY1 are operative in the T lineage, we will determine if YY1 loss promotes lineage plasticity of YY1-null DN3 cells. YY1 is also necessary in pro-B cells for Igk locus contraction required for rearrangement of distal Vk genes. It has been suggested that YY1 plays a structural role in regulating chromatin structures, but it is unclear if this requires the YY1 transcriptional activation, PcG, or self-association functions. We will utilize an established panel of YY1 mutants that are compromised in these functions to assess in parallel, the mechanisms of YY1 regulation of chromatin structures needed for B lineage commitment, and those needed for Igk locus contraction and Jk-Vk rearrangement. As YY1 is involved in embryogenesis and development of multiple tissue types, determining how YY1 controls genomic structures to specify B lineage commitment will provide a new paradigm for the function of a ubiquitous factor in lineage-specific development.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY Soft-tissue sarcomas (STS) are a diverse and often fatal set of malignancies arising from connective tissue with a 16% five-year survival rate for metastatic disease, reflecting the need for novel therapeutic strategies. One approach showing promise against multiple cancers is immune checkpoint blockade. However, clinical trials of immunotherapy in STS have produced disappointing results, likely due to immunosuppressive microenvironments characteristic of these diseases. The STS microenvironment is dominated by tumor- associated macrophages, which largely differentiate from tumor-infiltrating monocytes. These can also differentiate into anti-tumor monocyte-derived dendritic cells (Mo-DCs) in inflammatory conditions, but Mo-DC differentiation is inhibited in STS by as-yet unknown factors. We have recently shown that enhanced Mo-DC differentiation leads to synergy with immune checkpoint blockade. It is therefore critical to discover additional processes that regulate Mo-DC differentiation in order to improve the efficacy of immunotherapy against STS. One such mechanism may depend on glutamine metabolism. Glutamine is utilized as a metabolic fuel by several immune cell types, as it is involved in generating biosynthetic products via the rate-limiting enzyme glutaminase, glycosylation of proteins, and activation of mammalian target of rapamycin complex 1 (mTORC1) mediated signaling pathways. Additionally, recent work has shown that blocking glutamine metabolism can modulate the anti-tumor activity of tumor-infiltrating immune cells. Using an in vitro model of Mo-DC differentiation, we found that glutamine deprivation blocked Mo-DC differentiation, surprisingly independent of glutaminase activity. This finding suggests Mo-DC differentiation requires glutamine flux through separate pathway(s), such as the hexosamine biosynthetic pathway (HBP) or glutamine-leucine antiport leading to mTORC1 activation. I hypothesize that glutamine metabolism regulates monocyte-derived dendritic cell differentiation in the soft tissue sarcoma microenvironment through either the hexosamine biosynthetic pathway or leucine-dependent mTORC1 activation. Aim 1 will identify the mechanisms by which glutamine metabolism regulates the differentiation of monocytes into Mo-DCs. Isotopic labeling and mass spectrometry will be used to assess the incorporation of glutamine into downstream metabolites of glutaminase as well as HBP intermediates and export from the cell. Conditional knockout mice will be used to assess the contributions of HBP flux and mTORC1 activation to Mo- DC differentiation. Aim 2 will test the impact of glutamine availability and inhibition of glutamine metabolism on Mo-DC differentiation and function in the STS microenvironment, as well as synergy with immune checkpoint blockade. Together, these approaches will elucidate the role of glutamine metabolism in Mo-DC differentiation and function in the STS microenvironment.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract. An eradicative HIV cure requires safe and effective clearance of replication competent virus from all reservoirs, including the brain. Characterization of the CNS reservoir and empiric testing of novel cure strategies require physiologically relevant animal models of HIV persistence in the brain. Here, we propose to integrate major advances from our groups in SHIV NHP models and AAV-delivered CRISPR/Cas9 editing to delineate key features of the CNS reservoir and determine the efficacy of CRISPR-based eradication in the brain. Transmitted/founder (TF) SHIVs, which encode minimally adapted TF HIV-1 Envs, represent a major advance in biologically relevant NHP models. TF SHIVs have demonstrated robust replication in rhesus macaques, with viral kinetics, cell tropism, and pathogenesis that mirror HIV-1 infection of humans. Further, they recently been shown to faithfully recapitulate virus – host interactions, persist through suppressive ART, and rebound with similar kinetics and clonality as HIV-1. Here, we will employ a novel, genetically barcoded TF SHIV model of CNS pathogenesis and persistence, based on TF SHIV.D.191859 (SHIV.D), which encodes a clade D TF HIV- 1 Env that is CCR5-tropic, efficiently replicates in CD4 T cells and monocyte-derived macrophages, and demonstrates consistent CNS replication, pathogenesis and persistence. Using this barcoded TF SHIV.D model CNS persistence, we will test a novel all-in-one AAV9-mediated CRISPR/Cas9 gene editing system. A recent first-in nonhuman-primate study of SIV-infected rhesus macaques demonstrated the tolerability and efficacy of this approach. The AAV9-CRISPR-Cas9 was broadly distributed across tissues, leading to cleavage and excision of the SIV genome and substantial reductions in the size of the proviral reservoir across tissues. Notably, the AAV was well distributed within CNS resulting in excision of provirus across brain regions. In this application, we will leverage advances in the macrophage-tropic barcoded SHIV.D model and AAV-delivered CRISPR/Cas9 editing to gain insight on CNS neuropathogenesis and persistence. Our hypothesis is that by (i) characterizing SHIV.D persistence in key CNS cells and tissues, (ii) optimizing AAV-delivered CRISPR-Cas9 approaches for SHIV.D persistence in the brain, and (iii) testing the effects of global and myeloid-targeting CRISPR approaches on CNS reservoir reduction in vivo, we will advance prospects for eradicating HIV from the brain. If this hypothesis is affirmed, the significance to HIV cure field would be substantial, since it would improve our understanding of the CNS reservoir, develop a robust model for HIV pathogenesis and persistence in the brain, and provide key pre-clinical data on the safety and efficacy of a promising CRISPR-based cure strategy.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Over 13 million people develop acute kidney injury (AKI) each year. AKI increases the risk of incident chronic kidney disease, end-stage kidney disease, and death. Fluid removal (ultrafiltration) is an important component of managing dialysis-requiring AKI (AKI-D), but safe achievement of optimal volume status involves a delicate balance between preventing fluid overload and avoiding circulatory compromise. Acuity of illness and comorbid conditions render patients with AKI highly susceptible to even modest hemodynamic changes, and routine blood pressure monitoring may be inadequate to detect subtle, yet clinically meaningful, perfusion changes during hemodialysis. Continuous monitoring of systemic or local tissue perfusion is feasible using a variety of near-infrared optical technologies, but there has been limited application of these devices during hemodialysis particularly in the setting of AKI. The overarching premise of this proposal is that optimizing fluid management in an already vulnerable AKI population requires (1) a marker that captures an individualized hemodynamic response to fluid removal throughout hemodialysis treatments, and (2) more sensitive techniques to detect hypoperfusion. Funded by an F32 award, Dr. Wang previously leveraged continuous hematocrit monitoring (CHM) during maintenance hemodialysis treatments to calculate a semi-instantaneous plasma refill rate (PRR), which refers to the rate of refilling of the vascular space from the interstitial space during ultrafiltration. Through this K23 proposal, Dr. Wang will extend her investigations of PRR to the AKI setting and will evaluate novel approaches to assessing perfusion during hemodialysis. Specifically, Dr. Wang will perform mediation analysis using data from a large cohort of patients on maintenance hemodialysis to determine whether PRR mediates the effects of treatment-related factors on intradialytic hypotension (Aim 1); and, in a prospective cohort of patients with AKI-D, she will combine continuous hematocrit monitoring with two noninvasive techniques – diffuse correlation spectroscopy and diffuse optic spectroscopy ‒ to simultaneously measure changes in systemic perfusion (Aim 2) and cerebral perfusion and oxygenation (Aim 3) as a novel approach to elucidate the physiology underlying both overt and subtle hemodynamic effects of hemodialysis. Additionally, she will use bedside testing to link spatiotemporal changes in tissue physiology with changes in cognitive function. To conduct this work, Dr. Wang will utilize the formal methodologic training and practical experience in epidemiology, study design, and longitudinal analysis that she acquired through the Master of Science in Clinical Epidemiology Program. Through her K23 career development plan, she will gain (1) skills in designing and implementing prospective studies in dialysis, (2) experience studying outcomes in acute care, and (3) expertise in advanced methods for analyzing multidimensional data. Upon completion of this work, Dr. Wang will be well-positioned to obtain R01 funding to continue working toward her long-term goal of developing novel approaches that will increase the precision of renal replacement therapy and improve patient outcomes.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Cocaine addiction is characterized by compulsive drug seeking and high vulnerability to relapse even after prolonged abstinence. A major focus of the field of addiction research has therefore been to identify stable, cocaine-induced neuroadaptations occurring in brain reward circuits. Transcriptional changes are known to persist throughout abstinence, yet the underlying molecular mechanisms of such persistence remain elusive. We recently discovered that the transcription factor, Nr4a1 (nuclear receptor subfamily 4) represses cocaine reward and seeking behavior. Our preliminary data show that Nr4a1 is a central regulator of cocaine-induced transcription, including target gene expression in late abstinence. The significance of this study is strengthened by the utility of therapeutic agents that regulate Nr4a1 and block mouse cocaine self-administration, underscoring the enormous potential of this basic research program in combating drug addiction. Given that histone posttranslational modifications (hPTMs) confer long-lasting changes in gene expression necessary for stable cellular phenotypes, histone modifications acquired during abstinence may explain how individual genes “remember” prior drug exposure. We have previously found that Nr4a1 regulates hPTMs at individual target genes at late abstinence. This proposal aims to define the mechanism(s) of persistent gene expression in the nucleus accumbens (NAc) of male and female mice following volitional cocaine self-administration. We apply novel methods for cell-type specific quantification of both chromatin and gene expression in a single sample. We then validate the causal mechanism of Nr4a1 action using epigenetic editing in vivo. At the conclusion of this study we will have defined the cell-type specific mechanism by which Nr4a1 regulates stable gene expression across cocaine abstinence. Beyond this, we will apply machine learning to identify novel regulators of persistent gene expression relevant to cocaine addiction.

NIH Research Projects · FY 2025 · 2021-07

Modified Project Summary/Abstract Section Oral diseases and craniofacial disorders devastatingly afflict susceptible populations, particularly impoverished families and medically or physically compromised persons. Novel engineering approaches can yield new paradigms to reveal disease mechanism, new strategies for disease mitigation, and new approaches for affordable, personalized therapies for dental caries, periodontal diseases, and oral cancer. To achieve this vision, dentist-scientists must be adept in engineering concepts and engineers must be educated in oral and craniofacial sciences and needs; all must be aware of the complex clinical and regulatory landscapes. To address this urgent need, we propose a multidisciplinary training program in Penn’s School of Dental Medicine (PDM) and School of Engineering & Applied Sciences (SEAS) focused on training dentist-scientists and engineers for academic careers dedicated to precision oral healthcare innovation. Postdoctoral trainees will adopt cutting-edge approaches at the forefront of engineering and computational sciences to advance biofilm microbiome, host immunity and tissue regeneration, drawing on approaches including artificial intelligence, robotics, nanotechnology, and materials sciences, aware of developmental hurdles and requirements to safely bring new approaches to patients. Trainees will engage in interactive and tailored academic experiences with intensive mentored research via co-mentoring (one advisor from each school), and interaction with a career mentoring committee (CMC) that includes at least one clinician. Co-mentors and CMC members will be drawn from the Program Faculty in PDM and SEAS with significant records of mentorship of multidisciplinary and translational research. Training will include: (1) engineering fundamentals for dental trainees and oral & craniofacial biology principles for engineering trainees, tailored to match their research project and academic needs, (2) clinical research and regulatory affairs, (3) principles of scientific rigor and reproducibility, (4) scientific writing and (5) workshops on professional/career development. Trainees will engage in academic and industry interactions, journal club, give symposia presentations, and participate in AADOCR activities. Training will culminate in F/K award applications focused on advancing oral health at the dental-engineering interface. The applicant pool at the University of Pennsylvania is exceptionally strong, and we plan to enroll 4 trainees per year. Importantly, this effort will be integrated into a newly formed center between PDM and SEAS with shared funding. The proposed program, unique to Penn, leverages a superb research and training environment within a compact campus where resources for both schools are united through Penn’s new Center for Innovation and Precision Dentistry.

NIH Research Projects · FY 2024 · 2021-07

Project summary Post-acute care (PAC) is common, and costly, and may not lead to optimal health outcomes for older adults. However, it is unknown how to improve outcomes and/or lower costs, or value, of PAC. More than 40% of Medicare fee-for-service (FFS) beneficiaries receive PAC after hospitalization, predominantly in skilled nursing facilities (SNFs) and by home health (HH) agencies, at a cost of more than $60 billion annually. Unfortunately, around 1 in 4 is readmitted to the hospital within 30 days of discharge to PAC, and nearly half of beneficiaries in SNF fail to return to the community within 100 days of hospital discharge. The rapid expansion of Medicare Advantage (MA) provides an opportunity to evaluate a different approach to PAC utilization. More than one- third of all Medicare beneficiaries are now enrolled in Medicare Advantage (MA) plans, which receive capitated payments and take financial risk for the care needs of beneficiaries. MA plan directors confirm in interviews that PAC is a major focus of efforts to reduce care utilization and costs through four mechanisms: limiting use of PAC overall; steering beneficiaries to less expensive forms (HH) instead of more expensive forms (SNF); restricting choice of providers; and limiting PAC length of stay. Early reports suggest MA plans may strongly influence PAC utilization. Whether reductions in PAC utilization in MA improve PAC value is unknown. The limited existing literature has two main gaps: first, it does not adequately account for the substantial underlying differences in the MA and FFS populations. Second, it has focused on short-term outcomes, while PAC likely has a substantial impact on longer-term functional status and independence. Our overall goal is to inform patients, providers, and policymakers about ways to improve the value of PAC for all Medicare beneficiaries. To achieve this goal, we propose innovative analytic strategies that address limitations in the prior literature and allow accurate assessment of the use and outcomes of PAC in similar MA and FFS beneficiaries. Our aims are to: 1) Compare use of SNF and HH in similar MA and FFS beneficiaries after hospital discharge, and the impact of different mechanisms for limiting PAC utilization; 2) Compare PAC outcomes (community days, long-term institutionalization, rehospitalization, mortality) at 100 days and 1 year after hospital discharge in MA and FFS; and 3) Evaluate the effects of MA versus FFS enrollment on specific subpopulations of patients known to be at high risk for poor PAC outcomes. These results will provide novel insights into the effect of MA plans on PAC use and outcomes, identifying potential benefits or unintended consequences that can shape policy.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract Cardiovascular associated Advances the sensor exposures. easily analyzing conducting integrate issues focus and who and graduate, objectives workforce.” ability K24 data phenotypes health (CV) disease affects nearly half of all adult Americans. Although achieving “ideal CV health” i s with lower mortality nd morbidity, many individuals struggle with modifying CV health behaviors. in precision medicine offer promise in better targeting these behaviors to improve CV health. Over past decade, there has been exponential growth in digital data (e.g. social media, online search, remote measures) generated by individuals which reflects their health behaviors, attitudes, and environmental These data sources however are often not linked with validated measures, unprocessed, and not interpretable for use in research or clinical practice. My research portfolio has focused on collecting and CV focused digital data for insights, prediction, and clinical applications. My career focus is on high-impact atient-oriented research (POR) t o better understand how to best access, analyze and these data in a way that is patient-centered and highly applied. This includes work which focuses on related to privacy, security, and patient preferences. This K24 award will support protected time for me i ntently on: 1) implementing a research program to advance POR in CV health and digital data science 2) training a pipeline of investigators who are well trained in methodologies for conducting this work and will be future POR leaders i n this area of study. This proposal focuses on necessary steps for developing executing a comprehensive and structured mentorship program that will support mentees (undergraduate, fellow, faculty) for research and career development. This focus aligns with NHLBIs overarching in “data science” and “further developing, diversifying and sustaining a NHLBI focused scientific This proposal builds on my current NHLBI R01, R21, and Foundation grants and will support my to pursue career-oriented training in clinical trials, social media ethics, and academic leadership. This will also support two new areas of research: 1) building computational models using large digital media sets for population-level surveillance of modifiable CV health behaviors in real time, 2) developing patient reflecting use and engagement which can inform digital interventions to address modifiable CV behaviors. a p , The environment for this K24 is the University of Pennsylvania-a world renowned institution with extensive resources to support research at the intersection of medicine, computer science, communication, and engineering—fields critical for advancing this research and supporting interdisciplinary trainees. The areas of focus for this K24 represent new frontiers in precision medicine and digital phenotyping for CV health that can be advanced and led by well-trained interdisciplinary POR researchers.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT The goal of the proposed five-year training plan is the development of my independent research career as an academic adult hematology physician-scientist studying red cell biology and hemoglobin regulation. I have completed internal medicine residency and hematology/oncology fellowship training at the University of Pennsylvania, and I am currently a Senior Fellow/Program Scholar who will transition in July 2021 to an Instructor and attending physician in the Division of Hematology/Oncology at UPenn. I am specifically seeking to develop and refine the skills that will be required for a successful career as an independent investigator, including expertise in gene regulation, signaling pathways, functional genomics, and bioinformatics. My overarching goal is to improve therapeutic approaches for sickle cell disease (SCD) via study of key signaling pathways that regulate expression of the fetal form of hemoglobin. My mentor for this award is Dr. Gerd Blobel, an internationally recognized leader in erythroid gene regulation and hemoglobin switching. To add depth and breadth to my scientific and career guidance, I have assembled a Mentoring Committee composed of physician-scientists from diverse and complementary fields. I will have the full resources of UPenn and the Children’s Hospital of Philadelphia available for the completion of my research and career development goals. The goal of this proposal is to elucidate the molecular mechanisms of PP6C, a novel regulator of fetal hemoglobin, to improve upon current treatments for SCD and beta-thalassemia. SCD afflicts millions of people worldwide and can lead to severe complications including acute chest syndrome, stroke, avascular necrosis of bone, and nephropathy. Although increasing levels of fetal hemoglobin (HbF) significantly reduces cell sickling and SCD-related morbidity and mortality, effective HbF pharmacologic induction has been an elusive goal. To this end, I recently carried out a CRISPR-Cas9 based screen to identify additional potentially druggable molecules to increase HbF production; this screen uncovered the protein phosphatase PP6C as a novel HbF regulator. This proposal will explore PP6C-regulated pathways with both hypothesis-driven and unbiased approaches and will investigate suitability of PP6C as a target for HbF induction. These objectives will be achieved via three Specific Aims: to elucidate key mechanistic pathways in the regulation of HbF by PP6C, to explore potential cooperativities of PP6C with other HbF regulatory pathways utilizing CRISPR-Cas12a-based techniques; and to test the role of PP6C-mediated HbF regulation in SCD and in vivo models. The outcome of these studies will deepen our understanding of signaling pathways that govern HbF expression and unveil new therapeutic opportunities in SCD. Completion of these aims will consolidate my experience in models of hemoglobin switching, further my training in bioinformatics and functional genomics, and provide the basis for a future R01 funding proposal. These studies will leave me uniquely prepared for an independent career as a physician-scientist with a focus on red cell biology and hemoglobinopathies.

NIH Research Projects · FY 2024 · 2021-07

Project Summary Accurate segregation of chromosomes during cell division is one of the most fundamental requirements in biology. Without proper chromosomal segregation, genetic information cannot be faithfully transmitted across cell and organismal generations, leading to severe consequences including cell death, developmental defects, or progression of cancer. Furthermore, improper chromosome segregation in cancer cells has been shown to lead to anti-tumor inflammatory responses. Central to the process of chromosome segregation is the centromere, the chromosomal locus at which spindle microtubules bind. The centromere is defined epigenetically by the presence of nucleosomes containing the histone variant CENP-A. Centromeric chromatin serves as the foundation of the kinetochore, a large protein complex which assembles on CENP-A nucleosomes and mediates microtubule binding. Research into the centromere is necessary to better understand the processes that underlie chromosome segregation in both health and disease, but our understanding of the human centromere remains largely incomplete. This proposal aims to answer fundamental questions about the structure and function of the centromere and its associated proteins. Recent advances in reconstitution of large centromeric protein complexes have increased our understanding of the structure of the human kinetochore, but reconstituted complexes can only approximate in vivo structures, and currently there are multiple competing models for the structure and organization of the centromere and kinetochore. The emerging technology of cryo-electron tomography (cryo-ET) provides the opportunity to interrogate the structure of the centromere and kinetochore in their native context within vitreous hydrated cells. To this end, in Aim 1 cryo-ET will be used to obtain the first in situ structures of the human centromere and kinetochore in the interphase and mitosis stages of the cell cycle. The second focus of this proposal is to elucidate the interactions among centromeric proteins that are required for the essential functions of the centromere, including formation of microtubule attachments and maintenance of centromeric identity. Two kinetochore proteins, Ndc80 and CENP-Q, have both been shown to contribute to microtubule binding in vitro and in vivo, but their respective roles in microtubule binding in vivo have not been fully characterized. Multiple proteins within the constitutive centromere-associated network (CCAN) have similarly been shown to contribute to maintenance and deposition of CENP-A at the centromere, but the CCAN contains multiple interconnected subcomplexes whose contributions have never been systematically tested. In Aim 2 mutagenesis of the respective endogenous gene loci (and rapid depletion of the respective wild type gene products) will be used to elucidate in vivo and with temporal accuracy the interactions that underlie spindle attachment and maintenance of centromeric identity. These experiments will provide important insights into the structure and function of the human centromere and kinetochore which are essential for proper chromosome segregation and genomic fidelity across generations.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract α-Synuclein is a small, soluble neuronal protein that is the primary component of the Lewy body aggregates that are the hallmark of Parkinson's disease. While the mechanistic details are not yet well-understood, emerging evidence suggests that cell-to-cell transmission of toxic forms of α-Synuclein is the basis of disease propagation. Our lab has recently identified complex N-linked glycans as mediators of cellular internalization of both monomer and aggregate forms of α-Synuclein bearing an N-terminal acetyl group, a physiological modification of the protein. We specifically identified the neuronal glycoprotein neurexin 1β as capable of driving internalization of α-Synuclein in a glycan-dependent manner. The goal of our proposed research is to characterize the structural basis of α-Synuclein binding to neurexin 1β, the role of both N-terminal acetylation and glycosylation in conferring specificity in this interaction, and determine the molecular mechanisms resulting cellular internalization of α-Synuclein following binding neurexin 1β. Our hypothesis is that cell-to-cell transmission of αS is dependent on interactions with neurexin 1β and that the selectivity in these interactions is dependent on transient structural changes in α-Synuclein conferred by the N-terminal acetyl group. To investigate this hypothesis, we have developed three specific aims with the following goals: determine the structural features of α-Synuclein bound to neurexin 1β, including defining a minimal α-Synuclein construct required for binding (Aim 1); determine the mechanisms by which binding to neurexin 1β results in cellular internalization of α-Synuclein (Aim 2); and understand the functional impact of α-Synuclein binding to neurexin 1β (Aim 3). To achieve these goals, we will carry out in vitro coarse grain and high resolution structural characterization of α-Synuclein:neurexin 1β complexes and use live-cell imaging to quantify internalization of α-Synuclein and the ability of internalized α-Synuclein to seed aggregation of endogenous α-Synuclein. We will contrast WT monomer, PD-associated point mutants and fibrillar forms of α-Synuclein. Through this research we expect to characterize key interactions involved in propagation of α-Synuclein pathology in Parkinson's disease, as well as to gain insight into the structural features of α-Synuclein:neurexin 1β complexes. Ultimately, the characterization of α-Synuclein: neurexin 1β interactions carried out through our studies may provide a new target for Parkinson's disease treatment and serve as the basis identifying novel small molecule therapeutics.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT Injury to the cerebral cortex occurs frequently across the spectrum of severity in traumatic brain injury (TBI). No therapies exist to counter the neurological and cognitive deficits caused by these injuries, which are responsible for substantial disability after TBI. A promising strategy for restoring brain function after injury is cell replacement. Neural tissues that connect with host cortex locally and function as supplementary cortical processing modules are especially intriguing candidates for this approach. Currently available tissue substrates that are suitable for translation, including human brain organoids derived from patient-matched stem cell lines, do not fully recapitulate the architecture or micro-circuitry of cortex. However, they can still be used to investigate outstanding questions regarding neural tissue integration with the host brain. One essential issue that has not been examined systemically is the optimal timing of cell replacement after TBI. The overall objective of the current proposal is to evaluate how the interplay between the timing of neural tissue transplantation after TBI and the state of the cortical microenvironment affects anatomic and functional outcomes. Our central hypothesis is that acute neural tissue transplantation after TBI and removal of the injury perimeter will improve outcomes as a result of enhanced integration of graft neurons with host brain networks and maintenance of host cortex integrity. To test this hypothesis, we will transplant human cortical organoids into rat visual cortex in the chronic or acute setting after a controlled cortical impact injury and assess anatomic and functional outcome measures. In Aim 1, organoids will be transplanted directly into a chronic injury cavity or after resection of the glial scar at the border of the cavity. In Aim 2, organoid grafts will be inserted directly into an acute injury cavity or after the injury margin as been removed. In both of these Aims, organoid health and cell composition as well as host cortex integrity will be assessed histologically. The extent of formation of graft efferents (green fluorescent protein tracing) and afferents (modified rabies virus system for retrograde trans-synaptic tracing) also will be determined. Functional integration of organoid grafts with the host cortex will be investigated using in vivo techniques for recording extracellular neural activity and visual stimulation of the host animal. In Aim 3, we will examine how modulating the activity of organoids using optogenetic stimulation impacts their connectivity and integration with the chronically or acutely injured brain. The proposed research is innovative in its use of human brain organoids as structured neural tissues for cortical repair after TBI and because it explicitly assesses how the timing of transplantation affects outcomes. We expect that the proposed studies will elucidate conditions that result in improved outcomes after organoid transplantation while also identifying the limitations of currently available neural tissue substrates. These expected outcomes will advance the field of cortical repair after TBI by reinvigorating the concept of cell replacement therapy and inspiring novel strategies for modulating graft integration with the brain to achieve specific therapeutic goals.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Patients with cirrhosis have increased surgical risk relative to the general population Several risk factors have been established to predict cirrhosis surgical risk. These are reflected in the primary clinical tools used for risk prediction—the Model for End-stage Liver Disease-sodium (MELD-Na), Child-Turcotte-Pugh (CTP) score, and the Mayo surgical risk score—which rely on age, cirrhosis severity, ASA physical status score, and etiology of liver disease. However, significant heterogeneity in post-operative mortality by surgery type (e.g., cardiac versus orthopedic) suggests that these tools are inadequate. The literature on cirrhosis surgical risk prediction is further limited by: 1) single-center designs with small sample sizes, 2) lack of granular data for risk prediction, 3) evidence of poor prediction score calibration, 4) lack of key stakeholder involvement to inform real-world implementation of prediction tools, and 5) no incorporation of decision analysis methods to compare surgery to non-operative management. The impact of these shortcomings is that many patients with cirrhosis are denied necessary surgery due to overestimates of risk, and others receive surgery with inaccurate prognostic counseling or inadequate consideration of non-operative options. Granular, population-level data are needed to address the above gaps. By using national Veterans Health Administration (VHA) and University of Pennsylvania Hospital System (UPHS) data, we hypothesize that we will be able to create and implement an accurate, well-calibrated cirrhosis surgical risk calculator with broad clinical utility. The primary aims of this proposal are as follows: Aim 1 – derive, internally validate, and externally validate cirrhosis surgical risk models for short- and intermediate-term post-operative mortality among diverse patients with cirrhosis.; Aim 2 – create a web application for surgical risk prediction informed by key stakeholder input.; Aim 3 – use Markov modeling to compare operative to non-operative management pathways and determine optimal clinical decisions for a common clinical scenario: acute cholecystitis. This proposal will foster Dr. Nadim Mahmud's development as an independent, NIH-funded clinical researcher with a focus on improving risk prediction for patients with chronic liver diseases, as well as specific expertise in advanced prediction modeling, qualitative methods, and decision analysis. This will be facilitated through a comprehensive mentorship plan consisting of: 1) biweekly to monthly meetings with his mentorship team, 2) formal coursework in advanced prediction modeling, qualitative research methods, and decision analysis through the Center for Clinical Epidemiology and Biostatistics (CCEB), Wharton School, Department of Health Policy Research (HPR), Operations, Information, and Decisions Department (OIDD), and Department of Statistics (STAT) at the University of Pennsylvania, 3) structured research workshops and national conferences, and 4) conception, development, and submission of future grants during the latter portion of the award period to further explore issues related to surgical risk prediction among patients with cirrhosis.

NIH Research Projects · FY 2025 · 2021-07

Adoptive cellular therapy (ACT) has revolutionized the treatment of certain malignancies and responses in refractory B cell tumors treated with chimeric antibody receptor (CAR)-expressing T cells have been remarkable. However, ACT is not consistently curative even in these particularly responsive cancers, highlighting the critical need for innovative approaches to improve this powerful therapeutic approach. Type I interferon (IFN) signaling through the type I interferon receptor (IFNAR) plays a key role in the activation, differentiation and function of T cells. Importantly, degradation of the type I interferon receptor chain subunit 1 (IFNAR1) in anti-tumor T cells favors tumor progression whereas its genetic or pharmacologic (by p38 inhibition) stabilization improves anti-tumor T cell activity in mouse models. While rodent studies have yielded much preclinical insight into CAR T cells, they fail to accurately predict clinical safety and efficacy. However, genetically outbred and immunologically intact canine cancer patients that develop tumors spontaneously are rapidly gaining traction as an invaluable preclinical model. In exciting new preliminary data, we infused CAR T cells treated with the IFNAR1 stabilizing p38 inhibitor ralimetinib into a canine B cell lymphoma patient. Following treatment, we observed signs associated with CAR T cell mediated anti-tumor activity that have not been previously observed in canine patients treated with CAR T cells. Furthermore, in human chronic lymphocytic leukemia patients, an active type I IFN gene signature was associated with improved outcomes following CAR T cell therapy. Together, these data support the hypothesis that stabilization of IFNAR1 on the surface of CAR T cells will improve their therapeutic efficacy for the treatment of B cell malignancies. We will perform the following studies to test this: 1. Canine cancer patients with spontaneous diffuse large B cell lymphoma currently being enrolled in a pilot trial will be used to determine the safety and efficacy of IFNAR1-stabilized CART cells. 2. CART cells derived from multiple species will be evaluated in vitro and in vivo to ascertain the mechanism by which genetic and pharmacologic IFNAR1 stabilization enhances the anti-tumor activity of CART cells. 3. The prognostic significance of IFNAR1 and downstream signaling in T cell apheresis products used to manufacture CAR T cells and CAR T cells themselves will be evaluated in patients with B cell malignancies. We anticipate that IFNAR1 stabilization will safely enhance the activity of CAR T cells. As a veterinary oncologist with doctoral training in immunology I have a solid foundation of the knowledge and skillsets required to undertake these studies. The proposed research on the application of CART cell therapy for the treatment of B cell neoplasia will be performed under the expert guidance of Ors. Fuchs and Mason and represents a field for which the University of Pennsylvania is globally renowned.

NIH Research Projects · FY 2025 · 2021-07

Oral diseases and craniofacial disorders devastatingly afflict susceptible populations, particularly impoverished families and medically or physically compromised persons. Novel engineering approaches can yield new paradigms to reveal disease mechanism, new strategies for disease mitigation, and new approaches for affordable, personalized therapies for dental caries, periodontal diseases, and oral cancer. To achieve this vision, dentist-scientists must be adept in engineering concepts and engineers must be educated in oral and craniofacial sciences and needs; all must be aware of the complex clinical and regulatory landscapes. To address this urgent need, we propose a multidisciplinary training program in Penn’s School of Dental Medicine (PDM) and School of Engineering & Applied Sciences (SEAS) focused on training dentist-scientists and engineers for academic careers dedicated to precision oral healthcare innovation. Postdoctoral trainees will adopt cutting-edge approaches at the forefront of engineering and computational sciences to advance biofilm microbiome, host immunity and tissue regeneration, drawing on approaches including artificial intelligence, robotics, nanotechnology, and materials sciences, aware of developmental hurdles and requirements to safely bring new approaches to patients. Trainees will engage in interactive and tailored academic experiences with intensive mentored research via co-mentoring (one advisor from each school), and interaction with a career mentoring committee (CMC) that includes at least one clinician. Co-mentors and CMC members will be drawn from the Program Faculty in PDM and SEAS with significant records of mentorship of multidisciplinary and translational research. Training will include: (1) engineering fundamentals for dental trainees and oral & craniofacial biology principles for engineering trainees, tailored to match their research project and academic needs, (2) clinical research and regulatory affairs, (3) principles of scientific rigor and reproducibility, (4) scientific writing and (5) workshops on professional/career development. Trainees will engage in academic and industry interactions, journal club, give symposia presentations, and participate in AADOCR activities. Training will culminate in F/K award applications focused on advancing oral health at the dental-engineering interface. The applicant pool at the University of Pennsylvania is exceptionally strong, and we plan to enroll 4 trainees per year. Importantly, this effort will be integrated into a newly formed center between PDM and SEAS with shared funding. The proposed program, unique to Penn, leverages a superb research and training environment within a compact campus where resources for both schools are united through Penn’s new Center for Innovation and Precision Dentistry.

NIH Research Projects · FY 2024 · 2021-07

Project Summary Lymphangioleiomyomatosis (LAM) is a rare fatal cystic lung disease due to bi-allelic inactivating mutations in tuberous sclerosis complex (TSC1/TSC2) genes coding for suppressors of the mechanistic target of rapamycin complex 1 (mTORC1). The origin of LAM cells is still unknown. We profiled a LAM lung compared to an age- and sex-matched healthy control lung as a hypothesis-generating approach to identify cell subtypes that are specific to LAM. Our single-cell RNA sequencing analysis reveals novel mesenchymal and transitional alveolar epithelial states unique to LAM lung. This analysis identifies a mesenchymal cell hub coordinating the LAM disease phenotype. Mesenchymal-restricted deletion of Tsc2 in the mouse lung produces a mTORC1-driven pulmonary phenotype, with a progressive disruption of alveolar structure, a decline in pulmonary function, increase of WNT ligands, and profound female-specific changes in mesenchymal and epithelial lung cell gene expression. Genetic inactivation of WNT signaling reverses age-dependent changes of mTORC1-driven lung phenotype, but WNT activation alone in lung mesenchyme is not sufficient for the development of mouse LAM- like phenotype. Our study identifies sex- and age-specific gene changes in the mTORC1-activated lung mesenchyme and establishes the importance of the WNT signaling pathway in the mTORC1-driven lung phenotype. Based on these data, we propose to test our hypothesis that mTORC1 hyperactivation in LAM cells dysregulates mTORC1-WNT signaling crosstalk which drives cystic airspace enlargement due to chronic activation of resident mesenchymal and alveolar epithelial cells. Our translational hypothesis states that dampening hyperactive mTORC1 and WNT signaling to basal physiological levels represents a potential opportunity to enhance the efficacy of rapalogs and potentially provide novel therapy for LAM patients. To test our hypothesis, we will specifically ask in: Aim 1: How does the small subset of LAM cells induce global pathological changes in the lung centered around lung mesenchymal cell hub? Aim 2: Does activation of mTORC1-WNT/?-catenin pathways in LAM lung mesenchyme dysregulate alveolar epithelial cell fitness? Aim 3: Will combined therapeutic targeting of the mTORC1-WNT/?-catenin pathways provide new treatment opportunities for LAM? The proposed studies will yield not only essential new insights into the role of TSC2-dependent mTORC1 activation and WNT signaling in LAM but also advance our understanding of how LAM lung mesenchymal cells orchestrate cystic lung damage. Our translational studies may also provide novel strategies for therapeutic intervention targeting hyperactive mTORC1 and WNT signaling pathways, which have not been tested for treatment of LAM.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY. Sexual and gender minority (SGM) youth, inclusive of lesbian, gay, bisexual, transgender, and queer are more likely to initiate vaping and currently vape than non-SGM youth in the United States. Vaping significantly increases the risks of initiating cigarette smoking and poly-tobacco use, and consequently tobacco-related illnesses. Higher prevalence of vaping among SGM youth will therefore widen tobacco-related health disparities later in life. anti-vaping campaigns designed for the general youth population may not fully address SGM youths' underlying beliefs and attitudes toward vaping. There is a critical gap in research on evidence- based and culturally tailored interventions to reduce vaping initiation in the SGM youth population. Our long- term goal is to reduce tobacco use and tobacco-related health disparities among SGM populations. The objective of Project SMART (Social Media Anti-vaping Messages to Reduce ENDS Use Among Sexual and Gender Minority Teens) is to evaluate the effectiveness of an SGM-tailored social media intervention to prevent vaping initiation among SGM youth ages 13-18 years. Our central hypothesis is that culturally tailored anti-vaping social media messages will be more effective than non-tailored messages to prevent vaping initiation among SGM youth. The scientific premise for this work is based on the principles of cultural tailoring in health communication for vulnerable populations, the Health Equity Promotion Model, and the Message Impact Framework. We are developing and evaluating a social media intervention because SGM youth have a high rate of social media use and are more likely to go online for health information than non-SGM youth. Social media, moreover, are increasingly used for health promotion to address health disparities and well- being of SGM populations. Our specific aims are: 1) Explore salient beliefs and cultural tailoring preferences related to vaping initiation among SGM youth to inform the development of social media anti-vaping messages, 2) Identify promising anti-vaping messages and cultural tailoring strategies to reduce vaping initiation among SGM youth, and 3) Evaluate the effectiveness of repeated exposure to SGM-tailored anti-vaping social media messages on subsequent vaping initiation among SGM youth. We are developing and evaluating a culturally tailored social media intervention using qualitative research methods and survey experiments. We will conduct rapid-cycle feedback with stakeholders including SGM organization leaders to provide input on the message design, testing, and intervention implementation to ensure feasibility and acceptability of the intervention. Impact: Findings will provide evidence for the comparative effectiveness of an SGM-tailored anti-vaping social media intervention to reduce vaping initiation among SGM youth versus non-tailored messages. The study findings and approach will inform efforts to reduce disparities in vaping among SGM and other vulnerable youth populations.

NIH Research Projects · FY 2025 · 2021-07

Project Summary One in six children in the U.S. and Canada have one or more learning or behavioral problems, such as learning disability, anxiety, autism spectrum disorder, conduct disorder, depression, or attention deficit hyperactivity disorder (ADHD). Early brain development is sensitive to toxicant exposures, including heavy metals, persistent organic pollutants, and endocrine disrupting chemicals. Exposure to mixtures of environmental chemicals is a reality in children, and chemical mixtures may have different modes of action affecting neuronal proliferation, migration, differentiations, synaptic formation/trimming/plasticity, myelination, and neurotransmitters, resulting in adverse impact on the central nervous system. Majority of environmental epidemiologic studies have only examined the impact of a single chemical on neurobehavioral outcomes. Recent development and application of mixture statistical methods will provide great potential to reveal the impact of an individual chemical, interactions between chemicals, and cumulative exposure. These methods have only been applied in limited studies of child neurobehavior and none has been used for neuroimaging outcomes. We will use two existing birth cohorts to examine the impact of both pre- and postnatal exposures to chemical mixtures on child neurobehavior. The Health Outcomes and Measures of the Environment (HOME) Study is a Cincinnati-based birth cohort of 400 pregnant women with children followed up to age 12 years, and the Maternal-Infant Research on Environmental Chemicals (MIREC) is a Canadian study of 1983 pregnant women with children followed up to age 9-11 years. The two North American birth cohorts both measured over 60 environmental contaminants, including lead, mercury, cadmium, arsenic, polybrominated diphenyl ethers, polychlorinated biphenyls, perfluoroalkyl substances, organochlorine and organophosphate pesticides, bisphenol A, phthalates, triclosan, and organophosphate flame retardants, as well as child cognitive abilities (n>1000), behavior (n>1000), and neuroimaging (n=390). We will utilize advanced statistical methods for chemical mixtures, including Elastic Net (ENET) for variable selection, Sparse Partial Least Squares (SPLS) regression for individual chemical effect estimation, and Bayesian Kernel Machine Regression (BKMR) for interactions, nonlinearities, and joint effects. This project will be among the first to test and quantify the potential impact of prenatal and postnatal exposures to chemical mixtures on neurobehavioral and neuroimaging outcomes in well-established cohorts. The results have the potential to greatly increase our understanding of developmental neurotoxicity of chemical mixtures in children and affect environmental health policy making.

NIH Research Projects · FY 2026 · 2021-07

Project Summary/Abstract: The vital functions of the liver—detoxification, serum protein synthesis, and bile production—critically depend on the establishment of a unique hepatic polarity and the formation of bile canaliculi (BC). Defects in these processes contribute to serious liver diseases, including cholestasis and hepatocarcinoma. Using the rat hepatocyte line Can 10, the only known cells that can proliferate and form “tubular” BCs in vitro resembling those in vivo, we discovered that hepatocyte polarization and “primordial” BC formation are linked to cytokinesis. Our collaborative work suggests that this division-linked mechanism underlies BC biogenesis in mice during liver development and regeneration. However, it remains unclear how hepatic polarity is established and maintained at the molecular level, and how a primordial BC, formed between daughter cells at the division site, is remodeled into a tubular BC nestled between aligned hepatocytes produced by oriented divisions. We hypothesize that hepatocyte polarization and BC morphogenesis require the spatiotemporal coordination of cytokinesis with adherens junction and tight junction assembly and remodeling, spindle orientation, and polarized membrane trafficking. Within this conceptual framework, we address three interrelated questions, each guided by a specific hypothesis involving key players identified from our genomic and proteomic screens. In Aim 1, we will investigate the mechanisms underlying our surprising finding that knockdown of Epithelial(E)-cadherin impairs BC elongation, whereas knockdown of Neural(N)-cadherin causes a switch from hepatic polarity to columnar polarity. While both cadherins are required for establishing hepatic polarity at the division site, E-cadherin promotes BC elongation via GEF-mediated Rho activation, whereas N- cadherin maintains hepatic polarity via GAP-mediated Rho inactivation. In Aim 2, we will determine how the actin-binding LIM domain proteins Ablim1 and Alblim3 function independently and cooperatively with the small GTPase Arf6 to regulate junction assembly and remodeling during BC morphogenesis. In Aim 3, we will determine whether the key polarity regulator Crb3, the membrane-actin linker radixin, and the motor protein myosin-5A form a functional unit that stabilizes actin filaments near the BC membrane to enable apical trafficking. We will pursue these aims using cutting-edge technologies and three complementary models: the Can 10 model for discovery and mechanistic analysis; the mouse model for validation and physiological relevance; and the “engineered” human HepG2 model to explore evolutionary conservation. These studies will uncover fundamental mechanisms underlying BC formation and elongation, providing insights into liver diseases, and advancing the broader understanding of epithelial tube formation.

NIH Research Projects · FY 2025 · 2021-07

SUMMARY: Heart Failure with Preserved Ejection Fraction (HFpEF) is on pace to become the dominant form of heart failure, yet we have no treatments to offer patients, leaving them limited in terms of exercise tolerance and quality of life. While much attention has been paid to the myocardium, data suggest that abnormalities in skeletal muscle (SkM) oxygen utilization also contribute to exertional intolerance in this condition. Moreover, decreased nitric oxide (NO) bioavailability has been demonstrated in HFpEF patients. NO augments SkM oxygen delivery and plays a key role in enhancing fatty acid oxidation (FAO), both of which are important for submaximal exercise endurance. Recently, sodium-glucose cotransporter-2 inhibitors such as empagliflozin (EMPA) have demonstrated remarkable benefits in other cardiovascular disease patients, though their use in HFpEF remains unclear. EMPA could be beneficial in HFpEF patients via multiple mechanisms, many of which target abnormalities identified specifically in HFpEF, including: (a) increasing mitochondrial biogenesis, (b) increasing FAO, (c) increasing plasma ketone bodies, providing an additional source of acetyl-CoA for energy production, and (d) increasing blood hemoglobin, augmenting oxygen delivery for any given blood flow. Moreover, because NO is essential for FAO and a key mediator of exercise SkM blood flow, we propose that combining EMPA with a NO-donor such as potassium nitrate (KNO3) will lead to improvements in exercise capacity in HFpEF patients, as compared to EMPA alone or active control. Our overarching hypothesis is that impaired SkM oxidative phosphorylation capacity (OxPhos) limits exercise tolerance in HFpEF. We focus on submaximal exercise endurance in this proposal as submaximal exercise better reflects the level of exertion reached by HFpEF patients during daily activities, is more dependent on FAO than maximal effort exercise, and is less likely to constrained by cardiac output limitations. We will test the impact of three interventions in 53 HFpEF participants in a randomized double-blind cross-over trial: (1) EMPA; (2) EMPA + KNO3; and (3) Potassium chloride (active control). In Aim 1: participants will undergo cycle ergometry exercise tests. The primary endpoint will be the change in submaximal exercise endurance. In Aim 2: We will test the impact of our 3 interventions on SkM OxPhos using MRI following plantar flexion exercise. Novel MRI sequences will also be employed that quantify intramuscular perfusion. In Aim 3: We will conduct SkM tissue biopsies to assess mitochondrial respiration, the SkM metabolome, and quantify the SkM proteome, providing in vitro assessments to support our exercise measurements. Our proposal will target SkM metabolism in HFpEF and comprehensively assess the relationship between SkM OxPhos and submaximal exercise endurance using complementary techniques. This proposal has the potential to identify SkM metabolism as an important therapeutic target in this disease for which we currently have no approved pharmacologic therapies.