University Of Pennsylvania

NSF Awards · FY 2025 · 2025-01

This project studies the design and implementation of a new class of smart contracts. Smart contracts are a kind of contract used in Blockchain, where a transaction is automatically triggered when the contract's conditions are met. Reliable and efficient smart contracts are essential for the robustness of any financial infrastructure and supply chains running on blockchain platforms. Errors in smart contracts have resulted in millions of dollars in loses. The project will enable global financial transactions to be carried out in a safe and efficient manner, benefiting mission-critical capabilities of secure supply chains and ensuring the delivery of goods such as medical products. The resulting tool will be open-sourced for widespread adoption. This project aims to develop DeSCO, a Declarative Smart Contract Optimizer that integrates novel optimization techniques and conventional database optimization strategies into designing and implementing efficient smart contracts. DeSCO will be based on Datalog, a declarative logic programming language. The declarative smart contract written in Datalog is then compiled into efficient Solidity programs for actual implementation. DeSCO is part of a trend toward adopting higher-level domain-specific languages with strong guarantees. Datalog frees programmers from low-level implementation details, allowing them to reason about the contract at the specification level via inference rules. The first thrust aims to extend the Datalog language to support domain-specific language features necessary for implementing complex smart contracts. Language extensions to be explored include support for complex functions, recursion and iterations, and event-based programming, amongst others. The second thrust explores techniques for synthesizing smart contracts from input/output example scenarios. The third thrust explores techniques for optimizing smart contracts via minimizing resource consumption. Cost models estimate resource consumption, inform novel single-query and multi-query optimization techniques, and reduce the likelihood of resource exhaustion attacks. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract Chronic pain is a pervasive burden affecting over 50 million US citizens. The experience of pain is subject to modulation based on context; however, the mechanism of this modulation is unknown. This project aims to unravel the intricate dynamics of prefrontal cortical microcircuits during expectation-driven endogenous pain relief, or placebo analgesia. Specifically, this research focuses on mu opioid receptor (MOR) expressing anterior cingulate cortex (ACC) neurons. This proposal integrates a preclinical placebo analgesia model in mice with cutting-edge techniques such as in vivo single-neuron resolution calcium imaging and the application of a novel opioid biosensor (δLight), to investigate the nuanced interplay between nociceptive signaling and the opioid system within the ACC during placebo analgesia. In Aim 1, viral expression of GCaMP8m under a MOR promoter (mMORp-GCaMP8m) enables visualization of nociceptive MOR+ neurons during placebo conditioning. In Aim 2, the role of enkephalin in the ACC during placebo analgesia will be investigated through CRISPR-mediated excision and biosensor-based monitoring. These aims strive not only to elucidate the neural correlates and potential mechanisms of placebo analgesia, but also to contribute to a broader understanding of how pain is endogenously regulated in a context-dependent manner by opioid systems in prefrontal cortex. As a pivotal component of this research endeavor, a training plan is outlined to foster the development of Ms. Oswell’s skills, preparing her for an independent research career. The successful execution of these aims not only contributes to the scientific understanding of placebo analgesia but also provides significant training opportunities, ensuring she is well-equipped with the requisite skills for a future independent research career. Through hands-on experience in state-of-the-art methodologies, she will acquire expertise that extends beyond the immediate scope of this project, cultivating a foundation for continued contributions to the broader field of pain neuroscience. The interdisciplinary nature of this research, combining molecular biology, neuroimaging, and behavioral analysis, provides a unique platform for skill diversification and the cultivation of a holistic research perspective. This project holds promise not only for advancing our understanding of pain modulation but also for shaping Ms. Oswell into a proficient and independent researcher poised to make substantial contributions to the scientific community.

NIH Research Projects · FY 2026 · 2025-01

Our previous groundbreaking research, published in 2020 and 2023, unveiled the remarkable synergy achieved by combining oral Vancomycin with radiation therapy (RT) or Chimeric Antigen Receptor T-cell therapy (CART) in enhancing the antitumor response. This innovative approach exploited Vancomycin's unique ability to selectively deplete specific gut microbiota while promoting the cross-presentation of tumor-associated antigens (TAAs) by dendritic cells (DCs) post-therapy. Notably, our preclinical studies demonstrated significant improvements in antitumor effects across multiple mouse models, including metastatic melanoma, lung cancer, and lymphoma. Building upon these groundbreaking findings, we embarked on a pioneering randomized pilot study in stage I NSCLC patients, which yielded compelling results, including extended Progression-Free Survival (PFS) and Overall Survival (OS) when Vancomycin was integrated with RT. Subsequent investigations further illuminated Vancomycin's profound impact on bacterial populations, along with notable alterations in gene expression related to TAA cross-presentation and reductions in short-chain fatty acid (SCFA) concentration in the stool. Our proposed research represents a significant advancement in cancer treatment strategies as we seek to optimize the combination therapy of RT, CART, and gut microbiome modulation for maximal antitumor effects. This novel approach involves a meticulous exploration of optimal RT parameters, including dose, fractionation, and treatment volume, in tandem with CART therapy. Moreover, we aim to unravel the intricate mechanisms underlying Vancomycin-mediated gut microbiome modulation and its role in augmenting the antitumor effects of the combination therapy. To validate the transformative potential of our findings, we are poised to conduct the first controlled clinical trial of antimicrobial/RT/CART combination in lymphoma patients. This landmark trial, comprising two arms—RT alone plus CART and RT plus Vancomycin plus CART—will serve as a pivotal step towards revolutionizing cancer treatment paradigms. The comprehensive evaluation will encompass feasibility, toxicity, CART expansion as primary endpoints, alongside secondary endpoints such as PFS, OS, gut microbiome signature, SCFA levels in the stool, and molecular characterization. In summary, our research endeavors to push the boundaries of cancer therapy by harnessing the synergistic benefits of RT, CART, and gut microbiome modulation. By pioneering this innovative treatment approach, we aim to redefine the landscape of cancer treatment, offering newfound hope to patients and paving the way for transformative advancements in oncology.

NIH Research Projects · FY 2025 · 2025-01

Project summary Chromatin and epigenetic information influences gene expression without changing the underlying genome and can provide cellular memory through cell division. Epigenetic mechanisms are under intense investigation for regulation of age-related changes in phenotype, including age-related changes in gene expression and disease states. Studies in animal models reveal that genetic differences underlie longevity, but that non- genetic contributions play a major role, such as, famously, calorie restriction. Numerous findings, including seminal observations from our lab, reveal numerous epigenetic alterations in chromatin as eukaryotes age, and, importantly, are drivers of aging. A major theme in our work is that the epigenome is maintained by an active process of chromatin homeostasis—that healthy aging involves efficient epigenome maintenance. This process is imperfect, hence, in aging chromatin disorganization underlies tissue deterioration and organismal death. Interventions to enhance epigenome maintenance are prominent in our research both to investigate function and to advance as future therapeutics. Here, we explore mechanisms of local and global epigenetic changes in driving senescence and aging- associated phenotypes. We show (1) acquisition of new strong regulatory enhancers (“super-enhancers”) and hyper-connected enhancer-promoter “cliques” drive gene expression during senescence; (2) enhancer-to- promoter conversion within gene introns leads to inappropriate “cryptic” transcriptional initiation within genes; (3) evidence that metabolic enzymes “moonlight” in the nucleus and intersect with epigenetic pathways. Our results broadly support the hypothesis that changes in chromatin regulation and prominently loss of chromatin homeostasis leads to dysregulation of the epigenome and the nucleus, accompanied by cellular and organismal functional decline. In Aim 1, we will investigate how enhancers are rewired during senescence and aging with a focus on large enhancer communities or cliques. We will decipher how cliques are formed and identify major transcription factors that drive their formation. In Aim 2, we will investigate how cryptic transcription is initiated within genes, and transcription factors and epigenetic enzymes that regulate this process in senescence and aging. Further, our data suggest a link between cryptic initiation and cliques, to be investigated in detail. In Aim 3 we focus on a nuclear metabolic enzyme, PHDX, that interacts with the histone acetyltransferase KAT7 to activate senescence-associated secretory phenotypes. Here we will uncover how PDHX rewires the epigenome to promote senescence phenotypes and will identify whether disruption of the PDHX/KAT7 interface can be an intervention for age-related diseases. Overall, our research will reveal chromatin mechanisms—connecting to metabolic mechanisms—underlying stability of the epigenome in longevity, with the potential to discover new therapeutic targets to disrupt age-related diseases and to extend healthy lifespan.

NIH Research Projects · FY 2025 · 2025-01

ABSTRACT There is a bidirectional relationship between sleep disturbances and Alzheimer's disease (AD). Cohort studies show that there are short sleep durations in individuals who are cognitively intact who go on to develop dementia. It has been argued that short sleep duration may be an early manifestation of disease. There is, however, an alternative explanation; there could be an effect on sleep duration due to genetic variants that confer risk for AD also having an effect on sleep (a pleotropic effect of these gene variants). The goal of this study is to assess this possibility. AD, sleep duration, and other aspects of sleep are the result of polygenetic variations coupled with environmental influences. The nature of the genetic variants conferring risk for AD as well as sleep duration and other aspects of sleep have been determined by genome-wide association studies (GWAS). While it is commonly assumed that the causative gene is the nearest gene to the locus identified by association, this is not a valid assumption. Our group has developed an approach based on determining chromatin interactions to identify putative causative genes. This is called variant-to-gene mapping. Since chromatin interactions vary in different cell types, we generated data for multiple relevant cell types. We have done this analysis for results of GWAS for AD and present these results in our proposal. We plan to build on this in two ways to address our global hypothesis of pleiotropy. First, we will conduct variant-to-gene mapping for AD, sleep duration and other relevant sleep phenotypes to look for colocalization, i.e., to determine if there is evidence for identified causative genes conferring risk for AD that also affect sleep duration and/or other sleep phenotypes. This aim is in silico and involves a bioinformatic approach. This will be complimented by studies in zebrafish. We will use CRISPR to create loss of function in zebrafish of genes that are found in the analysis of chromatin interactions in neurons and are likely to be causative genes for risk of AD. These fish will have high-throughput assessment of sleep amounts, microarchitecture of sleep (bout lengths and numbers), and arousal threshold as a measure of sleep depth. Thus, we will seek direct evidence that putative causative genes for risk of AD have an effect on sleep. These complimentary approaches will definitely address our global hypothesis.

NIH Research Projects · FY 2026 · 2025-01

Project Summary Fragile X Syndrome (FXS) is characterized by the unstable expansion of a CGG short tandem repeat (STR) located in the 5’ untranslated region of the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene. Expansion of the CGG tract from normal-length (WT, <55 CGG) to mutation-length (ML, >200 CGG) results in silencing of FMR1 transcription and severe loss of the protein it encodes. Textbook models of FXS have long attributed FMR1 silencing to local DNA methylation over the promoter and CGG tract, however removal of methylation is insufficient to reproducibly de-repress the gene in many experiments. Recently, my lab discovered a Megabase- scale domain of H3K9me3 on the X-chromosome and multiple autosomes in FXS induced pluripotent stem cell (iPSC) lines with a mutation-length CGG tract. The H3K9me3 domains encompass repressed synaptic genes, including FMR1, and severe misfolding of the 3D genome. The overall objective of my proposal is to investigate the interplay between cohesin-mediated loop extrusion, H3K9me3, and gene expression in FXS at the resolution of single-cells. Progress toward testing this goal has been limited by the paucity of single-cell approaches for mapping cohesin occupancy and chromatin folding at kilobase-resolution. My central hypothesis is that the FMR1 locus can undergo cohesin-mediated loop extrusion in WT-iPSCs. By contrast, I posit that ML-FXS iPSCs with H3K9me3 domains will exhibit depleted cohesin binding that is functionally linked to severely misfolded topologically associating domains (TADs), subTADs, and loops and silenced FMR1 expression. I generated my hypotheses based on (1) published work demonstrating that depletion of the cohesin unloading factor WAPL causes hyper-extrusion, stabilization of cohesin binding, and loss of H3K9me3 signal, and (2) my own preliminary data showing that loops are abolished at H3K9me3 domains in ML-FXS iPSCs and rescued when H3K9me3 signal is reversed. I will test my hypothesis by employing both single-cell imaging and genomics techniques to map genome folding, immunofluorescence staining of cohesin and H3K9me3, and single-cell TIPseq to assay cohesin binding and H3K9me3 in WT and ML-FXS iPSCs. I will also utilize WAPL degron iPSC lines established in my lab in ML genetic backgrounds to assay changes in genome folding due to loss and gain of cohesin extrusion at the FMR1 locus. Upon successful completion of my experiments, I will have elucidated mechanisms underlying the interplay between chromatin folding and H3K9me3 maintenance in FXS. My work is significant because knowledge generated in this proposal will have impact on our understanding of how heterochromatin disrupts gene expression in a wide range of neurodevelopmental and neurodegenerative disorders. In addition, I will create freely available algorithms to quantify cohesin-mediated loop extrusion from chromatin tracing data.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Computed tomography (CT) is an x-ray based medical imaging technique commonly used for non-invasive gastrointestinal tract (GIT) imaging. Iodine and barium based CT contrast agents are used in the clinic for GIT imaging, however, inflammatory bowel disease (IBD) imaging is challenging since iodinated and barium based CT agents are not specific for sites of inflammation. Cerium oxide nanoparticles (CeNP) produce strong x-ray attenuation due to cerium's k-edge at 40.6 keV, but have only recently begun to be explored for CT imaging. We hypothesized that the use of dextran as a coating material on cerium oxide nanoparticles would encourage accumulation in IBD inflammation sites in a similar fashion to other inflammatory diseases. We have therefore studied a novel CT contrast agent, i.e. dextran coated cerium oxide nanoparticles (Dex-CeNP) for imaging GIT with IBD. Dex-CeNP produced strong CT contrast and accumulated in the IBD area of large intestines. >97 % of oral doses (an exceptionally high value) were cleared from the body within 24 hrs and the agents were found to be safe from clinical blood chemistry measurements and histology. Moreover, current treatments for IBD have poor efficacy and lead to undesirable side effects. Cerium oxide nanoparticles have significant anti-inflammatory properties, as indicated by our preliminary data. We therefore also hypothesized that Dex-CeNP may prove to be an effective treatment for IBD. We herein propose to develop several variations of Dex-CeNP characterize them, test them for their potential for imaging IBD and as a novel therapeutic for this disease, as well as perform extensive safety testing. By assembling a complementary team of scientists and physicians at the University of Pennsylvania, we seek to develop a breakthrough imaging and therapeutic agent for IBD.

NIH Research Projects · FY 2026 · 2025-01

Malignant pleural mesothelioma (MPM) is an aggressive malignant neoplasm with a devastating prognosis. Median survival from the time of diagnosis is approximately 18 months and limited improvement has been gained in this expectation over the past 25 years. Disease progression is driven through a local inflammatory response, thus there is therapeutic potential in methods that combat this inflammation or its related effects on innate immunity. We have revealed a strong, immune-mediated response of murine mesothelioma tumors to the FDA- approved drug Photofrin. Generally employed as a photosensitizer for photodynamic therapy (PDT), we noted the antitumor activity of Photofrin to mesothelioma tumor was independent of its light activation for photodynamic therapy (i.e., “dark effects”). Other of our preliminary data show that Photofrin, again independent of activation by light, is associated with increased expression of the antioxidant enzyme, heme oxygenase (HO) -1 in both murine and human mesothelioma. Moreover, another drug used in PDT, the photosensitizing pro-drug 5- aminolevulinic acid (ALA), is clinically employed as a HO-1 inducer and found in our data to increase the survival of mice with orthotopic mesothelioma tumors (again, independent of light activation). Due to their antioxidant properties, HO-1 inducers are applied in inflammatory disease. Although not traditionally employed in cancer because they could impede oxidative-dependent therapeutics, it is noted that HO-1 expression correlates with better prognosis in some malignancies. This may be related to the prevalence of inflammation in a particular histology. Overall, HO-1 inducers may stimulate antitumor immunity via effects on immune cell populations. Photofrin data show it to establish CD8+ T cell dependent immunity, which could be transferred to new hosts. Accompanying actions could include anti-inflammatory activity that combats mesothelioma tumor growth, and antioxidative action in T cells, potentially re-invigorating these cells and associated antitumor immunity. Moreover, these effects could cooperate with immune checkpoint blockade. Based on the above described preliminary and published data, studies of this proposal are directed toward the hypothesis that FDA approved porphyrin derivative, Photofrin, is immunomodulatory in mesothelioma and can augment immune checkpoint blockade. Research will be conducted with HO-1 inducers, Photofrin, ALA, hemin and docosahexaenoic acid (DHA) in primarily orthotopic models of murine mesothelioma. The research aims to: 1) characterize the tumor control and antitumor immunity generated by Photofrin and other HO-1 inducing drugs and 2) utilize transcriptomics to inform mechanisms of therapeutic effect by HO-1 inducers and guide their combination with immune checkpoint blockade. Data produced by this proposal will provide insight on application of Photofrin, ALA and/or other HO-1 inducers for treatment of MPM; fill knowledge gaps in antitumor activity by HO-1 inducers; and provide the mechanistic foundation for clinical translation of Photofrin, ALA and/or other HO- 1 inducers in combination with immune checkpoint blockade for the treatment of MPM.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Black women are three times more likely to die as a result of pregnancy compared to White women in the US and are more likely to die from cardiovascular (CV) disease, including peripartum cardiomyopathy (PPCM). PPCM is a condition of new onset heart failure in the weeks to months after delivery in women without pre-existing CV disease. Black women are more likely to develop PPCM, less likely to recover, and have a higher mortality rate compared to White women. Strategies to improve early detection and treatment of PPCM hold promise to substantially reduce disparities in maternal deaths. Prior work has demonstrated that Black women are diagnosed with PPCM later in the postpartum period and that delayed PPCM diagnosis is associated with worse clinical outcomes. The primary purpose of this application is to determine the patient, provider, and practice (health system) factors that drive delays in PPCM diagnosis and to develop a pragmatic intervention to overcome these barriers. We will focus directly on the patient and provider experience to understand mechanisms of delays, which have not been researched in prior PPCM studies. We have the unique opportunity to collect patient-reported data from patients with recently diagnosed PPCM referred from the PPCM Consortium, a geographically diverse network of 60 sites across the US. We propose a mixed methods study to accomplish the following aims. First, we will determine the patient-reported factors contributing to delayed PPCM diagnosis using in-depth interviews and patient surveys and compare how these factors differ for Black and White women. Next, we will determine the provider and practice factors contributing to delayed diagnosis by conducting interviews and surveys with providers from specialties responsible for diagnosing PPCM, including obstetrics, primary care, and emergency medicine. We will then work with our stakeholder advisory board, consisting of patients with lived experience of PPCM, community members, cardiologists, obstetricians, and experts in public health and health equity to develop an intervention to reduce disparities in PPCM diagnosis and test the feasibility of the intervention among patients and providers at five clinical sites. Developing scalable interventions to improve timely diagnosis and treatment of PPCM has the potential to improve short and long-term maternal CV outcomes and may be generalizable to CV disease diagnoses in women unrelated to pregnancy.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY High-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) exhibit shared clinical and genomic characteristics, including poor prognosis, homologous recombination deficiencies, and potential immunoreactivity. These diseases present a pressing and unmet medical need for the identification of new therapeutic targets. Histone acetylation enzymes have emerged as compelling drug targets due to the demonstrated clinical success of histone deacetylase inhibitors in hematological malignancies, affirming the feasibility of this therapeutic approach. Imbalanced histone acetylation, a hallmark of epigenetic alteration in cancer, disrupts genome organization and gene transcription, thereby promoting tumorigenesis. While both histone acetyltransferases (HATs) and histone deacetylases (HDACs) are involved in regulated histone acetylation, HATs are generally associated with accessible chromatin and increased transcription activity, essential for hyperproliferating tumor cells. Consequently, targeting HATs holds promise as a more efficient strategy for cancer treatment. However, the development of HAT-targeting therapy in the clinic has lagged significantly behind HDACi. Although potent and selective HATis with in vivo efficacy in cancer models have been developed at the preclinical stage, the identification and prioritization of specific HAT targets among the 37 distinct HATs in humans have posed challenges. Utilizing a novel systems biology approach developed by our team, we conducted a comprehensive characterization of genes encoding HATs in cancers, leading to the identification of KAT6A as a promising clinical actionable drug target. Preclinical studies have shown the potential of KAT6i as a monotherapy and in combination with FDA-approved drugs in KAT6A-dependent tumors. Notably, potent and selective KAT6is have been successfully developed and are currently undergoing evaluation in phase 1 clinical trial. We hypothesize that KAT6A hyperactivation disrupts the delicate balance of histone acetylation, leading to aberrant genome accessibility and dysregulated transcriptional programs (KAT6A addiction). Therefore, KAT6A serves as a novel therapeutic target in a subset of tumors primarily driven by the recurrent amplification of the KAT6A gene. In this application, we aim to assess the therapeutic potential of newly developed KAT6is as monotherapy and in combination with targeted therapy drugs in preclinical models of HGSOC and TNBC. We have assembled a team of collaborators with added expertise and resources to address the following specific aims: Specific Aim 1: Characterizing the epigenetic changes induced by KAT6i treatment in KAT6A-dependent cancer. Specific Aim 2: Defining the mechanism of action of KAT6i treatment in KAT6A- dependent cancer. Specific Aim 3: Evaluating the therapeutic potential of KAT6i as mono- and combination therapy for cancer. Through these proposed studies, we anticipate providing a strong rationale for KAT6A as a novel drug target for the treatment of cancer.

NIH Research Projects · FY 2026 · 2025-01

Project Summary The introduction of general anesthesia for “painless” surgery is one of the largest advancements ever made in medicine and has transformed surgery from being a barbaric last resort to an event that occurs over 40 million times a year in US operating rooms (ORs). There is an irony that one of the greatest medical advancements is still one of the least understood and anesthetics are still said to be among the most dangerous drugs in clinical use due to their remarkably narrow therapeutic indices. Perhaps related to their toxicity, anesthetics act on many targets and are likely to have multiple mechanisms of action in addition to agonism of the GABAA receptor. Identification of a broader cache of anesthetic targets would allow a novel approach to rational design where multiple targets are actually required. But a challenge is differentiating the many “non-specific” anesthetic interactions from the mechanistically important (or desirable) interactions. This work seeks to uncover anesthetic mechanistic targets by exploiting a propofol derivative that was incidentally discovered to antagonize anesthesia, and use it as a novel approach to the “reverse engineering” of anesthetic action. My research has blossomed out of observations made with a fluorinated derivative of propofol, called propofluor (aka fropofol). This molecule, initially hypothesized to be an anesthetic, but instead elicited propofol antagonism in tadpoles and larval zebrafish. Further experiments strongly suggest competitive antagonism, and we have now identified multiple additional compounds that have increased antagonistic potency. We have shown that sensitivity to this antagonist changes throughout early development in parallel with sensitivity to propofol, but the antagonism only persists until the age of sexual differentiation in zebrafish (21-15 days post fertilization). Preliminary findings have recapitulated this developmental effect in mice. In addition to further pharmacologic investigations, we propose to use bifunctional photoaffinity derivatives of propofol and its antagonists to identify the molecular interactomes during development. The relevance of these targets will be further refined via comparison to large developmental transcriptomic databases (for mice and zebrafish) that have just recently become available. This analysis will truncate the list of potential targets so that functional validation in animal models can be most efficiently pursued. This multidisciplinary program to utilize anesthetic antagonists as research tools to reveal relevant anesthetic mechanisms represents an entirely innovative approach to the study of anesthetic mechanisms.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Highly processed foods have become increasingly prevalent in our diets over the past several decades. These foods often contain high levels of high fructose corn syrup (HFCS), and excess intake of HFCS is associated with obesity and a host of associated metabolic diseases, such as type II diabetes and cardiovascular disease. However, the gut-brain mechanisms through which fructose is sensed, and how it influences homeostatic feeding circuits in the brain, are largely unknown. My preliminary data suggest that fructose engages the vagus nerve to inhibit activity in hunger-promoting agouti-related protein (AgRP)-expressing neurons in the brain. Building on this finding, this proposal will test the hypothesis that Y2-expressing vagal afferents sense fructose in the gut and are sufficient and necessary for gut fructose-induced inhibition of AgRP neuron activity. Aim 1 will use single- cell resolution two-photon calcium imaging to determine the real-time activity dynamics of fructose-activated vagal afferents. In a complementary experiment, we will combine retrograde tracing to label gut-innervating vagal afferents, with activity-dependent gene labeling via in situ hybridization in mice, to determine the molecular identity of gut-innervating fructose-activated vagal afferents. Aim 2 will leverage an activity-dependent technique to gain genetic access to fructose-responsive neurons to determine their role in modulating AgRP neuron activity Through these experiments, we expect to reveal the gut-hypothalamic nutrient sensing pathway for fructose. These results will not only provide new insight for the field of gut-brain signaling, but will also have clinical applications by uncovering new potential drug targets for obesity treatment. My Sponsor, Dr. Amber Alhadeff, is an expert in the gut-to-AgRP neural pathways involved in nutrient sensing, and my Co-sponsor, Dr. Guillaume de Lartigue, has extensive experience with anatomical tracing and two-photon microscopy of vagal afferents. My prior research experience, combined with the expertise of both Sponsors, make me uniquely positioned to successfully complete all proposed experiments during my graduate training. Therefore, funding of this NRSA proposal to support my dissertation studies will set me on the trajectory toward my ultimate goal of becoming an independent investigator that explores gut-brain circuits that regulate feeding.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY/ABSTRACT Respiratory infectious diseases including pneumonia are an immediate public threat to the USA and global healthcare systems. We must gain a deeper understanding of the innate immune system’s response to pulmonary pathogens in order to develop host-directed therapeutic approaches that combat infections in the lung. To this end, my lab studies innate immune mechanisms of defense against the bacterial pathogen Legionella pneumophila (Lp), an important cause of community- and hospital-acquired pneumonia. Effective host defense against Lp is driven by a class of inflammasome-assembling innate immune sensor proteins, which detect bacterial motifs to trigger an inflammatory form of cell death termed pyroptosis. By driving the production of inflammatory interleukin (IL)-1 cytokines and restricting the replicative niche for intracellular bacterial pathogens, inflammasomes promote host protection against a broad array of respiratory infectious diseases. Despite this knowledge, achieving a mechanistic understanding of the specific dietary factors and metabolic signaling events that license inflammasome function to drive antimicrobial defense remains an overlooked – and yet critically important – arm of host immunity. To fill this gap in understanding, I study the branched-chain amino acids (BCAAs), a physiologically abundant and dietarily tunable (i.e., essential) class of amino acids that have important metabolic properties. In addition to catabolically supporting oxidative energy production, the BCAAs are key factors that drive cellular anabolic signaling mediated by mammalian target of rapamycin complex 1 (mTORC1) and metabolic stress adaptation orchestrated by the integrated stress response (ISR). As such, the disparate nodes of BCAA metabolism and sensing integrate to selectively shape transcriptional and translational processes in the cell. Despite this knowledge, a mechanistic understanding of the pulmonary host defense functions of the BCAAs remains unknown. This scientific question motivates my studies proposed herein. I have generated preliminary data demonstrating that the BCAAs promote IL-1 cytokine production and broadly license diverse triggers of inflammasome-mediated pyroptotic cell death in macrophages. These findings therefore provoke the conceptually novel hypothesis that the coordinated sensing of the BCAAs through mTORC1 and the ISR transcriptionally and translationally supports inflammasome-driven host defense against respiratory bacterial pathogens. In Aim 1, I will mechanistically dissect how BCAA catabolism and sensing converge to transcriptionally and translationally drive inflammasome function and IL-1 release. In Aim 2, I will determine if and how the BCAAs are required for inflammasome-driven restriction of Lp in vitro and in vivo. The major scientific goal of this fellowship is to unveil a novel role for essential nutrient sensing in functionally tuning inflammasome-mediated defense against pulmonary pathogens. Another goal is to advance my training in preparation for a career in leading my own independent research group. The strong mentorship of Dr. Sunny Shin and exceptional research environment at Penn will ensure successful completion of this fellowship.

NIH Research Projects · FY 2026 · 2025-01

Project summary – This project investigates the role of mechanical cues of extracellular matrix in regulating monocyte inflammation. Monocytes are recruited from the bone marrow to diverse tissues, where they shape the local inflammatory response and regulate tissue repair, regeneration, cancer, and infection. Tissues of inflammation and injury exhibit not only solid-like elasticity, but also fluid-like dissipation of stress by multiple length-scales of physical interactions. We previously developed biomaterial systems for modeling innate immune cell-matrix interaction with an interpenetrating hydrogel of polysaccharide and fibrillar type I collagen that mimics tissue architecture with tunable ionic and covalent crosslinking. These dual-network hydrogels allow one to independently control the elasticity and stress relaxation for mechanobiological studies. Our recent studies showed that viscoelasticity of stiff fibrotic hydrogels controls a mechanical checkpoint of monocyte fate. Stiff, elastic gels increased differentiation of naïve monocytes into inflammatory monocytes and activated dendritic cells. Stiff, viscous gels suppressed inflammatory signaling and maintained immature monocytes. There remains a significant gap in knowledge of how mechanical cues regulate innate inflammation of monocytes. Further, monocytes pose significant challenges for mechanobiology, due lack in overlap of human and mouse markers, lack of relevant naïve immature cell lines, limited half-life of primary isolated cells, and difficulty in transducing/transfecting primary naïve monocytes cells. Here, we apply multiple strategies to overcome these challenges to investigate the overall question of how immature monocytes are regulated by stiffness and viscoelasticity of ECM. We develop human stem cell-based culture systems for studying monocyte mechanobiology. We investigate the mechanical regulation of non-canonical NF-kappa-B in vitro and in vivo. We determine the role of nuclear mechanotransduction and epigenetics on monocyte fate. Together these studies will build fundamental understanding of physical regulation of innate immunity and will launch further highly impact research in the future.

NIH Research Projects · FY 2026 · 2025-01

Several risk factors including older age, low nadir CD4+ count, and metabolic syndrome are related to neurocognitive impairment (NCI) and mental health disruption with altered activities of daily living in 15% of people with HIV (PWH) despite effective viral suppression and immune recovery with antiretroviral therapy (ART). Although rapid ART initiation achieves the most beneficial outcomes, a subset of PWH remain at risk for NCI. Identification of predictive markers for NCI are needed not only to recognize those at risk but also to provide therapeutic opportunities for pharmacologic interventions in at-risk populations. We have identified significant associations between the endoplasmic reticulum kinase, PERK, one of the four kinases in the ubiquitous integrated stress response, with NCI in samples from the NNTC. Pathologic evidence indicates increased PERK activity in neurons and astrocytes in autopsy tissue of PWH with NCI. PERK-regulated signaling plays roles in orchestrating responses to protein misfolding, inflammation, oxidative stress, iron regulation, and multiorganellar response, such as those associated with HIV infection in the CNS, Further, PERK contributes to cell fate decisions under such conditions. PERK signaling through phosphorylation of translation initiation factor, eIF2α, and antioxidant response transcriptional regulator, NRF2, are linked to neuronal, astrocytic, and macrophage/microglial function, health, and stress response. Tight regulation of PERK kinase activity to a narrow window is necessary for stress tolerance without pathological effects, wherein constant, low-level, non-lethal ER stress preconditions and provides acquired resilience against endogenous and exogenous stresses by priming downstream signaling cascades such as antioxidant response, autophagy, and iron regulation. Despite its canonical role as a coordinator in mounting stress response involving multiple organelles, much needs to be learned about PERK’s role in cells of the CNS and their contribution to cognitive impairment and mental health disruption, including a) its function in stress tolerance, b) cell type-specific mechanisms modulating PERK activity, c) impact cell type-specific PERK-mediated stress response and stress tolerance, and d) association of PERK expression and activity with specific NC domain deficits and disease progression in PWH. We found i) differential and cell type-specific activity of PERK in primary rodent astrocytes and macrophages compared with neurons, ii) cell type-specific stress tolerance in astrocytes which was not observed in neurons and iii) differential outcomes of cells exhibiting stress tolerance. We posit that differential stress tolerance of PERK in specific cell types contributes to NCI and other CNS phenotypes in PWH. We will use biochemical, cellular, and pharmacologic approaches to assess the potential role of PERK in the development of a mechanistic risk score as a precision medicine tool to identify PWH who may benefit from adjunctive pharmacologic therapies such as modulators of PERK and other mediators of stress tolerance during the best therapeutic window, soon after HIV diagnosis.

NIH Research Projects · FY 2026 · 2024-12

Epitranscriptomics, analogous to the epigenetic code formed by DNA and histone modifications, is the study of more than 170 chemically distinct types of RNA modifications, which modulate nearly all aspects of RNA metabolism, such as splicing, translocation, decay, stability, and translation. The recent profound success of COVID19 mRNA vaccines utilizing the pseudo-uridine modification highlights the translational potential of epitranscriptomics. Emerging evidence suggests diverse roles and mechanisms of dynamic RNA modifications in the mammalian nervous system and the association of epitranscriptomic dysregulations with developmental, neurological, psychiatric, and degenerative brain disorders. The majority of recent epitranscriptomic studies used cultured immortalized cell lines and the physiological functions of various RNA modifications remain largely unexplored. Recent technical advances in human induced pluripotent stem cell (iPSC)-derived brain organoids and genome editing open doors to investigate epitranscriptomic regulation in human brain development processes and associated brain disorders. The overarching goal of this research program is to investigate roles and mechanisms of epitranscriptomic regulation in the development and function of the mammalian nervous system, and pathological consequences of disrupting these processes, using both mouse and human iPSC-derived 2D and 3D brain organoid models. There are three interrelated projects designed to test innovative hypotheses and generate foundational data for the field. In Project 1, we will focus on the development of the hypothalamus, an understudied brain region that regulates many key physiological functions, such as sleep, reproduction, and feeding, through its distinct nuclei. Based on our preliminary finding of adult-onset obesity of mice with defective m6A signaling, we will test the hypothesis that m6A signaling regulates the fate specification of neural stem cells in the arcuate nucleus for generating feeding-related neurons both in mice and human arcuate organoids. In Project 2, we will use novel sequencing technology to reveal the landscape of locally translated transcripts at synapses and investigate the role of m6A signaling in regulating activity-dependent local translation of these transcripts at synapses in the mouse hippocampus and human hippocampal organoids. In Project 3, we will focus on several risk genes associated with microcephaly that encode writer proteins for diverse epitranscriptomic modifications beyond m6A. We will generate isogenic iPSC lines and genetically modified animal models to test the functional roles and mechanisms of these RNA modifications in cortical neurogenesis. Together, we will use several orthogonal approaches to investigate functional roles and mechanisms of neuroepitranscriptomics in regulating the mammalian nervous system and its causal roles in mediating some forms of developmental pathology. The research program will also provide a platform to train the next generation of scientists at all career stages. FACILITIES & OTHER RESOURCES The University of Pennsylvania Penn is home to a diverse body of over 20,000 students and over 4,000 faculties in its 12 leading graduate and professional schools. Penn’s schools are located on a compact campus, the geographical unity of which supports and fosters its multidisciplinary approach to education, scholarship, and research. Research and research training are substantial and esteemed enterprises, bolstered by an annual University budget of $6 billion. Penn’s 165 research centers and institutes bring together researchers from multiple departments, schools, and disciplines, and interdisciplinary collaboration is a key theme for Penn’s academic enterprises. The Perelman School of Medicine (PSOM) The Perelman School of Medicine at the University of Pennsylvania has been ranked among the top five medical schools in the United States for the 18th year in a row. The PSOM prides itself on the vision that education should be oriented toward combining theory and practice for the betterment of humanity. The PSOM has an internationally renowned research faculty and programs in all fundamental areas of basic and clinical biomedical science. The PSOM boasts a long record of innovation in both clinical and basic science, resulting in numerous landmark achievements, and is supported by state-of-the-art research core facilities and major clinical research facilities. Research and clinical training programs at Penn Medicine span the full range of participants – from high school and undergraduate students, through MD, PhD and master’s-level trainees, to postdoctoral and clinical residents and fellows. The PSOM has the nation’s largest combined degree training program, which is supported by one of the nation’s oldest and largest NIH- MSTP grants. My lab is fortunate to have talented young individuals from all these programs. Institute for Regenerative Medicine (IRM) I am the Associate Director of IRM, an Institute at Penn and a community of scientists working to explore ways to use cells and tissues to repair, rebuild, and replace organs and body systems afflicted by disease. IRM encourages collaborations across different fields of biology, engineering, and medicine. IRM provides an enriched environment for over 100 core labs with monthly stem cell clubs (organized by Dr. Ming) and faculty lunches, Annual Ralph Brinster Symposium and themed IRM symposiums and retreats. Our initial collaboration with neurosurgeon Dr. Issac Chen was established through IRM sponsored activities. IRM also provides a platform for trainees to interact and present their data in poster and short talk sessions at annual and themed symposiums. The Epigenetic Institute I am a member of the Epigenetics Institute, which was established in 2017 to bring together the epigenetics community at Penn, providing a space where scientific endeavors could flourish. With over 38 core labs, the Institute has created an unparalleled environment for collaboration and cutting-edge research, which is often published in top-tier journals. Faculty regularly collaborate with clinical investigators to conduct translational research that advances medical breakthroughs. I have been collaborating with several members, including Drs. Hongjun Song and Kathy Liu, of the Institute, and will continue our productive collaboration on the work proposed here. The Epigenetics also provides a platform for trainees into interact and present their data in poster and short talk sessions at annual and themed symposiums. The Institute for Diabetes, Obesity & Metabolism (IDOM) The IDOM was established in 2005 to address the ever-increasing prevalence of diabetes and obesity. The mission of the IDOM is to enhance and support research aimed at understanding the genetic, biochemical, molecular, environmental, and behavioral mechanisms underlying diabetes and obesity. IDOM initiatives include critical and unique scientific core facilities, and pilot grants that support new investigators as well as interdisciplinary science involving investigators from Penn Medicine and throughout the University of Pennsylvania that are relevant to the IDOM mission. We are collaborating with Dr. Lazar, the founding director of IDOM on the proposed project. We are also using the IDOM Rodent Metabolic Phenotyping Core to characterize the obesity phenotype of our genetically modified mice. The Penn Institute for RNA Innovation I am a member of the Penn Institute for RNA Innovation, which was recently established by Dr. Drew Weissman and dedicated to the understanding and development of all things RNA and will help form collaborations that will unify and link all elements from RNA-based basic science through therapeutic activities. There is a specific interest in RNA modifications in the institute with many investigators working on topics from basic science to therapeutic. It provides an enriching environment for the proposed studies in the current proposal and excellent training environment for trainees. Additional Neuroscience-related Core Research Support Facilities There are many biomedical research core facilities at Penn that are managed in a centralized manner. As a faculty member of PSOM, I have full access to the following Cores (relevant to the work proposed): The Cell & Developmental Biology (CDB) Microscopy Core is a full-service facility serving the entire Penn community. The Core provides personalized assistance on all aspects of imaging from consultation on experiment design to assisted imaging or hands-on training. The CDB also provides resources to help with image data analysis. The facility currently houses three laser scanning confocals, two spinning disk confocals, a widefield deconvolution microscope, and two widefield microscopes for routine work. In addition, the facility also houses a scanning electron microscope (SEM) and offers SEM sample preparation services. The Flow Cytometry and Cell Sorting Resource Laboratory is currently recognized as one of the largest and most comprehensive flow cytometry laboratories in the US. In 2010 it was designated a laboratory of exceptional merit by the National Cancer Institute. Using state-of-the-art technology, the resource provides a broad array of instrumentation, support, education and consultation to the research community at the University of Pennsylvania. A wide variety of cell sorting applications are supported, from high-speed multicolor (up to 14 colors) cell sorting to low-speed, large nozzle, improved viability sorting. Additionally, a wide variety of cell analysis services (up to 20 parameters) are offered, from traditional analog, easier to use tabletop analyzers to many-laser, many-color, high-speed, fully-digital modern instrumentation. Currently the facility offers 6 cell sorters and 19 analytical instruments. The Vector Core facility is an important technological resource for investigators, both within the University of Pennsylvania investigators and those external to Penn, interested in the use of vectors for gene transfer. The main objective of this Core is to provide investigators with access to state-of-the-art vector technology for preclinical studies and other basic research applications. Such studies provide tools critical to the understanding of gene function and development of therapeutic vectors. The Next Generation Sequencing Core offers ultra-high throughput sequencing services for the PSOM research community. We offer library quality assessments, sequencing, and optional preliminary data analysis for a wide variety of experimental protocols including ChIP-seq, RNA-Seq, HITS-CLIP, miR-Seq, exome capture, and BIS-seq. The Penn Genomic Analysis Core is comprised of the DNA Sequencing Facility (DSF) and the Molecular Profiling Facility (MPF). The Molecular Profiling Facility provides an integrated set of services for DNA and RNA profiling. These services are delivered by experienced genomics professionals, including a focused bioinformatics support staff. PSOM faculty benefit from consultations and training available throughout their projects, including during experimental design and budget development, sample accrual, Facility quality control assays and lab work, data management and analyses, and manuscript preparation. The core supports quantitative RNA profiling by Affymetrix GeneChips, Illumina BeadChips, real-time PCR, Sequenom custom multiplex assays, Fluidigm, Luminex and deep sequencing. DNA profiling of custom panels of sequence polymorphisms are conducted by quantitative PCR, Sequenom assays, and Illumina GoldenGate genotyping, while whole-genome assays are available on Affymetrix SNP GeneChip and Illumina Infinium platforms. Whole-exome and targeted genomic regions can be resequenced on an Illumina Genome Analyzer deep sequencer. Several other services including microRNA profiling, epigenetic DNA assays, and translational molecular diagnostics for clinical research are offered using these platforms. The Transgenic and Chimeric Mouse Facility provides a centralized service to efficiently produce infection-free transgenic, chimeric, and genome-edited strains of mice. These mice carry randomly inserted transgenes and/or site-specific alterations in the mouse genome of specific interest to Penn researchers. The Facility offers services including DNA pronuclear injection into fertilized oocytes (along with genotyping of transgenic founders), ES cell injection into blastocysts, cytoplasmic/pronuclear injections into fertilized oocytes of CRISPR-Cas9 mix (gRNA, Cas9RNA, ssDNA/dsDNA templates), embryo and sperm cryopreservation, in vitro fertilization, and re-derivation of live and cryopreserved lines. The Core also oversees a cyropreservation facility for long-term storage of mouse embryos and sperm samples. We have used the core to generate several genetically modified mice and will continue to use it for the current projects.  The Neurobehavior Testing Core provides core facilities and services to test mice in state of the art assays of simple and complex behaviors, including the assessment of circadian rhythms and sleep, learning and memory, motor and sensory function, as well as behavioral assays relevant to translational studies of neurological, neurodevelopmental and psychiatric disorders. The core offers comprehensive behavior phenotyping of your mice or can train your lab personnel to perform the tests in the facility. In addition, we provide consultation on study design including appropriate tests, mouse line/strain, numbers of animals, control groups and breeding strategies. The core will also provide consultation regarding ULAR, IACUC and other regulatory issues. Assistance with data analysis is also available. We have used the core for characterizing of our genetically modified mice and will continue to use it for the current project. The Small Animal Imaging Facility (SAIF) provides multi-modality radiological imaging and image analysis for cells, tissues, and small animals, primarily mice and rats. The assets of the SAIF include state-of-the-art instrumentation and a nationally recognized staff. SAIF currently provides a comprehensive suite of imaging modalities including: magnetic resonance imaging (MRI) and spectroscopy (MRS), optical imaging (including near IR and bioluminescence imaging), computed tomography (CT), positron emission tomography (PET), single photon emission computed tomography (SPECT), and ultrasound (US). In addition, dedicate housing is available for mice and rats undergoing longitudinal imaging studies. Ancillary facilities and resources of the SAIF are devoted to chemistry, radiochemistry, image analysis and animal tumor models. Other Relevant Research Resources The Biomedical Library, housed within PSOM, has a large collection of print and electronic journals, as well as many other services. As of July 2010, the Biomedical Library had close to 100,000 volumes, and access to over 6,000 current serials in the health sciences, primarily electronic, and 1,300 e-books. In addition, faculty, students and staff can access all the collections of the Penn Libraries, which number more than 5,000,000 printed volumes, more than 40,000 online and print journals and thousands of databases, e-books and other digitized resources. The Library's holdings are supplemented by membership in the National Network/Libraries of Medicine and many other resource-sharing consortia, and electronic delivery of documents is standard. The Biomedical Library houses 80 public workstations, several printers and a scanner, a poster printing service, a 10-station training lab, a wireless network throughout the library, and sixteen lending-laptops. Group study rooms are outfitted with computers and large flat screen monitors. Biomedical Library staff can provide in- library and off-site training and individual research consults in searching life science databases (Medline, PubMed, Scopus, CINAHL, ISI Web of Science, etc.), use of bibliographic management software (RefWorks), and research and productivity skills (mobile resources, systematic reviews, retrieving full text articles, PowerPoint, Excel, molecular biology tools). The Research Instrumentation Shop is non-profit, shared resource machine shop of the University of Pennsylvania, Perelman School of Medicine. Its mission is to assist University faculty in the design and construction of both laboratory and clinical instrumentation. The staff is comprised of mechanical and optical specialists and is experienced with working with scientists to design and construct custom instrumentation and apparatus. Career development and support for trainees The Perelman School of Medicine and University of Pennsylvania have established offices, programs, and research opportunities for trainees. These resources provide additional support for all trainees and are accessible to undergraduates, graduate students and postdoctoral associates. Undergraduates may apply for internship funding through the Center for Undergraduate Research & Fellowships. Current trainees have access to the Trainee Advocacy Alliance, which provides broad support for trainees at all levels. Postdoctoral associates have access to institutional support offices, such as the Biomedical Postdoctoral Programs Office, the Faculty Professional Development Office, and the Office of Research Support Services, which support career development. The Biomedical Postdoctoral Programs Office provides responsible conduct in research training, lab management workshops, career services, and grant writing seminars. The Faculty Professional Development Office offers workshops focused on career topics, such as mentoring, tenure track tips and science communication, as well as online courses and resources. The Office of Research Support Services provides help with grant proposal preparation and post-award grant management, as well as online access to internal and external funding opportunities. Laboratory The Ming laboratory is assigned approximately 2500 sq. ft. space in the Clinical Research Building (CRB) with seven bays of bench space. The laboratory has six individual rooms that are fully equipped to support cell culture, molecular biology, bioinformatics, cell biology, biochemistry, immunohistology, stereology, electrophysiology, optogenetics, confocal imaging, and CNS histology. Office and administrative space: Dr. Ming has 230 sq. ft. of personal office space in CRB and ~300 sq. ft. of space for administrative support and meeting rooms.

NSF Awards · FY 2024 · 2024-12

This CAREER project aims to develop a new method to better understand how cells are organized in tissues and how cells interact with each other over time. Recent advances in a technology called spatial transcriptomics have allowed scientists to study gene activity in tissue samples and see how cells are distributed within their environment. However, this technology currently provides only a snapshot of what is happening in the cells at a single moment, which limits our understanding of how cells change and respond to different conditions over time. To address this gap, this project will focus on creating a system that can track molecular changes in live tissue over time. This work aims to develop a new technology using a nanostructure (e.g., nanowires) to extract important molecules from live cells with minimal disturbance, allowing for repeated sampling and enabling longitudinal measurements. This will allow for continuous monitoring of cell activity and interactions, which can help scientists learn how cells develop, change, and respond to diseases. By improving the understanding of cellular behavior, this research has the potential to advance medical treatments, particularly for diseases that involve complex interactions between cells. This project also supports education and workforce development by training students in cutting-edge research techniques. The ultimate goal is to use this technology to improve healthcare and contribute to advances in science that benefit society and national well-being. Recent advancements in spatial transcriptomics have transformed understanding of cellular heterogeneity, enabling the identification of new cellular interactions and functional dynamics between different cell types and their microenvironments that were previously elusive. This technology allows researchers to study gene expression in the context of tissue architecture, providing a more complete understanding of the cellular function and interactions between cells. Although this method preserves the spatial information, it only offers a snapshot of gene expression at a single point in time, providing a static view of the dynamic biological processes. To fully understand the complex and temporal nature of tissue function and disease progression, it is essential to monitor molecular changes over time. Integrating spatial transcriptomics with longitudinal studies will be crucial for unraveling the continuous and evolving nature of cellular behavior. To address this limitation, the overall objective of this CAREER project is to develop a new method for the spatiotemporal monitoring of live tissue gene expression. This work proposes to develop a nanowire-based molecular transfer technology that can extract molecules from live cells and transfer them to a hydrogel to achieve spatiotemporal transcriptomics. By monitoring molecular changes within specific regions of tissue over time, researchers can gain valuable insights into how different cell types interact and respond to changes in the tissue microenvironment. This platform represents a significant advancement in our understanding of cellular behavior, particularly regarding cell development, differentiation, and disease pathogenesis. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-12

Summary The goal of this proposal is to investigate how changes in transcription factor and heterochromatin states in embryos and the adult liver impact hepatocyte development, homeostasis, and regeneration. To do so we will use new in vivo imaging technologies and genetic perturbations of heterochromatin to elucidate the molecular interactions that drive cell fate specification. Previous work from the Zaret and Mir laboratories have revealed that the interplay between transcription factor kinetics and heterochromatin limits cell plasticity in model systems. We will use our new approaches to understand how ventral-lateral and -medial domains of the embryonic endoderm induce hepatic fates and the morphologic transitions that form the liver bud. We will similarly investigate, in wild type and heterochromatin mutant adult livers, how differential heterochromatin states and transcription factor kinetics in the periportal, midlobular, and pericentral liver zones impact homeostasis and regenerative responses. Although single-cell genomics have revealed heterogeneity of embryonic hepatocytes and of cells in each zone of the adult liver, the functional significance is unclear. We propose that quantifying regulatory protein and chromatin kinetics within living wild type and heterochromatin mutant embryos and adult liver will provide novel insight into the basis for two origins of the liver and the functional nature of zonal heterogeneity. The Zaret lab showed that heterochromatin promotes liver cell stability in mouse development and identified diverse heterochromatin proteins that repress liver genes in adult cells. Using single-molecule tracking in cultured liver cells they showed that the pioneer factors FOXA1/FOXA2 scan heterochromatin, whereas the liver differentiation factor HNF4 is restricted to open chromatin. The Mir lab engineered high- resolution light-sheet technologies to image transcription factor and chromatin kinetics within specific nuclear domains in live Drosophila and pre-implantation mouse embryos. Mir discovered how transient high- concentration hubs of transcription factors at target genes regulate zygotic gene expression in fly embryos. We will couple our laboratories’ expertise, as seen by our preliminary data showing that liver regulatory proteins and chromatin can be genetically perturbed and imaged at high resolution in live developing mouse embryos and in adult liver slices. We ask in Aim 1: How do changes in heterochromatin, and the molecular kinetics and sub- nuclear clustering of transcription factors and chromatin components that interact with it, dictate how fields of endoderm cells acquire liver fates? Aim 2: How do protein kinetics and heterochromatic features impart different homeostatic capacities in the three zones of the adult liver lobule and the abilities of the periportal and pericentral zones to respond to liver damage? By bringing together state-of-the-art live imaging of embryos and liver slices with genetic perturbation, we will reveal how heterochromatin states and chromatin and transcription factor kinetics impact development in two domains of endoderm, liver homeostasis, and regeneration. The work will inform new ways to make hepatic cells from pluripotent cells and paradigms for other gut-derived tissues.

NIH Research Projects · FY 2026 · 2024-12

ABSTRACT Our overall objective is to model and contrast the microscopic, mesoscopic and macroscopic regional progression of neuroinflammation, abnormal iron accumulation and neurodegeneration in human brain tissue from patients with frontotemporal lobar degeneration (FTLD) with tau (FTLD-TDP) vs. TDP-43 (FTLD-TDP) pathology. Frontotemporal dementia (FTD) is a spectrum of clinical dementia syndromes that comprise progressive changes in behavior and social cognition, language and executive functioning. FTD is understudied, yet FTD is as common as Alzheimer’s disease in individuals < 65 years of age. FTD is an incurable condition with no FDA-approved therapies and treatment relies on supportive care. Currently, a major obstacle to the study of the underlying biology of FTLD proteinopathies and the development of disease-mechanism targeted therapies in FTLD is the inability to reliably diagnose underlying FTLD-Tau vs FTLD-TDP pathology in a living FTD patient, necessitating neuropathological examination of human brain tissue. Moreover, the majority of postmortem studies in FTLD to date have focused on the proteinaceous inclusions that define FTLD, while detailed studies of glia and non-cell-autonomous mechanisms of neurodegeneration in FTLD are understudied. We recently discovered distinct microscopic cellular patterns of iron-rich gliosis in FTLD-Tau and FTLD-TDP using histopathologic sampling guided by iron-sensitive 7T-guided ex vivo MRI. To address these gaps and build on our previous work, we propose a unique multidisciplinary approach to comprehensively study glial activation and iron-dysregulation in the extensive Penn FTLD brain bank and translate these findings by integrating our digital histology data with antemortem imaging and detailed clinical data. Our central hypothesis is that FTLD- Tau and FTLD-TDP have distinct cellular laminar and regional patterns of iron-rich gliosis postmortem that relate to antemortem clinical and radiographic progression of disease. We leverage the complementary expertise of our investigator team and our infrastructure of productive collaboration to perform complex statistical modeling from deep spatially-resolved cellular data from multiplexed immunofluorescence imaging methods in human brain tissue. Moreover, using our established ex vivo 7T MRI guided digital histology approach, we will test for cellular patterns of iron dysregulation on the mesoscopic scale and relate these to antemortem clinical data on cognitive and behavioral functioning. Finally, we will integrate multiple digital measures of gliosis and iron- overload with longitudinal antemortem MRI using network science. Thus, our work will provide a fresh examination of the role of glia and iron-homeostasis in progressive spread of Tau vs TDP-43 proteinopathies and provide critical ground-truth human histopathology data to guide MRI biomarker development thereby addressing several key recommendations from the 2022 NIH ADRD summit for FTD research priorities.

NIH Research Projects · FY 2026 · 2024-12

Abstract Alzheimer's disease (AD) is a widespread global neurodegenerative disorder, accounting for the majority of dementia cases in the United States. With over 25 million people affected by the disease, this number is expected to double by 2050, primarily due to the growing aging population. The amyloid cascade hypothesis posits that the accumulation of amyloid-β in the brain triggers AD. However, the complexities of AD pathogenesis extend beyond this theory, with mounting evidence linking inflammatory markers and AD risk genes to innate immune functions, emphasizing the significant role of neuroinflammation. This study aims to develop and optimize noninvasive imaging biomarkers to track neuroinflammation in the early stages of AD and explore the intricate connections between neuroinflammation, brain glucose hypometabolism, and the progression of AD. The study proposes the use of deuterium-magnetic-resonance-spectroscopy (DMRS) and quantitative- exchanged-label-turnover MRS (qeltMRS) with deuterium-labeled acetate to track the temporal dynamics of neuroinflammation in AD. The research comprises three specific aims. Firstly, it focuses on investigating the relationship between astrocytic acetate metabolism and astrocyte reactivity in an adenovirus-induced reactive astrogliosis model, utilizing DMRS and qeltMRS. The hypothesis suggests a higher uptake and metabolism of acetate in reactive astrocytes, correlated with increased expression of the monocarboxylate transporter (MCT1) protein. Secondly, the study aims to characterize the spatiotemporal patterns of neuroinflammation and brain glucose hypometabolism in an AD mouse model, using DMRS and qeltMRS. The hypothesis proposes that in AD mice, reactive astrocytes and microglia will metabolize acetate predominantly over glucose, with upregulated MCT1 transporters and downregulated GLUT3 transporters compared to age-matched WT mice. Lastly, the research seeks to validate the efficiency of the methods by assessing the impact of a ketogenic diet on neuroinflammation in AD mice over a longitudinal study. The hypothesis anticipates a slower level of neuroinflammation in the treated AD mice compared to the untreated group. The innovation and potential impact of this research lie in providing a robust preclinical framework for understanding AD pathophysiology, enabling the detection of early pathological processes in vivo. Successful outcomes may contribute to the development of prognostic tools and personalized treatment strategies targeting neuroinflammation, ultimately translating findings into clinical applications for improved outcomes in human patients affected by AD.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY Candidate: I am an Assistant Professor of Dermatology and Medicine at the University of Pennsylvania and a physician-scientist with a Master of Science in Clinical Epidemiology as well as expertise caring for patients with GVHD. I have a strong track record of published research at the intersection of dermatology, cancer, and transplantation. With the support of this career development award, I plan to further develop my scientific career in two ways. First, I have been struck by the substantial health-related quality of life (HrQOL) impact that results from skin chronic GVHD (cGVHD). I plan to gain expertise in the science of measuring disease burden and the approaches used to conduct studies that improve patient-centered outcomes. Second, I will move from analysis of existing datasets towards the design and execution of prospective observational and interventional studies that improve HrQOL, morbidity, and mortality for patients with skin cGVHD. Background: Skin cGVHD is a major contributor to morbidity, mortality, and impaired HrQOL after allogeneic hematopoietic cell transplantation. However, patient-reported outcome measures (PROs) are not routinely used to develop, evaluate, or select treatments. PROs provide complementary information to clinician-reported measures, can predict clinical outcomes including survival, and are required by the FDA to support labeling claims of new therapies. The absence of a valid and reliable PRO for skin cGVHD presents a critical barrier to designing and executing prospective studies. Without such a measure, we cannot determine whether interventions improve the outcomes that are important to patients. Training: To achieve research independence, I require additional training in 1) mixed methods research and consensus formation; 2) psychometric methods; and 3) design, implementation, and analysis of prospective observational studies and clinical trials that incorporate patient- centered outcomes. Mentors: My training and research will be overseen by Dr. Joel Gelfand, who has extensive experience mentoring K23 awardees to successful independent research careers, and expertise in instrument development, patient-centered research, and prospective observational and interventional studies in chronic inflammatory skin disease. Dr. Stephanie Lee (PRO development, prospective observational studies and clinical trials in GVHD), and Dr. Marilyn Schapira (psychometric methods, patient-centered outcomes) will be additional co-mentors. Dr. Alison Loren (GVHD at UPenn), Dr. Eric Tkaczyk (multicenter studies in skin cGVHD) and Dr. Daniel Shin (biostatistics) will be advisors. Research: To achieve my objective of developing and validating a PRO for skin cGVHD, I will pursue 3 aims: 1) Generate and evaluate content validity of an item bank for a skin cGVHD PRO; 2) Develop a skin cGVHD PRO scale using state-of-the-art psychometric methods; and 3) Evaluate responsiveness of the skin cGVHD PRO to therapy in a single-center prospective cohort study. I will use the findings from this K23 as the basis for future research including an R01 or U01 proposal to conduct a clinical trial with the skin cGVHD PRO as a clinical endpoint.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY/ABSTRACT The as complication and disease glucocorticoids there unvalidated, biomarker improve vascular cranial MRI goal of this project is to establish orbital and cranial vessel wall magnetic resonance imaging (oVW-MRI) an imaging tool that assesses disease activity in giant cell arteritis (GCA), including the most feared of ocular involvement. GCA is a relapsing systemic vasculitis which frequently involves the orbital cranial arteries. Due to the fear of blindness, high rates of relapse, and lack of a reliable biomarker of ocular in GCA, all patients with GCA regardless of visual symptoms receive high doses and long duration of resulting in significant treatment-related adverse events. To minimize glucocorticoid exposure, are now an unprecedented number of t herapeutic trials in GCA. However, these trials all rely on poorly standardized, and subjective outcome measures. There is a critical need for an objective of disease activity, particularly ocular involvement, which can inform treatment decision-making and t he conduct of clinical trials in GCA. oVW-MRI has the potential to address this need by visualizing inflammation of multiple arteries in a single 35-minute scan . Prior studies of MRI in GCA f ocused on vessel wall imaging but little is known about the utility of adding dedicated orbital imaging to vessel wall to better assess orbital pathology in GCA.We hypothesize that combining orbital MRI with cranial vessel wall MRI (oVW-MRI) enables detection of orbital activity even in the absence of visual symptoms and can be used to longitudinally monitor disease activity in GCA. The premise of this study is supported by our preliminary data which found that oVW-MRI enhancement has a negative predictive value of 97% for ocular GCA, detects orbital disease in patients with and without visual symptoms, significantly decreases with treatment, and has additive value over existing measures of disease activity. We will now perform a multi-center prospective observational study which aims to: (1) and validate oVW-MRI enhancement as a diagnostic biomarker of ocular GCA, (2) determine the responsiveness and convergent validity of oVW-MRI enhancement in GCA. Patients with suspected GCA will be recruited from four sites with excellent track records in recruitment for GCA studies and expertise in vessel wall MRI. The study will leverage the clinical research infrastructure of the Vasculitis Clinical Research Consortium which has performed numerous multi-center, international studies in vasculitis. At completion a and will the f the proposed study, we will have the components needed for clinical t rials of oVW-MRI including validated and refined MRI score for GCA and ocular GCA, tandardized imaging and clinical data collection, data for power calculations. We anticipate that oVW-MRI will be additive to current clinical measures and provide a more quantitative and precise measurement of disease activity. o s Our long-term goals are to: (1) perform a clinical trial which definitively demonstrates that incorporation of MRI into treatment decision-making in GCA reduces both disease- and treatment-related morbidity; and (2) utilize MRI as a surrogate outcome measure in therapeutic trials of GCA. This proposal is fully responsive to the NIAMS FOA PAR-24-036.

NIH Research Projects · FY 2026 · 2024-12

Project Summary GLP-1 receptor agonists and GLP-1/GIP dual agonists profoundly decrease body weight in association with significant cardiometabolic benefits. These therapeutic agents are being widely adopted for obesity treatment in the U.S. Certainly, lowering fat mass is metabolically advantageous; however, reduction of lean mass, especially skeletal muscle mass, is associated with poor health outcomes including reduced energy expenditure that could contribute to weight regain and functional defects in certain patient populations. Lean mass loss during GLP-1 receptor agonism is as great or greater than dieting or bariatric surgery. Discontinuing therapy leads to rapid weight regain, favoring increased adiposity and limited muscle growth. Thus, a major goal of next generation anti-obesity agents should be to maximize quality weight loss by reducing fat mass while preserving (even increasing) lean muscle mass. Myostatin and other TGF-b-like ligands, such as activins, are potent negative regulators of skeletal muscle mass acting primarily via the activin type II receptors (ActRII). Blockade of ActRII in both rodents and humans drives increased muscle mass that is associated with reductions in adipose tissue. The holy grail for an obesity treatment is an approach that maximizes reductions in fat mass while sparing lean tissues such as skeletal muscle. Excitingly, recent published and preliminary data in this application show that bimagrumab co-treatment in animals receiving semaglutide (a GLP-1 agonist) leads to even greater reductions fat mass while preserving functional lean mass in mice. These findings indicate that ActRII blockade protects against muscle loss and drive increased fat loss during calorie deficit, potentially leading to more durable and sustained metabolic benefits and diminishing susceptibility to “rebound” weight gain. In Aim 1 of this proposal, the effects of ActRII on systemic energy balance and metabolism both during and following the cessation of GLP-1/GIP receptor agonism will be explored. In Aim 2, the tissue specific mechanisms of ActRII blockade in the control of muscle and adipose tissue physiology will be elucidated. Collectively the experiments in this proposal will address critical knowledge gaps regarding how ActRII blockade alters muscle, adipose tissue and whole-body energy metabolism when given as a mono-therapy or in combination with GLP-1/GIP receptor agonists laying the foundation for future therapeutic opportunities targeting body composition for enhanced weight loss quality in obesity.

NIH Research Projects · FY 2026 · 2024-12

Project Summary Progression to type 2 diabetes results from the inability of insulin secreting pancreatic  cells to compensate for increased insulin demand, usually in the setting of obesity. We have previously found that the homeodomain transcription factor and human diabetes gene Pdx1 coordinates islet compensation in the setting of diet-induced obesity and associated insulin resistance. We identified Gpt2 as a target of a PDX1- ATF transcriptional complex whose stress induction and PDX1 enrichment of regulatory CARE sites is conserved in both human and murine islets. Importantly, silencing of the transaminase Gpt2 ex vivo protects primary mouse β cells from stress induced apoptosis. GPT2 is a rapid equilibrium transaminase that catalyzes a bidirectional reaction converting glutamate and pyruvate to α-ketoglutarate (-KG) and alanine. Recent reports implicate Gpt2 upregulation as a key step in the reprogramming of glutamine metabolism of cancer cells. The production of -KG feeds into the tricarboxylic acid (TCA) cycle, resulting in synthesis of ATP which leads to closure of KATP channels, membrane depolarization, calcium influx and exocytosis. TCA cycle intermediates also act enzymes as a cofactors for several chromatin-modifying enzymes, including the Tet family of involved in DNA demethylation. Our preliminary findings support the exciting hypothesis that the induction of GPT2 during fuel excess reprograms beta cell metabolism, thereby causing beta cell dysfunction and death. This hypothesis and the underlying mechanisms will be tested in the following Aims: Aim 1: To uncover the mechanism whereby the transaminase Gpt2 promotes  cell dysfunction and death. We will (A) complete the in vivo characterization of Gpt2 deficiency in pancreatic  cells, (B) elucidate how Gpt2 deficiency affects mitochondrial morphology, function and metabolism, (C) explore the epigenetic impact of Gpt2 silencing and (D) assess the role of Gpt2 in human  cell function and survival during metabolic stress. Aim 2: To elucidate how Gpt2 influences sensitivity of the  cell to GLP1. We will (A) determine whether the sensitivity of GIP and other insulin secretagogues are also affected by Gpt2 expression, (B) investigate the molecular mechanism whereby GPT2 impacts GLP1 sensitivity, and (C) determine whether its impact on cell survival and function is linked to its effect on GLP1 sensitivity via intraislet paracrine signaling. These results will address an important gap in our understanding of how glucoliptoxicity promotes  cell dysfunction and death. Further we will then be poised to devise strategies to target Gpt2 activity or expression as a therapeutic means to protect beta cells during metabolic stress and to enhance sensitivity to Glp1 receptor agonists, an application of the proposed studies that is of the highest translational relevance.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY In virtually all cell types, heterochromatin is enriched at the nuclear periphery, where it contacts the nuclear lamina and forms lamina-associated domains (LADs). LADs tend to be transcriptionally inactive and enriched for repressive histone modifications. It has been suggested that one possible function of peripheral positioning is to sequester genes whose expression would be inappropriate in that cell type, thereby ensuring maintenance of cell identity. In support of this model, LAD organization has been linked to both human disease and normal development. However, due to the limited number of factors known to regulate LADs in human cells, it has been challenging to directly test these and other models for LAD function. To this end, our group completed a genome-wide screen to identify factors required for maintaining chromatin positioning at the nuclear periphery. The arginine methyltransferase PRMT1 emerged as a top hit from the screen, and subsequent work found that PRMT1 regulated the peripheral attachment of hundreds of LADs across the genome. Interestingly, we also observed a reduction in the heterochromatic mark H3K9me2 over a subset of PRMT1-sensitive LADs. Having characterized PRMT1 as a novel regulator of peripheral chromatin, we set out to elucidate the mechanism by which it maintains LAD positioning. Strikingly, a canonical PRMT1 target, the RNA-binding protein hnRNPK, also emerged as a top hit from our genome-wide screen. hnRNPK has previously been demonstrated to bind nuclear lamina components and heterochromatin, making it an attractive candidate for serving as a tether of peripheral chromatin. The goal of this proposal is to determine the mechanism by which PRMT1 and hnRNPK regulate peripheral chromatin organization and to elucidate the effect of disrupted LAD positioning on heterochromatin maintenance and transcriptional repression over time. I hypothesize that PRMT1-mediated methylation on hnRNPK allows hnRNPK to act as a physical bridge tethering heterochromatin to the nuclear lamina. I will first profile LAD positioning genome-wide following hnRNPK KD to determine if loss of hnRNPK recapitulates the impaired peripheral positioning of LADs observed upon PRMT1 KD. I will then determine using imaging and genomics approaches whether PRMT1 regulates the localization of hnRNPK to chromatin or to the lamina. Finally, I will specifically perturb arginine methylation on hnRNPK to test if this modification is required for maintenance of peripheral positioning. In parallel, these novel regulators will be used to disrupt LAD organization and investigate the impact on heterochromatin maintenance and transcriptional repression over time. Ultimately, the work proposed here will pave the way for future studies understanding how spatial chromatin organization contributes to normal development and human disease.