University Of Pennsylvania

NIH Research Projects · FY 2025 · 2023-03

Abstract Non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging public health risk. Approximately 35% of Americans have NAFLD, and if left untreated, NAFLD can progress to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. Although obesity and Type II diabetes have emerged as risk factors, disease pathogenesis is poorly understood. Accordingly, there are no FDA-approved pharmacotherapies currently available for patients with NAFLD/NASH. An ideal therapeutic would target multiple hepatic lipid homeostatic processes, including de novo lipogenesis (DNL), fatty acid oxidation (FAO), and very-low-density lipoprotein (VLDL)-triglyceride (TG) secretion, as attempts to modulate a single lipid homeostatic process have often led to undesirable compensation from the remaining pathways. Our lab has identified the protein folliculin (FLCN) as a potential therapeutic target. Mice with hepatic deletion of FLCN are robustly protected against steatosis when challenged with NAFLD-inducing diets via an FLCN-mTORC1-TFE3 signaling axis. TFE3 suppresses DNL and induces genes involved in FAO to decrease hepatic TG content in FLCN-null livers. Given that VLDL-TG secretion is heavily regulated by TG availability, we expected to see a compensatory decrease in VLDL-TG secretion rate in FLCN-null mice. Strikingly, however, we saw marked upregulation of VLDL-TG secretion in FLCN deficient mice, which makes FLCN even more appealing as a therapeutic target. I hypothesize that upregulated VLDL-TG secretion is critical to the robust protection against steatosis afforded by liver deletion of FLCN, and that activated TFE3 is necessary and sufficient for VLDL-TG secretion. To test this, I will generate FLCN knockout (KO), TFE3 KO, and FLCN/TFE3 double knockout (DKO) mice and assess VLDL-TG secretion rates. I will overexpress constitutively active TFE3 in wild-type mice to test if TFE3 is sufficient for VLDL-TG secretion. My preliminary data further lead me to hypothesize that the upregulated VLDL-TG secretion in FLCN- null livers is mediated by TFE3-dependent transcriptional activation of CTP:phosphocholine cytidylyltransferase alpha (CCTa). CCTa is the rate-limiting enzyme in the synthesis of phosphatidylcholine, a critical necessary component for VLDL-TG secretion. My preliminary data show increased CCTa mRNA and protein levels in FLCN knockout mice. I will generate FLCN KO, CCTa KO, FLCN/CCTa DKO mice. I will assess their susceptibility to NAFLD/NASH and perform VLDL-TG secretion assays. Completion of this work would provide insight into the regulation of hepatic lipid homeostatic processes affected in NAFLD/NASH, as well as demonstrate FLCN to be a promising therapeutic target for treatment.

NIH Research Projects · FY 2026 · 2023-03

Obesity is a multifactorial disease which results from complex interactions among multiple genes, behaviors, and environmental factors. A heightened genetic risk does not always translate to the development of obesity among children, which points to the presence of risk-modifying factors that likely relate to energy balance behaviors as key behavioral drivers. This study will build on our prior research to comprehensively examine the interplay between genotype and phenotype and, for the first time, systematically assess if energy balance behaviors in the context of the family environment, alone or in combination, can modify behavioral and genetic predispositions to childhood obesity. Specifically, this research aims to prospectively evaluate the extent to which associations of a heightened drive to eat (HDE) on 1-year changes in weight and adiposity outcomes are independent or additive to those of a genetic risk for obesity among children, ages 5 to 7, from families with low resource backgrounds. HED will be conceptualized as a broader behavioral phenotype which combines hyperphagic appetitive traits (i.e., the relative reinforcing value of food, a rapid eating rate, and eating in the absence of hunger). Children’s genotype will be assessed using a genetic risk score (GRS), which serves as an aggregate measure from multiple obesity risk single nucleotide polymorphisms (SNPs). In addition, the study aims to prospectively examine risk-modifying energy balance behaviors in areas of diet, physical activity, and sleep that may mitigate the detrimental impact of a HDE or high genetic risk on weight and adiposity outcomes and test whether these factors differ for HDE versus a high genetic risk of obesity on those outcomes. Lastly, the research aims to examine the impact of family-level influences in areas of family functioning and parent feeding on risk-modifying energy balance behaviors in children. Primary outcomes will include 1-year changes in children’s body mass index (BMI) z-score, percent body fat, and waist circumference. Identifying behavioral and family factors that may mitigate the impact of obesity risk on weight and adiposity outcomes is critical for moving the prevention of childhood obesity into a new direction. If successful, the knowledge gleaned from this research will offer new and powerful targets for addressing health disparities in obesity and formulating tailored family-based interventions in the fight against childhood obesity.

NIH Research Projects · FY 2026 · 2023-03

Aggregated microtubule-associated protein tau (tau) is the common lesion of a group of neurodegenerative diseases with heterogeneous clinical and neuropathological manifestations, collectively referred to as tauopathies. In line with the clinical and pathological diversity, pathological tau aggregates are biologically potent to induce normal tau into aggregations in a strain-dependent manner, suggesting an underlying mechanism of disease-specific tau pathogenesis. The long-term goal of the study is to elucidate the mechanism(s) of the strain-dependent tau transmission and search for therapeutic targets to inhibit the transmission procedure. The central hypothesis is that the imprinted properties of tau strain impair neuron biology via transmission activities. Our rationale is that understanding the mechanism by which tau strains differentially transmit will facilitate the development of therapeutic targets to selectively and efficiently treat different forms of tauopathy by inhibiting the critical transmission steps in tau pathogenesis. Our specific aims are to test the following hypotheses: (Aim 1) amyloid β plaques pathology interact with different forms of tau pathology and modulate of tau transmission; (Aim 2) the conversion of pathological conformations is the molecular basis of tau strain-dependent pathogenicity; (Aim 3) novel genes modulating tau transmission can be targeted as therapeutic strategies for tauopathy. This contribution is significant since the study will elucidate the molecular basis of the diversity of tauopathy and provide specific strategies to treat the diseases. The proposed research is innovative because we will use novel models of tauopathy to spatiotemporally investigate the mechanism of tau transmission and search for potential therapeutic targets with a high degree of disease relevance.

NIH Research Projects · FY 2026 · 2023-02

Project summary Antibody-secreting plasma cells play critical roles in health and disease, yet little is known about the biochemical mechanisms controlling antibody synthesis and secretion or how these pathways intersect with pathways controlling cell survival. Furthermore, due to the high metabolic demands inherent to robust antibody secretion and the tendency for many plasma cells to persist for decades, pathways restraining apoptosis in plasma cells are likely to be integrated in unique ways. This project centers on the hypothesis that long- lived plasma cells in bone marrow obtain requisite signals to maintain antibody secretion and avoid cell death by sensing extracellular ATP with a purinergic receptor known as P2rX4. We further hypothesize that eATP is produced locally in the bone marrow for plasma cells by osteoblastic cells via the gap junction protein known as Panx3. Thus, consistent with our preliminary data, inhibition of P2rX4 or Panx3 function is predicted to cause the depletion of long-lived plasma cells and reduce serum antibody titers including for disease-associated antibodies. To test our hypothesis, we will: 1) Define cell intrinsic P2rX4-regulated outcomes for newborn and long-lived plasma cells, and 2) Define cell extrinsic sources and outcomes for eATP/Panx3 regulation of long-lived plasma cells. These studies will provide unique and needed insights into the specialized survival mechanisms employed by long-lived plasma cells. This work supports our long-term objective of developing strategies to effectively and specifically disable or deplete problematic plasma cells.

NIH Research Projects · FY 2026 · 2023-02

Project Summary Meniscus Meniscus initiation. therapy basic novel Previous located maintain Using a Gli1-CreER driven Hh reporter mouse line, we recently found that Gli1-labeled cells contribute to the development of the meniscus horns from 2 weeks of age. In adult and aged mice, Gli1+ cells were localized to the superficial layer of anterior and posterior meniscal horns, and gradually decreased in number during aging. In vivo, these cells co-expressed known markers of mesenchymal progenitors as well as the lubricant Prg4. In culture, meniscal Gli1+ cells possessed high progenitor activities, such as proliferation, migration, and differentiation, under the control of Hh signal. Using an in vivo mouse meniscus injury model, we showed a rapid expansion of Gli1-lineage cells at the injury site of anterior horn. Ablation of these Gli1+ cells prior to injury slowed the meniscus repair process. Strikingly, intra-articular injection of the Hh activator, Purmorphamine (Pur), following injury accelerated meniscus repair and attenuated OA progression. Based on these data, we hypothesize that the Hh/Gli1 pathway can be therapeutically targeted to treat meniscus injury tears are the most common injury to the knee, affecting both young and old populations. healing is limited, however, and loss of function leads o cartilage loss and osteoarthritis OA) Current clinical treatment is palliative, and does not restore function, and no disease modifying drug is available for the t reatment of meniscus injury. Thus, there is a critical need to better understand the biologic mechanisms and pathways regulating meniscus homeostasis and repair in order to develop therapeutics. Mesenchymal progenitors play a critical role in tissue maintenance and regeneration. studies have demonstrated the existence of mesenchymal progenitors in the meniscus, with most within the superficial layer. Hedgehog (Hh) signaling is one of a few fundamental pathways that adult stem and progenitor cells in various organs and can activate these cells post injury. t ( and prevent OA development via the mobilization of endogenous meniscus progenitors. work design Aims evaluate scaffold-mediated The objectives of this are to understand the role of this novel signaling pathway in meniscus homeostasis and repair and to a novel drug delivery system to enhance meniscus repair by locally targeting t his pathway. Our specific are to: 1) determine the function of Hh signaling in meniscus development and repair; 2) synthesize and a nanoparticle (NP)-conjugated fibrous delivery system for meniscus repair; 3) Assess Pur-NP repair in a large animal model of meniscus injury.Small (mouse) and large (minipig) animal models, and complementary experimental tools will be utilized to develop and translate this new therapy. multidisciplinary successful, this work will healing Our team has worked together to generate the exciting preliminary data supporting this proposal. provide a novel therapeutic scaffold that mobilizes meniscus progenitors to improve of tears that are otherwise considered irreparable. If

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY In numerous experimental studies, mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin, prolong lifespan, prevent the progression of Alzheimer's disease and related dementias (AD/ADRD) and improve multiple other age-dependent processes. However, there are limited clinical data to know whether these therapies have anti-aging effects in humans. The potential role of mTOR inhibitors as disease-modifying treatment for AD/ADRD is of particular significance given the ongoing lack of clearly effective therapies and their immense healthcare and societal burden. Concern over drug safety, particularly in older patients, has remained a key reason as to why clinical trials investigating the potential benefits of mTOR inhibitors with respect to AD/ADRD and other aging-related outcomes have not been pursued. Yet, the significance of increasing age as a risk factor for mTOR inhibitor-associated adverse effects is not clearly established. Moreover, the majority of clinical trials of mTOR inhibitors suggest that side effects are largely reversible with dose modification and rarely severe. Further clinical investigation into the potential benefits and risks of mTOR inhibitors in the context of human aging is therefore needed. Among patients currently receiving this therapy, transplant recipients are the ideal population in whom to conduct a large and longitudinal observational study on the aging-related effects of mTOR inhibitors. Advantages of this group include their prolonged survival, increasing prevalence and frequent occurrence of common aging-related diseases (including AD/ADRD), among other reasons. The recent creation of a comprehensive database linking national transplant registry data to Medicare claims by the PI represents a welcome opportunity to study these critical knowledge gaps in a real-world cohort. In this study, we will leverage and further enhance this linked Medicare database to investigate the effect of mTOR inhibitors on the survival and healthcare utilization of older kidney and liver transplant recipients in Aim 1. We will subsequently evaluate the effect of mTOR inhibitor therapy and its interaction with age on the risk of AD/ADRD using this data source in Aim 2. Then, in Aim 3, we will establish the independent predictors of mTOR inhibitor adverse effects and perform a comprehensive assessment of real-world drug safety in older transplant recipients using detailed electronic medical record (EMR) data from the Veterans Health Administration (VA). In estimating mTOR inhibitor treatment effects, this proposal will employ modern statistical techniques that draw upon the multidimensional nature of Medicare claims data to strengthen confounder adjustment while applying a time-dependent framework, a novel application of this technique in this research area. Our findings will bring new and important evidence on the clinical effects and safety of mTOR inhibitors in older persons, which will subsequently establish the feasibility of future trials of mTOR inhibitors as treatment for AD/ADRD and as anti-aging therapeutics. Secondarily, the results generated will play a key role in developing consensus guidance that allow for an individualized treatment approach for older kidney and liver transplant recipients in the U.S.

NIH Research Projects · FY 2025 · 2023-02

PROJECT SUMMARY/ABSTRACT The US health care system leads the world in administrative complexity and its associated costs. Research on administrative costs has primarily focused on costs to the health care system and rarely includes patients, though patients often perform a lot of administrative work, meaning the time, effort, and stress of navigating the health system to obtain, pay for, and coordinate care. Cancer is a complex and often high-acuity condition with persistent disparities in health outcomes, making cancer care delivery a priority setting for assessing patient administrative burdens and identifying solutions. This program of research harnesses multiple methods to define, measure, and describe the impact of patient administrative burden in cancer care delivery on quality and care outcomes including oral anticancer agent adherence and guideline-recommended supportive care. This early K99/R00 will establish Michael Anne Kyle, PhD, RN as an independent investigator focused on improving the quality and value of cancer care delivery by reducing nonfinancial costs. Training goals in advanced survey and psychometric methods and cancer care delivery will support Dr. Kyle’s success in her research aims and transition to independence. An outstanding team of mentors will guide her training and career development. The K99 phase focuses on defining patient administrative burden in cancer care delivery and developing a survey instrument to measure it. It will engage patients or patient proxies in focus groups elucidating the patient-facing nonclinical tasks involved in managing cancer care (Aim 1a). Qualitative insights will inform the development and validation of a novel survey instrument measuring patient administrative burden and its psychometric properties (Aim 1b). The R00 phase, will administer the validated patient administrative burden survey to patients with cancer across a large NCI Comprehensive Cancer Center (Aim 2a). It will then link patient survey data with data on practice characteristics to examine the relationship between organizational characteristics and administrative burden. (Aim 2b). Finally, it will link the survey with patient-level utilization data burden to examine the relationship between administrative burden and cancer care quality, including adherence to oral anticancer agents and adherence to guideline-recommended supportive care (Aim 2c). Together, these results will enable the measurement and diagnosis of administrative burden across cancer care settings and produce evidence identifying and motivating actionable organizational or policy targets for clinical leaders and policymakers.

NIH Research Projects · FY 2026 · 2023-02

Project Summary This application proposes a five-year research and training plan with a scientific focus on the mechanisms through which PD-1 checkpoint inhibition leads to immune-related adverse events (irAEs). The hypothesis is that de novo loss of PD-1 signaling enhances the antigen sensitivity of CD4 T cells and leads to the activation of autoreactive CD4 T cells that mediate irAE development. To investigate this hypothesis, the candidate will analyze two complementary human cohorts of his mentors: a) a prospective irAE cohort of patients on PD-1 immunotherapy to compare the early changes of CD4 T cells in patients who do (irAE+) and do not (irAE-) develop irAEs, and b) a cohort of patients on PD-1 immunotherapy that receive seasonal influenza vaccination to test whether any underlying immunopathology in irAE+ patients, not otherwise observed with evaluation of basal states, can be revealed with the help of immunization. Using the prospective irAE cohort, Aim 1 will evaluate whether patients that develop irAEs have a different naïve CD4 T cell epigenetic and transcriptional profile at baseline that allows robust activation of autoreactive CD4 T cells following PD-1 inhibition. Further, this aim will analyze the transcriptional profile and clonal diversity of the activated CD4 T cells generated after PD-1 inhibition in irAE+ patients. Using the aPD-1 cohort of influenza vaccination, Aim 2 will answer the question of whether PD-1 signaling is required to finetune the TCR threshold and transcriptional profile of CD4 T cell responses to maintain immune fitness and homeostasis. These studies will define the path through which PD-1 inhibition leads to the activation and propagation of autoreactive CD4 T cells to cause immune-mediated pathology. They will also identify cellular and molecular targets that can be used for early diagnosis and treatment of patients with rheumatologic and other irAEs. Scientifically, the candidate’s career development goals are to gain expertise in the conduct of clinical and translational research, bioinformatic analyses, single-cell technologies, and transcriptional and epigenetic regulation of autoimmune pathways in patient samples. Professionally, the candidate aims to gain experience in scientific writing, grant preparation, communication, and data presentation culminating to an R01 submission. To facilitate the candidate’s growth as a physician-scientist, this proposal combines novel experimental single-cell approaches and a specific career development plan designed by the candidate and his mentors, Dr. Wherry and Dr. Laufer. The candidate’s long-term goal is to transition to a tenure- track faculty position and to develop an independent NIH-funded research program focusing on the mechanisms of autoimmunity and T cell dysfunction to ultimately prevent and treat rheumatologic diseases. The rich scientific and collaborative environment at the University of Pennsylvania will position the candidate to have a highly impactful, translational research career in human autoimmunity.

NIH Research Projects · FY 2026 · 2023-02

Abstract The Achilles Tendinopathy Center of Research Translation (AT-CORT) at the University of Pennsylvania will foster fundamental discoveries to guide clinical translation, as well as develop and employ novel translational resources, models and technologies, to address the highly significant research and clinical challenge of Achilles tendinopathy. Despite the high frequency and increasing prevalence of tendinopathy in young and old patients, and the significant pain and disability that arises from this condition, as well as the associated high cost to society, effective treatment modalities have stagnated over the last two decades. This is due to the lack of fundamental understanding of tendon disease etiology and pathogenesis, which limits development of novel treatment modalities. At present, beyond surgical intervention for late-stage disease, the only approved clinical therapy involves physical interventions via controlled rehabilitation mechanical loading of the tendon. While efficacious in some patients, outcomes of this intervention do not stem disease progression in most patients. Given the central mechanical role of the Achilles tendon, and the critical role of mechanical loading and mechanobiology on tendon cell homeostasis, it is critical that we develop and expand our understanding of the role of mechanical forces in disease onset and progression to optimize existing and inform new treatment strategies. Our proposed AT-CORT is uniquely positioned with a critical mass of multidisciplinary scientists and clinicians with strong interest and expertise in these and related areas. The Overall goal of the AT-CORT is to develop new insight and technologies that uncover the mechanobiologic basis of Achilles tendinopathy across length scales, from the nucleus, to the cell, to the tissue microenvironment to patients. We will assess these critical elements during disease onset and progression, informed by both vivo animal models that replicate disease processes and source material and real-world loading data from living human subjects. The AT-CORT is comprised of four independent and yet interactive elements, including an Administrative Core to oversee and guide interactions and primary Research Projects focused on the transfer of information from the external tendon cell microenvironment through the cytoskeleton (Project 1) and on chromatin remodeling and mechano-epigenetic regulation of tendon cell phenotype (Project 2). Using cells, tissue, and loading information derived from both human and animal tendinopathic models (Tissue Core), these research projects will advance our knowledge of the origins of tendinopathic disease and define new avenues for therapeutic intervention. Together, our highly interdisciplinary team, innovative tools, and outstanding and interactive Research Projects and Cores will dramatically advance knowledge, develop innovative tools and insight, and provide new directions for translation of novel therapies to treat Achilles tendinopathy.

NIH Research Projects · FY 2025 · 2023-02

Abstract The Orthopaedic Research Society (ORS) Spine Section is the premier professional body devoted to basic and translational spine research, and an indispensable scientific forum for investigators at all career stages. While Spine Section Symposia have been held successfully as sub-meetings of the main ORS Annual Meeting for several years, available funds have resulted in significant programmatic limitations. A recent COVID-19 impact survey led by Spine Section members highlighted the challenges faced by spine researchers, particularly by trainees, early investigators and minorities. We are requesting NIH funds to facilitate programmatic expansion of Spine Section Symposia for the next 3 years to specifically support initiatives designed to address these challenges. Specifically, the objectives of these annual, half day symposia will be to leverage and expand the mission of the ORS Spine Section in order to catalyze transformative basic and translational spine research through enhanced mentoring, diversity and collaboration across institutions and countries. Four Specific Aims reflecting these integrated themes are proposed for each of 3 annual half day meetings: 1) Enhancing spine research through precision mentoring; 2) Enhancing spine research by increasing opportunity and participation by individuals from diverse backgrounds at all career stages; 3) Enhancing spine research through multi-institution and international collaborations; and 4) Cutting edge basic and translational spine research to enhance patient outcomes. Specific scientific topics will be selected from recent and future Spine Section membership surveys, and will include: emerging diagnostic strategies for disc degeneration and back pain; innovative drug delivery techniques for the spine; and leveraging developmental and evolutionary biology, genomics and artificial intelligence to advance next generation spine therapeutics. Symposia will be held the day preceding the main ORS Annual Meeting in a hybrid format that will in able full, interactive participation by both in person and virtual attendees.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY The multiple tissue compartments or niches in the respiratory system display varying abilities to repair and regenerate after acute injury or in chronic disease states. The alveolar niche is critical for gas exchange as well as acting as a sentinel for environmental stimuli including infectious organisms and pollutants. Much of the regenerative power of the alveoli rests within the alveolar type 2 (AT2) cell, which is not only critical for surfactant production and innate immune responses, but also harbors the resident progenitor cell population. A building body of research has shown that subsets of AT2 cells can proliferate and differentiate into alveolar type 1 (AT1) cells after acute injury, which is critical for regenerating functional alveoli. These AT2 cells behaviors are regulated by signaling, transcriptional, and epigenetic mechanisms that have only recently started to be elucidated. To further our understanding of the role that epigenetic pathways play in lung alveolar regeneration, we performed a small molecule screen using an alveolar organoid assay to identify pathways that promote alveolar repair and regeneration. This screen identified multiple inhibitors of the Disruptor of Telomeric Silencing- 1 like (Dot1L) that regulate alveolar organoid size. Dot1L is the sole enzyme which is known to methylate H3K79 (H3K79me1/2/3 marks), and Dot1L has been demonstrated to play critical roles in promoting pluripotent stem cell reprogramming, and cellular responses to injury and tissue regeneration. Our data show that Dot1L inhibition increases alveolar organoid size in a dose dependent manner. To better understand the role of Dot1L in lung development and regeneration in vivo, we generated a Dot1L conditional knockout mouse allele and inactivated Dot1L during lung development and in multiple models of lung injury and regeneration. Loss of Dot1L during lung endoderm development results in the loss of H3K79 methylation and premature or enhanced expression of AT1 and AT2 marker genes, suggesting acceleration of AT1 and AT2 cell differentiation. In two models of lung alveolar regeneration, loss of Dot1L in AT2 cells results in dramatic acceleration of AT2-AT1 differentiation after lung injury. Single cell RNA-seq (scRNA-seq) combined with ChIP-seq analysis reveals that loss of Dot1L leads to the emergence of a new AT2 cell state characterized by a dramatic increase in the expression of the important transcriptional regulators Id1 and Id2 as well as an overall increase in expression of metabolism genes related to oxidative phosphorylation (OxPhos). Taken together, our data lead to the hypothesis that Dot1L plays an important role in regulating lung alveolar responses to acute lung injury by regulating the lineage barrier between AT2 and AT1 cells via de-repression of the critical transcriptional regulators Id1/Id2 and a switch to OxPhos metabolism, resulting in acceleration of AT2-AT1 differentiation.

NIH Research Projects · FY 2026 · 2023-02

Abstract: The diagnosis and management for women who are at risk for ectopic pregnancy (EP) and spontaneous abortion (SAB), has not changed substantially in decades. While ultrasound can diagnose a significant portion of women at presentation, because of its limited accuracy in abnormal and early gestation, a large number of women need serial tests and procedures to determine the final location and viability of an early pregnancy. Misdiagnosis and iatrogenic complications during this time are still too common. This conundrum results in great stress and uncertainty for women undergoing clinical care and their health care teams. To date, we have demonstrated (and validated) that multiplexed biomarkers from divergent biological pathways can be used to minimize both false positive and false negative discrimination (rather than balancing the two errors with lower accuracy). In parallel, we have discovered and screened more than 54 novel biomarkers to obtain 11 candidates that can predict early pregnancy outcome. Using machine learning we have demonstrated that as few as 3 of these markers can predict the location of an early gestation (IUP vs EP or SAB) with 95% accuracy. Additionally, an overlapping group of 3 markers can predict the viability of a gestation (IUP vs SAB or EP) with 94% accuracy. When both tests are used in combination the accuracy is 96.9%. We now plan to externally validate these companion diagnostic(s) in a separate population-based prospective study and to determine the optimal conditions of use. We will optimize performance in women presenting with a pregnancy of unknown location as well as assess predictive values for all women presenting at risk for early pregnancy loss using an independent population-based prospective study (SA1). We will determine if accuracy can be improved with the combination of presenting signs, symptoms, and ultrasound findings (SA2). Moreover, we will validate our biomarker tests in two distinct populations of interest: women with high and low risk for EP: a) asymptomatic women prior to presentation for care (EAGeR Trial) and b) women diagnosed with a persistent pregnancy of unknown location (ACTorNOT Trial) (SA3). Developing novel biomarkers that distinguish normal physiology from the presence of gynecologic disorders is an NICHD research priority area. Our productive and established team is proposing rigorous cross disciplinary, state of the art, and novel research with great scientific impact that has the potential to produce a paradigm shift in clinical care models for the diagnosis and management of women in early pregnancy. Our detailed plan for biomarker development is iterative and nimble and, importantly, includes validation. Our development plan is informed by methodology from successful biomarker development, is designed to minimize known pitfalls, and is developed with FDA guidance.

NIH Research Projects · FY 2026 · 2023-02

Abstract. In the United States alone, the number of proton therapy centers has increased to 41 sites, with many more currently under construction or in planning stage. While the investment for such centers is in the hundreds of millions of US dollars, research is ongoing to determine whether proton therapy improves treatment outcomes. A sensitive diagnostic tool for the evaluation of alveoli architecture in this active research area would not only enable early targeted treatment to slow down progression of radiation-induced lung fibrosis but also significantly benefit the ongoing preclinical evaluation. The imaging tools currently in use have a poor to moderate sensitivity that is insufficient for detecting early changes in the lungs and/or are proving impractical with respect to radiation dose and logistical complexity for longitudinal preclinical studies. To address this critical need, we introduce an imaging tool for early detection of lung microstructural changes by advancing the emerging field of x-ray darkfield imaging. In conventional x-ray, image contrast is formed by attenuation based on the interpretation of x-rays as particles. If sensing x-rays as electromagnetic waves, additional x-ray contrast mechanisms such as diffraction, phase-shift and small-angle scattering can be accessed. X-ray scattering on healthy, gas-filled pulmonary alveoli generates a strong darkfield signal, and the signal decreases when the integrity of the alveoli is affected. Preliminary in-vivo small animal experiments successfully demonstrated an on average ten-weeks-earlier detection of early onset of radiation-induced lung fibrosis from conventional photon therapy. A number of methods for acquiring x-ray darkfield images have been investigated in recent years. However, current solutions require complicated, shock-sensitive and expensive hardware implementations. A more practical method involves the use of filters consisting of random structures (so-called diffusers) to generate near-field interference speckle patterns for acquiring darkfield images. Our long-term goal is translating x-ray dark-field imaging from physics research laboratories into the preclinical imaging arena to provide the needed tool for longitudinal lung assessment. Our solution includes the design of novel deep- learning based speckle tracking in combination with a diffuser design based on nanoparticles which is inexpensive to fabricate compared to gratings. The following specific aims will be pursued: (1) to develop a software infrastructure for in-vivo small animal x-ray darkfield imaging, (2) to implement an x-ray darkfield prototype for detection of early pulmonary toxicity from radiotherapy, and (3) to evaluate x-ray darkfield prototype performance in phantoms and in-vivo longitudinal animal studies. This proposal will advance the field of speckle- based x-ray dark-field imaging by deepening the basic understanding and by translating it from physics research laboratories into the preclinical arena. Toward this end, we anticipate that our x-ray dark-field imaging concept will serve as a low-dose tool for longitudinal in-vivo small animal studies. The proposed solutions have the potential to drive the translation of x-ray dark-field imaging forward into the clinical routine.

NIH Research Projects · FY 2025 · 2023-02

PROJECT SUMMARY / ABSTRACT Abdominal aortic aneurysm (AAA) is a life-threatening condition in which progressive dilatation of the infrarenal aorta leads to rupture. With ~2.3 million prevalent cases in the United States, AAA afflicts ~4% of the population ≥ 65 years of age and is responsible for ~41,000 deaths annually. No medical therapies exist that prevent AAA, AAA growth, rupture, or aneurysm-related death. The only efficacious intervention is surgery. Genome-wide association studies (GWAS) and preliminary pharmacogenetic causal inference studies from AAA GWAS demonstrate that both LDL-C lowering and PCSK9 inhibition are protective of AAA. The greatest therapeutic opportunities for pharmacological treatment of AAA lie in prevention of aneurysm expansion in individuals with small AAA. Despite our team's robust evidence linking LDL-C and PCSK9 to the development of AAA, their causal role in AAA growth remains unknown and the data to support conducting a large-scale efficacy trial is lacking. This project will leverage two orthogonal approaches to generate data that supports the role of LDL-C in AAA growth and that intensive LDL-C lowering with PCSK9 inhibitors will protect against aneurysm expansion. First, we will perform a multi-ancestry meta-analysis of GWAS of AAA growth rate and leverage these data to conduct genetic causal inference experiments interrogating the role of PCSK9 and LDL-c in AAA expansion. Second, we will conduct a quadruple-blind, randomized, placebo-controlled mechanistic clinical trial to test the effect of intensive short-term LDL-C lowering with PCSK9 inhibition on inflammation in the aneurysmal aortic wall; the primary outcome will be the production of the inflammatory cytokine interleukin 6 (IL-6) monocytes/macrophages in the aortic wall as measured by single nucleus RNA sequencing and confirmed by bulk RNA sequencing tissue- based immunofluorescence. Key secondary outcomes include: 1) matrix metalloproteinase 9 (MMP-9) production and activity; 2) relative numbers, cell type distribution, and inflammatory state of infiltrating immune cells; and 3) the relative number and proliferative/contractile state of aortic smooth muscle cells in the aortic wall. Successfully completing the proposed research will establish causal evidence linking LDL-C and AAA growth, and the ability to modulate this with PCSK9 inhibition. It will provide human mechanistic evidence that PCSK9 inhibitors induce anti-inflammatory changes in aneurysmal aortic wall that protect from aneurysm expansion. These data will provide the justification for a future large-scale randomized controlled trial to assess the efficacy of PCSK9 inhibitors to treat AAA.

NIH Research Projects · FY 2026 · 2023-01

Phagocytosis by microglia, the brain's resident macrophages, is central to Alzheimer Disease (AD) risk and pathogenesis. Current β-amyloid (Aβ) clearing immunotherapies use monoclonal antibodies to indirectly elicit phagocytosis, in an attempt to reverse AD manifestations. This form of passive immunization induces microglial reactivity and neuroinflammation, two features abundant in AD neuropathologic specimens. Due to a dearth of tools, prior mechanistic study of microglia-Aβ interactions used loss of function strategies such as Trem2 KO or microglial depletion, leaving uncertain whether microglial Aβ phagocytosis itself is sufficient to alter neuropathology. To address this knowledge gap, we clearance engraftment test engineered macrophages to achieve specific amyloid with antigen receptors ( AβCAR) , and developed methods for efficient macrophage in the brain. This dual PI proposal unites experts in immune cell therapies and microglial biology to the overarching hypothesis that Aβ-chimeric AβCAR-engineered macrophages effectively clear amyloid in vivo and that associated neuroinflammation is prevented by manipulation of the inflammasome. In preliminary data we successfully engineered AβCAR engulf into determine engraft neuropathology and AβCAR exposure NLRP3 on transplant determine in Aim inflammasome close to expressing macrophages, found that they specifically amyloid chemotherapy-dependent and - independent methods to transplant macrophages the mouse brain at unprecedented efficiency, ensuring the feasibility of this proposal. In Aim 1 we will how active cell-based Aβ targeting affects amyloid pathology in murine models. To do so, we will AβCAR macrophages into the 5xFAD model of Aβ pathology and measure their effects on and behavior. Aim 1 will clarify the controversial relationship between microglial Aβ phagocytosis pathology using a cell engineering strategy with high t herapeutic potential. Where Aim 1 focuses on how macrophages affect the brain Aβ, Aim 2 addresses how brain Aβ in turn affects macrophages. Amyloid induces microglial state changes and neuroinflammation, with strong mechanistic data implicating inflammasome signaling as a major driver. Aim 2 will therefore elucidate the effects of AβCAR targeting microglial reactivity, neuroinflammation, and their degree of rescue by NLRP3 eficiency. To do so, we will AβCAR macrophages engineered from Nlrp3 KO or Nlrp3 WT donor mice into the 5xFAD model and a) the degree to which CAR-augmented Aβ uptake increases macrophage inflammatory profiles and turn neuroinflammation b) whether this is rescued by Nlrp3 loss, and c) the consequences on Aβ pathology. 2 will improve our understanding of how immunotherapies lead to neuroinflammation and determine whether inhibition is a strategy to enhance cell therapies for neurodegeneration. Together, these aims will knowledge gaps about the role of microglial phagocytosis in AD treatment and create a cell-based strategy specifically target proteins in the CNS, using principles our multi-PI team has already tested in human disease. , and developed d

NIH Research Projects · FY 2025 · 2023-01

Project Summary: A number of neurodevelopmental disorders including autism spectrum disorder, attention- deficit/hyperactivity disorder and Tourette syndrome exhibit characteristic motor-related symptoms such as repetitive or impulsive action patterns. It is possible that these behaviors are a result of dysregulated action control mechanisms mediated via cortico-basal-thalamic pathway disruption. However, it is not currently understood how alterations in these circuits give rise to the deficits in action control commonly observed in these disorders. Copy number variation of genes encoding for synaptic adhesion molecules, such as Neurexin1α (Νrxn1α), have been shown to confer a significantly increased risk for these disorders, however, the underlying neural etiopathology is currently unknown. Recent findings in acute striatal slices have revealed that loss of Νrxn1α function results in decreased synaptic strength of medial prefrontal cortical inputs to the indirect pathway of the dorsal striatum, providing a potential neural mechanism for irregular action control. However, it is unclear whether these synaptic deficits confer variations to larger scale neural dynamics related to action control dysfunction in vivo. I hypothesize that these Νrxn1α mutations drive action control deficits (Aim 1) via mutation-associated corticostriatal circuit alterations specific to the indirect pathway (Aim 2) in Nrxn1a KO mice. To assay multiple key aspects of action control, I will employ a novel treadmill-based operant task that allows for the comprehensive study of action initiation, suppression, and modulation. Using dual-site in vivo electrophysiological techniques, I plan to describe the underlying corticostriatal population recruitment related to task performance in two fronto-striatal circuits (mPFC→DMS and M2→DLS) in both Νrxn1α WT and Νrxn1α KO mice. These findings will provide valuable insight into the neural pathology involved in many neuropsychiatric and neurodevelopmental disorders as well as elucidate corticostriatal neural mechanisms involved in action control regulation.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY CD8 T cell responses to viral infections and tumors contribute significantly to the immune responses that dictate the clinical outcomes of such pathologies. The functional integrity of CD8 T cell responses depends on the characteristic properties of CD8 effector (Teff) and memory (Tmem) populations. However, during chronic viral infections and cancer, antigen persistence without clearance precludes effective Teff and Tmem development, instead biasing CD8 T cell differentiation towards an epigenetically distinct “exhausted” lineage (Tex). Tex exhibit progressive dysfunction and loss of effector properties, proliferation capacity, and memory potential, as well as a sustained increase in co-expression of PD1 and multiple other inhibitory receptors (IRs). Interrogating the fundamental mechanisms that initiate and maintain the Tex epigenetic state is of central importance to understanding Tex biology and identifying strategies to selectively target or modulate Tex. However, the field generally lacks a detailed mechanistic understanding of Tex-specific epigenetic processes. In models of exhaustion during chronic infection and of dysfunctional tumor-specific T cells, the transcription factor TOX is essential for the initiation of Tex development, repressing terminal Teff differentiation and potentiating epigenetic commitment to the Tex lineage. This proposal seeks to identify and interrogate the mechanistic details of Tex regulation by TOX that would be required to begin developing immunotherapy approaches to epigenetically reprogram Tex and improve immunotherapy clinical outcomes. The molecular transactions TOX employs to exert its effects remain largely unknown. Understanding the details of TOX activity remains limited by a lack of functional characterization of its N- and C-terminal domains (“NTD” and “CTD”) in relation to its HMG-box DNA binding domain. My preliminary data demonstrate in vitro that loss of either the TOX NTD or CTD is sufficient to abrogate the increase in surface PD1 expression that is characteristically driven by full-length (“FL”) TOX, suggesting important, as yet unknown roles for these domains. The central hypothesis of this proposal is that distinct features of TOX activity are attributable to its N- vs. C-terminal domains and that NTD- or CTD-specific perturbations will enable selective modulation of Tex responses to chronic viral infection. This proposal tests this hypothesis by interrogating features of TOX’s interactions and domain-level function at the Pdcd1 locus (encoding PD1), by defining the extent to which the NTD and CTD exhibit global differences in their Tex-specific roles, by defining how the NTD and CTD program the Tex epigenetic state, and by determining which NTD- and CTD-mediated protein interactions TOX uses to regulate Tex transcription. This proposal will thus advance fundamental knowledge of how the molecular processes regulating exhaustion may be manipulated to improve CD8 T cell responses during chronic viral infections and cancer.

NIH Research Projects · FY 2025 · 2023-01

Project Summary/Abstract This project proposes the development of new methods and data resources to integrate modern artificial intelligence (AI) techniques into predictive toxicology, as well as the application of those methods and resources to generate new hypotheses linking putative toxicants to specific clinical outcomes. The recent explosion of publicly available chemical and biomedical data provides an immensely valuable resource for computational toxicologists, but existing techniques for learning from these data perform poorly and fail to capture crucial patterns that span multiple levels of biological organization. For example, the US FDA maintains a computational toxicology database cataloguing over 875 thousand chemicals of toxicologic concern, yet only a small handful of these have been characterized in terms of their downstream clinical effects. However, informatics and machine learning (ML) provide specific tools that may solve this issue. This project focuses on 2 of those in particular: Graph machine learning (Graph ML) and semantic data analysis. Since both of these techniques allow for the integration of information from multiple otherwise incongruent sources, they have the capacity to outperform simpler traditional methods for pattern discovery, while increasing both inferential capacity and statistical power. Our central hypothesis is that inductive learning on semantic graph data provides an effective means for generating and validating translational and mechanistic conclusions from existing public toxicology data. In Aim 1 (K99), a new data infrastructure—driven by a large, ontology-controlled graph database aggregating public toxicology data—will be constructed and evaluated on several important tasks in computational toxicology. Together, these resources will be named `ComptoxAI'. Aim 2 (K99) will develop and apply a graph machine learning strategy to predict new adverse outcome pathways (AOPs) in the graph database. Importantly, this aim will use an automated machine learning (Auto ML) approach to discover optimized neural network architectures for this prediction task in a data-driven manner. This Auto ML strategy will use estimation of distribution algorithms (EDAs) to search for optimized network architectures in a probabilistic manner. An expected side effect of the Auto ML approach is increased model interpretability over existing applications of Graph ML. Aim 3 (R00) will use semantic data analysis via ontological inference to refine Aim 2's model outputs into meaningful knowledge, proposing specific mechanistic explanations for the newly proposed AOPs. Aim 4 (R00) will use the resources and outcomes of the previous Aims as a starting point to develop and disseminate new open-source data standards, software resources, and research reporting protocols, with the goal of creating a collaborative, cross-institutional research ecosystem for AI research in computational toxicology. Beyond the methodological and infrastructural contributions of this work, successful completion of the Specific Aims will yield a library of mechanistically-based hypotheses linking putative toxicants to specific clinical outcomes, addressing a major need in predictive toxicology. In supporting the goals of the open science movement, all research outcomes from this project—including papers, software, data, and other resources—will be made available for free public reuse.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Negative valence states can be modeled in mice using fear conditioning and reward seeking (an example of frustrative nonreward). Such states lead to transcriptional aberrations at the level of both individual genes and genome-wide; these changes are also sexually dimorphic. One gene of considerable interest is cyclin-dependent kinase 5 (Cdk5). While most prior studies have focused on the mechanisms of Cdk5 protein activation and signaling in stress-evoked behavior, we recently reported that cocaine exposure and fear conditioning lead to transcriptional regulation of Cdk5 in male mouse brain. This proposal will directly examine the connection between Cdk5 gene activation and Cdk5 protein activity. Furthermore, there is emerging evidence that Cdk5-associated histone modifications correlate with drug-, and fear-induced Cdk5 expression. The current proposal explores the functional relevance of stress-induced histone modifications at the murine Cdk5 locus across multiple behavioral paradigms using the innovative approach of locus-specific epigenetic editing. This approach allows us to recapitulate endogenous mechanisms of gene expression, avoiding non-physiologically relevant changes in protein expression resulting from traditional knockout or overexpression. Furthermore, we apply cell-type specific analyses to elucidate Cdk5 function in brain regions across the corticostriatal-limbic circuitry in the regulation of negative valence states. This proposal will examine the direct functional relevance of sex-specific, epigenetic regulation of Cdk5 by fear conditioning (Aim 1) and cocaine seeking during abstinence (Aim 2) using targeted epigenetic editing. Sex- and region- specific phosphoproteomic profiling will be applied to define relevant Cdk5 targets. Identification of the precise transcriptional and epigenetic mechanisms by which stress regulates specific gene expression is critical for the development of targeted antidepressant treatments.

NIH Research Projects · FY 2026 · 2023-01

SUMMARY Reduction in functional insulin-secreting β cells underlies the progression of all forms of diabetes, underscoring the translational relevance of deciphering molecular pathways regulating the formation, growth, and function of β cells. The transcriptional networks critical for the proper development, differentiation, and expansion of β cells work through islet enhancers, super enhancers, and active promoters that form 3D hubs. The homeodomain transcription factor Pdx1 is a critical member of the β-cell transcriptional network during development and in postnatal β-cell function. Pdx1 is mutated in monogenic forms of human diabetes and plays critical roles in early pancreas specification, regulation of organ size, and in β-cell formation, proliferation, and identity. Our preliminary data reveal that developing β cells exhibit altered subnuclear localization and reduced levels of Pdx1 protein as they advance through the cell cycle. Further, ectopically elevated levels of Pdx1 prevent cell cycle progression, suggesting that dynamic regulation of expression is required for effective β-cell expansion. We identify an intrinsically disordered protein region (IDPR) in the Pdx1 C-terminus (aa 207-223). IDPRs, commonly found within transcription factors, lack fixed secondary structure and are amenable to flexible conformations and phase separation. IDPRs promote protein-protein interactions and transcriptional hub formation at super enhancers necessary for coordinated gene regulation. We have found that the Pdx1 C-terminus mediates interaction with the one cut homeodomain transcription factor Oc1 in multipotent pancreatic progenitor cells to establish the endocrine gene program, with long-term impact on postnatal islet function and β-cell compensation. We previously identified the E3 ubiquitin ligase substrate adaptor SPOP as a PDX1 C-terminus partner (via aa224-238) that mediates ubiquitination and proteasomal degradation of PDX1. Our preliminary data suggest that SPOP and Oc1 compete for interaction with the Pdx1 C-terminus and that Oc1 protects Pdx1 from SPOP- mediated degradation. Notably, the C-terminus harbors several diabetes-associated human mutations, one of which we recently found disrupts the PDX1/SPOP interaction. Thus, we hypothesize that PDX1/OC1 interactions, in part mediated by their IDPRs, regulate Pdx1 stability, cell cycle progression, and pancreatic endocrine differentiation. This hypothesis will be tested in 3 Aims: (1) To determine the mechanisms whereby Pdx1 and Oc1 cooperate to establish a chromatin landscape permissive for endocrine differentiation and proliferation; (2) To define the roles of the Pdx1 and Oc1 IDPRs in protein-protein interaction and pancreas development; and (3) To define the molecular mechanisms by which the Pdx1 C-terminal domain regulates protein stability and function during pancreas organogenesis and endocrine differentiation. The impact of human diabetes-associated mutations will be investigated in this context. Our studies will determine a novel and cohesive role for unstudied structural features of the Pdx1 C-terminus in β-cell development. Results of our studies will inform therapeutic efforts to optimize β-cell expansion for cell-based therapies.

NIH Research Projects · FY 2025 · 2023-01

Project Summary/Abstract Opioid Use Disorder remains a dire public health problem, but opioid agonists such as fentanyl remain a first- line therapy for several pain conditions. As in recreational use settings, prolonged use of opioid agonists in pain management can produce physical dependence and a paradoxical decrease in pain thresholds and tolerance, which increase patients’ reliance on opioids and increase the likelihood of transitioning to Opioid Use Disorder. Continual stimulation of the inhibitory µ-opioid receptor (MOR), the primary mediator of the analgesic and rewarding effects of opioid agonists, induces counter-adaptive excitatory processes and hyperexcitability in MOR-expressing neurons. To discover new treatments that leverage the benefits of opioids but mitigate aversive and life-threatening side effects of prolonged opioid use, it is critical to determine the specific cell-types and neural circuits in the brain that are susceptible to the opioid-induced cellular maladaptations that underlie dependence and OIH. MORs are densely expressed throughout ascending pain pathways, including in the parabrachial nucleus of the pons (PBNMOR). PBNMOR neurons project to the capsular region of the central amygdala (CeC), which itself contains a pronociceptive population of neurons expressing Protein Kinase C-δ (CeCPKCδ) Activation of the PBNMOR®CeC pathway decreases pain tolerance and increases aversion-related responses, but its role in driving OIH and withdrawal, and the contribution of CeCPKCδ neurons in particular, has not been investigated. The goal of the proposal is to determine the impact of fentanyl dependence on the neural activity in the PBNMOR®CeCPKCδ pathway and whether such activity drives withdrawal and OIH-related behaviors. Aim 1 will investigate the effects of fentanyl dependence on PBNMOR®CeC projections and their role in driving OIH and withdrawal behavior by using in vivo population calcium imaging and chemogenetic manipulations during nociceptive assays and withdrawal. Aim 2 will image and manipulate the CeCPKCδ population during behavior to determine its contribution to OIH and withdrawal. Successful completion of these Aims will lay the foundation for future investigations of the pathophysiology of opioid dependence. Ideally, results from this work will suggest novel therapeutic avenues for reducing dependence mechanisms within specific cell-types. Ms. Wooldridge will receive expert training in chemogenetics, in vivo calcium imaging and its analysis, viral-mediated genetic targeting, and rigorous experimental design and statistics. The addition of this training will facilitate the applicant’s current and future research goals and enable her to have continual impact on basic neuroscience research throughout a future career as an independent academic researcher.

NIH Research Projects · FY 2025 · 2023-01

Abstract: Melanoma is the fifth most common skin cancer; however, it is the most lethal. A minority of patients achieve a durable complete response to current standard of care immune and targeted therapies. Clues to new therapeutics may be found in understanding why melanoma incidence and outcomes differ significantly between groups of people that differ based on sex, geographic ancestry, and environmental exposures beyond UV light. Numerous epidemiological studies identified an inverse relationship between caffeinated coffee consumption and cutaneous melanoma. Daily caffeine consumption of at least 350mg (3 cups of coffee) is associated with a 20% risk reduction of cutaneous melanoma. Caffeine is arguably the most widely and frequently consumed bioactive molecule in the world and is known to alter activity of several cellular proteins; however, its specific impact on melanoma is unknown. The best characterized caffeine targets are the adenosine receptor family (ADORAs) which are G Protein-Coupled Receptors (GPCRs). There are four ADORA subtypes, A1, A2A, A2B, A3. My preliminary data suggests that caffeine inhibits melanoma proliferation, and that this effect is mediated by A2A, as cells with genetically depleted A2A were insensitive to caffeine. Additionally, pharmacologic A2A activation increased melanoma proliferation, together suggesting that A2A promotes melanoma and may be a druggable target. In preliminary in vivo studies using mouse melanoma models, daily consumption of caffeinated water delayed tumor growth and extended animal survival. This proposed study aims to understand the mechanism by which caffeine displays an anti-melanoma effect and if it is through modulating tumor intrinsic A2A. The central hypothesis is that caffeine inhibits melanoma through a pro-proliferative G protein-coupled A2A receptor signaling pathway and that caffeine may be beneficial alone or in combination with standard of care immune and/or targeted therapeutics to improve melanoma outcomes. In Aim 1, I will determine whether caffeine inhibits melanoma proliferation via modulation of A2A signaling and elucidate which G proteins are coupled to A2A in melanoma. In Aim 2, I will determine if caffeine’s anti-melanoma effect is mediated by tumor- intrinsic expression of A2A and if caffeine can potentiate the effects of the current standard of care. This project will delineate the anti-melanoma effects of caffeine and provide mechanistic/biomolecular insight to the many epidemiological observations, potentially leading to new therapeutics.

NIH Research Projects · FY 2026 · 2023-01

Project Summary/Abstract Diabetic kidney disease is the leading cause of end-stage renal disease and a major contributor to morbidity and mortality. We have successfully performed single nucleus RNA sequencing (snRNAseq) and single nucleus ATAC sequencing (snATACseq) on five healthy control and eight diabetic kidney samples to measure the cell-type-specific transcriptional and chromatin conformational profile of early human diabetic kidney disease. The differentially expressed transcripts in the diabetic proximal tubule showed upregulation of gluconeogenic genes and enrichment of pathways involved in corticosteroid signaling. This proposal aims to integrate snRNAseq and snATACseq to determine whether there are changes in chromatin accessibility in the enhancer and promoter regions of corticosteroid-sensitive genes that regulate gluconeogenesis. We will subsequently validate our in vivo findings with an in vitro model of diabetic injury and a diabetic mouse model. This proposal builds on the principal investigator’s previous research experience and clinical training. Currently, Dr. Parker Wilson is spending 25% of his time on the renal and molecular pathology clinical services with the remaining 75% allocated to basic research in Dr. Benjamin Humphreys’ laboratory. Dr. Wilson has an established mentoring relationship with Dr. Humphreys and has published his analysis of snRNAseq data from human diabetic kidney as a first author in PNAS. In addition, Dr. Wilson has a recently-accepted co-first author manuscript describing multimodal snRNAseq and snATACseq integration in the healthy adult kidney in Nature Communications. These findings provide the foundation for this application, which will focus on expanding Dr. Wilson’s scientific skills in single cell methods, bioinformatics analysis and models of diabetic kidney injury. The career development goals will be achieved through mentoring by Dr. Humphreys and an advisory committee with expertise in regulation of chromatin conformation, bioinformatics and diabetic nephropathy. Dr. Wilson will undertake didactic coursework in research ethics, scientific communication and grant writing, molecular biology and advanced computer programming to further his existing knowledgebase. The work will take place at Washington University, which has a strong history of mentoring successful physician-scientists. Completion of this career development award will build a solid foundation for Dr. Wilson as he pursues independence and R01-level funding.

NIH Research Projects · FY 2025 · 2023-01

Project Summary/Abstract C9orf72 expansion mutations are the most common genetic cause of frontotemporal dementia (C9-FTD), a fatal and incurable neurodegenerative disease. C9-FTD is most commonly neuropathologically characterized by frontotemporal lobar degeneration (C9-FTLD) and the accumulation of phospho-TDP-43 (pTDP-43) inclusions in neurons and glia. In C9-FTLD, the medial orbitofrontal cortex (mOFC) is affected early in the disease course, and individuals with mOFC lesions phenocopy patients with FTD, suggesting that mOFC dysfunction impacts FTD clinical phenotypes. In disease, the mOFC exhibits pTDP-43 inclusions, neuronal degeneration, and neuroinflammation, including the development of pathologic microglia. In other diseases, pathologic microglia increase extracellular glutamate and induce death in excitatory neurons. However, these factors’ relative contributions to C9-FTLD pathogenesis are not well understood, and the molecular profiles of degenerating neurons (termed vulnerable neurons) and pathologic microglia in the mOFC are unknown. We hypothesize that in C9-FTLD, pathologic microglia contribute to the selective degeneration of vulnerable populations of excitatory neurons, resulting in dementia. This proposal aims to use transcriptomic methods to identify vulnerable neuron and pathologic microglia subtypes and characterize their molecular profiles, spatial distributions, and interactions that may be contributing to disease progression. To this end, Aim #1 will use single-nucleus RNA sequencing to identify and characterize the pathologic microglia subtypes that arise and the vulnerable neuron subtypes that degenerate in C9-FTLD. Cellular proximity is the basis for intercellular interactions, and Aim #2 will use spatial transcriptomics to identify where these pathologic microglia and vulnerable neurons are spatially distributed as well as their spatial proximities in relation to each other and to pTDP-43 inclusions. A better understanding of how C9-FTLD changes neurons’ and microglia’s gene expression patterns, their spatial distributions, and their interactions may lead to strategies to protect cells from disease and patients from dementia. These studies will emphasize how pathologic microglia can contribute to neurodegeneration, enabling the development of microglia-targeted therapies for C9-FTD and other neurodegenerative diseases. Through this project, I will develop expertise in the use of histological and computational techniques. My sponsor, Dr. Edward Lee, is committed to my training and success as a physician-scientist.

NIH Research Projects · FY 2025 · 2022-12

Project Abstract Tendons can withstand large forces due to a highly aligned, dense collagen matrix. However, their low cellularity and relative inability to recruit reparative cells post-injury, as well as susceptibility to excessive scarring, results in loss of tendon structure and mechanical function. Type I collagen (Col1) is the primary collagen of healthy tendon and type III collagen (Col3) is a minor constituent that increases in response to injury. Persistently increased Col3 contributes to persistent fibrovascular scarring and structural and functional deficits in the healing tendon. In perinatal tendons, Col3 is increased, similar to the injured state, compared to healthy mature tendons. Unlike the healing response, the process of neonatal tendon development yields a structurally and functionally superior tendon with a highly aligned Col1-dense matrix. Moreover, neonatal developing tendon demonstrates improved efficiency and quality of healing compared to healing mature tendon. Understanding the role of Col3 in the developmental and healing processes of the neonatal tendon will increase our ability to recapitulate tendon development with tissue engineering and improve tendon injury treatment. Therefore, our overall objective is to delineate the contribution of Col3 to development and healing in the neonatal tendon through modulation of matrix properties and cellular activity. Specifically, we will test the hypothesis that Col3 is crucial for early neonatal development but contributes less to regulation of development at later time points as relative Col3 in the tendon decreases. We also hypothesize that the neonatal tendon has enhanced capacity for a robust proliferative response to tendon injury which creates a Col3-dense healing matrix favorable for tendon progenitor migration and differentiation to ultimately deposit aligned, Col1 fibrils which restore tendon structure and function. To test these hypotheses, we generated a novel, inducible Col3 deficient mouse (i.e. Col3a1F/F) to temporally control Col3 reduction. The study aims are: Aim 1: Define the temporal dynamics of the regulatory function(s) of Col3 during phases of neonatal tendon development and Aim 2: Define the regulatory function(s) of Col3 during phases of neonatal healing. Viscoelastic mechanical testing, transmission electron microscopy, immunohistochemistry, gene expression, proteomics, and 11 integrin analyses will be used to assess the structural, mechanical, and compositional properties of tendons in both aims. Insights gleaned from this work will be relevant to a variety of conditions that reduce Col3 expression including vascular Ehlers Danlos syndrome, aging, smoking and menopause and will highlight therapeutic targets for enhancing tendon injury treatment. The proposed work will be carried out in a world- class training environment at the University of Pennsylvania’s McKay Orthopaedic Research Laboratory. This environment combined with an expert sponsorship team, including experts in studies of tendon structure and function as well as matrix biology, will fully support completion of this proposal and facilitate development into a competent independent investigator capable of producing rigorous and reproducible clinically relevant work.