Children'S Hosp Of Philadelphia

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Glycosylation is an essential, post-translational modification with complex and poorly understood roles in protein function. My lab’s long-term objective is to elucidate the neurobiological functions of glycosylation, including identifying the roles of critical glycosylation sites in neuronal protein function. The importance of glycosylation is emphasized by the congenital disorders of glycosylation (CDG), a group of genetic disorders that disrupt cellular glycosylation machinery. Affected patients exhibit severe neurological deficits, including epilepsy and developmental delay. The genetic basis of CDG provides an opportunity to identify the neurobiological functions of glycosylation using mouse models and human cells and organoids to identify and manipulate glycosylation events to determine the roles of individual glycans on specific glycoproteins. Understanding glycosylation in the nervous system will elucidate the pathophysiology of CDG and other neurological diseases, enable therapeutic advances targeting glycosylation pathways, and inform normal function of glycosylation. GALNT2-CDG is a new CDG type caused by biallelic mutations in GALNT2, which encodes a critical glycosyltransferase initiating the first step in mucin-type O-glycosylation. GALNT2-CDG patients suffer from epilepsy and global developmental delay. Galnt2 whole body knock-out and conditional pan-neuronal knock- out mice recapitulate many of the patient neurological deficits. My central hypothesis is that loss of site-specific O-glycosylation in neurons impairs protein function contributing to the neurological deficits observed in Galnt2 KO mice and GALNT2-CDG patients. The specific objective of this proposal is to identify the roles of GALNT2- mediated O-glycosylation in neurological function and disease. This will be achieved by genetically dissecting the neurological circuit dysfunction in GALNT2-CDG cKO mice, using state-of-the-art glycoproteomics advances in instrumentation, experimental methodologies, and computational approaches to identify O- glycoproteome disruption, and determining the pathogenic impacts of disrupted O-glycosylation events using human induced neurons (iNeurons) and brain organoids. Successful completion of these aims will establish critical normal functions of GALNT2-mediated O- glycosylation and elucidate glycosylation-related pathophysiology of human neurological diseases, potentially enabling therapeutic advances.

NIH Research Projects · FY 2025 · 2025-08

Melanins are pigments responsible for skin, hair, and eye color in mammals and function in visual acuity, ocular development, and protecting skin from harmful effects of ultraviolet light. Melanins are synthesized by skin melanocytes and eye pigment cells within membrane-bound organelles called melanosomes. Variation in the type or quantity of melanin synthesized underlies characteristic skin tone differences among humans, and impaired melanosome biogenesis and/or melanin synthesis result in oculocutaneous albinism (OCA) or ocular albinism (OA) with corresponding skin and visual defects. Among the nine genes that are mutated in non- syndromic OCA and OA patients, one of the least understood is SLC24A5. Inactivating mutations in SLC24A5 cause OCA type 6, and genetic variation at the SLC24A5 locus is a major cause of skin tone differences across human populations. SLC24A5 encodes NCKX5, a member of a family of potassium-dependent sodium/calcium exchangers that facilitate ion transport across membranes. While the ion antiporter activity of NCKX5 has been validated, neither the subcellular localization of NCKX5 nor its role in pigmentation are well understood. Competing published studies suggest that NCKX5 localizes to the Golgi, melanosomes, or mitochondria, and functional studies have suggested roles for NCKX5 in mitochondrial or Golgi function, control of melanosome protein levels, or regulation of melanosome pH – a key determinant of melanogenesis – with no mechanistic insights into how NCKX5 influences these parameters. Each of these studies lacks rigor and suffers from the use of non-validated and/or poorly designed reagents, overexpressed tagged NCKX5 transgenes, a lack of attention to protein or organelle maturation, and experiments that lack rigor and proper controls and fail to consider dynamic cellular processes. We propose to circumvent these concerns and to develop tools that will allow us to better define NCKX5 localization and function in melanocytes. We will generate a panel of congenic immortalized mouse melanocyte cell lines in which (i) epitope or fluorescent tags are inserted into the Slc24a5 gene locus at distinct sites so that tagged NCKX5 is expressed at endogenous levels, and (ii) the Slc24a5 gene is knocked out or mutagenized to variants corresponding to human OCA6 or light skin-associated alleles. These tools will be used to define NCKX5 localization, assess how SLC24A5 loss- of-function impacts melanosome and Golgi pH, protein content and trafficking, and ultimately to define how SLC24A5 function impacts melanogenesis. Given the importance to human health of SLC24A5 gene alterations, a clearer determination of NCKX5 localization and function in melanocytes will advance our basic understanding of pigmentation and inform strategies for novel OCA therapeutics and to mitigate the damaging effects of ultraviolet radiation in individuals with light skin-associated SLC24A5 alleles. The specific aims are: 1. Define the site of NCKX5 localization and function at endogenous levels of expression. 2. Assess the impact of impaired SLC24A5 function on organelle protein levels, trafficking, and pH.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The proposed research aims to advance clinical trial readiness for SYNGAP1-related disorder (SYNGAP1-RD), a rare monogenic epilepsy associated with severe neurodevelopmental differences including epilepsy, intellectual disability, and autism spectrum disorder. Currently, the absence of natural history data and validated outcome measures hinders the assessment of therapeutic efficacy in clinical trials for SYNGAP1-RD and similar rare genetic epilepsies. This project will bridge this gap by defining disease trajectories and establishing reliable clinical and biomarker-based outcome measures. This study builds on preliminary work to determine disease trajectories, identify developmental and seizure patterns, and explore quantitative EEG (qEEG) features as biomarkers in SYNGAP1-RD. The objectives of the current study will be addressed through two specific aims. In Aim #1, I will follow a cohort of 50 individuals aged 2-10 years with SYNGAP1-RD prospectively to characterize developmental progress and seizure patterns. Regular assessments using tools like the Bayley-4 and Peabody will track motor and cognitive domains to distinguish predictable disease courses from treatment effects in potential future trials. In Aim #2 of this project, I will examine qEEG features as potential biomarkers for SYNGAP1-RD, focusing on specific findings such as the occipital alpha/theta power ratio, which I previously identified to be distinct in SYNGAP1-RD from controls and other genetic epilepsies. qEEG assessments in the study cohort, with comparison against age-matched controls, will clarify whether these features correlate with clinical severity and can predict developmental outcomes, ultimately identifying qEEG-based markers suitable for clinical trials. In addition, I will explore novel qEEG features, using random forest models to select key features to identify novel qEEG markers correlated with SYNGAP1 severity. As an expected outcome, this NIH K23 career development award will support my training in longitudinal study design, outcome measure validation, and advanced qEEG analysis. These efforts aim to provide a foundation for future precision therapies and ensure my progression toward an independent R01-funded career in rare neurological diseases, focusing on tools to evaluate and implement treatments effectively. The anticipated results will address critical gaps in clinical trial readiness, with validated outcome measures and biomarkers that may serve as robust endpoints in SYNGAP1-RD and potentially in other genetic epilepsies.

NIH Research Projects · FY 2025 · 2025-08

(Supplement Application): Under the parent program, we assessed and returned polygenic risk scores (PRS) for 3,250 participants at the Children’s Hospital of Philadelphia (CHOP). All participants received a “genome-informed risk assessment” (GIRA) report that includes clinical PRS results in addition to clinical data from self-report and the electronic health record (EHR), family history, and clinical sequencing for a limited number of monogenic risks (adults only). We established a recruitment, engagement, and retention protocol that will include targeted recruitment, enhanced communication with participants and health care professionals, boosted analysis and EHR integration (EHRI), and a dynamic education program in order to achieve long-term success and improved health outcomes. The education program was informed by a study of the ethical, legal, and social implications (ELSI) of return of genomic risk estimates, specifically differences in risk perception and willingness to participate in risk reduction recommendations based on how risk is framed, disease severity, age of onset, and actionability. We successfully worked with the consortium to delineate best practices for returning genomic risk estimates and create an innovative return of results protocol. Our original goal was to recruit and engage 2,500 participants who have not previously received genomic risk information, which we increased to 3,250 – per discussions with the NIH – to offset difficulties among non-CHOP eMERGE sites in recruiting children for the study. To this end, recruitment was successfully accelerated, and this revised target was met. Risk reports from an additional 750 individuals were integrated into the CHOP EHR and made available to research participants through the patient portal (MyCHOP, CHOP’s branded MyChart). CHOP was the only eMERGE site able to return reports directly to the patient portal (MyCHOP) and similarly the only site with fully-implemented and functioning clinical decision support. Working with research analysts with expertise in the CHOP Epic electronic health record system, we developed a novel infrastructure for capturing phenotype data from traditionally under-utilized components of the EHR, including data-access logs, derived from a Clarity database that catalogs interactions between the healthcare practitioner and the Epic system. These includes time-stamped logs of reports viewed and actions taken, which are critical variables/covariates in local and network-wide analyses. The primary activities in this supplement will be 1) incorporation and maintenance of GIRA risk reports in the CHOP EHR integration of risk reports, including CDS with custom best practice advisory for all high-risk results, 2) assessment of the uptake of risk reduction recommendations by patients and physicians following integration of genomic risk estimates into the EHR as CDS. The last reports were returned toward the end of Year 5 and supplemental funding will allow us to assess relevant healthcare outcomes at six and/or twelve months post return for participants.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Hemophilia A is an X-linked bleeding disorder caused by a deficiency in clotting factor VIII (FVIII). Current adeno- associated viral (AAV) vector mediated gene therapy approaches for hemophilia A are gene addition approaches that target the liver using promoter elements that result in expression in hepatocytes. However, liver sinusoidal endothelial cells (LSEC) are the natural site of FVIII synthesis, whereas other coagulation factors are expressed in hepatocytes. While AAV delivery of factor IX to hepatocytes for hemophilia B has resulted in stable FIX levels for >10 years, there have been unexpected observations in hemophilia A studies including a lack of a dose response with variability among patients and a significant decline in FVIII expression in the in first three years after AAV delivery. We hypothesize that these unanticipated findings in the clinical studies may be related to the site of FVIII synthesis. Thus, while significant hurdles have been overcome in gene therapy for hemophilia A, there remain unexplored opportunities to improve hemophilia patient outcomes. The goal of this proposal is to target FVIII expression to LSECs and to define the biological consequences of targeting FVIII expression to LSECs compared to hepatocytes. An LSEC targeted genome editing approach (Specific Aim 1) will be compared to an LSEC targeted AAV gene addition approach (Specific Aim 2) using novel tools that we have developed— an engineered AAV capsid with improved LSEC targeting and a lipid nanoparticle conjugated to PECAM-1 that targets endothelial cells. Targeting expression to LSECs or hepatocytes using an AAV approach compared to a genome editing strategy will provide the opportunity to define of the biological differences of expressing FVIII in LSECs compared to hepatocytes (Specific Aim 2). The study of whether chronic liver disease or acute liver injury may affect FVIII expression and different cell types after gene delivery using a genome editing or gene addition approach will also be investigated (Specific Aim 3). Together, these studies will provide the basis for understanding the biology and efficacy of LSEC targeted FVIII expression to support the development of approaches to target FVIII to its endogenous site to overcome the challenges of hemophilia A gene therapy.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Advancements in the evidence-based care of critically ill children have been limited by recent clinical trials failing to show definitive statistical benefits across a variety of outcomes. Clinicians at the bedside, however, often interpret the results of these trials differently, believing the results are not necessarily applicable to their patient, that the results are equivocal, or that results suggest potential benefit. These differences are partly due to the currently used frequentist clinical trial design methodology that hypothesizes unrealistically high treatment efficacy to adequately power trials amongst the limited pool of critically ill children and generate a binary interpretation of definitively beneficial or not based on these factors. Additionally, the heterogeneity of treatment effect amongst the critically ill has often been ignored and subgroup analysis fails to fully address this important consideration. Thus, this proposal aims to compare heterogeneity of treatment effect (HTE) analysis methodologies in a trial of pediatric ARDS ventilator management. Additionally, the proposed study would examine the construct validity of commonly employed 28-day composite outcomes to predict late morbidity and mortality. Finally, Dr. Keim will develop skills necessary to become a clinical trialist through conducting a feasibility trial comparing positive end-expiratory pressure (PEEP) guided by electrical impedance tomography (EIT), a non-ionizing, non-invasive imaging tool highlighting lung aeration, to standardized PEEP/FiO2 table titration. In addition to feasibility data, Bayesian trial simulations will be conducted to enhance Dr. Keim’s methodological expertise and inform the future definitive trial sample size. Successful completion of these Aims will propel Dr. Keim towards his career goals of refining the methodology utilized in clinical trials of critically ill children to (1) improve the assessment of the feasibility of trials, (2) generate more usable and understandable interpretations for the treating clinician, and (3) highlight methods to understand HTE in clinical trials and develop trial planning to generate individualized treatment effect (ITE) estimates. The results of the proposed studies promise to enhance future pediatric ARDS trials by increasing the clinical relevance of trial outcomes and generating clinically usable probabilistic interpretations that are responsive to the heterogeneity omnipresent in critical care trials. The principal investigator of this study, Dr. Garrett Keim, is uniquely positioned to complete the aims of this proposal due to his training in epidemiologic and biostatistical methods and his research experience investigating morbidity in survivors of pediatric acute respiratory failure. Supported by his strong mentorship team and research environment, he will leverage the opportunities presented by this proposal to develop into an independent patient-oriented physician-scientist and clinical trialist to improve the evidence-based care of critically ill children.

NIH Research Projects · FY 2025 · 2025-08

Abstract The temporomandibular joint (TMJ) controls jaw movement and function during speech and mastication and comprises the mandibular condyle, articular disc, and glenoid fossa. These structures exhibit a fibrocartilaginous organization whereby the apical tissue layer, rich in stem cells and chondroprogenitor cells, regulates normal growth and remodeling by supplying chondrocytes to underlying cartilage. While these aspects of TMJ biology are well understood, other functions remain unclear, such as how the stem/chondroprogenitor cells are maintained, whether changes in these cells underlie TMJ osteoarthritis (OA), or if other factors instigate pathogenesis, including TMJ lubrication. Hyaluronan/hyaluronic acid (HA) lubricates the TMJ and is used to alleviate OA symptoms, providing only short-term relief. Studies in other fields indicate that HA has additional physiological roles, including cell signaling, which may also apply to the TMJ. Notably, Has1- and Has3-null mice exhibit no significant skeletal abnormalities, suggesting the limited roles of these isoforms in skeletal development. Our preliminary studies reveal that Has2 is prominently expressed in condylar chondroprogenitor cells and chondrocytes. Conditional ablation of Has2 leads to changes resembling TMJ OA, with neither Has1 nor Has3 compensating for its loss. Early phenotypic changes in Has2-mutant condyles include disrupted chondrogenesis, impaired chondrocyte maturation, increased expression of the HA-processing enzyme Tmem2, and elevated nuclear translocation of YAP. This is further supported by siRNA-mediated knockdown of Has2 in primary condylar chondroprogenitor cells, which results in increased YAP nuclear translocation and decreased proteoglycan synthesis compared to controls. This leads to our central hypotheses that (i) HA is not only a TMJ lubricant but is also required for the preservation and functioning of TMJ skeletal stem cells, chondroprogenitors, and their progenies and (ii) pathological HA decreases and fragmentation would alter the phenotype of those cells via aberrant activation of YAP signaling. In Aim 1, we will investigate the regulatory mechanisms of HA synthesis and processing in both healthy TMJ and those with OA using scRNAseq. We will assess the impact of HA depletion on cell function in Has2f/f;AgrCreER TMJs. To gain insights into tissue stiffness, elasticity, and lubrication, we will measure their mechanical properties at the nanoscale and surface boundary lubrication. Additionally, we will test whether HA fragments contribute to YAP signaling, leading to dysregulation of cell function in condylar chondroprogenitor cells isolated from porcine embryos. Aim 2 will test whether YAP ablation ameliorates TMJ OA. To expand treatment options, we will evaluate whether YAP inhibitors can prevent or inhibit TMJ OA. This project is based on new data and novel insights into roles played by HA in TMJ health and disease and will test the efficacy of drug therapy. Given its novelty, the project is in its early stages but will undoubtedly have significant implications for basic research and translational medicine for TMJs and other joint-related musculoskeletal diseases.

NIH Research Projects · FY 2025 · 2025-08

SUMMARY Our understanding of pathogen virulence has largely been established by studying organisms in isolation. However, enteric infections are polymicrobial by nature, as pathogens become exposed to a rich microbial ecosystem and complex metabolic environment during invasion of the gastrointestinal tract. Thus, the study of pathogen-microbiota interactions during infection is central to our understanding of susceptibility to, severity of, and treatment of enteric infections. One of the most significant enteric pathogens globally is Clostridioides difficile. C. difficile is a spore-forming bacterium that causes a wide range of gastrointestinal (GI) disorders varying in severity from mild diarrhea to fulminant colitis and/or death. Over the past decade, incidence, severity, and costs associated with C. difficile infection (CDI) have increased dramatically; however, the factors that govern this broad spectrum of disease remain unknown. The primary risk factor for CDI is antibiotic treatment, which reduces colonization resistance to C. difficile by disrupting the resident microbial community inhabiting the GI tract. Surprisingly, little is known about how C. difficile cooperates with the rich collection of microorganisms in the GI tract. Moreover, we still understand very little about the impact of microenvironments and distinct metabolic niches on CDI. The studies proposed in this application will provide a molecular blueprint of CDI through the application of advanced imaging mass spectrometry and provide in-depth mechanistic insights into the role of the microbiota in shaping C. difficile virulence. Our preliminary studies demonstrate that a group of opportunistic pathogens, the enterococci, dramatically remodel the metabolic environment in the CDI gut and are a source of amino acids for C. difficile energy production. Enterococcus-mediated metabolic shaping of the gut metabolome enhances C. difficile colonization and support fitness following disease manifestation. Based on these fundamental discoveries, we propose a series of studies aimed at understanding the role enterococci play in CDI and defining the molecular mechanisms of interspecies interactions during infection. Completion of this work will (i) define the impact of enterococci on the metabolic environment during CDI and (ii) determine molecular mechanisms of enterococcal-mediated control of C. difficile virulence. Together, this work will define the supportive role that pathogenic microbiota play in the outcome of CDI and shed light on the importance of integration of metabolic signals from the microbiota in pathogen virulence.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Each year, over 1.4 million US adolescents undergo surgery. Between 3-5% of opioid-naïve adolescents continue to fill opioid prescriptions 3-6 months after surgery, which is long after acute surgical pain has resolved and may signify a new opioid use disorder. Due to complex biopsychosocial changes, there is significant concern among experts that adolescents may be at increased risk of perioperative opioid-related adverse outcomes, including postoperative opioid misuse (POM), defined as use after severe surgical pain resolves (>14 days after surgery), opioid use disorder and overdose. Anticipatory guidance is endorsed by the American Academy of Pediatrics for risk prevention among adolescents. It is routinely utilized in outpatient pediatric settings to prevent high-risk behaviors, including those related to opioid and substance use. However, anticipatory guidance, with its educational and behavioral components, has not been studied in the perioperative setting. To gather preliminary data, the principal investigator, Dr. Sutherland, completed an observational study of 501 adolescents with six months of follow-up at the Children’s Hospital of Philadelphia (CHOP). Among patients undergoing reconstructive knee surgery (ligament repair), 8% used any opioids in Week 2 after surgery, but 44% endorsed opioid use (median 5 days) in Weeks 3 and 4, consistent with unintended opioid misuse; interviews suggest use was associated with anxiety and uncertainty over recovery. The central objective for this proposal is to improve knowledge of effective anticipatory guidance techniques and design an intervention to prevent POM that is both adolescent-centered and reflective of perioperative considerations that will be evaluated in a subsequent R01- funded trial. This proposal includes: (1) a retrospective cohort study to establish contemporary (pandemic-era) dispensing trends and determine the association between patient characteristics and adverse opioid-related outcomes, (2) semi-structured interviews with surgeons, who vary in opioid dispensing practices, and adolescent-caregiver dyads to characterize effective surgical anticipatory guidance and POM themes to inform an adolescent-centered intervention, and (3) a single-arm feasibility study to measure acceptability of an anticipatory guidance intervention to prevent POM. With this proposal, Dr. Sutherland’s overarching career goal is to develop as a clinical trialist focused on prevention trials. To accomplish this, she seeks to gain additional training in the following domains: (1) use of population data to inform trial design (2) qualitative methodology to inform intervention design and (3) foundation in clinical trial design and conduct. Dr. Sutherland will conduct the proposed research at the Children’s Hospital of Philadelphia within the University of Pennsylvania and is supported by mentors, advisors and consultants with significant experience in trial design, specifically pragmatic and prevention trials, advanced biostatistical analysis, application of qualitative data to intervention design, adolescent psychology and addiction medicine.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY This proposal aims to investigate the role of the c-Jun N-terminal kinase (JNK) signaling pathway in neurodevelopmental disorders (NDDs). NDDs, such as autism and intellectual disability, affect individuals across the lifespan and present diagnostic and medical management challenges. Despite advancements in genomic diagnostics and precision therapies, their application to NDDs remains limited. A key obstacle in improving care is the inability to provide a molecular diagnosis for most affected individuals, hindering the understanding of pathomechanisms and development of precision therapies. Loss-of-function variants in several genes involved in the JNK pathway, including MAP2K4 and MAP4K4 identified by our group, were recently shown as causative in NDDs. While the JNK pathway is known to play critical roles in neurodevelopment, its relevance to NDDs remains underexplored. I hypothesize that disrupted JNK signaling is a feature shared across an emerging class of NDD, and plays a role in non-syndromic neurologic disorders across the lifespan. I will test this hypothesis through two main approaches: induced pluripotent stem cell (iPSC)-based disease models to understand the effects of MAP4K4 and MAP2K4 deficiencies on the JNK pathway in the neuronal context, and a biobank-scale investigation of individuals with rare variants among JNK-associated genes to test if novel gene-disease relationships are identified. The Aim 1 of this proposal will thoroughly examine neuronal cellular and organoid phenotypes in iPSC disease models, and evaluate whether JNK pathway disruption underlies the observed phenotypes, as suggested by my preliminary data. The experimental approach will include rescue experiments for each sub-Aim to further determine this relationship. The Aim 2 of the proposal will evaluate other genes integral to the JNK pathways as candidate disease genes, and will leverage biobank-scale data to test if novel gene-disease relationships could be determined. Completion of this project will lead to better understanding of genetics of NDDs and improve the diagnostic yield of genetic testing for cases of NDDs. The JNK pathway is heavily researched as a drug target in other contexts, including cancer therapeutics, and my project could nominate a subset of genetic NDDs as potential therapeutic targets. Lastly, the genotype-first disease discovery pipeline I am developing can be repurposed to investigate additional gene-disease relationships that extends beyond NDDs. Through the 5-year funding period, I plan to identify and describe 3 or more novel Mendelian NDDs, and comprehensively describe JNK-associated NDDs as a distinct disease group. I plan to emerge from this period as a leader in disease discovery with broad expertise from basic neuroscience to biobank-scale data management and analysis. I will use the experience and discoveries as the foundation for the next stage in my career as an independent R01-funded translational researcher with a focus on the genetics of NDDs, as well as on therapeutic approaches inspired by the data that I will generate throughout the funding period.

NIH Research Projects · FY 2025 · 2025-08

Project summary The gut microbiome is a dense, complex community of microbes associated with maintaining health in not only the gastrointestinal tract, but also other organs in the human body. Large shifts that disrupt microbial homeostasis are linked to digestive diseases, but diurnally rhythmic fluctuations in microbial populations are associated with good gut health. Therefore, it is of critical importance to delineate the microbial and temporal cues that control intestinal homeostasis to uncover the mechanisms that separate the beneficial and detrimental effects of microbial flux. These discoveries have the potential to reveal novel targets urgently needed for treating colitis, a digestive disease for which a cure remains unknown. We performed a time-course transcriptomics screen and single-cell transcriptomic analyses from mouse colon and identified a new intestinal gene associated with the secretory epithelial lineage that exhibited both rhythmic and microbiota-dependent expression. Mice lacking this protein had a thinner mucus layer compared to wild-type mice, and were not only more susceptible to DSS colitis, but also displayed delayed recovery after DSS cessation. Our preliminary results implicate host circadian clock and gut microbiome in this phenotype, but specifics of the upstream temporal and microbial signals that activate this gene, as well its downstream function in mucus production, remain completely unknown. Therefore, we hypothesize that the host circadian clock and gut microbiome regulates mucus production in the large intestine through a newly identified temporal and microbially regulated gene to protect the host from infectious and chemical colitis. To test this hypothesis, two specific aims are proposed. In Aim 1, we will identify specific microbiota members and metabolites and investigate circadian clock-dependent regulation that activate mucus production. In Aim 2, we will characterize the function of our novel protein plays in mucus production by comparing mucus secretion, permeability, and composition in wild-type and knockout mice. Successful completion of this proposal will identify a previously undefined mucosal protein that integrates temporal and microbial cues to regulate mucus production and protects the host against chemical and infectious colitis. This innovative project will additionally expand my training to include key methods and concepts in microbiota, chronobiology, and mucosal immunology. Altogether, the research and training plan proposed will facilitate a better understanding of the role the microbiome and circadian clock in modulating host immunity, while preparing me for a future career as an independent investigator studying the role the circadian clock and microbiome play in aggravating digestive diseases.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Over 120,000 children are identified as physical abuse victims in the U.S. annually, which is an underestimate as medical providers frequently fail to identify the abusive origin of injuries. In response to the critical need for evidence-based research to inform equitable recognition, evaluation, and care of victims of suspected physical abuse, CAPNET launched in February 2021 as the first multicenter child physical abuse research network designed to support multiple concurrent studies. CAPNET's mission is to make the care of potentially abused children more effective, safe, and fair. CAPNET collected detailed clinical data on >12,000 children undergoing evaluations for physical abuse at 10 pediatric centers. The goal of this proposal is to archive CAPNET's rich, complex data and increase its usability, generalizability, and impact through investigator support tools, linkage with external datasets, and dissemination of CAPNET data and research products. Increasing the usability of CAPNET's complex data is necessary to support ongoing high-quality research. With NICHD R24 funding, the CAPNET Data Coordinating Center (DCC) provided tailored, resource-intensive guidance to individual study investigators. Given high-demand for CAPNET data and upcoming end of R24 funding, there is a need to streamline this process to increase sustainability by (1) making CAPNET data more user-friendly and by (2) ensuring resource-efficient development and distribution of analytic datasets. In addition, linkages of CAPNET data with hospital and community-based data are needed to broaden the impact and generalizability of CAPNET studies. CAPNET has created a rich, clinical data source, but CAPNET only collects information on children undergoing evaluations by child abuse pediatrics specialists (CAPs) and not the wider denominator of children presenting with similar injuries and not referred for CAP evaluation. The Pediatric Health Information System (PHIS) administrative dataset captures all children evaluated in the emergency and inpatient setting at 7 of 10 CAPNET hospitals. Linkage with PHIS will facilitate research on disproportionality in abuse evaluations as well as validation of use of ICD-10 codes in abuse epidemiology research. Finally, CAPNET data has primarily been used by CAPs; we seek for CAPNET to be a resource for the wider research community. We will address these needs through the following aims: (1) increase accessibility, efficiency, and usability of CAPNET data, (2) harmonize CAPNET data with hospital and community-level data to increase generalizability and impact, and (3) promote awareness and use of CAPNET data, research products, and resources among researchers and policy makers. Completion of these aims will maximize impact of NICHD's R24 investment in CAPNET through ongoing rigorous research. Products of this proposal will be (a) an archived CAPNET master dataset containing data from >12,000 children harmonized with hospital and community data from which the DCC will generate user-friendly, study-specific datasets, (b) technical support tools to inform the rigorous use of CAPNET data, and (c) dissemination products and engagement tools.

NIH Research Projects · FY 2025 · 2025-07

Multidrug-resistant bacteria (MDRB) contribute increasingly to morbidity, mortality, and healthcare costs. Extended-spectrum beta-lactam- and carbapenem-resistant Enterobacterales (ESBL-E and CRE) are MDRB of particular concern due to their demonstrated ability to evolve into highly transmissible clones and acquire and spread antibiotic resistance determinants. Traditional epidemiological surveillance typically focuses on outbreaks of MDRB causing clinical infections, underestimating the burden of these pathogens within hospital systems. Broader efforts that account for asymptomatic carriage and environmental and community reservoirs would be ideal to track and mitigate the spread of MDRB. Wastewater surveillance has proven an effective tool for public health pathogen monitoring, as shown with SARS-CoV-2, but has not been established in clinical settings. This proposal will develop new systems to leverage wastewater for clinically applicable, proactive, and readily deployable MDRB monitoring. In Aim 1, we will establish standardized longitudinal surveillance strategies to inform infection control responses. We will use long-read metagenomics and novel bioinformatic approaches to rapidly identify significant changes in relative or absolute abundance of ESBL-E or CRE compared to site-specific baselines. We will also establish methods to translate wastewater testing data into interpretable “action thresholds” for use by hospital and clinical teams. In Aim 2, we will identify factors enabling the emergence of novel ESBL-E and CRE genotypes in the wastewater environment. Wastewater sampling can identify novel resistant genotypes before detection of clinical infections. We will further develop our novel Metapore-C technique to link bacterial hosts with resistance gene-harboring mobile elements and will use this approach to identify environmental factors such as wastewater antibiotic levels and plumbing design associated with acquisition of resistance. Lastly, in Aim 3 we will devise wastewater testing methodologies suited to resource-limited clinical settings. Given the costs and infrastructure needed for comprehensive clinical surveillance, wastewater testing is better poised to aid in mitigation of MDRB under resource constraints. Yet, current wastewater surveillance approaches are often impractical in such settings. Our strategies for reducing per-sample costs and the analytical burden of wastewater data interpretation, as piloted at a pediatric hospital in Gaborone, Botswana, will serve as a proof-of-concept for wastewater MDRB testing in diverse contexts. Overall, this project will significantly broaden the ability of wastewater surveillance to inform hospital and clinical care efforts, while establishing best practices for global surveillance of antimicrobial resistance in hospital wastewater.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Understanding neural processes from the earliest time possible is of interest as neural activity is a readout of current brain processes as well as a driver of brain development. Specifically, the modification of neural circuits as a function of experience results in more complex neural circuits that then allow more complex behaviors. A K08 Award would support training in advanced brain imaging and advanced statistics. K08 research focuses on examining the similarity between resting-state (RS) and auditory electroencephalography (EEG) and magnetoencephalography (MEG) measures as well as their associations with age and brain structure. Forty typically developing infants (50% female) will undergo simultaneous EEG+MEG exams at 1 month + 1 week, 6 months + 1 month, and 12 months + 1 month, as well as a magnetic resonance imaging (MRI) exam and developmental milestone testing at the final 12-month time point. The proposed grant builds upon the candidate’s prior training in pediatric EEG and MEG research and speech-language pathology, extending this knowledge to whole brain source level analyses and advanced statistical methods. Filling gaps in the PI’s training as well as gaps in the literature, Aim 1 will determine optimal recording and analysis strategies for assessing RS and auditory neural function in infants. Hypothesis 1 predicts that EEG-MEG reliability will be higher at 12 months than 1 and 6 months due to less accurate EEG estimates of neural activity at the earlier ages given ongoing changes in head tissue conductivity and electrical field distortion due to open fontanels. Aim 2 will determine optimal recording and analysis strategies for assessing RS and auditory neural function in infants. Hypothesis 2 predicts that given the higher dimensionality of MEG data and the fewer assumptions required for MEG source localization, EEG and MEG comparisons will show that (1) regional differences in the RS aperiodic activity are best assessed using MEG, and (2) regional differences in the association between age and aperiodic activity are best identified using MEG. Finally, Aim 3 compares the ability of EEG and MEG to detect neural function and brain structure associations (white, gray matter) at 12 months. Hypothesis 3 predicts that local measures of neural activity will be more strongly associated with local brain structure for MEG than EEG. The research plan is feasible given the candidate’s background and institutional resources (scientists and technology). Study findings will inform Dr. Green’s future research program, provide the candidate with pilot data for a future R01 examining associations between behavioral development and brain function and structure, and provide the field with a better understanding of infant EEG and MEG measures. The award will also provide the candidate with the opportunity to obtain training in cutting-edge imaging techniques and advanced statistical analyses. The research and training provided by this award are critical to the candidate’s long-term goal of conducting longitudinal research assessing brain function and structure in typically developing infants and children and infants at-risk for developmental disabilities.

NIH Research Projects · FY 2025 · 2025-07

SUMMARY Despite reliable use of infection prevention bundles, hospital-acquired infection rates have plateaued in neonatal intensive care units (NICUs). Our preliminary data from whole genome pathogen sequencing at Children’s Hospital of Philadelphia (CHOP) suggests that many serious infections are traceable to strains (highly related clones) that reside in the ICU and are transmitted amongst patients over time. In this proposal, we aim to determine patient, pathogen, and environment-specific factors that may contribute to infection risk. In Aim 1, we aim to use whole genome sequencing (WGS) of bacteria identified from patient colonization, systemic infection, and colonization of the environment, along with diagnostic allele (DA) surveillance, to determine the transmission and persistence dynamics associated with bacterial infection within two NICUs with distinct clinical and demographic characteristics. We will also assess the impact of patient case-mix and the contribution of NICU layout and environmental characteristics on observed patterns of colonization and infection. We will use the data from WGS to test DA sequencing tests as a strategy that can be scaled for broad use as rapid tests in NICUs to measure strain transmission. In Aim 2 we will assess the impact of providing near real time DA sequencing to guide targeted deployment of infection prevention strategies by the NICU to diminish the persistence and transmission of ‘high risk’ strains. The results of this investigation will demonstrate the feasibility and efficacy of DA sequencing guided targeted interventions to decrease bacterial transmission and/or colonization and thereby reduce risk of invasive infection. The findings from this study will be critical to our overall efforts to decrease the risk of hospital-acquired infection, among our most vulnerable ICU patients.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY / ABSTRACT Children with kidney failure, either Chronic Kidney Disease Stage 5 requiring chronic dialysis (CKD5D) or acute kidney injury (AKI) requiring continuous veno-venous hemodiafiltration (CVVHDF) have high rates of morbidity and mortality. Complications are mediated by uremic toxins. All dialysis modalities have inherent efficacy limitations for removing various toxins. Understanding how toxins are derived and factors affecting their accumulation may identify novel targets for clinical intervention. The scientific goals are to: AIM 1 – characterize novel and known contributions of gut microbes to the biosynthetic pathways of selected uremic toxins using a multi-meta-omics approach, and AIM 2- identify differences in toxin accumulation and clearance between children with requiring CVVHDF vs children with CKD5D. The Paired pre/post-Dialysis Investigation of Global Metabolomics (P/paraDIGM) study is approved with existing study infrastructure and biobanked samples, which will support achievement of these AIMs. The candidate’s career development plan focuses on building three pillars of focus: PILLAR 1 - multi-omics science and technologies, PILLAR 2 – biostatistics, bioinformatics, and data science, PILLAR 3 – designing and conducting clinical trials. The candidate will develop these three pillars through a combination of experiential learning and structured coursework. The candidate will be guided by his primary mentor, Dr. Michelle Denburg, who has strong record mentoring junior investigators. The candidate will be advised by a committee with expertise in pediatric and adult nephrology, multi-omics, the gut microbiome, biostatistics, epidemiology, and computational biology. The candidate will transition from P/paraDIGM’s lead investigator to principal investigator. The candidate’s career goal is to become an independent translational multi-omics investigator with a program seeking to improve clinical monitoring and intervention on modifiable toxins in children with kidney failure. The K23’s research and career development advancement will support the candidate’s future R01 submission. The Children’s Hospital of Philadelphia (CHOP) and the Perelman School of Medicine at the University of Pennsylvania (PennSOM) provide a strong institutional environment for carrying out the proposed research and career development plan. CHOP is one of the highest-volume pediatric dialysis centers in the country. CHOP and PennSOM host robust training programs to support junior faculty career development. CHOP and PennSOM are committed to protecting at least 80% of the candidate’s effort to support research and career development activities.

NIH Research Projects · FY 2026 · 2025-07

Project Summary/Abstract Ambient air pollution (AAP) exposure is a trigger for pediatric asthma exacerbations, significantly contributing to asthma related morbidity. While reducing AAP exposure is a long-term public health imperative, there is an urgent need for strategies to predict and prevent asthma exacerbations in children exposed to AAP. To address this critical gap, this project will leverage emerging opportunities in data science, aligning with the National Heart, Lung, and Blood Institute’s (NHLBI) Objective 7 to open new frontiers in heart, lung, blood, and sleep research. By integrating clinical, genetic, social, and environmental data, this project will assess individual susceptibility to AAP and develop predictive models for asthma outcomes. Preliminary data from Dr. Jelte Kelchtermans, Instructor at the University of Pennsylvania and attending physician in Pulmonary and Sleep Medicine at the Children’s Hospital of Philadelphia, identified a subgroup of pediatric patients with heightened sensitivity to AAP who experience increased asthma morbidity. This subgroup demonstrates specific genetic variants associated with exacerbation risk and reduced lung function under high AAP exposure, highlighting the influence of genetic predisposition in shaping AAP sensitivity. Building on these findings, this project will address two primary aims. In Aim 1, Dr. Kelchtermans will investigate how clinical, genetic, and social variables interact with short-term AAP variations to influence exacerbation incidence, with a focus on refining a polygenic risk score for AAP sensitivity, exploring gene-gene interactions, and developing predictive models validated with the All of Us cohort. In Aim 2, he will examine how these variables shape long-term lung function attainment in children genetically predisposed to AAP sensitivity, using longitudinal data to model lung function trajectories and interactions with clinical and social factors. The Center for Applied Genomics and the Center of Excellence in Environmental Toxicology provide an exceptional environment for this research, with mentorship from experts, including primary mentors Dr. Hakon Hakonarson and Dr. Sharon McGrath-Morrow and advisory members Dr. Joseph Glessner, Dr. Kim Dokyoon, Dr. Blanca Himes, and Dr. David Hill. This project aligns with NHLBI’s objectives and represents a critical step toward actionable interventions in pediatric asthma care. In conclusion, the proposed research offers Dr. Kelchtermans a unique opportunity to advance toward scientific independence while transforming population-level associations into individualized clinical tools for proactive and preventive asthma management.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT This proposal focuses on how the Tropomyosin 1 gene product impacts the development of hematopoietic stem and progenitor cells (HSPCs) and their precursors. There is considerable interest in augmenting in vitro HSPC production from cultured induced pluripotent stem cells (iPSCs) to support research and development of blood cell-based therapies. Defining genetic determinants of hematopoiesis is also relevant for understanding benign and malignant blood disorders. We identified Tropomyosin 1 as a blood regulatory gene from human genome wide association study data. We found that Tropomyosin 1 deficiency enhanced production of HSPCs and their hemogenic endothelial precursor cells (HECs) in vitro and in vivo. Using transcriptomic and molecular approaches, we found that Tropomyosin 1 loss enhanced TNFa signaling in cultured iPSCs and further determined that TPM1-related TNFa signaling augmentation was responsible for increased hematopoietic output. In Aim 1 of this grant, we will determine molecular mechanisms for how Tropomyosin 1 and actin biology regulate the TNFa signaling pathway in HECs and HSPCs using translationally relevant iPSC culture models. In Aim 2, we will determine if TPM1 deficiency increases the production of transplantable HSPCs through TNFa-mediated effects on HECs. My goal is to run a laboratory that integrates computational and biochemical approaches to understand genetic mechanisms that enhance blood production, ultimately supporting cell therapeutics and regenerative medicine applications to enhance human health. This proposal directly resulted from completion of the specific aims and achievement of career development goals set forth in my K99 proposal, including rigorous training in data science and hematopoiesis modeling. The three-year professional development program proposed in this R00 application will further propel my research career as an academic pediatric physician-scientist investigating Tropomyosin 1 and other genetic mechanisms that regulate hematopoiesis. Having completed my clinical training and having obtained a tenure-track appointment as an Assistant Professor of Pediatrics at the University of Pennsylvania Perelman School of Medicine, I am well-supported at the institutional level. As I develop my independent research program in this ideal academic environment, I will benefit from the exceptional resources, core facilities, and collaboration with internationally recognized leaders in hematopoiesis, human genetics, bioinformatics, and iPSC technologies at the University of Pennsylvania and Children’s Hospital of Philadelphia for the duration of the R00 award period. After establishing my independent research program with R00 support, I will be uniquely poised to compete for R01 funding as a successful physician-scientist studying hematopoiesis genetics.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Eosinophilic esophagitis (EoE) is a chronic inflammatory condition of the esophagus triggered by an immune response to food antigens. Despite effective treatment that leads to histologic remission, there are persistent changes in mucosal gene expression, epithelial differentiation, and histologic anomalies, including basal cell hyperplasia. Recent findings from other tissue sites indicate that mucosal inflammation can induce chromatin remodeling via immune signaling, including the JAK/STAT pathway. However, the mechanisms governing chromatin remodeling and persistent remodeling in the esophagus remain unknown. IL-4/IL-13 signaling via signal transducer and activator of transcription 6 (STAT6) has been shown to cause epithelial barrier dysfunction in EoE. However, emerging data suggests that other STATs may be involved in EoE. These include STAT3, which is also downstream of the type II IL-4 receptor, and STAT1. We have recently shown there is upregulation of interferon signature genes in the esophageal epithelium during EoE, indicating that there is dysregulation of multiple STAT pathways within the epithelium during EoE. Here, we will examine the specific roles of STAT3 and STAT1 in regulating STAT6-driven epithelial barrier function. We will determine the extent to which (1) coordinate activation of STAT1 via IFN-g will antagonize STAT3 and STAT6-mediated effects, and (2) coordinate activation of STAT3 and STAT6 will have synergistic effects. We generate human epithelial knockout lines (STAT1KO, STAT3 KO) using CRISPR-Cas9 and will interrogate the specific interactions of STAT1 and STAT3 on STAT6-mediated gene expression, chromatin remodeling, and epithelial barrier dysfunction. We will create novel epithelial conditional STAT1 and STAT3 knockout mice to evaluate the respective role of each transcription factor within the complex inflammatory milieu of EoE. The proposed experiments will advance understanding of the crosstalk between T1 and T2 cytokine signaling and epithelium in the esophagus. We will examine regulatory networks that impact JAK/STAT signaling within the epithelium and tissue-specific transcription factor behavior and its impact on chromatin remodeling. This work will identify key molecular drivers of persistent symptoms, paving the way for novel therapies for patients with esophageal remodeling and persistent symptoms. By addressing mechanisms that lead to chronic tissue remodeling and dysfunction, this study could ultimately improve long- term health outcomes for EoE patients, contributing to the goal of enhancing the quality of life for those with immune-mediated diseases.

NIH Research Projects · FY 2025 · 2025-07

Project Summary/Abstract The malaria parasite Plasmodium falciparum is responsible for millions of cases of malaria and over 500,000 global deaths each year. Resistance to current first-line antimalarial regimens, artemisinin-based therapies, is spreading in Southeast Asia and Africa. This continued spread threatens current malaria control and eradication efforts, and new antimalarials with novel mechanisms of action are urgently needed. All currently approved antimalarials target parasite-specific enzymes and pathways. Host-directed therapies, which target host proteins or pathways that are essential to the parasite, could serve as a novel antimalarial strategy. Determining the role of host pathways in the parasite lifecycle will inform future host-targeting antimalarial drug development. This project aims to determine the role of erythrocyte glycolysis in the Plasmodium falciparum intraerythrocytic lifecycle. In Specific Aim 1, the candidate will inhibit glycolysis in terminal erythrocytes and determine how this disruption affects P. falciparum growth, progression through the lifecycle, and susceptibility to oxidative stress. In Specific Aim 2, the candidate will use gene editing to generate pyruvate kinase-deficient erythrocytes and examine how prolonged disruptions in glycolysis affect the parasite. The results of the proposed experiments will yield a comprehensive understanding of why erythrocyte glycolysis is essential to the P. falciparum intraerythrocytic lifecycle and establish its tractability as an antimalarial target. The candidate is a pediatric infectious diseases specialist committed to studying host-parasite interactions in malaria parasites. The proposed training and research project will be conducted at the Children’s Hospital of Philadelphia (CHOP) and the University of Pennsylvania (UPenn). These two institutions are leaders in the study of host-pathogen interactions and erythrocyte biology and development and provide an ideal environment for the proposed research and career development. The candidate will be mentored by Dr. Audrey John with the support of an experienced and diverse advisory committee, including Drs. Stella Chou, Stefano Rivella, Akhil Vaidya, and Sarah Tishkoff. The candidate’s long-term goal is to develop a research program that examines host-parasite interactions in malaria infection and leverages these interactions to identify potential antimalarial targets and develop therapies. Completion of the proposed research and career development will provide a strong foundation for the candidate’s future career.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Optimizing antibiotic use is a strategy for limiting toxicity risk and combating resistance. Maternally-administered intrapartum antibiotics expose the fetus via the placenta, and result in variable neonatal antibiotic concentrations at birth. In some cases, the same antibiotics administered intrapartum to mothers (ampicillin and gentamicin) are also administered to newborns after birth due to risk of neonatal sepsis. However, intrapartum antibiotic exposures are not integrated into postnatal antibiotic dosing recommendations, risking excessive cumulative exposures. Furthermore, pharmacokinetic (PK) data are lacking to inform the adequacy of some antibiotics used for intrapartum antibiotic prophylaxis (IAP) against neonatal Group B Streptococcus (GBS) disease. These lacking data influence current national sepsis risk stratification guidelines, and may lead to overestimation of overall sepsis risk and prompt unnecessary postnatal antibiotic use in newborns. The purpose of this study is to identify pharmacologically-based frameworks to safely optimize neonatal antibiotic use. Its specific aims are: (1A) to quantify maternally-administered ampicillin and gentamicin in maternal and umbilical cord blood; (1B) to characterize maternal and umbilical cord blood concentrations of second-line GBS IAP agents, vancomycin and clindamycin; and (2) to perform maternal-neonatal population PK modeling integrating intrapartum and postnatal ampicillin and gentamicin exposures. These Aims will focus on a newborn population lacking PK data, enriched with preterm infants, and will use both prospectively consented and opportunistically-scavenged samples. This study will directly address top NICHD research priorities: improving understanding of developmental pharmacokinetics in the perinatal period, and optimizing antibiotic use in order to promote neonatal therapeutic safety. These Aims will inform neonatal empiric antibiotic prescribing based on intrapartum antibiotic exposures – with potential to limit excessive antibiotic dosing and to optimize neonatal sepsis risk stratification. This career development award is designed to support the transition of Dr. Sarah Coggins, Instructor of Pediatrics the University of Pennsylvania and Attending Neonatologist at the Children’s Hospital of Philadelphia, into an independent clinician-investigator. This award will further assist her in achieving her long-term career goal of becoming an expert in antimicrobial pharmacology, and using her expertise to optimize the care of infants at risk for severe infections. In addition, this award will allow her to obtain the training, mentorship, and research experience necessary to successfully compete for future R01 grants in this arena. Dr. Coggins will receive advanced training and experiential learning in pharmacokinetic modeling, pharmacoepidemiology, and advanced statistical methods. The University of Pennsylvania and Children’s Hospital of Philadelphia provide a stellar research environment for this proposed study. This project will be guided by an outstanding mentorship team with a track record of successful collaboration and scholarship, including experts in neonatology, clinical pharmacology, infectious diseases, biostatistics, and epidemiology.

NIH Research Projects · FY 2025 · 2025-07

Project Summary/Abstract The goal of this project is to define the mechanisms by which impaired mitochondrial function leads to failure of neutrophil development, and how neutrophil precursors use somatic mutations to adapt to this cell- intrinsic stress. Severe Congenital Neutropenia(SCN) is an inborn disorder of granulopoiesis characterized by severe chronic neutropenia from birth, premature death secondary to infectious complications, and transformation to myeloid malignancy. Despite the significant morbidity and mortality associated with SCN, the mechanisms underlying survival, proliferation, and differentiation of neutrophil precursors remain incompletely understood. We recently identified heterozygous missense mutations in CLPB as a new and frequent cause of SCN. CLPB is a nuclear-encoded protein that resides within the mitochondrial intermembrane space where it functions as a molecular chaperone to disaggregate and facilitate re-folding of misfolded proteins. Our previous work has also demonstrated impaired mitochondrial oxidative phosphorylation in CLPB-mutant myeloid cells accompanied by increased cell death. Interestingly, these cells demonstrate an increased non-apoptotic cell death in response to mitochondria proteostasis stress and sustained activation of the mitochondrial unfolded protein response (UPRmt). Patients with SCN also develop clonal hematopoiesis that may help alleviate cell intrinsic or cell extrinsic stress; however, the mechanisms of adaptation to CLPB-mutated SCN are not defined. In this proposal, we will test the hypothesis that mutant CLPB granulocyte precursors undergo ferroptotic cell death, a form of non-apoptotic cell death that has been linked to prolonged ROS generation. We will also test the hypothesis that CLPB-induced mitochondrial stress is ameliorated by RUNX1 mutations leading to a competitive growth advantage. To this end, the following specific aims are proposed: Aim 1) To understand the link between sustained UPRmt signaling and cell death in CLPB-mutated granulocyte precursors; Aim 2) To identify the mechanisms by which RUNX1 mutations promote clonal advantage in CLPB-mutated SCN. The proposed studies will provide an understanding of the molecular pathophysiology of CLPB-mutant SCN and RUNX1-mediated clonal adaptation. Ultimately, a better understanding of normal and SCN-related granulopoiesis may suggest new therapeutic approaches to treat or prevent neutropenia in patients with SCN, and in patients receiving myeloablative chemotherapy. These studies will also provide critical knowledge on how mitochondrial stress impacts granulocyte development, and how maladaptive clonal hematopoiesis provides a growth advantage to these cells. Filling these knowledge gaps can lay the foundation for developing strategies to augment neutrophil precursor cell survival and differentiation without enhancing risk for malignant transformation.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Cartilage growth plates are the primary drivers of skeletal growth and patterning throughout development. Their highly organized structure and dynamic turnover are tightly regulated by complex molecular networks and involve a multi-step program of differentiation, proliferation and maturation of their resident cells, chondrocytes. Supporting this complexity is evidence that hundreds of chondrodysplasias exist in humans, in which growth plate chondrocytes are impaired one way or another. These conditions and many research studies have demonstrated the importance of various factors, including signaling pathways and transcription factors. However, each study was typically limited to one factor and a few targets, and used different experimental models than others. Many gaps thus remain in our definition of the molecular networks regulating growth plate chondrocytes and distinguishing them from articular chondrocytes. These gaps limit our ability to decode the molecular basis and mechanisms underlying not only chondrodysplasias, but also osteoarthritis, where articular chondrocytes often acquire growth plate chondrocyte features, and thus to find treatments direly needed for many of these conditions. Cutting-edge technologies, such as single-cell profiling of transcriptomes and accessible chromatin regions, and genome-wide occupancy of transcription factors are powerful means to make major research progress. This project focuses on RUNX2 and RUNX3, which have long been known to be required for growth plate chondrocyte maturation, but whose full spectrum of actions and interactions with other factors remains sparsely defined. Findings from previous studies and preliminary data support the hypothesis that RUNX2/RUNX3 control a much larger panel of genes than currently appreciated and that these genes are involved both in the multistep program of growth plate chondrocyte specification and differentiation and in skeletal patterning, and that the two factors do this in concert with many other cell type-specific transcription factors and signaling pathway mediators. To test this hypothesis, Aim 1 will thoroughly define the skeletal phenotypes of mice inactivating both genes before or after growth plate formation at the tissue, cellular and molecular levels through histology-based and single-cell transcriptomic assays. Aim 2 will identify the genes whose expressions are directly or indirectly controlled by RUNX2/RUNX3 through genome occupancy profiling and reporter assays. Aim 3 will start to build an atlas of genomic occupancy and functions of all transcription factors driving articular and growth plate chondrocyte fate and differentiation along with, upstream of, or downstream of RUNX2/RUNX3. Scientifically, this project should propel the field of skeletogenesis regulation forward. Translationally, it could help decipher the molecular basis of still unresolved skeletal dysplasias and identify tools for disease therapies. Professionally, it provides an outstanding platform to increase the scientific and technical skills of the postdoctoral fellow and help him transition into a successful independent investigator in skeletal biology research.

NIH Research Projects · FY 2025 · 2025-06

ABSTRACT Persistence of the HIV reservoir is a major barrier to cure. Many cure-directed clinical trials use novel interventions to reactivate the reservoir for immune targeting and reservoir reduction. Improving the targeting and elimination of HIV reservoir requires a cell-by-cell understanding of where reservoir is and how reservoir persists after cure interventions. However, current strategies to identify the multi-modal biology of cells with integrated provirus are not able to fully resolve the intact proviral reservoir: they cannot detect provirus in closed chromatin and cannot discern intact from defective provirus. This proposal will work to interrogate cellular programs of the total intact reservoir using two new sequencing approaches to address the problems of proviral intactness and integration in closed chromatin. In aim 1, we will test the hypothesis that CD4+ T cells with provirus in closed chromatin are transcriptionally distinct from cells with provirus in open chromatin. In aim 2, we will test the hypothesis that cells with intact provirus have distinct transcriptional, epigenetic, and clonal features. We will therefore work to identify the distinct features of the intact reservoir and to generate a technical and analytic pipeline for adjunctive use across multiple cure strategies.

NIH Research Projects · FY 2026 · 2025-06

ABSTRACT Inherited diseases frequently manifest with distinctive facial features, collectively referred to as the “facial gestalt.” However, the effectiveness of using facial information relies heavily upon the clinician's breadth of experience, and such reliance often leads to delays in diagnosing patients. Furthermore, 1 in 600 newborns have craniofacial anomalies, impacting the skull, jaws, ears and/or teeth. Improved therapeutic approaches require a deep understanding of the molecular and biological systems underlying these craniofacial conditions. Through an interdisciplinary and agile team, our study has two objectives: (1) We will develop an ethically focused and data-driven multimodal machine learning algorithm that integrates frontal facial photos, clinical notes, patient demographics, and structured phenotypes to improve diagnosis of genetic syndromes. (2) We will develop a multimodal algorithm that takes 4D videos (non-invasive laser scanning to map craniofacial surface morphology), facial photos, electronic health records, and omics information (genome, transcriptome and epigenome), to prioritize genes in key pathways that are relevant to phenotypic features of craniofacial abnormalities. We will adapt a patient/clinician engaged design, ensuring that the training data and outcome evaluation metrics align closely with real-world applications. We will develop a modularized open-source software to decouple the multimodal approach from the underlying pretrained models, ensuring that the final product is adaptable to rapid changes in AI technologies to be easily integrated into evolving healthcare landscapes. Our project addresses the social-technological challenges in clinical diagnosis of rare diseases, to advance understanding, interpretation and prediction of complex biological, behavioral, and health manifestations through the implementation of multimodal approaches.