Cincinnati Childrens Hosp Med Ctr

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Metabolic dysfunction–associated steatotic liver disease (MASLD) is an inflammatory liver disease associated with progressive fibrosis, cirrhosis, hepatocellular carcinoma, and death from cardiovascular disease. Despite knowledge of MASLD risk loci identified in genome wide association studies (GWAS), it is not yet possible to predict MASLD using DNA sequences alone, justifying an urgent requirement for additional research on genetic mechanisms of MASLD. One cell type that is instrumental to sensing and responding to tissue changes during MASLD are Kupffer cells, the predominant liver resident macrophage. Kupffer cells contribute to MASLD progression by promoting local and systemic inflammation, immune cell infiltration, hepatocellular damage, and fibrosis. Additional research on the function and recruitment of Kupffer cells is important for understanding MASLD because blocking macrophage infiltration or globally ablating macrophages protects against severe disease in experimental models. A major shortcoming in our understanding of MASLD and associated inflammation is that most preclinical studies assessing Kupffer cell biology occur in a single genetic background. This shortcoming is significant because disparate phenotypes exist in MASLD models between inbred mouse strains, similar to the varied outcomes in the genetically diverse human population. Importantly, more than 90% percent of trait linked genetic variants are found in noncoding regulatory regions that control gene expression in cell type– and context–restricted manners. Therefore, studies defining roles for genetic variation in controlling transcriptional regulatory mechanisms in Kupffer cells may unveil novel insights into inflammation and MASLD relevant to human disease. In our recent publication, we predicted the identities of many transcription factors and pathways linked to allele–specific regulation of Kupffer cell gene expression in response to acute inflammation. However, we still do not understand with cell type resolution how genetic variation contributes to MASLD. For this proposal, the central hypothesis is that genetic variation controls the environmental signals sensed by Kupffer cells and that knowledge on regulatory mechanisms will identify new pathways relevant to MASLD in human patients. In specific aim 1, we will define genetic mechanisms controlling Kupffer cell transcription during experimental MASLD. In specific aim 2, we will assess the feasibility of gene editing for defining strain-specific functions of in vivo liver macrophages using an innovative combination of CRISPR/Cas9 and bone marrow transplantation. These results will support feasibility for expanded functional studies assessing roles for genetic variation in Kupffer cells during disease and be extendable to tissue macrophages from other organs and disease contexts. This research may define novel pathways and mechanisms controlling macrophage participation in MASLD pathogenesis, which may be required for the development of novel therapeutics.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Normal heart valve structure and composition are established by valve remodeling, starting at late embryonic stages, and continuing postnatally. Valve remodeling results in a stratified extracellular matrix (ECM), decreased cell density and reduction in cell proliferation. Congenital valve malformations include abnormalities in valve remodeling such as ECM disruption and disorganization. Congenital heart valve abnormalities due to ECM gene mutations and defects, often lead to myxomatous valve disease (MVD). Progressive MVD is characterized by collagen fiber fragmentation, replacement of mucopolysaccharides and proteoglycans, leaflet thickening, and insufficiency, but the mechanisms mediating progressive valve degeneration remain unknown and there are no therapies to prevent or reverse MVD. Recently, our group reported that, in an Fbn1C1041G/+ Marfan syndrome (MFS) mouse model, the ECM mechanics and morphological alterations of the mitral valve occur before functional abnormalities are detectable. Surprisingly, by 6-12 months, collagen fiber remodeling is increased with abnormal fiber organization suggesting a compensatory fibrotic response. We also observed induction of matrifibrocyte gene expression associated with collagen-rich connective tissue such as in myocardial scarring after infarction. In preliminary studies we also observed increased expression of pFAK, a nonconical focal adhesion kinase (FAK), in MVD at the time of increased collagen production and matrifibrocyte gene induction, suggestive of a mechanically regulated myofibroblast response. The role of matrifibrocytes and FAK activation in heart valve disease progression and compensation is unknown. Therefore, we hypothesize that stress activation and collagen overproduction in MFS mitral valves induce matrifibrocytes causing an intrinsic reparative response via activated FAK. We propose three aims to elucidate the role and function of matrifibrocytes in ECM remodeling, maturation and disease. 1: Determine the contributions of fibrotic ECM in MVD progression in mitral valves, 2: Determine if valvular interstitial cell (VICs) activation and matrifibrocyte differentiation occur in response to collagen production and tissue stiffness in mitral valves and 3: To determine if the fibrotic response in the mitral valve during homeostasis and MVD progression is mediated through FAK activation. The K99 portion of this proposal will be carried out in the lab of Dr. Katherine E. Yutzey, an expert in heart valve disease, and co-mentor Dr. Jeffery D. Molkentin, an expert in fibrotic remodeling of the heart. Under their guidance, I will carry out my in vitro (Aims 1-3A) experiments during the K99 phase and start my in vivo (Aims 1-3B) murine model experiments during the K99 phase and continue during the R00 phase. Overall, the comprehensive Aims proposed will provide me with the opportunity to develop an independent research program that will be instrumental in my success as a scientist. Moreover, the data generated will have important implications for cardiovascular science, specifically in development of new therapeutic targets for heart valve disease.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The objective of this project is to generate single cell resolution information about how metabolism changes at single cell resolution over the course of embryonic development in embryos from normal and hyperglycemic mothers. This is relevant to understanding why structural birth defects are 2-3 times more likely in pregnancies of diabetic mothers. We will use the nematode C. elegans as a model in our studies and since many aspects of development and metabolism are conserved, our findings will likely further our understanding how metabolism affects morphogenesis in vertebrate embryonic development and human structural birth defects. C. elegans is an excellent model because its simplicity and accessibility will enable us to fully characterize the cellular metabolism of individual cells during embryonic development and determine how hyperglycemia impacts cell fate specification and morphogenesis. Our preliminary data show that cellular metabolism varies across cells and across developmental time. We hypothesize that maternal hyperglycemia increases oxidative stress in embryonic cells, resulting in insufficient cellular energy for essential migrations and inducing AMPK-mediated chromatin changes that inhibit the expression of cell fate TFs, resulting in developmental defects. To test this hypothesis, we will use complimentary approaches of quantitative time-lapse imaging of biosensors, and transcriptomics plus metabolic modeling. We will integrate the results from each approach to create a single cell resolution profile of embryonic metabolism across developmental time in control and maternal hyperglycemia embryos. This will enable us to link developmental defects to disruptions in metabolism at the cellular level. This work will generate the first comprehensive metabolic atlas of a metazoan embryo with the single cell resolution across developmental time and determine how embryonic cellular metabolism and developmental processes are impacted by hyperglycemia.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Declining immune function in the elderly leads to increased risk and severity of infection and impaired responses to vaccination. Our and others prior work have shown that control of immune responses in aged individuals is complex. Although immune responses to immunization are generated in the elderly, they are tempered by impaired intrinsic function of many immune cell types, accrual of immune suppressive and senescent cells, which results in inappropriate localization and function of B and T cells. Strikingly however, our and other’s data also show that age-related, decreased immune responsiveness to immunization is reversible by enhancing stimulatory signals or reducing inhibitory signals. Thus, understanding the mechanism(s) regulating age-related immune responses is critical to unlocking the potential for reinvigorating immune responses in aging. Recent data from our extensive single cell genomics analysis of aged memory CD4+ T cells show that CD153 is one of the most highly expressed genes in aged T follicular helper (Tfh) cells. Although the general consensus is that CD153 expression on CD4+ T cells is critical for its function this remains to be rigorously tested and the cell- specific role of CD30 remains controversial. Some data show both stimulatory and suppressive roles of CD30 on B cells; while other data shows a critical immune stimulatory role of CD30 on T cells. Thus, the cell-specific functions of CD153 and CD30 have yet to be cleanly defined in vivo. To begin to determine the role of CD153/CD30 signaling in regulation of immune responses in aged mice, we blocked CD153/CD30 signaling in aged, NP-KLH immunized mice using a non-depleting, blocking antibody. Strikingly, we saw a significant decrease in NP-specific (NP-sp.) IgG1 in the serum as well as a dramatic loss of NP-sp. GC B cells after CD153/CD30 blockade. However, we still lack understanding of the underlying biology of CD153/CD30 interactions. Based on robust preliminary data, we hypothesize that: CD4+ T cell expression of CD153 is sustained by IL-6 and is required to signal to CD30 expression of B cells; these interactions are critical to promote antibody responses in aged mice. We generated mice with conditional alleles for CD153 (CD153fl/fl) and CD30 (CD30fl/fl), and will use them to (i) define the temporal role of CD153 on CD4+ T cells in controlling antibody responses in aged mice; and (ii) define the role of CD30 and its potential for regulating B cell responses in aged mice. Overall Impact: This research fits the R21 definition of “high-risk” as the cell-specific roles of CD153 and CD30, may not be clear-cut. It is also ”high-reward” because, if successful, we will fill important gaps in knowledge regarding the cell-specific and temporal roles of CD30 and CD153 in control of B cell responses in aged mice. Further, it will enable future mechanistic studies to determine the potential for manipulating this axis to boost B cell responses in the aged.

NIH Research Projects · FY 2025 · 2025-08

Summary: There are no effective treatments for the majority of brain disorders. Even diseases like epilepsy, for which many powerful therapies exist, are intractable in about one third of affected individuals. One of the reasons that make treating brain disorders like epilepsy so difficult is the complexity of the underlying pathology that is tough to correct with single-target approaches and poorly understood. Recent preclinical studies have begun to address this challenge by exploring aberrant microRNA-induced silencing as a pathological mechanism contributing to epilepsy. MicroRNA-induced silencing is a powerful epigenetic mechanism that controls neuronal function and is dysregulated in epilepsy. MicroRNAs repress translation or initiate the degradation of hundreds of target mRNAs and can regulate several components of the same biological pathway. This makes them powerful regulators of biological processes but also imposes a substantial challenge to maintain functional specificity. MicroRNA specificity in the brain is most likely achieved through different molecular environments in specific cell types and brain circuits; yet, not much is known about these factors which prevents the field from fully understanding their biology as well as their potential and risks as drug targets. The proposed research will address this challenge by using novel adeno-associated virus (AAV)-assisted strategies to reveal the cell types and cell type-specific target mRNAs that mediate microRNAs’ control of brain function in the context of epilepsy. Based on preliminary data suggesting that effects of a specific microRNA on seizure control and neuronal morphology are mediated through different cell types, the proposed research will test the central hypothesis that microRNA specificity is achieved through cell type- and brain circuit-specific control of microRNA function, so that for select proconvulsant microRNAs, the effects on seizures and healthy brain function can be separated by cell type and mRNA targets. To test this hypothesis, a two-pronged approach will be followed to (1) assess cell type-specific control of brain function of key microRNAs critical in epilepsy, and (2) identify their cell type-specific target mRNAs to reveal the molecular pathways contributing to their diverse functions. Leveraging the synergistic expertise of a multidisciplinary team in microRNA biology, epilepsy, cognition, and gene network analysis, two aims are proposed. In aim 1, microRNAs will be inhibited either pan-neuronally, in excitatory neurons, in inhibitory neurons, or in astrocytes to identify the cell types that mediate control of seizure susceptibility and recurrent seizures in epilepsy, neuronal dendritic spine morphology, and cognition. In aim 2, cell type-specific inhibition of microRNAs combined with isolation of mRNAs silenced or actively translated only from cells in which the microRNA is inhibited, will be used to experimentally identify the cell type-specific mRNA targets of, and thus biological networks regulated by, the same microRNAs as tested in aim 1. This research will provide essential insight into fundamental function of microRNAs in the brain and will be a critical step towards fully understanding the potential of microRNAs as treatment targets in epilepsy and other brain disorders.

NIH Research Projects · FY 2025 · 2025-08

Summary Neuronopathic Gaucher disease (nGD) is a severe pediatric genetic disorder caused by mutations in GBA1, the gene that codes for lysosomal acid β-glucosidase (GCase). Defective GCase impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph) in the central nervous system (CNS), activating glial cells and contributing to neuroinflammation. The prevalence of GD is 1 in 500 among Ashkenazi Jews and 1 in 60,000 in the general population. Approximately 10% of GD patients in the US and Europe, and around 75% in Asian countries, are diagnosed with nGD. Patients with nGD often manifest early in life with high mortality. Emerging evidence implicates microglial dysfunction in the pathogenesis of various neurodegenerative disorders, including nGD, but the mechanisms by which GCase deficiency leads to microglial activation and neurodegeneration in nGD remain largely unknown. Current enzyme replacement therapy (ERT) and substrate reduction therapy (SRT) are ineffective for CNS involvement, and their safety for newborns is not well understood. GD is a hereditary lysosomal storage disorder that is included in newborn screening programs, allowing for early detection and timely intervention. Traditional models, including 2D cultures fail to replicate the complexity of human brain tissues. Utilizing induced pluripotent stem cells (iPSCs) derived from nGD patients, we have successfully developed nGD midbrain-like organoids (MLOs) model. These nGD organoid models exhibit midbrain characteristics and nGD-specific phenotypes, such as GCase deficiency and GluSph accumulation, providing a model system for studying nGD and a platform for drug evaluation. Furthermore, we have advanced MLOs by incorporating microglia to create microglia-containing MLOs (mcMLOs). Microglial differentiation was achieved by inducing the homeostatic transcription factor PU.1 in re-engineered iPSCs within a neural differentiation environment, leading to the formation of mcMLOs. In GD, GluCer and GluSph accumulation activates microglia, increasing cytokine release and inflammation via the C5a/C5aR1 pathway. We hypothesize that microglia within mcMLOs profoundly influence nGD-relevant phenotypes, including microglial activation, and that the complement C5a/C5a receptor system plays a role in exacerbating neuroinflammation. Specifically blocking C5aR1 signaling in GCase-deficient microglia may represent a therapeutic target to prevent microglia- mediated inflammation and neurodegeneration in nGD. To test this hypothesis, in Aim 1, we will generate nGD mcMLOs to investigate the impact of nGD microglia in MLO growth and disease phenotypes, and to evaluate therapeutic effects on this nGD mcMLO model. In Aim 2, we will determine the role of the microglial C5a/C5aR1 pathway in nGD and assess C5aR1 inhibition as a therapeutic approach. This advanced nGD mcMLO model offers a unique, physiologically relevant platform using patient iPSC-derived organoids, enabling mechanistic studies and the development of novel therapies for nGD.

NIH Research Projects · FY 2026 · 2025-08

Staphylococcus aureus is the leading cause of serious deep-seated osteoarticular and implant- associated infections. S. aureus orthopedic implant-associated infections are difficult to treat, requiring surgery and / or prolonged systemic antibiotics (weeks-months). They are also associated with extended disability and rehabilitation, contributing to worse overall outcomes. Although infection rates of orthopedic implant-associated infections have remained at 1-2% after primary and 3-6% after revision arthroplasty, inpatient costs average $25,000-$107,000 per case, and an annual healthcare burden of $3 billion in the U.S. alone. The increasing prevalence of methicillin-resistant S. aureus (MRSA) in the U.S. and worldwide, poses yet another challenge for the treatment of these infections, requiring prolonged intravenous antibiotics. We have developed animal models of S. aureus orthopedic implant-associated infections that replicate key pathological features of human disease as well as novel, clinically translatable positron emission tomography (PET) bioimaging for holistic, noninvasive longitudinal measurements in live subjects - bacteria-specific detection of S. aureus infections [11C-para-aminobenzoic acid (PABA), selectively metabolized via the bacterial folate pathway] and 11C-rifampin, 18F-linezolid, 18F-sutezolid, all chemically identical to the parent antibiotic, to measure antibiotic area under the curve (AUC). Recently, we conducted first-in-human 11C-PABA (Ordonez et al. JCI Insight 2022) and 11C-rifampin (Gordon et al. Sci Transl Med. 2021) PET studies in healthy volunteers and newly identified patients with S. aureus orthopedic implant infections, respectively. We show that rifampin bone exposures are substantially lower than previously thought (based on single time-point biopsy studies). Pharmacokinetic modeling of this rich PET data enabled the development of optimized, shorter rifampin-based treatments for S. aureus orthopedic implant infections, which ameliorated the development of antibiotic resistant bacteria, reduced mutations conferring bacterial persistence, and mitigated adverse bone remodeling. Here, we will leverage our expertise in bioimaging, pharmacology and animal model approaches to perform comprehensive proof-of-principle studies and gain mechanistic insights into the interplay of spatiotemporally compartmentalized antibiotic exposures (rifampin, linezolid and sutezolid) and bacterial evolution / acquired drug resistance (ADR) during antibiotic treatments, to establish relapse-free cure for S. aureus orthopedic implant infections. Integration of findings from animal and human studies will enable us to refine our models and address clinically relevant challenges. Knowledge gained from these studies will not only provide unique mechanistic insights into bacterial evolution and ADR and inform the development of novel, short, oral-only therapeutic (antibiotic) regimens for S. aureus orthopedic implant infections, but will also be a major stride towards developing precision medicine tools for at-risk patients with complicated S. aureus infections.

NIH Research Projects · FY 2025 · 2025-07

Summary Gaucher disease (GD) is caused by mutations in the GBA1 gene encoding a lysosomal enzyme, acid β- glucosidase (GCase). GD is classified as visceral (Type 1) or neuronopathic (Types 2 and 3, nGD) diseases. Defective GCase function results in the gradual buildup of its substrates glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to conditions such as hepatosplenomegaly, chronic anemia, thrombocytopenia, and osteopenia in Type 1, or the severe neuronopathic forms (Types 2 and 3, nGD) which often manifest early in life with high mortality. Prevalence of GD in Ashkenazi Jews is 1/500 and, in the general population, 1/60,000. Approximately 10% of GD patients in the US and Europe, and around 75% in Asian countries, are diagnosed with nGD. The outcomes of visceral manifestation in GD patients are improved by Enzyme Replacement Therapy (ERT), but ongoing limitations include treatment delivery regimen challenges and high costs in part due biological instability of GCase requiring high doses to maintain a therapeutic effect. Moreover, and pertinent to this proposal, ERT is ineffective for nGD due to the blood-brain barrier (BBB) blocking central nervous system (CNS) access. Consequently, the CNS disease in nGD patients is currently untreatable. To enable GCase delivery of a more biologically stable form through the BBB into the CNS, we are developing an innovative approach that 1) utilizes a distinctive and safe nanocarrier for GCase consisting of Saposin C (SapC) and dioleoylphosphatidylserine (DOPS) that has the capability to cross the BBB and 2) uses a novel GCase (named fGCase) with markedly improved stability. Our preliminary data have shown the following features supporting the therapeutic promise of SapC-DOPS-fGCase nanodrug: 1) increased plasma half-life activity, 2) enhanced stability in both cells and the brain after crossing the BBB, 3) generalized entry of fGCase throughout the brain, 4) sustained catalytical activity in the brain as evidenced by a ~29% reduction in GluCer and a ~44% reduction in GluSph levels after a single intravenous dose over five days. The preliminary results strongly indicate that SapC-DOPS-fGCase, exhibiting remarkable stability in the brain and extended catalytic activity, has the potential to provide sustained and functional enzymatic action that could effectively prevent phenotypic deficits in nGD patients who currently lack viable treatment options. To demonstrate in vivo efficacy of SapC-DOPS-fGCase for further development to treat human nGD in the R61 phase, in Aim 1, we will optimize the formulation of SapC-DOPS with fGCase, validate its stability, and, in Aim 2, characterize pharmacokinetic properties to establish a dosing regimen. Upon demonstration of sustainable stability and activity of SapC-DOPS- fGCase and establishing therapeutic dosing regimen, in the R33 phase, Aim 3, we will evaluate in vivo efficacy of SapC-DOPS-fGCase in a nGD mouse model for treating nGD. Development of the novel SapC-DOPS-fGCase nanodrug will radically improve the delivery of more stable longer-acting enzymes to the brain, providing a promising CNS-ERT for patients with nGD.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Abnormal timing of labor, especially preterm birth (PTB), is a major threat to maternal and infant health. Mothers with pre-pregnancy obesity have increased risk for pregnancy complications including PTB. However, cellular and molecular mechanisms underlying the induction of such adverse health outcomes in pregnancy remain unknown. Induction of labor is associated with hormone release (e.g., oxytocin), increase in systemic inflammatory mediators (e.g., fetal DNA, LPS) and proinflammatory cytokines (e.g., IL-6, TNF), and sympathetic nervous system (SNS) activation. Here, we aim to define non-dogmatic sources of pro-inflammatory cytokine production throughout pregnancy that may affect the timing of labor. Specifically, due to its expanded status in pregnancy and known role in various inflammatory diseases, we propose to study how white adipose tissue (WAT) inflammation is induced in pregnancy and how such inflammation contributes to the overall inflammatory balance. Using a mouse pregnancy model, we show that WAT temporally expands in pregnancy, and that such expansion positively correlates with higher pro-inflammatory cytokine production by WAT at baseline and uniquely in adipocytes after LPS sensing. However, whether and how hormonal and inflammatory signals unite to instruct the timing of labor in WAT remains unknown. Dogmatically, oxytocin is known as the major hormone responsible for uterine contraction during labor. Recent findings show the ability of oxytocin to induce lipolysis and change metabolic characteristics of adipocytes via direct SNS stimulation. We now show that oxytocin and LPS have a synergistic effect on adipocyte IL-6 production, and that oxytocin-expressing neurons are present in WAT of female mice. Thus, our preliminary data and published reports support the overarching hypothesis that SNS-derived oxytocin secretion in pregnancy amplifies WAT inflammation via adipocyte oxytocin receptor signaling. In this proposal, we aim define how oxytocinergic innervation of WAT affects tissue and cellular inflammation in pregnancy (Aim 1), and to examine the role of adipocyte oxytocin sensing for amplified inflammation in pregnancy (Aim 2). Together, these studies will lay novel groundwork for exploration of peripheral tissue neuro-inflammation in initiation of labor – a timely need given the increasing prevalence of preterm labor and pre-pregnancy obesity in this country. Further, the associated training plan will establish a foundation for a successful career as a physician scientist with a focus on academic obstetrics & gynecology research.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Antibiotic associated reactions (AAR) occur in 5-10% of children and cause substantial distress to families due to the occurrence of rashes, swelling of the face/hands/feet, fever, and gastrointestinal upset. When hives and joint pain are both present, reactions are labeled as serum sickness like reactions (SSLR), a bothersome reaction due to prolonged, uncomfortable symptoms and lack of response to allergy medications such as antihistamines. Recently, it has become apparent that AAR (including SSLR) are rarely reproduceable (<5%) with re-exposure to the antibiotic in an allergist office. Research studies are needed to identify what causes AAR to occur in the first place and whether antibiotics may play a role in reactions without evidence for classical allergic hypersensitivity reactions. We have recently observed that children may experience repeat episodes of hives and SSLR despite non allergic testing and despite interval tolerance of the antibiotic. The reactions are delayed in onset and unpredictable. We hypothesize that prolonged use of antibiotics induces gut dysbiosis which leads to autoinflammatory reactions in susceptible children. To test this hypothesis, we plan a multi-omics approach, evaluating inflammatory blood markers, immune transcriptomics, fecal microbiota populations and metabolic derangements. We will associate the multi-omics assessments with 2 cohorts of children- those experiencing typical, short lived, antihistamine responsive urticaria and those affected by hives that are atypical- poorly response to antihistamines, prolonged in duration, and associated with painful swelling of joints and extremities. To characterize these 2 urticarial reactions as distinct subsets (not currently defined clinically), we will collect additional clinical information to establish pattern recognition in each cohort. This will include patient recorded outcome measures of urticarial symptoms, medication usage and response, photo documentation of rashes with dermatology confirmation and ultrasound-based assessment of joints for evidence of synovitis. Using computational modeling, we will determine which biomarkers associate strongly to one cohort versus the other cohort. We will accomplish our aims by engaging a multidisciplinary team of allergy/immunology/ rheumatology/ dermatology/infectious disease physician investigators and computational and metabolomics experts. Many children will experience urticarial reactions while taking antibiotics and will presume they are allergic. This study will help identify the scientific basis for occurrence of hives without evidence for classically defined allergy responses, including the possibility that antibiotic disruption of healthy commensals leads to autoinflammatory reactions in susceptible hosts. By identifying novel mechanisms whereby antibiotics lead to urticarial reactions and associating them to well defined, clinical syndromes, we may prevent the accumulation of unnecessary allergy labels and identify effective treatment of children who experience severe reactions such as SSLR.

NIH Research Projects · FY 2025 · 2025-07

Mycobacterium tuberculosis remains the second leading cause of death from a single infectious agent globally, after COVID-19. The risk of developing tuberculosis (TB) is substantially higher in human immunodeficiency virus (HIV) coinfected individuals, with TB and HIV potentiating each other, causing substantial declines in immune function. Globally in 2022, there were an estimated 1.3 million deaths and 10.6 million new cases due to TB. Importantly, it is estimated that more than 58 million patients have survived TB in this century alone. However, unlike other respiratory infections, many patients with TB have permanently damaged tissues and successful TB treatments only transition these patients from harboring a communicable infectious disease, to a syndrome of chronic pulmonary morbidity. This syndrome, commonly referred as post- TB lung disease, affects ~50% of patients with pulmonary TB, who develop chronic adverse outcomes beyond the TB treatments, including bronchiectasis, poor lung function and respiratory symptoms. Although post-TB lung disease remains poorly characterized, it is primarily mediated by M. tuberculosis-induced tissue destruction (necrosis) and subsequent adverse lung tissue remodeling and fibrosis, which in addition to causing pulmonary disease, also impairs vascular supply and reduces antibiotic access to the infected TB lesions. Currently, there are no approved treatments to prevent post-TB lung disease. We have characterized lung disease in animal models of TB to develop adjunctive treatments that prevent post-TB lung disease. Importantly, we have also developed and utilized novel, clinically translatable positron emission tomography (PET)-based imaging to longitudinally profile lesional characteristics in animal models and TB patients: 11C-rifampin (chemically identical to rifampin) PET to measure tissue antibiotic area under the curve exposures (Ordonez . . . Jain. Nat Med. 2020) and 18F-FAPI-74 PET for monitoring fibrosis. Our central hypothesis is that 18F-FAPI-74 PET could be used as a noninvasive biomarker to assess post-TB lung disease and fibrosis in TB patients. Additionally, we hypothesize that lesions with significantly reduced 11C-rifampin exposures in TB patients within 6-weeks of treatment initiation (early time-point) will develop post-TB lung disease sequelae at those sites after treatment completion. Here, we will leverage our expertise in TB pathogenesis and molecular imaging to conduct longitudinal, observational studies to visualize lesional rifampin exposures and lung fibrosis in patients with recently diagnosed pulmonary TB, with or without HIV coinfection, within 6-weeks of initiation and after completion of TB treatments. Our goals are to better characterize post-TB lung disease sequelae and identify key factors, such as lesional antibiotic exposures in TB patients that could subsequently lead to post-TB lung disease. In the future, we anticipate that these imaging approaches could be used to noninvasively characterize post TB- lung disease and evaluate novel treatments that prevent or mitigate post TB-lung disease.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY/ABSTRACT Cholangiopathies are liver disorders that primarily injure the cholangiocytes, leading to cholestasis, biliary fibro- sis/cirrhosis, and cholangiocarcinoma. These conditions cause significant morbidity and mortality and are a major indication for liver transplantation. Developing effective treatments is challenging due to the heterogene- ous nature of cholangiopathies, many of which are orphan diseases. About 30% of patients with suspected in- herited cholestasis, including many with cholangiopathies, lack pathogenic variants in known disease-causing genes. This lack of molecular diagnosis hinders investigation into disease pathogenesis and development of personalized treatment. Our long-term goal is to understand the etiology and molecular genetics of chronic cholestatic liver diseases and improve patient diagnosis and treatment. We recently identified a homozygous deleterious variant in ABCC12, which encodes the ATP-binding cassette protein MRP9, in a patient with chronic intrahepatic cholestasis. Studies of the patient and two animal models indicate that the loss of MRP9 renders cholangiocytes more susceptible to cell death. The overall objective of this application is to delineate how MRP9 deficiency causes cholangiocyte injury. Our preliminary studies revealed mitochondrial damage in a human cholangiocyte cell line, as well as in the cholangiocytes of larval zebrafish and neonatal mice lacking MRP9. The mitochondrial phenotype occurred prior to changes in bile duct morphology or cholangiocyte cell number and was not observed in hepatocytes in the same animals. In abcc12-/- zebrafish, bile ducts accumu- lated the heme intermediate protoporphyrin IX (PPIX) ectopically. Restoring wild-type MRP9 in mutant cholan- giocytes, but not hepatocytes, partially suppressed PPIX accumulation and bile duct loss. Both neonatal Abcc12-/- mouse cholangiocytes and human H69 cholangiocyte cells carrying the patient variant showed in- creased PPIX accumulation and cell death when treated with porphyrin precursor aminolevulinic acid. Our overarching hypothesis is that MRP9 maintains cholangiocyte integrity by mediating mitochondrial metabolism. We propose two complementary specific aims: 1) Elucidate the molecular and cellular mechanisms underlying cholangiocyte death caused by MRP9 deficiency, and 2) Define the function of MRP9 in cholangiocyte metabo- lism. Aim 1 will test if MRP9 deficiency causes PPIX accumulation and mitochondrial damage within cholangio- cytes, resulting in their death. Aim 2 will determine if MRP9 transports porphyrins. We will identify PPIX-inter- acting proteins in human and mouse cholangiocytes and study how MRP9 cooperates with other porphyrin transporters to maintain cholangiocyte porphyrin homeostasis and health. The proposed study is highly innova- tive, because it integrates both in vitro and in vivo models to study the function of a completely new gene in cholangiocyte metabolism and reveals a novel etiology for cholestatic cholangiopathis. It also investigates an unexplored concept: porphyrin homeostasis in cholangiocytes. The proposed research is significant, because it will reveal new susceptible factors and therapeutic targets for cholestatic cholangiopathies.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY SHINE syndrome, a rare form of monogenic autism, is named for its key symptoms: sleep problems, hypotonia, intellectual disability, neurological disorders, and epilepsy. This condition arises from an autosomal-dominant mutation in the gene discs large MAGUK scaffold protein 4 (DLG4), which encodes postsynaptic density protein 95 (PSD-95). PSD-95 is crucial for scaffolding, localizing, and stabilizing receptors on excitatory postsynaptic membranes, thereby influencing synaptic strength and plasticity. The mechanism of sleep problems in SHINE syndrome may be due to disruption of the molecular circadian clock and/or in synaptic function. Previous work in our lab demonstrated that siRNA knockdown of DLG4 in cells results in a shortened circadian period, suggesting that PSD-95 modulates circadian rhythms and contributes to the sleep issues observed in SHINE syndrome. Our team has recently generated novel SHINE syndrome mice with a patient-derived mutation, and these mice exhibit locomotor and sleep abnormalities. It remains unknown how PSD-95, either through molecular or synaptic changes, impacts sleep and circadian processes. The overarching goal of this proposed work is to elucidate the mechanisms behind sleep and circadian disturbances in SHINE syndrome. With two related but independent aims, this proposal investigates: (i) whether PSD-95 modulates circadian rhythms through tropomyosin receptor kinase B (TRKB)/mechanistic target of rapamycin (MTOR) signaling, (ii) abnormalities in receptor levels and synaptic function in the hypothalamus, and (iii) possible rescue of abnormalities with ketamine. Aim 1 proposes to determine whether there is abnormal TRKB signaling in SHINE mice–resulting in subsequent abnormal MTOR activity–affecting the cycling of the circadian clock protein BMAL1 in the hypothalamus. This will be accomplished by measuring MTOR activity and BMAL1 cycling in the hypothalamus of SHINE mice at baseline, after chronic administration of TRKB modulators (including ketamine), and following a washout period. The synchrony of molecular rhythms will also be assessed. Aim 2 will then identify impairment in receptor composition and synaptic function in the hypothalamus of SHINE mice. Sleep and epilepsy will be monitored by EEG, and levels of AMPA and NMDA receptor subunits in the hypothalamus will be measured at baseline and after ketamine administration. Collectively, the knowledge gained from this proposal has the potential to uncover disruption caused by PSD-95 mutations and inform potential interventions to restore normal sleep and synaptic function in patients with SHINE syndrome.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT: Primary cilia are ubiquitous, microtubule-based extensions that transduce molecular signals within a cell. Defects in primary cilia result in ciliopathies, a pleiotropic group of debilitating, and sometimes life-threatening disorders. One third of ciliopathies are defined by severe craniofacial anomalies. Currently there are no recognized treatments for these patients, mostly because we have an incomplete understanding of how the cilium integrates cellular/molecular signals and influences cell behaviors. Oral-facial-digital syndrome 14 (OFD14) is a human ciliopathy that is caused by mutations in the centriolar protein, C2 Calcium-Dependent Domain Containing 3 (C2CD3) and is characterized by multiple craniofacial skeletal anomalies including cleft palate and micrognathia. Our previous work revealed hypoplastic cranial neural crest cell (NCC) derived skeletal elements in OFD14 models; however, the mechanism of how C2cd3 functions in this context is unknown. Based on the known role of C2cd3 in ciliogenesis, its localization to the centriole, and the presence of numerous C2 protein binding domains we hypothesize that the craniofacial phenotypes present in OFD14 are molecularly due to impaired to Hh/Gli-driven NCC skeletal differentiation and cellularly due to impaired association between the centriole and actin cytoskeleton. To test these hypotheses, we will utilize knockdown and conditional knockout transgenic murine lines (C2cd3Ex2; C2cd3Exon4-5fl/fl; C2cd3Exon9fl/fl) as well as human induced pluripotent stem cells (hIPSCs) that either lack C2CD3 (C2CD3-/-) or carry OFD14 causing C2CD3 variants (C2CD3Trp65Cys, C2CD3Ile477*). Experiments in this proposal will (Aim 1) determine if skeletal differentiation is impaired in hIPSC-derived NCCs carrying OFD14 causing C2CD3 variants, (Aim 2) determine if C2CD3 functions to anchor the centriole to the actin cytoskeleton and if OFD14 causing C2CD3 variants impair this association, and (Aim 3) test if C2cd3 mutations and OFD14 causing C2CD3 variants impair Gli3/DNA and Gli3/co-factor binding, and subsequent Gli3 transcription. Together these studies will address both the cellular and molecular etiology of the human ciliopathy OFD14 and identify possible areas of therapeutic intervention for craniofacial phenotypes that arise in all ciliopathies.

NIH Research Projects · FY 2026 · 2025-06

Project Summary (Abstract) Cranial neural crest cells (CNCCs) are a unique, multipotent, migratory population that give rise to both ectodermal/non-ectomesenchymal (neurons, glia) and mesodermal/ectomesenchymal (bone, cartilage) derivatives. The cellular and molecular mechanisms that allow for proper craniofacial development, including establishing multipotency, epithelial to mesenchymal transformation (EMT) and migration; however, remain controversial and heavily focus on transcription factor expression. BAF is one of the major protein complexes regulating chromatin accessibility. AT-rich interactive domain- containing proteins 1A and 1B (ARID1A, ARID1B) are the largest, mutually exclusive, subunits of BAF and mutations in these subunits result in Coffin-Siris Syndrome (CSS), a disorder characterized by craniofacial phenotypes. Our previous work revealed a dynamic requirement for ARID1A/B during CNCC development in which ARID1A was required for maintenance of pluripotency and then as iPSCs differentiated into neurosphere intermediates, was decommissioned, and replaced by ARID1B to initiate commitment to the neuroectodermal lineage via the repression of thousands of pluripotency enhancers and genes. Upon differentiation of neurospheres into CNCCs, ARID1B was downregulated and ARID1A was reactivated. We hypothesize that the return of ARID1A in CNCCs is essential for multipotency, EMT, and migration of CNCCs, and that as CNCCs differentiate, ARID1B is reactivated to ensure lineage commitment. Interestingly, there is potential integration of chromatin remodeling activity with key developmental signaling pathways whereby the induction of CNCCs is a Wnt-dependent process and phenotypes associated with loss of ARID1A/B are similar to mutations linked to the Hedgehog (Hh) pathway. Further, our preliminary data identified an enrichment for TCF/LEF and ZIC2/3 binding sites in ARID1A bound regions and proteomic studies highlighted physical interactions with the transcription factor, Gli3. Gli3 acts as a bimodal regulator (e.g., can function as both an activator and repressor) of the Hh signaling pathway and has not previously been ascribed chromatin remodeling capability. Thus, we hypothesize a novel mechanism of Gli regulation which utilizes Arid1 subunits. Herein, we propose three distinct Aims to test our hypotheses. Aim 1: will test if ARID1A regulates multipotency, induction, EMT, and migration of CNCCs, by cooperating with pluripotency factors (OCT4, SOX2, NANOG), Wnt signaling transcription factors (LEF1/TCF), and ZIC2. Aim 2: will determine if ARID1A/ARID1B mediated mechanisms (chromatin remodeling and/or RNA Pol II pausing) are necessary for CNCC differentiation. Aim 3: will determine if Gli3 cooperates with ARID1A and ARID1B to drive transcriptional activation and repression, respectively. The proposed studies will shed new light on cellular and molecular mechanisms required for CNCC induction, multipotency, and differentiation. Furthermore, they will be the first studies to reveal mechanisms by which the BAF complex contributes to transduction of signaling pathways (e.g., Hh and Wnt) essential for craniofacial development.

NIH Research Projects · FY 2025 · 2025-06

Summary of Proposed Research Demyelinating diseases of the central nervous system, such as multiple sclerosis (MS), are among the most devastating and disabling neurological disorders that lead to severe handicaps and even death. Although substantial efforts have been made centered on the suppression of the immune response that attacks myelin, it is becoming clear that this approach does not address the major problem of the disease: the loss of myelin. A common feature in the demyelinated lesions is the blockage of myelin production by oligodendrocytes. Thus, the identification of critical factors that promote myelin production from oligodendrocytes and block inhibitory signals impeding myelination will help devise effective strategies for improving myelin repair in demyelinating diseases. Epigenetic reprogramming has been shown to rejuvenate cellular activity and thus is a promising approach to enhance repair capacity after injury. Our unprecedented screen using CNP+ oligodendrocytes identified epigenetic compounds that potently stimulate for myelin production and myelinogenesis. The top hit compound promotes remyelination in animal models of MS and initiates de novo myelination of regenerated axons after optic nerve crush. Our preliminary data suggest that the histone deacetylase HDAC3 is a target of the hit compound. Expression of HDAC3 is detected in oligodendrocytes that fail to produce myelin in human demyelinated MS lesions. Moreover, silencing of HDAC3 led to ectopic myelinogenesis in the developing brain and promoted robust remyelination after injury. Since the cellular specificity and function of HDACs are dependent on subunit compositions and interacting factors in multi- protein complexes, we further identified an HDAC3-interacting protein, the zinc-finger protein Znhit1, which is a key subunit of SNF2-related SRCAP chromatin-remodeling enzyme. Our preliminary data show that Znhit1 is enriched in oligodendrocytes and critical for oligodendrocyte myelination, while overexpression of Znhit1 promotes myelin gene expression. Based on these observations, we will utilize spatio-temporally specific mutagenesis approaches to identify the critical time window for the function of class I histone deacetylase isoforms in remyelination in animal models of MS and identify the HDAC3-Znhit1 regulatory network in myelination and remyelination. Furthermore, we will determine the therapeutic benefits of HDAC3 inhibitors for myelin repair. The proposed studies will advance our understanding of the mechanisms of central nervous system myelination and will also identify factors that could be targeted to promote oligodendrocyte regeneration and myelin repair in patients with demyelinating diseases such as MS, aging, leukodystrophies, stroke, autism, and injury to the brain or spinal cord.

NIH Research Projects · FY 2025 · 2025-06

PROJECT SUMMARY The Inaugural Cholestatic Liver Disease Summit will be held June 26-27, 2025 at Children’s Hospital Colorado and the Hyatt Regency Hotel in Denver, CO. The concept for this Cholestatic Liver Disease Summit was developed through a collaboration between three 501c3 nonprofit patient advocacy organizations in the cholestatic liver disease space – BARE Inc. (biliary atresia), PFIC Network (progressive familial intrahepatic cholestasis) and ALGSA (Alagille Syndrome Alliance). This event will be held in connection with family conferences held by these 3 organizations, allowing for free interaction and exchange of ideas between clinicians, scientists, patients and families. The 2025 Cholestatic Liver Disease Summit will address an unmet need in the scientific and clinical communities, both academic and industrial, by focusing on the common pathophysiology, models for study, and therapeutic challenges met by academic and industry practitioners in the field. We plan to use the program of this meeting to mix speakers representing diverse areas of cholangiocyte and hepatocyte biology, in clinical, basic and translational research, to foster networking opportunities and generate a platform for open discussion regarding the scientific and therapeutic challenges unique to this field. The goal will be realized by bringing together 1) clinicians that treat patients with pediatric cholestatic liver diseases, 2) investigators that focus on mechanistic aspects of bile duct development, hepatocyte canalicular development, cholestasis and cholangiopathies, 3) industry partners interested in the rare disease space, and 4) patients, families, caretakers, and patient advocates. Our goal is to not only disseminate cutting edge information to patients, advocates, clinicians, scientists and industry representatives, but also to generate a forum in which cutting edge technologies, ideas and discoveries can be freely exchanged to stimulate new research. The Specific Aim for this interdisciplinary meeting is to bring together a diverse community of clinicians and scientists engaged in the care of patients with these rare disorders and discovery of possible avenues to new, safer and more effective treatments. The 2 keynote speakers, 12 invited speakers and 14 senior discussion leaders represent world leaders with vast knowledge and cutting-edge discoveries in their respective fields. This conference will provide education to everyone and stimulate development of young investigators with inclusion of 8 short talks and 10 flash talks selected from abstracts. Significantly, the environment and time set aside for formal and informal discussion will generate a forum in which cutting edge technologies, ideas and discoveries can be freely exchanged, stimulating new ideas and collaborations. Most importantly, this forum will provide the opportunity for academics, industry representatives, patients and their families to interact and learn from each other.

NIH Research Projects · FY 2026 · 2025-06

Understanding the functions of LTR retrotransposon activation in preimplantation development Abstract Accumulating evidence indicates that retrotransposons, especially long terminal repeats (LTRs), can act as cis- regulatory elements, such as promoters and enhancers, to regulate host gene expression. After fertilization, the mammalian zygote activates its own genome in a process called zygotic gene activation (ZGA). Hundreds of retrotransposon subfamilies—which are repetitive elements that constitute ~40% of our genomes—become activated as well. This wave of retrotransposon activation is considered critical for ZGA and early embryogenesis, with its disruption leading to embryonic arrest. Similarly, in human in vitro fertilization, ~40% of embryos arrest during ZGA, and this is associated with mis-expression of retrotransposons. Thus, understanding the functions of retrotransposon activation during ZGA has important implications for both basic biology and reproductive medicine, but how specific retrotransposon subfamilies and single copies regulate ZGA, and preimplantation development is not known. The overall goal of this proposal is to understand to what extent LTR retrotransposon activation regulates ZGA and early embryogenesis. Genetic manipulation of retrotransposons is technically challenging because each retrotransposon subfamily has hundreds to thousands of copies interspersed throughout the mouse genome. Indeed, only a single LTR-derived promoter has been characterized in mouse preimplantation embryos. To overcome this barrier to progress, our lab recently developed a CRISPR interference (CRISPRi) method in mouse embryos that can systematically evaluate cis-regulatory functions of retrotransposons at the entire subfamily level (i.e., thousands of copies simultaneously). Using this method, we determined cis-regulatory activities of ~3500 copies of the MT2 subfamily, a ZGA and totipotency marker, at the 2-cell stage (when ZGA occurs). By integrating functional perturbation and multi-omics analyses, we identified hundreds of MT2 copies that function as promoters and/or enhancers during ZGA. Building upon this work, we aim to identify MT2-derived promoters and enhancers that are important for ZGA and preimplantation development and systematically evaluate to what extent ZGA depends on LTR retrotransposon activation. These goals will be achieved through integrative approaches such as epigenetic editing and base editing in zygotes, genetically engineered mouse models, and ultra-sensitive epigenome profiling. Completion of the proposed study will identify retrotransposon subfamilies and single copies that are important for early embryogenesis and provide novel insights on ZGA regulation in preimplantation embryos. Our findings will set the stage for understanding how retrotransposons participate in gene regulation in human embryogenesis. Collectively, these studies will advance our fundamental understanding of mammalian early development, with broader implications for improving reproductive technologies.

NIH Research Projects · FY 2026 · 2025-06

ABSTRACT: An in-depth understanding of the mechanisms that regulate alveolar septation and septal wall maturation will be re- quired to develop therapies for premature infants with Bronchopulmonary Dysplasia (BPD). We and others have begun to define changes in PDGFRa myo, lipo and matrix fibroblast (FB) function during alveolarization, homeostasis, regenera- tion, and fibrosis. Our long-term goal is elucidation of molecular regulators of FB functions and how diverse FBs support the alveolar niche. Our objective herein is to identify molecular mechanisms that result in functional changes in alveolar matrix FBs that regulate extra cellular matrix (ECM) organization and capillary network formation. Integration of tran- scriptomic datasets identified MEOX2 as a transcription factor of non-contractile fetal and postnatal alveolar FBs. Our preliminary data show that conditional inactivation of Meox2 in perinatal alveolar matrix FBs resulted in alveolar simpli- fication, gain of contractile FB function, and thickened interstitial ECM, comparable to findings in BPD and animal models of hyperoxia. Meox2 inactivation in alveolar matrix FBs also resulted in impaired capillary endothelial network for- mation, demonstrating a thus far unidentified role of alveolar FBs in postnatal angiogenesis. Inactivation of Meox2 re- sulted in an increase of ductal myo FB differentiation and gene expression analyses predict aberrant matrix synthesizing function in matrix FBs that is associated with abnormal vascular development and cell migration. The central hypothesis is that Meox2 directly regulates genes for matrix and myo FB function and their subsequent capacity to support vascular capillary formation. The rationale for this research is a new understanding of the regulation of matrix FB function and how these functionally different FB stages modify ECM and the FB-endothelial crosstalk. Aim 1 will test the hypothesis that MEOX2 regulates differentiation of myo and matrix FBs from a common mesenchymal progenitor. Using a new Meox2CreERT transgenic mouse we will identify lineage relationships and activation stages of alveolar FBs during normal and hyperoxia impaired alveolarization and identify direct MEOX2 target genes that regulate FB function. Aim 2 will test the hypothesis that MEOX2 regulates ECM composition, which is important for vascular network formation. We will gen- erate in vitro cell derived ECM from control and Meox2 deficient FBs and determine ECM composition, topography, and support of angiogenesis. Aim 3 will test the hypothesis that increased CXCL14 signaling from Meox2 deficient FBs to its receptor CXCR4 on capillary endothelial cells impairs postnatal capillary development. We will use in vivo and in vitro gain and loss of function analysis to determine the role of paracrine signaling from FBs to endothelial cells that direct angiogenesis. The proposed research is conceptually innovative, because we ask questions regarding FB plasticity and lineage specificity and define molecular players of the functional switches. The proposed research is scientifically innova- tive, because little is known about their ECM organizing function or their role in supporting capillary network formation. This contribution will be significant because it addresses 1) lack of knowledge of the function of the alveolar matrix FB; 2) identifies and validates the transcriptional network around Meox2 that regulates FB function.

NIH Research Projects · FY 2026 · 2025-05

Project Summary. Central apnea in newborns results from dysregulation of neuronal breathing circuitry. In sudden infant death syndrome (SIDS), these circuits can function apparently normally at birth but fail at some point in the first year. It is imperative to better understand the molecular and genetic requirements of developing neuronal breathing circuitry to create neuronal targeted therapies for SIDS. Mice with homozygous null mutations in the homeobox transcription factor (TF) gene Gsx2 die within hours of birth after becoming cyanotic and ceasing to breathe. Our preliminary data demonstrate that Gsx2 is expressed in hindbrain neural progenitors of the “dA3” domain which give rise to dorsal respiratory group (dRG) glutamatergic neurons in the nucleus tractus solitarius (nTS) and their associated catecholaminergic neurons of the A1/C1 and A2/C2 groups. At birth, the Gsx2-null mutants show a nearly complete loss of glutamatergic nTS neurons and A1/C1, A2/C2 catecholamine neurons, consistent with their lack of viability. Furthermore, Gsx2 mutant dA3 progenitors exhibit a loss of the bHLH TF Ascl1, despite that Ascl1 mutants only phenocopy the complete loss of A1/C1, A2/C2 neurons suggesting that Gsx2 regulates other TFs for nTS neurogenesis. Functional analysis shows that Gsx2 null mice aspirate milk when fed and display fewer apneas than their control littermates, consistent with the nTS regulating beneficial apneas and gating between swallowing and breathing for airway protection. The central hypothesis for this proposal is that Gsx2 directly regulates Ascl1 to induce differentiation of early-born A1/C1, A2/C2 catecholamine neurons and glutamatergic nTS neurons, but additional direct target TF genes are required for the normal generation of glutamatergic nTS neurons. To test this hypothesis, 2 aims are proposed. In Aim 1a, Gsx2 will be knocked out during early (E9.5) and late (E11.5) dA3 nTS neurogenesis using a tamoxifen-inducible Olig3- CreERT2 mouse and conditional Gsx2Flx allele to assess the neuronal alterations and effect on swallowing and breathing. In Aim 1b, E10.5 dorsocaudal hindbrains containing Gsx2+ progenitors will be dissected, RNA-seq performed on Gsx2-nulls, and CUT&RUN performed on Gsx2Flag/Flag animals to identify direct transcriptional targets of Gsx2 in dA3 progenitors. In Aim 2a, Ascl1 will be conditionally re-expressed (with tetO-Ascl1) in the Gsx2-null background, and in Aim 2b, Ascl1 will be conditionally knocked out (with Ascl1Flx) in dA3 progenitors in both cases using Gsx2e-CIE to determine if Ascl1 is a) sufficient, and b) required for differentiation of dA3- derived A1/C1, A2/C2 neurons in breathing, airway protection, and survival. Successful completion of these aims will decipher the temporal requirements of Gsx2 in dA3-derived dRG neurogenesis as well as the gene regulatory network (GRN) controlled by Gsx2 during hindbrain neurogenesis, contributing to our understanding of central control of breathing. The training received at Cincinnati Children’s Medical Center under Drs. Kenneth Campbell (neural development) and Steven Crone (breathing physiology) will help the applicant build a novel hindbrain respiration control research program in the Campbell lab and transition into an independent investigator.

NIH Research Projects · FY 2025 · 2025-05

Project Summary/Abstract This is a request for funds to purchase Hamilton BiOS M10 Biobanking automated storage system for the Discover Together (DT) Biobank at Cincinnati Children’s Hospital Medical Center (CCHMC). The Hamilton BiOS M10 will dramatically upgrade DT Biobank’s ability to: 1. Increase space efficiency of our storage footprint needed for large scale Biobanking. 2. Dramatically improve DT Biobank staff efficiency in sample receiving and biospecimen request fulfillment by automating all biospecimen pulls from frozen storage and eliminating repetitive maintenance on a high number of stand-up freezer units. 3. Provide top level fidelity of biospecimens by avoiding temperature fluctuations associated with repeated freezer door openings and biospecimen storage box removals during the fulfillment process. 4. Augment our overall lab automation strategy to best position DT Biobank to support major programs with large sample sizes in pediatrics, rare diseases, and network studies both inside and outside CCHMC. All of the above will help DT Biobank, a shared facility at CCHMC, to support a wide variety and number of NIH funded research studies. The DT Biobank supports NIH funded research in two ways. First, DT Biobank is an institutionally subsidized resource that provides services for standardized and centralized acquisition, processing, storage, and distribution of biospecimens for many NIH investigator funded projects at CCHMC. Second, DT Biobank houses and distributes the Discover Together institutional cohort which consists of current and ongoing DT enrollment, the Better Outcomes for Children (BofC) cohort, and the Genomic Control Cohort (GCC). The DT institutional cohort contains DNA and other biospecimens from over 95,000 individuals, with associated clinical data available as a resource for research projects at CCHMC. In total, biospecimen storage for both CCHMC investigator projects and the DT institutional cohort amounts to roughly 900,000 frozen storage aliquots. DT Biobank will continue to accelerate biospecimen storage via ongoing enrollment into the DT institutional sample collection protocol as well as increasing the number of investigator research projects with the emphasis on larger research networks. Additionally, institutional incentives and policies are being created to push higher levels of sample storage and partnerships to DT Biobank. Upgrading efficiency in storage capacity and technician time management while improving the sample storage environment are necessities for planned growth of DT Biobank and our support of NIH funded research.

NIH Research Projects · FY 2026 · 2025-05

PROGRAM SUMMARY/ABSTRACT: The goal of my research program is to determine how homeodomain (HD) transcription factors (TFs) that bind highly similar DNA sequences in vitro specify different developmental outcomes. HD genes constitute one of the largest groups of TFs in humans with over 200 family members that regulate a wide variety of processes including anterior-posterior patterning, organogenesis, and cell fate specification. Highlighting their impact on health, HD genes encode the most disease-associated variants in the Human Gene Mutation Database in comparison to other TF families. Collectively, over 100 HD genes have variants associated with diseases that impact numerous organs and tissues. Unfortunately, the identification of disease variants has greatly outpaced our understanding of their functional impact as hundreds of HD missense alleles have been classified as variants of unknown significance. Thus, it is imperative to define the underlying mechanisms to elucidate rules governing human variation and disease. While biochemical and structural studies have provided a wealth of information about how the HD binds DNA, recent studies revealed many variants cause unexpected changes in DNA binding. These findings expose three critical knowledge gaps: (1) How do HD proteins with highly similar DNA binding domains increase their DNA binding specificity to accurately regulate target genes? (2) How do the hundreds of HD missense variants associated with disease alter HD function? (3) Once bound to DNA, how do HD TFs activate and repress target genes and what role does the type of binding site play in transcriptional outcomes? To address these questions, I have assembled a transdisciplinary research team with complementary expertise that includes biochemical, bioinformatics, structural, cellular, genetic, and genomic approaches. Our team has recently developed a combined computational and experimental approach to discover that subsets of HD TFs form cooperative complexes on composite DNA sites with distinct site spacing and orientation requirements. Over the next five years, our plan is to use an iterative approach of using computational approaches for composite motif discovery followed by molecular, biochemical, and structural approaches to define the mechanisms underlying each cooperative complex. Comparative studies between different HDs and disease variants on different site configurations will provide novel insights into how HD factors gain DNA binding specificity and how disease variants cause molecular defects that lead to pathogenesis. We will develop new research tools and applications of high-throughput (HT) DNA binding assays, massively parallel reporter assays, and proteomic approaches to provide a comprehensive understanding of how binding affinity and cooperativity impact gene regulatory outcomes and define the co-factors used by different HD proteins to mediate accurate outcomes. Lastly, we will develop Drosophila and human organoid models that have unique strengths in genetics and genomics, respectively, to study the DNA binding and transcriptional regulatory potential of HD factors and analogous disease-associated variants within relevant developmental cell types.

NIH Research Projects · FY 2026 · 2025-05

Molecular basis of circadian and sensory integration in neuronal clocks In living organisms, cellular and physiological processes are coordinated in time and space to ensure the proper functioning of body systems. In mammals, a central pacemaker housed in the hypothalamic suprachiasmatic nucleus (SCN) orchestrates the regulation of these time-keeping mechanisms. The molecular basis of time- keeping systems involve the expression of circadian clock genes, which generate feedback loops with daily expression patterns. Clock gene rhythms are intrinsic, i.e. they emerge even in the absence of external cues. However, failure to synchronize these endogenous rhythms with environmental cues can lead to diseases such as sleep disruption, metabolic syndrome, and mood disorders. Among environmental signals, changes in light represent the strongest time cue affecting rhythmic SCN gene expression. Recent work from our group and others has revealed that light signals reach multiple brain areas that rhythmically express clock genes. The discovery of such regions, known as brain oscillators, has transformed our understanding of how light regulates brain and organismal physiology, prompting research into the relationships between clock gene expression, brain oscillators, lighting conditions, and disease. Over the next five years, my group aims to address this knowledge gap. Specifically, we propose to investigate the mechanisms that harmonize clock gene expression in brain oscillators with rhythmic SCN signals. To this end, we will investigate how gene expression in brain oscillators is affected when clock gene rhythms are disrupted in SCN neurons, using genetically modified animals and viral strategies. Additionally, we will explore the direct impact of light signals on resetting brain oscillator rhythms in the absence of a functional central pacemaker. We anticipate that these experiments will identify prospective photo-entrainable brain oscillators, challenging the conventional view of the SCN as the exclusive light-sensing circadian clock. Furthermore, we propose to investigate the molecular mechanisms of circadian and sensory integration in clock brain cells. We hypothesize that the expression of the clock gene Period2 mechanistically integrates internal and external time cues, and such integration is crucial for organismal health. To test our hypotheses, we will generate datasets analyzing the transcriptomic landscape of the SCN and brain oscillators, including clock gene expression, and conduct functional in vivo and ex vivo analyses. Lastly, we aim to investigate how cellular mechanisms of circadian and sensory integration are disrupted when external and internal signals are desynchronized, as experienced under unnatural lighting conditions. We will investigate the sustained behavioral consequences associated with light-mediated stress and clock gene dysregulation in longitudinal studies. In summary, our proposal seeks to elucidate how internal and external signals are integrated to coordinate clock gene expression within brain centers that exert circadian control of physiology and behavior and investigate the long-lasting deficits on SCN function resulting from light-mediated stress.

NIH Research Projects · FY 2026 · 2025-05

Epilepsy is a common pediatric neurological condition affecting ~470,000 youth in the United States. Adolescents with epilepsy are at significant risk for neurobehavioral comorbidities (i.e., depressive/behavioral symptoms) and suboptimal social, academic, and quality of life outcomes. Research suggests that deficits in executive functioning (EF), defined as the skills necessary for goal-directed and complex activities, including problem-solving, initiation, monitoring, organization, planning, self-regulation and working memory, contribute to suboptimal functioning. EF deficits have been documented in up to 50% of youth with epilepsy, which is 3 times the prevalence in healthy youth. Evidence-based interventions to improve EF could play a critical role in preventing adverse outcomes and promoting optimal functioning in adolescents with epilepsy; however none exists for this vulnerable population. To fill this gap, we successfully developed and tested Epilepsy Journey (EJ), a comprehensive e-health behavioral multi-component problem-solving intervention that combines 10 self-guided learning modules with 10 telehealth sessions. The promising proof-of-concept trial (n=39) showed high feasibility, acceptability, patient satisfaction, and significant improvements in parent-reported EF behaviors, neurobehavioral functioning, and quality of life. The next logical phase of this research is to conduct a definitive randomized clinical trial to examine: 1) whether the two components of treatment (EJ modules and telehealth) are both essential, 2) if the intervention generalizes to a racially diverse sample, and 3) has a durable impact on improving parent-reported and performance-based EF behaviors. Thus, the aim of the current proposal is to conduct a multi-site Phase 3 randomized controlled clinical trial (RCT) using a 2x2 factorial design to examine the efficacy of separate (EJ modules and EJ telehealth) and combined components of EJ on EF. Participants positive for EF deficits (n=232) will be randomized to one of four arms: 1) EJ modules with telehealth sessions, 2) EJ modules alone, 3) EJ telehealth sessions alone, or 4) Usual Care (no EJ modules or telehealth sessions). Treatment participants will either independently review EJ modules focused on EF skills (~15-30 min.) and/or have weekly telehealth sessions (~30-45 min.) with a therapist for three months. The groups will learn and apply problem-solving strategies to their individual EF difficulties. Participants will complete measures at baseline, post-treatment, 3- and 12-months post-treatment to examine maintenance of effects. There is a critical need for evidence-based interventions to improve executive functioning behaviors in youth with epilepsy. If the aims of this UG3/UH3 are achieved, we will have definitive evidence for addressing EF deficits. We expect that EJ modules and EJ telehealth will demonstrate efficacy alone and in combination, which will allow patients to select the approach best suited to their specific situation. Consequently, we can improve long-term outcomes (e.g., neurobehavioral comorbidities, academic success, social relationships, and QOL) in adolescents with epilepsy, a high-risk population.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY Cellular and immunotherapies are increasingly emerging as front-line treatments in clinical oncology. However, current immune checkpoint blockade therapies improve outcome in only a subset of patients for a few cancers. While immunotherapy is typically reserved for cancer patients with a high mutational burden, neoantigens produced from post-transcriptional regulation provide an untapped reservoir of common immunogenic targets for new targeted cancer therapies. To identify and exploit alternative mRNAs for the design of shared therapies, our team of computational and cancer biologists developed an innovative workflow to define and experimentally validate diverse forms of splicing neoantigens (Science Translational Medicine 2024). Our computational workflow, called Splicing Neoantigen Finder (SNAF), employs innovative artificial intelligence approaches to accurately predict shared immunogenic splicing neoantigens that can serve as the basis for broadly used immunotherapies. In addition to MHC presented neoantigens, SNAF defines novel expressed cell surface full- length neo-peptides from long-read sequencing data. Our computational and experimental studies demonstrate the feasibility of highly shared splicing neoantigens as targets for therapy in multiple solid and hematological malignancies, find shared neoantigens that span diverse malignancies, nominate new mechanisms underlying their regulation (splicing failure) and suggest a subset of antigens specifically derive from the tumor microenvironment. To capitalize on these new findings, we propose to create a comprehensive splicing neoantigen atlas of 30 adult and pediatric malignancies that span both known and novel disease subtypes. This work will establish clear roles for splicing neoantigen burden and the tumor microenvironment across cancers, define key molecular and extrinsic regulators (e.g., viral infection), and nominate recurrent liabilities for future vaccine and immunotherapy development. These analyses are expected to reveal thousands of highly shared splicing neoantigens, derived from distinct regulatory mechanisms, that are both specific to and recurrent across malignancies. To interactively explore and interrogate this pancancer splicing neoantigen atlas, we will develop an advanced web portal called NeoXplorer. NeoXplorer will build upon our recently developed and intuitive pancancer splicing analysis web infrastructure, to facilitate comparative cancer and healthy tissue analyses of splicing neoantigens, clinical and genetic covariates, orthogonal genomics and long-read sequencing evidence, advanced visualizations of protein structure and neoantigen immunogenicity and putative splicing regulators. The proposed atlas and tools will establish a crucial knowledgebase and interfaces to define highly shared and recurrent therapeutic targets that will have long-lasting impact on the field of cancer biology.