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U.S. National Science Foundation

The EDU Core Research (ECR) program offers this ECR:Core solicitation and invites proposals for fundamental research (curiosity-driven basic research and use-inspired basic research) that contributes to the general, explanatory knowledge that underlies STEM education in one or more of the three broadly conceived Research Areas: Research onSTEM Learning and Learning Environments, Research on Broadening Participation in STEM fields, andResearch on STEM Workforce Development. Within this framework, the ECR program supports a wide range of fundamental STEM education research activities, aimed at learners of all groups and ages in formal and informal settings. Fundamental researchgenerates knowledge and understanding with the potential for broad relevance. The potential implications of ECR fundamental research for improving STEM education practice may be indirect and long-term rather than direct and immediate. Moreover, whether they include basic or use-inspired basic research, all successful ECR:Core proposals focus on the advancement or refinement of foundational knowledge for STEM education. The amount of funding and duration requested in proposals submitted to the ECR:Core solicitation should align with the maturity of the proposed work and the size and scope of the empirical effort. The solicitation has three levels of funding with a range of budget sizes, and proposals may request a duration of 3 to 5 years for any level: (1)Level I proposals may request up to $500,000; (2)Level II proposals may request up to $1,500,000; (3)Level III proposalsmay request up to $2,500,000. All proposals should justify the level of funding and duration in the project description.

U.S. National Science Foundation

The EDU Core Research (ECR) program offers this ECR:Core solicitation and invites proposals for fundamental research (curiosity-driven basic research and use-inspired basic research) that contributes to the general, explanatory knowledge that underlies STEM education in one or more of the three broadly conceived Research Areas: Research onSTEM Learning and Learning Environments, Research on Broadening Participation in STEM fields, andResearch on STEM Workforce Development. Within this framework, the ECR program supports a wide range of fundamental STEM education research activities, aimed at learners of all groups and ages in formal and informal settings. Fundamental researchgenerates knowledge and understanding with the potential for broad relevance. The potential implications of ECR fundamental research for improving STEM education practice may be indirect and long-term rather than direct and immediate. Moreover, whether they include basic or use-inspired basic research, all successful ECR:Core proposals focus on the advancement or refinement of foundational knowledge for STEM education. The amount of funding and duration requested in proposals submitted to the ECR:Core solicitation should align with the maturity of the proposed work and the size and scope of the empirical effort. The solicitation has three levels of funding with a range of budget sizes, and proposals may request a duration of 3 to 5 years for any level: (1)Level I proposals may request up to $500,000; (2)Level II proposals may request up to $1,500,000; (3)Level III proposalsmay request up to $2,500,000. All proposals should justify the level of funding and duration in the project description.

U.S. National Science Foundation

ECR s Building Capacity in STEM Education Research (ECR: BCSER) supports projects that build investigators capacity to carry out high-quality STEM education research that will enhance the nation s STEM education enterprise. In addition, ECR: BCSER seeks to broaden the pool of researchers who can advance knowledge regarding STEM learning and learning environments, broadening participation in STEM fields, and STEM workforce development. Researchers institution types are particularly encouraged to submit proposals. Specifically, ECR: BCSER supports activities that enable researchers to expand their areas of expertise and acquire the requisite knowledge and skills to conduct rigorous research in STEM education. Career development may be accomplished through investigator-initiated professional development and research projects or through institutes that enable researchers to integrate methodological strategies with theoretical and practical issues in STEM education.

U.S. Mission to Georgia

This funding opportunity supports the America First agenda by strengthening U.S. competitiveness, expanding American universities' international recruitment pipelines and building lasting ties between Georgian talent and U.S. institutions through two complementary initiatives: the EducationUSA Sports Pathways Initiative and the EducationUSA AI and Innovation Hubs. Both proposals under this funding opportunity have been approved for a combined total of $50,000, pending the availability of funds. EducationUSA AI/STEM and Innovation Hubs.The proposed "AI/STEM and Innovation Hubs" initiative will engage high school and university students, young entrepreneurs, computer programmers, startup founders, and emerging tech leaders in five Georgian cities Tbilisi, Batumi, Kutaisi, Akhaltsikhe, and Telavi through a coordinated series of AI and STEM-focused advising workshops, innovation talks, and university outreach activities led by advisers. All sessions will be hosted at local American Spaces, leveraging these trusted venues to attract new audiences, expand community engagement, and reinforce America's commitment to accessible, high-quality educational opportunities.EducationUSA Sports Pathways Initiative.The proposed "Sports Pathways Initiative" will connect Georgia's top athletic talent with U.S. higher education opportunities through specialized advising on sports scholarships and athletic programs at American schools, colleges, and universities. Led by experienced advisers, the program will conduct targeted outreach to local sports schools and athletic academies in five Georgian cities Tbilisi, Batumi, Kutaisi, Akhaltsikhe, and Telavi, focusing on sports where Georgia produces world-class talent: soccer, basketball, rugby, martial arts/MMA, judo, wrestling, and tennis.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

Project Summary Building a healthy adult brain requires the precise coordination of multiple developmental processes, including neural stem cell proliferation and differentiation, neuropil extension, synaptogenesis, and synaptic pruning. These processes are sensitive to genetic, environmental, and physiological conditions, and disruptions can have lasting consequences on brain structure, function, and behavior. Early-life immune activation, such as that triggered by infection, has been associated with impaired growth, cognitive deficits, and increased risk of metabolic syndrome later in life. However, the mechanisms linking immune activity during development to long- term neural outcomes remain poorly understood. This project investigates how systemic infection and innate immune signaling influence brain development and adult physiology. Neural stem cells depend on nutrient- responsive growth signaling pathways to support their proliferation and the production of neurons and glia. Preliminary data show that activation of a conserved innate immune pathway—whether through bacterial exposure or genetic manipulation—leads to reduced body and brain size and delays the reactivation of neural stem cells from quiescence. The central hypothesis is that early-life infection activates immune signaling that disrupts neurodevelopmental programs, leading to persistent changes in brain structure and function. This research will define how immune activation alters neural stem cell behavior, impacts brain growth, and shapes adult behavioral outcomes. The findings will provide insight into how developmental immune stressors influence long-term brain health and may inform our understanding of neurodevelopmental disorders associated with early-life inflammation.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

PROJECT SUMMARY Over 1 million women living with HIV (WLWH) give birth annually. With widespread use of combination antiretroviral therapy (cART), vertical transmission has been significantly reduced, resulting in ~16 million HIV- exposed uninfected (HEU) children as of 2023. Despite being HIV negative, these children face increased risks of poor growth, infection-related mortality, and respiratory disease. These outcomes are believed to result from maternal HIV-induced inflammation and/or cART toxicity, as many antiretrovirals cross the placenta and may disrupt fetal immune development. However, distinguishing the effects of HIV versus ART is difficult in clinical studies due to challenges of studying non-HIV infected women receiving ART. Limited access to fetal tissues further hampers mechanistic insight, creating a need for translational animal models. To address this critical knowledge gap, we propose to use a rhesus macaque model of simian immunodeficiency virus (SIV) infection to investigate how maternal HIV and long-acting ART (LA-ART) affect fetal immune development. We hypothesize that despite the absence of vertical transmission, maternal SIV and LA-ART exposure dysregulates immune ontogeny in the offspring via altered hematopoiesis. A novel LA-ART regimen of FDA-approved drugs Lenacapavir (LEN) and Cabotegravir (CAB), shown to provide effective viral suppression in preliminary macaque studies, will be given bimonthly by injection to female macaques that will then undergo time-mated breeding following viral suppression. Three experimental groups will be studied: [1] SIV-infected, LA- ART treated; [2] uninfected, LA-ART treated; and [3] uninfected, untreated controls. Offspring will be delivered naturally and monitored through six months of age. Specific Aim 1 will assess how maternal SIV/LA-ART versus LA-ART alone affects infant immune maturation and function in the periphery and in tissues using flow cytometry, single-cell RNA/ATAC-sequencing, and in vitro stimulation. We will evaluate vaccine responsiveness using Varivax™ and examine B/T cell responses and receptor repertoires. Specific Aim 2 will study the impact of maternal SIV/LA-ART versus LA-ART alone on hematopoiesis in the offspring. We hypothesize that SIV/LA-ART exposure impairs differentiation and maturation of hematopoietic stem and progenitor cells (HSPCs). Bone marrow will be analyzed via flow cytometry, differentiation assays, and single-cell RNA/ATAC-sequencing. Functional HSPC capacity will be tested via transplantation into immunodeficient mice. This study uses a clinically highly relevant primate model for HIV cure research and neonatal immunity, and advanced immunological tools to uncover how maternal HIV and LA-ART exposure alter infant immune development. Findings will guide future strategies to improve immune outcomes in HEU children.

NIDCR - National Institute of Dental and Craniofacial Research

ABSTRACT Periodontitis is a common oral inflammatory condition that is epidemiologically linked to systemic disorders such as cardiovascular disease, rheumatoid arthritis, and type-2 diabetes. The relationship between periodontitis and systemic comorbidities is bidirectional, as proinflammatory diseases can also predispose to and accelerate the progression of periodontitis. Nevertheless, the factors leading to the association between periodontitis and comorbidities remains unclear. The low-grade systemic inflammation caused by periodontitis may contribute to its connection with systemic diseases. Recent studies have shown periodontitis to rewire hematopoietic stem cell (HSC) transcriptional and epigenetic profile, which is the base for trained innate immunity. Further, our own pilot data suggest that periodontitis activates HSC, inducing cell cycle entry and loss of repopulating potential in a sex-dependent way. However, the long-term effects of recurrent periodontitis on HSC function and clonal complexity remain unknown. Importantly, HSCs have limited replicative potential, and repeated acute inflammatory episodes that drive HSC proliferation may contribute to their decline. This decline is believed to be a major factor in the development of age-related hematologic diseases resulting from dysfunctional HSCs, such as clonal hematopoiesis, myeloid leukemias, and anemia. Understanding how inflammation regulates HSC fate and influences the blood system during development, aging, chronic inflammatory diseases, and hematological malignancies is crucial for uncovering the mechanistic foundations of these processes and their potential connections. In this study, we will: 1) establish a model for chronic periodontitis that mimics its long-term effects on HSCs, and 2) define the functional consequences of chronic periodontitis on HSCs, considering sexual dimorphism and local versus systemic effects. The results from this proposal will enhance our understanding of chronic inflammation's impact on HSC function. Moreover, the research outlined here will identify periodontitis as a risk factor for the development of hematopoietic pathologies. This will enable us to expand our studies to interrogate the consequences of this pathology in other tissues, both in isolation and alongside comorbidities, highlighting the importance of oral health in preventing inflammatory diseases.

U.S. National Science Foundation

The EHR Core Research (ECR) program establishes a mechanism in the Directorate for Education and Human Resources to provide funding in foundational research areas that are broad, essential and enduring. EHR seeks proposals that will help synthesize, build and/or expand research foundations in the following core areas: STEM learning, STEM learning environments, workforce development, and broadening participation in STEM. We invite researchers to identify and conduct research on questions or issues in order to advance the improvement of STEM learning in general, or to address specific challenges of great importance. Two types of proposals are invited: Core Research Proposals (maximum 5 years, $1.5 million) that propose to study a foundational research question/issue designed to inform the transformation of STEM learning and education and Capacity Building Proposals (maximum 3 years, $300,000) intended to support groundwork necessary for advancing research within the four core areas.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary / Abstract This is a R01 application from Dr. Daniel Lingwood (PI) an Associate Professor of Medicine at the Ragon Institute of MGH, MIT and Harvard and Dr. Batista (co-PI), a Professor of Medicine at the same institution. These investigators define immunological decision making by B cells to inform vaccine design and have built humanized mouse systems that recapitulate human antibody responses. Dr. Andrew Ward (co-I) is Professor in the Department of Structural and Computational Biology at Scripps and specializes in high resolution cryoEM of antibody:antigen complexes. The application goal is universal influenza vaccine development, centered on their discovery of a human broadly neutralizing antibody (bnAb) pathway that the investigators can vaccine-elicit to protect against all influenza A viruses (IAV), the major source influenza disease and all pandemic events. Differences in N-glycosylation generally prevents antibodies from engaging the otherwise conserved hemagglutinin (HA) stem of group 1 versus group 2 IAV. To solve this issue, the investigators engineered nanoparticle immunogens that elicit cross-group IAV immunity by selectively triggering and maturing germline B cell receptors (BCRs) encoding VH1-18 QxxV class bnAbs, a rare but genetically reproducible or ‘public’ category of human pan-IAV bnAbs that accommodates N-glycan diversity on the HA stem. The investigators successfully elicit this cross-group bnAb response using a single shot within a humanized vaccine model containing the VH1-18 QxxV bnAb precursors at physiologically relevant human frequency within the naïve B cell pool. The immunogens select for key affinity enhancing mutations, including N55T in the CDRH2, a hallmark of VH1-18 QxxV bnAbs. The investigators show that N55T alone provides cross-group IAV protection by a novel antibody tilting mechanism that accommodates N-glycan diversity on the HA stem. The investigators will now test the central hypothesis that this ‘molecular switch’ endows humans with an exceptionally simple vaccine- expandable pathway for eliciting broad spectrum IAV immunity. In Aim 1, the investigators will apply their modular human vaccine model to define the number of naive VH1-18 QxxV B cells needed for pan-IAV vaccine protection; if these bnAb precursors are absent, the germline stimulating nanoparticles no longer elicit pan-IAV bnAbs, revealing a human B cell repertoire effect encoding for unprecedently broad IAV immunity. In Aim 2, the investigators will define whether their nanoparticle immunogens can co-expand multiple classes of cross-group IAV bnAbs within their human vaccine model. Critically, their engineered nanoparticle immunogens also bear germline stimulating affinity for the naïve BCRs encoding the other known classes of genetically reproducible pan-IAV bnAbs produced by humans, potentiating multiclass bnAb elicitation via pan-germline stimulation. In the Aim 3, the investigators will define how prior exposure to IAV modulates (and enhances) germline stimulation and vaccine elicitation of pan-IAV bnAbs via imprinting effects. Collectively, this proposal will exploit novel genetically hardcoded templates for eliciting cross-group IAV immunity in humans.

NHLBI - National Heart Lung and Blood Institute

Project Summary Respiratory viruses can acutely lead to high mortality from respiratory failure. However, respiratory viral infections also cause permanent morbidity long after recovery from the acute infection. Such chronic debilitation was best exemplified during the COVID-19 pandemic that left many patients with post-acute sequelae of SARS-CoV-2 (PASC; aka long COVID). One unfortunate consequence after a severe COVID-19 infection is the development of persistent pulmonary fibrosis (PASC-PF). However, this fibrotic remodeling of the lungs is not unique to infections by SARS-CoV-2 and has also been found with other viral infections such as influenza, MERS-CoV, SARS-CoV-1. Investigations into PASC-PF have revealed common features when compared to idiopathic pulmonary fibrosis (IPF), the archetype of progressive lung fibrosis. A fundamental feature in PASC-PF, IPF, and other types of lung fibrosis is the maladaptive repair of the lung epithelium that is an upstream driver of fibroproliferation. In particular, the type 2 alveolar epithelial cell (AT2) cells, which are the facultative epithelial stem cells of the alveoli, are exhausted and fail to properly repair the lungs after injury. Accordingly, damaged alveoli have a repair deficiency with a failure of AT2 differentiation into alveolar type 1 (AT1) cells that are necessary for lining the alveoli to facilitate gas exchange in the lungs. To that end, recent studies have found IFN-γ to be a key signaling node that is enriched in PASC-PF and IPF. Moreover, studies by our group and others have demonstrated IFN-γ blockade to reduce fibrosis and augment epithelial repair in a viral-induced lung fibrosis model. Accordingly, we have developed a central hypothesis that anti-viral immunity by CD8+ T cells directly (via IFN-γ) and indirectly (via MDM secretion of IL-1β) stalls alveolar regeneration thereby shifting the injury response toward maladaptive repair thereby activating fibroproliferative pathways. We will test the hypothesis in three aims: Aim 1. Evaluate viral-mediated maladaptive reprogramming of AT2 cell. Aim 2. Uncover the cellular and molecular mechanisms that mediate the abnormal immune-epithelial interactions contributing to chronic lung fibrosis following viral infection. Aim 3. Identify abnormal immune-epithelial interactions in human post-viral lung fibrosis . The successful completion of this study promises to elucidate the cellular and molecular etiology of lung fibrosis and have a broad impact on understanding fibroproliferative mechanisms in PASC-PF, IPF, and other forms of lung fibrosis.

NIGMS - National Institute of General Medical Sciences

Project Summary The broad, long-term objectives of this application are to characterize mechanisms that allow a competitive germline stem cell (GSC) and its descendants to dominate the GSC population and cause super-Mendelian inheritance. The proposal will determine how a GSC in the Drosophila testis remodels its niche and causes the selective loss of WT neighbor GSCs. To accomplish this, the proposal will utilize immunofluorescence, genetics, RNA interference, extended ex vivo live-cell imaging, transcriptomics, chromatin labelling, and innovative assays of GSC competition and allele inheritance in F1 offspring. We will capitalize upon the powerful genetics available in Drosophila, as well as the ability to unequivocally identify the niche, GSCs, differentiating germline cells, and somatic stem cells (CySCs) and their lineage in the Drosophila testes. This proposal is supported by our published results demonstrating that (1) loss of the transcription factor Chinmo in a GSC causes the ectopic secretion of the extracellular matrix (ECM) protein Perlecan (Pcan), (2) this Pcan accumulates around the endogenous niche resulting in an ectopic ECM termed the moat within the testis lumen; (3) the moat causes the selective loss of WT neighbor GSCs, which no longer have strong adhesion with niche cells; (4) chinmo-/- GSCs remain in the resculpted niche because they upregulate ECM-binding proteins. This proposal is also supported by our unpublished results showing that Chinmo protein expression is promoted by an RNA-binding protein (RBP) in GSCs and that a ZAD-ZNF protein likely acts as a Chinmo co-factor in GSCs. In the first goal, we will determine whether clonal loss of the RBP that promotes Chinmo expression imparts that GSC with a competitive advantage. We will also determine what regulates that RBP in GSCs and test whether loss of any regulators of the RBP imparts a competitive advantage to a mutant GSC. In the second goal, we will determine whether Chinmo and the ZAD-ZNF protein work together to repress Pcan by recruiting histone methyltransferases. We will also determine how niche cells promote the ectopic Pcan produced by chinmo-/- GSCs. In the third goal, we will test the role of somatic stem cells (CySCs) in GSC competition and assess whether they push out WT neighbors GSCs. We will also use live-cell imaging to determine the types of GSC division that occur in chinmo- /- GSCs. The studies in this proposal will increase the knowledge base about GSC competition and will foster new avenues of research into mechanisms and possible treatments for human paternal age effect disorders caused by competitive spermatogonial stem cells and for tumor cells which remodel their microenvironment to benefit themselves and disadvantage WT neighboring cells.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

ABSTRACT Understanding the fundamental processes that underscore hematopoiesis is necessary to generate therapeutics that can correct hematopoiesis during disease or support hematopoietic regeneration during stress. The bone marrow microenvironment, or niche, supports hematopoiesis by providing cellular and acellular cues that influence hematopoietic differentiation, hematopoietic stem cell self-renewal, and the functional integrity of the bone marrow niche itself. Inadequate or inappropriate hematopoietic differentiation can lead to disease or death by depleting hematopoietic cell populations needed for organismal survival. Additionally, commonly used therapeutics like radiation and chemotherapy cause hematopoietic cell death and damage the bone marrow niche, leaving patients in a vulnerable state of hematopoietic insufficiency. The inability to restore hematopoietic homeostasis after radiation exposure puts patients at a heightened risk for developing deadly complications, such as infection or hemorrhage. Therefore, understanding how hematopoiesis is maintained and restored is of the utmost importance. Our previous studies showed that syndecan-2 (a specific heparan sulfate proteoglycan) expressed by hematopoietic stem cells promotes long-term hematopoietic stem cell self-renewal ability by supporting quiescence. The bone marrow niche is also a rich source of proteoglycans. Our preliminary data indicate that bone marrow mesenchymal stromal cells (MSCs) also highly express syndecan-2. Genetic depletion of syndecan-2 in MSCs using transgenic mouse models caused significant hematopoietic system imbalances in the peripheral blood at steady-state and after hematologic stress. In this application, we propose to elucidate how MSC-derived syndecan-2 regulates hematopoiesis in vivo. We will use a multi-scale approach to test the function of syndecan-2 at the molecular, cellular, and systemic scales by combining transgenic knockout mouse models and in vivo injury models with high-resolution bone marrow imaging and super- resolution imaging of MSCs. We will test the role of MSC syndecan-2 in hematopoietic differentiation, growth factor organization, and signaling. Because hematopoietic demands increase during states of hematopoietic stress, we will also test the function of syndecan-2 from MSCs in hematologic and niche regeneration from radiation injury. Successful completion of these aims will define the role of MSC-derived syndecan-2 in hematopoietic homeostasis and regeneration, providing foundational knowledge needed to leverage proteoglycans to correct or boost hematopoiesis during states of imbalance or stress.

NHLBI - National Heart Lung and Blood Institute

Project Summary Smoking remains a leading cause of cardiovascular disease (CVD), responsible for millions of deaths worldwide. Despite its widespread impact, the precise mechanisms linking smoking to CVD risk, particularly in relation to genetic factors, remain incompletely understood. One such genetic variation, ALDH2*2, affects approximately 540 million individuals globally and may interact with tobacco smoke to exacerbate CVD risk. However, the addictive nature of smoking, compounded by social and cultural influences, complicates efforts to reduce smoking prevalence in this population. Consequently, understanding the role of ALDH2*2 in smoking-induced CVD is crucial for advancing precision medicine for individuals affected by this genetic variation. Our previous research, which utilized induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs) from individuals carrying the ALDH2*2 variant, revealed significant endothelial dysfunction. These cells demonstrated elevated oxidative stress and inflammation, along with reduced nitric oxide production and tube formation capacity (Guo et al., Science Translational Medicine, 2023). Our recent findings further suggest that the endothelial dysfunction associated with the ALDH2*2 variant is exacerbated by exposure to cigarette smoke in both human iPSC and transgenic mouse models. Despite these findings, the specific mechanisms by which tobacco consumption exacerbates CVD risk in individuals with the ALDH2*2 variant remain unclear, impeding the development of tailored approaches for ALDH2*2 smokers. The overarching goal of our proposal is to utilize a multidisciplinary approach that integrates stem cell biology, molecular biology, toxicology, vascular physiology, and endothelial mechanobiology to elucidate the molecular mechanisms underlying ALDH2*2- and smoking- induced endothelial dysfunction. We will pursue two specific aims. In Aim 1, we will examine the ROS-FOXO1- KLF5→IL-18/IL-1β signaling axis in modulating endothelial dysfunction in both human iPSC and mouse models carrying the ALDH2*2 variant. Additionally, we will screen small molecules targeting the ROS-FOXO1-KLF5 axis in cigarette smoke-exposed ALDH2*2 iPSC-ECs to evaluate their effects on endothelial function. In Aim 2, we will examine NUP210’s interaction with the LINC complex in mediating the shear stress response in ALDH2*2- and smoking-induced endothelial dysfunction. We will utilize RNA-seq, ATAC-seq, ChIP-seq, and single-cell RNA-seq to gain mechanistic insights into how NUP210 interacts with LINC complex and regulates the H3K27me3 modification of extracellular matrix genes in response to mechanical forces. Our proposal is supported by robust preliminary data, and the successful completion of this research will identify two novel molecular mechanisms—KLF5-mediated inflammation and NUP210-mediated shear stress response—through which smoking exacerbates CVD risk in the ALDH2*2 carriers. Additionally, the study will provide insights into potential prognostic biomarkers and therapeutic targets to mitigate CVD in smokers with the ALDH2*2 allele.

NHLBI - National Heart Lung and Blood Institute

The human body produces hundreds of billions of blood cells daily, replenished by hematopoietic stem cells (HSCs) in the bone marrow. Over time, HSC clones—populations derived from a single HSC—fluctuate in size, with some clones expanding while others dwindle. Clonal dynamics have been studied by reconstructing HSC phylogenic trees from somatic mutations they have accrued using whole-genome sequencing of single-cell- derived colonies. Our group pioneered the study of clonal dynamics in myeloproliferative neoplasms (MPNs), showing that driver mutations, such as JAK2, arise decades before diagnosis and confer a fitness advantage, enabling mutant clones to dominate the population. Strikingly, similar clonal dominance is observed in aging healthy individuals, though only ~20% of expansions can be attributed to known driver mutations. Understanding why certain HSC clones expand, especially in the absence of clear genetic causes, remains a fundamental unanswered question in hematopoiesis. Clonal expansion of HSCs may result from cell-intrinsic factors, as not all HSCs are equivalent. We would like to understand how during development a heterogenous population of HSCs is generated. Extrinsic factors, such as signals from the niche or systemic inflammation, may also drive clonal expansion. However, we lack basic knowledge of the drivers of clonal dynamics in native hematopoiesis because (1) reconstructing clonal history using single-cell phylogenies is not scalable—whole-genome sequencing of colonies is invasive, slow, and costly; and (2) mouse models, while useful for perturbing clonal dynamics, fail to recapitulate human clonal dynamics. This is because, despite the fitness advantages of certain HSC clones, the short lifespan of mice does not allow sufficient time for these clones to expand and dominate the stem cell population. To resolve clonal expansions in mice, we need scalable methods to reconstruct the phylogenetic history of all HSCs, not just a subset. We propose a comprehensive research program for developing new technologies to address these challenges and uncover the drivers of HSC clonal dynamics. First, we will create a non-invasive, rapid, and cost-effective method to reconstruct HSC clonal histories using long-read bulk sequencing of methylation patterns in blood cells, reducing the cost per sample from $100,000 to $1,000 and enabling large-scale human studies. Second, we will engineer mice to record lineage and key signaling histories of HSCs directly in their own DNA by extending lineage-recording mouse models we previously developed. Phylogenetic trees of all HSCs can then be reconstructed efficiently by sequencing specific target regions instead of entire genomes. By integrating signaling activity with lineage history, we will decorate tree branches with molecular events that drive clonal expansion. These engineered mice will enable mapping the developmental origins of HSC heterogeneity and quantifying the impact of extrinsic factors on clonal dynamics. Together, these approaches will address fundamental questions in stem cell regulation and aging, improve prognosis and treatment of hematological disorders, and provide transformative tools for studying blood.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Hirschsprung disease associated enterocolitis (HAEC) is the leading cause of death in children who lack enteric neurons in distal bowel, a birth defect called Hirschsprung disease. The etiology of HAEC is not well understood, but hypothesized disease mechanisms include altered gut microbes (“dysbiosis”), abnormal mucosal immune system and epithelial barrier defects. To date, there are no immune-targeted therapies to treat or prevent HAEC, but new treatments are needed. This proposal builds on the candidate’s preliminary data suggesting interleukin 22 (IL22) critically modulates HAEC risk and HAEC severity. The central hypothesis is that enteric nervous system (ENS) signaling induces IL22 release and facilitates IL22 epithelial responses to enhance mucosal immunity and strengthen epithelial barrier functions that prevent enterocolitis. The Piebald lethal (sl/sl) Hirschsprung disease mouse model of HAEC will be used, as survival of sl/sl mice is dramatically (> 3-fold) altered by diet (Tjaden et al, in BioRxiv and submitted) and IL22 mRNA is much higher in sl/sl fed a Protective diet that extends median survival (“late onset HAEC”). Aim 1 will define the cellular source(s) of IL22 from bowel regions of sl/sl model mice that develop early or late onset HAEC. In parallel, this aim tests the hypothesis that IL22 prevents HAEC, by using genetic and pharmacologic strategies to alter IL22 levels. Aim 2 will precisely define the role of IL22 on epithelial integrity, stem cell renewal and differentiation in organoids derived from sl/sl mice with early or late onset HAEC and from children with Hirschsprung disease with or without HAEC. Organoids facilitate studies of epithelial stem cell biology and IL22-epithelium interactions in the absence of microbes, neurons, or diffusible small molecules such as neurotransmitters. Collectively, these studies will determine cellular sources of IL22, the effect of ENS cells on IL22 secretion, the role of IL22 in enterocolitis, and the impact of Hirschsprung disease associated aganglionosis on epithelial cell biology. These studies build on the candidate’s training as a pediatric gastroenterologist, who has clinical exposure to the diagnosis and treatment of children with Hirschsprung disease and HAEC, as well as her basic science training in enteric nervous system biology. As the work proceeds, she will become an expert in mucosal immunology and epithelial biology with a focus on neuro-immune and neuro-epithelial interactions. The mentors, Dr. Robert Heuckeroth, and Dr. Kathryn Hamilton are experts in ENS biology and epithelial biology respectively. Both mentors have a strong commitment to mentorship and NIH funding track records. Experiments will be conducted at the Children’s Hospital of Philadelphia and Perelman School of Medicine at the University of Pennsylvania, a collegial, collaborative and state-of-the art institution. The professional development and training plan will position the candidate as a successful pediatrician-scientist, who is focused on the prevention and treatment of Hirschsprung associated enterocolitis. These studies should determine if IL22-based therapies would likely be successful in HAEC, and if a human clinical trial is appropriate.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Vascular calcification serves as the primary risk factor for predicting cardiovascular events. It involves arterial calcification, an actively regulated process mediated by cells, resembling physiological biomineralization but with impaired resorption. The vessel wall contains “calcifying vascular progenitor cells” that react to pro- calcific signals such as inflammation or infection. They then differentiate into osteoblast-like cells, which produce minerals and matrix in the vessel wall. The bulk of progenitor cells within the vasculature reside in the microanatomic progenitor niche, the tunica adventitia. Under normal conditions, these progenitors regulate vascular homeostasis and remodeling. The identity, location, function, and regulatory mechanism of adventitial progenitor cells in vascular calcification remain poorly studied. Further, uncovering the niche- specific role of adventitial progenitors in vessel calcification will guide the development of targeted therapeutic strategies. Recently, our integrated transcriptomic study on normal human blood vessel adventitia discovered a cell surface marker to typify adventitial progenitor cells. This previously undescribed marker in adventitial cell biology is Endothelial Protein C Receptor (CD201). CD201-expressing progenitor cells are spatially localized in the outer layer of the adventitia, and their expression level dictates the osteogenic potential of these cells. These recent observations raised questions regarding their role in the progression of vascular calcification and are comprehensively investigated in the present K99/R00 proposal. To achieve this, integrated spatial transcriptomics and single-cell RNA sequencing-based transcriptomic mapping of the human calcified vessel with implications of CD201+CD34+ adventitial cells in calcification will be initially performed. Additionally, reporter mice with nephrectomy-induced calcification will be also examined (Aim 1, K99 Phase). Secondly, a detailed in vitro functional characterization of FACS purified CD201High/Low cells from human calcified and healthy vessels will be performed, along with CD201 cell ablation in mouse calcification model to determine the functional role of the cell in calcification (Aim 2, K99 Phase). Lastly, the underlying signaling mechanism regulating CD201-expressing cells to be pro-calcific is investigated by CRISPR/Cas9 gene knockdowns, genetic mice calcification models, and integrated transcriptomics (Aim 3, R00 phase). Completing the proposal will greatly improve the knowledge of adventitial progenitor cell-mediated vessel calcification. The proposed research and training plan aligns with my long-term research objective. It will also significantly contribute to my scientific and career goals of establishing an independent research career in blood vessel resident stem cells and their role in vascular pathology.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Hematopoietic stem cells (HSCs) maintain blood production throughout an organism’s lifespan. Thus, HSCs must balance differentiation with self-renewal to protect stemness. To this end, HSCs are largely quiescent. Quiescence protects HSCs from genotoxic insults and functional exhaustion. Understanding the molecular mechanisms controlling HSC quiescence will yield valuable insights into mechanisms underlying hematopoietic disorders. While post-transcriptional regulation is increasingly recognized as important for hematopoietic cell fate specification, its role in HSC quiescence remains poorly understood. Preliminary data indicate that the post- transcriptional regulator DDX6 is important to maintain HSC quiescence. DDX6, an RNA helicase, orchestrates translational suppression and mRNA sequestration in cytoplasmic condensates known as P-bodies. Notably, Ddx6 knockout mice have normal mature blood cell populations but exhibit loss of HSC quiescence. Accordingly, Ddx6−⁄− HSCs exhibit increased proliferation and mitochondrial numbers, which results in diminished fitness during serial, competitive transplants. Mechanistically, initial analysis of DDX6-targeted transcripts in P-bodies revealed an enrichment for untranslated mRNAs encoding crucial regulators involved in exiting quiescence. Together, these data lead to our central hypothesis that Ddx6-mediated RNA processing is pivotal in protecting HSC quiescence and function. Aim 1 will test the hypothesis that Ddx6 is required for in situ stress hematopoiesis by challenging Ddx6−⁄− HSCs in vivo using regenerative and infectious stressors. Aim 2 will elucidate the mRNAs translationally suppressed by Ddx6 in HSCs and characterize the HSC translatome in situ both with and without Ddx6 deletion. Additionally, we will investigate the functional role of Ddx6 targeted transcripts in vivo, specifically Myc. The overall goal of this project is to elucidate a new mechanism controlling HSC function at the molecular and cellular levels and to advance strategies for treating hematologic diseases. This fellowship application is sponsored by Bruno Di Stefano, Ph.D., an expert in post-transcriptional gene regulation in stem cells, and Katherine King, M.D., Ph.D., a physician-scientist and expert in hematopoietic stem cells, who will provide close guidance throughout the fellowship period. The training plan includes strategies to 1) Learn from accomplished scientists and physician-scientists that will advise the applicant through her training goals; 2) Undergo rigorous scientific training in hematopoiesis and gene regulation; 3) Experience opportunities to improve scientific communication skills and expand professional networks; 4) Advance the applicant’s clinical training, especially in hematology. The clinical and scientific training environment at Baylor College of Medicine is within the Texas Medical Center, the largest medical research complex in the world. This environment is ideal to foster the applicant’s scientific and clinical growth toward a career as a physician-scientist investigating the role of post-transcriptional regulation in hemopoietic stem cell function and dysfunction.

Emerson Foundation

Supports STEM education, community building, and basic human needs in Emerson plant communities.

New York State Education Department

STEM education initiative grants for NY to expand science, technology, engineering, and mathematics programs in K-12 and postsecondary institutions.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY This proposal establishes a partnership between the California State Polytechnic University, Pomona (CPP) and the University of California, Los Angeles (UCLA) to create a pipeline for a resource limited institution (RLI) serving many first-generation college students to pursue PhD programs in neuroscience. This career development program provides these students with valuable research training and exposure to an R1 research environment, addressing a critical need for highly skilled labor force in neuroscience and STEM fields. Students will begin their training at CPP, where they will engage in foundational research skills, including techniques in adeno-associated virus (AAV) applications at Cal Poly’s Armamentarium Vector Core (ArmVC). Over the course of a year (summer + academic year), they will gain hands-on experience in AAV production, purification, and capsid characterization, building skills and confidence in a familiar and supportive setting. In the 2nd summer in the program, the students will transition to UCLA to participate in intensive research experiences, applying their training to cutting-edge neuroscience projects—ideally bringing gene tools that they have prepared at the CPP ArmVC. While at UCLA, they will collaboratively use AAVs and other tools to address questions critical to PIs at UCLA, thereby benefiting UCLA faculty and projects. Upon returning to Cal Poly Pomona, students will continue their UCLA-related projects in a supportive role, ensuring continuity in their research and strengthening ties between the institutions. This may include additional AAV production, characterization of tissue samples from ongoing projects, or design and validation of novel AAVs to continue to delve deeper into a gene therapy related project. This program equips students with technical expertise, mentorship, and the experience of working in a high-level research environment, empowering them to pursue advanced degrees. By fostering a well-trained cohort of neuroscience researchers, this partnership creates a sustainable model for promoting excellence in STEM education and research.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

ABSTRACT Rotator cuff (RC) tears are a leading cause of musculoskeletal impairment, affecting over 250,000 individuals annually in the United States. Despite surgical repair being the standard of care, failure rates remain alarmingly high, with up to 90% of cases experiencing re-tears or poor functional recovery. This failure is largely driven by irreversible muscle degeneration, including atrophy and fatty infiltration, which impair healing and compromise surgical outcomes. Current therapies primarily target the bone-tendon interface while neglecting muscle pathology, underscoring a critical need for regenerative strategies that directly address RC muscle degeneration. This proposal aims to develop a therapeutic approach leveraging lipid nanoparticles (LNPs) encapsulating WNT7a mRNA (W7a-LNP) to promote muscle regeneration and prevent degeneration following RC injury. WNT7a has been shown to increase muscle mass, enhance muscle stem cell (MuSC) expansion, and reduce fatty infiltration, but its recombinant protein form is limited by poor bioavailability and high production costs. W7a- LNP circumvents these limitations by enabling localized, sustained WNT7a production at the injury site, transforming muscle cells into `in vivo protein factories.' Our preliminary data demonstrate that W7a-LNP reduces fibro/adipogenic progenitor (FAP) adipogenesis and fatty infiltration in both in vitro and in vivo models. We will test the hypothesis that intramuscular delivery of W7a-LNP prevents and reverses RC muscle degeneration through three specific aims. Aim 1 will engineer and validate W7a-LNP as a targeted muscle regeneration platform by optimizing delivery, evaluating WNT7a expression kinetics, and assessing its effects on MuSC expansion, myofiber hypertrophy, and FAP adipogenesis in vitro and in vivo. Aim 2 will determine the efficacy of W7a-LNP in preventing RC muscle degeneration when administered at the time of injury using a clinically relevant delayed tendon repair model. Aim 3 will evaluate W7a-LNP's ability to reverse established muscle degeneration when delivered at later stages of injury, with or without mechanical loading, to assess potential synergistic effects. The impact of this research is the development of a scalable, translatable mRNA-LNP therapy that preserves and restores muscle quality in RC injuries. By addressing a critical gap in current treatment paradigms, this strategy has the potential to improve surgical outcomes, enhance functional recovery, and reduce the need for invasive salvage procedures, ultimately transforming the management of RC muscle degeneration.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Chronic idiopathic constipation is a functional gastrointestinal disorder that affects up to 1 in 5 children. As a pediatric surgeon, often the only effective treatments I can offer are intestinal resection or diversion. Thus, I strive to better understand the mechanisms that result in this cryptic disease. The K08 program has been an ideal foundation to develop the technical and scientific skills I need to make a translational impact for my patients. Through my career development award, I have been able to explore the effect of biomechanical forces on the enteric nervous system (ENS) as a driver of persistent bowel dysfunction in Hirschsprung disease. In the last few years, we have uncovered that the chronically distended, ganglionic intestine in Hirschsprung disease has altered tissue mechanical properties. We have shown that the biomechanical forces of stretch and stiffness impact enteric neuronal phenotype and function and we have tied these changes to the mechanoreceptor, Piezo1. Taken together, our preliminary findings suggest that bowel dysfunction may be driven by mechanically- induced changes in the ENS microenvironment. The concept that biomechanical forces regulate enteric neuronal behavior and intestinal function is applicable to many gastrointestinal diseases. However, it is difficult to discern the contribution of individual biomechanical forces and the response of different cell types to biomechanical force using in vivo models alone. Thus, we seek to leverage this R03 mechanism to develop new methods and new technology to study the effect of biomechanical forces on enteric neurons and glia in vitro. This proposal lays out a two-year plan to optimize a novel engineered three-dimensional (3-D) microfluidic, co-culture system to determine the impact of biomechanical forces on enteric neurons and glia. The current proposal builds on the premise of my K08, that biomechanical forces influence ENS behavior to affect intestinal function, with two aims: (1) to test the role of biomechanical forces (stretch and shear stress) on enteric neuronal morphology, neuroglial differentiation, and neuronal function; (2) to determine how neuroglial interactions shape the ENS response to biomechanical forces. This project will leverage an established organ- on-chip platform, enteric neuronal stem cell culture, in vitro modeling, and cutting-edge cellular and molecular biology techniques to achieve these goals. Completion of these aims will further my transition to independence as an investigator and lead to a new mechanistic understanding of gastrointestinal function with the potential to improve the quality of life for millions of adult and pediatric patients.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract Invasive fungal infection by Aspergillus species, most commonly by Aspergillus fumigatus, remains one of the leading causes of mortality in immunocompromised patients undergoing solid organ or HSC transplantation because antifungal agents currently used in clinics are poorly effective in treating Aspergillus infection in patients with impaired immunity and neutropenia due to drug toxicities, drug-drug interactions, and the emergence of drug-resistant strains. Neutrophils provide the first line of defense against fungal infection. They effectively kill fungi by fungicidal oxidative bursts, presentation of fungal antigens to T cells, and by increasing fungal clearance by folding their hyphae and decreasing the spreading of infection with extracellular traps (NETs). However, the efficacy of neutrophil transfusions for A. fumigatus has been limited. Therefore, enhancing the antifungal properties of neutrophils is essential for advancing adoptive neutrophil transfer for treating Aspergillus infections. In this application, we aim to develop a novel therapeutic approach to effectively target A. fumigatus infections using induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) armed with anti-Aspergillus CARs. The major goal of the R21 phase is to provide proof of principle that iNeutrophils equipped with anti-Aspergillus CARs possess superior fungicidal properties. In Aim 1, we will identify the single-chain variable fragment (scFv) that is most effective in iPSC-derived neutrophils for targeting A. fumigatus. In Aim 2, we will characterize the anti-fungal potential of anti-A. fumigatus CAR-iNeutrophils in vitro. If the R21 milestones are achieved, we will advance the development of CAR-iNeutrophil therapies into the R33 phase by enhancing their antifungal potential and demonstrating their efficacy and safety in vivo. In Aim 3, we will identify the most effective CAR configuration and genetic modifications that enhance the fungicidal properties of iNeutrophils. In Aim 4, we will assess the efficacy and toxicity of CAR-iNeutrophils in vivo using zebrafish larvae and invasive pulmonary Aspergillosis mouse models. Overall, generating CAR-iNeutrophils that directly target fungal species will enable the development of a new class of antifungal therapies. These therapies will employ the adoptive transfer of readily available neutrophils with enhanced antifungal functions to treat life-threatening drug-resistant A. fumigatus infections in patients with neutropenia or dysfunctional neutrophils.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Autoimmune diseases impact ~25 million people in the United States and are increasing in prevalence. Autoimmune diseases are driven by a failure of immunological tolerance that triggers aberrant immune responses against self-antigens that can impart debilitating morbidities and even death. There are no cures for autoimmune disease and current treatments non-specifically blunt immune responses against both self- and non-self-antigen, requiring life-long treatment compliance that leaves patients more susceptible to infection and malignancy. We propose to develop a develop and test a new strategy for targeted treatment of autoimmune diseases that harnesses the intrinsic immunoregulatory properties of extracellular vesicles (EVs). EVs are secreted by all cell types as a mechanism for promoting transfer of molecules between cells and have been implicated in the maintenance or induction of immunological tolerance through their ability to deliver diverse immunoregulatory cargo. We hypothesize that EVs derived from immunosuppressive cell sources and engineered to deliver autoantigens can be employed as a tolerogenic vaccine (i.e., inverse vaccine) that promotes antigen-specific T cell tolerance that abrogates autoimmune disease. Towards this end, we have devised strategies for exogenous loading of peptide antigens onto EV surfaces, thereby enabling coordinated delivery of antigens and immunosuppressive EV cargo to antigen presenting cells (APCs), resulting in the presentation of autoantigen in a potently tolerogenic context. While our EV-based tolerogenic vaccine platform – tolEVax – is amenable to EVs isolated any cell source and can be applied to several autoimmune diseases, we will focus on engineering of EVs derived from mesenchymal stem cells (MSC-EVs) and will test our approach in a model of multiple sclerosis. We propose to establish tolEVax as a promising strategy for promoting immune tolerance and treating autoimmunity through two Specific Aims. In Aim 1, we will load MSC- EVs with peptide antigens, evaluate effects on antigen biodistribution and uptake by APCs, and characterize effects on antigen-specific CD8+ and CD4+ T cell responses to model antigens. In Aim 2, we will evaluate the capacity of tolEVax to inhibit autoreactive T cell responses and self-antigen mediated inflammation and pathology in a model of multiple sclerosis. We expect these studies to identify MSC-EVs as potently tolerogenic antigen nanocarriers, to provide new insight into how EVs modulate adaptive immune responses, and to demonstrate the efficacy of tolEVax as a potential treatment for MS. Overall, this research will result in a platform technology that addresses the unmet need for effective antigen-specific immunotherapies for autoimmune disease by exploiting the inherent and multimodal immunosuppressive functions of EVs.