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NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Project summary/abstract In barrier tissues, developmental transcription factors are often reactivated in cases of disease or inflammation to drive expression of a suite of embryonic genes. While this reuse of pathways can prompt the increased proliferation or migration characteristic of both states, other times the reason for repeated activity is less clear. As an example, HIF1α activity during epidermal development within the hypoxic womb of most mammals does not contribute to generation of proper skin structure, but reactivation after wounding is critical for appropriate healing. One possible explanation for this bimodal activity is that the first wave of activation, during development, might regulate the second wave. Prior work has demonstrated that epithelial stem cells are capable of learning from their experiences, encoding epigenetic memories to respond to a stimulus more quickly the second time encountered. These epigenetic memories are evident at the chromatin level as maintained accessibility of stress-induced loci. If a similar phenomenon is active during development, it could serve to prime epidermal stem cells to respond to inflammatory cues later in life. This hypothesis will be tested by modulating HIF1α activity during embryonic epidermal development and carefully assessing resultant molecular changes. In utero lentiviral delivery of Cre to Hif1a fl/fl embryos will selectively prevent epidermal Hif1a induction in the post-gastrulation embryo. Preliminary work supports that HIF1α is dispensable for skin morphogenesis but drives substantial transcriptional changes in epidermal stem cells. Notably, expression of hypoxia-induced target genes and chromatin-modifying enzymes is altered in the absence of HIF1α. After further characterizing molecular changes, focusing on the chromatin landscape, next experiments will leverage inducible systems to selectively perturb the first wave of HIF1α activity, leaving postnatal activity intact. Inflammatory challenge of these animals will reveal whether embryonic HIF1α activity has lifelong effects on stem cell function, as hypothesized. Moreover, bioinformatic analysis will uncover loci throughout the adult genome which are poised in naïve homeostatic skin but are only expressed and essential in HIF1α-activated situations such as wounding. When compared with epigenetic changes occurring in Hif1a-null embryonic epidermis, I will learn if this poising occurs during embryogenesis and is reliant upon hypoxia. Hif1a has not yet been implicated in epigenetic regulation of the skin, and exploring this function could have important implications for wound healing and disease states of the skin where the protein is again stabilized. The proposed project will be conducted in the lab of Dr. Elaine Fuchs, PhD at the Rockefeller University. The Fuchs lab provides a supportive environment with ample resources to explore the connections between development and inflammation of the skin. Performing the above experiments will establish a robust foundation in cellular and molecular biology techniques with skills in data analysis, critical thinking, leadership, mentorship, and communication, providing strong support for a future independent scientific career in academia.

NIDCD - National Institute on Deafness and Other Communication Disorders

Project Summary/Abstract Objectives: Blood-Labyrinthine Barrier (BLB) dysfunction is implicated in a range of inner ear disorders (BLB- IEDs), including Meniere's disease, autoimmune inner ear disease, and sensorineural hearing loss. These conditions disrupt inner ear fluid homeostasis, leading to vertigo, hearing loss, tinnitus, and imbalance. The BLB comprises microvascular endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes (PVM/Ms), which are essential for auditory and vestibular function. This project aims to investigate the role of the BLB in inner ear homeostasis and disease pathophysiology while developing hiPSC-based models to identify therapeutic targets. Research Design: Using human induced pluripotent stem cell (hiPSC) technology, we will model BLB-specific microvascular interactions and explore novel therapeutic interventions. Our approach focuses on generating hiPSC-derived BLB pericyte spheroids by directing neural crest stem cells toward a pericyte fate using vestibular neuronal spheroid-conditioned medium (VNS-CM). Additionally, we will develop hiPSC-derived PVM/M spheroids by differentiating yolk sac macrophage-like cells into BLB-specific PVM/Ms. These models will be integrated into advanced microfluidic devices to create physiologically relevant 3D BLB spheroids, laying the groundwork for BLB assembloid development in future R01 studies. Methodology: We will characterize BLB-specific structural, molecular, and functional properties of hiPSC- derived pericytes and PVM/Ms using advanced imaging, molecular biology, and functional assays. Structural characterization will involve transmission electron microscopy (TEM) to examine ultrastructural features. Molecular profiling will be conducted through immunocytochemistry and RT-PCR to confirm BLB-specific gene and protein expression. Functional validation will include transepithelial electrical resistance (TEER) and dextran permeability assays to assess barrier integrity, as well as cytokine response assays to evaluate BLB-selective properties under inflammatory conditions. By integrating stem cell engineering and microfluidic technologies, we will construct 3D spheroids that replicate BLB molecular and functional characteristics, providing a robust platform for disease modeling, mechanistic studies, and therapeutic screening for BLB-IEDs. Clinical Relevance: By addressing a critical gap in BLB research, this project will advance our understanding of BLB dysfunction across multiple inner ear disorders. Our hiPSC-derived models will facilitate drug screening for patient-specific responses to treatments such as diuretics, histamine modulators, and corticosteroids, reducing the current trial-and-error approach. Additionally, these assembloids will enable disease modeling of BLB-IEDs, offering new insights into disease mechanisms and therapeutic development. This research aligns directly with the NIDCD's mission to support biomedical and behavioral research in hearing and balance disorders, ultimately improving public health and quality of life.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Epilepsy is a common neurological disorder that affects approximately 50 million people worldwide. Approximately 30% of patients with epilepsy have treatment-resistant (refractory) seizures, presenting a major clinical challenge and burden. The acquired and genetic forms of epilepsy represent the two major classes of epilepsy, and these arise mainly from neurological insults and genetic mutations, respectively. Temporal lobe epilepsy (TLE) is the most common form of acquired epilepsy, and mesial temporal lobe epilepsy (MTLE) is the most common subtype of TLE. Dravet syndrome (DS), the most common form of genetic epilepsy, is a catastrophic pediatric disorder which is most frequently caused by mutations in the SCN1A voltage-gated sodium channel. The mechanisms that contribute to the eventual development of seizures and associated comorbidities in MTLE and DS are still incompletely understood, and further research on the cellular and molecular changes that underlie these disorders is necessary in order to facilitate the development of improved treatments. Extracellular vesicles (EVs) are small, membranous particles that are naturally released by cells. EVs play an important role in intercellular communication and have been shown to possess anti-inflammatory and neuroprotective properties. Accordingly, the administration of EVs isolated from healthy, non-pathogenic cellular sources such as mesenchymal stem cells (MSEs) and neural stem cells have been demonstrated to reduce pathology in models of MTLE, stroke, TBI, and neurodegenerative disorders. Our preliminary data also suggests that endogenously-released EVs in the brain (i.e. brain derived EVs or BDEVs) from naïve wild-type mice have anti-inflammatory and cell protective properties. However, in certain disease states, BDEVs can become dysregulated and contribute to neuroinflammation and disease pathology. Little is known about the role of BDEVs (i.e., protective versus pathogenic) during the development of epilepsy. To date, only two studies have examined BDEVs in rodent MTLE models. While both studies identified changes in the expression of BDEV miRNAs following status epilepticus, neither study examined whether the functional properties of the BDEVs were altered. Furthermore, whether BDEVs are altered in genetic epilepsies and contribute to disease development is completely unknown. Hence, the objective of this exploratory R21 proposal is to establish whether BDEV properties are altered in mouse models of MTLE and DS. Importantly, the analysis of two models with distinct epileptogenic mechanisms will establish conserved and epilepsy subtype-specific BDEV contributions. The data generated in this study will provide new information on the role of BDEVs in the development of acquired and genetic forms of epilepsy, and may potentially identify novel targets for therapeutic intervention.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Summary Obesity, now at pandemic levels, significantly increases the risk of numerous comorbidities, including sepsis- a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection. A major contributor to the obesity epidemic is the widespread consumption of ultra-processed foods (UPFs), which promote excessive nutrient intake and are linked to chronic metabolic and inflammatory disturbances. These conditions activate nutrient-sensing pathways that connect nutrient excess with systemic inflammation, contributing to obesity-associated immune dysregulation. We hypothesize that obesity-associated metabolic stress enhances the production of pro-inflammatory small extracellular vesicles (sEVs), which in turn drive systemic inflammation and contribute to organ injury during sepsis. Small EVs are released by various cell types into the extracellular space and circulate in body fluids, where they are taken up by local or distant recipient cells. Our data from obese pediatric patients and healthy controls suggest that obesity imparts inflammatory traits to circulating sEVs. These vesicles, when internalized by immune cells such as macrophages, modulate inflammatory gene expression. Preliminary findings further indicate that the RNA cargo within sEVs plays a central role in regulating these immune responses. In this study, we will: 1. Define the upstream regulatory pathways that confer pro-inflammatory properties to liver-derived sEVs and evaluate their impact on sepsis outcomes in pre-clinical models. 2. Identify key molecular mediators in macrophages that drive sEV-induced inflammatory responses. Although sEV biology is rapidly evolving, the clinical implications of these vesicles remain largely unexplored. Leveraging our unique mouse models, human sEV samples, and human induced pluripotent stem cell (iPSC)- derived hepatocytes and macrophages, we aim to uncover how liver-derived sEVs shape systemic immune responses and drive organ dysfunction in sepsis. This work will clarify how obesity amplifies inflammation during critical illness and may identify novel biomarkers and therapeutic targets.

NHLBI - National Heart Lung and Blood Institute

Project Summary / Abstract Congenital diaphragmatic hernia (CDH) is a birth defect where the fetal diaphragm does not form properly and organs from the abdomen move into the chest, affecting lung development. As a result, one-third of U.S. infants with CDH dies, mainly due to the abnormal development of lung blood vessels (vascular remodeling) and high blood pressure after birth. Although surgery after birth can fix the defect in the diaphragm, it cannot reverse the damage already done to the lungs before birth. Experts agree that a therapy that promotes lung development and reverses vascular remodeling before birth is urgently needed, but currently no such treatment exists. Our team has established a new therapy for fetal lung regeneration using stem cell-derived nanoparticles, called extracellular vesicles (EVs). These particles contain bioactive molecules (proteins, lipids, and miRNAs) that are key for normal lung development. In experimental studies using models of CDH, we showed that prenatal EV treatment improves all aspects of lung development, including formation of lung vasculature. However, the mechanisms behind EV beneficial effects on fetal lung vessels in CDH remain undefined. The objectives of this proposal are to determine how EV therapy mediates lung vascular regeneration and which CDH babies are most likely to benefit from it. Our central hypothesis is that EVs reverse fetal lung vascular remodeling by delivering miRNAs that are critical for normal vessel development, benefiting those with the most severe disease. We will test our hypothesis with three specific aims: Aim1 will study how EVs promote the regeneration of CDH lung endothelial cells, the most critical component of blood vessels; Aim2 will employ cutting-edge technology (spatial transcriptomics and proteomics) to determine the molecular pathways that are modulated by EV treatment throughout the CDH lung; Aim3 will test the effects of prenatal EV treatment on varying degrees of CDH severity by assessing lung morphology with histology and advanced imaging (micro-CT), and lung function after birth with ventilation and echocardiography studies. This project combines the expertise of specialists in EV biology and regenerative medicine, lung development and CDH modeling, spatial transcriptomics and proteomics, fetal surgery and clinical outcomes. This team will advance the knowledge on CDH lung disease and lay the foundation for the clinical translation of a novel EV- based strategy that will improve survival and quality of life for CDH babies and their families.

ExxonMobil Foundation

Funds education initiatives and global health programs including malaria prevention, women's economic opportunity, and STEM education.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT The University of Puerto Rico at Aguadilla (UPR-Aguadilla) requests funding to acquire a Nikon AX Galvo Confocal Laser Scanning Microscope (LSCM) with a TI2-E inverted platform and a four- laser configuration (405/488/561/640 nm) to establish transformative imaging capabilities at our resource-limited institution serving 96% Pell Grant recipients. This state-of-the-art instrument addresses a critical infrastructure gap, enabling high-resolution fluorescence imaging, live-cell microscopy, and quantitative analysis essential for competitive biomedical research and undergraduate education. The LSCM will directly support four active research projects spanning parasitology (monogenean host-specificity studies), plant pathology (coffee biocontrol development), environmental chemistry (metalloprotein biomarkers), and neuroscience (astrocyte dysfunction in diabetic epilepsy) while integrating into core laboratory courses including Immunology (BIOL 4009) and Undergraduate research courses (BIOL 3108 and QUIM 4999). Our multidisciplinary faculty, in partnership with the Neuroimaging and Electrophysiology Facility (NIEF) Excellence Imaging Center, offers expertise in confocal microscopy, encompassing advanced imaging and specialized sample preparation techniques. This collaboration ensures effective implementation of the technology, sustained technical support, and high-quality training programs that will enhance research productivity and broaden educational impact. The broad, long-term objective is to transform UPR-Aguadilla from a primarily teaching institution into a research-active campus capable of producing graduate-school-ready students equipped with cutting-edge technical skills. Access to advanced confocal microscopy will stimulate new research collaborations, enhance faculty productivity, and provide 30-40 students annually with hands-on experience in modern imaging technologies currently absent from our curriculum. The instrument will strengthen our partnership with the emerging Natural History Museum of Puerto Rico for specimen digitization and support comprehensive outreach programs targeting 25-50 high school students annually through "Seeing Science Up Close" workshops. Expected outcomes include 1- 2 peer-reviewed publications within three years, establishment of 1-2 new institutional collaborations, and measurable enhancement of biomedical research capacity. This investment will significantly advance STEM education and research opportunities at UPR-Aguadilla while expanding access to cutting-edge scientific instrumentation for students pursuing biomedical careers and contributing to the development of skilled researchers in the biomedical sciences.

NCI - National Cancer Institute

Project Summary Pancreas cancer is now the third leading cause of cancer related deaths, due to late diagnosis and therapy resistance. While pancreatic ductal adenocarcinoma (PDAC) has been the main focus of the field, cells of different subtypes of disease can be mixed in PDAC tumors. Single-cell data demonstrate there are subclones within PDAC with a basal gene signature, which align with adenosquamous. The basal-squamous signature is associated with worse prognosis and resistance to gemcitabine. This resistance demonstrates the need to understand the establishment and maintenance of basal-squamous pancreas cancer so effective therapies may be developed. We completed RNA-sequencing analysis to find genes upregulated in basal-squamous patient samples compared to classical patient samples to identify candidates that may be involved in basal- squamous establishment and maintenance in a physiologically relevant model. We identified novel candidate genes, whose expression correlate with canonical basal-squamous genes, that we hypothesize may play a role in basal-squamous pancreas cancer growth and identity. The Reya lab previously defined Musashi2 (Msi2) as a functional marker for cancer stem cells in pancreas cancer and recently published a novel mouse model to study pancreas cancer development from a common precancerous pool of cells. This model induces the expression of stabilized MycT58A in Msi2+ cells (Msi2-Myc mice). Msi2-Myc mice can form multiple subtypes of pancreas cancer and reliably form basal-squamous tumors (68% of mice). We will use Msi2-Myc derived precancers, human PDAC cell lines, and PDAC patient samples to determine if candidate genes are sufficient for basal-squamous establishment. We will overexpress candidate genes in these models to determine if there is an emergence and/or acceleration of the basal-squamous state through in vitro and in vivo models. We will also determine if they are necessary to maintain the basal-squamous state and growth. We will knockdown candidate genes by shRNA in human squamous cell lines, Msi2-Myc derived tumor cells, and basal-squamous patient samples, and determine the impact on the basal-squamous state and growth in in vitro and in vivo models. We will conduct CLIP-sequencing to find direct targets and integrate with RNA-seq to elucidate the mechanism by which the basal-squamous state is maintained. Preliminary data show that knockdown of candidate genes in Msi2-Myc derived mouse cells and human cell lines significantly reduces growth in vitro and in vivo, suggesting their role in maintaining growth. By RNA-seq and qPCR analysis of human cell lines and histology and immunofluorescence of resultant Msi2-Myc tumors, we have preliminary evidence that suggest genes of interest may be necessary to maintain basal-squamous identity. Based on these data, the aims of this proposal are to test the hypotheses that candidate genes are (1) necessary for the maintenance of basal-squamous pancreas cancer growth and identity and (2) sufficient to drive the establishment of basal- squamous pancreas cancer.

FIRST Robotics

Funds robotics teams and STEM mentorship programs for K-12 students.

FirstEnergy Foundation

Supports STEM education, community improvement, and diversity and inclusion programs in FirstEnergy service territories.

Office of Naval Research

Key programmatic objectives of the DoN HBCU/MI Program are achieved through the implementation and performance of three program goals: enhancing the research and educational capabilities of HBCU/MIs in scientific and engineering disciplines critical to the defense mission of the U.S. Navy and U.S. Marine Corps, encouraging cross-institutional, collaborative efforts that explore innovative solutions to naval science and technology (S&T) challenges, and increasing the engagement of students in STEM fields important to the U.S. Navy and U.S. Marine Corps.This particular NOFO aims to enhance the research capacity and research infrastructure for HBCU/MIs. Competitive white papers and invited full proposals submitted to this NOFO must clearly and succinctly describe efforts that advance basic naval-relevant S&T, engage faculty and students in STEM discovery, and expand the research capacity of participant institutions.This announcement is only for research efforts that also promote student/faculty engagement, and expand the institution s research capacity. This announcement is not intended for projects that focus on non-research STEM activities.The technical content of any proposed effort must contribute to the S&T mission and vision of the DoN.DeadlinesWhite Paper Inquiries and Questions-Email: don_hbcufoa@navy.mil09 September 2026 (Wednesday)White Papers must be received no later than18 September 2026 (Friday) at 5:00 PM Eastern TimeApplication Inquiries and Questions04 December 2026 (Friday)

Florida Department of Education

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

U.S. National Science Foundation

Focus On Recruiting Emerging Climate and Adaptation Scientists and Transformers (FORECAST) seeks to facilitate the transition from status quo graduate career preparation to a student-centered model with a particular emphasis on building entrepreneurial and innovation capacity at emerging research institutions (ERIs). Transformers are scientists ready to tackle the challenges the nation and world are facing due to climate change. This opportunity will adopt the spirit of multiple directives for the research community; for example, the National Academies of Sciences, Engineering, and Medicine (NASEM) report on Earth System Scienceand the Advisory Committee for Environmental Research and Education report on Engaged Research. These directives call on the research enterprise to support the building of a robust scientific workforce ready to work with communities in addressing societal challenges. Through convergence research approaches to address societal challenges, the transdisciplinary researchers engaged in FORECAST will foster community resilience and the translation of research outcomes for societal benefits, as well as gain a broader understanding of the governmental context related to these issues. A new generation of scientists trained in "engaged research" will be expected to have a national impact in communities that may be disproportionately affected by climate change impacts. The program will build cohorts of innovative scholars from the full spectrum of diverse talent at emerging research institutions to include groups historically excluded in science, technology, engineering and mathematics (STEM). Participants, who are senior students in undergraduate programs and students who are in master's degree programs, will be supported through intentional professional development activities. FORECAST participants must be US citizens or permanent residents. FORECAST proposals will fall into three categories: Track 1, Track 2, and FORECAST Planning grants. Track 1 will support one Coordination Hub, to coordinate support for rising seniors from emerging research institutions (ERIs) or historically excluded and underserved groups as part of a national cohort to participate in structured professional development opportunities. Track 2 projects will support cohorts of Master's degree students at ERIs. Mentorship and capacity building should be central to the cohort approach. FORECAST Planning grant proposals will build capacity at ERI institutions and with the appropriate partners to undertake the activities necessary to establish a future FORECAST track 2 cohort.

European Commission - HORIZON

Fostering cooperation and integration between SSH and STEM research and innovation in the EU. Call: Culture, Creativity and Inclusive Society 2026. Programme: HORIZON. Keywords: Innovation methodologies, Knowledge management, Project management and coordination, Types of innovation. Open call from the EU Funding & Tenders Portal.

OD - NIH Office of the Director

Project Summary The discovery of embryonic stem cells (ESCs) and the development of genetic tools for cell type-specific manipulation, lineage tracing, and functional analysis have elevated the mouse to a foundational role in biomedical research. However, limitations inherent to mice prevent them from serving as “universal” models. Large animal “bridge” models, such as pigs that share a close similarity in size, anatomy, and physiology with humans, combined with the feasibility of genetic engineering (GE), offer a transformative alternative. These attributes make pigs uniquely suited for xenotransplantation research and for modeling chronic diseases that account for over 90% of the $4.1 trillion annual U.S. healthcare costs. Despite this potential, the utility and broad adoption of pigs have been limited by the lack of authentic ESCs that enable iterative, complex genetic modifications required for generating Cre-driver and Cre-responder lines, developing sophisticated models for disease research, and for xenotransplantation. This R24 resource proposal aims to close this critical gap through three Specific Aims: • Aim 1: Validate a site-specific serine recombinase-based porcine embryonic stem cell (pESC) platform. A landing pad compatible with Bxb1, PA01, and KP03 recombinases has been integrated into the pROSA26 safe harbor locus, enabling the scalable and seamless insertion of large transgenes. As a proof-of-concept, we will generate three pESC-derived pig models of increasing complexity: (i) a dual fluorescent/PET reporter (~5 kb) for lineage tracing, non-invasive imaging, and conditional ablation; (ii) an ~20 kb transgene designed to overcome innate immune barriers to xenotransplantation; and (iii) a human cholesteryl ester transfer protein (CETP) BAC (~50 kb) to model human lipid metabolism. • Aim 2: Generate and characterize lineage-specific, tamoxifen-inducible CreERT2 driver lines targeting alveolar cells, cardiomyocytes, endothelial cells, enterocytes, hepatocytes, and pancreatic β-cells, chosen for their relevance to vascular biology, metabolism, and xenotransplantation. Additional drivers will be developed in response to community needs. • Aim 3: Implement transparent prioritization, access, and resource sharing via the National Swine Resource and Research Center (NSRRC), supported by standardized MTAs, cost-recovery mechanisms, and community outreach. In summary, this project will deliver: (i) the first standardized pESC-based GE platform for pigs, (ii) a suite of lineage-specific, temporally inducible Cre-driver lines, and (iii) a transparent distribution framework to ensure broad access. By filling critical infrastructure gaps and ensuring rigorous validation and dissemination, this proposal aims to transform the utility of pigs as a genetically tractable model for biomedical research.

Hunger Intervention

Hunger Intervention Program (HIP) is partnering with Lake City Farmers Market to offer free, fun activities for children. These include arts and crafts, science exhibits, and other STEM activities. In addition, HIP will also offer cooking demos and nutrition tips at the farmers' market to showcase fresh, seasonal produce from the market. The activities are in conjunction with HIP's Summer Meals program, which addresses food insecurity and promotes healthy eating for children under the age of 18 who are at risk of going hungry when schools are closed for the summer. This year, HIP is serving summer meals at twelve locations, including the Lake City Farmers Market, which runs once a week on Thursdays. This funding request is specifically for activities at the Lake City Farmers Market for the month of August, September, and October (up to ten market days). This funding will not be used for the summer meals program.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY The conventional approach to structural-knowledge driven drug discovery for protein targets often couples high-res- olution structural data (such as crystallography or electron microscopy) to identify potential binding sites with high- throughput computational docking simulations to optimize molecular design. While this approach, developed over decades, has been extremely productive for protein targets, it has limited applicability to unconventional targets like RNA and disordered proteins that are highly dynamic, sample rare and transient states that may contribute to bind- ing, and lack identifiable structure features that serve as high-aƯinity binding regions. These targets therefore lay outside the current druggable landscape. Progress extending this landscape requires the development of a compre- hensive understanding of the biophysics of currently undruggable biomolecules and new methodologies for identi- fying and characterizing unconventional targets. This proposal seeks to apply novel single-molecule spectroscopic methods, non-perturbative labeling, and computational modeling to help establish RNA stem loops, small RNA hair- pins that are critical regulatory molecules, as a viable target for peptide therapeutics. Using the trans-activation re- gion (TAR) RNA stem loop of the HIV1 virus as a model system, we seek to (Specific Aim 1) characterize the equilib- rium ensemble of the TAR molecule, (Specific Aim 2) resolve the binding mechanism between TAR and the native HIV1 polymerase as well as synthetic peptide mimics, and (Specific Aim 3) quantify state and nucleotide-resolved binding free energies to overcome the ensemble averaging limitation of conventional measurements of binding af- finities. Success of these aims is predicated on the unique information content of single-molecule spectroscopic data processed with multidimensional correlation function analyses, which has been shown to drastically expand the dynamic range of the spectroscopy (10-6-101s) and to be sensitive to ultra-rare conformations (<0.01% of an en- semble). Combining this new methodology with non-perturbative labeling provides a route to address the Specific Aims outlined above. Success in this project will provide a quantitative (thermodynamics and kinetics) mapping of protein-RNA interactions that can serve as the foundation for structure-knowledge based drug discovery. Further- more, comparing the binding mechanisms of HIV1 native polymerase protein with known cyclical peptides that are believed to mimic the binding will provide a productive road map for the further development and refinement of pep- tide therapeutics. In addition to what is learned about the specific system of study, the technical methodologies and scientific approach developed over the course of this project will be completely generalizable to any RNA stem loop system. We anticipate that the experimental technique will be extended to studying the binding of RNA to any pro- tein, peptide, or small molecule binding partner.

NIAID - National Institute of Allergy and Infectious Diseases

1 PROJECT SUMMARY and ABSTRACT 2 Hematopoietic stem cell transplantation (HCT) cures hematologic malignancies, but infection-related mortality 3 remains high, with bloodstream infections (BSIs) accounting for up to one-quarter of deaths in the first year. 4 Enterobacterales translocation from the gut is the primary source of these infections. While antibiotic (ABX) 5 prophylaxis reduces BSI risk, incomplete clearance of potential pathogens in the gut allows susceptible strains 6 to persist and acquire AMR under ongoing pressure, further limiting ABX effectiveness. Gut Enterobacterales 7 often persist despite in vitro susceptibility, suggesting a survival mechanism beyond resistance that remains 8 underexplored. 9 We hypothesize that antibiotic tolerance—the ability of bacteria to survive lethal antibiotic concentrations without 10 a change in minimum inhibitory concentration—is the key driver of Enterobacterales persistence in the gut and 11 a precursor to AMR. Preliminary data show that gut-resident E. coli and K. pneumoniae persist in almost two- 12 thirds of HCT patients despite antibiotic use and that tolerance levels rise during ABX and decrease after 13 withdrawal. We find that recurrent mutations in tolerance loci such as relA, hipA, and ptsI occur during ABX 14 treatment, and that tolerant strains acquire resistance more rapidly under antibiotic pressure in vitro. 15 To test this hypothesis, we will combine culture-based and genomic approaches across two large HCT cohorts. 16 In Aim 1 we will selectively culture E. coli and K. pneumoniae from stool samples and quantify tolerance using 17 high-throughput screening (TD test) and standardized time-kill assays with multiple antibiotics to measure both 18 isolate- and population-level survival. In Aim 2 we will identify genetic determinants of tolerance by sequencing 19 paired stool metagenomes and isolates, tracking the emergence of single-nucleotide variants in known 20 tolerance genes, and performing bacterial genome-wide association studies (GWAS) to discover novel loci. 21 Candidate genes will be validated through plasmid complementation and functional assays. In Aim 3 we will 22 link tolerance to clinical outcomes by integrating stool and bloodstream isolate sequencing with longitudinal 23 antibiotic exposure data in order to determine whether tolerant strains predict BSIs and accelerate acquisition 24 of phenotypic or genotypic AMR. 25 This project will be the first to investigate the reservoir of antibiotic tolerance in the human gut microbiome of 26 immunocompromised patients. Using complementary microbiology and isolate/stool genomics, we will directly 27 link in vivo tolerance phenotypes to genetic mechanisms and clinical outcomes. Establishing how tolerance 28 enables Enterobacterales to persist in the gut, seed bloodstream infections, and accelerate resistance in HCT 29 patients likely has impact for other vulnerable groups. By identifying tolerance as a critical determinant of infection 30 and AMR risk this work will identify novel strategies in overcoming tolerance to improve pathogen clearance, limit 31 multidrug-resistant transmission, and reduce infection-related mortality in immunocompromised patients.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary The long term goal of this project is to characterize the molecular steps that allow a post-mitotic neuron to correctly integrate into a neural circuit, with both upstream and downstream targets. For this project, the primary focus is the development of a subset of the motor neurons that target the adult leg muscles in Drosophila melanogaster. Previous work established much of the ground-work on this system that will be built upon here. In particular, the specific neuroblast stem cells, their motor neuron progeny and birthdates, and all single neuron morphologies are known from previous work. A handful of key cell surface molecules of the immunoglobulin superfamily (IgSF) have been identified and characterized in detail in single motor neurons. These proteins are essential for some, but not all, motor neurons to make the correct connections. Finally, single cell RNA sequencing of motor neurons has identified additional cell surface proteins that also potentially contribute to target recognition and synapse formation. Building on these advances, we will use a powerful tool that was developed by us in collaboration with a computational biologist. This tool leverages both single cell RNA sequencing data with the known electron micrograph ‘connectome’ to predict molecules that interact across synapses. A second tool allows the visualization of cell surface proteins in vivo, in both in fixed and in live tissues. Together, these approaches will help to reveal how post-mitotic motor neurons establish their specific morphologies and connections with both upstream neurons and target muscles. Importantly, the principles and tools developed here will be broadly applicable to many problems currently being studied by both the development and neuroscience communities.

NIA - National Institute on Aging

PROJECT SUMMARY Maintaining healthy adipose tissue function is essential for metabolic homeostasis and the prevention of metabolic diseases. As potent endocrine cells, adipocytes secret various bioactive molecules and extracellular vesicles that influence the function of tissues and organs throughout the body. Besides adipocytes, multipotent stem and progenitor cells in adipose tissue are crucial for tissue maintenance and repair throughout life. With aging, adipose tissue undergoes species-conserved changes, including decreased subcutaneous adiposity, increased visceral adiposity, and a decline in the thermogenic capacity of brown and beige adipose tissue. In contrast to the detrimental effects of adipocyte hypertrophy, hyperplasia, a process known as adipogenesis, supports tissue development, repair, and metabolic health. However, adipogenesis is impaired during aging, which has been linked to adipose progenitor cell senescence, potentially contributing to the development of metabolic diseases. Recent studies indicate that extracellular vehicles (EVs), particularly adipocyte-derived EVs (Ad-EVs) play a role in intercellular communication within adipose tissue, regulating its function. Ad-EVs exhibit heterogeneity, with large and small Ad-EVs differing in protein and lipid composition, suggesting functional diversity. However, the specific subtypes of Ad-EVs secreted by adipocytes and their distinct roles in local and systemic metabolic regulation remain unexplored. Our preliminary studies indicate that Lipocalin 2 (LCN2), a novel phosphatidic acid (PA) binding protein, plays a potential role in senescence and adipogenesis of adipose stem and progenitor cells (ASPCs) through EV-mediated intercellular communication. Lcn2 deficiency impairs adipogenesis and results in hypertrophic obesity. Stromal- vascular (SV) cells from the brown and white adipose tissue of Lcn2 knockout mice exhibit increased senescence and decreased adipogenesis. Importantly, we have identified LCN2 in a distinct subpopulation of Ad-EVs that is separate from adiponectin-containing Ad-EVs. In this proposal, we aim to characterize the cargo composition and function of LCN2-containing EVs (LCN2+EVs) released from adipocytes, examining their role in ASPC senescence and adipogenesis during aging. We hypothesize that adipocyte-derived LCN2+EVs possess anti-senescence properties that maintain ASPC health and adipogenic capacity through adipocyte-to-ASPC communication within adipose tissue, and this effect is context-dependent. We propose two aims to characterize the cargo composition of LCN2+EVs released from adipocytes upon metabolic and inflammatory stress, and 2) determine the role of adipocyte-derived LCN2+EVs in ASPC senescence and adipogenesis during aging. The project outcomes are expect to provide new perspectives on the pathogenesis of aging-related metabolic disorders and pave the way for developing new therapeutic strategies targeting adipose tissue function.

NIMH - National Institute of Mental Health

PROJECT SUMMARY Somatic mosaicism, the genomic differences among the billions of cells in the human brain, may explain the incomplete penetrance and variable expressivity in highly heritable neurodevelopmental disorders. Thousands of clonal somatic mosaic variants (SMVs) in subpopulations of neurons have been discovered in brains of schizophrenia and autism patients, necessitating an urgent, unmet demand to determine if these diverse somatic mutations have a causal role in disease. Major challenges include (1) the inability of using conventional statistical methods for common variants to associate disease status with risk variant, (2) the vast space of non-coding candidates with unknown function, and (3) the unresolved relevant cell types and developmental stages linking mutations to phenotypes. Just as integrating high-throughput genomic-, CRISPR, and stem cell-based technologies resulted in significant progress in understanding germline risk variants, they represent a novel approach to uniquely address the major challenges in the field of somatic mosaicism. As a co-mentored computational and experimental biologist, I will leverage state-of-the-art functional genomic technologies and bioinformatic pipelines to systematically characterize all brain non-coding SMVs discovered to date, resolving their causal roles in neurodevelopmental disorders. From all SMVs identified in case and control brains, I will first create a functional catalog of expression-modulated SMVs in a developmental- and cell-type-specific manner by applying massively parallel reporter assays in human induced pluripotent stem cells (hiPSCs)-derived neural progenitor cells (NPCs) and post-mitotic neurons. By doing so, I will be able to interrogate whether differences in patterns of expression-modulated SMVs exist between cases and controls. Second, I will compare the somatic and germline genetic architectures across neurodevelopmental disorders, determining whether somatic mutations act via the same pathways as germline mutations, or affect genes relevant to diseases, indicating a causal role. By simultaneously uncovering the downstream transcriptomic profiles of hundreds of regulatory elements harboring SMVs with CRISPR screen, I will be able to pinpoint putative disease-causal SMVs. Finally, I will validate the phenotypic impact of putative causal SMVs in physiologically complex and relevant models including 3D brain organoids and “mosaicism-in-a-dish”, testing both cell-autonomous and non-autonomous mechanisms of SMVs. Overall, this work, representing a novel application of scalable functional genomic technologies to SMVs, provides a framework to identify SMVs with putative causal effects in neurodevelopmental diseases, advancing our understanding of a poorly understood disease mechanism. This fellowship will provide me with training encompassing computational genomics, stem cell models and broadly applicable phenotyping techniques, setting a foundation for me to launch an independent research program distinct from my mentors', querying somatic mosaicism's impact into novel cell types, contexts and diseases towards discovering novel therapeutic targets.

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

Orofacial cleft (OFC, primarily cleft lip and/or cleft palate) is a relatively common structural birth defect with environmental and genetic contributions to etiology. Genome wide association studies (GWAS) and linkage studies have identified many gene variants, most in non-coding DNA, that are associated with elevated risk for isolated OFC. However, our understanding of the pathogenic mechanisms underlying this disease remains poor because, one, only a fraction of the heritable risk lies is derived from common variants, two, we have yet to distinguish the non- coding variants that directly influence risk for OFC (i.e., causal or functional variants) from those that are merely in linkage disequilibrium with them, three, it has never been directly shown that a common variant can affect the gross phenotype of an embryo. In Aim 1 we will conduct statistical analyses to identify de novo non-coding mutations that are likely to be functional. In Aim 2 we propose to identify the OFC-associated SNPs that are functional by filtering them against enhancer marks, testing them for allele-specific effects in reporter assays in vitro, and finally by engineering them singly or in combination into induced pluriopotent stem cells, differentiating the cells in to embryonic oral epithelium, and assessing allele-specific effects on gene expression and transcription factor binding. In Aim 3, we will engineer the genome of mouse strains that are genetically predisposed to cleft lip, cleft plate, or both, to be homozygous for risk or non-risk alleles of proven functional SNPs that are conserved in mice and humans, expecting the risk allele to increase the penetrance or expressivity of the cleft phenotype. The expected outcome of the proposed experiments is identification of the mechanisms by which genetic risk variants cause a common birth defect.

NCI - National Cancer Institute

PROJECT SUMMARY Trained immunity is a process that is activated in hematopoietic stem and progenitor cells (HSPCs) in response to inflammatory stimuli such as severe infection and cancer. Evidence has now shown that following the resolution of inflammation, HSPCs maintain an altered epigenetic program over time. This process, termed “trained immunity” is a type of epigenetic memory that can result in robust immune responses to subsequent inflammatory challenges. Despite growing evidence of its importance in regulating responses to inflammation, the cellular players and molecular pathways involved in controlling the epigenetic responses that lead to trained immunity are poorly understood. In preliminary studies, we performed scRNA-seq in sorted bone marrow immune cells following challenge with β-glucan, a stimulus which induces trained immunity and robust secondary protection against tumor challenge. This analysis identified a population of bone marrow ILC2s that upregulated cytokine expression following β-glucan challenge, suggesting these cells may play a key role in trained immunity. Strikingly, depletion of ILC2s abolished the protective phenotype of β-glucan in tumor challenge, confirming that ILC2s play an important role in this system. Based on this data, we propose that ILC2s play a critical role in the establishment of trained immunity. While ILC2s were not found within the tumor, they regulated neutrophil differentiation in the tumor microenvironment, blocking acquisition of a pro-tumor phenotype, and maintaining an anti-tumor phenotype. However, how neutrophils are functionally altered by ILC2s in β-glucan training and which of these functions culminates in protection from tumor challenge is not known. Further, whether ILC2s modulate responses in monocytes and macrophages or respond to additional proinflammatory microbial ligands to initiate trained immunity has not been studied. Here we interrogate these questions by (i) defining the functional alterations dependent on ILC2s in tumor-infiltrating myeloid cells in trained immunity; (ii) determining the epigenetic and transcriptomic mechanisms in bone marrow progenitors and mature neutrophils in trained immunity; and (iii) interrogation of the gene expression pathways regulated in ILC2s by inflammatory challenges. Altogether, completion of these studies will fundamentally advance our understanding of the regulation of trained immunity. Furthermore, as trained immunity is actively investigated as an intervention in the clinic, modulation of the ILC2-neutrophil pathway could be a novel therapeutic target for the treatment of inflammatory diseases and cancer. Completion of these studies will establish the groundwork for future efforts to identify key cellular and molecular pathways involved in regulating trained immunity in vivo. Finally, while we considered alternatives to animal models for this work, trained immunity involves integrated multiorgan processes, such as activation of ILC2s, reprogramming of HSPCs in the bone marrow, and skewing of neutrophil responses in the tumor microenvironment, all of which depend on intact systemic physiology and cellular crosstalk that cannot be recapitulated in vitro, necessitating the use of animal models in our studies.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Building a nervous system requires both the specification and maturation of diverse neuron types. Most post-mitotic neurons are specified during embryonic development, but their synaptic connections and electrophysiological properties continue to mature during postnatal life. Compared to our understanding of embryonic neuronal specification, the regulatory mechanisms that orchestrate functional maturation of neurons in postnatal life remain poorly understood. The goal of this proposal is to fill this gap in our understanding of skeletal motor neurons. Skeletal motor neurons innervate muscles throughout the body to control movement throughout life. To accomplish this, adult skeletal motor neurons are found in three subtypes: alpha, gamma, and type3/beta, which have different morphology, electrophysiological properties, and circuit components. Importantly, these subtypes also show differential susceptibility to degeneration in the lethal disease Amyotrophic Lateral Sclerosis (ALS): alpha motor neurons die, gammas survive, and the fate of type3/betas is not known. Despite the fact that embryonic motor neuron development has been intensely studied for many years now, we do not understand how these functionally important and disease relevant adult subtypes are generated. In this proposal, we aim to fill this gap by utilizing novel single nuclei sequencing datasets and subtype-specific AAV tools that we have generated. By performing single nuclei RNA- and ATAC-sequencing on mouse motor neurons from embryonic, neonatal, juvenile and adult stages, we have found that adult alpha, gamma, and type3 subtype identities are established during the process of maturation. We have also identified candidate regulators that include subtype-specific, activity-independent transcription factors, and shared, activity- dependent transcription factors. In Aim 1 of this proposal, we will delineate the temporal maturation trajectory of all three subtypes by performing RNA-FISH against temporally activated genes. We will also use subtype- specific enhancer-AAV reporters to determine when subtype-specific muscle innervation patterns are established. In Aim 2, we will functionally interrogate the role of subtype-specific transcription factors in controlling mature gene expression, muscle innervation patterns, and motor behaviors. In Aim 3, we will functionally test the role of neural activity in controlling maturation of all subtypes. We will also map the binding profiles of subtype-specific and activity-dependent transcription factors to understand how they work together to generate mature and functionally distinct subtype identities. This work will lead to a ground-breaking understanding of adult motor neuron development, identify molecular and functional differences between disease relevant cell types, and inform methods to generate adult-like motor neurons from stem cells. The conceptual advances and AAV tools generated by this study will lead to improvements in treating diseases like ALS and spinal cord injury.