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NIMH - National Institute of Mental Health

Abstract Despite antiretroviral therapy (ART), HIV-associated brain injury (HABI) persists in over half of people with HIV (PWH), manifesting as chronic cognitive impairment. While HIV-1 primarily infects microglia, driving central nervous system (CNS) neuroinflammation, current preclinical models do not recapitulate the chronic, suppressed infection characteristic of the ART era. Furthermore, they do not capture complex patient genetics and multicellular, glial and neuronal, interactions in a scalable and efficient manner. To address this need for more physiologically relevant models, we propose the development of an AI-guided triculture platform comprising major CNS cell types. This platform will use induced pluripotent stem cell (iPSC)-derived microglia, astrocytes, and neurons, using both morphological profiling and other omics-based profiling to model HABI under ART suppression. AI/machine learning (ML)-driven analysis of cellular morphology, combined with multi-omic data integration, will facilitate rapid classification and prediction of microglial functional states and their impact on neuronal health. Leveraging Modulo's established triculture system, previously successful in yielding therapeutic candidates for amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) currently in Investigational New Drug (IND)-enabling studies, we will construct a scalable HABI model under ART suppression. Our objectives are to (1) develop and validate an HIV-infected, ART-suppressed triculture platform, utilizing AI/ML-driven morphological profiling to classify HABI-specific microglial states; and (2) comprehensively characterize this model through neuroinflammatory profiling, behavioral correlates, and integration with publicly available HABI patient datasets. We hypothesize that our combined computational lab-based triculture system can effectively model HABI pathophysiology under ART conditions, enabling both rapid disease state classification and identification of therapeutic targets. Through the integration of experimental and computational approaches, this platform will provide insights into HABI mechanisms and accelerate therapeutic development. We will disseminate this model to the scientific community through publication and collaboration. Connecting in vitro modeling with patient outcomes offers a powerful tool for investigating neuroimmune dysfunction in HIV and related neurological disorders. Successful implementation will yield a platform for modeling neuroHIV under ART suppression, advancing our understanding of disease mechanisms and facilitating the discovery of novel therapeutic strategies for PWH with cognitive impairment.

NIDCR - National Institute of Dental and Craniofacial Research

SUMMARY. The programmed death receptor 1 (PD-1) drives immune escape in head and neck squamous cell cancer (HNSCC), whose incidence is rising due to human papillomavirus (HPV). PD-1 inhibitors (PD-1i) are in use in many cancers including both resectable HPV-neg and recurrent/metastatic (R/M) HPV-pos and -neg HSNCC, but most patients do not respond. Limitations in studying immunotherapy include the scarcity of models providing key aspects of human adaptive immunity, such as 1) systems where the tumor and immune system are partially or fully matched, and 2) thymic education necessary for T cell recognition to occur. In recently reported work we reprogrammed peripheral blood mononuclear cells (PBMCs) and cancer-associated fibroblasts (CAFs) from melanoma and HNSCC patients into induced pluripotent stem cells (iPSC). Next, we developed a novel protocol to differentiate patient iPSCs into functional isogenic multicellular stem cell derived thymic organoids (sTOs) by combining TEPs, mesenchymal cells and hematopoietic lymphoid progenitor cells generated from the same iPSC line. sTOs can educate developing T cells, both via positive and negative selection. The key advantage of sTOs is that they can be used both as organoids to generate T cells for testing in vitro and can also be implanted in humanized mice (HM) to test immunotherapies in vivo. The sTO produces T cells that are educated isogenically; after activation and exposure to PD-1i and other immune checkpoint inhibitors (ICI) these T cells can be co-cultured with autologous cancer cells to generate organoids (iSpheres). This cancer and T cell interaction tests immune-directed therapies in a patient specific manner, and by using controls that are not autologous (both cancer and T cells) the contribution of thymic education can be accurately dissected. Then we will produce cohorts of thymectomized HM, with HLA-matched cord blood derived HSCs, that will be transplanted with sTOs and patient-derived xenografts (PDX) from the same patient. This results in functional maturation of thymic tissue in vivo, education of developing T cells, peripheral migration, and tumor infiltration in an isogenic manner. We recently published results showing a delay in tumor growth in HM bearing sTO, but not in HM with mouse thymus, which we hypothesize is due to improved T cell education in matched thymi, a proof of principle observation. We have a large, developing collection of patient-matched PBMCs, CAFs, iPSC, cancer cell lines and PDX from HNSCC patients that will be used to develop a first set of ~10 in vitro and in vivo testing tools. We will then test this approach prospectively on models generated from ~10 patients receiving PD-1i. This plan has the potential of identify rational ICI selection strategy and allow testing the value of thymic education in cancer immunology. Neoantigen (neoAg) profiling of PD-1i-susceptible vs resistant cases to identify neoAg that are immunogenic adds a mechanistic layer. This project will focus on creating in vitro and in vivo patient-derived tools with T cell education to test mechanisms of therapy response in HNSCC, and will provide innovative mechanistic insights at the immunologic and molecular level of immune response.

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

ABSTRACT Early stage pediatric faculty members, even those who have potential for success as academic investigators based upon substantial experience in basic research, will require further intensive training and mentoring in basic research to successfully embark upon an independent research career. Our proposed Child Health Research Career Development Award (CHRCDA) Program entitled “Developing Diverse Physician-Investigator Leaders for the Future of Child Health” will provide a cutting-edge research opportunities, combined with a carefully constructed mentoring and career development pathway, to enable our Scholars to emerge as leaders among the ranks of pediatric academic investigators. The basic biomedical research training community at the University of California San Diego (UCSD) has a long-established reputation of excellence, and UCSD Pediatrics now ranks in the top five in NIH research funding of all Pediatric Departments in the country. Notably, the last 5-10 years have witnessed an impressive academic expansion within of the UCSD Department of Pediatrics and the research it conducts, coupled with the formalization of its partnership with Rady Children's Hospital San Diego, the largest Children's Hospital in California. The key objectives of our CHRCDA are as follows: (1) To increase the number Pediatrician-Scientists engaged in basic research as applied to children's health; (2) to attract outstanding young pediatricians to UCSD and to facilitate their career development under the guidance of world class, established investigator-faculty mentors; and (3) to cultivate the early careers of women and minority investigators in children's health. With close input from the program pioneers in our Department, CHRCDA Scholars will participate fully in the program of UCSD National Center for Leadership in Academic Medicine (NCLAM), a longstanding and highly successful junior faculty mentoring program in UC Health Sciences that provides workshops and longitudinal mentoring in all facets required for successful advancement in academic medicine, and diversity enrichment modules including the Border Health and Doc-for-A-Day programs. All CHRCDA program faculty mentors are highly regarded scientific investigators, each leading a vibrant and cutting-edge basic or basic/translational research program of strong relevance to pediatric medicine. Research training opportunities for CHRCDA Scholars are organized into six research themes of five members each, which an integral role in the program structure, curriculum and mentorship approach: (1) Genomics, Big Data & Systems Biology; (2) Infection, Immunity & Inflammation; (3) Organ Physiology & Metabolism; (4) Neuroscience & Brain Development; (5) Human Microbiome & Child Health; and (6) Developmental & Stem Cell Biology. A guiding philosophy of this CHRCDA will be to support the greatest possible number of young physician-scientists within this Program, and an additional year of Department-funded support has been added to three years of K12 support to create a vibrant program with one new Scholar per year and four fellows total in the steady state.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

PROJECT SUMMARY Chemical vesicants such as sulfur mustard (SM) and nitrogen mustard (NM) act as alkylating agents causing severe skin injuries characterized by blistering, necrosis, and persistent pathology. Both pose significant threats to civilians and warfighters, yet no approved therapeutics exist for managing vesicant-induced skin injuries. This project addresses this critical medical gap by developing innovative multifunctional wound dressings engineered to simultaneously target cell membrane damage, chronic inflammation, and secondary infection—the primary pathological mechanisms underlying vesicant-induced skin trauma. Our therapeutic approach centers on MG53, a TRIM family protein with demonstrated efficacy in cell membrane repair and wound healing. Beyond its membrane-protective functions, MG53 enhances regenerative capacity in diabetic wounds by revitalizing hair follicle stem cell activity and exerts potent anti-inflammatory effects through NF-κB pathway modulation. Building on these mechanistic insights, we will develop transformative wound dressings that integrate a novel rapid-gelling antimicrobial hydrogel with recombinant human MG53 (rhMG53) protein. We hypothesize that this synergistic combination will dramatically accelerate wound healing and tissue regeneration following chemical vesicant exposure. The goal of this project is to engineer and optimize multifunctional wound dressings combining rapid-gelation antimicrobial hydrogel technology with rhMG53 protein for vesicant-induced cutaneous injuries. Successful completion will yield breakthrough multifunctional wound dressings with integrated tissue repair, anti-inflammatory, and antimicrobial capabilities. These shelf-stable therapeutic platforms can be strategically stockpiled and rapidly deployed as essential medical countermeasures against chemical vesicant exposure, addressing both immediate clinical needs and national security preparedness requirements.

NEI - National Eye Institute

Project Summary/Abstract Vision loss is a devastating medical problem as it can lead to reduced productivity, lower quality of life, and loss of independence. Glaucoma affects over four million Americans and 76.0 million people worldwide, making it the second leading cause of irreversible blindness globally. The disease is characterized by retinal ganglion cell degeneration with consequent loss of the axons that connect the eye to the brain and progressive damage to the optic nerve. Pharmacological and surgical interventions that lower intraocular pressure can slow or even stop retinal ganglion cell degeneration. However, many patients do not seek medical attention until the disease is advanced and others continue to experience disease progression despite treatment, ultimately resulting in the widespread loss of retinal ganglion cells and profound vision loss. Unfortunately, the human retina has minimal regenerative capacity and cannot replace lost retinal ganglion cells, making vision loss in these patients permanent. The candidate’s long-term career goals are to advance our understanding of gene regulatory networks directing retinal ganglion cell differentiation during development and to apply these insights to formulate strategies for regenerating retinal ganglion cells from dormant progenitor cells in the adult retina. The proposed career development and training plans will allow the candidate to acquire further expertise in retinal development and regeneration. By learning additional cutting-edge experimental techniques, the candidate will also enhance the scientific rigor and impact of their research program. In the first specific aim, the candidate will investigate the role of transcription factor Pou2f2 in retinal ganglion cell development using conditional gene deletion, as well as gain-of-function by in vivo electroporation of postnatal progenitors. In the second and third specific aims, the candidate will perform an in vivo screen of more than 40 candidate transcription factors to identify a combination capable of reprogramming Müller glia into retinal ganglion cells. Further studies will focus on characterizing induced retinal ganglion cell morphology, laminar position, axon extension, electrophysiology and gene expression. Adaptive optics will be used to longitudinally image the reprogramming process in vivo. Lastly, the candidate will investigate survival and circuit integration of these newly generated retinal ganglion cells in mouse models of glaucoma. Because retinal ganglion cells are widely used as a model for axonal regeneration in vertebrates, these studies have broader implications for regeneration of central nervous system neurons and pathways. The candidate will conduct the proposed research in collaboration with co-mentors Dr. Yvonne Ou and Dr. Xin Duan, and the other members of the advisory committee. Experiments will take place in Rock Hall, where the candidate has dedicated laboratory space in close proximity to all collaborators. The UCSF Department of Ophthalmology is a leading center for vision science research, providing the candidate with access to NEI P30 funded core facilities and extensive university-wide resources. The candidate will also benefit from interactions with the broader neuroscience and stem cell research communities at UCSF.

OD - NIH Office of the Director

Project Summary Accidental exposure to ionizing radiation (IR) poses a significant risk for cardiovascular morbidity, a leading cause of mortality among irradiated populations. However, the mechanisms driving long-term cardiovascular risks remain poorly understood. IR disrupts immune homeostasis, exacerbating chronic inflammation and accelerating pathological remodeling. The current multi-PI U01 proposal seeks to address this knowledge gap by developing an innovative extracorporeal system composed of human stem cell derivatives to identify biomarkers and medical countermeasures (MCMs) for radiation-induced delayed cardiac remodeling (RidCR). In Milestone 1, we will establish a 3D cardiac-immune co-culture system using iPSC-derived cardiomyocytes, endothelial cells, fibroblasts, and macrophages to simulate the immune-competent 3D microenvironment. Optimized culture conditions will be validated in 3D engineered cardiac tissues (EHTs) for physiological and inflammatory responses. In Milestone 2, we will perform multi-omics analyses on irradiated EHTs and parallel animal models to identify molecular signatures of RidCR via multi-omics and functional assessments of tissue contractility. In Milestone 3, AI/ML will integrate the generated datasets to predict candidate MCMs, which will be validated in vitro and tested in vivo using a protracted irradiation mouse model. Comprehensive evaluations will assess the efficacy of the lead MCM in mitigating RidCR.

NINDS - National Institute of Neurological Disorders and Stroke

Project summary/Abstract Variants in the ubiquitin like modifier activating enzyme 5 (UBA5) result in an ultra-rare autosomal recessive disease with neurological presentations. UBA5 patients present with infantile spasms, failure to thrive, hypotonia, developmental delay, microcephaly, intellectual deficit, loss of motor skills and seizures. Most of the patients die in childhood. Current standard of care for UBA5 deficiency is focused on managing the clinical signs with standard anti-seizure medications or surgical procedures and physical therapies, but there is no treatment. Compound heterozygous mutations in UBA5 causes impairment in a ubiquitin-like post-translational modification pathway called Ubiquitin-fold modifier 1 (UFM1). UBA5 is an E1 activating enzyme on UFM1 pathway. The role of the UBA5 and UFM1 system in the central nervous system (CNS) has not been studied. This stems from lack of a viable mammalian model for UBA5 deficiency. Our team has identified the first viable Uba5 mouse model that carries patient mutation, exhibits an overt phenotype, and recapitulates presentations of UBA5 deficiency in patients including smaller body size, motor, cognitive and gait abnormalities. Our team has postmortem tissues of UAB5 patients, their clinical course, MRI and EEG records. The first neuropathological characterization of postmortem UBA5 patient brain indicates the shared features with Uba5 mice. To determine the top adeno associated virus (AAV) vector candidate for efficacy studies in Uba5 mouse, we developed four UBA5 expressing constructs and showed their 1) efficacy in restoration of expression and function of UBA5 in UBA5 Knockout HEK293T cells and 2) durability, safety, and cell type tropism in a one-year study in wild type mouse. The top candidate, AAV9-JeT-UBA5, restored motor, cognitive and most aspect of gait abnormalities in Uba5 mouse model treated by neonatal intracerebroventricular (ICV) treatment. However, weight of treated Uba5 mice did not get normalized. We hypothesize that gradual loss of transduced cells in liver prevented long term weight gain normalization. Since the overarching goal of this project is to develop a transformational AAV gene therapy to treat our symptomatic UBA5 patient cohort at UMass Chan, we need to address the therapeutic imperfections and develop biomarkers. We will perform CNS and periphery wide gene therapy of JeT-UBA5 in pre and post symptomatic Uba5 mouse to determine the therapeutic window and feasibility of gene therapy to rescue or modify disease course. We will use 1) AAV9 capsid for combined CSF and periphery wide gene delivery in Uba5 mouse and 2) a new blood-brain barrier penetrant capsid (BI-hTFR1; interact with human Transferrin Receptor (TFRC)) for very efficient gene delivery to CNS and periphery by systemic injection. We will perform an in-depth characterization of the Uba5 mouse model and its humanized version, expressing TFRC, with clinically relevant outcomes measure (MRI and EEG) and compare them with patient findings (Aim 1). Four gene therapy approaches will be performed in Aim 2. In Aim 3 metabolomic based biomarker discovery will be performed and the best gene therapy approach to normalize transcriptomic profile of Uba5 mouse of will be determined.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that establishes lifelong latency and poses a serious threat to immunocompromised individuals and developing fetuses. A major barrier to developing effective antiviral therapies is our limited understanding of the mechanisms governing HCMV latency, due in part to challenges in modeling this process in physiologically relevant human cells. This R03 project proposes to establish adipose-derived stem cells (ADSCs) as a novel, scalable, and biologically relevant in vitro model of HCMV latency. We will define the conditions under which ADSCs support latent infection and compare their transcriptional program to established latency models using RNA sequencing. This research will advance our understanding of HCMV latency and provide a critical tool for future studies aimed at developing therapies to prevent viral reactivation in vulnerable populations.

NCI - National Cancer Institute

PROJECT SUMMARY Immunotherapies such as bispecific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR) T cells have shown significant promise in treating hematologic malignancies. However, therapeutic responses vary between patients and across the different subtypes of leukemia and lymphoma. A key challenge in optimizing these therapies is the lack of in vivo preclinical models that accurately reflect both the patient's immune and cancer cell biology. Current patient-derived xenograft (PDX) models lack functional immune systems, while humanized mouse models typically involve healthy donor immune cells paired with cancer cells from a different donor. These immunologically mismatched, or allogeneic, models fail to replicate autologous immune-cancer cell dynamics and the effect the cancer microenvironment and therapy have on immune cell function. To overcome these limitations, we aim to develop innovative autologous humanized PDX models using leukemia and immune cells derived from the same patient. We will collect paired bone marrow (BM) samples from pre-B acute lymphoblastic leukemia (B-ALL) patients at diagnosis and remission. Hematopoietic stem and progenitor cells (HSPCs) from the remission BM will be expanded and transplanted into immunodeficient mice to generate humanized mice with intact immune systems. These mice will then be engrafted with diagnostic leukemia cells, creating PDX models with autologous immune and leukemia cells. In parallel, a second cohort of models will be developed using autologous peripheral blood mononuclear cells (PBMCs) and leukemia cells from the same patient. We hypothesize that these fully patient-derived autologous models will provide deeper insights into immune-leukemia interactions and enhance preclinical testing of immunotherapies. To test this hypothesis, we will pursue three specific aims. First, we will establish autologous PBMC- and HSPC- humanized models using samples from B-ALL patients with diverse genetic and risk subtypes. We will evaluate disease progression and immune responses longitudinally in these models. Second, we will assess the efficacy of a CD3/CD19 BiTE and CD19 CAR-T cells in these autologous models. Third, we will analyze the immunophenotypic and transcriptional profiles of cells from autologous models, allogeneic models, and patient samples before and after immunotherapy treatment to validate the translational relevance of the models. These studies aim to create robust, patient-specific models that can be used to test and optimize immunotherapies, ultimately improving their clinical impact in the treatment of hematologic malignancies.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Immunotherapy has revolutionized the treatment of many tumors. However, most GBM patients have not, so far, benefited from immunotherapeutic treatment. With the goal of exploring ways to boost anti-GBM immunity, we’ve developed a B-cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism, BAFF and IFNγ stimulation. BVax migrate to key secondary lymphoid organs and are proficient at antigen cross-presentation, which promotes both the survival and functionality of CD8+ T cells. A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals. We have been successful at generating GBM patient-derived BVax that activated autologous CD8+ T cells, which shows a strong ability to kill autologous glioma cells. This demonstrates that BVax can be produced from patient’s peripheral blood. Our preliminary data obtained under the parental 5R37CA258426 proposal showed that BVax promotes the expansion of clones that differ from CD8 T cells activated by dendritic cells (DC) and the proliferation of stem-like TCF-1+ CD8 T cells. In addition, we provided solid evidence that BVax produces antibodies that react to tumor-associated antigens and inhibit tumor growth. Our central hypothesis is that the BVax have unique properties as antigen-presenting and antibody- producing cells. More specifically, BVax might present a different set of antigens to CD8 T cells. In addition, BVax monoclonal antibodies (mAbs) might have a potential therapeutic effect. This research proposal aims to deep-dive into the immune mechanisms underlying this protection and prevention of tumor growth. We will focus on two processes: antigen presentation and activation of CD8+ T-cell memory formation (Aim 1) and the characterization (sequencing and cloning) of single-BVax monoclonal Ab production (Aim 2). Overall, our study provides a novel alternative to current immunotherapeutic approaches that can be readily translated to the clinic.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) remains a lethal disease despite recent therapeutic advances, including the BCL2 inhibitor venetoclax. Most patients ultimately develop relapsed or refractory disease, underscoring the urgent need for new therapies, particularly agents that synergize with venetoclax. One promising strategy is to target the integrated stress response (ISR), a conserved pathway that modulates protein synthesis through phosphorylation of eIF2á by one of four stress-sensing kinases: GCN2, PKR, PERK, or HRI. This phosphorylation reduces global cap-dependent translation while selectively increasing translation of transcripts such as ATF4, which drive adaptive or pro-apoptotic programs depending on context. Leukemia stem cells rely on chronic ISR activity to withstand metabolic stress, suggesting that further ISR activation could tip the balance toward apoptosis. Consistent with this idea, we found that venetoclax itself activates the ISR via HRI, and its efficacy in preclinical models depends on this mechanism. We discovered novel ISR modulators using our integrated platform that combines high-throughput phenotypic screening with rapid target deconvolution. Through this approach, we identified ligands of RPS23, a 40S ribosomal subunit protein, that activate the ISR through GCN2 by a mechanism distinct from venetoclax and known ribosome binders. These ligands trigger apoptosis in leukemia cells and prolong survival in aggressive AML mouse models with minimal toxicity to normal hematopoietic cells. We hypothesize that RPS23 ligands represent a novel therapeutic strategy for AML and may act synergistically with venetoclax to overcome resistance. The discovery of this ISR-inducing target, together with a bioavailable compound showing preclinical efficacy, highlights the significance and innovation of our approach and provides a strong foundation for clinical translation. To advance this therapeutic strategy, our Specific Aims will define the mechanism by which RPS23 ligands activate the ISR and evaluate their on-target toxicities and efficacy alone and in combination with venetoclax in disease-relevant models of AML. No validated in vitro or computational model currently recapitulates the integrated immune, vascular, and metabolic interactions required to evaluate therapeutic efficacy and toxicity in vivo.

OD - NIH Office of the Director

PROJECT SUMMARY Rodent models have been foundational for biomedical discovery for decades. Yet the limitations of mice as a “universal” biomedical model are becoming increasingly apparent. Large animal models like minipigs offer a transformative opportunity to bridge this gap. Among the available minipig breeds, the naturally obesogenic Ossabaw minipig is increasingly becoming a model of choice for modeling the metabolic and chronic disease burdens that account for over 90% of U.S. healthcare costs. However, the generation of genetically engineered minipigs (GEMPs) remains inefficient and poorly scalable, limiting their utility in disease modeling and translational research. This proposal aims to overcome these limitations by establishing a precision genetic engineering (GE) and chimera generation platform using porcine induced pluripotent stem cells (piPSCs). The piPSC confer many advantages for GE efforts including the lack of senescence and maintenance over multiple generations, ease for genetic modification, potential for serial and multiplex editing, high throughput mutational screens, and contribution to soma and germline in the chimeric offspring. These attributes are eminently desirable for this animal resource. Our central hypothesis is that chimera-competent piPSC are a superior platform for programmable, high-efficiency GE and for generating functional chimeras via embryo complementation. To test this hypothesis, we propose the following two Specific Aims: Aim 1 will develop and validate a high-efficiency, site-specific genome engineering platform in Ossabaw piPSC. We will develop a high-fidelity piPSC based serine recombinases (Bxb1, PA01, KP03) platform for robust and modular GE. By targeting a universal landing pad to the pROSA26 safe harbor locus via CRISPR/Cas9, we will enable site-specific, scalable, and reproducible transgene integration, including large constructs like BACs. As a proof-of-concept, we will generate two novel models: (i) Cre-inducible dual fluorescent-PET reporter pigs for in vivo tracking and imaging, and (ii) a humanized CETP-transgenic minipig that addresses a key interspecies gap in lipoprotein metabolism and cardiovascular disease research. Aim 2 will enable somatic and germline chimerism using piPSC in lineage-deficient embryos. By modulating pluripotency state, injection parameters, and using lineage-deficient host embryos (e.g., lacking IGFR1, HHEX, PAX4, or NANOS3), we will demonstrate donor cell integration, niche occupancy, and rescue of organogenesis or gametogenesis. Donor contribution will be validated using advanced tools such as spatial transcriptomics, immunohistochemistry, and ddPCR, and functionality validated by phenotyping the founder animals. In summary, the proposal will establish and validate a high-efficiency, precision pipeline for producing GEMPs and functional chimeras. The stem cell tools and resources developed in this proposal will firmly establish Ossabaw minipigs as one of the models of choice for preclinical research.

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

PROJECT SUMMARY/ABSTRACT Development of novel contraceptive strategies is central to the mission of the Contraceptive Research Branch of the NICHD. This goal is driven by a global need for effective contraceptive methods to address: 1) the glut of unintended pregnancies (~45% of US pregnancies in 2011); 2) the high rate of elective abortion (1.15M unintended pregnancies ended in abortion in 2011 in the US); and 3) the high risk of maternal mortality (~830 women/day worldwide die due to pregnancy or childbirth complications). In a search for novel male contraceptive drug targets, we identified RBM46, which is a germ cell-specific RNA binding protein expressed by germ cells on the basement membrane of seminiferous tubules (outside the blood-testis-barrier), and is essential for spermatogenesis. Indeed, Rbm46 knockout mice are sterile and have no other phenotype, raising the distinct possibility that targeting RBM46 could lead to safe and effective male contraception by blocking spermatogenesis at the differentiating spermatogonial stage. Thus, we propose to develop drugs that target degradation of RBM46 as a means of oral, non-hormonal male contraception, which will significantly advance additional safe and reversible options for male contraception towards the clinic. Specifically, we will combine: 1) exceptional expertise in drug screening and development at UTSA and UT Health San Antonio; 2) leading expertise in male reproduction, spermatogenesis, and infertility at UTSA and ECU; 3) close proximity to one of two NIH-designated Marmoset Breeding Colonies, maintained at the Southwest National Primate Research Center; 4) growing and ongoing experience collecting and assessing marmoset sperm; 5) published experience in the use of cutting- edge single-cell genomics to assess normality of spermatogenic cell types; and 6) documented expertise with spermatogonial stem cell (SSC) transplantation. In Aim 1, we will identify small molecules that bind RBM46 and could be used to develop PROTACs. In Aim 2, we will produce initial RBM46 PROTACs and validate that they degrade the protein in vitro. In Aim 3, we will use medicinal chemistry to optimize the drug-like characteristics of top validated RBM46 PROTACs. In Aims 4 and 5, we will determine whether optimized RBM46 PROTACs induce reversible contraception in vivo using mice and marmosets, respectively. Together, these Aims are designed to advance RBM46 degradation as a novel strategy to achieve reversible, non-hormonal male contraception and provide key results to justify further preclinical investigation and eventual commercialization.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Atopic dermatitis (AD) is a chronic skin condition characterized by T cell-driven Type 2 inflammation. The MC903- induced dermatitis model has enabled dissection of pathways driving acute AD-like disease such as keratinocyte TSLP production and Th2 signaling; however, its ability to model persistent disease states remains unexplored. Using a repeated-challenge protocol with MC903, we found that adult mice undergoing a primary bout of AD-like inflammation develop persistent, tissue-specific inflammatory memories in skin that manifest as exaggerated pathologic responses during secondary MC903 challenge. This aligns with the emerging idea that AD chronicity stems from local memories of inflammation that persist in healed lesions and exacerbate future disease flares. However, AD in childhood follows a distinct course, with a unique immune composition and lower incidence of chronic disease. We thus tested our model in neonatal mice, and despite observing similar primary response kinetics to MC903-treated adults, we strikingly saw no signs of aggravated pathology during secondary MC903 challenge. This suggests that AD-like inflammatory memory fails in early life. Characterizing the cellular and molecular mediators of these divergent skin phenotypes will be the focus of our proposed work. Our data suggests that inflammatory memory in adults is driven by the emergence of Type 1 (T1) immune features in skin such as T1 tissue-resident memory T cells (Trms), mirroring recent findings in human AD. Transcriptional profiling suggests fibroblasts help to organize these networks by supporting T cell positioning and Trm development within inflamed adult skin. Given emerging data to suggest that neonatal T cells and fibroblasts exhibit distinct inflammatory behaviors from their adult counterparts, we hypothesize that inflammatory memory is impaired in neonatal skin due to age-related differences in T cell and fibroblast function. To test this, we will first use lineage tracing to define the fates and phenotypes of adult and neonatal T cells during AD-like inflammation in developing and adult skin (Aim 1). Subsequently, we will interrogate fibroblast-T cell interactions in the atopic skin of adult and neonatal mice using in vivo profiling via scRNA-seq and immunofluorescence staining as well as in vitro functional assays involving fibroblast-T cell co-cultures. Our work is conceptually innovative and clinically relevant, especially given the growing incidence of chronic AD in adults. Completion of the proposed work will clarify the basic mechanisms by which neonatal skin escapes inflammatory memory, potentially aiding in the identification of new therapeutic interventions for chronic AD.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Metabolic-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease affecting adults and up to 40% of children with obesity. A growing body of evidence supports the role of early life stressors in the etiology of MASLD. In humans and animal models, exposure to maternal Western diet (mWD) consumption and obesity during gestation and lactation increases offspring risk for steatosis and more severe metabolic- associated steatohepatitis (MASH). MASH involves the recruitment of bone marrow derived monocytes to the liver, which give rise to macrophages (Mφ) that are unable to resolve inflammation or repair damage, accelerating liver fibrosis. Previous studies show mWD skews bone marrow (BM) derived Mφ (BMDMs) toward a proinflammatory phenotype by remodeling fetal hematopoietic stem and progenitor cells (HSPCs). Importantly, HSPC programming persists long-term, as BMDMs from juvenile offspring exposed to mWD in early life are similarly primed for inflammation by an as-yet characterized mechanism that drives inflammation long term and may contribute to ongoing fibrosis in the juvenile liver. Mitochondrial dysfunction, oxidative stress, and inflammation are hallmarks of Mφ trained immunity but crosstalk between these processes due to mWD and their impact on MASLD are not well understood. Our preliminary data shows that mononuclear cells (MNCs) from BM of 3-week-old mice exposed to mWD have decreased oxidative capacity relative to MNCs from BM of control offspring from chow-fed dams. Therefore, we hypothesize that mWD alters mitochondrial function in HSPCs during critical windows of early development to promote BMDM activation and hepatic inflammation, thereby increasing susceptibility to MASH. The overall goal of this project is to understand the mechanisms for how mitochondria are maladaptive to maternal WD during gestation and/or lactation and how specific pathways contribute to the pathogenesis of inflammation through HSPC remodeling of BMDMs. This fellowship has two aims: 1) determine the impact of mWD exposure during pregnancy and lactation on HSPC and BMDM mitochondrial metabolism in mice at weaning and 2) determine the long-term effect of mWD during gestation or lactation on hepatic Mφ populations, Mφ function, and liver fibrosis in adult offspring following WD challenge. Our approach utilizes metabolomics and proteomics, fluorometry, and respirometry to assess mitochondrial physiology and single cell RNA-sequencing to identify unique populations of hepatic Mφ, capacity for liver repair, and fibrosis in livers of adult mice from the same early life mWD exposures. Completion of this work will provide me with training in mouse research, immune cell metabolism, deeper analysis of mitochondrial physiology, bioinformatics, and use of ‘Omics technologies to use in my next steps in career development. Impact: the work from this fellowship will fill a gap in our understanding of maternal diet’s impact on the pathogenesis of pediatric MASLD.

NCCIH - National Center for Complementary and Integrative Health

PROJECT SUMMARY: Human clinical trials worldwide evaluated probiotic effects in a variety of diseases, resulting in confounding results about the therapeutic efficacy. The uncertainty of probiotic benefits stem from the vastly different patterns in diets, genetics, microbiota, and lifestyle in the human population. In addition, side effects associated with live bacteria have been reported, raising concerns about the safety of probiotics. Thus, the rationale for the mechanistic research into probiotic interactions with host diet, digestive, metabolism, and the microbial system is to identify the group of patients who may receive the most benefit from the probiotic intervention. This MPI research team, consisting of a nutrient physiologist, metabolomic biochemist and a gut biologist, has taken a chemical biology and reductionist approach, and has uncovered two probiotic metabolic pathways that regulate the host gut mucosal homeostasis and tissue repair after injury. In the first pathway, the probiotic bacterium directly produces two structural isomers of nicotinamide metabolites, one of which can be uptake by host cells and used for synthesis of nicotinamide adenine dinucleotide (NAD), a critical cellular coenzyme involved in energy production, DNA repair, and gene expression. In the second pathway, the probiotic species facilitates the degradation and removal of a serum arginine metabolite that is barrier-impairing. These pathways are interlinked as both are driven by the probiotic colonization in mice and by a tryptophan-sufficient diet and both impact the gut barrier integrity. Functional evaluations across mice, human cells and fruit fly demonstrate a conserved protection of gut barrier integrity through these mechanisms. Two aims will use stable isotope tracing to define probiotic’s metabolic impact on NAD turnover and a key NAD-dependent enzyme that has been revealed as a promising target mediating the protection; will employ fly and mouse genetics to dissect regulatory nodes and the cell types mediating the probiotic’s protection; and will utilize mouse disease models, human cell and organoid cultures to elucidate molecular basis on the regulation of intestinal stem cell renewal and epithelial healing. This research is significant as the metabolites under investigation are perturbed in inflammatory bowel disease patients, whose serum or fecal profiles of these molecules can be utilized to guide probiotic intervention. The three PIs have over a decade collaborative record with complementary expertise indispensable for this multidisciplinary project that intersects nutrition, metabolite, microbiota and gut mucosal biology.

NIA - National Institute on Aging

PROJECT SUMMARY Dementias are projected to become the most burdening group of diseases, expected to affect over 150 million people worldwide by 2050, and Alzheimer’s disease (AD) is the most common among them. Currently, over 6 million Americans are living with AD. While there have been several recently-approved drugs for AD, these drugs target amyloid beta plaques – just one facet of the disease – and have not proven effective in all patients. Non-neuronal cell types in the brain such as microglia (the brain’s immune cells) and astrocytes (star- shaped helper cells) play a critical role in disease progression but have been traditionally understudied. Microglia are known to be activated by amyloid beta plaques and they were ascribed both a protective and a detrimental role. They were shown to induce a toxic state in astrocytes. The microglia’s behavior is likely influenced by patient genetics, which might explain why the majority of AD risk genes are expressed in microglia. To determine which of the 90 known AD-associated genetic variants exert their effect through microglia, we need a better understanding of the functional links between a variant and the disease. This requires large-scale studies of diseased, human cells from genetically diverse patients, as there is a wide variety of genetic variants that can influence AD risk. This project proposes to develop an automatable protocol for the creation of human induced pluripotent stem cell (iPSC)-derived microglia (iMG) and their co-culture with primary astrocytes. Standardized co-cultures of iMGs from AD patients and primary astrocytes will allow scientists to observe how these cells interact in a diseased environment, better understand known variants, and possibly identify new risk variants. Attaining sufficient statistical power for the identification of novel variants requires many cell lines, which can only be achieved with robotic automation. This study will serve as a proof of concept to demonstrate that such co-culture systems be automated and will later be scaled up to include additional cell lines. It will furthermore help to understand how certain genetic variants contribute to AD, which might inspire new therapeutic approaches.

U.S. National Science Foundation

The Discovery Research K-12 (DR K-12) program seeks to enable significant advances in preK-12 student and teacher learning of the STEM disciplines through development, study, and implementation of resources, models, and technologies for use by students, teachers, and policymakers. Projects funded under this solicitation begin with a research question or a hypothesis about how to improve preK-12 STEM learning and teaching. Projects create or adapt and study innovative resources, models, or technologies and determine how and why implementation affects STEM learning.DR K-12 invites proposals that meet a variety of educational needs, from those that address immediate and pressing challenges facing preK-12 STEM education to those that anticipate opportunities for the future. DR K-12 especially encourages proposals that challenge existing assumptions about learning and teaching within or across STEM fields, envision needs of learners in 10-15 years, and consider new and innovative ways to educate students and teachers. Project goals, designs, and working strategies should be informed by prior research and practical experience drawn from all relevant disciplines, while focusing on concepts and skills that are central to STEM education. The DR K-12 program is primarily concerned with improving education of students and teachers in formal settings. As appropriate, the program encourages projects also to draw from knowledge and practice of learning in informal settings. While many projects supported under this solicitation will focus on exploratory development and testing of innovative ideas for some specific facet of STEM education, all proposals must explain how the work can lead ultimately to successful adoption of findings or products in the K-12 enterprise on a national scale.The DR K-12 program accepts proposals for exploratory projects, full research and development projects, and synthesis projects, as well as for conferences and workshops related to the mission of the program.

U.S. National Science Foundation

The Discovery Research PreK-12 program (DRK-12) seeks to significantly enhance the learning and teaching of science, technology, engineering, mathematics and computer science (STEM) by preK-12 students and teachers, through research and development of STEM education innovations and approaches. Projects in the DRK-12 program build on fundamental research in STEM education and prior research and development efforts that provide theoretical and empirical justification for proposed projects. Projects should result in research-informed and field-tested outcomes and products that inform teaching and learning. Teachers and students who participate in DRK-12 studies are expected to enhance their understanding and use of STEM content, practices and skills. The DRK-12 program invites proposals that address immediate challenges that are facing preK-12 STEM education as well as those that anticipate radically different structures and functions of preK-12 teaching and learning. The DRK-12 program has three major research and development strands: (1) Assessment; (2) Learning; and (3) Teaching. The program recognizes the synergy among the three strands and that there is some overlap and interdependence among them. However, proposals should identify a clear focus of the proposed research efforts (i.e., assessment, learning, or teaching) consistent with the proposal s main objectives and research questions. The program supportssix types of projects: (1) Exploratory, (2) Design and Development, (3) Impact, (4) Implementation and Improvement, (5) Syntheses, and (6) Conferences. Allsix types of projects apply to each of the three DRK-12 program strands.

NHLBI - National Heart Lung and Blood Institute

Progressive telomere shortening, which occurs over humans' natural lifespan, is a primary molecular cause of the functional decline of stem cells in high-turnover tissues, including hematopoietic stem cells (HSCs). However, how telomere damage compromises HSCs’ functions is largely unknown. Here, we propose investigating the molecular mechanisms behind telomere damage–induced functional HSCs’ decline to uncover therapeutic strategies to ameliorate bone marrow (BM) failure disorders. In preliminary studies, we demonstrated that telomere damage does not activate programs of apoptosis or senescence in HSCs but instead induces their aberrant activation and differentiation towards the megakaryocytic lineage through the cell-intrinsic upregulation of Ifi20x/IFI16-mediated innate immune signaling response, which directly compromises HSCs’ self-renewal capabilities and eventually leads to their exhaustion. Given that HSCs’ exhaustion in the context of BM failure disorders predisposes to clonal selection, we evaluated whether telomere shortening–induced DNA damage in patients with germline mutations affecting telomere maintenance genes who developed telomere biology disorders (TBDs) is associated with clonal hematopoiesis (CH). We studied the architecture, trajectories, and impact of CH in a cohort of 207 TBD patients. CH was rare in asymptomatic patients but present in 46% of symptomatic patients and involved chromosome 1q aberrations (mainly chromosome 1q gain [Chr1q+]) and recurrent mutations in PPM1D, POT1, TERT promoter, and U2AF1S34. Compared with age-matched healthy controls, patients with TBDs had a significantly higher CH frequency, which increased with age. Regardless of allele burden, Chr1q+ and mutations in U2AF1S34 or TP53 increased the risk of developing myelodysplastic syndromes and acute myeloid leukemia. Further functional studies demonstrated that the U2AF1S34 mutation compensated for the aberrant activation of the TP53 and interferon pathways, which contribute to HSC exhaustion in patients with TBDs. These results suggest that the acquisition of U2AF1S34 mutations in TBDs compensates for the restricted cell fitness caused by germline mutations in telomere maintenance genes, which underscores the importance of understanding the molecular mechanisms of U2AF1S34 mutation–induced tumorigenesis. In this proposal, we will use innovative technologies, such as organoid and induced pluripotent stem cell systems and the MISTRG mouse model to 1) Dissect the IFI16-mediated signaling pathway under telomere attrition and determine the feasibility of targeting IFI16 in humans to rescue telomere-dysfunctional HSC function and 2) Dissect the mechanisms of U2AF1S34 mutation–induced tumorigenesis under telomere stress. The proposed study will expand our understanding of the contribution of telomere damage to HSCs’ functional decline and CH and provide new opportunities for developing strategies to improve the prevention and treatment of hematological disorders associated with telomere dysfunction.

NIDA - National Institute on Drug Abuse

PROJECT SUMMARY Opioid use disorder (OUD) is a devastating neuropsychiatric condition and a major public health crisis in the United States, with fentanyl, a synthetic opioid 100 times more potent than morphine, driving a sharp rise in overdose deaths. Although many individuals are exposed to opioids, only a subset develop OUD. This is especially evident in family studies, which estimate OUD heritability at 40–60%, suggesting that genetics shape individual risk. Genome-wide association studies (GWAS) have identified common variants associated with OUD. Over 90% of these GWAS variants are located in non-coding regions of the genome—regions that are often regulatory and whose activity varies by context (e.g., cell type, tissue, exposure). Postmortem studies have been invaluable in detecting dysregulated genes in the brains of OUD donors. However, these studies cannot distinguish inherited regulatory effects from changes caused by opioid exposure or the disease itself. For this reason, the genes and specific regulatory non-coding variants that contribute to OUD risk remain unknown. This project addresses this gap by identifying the genes that causally mediate OUD risk and by experimentally testing the effects of associated regulatory variants in human neurons exposed to fentanyl or vehicle. In Aim 1, I will apply a multi-tissue integrative Mendelian randomization framework (mintMR) to determine how inherited variation influences gene regulation (expression and DNA methylation) across human brain regions, allowing me to identify causal genes and the tissues in which they act. In Aim 2, I will use Massively Parallel Reporter Assays (MPRA) to functionally test thousands of fine-mapped non-coding variants associated with OUD, integrating these results with enhancer–gene interaction models to identify the putative target genes of regulatory variants. Together, these studies will identify the genes and brain regions that causally contribute to OUD risk and determine which non-coding OUD-associated variants affect gene regulation in unexposed and opioid- exposed human neurons. Through this project, I will receive rigorous training in computational biology, functional genomics, stem cell neuroscience, and addiction biology, preparing me for a career leading research on the molecular mechanisms of OUD.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Induced pluripotent stem cells (iPSCs) offer a promising platform for regenerative medicine, with their ability to self-renew indefinitely and differentiate into vascular cells. However, their clinical translation in vascular diseases is limited by major challenges: the impracticality of autologous therapy, immune rejection in allogeneic transplantation, and inefficient differentiation. Allogeneic iPSC-derived cells are rapidly recognized and eliminated by the host immune system, triggering both innate and adaptive immune responses. Additionally, current differentiation protocols yield vascular cells with low and inconsistent efficiency (~20%), limiting scalability and therapeutic viability. To overcome these challenges, this study aims to engineer hypoimmunogenic iPSC-derived vascular cells that evade immune detection while efficiently integrating into host vasculature. Simultaneously, spatial transcriptomics will be utilized to map immune- vascular interactions and uncover pathways that drive immune tolerance and vascular remodeling. To overcome differentiation inefficiencies, we have developed a transcription factor-driven strategy using ETV2 and NKX3.1, enabling >95% efficiency in generating endothelial and mural progenitor cells, respectively. Their ability to integrate and enhance perfusion will be tested in ischemic hindlimb models. The project comprises three aims. First, we will engineer hypoimmunogenic vascular cells by knocking out B2M/CIITA to eliminate highly polymorphic MHC expression, preventing adaptive immune recognition and overexpressing CD47 to prevent innate immune clearance. Second, we will assess vascular integration and perfusion enhancement in ischemic models by transplanting the engineered vascular cells. Third, we will apply multiplexed spatial transcriptomics to map immune-vascular interactions, identifying pathways that regulate immune evasion and vascular remodeling. This study will provide critical insights into immune-vascular dynamics, establishing a foundation for hypoimmunogenic iPSC-based therapies that achieve long-term immune tolerance and functional vascular regeneration in ischemic diseases. The training will occur under the mentorship of Dr. Juan Melero-Martin at Boston Children's Hospital/Harvard Medical School. I will be co-mentored by an extraordinary team of scientists on my advisory committee, including Dr. Torsten Meissner and Dr. Kaifu Chen, for new training goals in immune biology, vascular biology, transcriptomics as well as career guidance. Through this training, I will acquire advanced conceptual, technical, and professional skills, preparing me for an independent research career in translational regenerative medicine.

NHLBI - National Heart Lung and Blood Institute

Abstract The human sinoatrial node (SAN) is the heart’s natural pacemaker, initiating electrical impulses that maintain rhythmic contraction and coordinate blood flow. While its role is essential, our understanding of SAN development—particularly in humans—remains limited. Sinus node dysfunction (SND), often caused by SAN cell loss or fibrosis, is a major cause of arrhythmia and the primary reason for up to 50% of artificial pacemaker implants in the US. There are no approved cellular or pharmacological treatments. Compared to chamber muscle formation, the mechanisms underlying SAN lineage specification and subpopulation diversity are poorly defined, representing a critical gap in knowledge. In preliminary studies, we used single-nucleus multiomics and spatial transcriptomics to analyze the human fetal SAN and identified candidate regulatory networks. We also developed a dual knock-in human pluripotent stem cell (hPSC) reporter line (SHOX2:eGFP; MYH6:mCherry) and established protocols to generate functional pacemaker cells, SAN organoids (Sinoids) and SAN-paced cardioids (SAN-PCOs). These tools enable real-time visualization, isolation, and functional analysis of human pacemaker cells and tissues. Three Aims are proposed to 1) identify key transcriptional regulators of SAN differentiation using inducible CRISPR interference (CRISPRi) and activation (CRISPRa) in hPSCs. Validated genes will be tested in SAN-PCO models using molecular and electrophysiological assays to assess their role in pacemaker function; 2) dissect the molecular programs guiding SAN subpopulation specification, including head, tail, and transitional zone domains. Using CRISPR-based perturbation and single-cell RNA sequencing, we will evaluate how candidate regulators shape subpopulation identity and influence SAN-cardiomyocyte interactions; and 3) assess how genetic variants associated with SND affect SAN development and function. We will generate isogenic SNP knock-in and gene knockout hPSC lines based on top candidate GWAS hits and evaluate the variant–gene–cell axis using multiomics and functional assays. By integrating single-cell genomics, gene editing, and human stem cell-derived SAN models, this study will define the molecular architecture of human pacemaker development and identify genetic mechanisms underlying SND. These findings will advance our understanding of cardiac conduction system biology and support future therapeutic strategies.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY / ABSTRACT Orofacial neuropathic pain, often stemming from somatosensory nervous system dysregulation, frequently manifests in conditions such as chemotherapy-induced neuropathy and trigeminal neuralgia. This pain commonly results in altered somatosensory experiences, including orofacial cold allodynia (pain from normally innocuous cool stimuli) and cold hyperalgesia (exaggerated pain in response to noxious cold). Current treatments often fail to alleviate these cold-sensation symptoms because the underlying cellular and molecular mechanisms for low temperature sensation are not well defined. Recent single-cell sequencing has revealed new layers of molecularly distinct somatosensory neuron populations, yet how the coding logic underlying low temperature sensation and cold-nociception is distributed across these neurons remains severely understudied. This proposal aims to delineate the neural and molecular logic underlying low temperature sensation, revealing fundamental principles essential for informing development of new strategies for cold allodynia and neuropathic pain. To accomplish this, we will apply an intersectional genetic strategy in mice, combining state-of-the-art thermoelectric cooling (TEC) probes, in vivo trigeminal (TG) functional imaging, multiplexed in situ hybridization, neural tracing, and advanced AI-based behavioral analyses. Preliminary data reveal Chrna7+ TG Aδ-nociceptors encode noxious cold sensation in teeth independent of the canonical cool receptor Trpm8, providing direct evidence that low temperature encoding is distributed across several TG classes. This proposal will 1) define molecular and neural mechanisms underlying orofacial low temperature sensation and 2) characterize behavioral outputs and central projections of low temperature encoding TG populations using optogenetics and AI-based machine learning, and mapping associated afferent brainstem nuclei.This research will launch the candidate’s independent research program aimed at elucidating low temperature encoding mechanisms throughout the periphery. This will set the stage for future directions examining higher-order circuit mechanisms of cold sensation versus pain, and how sensation is transformed in disease/pain models. This work will bring new perspectives and hypotheses towards the development of pain treatment strategies, including those for neuropathic cold allodynia and hyperalgesia. During the mentored stage, the candidate will gain valuable training in: 1) intersectional genetic techniques for neuroscience studies, 2) advanced computational analysis for calcium imaging and behavior, and 3) guidance from the formal advisory committee in lab leadership, grant writing, networking, and presentation skills. This award will set the candidate up for success in transitioning to independence and making significant discoveries towards understanding and treating pain.