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U.S. National Science Foundation

The Tribal Colleges and Universities Program (TCUP) provides awards to federally recognized1 Tribal Colleges and Universities, Alaska Native-serving institutions, and Native Hawaiian-serving institutions to promote high quality science (including sociology, psychology, anthropology, linguistics, economics and bioeconomics, statistics, and other social and behavioral sciences; natural sciences; computer science, including, but not limited to, artificial intelligence, quantum information science, and cybersecurity), technology, engineering and mathematics (STEM), STEM education, research, and outreach. Support is available to TCUP-eligible institutions (see the Additional Eligibility subsection of Section IV of this solicitation) for transformative capacity-building or community engagement projects through Instructional Capacity Excellence in TCUP Institutions (ICE-TI), Targeted STEM Infusion Projects (TSIP),TCUP for Secondary and Elementary Teachers in STEM (TSETS), TCU Enterprise Advancement Centers (TEA Centers), Cyberinfrastructure Health, Assistance, and Improvements (CHAI), and Preparing for TCUP Implementation (Pre-TI). Collaborations led by TCUP institutions that involve non-TCUP institutions of higher education are supported through TCUP Partnerships, with the participation of other NSF programs to support the work of non-TCUP institutions. Finally, research studies that further the scholarly activity of individual faculty members are supported through Small Grants for Research (SGR). Through the opportunities highlighted above, as well as collaborations with other National Science Foundation (NSF) divisions and directorates, and other organizations,TCUPaims toincrease Native individuals' participation in STEM careers, improve the quality of STEM programs atTCUP-eligible institutions, and facilitate the development of a strong STEM enterprise in TCUP institutions' service areas. TCUP supports transformative capacity-building, community engagement, or research projects at TCUP-eligible institutions through the following funding tracks: Instructional Capacity Excellence in TCUP Institutions (ICE-TI) projects provide support to design, implement, and assess comprehensive institutional improvements in STEM education and research capacity at TCUP-eligible institutions of higher education. By strengthening STEM education and STEM education research, successful projects will increase the number of STEM students and improve the quality of their preparation. ICE-TI projects create and/or adapt and assess innovative models and materials for teaching and learning in STEM, embody knowledge about how students learn most effectively in STEM teaching and learning activities, and bring STEM disciplinary advances into the undergraduate or graduate experience. The objective of this strand is to expand STEM degrees offered by TCUP-eligible institutions or significantly enhance instructional approaches. Targeted STEM Infusion Projects (TSIP) support the attainment of a short-term, well-defined goal to improve the quality of STEM education at an eligible institution. Targeted STEM Infusion Projects could, for example, enhance academic infrastructure by systematically adding traditional knowledge to the scope or content of a STEM course, updating curricula, modernizing laboratory research equipment, developing and delivering professional development for K-12 STEM educators, or improving the computational infrastructure.The objective of this strand is to expand STEM degrees or significantly enhance instructional approaches. TCUP for Secondary & Elementary Teachers in STEM (TSETS) supports in-service professional development in STEM disciplinary or STEM education content and/or research for K-12 STEM teachers in the relevant service area. Examples of project activities include, but are not limited to, professional development involving seminar series and engagement in STEM instruction and content during the academic year, structured series of summer intensive workshops and trainings, and summer research opportunities. The objective of this strand is to broaden the instructional capacity for STEM in the K-12 workforce and thereby to the entire community, and to build the capacity for STEM disciplinary or education research among participating educational professionals. TCU Enterprise Advancement Centers (TEA Centers) coalesce the STEM and/or STEM education expertise into a team, designed to support and promote the STEM goals, needs, aspirations, or interests of the chartering reservation or tribe(s). TEA Centers may address a critical tribal or community need or focus on a realm of research or design that is beyond the scope of individual research grants or that is of interest to multiple tribes. The objective of this strand is to build on the capacity developed through prior TCUP support and apply expertise to collaborations with communities in the institution s service area, or nationally. The Cyberinfrastructure Health, Assistance, and Improvements (CHAI) strand supports projects at TCUP-eligible institutions of higher education to upgrade the cyberinfrastructure necessary to conduct, expand, manage and administer STEM programs of study, including research. The objective of this strand is to equip TCUP institutions to meet the demands of virtual instruction, advanced computing, and data science opportunities. Preparing for TCUP Implementation (Pre-TI) provides support for activities that prepare an institution for Implementation-level projects.Consequently, they are available only to TCUP-eligible institutions of higher education that have never received TCUP support, have not received TCUP support within the previous five years, or are embarking on a significantly novel STEM strategic plan. Examples of supported activities include completing an institutional assessment of its current STEM instructional capacity, or engaging in conversations necessary to formulate a shared vision of what that capacity should be and how to achieve it. Pre-TI awards can support staff and faculty release time, travel, stakeholder gatherings, and associated administrative costs.The objective of this strand is to conduct self-studies and formulate strategic plans for the development of STEM instructional programs of study. The TCUP Partnerships strand provides support for collaborations that will improve TCUP institutions' instructional and research capacity in STEM fields supported by NSF; attract, retain, and support TCUP students in internships and research endeavors deemed to be necessary for a complete curriculum offering; and engage partner universities to provide an academic grounding and a successful transition for students who wish to study or attain degrees in STEM fields supported by NSF. TCUP Partnerships broaden the number of scientific disciplines available to students at TCUP institution through collaborations with non-TCUP institutions. Active Pre-Engineering Education Collaboratives or Partnerships in Geoscience Education awards are not affected by this revision. The objective of this strand is the development, through instructional and research capacity-building, of academic and career pathways for TCUP students through supporting collaborative projects between and among TCUP and non-TCUP institutions. Interested teams of collaborators for which a TCUP institution serves as lead should contact the TCUP program directors. Support for non-TCUP partners must be obtained from other NSF programs, which follows the procedures of the prior Partnership strands. Small Grants for Research (SGR) strand support STEM or STEM Education faculty members at TCUP-eligible institutions to initiate or pursue research projects or programs that may include undergraduate or graduate student engagement. Awards are intended to help further the faculty member's research capability and effectiveness; improve research and teaching at his or her home institution and create and study new models and innovations in STEM teaching and learning. International research or collaborations are strongly encouraged. TCUP students may seek support for international research opportunities under the guidance of a TCUP STEM or STEM education faculty member and an international research collaborator. These awards are particularly appropriate as a means of recruiting and retaining highly qualified scientists, engineers, and educators at TCUP-eligible institutions. The objective of this strand is to support faculty research and professional development that build research capacity at TCUP institutions. [1] Executive Order 13021 defines Tribal Colleges and Universities ("tribal colleges") as those institutions cited in section 532 of the Equity in Educational Land-Grant Status Act of 1994 (7 U.S.C. 301 note), and other institutions that qualify for funding under the Tribally Controlled Community College Assistance Act of 1978, (25 U.S.C. 1801 et seq.), as well as Navajo Community College as authorized in the Navajo Community College Assistance Act of 1978, Public Law 95-471, Title II (25 U.S.C. 640a note). The term "Alaska Native-serving institution" means an institution of higher education that is an eligible institution under section 1058(b) of the Higher Education Act; and that, at the time of submission, has an undergraduate enrollment that is at least 20 percent Alaska Native students. The term "Native Hawaiian-serving institution" means an institution of higher education that is an eligible institution under section 1058(b) of the Higher Education Act; and that, at the time of submission, has an undergraduate enrollment that is at least 10 percent Native Hawaiian students. Most TCUP-eligible institutions of higher education are two-year or community colleges. See the Who May Submit Proposals section in this solicitation for further details.

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

PROJECT SUMMARY Placental aging is the programmed process of progressive functional decline and tissue maturation that occurs throughout the course of pregnancy, particularly in the later stages. While this is a normal feature of development, accelerated or pathological placental aging triggered by stress can impair placental function and lead to serious complications, including stillbirth, preeclampsia, fetal growth restriction, and preterm birth. Understanding mechanisms driving premature placental aging is critical for developing strategies to improve pregnancy outcomes. This proposal tests the hypothesis that precocious trophoblast differentiation leads to exhaustion of the trophoblast stem cell (TSC) pool, triggering premature placental aging and disease. TSCs maintain placental homeostasis by regenerating the syncytiotrophoblast – the multinucleated epithelial layer that mediates nutrient and gas exchange at the maternal-fetal interface. Loss of TSC self-renewal or differentiation under stress may compromise placental regenerative capacity and contribute to aging and failure. Our preliminary data identify the transcription factor CEBPB as a key regulator of the stress response and TSC differentiation. Conversely, trophoblast-associated microRNAs, including the murine miR-290~295 cluster and its human ortholog miR- 371~373, help preserve TSC identity by supporting stem cell self-renewal and metabolism. Loss of miR-290 in mice leads to premature depletion of the TSC pool, the accumulation of aging markers in the placenta, and stillbirth – linking early stem cell dysfunction with placental failure. The overall goal of this project is to define the molecular and metabolic pathways that govern TSC differentiation and placental aging. Aim 1 will characterize stress-induced TSC differentiation and senescence, focusing on the regulatory roles of CEBPB and miR- 371~373. Aim 2 will determine how TSC depletion contributes to placental aging and stillbirth in miR-290 knockout mice, using transcriptomic and histologic approaches. Aim 3 will investigate the role of metabolic reprogramming in TSC fate and aging, and test whether metabolic interventions can preserve stemness under stress. By linking TSC biology with placental aging and adverse outcomes, this work provides a conceptual framework that opens avenues for therapeutic innovation. Applying principles from aging biology to the placenta may enable repurposing of existing anti-aging strategies to reduce placental dysfunction and prevent the most devastating consequence—stillbirth. This project directly addresses the goals of NOSI NOT-HD-23-021: The Road to Prevention of Stillbirth.

NIGMS - National Institute of General Medical Sciences

Primary cilia play a pivotal role in human health, acting as signaling hubs to sense extracellular cues such as odorants, metabolites, light, neurotransmitters, and more. Defects or failure of these signaling hubs to function leads to developmental disorders, immune dysfunction, diabetes, obesity, Parkinson’s disease, cancer, and other pathologies. An early focus of this grant is the generation of better tools to dissect primary cilia function and mechanism, such as a protocol for synchronized ciliogenesis in human retinal pigmented epithelial (RPE) cells coupled with microscopy and shotgun/phosphoproteomic mass spectrometry, to dissect signaling events temporally and spatially. While mapping ciliogenesis via immunofluorescence with known markers of the stages, one protein that emerged as a strong candidate for regulating interpathway communication was TULP3, a 50kDa ciliary protein whose primary known function is in driving the import of ciliary GPCRs through unclear mechanisms. A combination of synchronization and classical cell biological approaches were used to uncover novel phenotypes revealing temporal and spatial timing of TULP3’s function in GPCR traffic as well as new functions in ciliogenesis, downstream of TTBK2 recruitment but before axoneme protrusion. Patient mutations were identified from the use of GWAS databases to probe for links between protein function and human health. Constitutive expression of these TULP3 mutants in TULP3 KO background generated new tools to perturb select functions for TULP3, which is especially powerful for probing function-specific binding domains and partners. This proposal tests the following hypotheses: TULP3 (i) regulates receptor traffic prior to cargo arrival at the basal body and coupling to IFT machinery to mediate receptor entry into cilia and (ii) licenses axonemogenesis. Furthermore, primary cilia perform two different functions in lymphoid tissues: regulating fate change of hematopoietic stem cells into lymphocytes in the bone marrow, and in the lymph node facilitating lymphocyte maturation. These hypotheses will be tested in the following aims: Aim 1- Determine the mechanism by which TULP3 mediates membrane receptor transport into cilia. Aim 2- Identify the function and mechanism of TULP3 in ciliogenesis. Aim 3- Uncover the function of primary cilia and TULP3 in the generation of white blood cells. These aims will be addressed using cell culture and mouse model systems to probe TULP3 function and its role in immunity in both in vitro and in vivo contexts. The success of any and all of these aims will provide novel insight into key mechanisms driving ciliary function and reveal cellular contexts for how disrupted immune function arises. The study of primary cilia in the lymphatic system is largely uncharted territory. As a result, these studies are expected to open new fields of investigation into mechanisms of immune regulation.

NCI - National Cancer Institute

PROJECT SUMMARY Metastasis is the leading cause of mortality among patients diagnosed with solid tumors. In this regard, identifying common mechanisms within primary, premetastatic and metastatic niches that contribute to progression of solid tumors represents a significant need. The Lipocalin-2 gene (LCN2, neutrophil gelatinase- associated lipocalin or NGAL) encodes a cytosolic and secreted protein (Lcn2) that regulates receptor trafficking, innate immune responses, inflammation, microbiome dynamics and iron homeostasis. Previous work from our group and others has shown that Lcn2 can promote stemness and tumorigenesis in models of solid tumor progression. However, uncertainty persists about the stage or stages at which LCN2 exerts tumorigenic effects and whether the mechanisms of LCN2 action vary based upon stage or tumor location. In this regard, we have recently performed unbiased single-cell spatial proteomic and transcriptomic screens of breast and pancreatic cancer patient samples leading us to define tumor cell autonomous and non-atonomous roles for Lcn2/LCN2 in promoting solid tumor progression. Based upon these findings, the central hypothesis of this proposal is that Lcn2 promotes FGFR2 signaling and adaptive anti-inflammatory immunity to support solid tumor progression. The overall objective of this proposal is to determine the spatiotemporal dynamics of Lcn2- governed tumor cell autonomous and non-autonomous mechanisms driving metastatic progression of solid tumors. Our rationale for pursing this work is that understanding these mechanisms will position Lcn2 as a biomarker for immune therapy resistance and target for improving immune therapy success in immunologically cold tumors. To test the central hypothesis, we propose to specific aims that will (I) identify the tumor cell autonomous signaling mechanisms governing Lcn2-dependent FGF2-induced tumor cell invasion and (II) define the intercellular communication mechanisms by which Lcn2 reduces inflammation and enriches regulatory T cells within the metastatic niche. The basis of this project is conceptually innovative and employs technically innovative transgenic, pharmacology, cell cycle reporter, protein reporter tagging, single-cell spatial omics, spinning disc time-lapse confocal and CRISPR methods in combination with preclinical allo- graft/xenograft models of solid tumors and hetero-multicellular cancer spheroid cultures. This work is expected to (1) provide exceptional research opportunities for undergraduates while enhancing the research environ- ment at Baylor University and (2) elucidate targetable mechanisms that govern solid tumor progres- sion/metastasis. As such, the proposed studies will have a positive impact on Baylor student success and identify novel treatment strategies to improve cancer outcomes.

NCI - National Cancer Institute

PROJECT SUMMARY Acute myeloid leukemia (AML) is an aggressive, devastating cancer with limited treatment options. AML progresses rapidly and presents significant treatment challenges due to its immunosuppressive tumor microenvironment , which impairs immune cell function. Group 1 innate lymphoid cells (ILCs), including natural killer (NK) cells and ILC1s, play key roles in immunity. ILC1s reside in tissues and were initially believed to function primarily by secreting cytokines such as IFN-γ, TNF-α, and GM-CSF. However, their anti-tumor activity has been largely unknown. In 2022, we addressed this gap and published our discovery in Nature Immunology as a cover story. We found that ILC1s isolated from AML patients are functionally impaired, whereas ILC1s from healthy mice are significantly more potent. Healthy ILC1s induce the death of leukemia stem cells (LSCs), block LSC differentiation into leukemia progenitor cells, and promote the transition of LSCs into non-leukemic lymphoid progenitors. Mechanistically, normal ILC1s target LSCs by secreting IFN-γ and engaging receptor-ligand interactions (e.g., DNAM-1–CD155 and IL-7 receptor–IL-7). Despite identifying key features of ILC1s and their role in inhibiting LSCs, important questions remain unanswered. It is still unclear how ILC1s develop in vivo under normal or AML conditions, and the mechanisms through which ILC1s induce LSC death and differentiation in humans are largely unexplored. Moreover, the therapeutic potential of ILC1s remains unknown. We hypothesize that ILC1s possess strong anti-LSC activity and unique developmental pathways, offering a novel approach to control or treat AML and potentially prevent its relapse. The goals of this project are to elucidate the mechanisms of ILC1 anti-tumor activity, characterize their developmental pathways, and explore their therapeutic applications. In Aim 1, we will dissect the mechanisms by which ILC1s induce LSC death (e.g., via pyroptosis) and drive M1 polarization of LSC-differentiated myeloid cells in humans. In Aim 2, we will characterize ILC1 developmental pathways in both normal and AML conditions. Leveraging our expertise in developing adoptive cellular therapies, including chimeric antigen receptor (CAR) NK cells for AML, in Aim 3, we will study novel FLT3-targeting CAR ILC1s that we generated. FLT3 is highly and selectively expressed on AML blasts and LSCs, making it an ideal target. We will generate allogeneic, off-the-shelf, ready-to-use FLT3- CAR ILC1s from umbilical cord blood CD34⁺ cells or by converting NK cells into ILC1s, which we demonstrated. These CAR ILC1s will be tested for their anti-AML efficacy in preclinical models and compared to unmodified ILC1s. Additionally, we will combine ILC1s or FLT3-CAR ILC1s with NK cells and an FDA-approved tyrosine kinase inhibitor, which upregulates FLT3 expression on AML cells. Finally, we propose to reprogram endogenous ILC1s by treating them with IL-7 to enhance their activity. A deeper understanding of ILC1 development and function, anticipated through the completion of this study, holds significant promise. The knowledge gained could lay the groundwork for diverse therapeutic strategies that have the potential to reduce mortality in AML patients.

U.S. National Science Foundation

The NSF Research Traineeship (NRT) program seeks proposals that explore ways for graduate students in research-based master s and doctoral degree programs to develop the skills, knowledge, and competencies needed to pursue a range of STEM careers. The program is dedicated to effective training of STEM graduate students in high priority interdisciplinary or convergent research areas, through a comprehensive traineeship model that is innovative, evidence-based, and aligned with changing workforce and research needs. Proposals are requested that address any interdisciplinary or convergent research theme of national priority, as described in section II.D below. The NRT program addresses workforce development, emphasizing broad participation, and institutional capacity building needs in graduate education. The program encourages proposals that involve strategic collaborations with the private sector, non-governmental organizations (NGOs), government agencies, national laboratories, field stations, teaching and learning centers, informal science centers, and academic partners. NRT especially welcomes proposals that reflect collaborations between NRT proposals and existing NSF Eddie Bernice Johnson Inclusion across the Nation of Communities of Learners of Underrepresented Discoverers in Engineering and Science (INCLUDES) Initiative, Research Experiences for Undergraduates (REU), Louis Stokes Alliances for Minority Participation (LSAMP), NSF Scholarships in Science, Technology, Engineering, and Mathematics (S-STEM), and NSF STEM Ed Organizational Postdoctoral Fellowship program (STEM Ed OPRF) projects, provided the collaboration will strengthen both projects. Researchers at minority serving institutions and emerging research institutions are strongly encouraged to submit proposals. Collaborations between NRT proposals and existing NSF INCLUDES projects should strengthen both NRT and INCLUDES projects.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

ABSTRACT Epidermis of the skin undergoes continuous self-renewal through a tightly regulated balance of keratinocyte proliferation and terminal differentiation. Disruption of this balance is characteristic of inflammatory skin disorders such a psoriasis, atopic dermatitis, and neutrophilic dermatoses. The etiologies of these skin disorders are complex and heterogeneous, as are the needs for treatments. Our long-term goal is to elucidate how dysregulation of K63-Ub-mediated signal transduction pathways in keratinocytes contribute to skin inflammation. Towards this end, our recent studies have focused on UBE2N, a ubiquitin conjugase that forms heterodimers with an essential noncatalytic partner, UBE2V1 or UBE2V2, to specifically catalyze K63-Ub of target proteins. We demonstrate that conditional knockout of Ube2n in mouse keratinocytes induces psoriasis- like inflammatory skin lesions with a raised and scaly appearance. Transcriptomic and histological analyses identified a diminished epidermal stem cell compartment, a thickened epidermal spinous layer, and an increased infiltration of myeloid-skewed immune cells. This is correlated with increased expressions of myeloid cell chemokines such as CXCL1 and CXCL2 and IL1 family cytokines in keratinocytes and infiltrating myeloid cells. Oral delivery of the small molecule inhibitor of IRAK1/4, common mediators of the IL1R and TLR signaling pathways, alleviated immune infiltration and epidermal defects of the mutant skin. These data highlight a key role for UBE2N in regulation of epidermal and cutaneous immune homeostasis. In line with these animal data, recent GWS studies demonstrate a causal association between UBE2V1 polymorphism and psoriasis. Together, these data support the hypothesis that UBE2N partners with UBE2V1 to restrain keratinocyte recruitment of myeloid cells through suppression of IL1 and CXCL1/2-mediated inflammatory crosstalk between keratinocytes and myeloid cells. We propose 3 specific aims to: 1) validate the importance of UBE2N catalytic function and the role of UBE2V1 in epidermal homeostasis and cutaneous immune homeostasis, 2) determine the contribution of the IL1 signaling pathway in UBE2N-null skin inflammation, and 3) assess the utility of CXCL1/2 receptor antagonists in mitigating neutrophilic dermatosis. We will utilize conditional genetic animal models along with the cutting-edge techniques of single cell transcriptomics and global proteomics to comprehensively analyze mechanistic aspects of UBE2N/UBE2V1-mediated K63-Ub in cutaneous inflammation and therapeutic targeting. Results of these studies will reveal novel mechanisms of epidermal and cutaneous immune homeostasis, as well as insights for therapeutic development.

NIDA - National Institute on Drug Abuse

PROJECT SUMMARY Cocaine use disorder (CUD) is highly comorbid in people with HIV (PWH) and can accelerate infection, alter neuropathology, and exacerbate cognitive decline despite antiretroviral therapy (ART). Many of these effects are due to the infection and dysregulation of CNS-associated myeloid cells, especially microglia, which comprise a significant reservoir in this compartment. However, the precise mechanisms by which cocaine (Coc) dysregulates microglia to enhance HIV infection are unclear, partly due to the lack of translationally relevant human microglial models suitable for mechanistic evaluation of Coc-mediated changes in viral dynamics. Classically, Coc has been thought to act by blocking dopamine transporter (DAT) activity, exposing microglia to aberrantly high dopamine concentrations. Our data show that dopamine can increase HIV infection and inflammation in microglia and other myeloid cells. However, recent data show that Coc has other mechanisms of action beyond the modulation of dopaminergic tone, involving the ER protein sigma1 (σ1), which has diverse cellular functions including the modulation of cellular stress pathways such as the unfolded protein response (UPR). Viruses, including HIV, can exploit the UPR to amplify stress-induced protein production in the host cell, enhancing viral replication. Our preliminary studies indicate that Coc’s effects on σ1 may drive a Coc-mediated increase in HIV infection in microglia, potentially through increased stress response and independent of dopamine’s effects. My preliminary data show that both Coc and σ1 agonists increase HIV replication in human inducible pluripotent stem cell (iPSC)-derived microglia (iMg). These effects are blocked by σ1 antagonism but not by inhibition of DAT or dopamine receptors. We also show increased σ1 protein expression and recruitment to the ER/nuclear envelope space in HIV-infected iMg treated with Coc, and preliminary single-cell RNAseq data suggest changes in the UPR. Therefore, we hypothesize that Coc-mediated activation of σ1 increases HIV infection of microglia via activation of the UPR. In Aim 1, we will test the involvement of σ1 in driving Coc-mediated changes in HIV infection of iMg using pharmacological and genetic modulation, and we will also confirm the absence of dopaminergic involvement. We will assess changes in viral dynamics using AlphaLISA and immunofluorescence (IF) high-content imaging. In Aim 2, we will test the hypothesis that Coc induces greater σ1 activity in the presence of HIV infection utilizing confocal and high-content IF imaging of σ1 subcellular localization in cellular compartments like the nuclear envelope, ER, and mitochondria-associated ER membrane. Movement of σ1 to these compartments is a feature of σ1 activation. In Aim 3, we will use single-cell RNAseq to test the hypothesis that Coc-induced σ1 activity drives increased HIV infection in iMg via upregulation of UPR genes. The results from these experiments will not only define novel interactions between HIV and σ1 that could reveal new antiretroviral targets but will also broadly inform on the role of σ1 in microglia and potentially identify biomarkers for prevention strategies against CUD and its associated comorbid diseases.

NHLBI - National Heart Lung and Blood Institute

Abstract: The adult zebrafish is capable of rapid regenerative processes in response to cardiac damage that result in a fully functional and scarless heart. This robust regenerative response is controlled by pre-existing populations of cardiomyocytes that de-differentiate prior to proliferation in response to injury; however, the full extent of intercellular signaling that facilitates the injury-response of these cardiomyocytes remains unknown. A subpopulation of cardiomyocytes in the zebrafish heart are derived from the cardiac neural crest (CNC), a highly migratory stem cell population in the early vertebrate embryo. These neural crest-derived cardiomyocytes (NCCMs) may contribute extensively to regeneration in the adult heart by reactivating developmental gene networks, including those controlling morphogenesis in the early embryo. In particular, the cxcr4b/cxcl12a chemokine signaling pathway is known to be essential for CNC migration in the developing embryo and regulates aspects of migration in the regenerating heart. To assess the roles of cxcr4b/cxcl12a in relation to the CNC during cardiac regeneration in the adult, I will conduct mRNA expression and localization analyses via hybridization chain reaction (HCR), in vitro migration assays of primary cardiomyocyte cultures, and create transgenic fish lines for the conditional overexpression and knockout of cxcl12a and cxcr4b, respectively, during cardiac regeneration in the adult. I will also analyze existing cardiac ATAC-seq data to identify and validate putative damage-responsive enhancer regions that drive chemokine gene expression. Identifying how migratory mechanisms guide cardiomyocytes will assist in discovering therapeutic approaches aimed at stimulating human hearts with a regenerative capacity.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Mutations in telomerase cause bone marrow failure in patients suffering with dyskeratosis congenita and other associated telomere biology disorders. While mutations in these patients are found in different components of telomerase, mutations in genes that regulate the processing of the RNA component of telomerase, TERC, are the most prevalent. Due to a lack of adequate models and intrinsic difficulties in studying human telomerase in physiologically relevant cells, the molecular pathways that control TERC biogenesis and decay during hematopoiesis remain largely unknown. Progress in the field has been hampered by species and even cell-type specific differences in telomerase biology that limit our understanding of the molecular mechanisms leading to the disproportionate role of TERC in hematopoietic failure when compared to other components of telomerase. A better understanding of the molecular regulation of TERC biogenesis and function in hematopoietic cells is essential for development of novel alternatives for patients, which remain without a cure. The focus of this proposal is to use different in vitro and in vivo approaches to decipher molecular pathways controlling TERC biogenesis and decay in blood cells, as well as the function of TERC during erythroid, myeloid and lymphoid development. We have developed unique models, including targeted hematopoietic differentiation of human pluripotent stem cells, transplantation of primary CD34+ human stem cells into sub-lethally irradiated mice, and studies in primary patient samples, that will allow a complete analysis of the pathways regulating TERC decay and function during hematopoietic development. For that, two specific aims are proposed that will both identify novel regulators of TERC decay in blood cells, as well as specific functions of TERC in the hematopoietic system. Aim 1 will determine the role of novel, recently identified 3'- end RNA deadenylases to TERC processing in the blood, and to which extent different RNA deadenylases prevent TERC degradation by the exosome. We will complement these experiments with the identification of the molecular effectors of a novel route for TERC decay, triggered by differential TERC capping on its 5'- end, and mediated by trafficking to the cytoplasm. We will investigate if modulation of these different pathways can rescue hematopoietic development in telomerase mutants. Aim 2 will investigate novel functions of TERC outside telomerase that can explain the disproportionate role that mutations that affect TERC levels show in bone marrow failure. We have created unique cellular systems where we can uncouple TERC expression from telomere length, and will utilize them during hematopoietic differentiation to study direct functions of TERC on DNA damage and regulation of hematopoietic gene expression programs. These studies will determine the molecular mechanisms controlling TERC decay and function in hematopoietic cells. Our unique cellular tools, combined with our expertise in telomerase, RNA decay, and stem cell biology puts us in an ideal position to make a significant impact in this field.

NIA - National Institute on Aging

PROJECT SUMMARY This NIH F30 grant proposal investigates how somatic mutations in hematopoietic stem cells (HSCs) contribute to aging-related diseases through clonal expansion of mutated blood cells. This phenomenon, called clonal hematopoiesis, becomes more common as individuals age, affecting over 10% of in people over 60 years old. This study focuses on mosaic chromosomal alterations (mCAs), large-scale mutations that are under-studied compared to smaller mutations like clonal hematopoiesis of indeterminate potential (CHIP). mCAs are linked to lymphoid malignancies and infection susceptibility due to lymphoid-biased differentiation, while CHIP correlates with cardiovascular diseases and myeloid malignancies through myeloid-biased differentiation. I hypothesize that mCA expansion is determined by individual factors rather than mCA genetic change and that clones with greater expansion rates will have increased disease risk. Aim 1 examines the influence of mCA characteristics and environmental factors on clonal expansion rates using longitudinal blood samples from 30,000 individuals in Vanderbilt’s BioVU genomic and clinical biobank. Using longitudinal mCA trajectories, I will quantify the contribution of the mCA mutation and individual characteristics (e.g., age, sex, BMI, smoking, type 2 diabetes, lipoprotein levels) to clonal expansion rate and build a predictive model for mCA clonal expansion. My working hypothesis for Aim 1 is that mCA expansion varies widely among individuals with the same mCA and thus modifiable lifestyle exposures are major contributors to clonal expansion rate. The longitudinal samples in BioVU will not be sufficient to test genetic and phenotypic associations with clonal expansion rate. Therefore, Aim 2 expands the study to detect mCAs in > 1 million individuals across various genomic biobanks with single blood draws (i.e., NHLBI TOPMed, NIH All of Us, UK Biobank, and BioVU). To determine mCA clonal expansion rate from a single timepoint, I will apply Passenger-Approximated Clonal Expansion Rate (PACER), which estimates mCA expansion rate from a single blood draw to build upon my measured mCA analysis by two orders of magnitude. A genome-wide association study and a phenome-wide association study will be conducted to identify germline variants and phenotypic correlations related to mCA clonal expansion rates. My working hypothesis for Aim 2 is that 1) certain germline variants predispose individuals to faster mCA growth and 2) specific disease phenotypes, including chronic lymphocytic leukemia and infection susceptibility, are associated with faster mCA clonal expansion rate. This research aims to significantly enhance our understanding of mCA clonal expansion, addressing a fundamental biological mechanism of aging to prevent multiple diseases. Collectively, these insights will contribute to mCA risk prediction models and highlight potential biological pathways or lifestyle strategies to slow mCA expansion.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract CD8+ T cells are unique in effective sensing and killing of intracellular pathogen-infected cells and tumor cells. Because current vaccines are designed to induce high titer pathogen-specific antibodies for host protection, new vaccines focused on promoting effective memory CD8+ T cells are needed. Since a single naive CD8+ T cell has the potential to give rise to multiple types of progenies, it is essential to understand how naive T cells are primed to form distinct effector and memory cells. It is generally accepted that the strength of cognate antigen (Ag) stimulation determines the size of the primary response and of the memory cell pool, and that strong cognate Ag signals coupled with robust co-stimulation and cytokines altogether drive naive CD8+ T cells towards an effector rather than a memory cell fate. The current dogma also states that cognate Ag stimulation does not lead to functionally distinct subsets of memory CD8+ T cells. In contrast, however, we recently discovered that the strength and the stability of cognate Ag/MHC interactions with the T cell receptor (TCR) determine the development of memory cell functional characteristics, in particular stem-cell associated characteristics, through epigenetic imprinting. Stem cell memory CD8+ T (TSCM) cells have been shown to exhibit superior functional features, progeny potential, self-renewal capacity and longevity. Using state of the art conditional mouse models, high dimensional spectral flow cytometry, lentiviral-based inducible gain or loss of function experiments, and computational modeling approaches, we will define the features of T cell epitopes, key TCR structural modes of recognition, TCR signaling pathways, genetic and epigenetic regulators that enhance the differentiation of TSCM cells in vivo. We will validate our findings in models of chronic infections and tumors. This research directly impacts the rational design of more effective vaccines and adoptive T cell transfer therapies.

NCI - National Cancer Institute

Project Summary/Abstract Somatically acquired mutation in the E1 ubiquitin-activating enzyme UBA1 within hematopoietic stem and progenitor cells (HSPCs) was recently identified as the cause of the adult-onset autoinflammatory syndrome VEXAS (vacuoles, E1 enzyme, X linked, autoinflammatory, somatic). Most VEXAS-associated mutations occur at Met41 in UBA1 and lead to clonal expansion within the HSPC and myeloid compartments, and frequently cause myelodysplastic syndrome (MDS) in addition to multi-organ inflammation. Despite the severity and prevalence of VEXAS, the mechanisms whereby UBA1 mutations cause multiorgan autoinflammation and myeloid malignancy are unknown. Our preliminary work utilizes newly developed and genetically controlled primary cell-based models of VEXAS to show that UBA1 mutant HSCs are primed towards myeloid differentiation and that their myeloid progeny undergo aberrant inflammatory cell death upon engagement of innate immune receptors. We hypothesize that aberrant inflammatory cell death underlies VEXAS pathogenesis. To build upon our preliminary work, this career development program will address 2 specific aims: (1) to determine cell-intrinsic and -extrinsic factors that promote clonal expansion and myeloid skew of UBA1 mutant HSPCs, and (2) to evaluate the role of inflammatory cell death as a therapeutic target in VEXAS. The proposed studies are part of the candidate’s developing research program at the intersection of myeloid malignancy and innate immune signaling and build upon his clinical expertise in MDS and AML. Dr. Narendra will develop a research program under the close supervision of his mentor Dr. Alexander Gitlin, an expert in the molecular basis of innate immune signaling, and co-mentor Dr. Scott Lowe, an expert in cancer biology, mouse models and functional genetics. Dr. Narendra will additionally be supported by members of his advisory committee including Drs. Omar Abdel- Wahab, Caleb Lareau, and Alexander Rudensky, experts in experimental and computational approaches to the study of hematopoiesis and immunity. Under the guidance of his mentors and advisory committee, the candidate will continue to gain expertise in immunology, malignant hematopoiesis, and single cell analytics. Dr. Narendra’s training environment will ideally position him to achieve his goal of becoming an independent physician-scientist, working at the convergence of innate immunity and cancer.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract. In the last 10 years, the field of induced pluripotent stem cells (iPSCs) has come far, but the reprogramming process is still sub-inefficient; around 1-3% of the transduced cells reprogram, and the iPSCs are just starting to be translated into a clinical trial. Therefore, there is a need for an efficient reprogramming system that would allow the in-depth analysis of the different phases of the reprogramming process and the role of the innate immune response. We have developed a new vector system, based on a human Paramyxovirus, measles virus (MeV). From this virus, we have shown that the measles virus can be modified into a “one cycle” reprogramming vector expressing the four reprogramming factors (RFs, OCT4, KLF4, SOX2 and cMYC) in one single genome and successfully generate genomic modification-free iPSCs from human fibroblasts. We showed that the MeV vector is equivalent to or superior to the Sendai (SeV) vector and that the MeV-derived iPSC clones present all the characteristics of iPSC clones. The long-term goal is to understand the process of reprogramming by MeV vectors to produce better human iPSCs for the treatment of degenerative diseases. The central hypothesis is that because the MeV reprogramming vector expresses the four reprogramming factors in one single vector, it can be used as a tool to understand the underlying process of iPSC reprogramming. The objectives of this particular application are to (1) identify the difference in the state of the innate immune response activation during MeV vector reprogramming, (2) understand how the control of the innate immunity by MeV affects reprogramming efficiency, and (3) understand the role of exogenous RFs expression in the reprogramming process. The proposed work is innovative because it capitalizes on a new MeV vector expressing the four RFs for the reprogramming of somatic cells into iPSC, and our group developed this technology. Also, the RFs expression can be further modulated in an innovative way by either post-transcriptional regulation, using miRNA that are naturally up-regulated or down-regulated during the reprogramming process or by changing their position in the genome. The proposed work is significant because it will collectively validate MV vector as a platform to produce clinically relevant iPSCs, but also as a new tool to study the reprogramming process. This work will lead to the identification of pathways important in MeV reprogramming, but these pathways could be investigated in the context of the related SeV vector and other reprogramming technologies.

NCI - National Cancer Institute

SUMMARY Basal-like breast cancer (BLBC) is an aggressive cancer subtype that disproportionally affects younger women and women of African ancestry, contributing to cancer disparity. Additionally, women with BRCA1 germline mutations have an extremely high risk of developing BLBC. BLBC precancer tends to be high-grade and likely to progress to malignant cancer. Understanding immune escape mechanisms in BLBC precancer is crucial for developing immunoprevention strategies to intercept its progression to aggressive breast cancer. Recent studies have showed that the generation of embryonic multipotent stem-like cells (referred to as pre-CSCs) through de- differentiation is critical for BLBC precancer progression in mouse models. Importantly, de-differentiation to multipotent cells has also been observed in patients with germline BRCA1 mutations. Similar de-differentiation processes occur in other breast cancer subtypes, suggesting a broad strategy for cancer prevention by targeting pre-CSCs. However, how pre-CSCs escape immunosurveillance remains poorly understood. In our preliminary studies, we have discovered that BLBC pre-CSCs express high levels of the stem cell transcription factor SOX9, which greatly induce the expression of the immune checkpoint B7x (also known as B7-H4, B7S1 or VTCN1). These cells also upregulate multiple cancer testis antigens. We found that the SOX9- B7x pathway is necessary for inhibiting T cell infiltration and protecting pre-CSCs from T cell-mediated elimination. SOX9 or B7x knockout blocks the progression of BLBC precancer. These findings strongly support the hypothesis that BLBC pre-CSCs are unexpectedly immunogenic due to upregulation of cancer testis antigens, and the upregulation of immune checkpoint B7x by stem cell factor SOX9 in these cells are required for establishing an immuno-suppressive microenvironment crucial for safeguarding pre-CSCs from immune elimination. This hypothesis will be tested by pursuing the following specific aims: (1) Dissect the mechanisms by which SOX9 regulates B7x and other immunosuppressive pathways in in precancer stem cells; (2) Determine the mechanistic interactions between precancer stem cells and their reactive immune cells; and (3) Develop anti- B7x therapy, either alone or in combination, as new immunoprevention strategies targeting pre-cancer stem cells in breast cancer. The most effective strategies will be further tested in mouse models of other breast cancer subtypes. To accomplish our goals, we have generated a number of novel tools and resources. The outcomes of this project will elucidate new immunosuppressive mechanisms of pre-CSCs and develop anti-B7x therapy, either alone or in combination, as immunoprevention approaches against breast cancer.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary/Abstract Mutations in the myelin protein zero (MPZ) gene cause one of the most common forms of hereditary neuropathy known as Charcot-Marie-Tooth type 1B (CMT1B) disease with phenotypes ranging from severe early-onset demyelinating to late-onset axonal neuropathy. CMT1B is a debilitating disorder with patients showing progressive distal weakness and atrophy, sensory loss, classical steppage gait, and painful and disfiguring lower limb contractures. MPZ is expressed mainly in myelinating Schwann cells and functions to compact myelin sheath in peripheral nerves. Here we propose two aims to elucidate the cellular and molecular disease mechanism and develop a novel gene therapy approach. In Aim 1, we will determine molecular and cellular mechanisms by which MPZ variants lead to a spectrum of phenotype from demyelination to axonal form using patient-derived peripheral nerve organoids (PNOs). Our team has the largest known cohort of patient Fibroblasts with various MPZ mutations where we are developing induced pluripotent stem cell (iPSC) lines. We demonstrate that CMT1B PNOs display reduced myelination, elevated ER stress and UPR, and Schwann cell survival, emphasizing the potential of PNOs to mimic CMT1B patient pathology. We also found that PNOs replicate disease severity, as R98C mutant PNOs show greater demyelination, axonal loss, and lower MPZ expression than S63del, mirroring patient phenotypes. We will use various cutting-edge technologies including single-cell RNAseq, MEA array, high-resolution imaging, and CRISPR gene correction to investigate disease mechanisms in patient PNOs. In Aim 2, we will develop a novel gene therapy approach for CMT1B and evaluate its efficacy in rescuing disease phenotypes in MPZR98C mice and our newly established patient-derived PNOs toward establishing essential preclinical data for translation. Considering that MPZ is an essential protein, and many MPZ mutations act through dominant negative, toxic-gain-of-function mechanisms, we hypothesize that a successful therapeutic strategy will eliminate the mutant MPZ while replacing it with wild-type form to maintain healthy myelination. Therefore, here we propose to develop a universal knockdown-and-replace gene therapy strategy that can be applied to virtually all patients with a dominant MPZ-associated neuropathy, regardless of the causative mutation. To do this, we will generate bi-functional AAV vectors designed to: (1)_knock down both wild-type and mutant MPZ using designed artificial miRNAs, and (2) deliver a miRNA-resistant wild-type MPZ replacement gene within the same vector. Our goal is to restore normal MPZ to Schwann cells, improving myelination and Schwann-axon interaction. This multidisciplinary research will uncover the pathomechanisms of MPZ-related neuropathy by pioneering the patient-specific CMT1B PNO model and generating preclinical data to support the translation of a novel AAV-based knockdown-and-replace gene therapy with strong clinical potential. The methods and findings from this study may also broaden the application of this technology to other inherited diseases.

NCI - National Cancer Institute

This application is focused on the study of bioengineered plant virus-based adjuvant and vaccine technology. We discovered that some plant viruses serve as potent adjuvants in the context of infectious disease and cancer vaccines/immunotherapy. Cowpea mosaic virus (CPMV) was identified as a uniquely potent adjuvant with distinct mechanism of immunomodulation compared to small molecule agonists, other plant viruses, or oncolytic viruses. Recently we discovered that systemic CPMV administration prior to tumor challenge protects mice from onset of tumor growth. Data indicate that the innate immune stimulation by CPMV is durable and lasts for weeks after CPMV exposure when innate cells would have returned to a homeostasis state – therefore data are consistent with induction of trained immunity. Single cell sequencing analysis of human PBMCs after CPMV adjuvant exposure indicates stimulation of interferon signaling pathways along with metabolic changes, and epigenetic rewiring – also consistent with a mechanism involving trained immunity. Together our data suggest that CPMV could act as an inducer of trained immunity – to date there are no reports on the study of plant viruses in trained immunity. Proposed studies will help elucidate the foundational principles that make CPMV a uniquely potent immunomodulator. We will fulfil the following specific aims: (1) We will establish the mechanism of CPMV as a training agent in vitro using immune cells followed by LPS challenge; longitudinal studies will be carried out and analysis will include measure of pro- inflammatory cytokine as well as CHiP and ATAC sequencing to confirm epigenetic rewiring and metabolic cell changes. Structure-function studies of bioengineered viruses will provide foundational insights into differential potency. (2) We will establish the mechanism of CPMV as a training agent using the B16F10 tumor model using WT and Rag 1 vs CCR2 knockout (KO) mice to delineate the role of adaptative vs. innate immune cells (β-glucan will serve as benchmark). Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) will be analyzed by single cell sequencing, CHiP and ATAC sequencing to delineate the mechanism of action. Studies will be paralleled with safety and biodistribution studies. (3) We will test the ability of the CPMV training agent to facilitate protection from influenza virus challenge; protection from influenza virus infection and pathology from CPMV will be benchmarked against FLUMIST and β-glucan. These studies could lay the foundation for continued and deeper studies of CPMV as an adjuvant technology towards the development of more broadly protective and efficacious vaccine formulations and immunoprevention strategies.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY BCL11A is a zinc-finger transcription factor that has been well-studied in erythroid biology, but evidence for an important role in the brain is beginning to emerge. Patients with heterozygous loss-of-function BCL11A mutations present with clinical features that can include intellectual disability (ID), autism spectrum disorder (ASD), and epilepsy. BCL11A is identified as a high confidence ASD risk gene in the SFARI database, and multiple lines of evidence also support potential roles in the etiology of schizophrenia (SZ) and Alzheimer’s disease (AD). However, the mechanisms that link BCL11A to these clinically challenging disorders are poorly understood. To better understand the neuronal function of BCL11A, we performed a series of preliminary behavioral analyses following the selective heterozygous deletion of Bcl11a from excitatory and/or inhibitory neurons in the mouse brain. Surprisingly, we found that Bcl11a deletion from inhibitory GABAergic interneurons (GINs) resulted in social deficits, hyperactivity, and increased seizure susceptibility. Furthermore, we observed increasing levels of BCL11A expression and physical occupation at predicted binding motifs during differentiation and maturation of GINs derived from human induced pluripotent stem cells (iPSCs). Additionally, we found that GIN-enriched ventral forebrain organoids derived from BCL11A-null iPSCs display differential gene expression signatures that overlap with pathological changes in the prefrontal cortex of postmortem brains of individuals with ASD and SZ. Taking these observations together, we hypothesize that the clinically challenging neurological phenotypes associated with BCL11A mutations likely reflect the specific impact of altered BCL11A function on different classes of neurons, with GINs being particularly vulnerable. We will test this hypothesis through a comprehensive series of in vitro (Aim 1) and in vivo (Aim 2) approaches. In Aim 1, we will identify and compare the gene targets of BCL11A in human iPSC-derived excitatory neurons and GINs. We will also employ single-nucleus (sn)RNA-seq and snATAC-seq along with whole-cell patch clamp electrophysiology and histology to establish the overlapping and distinct roles of BCL11A in human neuron populations. In Aim 2, we will further explore the in vivo function of Bcl11a by determining the behavioral and physiological effects of deleting Bcl11a in a neuron type-specific manner in mice. We will also use a chemogenetic approach to further interrogate GIN subtype-specific contributions to BCL11A disease mechanisms. Our long-term goal is to translate these findings into a better understanding of the role of BCL11A in the brain, which will help guide treatment development for patients with BCL11A dysfunction and other GIN-associated disorders.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Abstract Intestinal stem cells (ISCs) play pivotal roles in intestinal epithelium renewal during homeostasis and after injury. The metabolic demands faced by ISCs require high mitochondrial oxidative phosphorylation (OXPHOS) activity compared to other differentiated cells. ISCs’ mitochondrial dysfunction has been implicated in the etiopathogenesis of intestinal bowel diseases (IBD), which afflicts over 2 million people in the US. The carbon sources that fuel ISC OXPHOS have been broadly described in the small intestine (SI) but not in the colon. I seek to understand how ISC metabolic demands are met in the colon. The colonic epithelium is organized into the colonic crypt. ISCs localize to the base of the crypt (base-crypt), and this protects them from microbial metabolites and microbe-associated molecular patterns (MAMPs). Top-crypt differentiated colonic epithelial cells (CECs) oxidize microbial-derived short-chain fatty acid (SCFA) butyrate making it inaccessible to base-crypt cells. This shields ISCs, as butyrate suppresses ISC proliferation. This metabolic interaction between CECs and ISCs has focused my interest in CEC-ISC metabolic cross-talk. Ketones (acetoacetate, β-hydroxybutyric acid (βHB), and acetone) are important metabolic substrates. I hypothesize that CECs generate ketones that are used by ISCs as their principal energy source. This hypothesis is supported by the localization of rate-limiting enzymes (RLE) for the generation of ketones to the CECs and my preliminary data demonstrating that loss of these enzymes in CECs compromises ISC self-renewal and differentiation. My proposal focuses on this metabolic cooperation between epithelial cells within the crypt where top-crypt CECs shuttle ketones to base-crypt ISCs thus maintaining their turnover capacity. Understanding colonic ketone biosynthesis and function could lead to new treatments and therapeutic targets for IBD. Beyond defining this proposed metabolic crosstalk between CEC and ISC, I am interested in factors regulating CEC metabolic enzymes. I have identified microbial features that regulate the expression of the CEC RLE for ketone generation. I will determine the receptor and pathways downstream of this receptor by which they regulate the expression of this RLE. This research will help to decipher how microbial signals and metabolites contribute to epithelial repair and regeneration.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Stem cells and progenitors are integral for tissue development, homeostasis and regeneration, and their dysfunction underlie development disorders, defective tissue regeneration, aging and cancer. In addition to intrinsic mechanisms, diverse niche cells influence stem/progenitor cell fate. Adult tissues harbor heterogenous stem/progenitor cell subsets which are intricately involved in tissue regeneration and disease. Yet, the fundamental cell types and molecular mechanisms that regulate distinct tissue-specific stem/progenitor cell populations remain poorly understood. Using the murine mammary gland as a model, we were the first to uncover primitive mesenchymal progenitor niche cells that have a remarkable capacity to contribute to a major subset of tissue-specific epithelial progenitors, indicating their potential role in replenishing progenitor cells central to tissue regeneration. We have also observed that sympathetic neurons modulate progenitor subsets in the niche and epithelium to orchestrate tissue regeneration. These niche cell types permeate diverse somatic tissues and thus, understanding their control of tissue-specific progenitors is critical for advancing progenitor- targeted therapies in regenerative medicine and malignancy. The proposed research will investigate how tissue-specific progenitor cell fate is dictated by mesenchymal progenitors and sympathetic neurons during adult tissue regeneration. We will utilize the postnatal mouse mammary gland as a tractable model to study tissue-specific progenitors given that it undergoes extensive morphogenesis directed by progenitors. We will explore niche cell-mediated modulation of progenitors and identify underlying molecular signals and mechanisms. We will employ a variety of strategies that include in vivo genetic reporter, lineage tracing and cell ablation mouse models and ex vivo organoids combined with phenotypic, functional assays and next-generation sequencing. Our findings will bridge key gaps in knowledge of the niche-driven circuitry that impacts tissue progenitors and provide a framework for improving the outcome of diseases attributed to aberrant progenitors.

U.S. National Science Foundation

This program will advance scientific community datasets to enable scientific discovery and innovation using artificial intelligence (AI) and other methods. Its goal is to increase the value that can be derived from existing scientific datasets by leveraging novel methods and artificial intelligence (AI). This will unlock new AI-driven insights. It can enable interdisciplinary research. It also enables investigations outside of the original motivation for data collection and analysis.The program seeks to 1) apply AI-based capabilities to feature extraction and metadata generation, and the integration of multiple datasets. 2) Develop robust data pipelines necessary for automated analysis of existing datasets and similar use-cases by AI tools and systems. 3) Augment and/or harmonize existing datasets to better enable use by AI data pipelines and automated analysis. Proposals should address dataset security and integrity. Governance and the process for scientific communities to contribute to the datasets should also be addressed. Proposals are encouraged to leverage existing resources. These may include NSF data platforms, the NSF Integrated Data Systems and Services program, the NSF-led National AI Research Resource, the Genesis Mission platform, or other national infrastructure.NSF is open to exploring partnerships with philanthropy, private industry, or the non-profit sector to support additional proposals or collaborative opportunities that will advance AI-driven scientific discovery through unlocking the value of high-impact scientific datasets.Expanding Participation in STEM, NSF Priorities, and Gold Standard Science:NSF prioritizes cutting-edge discovery science and engineering research, advancing technology and innovation, and creating opportunities for all Americans. NSF has established priorities set forth by Congress, the administration and the NSF director to promote NSF's mission. Proposers should review the list of NSF priorities and are encouraged to align their proposals with them, where appropriate. NSF also expects the highest standards of scientific rigor, integrity and adherence tenets of Gold Standard Science in proposals, as appropriate for the field of science and research modality.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Type 1 diabetes (T1D) results from immune-mediated destruction of pancreatic beta cells. Increasing evidence suggests that beta cells actively contribute to their own demise through heightened immunogenicity, stress-induced neoantigen formation, and impaired immune tolerance. Enhancing beta cell resilience under inflammatory conditions remains a major challenge to the success of durable therapies, especially for cell replacement approaches. Our in vivo CRISPR/Cas9 screen identified ZBED3 — a transcription factor previously implicated in type 2 diabetes (T2D) — as a modulator of beta cell survival under autoimmune attack. Analysis of human islet single-cell RNA sequencing data revealed increased ZBED3 expression in beta cell subclusters associated with both T1D and T2D, whereas ZBED2, a related transcription factor with anti-inflammatory properties, is selectively absent in beta cells. Integration of public ZBED2 chromatin immunoprecipitation sequencing (ChIP-seq) and our in-house ZBED3 ChIP-seq datasets identified shared transcriptional targets involved in interferon signaling, FOXO-mediated transcription, and beta cell function. These findings suggest that ZBED family members may exert opposing regulatory effects on beta cell fate during autoimmune diabetes. This proposal aims to elucidate the transcriptional mechanisms underlying beta cell vulnerability by testing the hypothesis that ZBED2 and ZBED3 govern the balance between beta cell function, immune tolerance, and stress adaptation. Using genetically engineered beta cells derived from human induced pluripotent stem cells (iPSCs) and primary islets, we will pursue two specific aims: Aim 1. Define the role of ZBED3 in regulating beta cell immunogenicity, functional maturation, and stress response. Aim 2. Enhance immune tolerance by inducing ZBED2 and investigating its antagonistic effects on inflammatory signaling. This study combines gene editing, transcriptomic profiling, and T cell cytotoxicity assays to elucidate regulatory pathways governing beta cell survival. By establishing ZBED2 and ZBED3 as central regulators of beta cell fate, this work addresses a critical gap in T1D therapy and supports the development of transcription-based strategies to improve the durability of cell replacement interventions.

NIMH - National Institute of Mental Health

Project Summary Uncertainty-related cognitive dysfunctions are central to anxiety disorders, behavioral addictions, attention- deficit hyperactivity disorder, and schizophrenia, yet remain poorly understood. Anxious individuals, for example, are highly intolerant of uncertain situations, while those with gambling addictions often seek uncertainty, leading to loss-chasing behaviors. These diverse clinical presentations may stem from a fundamental computational challenge: the brain must simultaneously distinguish between two types of uncertainty—moment-to-moment stochasticity of observations and environmental volatility (how quickly underlying causes change)—that require opposite learning strategies. Previous work has focused on one factor or the other, but in reality, both volatility and stochasticity are unknown and potentially changing. Importantly, while they both increase experienced noise, they require opposite behavioral responses, making their dissociation both critical and computationally difficult and prone to systematic errors. Our recent work provides a computational framework for how the brain solves this challenge and how this process breaks down in psychiatric illness. In a large-scale neuroimaging program spanning three aims, we combine behavior, computational modeling, simultaneous fMRI-pupillometry, and causal arousal manipulation to elucidate neural mechanisms processing uncertainty while systematically manipulating both volatility and stochasticity across different outcome types. We will test specific hypotheses about the neurocomputational mechanisms of uncertainty processing (Aim 1), determine whether they are causally linked to arousal mediated by the locus coeruleus–norepinephrine system (Aim 2), and examine how these processes differ across outcome types (Aim 3), providing a mechanistic foundation for understanding uncertainty-related symptoms across psychiatric disorders.

NCI - National Cancer Institute

The incidence and mortality rate of penile squamous cell carcinoma (PSCC) is significantly higher in Puerto Rico compared to the continental United States (US). Puerto Rican men have approximately a three-fold higher incidence rate than non-Hispanic white men in the continental US. Our proposal will compare molecular profiles across 350 primary tumors representing distinct geographic areas to study the spectrum of disease prevalence and discover the possible biological cause of this health disparity. High-risk human papillomavirus genotypes (HR-HPV) are implicated in the carcinogenesis of PSCC through the action of viral E6 and E7 oncoproteins. E6 disrupts the p53 tumor suppressor pathway, while E7 inhibits the retinoblastoma protein (Rb) pathway, leading to increased expression of the p16 protein. Due to the ease of detecting p16 overexpression by immunohistochemistry (IHC), it serves as a marker for HR-HPV infection in PSCC. The World Health Organization recently recommended that p16 status be reported as a “requirement” for PSCC pathology, as it is a recognized biomarker for HR-HPV infection. However, various studies show that the expected concordance between p16 expression and HR-HPV infection may not hold true in high-incidence populations. We suspect that these discrepancies in concordance within such populations could stem from variations in the intratumoral microbiome or DNA methylation patterns. We hypothesize that microbiome profiles and DNA methylation patterns reflect stable reservoirs of biomarkers that indicate how HR-HPV influences the development and progression of PSCC. Our proposal seeks to utilize these markers as prognostic indicators of outcomes in populations with high rates of HR-HPV/p16 discordance. We will evaluate: Aim 1a. HR-HPV subtype using the Roche Cobas system; Aim 1b. p16 expression by IHC; Aim 1c. DNA methylation using reduced representation bisulfite sequencing (RRBS) and Aim 1d. microbiome using 16S rRNA sequencing. We will determine how the overall diversity of the tumor microbiome affects prognosis and how specific microbes correlate with p16 expression, HR-HPV infection, and clinical characteristics such as tumor stage, histological subtype, nodal stage, cancer-specific survival, disease-free survival, and overall survival. Similar correlations will be determined with DNA methylation patterns and specific loci. We are specifically interested in how these correlations differ between geographic areas to identify biological factors associated with PSCC health disparities. Our proposal could significantly change the biomarker used for determining HR-HPV infection in PSCC and could find relevant biomarkers for overall prognosis of PSCC in high incidence regions of the world.