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NIA - National Institute on Aging

Myelodysplastic Syndromes (MDS) are a group of bone marrow failure disorders caused by clonal expansion of hematopoietic stem cells (HSC) that fail to produce mature blood cells of sufficient quality and quantity. The disease-causing HSC bear acquired mutations that confer a selective advantage compared to other HSC but are detrimental to hematopoiesis. Typically, the mutations confer increased proliferation and survival upon HSC and their progeny, creating a hypercellular bone marrow with increased proportions of immature myeloid cells. In contrast, mutations in DDX41, an essential RNA helicase, cause reduced proliferation and survival of hematopoietic progenitors and yet contribute to about 4% of MDS cases. DDX41-mutated MDS most often occurs in individuals with inherited heterozygous mutations in the gene, 50-70% of which are truncating (frameshift or loss of translation start) and are thus considered loss-of-function. The other 30-50% of these are missense mutations, whose effect on protein function is largely unknown. The most common acquired mutation occurring in these patients is a second-hit mutation affecting the other allele of DDX41, typically causing the amino acid change R525H. Unique features of DDX41-mutated MDS include a hypocellular bone marrow, few co-mutations, and relatively slower disease progression. Our published and preliminary data indicate that the most common combination of DDX41 mutations observed in patients (truncating/R525H) causes a profound defect in hematopoietic progenitor cell proliferation and survival. Remarkably, our patient sequencing studies demonstrate that HSC bearing biallelic DDX41 mutations clonally expand and dominate the HSC pool, accounting for over 90% of HSCs in 11 out of 11 patients analyzed but only 5-25% of total bone marrow cells. These data and our published mouse models indicate that biallelic DDX41 mutations are favored in HSC but strongly detrimental to progenitor cells. Mechanistically, we found that DDX41 is required for ribosome biogenesis through its function in splicing at small nucleolar RNA (snoRNA) genes. Thus, biallelic DDX41 mutations confer reduced protein synthesis, which is a cause of the progenitor cell viability defect. HSC maintain a lower protein synthesis rate than progenitors, even when cycling, likely for protection from proteotoxic stress, which contributes to aging-associated decline of HSC and other tissue-specific stem cells. We hypothesize that biallelic DDX41 mutations are positively selected in aging HSC pools due to a reduction in proteotoxic stress. In the case of germline missense mutations, this requires dominant negative effects by the acquired R525H mutation to cause reduced protein synthesis and the associated stem cell expansion and progenitor cell defect. To test these hypotheses, we propose to determine the effect of combined germline and acquired missense DDX41 mutations on HSC function through analysis of MDS patient samples and mouse models, and then we propose to determine if proteotoxic stress is the driver of the clonal advantage of biallelic DDX41-mutated HSC in aging bone marrow through analysis of genetically and temporally precise mouse models of the disease.

NIAID - National Institute of Allergy and Infectious Diseases

Summary T cells are central to transplant rejection, driving allograft destruction through differentiation into effector cells. However, the mechanisms by which effector T cells sustain persistent alloimmune responses remain unclear. Our recent studies have identified a subset of “stem-like” T cells within the alloreactive pool. These stem-like T cells possess two fundamental features: self-renewal and the capacity for continuous differentiation into effector T cells. Importantly, terminal effector T cells, despite having all the cardinal features of effector activity, rapidly undergo apoptosis and fail to sustain graft rejection in vivo. This underscores the critical role of stem-like T cells, which continuously generate effector T cells to drive allograft rejection. Understanding the fundamental mechanisms regulating T cell stemness is a key question with significant therapeutic implications. Our preliminary data reveal that T cell stemness is epigenetically regulated by enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2). EZH2 functions as a histone methyltransferase, catalyzing the trimethylation of histone H3 at lysine 27 (H3K27me3), a key marker of gene repression. Deletion of Ezh2 in T cells completely abrogates their differentiation into effector cells. Furthermore, mice with T cell-specific EZH2 deletion (Ezh2fl/flCd4-Cre) or WT mice transiently treated with an EZH2 inhibitor (DZNep) accepted heart allografts long term (>100 days). These results suggest that EZH2 deletion/inhibition disrupts the stemness of alloreactive T cells, rendering them unable to sustain graft rejection. The central goal of this proposal is to elucidate how EZH2, an epigenetic repressor, regulates T cell stemness. We hypothesize that EZH2 preserves two key features of stem-like T cells: maintaining their long-term functional persistence and enabling their differentiation into effector cells. This hypothesis will be tested through two aims: Aim 1: Investigate whether EZH2-mediated repression of key transcription regulators is required for the differentiation of stem-like T cells into effector cells. Aim 2: Investigate whether EZH2 preserves T cell stemness by epigenetically repressing genes involved in apoptosis, cell cycle arrest, and functional exhaustion. Successful completion of these studies will uncover the epigenetic mechanisms governing stem-like T cell persistence and effector differentiation, providing a foundation for novel therapeutic strategies to improve transplant outcomes.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Clostridioides difficile infection (CDI) poses a significant public health burden, with approximately half a million cases annually in the United States and recurrence rates of up to 25%. CDI is primarily driven by disruptions to the gut microbiome, often due to antibiotic treatment, which creates an environment conducive to C. difficile (Cd) colonization. Current microbiome-based therapeutic strategies, such as fecal microbiota transplants (FMTs), have shown promise in restoring colonization resistance; however, their safety, reproducibility, and regulatory challenges limit widespread clinical application. While defined microbial consortia represent a promising alternative, their efficacy remains suboptimal. We hypothesize that these failures stem at least in part from an over-reliance on high-abundance (HA) species and an incomplete understanding of how low- abundance (LA) species contribute to microbiome stability and pathogen resistance. This project seeks to systematically evaluate the role of LA species in microbiome assembly and in conferring colonization resistance using a combination of synthetic microbial communities, metabolomics, and gnotobiotic mouse models. We hypothesize that LA species play crucial metabolic and ecological roles, through direct competition with Cd and by reinforcing community resilience under antibiotic-induced perturbations. To test this hypothesis, we will employ a defined yet complex synthetic community, mhCom, that encompasses both HA and LA species and assembles reproducibly in vitro and in vivo. Leveraging high-resolution metabolomics, we will (i) characterize the metabolic niches and functional redundancies of LA species and (ii) determine their role in resistance to Cd colonization and microbiome recovery and Cd suppression following antibiotic-induced CDI. In Aim 1, we will map the metabolic functions of LA species within mhCom in vitro, identifying privileged metabolic niches and cross-feeding interactions that contribute to community stability. We will use untargeted metabolomics and species dropouts to establish whether LA species and/or Cd fill metabolic voids when HA species are lost. In Aim 2, we will use gnotobiotic mice to assess the impact of LA species on C. difficile colonization resistance, determining whether the inclusion of LA species enhances pathogen exclusion both before and after antibiotic-induced microbiome disruption. By addressing a critical knowledge gap in microbiome ecology, this study has the potential to redefine microbiome-based therapeutics, either by demonstrating that LA species are not merely passive members of the gut community but essential contributors to microbiome resilience or by affirming strategies focused on HA species. The outcomes of this research will inform the rational design of next-generation microbial therapeutics with enhanced robustness against CDI and will have direct impact for other microbiome-related diseases, providing a foundation for safer, more effective, and precision-targeted microbiome interventions.

NCI - National Cancer Institute

Abstract The ability of tumor cells to adapt and survive endogenous and environmental stress is essential for tumor initiation and development. Glioblastoma (GBM) is the most malignant brain tumor, with high mortality and resistance to therapy. Within the heterogeneous GBM tumors, a highly tumorigenic subpopulation of Glioma Stem Cells (GSCs) drives tumor growth and promotes recurrence. GSCs can survive and proliferate in a relatively hostile tumor microenvironment, which triggers adaptive stress response mechanisms to restore protein homeostasis and promote tumor cell survival under abnormal conditions. Most forms of stress converge on one signaling pathway termed the integrated stress response (ISR), which signals through phosphorylating eIF2α (p-eIF2α; eukaryotic translation initiation factor 2α). Activation of the ISR causes a temporary shutdown of global protein translation and selective translation of cytoprotective transcripts. However, under prolonged stress, p-eIF2α promotes apoptosis. p-eIF2α halts protein synthesis by inhibiting eIF2B, which plays a key role in regulating mRNA translation and balancing the pro- and anti-survival effects of p-eIF2α. Our studies revealed a direct link between ISR signaling and response to several GBM therapeutics. Our central hypothesis is that the interplay between p-eIF2α and eIF2B determines therapeutic sensitivity and translation potential that drives tumor growth. We propose that while high levels of ISR effectively block global protein translation, mild ISR signaling, such as one caused by therapy, reprograms translation and selectively enhances the translation of a subset of mRNAs to confer cytoprotection and promote survival. To test our hypotheses and to address how conventional GBM therapies or the abnormal tumor microenvironment supports tumor progression and confers therapeutic resistance, we propose to carry out the following aims: Aim 1. Defining the role of eIF2B in translational regulation, tumor growth, and therapy response. Aim 2. Investigating the role and mechanism of translational reprogramming in GBM therapy response. Aim 3. Selective Targeting of aminoacyl-tRNA synthetases to enhance the efficacy of GBM Therapies. The proposed work will determine whether modulating the ISR could impair tumor growth and increase the efficacy of targeted therapeutics currently used in the clinic. Cell culture or in silico models cannot closely mimic the tumor environment, compounds pharmacokinetics and brain penetrance of small molecules, therefore the use of animals is warranted in our study.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary The incidence of stroke increases in women after their menopause, and stroke outcomes also become worse with aging in female stroke patients. It has been hypothesized that the exacerbated stroke phenotypes seen in elderly women are due to the loss of E2’s neuroprotection after menopause; however, the Women’s Health Initiative trial (WHI) has found E2 replacement therapy (ERT) had detrimental effects on stroke. Stroke scientists have argued that the women involved in the WHI trial were well passed their menopause when they received the ERT (late ERT or lERT), and have now shown ERT administered early after the menopause (early ERT or eERT) confers neuroprotection. Interestingly, for the past decade the increasing data have suggested that the second X chromosome (chromosomal effect) contributes to the worsened stroke outcomes in elderly women in addition to the decline of E2 levels (hormonal effect). Specifically, the X chromosome linked gene Kdm6a that escapes from X chromosome inactivation (XCI) in microglia has been found to up-regulate the histone H3K27me1 activational effect on the transcription of the pro-inflammatory IRF5 (interferon regulatory factor 5; responsible for microglial pro-inflammatory activation), leading to exacerbated stroke outcomes. In this proposal, we hypothesize that eERT in postmenopausal females inhibits Kdm6a gene escape from XCI, and suppresses the Kdm6a-H3K27-IRF5 signaling to ameliorate post-stroke inflammation and improve stroke outcomes. Aim 1 will use aged WT mice (in vivo) and in vitro assays (human induced pluripotent stem cells, hiPSCs), and test the hypothesis that the escape of Kdm6a from XCI and the Kdm6a-IRF5 signaling in aged females are repressed by eERT; whereas lERT will not have the same effects. Aim 2 will Test the hypothesis that eERT’s effect on post-stroke inflammation and outcomes is not Kdm6a allele dosage dependent but lERT's is. Microglial and astrocytic Kdm6a conditional knockout (CKO) female mouse models will be used to produce the knockout of one allele of Kdm6a in these cells. Kdm6a floxed females (two alleles of Kdm6a) will be included as a comparison. Aim 3 will examine if surgical menopause promotes Kdm6a escape from XCI in microglia. Ovariectomy will be performed in young and adult female mice to generate surgical menopause model. Kdm6a escape from XCI, Kdm6a-H3K27-IRF5 signaling, post-stroke inflammation and outcomes will be investigated in Aim 3. The goal of this project is to target XCI gene escape to suppress neuroinflammation after stroke.

NCI - National Cancer Institute

Project Summary Since 1990, the biennial International Workshop on Molecular Aspects of Myeloid Stem Cell Development and Leukemia has been the premier global meeting which spans fundamental stem cell biology and function, myeloid cell differentiation, marrow failure syndromes and myeloid cell malignancies. At the 2026 installment of the meeting (“MYELOID2026”), scientists with expertise in normal and abnormal hematopoiesis and clinicians who treat patients with myeloid leukemias, malignancies, and pre-leukemic disorders and also have active research programs in these diseases will achieve a better understanding of critical steps/factors that regulate hematopoiesis, their impact on transformation and disease resistance, and their potential relevance in clinical settings. MYELOID2026 will stimulate the community with collaborations on active projects, educate trainees, and “cross pollinate” critical and influential sectors of multiple myeloid biology fields. Moreover, the meeting size, meeting program, and ratio of trainees to faculty at the MYELOID meetings, provide trainee attendees ample opportunities for networking and faculty engagement for project discussions. We expect several landmark studies for the respective fields of normal hematopoiesis, stem cell biology, and myeloid malignancies to result from presentations by attendees. MYELOID2026 will bring together world renowned scientists, clinicians, and trainees to improve our understanding of hematopoietic development and differentiation, stem cells, and the evolution of myeloid malignancies.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT No changes are being made in this proposed supplement to the stated project summary. With the evolution of the Experimental Therapeutics Clinical Trials Network (ETCTN), the Johns Hopkins Translational Science Team (JHTST) supports 8 Affiliate Organizations (AO) under our Lead Academic Organization (LAO) with the goal to enhance and accelerate our collective contribution to the drug development efforts of the NCI. We have organized a comprehensive and cohesive infrastructure that can conduct high-quality clinical trials evaluating novel anticancer agents, in combinations, in molecularly selected patient populations, or rare tumor sites. Our infrastructure stems from the need to be clinically efficient, regulatory compliant and scientifically rigorous in our approach as we collaborate as network members within the ETCTN. Our infrastructure consists of seven experienced NCI-designated Cancer Centers: Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Emory Winship Cancer Institute, Georgetown Lombardi Comprehensive Cancer Center, Sidney Kimmel Cancer Center at Thomas Jefferson University (withdrawn), Memorial Sloan Kettering Cancer Center, University of Virginia Cancer Center, University of Wisconsin Carbone Cancer Center, University of Colorado Denver, University of Maryland Greenbaum Cancer Center, and most recently Wake Forest University. This collection of Centers brings together many unique discovery strengths, diverse patient volumes, and locations both urban and rural. We will focus on 4 specific aims: Aim 1- To lead and maintain a clinical trial consortium that will support cancer clinical trials from Phase 1 initiation through proof-of-activity and foster seamless Phase 2 clinical development of NCI CTEP IND agents; Aim 2- To actively participate and engage disease-focused clinical investigators in the ETCTN by promoting accrual to a range of ETCTN studies led by other LAO/AO members; Aim 3- To incorporate and implement innovative correlative and biological laboratory studies in the context of or as eligibility for participation in early phase clinical studies that enhance our understanding of determinants of toxicity and response that will be used for further definitive practice-changing clinical trial evaluation; and Aim 4- To train the next generation of investigators in drug development. With 9 centers within our consortium, we anticipate no problem in meeting the grant metrics of 100 accruals per year, submitting enough LOI concepts to have 6 new studies approved each year, functioning as a network by having Disease-Focused Clinical Investigators champion ETCTN studies at each site across disease sites, and to provide opportunities to engage and train early career investigators in drug development. We anticipate that our contributions within the ETCTN will impact the clinical care of cancer patients.

OD - NIH Office of the Director

PROJECT SUMMARY Aging impacts many functions within cells, from genomic instability to oxidative damage to shifted cellular metabolism. Similarly, collections of cells change in their relative composition with age—a phenomenon mainly driven by clonal selection; this process has been extensively studied in the hematopoietic system, where a clear correlation between clonality changes and age has been described. While these changes are sometimes precursors to blood malignancies, they are much more commonly observed in their absence and have been correlated with changes in inflammation status, as well as cardiovascular, liver, and Alzheimer’s disease. Counterintuitively, this so-called ‘clonal hematopoiesis of indeterminate potential’ (CHIP) has been associated with both adverse and beneficial outcomes, suggesting a currently unresolved dichotomy. Individuals with Down syndrome (DS) exhibit global changes in immune activation and altered hematopoiesis that frequently result in transient preleukemia and elevated leukemia risk. Connecting clonal hematopoiesis to hematological malignancies, increased levels of CHIP have been observed in individuals with DS; critically, the frequency is both increased and significantly earlier in life than expected. One limitation of current CHIP analysis is its phenotype based on the presence or absence of known driver mutations or large-scale structural variants. However, changes in clonality may presage or be independent of CHIP and are available through the genome-wide detection of ‘neutral’ mosaic variants. Thus, we hypothesize that clonal mosaicism of the hematopoietic system (CM-HS) represents a quantitative trait with increased sensitivity to detect blood clonality alterations for individuals with DS. To test this hypothesis and implement this framework, we plan to leverage local resources through the Linda Crnic Institute, which harbors a biobank of samples from individuals with DS with matched genome sequencing data through the Gabriella Miller Kids First (GMKF) program. We will first analyze ~450 readily available 60× coverage whole- genome data sets employing our novel approaches for mosaicism detection. Our team also has access to detailed clinical and multi-omics data; this will allow the correlation of CM-HS with clinical and functional phenotypes and establish a biosignature of blood clonality changes in DS (Aim 1). Subsequently, we will migrate our bioinformatic toolkit to the CAVATICA platform to perform analyses across the available Down syndrome data (~1,700 cases) and matched controls through GMKF (Aim 2). Together, these analyses will provide novel insight into blood clonality changes in DS through CM-HS detection. Based on our expectations and preliminary data, we expect that CM-HS can reflect and possibly predict alterations in the hematopoietic system—be it through inflammation or stem cell selection. The work proposed here will allow us to develop the analytical framework for CM-HS analysis, employ it across a deeply phenotyped smaller cohort to determine associations, as well as a population-scale cohort.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

ABSTRACT Systemic autoimmune diseases such as systemic lupus erythematosus (SLE) are mediated by autoantibodies against key tissue constituents, accompanied by the activation of innate immune system. In addition, systemic autoimmunity is frequently associated with hematological complications such as lymphopenia, anemia and/or thrombocytopenia, which can be debilitating and even life-threatening. These abnormalities are typically considered as isolated symptoms caused by autoantibodies against the respective blood cell types, and are treated by immunosuppressive therapies. On the other hand, it is possible that frequent hematopoietic abnormalities in SLE may reflect a defect in the source of hematopoiesis, i.e. hematopoietic stem cells (HSC) and/or progenitors. This model has important implications for the pathogenesis and treatment of SLE; however, it remains to be supported by genetic and mechanistic evidence. Our preliminary studies suggest that the bone marrow from mice with SLE-like disease showed impaired ability to reconstitute irradiated recipients. Moreover, HSCs in moribund mice showed increased proliferation and upregulation of transcripts associated with stem cell exhaustion. We therefore hypothesize that clinical SLE-like disease impairs the activity of HSC, which may further exacerbate hematological abnormalities and inflammation. This hypothesis will be tested using two Specific Aims. In Aim 1, we will characterize HSC impairment in a mouse model of SLE-like disease, including its cell-extrinsic nature and relevance to endogenous hematopoiesis. In Aim 2, we will characterize the epigenome of disease-affected HSC, as well as molecular pathways that cause HSC impairment in this model. Collectively, these studies may support the paradigm of impaired HSC activity as a source of hematopoietic abnormalities and "trained autoimmunity" in SLE. As such, they may pave the way for future studies of stem/progenitor function in human SLE and of its potential therapeutic modulation.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY The National Summer Undergraduate Research Project (NSURP) was created as an eight-week virtual summer research program in the microbial sciences that matches underrepresented minorities in STEM undergraduate students nationwide with faculty mentors worldwide to address the barriers presented by COVID-19 cancelations. NSURP mentees conduct research from any location with an internet connection, allowing students to learn how to conduct impactful research by forming and testing hypotheses. As individuals returned to in-person programs, NSURP found a population unable to travel, independent of COVID-19, due to financial, familial, geography, and health constraints. As we see an increased demand from these individuals, post-pandemic, who can’t travel to in-person opportunities, we seek to expand our ability to serve these undergraduates with research opportunities in our program and provide them with year-long mentoring and professional development. NSURP seeks to apply its virtual research model to human health-related topics, such as the basic and applied disease research conducted within the purview of the NIAID, both in microbiology and immunology. Implementing NSURP within the NIAID infrastructure will create more opportunities for URM scientists and ultimately aid in developing a diverse biomedical research workforce. The central premise of NSURP is to meet undergraduates where they are, facilitate exposure and pursuit of a scientific opportunity, and give them the tools and confidence to continue in their scientific careers. To maximize the contribution of NSURP within the NIAID mission, we will 1) provide virtual biomedical-specific summer research opportunities within the NIAID mission for minoritized undergraduates, 2) facilitate synergistic virtual scientific and career multi-level mentoring relationships, and 3) assess program impact by evaluating educational outcomes of NSURP participants and conducting longitudinal studies on the effects of culturally-responsive virtual mentorship training.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Infectious stress and damage are important concepts; however, we still lack understanding of how immunity and microbial virulence result in cellular stress, and how this stress in turn influences the immune response. Using a powerful model system, Drosophila melanogaster, we have recently found that phosphorylation of histone H2a (γH2av) is a hallmark of microbial infection (oral infection, septic injury). γH2av is considered a marker of DNA damage response (DDR) downstream of DDR kinases (ATM, ATR, DNA-PKc). We found that pathogens induce host γH2av accumulation in a virulence-, NOX- and ATM kinase-dependent manner, but without DNA damage. We have also found that ATM-mediated signaling promotes inflammatory cytokine production, alters cell dynamics and strongly promotes survival to infection. Based on these results, we hypothesize that ATM- mediated signaling is a central stress response upon infection, altering cytokine production and modulating cell death and survival. To test these hypotheses, we propose the following specific aims: Aim1: We will determine the microbial characteristics (immunogenicity, virulence/tissue damage) that induce γH2av accumulation in midgut enterocytes (ECs). In parallel, we will analyze the role of NADPH oxidases (Duox, NOX) on triggering the ATM pathway. Finally, we will elucidate how cellular age influences the ability of a cell to trigger ATM signaling upon infection. Aim2: We will analyze the impact of ATM signaling in enterocytes where it regulates inflammatory cytokine production, cell dynamics and tissue repair. We will clarify the cell-autonomous requirement of ATM signaling for enterocyte elimination, cytokine induction and tissue repair using a combination of FACS, RNA sequencing, functional genetics, cell tracing and pharmacological treatments. Aim3: We will investigate how ATM signaling decreases host survival upon septic injury. We will first elucidate and characterize spatial-temporal activation of ATM signaling in response to systemic infection and identify key regulators involved. We will determine the mechanisms leading to host lethality downstream of ATM signaling. Finally, we will investigate whether γH2av itself mediates some of the influence of ATM signaling upon infection. This project will demonstrate that NOX-ATM signaling acts as a new central regulator of infectious stress and immunity. Our multi-omics approach coupled with tissue-specific functional genetics and unparalleled Drosophila lineage tracing and clonal systems will help clarify both the upstream regulators and downstream effects of this pathway. Importantly, our work will also show how a stress response can increase or decrease survival to infection in different tissue and infection contexts. We will demonstrate that NOX-ATM is not only central for the control of cytokine production and innate immunity, but also a key regulator of cell death and stem cell-mediated repair. Finally, our research may open new avenues of research on cellular aging and immune stress responsiveness. The key characteristics by which pathogens influence innate immunity are largely conserved, therefore we believe that our studies pave the way to develop strategies aiming to improve the outcome of microbial infections. 1

U.S. National Science Foundation

NSF is committed to securing the nation's research enterprise as part of its core mission. The Research on Research Security (RoRS) program will advance the understanding of the full scope, potential, challenges, and nature of the research on research security field through scholarly evidence. Background The following activities provide background and context for developing proposals to submit to the RoRS program. <ul> <li>The foundational legislative and policy documents include <a href="https://trumpwhitehouse.archives.gov/presidential-actions/presidential-memorandum-united-states-government-supported-research-development-national-security-policy/">National Security Presidential Memorandum-33 (NSPM-33)</a>and its associated supporting documents, as well as research security provisions in CHIPS and Science Act 2022.</li> <li>In 2022 NSF asked JASON to consider what a research program on research security might entail and how it would be defined.The findings are summarized in the report (JSR-22-08), <a href="https://nsf-gov-resources.nsf.gov/2023-03/JSR-22-08%20NSF%20Research%20Program%20on%20Research%20Security_03152023_FINAL_1.pdf?VersionId=lwtxqUjbqGNmbtJ7E66IqQBbt9gzCV8A">Research Program on Research Security</a>.</li> <li>The 2024 NSF-funded workshop, <a href="https://www.nsf.gov/cgi-bin/good-bye?https://www.bakerinstitute.org/research/responsible-collaboration-through-appropriate-research-security">Responsible Collaboration Through Appropriate Research Security</a>: A Workshop To Discuss and Study the Emergent Discipline of Research on Research Security, identified current themes, major issues, and challenges in research security.</li> </ul> Program Description Collectively, the research that RoRS funds will foster a broad community that builds collaborations between the STEM research community, research security researchers, and research security practitioners. Interdisciplinary approaches are encouraged, and proposers should address how they will leverage the range of expertise, theories, and methods of the team to engage in evidence-based research on research security. Proposers are encouraged to identify collaborators across a wide range of sectors, and to consider projects in collaboration with international partners that share U.S. concerns with research security, when appropriate. RoRS encourages the following types of proposals to help build the emerging field of research on research security. (See the <a href="https://www.nsf.gov/policies/pappg">PAPPG</a> for guidance on preparing specific proposal types.) <ul> <li>Conferences and Workshops</li> <li>Planning Grants</li> <li>Early-concept Grants for Exploratory Research (EAGER)</li> </ul> Proposal topics may include, but are not limited to, the following: <ul> <li>The nature and pervasiveness of research security threats.</li> <li>Methods for identifying research security risks, and strategies for preventing and mitigating them.</li> <li>Methods for strengthening research security protocol and approaches.</li> <li>The complex relationships between human behavior and research security policies.</li> <li>Research security policies and their implications.</li> <li>Research on organizational change around systemic and cultural factors as they pertain to research security.</li> <li>Research on research security in the context of a particular field or discipline, especially in high-risk areas.</li> <li>The international dimensions of research security.</li> </ul> Collectively, RoRS seeks to fund research projects with the following characteristics: <ol> <li>Produce data, analysis, theory, and tools that inform current and future decision-making on U.S. research security.</li> <li>Use rigorous empirical methods to advance understanding of the factors that influence research security.</li> <li>Build upon established methodologies from diverse fields of study to ensure that RoRS develops quickly and efficiently into a robust, mature discipline with its own novel approaches.</li> <li>Develop innovative strategies to leverage previously unidentified, unconnected, and/or inaccessible sources of data.</li> </ol> Prospective PIs are strongly encouraged review <a href="https://new.nsf.gov/research-security">NSF Research Security</a> resources and to contact the cognizant RoRS program director(s) prior to submission. Proposals should be prepared and submitted following the guidance in the <a href="https://new.nsf.gov/policies/pappg">NSF Proposal &amp; Award Policies &amp; Procedures Guide (PAPPG)</a>.

U.S. National Science Foundation

NSF is committed to securing the nation's research enterprise as part of its core mission. The Research on Research Security (RoRS) program will advance the understanding of the full scope, potential, challenges, and nature of the research on research security field through scholarly evidence. Background The following activities provide background and context for developing proposals to submit to the RoRS program. The foundational legislative and policy documents include National Security Presidential Memorandum-33 (NSPM-33)and its associated supporting documents, as well as research security provisions in CHIPS and Science Act 2022. In 2022 NSF asked JASON to consider what a research program on research security might entail and how it would be defined.The findings are summarized in the report (JSR-22-08), Research Program on Research Security. The 2024 NSF-funded workshop, Responsible Collaboration Through Appropriate Research Security: A Workshop To Discuss and Study the Emergent Discipline of Research on Research Security, identified current themes, major issues, and challenges in research security. Program Description Collectively, the research that RoRS funds will foster a broad community that builds collaborations between the STEM research community, research security researchers, and research security practitioners. Interdisciplinary approaches are encouraged, and proposers should address how they will leverage the range of expertise, theories, and methods of the team to engage in evidence-based research on research security. Proposers are encouraged to identify collaborators across a wide range of sectors, and to consider projects in collaboration with international partners that share U.S. concerns with research security, when appropriate. RoRS encourages the following types of proposals to help build the emerging field of research on research security. (See the PAPPG for guidance on preparing specific proposal types.) Conferences and Workshops Planning Grants Early-concept Grants for Exploratory Research (EAGER) Proposal topics may include, but are not limited to, the following: The nature and pervasiveness of research security threats. Methods for identifying research security risks, and strategies for preventing and mitigating them. Methods for strengthening research security protocol and approaches. The complex relationships between human behavior and research security policies. Research security policies and their implications. Research on organizational change around systemic and cultural factors as they pertain to research security. Research on research security in the context of a particular field or discipline, especially in high-risk areas. The international dimensions of research security. Collectively, RoRS seeks to fund research projects with the following characteristics: Produce data, analysis, theory, and tools that inform current and future decision-making on U.S. research security. Use rigorous empirical methods to advance understanding of the factors that influence research security. Build upon established methodologies from diverse fields of study to ensure that RoRS develops quickly and efficiently into a robust, mature discipline with its own novel approaches. Develop innovative strategies to leverage previously unidentified, unconnected, and/or inaccessible sources of data. Prospective PIs are strongly encouraged review NSF Research Security resources and to contact the cognizant RoRS program director(s) prior to submission. Proposals should be prepared and submitted following the guidance in the NSF Proposal &amp; Award Policies &amp; Procedures Guide (PAPPG).

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Pancreatic islets are essential for glucagon and insulin secretion from - and -cells, respectively, to maintain glucose homeostasis. During diabetes mellitus, chronic dysglycemia results from autoimmune destruction (Type 1) or dysregulation (Type 2) of pancreatic β-cells. While treatments such as exogenous insulin manage disease symptoms, there is currently no cure. Future therapies may include the transplantation of stem cell-derived islet- like cells, but success requires a deeper understanding of pancreas development. Pancreatic organogenesis occurs in three phases – the primary, secondary, and tertiary transitions – to drive multipotent progenitor cell initiation, cellular differentiation, and islet maturation, respectively. Notably, many studies identified transcription factors (TFs) required for pancreatic organogenesis and differentiation. TF activities also require interacting transcriptional co-regulators, but the role of co-regulators in pancreas development is largely undescribed. We study the Islet-1 (Isl1) TF, which is a regulator of islet development and function. Previously, we found that Isl1 interacts with co-regulators Ring finger (Rnf)20 and Rnf40 in β-cells. RNF20 and, less robustly, Rnf40 are ubiquitin ligases that act as homo- or heterodimers required for the monoubiquitination of histone 2B, termed H2Bub1. H2Bub1 precedes H3K4 trimethylation to generally activate gene targets. Upon the deletion of Rnf20 in adult -cells, we observed -cell dysfunction, including glucose intolerance and fasting hyperglycemia. Because of the known roles for Isl1 in embryonic pancreas development and its interaction with Rnf20 in adult -cells, we tested for the requirement of Rnf20 and downstream H2Bub1 in the early embryonic mouse pancreas. Strikingly, we observed severe pancreatic dysfunction in pancreas-specific Rnf20-deficient neonates (Rnf20Flox;Pdx1-Cre - termed Rnf20panc) as indicated by hyperglycemia and loss of plasma insulin. Overall, these observations suggest a novel requirement for Rnf20 (and H2Bub1) in pancreatic organogenesis and neonatal islet function. For this training plan, I propose to investigate the role of Rnf20 during pancreas development and identify the genes directly or indirectly impacted by Rnf20. My overarching hypothesis is that Rnf20 promotes pancreatic development beginning after the initial budding stage and establishes the proliferative and morphological dynamics in the early secondary transition. I will assess pancreatic development and endocrine cell identity (e.g., insulin and glucagon) markers by immunofluorescence, as well as proliferation and apoptosis, and assess neonatal glucose homeostasis. In Aim 2, I will characterize Rnf20, Isl1, and H2Bub1 loci occupancy using CUT&RUN and assess gene expression changes impacted by Rnf20 loss via scRNA-seq. Together, I will evaluate the transcriptional consequences of pancreas-specific Rnf20 loss. The results of this research will increase our understanding of mammalian pancreas development and advance future diabetes treatments, like stem-cell therapy.

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

PROJECT ABSTRACT The development of the human placenta depends on proper differentiation of the epithelial trophoblasts. Defects in trophoblast differentiation, particularly toward the extravillous trophoblast (EVT) lineage, are associated with adverse pregnancy outcomes. However, the mechanisms that regulate the differentiation of the germinative cytotrophoblasts to EVTs remain unknown. The imprinting disorder Beckwith-Wiedemann Syndrome (BWS) is characterized by abnormal EVT morphology and can offer insights into candidate regulators of EVT differentiation. BWS is most often caused by dysregulation of a cluster of imprinted genes whose expression from the maternal allele is controlled by the imprinting control region IC2. In the human placenta, the IC2- regulated genes are CDKN1C, PHLDA2, SLC22A18, and KCNQ1. Both CDKN1C and PHLDA2 are upregulated over the course of EVT differentiation, are associated with placental phenotypes in knockout mice, and have been previously shown to influence the activity of the transcription factor ASCL2, a critical regulator of EVT differentiation. Therefore, CDKN1C and PHLDA2 most likely drive the EVT phenotypes in BWS. However, the mechanisms underlying the role of the IC2 genes in EVT differentiation and the interactions between these genes remain unexplored. In Aim 1, I will establish the role of the IC2 genes in EVT differentiation. I hypothesize that CDKN1C and PHLDA2 act synergistically to promote EVT differentiation by altering the activity of the transcription factor ASCL2. I will investigate this hypothesis by knockdown of the IC2 genes in human trophoblast stem cells, which can be differentiated into EVTs in vitro, and by characterizing EVTs in BWS patient placentas. Aside from their role in EVT differentiation, the factors that regulate the expression of CDKN1C and PHLDA2 in this process are unknown. Both genes are upregulated in EVT differentiation without changes in methylation at their promoters or the imprinting control region IC2. This implies that there are uncharacterized enhancers regulating the expression of CDKN1C and PHLDA2. For many imprinted genes, promoter-enhancers interactions are mediated by CTCF-dependent contacts with their imprinting control region; however, the CTCF landscape of the IC2 region during EVT differentiation has not been explored. In Aim 2, I will establish the mechanism regulating CDKN1C and PHLDA2 expression in EVT differentiation. I hypothesize that CDKN1C and PHLDA2 are regulated in EVT differentiation by uncharacterized enhancers and that contact with these enhancers depends on contact with IC2 through CTCF-mediated chromatin looping. To test this hypothesis, I will investigate the impact of maternal IC2 deletion and CTCF depletion on IC2 gene expression and EVT differentiation efficiency in iPSC-derived human trophoblast stem-like cells. I aim to establish the role and regulation of the IC2 imprinted cluster in EVT differentiation. By identifying novel regulators of EVT differentiation, I will improve our understanding of this fundamental process in placental development.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Abstract Myelodysplastic syndromes (MDS) are a group of diverse malignant hematological disorders that originate from hematopoietic stem cells (HSCs). Increased levels of reactive oxygen species (ROS) and DNA damage are commonly detected in hematopoietic cells from MDS patients. An elevated level of ROS, generated from either endogenous or exogenous sources including oncogene activation, leads to loss of quiescence and self-renewal of HSCs. ROS-induced DNA damage speeds up the aging process of stem cells and contributes to the mutagenesis associated with cancer development. m6A RNA methylation plays a significant role in multiple biological processes by introducing another layer of post-transcriptional regulation of gene expression within cells. The goal of this project is to elucidate the significant role of ALKBH5-mediated epigenetic regulation in the maintenance of genomic stability in hematopoietic stem/progenitor cell (HSPCs) during oxidative stress, and how deregulation of ALKBH5 contributes to promotion of leukemic transformation of HSPCs in the initiation and development of MDS. We found that ROS significantly increased global m6A RNA methylation in human cell lines, and that the elevation of m6A mRNA methylation is required for rapidly repairing ROS-induced DNA lesions and preventing cell death. Interestingly, we found that ALKBH5, the m6A RNA demethylase, is responsible for ROS-induced elevation of m6A mRNA methylation. ROS induced post- translational modification of ALKBH5, and inhibited the demethylase activity of ALKBH5. We showed that forced expression of ALKBH5 inhibited ROS-induced m6A mRNA methylation and significantly delayed repair of ROS-induced DNA damage. Thus, we hypothesize that aberrant expression of ALKBH5 disrupts HSPC functions by negatively influencing genome integrity and survival of HSPCs, thereby contributing to leukemic transformation of HSPCs during the initiation and development of MDS. In this proposal, we will determine 1) the role and underlying mechanism of ALKBH5 in the maintenance of genomic stability in HSPCs in response to oxidative stress; 2) the effects of ALKBH5/Alkbh5 overexpression on the maintenance of mouse and human primary HSPCs during ROS stress in vivo; and 3) whether ALKBH5/Alkbh5 is required for the maintenance of pre-leukemic stem cells (pre-LSCs) in MDS. Our study will provide new insights into novel mechanisms of MDS development and epitranscriptional regulation of gene expression in HSPCs in response to oxidative stress. Additionally, our study will provide the first set of evidence to support a significant role of ALKBH5- mediated m6A mRNA demethylation in the maintenance of normal HSPCs and pre-leukemic stem cell (pre- LSCs).

NCI - National Cancer Institute

Breast tissue is dynamic and undergoes significant structural changes throughout a woman's life. The breast tissue architecture is maintained by a population of stem cells with self-renewal capacity which are essential for tissue repair and remodeling. Recently, the role of stem cells in breast carcinogenesis has been recognized as a high priority for translational breast cancer (BCa) research. The stem cell hypothesis of breast carcinogenesis suggests that breast cancer development might be directly related to the size of the stem cell pool and its mitotic activity. Further, in the mammary gland, stem cells are the only cell subpopulation that has capacity to accumulate all the oncogenic alterations. This project will fill gaps in our understanding of the role of stem cells in the early stages of breast carcinogenesis and their interplay with BCa risk factors by focusing on co-stained stem cell marker combinations that are reflective of high-risk stem cell lineages. Specifically, we will explore: 1) the associations of high-risk stem cell lineages (CD44+/CD24-/low, EpCAM-/low/CD49fhigh and ALDH1A1high/CD44+/CD133+) in histologically normal breast tissue with mammographic percent density and AI- based mammographic features (n=1,290); 2) the associations of high-risk stem cell lineages with subsequent BCa risk in women with a previous benign biopsy (444 cases/1,003 controls); and 3) the associations of hormone-related factors (oral contraceptives, menopausal hormone therapy, alcohol use, body mass index, weight gain since age 18, and reproductive factors) with high-risk stem cell lineages (n=1,834). We will use prospectively collected data/samples/ mammograms from cancer-free women with benign breast disease (BBD) within the Nurses' Health Study (NHS), NHS II, Washington University's Women's Health Repository (WHR), and a unique collection of data/samples/mammograms from healthy women in the Komen Tissue Bank, with the final study population representative of US demographics. Stem cell markers will be stained on study tissue microarrays (specifically constructed from histologically normal breast tissue regions) with commercially available antibodies using multiplex immunofluorescence, and the staining results will be evaluated with automated image analysis. We propose a highly novel investigation that will comprehensively examine the role of stem cell markers in breast carcinogenesis. The study aims to shed light on molecular pathways behind the observed associations of risk factors with BCa risk as well as to identify markers that could advance future risk prediction in a large segment of high-risk women undergoing routine breast biopsies and those with high-risk mammographic features. This proposal could pave the way for novel personalized breast cancer prevention and surveillance strategies. As stem cell activity is potentially modifiable via a variety of targeted therapies, the findings could translate into stem cell -directed pharmaceutical interventions aimed at BCa risk reduction in high-risk women with BBD and/or high mammographic breast density in whom novel prevention strategies are urgently needed.

NIA - National Institute on Aging

ABSTRACT The motor symptoms associated with Parkinson’s Disease (PD) are caused by a progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), which act as key modulators in the nigrostriatal circuitry. Currently, there is no explanation for what causes this cell vulnerability. A key physiological feature of DA neurons is that they pacemake – they fire action potentials (AP) autonomously without the need for synaptic input. Pacemaking is essential for their functionality but is also energetically demanding, which puts them on an ‘energetic cliff’ that may increase their likelihood of cell death. KCNS3 is a high-risk PD gene that is highly expressed in the highly vulnerable SNpc DA neurons. KCNS3 encodes KCNS3, a K+ channel modulatory subunit. In other brain regions, KCNS3 has been shown to regulate action potential firing, but its role in SNpc DA neurons and how it might contribute to SNpc vulnerability is completely unknown. My preliminary data in stem-cell derived midbrain DA neurons indicate that in the absence of KCNS3, these cells have changes in action potential shape and pacemaking frequency. Additionally, these DA neurons experience greater cell death. Based on these results, my central hypothesis is that KCNS3 is a key regulator of SNpc DA neuron pacemaking and that its downregulation increases their vulnerability and PD pathophysiology. To test this hypothesis, I will carry out two Aims. In Aim 1, I will determine how KCNS3 affects DA neuron intrinsic excitability and vulnerability. Using in vitro human embryonic stem cell (hESC) derived midbrain DA neurons, I have generated a knockout of KCNS3, and I will use this construct, along with wild type DA neurons, to define the role of KCNS3 in regulating pacemaking using patch clamp electrophysiology, cell vulnerability using cell death assays, and pacemaking-driven Ca2+ influx using 2-photon calcium imaging. I will also test comparable factors in native SNpc neurons in mice, using stereotaxic injections to virally knock down KCNS3 expression in SNpc DA neurons. In Aim 2, I will look at how the loss of KCNS3 in SNpc DA neurons affects downstream nigrostriatal circuitry and the generation of a parkinsonian behavioral phenotype. For this, I will use the in vivo mouse model mentioned above to look at changes in basal dopamine release and subsequent changes to striatal circuitry. In addition, I will conduct motor behavioral paradigms in these mice to study KCNS3’s impact on motor control. This proposed research will uncover crucial insights into how the functionality of SNpc DA neurons influence the pathophysiology of PD.

NINDS - National Institute of Neurological Disorders and Stroke

ABSTRACT The motor symptoms associated with Parkinson’s Disease (PD) are caused by progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Currently, there is no explanation for what causes this selective cell vulnerability. In addition to age, environmental factors (e.g. exposure to neurotoxins) also increase the risk for PD, but the mechanisms contributing to vulnerability are poorly understood. Further, there are no available treatment options that effectively prevent or slow DA neuron loss or the progression of related symptoms. Thus, there is a significant need for molecular dissection of the key pathways involved in regulating susceptibility to degeneration in order to identify novel potential drug targets and develop improved treatment options for PD patients. Myocyte enhancer factor 2A (MEF2A) was identified as a genetic master regulator whose activity is decreased in the DA neurons of MPTP treated mice. Dysregulation of MEF2A activity is thought to underlie DA neuron vulnerability. Inactivation of MEF2A can occur through phosphorylation of serine 408 (S408) in the carboxy terminal of the protein. The kinases involved in this phosphorylation are known to be activated by environmental exposure to neurotoxins which disrupt mitochondrial function and energy production. My preliminary data in stem cell-derived midbrain DA neurons indicates that in the absence of MEF2A, these cells experience significant changes in the expression of genes associated with synaptic signaling, maintenance of membrane potential, regulation of cell cycle processes, and DNA metabolic processes. My central hypothesis is that MEF2A is a critical regulator of vulnerability because it regulates the expression of other genes necessary for proper DA neuronal function, and that dysregulation of MEF2A activity underlies the DA neuron vulnerability associated with PD pathogenesis. To test this hypothesis, I will carry out two Aims. In Aim 1, I will determine how MEF2A affects intrinsic DA neuron vulnerability. Using in vitro human embryonic stem cell (hESC)-derived midbrain DA neurons, I have generated an inducible MEF2A knockout cell line. I will use this line, along with wild type (WT) DA neurons, to define the role of MEF2A in cell vulnerability using cell death assays, immunohistochemistry, senescence assays, patch clamp electrophysiology, live Ca2+ imaging, and fluorescent mitochondrial ROS assays. In Aim 2, I will look at how environmental factors contribute to MEF2A activity dysregulation and SNpc DA neuron vulnerability. For this, I will use in vitro neurotoxin and kinase inhibitor treatments along with cell death assays and quantitative western blotting to look at changes in survival as well as levels of phosphorylated MEF2A (p-MEF2A) in both WT and MEF2A-KO DA neurons. This proposed research will provide crucial insights into the molecular and cellular mechanism underlying SNpc DA neuron vulnerability and inform future avenues of investigation for the development of preventative treatments for PD patients.

NCI - National Cancer Institute

Project summary/abstract Richter Transformation (RT) is an aggressive, therapy-refractory large cell lymphoma that arises in up to 10% of patients with chronic lymphocytic leukemia (CLL). RT represents a major unmet need in CLL management, as patients are largely refractory to existing therapies and have a median overall survival of less than 12 months. Immunotherapies, including immune checkpoint blockade (ICB), have shown promise in RT, but responses often display limited duration. The objective of this study is to elucidate the molecular function of the MAX dimerization protein MGA, a gene that is disrupted by inactivating mutations leading to loss-of-function (LOF) in ~29% of RT cases. Our long-term goal is to improve the efficacy of immunotherapy-based combination treatments for this currently incurable malignancy, tailored to the molecular characteristics of patients. We hypothesize that MGALOF regulates tumor cell fitness by controlling growth programs and inflammatory signaling, thereby promoting B-cell clonal expansion and immune evasion. This hypothesis is supported by preliminary data showing that MGA-LOF leads to de-repression of transcription factors involved in proliferation and inflammatory cytokine production in both human tumors and murine models. In murine models faithful to the CLL-to-RT progression, Mga-LOF drives clonal selection of malignant B cells and promotes the formation of a PD-1+ T-cell rich tumor microenvironment (TME). In this study, we will pursue two specific aims: 1) define the mechanisms through which Mga-LOF favors B-cell fitness during the CLL transformation into RT; 2) determine how Mga-LOF driven inflammatory signaling rewires the RT-TME, predisposing Mga-LOF RT to anti-PD-1 ICB-based combination therapies. These questions will be addressed through integrated studies of faithful murine models of the CLL-to- RT evolution, isogenic human cell lines, and genetically annotated patient samples collected longitudinally during CLL progression or at time of RT. As primary RT samples are generally scarce and mostly available as fixed, non-viable, tissue, they preclude ex vivo manipulation and longitudinal functional studies. Our novel immunocompetent murine models provide a necessary and physiologically relevant platform to investigate the molecular mechanisms underlying the stepwise CLL-to-RT evolution, enabling the preclinical evaluation of novel therapeutic strategies. We have assembled a multidisciplinary team to support this work, including experts in computational biology (Landau), stem cell biology and epigenetics (Apostolou), and tumor immunology (Zappasodi). Collaborations with clinicians (Furman, Thompson, Inghirami, Parry) will provide access to genetically annotated patient specimens and spatial transcriptomics datasets from ongoing immunotherapy clinical trials. This research is significant because it will reveal critical biological mechanisms underlying CLL transformation and provide a preclinical foundation for personalized therapies for RT patients.

NIA - National Institute on Aging

Project Summary: Hutchinson-Gilford progeria syndrome (HGPS) is a rapid, progressive aging disease caused be a de novo point mutation in the LMNA gene. The mutation causes missplicing of LMNA and production of a mutant mRNA and truncated protein called progerin. Progerin accumulation exerts a dominant negative effect on wild type LMNA, leading to DNA damage, proinflammatory gene activation, premature senescence and ultimately cell death. Children with HGPS die of accelerated atherosclerotic vascular disease. A notable pathologic feature of HGPS vascular disease is progressive loss of vascular smooth muscle cells with a simultaneous increase in inflammatory cells in the tunica adventitia and extensive periadventitial fibrosis. Some of these features are shared with other aging-associated cardiovascular diseases including hypertension and arteriosclerosis. Thus, a better understanding of how adventitial inflammation amplified vascular disease in HGPS may have implications for more common forms of aging in the cardiovascular system. In preliminary data we have found that HGPG aortas from a mouse model of disease harbor accumulation of Mac2 positive macrophage cells in the adventitia. On single-cell RNA sequencing we observe a significant increase in proinflammatory macrophage gene expression with enrichment for pathways involving interferon and tumor necrosis alpha pathways. Our central hypothesis is that progerin expression in bone-marrow derived myeloid cells promote vascular inflammation in HGPS. To test this hypothesis we will pursue the following two Specific Aims: 1) determine the contribution of myeloid-expressed mutant Lamin A (i.e. progerin) in adventitial fibrosis and vascular disease; 2) develop in vitro human stem cell models of HGPS to dissect the impact of Lamin dysfunction in myeloid cell differentiation and function. This project will explore a novel mechanism in HGPS- associate vascular disease, which has broader implications for how adventitial inflammation derived from bone- marrow cells may promote more common forms of atherosclerosis in humans.

NHLBI - National Heart Lung and Blood Institute

Abstract Hematopoietic stem cell (HSC) transplantation is a potent therapeutic strategy for the treatment of many blood disorders. Therefore, the development of methods for deriving large quantities of HSCs is a major focus in regenerative medicine. Unlike HSCs, human induced pluripotent stem cells (hiPSCs) can be grown indefinitely. Despite hiPSCs being an extremely scalable source, HSCs derived from hiPSCs using current protocols are largely devoid of long-term blood reconstitution potential. These shortcomings are suggestive of knowledge gaps surrounding HSC biology. In the Camargo lab we leveraged our in-vivo barcoding mouse model to show definitively that HSCs arise from both intraembryonic (aorta) and extraembryonic (umbilical and vitelline arteries) sites during native hematopoiesis, and that these sites play differential roles in blood production. Our findings suggest for the first time that umbilical and vitelline (UV) artery HSCs are more short-lived than aortic HSCs, and that the UV artery engages potently in embryonic lymphopoiesis. By performing single cell RNA-seq on murine blood producing endothelial cells, I observed differential NOTCH signaling strength and the presence of NOTCH inhibitors GPR183 and DLK1 in the aorta and UV arteries respectively. While it is vastly appreciated that a transient reduction in NOTCH signaling strength is required for hematopoiesis to occur, no study has detailed differential mechanisms of NOTCH inhibition at spatially distinct HSC-producing sites. Cross-referencing this site-specific data with scRNA-seq on a commonly used hiPSC hematopoietic differentiation protocol, I identified for the first time the exclusive prevalence of the UV-like hemogenic endothelium in vitro. To develop methods of producing long-lived HSCs with adult-like lymphoid potential, we plan to study the Notch pathway as a regulator of site-specific hematopoiesis and modulate NOTCH signaling strength to produce more aortic-like hemogenic endothelium from hiPSCs. From this preliminary data, we hypothesize that differential NOTCH signaling strength is crucial for producing distinct hematopoietic programs in the UV arteries and the aorta. To test this central hypothesis, we plan to pursue the following specific aims: (1) characterize the role of GPR183 in aortic hematopoiesis through murine loss of function studies, (2) describe the role of DLK1 in UV hematopoiesis by murine loss of function, and (3) determine the function of DLK1 in controlling the hiPSC spatial hematopoietic program. From these experiments we expect to elucidate the role of these NOTCH inhibitors in spatially defined hematopoiesis. By leveraging our site-specific scRNA-seq dataset, we are uniquely positioned to produce methods of deriving aortic-like blood cells. These novel blood populations have the potential to revolutionize the therapeutics landscape.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY The genes associated with epilepsy, a major neurological disorder, are critical to the development of both neural progenitor cells (NPCs) and the early stages of human brains. However, the molecular mechanism of how NPCs shape the developing cortex and contribute to epilepsy and other neurodevelopmental disorders remains unclear. In particular, it is crucial to determine how the epilepsy associated gene variants disrupt NPC functions and alter both the complexity and expansion of human brains. Protocadherin-19 (PCDH19) Clustering Epilepsy (PCE) is one of the most common monogenic epilepsies, caused by loss-of-function mutations in the X-linked gene PCDH19. Unique among the epilepsy associated genes, PCE affects only heterozygous females and mosaic males. This is presumedly due to random X-inactivation (RXI), which generates mosaic PCDH19 expression. The prevailing theory hypothesize that this mosaicism causes abnormal cell–cell interactions, or “cellular interference,” between PCDH19 wild-type (WT) and mutant cells. Yet, how this cellular interference arises during cortical development and leads to epilepsy remains unknown. Here, we have developed a novel human pluripotent stem cell (hPSC)-derived cortical organoid (hCO) model that faithfully recapitulates mosaic PCDH19 expression during human neurodevelopment. Using this model, we discovered abnormal cell segregation in the ventricular/subventricular zone (VZ/SVZ), where WT and mutant cells form distinct stripes, accompanied by altered expression of PCDH19 and N-cadherin (NCAD) and abnormal radial glial (RG) cell morphology. PCDH19 expression labels dividing RG cells at the apical junction, implicating its role in progenitor polarity and neurogenesis. These observations let us to propose that PCDH19 regulates RG cell polarity and lineage progression through the NCAD-mediated adherent junction complex. Based on this hypothesis, mosaic PCDH19 loss disrupts early human cortical development, leading to epilepsy-associated neurodevelopmental abnormalities. Our proposed study will uncover novel mechanisms of PCDH19 function in human cortical development, providing critical insights into the pathogenesis of PCE. This project establishes the first human brain organoid research platform for undergraduate education at The University of Toledo, offering the students hands-on training in cutting-edge neuroscience techniques, including human stem cell culture, CRISPR/Cas9 genome editing, confocal imaging, and bioinformatics analysis. Through this research, the students will gain interdisciplinary research experience that will prepare them for advanced graduate and professional programs, directly supported by the educational mission of the NIH R15 AREA mechanism.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY The intestinal microbiota is a complex community that modulates immune responses and host metabolism. However, colonization of this niche by pathobionts, particularly early in life, can perturb this homeostasis and cause intestinal damage and inflammation. Further, because the neonatal intestinal microbiota, immunity, and barriers are not fully developed, infants are particularly susceptible to pathogen barrier breach and systemic bloodstream infection. Group B Streptococcus (GBS) is one such pathobiont that colonizes the neonatal gastrointestinal tract following aspiration of infected amniotic fluid and/or breast milk. This intestinal colonization serves as a reservoir for “late-onset” GBS systemic disease (LOD), which presents at least one week following birth (typically as bacteremia and/or meningitis) and causes ~15% mortality regardless of antibiotic treatment. Despite this, the mechanisms underlying GBS infant intestinal colonization and resulting systemic neonatal disease remain largely unknown. Type VII secretion systems (T7SS) export effector proteins with functions in virulence, toxicity, or interbacterial killing, and we recently characterized this system in GBS. Our previous work showed that GBS T7SS and its effectors promote virulence in murine models of meningitis by pore-forming effectors as well as female genital tract colonization by toxin-mediated interbacterial competition and immune evasion. We recently found that GBS T7SS is also important for neonatal systemic infection stemming from intestinal colonization and that GBS T7SS effectors are toxic to intestinal epithelium. Based on these data, this proposal will investigate the role of the GBS T7SS in neonatal intestinal dysbiosis, including disruption of the microbiota, impairment of immune responses, barrier breakdown, and ultimately systemic bacterial spread/bacteremia. These questions will be addressed with human cohort data as well as in vitro and in vivo models of GBS intestinal colonization and systemic dissemination in the following aims: AIM 1: Investigate GBS perturbation of the intestinal microbiota during infant intestinal colonization. AIM 2: Evaluate anti-GBS mucosal immune responses during infant intestinal colonization. AIM 3: Determine mechanisms of GBS-mediated inflammation and barrier damage in intestinal epithelium in vitro and during infant intestinal colonization in vivo. This proposal will investigate the role of GBS T7SS effectors on newborn intestinal colonization and barrier loss, which may afford novel targets and alternative therapeutic strategies to treat and prevent neonatal infections.