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NCI - National Cancer Institute

The purpose of this proposal Is to elucidate the rate-limiting non-mutational events underlying benign to malignant transition of colorectal cancer (CRC). Colon polyps are extremely common, occurring in up to 40% of adults Over 50, and are the precursors to CRC. While the causative mutations and their stepwise acquisition In this polyp-to-c:arcinoma sequence are well understood, the non-mutational mechanisms are not. In this proposal, we will use a new mouse model that emulates, for the first lime, the stepwise acquisition of mutations In the distal colon to accurately model benign-to-malignant transition In human CRC. Benign polyps are initialed via knockout of the moat commonly mutated gene in human colon polyps (Apc). The most common mutational events In advanced CRC (Kras G120, Trp53 loss) are then Induced In rare cells of established polyps wl1h spatial and temporal precision. In our preliminary studies, we have found 1hat this model accurately emulates the histopathological progression of benign-to-malignant transition of human CRC, but not in mice that are lacking T c:ells-these tumors do not progress beyond adenoma. Spatial transcriptomic (S1) analyses Of these tumors revealed that KrasG120 induces loss of signatures Of homeostatic regeneration and gain of a fetal intestinal- like stem cell state, which Is known to play a critical role In intestinal repair following injury and inflammation, KrasG120 regions within polyps were also less proliferative and depleted over time, consistent wi1h the slow cycling nature of injury-responsive stem cells in the c:olon. These findings establish that mutations, while necessary, are not sufficient to drive progression to CRC, at least when acquired In the stepwise sequence characteristic of human CRC. We hypothesize that malignant precursors in benign polyps despite having the necessary genetic mutations, require inflammatory signals to progress to cancer. These signals enable this transition by shifting the fitness landscape of the premalignant niche in favor of a fetal intestinal wound healing response over homeostatic regeneration. In Aim 1, we will leverage our innovative model to functionally interrogate the role of the fetal Intestinal state In benign-to-malignant transition by Inducing 1h18 slate via 1) wounding or non-specific T cell activation, 2) knocking out Its key transcriptional coordinator ( Yap), and 3) ablating cells in this Slate. we will use ST data from the model to define the transcriptional regulatory networks underlying this state In progressing versus non-progressing lesions. In Aim 2, we will identify rate-limiting microenvironmental factors that preferentially select for malignant precursor cells, and define the tranSC11pllonal mechanism by which these factors cooperates with, oncogenic mutations. Results will be validated against ST data we have generated on human polyps with early malignancy. Impact: completion Of !hie proposal will provide a mechanistic foundation for understanding why some benign polyps progress to cancer while most do not. This knowledge may nominate new and mare specific biomarkers for early cancer detection, as well as rate-limiting events that could be targeted fur cancer prevention.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY / ABSTRACT Hematopoietic aging is characterized by chronic inflammation, hematopoietic stem cell (HSC) dysfunction, and hematopoietic bias toward myeloid lineages, leading to less efficient immune responses and increasing incidence of hematologic neoplasia. Emerging evidence from our group and others shows that NFκB-mediated chronic inflammatory responses affect HSCs and their cellular niches in different ways. Elevated NFκB activity within HSCs leads to loss of replicative capacity, while in contrast, elevated NFκB within the bone marrow niche drives epigenomic reprogramming of HSCs and myeloid bias of their progeny. These observations lead to the central hypothesis of this proposal, that unique NFκB responses in HSCs and their niches together form a biologic buffering mechanism balancing inflammatory hematopoietic responses with HSC preservation to maintain lifelong blood production. Importantly, chronic and dysregulated inflammation is implicated in a wide range of blood disorders, yet the mechanisms governing HSC and niche cell responses remain unclear. Thus, the proposed studies are expected to open avenues of inquiry with broad relevance to hematopoietic aging, clonal hematopoiesis, myeloid neoplasia, aplastic anemia, iatrogenic myelosuppression, and more. This mentored career development award will support the advanced research training of Dr. Jennifer Chia, a hematopathologist at UCLA, to become an independent academic physician-scientist in the field of hematopoiesis. Dr. Chia received her MD and PhD degrees from the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD program, and completed her Anatomic Pathology residency and Hematopathology fellowship at UCLA. She is now pursuing post-doctoral research training in the laboratory of Dr. Alexander Hoffmann, an internationally recognized expert in NFκB and the signaling systems that control immune cell function and fate decisions. The specific training objectives detailed in this proposal are to develop expertise in 1) inflammatory signaling and animal models of immune dysregulation; 2) HSC biology; 3) bioinformatics; and 4) the bone marrow niche. Training will be provided through mentorship by senior faculty members who are leaders in their fields, coursework, seminars, and conferences, all in the context of UCLA’s extensive resources and strong institutional commitment to the development of physician-scientists.

NIA - National Institute on Aging

Project Summary Alzheimer’s disease (AD) is a progressive neurodegenerative disease affecting more than 50 million people worldwide, but the mechanisms involved in disease development remain poorly understood. Genome-wide association studies have uncovered numerous loci associated with disease, but identifying causal variants and genes remains a challenge. Studies have revealed that AD risk variants are enriched in active enhancers of human monocytes, macrophages, and microglia, suggesting many of these variants act by disrupting gene expression specifically in myeloid cells. In addition, many genes implicated in AD risk are highly expressed in myeloid cells, strongly implicating these cell types in the etiology of AD. By integrating human genetics with epigenomic and transcriptomic datasets from human myeloid cells, we identified a myeloid cell enhancer containing an AD-associated functional variant on chromosome 11, and two putative target genes of this enhancer, embryonic ectoderm development (EED) and phosphatidylinositol binding clathrin assembly protein (PICALM). EED is an essential subunit of the polycomb repressive complex 2 (PRC2) known to function in regulation of gene expression and clearance behavior in mouse microglia, but it remains relatively unstudied in the context of AD in human cells. PICALM is an adaptor protein known to function in endolysosomal pathway and autophagy, but its role in microglia remains unknown. The overall goal of this proposal is to understand how our nominated AD risk enhancer influences the expression of its two target genes PICALM and EED, and to test the hypothesis that these two potential causal genes regulate microglia functions downstream of TREM2. In Aim 1, I will determine the role of the candidate AD risk enhancer in regulating the expression of PICALM and EED and human microglial cell function by combining CRISPR gene editing in human induced pluripotent stem cells (iPSCs), microglial differentiation protocols, and xenotransplantation methods involving direct injection of microglia precursor cells into the mouse brain. I will delete the candidate enhancer region and perform transcriptomic and epigenetic profile of edited microglia via RNAseq and ATACseq in addition to microglia functional assays. To assess the in vivo consequences of deleting the risk enhancer in microglia, I will transplant the edited microglia into the brains of wildtype and 5xFAD humanized mice and characterize transcriptomic profile of these microglia via snRNAseq analysis and perform immunohistochemistry. In Aim 2, I will investigate how PICALM and EED regulate human microglia function, namely TREM2 mediated efferocytosis, or phagocytic pathway. I will determine the subcellular localization of PICALM and EED using biochemical and imaging techniques and test the role of these two genes in microglial functions downstream of TREM2 signaling such as actin rearrangement, phagosome formation, and downstream kinase signaling. My proposed aims will help understand the mechanisms in which the causal variants and target genes drive the AD risk, and enhance our understanding of PICALM and EED in microglia and in AD.

NIAID - National Institute of Allergy and Infectious Diseases

SARS-CoV-2 protein nonstructural protein 1 (Nsp1) induces a global translation shutdown in host cells upon infection. Irrespective of mechanism, the Nsp1 imparted translation shutdown is required for efficient viral replication and to suppress the host immune response. Thus, fully understanding this protein involves understanding how it interacts with viral components and host machinery. The viral genome can escape the translational shutdown via secondary structure in its 5’ untranslated region (UTR). The first hairpin structure, stem-loop 1 (SL1), has been identified as necessary and sufficient to evade Nsp1-mediated translation shutdown. Previous reports show that host genes are suppressed differently by Nsp1. For example, translation- related genes, especially those with terminal oligopyrimidine (TOP) motifs, are translated more efficiently in the presence of Nsp1, while immune response genes are suppressed. Despite proven significant impacts of the 5’ UTR of SARS-CoV-2 and host genes, other elements remain understudied in this interaction with Nsp1. Therefore, this work examines if the 5’ UTR has other functional regions that might influence translational control and evasion of the translational shutdown. This research utilizes a recently developed method called direct analysis of ribosome targeting (DART), a high throughput method that tests the ribosome recruitment ability of thousands of 5’ UTRs. To analyze RNA features of SARS-CoV-2 and host genes that impact ribosome recruitment, a diverse pool of viral and host sequences was generated to allow thorough examination of each region of the 5’ UTR and its role in translation and evasion of host shutdown. The pool includes all known natural mutations reported in the NCBI virus sequence repository, along with systematic scanning, structural disrupting and compensatory mutations. Completing DART with and without Nsp1 will elucidate what elements facilitate translation and the evasion of Nsp1-mediated translational shutdown. The translation shutdown mechanism is thought to function through a two-pronged approach where the C-terminal domain binds the ribosome at the mRNA entry channel and sterically blocks RNAs from loading onto the ribosome, and the N-terminal domain (NTD) cleaves RNAs while bound to the ribosome, both activities preventing RNAs from being translated. However, it is unclear whether channel exclusion and cleavage are linked activities of Nsp1, or if different RNA features can mediate mRNA channel entry or escape of RNA cleavage. To address this, a high throughput cleavage experiment will be completed on pools of diverse RNAs to examine what host or viral features mediate resistance or susceptibility to cleavage, in ribosome-containing or depleted lysate. These cleavage experiments will be completed using the previously described RNA pool containing SARS-CoV-2 and immune related genes, along with another RNA pool of 24,000 sequences comprised of human genes, including translation-related sequences. This work will be instrumental for understanding the role of Nsp1 in coronavirus pathogenesis and to inform the design of future therapeutics.

NCI - National Cancer Institute

Project Summary/Abstract Acute myeloid leukemia (AML) is a lethal hematologic malignancy caused by mutations in hematopoietic stem cells. Normal karyotype AML (NK-AML) accounts for ~50% of cases and presents unique challenges due to its clinical and genetic heterogeneity. Prior research has shown that NK-AML develops from pre-existing clonal bone marrow diseases, including clonal hematopoiesis (CH) and myelodysplastic syndromes (MDS). In some CH and MDS patients, transformation to AML occurs in the absence of new mutations, indicating that epigenetic factors play a role in AML development. Recent studies by our lab using long-read DNA sequencing investigated DNA methylation as a potential source of novel contributing factors to AML development. These studies discovered thousands of discrete blocks with Allele-Specific DNA methylation (ASM) in primary AML samples that are sequence-independent and distinct from imprinted genes. These allele-specific “epimutations” are remarkably stable and are conserved in patient-matched samples obtained at presentation and relapse. Epimutations were also identified in remission samples from patients with genetic evidence of clonal disease and in single-cell clones expanded from normal hematopoietic stem cells. Furthermore, recurrent epimutations are associated with differential chromatin accessibility at genes that regulate stem cell differentiation and self-renewal, including GATA2 and genes in the HOXB cluster. Based on this evidence, we hypothesize that these allele-specific DNA methylation epimutations are clonal events that arise in normal cells and can disrupt gene regulation and be selected for during leukemogenesis. In this proposal we will investigate the role of DNA methylation epimutations in NK-AML using primary human AML samples, normal hematopoietic stem cells, and model systems via 3 aims. Aim 1 will define the landscape of recurrent epimutations in NK-AML using long-read native DNA sequencing. We will assess epimutation frequency and stability and explore their functional impact via RNA-seq, histone modification profiling, and Fiber-seq chromatin accessibility profiling. Aim 2 will use high resolution chromatin structure analysis and targeted epigenome editing to investigate the mechanisms of allele-specific gene regulation at the GATA2 locus. This aim will then test the functional consequences of allele-specific GATA2 activity by generating human AML cells with allele-specific silencing of GATA2 and engrafting them in humanized mice. These critical experiments will directly test whether epigenetic allele-specific expression of GATA2 confers a selective advantage for human AML cells in vivo. Finally, we will develop a novel targeted sequencing assay capable of detecting both mutations and epimutations and determine whether this approach improves the identification of persistent AML-related clones predictive of relapse in NK-AML patients in remission after chemotherapy. Together these studies will define the functional role of DNA methylation epimutations in AML and provide a novel molecular tool for monitoring AML patients and predicting relapse risk.

NIDA - National Institute on Drug Abuse

Neurologic complications remain prevalent in nearly 50% of people with HIV (PWH) and persist despite viral suppression with antiretroviral therapy (ART). Though the exact processes mediating HIV neuropathogenesis are not well understood, co-morbidities such as substance use disorders (SUD), which are higher in PWH compared to the general population, exacerbate neuropathogenesis of HIV and worsen outcomes. Multiple substances of misuse are reported to increase HIV replication, induce inflammatory signaling, and amplify neurodegenerative phenotypes. Thus, there is a significant need to understand the intersection between SUD and NeuroHIV to improve longitudinal care and inform the public. The overlapping effects of distinct substances of misuse on HIV pathogenesis in the CNS suggest that a common pathway may be involved through presently undefined mechanisms. All addictive substances increase extracellular dopamine in the central nervous system (CNS), which signals neurons and other nearby glial cells expressing dopamine receptors. Our lab has shown that myeloid cells such as macrophages and microglia, which are major HIV reservoirs in the brain, express dopamine receptors more D1-like receptors (D1 and D5) than D2-like receptors (D2, D3, D4). Treatment of macrophages and microglia with micromolar concentrations of dopamine increased pro-inflammatory signaling, increased viral entry, and potentiated viral secretion in vitro. We recently found that a higher D1-like to D2-like ratio is associated with a more pro-inflammatory response in microglia. Further, we showed that dopamine increases activation of nuclear factor-kappa B (NF-κB) in macrophages, and that inhibition of NF-κB can block the pro-inflammatory effects of dopamine. Together, these data suggest that dopamine-enriched brain regions, such as the cortex and striatum, may be especially vulnerable to HIV and neuroinflammation in PWH and co-morbid addiction through the action of dopamine on microglia. Therefore, the central hypothesis of this proposal is that dopamine D1-like receptor activation promotes HIV infection and NF-κB-mediated inflammation in microglia to worsen neurodegeneration. This hypothesis will be tested using human induced pluripotent stem cell (iPSC)-derived brain human cortical assembloids and several orthogonal assays to explore the dopamine-mediated pathways that modulate HIV neuroimmune pathogenesis. We will use pharmacologic activation of dopamine receptors in cortical assembloids to assess viral kinetics (Aim 1), neuroinflammation (Aim 2), and neuronal degeneration of synapses and dendrites (Aim 3). Together, these studies will significantly advance our understanding of dopamine as an immunomodulatory signaling molecule in the context of substance use and HIV, as well as expand the approaches to studying neuroimmune pharmacology using human micro-physiological systems.

NCI - National Cancer Institute

SCIENTIFIC ABSTRACT Acute myeloid leukemia (AML) is an aggressive clonal hematologic malignancy characterized by the defects in differentiation of the myeloid lineage, heightened proliferation, and resilience to cell death. Despite recent FDA approvals of several targeted therapies including venetoclax-based regimens, most patients relapse due to the survival and expansion of residual leukemia blasts and leukemia stem cells (LSCs) evading therapy. Eradiation of these cells through novel therapeutic approaches is critical for curing AML. CD123 is a surface marker strongly expressed on AML blasts and LSCs but largely sparing normal cells including hematopoietic stem cells (HSCs). AFM28 is a novel bispecific Innate Cell Engager (ICE ®)) that in pre-clinical studies effectively depleted CD123+ leukemic cells and LSC through NK cell engagement and recently showed encouraging activity in Phase I monotherapy trial in relapsed/refractory AML. NK-cell-mediated cytotoxicity can be sensitized through BCL-2 inhibition with venetoclax (Ven). Our preliminary data demonstrate that co- targeting of CD123+ AML by NK cells and of BCL2 by Ven translates into apoptosis of both, phenotypically defined AML stem/progenitor cells and AML blasts. We hypothesize that co-targeting AML by CD123- directed NK cells and BCL-2 inhibition harnessing apoptotic machinery will elicit AML cell kill through synergistic mitochondrial apoptotic priming. We will test our hypothesis in Specific Aims: In Aim 1, we will investigate combinatorial efficacy and molecular mechanisms of co-targeting CD123+ AML by NK-cell engager and BCL2 inhibition by Ven. We will perform dynamic BH3 profiling to probe modulation of mitochondrial priming, focusing on mitochondrial membrane integrity, induction of pro-apoptotic proteins, reprogramming of mitochondrial metabolism and co-dependency on mitochondrial pathway in AML and NK cells. In Aim 2, we will first determine the safety of the combination utilizing humanized NSGS mice producing human IL15 that provides support for NK cells maintenance, engrafted with CD123+ AML cell line and treated with AMF28-NK_Ven/Aza. Next, we will study the combinatorial efficacy of AMF28-NK_Ven/Aza in vivo in patient-derived xenograft (PDX) models generated from Ven/Aza-sensitive or -resistant AML. Molecular signatures of each therapeutic arm and combinations will be determined by flow cytometry, immunochemistry, immunophenotypic profiling, methylation assays and scRNAseq. Results of this proposed work will lay the foundation and provide rationale for successfully translating the combination of potent BCL-2 inhibitor with a novel engager AFM28-directed NK cell therapy into curative targeted therapy approach for AML patients.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract: Chronic inflammation and fibrosis are hallmark pathologies of Duchenne Muscular Dystrophy (DMD), yet current therapies focus on restoring dystrophin in muscle cells and largely overlook the pathological microenvironment. This niche—sustained by pro-inflammatory macrophages and fibrogenic fibro-adipogenic progenitors (FAPs)—impairs regeneration and reduces therapeutic efficacy. Existing gene therapies perform poorly in fibrotic muscle and require high doses that have caused toxicity and patient deaths. To address this critical therapeutic gap, we propose a first-in-class chimeric antigen receptor T cell (CAR-T) therapy for DMD that dually targets both inflammatory macrophages and fibrogenic FAPs to reprogram the dystrophic niche. While CAR-T therapies have revolutionized cancer treatment, they have not been applied to DMD or regenerative medicine. Supported by preliminary data, our goal is to develop a CAR-T cell therapy that can eliminate pro-inflammatory macrophages and fibrogenic FAPs, restore muscle stem cell (MuSC) regenerative potential, and improve both limb and diaphragm function in pre-clinical murine models of DMD. Specifically, we will: (Aim 1) Evaluate the effect of CAR-T therapy on inflammation and fibrosis; (Aim 2) Assess its impact on muscle regeneration and function. This paradigm-shifting approach addresses a major unmet need by targeting the inflamed-fibrotic niche and may enhance the effectiveness of current gene and cell therapies. If successful, it will establish a new therapeutic framework for DMD and other muscle diseases marked by chronic inflammation and fibrosis.

NEI - National Eye Institute

PROJECT ABSTRACT Cataracts and glaucoma are the two leading causes of blindness worldwide. Crucial ophthalmic procedures to treat cataract, glaucoma, and other vision conditions require precise visualization of anatomy and microsurgical instruments. Visualization in such surgeries has been limited to stereo optical microscopes since the early 20th century. With advancements in optical coherence tomography (OCT), we can now obtain real-time 3D visualization within the eye. Over the past decade, intraoperative OCT (iOCT) systems have become widely researched and integrated into the latest ophthalmic microscopes built by companies such as Zeiss and Leica. These iOCT systems come with the potential to revolutionize ophthalmic surgery, with an unparalleled ability to resolve key anatomic features at micron-level precision. However, there is a crucial challenge that hampers the clinical utility of iOCT. This challenge stems from the fundamental tradeoff between OCT field-of-view and imaging speed. This tradeoff constrains state-of-the-art systems to operate with a relatively small (e.g. 5x5 mm) field of view to achieve the volume update speeds (~10-15 Hz) required for surgical visualization. Consequently, a trained operator on the surgical team must manually reposition the OCT scan throughout the surgery. The current implementation of iOCT results in a “point-and-shoot” approach to imaging, i.e. using OCT as an intermittent snapshot tool, rather than as a continuous surgical visualization technology. With even small movements of the surgical instruments, the OCT image can quickly lose sight of the surgical region of interest (ROI). Manual tracking of iOCT discards a key advantage of OCT, which is real-time 3D data collection. With advances in deep learning methods for image processing and object recognition, there are new opportunities to tackle this problem. The goal of this project is to engineer a novel computational system for automatic, real- time tracking of the surgical ROI in a clinical iOCT system. Our vision is to develop a system that can be readily applied to existing clinical microscopes, and adaptable to future robotic surgical systems. As part of our preliminary work, we have created a lateral tool tracking OCT system using deep learning models applied to the microscope feed. Our current system utilizes a novel synthetic data approach, making use of 3D-rendered models of eyes and tools to accelerate deep learning model development. In the proposed project, we expand on this preliminary work by developing a system for 3D multimodal surgical ROI tracking of iOCT that can be applied to many different types of ophthalmic surgeries. We will then evaluate our platform via ex-vivo porcine and human cadaver eye studies with wet-lab benchmarking and simulated surgeries with our clinical collaborators. Our immediate application is ophthalmic surgery, but the methodology has relevance to a wide range of 3D imaging systems for microsurgical procedures. By developing this system for dynamic OCT surgical tracking, we hope to improve ophthalmic visualization in both training and surgical practice.

NHLBI - National Heart Lung and Blood Institute

Project summary/Abstract Hematopoiesis is a highly regulated process fueled by hematopoietic stem cells (HSCs) and progenitors in response to physiological and pathological changes throughout life. During development, the system quickly expands to provide increasing numbers of blood cells for the growing tissues and organs. In regeneration, HSCs re-establish the hematopoietic hierarchy and supply lost blood cells to restore tissue function. In pathological conditions, dysregulated hematopoiesis drives disease progression. A detailed cellular and molecular understanding of the mechanisms of dynamic hematopoiesis is key to intervening in these processes for therapeutic benefits. Although the niche critically regulates HSCs and hematopoiesis, how the niche is dynamically regulated to adapt to the distinct demands in the ever-changing conditions is not clear. Our previous work has identified key cellular components of the niche in the bone marrow and developing liver, allowing precise studies of niche dynamics in these organs. Our recent work has also revealed surprising cell fate plasticity in the bone marrow niche. The proposed work in this application builds on these findings to 1) define niche dynamics and plasticity in development, regeneration, and hematological disease, 2) uncover the mechanisms that regulate these processes, and 3) harness the mechanisms to enhance niche function and boost blood cell production. We will use several novel mouse models generated in the lab to study the function of key pathways in regulating niche dynamics and cell fate plasticity. We will employ single-cell transcriptomics, imaging, metabolic analysis, functional studies, and other cutting-edge approaches to understand how these key pathways regulate niche dynamics and plasticity. Collectively, these experiments will uncover novel mechanisms that regulate niche cell dynamics and plasticity with broad implications for better treatment of blood diseases. They may lead to transformative strategies for boosting blood cell production by enhancing niche function.

NIGMS - National Institute of General Medical Sciences

Abstract Flagella—motile, hair-like appendages extending from the surface of cells—are ubiquitously present across all three domains of life. These organelles carry out diverse functions of cells, including motility, sensory perception, and fluid transport, through their primary ability to move ambient fluids relative to the cells. Thus, understanding the interaction between flagella and their surrounding fluid, particularly their fluid-transport capability, is crucial to addressing a wide range of fundamental biological questions. While advances in electron microscopy and X-ray crystallography have illuminated the ultrastructures of bacterial and eukaryotic flagella, the dynamics of motile flagella in fluid environments remain poorly understood. The challenges in studying flagellar dynamics in fluid media stem from the lack of suitable experimental tools capable of imaging collective flagellar motions in real time at small length scales and mapping the three-dimensional (3D) fluid flow around rapidly beating flagella with high spatial and temporal resolution. Drawing on my unique training and career path, I lead a research group that develops new physical model systems and advanced novel imaging techniques to elucidate fluid-mediated flagellar dynamics in key biological processes. Specifically, we aim to address two critical questions on flagellar dynamics in this R35 MIRA proposal. 1) Resolving the synchronized dynamics of prokaryotic flagella that enable the formation of a bacterial flagellar bundle, a process essential for bacterial motility and chemotaxis. 2) Imaging the 3D fluid flow generated by beating eukaryotic flagella and their various mutants, a long-standing challenge that is central to the understanding of the functional consequences of normal and dysfunctional flagella and the key step towards the development of treatments for ciliopathies. Specifically, in Goal 1 of our proposed research, we will integrate experiments on peritrichous bacteria Escherichia coli and their genetically engineered mutants with a scaled physical model of a bacterial flagellar bundle constructed in my lab. This unique approach will help to reveal the detailed mechanisms, through which different physical factors, such as hydrodynamic interactions, the elastic properties of flagellar hooks, and motor torque fluctuations, control the synchronization and formation of bacterial flagellar bundles. In Goal 2, we will develop a new imaging technique—high-speed tracking holographic microscopy—to measure the temporal variations of the three-dimensional flow around the beating flagella of a green alga, Chlamydomonas reinhardtii, which serves as a premier model for eukaryotic flagella. Our research will deliver the first comprehensive characterization of the 3D flow field generated by isolated motile eukaryotic flagella in their natural, unperturbed state and directly correlate abnormal flagellar structures with their functional deficiencies in fluid transport. Thus, through the innovative model system and the advanced experimental techniques pioneered in our lab, our study will address crucial open questions on the dynamics of motile prokaryotic and eukaryotic flagella in fluid media.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Radiation-induced intestinal injury is a frequent and debilitating complication of abdomino-pelvic cancer therapy, often resulting in malabsorption, infection, and chronic gastrointestinal dysfunction. The intestinal epithelium regenerates through dedifferentiation events that give rise to revival stem cells (RevSCs), which replenish lost LGR5⁺ intestinal stem cells (ISCs) following injury. While the identity and regenerative potential of RevSCs are increasingly well understood, the earliest chromatin remodeling events and mechanisms that initiate their formation are not. Preliminary scRNA-seq data from our lab show that Bmi1-YFP⁺ epithelial cells, derived from Bmi1-CreERT2; Rosa26eYFP mice, form a distinct RevSC cluster as early as 24 hours post-irradiation, a timepoint significantly earlier than previous reports of RevSCs emerging from 48-96h. Thus, employing this mouse model provides a unique mechanistic window for studying the early emergence and regulation of RevSCs. We observed widespread chromatin accessibility changes at RevSC loci within 3 hours following injury from ATAC-seq data on intestinal epithelial cells (IECs), indicating promoter and enhancer remodeling as a key early event. Given the role of CBP/p300 histone acetyltransferases in maintaining enhancer activity, I hypothesize that injury-induced chromatin remodeling and a transient reduction in CBP/p300-dependent acetylation following injury, direct IECs toward a “pre-RevSC” state, which can be investigated through the lens of the Bmi1-lineage, enabling subsequent RevSC formation. I will test my hypothesis with two SPECIFIC AIMS. Aim 1 will define the transcriptional and chromatin dynamics that drive RevSC emergence by combining single-nucleus multi-ome (snRNA+snATAC-seq) and CUT&RUN profiling in both whole intestinal tissues, capturing broad, lineage- independent changes, and Bmi1-YFP⁺ IECs to investigate drivers of RevSC emergence. Aim 2 will determine how CBP/p300 inhibition alters enhancer accessibility and promotes RevSC formation and lineage plasticity using in vitro organoid models and in vivo pharmacologic and genetic perturbations. This work will generate a high-resolution atlas of early regenerative chromatin states and uncover enhancer-centric mechanisms that govern epithelial reprogramming. Importantly, it will elucidate how enhancer accessibility and histone modifications orchestrate cell fate transitions during intestinal regeneration, uncovering early epigenetic regulators of RevSC formation. These findings will identify molecular markers of regenerative potential and inform strategies to enhance mucosal repair in radiation enteropathy and inflammatory bowel disease. As part of a structured training plan, I will receive hands-on instruction in single-nucleus multi-omics, enhancer mapping, and organoid-based assays, complemented by formal coursework, clinical shadowing, and mentorship from physician-scientists. This integrated approach will provide the technical, analytical, and professional foundation necessary for a successful career as a physician-scientist in gastroenterology.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Early life stress (ELS), particularly during fetal development, is a critical risk factor for long-term health, including obesity and metabolic disorders. This project investigates how prenatal stress exposure is biologically embedded, leading to increased vulnerability to abdominal adiposity and metabolic dysfunction. Our long-term goal is to eluci- date cellular and molecular pathways that mediate the developmental origins of metabolic disease, supporting early identification and prevention strategies for at-risk children. Despite known associations between ELS and adult dis- ease, current research is limited by inconsistent findings in early life, inadequate biomarkers of fetal stress exposure, and poor measurement of adiposity in infants. Traditional reliance on weight-based metrics fails to capture fat dis- tribution, which is key to metabolic risk. Moreover, stress exposure during pregnancy is typically estimated from basal circulating biomarkers, neglecting dynamic physiological stress responses. To address these gaps, we employ a translational, multi-level design integrating basic science and clinical research. Using umbilical-derived mesenchy- mal stromal cells (MSCs) from human newborns, we will model individualized cellular vulnerability to ELS. In par- allel, we will track in vivo adipose development using serial MRI assessments and metabolic profiling in infants. Our specific aims are: Aim 1: Determine if biological stress during pregnancy predicts infant adiposity, distribution, and metabolic function using state-of-the-art MR imaging at birth and 5–6 months. Aim 2: Test whether MSCs from high-stress exposed infants exhibit greater cellular vulnerability under in vitro adi- pogenic challenge conditions. Stress exposure will be comprehensively quantified using ex vivo glucocorticoid-cytokine stimulation, diurnal sali- vary cortisol sampling, and maternal blood assays during early and late pregnancy. These data will be synthesized into a composite (PCA) biological stress exposure score. We hypothesize that dynamic, functionally derived measures of maternal stress will better predict infant abdominal adiposity and metabolic function than static bi- omarkers, and that stem cells from high-stress-exposed infants will exhibit greater vulnerability—reflected by in- creased lipid accumulation and hypertrophy—especially under in vitro challenge conditions. This integrated ap- proach will illuminate mechanisms of biological embedding and identify novel markers of metabolic risk. Findings will advance precision health by enabling targeted early-life interventions. This project will also establish a scalable human newborn stem cell biobank for future studies of stress-related disease pathways.

U.S. National Science Foundation

Plasma science is a transdisciplinary field of research where fundamental studies in many disciplines, including plasma physics, plasma chemistry, materials science, and space science, come together to advance knowledge for discovery and technological innovation. The primary goal of the <span style="text-decoration: underline;">EC</span>osystem for<span style="text-decoration: underline;">L</span>eading<span style="text-decoration: underline;">I</span>nnovation in<span style="text-decoration: underline;">P</span>lasma<span style="text-decoration: underline;">S</span>cience and<span style="text-decoration: underline;">E</span>ngineering (ECLIPSE) program is to identify and capitalize on opportunities for bringing fundamental plasma science investigations to bear on problems of societal and technological need within the scope of science and engineering supported by the participating NSF programs. The ECLIPSE meta-program has been created to foster an inclusive community of scientists and engineers, an ecosystem spanning multiple NSF Directorates, in the pursuit of translational research at the interface of fundamental plasma science and technological innovation. The ECLIPSE program builds on the long history of NSF leadership in supporting multi-disciplinary research in plasma science and engineering, and is intended to enhance organizational unity within NSF, and potentially with other funding agencies, in considering proposals and supporting projects that may otherwise struggle to find a natural home within the existing hierarchy of Directorates, Divisions, and programs within the Foundation. Examples of topical areas within the scope of the ECLIPSE program include but are not limited to: <ul type="disc"> <li>Plasma surface interactions, with applications to, e.g., advanced manufacturing, materials processing, and catalysis.</li> <li>Atmospheric pressure plasmas and microplasmas with applications to, e.g., microelectronics, plasma agriculture, environmental remediation, and other clean and decarbonized energy goals enabled by electrification of the chemical industry.</li> <li>Dusty plasmas with applications to, e.g., development of nanomaterials, aerosols, and functionalized surface coatings.</li> <li>Novel sensor development for highly non-equilibrium plasmas with applications to, e.g., cubesat-based geospace measurements and industrial plasma diagnostics.</li> <li>Novel computational modeling for multi-component and/or multi-phase plasma systems with applications to, e.g., space weather prediction and plasma reactor design.</li> <li>Novel studies of plasmons in nano-photonics and nano-optics with applications to, e.g., sub-THz wireless communication and photocatalytic chemical processes.</li> <li>New chemical measurement science for characterizing processes occurring in plasmas and using plasmas as part of measurement systems with applications to, e.g., analysis of environmental contaminants or identification of forensic evidence.</li> <li>Study of fundamental chemical reactions and mechanisms in plasmas with applications to, e.g., novel chemical synthesis.</li> </ul> Proposals submitted for consideration by this program should address societal or technological needs within the scope of science and engineering supported by the National Science Foundation. Proposals addressing technology development primarily supported by other US government funding agencies are not eligible for consideration and may be returned without review. Proposers are strongly encouraged to contact the cognizant Program Officers if they are unsure of the suitability of a project to this program. Proposals submitted for consideration by the ECLIPSE program should satisfy the following criteria: (1) clearly articulate the fundamental scientific and/or engineering challenge in plasma science and engineering that may be relevant to more than one NSF program;<ins cite="mailto:Mangala%20Sharma" datetime="2021-06-04T14:45"></ins> and (2) provide a substantive discussion of how a resolution of the stated scientific and/or engineering challenge will address specific societal and/or technological needs identified as priorities by the research communities, policymakers and/or other stakeholders. Depending on the nature of the proposal, the latter may be described as the Intellectual Merit or the Broader Impact of the proposed activity. The program encourages inclusion of specific efforts to increase the diversity of the ECLIPSE community and to broaden participation of under-represented groups in Science, Technology, Engineering, and Mathematics (STEM) as Broader Impacts of proposed work. The program welcomes proposals from Historically Black Colleges and Universities (HBCUs), other Minority Serving Institutions (MSIs), and institutions in <a href="https://new.nsf.gov/funding/initiatives/epscor/epscor-criteria-eligibility" target="_blank">EPSCoR-eligible jurisdictions</a>, along with collaborations between these institutions. Proposers are also encouraged to address how the proposed efforts may enhance workforce development towards STEM careers associated with the field of plasma science and engineering. The ECLIPSE program is not intended to replace existing programs. A proposal that is requesting consideration within the context of ECLIPSE should begin the title with the identifying acronym "ECLIPSE:" and should be submitted to one of the "Related Programs" listed below. In choosing the most relevant program, proposers are advised to read program descriptions and solicitations carefully and to consult with cognizant Program Officers in advance of proposal preparation. Proposal submissions outside of the scientific scope of the receiving program may be transferred to a different program or may be returned without review. Proposers should ask for consideration and review as an ECLIPSE proposal only if the proposal addresses both of the criteria listed above. Proposals marked for consideration by the ECLIPSE program that do not address both of these criteria may be returned without review or reviewed within the context of an individual program. Supplement requests to existing awards within a program that address both of the above criteria may also be considered. Information Sharing with other Funding Agencies When permitted under an MOU between NSF and another funding agency, NSF may share information from proposals for consideration of joint funding and may invite employees of such organizations to attend merit review panels as observers.

U.S. National Science Foundation

Plasma science is a transdisciplinary field of research where fundamental studies in many disciplines, including plasma physics, plasma chemistry, materials science, and space science, come together to advance knowledge for discovery and technological innovation. The primary goal of the ECosystem forLeadingInnovation inPlasmaScience andEngineering (ECLIPSE) program is to identify and capitalize on opportunities for bringing fundamental plasma science investigations to bear on problems of societal and technological need within the scope of science and engineering supported by the participating NSF programs. The ECLIPSE meta-program has been created to foster an inclusive community of scientists and engineers, an ecosystem spanning multiple NSF Directorates, in the pursuit of translational research at the interface of fundamental plasma science and technological innovation. The ECLIPSE program builds on the long history of NSF leadership in supporting multi-disciplinary research in plasma science and engineering, and is intended to enhance organizational unity within NSF, and potentially with other funding agencies, in considering proposals and supporting projects that may otherwise struggle to find a natural home within the existing hierarchy of Directorates, Divisions, and programs within the Foundation. Examples of topical areas within the scope of the ECLIPSE program include but are not limited to: Plasma surface interactions, with applications to, e.g., advanced manufacturing, materials processing, and catalysis. Atmospheric pressure plasmas and microplasmas with applications to, e.g., microelectronics, plasma agriculture, environmental remediation, and other clean and decarbonized energy goals enabled by electrification of the chemical industry. Dusty plasmas with applications to, e.g., development of nanomaterials, aerosols, and functionalized surface coatings. Novel sensor development for highly non-equilibrium plasmas with applications to, e.g., cubesat-based geospace measurements and industrial plasma diagnostics. Novel computational modeling for multi-component and/or multi-phase plasma systems with applications to, e.g., space weather prediction and plasma reactor design. Novel studies of plasmons in nano-photonics and nano-optics with applications to, e.g., sub-THz wireless communication and photocatalytic chemical processes. New chemical measurement science for characterizing processes occurring in plasmas and using plasmas as part of measurement systems with applications to, e.g., analysis of environmental contaminants or identification of forensic evidence. Study of fundamental chemical reactions and mechanisms in plasmas with applications to, e.g., novel chemical synthesis. Proposals submitted for consideration by this program should address societal or technological needs within the scope of science and engineering supported by the National Science Foundation. Proposals addressing technology development primarily supported by other US government funding agencies are not eligible for consideration and may be returned without review. Proposers are strongly encouraged to contact the cognizant Program Officers if they are unsure of the suitability of a project to this program. Proposals submitted for consideration by the ECLIPSE program should satisfy the following criteria: (1) clearly articulate the fundamental scientific and/or engineering challenge in plasma science and engineering that may be relevant to more than one NSF program; and (2) provide a substantive discussion of how a resolution of the stated scientific and/or engineering challenge will address specific societal and/or technological needs identified as priorities by the research communities, policymakers and/or other stakeholders. Depending on the nature of the proposal, the latter may be described as the Intellectual Merit or the Broader Impact of the proposed activity. The program encourages inclusion of specific efforts to increase the diversity of the ECLIPSE community and to broaden participation of under-represented groups in Science, Technology, Engineering, and Mathematics (STEM) as Broader Impacts of proposed work. The program welcomes proposals from Historically Black Colleges and Universities (HBCUs), other Minority Serving Institutions (MSIs), and institutions in EPSCoR-eligible jurisdictions, along with collaborations between these institutions. Proposers are also encouraged to address how the proposed efforts may enhance workforce development towards STEM careers associated with the field of plasma science and engineering. The ECLIPSE program is not intended to replace existing programs. A proposal that is requesting consideration within the context of ECLIPSE should begin the title with the identifying acronym "ECLIPSE:" and should be submitted to one of the "Related Programs" listed below. In choosing the most relevant program, proposers are advised to read program descriptions and solicitations carefully and to consult with cognizant Program Officers in advance of proposal preparation. Proposal submissions outside of the scientific scope of the receiving program may be transferred to a different program or may be returned without review. Proposers should ask for consideration and review as an ECLIPSE proposal only if the proposal addresses both of the criteria listed above. Proposals marked for consideration by the ECLIPSE program that do not address both of these criteria may be returned without review or reviewed within the context of an individual program. Supplement requests to existing awards within a program that address both of the above criteria may also be considered. Information Sharing with other Funding Agencies When permitted under an MOU between NSF and another funding agency, NSF may share information from proposals for consideration of joint funding and may invite employees of such organizations to attend merit review panels as observers.

NIEHS - National Institute of Environmental Health Sciences

Identifier: 1908301 EcoWell: Smart Incubation Technology for Next-Generation Environmental Science Labs Project Summary Overview: The EcoWell™ platform addresses a critical gap in environmental science education by introducing a novel, affordable, and accessible classroom tool that models environmental conditions in real time. Built around a modular micro-incubation system, the platform empowers students to explore the biological effects of environmental pollutants and measure related biological endpoints. These environmental stressors are increasingly prevalent due to systemic pollution, habitat degradation, and industrial activity, yet remain underrepresented in K–12 science curricula. The Need: Systemic pollution has become a significant threat to public and environmental health. Despite the urgency of these issues, educational infrastructure has not kept pace with the tools needed to effectively engage students in hands-on learning about toxicological processes, bioremediation, and environmental monitoring. Traditional science classroom kits are often linear, rigid, and fail to replicate real-world complexity or generate meaningful data. Moreover, students frequently lack the tools to interpret experimental results, assess sources, or connect local phenomena to regional, national, and global systems. Innovation and Impact: EcoWell™ is a transformative tool that enables students to simulate, manipulate, and analyze complex environmental scenarios using compact, programmable six-well incubation chambers. Each chamber is capable of independently controlling and measuring variables such as temperature, ultraviolet light and CO₂ levels, and gas exchange—parameters critical to understanding pollution’s biological impact. This modularity supports a wide variety of experimental applications: students can assess bacterial growth under UV-induced DNA stress, explore algal blooms in nutrient-loaded water, or measure plant response to synthetic pollutants. The platform is paired with a suite of NGSS-aligned educational kits covering a range of NIEHS-relevant topics such as: Bioremediation using duckweed to extract nitrates and heavy metals, Water sterilization and pathogen load mitigation using UV radiation, Gas exchange and oxidative stress modeling, Growth response of model organisms under pollutant and light stress conditions. By offering customizable experimentation and quantifiable outputs, EcoWell™ bridges the gap between basic science education and environmental toxicology, preparing students for STEM careers while improving environmental literacy. Alignment with NIEHS Goals: EcoWell™ aligns with NIEHS's mission to understand how environmental exposures affect human health by educating future generations in exposure biology, toxicology, and environmental systems thinking. The system gives students the skills to explore real-world questions with real-time data, and to connect experimental results to ongoing public health and environmental challenges.

Edison International

Supports STEM education, workforce development, environment, and civic engagement through Southern California Edison's parent foundation.

U.S. National Science Foundation

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

U.S. National Science Foundation

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

U.S. National Science Foundation

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

U.S. Mission to Georgia

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

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

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

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

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

NIDCR - National Institute of Dental and Craniofacial Research

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