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NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Alloreactive T cells initiate effector cell and support alloantibody (alloAb) responses in patients after allogeneic transplantation. Current approaches are not uniformly efficacious in preventing or ameliorating T effector cell and alloAb mediated kidney rejection. Using fate-mapper and reporter strains, we show that Tfh cells in kidney allografts sequentially differentiate through stem-like Tfh progenitor (TfhProg), effector Tfh, and dysfunctional TfhEx stages which is supported by kidney proximal tubule epithelial cells (PTECs). Moreover, kidney PTECs sense and respond to Tfh-induced stimuli leading to local injury. We hypothesize that bidirectional communication between Tfh cells and PTEC provide a feedback loop that exacerbates kidney injury and inflammation leading to graft rejection. In Aim 1, we will identify the kidney microniches promoting Tfh reprogramming in grafts. In Aim 2, we will assess the roles of kidney epithelial-derived signals in Tfh differentiation and reprogramming. In Aim 3, we will assess how Tfh-derived cytokine signals promote PTEC activation and injury. We will pursue these aims using innovative strategies to identify, fate map, and perturb Tfh cell differentiation as well as PTEC pathways in vivo during murine kidney transplantation. We will combine these innovative tools with human kidney biopsy and gene perturbation studies to validate findings. The expected outcome of these studies is to determine the fundamental role of Tfh cells in kidney allograft injury along with precise mechanisms used by Tfh and PTEC to promote disease. These studies are significant because they will provide new strategies to prevent and treat kidney allograft rejection.

NIAID - National Institute of Allergy and Infectious Diseases

SUMMARY Chronic postsurgical pain (CPSP), pain persisting for more than 3 months post-surgery, affects 14.5-38% of children and adolescents in the U.S. undergoing major surgery. CPSP has significant consequences, including depression, disability, opioid misuse and healthcare costs. Preliminary data suggest that immune dysregulation and neuroimmune interactions play a role in CPSP pathogenesis, with proinflammatory cytokines contributing to the chronification of pain while IL-6, IL-8 and IL-13 may have protective effects, with potential for development of management strategies. However, cellular immune mechanisms that underly CPSP are not well understood and lack of a translational immune ex-vivo model presents a critical gap in translational research, hindering prevention and treatment of CPSP. This study aims to elucidate the mechanistic links between immune signatures and sensory neuron excitability in CPSP pathogenesis. This research will leverage banked serum and peripheral blood mononuclear cell samples from pediatric surgical patients and will be the first to employ induced pluripotent stem cell (iPSC)-derived sensory neurons, to model immune- neural interactions in CPSP. This will enable novel investigation of immune-neural interactions involved in CPSP development, integrating experimental, clinical, immunology and neurobiology data through rigorous analyses. The central hypothesis is that serum from CPSP patients is inherently proinflammatory, promoting immune cell dysregulation and sensitizing nociceptive sensory neurons. This hypothesis will be tested by the following specific aims: Aim 1 will examine immune mechanisms contributing to CPSP by comparing in vitro cytokine responses and immune cell differentiation of control peripheral blood mononuclear cells PBMCs exposed to serum from CPSP vs. non-CPSP patients. We will correlate these with in vivo longitudinal immune profiles from surgical subjects. Aim 2 will investigate how CPSP vs. non-CPSP patient serum modulates excitability of ex vivo iPSC-derived sensory neurons. Using calcium imaging, we will assess responses to noxious stimuli in and correlate excitability with experimental (pressure pain threshold) and clinical pain phenotypes. Combining in vivo and in vitro data to elucidate immune-sensory neuron interactions with patient- banked samples offers a unique opportunity to gain insight into cellular models of CPSP, providing a robust, powerful, human-specific platform for linking immune signals to pain mechanisms. Identifying key cytokine and immune cell signatures that differentiate CPSP patients will guide future development of immune-targeted treatments and personalized pain management strategies. This translational study will provide foundational insights into the immune-neural mechanisms of CPSP, enabling the development of predictive biomarkers and novel therapeutic strategies. The proposed research will establish a platform for future R01 studies focused on iPSC-based CPSP modeling and mechanism based therapeutic interventions.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT: Sepsis results from a dysregulated host response to infection leading to life-threatening organ dysfunction. It is a complex and dynamic disease process, and a leading cause of morbidity and mortality in intensive care units (ICUs). Due to the therapeutic challenges of patients with sepsis, and the fact that management remains predominantly supportive, there is an undeniable need to develop new treatment strategies for sepsis. New advances being explored include cell-based therapies. For more than 25 years my laboratory has explored the pathobiology of sepsis, and related organ injuries, including the lung. Our approach is to investigate mechanisms of disease, starting at the cellular level in vitro and translating these findings into models of disease ex vivo and in vivo. To complement our work further in sepsis, with an interest in therapy, we became interested in stem/stromal cells. My laboratory has explored the use of mesenchymal stem/stromal cells (MSCs) for therapeutic intervention in pre-clinical models of sepsis and lung injury. We investigate mechanisms responsible for the biological activity of MSCs, including paracrine actions via their conditioned medium and the impact of MSC-derived extracellular vesicles (EVs) and their cargo (miRNAs). To advance our understanding of cellular therapy for sepsis, we recently began to explore a new source of cells – placenta-derived trophoblast stem cells (TSCs). Our laboratory was the first to isolate murine (m) TSCs, using CD117 as a cell surface marker of stem/progenitor cells. Beyond paracrine actions, these cells can engraft and differentiate into parenchymal cells. We now propose to advance our investigation of TSCs harvested from human(h) term placentas. hMSCs are immune evasive, and hTSCs are immune privileged, allowing the use of both cells for allogeneic therapy. We will investigate hMSCs and hTSCs to modify the pathobiology of sepsis and provide insight into the immune response to eradicate microbes, resolve inflammation, and decrease organ injury along with promoting repair. Sepsis and organ injury, such as acute respiratory distress syndrome (ARDS), are very heterogeneous clinical processes, thus targeting a specific biological pathway is challenging. We propose that viable therapeutic cells will sense the underlying septic environment, and respond accordingly with varied paracrine actions. Plasma and immune cells from patients with sepsis ± ARDS, compared with ICU control (non-infected) patients, will allow us to explore a personalized approach using hMSCs and hTSCs. Moreover, due to differences that exist between human and mouse lungs, we propose to evaluate the actions of hMSC and hTSCs using human lung organoids and precision-cut lung slices (PSLS) as human models of lung/alveolar injury, and transcriptomic approaches to identify pathways critical for disease modification. Thus, our vision is to advance the insight into therapies for sepsis using hMSCs and hTSC, and that using human models of disease in vitro, ex vivo, and with confirmation in a pre-clinical model of pneumosepsis will provide insight into critical sepsis pathways and advance our approach to the therapy of sepsis and ARDS.

NIAID - National Institute of Allergy and Infectious Diseases

Summary The transformative advancements in cell and gene therapy have significantly enhanced our understanding and treatment of congenital diseases and regenerative medicine. As we approach the 10th annual Center for Definitive and Curative Medicine (CDCM) symposium, scheduled for March 30-31, 2026, at Stanford University's Li Ka Shing Learning and Knowledge Center, we seek financial support to facilitate this pivotal event. This symposium will serve as a platform for scientific discourse on the latest discoveries and developments in the field, inviting participation from experts and trainees across academia, non-profits, government, and industry. The two-day event will focus on the theme “Past, Present, and Future of Cell and Gene Therapy,” addressing critical challenges in translating laboratory discoveries into clinical applications. Day 1 will feature a Clinical Trial Bootcamp, workshops on relevant topics, a poster session for early- stage investigators, and an evening networking event. Day 2 will showcase luminary speakers discussing breakthroughs in lentiviral and AAV gene therapy, CAR-T, gene editing, regenerative medicine, and hematopoietic stem cell transplantation. Specific aims of the symposium include: (1) elucidating the bench-to-bedside journey through real-world case studies presented by the Stanford CIRM-funded Alpha Clinic; (2) providing a platform for graduate students and early-career researchers to present their findings; (3) facilitating workshops that address community engagement in clinical trials, the role of Artificial Intelligence in healthcare, and career opportunities in health sciences; and (4) fostering collaboration through platform sessions that expose participants to emerging research areas. This symposium has been a cornerstone of the Cell and Gene Therapy Community for the past nine years, celebrating past achievements while catalyzing future innovations. The 2026 meeting promises to be a significant milestone, driving forward the dialogue and collaboration necessary to tackle the complexities of cell and gene therapy and improve patient outcomes.

U.S. National Science Foundation

CREST Center awards provide support to enhance the research capabilities of Minority-serving institutions (MSIs) through the establishment of centers that effectively integrate education and research. CREST Center awards promote the development of new knowledge, enhancements of the research productivity of individual faculty, and an expanded presence of students historically underrepresented in science, technology, engineering, and mathematics (STEM) disciplines. Successful CREST Center proposals will demonstrate a clear vision and integration of STEM research and education and will align with the mission of the Division of Equity for Excellence in STEM (EES) with respect to the development of a diverse STEM workforce. CREST Centers are also expected to provide leadership by meaningfully involving the efforts of those faculty, students, and postdoctoral researchers who are traditionally underrepresented in STEM at all levels. Centers are required to use evidence-based and innovative strategies to address salient broadening participation and workforce development issues, such as recruitment, retention, and mentorship of participants from underrepresented groups. Successful proposals are expected to achieve national research competitiveness, broaden participation in STEM, and generate sustained, non-CREST funding from federal, state, and/or private-sector sources. PhaseI and Phase II CREST Center Awards Preliminary proposals are required for Phase I and Phase II projects. Thus, an invitation from NSF must be received before submitting a full proposal. Both Phase I and Phase II CREST Center awards provide multi-year support for institutions that demonstrate a strong research base. Phase I CREST Center awards provide funding for five years of research on a specific NSF-supported topic. If invited, institutions may submit a Phase II CREST Center proposal requesting funding to continue research in the same disciplinary area as the Phase I Center or may submit a Phase I proposal focused on a disciplinary area that is significantly different from those of the previous award(s). CREST Partnership Supplements CREST Partnership Supplemental funding requests are invited from current CREST Center awardees. Supplements support the establishment or strengthening of partnerships and collaborations with active CREST Centers and other nationally or internationally recognized research centers (including NSF-supported research centers), private sector research laboratories, K-12 schools, and/or informal science entities, including museums and science centers, as appropriate. Such partnerships and collaborations should aid CREST Centers quest in advancing knowledge and education on a research theme of national significance.

U.S. National Science Foundation

The Centers of Research Excellence in Science and Technology (CREST) program provides support to enhance the research capabilities of minority-serving institutions (MSIs) as defined in this solicitation s Eligibility section, through effective integration of education and research. The CREST program, composed of the CREST Centers, the CREST Postdoctoral Research Program, and the projects supported by this CREST-RISE solicitation, promotes the development of new knowledge, enhancements of the research productivity of individual faculty and postdoctoral scholars, and an expanded presence of research doctoral students in science, technology, engineering, and mathematics (STEM) disciplines, especially those from underrepresented groups.CREST-RISE is the component of the CREST program that supports the expansion of institutional research capacity by increasing the strength of institutional graduate programs and the successful production of research doctoral students, especially those from groups underrepresented in STEM.The CREST-RISE component supports STEM research doctoral programs in all NSF supported areas and encourages proposals in areas of national interest, such as artificial intelligence, data science and analytics; advanced materials, manufacturing, robotics; cybersecurity; plant genetics/agricultural technologies; quantum information sciences; nanotechnology, semiconductors/microelectronics technologies; climate change and clean energy.CREST-RISE projects must have a direct connection to the long-term plans of the host department(s) and the institution s strategic plan and mission. Project plans should emphasize activities designed to increase the production of research doctoral students, especially those underrepresented in STEM as well as expand institutional research capacity.The goals of CREST-RISE are to increase: 1) the number of STEM research doctoral programs at MSIs (as defined in the Eligibility section), 2) the number of STEM research doctoral students graduating from MSIs, especially those from groups underrepresented in STEM, and 3) institutional research capacity to increase doctoral students graduation rates.To achieve these goals, the CREST-RISE program includes three tracks as follows:CREST-RISE STEM Doctoral Programs Support Initiative (CREST-RISE DPSI)CREST-RISE Research Advancement and Development (CREST-RISE RAD)CREST-RISE Equipment & Instrumentation (CREST-RISE E&I)

U.S. National Science Foundation

The Centers of Research Excellence in Science and Technology (CREST) program provides support to enhance the research capabilities of minority-serving institutions (MSIs) through the establishment of centers that effectively integrate education and research. CREST promotes the development of new knowledge, enhancements of the research productivity of individual faculty, and an expanded presence of students who are members of groups underrepresented in science, technology, engineering, and mathematics (STEM) disciplines. The CREST Postdoctoral Research Program (CREST-PRP) awards are part of the overarching CREST program and provide two years of support for research experience and training for early career scientists at active CREST Centers. The goal of the CREST-PRP awards isto increase the workforce presence of individuals from groups underrepresented in STEM fields. CREST-PRP awards recognize investigators with significant potential and provide them with research experiences that broaden perspectives, facilitate interdisciplinary interactions, and prepare CREST-PRP scholars for positions of leadership within the scientific community. Postdoctoral scholars conduct research on topics aligned with the research focus of the host CREST Center. The awards are also designed to provide active mentoring to the postdoctoral scholars by thescientific mentorwho, in turn, will benefit from the incorporation of these talented scientists into their research groups. Proposals must be submitted by individual postdoctoral candidates. However, if an award is recommended, the award will be transferred to the host institution where the postdoctoral scholar will be named as the PI. The award will be issued to the host institution as a regular research award, and the award will be administered by the host institution. Women, veterans, persons with disabilities, and members of groups underrepresented in STEM are especially encouraged to apply.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Solid organ transplantation has proven effective at extending and improving the quality of life of patients with end-stage organ failure-associated diseases. However, the long-term outcomes after solid organ transplantation (Tx) are suboptimal, with only 60% graft survival by 10 years post-Tx due to chronic rejection (CR). Donor- specific anti-HLA antibodies (DSA) and antibody-mediated rejection (ABMR) are among the most important causes of CR, but the immune mechanisms that dictate which DSA+ABMR patients fair well and which are refractory to treatments and progress to CR and graft loss are still not understood. Therefore, there is a high priority to decipher the immune mechanisms responsible for this heterogeneous clinical behavior, and to identify novel targeted strategies to overcome these unwanted outcomes. We have recently discovered that unlike DSA+ABMR KTx patients with better clinical outcomes, those patients that progressed to CR and graft loss preferentially expanded a unique, previously not described central memory (CM) stem-like cTFH subset, that was highly correlated with durable DSA of IgG3 isotype, activated memory B (memB) cells and plasma blasts (PB), as well as with graft injury and graft loss post-KTx. Based on these novel observations we hypothesize that when present and significantly expanded, stem-like cTFH cells proliferate, self-renew and support a potent, durable humoral response, leading to more aggressive ABMR. We further postulate that persistence of stem-like cTFH contributes to the development of CR and graft loss. Therefore, we will aim to (1) Decipher the longitudinal (pre- Tx, 1, 3, 12, 36 mo) differentiation fate, function, allo-specificity, transcriptomic profiles of stem-like TFH (2) Define signaling and metabolic characteristics linked to the stability and function of stem-like cTFH cells (3) Determine the relationship between tissue-infiltrating stem-like TFH cell profile during chronic DSA+ABMR and subsequent kidney allograft loss. We will also assess whether we can disrupt stem-like TFH generation or drive these cells to become terminally exhausted. We believe this proposal is innovative because it investigates the concept of cTFH stemness fueling humoral rejection, a concept that has not been explored in human organ transplantation. The proposal is significant because (i) understanding the biology and clinical implications of stem-like cTFH cells would provide valuable mechanistic insights into how to optimally monitor to risk-stratify patients with DSA+ABMR and predict poor allograft outcomes; (ii) identify biologic or pharmacologic tools to disrupt or re-program stem-like cTFH leading to novel therapeutic opportunities for CR; (iii) open new avenues of research applicable to recipients of other types of organ transplants (e.g. heart and lung), or to patients with autoimmunity.

NIH

Background and Innovation: The proposed work is focused upon enhancing our understanding of CD34+ melanocyte stem cells (McSCs). These are cells previously described by us which localize to the bulge area of the hair follicle (HF) during its resting stage. Unlike their counterparts, CD34- McSCs which reside at the base of the resting HF and principally undergo melanocytic differentiation, CD34+ McSCs resemble a neural crest stem cell; and differentiate principally as neural crest derivatives other than melanocytes- including glia. The prospect of isolating and expanding a skin-derived stem cell type to treat demyelinating diseases and restore nerve function is enticing and may represent one of the most innovative solutions to these problems faced by Veterans. VA has an interest in treating Veterans with demyelinating diseases through its support of centers such as the Multiple Sclerosis Centers of Excellence. Rehabilitation and restoration of function following neurotrauma has consistently been a strong interest in VA healthcare. Seemingly unrelated, but nonetheless linked to the ability of melanocytes and McSCs to transition between discrete differentiation states, is the relevance of CD34+ McSCs to melanoma therapeutic resistance. The less differentiated CD34+ McSC subtype has been correlated with relative resistance to immunotherapy in murine melanoma models. Understanding the molecular and cellular basis of McSC dedifferentiation could inform strategies designed to block melanoma cell dedifferentiation as a novel strategy to maintain therapeutic responsiveness. Scientific efforts in this direction are synergistic with VA’s interest in Precision Oncology, both with the new Precision Oncology Actively-Managed Research Portfolio and with its clinical support of precision oncology programs. Significance and Impact to Veterans Healthcare: The proposed work is relevant to Veteran healthcare and disease as follows: (1) Developing stem cells easily accessible from autologous skin that have the potential to undergo glial differentiation represents a potential cellular therapy solution to diseases that affect Veterans. These include demyelinating diseases such as multiple sclerosis and recovery from neurotrauma, since stem cells with glial differentiation properties introduced into a neural injury environment facilitate recovery of nerve function. Both multiple sclerosis and recovery from neurotrauma are priorities for Veteran health care. (2) Dedifferentiation enables melanoma cells to escape as targets for immunotherapy. Understanding the mechanistic underpinnings of McSC dedifferentiation, which are likely to share a molecular basis with melanoma cells, may illuminate strategies designed to block dedifferentiation in cancer and enhance therapeutic responsiveness. Novel strategies to treat cancer are relevant to VA health priorities, given the recent emphasis on developing Precision Oncology clinical care pathways and research initiatives within the Veterans Health Administration. Path to translation/implementation: We need to refine the identification of the CD34+ McSC subtype in murine skin to maximize the opportunity to identify a similar cell in human skin. Optimizing techniques to expand CD34+ McSCs that have glial differentiation potential will be important for obtaining sufficient cells that maintain the glial differentiation phenotype to evaluate for cellular therapy. Identifying the master regulators of melanocyte dedifferentiation will be important. Those regulators, or critical effector genes of those regulators, could represent therapeutic targets to be tested in clinical trials of advanced melanoma refractory to standard therapy, as part of VA’s Precision Oncology initiative.

NCI - National Cancer Institute

PROJECT SUMMARY Acute leukemias are the most common pediatric cancers, exhibiting significant variability in treatment response and relapse rates among genomic subtypes. A major challenge in treatment is the presence of leukemic-initiating cells that are resistant to therapy. While these cells were first discovered in acute myeloid leukemia, their role in other types of acute leukemia remains poorly understood. Recent studies have uncovered a neoplastic population of hematopoietic stem and progenitor cell-like (HSPC-like) blast in high-risk subtypes of acute lymphoblastic leukemia. These HSPC-like blasts are molecularly heterogeneous and linked to treatment resistance and poor outcomes. This study aims to investigate the molecular characteristics and clinical implications of HSPC-like blasts across major high-risk leukemia subtypes, including myeloid, lymphoblastic, and mixed-lineage leukemias. Our goal is to uncover common, targetable mechanisms of treatment failure and to develop therapeutic strategies that specifically target these cells. Using single-cell multiomics and patient samples, we will identify the mutational and transcriptomic signatures and define gene regulatory networks using novel network biology algorithms. We will determine the prognostic significance of HSPC-like signatures and explore therapeutic strategies through ex vivo drug screening and in vivo validation in PDX models. Because primary human leukemic blasts are a scarce, patient-limited resource, expansion in immunodeficient mice yields the millions of cells needed for ex vivo drug testing, in vivo therapeutic studies, and mechanistic analyses.

NIDCD - National Institute on Deafness and Other Communication Disorders

Project Summary Olfactory sensory neurons (OSNs) facilitate our sense of smell but constantly need to be replaced, likely because they are in direct contact with the external environment. The olfactory epithelium (OE) houses OSNs, and maintains our ability to smell throughout adulthood through nearly life-long neurogenesis. This remarkable ability for adult neurogenesis is not limitless, however. With increasing lifespan and a hostile external environment, a near majority of people develop hyposmia or anosmia by the age of 80. This is correlated with reduced quality of life, a slew of mental disorders, and malnutrition. Pathologic examination of aged human patient tissue suggests that olfactory neurogenic stem cells exhaust with age, and previously neuronal olfactory epithelium gradually becomes a-neuronal potentially even becoming respiratory epithelium. Unfortunately, no facile preclinical model that closely mimics this human OE pathology exists, hampering research and therapeutic development. Previous models were slow and poorly penetrant. Here, we describe a new model using an engineered nitroreductase enzyme (OMP-NTR2.0) that is highly effective at accelerating OSN turnover, can strikingly mimic aged human olfactory epithelium in as little as 12 weeks time, and could be used as the first platform for testing therapeutic approaches. In this grant, we propose to (Aim 1) extensively characterize this new model of accelerated aging in the OE, stage and compare it to human biopsy and donor tissue, (Aim 2) test the hypothesis that respiratory metaplasia results from conversion of exhausted olfactory epithelium as well as invasion from the surrounding respiratory epithelium, and finally, (Aim 3) test targeted therapies developed on our knowledge of olfactory epithelial stem cell dynamics. The objective of this proposal is to establish the OMP-NTR2.0 model as the viable preclinical model of age-associated olfactory dysfunction and use it to test first-generation therapeutic approaches. Our approach is innovative because it leverages a novel mouse model that we generated de novo that incorporates an engineered enzyme, which effectively accelerates aging of the olfactory epithelium and creates a platform for drug testing. Our long-term goal of our research is to develop prophylactic or curative treatments for age-associated anosmia.

NIAID - National Institute of Allergy and Infectious Diseases

New treatments are desperately needed to control the ongoing tuberculosis (TB) pandemic. Newly emergent antibiotic-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB, are hampering control efforts. Mtb is an unusual pathogen with the remarkable ability to cause both acute life-threatening disease and symptomless latent infections that can last a lifetime. It is estimated that 25% of the world has latent tuberculosis, and in 2023 alone, there were more than 8.2 million TB cases and 1.3 million deaths, making Mtb the leading cause of infectious disease world- wide. Mtb is such a successful pathogen partly due to its extraordinary metabolism; part of its virulence stems from the metabolic flexibility to utilize and scavenge a range of nutrients derived from its human host. A fundamental and currently unanswered question regards how the bacterium regulates its metabolism to cause infection. Answering this question is of therapeutic significance as targeted dysregulation would both starve the bacteria and prevent it from successfully colonizing the human host, effectively killing the bacteria and stalling pathogenesis. We have discovered a novel regulatory system that Mtb requires to consume essential energy sources for its survival in the host and to cause disease. Our hypothesis is that Virulence Associated Dikinase (VadK) coordinates metabolism and virulence by interacting with partner proteins. Importantly, VadK also represents a novel drug target. We will test this hypothesis using a combination of biochemistry, structural biology and microbiology. We will investigate how VadK physically and functionally interacts with its partner proteins to gain insights into how this novel histidine kinase system functions, while also unraveling the mechanism of action of VadK.

NIGMS - National Institute of General Medical Sciences

Glycosylation is a ubiquitous post-translational modification in mammalian systems that fine tune or fully control every aspect of cellular function. This process involves the biosynthesis of glycans by the concerted action of ~400 genes that are called ‘glycogenes’. Because of the importance of glycosylation in normal physiology and pathophysiology, understanding how glycans are regulated is of the utmost biomedical importance. The factors that regulate glycan biosynthesis in mammals remain incompletely known because systems-level characterization of glycosylation regulatory network is absent. This project brings together synergistic expertise in systems biology and bioinformatics (Gunawan), glycobiology and biomolecular engineering (Neelamegham), and machine and deep learning (Chen), to address this knowledge gap. We hypothesize that single-cell profiling coupled with mechanistic and deep learning-based modeling and analysis can reveal the key regulators and regulatory networks of glycosylation. The specific aims are: 1) Generate single-cell epigenomics, transcriptomics, and glycomics profiles in hematopoietic stem cell (HSC) differentiation. We choose blood cell system due to their broad biological importance and ease of access. This aim produces single-cell multi-omics data related to glycosylation that will be computationally analyzed in subsequent aims using mathematical and deep learning (DL) modeling. 2) Curate transcriptional regulators and reconstruct gene regulatory networks of glycosylation using data mining and integrative bioinformatics analysis of single-cell data. This aim focuses on transcriptional regulation of glycogenes. We will catalog transcriptional regulators (TRs) and reconstruct and experimentally validate gene regulatory networks of glycosylation in HSCs using single-cell epigenomics and transcriptomics data. 3) Bridge the expression of glycogenes and glycans using flux analysis and deep learning to elucidate regulatory factors of glycosylation. This aim employs first-principle and deep learning models of glycosylation reaction networks to learn the complex, non-linear mapping from glycogene expression to glycoenzyme activity to glycosylation fluxes and glycan abundances. A novel DL model using a combination of Representation Learning and Graph Attention Network will be developed. Systems analysis of the model using Metabolic Control Analysis will provide network-level insights on the regulators of glycosylation. Model-derived glycosylation regulators will be experimentally validated in HSCs, the data from which will be used to fine-tune the model. Overall, this project will generate systems-oriented methods for the Glycosciences that will enable linking cellular epigenetics, transcriptomics, glycoenzyme activity, glycosylation network, and glycan structures. By iterating modeling, systems analysis, and experiments, we will generate insights into gene-level and network-level regulation of glycosylation. Given the importance of glycosylation in human biology, such insights will have broad impact on basic science and disease studies, and in the development of related protein therapeutics.

NIMH - National Institute of Mental Health

Project Summary Autism spectrum disorder (ASD) is a neurodevelopmental disorder long associated with functional connectivity abnormalities that are widespread throughout the brain. Despite countless studies on the topic, no single unifying model of functional connectivity abnormalities in ASD has emerged to date. Enthusiasm for studying functional connectivity differences in ASD has subsided in light of evidence of alterations that are more heterogeneous than some of the earlier hypotheses suggested, and because the links of these alterations to neural mechanisms of ASD have been challenging to map. Yet, one related theme that has garnered support has been that functional connectivity in ASD is increased in the bottom-up (or "feedforward") direction and decreased in the top-down (or "feedback) direction. Here, we propose to test a hypothesis that the functional characteristics of ASD are rooted in a fundamental imbalance between feedforward and feedback influences. This hypothesis stems from our preliminary data and prior studies, and it is motivated by many aspects of the ASD phenotype: These include increased perceived salience of sensory stimuli and evidence of reduced top-down control in ASD, which manifest across a range of atypical behaviors characteristic of the disorder. Thus far, mapping feedforward and feedback inputs non-invasively in the human brain has been methodologically challenging. Here we propose a multimodal neuroimaging approach, which combines (a) effective connectivity measures using millisecond temporal resolution magnetoencephalography (MEG) with (b) highly novel submillimeter-resolution layer- specific 7T functional MRI. We will use these advanced techniques to characterize feedforward and feedback flow of information along the auditory cortical hierarchy, in 90 ASD and 60 neurotypical (NT) adults, ages 21- 35, with average or above average IQ. Using our multimodal research design, which is firmly rooted in laminar neurophysiological recordings in non- human primates, we will pursue the following Specific Aims: (1) Test the hypothesis that feedforward inputs are abnormally increased in ASD relative to NT individuals; (2) Test the hypothesis that feedback inputs are abnormally decreased in ASD relative to NT individuals; (3) Test the hypothesis that the extent to which feedforward and feedback inputs in the ASD group are indeed different, is predictive of ASD severity and the extent of auditory processing deficits, assessed behaviorally. We expect that the results of this study will lead to a substantially more detailed, comprehensive, and mechanistically motivated framework for the wide range of functional connectivity abnormalities observed in ASD.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Mature cell types must be properly differentiated and distributed in tissues during development in all organisms, including humans. Some examples of cells that are broadly distributed include mucus cells (also called goblet cells) and ionocytes, which populate many human mucosal tissues, including the lung. Mucus cells produce mucus to lubricate and protect the epithelial surface; ionocytes regulate the ion balance on either side of the epithelial barrier. Dysregulation of either of these cell types causes severe human disease in many organ systems. Analogous cell types are also found in embryonic zebrafish skin, a highly tractable model system in which live imaging can be easily used to study development. This proposal will characterize the development of recently discovered intraepithelial migratory cells in embryonic zebrafish skin that are precursors to mucus cells and ionocytes, dissecting their mechanism of migration as well as the gene expression patterns responsible for their differentiation. The Sagasti lab has found that these cells derive from stem-like tp63+ basal cells in zebrafish skin during the first day of development, migrate between the basal and periderm layers of skin throughout the body for several hours, and eventually halt and intercalate into the periderm, where they become differentiated mucus cells or ionocytes. We propose that this process functions to spatially distribute mature ionocytes and mucus cells throughout the skin and may be conserved in human mucosal tissues. In this proposal, I will use live imaging and antibody staining to determine whether these migratory cells exhibit mechanisms of amoeboid or mesenchymal migration, as well as pharmacologic and genetic inhibitions to functionally characterize these pathways. Additionally, I will use single-cell RNA sequencing and pseudotime analysis to describe the process by which these cells differentiate into mature cell types and generate candidate genes that are involved in migration. Finally, I will use a CRISPR screen to find genes that are required for migratory cell motility and distribution, and create knockout lines for future study. Taken together, this project will investigate a method of simultaneous cell migration and differentiation within a developing epithelial tissue, resulting in the proper distribution of mature cells. My training goal is to be an independent investigator in the field of skin developmental biology. The proposed fellowship training is an important step towards this career. The research proposed here will give me an excellent background in zebrafish research and epithelial development, training me in impactful techniques, including live imaging, genetic manipulation, and bioinformatics. Further, my training plan includes the IRACDA program at UCLA, which will train me in teaching pedagogy and provide experience designing and delivering lectures at CSULA under the mentorship of experienced professors. This experience will prepare me for the multifaceted career of a principal investigator by gaining experience with zebrafish epithelial research, teaching, and mentorship.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Tuft cells are rare chemosensory cells, which reside in the epithelial lining of the small intestine (SI) where they serve as key intestinal sentinels by detecting luminal cues and triggering type II immune responses to infections. Specifically, tuft cells signal to and activate type 2 innate lymphoid cells (ILC2s) to mount an immune response and enhance tuft cell production from undifferentiated epithelial cells through a feed forward mechanism. Recent work has demonstrated that fully differentiated tuft cells can dedifferentiation and regenerate all intestinal lineages under damage conditions, highlighting a new facet of epithelial regeneration; however, the molecular signals that balance tuft cell specification and epithelial plasticity remain incompletely understood. Previous work from the Roose lab has identified the Ras Guanine Nucleotide Exchange Factor, RasGRP1, as a suppressor of proliferative EGFR/Ras signals in colorectal cancer. My foundational studies, using a novel multi-antibody spectral flow panel capable, show that mice deficient for Rasgrp1 (Rasgrp1-/-) have significantly fewer tuft cells and clear helminth infections less efficiently than wild-type controls. Single-cell RNA sequencing of sorted tuft cells reveals a reduction in immature tuft cell populations in Rasgrp1-/- mice, which also show increased proliferative activity compared to wild-type controls. Strikingly, I recently discovered that intestinal stem cell (ISC) ablation in Rasgrp1-/- mice results in lethality, suggesting that RasGRP1 is required for the dedifferentiation process that normally restores the ISC pool after injury. These findings support my central hypothesis that RasGRP1 coordinates both forward differentiation and dedifferentiation programs in response to environmental cues. In Aim 1, I will test whether RasGRP1 promotes commitment to the tuft cell lineage by combining transgenic overexpression models, multiplexed spectral flow cytometry, and organoid-based pharmacological assays to mechanistically dissect how RasGRP1 modulates progenitor proliferation and tuft cell generation during homeostasis and infection. I will also determine whether RasGRP1-driven tuft cell hyperplasia spontaneously activates the tuft–ILC2 circuit and alters local immune composition. In Aim 2, I will define whether RasGRP1 enables progenitor-to-ISC dedifferentiation during epithelial injury. I will map RasGRP1 expression patterns in homeostasis and post-injury, use ISC ablation and conditional knockout models to test whether RasGRP1 loss impairs ISC recovery. I will apply innovative real-time lineage tracing and live imaging of organoids to directly visualize RasGRP1+ cells regenerating ISCs. Together, these integrated approaches will reveal how RasGRP1 governs epithelial plasticity and the balance between differentiation and regeneration. Insights gained from this proposal will have broad implications for understanding intestinal homeostasis, type II immunity, and epithelial repair. Furthermore, I anticipate these findings to have implications for future therapeutic strategies to modulate tuft cell proportions in diseases characterized by disrupted epithelial homeostasis or impaired immune responses, such as chronic infections, inflammatory disorders, obesity, or cancer.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary: Annually, millions of patients are diagnosed with autoimmune disorders. The majority of these are treated with global immune suppressants. These drugs often tip the balance from autoimmunity to immune suppression, triggering undesirable, systemic side effects. Previously, it had been established in literature that Exportin-1 (XPO1) is a novel target for the inhibition of T cell activation. XPO1 is canonically recognized as a nuclear cargo export protein where it shuttles cargo from the nucleus into the cytoplasm. Recently, it has been reported that XPO1 also plays a critical role as a chromatin factor. XPO1’s chromatin function can be inhibited with novel molecules coined “Selective Inhibitors of Transcriptional Activation” (SITAs). Mechanistically, it has been determined that these SITAs covalently bind to the C528 position of XPO1 in the same binding pocket as known nuclear export inhibitors called “Selective Inhibitors of Nuclear Export” (SINEs), as exemplified by the FDA approved inhibitor, Selinexor. While SITAs and SINEs covalently engage the same pocket, it is mechanistically unclear how and why these reported SITAs have the disparate phenotypic effect of inhibiting T cell activation compared to established SINEs which inhibit nuclear export. This proposal seeks to test the central hypothesis that the SITAs’ phenotypic deviation from SINEs stems from the rapid reversion of the covalent bond formed by SITAs in the C528 pocket.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Prostate cancer arises as an androgen driven disease, and androgen receptor (AR)-targeted therapies are the mainstay of treatment for men with advanced disease. One mechanism of resistance is ahistologic transformation from an AR-driven prostate adenocarcinoma to an AR-independent small cell neuroendocrine carcinoma, often referred to as neuroendocrine prostate cancer (NEPC). NEPC is clinically aggressive, frequently metastasizes to visceral organs, and carries a poor prognosis. A thorough molecular understanding of NEPC progression is needed for the development of strategies to treat, prevent, or reverse the development of this lethal disease. Although NEPC tumors arise clonally from prostate adenocarcinoma, there is significant epigenetic and transcriptomic dysregulation that occurs during the lineage plasticity process. Mechanistically, we still do not know how these alterations arise and how best to leverage these alterations as a therapeutic opportunity. Based on published reports and on our preliminary data, E2F1 is over-expressed in the majority of NEPC cases and in a subset of CRPC cases and is associated with a poorer prognosis compared to CRPC with low to no E2F1 expression. However, little is known about the role of E2F1 in the progression from CRPC to NEPC. Our preliminary and published data from patient tumors and in vivo, in vitro and ex vivo models (patient- derived organoids) suggest that E2F1 drives a reprogramming of chromatin accessibility which in turn, results in a NEPC-associated change in gene expression and that this is potentially mediated through a physically interaction with specific NEPC-associated co-factors and transcription factors. Our long-term goal is to develop new biomarker-driven therapeutic strategies for treating patients with advanced prostate cancer and untreatable NEPC. The objective here is to identify the key molecular events and mechanisms underlying lineage plasticity in prostate cancer. This would allow for early therapeutic intervention and improve patient outcome. Our over- arching hypothesis, which is based on our published and preliminary data, is that specific molecular alterations (e.g. RB1 loss and E2F1 induction) in prostate cancer cells drive the progression of CRPC tumors towards NEPC resulting in changes to chromatin accessibility and interactions with specific pro-stem cell- and neural lineage- associated transcription factors to drive a NEPC-associated change in gene expression. To address this hypothesis, we will define essential E2F1-transcriptional complex proteins that mediate the gene expression program driving and maintaining NEPC-progression (Aim 1) and determine if E2F1 is essential in mediating the gene expression program associated with the transition from CRPC towards NEPC (Aim 2). Successful completion of these Aims will provide unique insights into NEPC development, identify potential targetable mediators of lineage plasticity, and a timely and unique opportunity for the early detection of patients with E2F1- expressing CRPC that are evolving towards NEPC that may not respond to standard-of-care anti-AR therapy.

Chevron Foundation

Supports STEM education, workforce development, and community vitality in regions where Chevron operates.

NIEHS - National Institute of Environmental Health Sciences

Project Summary/Abstract N6-methyladenosine (m6A) is the most abundant messenger RNA (mRNA) modification and also occurs on chromatin-associated RNA in mammals. m6A significantly affects messenger RNA (mRNA) processing, export, localization, translation, and stability, thus playing critical roles in a diverse range of biological processes including stress responses. We identified the first two m6A RNA demethylases, FTO and ALKBH1, in 2011 and 2013 respectively, revealing that m6A RNA methylation is reversible and can broadly affect gene expression. Our previous research also revealed upregulation of FTO in human and mouse keratinocyte cells upon exposure to arsenic. The upregulated FTO may mediate mRNA m6A demethylation and affect cellular homeostasis, DNA damage response, and tumorigenesis induced by arsenic exposure and UV irradiation. We have recently discovered an additional layer of RNA m6A methylation regulation: we identified prevalent m6A methylation of mammalian promoter-associated RNA (paRNA), enhancer RNA (eRNA), and RNA transcribed from transposable elements (repeat RNA), which we collectively termed chromatin-associated regulatory RNAs (carRNAs). We showed that these carRNA m6A modifications could be recognized by binding proteins (readers), which recruit either the nuclear degradation exosome complex to degrade the methylated RNA or histone modification proteins to alter local histone mark deposition, thereby modulating the chromatin state and downstream transcription. Our preliminary results suggest that nuclear-localized FTO in keratinocyte cells also mediates carRNA demethylation, which can induce chromatin state and transcription changes in response to arsenic stimulation. In Aim 1, we plan to identify the exact carRNA and carRNA m6A sites affected by FTO and uncover the underlying regulatory pathways that contribute to stress response and tumorigenesis in response to arsenic exposure and UVB irradiation. We have also discovered carRNA m6A demethylation by ALKBH5, which was not previously known, in mouse embryonic stem cells (mESCs). ALKBH5 undergoes SUMOylation in cells treated with reactive oxygen species (ROS), which inhibits its demethylation activity and protects cells from DNA damage. In Aim 2, we plan to establish carRNA m6A demethylation by ALKBH5 and elucidate the underlying chromatin regulation pathways and mechanisms using mESCs as the mode system. We will also investigate how ROS-induced ALKBH5 SUMOylation may affect the chromatin state and DNA damage response in mESCs and keratinocyte cells under H2O2 treatment or arsenic exposure. Collectively, the proposed studies will determine the epitranscriptomic-epigenetic interactions in environmental stress responses and tumorigenesis for the first time. New mechanistic insights and new molecular targets for improving human health may emerge.

NIA - National Institute on Aging

ABSTRACT Our broad research goal is to understand chromatin regulatory mechanisms in nuclear and epigenetic programs and how these mechanisms are deregulated in aging and disease. A fundamental mechanism for regulating chromatin involves the reversible modification of histones by chemical moieties such as acetyl-, methyl-, and phospho- groups. These different histone marks are linked to discrete chromatin states and regulate the accessibility of DNA to transacting factors. In budding yeast, histone deacetylation by the chromatin silencing factor Sir2 prevents genomic instability and aging, and in mammals, de-regulation of histone acetylation is linked to cellular senescence and aging-related pathologies from neurodegeneration to cancer. Here, we focus on the mammalian Sir2 family member SIRT7, a chromatin regulatory, highly selective, lysine deacetylase enzyme. Previous studies reported that loss of SIRT7 function in mice leads to genomic instability, shortened lifespan and aging-related phenotypes including fatty liver, cardiac disease, and hematopoietic stem cell dysfunction. This project will study new roles of SIRT7-dependent histone deacetylation in chromatin regulatory mechanisms that are deregulated in aging-associated metabolic pathologies. It employs biochemical, cellular, and genomic approaches, and leverages SIRT7 knockout (SIRT7-KO) mice and new mouse models in which SIRT7 is overexpressed (SIRT7-OE). A central hypothesis is that SIRT7 protects against aging and metabolic disease processes and attenuates metabolic pathologies when overexpressed in mice. The project also hypothesizes that a novel histone substrate of SIRT7, H3K36, contributes to functions of SIRT7 in aging and metabolic pathways. Little is known about acetylation of H3K36, but di-methylation of H3K36 (a histone modification linked to gene regulation) is implicated in many human cancers, developmental disorders, and recently, metabolic disease. We hypothesize that deacetylation of H3K36 by SIRT7 is coupled to methylation by lysine methyltransferase (KMT) enzymes. We will test the model that a key mechanistic function of SIRT7 is to clear H3K36 acetylation from large swaths of DNA across the genome to enable methylation by KMTs, and that such a H3K36 acetyl-methyl switch mechanism is essential for preventing aging-associated gene expression reprograming in metabolic tissues.

NIH

Significance to VA: Type 2 diabetes (T2D) is a chronic, adult-onset metabolic disease with no cure to date. Alarmingly 1 in 4 (25%) Veterans are diabetic, significantly higher percent compared to civilians. Diabetes is the leading cause of renal and cardiovascular diseases, blindness, and amputations in Veterans. The annual mortality rate of Veterans with diabetes is nearly twice the rate compared to Veterans without diabetes (VA Fact Sheet 2019). The predominant risk factors for T2D advent in Veterans are military exposures and PTSD. Relevance to VA Military Exposures Research Program Mission: Vietnam War (VW) Veterans were exposed to Agent Orange (AO). Evaluating the effects of military exposures, it is established that tetra- chloro-dibenzo-p-dioxin (TCDD), contained in AO caused T2D pathology. T2D is a presumptive condition in VW Veterans. In the recent Global War on Terrorism (GWOT) conflicts, 85% of military personnel were exposed to open burn pits which emitted large amounts of TCDD. Measuring TCDD levels in the blood involves a long, cumbersome procedure requiring specific equipment and highly trained personnel, thereby deterring routine evaluation of TCDD exposures. Further, little is known about the chronic metabolic outcomes, specifically T2D, in GWOT Veterans. There is an immediate and dire need to identify Veterans with increased susceptibility to advent of T2D due to service-related TCDD exposures, independent of their genetic makeup, aging, or obesity. The proposal fills this gap with our overarching goal to establish a rapid, blood genetic test to identify Veterans vulnerable to chronic health sequelae of T2D, years after TCDD exposures. Innovation and Impact: We have established that low GAS5 levels are causal to T2D (US Patent Nos. 10,724,097; 11,214,835, 11,278,521. All with VA-asserted rights). The data demonstrates that GAS5, a noncoding regulatory RNA, is the genetic target of TCDD in humans, which unknown thus far. The project will establish GAS5 as a surrogate biomarker of TCDD-exposure related advent of T2D in Veterans. Importantly, to integrate into clinic, we developed a rapid blood droplet test for reproducible, consistent measurements of GAS5 levels requiring minimal technical expertise. Successful completion of the proposal will establish GAS5 as a surrogate biomarker of TCDD-exposure related advent of T2D in Veterans. This determination will significantly reduce the advent of T2D as the most preventable cause of death in US Veterans. Specific Aims: Our compelling data shows TCDD decreases GAS5. We hypothesize that low GAS5 levels in the blood correlate with past military associated TCDD exposures and the advent of T2D in Veterans. Specific Aim 1: Longitudinal study evaluating the correlation of blood GAS5 with TCDD levels in Veterans. Specific Aim 2: Determine the TCDD-regulated molecular mechanisms affecting GAS5 in human adipocytes. Methodology: For SA1, we will leverage our existing JAHVA Research Biospecimen Repository and obtain de-identified blood samples (IRB determination: Not Human Subject Research). The levels of TCDD, GAS5 will be measured and correlated to T2D by a sample-blinded biostatistician. For SA2, we will use human adipose stem cells differentiated to adipocytes. We will elucidate the underlying molecular and cellular mechanisms to understand TCDD-regulation of GAS5 levels, thereby providing unequivocable support complementary to SA1. NPC86, a therapeutic which increases GAS5, will be used as a molecular tool. Path to Translation/Implementation: We have developed a minimally invasive, minimal technical expertise requirement and ease-of-use, rapid (2-hour turn around) blood droplet test to measure GAS5 levels that can be easily incorporated into clinic. The results can be evaluated along with the Veterans’ TCDD exposure history to implement a personalized, time-sensitive treatment plan to prevent the advent of T2D.

NCI - National Cancer Institute

SUMMARY. Regulating the timing of meals and snacks to re-align the body’s circadian clock and improve metabolic health is emerging as a promising approach for cancer prevention in early animal and small clinical studies. Yet, a major barrier to studying meal timing and cancer is that large population studies rarely measure meal timing, which makes it impossible to conduct epidemiological studies of meal timing and cancer risk on a large-scale and across individuals with different biological and environmental characteristics and varied meal timing practices. Although obesity and its related metabolic dysregulation are important risk factors for at least 13 cancer types, weight management is notoriously difficult in the long-term. Behavioral strategies are needed that can improve metabolic risk factors for cancer but that do not necessarily rely on weight loss. Herein, we propose to discover and then externally validate novel objective biomarkers of meal timing practices, then test their relationship with incidence of breast (BC), endometrial (EC), and colorectal (CRC) cancers, three of the most prevalent obesity-linked cancers. Our central hypothesis is that meal timing is associated with perturbations in blood metabolomic profile, and with obesity-related cancer incidence in free-living humans. We will test our hypothesis with unique data from large-scale cohorts with validated measures of meal timing and sleep and longitudinal metabolomics data measured on the same metabolomics platform to facilitate data harmonization. In Aim 1, we will Identify biomarkers of meal timing patterns using a discovery and external validation design in the Cancer Prevention Study 3 Diet Assessment Sub-study (DAS) (n=750) and the Interactive Diet and Activity Tracking in AARP (IDATA) Study (n=718) and measure their association with risk of obesity-related cancer in the Cancer Prevention Study 2 (CPS-2, 782 BC matched sets; 517 CRC matched sets with 16-year follow-up time) and CPS-3 cohorts (1695 BC patients, 1983 controls, 3-year follow-up). In Aim 2, we will examine whether there is large-scale, real-world evidence that meal timing patterns are associated with obesity-related cancer risk among 185,000 US adults in the CPS-3 cohort. The proposed study will answer critical, outstanding questions about which meal timing practices are associated with cancer- relevant metabolic factors and risk of obesity-related cancers in a real-world scenario and identify objective biomarkers of meal timing behaviors that will facilitate large-scale investigations of meal timing and cancer risk at the population level. These meal timing biomarkers could also be used to assess response to meal timing interventions in clinical studies. Following successful completion of this project, we plan to apply the resulting biomarker profiles to study meal timing and cancer risk across international cohorts in the Consortium of Metabolomics Studies (COMETS). Epidemiological research stemming from our study findings will be vitally important prior to issuing public health guidance on meal timing for cancer prevention.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT (30 lines or less) The premise for the proposed research stems from precedence in other diseases, such as cancer, cardiovascular, neurodegenerative, and most relevant to the current proposal, autoimmune diseases, in which circulating extracellular vesicles (cEVs) play a role in pathophysiology and are important biomarkers for early detection. However, little is known concerning the role of cEVs in human type 1 diabetes (T1D). Our overall hypothesis is that cEVs have the potential to be used as biomarkers for early pre-disease detection of T1D, based on their distinct molecular and functional phenotype in T1D and pre-disease stages, compared to healthy or low risk individuals. Our specific aims are: 1) To identify the distinct protein and RNA cargo unique to cEVs at different stages of T1D disease development; and 2) To investigate the effect of cEVs from subjects at different stages of disease progression on immune and beta cell phenotypes and elucidate the functional relevance of their distinct molecular cargo. To address these aims, we have assembled a team of investigators with highly relevant expertise and techniques. We propose to use longitudinal samples from The Environmental Determinants of Diabetes in the Young (TEDDY) study of children with T1D associated genetic risk to identify critical timepoints and underlying mechanisms mediating, A) the earliest stages of pathogenesis preceding AAb appearance of the first islet autoantibody, and B) the period of seroconversion from single to multipe AAb+ or remaining single AAb+ and C) the period after multiple AAb appearance with a highly variable rate of progression to hyperglycemia. We have the expertise and technical ability to isolate cEVs from plasma, perform proteomic and RNAseq analysis on EVs, and perform immune and beta cell related functional assays. Our Research Plan is to generate cEVs from donors at different stages of T1D disease progression, to identify the uniquely packaged protein and RNA cargo from these cEVs, to evaluate the effects of the cEVs on immune cell functional phenotype and islet health and elucidate the functional relevance of the distinct molecular cargo targets. These studies will yield novel mechanistic insights into early disease pathogenesis and identify potential novel biomarkers for T1D initiation and progression.