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NIBIB - National Institute of Biomedical Imaging and Bioengineering

TITLE: PRAISE (Pressure Relief Assessment Information System): A Paradigm-shifting Mobile Health Platform for Pressure Relief Adherence in Manual Wheelchair Users PROJECT SUMMARY: The proposed project aims to create a pressure relief assessment information system (PRAISE) to enhance the adherence of manual wheelchair users to Clinical Practice Guidelines (CPGs) designed to prevent pressure ulcers. The motivation of this research stems from two core challenges. First, pressure ulcers pose a serious threat to manual wheelchair users with spinal cord injuries, frequently leading to painful complications, infections, and even premature death. To reduce pressure ulcer risks, CPGs recommend that wheelchair users perform pressure relief activities (i.e., vertical pushups, lateral, and forward leans) every 15 to 30 minutes. However, research reveals that wheelchair users may not adhere to CPGs in everyday life. Second, no universally adopted tools currently exist to monitor CPG adherence, nor is the understanding of factors leading to non-adherence. As a result, the prevalence of pressure ulcers among wheelchair users with spinal cord injuries remains high. Built upon the International Classification of Functioning, Disability and Health (ICF) model, PRAISE will shift from the conventional singular focus on adherence to a holistic approach, which will cohesively integrate a user's health, personal, and environmental factors through its multidimensional design. First, PRAISE will enable users to create profiles, including demographics, wheelchair usage patterns, and medical records related to pressure ulcers. Second, this foundational data will be augmented by a spectrum of sensor data (i.e., accelerometer, heart rate, GPS, and battery life) from a smartwatch, critical for ecological momentary assessments (EMAs). Third, our novel distributed algorithm can accurately detect pressure relief activities without relying on frequent, costly internet connections. It achieves this through lightweight processing on mobile devices to capture patterns intrinsic to pressure relief activities, hence transmitting only relevant data segments to the server for fine-grained recognition. Fourth, grounded in the ICF framework, PRAISE will dynamically integrate user-specific health, personal, and environmental factors to deliver context-aware feedback and personalized guidance. Through reinforcement learning, PRAISE will continuously evolve its guidance by learning from user responses and behavior, ensuring that interventions remain effective and tailored to individual needs over time. In collaboration with a diverse advisory team, PRAISE's development will prioritize robust security, user- friendliness, advanced analytics, and customizable assessment modules. Once the advisory team completes the initial validation, a feasibility and acceptability assessment will be conducted by involving 15 manual wheelchair users for two weeks. To gain a deeper understanding of user experiences, we will employ multifaceted approaches to gather and analyze user feedback. As PRAISE strives to make pressure ulcer prevention more accessible and personalized for wheelchair users, it will help reduce health disparities, particularly for those who may not have easy access to traditional healthcare resources. Therefore, PRAISE will revolutionize care for the manual wheelchair users to achieve patient-centric, evidence-based interventions.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT A lack of proven interventions to prevent leukemia in aged populations leaves a growing demographic vulnerable to this devastating disease. While leukemic cells are subject to immune selection which influences disease progression, we lack knowledge of the stages at which T cells shape the hematopoietic stem and progenitor cell (HSPC) pool from the initiation of clonal hematopoiesis (CH) through to the progression to leukemia, which limits our ability to intervene in this process. The long-term goal of this project is to identify immuno-preventative strategies to intercept leukemogenesis at its earliest stages. The overall objective of this application is to determine the mechanisms by which, and at which stages of pre- leukemic development, HSPC clones are detected and selected by the adaptive immune system. The central hypothesis is that reduced IFNγ responsiveness enables immune evasion of CH-mutant (Dnmt3amut) HSPCs thereby promoting clonal expansion and pre-leukemic evolution. The rationale is grounded in the observation that HSPCs from humans and mice with recurrent CH driver mutations in Dnmt3a have reduced transcript and protein expression of MHC-II machinery, and reduced presentation of exogenous and endogenous antigens via MHC-II. Mechanistically, MHC-II is potently induced by IFNγ on wild-type HSPCs but to a lesser extent on Dnmt3amut HSPCs. In vitro and in vivo, we observe less activation and proliferation of CD4+ T cells by Dnmt3amut HSPCs compared to control HSPCs, supporting that Dnmt3amut HSPCs have reduced immunogenicity. The central hypothesis will be tested by pursuing two specific aims: 1) to define the stages of pre-leukemic HSPC selection that are controlled by CD4+ T cells, and 2) to evaluate decreased IFNγ response of pre-leukemic Dnmt3amut HSPCs as a mechanism of immune evasion. This research is innovative because it introduces a novel framework for understanding how adaptive immunity shapes clonal evolution of pre-leukemic HSPCs. While considerable attention has been given to genetic and cell-intrinsic drivers of CH, the role of immune surveillance—particularly adaptive immune selection—in governing HSPC clonality remains largely unexplored. Ultimately, the proposed work is significant because it will define the role of CD4⁺ T cells in HSPC clone selection during early disease phases in CH and pre-leukemia which has major therapeutic implications for immunoprevention of leukemia.

NINDS - National Institute of Neurological Disorders and Stroke

Abstract The ultimate success of immunotherapy for brain malignancies, such as malignant glioma, will require integration of in-depth understanding of immunology with solutions for the following long-standing challenges: 1) paucity and heterogeneous expression of glioma-specific antigens; 2) on-target off-tumor toxicity and exhaustion of therapeutic T lymphocytes, such as chimeric antigen receptor (CAR) T-cells; 3) immunological privilege of the CNS and 4) immunosuppression involving tumor, neuronal, and immune cells. My laboratory has contributed to critical discoveries in these areas and integrated our findings into novel immunotherapy clinical trials for glioma patients. In the current proposal, I will enhance my research by mobilizing multiple immune mechanisms. To this end, I will collaborate with an outstanding group of investigators whose diverse expertise in multi-disciplinary areas complements my own in brain tumor immunology as the central component and apply a wide variety of resources available at UCSF and collaborators to one overarching program. I will evaluate the overarching hypothesis that the integration of novel cell-engineering and antigen-targeting approaches will allow us to develop safer and more effective immunotherapy strategies by overcoming heterogeneous expression of antigens and unique challenges in brain immunology. I will evaluate the following strategies: 1. Develop neo- junction-targeting T-cell receptor (TCR)-T cell-based immunotherapy. We will leverage our highly reliable and valuable pipeline for T-cell epitope prediction, which we established during the current funding cycle, to discover novel neoepitopes derived from tumor-specific alternative splicing events (neojunctions). 2. Develop novel cell therapies using allogeneic induced pluripotent stem cells (iPSCs) and in vivo transduction approaches. While my current NINDS R35 award allowed me to implement the first-in-human phase I study of Synthetic Notch (synNotch)-CAR T-cell therapy in patients with glioblastoma, inherent and logistical challenges associated with the use of autologous T-cells motivate us to develop these novel and alternative approaches. 3. Enhance “epitope spreading” to overcome the antigen heterogeneity. While the novel synNotch-CAR approaches are promising, one major inherent challenge is that targeting a few or several antigens by CARs or TCRs may not adequately cover the marked antigenic heterogeneity of tumors. We will enhance the effects of low-intensity pulsed ultrasound with microbubbles (LIPU/MB) to induce adaptive immune responses against heterogeneous tumor antigens. 4. Investigate the glioma-neuronal circuit-induced immune regulation. We will delineate essential mechanisms on our recent discovery of neuronal activity-driven immunosuppression as a previously unrecognized resistance mechanism of cancer immunotherapy for gliomas. These 4 strategies will be logically integrated into combination approaches. As expected per the purpose of the NINDS R35 mechanism, these strategies may involve high risks. However, based on our preliminary proof-of-principle data, we will persistently pursue our goals with long-term support from the R35 mechanism and adopt new technologies flexibly and swiftly.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Patients with obstructive vascular diseases, such as atherosclerosis or peripheral arterial disease, or acute peripheral injuries require vascular grafts to restore blood flow to areas of the body. While the use of autologous vessels is considered the gold standard of treatment, many patients lack suitable vessels due to either vascular disease, previous usage, or have a size mismatch to the injured vessel. Therefore, clinicians turn towards synthetic grafts, such as expanded polytetrafluoroethylene (ePTFE) or Dacron, for large diameter vessel reconstruction. However, these synthetic materials fail when used in clinical small-diameter vascular applications, requiring the development of novel, hemocompatible vascular grafts for these clinical needs. Previous clinical trials have investigated acellular tissue-engineered vascular grafts (TEVGs) developed using human primary smooth muscle cells seeded on biodegradable scaffolds. After robust extracellular matrix (ECM) deposition, these TEVGs were subsequently decellularized and directly investigated for vascular treatment. While promising, the acellular TEVGs lacked an endothelium, and resulted in significant occlusion and suboptimal function within patients. Therefore, developing a novel TEVG with a functional endothelium that is immunocompatible to any recipient is of great clinical need. To address this issue, we propose using human induced pluripotent stem cells (hiPSCs) to fabricate a robust TEVG lined with an endothelium that is universally accepted by any patient, mitigating allogeneic immunorejection. In this proposal, hiPSCs will be differentiated into vascular smooth muscle cells (VSMCs) and subsequently used to generate a robust TEVG in our bioreactors that is then decellularized. Of novelty, we will then endothelialize the TEVGs with hypoimmunogenic, “universal” endothelial cells (ECs) that have been previously developed in our lab by modulating human leukocyte antigens (HLA) expression. To avoid xenograft immunorejection, this proposal will develop and characterize universal pig iPSCs (piPSCs) to endothelialize the TEVGs through downregulating expression of MHC I and II molecules and upregulating expression of CD47 via CRISPR-Cas9. The aims of this grant are to (1) characterize hypoimmunogenic universal piPSC lines for vascular graft engineering and (2) to generate universal iPSC- TEVGs and investigate their hemocompatibility in a preclinical porcine carotid bypass model in vivo. By investigating the universal iPSC technology in a preclinical porcine model, future studies will investigate human universal iPSCs for vascular tissue engineering purposes, furthering our goal towards developing a universal vascular conduit accepted by any patient.

NIGMS - National Institute of General Medical Sciences

Convergent molecular evolution, especially among distantly related species, is a hallmark of adaptation, yet the drivers of such convergence (or lack thereof) are typically unknown. Variation in molecular convergence may stem from constraints on evolutionary trajectories, such as how intramolecular epistasis and broader scale interactions among genes differ across lineages. While substantial progress has been made in understanding the prevalence of epistasis for fitness-related phenotypes, particularly in microbial systems, empirical tests of the role of epistasis in convergent molecular evolution are rare, especially in metazoans. A key obstacle is the lack of tractable, highly replicated systems to investigate the extent and generality in the causes of molecular convergence. To meet this need, we have been studying a diverse group of insects which have adapted to cardenolides, a class of steroidal plant toxins that disrupts the biomedically-relevant animal protein, Na/K-ATPase. We recently documented a remarkable 30 independent origins of cardenolide-specialization in insects, spanning 350 million years of evolution (in six taxonomic orders, spanning beetles and flies to grasshoppers). Although a handful of substitutions did indeed convergently evolve in all orders, some species lack these substitutions and others have taken alternative paths. Our findings, which also show distinct patterns among groups (e.g., Coleoptera vs. Lepidoptera, each with multiple origins) suggests lineage-specific constraints of genomic background. This group of insects thus presents a treasure trove of opportunity to decipher the drivers of molecular convergence. How variable are the epistatic interactions between lineages, and do these differences drive alternative outcomes in molecular evolution? Do multiple genes coevolve, shaping patterns of convergence? For example, have ABC transporter genes involved in excretion and storage, which complement resistance to cardenolides, evolved in parallel to Na/K-ATPase substitutions? And finally, do molecular substitutions predictably track the evolution of specific toxins coevolving in host plants? This system allows for some of the strongest general tests of why adaptive phenotypic outcomes have a similar genetic basis. Beyond comparative genomics, which will reveal distinct evolutionary outcomes and genetic associations, we will integrate the power of transcriptomics, in silico models, and functional assays to directly test our hypotheses. Our five-year program is expected to reveal general rules governing when intramolecular epistasis versus broader interactions among genes drive molecular convergence.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY AND ABSTRACT Patients with inflammatory bowel disease (IBD) often require a restorative proctocolectomy and ileal pouch- anal anastomosis (IPAA) for medically refractory ulcerative colitis (UC) or colonic Crohn’s disease (CD), which involves removal of the entire colon and rectum while preserving the anal sphincter with the creation of a pouch organ by looping healthy ileum into a J-shape that serves as an internal pelvic reservoir. While an IPAA preserves continence and avoids the requirement for a permanent ileostomy, approximately 50% of patients develop pouchitis— a de novo inflammatory condition of the ileal reservoir—resulting in substantial morbidity. The pathogenesis of pouchitis remains poorly understood. Our preliminary data using ExCITE-seq, a multi- modal single-cell sequencing platform, revealed that patients who later develop pouchitis exhibit a distinct immune-epithelial signature in the pre-IPAA ileum, including enrichment of Th17 cells, epithelial remodeling, and persistent clonal T cell expansion. Additionally, stem cells derived from inflamed pouch tissue demonstrated epigenetic alterations associated with impaired viability and secretory lineage differentiation. We hypothesize that pre-existing Th17-driven immune dysregulation and aberrant epithelial responses contribute to the development of pouchitis. To test this hypothesis, in Aim 1, we will evaluate the relationship between the immune microenvironment in the pre-IPAA ileum and subsequent pouchitis. In an existing cohort of patients with longitudinal follow-up we will assess pre-IPAA ileal tissue for Th17-associated immune infiltration, epithelial apoptosis, and spatial transcriptomic profiles, and associating these findings with clinical pouch outcomes. Aim 2 will define how pouchitis-associated immune dysregulation impacts epithelial biology. We will determine the impact of Th17-mediated cytokines on epithelial cell fate and viability using organoid models derived from pre-IPAA ileum and investigate the role of the microbiome in modulating this immune-epithelial axis through integrated metagenomic analyses of pre-IPAA ileostomy stool. Collectively, this work will identify predictive immune, epithelial, and microbial biomarkers and mechanisms of pouchitis, with the potential to inform IPAA eligibility and guide preventive therapeutic strategies in pouchitis.

NIDA - National Institute on Drug Abuse

PROJECT SUMMARY Cannabis use in pregnancy is becoming increasingly common, with up to 23% of pregnant people testing positive for delta 9-tetrahydrocannabinol (Δ9-THC; the most common cannabinoid) at delivery. Prenatal exposure to cannabis has been linked to low birthweight. Our pilot data further suggests that prenatal cannabis is associated with rapid infant growth followed by higher adiposity and glucose at 5 years. While compelling, the epidemiologic data is often limited by self-report of whole cannabis use or bioanalytic testing of Δ9-THC. As such, there is a paucity of data on the potential health effects of cannabidiol (CBD). Emerging evidence suggests that one in five pregnant people use CBD products, yet we know shockingly little about its health impacts in pregnancy. Furthermore, human studies may be confounded by socioeconomic status, polysubstance use, and other lifestyle factors, which limits our ability to establish causality. Therefore, exploring the direct effects of prenatal exposure to cannabis on perinatal tissues may help to provide evidence of a causal relationship. Both Δ9-THC and CBD interact with receptors in the endocannabinoid system, as well as non-canonical metabolic receptors like peroxisome proliferator-activated receptor (PPAR)-γ. These pathways are relevant to two perinatal tissue types: placental microvascular endothelial cells (ECs) and umbilical cord tissue-derived mesenchymal stem cells (MSCs). To address these gaps in knowledge, we have designed a translational study that will combine large-scale epidemiologic investigation with in vitro experiments. We will leverage two ongoing racially and ethnically diverse Colorado-based cohorts: Healthy Start and Mile High ECHO. Our overarching goal is to assess the impact of in utero and in vitro exposure to Δ9-THC and CBD on angiogenic and adipogenic pathways in primary placenta EC and MSC cell lines derived from our participants. We will also explore the extent to which these phenotypes mediate the associations between prenatal exposure to cannabis and child adiposity and metabolic health. Finally, we will explore whether other individual-level factors (diet, tobacco) alter risk, and whether there are prenatal windows of heightened suscepticilty. Drs. Moore (contact MPI; environmental epidemiologist with established expertise on health effects of prenatal cannabis exposure) and Boyle (MPI; basic scientist with a strong focus on molecular metabolism and stem cell biology) will carry out this research. They will lead a talented, multidisciplinary team of investigators with complementary and integrated research expertise with expertise in molecular mechanisms underlying placental insufficiency (Dr. Su), cannabinoid exposure assessment (Dr. Klawitter), and community- based dissemination (Dr. Rinehart). The findings have great potential to identify biological pathways and will support a community-based dissemination plan that is designed to educate providers and empower pregnant people about the potential health risks and legal consequences of cannabis use.

NIA - National Institute on Aging

PROJECT SUMMARY The mission of our laboratory is to pursue answers to essential questions in the field vascular aging that will advance our basic understanding and translate into more effective treatments to optimize human vascular healthspan. The central thesis of this project is that endothelial cells differentiated from hiPSCs, obtained from a diverse group of healthy adults and those with vascular contributions to cognitive impairment and dementia (VCID), can be leveraged to study endothelial aging in dementia. Using a computational model to identify biosignatures that predict endothelial cell aging, we will leverage this information to probe mechanisms relevant to dementia. Our research bridges the fields of vascular biology, stem cell biology, epigenetic clocks, multi -omics, and computational modeling to close the gap in the availability of models for the study of endothelial aging in dementia. There is a tremendous opportunity to address outstanding questions in this field using the novel human induced PlurIPotent stem cell-endothELIal cell model of aging for the study of vascular coNtributIoNs to coGnitive impairment and dementia (PIPELINING) described in this application. We will (1) passage human induced pluripotent stem cells differentiated to endothelial cells (hiPSC-ECs) and identify aging endpoints modeled in vitro (mitochondrial function, senescence, and angiogenesis). (2) A computational multi-scale model will be developed to predict the aging endpoints using multi -omic biosignatures for each human donor and passage. (3) Biosignature covariates judged to be critical contributors to the PIPELINING model will be selected for further mechanistic study. Achievement of the PIPELINING model would represent a significant advance in the application of contemporary technologies (iPSCs, epigenetic clocks, -omics, computational multi-scale modeling) to VCID.

NIMH - National Institute of Mental Health

Project Summary Fragile X syndrome (FXS) stands as a prominent contributor to intellectual disability and autism spectrum disorders, stemming from mutations within the FMR1 gene. These mutations lead to severe reduction or absence of the FMRP protein. Despite extensive research, effective medical interventions for FXS remain elusive, hindered by a limited understanding of its underlying mechanisms. Biochemical investigations have consistently highlighted FMRP's role in modulating mRNA translation, with its absence correlating with increased translation levels of select FMRP- interacting mRNA targets. However, emerging evidence suggests broader dysregulation, as FXS neurons exhibit heightened overall protein synthesis, hinting at elevated translation of non-FMRP interacting mRNAs. This intriguing phenomenon underscores the need for a deeper exploration into the cellular dysfunctions characterizing FXS. This research initiative aims to unravel a novel facet of FXS pathology—nucleolar hyper-function. We propose that this hyper-function contributes to aberrant ribosome biogenesis, thus augmenting the cellular capacity for translation and driving the observed global increase in protein synthesis in FXS. Aim 1 will assess neuronal and glial nucleolar function in wild-type (WT) and Fmr1 knockout (KO) mice. Aim 2 will conduct a comparative analysis of genome-wide proteomic data encompassing nucleolar proteins in WT and Fmr1 KO samples, discerning molecular alterations integral to ribosome biogenesis and assembly. Aim 3 will assess nucleolar function in the peripheral tissue in Fmr1 KO mice, establishing the hyper-functional pathological outcome as a potential clinical biomarker. The successful execution of this exploratory R21 project promises to unveil previously unexplored cellular mechanisms underlying FXS pathology. This study will also suggest nucleolus-associated abnormalities as novel molecular/cellular measures and potential biomarkers.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary The parathyroid glands are essential endocrine organs that regulate calcium and phosphate balance through secretion of parathyroid hormone (PTH). Loss or dysfunction of parathyroid tissue—commonly occurring after thyroid or neck surgery—leads to hypoparathyroidism, a debilitating condition for which current treatments rely on chronic calcium supplementation or hormone replacement. These therapies do not achieve the precise feedback regulation of calcium levels that native parathyroid tissue provides. Stem cell-derived parathyroid cells represent a potentially curative alternative. This project seeks to generate functional parathyroid-like cells from human induced pluripotent stem cells (iPSCs) through directed differentiation informed by principles of developmental biology and organogenesis. Our approach leverages recent progress in guiding iPSCs through definitive endoderm (DE), anterior foregut endoderm (AFE), and pharyngeal endoderm (PE) stages, alongside scRNA-seq data I have collected and analyzed from developing parathyroids to identify strategies to drive specification toward a parathyroid phenotype. In parallel, we will test the inductive capacity of transcription factor modules to forward program stem cells at pluripotent, DE, AFE, and PE stages to parathyroid identity. The differentiation protocol integrates small molecule modulation of key signaling pathways, such as BMP, WNT, and SHH, augmented by transcriptional cues derived from embryonic development. To validate lineage fidelity and functional capacity, we will assess expression of parathyroid-specific markers—including GCM2, PTH, and CASR—as well as calcium-responsive PTH secretion in vitro. Single-cell RNA sequencing will map lineage trajectories and dissect genetic programs governing parathyroid fate decisions. This work is enabled by the collaborative and resource-rich environment at Yale University. The project benefits interdisciplinary supervision of both Dr. Diane S. Krause, a leader in hematopoiesis as well as iPSC to parathyroid differentiation, and Dr. Zachary D. Smith. Together, their labs offer expertise in iPSC culture and parathyroid functional assessment embryonic development, germ layer specification, and embryo manipulation, offering key insight into developmental timing and patterning. Yale's core facilities for stem cell cultivation, flow cytometry, imaging, cluster computing, and high throughput sequencing will further accelerate progress and ensure technical rigor. By bridging stem cell biology with developmental genetics, this project aims to elucidate the regulatory networks that specify parathyroid identity and establish a platform for patient-specific cell therapies. The ultimate goal is to provide an autologous source of transplantable, functional parathyroid tissue to restore calcium homeostasis in individuals with hypoparathyroidism.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary In the human brain, a large repertoire of ion channels regulates the excitability of neurons, circuits, and networks, generating complex human cognition and behavior. Most ion channels are encoded by long, multi- exon genes which undergo extensive alternative splicing. Dysregulated splicing can alter ion channel function and is implicated in disorders ranging from autism to epileptic encephalopathy. Specific splicing events in ion channels have been shown to be critical for brain development and homeostasis, such as the neonatal-to-adult splicing switch in voltage-gated sodium channels. Splice-modulating therapeutics, such as antisense oligonucleotides, have shown efficacy for multiple neurologic disorders including spinal muscular atrophy, Duchenne muscular dystrophy, and Dravet syndrome. As potential targets for both small molecules and RNA therapeutics, ion channels are particularly important candidates for the treatment of neurologic disorders. However, there has never been a systematic study of alternative splicing of ion channels to-date. This proposal applies two innovative methods to profile the ion channel transcriptome and study the functional impact of alternative splicing on neuronal physiology. Aim 1 combines long-read RNA-sequencing and transcript capture technology to comprehensively identify and annotate channel isoforms in the human cerebral cortex. Bioinformatic tools will be used to uncover ion channel isoforms which are differentially regulated during postnatal brain development. Aim 2 employs splice modulation technology to study the functional impact of alternative splicing on neuronal physiology, using the epilepsy-associated KCNMA1 gene as proof of concept. A Cas-based toolkit is developed for human stem cell-derived neurons to manipulate the splicing of KCNMA1, and whole-cell patch-clamp physiology measures the impact of splice modulation on neuronal excitability. Taken together, this proposal will generate the most comprehensive profile of ion channel isoforms to-date, uncover developmentally regulated splicing events that can be potentially targeted by RNA therapeutics, and demonstrate proof of concept for transcriptome engineering in human neurons. These skills and resources will catalyze my career as an independent researcher, which will focus on the study of alternative splicing and the development of splice-modulating therapeutics for epilepsy and other neurologic disorders.

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

PROJECT SUMMARY: The mechanisms by which stem cells orchestrate their program to become functional differentiated cells require accurate temporal regulation of specific gene expression programs. This complex network requires precise temporal regulation of transcription and degradation processes to activate specific programs in a coordinated manner. So far, most of the studies have explored the regulation of transcriptional pathways and chromatin remodeling events during the differentiation process. mRNA degradation processes may present an attractive and still poorly explored opportunity for enhancing our understanding of the differentiation process. However, the lack of technologies that can capture rapid mRNA degradation events over highly dynamic processes, such as differentiation, and the heterogeneity of the mRNA degradation machinery in composition and expression patterns during differentiation have presented major technical limitations to further exploring the role of mRNA degradation across the continuum of the differentiation program. Here I propose to explore the existence of specialized RNA degradation complexes that control the decay of specific mRNA subclasses at precise timeframes of the differentiation process. To test this, we will introduce a new platform that uses cutting-edge technologies integrated in an innovative way to interrogate the continuum of the differentiation process at an unprecedented resolution. Our programmable depletion and rescue strategy will allow us to control the expression level of each subunit of complex mRNA degradation machinery robustly and with a precise time resolution of hours. By combining this technology with a high-content imaging system, we can record phenotype changes and accurately determine the specific impact of any perturbed protein on differentiation. Additionally, the use of this platform will guide us to understand the exact gene regulatory network controlled by the machinery at the transcriptional and stability level. The conceptualization and development of this workflow have the potential to impact a broader scientific audience; due to its extremely high flexibility, it could be applied to the study of unlimited biological processes or proteins. In this essay, the application of our proposed platform has the potential to fundamentally overturn the current view of how mRNA decay is dynamically regulated, providing a definite understanding of the function of the degradation machinery on mRNAs and, at the same time, revealing the broader impact of the degradation process on differentiation.

U.S. National Science Foundation

The Promoting Research and Innovation in Methodologies for Evaluation (PRIME) program seeks to support research on evaluation with special emphasis on exploring innovative approaches for determining the impacts and usefulness of STEM education projects and programs; building on and expanding the theoretical foundations for evaluating STEM education and workforce development initiatives, including translating and adapting approaches from other fields; and growing the capacity and infrastructure of the evaluation field. Two types of proposals will be supported by the program: Exploratory Projects that include proof-of-concept and feasibility studies and more extensive Full-Scale Projects.

NCI - National Cancer Institute

Project Summary The goal of this project is to develop a strategy to effectively eradicate leukemia-initiating cells. Leukemia- initiating cells are responsible for tumor initiation and recurrence in acute myeloid leukemia (AML), making it critically important to understand and target the biology required for leukemia-initiating cell survival. LICs are characterized by their self-renewal capacity, block in differentiation, and quiescent nature making them therapy resistant. A well characterized vulnerability of leukemia-initiating cells is oxidative phosphorylation (OxPhos) a pathway responsible for energy production. Direct OxPhos inhibition has been toxic in cancer patients. Thus, the development of approaches to target processes that regulate OxPhos in leukemia-initiating cells that are dispensable in normal cells is required. Our preliminary data shows that OxPhos is regulated by a post- translational modification called protein glutathionylation in AML cells and leukemia-initiating cells but not in normal hematopoietic stem and progenitor cells (HSPCs). These data indicate that protein glutathionylation regulation may represent a mechanism for decreasing OxPhos that could be LIC/AML specific and therefore targeting protein glutathionylation may be an approach to kill LICs with a more favorable therapeutic window than other approaches. Importantly, our data suggests that depletion of mitochondrial proteins that regulate protein glutathionylation results in reduced LIC function, induction of myeloid cell differentiation and sensitizes primary human AML cells to commonly used AML therapies but does not impact HSPCs. These data further support the potential for a therapeutic window may exist to target protein glutathionylation in AML. Based on these findings, we hypothesize that the regulation of mitochondrial protein glutathionylation is essential for LIC function by regulating OxPhos. We will examine this hypothesis by determining the molecular and biological role of protein glutathionylation in regulating leukemia-initiating cells and HSPC function using primary AML specimens, patient derived xenograft (PDX) models, and normal bone marrow specimens from healthy donors. Specifically, we will quantify leukemia-initiating cell phenotypes and function upon genetic depletion of proteins that regulate glutathionylation. Further, we will interrogate the mechanism(s) by which protein glutathionylation regulates mitochondrial energy production in leukemia-initiating cells. Taken together, our studies will be the first to establish protein glutathionylation as a novel regulator of 1) leukemia-initiating cells function and 2) OxPhos in cancer.

NIBIB - National Institute of Biomedical Imaging and Bioengineering

Project Summary/Abstract Many cells are responsive to electrically conductive materials; however, to date electrical conductivity is mostly achieved through graphene or synthetic polymers. These materials have limited translational use due to a lack of biodegradability and rigid mechanical properties. To overcome these challenges, we propose the design of a recombinant engineered, conductive, injectable, and biodegradable hydrogel that has the potential to induce regeneration across a wide range of tissues. We have recently pioneered the synthesis of a fully recombinant gel that incorporates electrically-conductive protein nanowires (ePN), an engineered matrix-like protein, and the polysaccharide hyaluronic acid (HA). While the ePN provides conductivity, the engineered matrix-like protein and HA provide biochemical ligands that promote cell adhesion. The hydrogel material is crosslinked through dynamic covalent chemistry, allowing for tunable viscoelastic properties and injectability. The resulting gel supports three-dimensional cell culture and biodegrades in response to cell-secreted enzymes. As the spinal cord is an electrically conductive tissue, we will demonstrate the efficacy of our technology in a cell-based therapy for spinal cord injury (SCI). Less than 1% of SCI patients have full neurological recovery by the time of hospital discharge. We previously demonstrated with non-conductive hydrogels that intraspinal transplantation of neural progenitor cells (NPCs) can significantly improve function in a rodent SCI model, but only when they are sufficiently matured into a neuronal phenotype. We have also demonstrated that NPCs enhance their neuronal maturation in vitro when grown on conductive biomaterials that were rigid and non-biodegradable. Thus, we hypothesize that our new hydrogel will facilitate the intraspinal injection of NPCs and significantly promote their neuronal maturation, thus resulting in significant functional and histological improvements. In Aim 1, we identify the gel formulation that best promotes neuronal differentiation and maturation of human induced pluripotent stem cell-derived NPCs in vitro. Specifically, we will tune the bulk conductivity of the fabricated gels through altering the ePN concentration and amino acid sequence. Recombinant engineering of ePN allows for tunability of the electrical conductivity along a single protein wire. The cell morphology, gene expression, and protein expression of encapsulated NPCs in the gels without and with varying levels of conductivity will be quantified. In Aim 2, we will select the gel variant that provides the best in vitro results for assessment in a preclinical, rat model of cervical SCI. NPCs will be transplanted within the conductive, biodegradable gel and evaluated for functional behavior over 6 weeks. Histological outcomes include transplanted cell survival and neurite outgrowth. Controls include conductive gels without cells and non-conductive gels with cells. This study would represent the first use of conductive, biodegradable, recombinant nanowires in tissue engineering, which can have broad application in conductive tissues including brain, cardiac muscle, skeletal muscle, and skin.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary Neutropenia, defined by abnormally low neutrophil counts, compromises innate immunity and increases susceptibility to life-threatening infections. Although most cases are acquired—resulting from malignancy, chemotherapy, infections, medications, or autoimmune disease—the study of inherited forms, though rarer, offers critical insights into the core mechanisms of myelopoiesis and granulocytic differentiation. Among these, autosomal dominant, heterozygous mutations in ELANE (formerly ELA2), which encodes the neutrophil granule serine protease neutrophil elastase (NE), represent the most common cause of severe congenital neutropenia (SCN) and the primary cause of cyclic neutropenia. SCN presents at birth with lifelong neutropenia, bone marrow maturation arrest, and elevated risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In cyclic neutropenia, neutrophil counts fluctuate between zero and near-normal with a striking 21-day periodicity. Despite their clinical importance, the pathogenic mechanisms of ELANE mutations remain poorly understood, and curative treatment is currently limited to hematopoietic stem cell transplantation. Mouse models fail to recapitulate the human phenotype, highlighting the need for human systems to investigate disease biology. All known pathogenic ELANE mutations result in production of a variant NE polypeptide, which may bypass key steps of proteolytic maturation and mislocalize within developing cells. This project tests the hypothesis that ELANE mutations cause disease by disrupting the spatial or temporal control of NE activity during granulopoiesis. Using isogenic, gene-targeted human induced pluripotent stem cells (iPSCs), the proposed research will: (1) define the spatial and temporal determinants of NE pathogenicity by introducing cis-acting suppressor mutations that disrupt its processing, trafficking, and catalytic activity; (2) determine whether the NE paralogs proteinase 3 and cathepsin G function as trans-acting modifiers; and (3) test whether CD34, a critical hematopoietic surface protein with distinct properties differing between mouse and human, is an NE substrate, and whether cleavage-resistant CD34 variants can restore granulopoiesis in ELANE-mutant cells. These studies will elucidate mechanisms of protease regulation in human neutrophil development, clarify the pathogenesis of both inherited and acquired neutropenia, and identify molecular targets for potential therapeutic intervention. The proposed work aligns directly with the NIH mission to advance understanding and treatment of hematologic and immune disorders.

PSEG Foundation

Supports STEM education, workforce development, and community sustainability programs in PSE&G's New Jersey service area.

National Park Service

Linking college students and recent graduates to National Park Service sites and units for the purpose of completing STEM (science, technology, engineering, and math) internships in National Park Service units across the country.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT Sequence-specific transcription factors (TFs) drive precise and timely control of gene expression during development. Changes in TF activity underlie much of the phenotypic divergence between species, and dysregulation is increasingly recognized as a cause of diverse human diseases. Despite the importance of TFs in cellular and organismal health and disease, most tools for studying TF function and conceptual models are binary in nature. The binary nature of TF models suggest an on-off switch, yet many disease-associated TFs operate in an analog fashion, where dosage is correlated with phenotypic outcome. Molecular tools to study and manipulate TF activities in an analog fashion are minimal, hindering quantitative modeling of TF function. This proposal seeks to fill this knowledge gap using an innovative chemical genetic approach that can precisely modulate TF dosage in human pluripotent stem cell (hPSC)-derived cell types. We will use this system to define how dosage of the key TFs SOX2, SOX9, and TWIST1 modulates chromatin state, gene expression, and cellular phenotypes and how this dosage-dependent regulation varies across cell types and evolutionary time. These TFs are ideal models to address our questions as they are associated with dosage-sensitive developmental disorders, function in multiple cell types, and have been associated with regulatory evolution. Our preliminary datasets validate our approach, revealing substantial variation in the dosage response of TF-dependent regulatory elements (REs) and genes, from highly sensitive to robust and buffered against quantitative TF dosage changes. We also recently applied cutting-edge deep learning models to learn sequence features underlying variation in RE dosage responses, revealing a surprising contribution of low-affinity binding motifs not apparent in unperturbed states. These results hint at a new layer of the cis-regulatory code, where certain sequence features are only apparent when studying transcriptional regulation in a quantitative regime. Our goal for this Program is to comprehensively characterize the underlying logic of these previously undefined additional regulatory layers and understand how they vary between cell types and species. To reach this goal, 3 directions will be pursued. 1) We will use multi-lineage differentiation of our unique hPSC reagents to investigate while certain cell types are sensitive to SOX2 and SOX9 dosage while others are buffered against quantitative changes. 2) We will apply chemical genetics and deep learning in chimpanzee PSCs to test whether the cis- regulatory logic defined in human cells generalizes across species and to distinguish cis from trans contributions. 3) Tool development, both experimentally where we will expand our system to allow bi-directional TF dosage control, and computationally, where we will develop improved deep learning models for predicting how REs respond to TF dosage. Together, these directions provide orthogonal but complementary approaches and tools that will converge on a unified model of how TF dosage impacts development and evolution, ultimately ushering our understanding of transcriptional regulation into a more mechanistic and quantitative realm.

U.S. National Science Foundation

Racial Equity in STEM Education Program Description (EHR Racial Equity) Persistent racial injustices and inequalities in the United States have led to renewed concern and interest in addressing systemic racism. The National Science Foundation (NSF) Directorate for Education and Human Resources (EHR) seeks to support bold,ground-breaking,andpotentiallytransformativeprojectsaddressing systemic racismin STEM.Proposalsshouldadvanceracial equity in science, technology, engineering, and mathematics (STEM) education and workforce developmentthroughresearch(both fundamental andapplied)andpractice. Core to this funding opportunity is thatproposalsare led by, or developed and led in authentic partnership with, individuals and communities most impacted bytheinequities caused by systemic racism. The voices, knowledge, and experiences of those who have been impacted by enduring racial inequities should be at the center oftheseproposals,includingin, for example: project leadership and research positions,conceptualization of theproposal,decision-making processes, and the interpretationand disseminationof evidence and research results. Theproposed workshould provide positive outcomes fortheindividuals and communities engaged and should recognize peoples humanity, experiences, and resilience.Proposalsneed to considersystemic barriers to opportunities and benefits, and how these barriersimpact access to, retentionin,and success in STEM education, research,and workforce development.Competitiveproposalswillbe clear with respect to how the workadvancesracial equity andaddressessystemic racism, as these constructs may have different meanings in different settings. Proposals should articulate a rigorous plan to generate knowledgethroughresearch (both fundamental and applied) and practice, such as, but not limited to: buildingtheory; developingmethods; testing approaches andinterventions; assessing the potential, efficacy, effectiveness, and scalability of approaches andinterventions; establishing,cultivatingand assessingauthenticpartnerships; changing institutional, organizational, and structural practices and policies; and/or focusing on affective, behavioral, cultural, social components,and implications. Contexts may include, but are not limited to: preK-12,two- andfour-year undergraduate, and graduate institutions; municipal organizations;STEM workplaces;andinformal STEM contexts, such as museums, community organizations, and media. In addition, proposals should include adissemination plan to proactively share what is learned with individuals and communities most impacted,as well asrelevant leaders, policy makers, and other stakeholders.Proposal budgets and project durations should be determined by the scope of the activities and in accordance with theNSF Proposal & Award Policies & Procedures Guide (PAPPG).PIs should include Racial Equity: at the beginning of the proposal title.

NIA - National Institute on Aging

The population of older adults (OA) is rapidly rising and anticipated to exceed 2 billion by 2050 causing an exponential rise in age-related comorbidities and healthcare costs. Age-related defects include mitochondrial dysfunction, inflammation, oxidative stress (OxS), insulin resistance (IR), genomic damage and endothelial dysfunction and result in declining physical function (gait speed and muscle strength), elevated blood pressure (BP) and higher waist circumferences. Via studies in OA and old mice (OM), we identified that deficiency of the body’s most abundant antioxidant Glutathione (GSH) plays a key contributory role for these defects in aging. GSH is an intracellular tripeptide composed of glycine, cysteine and glutamic acid, and declines with age. We found that GSH deficiency in OA occurs due to diminished synthesis caused by deficiency of glycine and cysteine (and not glutamic acid), and that GSH deficiency can be corrected by supplementing GlyNAC (combination of oral glycine, and N-acetyl-cysteine (NAC) as a cysteine donor because oral cysteine is absorbed poorly). In OM and OA, we discovered that GSH adequacy is critically necessary for efficient mitochondrial fuel (fatty-acid) oxidation (MFO) and for lowering OxS. In a small NIH-funded double-blinded, placebo-controlled, proof-of-concept pilot randomized clinical trial (RCT) in 24 highly selected, healthy OA and 12 young adults (YA) we reported that OA had (a) GSH deficiency in muscle and red blood cells; (b) impaired mitochondrial function; (c) deficient nutrient sensing; (d) increased inflammation; (e) elevated IR; (f) endothelial dysfunction; (g) genomic damage; (h) stem cell fatigue; and (i) cellular senescence. These abnormalities were associated with: (i) physical decline in gait speed, strength and exercise capacity; (ii) increased waist circumference; and (iii) higher blood pressure. GlyNAC (and not placebo) supplementation: (a) normalized RBC GSH concentrations, mitochondrial fuel oxidation, molecular regulators of energy metabolism, nutrient sensors, genomic damage, stem cells and cellular senescence; (b) lowered OxS, proinflammatory cytokines (IL6, TNFa, hsCRP); IR; endothelial dysfunction; (c) improved gait speed, strength, exercise capacity, body composition and systolic BP. GlyNAC supplementation in young humans had no impact. These data provide proof-of-concept that supplementing GlyNAC in OA corrects GSH deficiency and improves 7 aging hallmarks, and was not associated with any adverse effects. Could GlyNAC supplementation introduce a transformational change to improve the health of aging humans by promoting healthy aging? Although our completed RCT provides proof-of-concept for this, the sample size was small. Critically, the RCT was conducted in a rigorously screened cohort of healthy OA, using a high dose of GlyNAC. Therefore, it is important to definitively establish the validity and effectiveness of GlyNAC supplementation in a larger RCT conducted in a more typical population of OA, and also determine whether a lower GlyNAC dose, with lesser pill burden, could be effective. We propose a less invasive, less restrictive RCT in 150 more typical OA to determine the effects of supplementing GlyNAC on intracellular GSH, OxS, mitochondrial function, inflammation, IR, endothelial function, genomic damage, physical function, body composition and QoL. The proposed RCT will also test and compare two doses of GlyNAC to determine whether a lower dose of GlyNAC can be as effective as a higher dose on measured outcomes after 24-weeks.

Raytheon Foundation

Major funder of STEM education programs for K-12 students and veterans transitioning to tech careers.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

ABSTRACT Intestinal stem cells (ISCs) maintain and regenerate the intestinal epithelium within a specialized niche known as the crypt. In mouse models, ISCs have been shown to activate a gene expression program similar to the developing intestine, referred to as ‘fetal reversion’ or ‘developmental reprogramming’ during injury repair/regeneration. Reactivation of this program is emerging as a critical process for effective regeneration. However, whether a similar developmental program is reactivated during human intestinal repair/regeneration remains unknown. The current proposal provides preliminary data that developmental reprogramming takes place during human intestinal repair/regeneration, and aims to interrogate the functional importance of development and repair mechanisms. As preliminary data, we have identified the EGF family member EPIREGULIN (EREG) as a developmentally expressed gene/protein that is re-activated after various forms of injury in vitro. In addition, we have developed novel iPSC-derived human intestinal organoid (HIO) injury models following transplantation into immunocompromised mice to create an in vivo-like injury and observe that EREG influences growth/proliferation in this transplanted HIO (tHIO) model. Based on our preliminary data, this proposal will test the hypothesis that the human intestine reactivates a developmental gene expression program following injury, including reactivation of EREG, which is an essential regenerative pathway. Using adult tissue-derived organoids and genetically engineered iPSC-derived HIO models, we propose three aims: (1) Define the developmental role of EREG through overexpression and knockout experiments in tHIOs; (2) Investigate the role of EREG during injury repair in EREG-modified tHIOs challenged with irradiation, 5-fluorouracil and Oxaliplatin; and (3) Elucidate the mechanistic pathway of EREG action, specifically evaluating downstream targets including c-MYC activation, that are crucial for the regenerative response.

NIGMS - National Institute of General Medical Sciences

Mechanical forces drive tissue function and pathophysiology, yet current high-throughput systems for drug development rarely incorporate mechanical forces, and those that do typically do not allow dynamic, feedback- based control over the forces acting on cells and/or engineered tissues. We propose to integrate key technologies developed by our team members: 1) rapid algorithms for directly estimating contractility of excitable tissues; 2) GPU-acceleration approaches for rapid computing; 3) externally triggered smart materials that can change their mechanical properties in response to magnetic fields; and 4) high-throughput engineered tissue platforms. This integration will allow us to create a high-throughput system that allows for real-time control over tissue mechanical loading based on the mechanical forces produced by the tissue. For this technology-development application, we propose milestone-driven efforts to optimize, validate, and integrate these technologies into a user-friendly, graphical-user-interface (GUI) supported platform. The approach we propose is unique in that the software-to-hardware interfacing, driven by imaging, can readily be adapted in the future by the research community, without requiring costly, user-dependent, one-time-use pure hardware-based approaches. The ability to parallelize the algorithm for computing tissue deformation, direct deformation estimation (DDE), will allow for dramatic acceleration of computing deformation, to the point that it can be computed in real-time, thereby allowing for magnetically-responsive biomaterials to be triggered in response to image-based data on contractility. We will demonstrate integration of our software-hardware interfacing based feedback approach by performing mechano-pharmacologic screens in skeletal muscle engineered from murine myoblasts and cardiac muscle engineered from human induced pluripotent stem cells. We will apply diverse loading regimes to the tissues, in combination with drugs known to have differential effects in mechanically loaded skeletal and heart muscle. We will also create tools for mining the resulting mechano-pharmacologic data. We envision that this technology will be broadly enabling for studies in mechanobiology and for improving translation of drug screens.