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NIDA - National Institute on Drug Abuse

PROJECT SUMMARY This proposal responds to NIDA RFA-DA-26-009 and aims to characterize the mitochondrial regulation of inflammaging in immune cells and neurons, along with their translational effects on cognitive function among people with HIV (PWH) who use substances (SU). It is estimated that 30-60% of PWH will develop cognitive impairment, which is associated with numerous critical adverse outcomes such as frailty, increased hospitalization rates, and elevated mortality. Among PWH, persons who inject drugs (PWID) represent one of the most vulnerable groups for cognitive impairment, as substances like cocaine and heroin exhibit pronounced neurotoxicity and are directly linked to cognitive decline. A primary cause of cognitive impairment among PWH is inflammaging, characterized by persistent low-grade inflammation and immune activation. Mitochondria, central to adenosine triphosphate (ATP) production, regulate bioenergetic processes and signaling pathways across nearly all organ systems but are susceptible to oxidative stress and inflammation. Our ongoing research indicates that mitochondrial DNA copy number (mtDNA-CN), a marker of mitochondrial integrity, is associated with HIV infection, T-cell senescence, and cognitive impairment, and is further reduced by active heroin and cocaine use. Additionally, mitochondrial genetic variation has been closely linked with multiple aging-related outcomes specifically in PWH, suggesting interactions between mitochondrial genetics and HIV infection. Therefore, we hypothesize that mitochondrial function and genetics independently and jointly influence immune dysfunction and cognitive impairment following HIV infection and substance use. Clarifying the interplay between these factors may yield therapeutic insights capable of improving brain health among PWH. Our study will address the following aims: 1) To characterize the association of cell type-specific MT function with immune dysfunction and cognitive impairment as influenced by HIV and SU; 2) To identify mtDNA genetic variation associated with immune dysfunction and cognitive impairment, and whether HIV and SU modify these associations; 3) To quantify the effects of HIV and SU on MT function, inflammaging, and nuclear DNA methylation in induced pluripotent stem cell (iPSC)-derived cortical neurons from PWIDs.

NINDS - National Institute of Neurological Disorders and Stroke

Repeat-expansion disorders myotonic dystrophy 1 and 2 (DM1, DM2) produce disabling cognitive and behavioral deficits, yet the molecular drivers of central-nervous-system (CNS) pathology remain undefined. DM1 is caused by expanded CTG repeats in the dystrophia-myotonica protein kinase (DMPK) gene that produces toxic CUG RNAs, whereas DM2 is caused by expanded CCTG repeats in the cellular nucleic-acid–binding protein (CNBP) gene that yield toxic CCUG RNAs. These expanded RNAs sequester the splicing regulator muscleblind-like protein 1 (MBNL1), disrupt microtubule-associated protein tau (MAPT) processing, and may provoke excitotoxicity via hyperactive CUG-binding protein ELAV-like family 2 (CELF2). The long-term objective is to enable CNS-directed therapies for myotonic dystrophy. The central hypothesis is that repeat-expansion RNA toxicity mis-splices MAPT and activates CELF2-mediated glutamatergic hyperexcitability, jointly driving tauopathy and neuronal loss. This project will delineate convergent and divergent mechanisms of cortical dysfunction in DM1 and DM2 using human induced-pluripotent-stem-cell (iPSC) cortical organoids produced by an optimized protocol validated in more than sixty lines. Organoids derived from this protocol retain physiological DMPK1, CNBP, and MBNL1 expression overcoming the low expression of these genes observed in other organoid models. The optimized protocol will be applied to DM1 iPSC lines (238–1,600 CTG repeats), DM2 lines (8.8–11.9 kb CCTG repeats), and healthy control lines to generate side-by-side disease and reference cortical organoids. Aim 1 will track RNA- foci formation, splice defects, and tau aggregation at 2, 4, and 6 months in DM1, DM2, and control organoids, combining long-read RNA-seq with quantitative neuropathology. Aim 2 will test whether hyper-phosphorylated CELF2 drives glutamatergic mis-splicing, network hyperexcitability, and neuronal loss across DM1 and DM2 organoids, and whether inducible shRNA knock-down of CELF2 restores receptor splicing, electrophysiological balance, and tau status relative to controls. High-density multielectrode-array recordings, long-read transcriptomics, and tau biochemistry will integrate molecular and functional endpoints throughout the project. The study is expected to (i) establish mechanistic links between repeat RNA toxicity and tauopathy, (ii) identify CELF2 as a modifiable driver of excitotoxicity, and (iii) deliver a scalable, biomarker-rich organoid platform for therapeutic screening. By clarifying disease pathways and providing human assay systems, the work will accelerate the development of tau-lowering and synapse-stabilizing strategies, directly advancing NIH priorities to translate mechanistic insight into treatments for rare neurodegenerative diseases.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT African American individuals face a disproportionately higher risk of tobacco-related cardiovascular diseases (CVD) than other races, a disparity not fully explained by traditional and socioeconomic risk factors. Despite lacking approval from the U.S. Food and Drug Administration (FDA) for their safety, e-cigarettes (e-cigs) have become increasingly popular, particularly among youth, and are now among the most commonly used tobacco products alongside traditional cigarettes. Approximately half of African American individuals carry at least one of two genetic variants (G1 and G2) of the apolipoprotein L1 (APOL1) gene, which are exceedingly rare in other populations. APOL1 is widely expressed, particularly in the vasculature. We have shown that carriers of APOL1 G1 and G2 variants have increased susceptibility to tobacco-related CVD, including stroke and coronary heart disease. Dysfunction of vascular endothelial cells (ECs) is a critical precursor to CVD. EC dysfunction also plays a key role in APOL1-associated pathology, including exacerbated renal issues and increased susceptibility to sepsis and severe COVID-19. Recent research indicates that, similar to cigarettes, both e-cigs and menthol—a flavor popular in the African American community—independently impair endothelial function. While studies, including those using induced pluripotent stem cell (iPSC)-derived ECs, demonstrate these effects, the specific impact of APOL1 risk variants on vascular health in African American tobacco product users remains unknown. The goal of the proposed research is to determine the effects of e-cigs, with and without menthol, on endothelial health, compare them to the effects of cigarettes, and identify potential molecular markers and pathways associated with CVD in African American users, with a focus on the APOL1 genotype. As such, this application aims to expand my background and expertise in modeling the CVD risk from tobacco products and to provide specific training in tobacco product-related in vitro assays, iPSC methodology, gene editing, and computational techniques. Building on my prior work in human studies, this research extends to the cellular level to address significant gaps in knowledge regarding the adverse effects of the most popular tobacco products, with and without menthol, among the African American population—a demographic long targeted by the tobacco industry marketing. To achieve this goal, I will use a robust in vitro platform of human iPSC-ECs to address the following aims: Aim K1) to determine the effect of e-cigs and cigarettes on markers of EC dysfunction in G1/G1 iPSC-ECs, Aim R1) to determine the effect of e-cigs and cigarettes on endothelial function in G2/G2 iPSC-ECs, and Aim R2) to determine the effect of e-cigs and cigarettes on inflammatory markers and lipid mediators of inflammation in G1/G1 and G2/G2 iPSC-ECs. This project will deepen our understanding of the adverse effects of widely used tobacco products on vascular health in the CVD-burdened African American population. It also aims to identify molecular markers of cardiovascular injury in this high-risk group, providing insights into the mechanisms of tobacco-related cardiovascular damage and supporting the development of targeted interventions.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary The goal of this project is to understand the mechanisms and potential therapeutic targets of a novel inborn error of immunity (IEI) caused by variants in toll like receptor 8 (TLR8). We recently identified genetic variants in TLR8 leading to gain-of-function (GOF) of the encoded protein as the cause of a new IEI presenting with profound neutropenia with recurrent infections, lymphoproliferation, T and B cell abnormalities, and bone marrow failure. Toll-like receptor 8 (TLR8) is an endosomal TLR encoded on the X chromosome that recognizes single-stranded RNA (ssRNA) and is expressed in neutrophils and other myeloid cells, including myeloid progenitors. Relatively little is known about human TLR8, largely because the murine equivalent differs in structure and ability to sense ssRNA. There is no available targeted therapy for patients with TLR8 GOF; however, patients have benefited from hematopoietic stem cell therapy, confirming that disease is driven by the immune system. There is a gap in our understanding of how GOF in TLR8 protein function leads to the clinical features of this newly recognized disease. As TLR8 expression is myeloid-specific and, in most cases of TLR8 GOF disease, expressed in a mosaic fashion (with approximately 10-20% of cells harboring TLR8 variants), we hypothesize that myeloid cells expressing mutant TLR8 produce inflammatory cytokines leading to T cell activation, and this inflammatory state and dysfunctional T cells contribute to the disease in patients including lymphoproliferation and bone marrow failure. Given the differences between mouse and human TLR8, we recently generated transgenic mice that conditionally express either WT or GOF human TLR8. Herein, we will use these novel models to address several gaps in our knowledge of TLR8 biology and TLR8 GOF disease, as well as to test potential therapies. In Aim 1, we will identify mechanisms of TLR8 GOF disease, using our transgenic mouse models and human cell xenografts to determine how TLR8 GOF myeloid cells influence hematopoiesis and T cell development and identify putative disease-driving cytokines. In addition, we will formally test the role of T cells in the disease pathogenesis by crossing TLR8 GOF mice to T-cell deficient mice and determining the effects on hematopoiesis, cytokine production and disease progression. In Aim 2, we will identify therapeutic targets of TLR8 GOF disease using inhibitors of TLR8 signaling and blocking candidate cytokines in our transgenic mice. Together, these studies will lead to an increased understanding of the pathology of TLR8 GOF and will identify therapeutic targets. The long-term goal of this application and our work is to develop a mechanistic understanding of how TLR8 GOF alters the immune response to enable improved therapies for patients.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Self-reactive B cells must distinguish self from foreign antigens (Ag), mounting immunogenic responses to the latter but not the former. However, the mechanisms they employ to do so are not fully understood. Long-term, we aim to exploit new molecular insight gained by filling this knowledge gap to restore B cell tolerance in autoimmunity, enhance anti-tumor B cell responses to self-like Ag, and optimize host defense. In this proposal, we leverage a unique and modular platform of liposomes decorated with model Ag, enabling independent control of epitope affinity and density. Using this system, we recently demonstrated that such particulate Ag are much more potent activators of B cells than identical Ag in soluble, monovalent form. We found that this stems not merely from avidity but from the ability of particulate Ag to evade inhibitory signaling pathways normally engaged by soluble Ag and mediated by the Src family tyrosine kinase Lyn. Moreover, we discovered that transcriptional programs and B cell functional responses to particulate and soluble Ag diverge markedly; despite an identical epitope recognized by the BCR, we found that particulate Ag produce robust NF- kB activation even in the absence of T cell help, while soluble Ag drive an NFAT-associated anergy program. We propose that biophysical characteristics of particulate Ag display serves as a stand-alone danger signal that evades tolerogenic transcriptional programs and elicits immunogenic responses by B cells. In this grant, we propose to: (1) Identify proximal biochemical pathways downstream of the Lyn Src family tyrosine kinase that enable B cells to distinguish tolerogenic and immunogenic Ag display. We will edit mouse and human B cells in vitro and in vivo to test candidate substrates of Lyn. We will complement this with an unbiased proteomics approach to identify novel negative regulators of B cell responses to self-like soluble Ag. (2) Elucidate how early signaling events triggered by particulate Ag are transformed into immunogenic transcriptional programming of B cell fate through robust activation of NF-kB but not NFAT pathways. We will take a genetic approach to manipulate the second messenger diacylglycerol (DAG) which we hypothesize toggles between these immunogenic and tolerogenic B cell transcriptional responses. We will test this in human B cells. We will pursue a genomic strategy to survey the epigenetic landscape of B cells in order to define the transcriptional architecture that translates pattern of Ag display into B cell fate. (3) Systematically map the lower boundary of Ag affinity, density, and particle dose, and test the biophysical properties (particle size, membrane fluidity, linker length) required to activate B cells, including anergic self- reactive cells. We will test the hypothesis that these thresholds are controlled by Lyn-dependent inhibitory signaling pathways.

NINDS - National Institute of Neurological Disorders and Stroke

SUMMARY Diffuse dissemination of tumor cells into the brain is one of the key problems impacting optimal treatment of gliomas. We hypothesize that distinct types of tumor cells harbor characteristic diffusion patterns. Specifically, we suspect that NPC-like cells predominate in the cortical layers and interact with the laminar extension of neuronal cells in the cortex, while OPC-like cells dominate subcortically and associate with axons and APC-like cells associate with vascular and immune cells. In order to provide four-dimensional reconstructions of tumor development and test our hypotheses, we will perform spatial transcriptomics on n = 254 image-mapped samples from n = 22 distinct tumors (aim 1). Tumor-microenvironment interactions will be evaluated separately for each patient and by comparing distinct locations across patients. Finally, we will develop an agent-based model using n = 179 samples from n = 20 individual with integrated imaging, DNA methylation and DNA sequencing information to reconstruct historical and forecast future tumor development in space and time (aim 2). The findings of these studies will allow for a high-resolution dissection of the identity and location of ancestral stem- like tumor cell populations, as well as the identification of sites of diffuse infiltration. A four-dimensional model of tumor cell dissemination will enable individualized local treatment planning and prognostication.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

ABSTRACT Islet transplantation is a promising treatment for type 1 diabetes but suffers from significant islet loss with the standard intraportal liver infusion, consequently requiring large numbers of costly islets, often multiple transplants, and suboptimal achievement of long-term insulin independence. This is because islet isolation protocols result in regression of islet-specific endothelial cells (ISECs), with the loss of the critical, supportive islet vascular niche resulting in islet death. Furthermore, strategies to achieve islet engraftment in an extrahepatic site have been stymied by poor vascularization and lack of knowledge of how β-cell interaction with ISECs contributes to β-cell homeostasis. The overall goal of this project is to engineer ISECs to uncover the cellular cross-talk between -cells and their specialized vascular niche, to ultimately augment transplanted islet engraftment in the subcutaneous space. Each organ is vascularized by unique, specialized endothelial cells (ECs) that provide a tissue-specific vascular niche that supplies angiocrine factors key in choreographing organ homeostasis and repair. Indeed, there is growing evidence that a functional and physical interplay exists between specialized ISECs and -cells. Employing single cell analyses, we obtained a molecular signature of ISECs, identifying the novel transcription factor NKX2-3. This proposal will explore if vascularization of islets with NKX2-3+ ECs will facilitate engraftment, function, and survival of subcutaneously transplanted islets. ISECs have the additional critical feature of modulating the expression and migration of immune mediators, with macrophages being the most abundant pancreatic immune cell. In turn, these macrophages supply growth and immunomodulatory factors to sustain the integrity of -cells. Therefore, we hypothesize that induction of NKX2-3 in ECs confers these cells with the specialized properties of ISECs which, by supplying defined angiocrine factors and proper polarization of pro-reparative macrophages, support islet function. Accordingly, we will test the hypothesis that NKX2-3 is necessary for the specification of ISECs, which have the capacity to support islets in vitro and in vivo, through the following aims: Aim 1: Assess the impact on islet function of selective and conditional loss of NKX2-3 in pancreatic ECs. Aim 2: Examine the efficacy of enforced NKX2-3 expression in human umbilical vein endothelial cells in augmenting durable subcutaneous engraftment of transplanted islets. Aim 3: Decipher the mechanism by which NKX2-3, through macrophage polarization, coordinates pro-regenerative and anti-fibrotic islet innate inflammatory responses within the pancreas. The proposed training will guide and enhance my development in core competencies, including immunology, bioinformatics, and stem cell-derived islets, that will enable me to transition to research independence as a surgeon-scientist dedicated to improving islet transplantation outcomes. Weill Cornell Medicine is an ideal environment to execute this training plan due to its outstanding physical resources and its robust intellectual community of researchers with strong records of mentorship of early-stage investigators.

OD - NIH Office of the Director

Project Summary/Abstract Down syndrome (DS), caused by trisomy of chromosome 21, affects approximately 1 in 700 live births worldwide. Despite advances in prenatal diagnosis and maternal screening, the incidence of DS has not declined, and its prevalence continues to rise due to increased maternal age and longer life expectancy. Nearly all individuals with DS develop Alzheimer’s disease (AD)–like pathology and dementia (DSAD) by middle age, primarily due to triplication of the amyloid precursor protein (APP) gene on chromosome 21. Overexpression of APP leads to elevated amyloid-β (Aβ) production, aggregation into plaques, and progressive neurodegeneration and dementia. Recently, Aβ-targeted monoclonal antibodies such as donanemab have been approved by the FDA for early-stage AD, and clinical trials are underway to evaluate their efficacy in DS. However, antibody therapies face major challenges in crossing the blood–brain barrier (BBB) and are associated with serious adverse effects, including cerebral hemorrhage. Thus, there remains a critical unmet need for effective strategies that directly and selectively target APP in DS. To address this need, we have developed a targeted protein degradation (TPD) approach and discovered molecular glue degraders that selectively promote lysosomal degradation of APP and reduce Aβ production in induced pluripotent stem cell (iPSC)–derived neurons from AD patients. These degraders bind at the interface between APP and CAPRIN1 (cytoplasmic activation/proliferation–associated protein 1), stabilizing their interaction and triggering CAPRIN1-mediated lysosomal degradation of APP. Our lead compound crosses the BBB, is orally bioavailable, and significantly reduces APP levels and Aβ plaque burden in AD mouse models. Using AI-assisted ligand- and structure-based drug design, we have generated a focused library of optimized lead derivatives. Building on this breakthrough, the objective of this exploratory project is to evaluate the therapeutic potential of APP molecular glue degraders for treating DS. In Aim 1, we will screen our AI-designed compound library in DS patient–derived iPSC neurons to identify candidate compounds that selectively degrade APP via the CAPRIN1-dependent lysosomal pathway. Promising compounds will be further characterized for ADME (absorption, distribution, metabolism, and excretion) properties to identify potent, metabolically stable candidates for in vivo testing. In Aim 2, the top optimized compounds will be evaluated for pharmacokinetics (PK), BBB permeability, off-target activity, and toxicity using standard repeated-dose studies in euploid mice. One optimized compound will then be tested for therapeutic efficacy in the Ts65Dn DS mouse model to determine if oral administration reduces APP, Aβ40, phosphorylated tau, and neuroinflammation, and preserves or improves cognitive function. Successful completion of this project will provide proof of concept for APP-targeted molecular glue degraders as first-in-class therapeutics to prevent the progression of DS to DSAD and will establish a powerful TPD and AI-integrated framework for drug discovery in DS.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Bacteria shield themselves from the exterior environment with a rigid cell wall. The major structural component of this exoskeleton is the mesh-like peptidoglycan (PG), a heteropolymer composed of glycan strands and crosslinked stem peptides. Glycan strand polymerization and transpeptidation are catalyzed by penicillin-binding proteins (PBPs). Organisms express multiple PBP isoforms with varied spatiotemporal activity. Transpeptidation is inhibited by the b-lactams, a family of covalent inhibitors that mimic the terminal dipeptide structure of the nascent PG monomer; PBP inhibition results in growth abnormalities and cell lysis. Despite the therapeutic successes of b-lactam antibiotics, the current antimicrobial resistance crisis underscores the importance of understanding of PBP activity and inhibition, and cell wall composition more broadly. The Carlson Group studies PBP activity with chemical probes; however, achieving isoform selectivity is difficult due to spatial conservation of the transpeptidase active site. Specifically, the field lacks a comprehensive description of how conserved motifs in this active site mediate substrate and inhibitor binding. There is also a lack of understanding of how changes in the PBP activity profile alter PG composition. While nonuniformity of PG composition has been demonstrated in the context of bacterial resistance and host immune response to infection, a lack of bioinformatic methods for the unbiased identification of PG fragments impedes analyses of cell wall digests using liquid chromatography coupled to mass spectrometry (LC-MS). To address these outstanding questions in PBP activity and cell wall maintenance, the proposed research will use an alanine scan to map and quantify the contribution of conserved active site motifs in PBP2x from the Gram-positive pathogen Streptococcus pneumoniae to native substrate processing and b-lactam inhibition. Following initial assessment with a commercially available probe, this approach will be extended to a chemically diverse suite of b-lactams in live cell, in vitro, and in silico experiments to clarify how the active site permits occupancy of multiple inhibitor classes. Additionally, this project will use activity-based protein profiling and LC- MS/MS analyses to investigate how conditions mimicking the acidic infection microenvironment perturb PBP activity and PG composition. This work will be coupled to experiments probing the effects of transpeptidase inhibition and PBP deletion on PG structural diversity. Bioinformatic analyses of LC-MS/MS data will serve as crucial validation of the application of -omics strategies to interrogate PG composition. Ultimately, these aims will provide insight into key mediators of cell wall synthesis and maintenance, inform understanding of how bacteria leverage cell wall perturbations as defense mechanisms to evade antimicrobial threats, and inspire future probe design. The training plan will facilitate instruction in chemical biology, computational biochemistry, and bioinformatic -omics analyses through education in the group of Erin Carlson, and through collaborations at the University of Minnesota.

NIGMS - National Institute of General Medical Sciences

Myosin-X (Myo10) is a molecular motor crucial for the formation and function of filopodia, finger-like protrusions cells use interact with their surroundings in processes such as brain development, blood vessel formation, and the spread of cancer cells. We have shown that Myo10 localizes to the tips of filopodia, increases the number and length of filopodia, and moves within filopodia in a process known as intrafilopodial motility. Our generation of Myo10 knock-out mice showed that loss of Myo10 causes developmental defects in brain, eye, and blood vessels, but is not essential for survival of adult mice. Myo10 promotes tumor growth and invasion in many cancers, including breast, lung, and melanoma. Myo10 also has important functions in cell division, where it is required for spindle orientation and for clustering the excess centrosomes that are a hallmark of cancer cells. Myo10’s important roles in biology, plus the need to understand the fundamental cell biology of filopodia, make it essential to investigate the molecular mechanisms of Myo10 and filopodial function at the cellular and organismal levels. To fill these knowledge gaps, we will address the following: -How does Myo10 promote filopodia and what are its molecular cargos? -What are the functions of headless Myo10, a form of Myo10 that lacks the motor domain and is expressed in brain and stem cells? -Purify filopodia and use modern proteomics approaches to identify and quantify the full set of their molecular components, including the filopodial cytoskeleton, tip, and plasma membrane. -What are Myo10’s organismal functions in epithelial tissues such as kidney where it localizes basolaterally and in eye where KO results in major developmental defects. Because filopodia are a major cellular organelle whose purification has not been reported, we will combine our recent progress purifying filopodia with quantitative proteomics to identify the molecular components of filopodia. We will also take advantage of the extensive set of tools and techniques we have established to investigate Myo10 and filopodia, including Myo10 floxed and knock-out mice, KO and stable cell lines, and deletion and point mutant constructs. Although the other members of the MyTH4-FERM family of myosins in vertebrates have important roles in human physiology and disease at the tips of other protrusions based on actin bundles like epithelial microvilli and inner ear stereocilia, Myo10 is the MyTH4-FERM myosin present in filopodia and most mammalian cells and tissues. This research will answer fundamental questions about Myo10 and filopodia as well as investigating Myo10 functions at the organismal level in health and disease.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Project Summary/Abstract Tissue restoration is essential to maintain skeletal muscles after injury, due to overuse, aging or disease. Repeated cycles of muscle damage and repair are associated with muscle stem cell (MuSC) dysfunction and impaired myogenesis. Telomeres protect and stability and telomeric proteins do not only the end of our chromosomes from deterioration but are main components of the stem cell progenitor cells `ignition' mechanism, which maintain tissue homeostasis and genome by repairing damage throughout life .Although the telomere protective machinery has been primarily established during carcinogenesis and aging; its importance during regeneration and particularly in muscle injuries, a tissue known for its high regenerative capacity and low propensity for carcinogenesis, is not well understood. We previously demonstrated that telomere attrition is a distinct feature of dystrophic MuSCs in both mice and patients, even at very young ages. More recently, we discovered that TRF2, a key telomere-capping protein, is dynamically regulated in skeletal muscles and has distinct functions, independent of its conventional telomeric role. We also developed genetic tools to define how this protein operates in uninjured, injured, and diseased skeletal muscles. The studies proposed here will determine the extent of previously unknown extra-telomeric functions of TRF2 in muscle stem cells (Aim 1), they will define its new role in regenerating myofibers upon acute and chronic injuries and will uncover new interacting proteins during this process (Aim 2). We expect that this project will fundamentally advance our understanding of the molecular mechanisms by which TRF2 maintains stem cell identity versus how it regulates reparative myogenesis and could effectively guide ways of promoting regeneration and function in healthy and diseased conditions.

NIH

Project Summary/Abstract Significance to VA During deployment to Operation Desert Storm as an Army Medical Officer the Pl of this project cared for many soldiers with severe diarrhea, later shown to be caused primarily by enterotoxigenic E. coli (ETEC)1. These infections remain a major cause of acute diarrheal illness worldwide, have been responsible for major outbreaks in the United States, and fuel the global spread of antibiotic resistance determinants. In addition, these pathogens are closely associated with long-term sequelae including malnutrition and wasting in young children, tropical sprue in adults, and Irritable Bowel Syndrome (IBS) in returning Veteran service members. Gaps addressed by these studies Our understanding of the molecular pathogenesis of gastrointestinal pathogens as well as the composition of the commensal microflora is exceedingly biased by models that fail to faithfully recapitulate key features of the human intestine, particularly the major immunoglobulin superfamily glycoproteins known as CEACAMs. The proposed studies seek to overcome these deficiencies using transgenic mice and human stem cell-derived gastrointestinal organoids that accurately reflect the molecular organization of human small intestine. Innovation and Impact: These studies center on the interaction of ETEC and other intestinal bacteria (including commensals) with Carcinoembryonic Antigen-related Cell Adhesion Molecules (CEACAMs), which we have recently shown in novel human intestinal organoid models and a transgenic mouse model to play essential roles in pathogen­ host interactions, as well as innate response to ETEC. While thousands of intestinal microbiome studies have been performed in conventional mice, they lack CEACAM genes normally expressed in the human gastrointestinal tract. We therefore anticipate that the proposed studies will significantly impact our understanding of intestinal bacteriology and approach to care. Specific Aims: 1. Define molecular interactions between ETEC, EV and CEACAMs. 2. Examine the interplay of CEACAMs, intestinal microbiota and ETEC 3. Examine the impact of ETEC pathogen-host interactions on gene expression. Methodology: Aim 1 we will use recombinant CEACAMs to investigate interactions between the bacteria and these receptors. In addition, we will map glycosylation sites on CEACAM6 before and after treatment with ETEC heat labile toxin by mass spectrometry, mutate the respective sites, and examine their impact as recombinant proteins and in transformed cells. Aim 2: Here we will examine the impact of CEACAM expression and the introduction of ETEC on defined microbiota in a gnotobiotic core facility by next generation metagenomic sequencing. Aim 3: To examine the impact of EV- interactions with ETEC we plan to perform RNA sequencing (RNAseq) of bacterial RNA following contact with CEACAM-laden extracellular vesicles (EV). In addition, we will explore the impact of heat-labile toxin on activation of gene expression pathways that lead to CEACAM upregulation by this toxin. Path to Translation/Implementation: Although basic in nature, we anticipate that these studies will provide a more detailed understanding of both host-pathogen interactions and the intestinal microbiota that can readily be applied to improving preclinical testing of orally delivered drugs, vaccines, and other therapeutics.

National Park Service

The Mosaics in Science (MIS) internships provide college students and recent graduates opportunities to work at National Park Service sites and units for the purpose of completing STEM (science, technology, engineering, and math) internship projects in NPS units across the country. The MIS Program provides young adults with structured, safe and challenging work and educational opportunities through employment projects that promote personal growth, the development of life skills, and an ethic of natural resource stewardship.

National Park Service

The purpose of the Mosaics in Science Internship Program is to link 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: Students from historically minoritized backgrounds with a variety of lived experiences often struggle to navigate the hidden curriculum of academia towards achieving careers in biomedical science. Additionally, the transfer student experience can be isolating in that students develop a cohort and near peer network in an intimate community college setting and then must reestablish a support network while taking on challenging upper division coursework. With support and community building, these barriers can be overcome. Therefore, we are building off our historic success established by our previous R25 B2B ACCESS program (1994-2024) to build the T34 B2B program (MPC2SC) at the University of California Santa Cruz (UCSC) which aims to recruit, retain, and prepare historically minoritized students from Monterey Peninsula College (MPC) for biomedical bachelor’s degrees and entry into PhD programs or the biomedical research workforce. A major change to our program will be the integration with the UCSC STEM Diversity office which houses established programs such as MARC, CAMP, and UCLEADS. These undergraduate focused programs allow us to leverage a strong, established community of minoritized scholars with a focus and celebration of the intersectionality of their identities. We will systematically guide our B2B scholars through the MPC to UCSC transition by offering seminars, workshops, and 8-week summer research experiences (SRE) at UCSC to develop essential skills and conduct research and demystifying the ‘hidden curriculum’. We have run a R25 program (ACCESS) for 30 years, so we are excited to leverage its successes for our new T34 program. Our objectives are to: 1. Strengthen the partnership with MPC to increase acceptance and retention rates by supporting an annual cohort of ten MPC2SC students, targeting a 100% retention rate from MPC to UCSC. 2. Ensure 100% of MPC2SC fellows develop independent research skills through placements in UCSC labs committed to undergraduate training and requiring each student will be highly encouraged to apply for an NSF Graduate Research Fellowship (GRFP) application. 3. Achieve a 100% completion rate for biomedical 5-year bachelor’s degrees among fellows and ensure at least 75% will apply to a biomedical graduate program. We anticipate that the other 25% of the students will pursue biomedical research careers or further postbaccalaureate training opportunities. Our core leadership team comprises PI Holman (Co-Director of ACCESS 2019-2024), MPI Sanchez (Advisory role, ACCESS 2021-2024), MPI Turner (ACCESS faculty, 2014-2024), MPI Gilbert (ACCESS faculty, 2020-2024) and Yulianna Ortega (STEM diversity director), who work closely to transition the R25 to the T34 program, ensuring these goals are met.

NINDS - National Institute of Neurological Disorders and Stroke

Abstract Brain organoids (BOs) derived from human stem cells bridge the gap between monolayer cell culture studies and animal models, which have well-documented limitations. Monolayer cell culture models fail to accurately replicate the 3D interconnectivity in the brain; animal models, while helpful, are limited due to interspecies differences, with most research focusing on rather phenotypical rather than mechanistic aspects. Concurrent with the advancement of BO models is the urgent need to develop 3D micro instrumentation supporting these organoids to investigate brain development and disease in their accurate physiological environment. Conventional microelectrode arrays (MEAs) used for neuronal cell culture studies are planar, which limits recording access to a small fraction of cells on the bottom side of the organoid. Also, conventional microfluidics is inherently planar, and while recent advances in 3D MEAs and 3D microfluidics have enabled electrical and chemical interrogation in 3D, combining both features with tunability and precision to allow independent and simultaneous control is challenging. Recently, we reported new 3D micro instrumentation in the form of 3D shell MEAs and demonstrated its applicability for electrical recording from BOs. They feature lithographically patterned and chip-integrated electrodes and self-folding polymer shells that can be triggered to wrap around BOs to measure electrical activity from the entire organoid surface. The 3D MEA shell system is modeled on and resembles a miniaturized electroencephalography (EEG) cap; the process used to make them is size-scalable, chip-integrated, and mass- producible. In the research, we aim to develop and validate 3D Micro Electrode Fluidic Array (MEFA) shells with multi-modal electrical recording and biochemical control capabilities, offering high spatiotemporal resolution, tunability, and scalability. Since 3D spatiotemporal patterns of neurochemicals play a critical role in molecular and cellular events of neural development and disease, we propose to apply and validate the MEFA shells in two studies that mimic neurodevelopment and monitor the spatiotemporal effects in neurological disorders and their treatments in vitro. We anticipate that the proposed 3D MEFAs would revolutionize brain sciences by permitting real-time, in-situ studies of electrical and chemical stimulation and interrogation of BOs in a high- throughput manner. The proposed 3D scalable, reproducible, and tunable 3D micro instrumentation for BOs has broad relevance to understanding brain development in utero and the development of anatomically accurate drug and toxicity screening platforms for brain sciences and neurological disorders.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Animals depend critically on their capacity to choose the appropriate time to act. In the absence of explicit cues, these decisions are thought to depend on internal deliberation that dictates action timing to maximize outcomes. A broad range of previous observations suggest that internally driven timing decisions involve a distributed network of brain regions. However, it remains unclear how these regions interact during decision-making; mechanistic understanding is very limited and hypothesized interactions between brain regions remain untested. This critical knowledge gap stems from two main factors. First, the absence of explicit cues makes decision timing unpredictable, which historically has presented problems for experimental design. Second, previous methods for measuring and perturbing neural activity, and for quantifying the dependence relations between neural activity patterns, have been ill-suited for probing interactions between brain areas on the relevant timescales. To overcome these barriers, our collaboration combines a novel behavioral paradigm, multi-region spike- resolution neural recording, rapid neural activity perturbation, and a range of model-based computational approaches. In recent work, our analysis of activity on individual decision trials has demonstrated the prominent involvement of a deterministic process, in contrast to recent models that emphasize stochasticity. We have also developed an approach for analyzing multi-region recordings that has revealed a modular structure in the influence of several frontal cortical regions on the striatum, an influence thought to be central to timing decisions. Our preliminary results for this proposal point to a revised decision model that involves an urgency-like signal and a source of unpredictability distinct from that of prevailing models. We have also begun to examine interactions between prefrontal and somatomotor circuits that are also thought to be central to timing decisions. Here we have found evidence of an interaction mediated primarily by corticocortical connections, and one that has a modular structure. Our proposed work would build on these results to test our new model and identify its neural substrates (Aim 1), and test existing ideas about prefrontal-somatomotor interactions (Aim 2). Here we will use multi-region Neuropixels recordings, and an approach we have recently demonstrated for fast optogenetic silencing during internally driven timing decisions. We will compare our new model to others using rigorous statistical methods for model selection. We will identify neural substrates by analyzing best-fit models and perturbation results. We will also continue to develop our new methods for quantifying interregional interactions from activity recordings. Collectively, our work will quantify relevant interactions between brain regions that could not be resolved with previous approaches, leading to improved models. This will provide a new foundation for understanding the neural mechanisms of a basic aspect of natural behavior with relevance to cognition.

NIGMS - National Institute of General Medical Sciences

Project summary Understanding the dynamic change of cell-type composition, functional evolution, migration, and interaction of single cells within complex tissue or in vivo environments is essential for advancing our knowledge of developmental, pathological, and therapeutic processes. Yet current single-cell analytical methods such as flow cytometry, histological staining, sequencing, and mass cytometry are inherently destructive and provide only static views of dynamic cellular processes. While intravital microscopy with multiphoton technology enables nondestructive real-time imaging of single-cell behavior in three- dimension (3D) tissue environment, its multiplexing capacity is constrained by reliance on a narrow range of spectrally distinct colors. As a result, we remain unable to continuously track live cells and their functional evolution in physiologically relevant environments. My research program addresses this critical gap by developing Spatiotemporal Multiplexed Antigen Revealing Technology (SMART), an innovative platform for multiplexed, longitudinal imaging of live cells. SMART enables repeated cycles of labeling and erasing of fluorescent signals on cell surface, allowing continuous tracking of dozens of extracellular markers on the same cells over time. By reconstructing these signals across cycles, SMART provides a dynamic view of cell phenotypes, migration, differentiation, and communication that current methods cannot achieve. Over the next five years, my laboratory will develop and establish SMART as a versatile and reliable tool through applications in PBMCs (peripheral blood mononuclear cells) phenotyping, immune cell migration and function, stem cell differentiation, and neuron–astrocyte interactions. These studies will demonstrate SMART’s ability to uncover how cellular states evolve and how microenvironmental interactions shape biological outcomes. Looking ahead, my long-term vision is to extend SMART into living organisms, enabling noninvasive, real-time, multiplexed imaging of cellular dynamics in vivo. This advance would transform our ability to study immune responses, stem cell fate, and neural development directly in their native environments, providing insights that cannot be obtained with current technology. Ultimately, SMART has the potential to redefine how biomedical research observes and understands cellular behavior across diverse biological systems.

NIGMS - National Institute of General Medical Sciences

ABSTRACT / SUMMARY Reactive oxygen species (ROS) and regulation of redox pathways are critical to human health and disease, as they influence cellular metabolism, signaling, and stress responses. Disruptions in redox homeostasis contribute to the pathophysiology of numerous disorders, including neurodegenerative diseases, muscular degeneration, and drug-induced cardiotoxicity. However, the tools for monitoring redox dynamics in living human cells remain limited in dimensionality, sensitivity, and applicability to disease-relevant models. To overcome these challenges, my research program aims to develop a next-generation, multiplexed optical platform for quantitative redox phenotyping and apply it to disease modeling and drug screening in human induced pluripotent stem cell (iPSC)- derived systems. Over the past five years, my lab has engineered two advanced genetically encoded hydrogen peroxide (H₂O₂) sensors, oROS-G and oROS-HT, exhibiting improved dynamic range, kinetics, and spectral flexibility. We established a high-throughput optical screening platform and integrated machine learning approaches to accelerate protein sensor engineering. These sensors have been applied in diverse host systems, including iPSC-derived neurons and cardiomyocytes, and have revealed new aspects of redox signaling in cell health. Building on this foundation, our future research will continue along three complementary directions. First, we will complete the development of a fully multiplexed, intensity-based TreDox sensor suite to simultaneously monitor oxidative pressure and antioxidant capacity with single-cell resolution in real time. Second, we will engineer lifetime-resolved redox biosensors and use fluorescence lifetime imaging microscopy (FLIM) to enable robust, expression-independent quantification of intracellular redox states. Third, using single-cell optical phenotyping, we will apply these tools to profile redox imbalances and early cytotoxicity signals in human iPSC- derived cardiomyocytes, neurons, and skeletal muscle cells. We aim to detect subtle cellular imbalances in redox pathways that precede cellular dysfunction and are often missed by traditional high throughput assays. This research program will fill critical gaps in our ability to study redox biology in human-derived host systems by integrating state-of-the-art protein engineering, advanced imaging, and human stem cell models. The tools and knowledge generated will improve our understanding of redox-linked disease mechanisms, enhance the predictive power of preclinical drug testing, and establish a flexible, generalizable platform for functional phenotyping at single-cell resolution.

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

Project Summary Pelvic floor disorders (PFDs) are common conditions that affect -25% of U.S. women. PFDs are morbid, with more than 50% of afflicted patients rating them as "worse than death·. As vaginal delivery is the greatest epidemiologic risk factor for PFDs-likely due to the pelvic floor muscle (PFM) dysfunction it often incites greater understanding of vaginal delivery biomechanics and birth injury is needed. But in order to accurately study the mechanical behavior of the PFMs during childbirth, their structure, function, and integrity just before childbirth must be known. Acquiring this knowledge is not trivial given the degree of pregnancy-induced remodeling that soft tissues undergo during gestation. Thus, this study aims to implement multiscale experimental and computational methods to characterize and simulate pregnancy-induced remodeling of PFMs. Phase 1 (K99) is the experimental arm of this proposal. In Alm 1 cell culture of primary 1) skeletal muscle stem cells and 2) fibro-adipogenic progenitors isolated from female rat PFMs will be used to determine the impact of sex hormones (e.g., estrogen) and mechanical stretch on resulting 1) myotube growth and 2) collagen secretion by fibroblasts, respectively. After the cultured cells have differentiated, myotube size, fusion index, and the amount of collagen secreted (quantified as a percentage of the sampled area) will be quantified via bright field (myotubes) and fluorescence (fibroblasts) microscopy. These will serve as proxies for muscle fiber growth and collagen deposition in vivo, allowing for the determination of the effect of sex hormones and mechanical stretch on the contractile and extracellular matrix (ECM) components of the PFMs. Meanwhile, Alm 2 will define changes in whole PFM active and passive mechanics across the nonpregnant-postpartum continuum. Whole PFMs will be harvested from rats at various stages throughout the pregnancy and postpartum, and then ex vivo active and passive mechanical testing will be performed. This will establish changes in force generating capacity (active properties) and load bearing capacity (passive properties) across the continuum, revealing how the function of both the contractile (active) and ECM (passive) components of PFMs are altered by pregnancy and childbirth. Phase 2 (ROO) is the computational arm of this proposal. Aim 3 will generate intracellular signaling network (cell level) and finite element (whole muscle level) models, calibrate and validate those models using literature and Phase 1 data, and then couple those models; resulting in a multiscale computational model of pregnancy-induced PFM remodeling. This coupled model will consider sex hormone levels, the degree of mechanical stretch acting on myofibers and the ECM, myofiber growth, and collagen deposition collectively while simulating their impact on whole PFM active and passive function. Together, these aims will characterize the multiscale (intracellular and whole muscle) mechanisms of pregnancy-induced PFM remodeling and identify the most influential sex hormones and mechanical properties driving these adaptations; thus, promoting translational studies evaluating the PFMs' ability to withstand vaginal birth and avoid injury.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract Support is requested for a Keystone Symposia conference entitled “Myeloid Cells: Functional Heterogeneity with Therapeutic Promise,” organized by Drs. Charlotte L. Scott, Shalin H. Naik and Thomas Fabre, with scientific programming input from Keystone Symposia. The meeting will take place February 23–26, 2026 at the Keystone Resort in Keystone, Colorado, USA. Myeloid cells play crucial roles in the innate immune system, responding to infections and maintaining tissue homeostasis. Despite their significant therapeutic promise, the potential of myeloid cells is yet to be fully realized. This Keystone Symposia meeting aims to bring together key leaders in academia and industry to discuss recent insights regarding myeloid cell functional heterogeneity and how to target these cells for therapeutic interventions. This conference will highlight recent advances in our understanding of the role of myeloid cells in different disease settings, including cancer, infection and other immune-mediated disorders, which will enable new translational perspectives for understanding, treating, and preventing infectious and immunologic diseases. The meeting program will provide opportunities for attendees to gain a deeper understanding of unique and conserved myeloid cell populations across tissues and diseases and explore how these might be leveraged therapeutically. Through rigorous discussions, this meeting aims to outline key questions for future research that will harness the power of myeloid cells and showcase current and emerging technologies. A key feature of this meeting is that it will be co-located with another Keystone Symposia conference, “Hematopoiesis.” This partnership will provide valuable insights into the interconnected roles of hematopoietic stem cells and myeloid lineages in both health and disease. Inclusive poster sessions, panel discussions, shared meals and social activities will promote networking, encourage the sharing of cross-disciplinary insights and provide broader scientific perspectives important for future research collaborations towards the development of successful therapeutic strategies.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Oxidative stress caused by dysregulated reactive oxygen species (ROS) plays a critical role in neurodegenerative diseases, cancer, and aging. Despite its importance, the boundary between ROS beneficial signaling and harmful oxidative stress remains poorly defined, in part due to limited tools for precise, real- time measurement of redox dynamics in live cells. Clarifying how cells respond to different ROS levels is essential to understanding redox regulation and developing strategies to mitigate oxidative damage. A better grasp of these mechanisms could impact diverse fields including neuroscience, cancer biology, and metabolic disease. The Usselman Lab at Florida Tech brings a unique combination of redox biology, hyperspectral imaging, and quantum sensing expertise. As a teaching-intensive institution, we also engage undergraduates directly in hands-on research, building the next generation of STEM leaders through immersive, high-impact training. We will engineer chemogenetic systems using D-amino acid oxidase (DAAO) to generate H₂O₂ with spatial and dose control in mammalian cells. Redox dynamics will be quantified using ratiometric biosensors, Seahorse metabolic assays, and a novel optically detected magnetic resonance microscopy (ODMRM) platform with nanodiamond quantum sensors. These tools will allow us to define oxidative thresholds, measure mitochondrial responses, and track real-time redox changes with subcellular resolution. This multidisciplinary approach offers a powerful framework for understanding redox adaptation and stress, with broad implications for human health.

NIGMS - National Institute of General Medical Sciences

ABSTRACT Biomolecular delivery is foundational to modern biology and medicine, offering precise control over cell behavior through surface receptor engagement and intracellular delivery of genes and proteins. Yet this potential is fundamentally limited by biological barriers, including tight junctions, plasma membranes, and endosomal compartments, that restrict transport, uptake, and cytosolic access. My lab specializes in modular biomaterial platforms for programmable ligand display and light-triggered intracellular release. We will leverage these precision tools to investigate how nanoscale features influence delivery mechanisms at the molecular and cellular scales. Theme 1: Can Nanoscale Engineering of Integrin Clustering Induce Transient Tight Junction Opening? Tight junctions are a major barrier to tissue-level delivery of large biologics, particularly across epithelial and endothelial interfaces. Previous work from our group demonstrated that nanotopographic cues remodel tight junctions via integrin signaling. Here, we hypothesize that spherical particles displaying nanoscale-clustered RGD ligands or integrin-targeting antibodies can recapitulate this remodeling effect without physical topography. Using our DNA-scaffolded ligand display system, we will systematically vary density, spacing, and flexibility to identify design rules for non-toxic, reversible tight junction modulation. This project will establish foundational principles for enhancing paracellular transport using spherical biomaterials. Theme 2: Can Ligand Architecture and Carrier Design Override Receptor-Intrinsic Internalization? While receptor- mediated endocytosis is often described as intrinsic to each receptor, growing evidence suggests that external presentation cues can redirect uptake pathways. We will apply our modular surface-engineering system to dissect how ligand architecture influences internalization for three distinct receptor systems: CD40 (clustering- driven), c-Kit (signal-dependent), and integrins (mechanosensitive). We hypothesize that tuning ligand density and flexibility on synthetic carriers can override receptor preferences, reprogramming trafficking and cargo fate. This project aims to uncover generalizable design strategies for targeted, receptor-agnostic intracellular delivery. Theme 3: Can NIR-Stimulated Nanoparticles Over Endosomal Barriers Across Cell Type and Cargos? Endosomal escape remains a major bottleneck for the intracellular delivery of therapeutic macromolecules. We previously demonstrated that hollow gold nanoshells enable siRNA release via NIR-triggered photothermal disruption. Here, we extend this strategy to mRNA and Cas9/gRNA delivery across challenging primary cell types, including primary T cells, macrophages, dendritic cells, and hematopoietic stem cells. We hypothesize that optimal escape thresholds depend on both cargo physicochemical properties and cell-intrinsic membrane repair capacity. By mapping delivery efficiency across cargo and cell types, we aim to define tunable parameters for safe and efficient endosomal release. Altogether, this program will generate broadly applicable principles for biologic delivery while training students across biomaterials, cellular analysis, and advanced imaging.

NCI - National Cancer Institute

PROJECT SUMMARY Colorectal cancer (CRC) is the second leading cause of cancer deaths in the U.S. despite being preventable by colonoscopy through removal of premalignant lesions, such as advanced adenomas, the clinically significant precursor of CRC. While colonoscopy is recommended for all patients over the age of 45, many patients are not screened due to poor compliance, cost, and limited capacity to screen over 90 million eligible Americans. Attempting to perform colonoscopy on all average-risk patients is also inefficient: only 7% of patients have advanced adenomas. While current and emerging non-endoscopic CRC screening tests, including fecal and blood tests, might show sensitivity to cancers, they consistently fail to detect advanced adenomas. This failure is biologically fundamental and stems from the low yield of tumor biomarkers shed or secreted into the stool or blood by early lesions and tumor heterogeneity. We propose an alternate strategy that addresses these failures by integrating three key innovations. First, instead of sampling tumor secretions, we leverage the phenomenon of CRC field carcinogenesis: a histologically normal, preconditioned "field" that includes the entire colon and rectum and carries systemic transcriptional and epigenetic alterations predisposing the patient to neoplasia. This approach maintains high sensitivity regardless of lesion size. Second, we target chromatin-regulated transcriptional plasticity as a new class of biomarkers. We propose to integrate the measurements of alterations in nanoscopic, sub-Mbp chromatin packing domains—key elements of transcriptional memory that drive transcriptional plasticity necessary for pre-neoplastic cells to acquire malignant traits—and their transcriptomic consequences. This choice of biomarkers circumvents the problem with tumor heterogeneity. Third, we will develop chromatinomics: a new CRC screening platform integrating chromatin-sensitive optical spectroscopic nanosensing and global miRNA/mRNA transcriptomics, enhanced by highly multiplexed spectroscopic single molecule localization microscopy and mechanistically-guided AI analysis, and informed by chromatin-specific 3D electron tomography and predictive molecular modeling to detect transcriptional plasticity as a biomarker of CRC risk. This platform will be validated in a prospective, cross-sectional clinical study of a 500-patient average-risk cohort. Our long-term vision is to develop chromatinomics analysis of a simple, low-cost rectal swab, performed in primary care, as a high-accuracy, first-line prescreen to efficiently prioritize colonoscopy for patients harboring advanced adenomas. The ultimate goal is to significantly increase CRC screening uptake while reducing unnecessary procedures.