University Of Connecticut Sch Of Med/Dnt

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT In all animals, the maternal-to-zygotic transition allows the transfer of information required for a single zygote to develop into a mature organism. After fertilization, the maternal program, composed of maternally-inherited mRNAs and proteins, drives cellular development and is replaced by the zygotic program. Because this transition occurs primarily in a transcriptionally silent embryo, it relies heavily on post-transcriptional regulation. Failure to properly regulate maternally-inherited mRNAs generally leads to developmental arrest or abnormalities. Recent studies have shown that maternal mRNAs are decorated with RNA modifications, collectively known as the ‘epitranscriptome’, that correlate with different mRNA fates. Moreover, we and others performed global analyses of mRNA structure dynamics during the maternal-to-zygotic transition and identified numerous regions that are structurally remodeled during this fundamental process, many of which impact mRNA decay. These studies suggest that RNA modifications and dynamic RNA structures are emerging as key regulators of gene expression during the maternal-to-zygotic transition. However, the detailed landscape of the epitranscriptome and dynamic RNA structures, and their roles in gene regulation during the maternal-to- zygotic transition remain poorly understood. Furthermore, RNA modifications and structures affect one another to regulate RNA functions, but little is known about how they interact to control gene expression during vertebrate development. The primary goal of my lab is to understand how the epitranscriptome and RNA structures mediate gene regulatory networks, separately and cooperatively, during vertebrate development and how their dysfunction promotes developmental defects or diseases. Here, we hypothesize that RNA modifications and structures interact with trans-factors to participate in the post-transcriptional regulatory landscape driving vertebrate development. To test this hypothesis, we will combine zebrafish —an in vivo model of vertebrate development— and its genetic toolbox with innovative multi-omics approaches. Over the next five years, we will inspect the native transcriptome to generate global, yet detailed, maps of the epitranscriptome during the maternal-to-zygotic transition, and study how specific RNA modifications impact gene expression. We will also decipher the RNA folds formed by dynamic regions of the transcriptome and analyze their effect on RNA regulation. We will identify trans-factors interacting with RNA modifications and structures of interest and study the consequences of their loss-of-function on gene expression and vertebrate development. Finally, we will examine how RNA modifications and structures cooperate to modulate post- transcriptional regulation. Successful completion of these investigations will greatly increase the existing knowledge of how RNA modifications and structures orchestrate post-transcriptional regulation, and will expand our understanding of the molecular mechanisms shaping vertebrate development.

NIH Research Projects · FY 2026 · 2022-07

The focus of this project will be to investigate the role of the myostatin/GDF-11/activin branch of the transforming growth factor-ß (TGF-ß) superfamily of secreted signaling molecules in regulating bone mass and density. The important role that this signaling pathway plays in regulating bone homeostasis has been documented by both pharmacologic and genetic studies targeting receptors for this group of ligands. Work from several groups, including ours, has shown that systemic administration of soluble forms of either of the activin type 2 receptors, ACVR2 and ACVR2B, is capable of inducing significant increases in bone density. By genetically targeting these receptors in osteoblasts, we showed that at least part of this effect is due to inhibition of direct signaling to bone. Strikingly, however, we very recently showed that targeting the type 1 receptors, ALK4 and ALK5, in osteoblasts led to much more substantial effects, resulting in increases in bone mass and density by approximately 10-fold. These findings revealed the extraordinary capacity for bone accrual that is normally kept in check by this regulatory system and suggest that the potential for increasing bone mass and density by targeting this signaling pathway is substantially greater than previously appreciated. As a starting point for developing the most effective strategies to harness the potential of targeting this pathway for bone applications, we will elucidate the extracellular components that play key roles in this regulatory network in bone. In Specific Aim 1, we will examine the roles of known inhibitory binding proteins for this group of ligands in regulating bone structure. In our recent study, we carried out an extensive analysis of the role of one binding protein, namely follistatin (FST), using genetically-targeted mouse lines in which expression levels of FST were either up- or down-regulated. Here, we will examine the roles of the three other known binding proteins, FSTL-3, GASP-1, and GASP-2, utilizing targeted mouse lines that we have generated carrying both deletion and floxed alleles for each of these components. In Specific Aim 2, we will examine the roles of specific ligands in this subgroup of the TGF-ß superfamily in regulating bone structure. In our recent study, we showed that targeting two ligands simultaneously, namely myostatin and activin A, led to substantial increases in bone mass and density but that these increases were significantly less pronounced than the approximately 10-fold effects that we observed upon targeting their type 1 receptors. Here, we will use genetic approaches to examine the roles of a wider spectrum of ligands in this subgroup of the TGF-ß superfamily in regulating bone structure. The overall goal of this project will be to elucidate the specific extracellular signaling components that play key roles in regulating bone homeostasis with the long-term goal of developing the most effective strategies to target this signaling pathway to treat bone loss.

NIH Research Projects · FY 2026 · 2022-07

Abstract The goal of this new application is to define the role of the chromatin modifier Dot1L (Disruptor of telomeric silencing-1 like) in normal skeletal growth and development. Dot1L is the only enzyme that catalyzes the methylation of lysine 79 in histone 3 (H3K79), which plays an important role in the epigenetic regulation of gene expression. The only molecular function that has been demonstrated for Dot1L resides in its catalytic or methyltransferase (MT) domain, however new studies indicate that non-catalytic functions of Dot1L also contributes to the regulation of gene expression and cell differentiation. Currently, very little is known about how Dot1L regulates skeletal growth and development. This represents a critical gap in our knowledge because Dot1L targeted approaches are being studied as therapies for pediatric cancers. We recently reported that the conditional loss of Dot1L expression in limb mesenchyme induced an aberrant skeletal phenotype characterized by long bone shortening, and defects in growth plate (GP) chondrocyte proliferation. Interestingly, small molecule inhibition of Dot1L catalytic activity did not impair chondrocyte proliferation in vitro, suggesting an underlying but critical role for non-catalytic functions of Dot1L in these complex processes. Chemical inhibition of Dot1L in chondrogenic limb bud micromass assays resulted in premature chondrocyte hypertrophy through mis-regulation of the Bone morphogenetic protein (Bmp) signaling pathway. New in vivo data from our lab provides compelling evidence that a catalytic inactive Dot1L mutant protein can restore the cartilage GP dysfunction and long bone growth deficits in mice with conditional loss of Dot1L function in limb mesenchyme. Together, these data, support our novel central hypothesis that Dot1L regulates long bone growth at the GP through: i) non-catalytic activities that support chondrocyte proliferation; and ii) MT-dependent activities which restrict chondrocyte maturation. We have assembled a strong team of investigators with expertise in skeletal biology, epigenetics, and Dot1L biology to address this novel hypothesis. In Aim 1, we will establish the functional requirement for Dot1L catalytic activity in endochondral bone growth in vivo, using novel Dot1L MT mutant mice. Our studies will determine whether a catalytic-dead Dot1L mutant can rescue skeletal defects in Dot1L cKOPrrx1 mice. We will apply single cell transcriptomic analyses to identify novel Dot1L-regulated genes and pathways. In Aim 2, we will define the regulatory functions of Dot1L that provide stage-specific control of chondrogenic differentiation. Using Dot1L knockout versus MT mutant cells, mechanistic studies will assess the direct contribution of Dot1L catalytic versus non-catalytic functions to chondrocyte proliferation versus hypertrophy. Lastly, ChIPseq experiments will identify genome-wide methylation patterns (H3K79me2) associated with chondrocyte proliferation and maturation. Outcomes from these studies are expected to generate new knowledge on Dot1L during normal skeletal growth and development, with implications for targeting Dot1L therapeutically.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Cardiomyopathies occur in ~1:200 individuals and are commonly caused by inheritance of variants in genes that encode proteins that regulate the sarcomere, the force-producing organelle of heart cells. Due to an incomplete understanding of variant pathogenicity and cardiomyopathy pathogenesis, physicians are currently limited in their ability to provide diagnoses, prognoses, and therapeutic options for cardiomyopathy patients. Variants in the TTN gene, which encodes the sarcomere protein titin, are the most frequently identified genetic lesion in dilated cardiomyopathy (DCM), which is characterized by heart chamber dilation, reduced contractile function, risk of sudden death, and progressive heart failure. The most frequent type of TTN variant identified in DCM is a truncation mutation that would be predicted to shorten TTN protein length and to reduce TTN protein quantities. Significantly, truncation variants localized to distal TTN structural domains are more pathogenic than those localized to proximal structural domains, but the mechanistic basis for this relationship is uncertain. It remains incompletely understood how TTN truncation variants cause DCM generally, which is compounded by our lack of understanding of the ‘length dependence’ of TTN variant pathogenicity. These knowledge gaps limit disease prognostication, biomarker identification, and therapeutic development for DCM patients. The central goal of our study is to define how disruptions in TTN length and dosage by TTN variants cause DCM, and exploit this knowledge to develop DCM therapeutics for TTN variant carriers. We hypothesize that healthy cardiac contractile function and structure depends on the regulation of TTN length and dosage, and that varying pathogenicity of TTN truncation can be explained by distinct structural and functional consequences associated with the specific site of truncation. In Aim 1, we will determine the functional consequences of TTN truncations across structural domains by harnessing 3-dimensional heart tissue models composed of human cardiomyocytes differentiated from induced pluripotent stem cells in which variants have been introduced by CRISPR-mediated genome editing. We will interrogate these models for tissue mechanical phenotypes (such as passive tension and Frank-Starling behavior), TTN protein length and levels (using specialized methods), proteostasis stress pathway responses (using immunoblotting), and mechanotransduction signaling and alternative splicing (using expression analysis and transcriptomics, respectively). In Aim 2, we will restore TTN protein levels using the recently developed method of CRISPR activation applied to DCM engineered heart tissue models for both evaluating the function of TTN isoforms generally and as a DCM proof-of-concept therapeutic. Through these Aims, we will gain critical new insights into the pathophysiology of DCM-associated TTN truncation variants, uncover features to explain the variable pathogenicity identified in DCM patients, and develop a therapeutic to target TTN directly. We anticipate this new knowledge will improve physicians’ capacity to diagnose, prognose, and treat patients with DCM due to TTN variants.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract Investigating pathogenic mechanisms for rare Mendelian disorders is important not only to identify therapeutic strategies for lifelong debilitating diseases but also to understand fundamental biological mechanisms. In this renewal application, we propose mechanistic and translational studies for craniometaphyseal dysplasia (CMD), an understudied craniotubular bone disorder characterized by lifelong progressing hyperostosis of craniofacial bones and abnormal shape of long bones. Continued bone accrual in CMD can lead to excruciating headaches, blindness, deafness, and facial palsy. Severe cases can be life-threatening. CMD patients are treated with repetitive, costly and risky surgeries when corrections of facial deformity are needed or severe neurological symptoms occur. Mutations in the progressive ankylosis protein (ANKH) and connexin 43 (Cx43) have been identified as causes for autosomal dominant and recessive CMD, respectively. To study CMD, we have generated state-of-the-art research tools, which include mouse models carrying CMD mutations, isogenic human induced pluripotent stem cells (hiPSCs) with or without CMD mutations, and bone resorbing cells (osteoclasts) derived from these hiPSCs. In the past funding period, we have discovered the rapid degradation of mutant ANKH(Human)/ANK(Mouse) protein and studied negative effects of mutant ANKH/ANK on the cytoskeleton, which determinates cell shape, size, and polarity. We also identified differentially expressed proteins in CMD osteoclasts and preferential binding partners for mutant ANK protein. However, CMD pathogenesis is not fully understood and potential therapeutics have not been explored. Our long-term goal is to utilize our research findings for identifying potential therapeutic targets to reduce or prevent the lifelong bone deposition in craniofacial bones. In the next 5 years, we will use animal models and molecular and cellular methodologies that we have developed to focus on mechanistic investigations and prepare for future clinical studies. Based on our preliminary data and previous publications we propose three specific aims. We will study the impact of CMD-mutant ANK on cellular acidification of osteoclasts (Aim 1) and on the bi-directional regulation between the cytoskeleton and an energy metabolism regulator in CMD (Aim 2). These are likely novel dominant functions of mutant ANK leading to CMD. In Aim 3 we will identify biomarkers that can be used to monitor the disease progression in patients and mouse models. We will also evaluate shifts in biomarker expression in response to experimental treatment regimen in our model systems. We expect that the proposed studies will give deeper insight into pathogenic mechanisms of CMD, knowledge needed to discover candidate targets for therapeutics. Biomarkers that correspond to disease progression or treatment efficacy will be the basis for future clinical studies.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Globoid Cell Leukodystrophy (GLD) is a demyelinating central nervous system (CNS) disease that results in death in 99% of children before the age of 5 years old. Loss of function mutation in galactocerebrosidase in GLD leads to a toxic build-up of the lipid psychosine, which is currently thought to underlie the development of this disease. The rapid progression of behavioral and cognitive deficits present in GLD is devastating for both patients and families, however current treatments have limited success at modulating these symptoms. Therefore, it is critical to further understand the complex cellular changes associated with the pathology of this disease to develop successful therapies for these patients. Our lab has recently identified a novel role for CD8+ T cells in the pathology of GLD, however the mechanism underlying the recruitment and activation of these CD8+ T-cells is unknown. It is known that microgliosis is a prominent feature of GLD neuropathology and our preliminary data indicate that activated CD68+ microglia are anatomically clustered with CD8+ T-cells in demyelinated lesions in GLD. We also find that microglia upregulate major histocompatibility complex class I (MHC I) when exposed to psychosine. Together, these findings indicate that microglia are a plausible antigen presenting cell type contributing to CD8+ T-cell activation and neuropathology in GLD. We hypothesize that microglial MHC I expression contributes CD8+ T-cell activation in GLD. Accordingly, in this proposal, we will define and test the role of microglial MHC I on GLD neuropathology and CD8+ T-cells activation. In Aim 1, we will define the temporal and anatomical expression patterns of microglial MHC I expression in a GLD mouse model over the time course of disease. We will also use imaging mass cytometry to corroborate these findings using human GLD neurospecimens to translate our findings. In Aim 2, we will determine the role of microglial MHC I function to neuropathology and CD8+ T cell responses in twitcher mice using microglial cell-specific MHC I knockout mice in the GLD mouse model to investigate effects on disease course, neuropathology and CD8+ T-cell responses. We will also functionally examine the capability of psychosine to direct microglial MHC I antigen presentation and CD8+ T-cell activation. The long-term goal of this project is to understand the novel role of microglial antigen presentation in GLD neuropathology. The expected impact of this project may be a shift in our thinking on the nature of demyelination in this untreatable disease and the role of microglia in neuropathology. The training goals of this application will provide both skills and training in neuroimmunology and to enhance clinical, and professional goals directed toward my future as a physician scientist. Toward these goals, this highly translational project will take advantage of a strong intellectual environment and unique opportunities available at UConn School of Medicine and in the sponsor's lab. Collectively, the expected outcomes of this proposal will foster and support my career aspirations to achieve training in neurological and immunological interactions and to contribute to the field of neurological disease.

NIH Research Projects · FY 2026 · 2022-06

Summary: Stroke remains a leading cause of disability in the United States. Stroke is a heterogeneous multifactorial disorder. Prior attempts at developing new therapies have failed in clinics due to imperfect target validation, unrealistic therapeutic windows and lack of age appropriate models. Thus, there is an opportunity and a need to identify new medical treatment for stroke. The immune cells move to stroke area in the brain and contribute to damage. We have recently shown that the purinergic P2X4 receptor is excessively stimulated by adenosine triphosphate (ATP) released by dying brain cells during stroke. This receptor protein then causes activation of the immune cells and results in greater stroke injury. We have assembled the necessary tools such as animals lacking P2X4 receptors as well as blockers that selectively inhibit the receptor. The proposal, using these new tools, aims to investigate whether this receptor protein is a novel target to develop treatment for subjects with stroke. Our initial data suggest that blocking or deleting this receptor protein during the acute phase of stroke is beneficial. However, it is unknown how this protein works and what its potential adverse effects might be. Answering these questions is the overall goal here and should define the correct approach in pursuing a new stroke treatment

NIH Research Projects · FY 2026 · 2022-05

Abstract Cervical cancer is among the leading causes of cancer death in women worldwide, especially in low- and middle- income countries (LMICs). High-risk human papillomaviruses (HPV) are the main causative agents of cervical cancer and its precursor lesions; therefore, the World Health Organization (WHO) has recommended HPV DNA testing for cervical cancer screening in LMICs. Although polymerase chain reaction (PCR) methods have been widely used for HPV DNA detection, they are restricted to centralized clinical laboratories due to the need for labor-intensive procedures and expensive equipment. Here, we propose to develop a simple, rapid, highly sensitive, and specific CRISPR-on-paper diagnostic platform to simultaneously detect multiple high-risk HPV genotypes for cervical cancer screening at the point of care. This innovative diagnostic system is based on our recently developed all-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay, which combines the simplicity and high sensitivity of isothermal nucleic acid amplification with the high specificity of CRISPR detection. To develop a low-cost, multiplexed molecular detection technology, we will incorporate the AIOD-CRISPR assay into a paper-based microfluidics platform. To eliminate the need for complex electronic instruments, we will take advantage of an exothermic reaction to generate chemical heat for the CRISPR-on-paper system by using a disposable hand warmer, thus enabling instrument-free cervical cancer screening. The detection results can be read by the naked eye or reported by a programmed smartphone without the need for an expensive optical detector. We will rigorously evaluate and validate the clinical applications of our CRISPR-on-paper diagnostic system by testing clinical samples in collaboration with clinicians and healthcare workers in UConn Health and Zambia. If successful, the proposed project has an important impact on global health by providing a simple, affordable, and sensitive method for rapid screening of cervical cancer in resource-poor settings. As a platform technology, the proposed CRISPR-on-paper diagnostic system can be easily adapted to detect other emerging pathogens.

NIH Research Projects · FY 2025 · 2022-04

Summary Sickle cell disease (SCD) is a hemoglobinopathy associated with severe bone abnormalities including osteoporosis. Eighty percent of SCD adults have low bone mineral density (BMD) that is independent of risk factors such as age, gender, and menopausal status, suggesting the etiology of osteoporosis in SCD differs from the general population. Proposed contributing factors to bone loss in SCD include marrow hyperplasia secondary to chronic anemia, inflammation, ischemia, and vitamin D deficiency. However, the mechanisms of bone loss in SCD subjects has not been fully investigated, and there are no targeted therapies. Hormonal fibroblast growth factor 23 (FGF23), which controls phosphate homeostasis and has direct and indirect effects on bone mineralization, is reported to be increased in human anemia. Based on our exciting preliminary data showing that increased serum FGF23 and hypophosphatemia in humanized Townes SCD mice, which are anemic but not in renal failure, and that in vitro and in vivo FGF23 blockade partially rescues impaired mineralization and improved reduced BMD in SCD mice, we posit that cross-talk involving bone marrow erythropoiesis, kidney, and bone contributes to osteoporosis in SCD mice. Specifically, we posit that 1) sickling of red blood cells and the resulting anemia causes increased erythropoietin production by the kidney, which increases bone FGF23 production that impairs phosphate reabsorption; and 2) anemia-induced FGF23 results in impaired osteoblast differentiation, mineralization, and bone strength in SCD mice due to hypophosphatemia and pyrophosphate abnormalities via impaired sodium phosphate transporters PIT1 and PIT2 signaling in bone. Furthermore, increased FGF23 reduces PIT1 signaling that can interfere with erythrocyte differentiation, further perpetuating the anemic state. To test our hypotheses, we propose the following Specific Aims: Aim 1: Examine the molecular mechanisms by which FGF23 contributes to phosphate wasting in SCD Mice; Aim 2: Assess the molecular mechanism by which FGF23 contributes to impaired bone mineralization in SCD mice; and Aim 3: Determine whether FGF23 neutralizing antibody modulates the anemia phenotype of SCD mice. Our proposed studies may identify FGF23 as a novel contributor to the pathogenesis of bone loss and anemia in SCD mice. Since the FGF23Ab is now FDA approved for the treatment of X-linked hypophosphatemia, it may also be a useful therapy to prevent bone loss and improve anemia in human SCD in the future.

NIH Research Projects · FY 2025 · 2022-03

PROJECT SUMMARY Type 2 Diabetes (T2D) is a complex disease caused by both genetic and environmental factors. Genome-wide association studies (GWAS) have identified 403 association signals at 243 loci (T2D variants) that increase T2D genetic risk. Functional (epi)genomic analyses strongly suggest that non-coding T2D variants alter transcriptional regulation and target gene expression in pancreatic islets, but only ~20% of T2D variants elicit changes in islet cis-regulatory element (CRE) use or gene expression under steady state conditions. Environmental factors such as endoplasmic reticulum (ER) stress have been implicated in islet dysfunction. However, studies to date have not assessed if or how T2D variants modulate the response of islets to ER stress. I hypothesize that these T2D variants alter islet ER stress-responsive CRE use or activity and target gene expression to contribute to islet β cell dysfunction or death in T2D. In my preliminary data analysis, I have identified ER stress-responsive CREs that overlap 407 T2D variants in islets and connected these CREs to 22 putative target gene promoters using islet promoter capture Hi-C maps. In Aim 1, I will comprehensively assess the effects of T2D variants on the use or activity of ER stress-responsive CREs using chromatin accessibility quantitative trait locus (caQTL) and massively parallel reporter assays (MPRA), respectively. In Aim 2, I will determine if the putative CRE- targeted genes are required in human islet cells for their ER stress response and survival by altering their expression using CRISPR/Cas9 epigenomic editing platforms. Successful completion of these Aims will yield a functionally characterized set of T2D variants and a validated set of downstream 'T2D target genes’ that modulate islet function and survival in response to a central (patho)physiologic stressor. More broadly, I anticipate that the principles and framework I employ in this mechanistic variant-to-function project could be applied to characterize the effects of T2D variants in the context of other environmental stressors and metabolic tissues. Importantly, completion of this project will help me master current concepts and state-of-the-art techniques in genetics and functional genomics and increase my scientific communication skills through extensive opportunities to present and publish my studies. Finally, my position as an MD/PhD student at UConn Health and The Jackson Laboratory for Genomic Medicine will not only allow me to be mentored in a world- class, highly collaborative environment, but it will provide me with opportunities to continue honing my clinical skills and gain specialized experience during and after my research phase. Fulfilling my training and development plan will be a crucial step toward my future career as a physician-scientist studying the genetic and (epi)genomic mechanisms of disease-associated variants in patients.

NIH Research Projects · FY 2025 · 2021-09

Abstract This Administrative Supplement to KAPP-Sen Tissue Mapping Center (TMC) award (U54AG075941) funded by National Institute of Aging to leverage recent advances in the development of technologies used for the detection of senescence markers within tissue-specific extracellular vesicles (EVs). The PA-20-272 (Administrative Supplements to Existing NIH Grants and Cooperative Agreements) notes that this funding mechanism be used to cover cost increases that are associated with achieving certain new research objectives, as long as the research objectives are within the original scope of the peer-reviewed and approved project. Specific Aims of U54AG075941 include Aim 1: Coordinate research activities across KAPP-Sen TMC Collaborative sites in support of Sen-Net goals towards mapping cellular senescence and its associated secretory phenotype in the healthy human kidney, adipose tissues, pancreas and placenta. Aim 2: Obtain tissues from healthy kidney transplant donors (kidneys, fat, skin), C-section pregnancies (placenta, cord, fat, skin), outpatient healthy donor biopsies (fat, skin), beating heart brain dead donors (full pancreas) and IIPD/Prodo (dispersed pancreas). Aim 3: Generate highest-quality data pertaining to cellular senescence including scRNA- Seq, snRNA-Seq, spatial transcriptomics, immunohistochemistry and Telomere-associated DDR foci (TAFs) in all tissues. Aim 4: Perform high-level integrative data analysis required for the creation of aUases of human cellular senescence in collaboration with other TMCs, CODDC and NIH staff. Our Administrative Supplement proposal seeks to leverage ongoing work conducted by our KAPP- Sen TMC to create maps of senescent cells in Kidneys, Adipose tissues, Pancreas, and Placenta with advances involving new techniques designed to isolate, identify, characterize, and quantify tissue-specific EVs from blood and urine, combined with senescent biomarkers. Our Overarching Hypothesis raises the rather intriguing possibility that EV subsets and their protein cargo components may function as a liquid biopsy, thus permitting the non-invasive qualitative and quantitative evaluation of the senescent cell burden residing within the tissues from which those EVs likely originate. Furthermore, suppose future evidence was to support this postulate. In that case, such a development might permit clinicians to one day link peripheral blood- or urine- borne EV biomarker data to information residing in curated high-resolution senescent cell data obtained via SenNET.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Anti-PD-1 antibody has been approved to treat distinct cancers including hepatocellular cancer (HCC); however, the objective therapeutic response in human HCC patients is only about 14%. Microbes are now widely accepted to play critical roles in cancer pathology, and targeting them to improve cancer treatment is an active research area. To investigate the role of gut microbiota in HCC, a novel murine model was created which reflects the typical features in human disease and expresses SV40 T antigen (TAg) as a trackable tumor specific antigen (TSA). This model was used to study the influence of gut microbiota on HCC initiation and progression by treating pre- or post-malignant mice with an antibiotic cocktail (ABX) that contains three types of antibiotics. ABX treatment restored TSA CD8+ T-cell function, retarded hepatocarcinogenesis, and therapeutically slowed HCC growth. Metagenomic assay demonstrated ABX treatment mediated an enrichment of Bacteroides. Supplementation of Bacteroides thetaiotaomicron (B.th), one member of genus Bacteroides, acted similarly to ABX in suppressing tumor growth and activating anti-tumor immune response, associating with the intratumoral accumulation of CpG-rich genomic DNAs and increased expression of TLR9 in intratumoral dendritic cells (DCs) and macrophages (MΦs). In particular, complete gut sterilization of HCC-bearing mice with five types of antibiotics followed by B.th repopulation markedly improved the therapeutic efficacy of αPD1 Abs. Single cell RNA sequencing (scRNA-seq) revealed that B.th repopulation was associated with significant suppression of Kruppel-like factor 2 (KLF2) and significant increase of TLR9. Previous studies have demonstrated that KLF2 is a transcription factor which negatively controls expression of TLR9, phagocytosis in MФs and DCs, and function of T cells. Together, these results imply that B.th suppresses KLF2 expression, which abrogates its suppressive effect on TLR9, a pattern recognition receptor (PRR). The resultant increased TLR9 on sentinel MФs and DCs recognizes B.th-derived CpG-rich DNAs in tumors to activate TSA effector CD8+ T cells against HCC. Thus, this study will test the hypothesis that gut Bacteroides activate anti-HCC immune responses and improve anti-HCC immunotherapy by modulating immunological function of DCs and MФs via KLF2/TLR9/CpG molecular pathways. Aim 1 will dissect the molecular mechanisms by which B.th modulates DCs and MΦs to improve anti- HCC immunity and therapeutic effect through KLF2 and TLR9 pathways; aim 2 will dissect the cellular mechanisms by which B.th modulates DCs and MΦs to improve anti-HCC immunity and therapeutic effect; and aim 3 will investigate therapeutic and immune regulatory effect of gut microbiota in human HCC patient response to αPD-1 Ab treatment. Successful completion of the proposed studies will provide insight into the cellular and molecular mechanisms underlying B.th-activated anti-HCC immune response and advance gut microbiota- integrated immunotherapy to clinical application.

NIH Research Projects · FY 2024 · 2021-09

Abstract: The discovery of antibiotics in the early 20th century has transformed modern medicine; yet decades of use, overuse, and misuse have culminated in the rapid rise in pathogens that are refractory to our existing drugs. Antibiotic resistance is not the only reason for treatment failure. Within antibiotic-sensitive cultures, subpopulations of bacteria can transiently reprogram their phenotype, which enable them to survive lethal antibiotic doses. These bacterial persisters are thought to underlie recurrent and chronic infections, and they can fuel the development of resistance. Mounting evidence shows that environmental factors modulate phenotypic changes that lead to antibiotic persistence before, during, and after treatment. As such, achieving a deeper understanding of the interplay between environmental cues, bacterial phenotypic responses, and antibiotic susceptibility will improve our ability to devise more effective treatment regimens. When pathogens colonize and infect different parts of the host, they are often exposed to other pathogens and constituents of the host microbiome. The extent to which microbial interactions modulate a pathogen's phenotype and antibiotic persistence remains largely unexplored. Our overarching objective for this project is to systematically investigate the impact of microbiome constituents on Staphylococcus aureus's phenotypic response and persistence to antibiotics. To achieve this goal, we will develop a co-culturing phenotypic screen to identify bacterial strains and communities that impact S. aureus antibiotic persistence toward distinct classes of antibiotics. Using a combination of transcriptomics, metabolomics, and single-cell approaches, we will determine how these microbial interactions modulate S. aureus phenotypes (on the population- and single- cell level) before, during, and after antibiotic treatment. We will also use analytical techniques to identify molecular determinants that mediate microbial interactions responsible for the strongest effect on S. aureus persistence. We envision that the outcome of this project will expand our knowledge of the persister phenotype, contribute to the discovery of novel antimicrobial adjuvants, and guide the development of innovative treatment strategies to tackle chronic infections.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY (Overall section) The mission of the CPH-NEW Total Worker Health® (TWH) Center of Excellence is to be a national leader in research, policy, practice, and workforce development to achieve our vision of health, safety, and well-being for all working people. We will achieve this by: (1) conducting research on challenges, opportunities, strategies, benefits, and costs of integrated programs for workforce health, safety, and well-being; (2) disseminating cutting-edge TWH concepts, research findings, and program materials in multiple formats to advance workplace practice; (3) developing policy recommendations; (4) building the capacity of professionals and organizations to recognize the value of a TWH program and to strengthen the skills and organizational resources needed to achieve it; and (5) educating graduate students from multiple disciplines in TWH through their involvement in research and dissemination activities. Our proposed Center activities include two large intervention research projects, one smaller exploratory research project, an Outreach core for research translation and dissemination, and an Evaluation & Planning core to provide centralized administration and support. Our research aims to integrate program models of TWH to advance workforce health, safety, and well-being and will include: (1) a large intervention study to develop and evaluate TWH interventions designed to improve the mental well-being of teachers, with a focus on work-life balance, burnout, and work engagement (“Total Teacher Health”); (2) a large intervention study to develop and evaluate TWH interventions designed to promote the health and safety of healthcare workers, with a focus on sustainability and union involvement in implementation processes (the “SHIFT-II” study); and (3) a smaller exploratory study to develop and evaluate professional education programs designed to integrate TWH concepts into employer crisis preparation and planning efforts (the “TWH-ECP” study). Innovative aspects of our Center evaluation and planning methods include the use of cross-project methodological teams, a transdisciplinary framework for sharing knowledge, and a unique three-campus collaboration involving experts from engineering, medicine, epidemiology, public health, nursing, psychology, education, and management. Our experience shows that a worker-centered approach to TWH that involves workers in the design, implementation, and evaluation of interventions is an impactful, innovative, and efficient method for improving the safety, health, and well-being of the U.S. workforce. With this application, we propose to extend this model in new occupational settings and for new health challenges to better engage workers in organizational decision-making that advances worker well-being.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY OVERALL This proposal seeks to establish the Claude D Pepper Older Americans Independence Center at UConn (UConn OAIC) with the overarching goal of developing clinical strategies for enhancing function and independence in older adults that are more precise, personalized, and effective. Our focus on Precision Gerontology has emerged through the collective experiences and insights of our multidisciplinary team of accomplished investigators in the UConn Center on Aging – all united by a shared commitment to improving the lives of older adults through research, education, and clinical care. Our emphasis on Precision Gerontology has been guided by three principles: 1. A growing heterogeneity or variability over time among individuals involving all aspects of aging that therefore necessitates a multidisciplinary team-based approach to aging research; 2. Increased heterogeneity or variability with aging in older adults’ responses to treatment, highlighting the need for better targeting; and 3. Recognition of a need to incorporate these considerations into the design and testing of novel geroscience-guided therapies. These objectives will be achieved by establishing a UConn OAIC through the coordinated activities of a Leadership and Administration Core (LAC), Recruitment and Community Engagement Core (RC1), Data Resource Core (RC2), Biomarker and Preclinical Research Core (RC3), Pilot and Exploratory Studies Core (PESC), and Research Education Component (REC). We will seek to address these goals through the following aims: Aim 1: To develop and refine new approaches to the study and clinical translation of Precision Gerontology. Aim 2: To develop and evaluate new research methods, techniques, and collaborative approaches required to advance the goals of Precision Gerontology. Aim 3: To identify, develop, and learn from experiences of the next generation of researchers poised to become leaders in geriatrics research related to the UConn OAIC focus. Aim 4: To select and support innovative pilot studies that show promise in helping to guide the design, funding, and completion of more definitive future research studies related to the UConn OAIC focus.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT Notch receptors play a critical role in cell fate decisions and in the regulation of bone remodeling, either directly or through the induction of their target genes, namely Hairy Enhancer of Split (Hes) and Hes-related with YRPW motif (Hey). Hajdu Cheney Syndrome (HCS) is a devastating disease characterized by developmental abnormalities, acroosteolysis and bone loss with fractures. HCS is associated with mutations in exon 34 of NOTCH2 upstream of the PEST domain leading to NOTCH2 stabilization and gain-of-function. We created a mouse model of HCS (Notch2tm1.1Ecan) that presents with osteopenia due to enhanced osteoclastogenesis and bone resorption. These events are secondary to an increase in receptor activator of nuclear factor Kappa B ligand (RANKL) by cells of the osteoblast lineage, and to direct effects of NOTCH2 in cells of the myeloid lineage. In this lineage, the expression of HES1 is induced by NOTCH2 and the inactivation of Hes1 in the osteoclast lineage reverses the in vitro and in vivo phenotype of HCS mutants. Moreover, HES1 induces osteoclastogenesis directly and as a result causes osteopenia in vivo. This reveals a previously unrecognized function of HES1 in osteoclast differentiation and function that will be explored as part of the proposed research. An additional goal of the proposed work is to develop ways to correct the skeletal manifestations of HCS by targeting the mutation with Notch2 antisense oligonucleotides (ASO), a strategy that would be applicable to other genetic disorders of the skeleton. Our specific aims are: Aim 1) To determine the role of HES1 in osteoclastogenesis. Our goals are to induce and inactivate Hes1 specifically in cells of the osteoclast lineage to determine its contribution to osteoclast differentiation and bone remodeling as determined by microcomputed tomography and histomorphometry; Aim 2) To establish that the Notch2tm1.1Ecan mutation can be targeted. We will determine whether the Notch2tm1.1Ecan mutation can be downregulated specifically and the Notch2tm1.1Ecan skeletal phenotype ameliorated by the administration of antisense oligonucleotides targeting the Notch26955C>T mutation; and Aim 3) To validate the mechanisms of the HCS phenotype and ASO approach in NOTCH2 mutant-induced pluripotent (iPS) cells. To this end, we created NOTCH2HCS mutant iPS cell lines to study the impact of the mutation on osteoclastogenesis and the efficacy of ASOs in downregulating NOTCH2 mutant alleles. The goals of the proposed work are to understand the mechanisms and develop specific antisense technology to treat the skeletal manifestations of a devastating NOTCH2-associated disease.

NIH Research Projects · FY 2025 · 2021-07

We propose a novel NIAMS doctoral T32 Program, Regenerative Engineering of Musculoskeletal Tissues: A Convergence Doctoral Training Program, which offers interdisciplinary research areas at the University of Connecticut (UConn) combining biomedical science and engineering faculties. Regenerative Engineering is defined as the Convergence of advanced materials science, stem cell science, physics, developmental biology and clinical translation for the regeneration of complex tissues and organ systems. They will all receive their Ph.D. from the Graduate School at UConn. The T32 Program will offer trainees a broad level of expertise in research and instruction based on the research, educational, and clinical experiences of the biomedical and engineering faculty who serve as preceptors. Trainees will become experts in regenerative engineering and its foundations to work towards the alleviation of human disease and musculoskeletal injuries by means of tissue regeneration. Musculoskeletal regeneration is a field ripe for an inventive approach based on convergence to address challenging issues, advance technology and further fundamental knowledge for therapeutic applications. At the center of the Convergence approach is the understanding that new solutions in regeneration will take place through an ‘un-siloed’ approach. Thus, Regenerative Engineering welcomes ideas and research across a gamut of disciplines. The T32 Program has preceptorship commitments from 20 distinguished faculty at UConn (representing Biomedical Engineering, Cell Biology, Computer Science, Genetics and Genome Sciences, Materials Science, Mechanical Engineering, Molecular Biology and Biophysics, Oral Health, Orthopedic Surgery). This eminent group of investigators is well funded and published to provide the primary research training and serve as role models for doctoral trainees. The T32 Program goals are to educate, support and enhance the training of individuals dedicated to careers as independent clinical translational and basic scientists in regenerative engineering. Our Program strengths include its interdisciplinary and collaborative research in biomedical science and engineering, training in contemporary research methodologies, and successful preceptors. T32 Program administration through The Cato T. Laurencin Institute for Regenerative Engineering will provide the experience to recruit talented trainees, implement the curriculum, and train a new cadre of convergence scientists.

NIH Research Projects · FY 2025 · 2021-06

Although, we know much about the molecular signals that regulate OC function, we know relatively little about the lineage and mechanisms that OC use to develop from progenitors. The goal of this application is to better define OC progenitor (OCP) maturation and trafficking and the mechanisms regulating this process in health and disease so that we can identify potential drug targets to develop superior therapies for bone diseases. The central hypotheses are: 1) During homeostasis marrow-resident cells are the principal OCP source, while during inflammation or fracture repair, circulating cells become a significant source of OCP. 2) The mechanisms regulating OCP migration, engraftment and maturation to OC in bone differ between healthy and disease states. To test these hypotheses, we propose the following aims: 1. Define the role that CX3CR1+ OCP have in OC development during homeostasis and identify mechanisms regulating their homing, engraftment and maturation to OC: 1A) Perform time course studies in CX3CR1-CreERT2-Ai14 mice at various ages to examine labeled OC formation kinetics. We will also monitor the kinetics of labeled OCP in the bone marrow, blood and spleen. 1B) The chemokine receptor CX3CR1 is expressed on OCP and has previously been implicated to influence OCP homing, engraftment and maturation. We will determine its role in OCP lineage development and trafficking in vivo under homeostatic conditions (when marrow-derived cells predominate as the OCP source) using CX3CR1-CreERT2-Ai14 mice to generate Cx3cr1 gene deletions. 2. Examine OCP homing from the circulation during bone inflammation and fracture repair. These studies will examine two disease models in which we previously demonstrated that circulating OCP are recruited to engraft in bone: TNFa-induced bone inflammation and a repairing fracture. 2A) Study a TNFa-induced inflammatory bone model (a WT parabiont has TNFα injected over its calvaria; the other parabiont is a CX3CR1-EGFP; TRAP-tdTomato mouse) and determine the rate that circulating labeled cells home to the inflammatory site, form OC and disappear. 2B) Study a parabiosis fracture model (a WT parabiont receives a femur fracture; the other parabiont is a CX3CR1-EGFP; TRAP-tdTomato mouse) and determine the rate that circulating labeled cells home to the repairing callus, form OC and disappear. 2C) Determine the phenotype and kinetics of circulating OCP that home, mature and engraft in bone with TNFa-induced inflammation or fracture repair by injecting selected populations of OCP from CX3CR1-EGFP; TRAP-tdTomato mice and monitoring the rates that labeled OC appear and disappear in bone. 2D) Determine the effect that deletion of Cx3cr1 has on the ability of circulating OCP to home, engraft and mature in bone with TNFa-induced inflammation or a fracture repair using parabiosis and adoptive transfer.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract: IL-17 family cytokines promote inflammation that drives the development of colorectal neoplasia, which eventually lead to colorectal cancer (CRC). Thus far, the underlying mechanism has largely been attributed to IL-17’s role in myeloid cell recruitment. Whether IL-17 also signals to adaptive immune cells, in particular CD4+ regulatory T cells (Tregs), and whether this signaling plays a role in colorectal tumorigenesis, remains unknown. Our preliminary studies show that targeted ablation of IL-17 signaling on Treg cells increased colonic tumor development in mice, demonstrating a previously unknown protective role of IL-17 in CRC. We also found that IL-17 directly inhibits Treg accumulation in tumors. Further, IL-17 inhibits the expression of genes that facilitate Treg migration, proliferation, and immune suppressive function. Importantly, these effects are only observed in tumor-infiltrating Tregs, suggesting a site-specific inhibition of Tregs by IL-17. Consistent with this notion, only tumor-infiltrating Tregs express IL-17RC and RE – co-receptors for IL-17A, C, and F cytokines. Stimulation of Tregs with IL-6 and IL-1β, two cytokines that are abundant in the tumor environment, upregulates IL-17RE, suggesting that the tumor microenvironment renders Tregs susceptible to IL-17-mediated inhibition. On the other hand, we also found that IL-17 signals to tumor cells to downregulate the expression of CXCL9 and 10, which signal through their cognate receptor CXCR3 to attract CD8+ CTLs to the tumor. These preliminary findings support our hypothesis that IL-17 regulates colorectal tumor development through two opposing mechanisms: 1) IL-17 directly inhibits Tregs that would otherwise suppress cancer immunosurveillance; and 2) IL-17 inhibits the attraction of CD8+ CTLs into the tumor environment by downregulating CXCL9/10 production. Given the critical roles of both Tregs and Th17 cells in tumor development, along with the knowledge gap relating to the impact of IL-17 on Treg biology, we propose the following studies: 1) Delineate how IL-17 mediates direct inhibition of Tregs in colorectal tumors; 2) Elucidate the molecular mechanism(s) underlying IL-17-mediated inhibition of Tregs; and 3) Interrogate how IL-17 inhibits T cell attraction through the regulation of CXCL9/10, and test the importance of IL-17-Treg circuitry in colorectal tumor development and therapy. These investigations will provide new insights into the mechanisms by which IL-17 impacts colorectal tumorigenesis, as well as guide the invention and use of novel therapies for the treatment of CRC in humans. For example, based on a role for IL- 17 in inhibiting CD8+ CTL attraction to tumor, we may employ currently available IL-17A and IL-17RA antibodies as adjuvant agents for cancer immunotherapy. However, for tumors that are abundant with IL-17 and Tregs, neutralizing IL-17 may further enhance Treg’s immune suppression and worsen treatment outcome. Uncoupling IL-17-Treg interactions may also be important for the treatment of autoimmunity and bacterial infections, and will be explored in subsequent studies.

NIH Research Projects · FY 2026 · 2021-05

Endogenous glucocorticoids are critical for normal bone physiology. However, glucocorticoid excess, due to systemic administration or syndromes such as Cushing’s disease, causes osteopenia and metabolic disorder. Overall, glucocorticoid receptor signaling must be tightly controlled for optimal bone health. We identified microRNA-433 (miR-433) as negative regulator of glucocorticoid signaling in the osteoblast lineage. In vitro, inhibition of miR-433 activity made mesenchymal cells more responsive to glucocorticoids and increased glucocorticoid receptor residence in the nucleus, suggesting that miR-433 may target mechanisms designed to limit the responsiveness of cells to glucocorticoid signaling. In addition to effects on glucocorticoid signaling, miR-433 is a negative regulator of osteoblastic differentiation. miR-433 decreases as osteoblast differentiation progresses, and inhibition of miR-433 activity increases osteoblastic maker gene expression. To better understand the function of miR-433 in bone, we generated transgenic mice expressing a miR-433 competitive inhibitor (tough decoy) in osteoblastic cells. Calvarial bone from the miR-433 decoy mice has increased mRNA for osteocalcin and the direct miR-433 target Runx2. Further, miR-433 decoy mice display increased trabecular and cortical bone thickness due to increased bone formation, although molecular mechanisms remain to be identified. We propose to test the hypothesis that miR-433 targets genes and pathways critical for osteoblastogenesis and for limiting glucocorticoid receptor signaling. In Aim 1, we will comprehensively characterize the skeletal phenotype of both male and female miR-433 decoy mice in maturity and aging. We will also identify miR-433 targets using a non-biased approach, to better understand how miR-433 regulates osteoblast biology. In Aim 2, we will determine the mechanisms by which miR-433 limits glucocorticoid responsiveness at a molecular level, as well as determining the impact of miR- 433 on the response of bone to exogenous glucocorticoids excess. Overall Impact: miR-433 is a novel negative of both glucocorticoid responsiveness and osteoblast differentiation. Glucocorticoid excess is the most common secondary cause of osteopenia, and tissue sensitivity to glucocorticoids is regulated by multiple mechanisms. Understanding the interaction between osteoblastogenesis and glucocorticoid signaling is critical for the design of novel strategies to limit the adverse effects of glucocorticoid excess on the skeleton.

NIH Research Projects · FY 2025 · 2021-05

Cilia are microtubule-based cellular extensions that play key roles in sensing the extracellular environment, processing developmental signals and generating propulsive force and fluid flow. They also act as secretory organelles releasing bioactive vesicular ectosomes involved in cell-cell communication and other processes. Cilia are ancient and complex; in humans, ~5% of all genes are involved in their formation/activity and defects result in complex syndromes or ciliopathies. For many years, my laboratory has been broadly interested in the assembly and function of motile cilia, and has a strong record of identifying new opportunities and pursuing them to reveal novel aspects of ciliary biology – most recently we demonstrated that cilia act as a source of peptidergic signals. For most studies, we utilize the biciliate unicellular green alga Chlamydomonas as a model due to the ease of biochemical analysis and large array of molecular genetic approaches available. Over the next five years, we will pursue two broad areas of focus to address what I consider key questions in ciliary biology. Although superficially distinct, these two areas are intimately connected, and I anticipate we will be able to integrate them to yield novel insights into conserved and essential cilia-based pathways. 1) Ciliary Motility: dissecting the dynein motors and control systems that generate ciliary beating and power retrograde intraflagellar transport (IFT). We plan to focus on three major issues. We will dissect the complex pathways by which axonemal and IFT dyneins are synthesized and assembled in cytoplasm employing our newly devised biochemical fractionation methods. Building a cilium is an immensely complex problem in macromolecular assembly and we will examine how assembly factors control the axonemal incorporation of outer dynein arms at precise locations on doublet microtubules. We will also study axonemal dynein motor regulation to a) determine how responses to alterations in Ca2+ and redox poise are combined with curvature sensing to yield integrated changes in motility, and b) assess how cells sense imposed changes in ciliary beating and respond by increasing intraciliary levels of the dynein regulator Lis1. 2) Cilia Formation and Peptidergic Signaling: studying the peptide amidating enzyme (peptidylglycine - amidating monooxygenase; PAM) and its amidated bioactive products in ciliary assembly and cilia-based cell- cell communication. We recently demonstrated that active PAM occurs in cilia and that PAM loss leads to the failure of ciliogenesis and disrupts dynein-driven retrograde IFT. Furthermore, PAM-generated amidated bioactive products are released in cilia-derived vesicular ectosomes and one acts as a chemotactic modulator. We will build on these observations to identify novel amidated PAM products involved in cilia formation. We will dissect the pathways leading to regulated amidated product release in ciliary ectosomes and determine where/when processing of the precursors occurs. We will also pursue the amidated product receptors and their downstream signaling pathways, which lead to differential regulation of the two motile cilia and chemotaxis.

NIH Research Projects · FY 2025 · 2021-05

Project Summary The human diet can positively or negatively impact cancer incidence, with plant-derived compounds – such as polyphenols – often exhibiting antioxidant cancer-preventive properties. Walnuts are an exceptional source of polyphenolic ellagitannins (ETs) that are converted to ellagic acid and various urolithins by gut microbiota in the colon. Urolithin A (UroA) is of particular interest for its potent anti-cancer, anti-inflammatory, and prebiotic activities. However, UroA production in individuals can vary significantly, likely based on differences in gut microbiota. We will substantiate the anti-cancer benefits of a prebiotic/probiotic complex derived from consuming walnuts and determine the basis of human inter-individual variability in UroA formation. Our overall hypothesis is that walnut supplementation improves colonic health and lowers colorectal cancer (CRC) risk through UroA formation. This leads to several working hypotheses guiding our Aims: Working Hypothesis 1. UroA producers are at a lower risk of having an advanced colonic lesion; Working Hypothesis 2. Walnut supplementation will increase urinary UroA levels; and Working Hypothesis 3. CRC prevention by walnuts will be greater in UroA-producers than in non-producers. In Aim 1, we propose a randomized, controlled crossover trial in 69 patients (45-75 y) to examine walnut effects on CRC risk factors. We will associate an individual's ability to produce UroA with biomarkers of inflammation and CRC risk, and identify the bacterial species responsible for urolithin metabolism. In Aim 2, we will investigate prebiotic effects of ET-containing walnuts in two conditional mouse CRC models, focusing on important processes in CRC and inflammation, including bile acid metabolism, inflammation, and short chain fatty acid production. In Aim 3, we will test the probiotic effects of human UroA-producing microbiota in a mouse fecal microbiota transplant (MT) study and demonstrate a causal role for specific microbes in UroA formation. This will enable us to validate the concept that important protective effects of walnuts and other ET-rich foods occur through specific microbiota-derived metabolites. This will also define biomarkers and probiotics that highlight the benefits of these foods. Our approach incorporates personalized nutrition with a focus on UroA producers and non-producers in colonic health. Ultimately, our human and pre-clinical mouse studies may lead to prebiotics and probiotics that increase protective urolithins for CRC prevention. These highly significant studies will test the ability of the microbiota to generate colonic mucosa-protective agents (e.g., UroA). It is possible that high-risk patients can be efficiently converted to a protective state by taking probiotics to realize the full benefits of ET-rich foods.

NIH Research Projects · FY 2026 · 2021-04

ABSTRACT Emerging adults (EAs; ages 18-25) have higher rates of substance use disorders than any other age group and have been hit particularly hard by the opioid crisis. EAs also demonstrate poor adherence to healthcare regimens associated with substance use services, with higher dropout rates and lower service utilization than any other age group. This poor adherence leads to devastating outcomes, including continued substance use, incarceration, and overdose. In addition, high dropout rates contribute to skyrocketing costs to treatment systems as a result of more acute service needs, expensive service utilization, and long waitlists. Cost- effective strategies that are aimed at improving treatment adherence to substance use services and tailored to meet the unique developmental needs of EAs are an imminent need. Further, little is known about risk factors for dropout specific to this age group, hindering effective system responses to this significant problem. At the same time, substance use service systems are increasingly using peer recovery supports (PRS; i.e., paraprofessionals who have “lived experience” with substance use problems) to bolster treatment outcomes without incurring considerable additional costs. However, services delivered by PRS have not been tailored specifically to reduce EA dropout, and few have been rigorously tested at all. The current study will evaluate an innovative EA-specific dropout prevention enhancement to usual treatment services, delivered by PRS in community-based substance use treatment clinics (Aim 1). We will employ a stepped-wedge cluster randomized design, resulting in each clinic having a longitudinal usual services phase and a longitudinal dropout prevention phase. The two phases will be compared on rates of EA dropout and service utilization using objective data from clinical charts. We will also evaluate cost-effectiveness and employ a qualitative approach to understanding the varied financial factors that influence potential sustainability of such a dropout prevention enhancement (Aim 2). In addition, we will leverage the stepped-wedge design to investigate factors purported to predict EA dropout from substance use services and preliminarily investigate whether factors moderate dropout prevention (Aim 3). These key variables include executive functioning, identity formation, motivation, substance use severity, comorbid mental health symptoms, social support, and treatment-related cognitions. In particular, this study will be the first to use a comprehensive assessment of executive functioning, including event-related potential and behavioral data collected during computerized tasks, as a predictor of dropout from substance use services. Results will greatly advance our knowledge of EA dropout and a potential enhancement specifically aimed at reducing EA dropout, which has high potential to be cost-effective and easily disseminated. Answering these key questions is a crucial next step in improving patient adherence for EAs in substance use services and, ultimately, promoting positive outcomes for this high-risk group.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Lynch syndrome (LS) is a hereditary disease that predisposes patients to colorectal, endometrial, ovarian and other cancers. LS is caused by inherited mutations in the DNA mismatch repair (MMR) genes, defects in which also underlie 15-30% of sporadic colorectal cancers. Loss of MMR function is associated with a 1,000-fold increase in mutation rate likely increasing the risk of mutation to important oncogenes and tumor suppressors. The MMR pathway also activates cell cycle checkpoints and cell death in response to exogenous DNA damage, however, the role of this damage response in preventing tumorigenesis is not known. We hypothesize that colonic stem cells (CSCs) that lose this MMR-dependent damage response will gain a selective advantage over neighboring MMR-proficient cells, particularly in a mutagenic environment such as may be found in the colon. We predict that loss of MMR will enhance survival under conditions of increased DNA damage, favoring these cells in a competition for stem cell niche occupancy. Ultimately, this will lead to the production of more hypermutable intestinal cells, increasing the penetrance of the cancer phenotype. Testing this hypothesis has been problematic previously due to lack of a suitable model system. However, the recent development of human colonic organoid and enteroid models now allow us to study the effects of MMR loss on CSC dynamics via the following aims: 1) Determine whether loss of MMR function in human embryonic stem cells (hESCs) leads to an immediate advantage in the absence or presence of exogenous DNA damaging agents. We will use CRISPR/Cas9-mediated gene editing to knock out the MMR genes in hESCs and examine their growth and damage response as well as determine the mechanism underlying those responses. 2) Determine whether MMR-deficient CSCs have a selective advantage in colonic organoids and colonic enteroids. For this purpose, we will differentiate MMR-proficient and deficient hESCs into colonic organoids. As a complementary model, we will also create MMR knock out enteroids from human adult colon tissue samples. Using both systems, we will compare the response to exogenous DNA damaging agents or oncogenic stress. We will also create mixed organoids containing MMR-proficient and deficient cells and test whether the MMR-deficient cells have a growth or survival advantage over time in the presence or absence of DNA damage. 3) Determine whether MMR loss leads to a selective advantage for CSCs in vivo. We will use an inducible, stem cell specific knockout mouse model of Msh2 to create mosaic intestinal crypts and test whether MMR-deficient CSCs outcompete wild-type CSCs. Together, these aims utilize novel approaches to study the mechanism by which loss of MMR function contributes to tumorigenesis providing information that may help explain disease penetrance while guiding the diagnosis, prevention and treatment of LS-associated cancers.

NIH Research Projects · FY 2025 · 2021-04

SUMMARY Abnormalities in cerebellar development, especially pathology and dysfunction of Purkinje cells, have been implicated in a wide variety of neurodevelopmental diseases, including ataxia, autism spectrum disorder, schizophrenia, and language impairment. Being one of the earliest-born cerebellar cell groups, Purkinje cells are believed instrumental in the development, function, and pathogenesis of the cerebellum. Evidence suggests the existence of Purkinje cell subtypes with distinct molecular features. However, the molecular mechanisms underlying the diversification of Purkinje cells remain poorly understood. Consequently, we lack an entry to assess the role of individual Purkinje cell subtypes. Through single-cell RNA and chromatin accessibility analyses, we uncovered at least nine molecularly distinct subtypes of Purkinje cells in the developing mouse cerebellum. These Purkinje cell subtypes contribute to different compartments in the developing cerebellum. Remarkably, the Purkinje cell subtypes display a characteristic combinatorial expression of Foxp1, Foxp2, and Foxp4, which belong to a subgroup of the forkhead-box transcription factor family. Mutations of human FOXP1 or FOXP2 are linked to speech disorders, autism spectrum disorder, and intellectual disability, indicating that these proteins coordinate the development of the neural circuits related to cognitive diseases. In vitro evidence shows that FoxP proteins form dimers or oligomers with variable transcriptional targets and actives depending on the binding partner. We hypothesize that Foxp1/2/4 form combinatorial “FoxP codes” to specify distinct Purkinje cell subtypes, which in turn control the morphogenesis of the cerebellum. Aim 1 will combine conventional expression analysis, spatial transcriptomics, and volume imaging to determine the development of PC subtypes in relation to the morphogenesis of the cerebellum. Aim 2 will delete Foxp1/2/4, individually and in combinations, from the mouse cerebellum. We will use histology, single-cell RNA-seq, and behavioral studies to evaluate the impacts of single and compound Foxp1/2/4 mutations on cerebellar development and behavioral function. Aim 3 will use a multi- omic approach to study the molecular mechanism by which combinatorial FoxP genes regulate the transcription program for Purkinje cell differentiation. At the completion of this project, we expect to have identified the individual and combinatorial roles of Foxp1/2/4 in cerebellar development. This study will have a significant positive impact, not only on the basic knowledge of cerebellar development but also on the understanding of the molecular basis of the vast number of unexplored cerebellum-related diseases.